Citation
Toxicity and hormonal activity in leachates from municipal solid waste (msw) landfills in Florida

Material Information

Title:
Toxicity and hormonal activity in leachates from municipal solid waste (msw) landfills in Florida
Creator:
Ward, Marnie Lynn
Publication Date:
Language:
English
Physical Description:
xx, 288 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Alkalinity ( jstor )
Ammonia ( jstor )
Chemicals ( jstor )
Hardness ( jstor )
Heavy metals ( jstor )
Landfills ( jstor )
Phthalates ( jstor )
Toxicity ( jstor )
Toxicology ( jstor )
Yeasts ( jstor )
Dissertations, Academic -- Environmental Engineering Sciences -- UF ( lcsh )
Environmental Engineering Sciences thesis, Ph.D ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references.
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by Marnie Lynn Ward.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030590868 ( ALEPH )
880438737 ( OCLC )

Downloads

This item has the following downloads:


Full Text










TOXICITY AND HORMONAL ACTIVITY IN MUNICIPAL SOLID WASTE (MSW)
LEACHATES FROM FLORIDA LANDFILLS













By

MARNIE LYNN WARD


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2003






























Copyright 2003

by

Marnie Ward






























This dissertation is dedicated to my family. To my husband, Bill, and my
daughter, Diana Mary, whose love and support fortified me on many long days
and nights. To my parents Nanny and Poppy, my sister Lisa, my brother
Jonathan and my nieces; Deonna, Megan, and Rebecca, for their presence and
guidance in my life.














ACKNOWLEDGMENTS

I wish to thank Dr. Gabriel Bitton for his guidance, patience, humor, and

unwavering steadfastness during the past eight years. He has inspired me to

investigate new ideas, question my theories, and to always ask why. I have been

awed by his wealth of knowledge and experience, while simultaneously humbled

by his understated persona. Dr. Bitton has been my mentor, my teacher, and a

model of all that I could hope to become. He continues to exemplify the strong

values and principles that are the foundation of graduate education.

Special thanks are extended to members of my doctoral committee for their

time and interest in my education and academic growth. Dr. Timothy Townsend

has been a continuous source of support and guidance. Dr. Matthew Booth

provided assistance with analytical questions and performed GC/MS analysis.

Dr. Angela Lindner maintained an "open-door" policy and was always interested

to hear updates on my research. Dr. Nancy Denslow made available

opportunities for further study and has been a reference for many research

questions.

I also extend my gratitude to my fellow students in the Department of

Environmental Engineering Sciences; both past and present. They were always

a source of support and inspiration to me, through many long nights in the lab

and even during times of personal crisis. I specifically wish to recognize the








following students; Kristin Stook, Roi Dagan, Thabet Toylamet, Diana Lee, Libby

Schmidt, Jenna Jambeck, Pradeep, Dubey, Jeff, Jenn, Tracy, and Marissa.

My heartfelt appreciation is extended to my dear friend and personal

mentor, Linda Tyson. Her house became my second home, which was a

welcome respite during my qualifying exams. I first met Linda in the early 1990s

when I was a student at Central Florida Community College and she was an

instructor. It was after listening to her talk about her graduate work that I became

inspired to attend the University of Florida. She has been an unwavering source

of inspiration and wisdom.

I owe a deep appreciation to Peter Meyers and Craig Watts at Hydrosphere

Research. They provided the starter cultures of Ceriodaphnia dubia, Daphnia

pulex, and Pseudokirchneriella subcapitata, which were subcultured to supply in-

house test organisms. Peter Meyers was a reliable and experienced source for

information concerning techniques and methods for culturing aquatic test

organisms and for conducting acute and chronic assays.

I also extend appreciation to the operators of the landfills at which the

leachates were collected. I specifically thank Jim Brunswick for his friendship

and guidance.














TABLE OF CONTENTS
Paae

ACKNOW LEDGM ENTS ................................................... ............................. iv

LIST O F TABLES................................. ..................................................... .... x

LIST O F FIG URES ................................... ....... ............................. xiv

A B ST R A C T ......................................................................... ...................... xviii

CHAPTER

1 INTRODUCTION ..................................... ......................... 1

2 LITERATURE REVIEW ............................ .................. ...................... 6

M SW Landfills ............................................................. ......... ............ 6
Modern MSW Landfills ......................................... ................................ 6
Characterization of the Chemical and Physical Composition of MSW
Landfill Leachates............................. ................. ...................... 9
Bioassays for the Evaluation of Toxicity in the Environment ...................... 14
Toxicity of MSW Landfill Leachates...................... ....................... 16
Hormonally Active Agents in the Environment......................................21
Phytoestrogens.................................................................. 23
Phthalates ..................... .................................... 24
Alkylphenols.......................... .......... ...................... 26
Natural and Synthetic Estrogens......................................................... 28
Effects of Hormonally Active Compounds on Humans.......................31
Bioassays to Identify Hormonal Activity ............................................... 33
In vivo assays for the determination of hormonal activity ................36
In vitro assays for the determination of hormonal activity ................37
A test battery to determine hormonal activity.................................43
Characterizing Hormonal Activity in MSW Landfill Leachates and Other
Environmental Samples................................................................44

3 TOXICITY OF LEACHATES FROM FLORIDA MUNICIPAL SOLID WASTE
(MSW) LANDFILLS USING A BATTERY OF TESTS APPROACH..............54

Introduction...................... ..... ..........................................................54
Materials and Methods ................... .... ..............................57
Leachate Collection ............................................ 57








Chemical and Physical Characterization of Leachates ........................58
Maintenance of Test Organisms ............................................58
Pseudokirchneriella subcapitata..........................................58
Ceriodaphnia dubia and Daphnia pulex................................. ..61
Preparation of aquatic invertebrate food........................................ 62
Maintenance of aquatic invertebrate cultures..................................63
Toxicity Assays ................................... .... ..................... 65
Pseudokirchneriella subcapitata..........................................65
Ceriodaphnia dubia and Daphnia pulex................................. ..67
M icrotoxT .............. ......................................................70
Data A analysis ..................................................... ........................ 72
Results and Discussion .............................................................................74
Chemical Analysis of MSW Leachates .......................................74
Toxicity of MSW Landfill Leachates .............................. ........... 81
Regression Analysis ........................................... .......................88
Ammonia Toxicity........................................................... ..................... 92
Influence of Site-Specific Factors on Leachate Toxicity ......................... 93

4 A SURVEY TO ASSESS THE ACUTE AND CHRONIC TOXICITY OF
LEACHATES FROM MSW LANDFILLS IN FLORIDA: ................................95

Introd auction ................................................................ ............................. 9 5
Materials and Methods ................... ................................98
Sampling Sites ................................... ...... ....................... 98
Collection of MSW Landfill Leachates....................................... 100
Chemical and Physical Characterization of MSW Leachates............... 101
Toxicity Assays .................................................. ...................... 101
Data Analysis............................................ 103
Results and Discussion .......................................................................... 104
Chemical and Physical Characteristics of the MSW Leachates........... 104
Toxicity of MSW Landfill Leachates .................................................. 117
Toxicity of MSW leachates to aquatic invertebrates...................... 117
Toxicity of MSW leachates with algae........................................... 121
Toxicity of MSW landfill leachates with Microtox ......................... 125
Heavy metal toxicity of MSW leachates using MetPLATE .............126
NOEC/LOEC vs. ECso or TU results........................................... 126
Monitoring MSW landfill leachate toxicity over time..................... 130
Comparative sensitivity of the bioassays..................................... 134
Relationship Between Chemical/Physical Leachate Characteristics and
Leachate Toxicity.................................. .............. 138

5 HEAVY METAL BINDING CAPACITY (HMBC) OF MUNICIPAL SOLID
W ASTE LANDFILL LEACHATES............................................................... 140

Introduction............................ ................................. ............. 140
Materials and Methods .......................................................... 143
Sam ple Sites..................................... ............... 143








Leachate Collection .......................... ............. ................... 143
Chemicals and Reagents.... ....... .......................................... 144
Chemical Analysis..........................................................................144
Determination of Heavy Metal Toxicity.............................................. 146
Determination of HMBC .................................................................... 146
Influence of Some Leachate Parameters on HMBC..........................149
Data Analysis.......................................................... 150
Results and Discussion .................................... ....................... 151
Heavy Metal Toxicity of Landfill Leachates........................................ 151
HMBC of MSW Landfill Leachates..................... .......... 157
Leachate toxicity as a function of time .............................................. 163
Influence of Selected Leachate Parameters on HMBC...................... 164

6 IDENTIFYING TOXICITY IN FLORIDA MSW LANDFILL LEACHATES WITH
A TOXICITY IDENTIFICATION AND EVALUATION (TIE) PROCEDURE .179

Introduction..................................... ..... 179
Material and Methods............................ .... ........................182
Sam ple C collection .......................................... ...... ..........................182
Chemical Analysis of MSW Landfill Leachates.................................... 183
TIE Procedure: Phase I..................................................................... 185
pH-adjustment of the MSW landfill leachates.............................. 185
Filtration of the MSW landfill leachates......................................... 186
Solid phase extraction of the MSW landfill leachates.................... 186
Aeration of the MSW landfill leachates .......................................... 187
Blank preparation ...................................................... 188
Zeolite test............................. ......................... 189
Toxicity Assays ................. ......................... ... ........................ 189
Initial toxicity assays...................... .......... ...................... 189
Baseline toxicity assays............................ ........................ 191
Post-manipulation toxicity assays............................................... 192
Data Analysis .... ..................................................... 192
Results and Discussion ............................................... 194
Chemical/Physical Characterization..................................................... 194
Determination of Heavy Metal Bioavailability ....................................... 195
Initial Toxicity ............................................................. 196
Blanks and Controls........................... .... ....................... 197
Baseline Toxicity .................................... ........................ 197
Effect of Zeolite Treatment................................ ............. 197
Post-manipulation Toxicity ..................................................................199
Site 7 ............................................................ 199
Site 8 ..................... .... ....... ...................... 202
Site 14 .................... ................................... 206






viii








7 HORMONAL ACTIVITY OF MUNICIPAL SOLID WASTE (MSW)
LEACHATES FROM FLORIDA LANDFILLS ..............................................209

Introduction ...................................... ...... 209
Materials and Methods .........................................................211
C hem icals ............................................ .......... ..................... 211
MSW Landfills and Leachate Collection.............................. ..........212
MSW Landfill Leachate Treatment Facility..........................................212
Solid Phase Extraction (SPE) of MSW Landfill Leachates................... 214
YES Assay for Determining Hormonal Activity.............................. 215
Toxicity of MSW Leachates to Yeast Cells...........................................217
GC/MS Analysis........ ...... ........................218
R results .............................................. ................ ........................ 2 19
Hormonal Activity of MSW Landfill Leachates....... ..................219
Effect of Biological Treatment on Hormonal Activity ..........................224
GC/MS Analysis of MSW Landfill Leachates ......................... ...........226
Influence of Concentration Factor on Hormonal Activity of Leachates.229
E2 Recovery in Spiked Methanol Extracts of Leachates....................232
Interpretation of GC/MS Results with the Hormonal Activity of MSW
Landfill Leachates...................................................234
Isolation of Hormonal Activity at LF 12................................. ... ..241
Issues Raised When Analyzing MSW Landfill Leachates for Hormonal
Activity .............................................................. ........ 242
Toxicity of MSW leachates to yeast cells.................................243
Coliform bacteria ................................... .............. .............. 246
CPRG activity issue............................. ... ..................248
Assessment of organic solvents ................... .......... ..........249

8 CONCLUSIONS ............................................. ....... 250

LIST OF REFERENCES.............................. ..... ..... .............253

BIOGRAPHICAL SKETCH....................... ..... .................. 287














LIST OF TABLES


Table page

1-1. Frequently used acronyms........................... .... .. .................... 2

2-1. Range of selected chemical and physical characteristics reported
in the literature for domestic wastewater and MSW landfill
leachates in Florida and internationally ........................................ ..... 10

2-2. Toxicity of individual constituents identified in MSW landfill leachates .......13

2-3. Reported toxicity in the literature for MSW landfill leachates.................... 17

2-4. Select phthalate compounds and their common usage ............................24

2-5. Concentrations of nonylphenols in food items in Germany.......................26

2-6. Rates for the urinary excretion of natural estrogens from men
and women.............................. ......... ... ......................29

2-7. Advantages and disadvantages associated with the use of in vivo and
in vitro assays for identifying hormonal activity ........................................ 33

2-8. In vivo assays for the determination of hormonal activity.........................34

2-9. Threshold dose for the induction of hormonal effects following
exposure of fish to natural and synthetic estrogens .................................35

2-10. In vitro assays for the determination of hormonal activity .......................37

2-11. Relative sensitivity of in vitro assays to 17 p-estradiol (E2) ....................... 38

2-12. The hormonal activity of selected metal species.......................................46

2-13. Reported phthalate concentrations in landfill leachates..........................47

2-14. Concentrations (ng/L) of natural and synthetic hormones in
wastewater treatment plants(WWTPs)...........................................49

2-15. Reported concentrations (ng/L) of natural and synthetic
estrogens in surface waters ........................ ...........................52








3-1. Amount of MSW generated and landfilled at six landfill
sites in Florida .................... .................................57

3-2. Components of the preliminary algal assay procedure (PAAP)
m edium .............. .................................... ........................ .......... 59

3-3. Physical and chemical characteristics of MSW landfill leachates
at six sites in Florida............................................. ................................ 73

3-4. Correlative analysis with the C. dubia, P. subcapitata, and MicrotoxTm
assay results versus leachate chemical characteristics ...........................87

4-1. Description of 14 MSW landfill sites where leachates were
collected .......................................................................... .......... ........ 99

4-2. Physical and chemical characteristics of MSW leachates collected
from 14 lined landfills in Florida............................................................. 107

4-3. Distribution of major ions in leachates from 14 lined MSW
landfills in Florida.......................... ...................... ..................... 110

4-4. Mean concentrations (mg/L) of total (NH4/NH3) and un-ionized (NH3)
ammonia in leachates from fourteen MSW landfills in Florida................113

4-5. Metal concentrations in leachates from fourteen MSW landfills
in Florida ............................................................... ................... 114

4-6. Toxicity of leachates collected from 14 lined MSW landfills with
C. dubia, D. pulex, and P. subcapitata.................................................. 120

4-7. Toxicity of the MSW landfill leachates from 14 sites in Florida
using the 15-minute Microtox acute assay ........................................... 124

4-8. Relationship between the toxic endpoints of ICso (%),NOEC (%)
and LOEC (%) with the results of the P. subcapitata assay
with leachate from site 1............................ ............................... 127

4-9. Coefficients of variation (CV)(%) for the P. subcapitata, C. dubia,
and D. pulex assays ....................... ...................................... 35

4-10. Classification system for ranking the toxicity of MSW landfill
leachates from 16 sites in Florida................................... ..................... 137

5-1. ECs5for Cu+2, Zn+2, and Hg*2 determined with the MetPLATE
assay ................................................................ ............................ 15 1

5-2. Toxicity of leachates from 16 lined MSW landfills using
MetPLATE............................................................................... ....... ... .....152








5-3. Physical and chemical characteristics of leachates collected
from 16 lined MSW landfills in Florida. .................................... ....... 154

5-4. Heavy metal binding capacity (HMBC) (unitless) of leachates
from 16 MSW landfills with copper, zinc, and mercury ....................... 158

5-5. MetPLATE and HMBC results with MSW landfill leachates
collected from sites 1, 4, 5, and 8........................... ...... ................ 164

5-6. HMBC of MSW landfill leachates with copper, zinc, and mercury
following fractionation.............................................................................. 165

5-7. Changes in physical and chemical characteristics during fractionation
of the MSW landfill leachates from site 1, 4, 5, and 8............................... 166

5-8. Coefficients of determination (R2) obtained between MSW landfill
leachate characteristics and the heavy metal binding capacity
(HMBC) for copper, mercury, and zinc.................................................... 175

6-1. Population served and amount of waste landfilled, as a percent
of total waste generated, at sites 7, 8, and 14.......................................... 182

6-2. Manipulations to identify suspected toxicants...........................................184

6-3. Chemical and physical characteristics of the MSW landfill leachates
from sites 7, 8, and 14........................................................ 193

6-4. The initial (day 1) and baseline (day 2) acute and chronic toxicity
of the whole MSW landfill leachates from sites 7, 8, and 14 prior
to fractionation.......................................... ........................................ 196

6-5. Ammonia concentrations in MSW landfill leachates before and after
treatment on a Zeolite cation exchange column....................................... 198

6-6. Acute toxicity of the whole and post-Zeolite MSW landfill
leachates to C. dubia neonates............................................................... 199

6-7. Summary of TIE results with MSW landfill leachates from
sites 7, 8, and 14 ......................................................... ........... ........ 208

7-1. Hormonal activity of raw MSW landfill leachates and their
methanol extracts............................................................. 220

7-2. Hormonal activity of raw MSW landfill leachates from LF 8
before (influent) and after (effluent) treatment in a powdered
activated carbon treatment (PACT) facility............................................ 224








7-3. Organic compounds tentatively identified in MSW landfill leachates
by GC/MS analysis in full scan mode................................ ..........226

7-4. Recovery (%) of 17 p-estradiol (E2) from the E2 spiked methanol
extracts of MSW landfill leachates....................... ............................232

7-5. Categories of hormonal activity in MSW landfill leachates......................234

7-6. Presence of hormonal activity in the raw leachates and methanol
extracts of MSW landfill leachates with identified hormonally active
compounds in parenthesis .................................................235

7-7. Effect of extraction procedures on the hormonal activity of leachates
from LF 12 (March 2002).......................... ............................240

7-8. Total and fecal coliform bacteria determined in MSW landfill
leachates with results expressed as the most probable
number (MPN) of bacteria/100 ml of leachate........................................ 247














LIST OF FIGURES


Figure page

1-1. Pathways for the characterization of the biological effects of MSW
landfill leachates............... ............... ...................... 3

2-1. Representation of the vertebrate endocrine system and the possible
influences of hormonally active compounds on various system
and organs .................... ..... ... .... ...................32

3-1. Locations of the MSW landfills for the collection of leachates
in Florida ................................................................. ....................... 56

3-2. Flowchart for the P. subcapitata assay .................... ......................64

3-3. Flowchart for the C. dubia assay ...................... .........................67

3-4. Flowchart for the MicrotoxT assay .............................................................69

3-5. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NHa) (diamonds) in MSW landfill leachates from site 1 over a 6-month
sampling interval ................................................... 76

3-6. Concentrations of total (NH4+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 2 over a 6-month
sam pling interval ................................................. .......................... ...... 77

3-7. Concentrations of total (NH4'+NH3) (bars) and un-ionized ammonia
(NHa) (diamonds) in MSW landfill leachates from site 3 over a 6-month
sampling interval ..................... ................................... 78

3-8. Concentrations of total (NH4'+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 4 over a 6-month
sampling interval ..................... ............................. 79

3-9. Concentrations of total (NH4'+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 5 over a 6-month
sam pling interval ........................ ... ................. ..................... 80








3-10. Concentrations of total (NH4+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 6 over a 6-month
sam pling interval .............................................. ... .................................81

3-11. The mean toxicity of MSW leachates collected from six
landfill sites.................................................... .......................................82

3-12. Toxicity of MSW landfill leachates from site 1 over time......................... 82

3-13. Toxicity of MSW landfill leachates from site 2 over time........................... 83

3-14. Toxicity of MSW landfill leachates from site 3 over time......................... 83

3-15. Toxicity of MSW landfill leachates from site 4 over time .........................85

3-16. Toxicity of MSW landfill leachates from site 5 over time........................... 85

3-17. Toxicity of MSW landfill leachates from site 6 over time ...........................86

3-18. Relationship between the P. subcapitata (EC5o) and C. dubia (EC5a)
assay results with MSW landfill leachates.................................................89

3-19. Toxicity fluctuations in the leachates collected from the MSW
landfill at site 5 during February 2000 ...................... .......................90

4-1. Locations of the MSW landfills for the collection of leachates
in Florida .................................................................... .................. 98

4-2. Acute (48-hr C. dubia) toxicity of MSW landfill leachates collected
from 14 landfill sites in Florida.......................................... 116

4-3. Correlation between the 48-hour acute toxicity assays using
C. dubia and D. pulex assays with MSW leachates collected
from 14 landfill sites in Florida............................................................ 118

4-4. Chronic (96-hour P. subcapitata) toxicity of MSW landfill leachates
collected from 14 landfill sites in Florida.......................................121

4-5. Correlation between the results of the standard (125-ml) and
modified (25-ml) P. subcapitata chronic 96-hour assays.........................122

4-6. Influence of time on A.) conductivity, B.) chemical oxygen demand,
C.) total organic carbon of the MSW landfill leachates from site 1 ...........128

4-7. Influence of time on A.) conductivity, B.) chemical oxygen demand,
C.) total organic carbon of the MSW landfill leachates from site 5 ...........129








4-8. Acute (C. dubia and Microtox) and chronic (P. subcapitata) toxicity
of MSW landfill leachates collected from site 1 between February
2000 and May 2001 ........................................................... 132

4-9. Acute (C. dubia and Microtox) and chronic (P. subcapitata) toxicity
of MSW landfill leachates collected from site 5 between February
2000 and March 2001 ........................................................132

4-10. Relationship between the results of the chronic 96-hour P. subcapitata
(IC5o) and the acute 48-hour C. dubia (LCso) assays with MSW
landfill leachates from fourteen sites in Florida ...................................... 134

4-11. Ranking of sixteen MSW leachates with the results of the
Microtox (MT), P. subcapitata (P. sub), D. pulex (D. p.),
and Ceriodaphnia dubia (C.d.) assays.................................................. 138

5-1. The MetPLATE assay protocol for determining the heavy metal
toxicity of MSW landfill leachates.................................... ................... 145

5-2. The protocol for determining HMBC of MSW landfill leachates ..............147

5-3. The protocol used for fractionation of HMBC........................................ 149

5-4. MetPLATE results for MSW landfill leachates collected from site 1
and site 5 over time ................................ ... ... .................. 163

5-5. Effect of leachate treatment by filtration (Solids), DEAE resin
(Organics), and Dowex resin (Hardness) on the HMBC......................... 177

6-1. Results of the Phase 1 toxicity fractionation with the 24-hour
C. dubia assay for MSW landfill leachates collected from site 7 ............200

6-2. Results of the Phase 1 toxicity fractionation with the MicrotoxT
assay for MSW landfill leachates collected from site 7............................. 201

6-3. Results of the Phase 1 toxicity fractionation with the 24-hour
C. dubia assay for MSW landfill leachates collected from site 8 ............202

6-4. Results of the Phase 1 toxicity fractionation with the MicrotoxT
assay for MSW landfill leachates collected from site 8...........................204

6-5. Results of the Phase 1 toxicity fractionation with the 24-hour
C. dubia assay for MSW landfill leachates collected from site 14 ............205

6-6. Results of the Phase 1 toxicity fractionation with the MicrotoxT
assay for MSW landfill leachates collected from site 14........................... 207








7-1. Procedure for preparing MSW leachates and methanol extracts
of leachates for analysis of hormonal activity ......................................... 213

7-2. Response of the YES assay to 17-p estradiol (E2) ...................................219

7-3. Total ion chromatogram of MSW leachates from LF 8 (Feb. '02#1)
in the full scan mode ..................................... ... ..................... 228

7-4. Dose-response of the LF 1 (Nov. '01) methanol extracts versus
concentration factor................................................. ........ .................. 230

7-5. Dose-response of the LF 8 (Dec. '01) methanol extracts versus
concentration factor................................ ..................... 231

7-6. Dose-response of the LF 12 (Nov. '01) methanol extracts versus
concentration factor...................... .... .. .......................231

7-7. The solid phase extraction protocol used with MSW landfill
leachates........................ ......... ... ....................... 241

7-8. The toxicity of MSW landfill leachates to yeast cells according
to the INT procedure .............................................................................244














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

AN INVESTIGATION OF TOXICITY AND HORMONAL ACTIVITY IN
LEACHATES FROM MUNICIPAL SOLID WASTE (MSW) LANDFILLS IN
FLORIDA

By

Marnie Lynn Ward

December, 2003

Chair: Dr. Gabriel Bitton
Major Department: Environmental Engineering Sciences

The purpose of this research was to characterize the chemical composition

and biological effects of leachates from MSW landfills in Florida. Samples were

collected from 16 engineered landfills to encompass a cross-section of leachate

quality and characteristics. The MSW landfill leachates were tested using a suite

of bioassays, which included the chronic Pseudokirchneriella subcapitata and the

acute Ceriodaphnia dubia, D. pulex, and MicrotoxTM. Leachates were tested with

MetPLATE, a heavy metal specific assay. Additionally, using a yeast reporter

assay, the leachates were tested for hormonal activity.

Landfill leachates are complex mixtures of organic and inorganic

contaminants with compositions heavily influenced by site-specific parameters

(e.g. waste composition and age). The chemical composition of the Florida

landfill leachates varied widely. In some leachates, high levels of un-ionized








ammonia, inorganic components, CBOD, and COD were recorded. The

corresponding toxicity at these sites was high. Significant relationships were

shown between the ammonia content of the leachates and toxicity as determined

by the C. dubia (R2 = 0.62) and P. subcapitata (R2 = 0.69). The assays were

ranked for their sensitivities to the MSW landfill leachates as follows: C. dubia

D. pulex >P. subcapitata (125-ml) P. subcapitata (25-ml) > MicrotoxT.

The heavy metal toxicity/bioavailability and heavy metal binding capacity

(HMBC) of landfill leachates was determined with MetPLATE. The heavy metal

toxicity was low, which was attributed to the presence of complex-forming

ligands. The magnitude of the HMBC was investigated with the metals copper,

zinc, and mercury. The results showed that the HMBC ranged from 3 to 115, 5

to 93 and 4 to 101 for HMBC-Cu+2, HMBC-Zn*2, and HMBC-Hg*2, respectively.

The leachates were chemically/physically treated to reduce or fractionate their

complexity. Fractionation of selected leachates revealed that HMBC was

influenced by the solid, organic, and hardness content of the tested leachates.

Additionally, other unidentified components influenced the HMBC of the landfill

leachates.

The leachates were evaluated for their hormonal activity using a yeast

reporter assay. The hormonal activity of the raw MSW landfill leachates was

highly variable, with an E2 equivalent range from 2.6 to 45.7 ng E2/L. A similar

range from 6.2 to 59.7 ng E2/L was reported for the methanol extracts of the

leachates. The presence of unidentified substances in the leachates reduced the








recovery of hormonal activity. Treatment processes utilizing powdered activated

carbon (PAC) removed hormonal activity.













CHAPTER 1
INTRODUCTION


Historically, domestic wastes were disposed in open pits or surface piles,

and these sites often posed a significant risk to the surrounding environments

(Reinhart and Townsend, 1998). Concerns with the environmental fate of

discarded materials led to the construction of engineered systems for long-term

storage and management of waste materials and their degradation products.

These engineered systems included lined cells for the disposal of waste and

collection systems for leachate recovery. Government regulations prohibit the

disposal of waste materials except at regulated municipal solid waste (MSW)

disposal facilities. Although MSW landfills may contain small quantities of

hazardous waste materials, they are primarily designed to receive domestic

wastes. These items may include packaging materials, food scraps, furniture,

clothing, and grass clippings (USEPA, 2002).

The acute and chronic toxicity of MSW landfill leachates has been

extensively studied using various bioassays (Rutherford et al., 2000; Clement et

al., 1997; Kaur et al., 1996; Plotkin and Ram, 1984); however, little information is

available concerning the biological effects of MSW leachates collected from

landfills in Florida. Reinhart and Grosh (1998) have reported a lower chemical

strength in Florida MSW leachates, resulting from the predominant environmental

conditions, e.g., abundant rainfall and warm temperatures. Therefore, the basis








Table 1-1. Frequently used acronyms
Acronym Definition Acronym Definition
Carbonaceous Hormonally active
CBOD biochemical oxygen HAA agent
demandagent
COD Chemical oxygen HAC Hormonally active
demand compound
CPRG Chlorophenol red HMBC Heavy metal binding
galactopyranoside capacity
Sample concentration
DOC Dissolved organic IC responsible for 50 %
carbon inhibition in test
organism
Sample concentration
DOM Dissolved organic LC5 lethal to 50 % of the
matertest organisms
E1 Estrone MSW Municipal solid waste
E2 17 p-estradiol MHW Moderately hard water
E3 Estriol ONPG Ortho-nitrophenyl
galactopyranoside
EE2 17 a-ethinyl estradiol TDS Total dissolved solids
Sample concentrationcity identification
ECso responsible for a 50 % TIE To identification
effect in test organism
ED Endocrine disrupter TRE Toxicity reduction
evaluation
ERa Estrogen receptor alpha TS Total solids
United States
ERp Estrogen receptor beta USEPA Environmental
Protection Agency
Estrogen receptor -
ER-CALUX chemically activated YES Yeast estrogen
luciferase reporter gene sc


of this investigation was to determine the biological affects associated with


exposure to MSW leachates from Florida landfills.








ACUTE CHRONIC
TOXICITY TOXICITY




LEACHATE




I ME-AL
HORMONE BINDING
ACTIVITY ME
METAL
BIOAVAILABILITY




Figure 1-1. Pathways for the characterization of the biological effects of MSW
landfill leachates

In recent years, the number of operating landfills in the U.S. has

decreased; however, the overall size of the remaining landfills has increased

(USEPA, 2002). Landfills continue to be the most economically feasible and

least environmentally intrusive method for the disposal of discarded waste

materials. In Florida, more than 14 million tons (total 24.8 million tons) of MSW

were landfilled in 1998, the most recent year data were available, with a per

capital generation rate of 9.1 pounds/person/day (FDEP, 2000).

While the chemical and physical composition of MSW landfill leachates in

Florida have been summarized (Reinhart and Grosh, 1998), only one study has

evaluated the toxicity of Florida leachates (Ward et al., 2000) and then only on a

very limited scale. The scientific community, environmental regulators, landfill

operators, and the operators of facilities treating landfill leachates require








comprehensive databases to provide information relative to biological effects and

chemical constituents of Florida MSW landfill leachates.

Numerous acronyms are frequently used in the scientific research

community, and many are commonly recognized; however, some are relatively

new and discipline-specific. Therefore, a table of the acronyms used in the

following chapters has been included to aid the reader as a quick reference

(Table 1-1).

The purpose of this research was to evaluate MSW landfill leachates in

Florida, for their toxicity, hormonal activity, and chemical characteristics. To

date, there have been no reported investigations of MSW landfills of this scope or

magnitude. Most investigations have focused on leachate from one or several

landfills (Kaur et al., 1996; Wong, 1989), but few evaluated multiple leachates

(Clement et al., 1996, 1997), and none have tracked patterns over time. The

toxicity of leachates from Florida landfills has received little attention. Ward et al.

(2000) evaluated the toxicity of landfill leachates from three Florida landfills;

however, leachates were evaluated with only one acute toxicity assay.

The basis of this research project was to provide a statewide database

for landfill operators, regulators, and other research investigators, that

established a range of biological effects from exposure to MSW landfill leachates.

The overall objectives of this research project were as follows (Figure 1-1):

1. To characterize the toxicity in the MSW landfill leachates using a battery of

toxicity assays including the chronic 96-hr Selenastrum capricomutum, and





5

the acute 48-hour Ceriodaphnia dubia, 48-hour Daphnia pulex, and

MicrotoxTM assay (Chapters 3 and 4).


2. To characterize the chemical and physical composition of MSW landfill

leachates (Chapters 3, 4, 5, 6, and 7).


3. To determine the toxicity of heavy metals in MSW landfill leachates with the

heavy metal specific MetPLATE assay and to quantify the ability of MSW

leachates to reduce the bioavailability of heavy metals with a heavy metal

binding capacity (HMBC) assay (Chapter 5).


4. To conduct a toxicity identification evaluation (TIE) with selected MSW

landfill leachates (Chapter 6).


5. To determine the hormonal activity of MSW landfill leachates with a yeast

estrogen screen (YES) and identify by gas chromatography/mass

spectrometry (GC/MS) organic compounds responsible for hormonal

activity (Chapter 7).


6. To determine the effect of powdered activated carbon treatment on

hormonal activity in landfill leachates (Chapter 7).


7. The results obtained during this research investigation are summarized

(Chapter 8).













CHAPTER 2
LITERATURE REVIEW

Forty years after Rachel Carson first revealed the risks to humans and the

environment posed by a diverse array of man-made substances, the threat from

these anthropogenic chemicals persists (Carson, 1962). Over 100,000 synthetic

chemicals including pesticides, solvents, domestic cleaners, plasticizers, and

flame-retardants are produced yearly for domestic and industrial usage; however,

little is known about the biological effects from long-term exposure to these

compounds (Darnerud et al., 2001; Hale et al., 2001; Lyytikainen et al., 2001). In

the US, the Resource Conservation and Recovery Act (RCRA), Toxic

Substances Control Act (ToSCA), and the Clean Water Act (CWA) and their

amendments have been effective in the control of point source pollution.

However, non-point sources of pollution continue to pose significant threats to

the environment.

MSW Landfills

Modem MSW Landfills

MSW landfills are engineered systems that are designed through the use

of heavy-duty plastic liners and/or low permeability clay barriers to retard the

escape of pollutants to surrounding environments and for the recovery of

leachates. Leachates are formed following rainwater infiltration. Percolation of

this water through the waste materials mobilizes soluble substances (Ross,

1990). These leachates represent the mobile fraction of landfill toxicants, and








they contain high concentrations of inorganic and organic compounds (Bozkurt et

al., 2000). The majority of MSW landfill leachates are closely regulated;

therefore, their release of leachates from modern landfills is unlikely (Barlaz et

al., 2002). Accidental leachate releases may occur, e.g. during periods of high

rainfall when leachates are contaminated by stormwater or from the improper or

faulty installation of leachate collection systems. Additional sources for the

unintentional release of MSW landfill leachates exist. Prior to 1990, federal

regulations did not require landfill liners, therefore, the escape of leachates from

these sites represents a potential adverse environmental impact (Assmuth,

1996). Florida was more proactive and required landfill liners for MSW landfill in

the Solid Waste Act of 1988.

Landfilling remains the predominant management method for municipal

solid waste (MSW), accounting for more than 50% (128.3 million tons) of the

MSW generated in the United States (USEPA, 2002). Under authority granted by

RCRA, subtitle D, the U.S. Environmental Protection Agency (USEPA) regulates

the construction, operation, and post-closure of municipal solid waste (MSW)

landfills (CFR 258, 1996). Strict regulations require landfill operators to minimize,

recover, and treat the leachates generated in MSW landfills. Landfill liners are

used to restrict the flow of leachates to ground and surface waters, and they may

be composed of clay, high or low-density polyethylene, or concrete, depending

on the local conditions. In some landfills, leachates are recycled through the

waste to encourage waste stabilization and improve leachate quality (Reinhart

and Townsend, 1998; Nopharatana et al., 1998).








The chemical characteristics of MSW leachates are dependent on the type

and amount of waste landfilled, the landfill age and various environmental

conditions, e.g. temperature and rainfall. Environmental regulations in the U.S.

greatly limit the disposal of hazardous wastes in municipal landfills, but waste

materials containing toxic chemicals may enter many landfills. Some sources of

these substances include generators of small amounts of hazardous waste, non-

hazardous industrial wastes, and household hazardous wastes (HHW), such as

electrical devices, fluorescent light bulbs, thermometers, batteries, pesticides,

and other chemical products (Boyle and Baetz, 1993). Using a risk-based

assessment, comparable health risks were associated with MSW landfill

leachates and industrial waste leachates (Brown and Donnelly, 1988). Their

assessment was based on the individual leachate constituents, while not

accounting for potential synergistic and/or antagonistic responses (Brown and

Donnelly, 1988).

Landfills are classified based on the composition of the waste materials

landfilled. Class I and II landfills are designated for the disposal of non-

hazardous household wastes and some commercial, industrial and agricultural

wastes, while Class III landfills are designed for yard waste, construction and

demolition debris, carpet, furniture, and similar non-putrescible waste materials.

The basis for the distinction between Class I and Class II landfills is the volume

of MSW landfilled daily. Class I landfills receive 20 or more tons of MSW per

day, while Class II landfills less than 20 tons of MSW per day.








Characterization of the Chemical and Physical Composition of MSW
Landfill Leachates

Waste stabilization is based on microbial degradation processes, with the

conversion of organic matter to methane gas occurring predominately under

anaerobic conditions (Barlaz, 1997). Initially, aerobic conditions dominate in

landfills, but oxygen is rapidly depleted with the continuous addition of waste.

The phases of waste degradation are loosely defined by the predominant

chemical characteristics. Early phases are defined by the transition from aerobic

to anoxic and finally strictly anaerobic conditions within the waste.

Concomitantly, microbial degradation converts the large organic molecules to

smaller organic acids, which are further degraded to hydrogen and acetate.

Methanogenesis, the conversion of these small molecules to methane gas by

methanogenic bacteria, is a strictly anaerobic process. Researchers theorize

that landfills may revert to aerobic conditions as oxidized micro-environments

begin to form at the boundaries of the waste. However, no landfill currently

under study has reached this stage of decomposition (Bozkurt et al., 2000;

Kjeldsen et al., 2002). As landfilling is a continuous and on-going process,

various stages of waste decomposition occur simultaneously, and this may be

reflected in the chemical characteristics of the leachates.

MSW landfill leachates are complex mixtures, with a wide ranging

chemical strength (Table 2-1). In a recent review, Kjeldsen et al. (2002)

summarized the chemical and physical characteristics of MSW landfill leachates.








Table 2-1. Range of selected chemical and physical characteristics reported in
the literature for domestic wastewater and MSW landfill leachates in
Florida and internationally
Florida MSW International MSW
Parameter Raw Wastewater Landfill Leachate Landfill Leachate
BODs 110-400c 0.3-4800e 42-10,900a
(mg/L)
COD 250-1,000c 7-50,000e 40-90,000a
(mg/L)
Alkalinity 50-200c 350-8775' 1,350-3,510b
(mg/L)
pH -6.2-9.7e 3-7.9a
NH-N 12-50c 0-4110e <0.3b-13,000d
(mg/L)
Chloride 20-500 1.9-2720e 125-2,400b
(mg/L)
Sulfide <0.01-3.8e <0.02-30b
(mg(L)
TDS 250-850c 1,800-31,700' 2,000-60,000g
(mgfL)
Na* 40-70c 25.6-1963' 128-840b
(mg/L)
Ca+2 45-4,400' 10-7,200g
(mg/L)
Mg+2 25-122' 30-15,0009
(mg/L)
Total phosphorus 4-15c 0.1-39.6' <0.01-2.7.
(mg/L as PO42)
Conductivity 3.4-39.6' 1,200-16,000a
(mS/cm)
References: aKadlec and Knight, 1996; 'Cameron and Koch, 1980; "Metcalf and Eddy;1991; 0Lo,
1996; eReinhart and Grosh, 1998; 'this research; gKjeldsen et al., 2002.

They stressed four primary categories of contaminants to consider in discussions

of leachate quality, and these were dissolved organic matter, organic xenobiotics,

inorganic components, and heavy metals (Christensen et al., 1994).

In MSW landfill leachates, dissolved organic matter (DOM) includes the

dissolved and colloidal particles (Gounaris et al., 1993). The molecular structure

and elemental composition of dissolved organic matter in MSW landfill leachates

is strongly influenced by microbial degradative processes. Calace et al. (2001)








reported a narrow distribution of organic molecular weight groups in young

landfills (< 5 years old), with primarily low molecular weight constituents (<500

Dalton). This contrasted with their findings in older landfills (>10 years old)

with an increased distribution of molecular weight fractions and high molecular

weight constituents (> 10,000 Dalton) (Calace et al., 2001). These high

molecular weight fractions contain structurally complex humic materials (Croue et

al., 2003). Kang et al. (2002) reported an increased presence of humic

substances with increasing landfill age and a decrease in the easily degraded

lower molecular weight organic materials. Attributed to the high ammonia

concentrations in the leachate, the humic materials contained a large distribution

of nitrogen functional groups (Kang et al., 2002). This is significant when

considering the strong metal complexes that are formed with organic ligands

containing nitrogen functional groups (Croue et al., 2003; Stumm and Morgan,

1995).

Xenobiotics are frequently detected at low levels in MSW landfill leachates

(Schwarzbauer et al., 2003; Kawagoshi et al., 2002; Yasuhara et al., 1999).

Some xenobiotics of significant environmental concern, relative to reproductive

effects, have been identified in MSW landfill leachates. Wintgens et al. (2003)

reported nonylphenol, a surfactant, and Bisphenol A, a plasticizer, at 60 and 37.5

ig/L, respectively, in MSW landfill leachates. In a Japanese landfill, xenobiotics

identified included Bisphenol A, nonylphenol, octylphenol, and some dioxin-like

substances (Behnisch et al., 2001). The organic contaminants in MSW landfill

leachates have been summarized (Kjeldsen et al., 2002).








Inorganic contaminants in MSW landfill leachates include anionic and

cationic species, and some of the most widespread are NH4*, Ca+2, Mg'2, Na1,

ClI', HCO3-1, S04-2. Typically, the ammonia in MSW landfill leachates occurs as

the ionized ammonium (NH4*) species. Ammonia speciation is pH dependent,

with a pKa of 9.3. Therefore, the dominant species is ammonium, rather than the

highly toxic ammonia (NH3) form (McBean et al., 1995). Generally, total

ammonia concentrations are high, with reports of up to 740 mg/L (Kjeldsen et al.,

2002). These high ammonia levels are often attributed to the absence of

degradative pathways for the removal of ammonia from landfills (Burton and

Watson-Craik, 1998). Also found at high concentrations are the hardness

cations. Hardness is a measure of multivalent metallic cations; e.g. Ca *2, Mg2,

Sr'2, Fe.2, Mn+2, but mainly Ca2 and Mg+2(Stumm and Morgan, 1995).

Hardness has a strong influence on heavy metal bioavailability in landfill

leachates (Heijerick et al., 2003).

Heavy metals are generally reported in the low mg/L range (Reinhart and

Grosh, 1998). However, this represents only a fraction of the total metal

associated with the waste. In some cases, metals that are strongly associated

with waste materials are not easily leached under landfill conditions. Flyhammer

(1995), in a mass balance on cadmium in Swedish landfills, concluded that the

total concentrations associated with the landfilled wastes were up to four orders

of magnitude greater than leached concentrations (Flyhammer, 1995).

Kjeldsen et al. (2002) discussed leachate characteristics in MSW landfills

with a primarily organic composition and the influences of time on these leachate








Table 2-2. Toxicity of individual constituents identified in MSW landfill leachates
S C dubia S. capricomutum MicrotoxT D. magna MetPLATET
Compound (L) (mg) (mL) (mgL) (mgL)
Copper 0.01b 0.04 7.4 0.02 0.22
5-min ECo 48-hr LC5o

Cadmium 0.05c 0.34- 0.03

Zinc -0.18h 12' 5.1 0.11J
15-min EC5o 48-hr LC5o
Total 3607'
ammonia 5-min ECo
Unionized 1.1a 1.7' 3.3e
ammonia 15-min ECso 24-hr ECso
14.5d
Manganese (MHW)
Alkalinity 7819 9219
(HCO3)
Chloride -

Sodium
renamed Pseudokerchneriella subcapitata, References: 'Andersen and Buckley, 1998; Jung,
1995; CRhodes,1992; dLassier et al., 2000; "Clement and Merlin, 1995; 'Qureshi et al., 1982;
"Hoke et al., 1992; hChen et al., 1997;'Doherty et al., 1999; 'Bitton et ., 1994.


characteristics. In earlier work, Bozkurt et al. (2000) discussed leachates

generated in MSW landfills with either a primarily organic or inorganic waste

composition. The latter case represented ash monofills or co-disposal facilities

for MSW and ash. The focus of their investigation was to predict the long-term

fate of heavy metals using a conceptual model, which included influences from

various organic and inorganic ligands (Bozkurt et al., 2000).

The toxicity of MSW landfill leachates is heavily influenced by chemical

and physical interactions (Table 2-2). In this regard, complexation reactions

have a mitigating influence on toxicity and are frequently underestimated and

poorly understood, due to the sheer number of possible ligands in solution

(Martensson et al., 1999; Stumm and Morgan, 1995). Some of the typical








inorganic ligands in leachates include carbonate (Sletten et al., 1995), chloride

(Bolton and Evans, 1991), and sulfide ions (Bozkurt et al., 2000). These

inorganic ligands can form insoluble precipitates with heavy metals (Majone et

al., 1996). Other ligands present are dissolved organic matter (Kaschl et al.,

2002) and colloidal solids (Gounaris et al., 1993). These complexes exert a

strong influence on heavy metal toxicity (Heijerick et al., 2003), with up to 98 % of

metals in some landfills present as organo-metallic complexes (Kang et al., 2002;

Weng et al., 2002). However, there have been some questions concerning the

toxicity of these organo-metallic complexes (Palmer et al., 1998). Fraser et al.

(2000) suggested low-level toxicity associated with complexes between copper

and dissolved organic materials (DOM).

Bioassays for the Evaluation of Toxicity in the Environment

Luoma (1995) described bioassays as tools for investigating the complex

continuum of biochemical, physiological, and reproductive responses that occur

in organisms following exposures to suspect toxicants. Traditionally, bioassays

with various vertebrate and invertebrate organisms were used to monitor and

track environmental perturbations (USEPA, 1993a, 1994a). These bioassays

measured chronic effects in low-level, long-term exposures and acute toxicity in

high-level, short-term exposures. Critically important to all environmental

researchers was how best to reconcile chronic low-level environmental exposure

with laboratory investigations utilizing high dose acute substances (Mowat and

Bundy, 2001; Degen and Bolt, 2000). Some of the most common bioassays

have used algae (Eullaffroy and Vemet, 2003; van der Heever and Grobbelaar,

1998), aquatic plants (Mohan and Hosetti, 1999; Klaine and Lewis, 1995),








invertebrates (Heijerick et al., 2003; Kim et al., 2003; Preston and Snell, 2001;

Pereira et al., 1999), or microorganisms (LeBlond et al., 2001; Doherty et al.,

1999; Jung et al., 1997).

While most bioassays have been extensively validated, each has intrinsic

advantages and disadvantages that are specific to the method. For example, in

some algal assays, cell exudates extracellularr organic material) mitigate metal

toxicity by acting as ligands that form complexes with free metal ions. In

microplate assays, these and similar problems are controlled, which may explain

the recent increase in the use of microbiotests. Additionally, these microbiotests

offer an increased affordability, portability, and the availability of results in a short

interval of time (Chial and Persoone, 2002; Bitton et al., 1994). Gabrielson et al.

(2003) recently developed a microplate assay, referred to by the acronym MARA

(microplate assay risk assessment), that utilized 11 lyophilized microbial strains

for determining the toxic fingerprint of a chemical. This assay allows for the

testing of multiple species simultaneously; however, it is not sensitive to any

specific class of toxicants. There are microplate assays (e.g., MetPLATE and

MetPAD) that are designed specifically for the detection of heavy metal toxicity

(Bitton et al., 1992b, 1994).

Developments in the field of environmental chemistry have produced

analytical methodologies and techniques that are highly successful at identifying

and quantifying contaminants, even in highly complex matrices (Richardson,

2001). They use a suite of analytical tools that have in common an electrode,

which senses changes based on electronic signals. Some typical electrodes








measure dissolved oxygen (DO), conductivity, pH, select ions (e.g. ionized

metals), and oxidative potential. Parallel techniques for application in the field of

environmental toxicology would allow for the rapid identification of toxicity, while

simultaneously reducing the time and cost involved in continuous monitoring

programs (Arikawa et al., 1998). Biosensors are a rapid and convenient

monitoring tool, which incorporate biological tissues in a system highly sensitive

to a broad spectrum of toxic substances (Botre et al., 2000; Buffle and Horvai,

1998; Argese et al., 1996). Although their use is currently limited, biosensors

have been successfully applied to the evaluation of wastewater toxicity (Farre

and Barcello, 2003).

Toxicity of MSW Landfill Leachates

Research investigations that simultaneously combine bioassays with

methods for chemical characterization are highly valued, but the associated

expenses and labor demands constrain their extensive utilization (Ferrari et al.,

1999; Atwater et al., 1983). To date, the most extensive study of waste

leachates was conducted in France. Clement et al. (1996) investigated the

toxicity of ten domestic landfill leachates and various other hazardous and non-

hazardous waste leachates. Bioassay results with protozoa, bacteria, algae, and

invertebrates demonstrated that the toxicity of the domestic waste leachate was

higher than the industrial or hazardous waste leachates (Clement et al., 1996).

Furthermore, the chemical characterization of the domestic leachates revealed

that ammonia, alkalinity, conductivity, and COD were highly associated with

increased leachate toxicity (Clement et al., 1997). In earlier work, a significant

contribution of ammonia to the acute toxicity of landfill leachate to duckweed








Table 2-3. Reported toxicity in the literature for MSW landfill leachates
Leachate Origin Species Endpoint Reference
Solid waste landfill Tilapia
Soidnwat aditio) (Sarotherodon 96-hr LC = 1.4 to 12% Wong,
(unknown composition) mossambicus) 1989
mossambicus)
Solid waste landfill
(40 % household and Fathead minow Plotkin and
60 % (Pimephales 96-hr LC50 = 100 % Ram, 1984
industrial/commercial) promelas)
Solid waste landfill
(40 % household and Plotkin and
60 % Daphnia magna 48-hr LCo = 62 to 66 % Ram, 1984
industrial/commercial)
Solid waste landfill
(40 % household and Selenastrum Plotkin and
60% capricomutum 13 day EC5= to 10 % Ram, 1984
industrial/commercial)
Solid waste landfill
(40 % household and Plotkin and
(nnw60 % Microtox 5 min. ECso= 17 % Ram, 1984
60 % Ram, 1984
industrial/commercial)
Devare
Solid waste landfill and
(unknown composition) Aquaticplants ECo = 10% Bahadir,
1994
Devare
Solid waste landfill and
(unknown composition) Microtox ECo= 18-35% Bahadir,
1994


(Lemna sp.) was reported (Clement and Merlin, 1995). Clement et al. (1997)

performed correlative analyses between their bioassay results and various

chemical characteristics and revealed a strong correlation (R2 = 0.92) between

Daphnia magna and combined ammonia and alkalinity concentrations. Similar

relationships were shown between the bioassay results with aquatic plants,

algae, and other crustaceans and ammonia and alkalinity (Clement et al., 1997).

Using the MicrotoxTm assay, the relationship between COD and toxicity was








significant (p< 0.01) but weaker (R2 = 0.58). This was the only assay sensitive to

toxicity associated with increasing organic content (Clement et al., 1997).

The toxicity of MSW landfill leachates have been well characterized

around the world (Ernst et al., 1994; Lambolez et al., 1994; Devare and Bahadir,

1994; Cheung et al., 1993; Wong, 1989; Radi et al., 1987; Plotkin and Ram,

1984; Atwater et al., 1983; Millemann and Parkhurst, 1980; Cameron and Koch,

1980), but the toxicity of Florida landfill leachates remains poorly characterized

(Table 2-3). Ward et al. (2002) studied the leachates from six MSW landfill

leachates in Florida and concluded that the leachates were highly toxic. The

toxicity of the MSW landfill leachates varied widely, due to site-specific chemical

characteristics of the leachates. Furthermore, on a monthly basis, fluctuations in

leachate toxicity indicated the heterogenous composition of the waste materials

and local conditions (Ward et al., 2002).

Recently, investigations of waste leachates in countries that do not require

landfill liners, minimization of leachate generation, or leachate collection and

treatment have been reported. These studies offer insight to researchers

concerned with the potential for leachate release to the environment. Magdaleno

and De Rosa (2000) characterized leachates from a waste dump in Argentina

with an algal assay using Selenastrum capricomutum (renamed

Pseudokirchneriella subcapitata), while Sisinno et al. (2000) evaluated waste

leachates in Brazil with the Zebrafish (Brachydanio rerio). The chemical strength

of these leachates were comparable to reports of others (Kjeldsen et al., 2002;

see Table 2-1).








The chemical strength of the Argentinian leachates was demonstrated by

COD concentrations from 502 to 4640 mg/L, ammonia from 26.5 to 35 mg/L, and

pH from 7 to 7.3 (Magdaleno and De Rosa, 2000). Similar chemical

characteristics were reported with the leachates from Brazil, with conductivity

values from 3.1 to 6.2 mS/cm, alkalinity from 212 to 372 mg/L as CaCO3, and

COD from 5,200 to 11,500 mg/L (Sisinno et al., 2000). Overall, the toxicity of the

Argentinian leachates was low with TU (toxicity unit) values that ranged from 1 to

2.1 (Magdaleno and De Rosa, 2000). Higher toxicity was reported in the

Brazilian leachates and ranged from 17.5 to 45.5 TU (Sisinno et al., 2000). The

leachates from Brazil demonstrated a toxicity similar to that reported with Florida

leachates (Ward et al., 2002). However, the leachates from Argentina displayed

reduced toxicity. The predominance of plastics and other disposable materials in

US MSW landfills may be a factor contributing to their higher toxicity.

The contamination of groundwater by waste leachates is a primary

concern, relative to the escape of leachates into the environment. Baun et al.

(1999) investigated the toxicity of groundwater contaminated by MSW leachates

in Denmark with an algae assay, a crustacean assay, and a bacterial

genotoxicity assay. Using the algae assay, the leachate-contaminated

groundwater sample displayed an EC20 of 17 %; however, the toxicity decreased

by 75 % at twice the distance from the landfill. Similar toxicity was demonstrated

by the crustacean, Daphnia magna, with an EC20 of 18 % at the landfill, but,

further downstream, no toxicity was reported. Further analysis of this

contaminated groundwater revealed that organic contaminants were responsible








for the high toxicity, and with increasing distance from the landfill the organic

toxicity decreased, suggesting metabolic degradation or dilution effects (Baun et

al., 2000). Additional bioassays with the organic fraction revealed a low

sensitivity of the D. magna assay to organic toxicants, which contrasted with the

algal and MicrotoxT assay results (Baun et al., 2000). Other researchers have

reported the higher sensitivity of MicrotoxTM to organic contaminants (Bitton et al.,

1994).

In 1988, reports of comparable carcinogenic risk associated with exposure

to MSW landfill leachates or hazardous waste leachates raised concerns in the

regulatory community (Brown and Donnelly, 1988). Subsequent investigations,

to determine the genotoxic potential of MSW landfill leachates, have revealed

conflicting results. Beg and AI-Muzaini (1998) investigated the genotoxicity of

MSW landfill leachates in Kuwait using a dark mutant strain (nonluminescent) of

Vibrio fisher, a bioluminescent bacterium. In the presence of a mutagen, the

dark strain reverts to the luminescent state and this response is quantified by

increased light intensity. These results suggested, in some of the Kuwaiti

leachates there was a high degree of genotoxicity, and this was dependent on

the type of waste landfilled and seasonal conditions (Beg and AI-Muzaini, 1998).

Helma et al. (1996) used four bacterial assays to characterize genotoxicity in

landfill leachates, wastewater effluents, pulp and papermill effluents, and

contaminated groundwater. Overall, the highest genotoxicity was displayed by

the MSW landfill leachates with more than 35,000 revertants/L of leachate. This

was comparable to the genotoxicity of the leachates produced by mixed industrial








and domestic wastes, which were reported as approximately 40,000 revertants/L

(Helma et al., 1996). Baun et al. (1999) using the umuC strain of Salmonella

typhimurium showed that leachate-contaminated groundwater was not genotoxic

at concentrations up to 25% by volume. However, bacterial toxicity at higher

concentrations prevented the evaluation of genotoxic effects. After isolation of

the organic fraction of these contaminated groundwaters, a similar mutagenicity

was identified. These results suggested that the organic fraction contained the

mutagens (Baun et al., 2000).

Hormonally Active Agents in the Environment

Some natural and anthropogenic substances may interact or interfere with

the nuclear receptors and chemical messengers of the endocrine system and

have been identified as a threat to the environment and wildlife (McLachlan,

2001). Alterations in both the developmental and reproductive functions of cells

and whole organisms are increasingly documented and attributed to exposure to

these exogenous substances (NRC, 1999). These substances are ubiquitous

environmental contaminants, which are commonly referred to as hormonally

active agents (HAAs) or compounds (HACs), xenoestrogens, estrogen-like

compounds, endocrine disruptors, estrogen-mimics, or estrogen

agonists/antagonists. Substances are labeled based on their interaction with

and/or displacement of an endogenous hormone from its conservative function

(McLachlan, 2001). Recent congressional mandates in the Safe Drinking Water

Act (1996) (Bill No.S.1316) and the Food Quality Protection Act (1996)(Bill

No.P.L.104-170) have required the USEPA to evaluate the hormonal activity of

all chemicals produced in the U.S.








In a recent survey of 139 contaminated U.S. surface waters, alkylphenols,

phthalate compounds, and natural and synthetic estrogens were shown to

comprise roughly 75% of the organic contaminant load (Kolpin et al., 2002). Of

critical concern is the exposure to these three classes of compounds, because of

reports of hormone-like effects. Their origin may be due in part to agricultural

non-point source runoffs (Casey et al., 2003), but the majority are discharged

from domestic and industrial wastewater treatment plants (WWTPs) (Sheahan et

al., 2002b; Snyder et al., 2001; Baronti et al., 2000; Rudel et al., 1998).

Environmentally relevant concentrations of these compounds have been linked to

altered sexual characteristics (Jobling et al., 1995) and elevated tissue levels of

hormonally active compounds (Sheahan et al., 2002b) in fish. However, links are

not easily established in some situations (Jacobsen and Guildal, 2000; Fawell et

al., 2001; Sepulveda et al., 2002).

According to the European Union Scientific Committee, hormonally active

agents (HAAs) (referred to as endocrine disruptors) are "exogenous substances

or mixtures that alter functions) of the endocrine system and consequently

cause adverse health effects in an intact organism, or its progeny, or sub-

populations" (Baker, 2001). Sweeping in its brevity, this definition fails to address

some concerns (McLachlan, 2001; Ashby, 2000), specifically, altered cellular

functions in relation to the overall health of the organism. Although, the National

Research Council (1999) was even less direct when they defined hormonally

active compounds as any "substance that possesses hormone-like activity,








regardless of the mechanism" of action. In light of new research (Wu et al.,

2003), changes to the definition may read, "any substance or influencing factor."

Hormonally active compounds are arranged in three groups; the natural

and synthetic estrogens, anthropogenic chemicals, and phytoestrogens (naturally

produced substances in plants). The effects of these substances may be

agonistic or antagonistic. Agonistic hormonally active compounds act in a

manner similar to an endogenous hormone, while antagonists block the activity

of endogenous substances. Beginning with reports of the estrogen-like effects

following exposure to the insecticide DDT (Burlington and Lindeman, 1950),

researchers continue to study the interaction of non-steroidal compounds with

the estrogen receptor (Miksicek, 1994).

Phytoestrogens

Phytoestrogens are naturally occurring compounds in plants and plant-

derived products (Nilsson, 2000) and include genistein, equol, formononetin,

biochanin A (Latonelle et al., 2000). The hormonal activity of phytoestrogens has

been reviewed, with special emphasis on environmentally relevant dosages

(Nilsson, 2000). One source of phytoestrogens is the urine of vegetarians (Fotsis

and Adlercreutz, 1987). Phytoestrogens impact the reproduction and sexual

health of wildlife (Hughes, 1988), but there is no evidence in humans (Strauss et

al., 1998). In fact, limited evidence suggests phytoestrogens are beneficial in

treating some types of human cancers (DiPaola et al., 1998). Ju et al. (2000)








Table 2-4. Select phthalate compounds and their common usage
COMPOUND USES
Rain gear, footwear, upholstery materials, I.V.
Di-ethylhexyl phthalate fluid bags, waterproof gloves
(DEHP) Heat seal coating on metal foils used on portioned
food items.
Dispersant in insect repellants and perfumes
Butyl benyzl phthalate Component of cellulose plastics
(BBP) Floor tiles
Di-butyl p e Coatings on cellophane, insect repellants,
Di-butyl phthalate Hair spray
(DBP) Carpet backing
Cellulose acetate plastic films- used as
Di-ethyl phthalate carton windows to display foods
(DEP) Molded plastics, i.e. toothbrushes, car
components and children's toys
Di-isononyl phthalate Vinyl wall coverings, toys, and medical devices
(DINP)


showed low concentrations of some plant substances reduce estrogenic effects,

while at high doses estrogenic effects may be increased. The contamination of

foodstuff by zearalenone, a fungal phytoestrogen, is common and human

consumption is estimated at 3 pg/person/day in North America (McLachlan,

2001).

Phthalates

Phthalates are plasticizers that are commonly used as softeners in the

production of paints, inks, adhesives, and various plastic goods (Table 2-4). In

an extensive study of phthalate compounds, the National Institute for Health

(NIH) concluded that benzyl butyl phthalate (BBP) was both a developmental and

reproductive toxicant (NIH, 2003). Additionally, extensive phthalate

contamination has been reported for over-the-counter beauty products

(Environmental Working Group, 2002). With concem, researchers have shown








that body burdens of phthalate compounds in women of child-bearing age (20-40

years) are higher than males and any other age group (Blount et al., 2000), and

the long-term consequences of this are unknown.

Phthalate compounds are ubiquitous contaminants of both terrestrial and

aquatic environments. Freshwater levels of di-ethyl hexyl phthalate (DEHP) and

di-butyl benzyl phthalate (DBP) ranged from 4.6 to 90.5 1pg/L and 0.1 to 75.6

pg/L, respectively, while marine concentrations of DEHP and DBP ranged 0.1 to

2306.8 ig/L and 1.0 to 1028.1 ig/L, respectively (Fatoki and Noma, 2002).

Additionally, contamination of raw drinking water by di-ethyl phthalate (DEP) has

been reported (USEPA, 2001). Some phthalate contamination in the

environment may be traced back to WWTP discharges. Fromme et al. (2002)

surveyed 39 German wastewater treatment plants and showed the

concentrations of DEHP and DBP were highly variable in the effluents and

ranged from 1.7 to 182 [ig/L and 0.2 to 10.4 pg/L, respectively. Furthermore, the

concentrations of phthalate esters in the WWTP sludge ranged from 27.9 to 154

mg/kg dry weight for DEHP and 0.2 to 1.7 mg/kg dry weight for DBP (Fromme et

al., 2002).

Phthalate esters, including the commonly used di-ethyl phthalate (DEP),

DBP, BBP, and di-isobutyl phthalate (DIBP), are capable of inducing an

estrogenic response in reporter assays, but their potency was one millionth that

of 17 p-estradiol (Jobling et al., 1998; Harris et al., 1997). Legler et al. (2002)

identified the hormonal activity of BBP using an estrogen receptor-chemically

activated luciferase reporter gene (ER-CALUX) construct; however, the








Table 2-5. Concentrations of nonylphenols in food items in Germany
Concentrationa
Food Item (ig/kg)
Peanut butter 5.2
Marmalade 7.3
Butter 14.4
Tomatoes 18.5
Apples 19.4
Breast milk 0.3
Infant formula 1.6 2.1
a from Guenther et al. (2002).

responses of other phthalates, e.g., DEP and DBP, were weaker. Researchers

continue to investigate the hormonal activity of phthalate compounds; in fact,

there is still a debate surrounding the hormonal activity of the most commonly

used phthalate, DEHP (Metcalfe et al., 2001).

In humans, the main pathway for the conjugation of phthalates prior to

excretion is via glucuronidation (Albro et al., 1982). Evidence for the reduced

hormonal activity of phthalate conjugates comes from rodent assays (Foster et

al., 2000). Although the conjugated phthalates are excreted at concentrations in

the microgram per liter range, they may be rapidly deconjugated in the presence

of the glucuronidase enzymes (Blount et al., 2000). These glucuronidase

enzymes are present in high concentrations in domestic wastewaters.

Alkylphenols

Alkylphenol polyethoxylates (APEs) are one class of non-ionic surfactants,

with numerous industrial and domestic uses (Talmage, 1994). These hydrophilic

APEs are rapidly degraded during biological treatment, e.g. in wastewater

treatment plant (WWTP), to hydrophobic and recalcitrant alkylphenols (AP). Due

to the high degree of ethoxylation, APEs are not estrogenic; however, activity has








been reported in the degradation products nonylphenol (NP) and octylphenol

(OP) (Routledge and Sumpter, 1996a). Nonylphenols are ubiquitous

contaminants of commercially available food items (Guenther et al., 2002) (Table

2-5).

The affinity of the hydrophobic APs to sediment increases with organic

content (Lye et al., 1999). As a result, the reported half-life of sediment-

associated alkylphenols is roughly 60 years (Shang et al., 1999). In the outfall of

WWTPs, reported concentrations of APs in the sediments range from 2 to 9,050

ng/g dry weight in freshwater environments (Lye et al., 1999) and from 1370 to

1630 ng/g in marine environments (Shang et al., 1999).

Humans excrete AP compounds as glucuronide conjugates (Muller et al.,

1998), and these conjugates are then subjected to biological degradation

processes. Current wastewater treatment technologies are not effective for the

complete removal of APs (Sheahan et al., 2002b); hence, pg/L levels are

discharged to receiving waters and induce hormonal responses in fish (Sheahan

et al., 2002a). In aquatic environments, one of the biomarkers for exposure to

hormonally active compounds is the presence of vitellogenin (Vtg), a fish egg

yolk protein. Jobling and Sumpter (1993) reported a 20- to 90-fold increase in

Vtg production in Rainbow trout (Oncorhynchus mykiss) exposed to various

concentrations of alkylphenols (1 to 100 jpM) in a laboratory study. Alkylphenols

bioaccumulate (Sheahan et al., 2002b); in fact, 10-30 ng NP/g of liver (wet

weight) was reported in male flounder (Platichthys flesus) (Lye et al., 1999).

Although WWTP effluent concentrations of the hydrophobic NP and OP have








been reported in the mid ng/L range (Snyder et al., 1999), higher concentrations

may be found in the wasted sludge due to partitioning (Ejlertsson et al., 1999; La

Guardia et al., 2001). While octylphenol and nonylphenol are weakly estrogenic

(Legler et al., 2002), their conjugated forms are not capable of inducing estrogen-

like responses (Moffat et al., 2001).

Natural and Synthetic Estrogens

Research indicates that estrogens, both natural and synthetic, represent

the predominant fraction of organic wastewater contaminants and concurrently

induce the highest hormone activity (Metcalfe et al., 2001; Snyder et al., 2001;

Rodgers-Gray et al., 2000; Desbrow et al., 1998). The vertebrate endocrine

system produces chemical messages, called hormones, which regulate body

functions, e.g., reproduction, growth, and homeostasis (Figure 2-2). Estrogens

are the hormones produced by the ovaries and they are responsible for the

development and regulation of female secondary sexual characteristics. The

endogenous estrogens, 17 p-estradiol (E2) and estrone (E1), together with their

degradation product estriol (E3) are rapidly conjugated and excreted from the

body. This made their use in hormone therapies ineffective and led to the

development of synthetic hormones (Bolt, 1979). Although the synthetic

hormones are rapidly absorbed in the bloodstream, they are slowly metabolized

and are; therefore, better suited for drug therapies (Guengerich, 1990). The

most commonly prescribed synthetic hormones are 17a-ethynylestradiol (EE2)

and mestranol, which are both utilized in the production of birth control pills

(BCP) and as inhibitors of ovulation (Ranney, 1977). Although E1 and E3 are the








Table 2-6. Rates for the urinary excretion of natural estrogens from men and
women
Women

Estrogen Pre- Pre- Post- Menc
Menopausala menopausalb menopausala (n=2)
(n=114) (n=25) (n=146)
17p-Estradiol (E2) 3.5 1.1-2.8 0.7 1.5
( g/day)
Estrone (Ei) 7.0 2.6-7.8 1.4 3.9
(gg/day)
Estriol (E3) 8.7 4.7-5.6 1.6 1.5
(lg/day)
Key et al., 1996, reported as geometric mean, "Adlercreutz et al., 1994, reported as range, and
CFotsis and Adlercreutz, 1987 reported as mean. Standard deviations were not reported.

main metabolites of E2, there is also a group of minor metabolites with

inconsequential hormonal activity.

The metabolic pathways for natural and synthetic estrogens have been

extensively reviewed (Bolt, 1979; Guengerich, 1990). Generally, natural and

synthetic hormones in the human body are metabolized to inactive glucuronide or

sulfonide conjugates before excretion (Bolt, 1979). The age distribution and;

hence, the reproductive conditions of women in a population determines the total

concentration of excreted estrogens. Table 2-6 summarizes reported excretion

rates of natural estrogens from both men and women. Keys et al. (1996) showed

that pre-menopausal women excreted 3.5 pg/day of E2, 7.0 ig/day El, and 8.7

pg/day E3. In a separate study with pre-menopausal women, similar estrogen

concentrations in urine were reported by Adlercreutz et al. (1994). The slight

variation in estrogen excretion reported by Key et al. (1996) and Adlercreutz et

al. (1994) was probably due to the menstrual phase during urine collection.








Adlercreutz et al. (1994) collected urine samples during the mid-follicular phase

(3-11 days after the onset of the last menstruation), while Key et al. (1996)

analyzed urine collected throughout the entire menstrual cycle.

Overall, pregnant women excrete the highest concentrations of natural

hormones at 600 jig/day, 259 pg/day, and 6000 pg/day for Ei, E2, and E3,

respectively (Fotsis et al., 1980). The estrogen concentrations reported for post-

menopausal women were 0.7 jig/day for E2, 1.4 pg/day for El, and 1.6 pg/day for

E3 (Key et al., 1996) and were comparable to male estrogen excretion rates.

Male (n=2) excretion was reported for El, E2, and Esat 3.9 pg/day, 1.5 ig/day,

and 1.5 ig/day, respectively (Fotsis and Adlercreutz, 1987). Predicting the

excretion rates of synthetic hormones is more difficult and depends on the

number of pre-menopausal females in a population, cultural mores and the brand

of birth control pill used (Johnson et al., 2000). A search of pharmaceutical

information on the Internet showed a range of 30 40 j.g EE2I tablet, with a

typical dosing regime of 21-28 days, followed by 7 days of inactive tablets.

Larsson et al. (1999) estimated EE2 excretion rates at 4 jg/day per female

consuming oral contraceptive pills in Sweden. The excretion of endogenous

hormones is predominantly via the urine, while fecal elimination generally

exhibits a minor secondary role; however, for the excretion of synthetic hormones

the fecal route is primary (Ranney, 1977).

Fecal excretion rates of endogenous estrogens from pre-menopausal

women (n=25) were reported as 0.5 ng/day for E1, 0.4 ng/day for E2, and 0.8

ng/day for E3 (Adlercreutz et al., 1994). Daily excretory rates for feces and urine








have been reported at 100-400 grams and 1-1.3 kg wet volume/person/day,

respectively. These ranges generally apply to men, with excretion rates for

women generally at the lower limit of this range (Polprasert, 1989 as cited in

Bitton, 1994).

Effects of Hormonally Active Compounds on Humans

Over the past 30 years, what began as anecdotal observations of altered

reproductive and sexual development in humans have coalesced into concem for

long-term species survival (McLachlan, 2001). The early onset of middle-age

vaginal carcinomas and deformed uteri in young women have been linked to the

potent synthetic estrogen diethylstilbesterol, widely prescribed to pregnant

women throughout the 1950's and 60's (Colbum et al., 1996). During the first

trimester of pregnancy, human fetuses are highly sensitive to exposures from

hormonally active compounds.

Industrialized nations, including the U.S., Scandinavia, and Japan, have

reported an increased incidence of hypospadias (displacement of the urethral

opening toward the scrotum) and cryptorchidism (failure of the testicles to

descend into the scrotum) in males (Paulozzi, 1999). Some researchers have

questioned this conclusion and instead cite increased reporting and stricter

definitions as factors artificially inflating the data. Widespread trends are difficult

to establish, but adverse sexual effects from exogenous substances have been

confirmed. The feminization of males has been attributed to work place

exposure to formaldehyde (Finkelstein et al., 1988) and therapeutic treatments

with herbal supplements (DiPaola et al., 1998). Gray (1998a) showed that

sexual differentiation in male rats was altered after exposure to hormonally active








HORMONALLY ACTIVE COMPOUNDS


CENTRAL NERVOUS SYSTEM


HYPOTHALAMUS

PITUITARY GLAND


N PANCREAS OVARY
Jl 1t
ggn i" ""


I

]


GLTESTESND
J TESTES PNA GLAND


MUSCLE. NERVOUS LIVER, I CIRCADIAN
[IVERSYSTEMRLE SYSTEM RYTHYMS
LYMPH TISSUE MKDNEYS MUSCLES REPRODUCTIVE ORGANS R
KIDNEYS U C

Figure 2-1. Representation of the vertebrate endocrine system and the possible influences of hormonally active
compounds on various systems and organs. (adapted from Mathews and van Holde, 1996)


L

I


THYMUS


I


-U
THYROID








Table 2-7. Advantages and disadvantages associated with the use of in vivo and
in vitro assays for identifying hormonal activity
Advantages Disadvantages
In Vivo
Metabolic capability High cost

Multiple nuclear receptors Assay duration (weeks to months)
Non-standardized protocols (dosing
Established assays regime, food, endpoint)

Sensitivity to non-hormonal effects
In Vitro
Low cost No metabolic capability
Rapid (hours to days) Predominately measure ER mediated
Rapid (hours to days) effects
Simplified culture techniques Lack of pathways to clear hormones

Minimize endocrine system complexity


compounds and pesticides. The human reproductive system is regulated by a

plethora of chemical messengers in a complex relay of signals that control

gametogenesis, ovulation, fertilization and sexual differentiation (Thomas, 1997).

The vertebrate endocrine system produces chemical messages, called

hormones, which regulate body functions, e.g. reproduction, growth, and

homeostasis (Figure 2-1). Numerous reviews have been published that discuss

the effects of endocrine-disrupting compounds on humans (Sultan et al., 2001;

Degen and Bolt, 2000; Paulozzi, 1999; Neubert, 1997).

Bioassays to Identify Hormonal Activity

Pursuant to congressional mandates, the US environmental protection

agency (USEPA, 1998) developed a framework for a tiered screening program








Table 2-8. In vivo assays for the determination of hormonal activity
ASSAY ENDPOINT
Measure uterine weight of ovariectomised
Rodent rodents
Measure vaginal cornification" of
Rodent ovariectomisedb rodents
Measures androgen sensitive tissue weight of
Hershberger Castrated Rat castrated male rodents

Fish Gonadosomatic index, Vtgc induction

Turtles Vtg induction
vaginal lesions, ovaries removed surgically, CVtg, vitellogenin (a female egg yolk protein)

that integrated in vivo and in vitro bioassays for the identification and

quantification of hormonally active agents in the environment (Gray, 1998b).

Researchers continue to investigate novel approaches for identifying hormonal

activity, and, although these new methods increase the knowledge base of

hormonal effects, more work is needed in establish the foundation of adverse

hormonal effects. Primarily, increased validation of the more widely utilized

assays, e.g., the yeast estrogen screen (Routledge and Sumpter, 1996b) and

rodent assays (Ashby, 2000) and inter-laboratory comparison of these

established assays (Ashby, 2003) are needed. In vitro and in vivo assays each

have their own advantages and disadvantages; therefore, a battery of assays

utilizing both types of assays has the greatest value (Table 2-7).

Endogenous estrogen ligands bind with estrogen receptors at the cellular

level in a well-defined cascade of cellular events (Okamura and Nakahara,

1999). The endogenous ligand enters the cell via active transport mechanisms

or diffusion. Once inside the cell, the ligand enters the nucleus and binds with

the estrogen receptor displacing the heat shock proteins (e.g., Hsp90) associated








Table 2-9. Threshold dose for the induction of hormonal effects following
exposure of fish to natural and synthetic estrogens
Conc.
Species Hormone on. Response Source
(ng/L)

Oncorhynchus
mykiss Ei 25-50 Vtg induction Routledge et al.,
(Rainbow E2 1-10 Vtg induction 1998
trout)
Oncorhynchus
mykiss EE2 1.5 Vtg induction Larsson et al., 1999
(Rainbow
trout)
Rutilus
Rutilus 1-10 Routledge et al.,
rutilus E2 Vtg induction 1998
(Roach)

Oryzias EE2 0.0b Alteration in
latipes E2 8b reproductive Metcalfe et al., 2001
(Medaka) E3 750b characteristics

Danio Vtg induction; a n B l.
rerio EE2 5-10 Erratic
(Zebrafish) spawning
Ictalurus
punctatus E2 2.7 Vtg induction Monteverdi et al.,
(Channel E2 2.7 Vtg induction 1999
(Channel 1999
catfish)
Platichthys
flesus EE2 10 Vtg induction Allen etal., 1999
(Flounder)
aAbbreviations: El, estrone; E2, 17p-estradiol; E3, estriol; EE2, ethynyl estradiol; Vtg, vitellogenin
lowest observed effect concentration


with the receptor. These proteins maintain the structural conformation of the

estrogen receptor (Fliss et al., 2000). The receptor-ligand complex then binds to

a specific ligand-binding domain on the nuclear DNA (Massaad et al., 1998),

which codes for the transcription of messenger RNA (mRNA). In the cellular








machinery, the genomic message on the mRNA is translated into protein. This

suite of events is initiated in response to the estrogenic ligand. Estrogen

receptors are part of a "superfamily" of nuclear receptors and include a large

number of orphan receptors, with no recognized ligands (McLachlan, 2001).

There are two forms of the estrogen receptor, estrogen receptor a (ERa) and

estrogen receptor p (ERp). Although, the tissue distribution of ERa and ERp

differ based on sex (male or female) and organ type, they display similar

sensitivities to the endogenous estrogen 17 p-estradiol (Couse et al., 1997).

While the majority of hormonally active substances exert their influence via

ligand-dependent activation of the estrogen receptor, some substances do not

act via receptor interactions (EI-Tanani and Green, 1997).

In vivo assays for the determination of hormonal activity

Traditionally, the potential for hormonal activity was assessed with in vivo

assays (Table 2-8). Typical endpoints measured are increased uterine weight,

altered sex ratios, skewed gonado-somatic index, and induction of vitellogenin

(Vtg) (an egg yolk protein in vertebrates). In vivo assays using various

crustacean species (Andersen et al., 1999; Fingerman et al., 1998), including

Daphnia magna (Shurin and Dodson, 1997; Baldwin et al., 1997; Baldwin et al.,

1995) have been used to evaluate hormonal activity as decreased steroid

metabolism and developmental abnormalities.

Common in vivo assays use rodents (Prinsen and Gouko, 2001), fish

(Sepulveda et al., 2002; Bowman et al., 2000) and some invertebrates (Gagne et

al., 2001; Blaise et al., 1999), but these assays are expensive, labor-intensive








Table 2-10. In vitro assays for the determination of hormonal activity
ASSAY MODE OF ACTION
measures the ability of a substance to
stimulate proliferation in estrogen sensitive
Cell Proliferation Assays cells
Ex. E-Screen (Soto et al., 1992)
measures the affinity between a substance
Receptor Binding Assays and the estrogen receptor
Ex. hER a or p (Gutendorf and
Westendorf, 2001)
measures the ability of a substance to induce
Reporter Gene Assays the reporter gene
Ex. YES (Routledge and Sumpter, 1996b)
measures the induction of a specific proteins
Cell Line Assays or enzymes by a substance
Ex. Liver cells (Monteverdi et al., 1999)
measures the ability of substance to stimulate
Cell Prolifer Ge cell proliferation and induce reporter gene
Cell Proliferation/Reporter Gene transcription
transcription
Ex. ER-CALUX (Legler et al., 1999)


and, in some cases, raise ethical concerns. One of the advantages of in vivo

assays is the cellular machinery for the metabolism and/or conjugation

ofhormonally active compounds (HAC). The degradation of the parent HAC may

produce a metabolite with no hormonal activity (Harris et al., 1997). Table 2-9

summarizes literature reports for the threshold dose of natural or synthetic

estrogens required for the induction of a hormonal response in various fish

species.

In vitro assays for the determination of hormonal activity

Generally, the premise on which in vitro assays are based is the defined

mechanism of action between hormonally active ligands and nuclear receptors,

usually the estrogen receptor. Due to the lack of metabolic pathways in the in

vitro assays, the hormonal activity may be over-predicted. In most cases, in vitro









Table 2-11. Relative sensitivity of in vitro assays to 17 p-estradiol (E2)
MDLa EC5o Source
Assay (ng/L) (ng/L)ource
RCBAb .03 Coldham et al., 1997

RCBA .02 Klein et al., 1994

YES 3 Routledge and Sumpter, 1996b

YES 2.7 27 Murk et al., 2002

YES 19 Beresford et al., 2000

YES 27 Tanaka et al., 2001

YES 13 Elsby et al., 2001

YES 5.4 Vinggaard et al., 2000

YES 22.8 Layton et al., 2002

YES 2.7 27 Legler et al., 2002


E-SCREEN

E-SCREEN

E-SCREEN
Cell line/reporter
(MVLN)
Cell line/reporter
(HGELN)
Cell line/reporter
(ER-CALUX)
Competitive
binding (ER a)
Competitive
binding (ER p)
Competitive
binding (ER (?))


1.4

2.7

1.7

1.4

10.9

0.1 1.6

900

17700


Gutendorf and Westendorf, 2001

Soto et al., 1992

Behnisch et al., 2001

Gutendorf and Westendorf, 2001

Gutendorf and Westendorf, 2001

Legler et al., 2002

Gutendorf and Westendorf, 2001

Gutendorf and Westendorf, 2001


272 1360 Murk et al., 2002


Abbreviations: MDL, minimum detection limit; "RCBA, recombinant cell bioassay; 'estrogen
receptor form not indicated








assays are constructed with recombinant molecules or cells from either

mammalian or fish tissues (Zacharewski, 1997; Diel et al., 1999). Most

frequently, yeast cells (Saccharomyces cerevisiae) are used as carriers for the

hormone receptors. Early in vitro assays incorporated an estrogen receptor (ER)

and reported only ER-mediated hormonal activity. Recently, in vitro assays have

been designed with other nuclear receptors, including androgen receptors (AR)

and progesterone receptors (PR) (Nishikawa et al., 1999). Table 2-10

summarizes the general types of in vitro assays currently available.

The hallmark of an in vitro assay is the sensitivity of the assay to the

endogenous estrogen E2. Table 2-11 summarizes the sensitivities of various in

vitro assays. Advantages afforded by in vitro assays include the rapid

identification of hormonal effects, relatively lower cost, and the ability to screen

numerous samples simultaneously. Typical endpoints for in vitro assays include

cell proliferation, enzyme expression (e.g. p-galactosidase), and protein

synthesis. Some of the limitations inherent in in vitro assays are the absence of

metabolic pathways, the over-estimation of binding in a single receptor systems

(Jobling et al., 2002), and reliance on estrogen mediated effects, while ignoring

other receptors (Diel et al., 1999). Despite these concerns in vitro

assayscontinue to be widely used for the identification of hormonal activity and

for the quantification of the contributions from individual chemicals to overall

activity (Degen and Bolt, 2000; Gutendorf and Westendorf, 2001).

The most widely used reporter gene assay incorporates the human

estrogen receptor (hER) into the genome of the yeast Saccharomyces cerevisiae








(Rehmann et al., 1999; Coldham et al., 1997; Gaido et al., 1997; Routledge and

Sumpter, 1996b). Yeast cells are stably transfected with the human ER (hER)

and expression plasmids for a reporter gene, usually lac-Z (codes for the enzyme

p- galactosidase). When estrogenic ligands enter the cell, they bind with the ER

to form a ligand-ER complex. This complex then interacts with the estrogen

responsive element (ERE) on the plasmid and initiates transcription of the

reporter gene. The reporter gene product is quantified by the addition of a

suitable substrate. Due to their easy quantification by spectrophotometers,

chromogenic substrates are typically used, e.g., chlorophenol red

galactopyranoside (CPRG) (Routledge and Sumpter, 1996b) or ortho-nitrophenol

galactopyranoside (ONPG) (Lascombe et al., 2000; Klein et al., 1994). These

estrogen receptor/reporter assays are rapid, reproducible, and have

demonstrated a high degree of sensitivity to hormonally active compounds.

The YES (Routledge and Sumpter, 1996b) has been widely used to

identify hormonal activity in pure compounds (Beresford et al., 2000), wastewater

treatment plant influents and effluents (Holbrook et al., 2002), flue gases

(Muthumbi et al., 2002) and recycled materials (Vinggaard et al., 2000). This

assay has also been adapted to include the androgen receptor and thus quantify

androgenic effects (Beresford et al., 2000).

Other yeast-based assays have been developed, and some are gaining

wide acceptance due to their use of multiple nuclear receptors. A novel ligand-

receptor binding assay was constructed in a yeast (Y190) two-hybrid assay with

expression plasmids (pGBT9 and pGAD424) to determine the interaction








between selected hormone receptors (ER, AR, PR, MR, TR) and co-activators

(TIF2, SRC1, TIF1, RIP140) in the presence of HACs (Nishikawa et al., 1999).

This assay is highly sensitive to phytoestrogens and nonylphenol (Nakano et al.,

2002) and environmentally relevant concentrations of HACs (Kawagoshi et al.,

2003). The role of the co-activator is poorly understood, but, after binding of the

ligand to the nuclear receptor, it appears to influence processes that initiate gene

transcription (Nishikawa et al., 1999). The TIF 2 co-activator had the greatest

influence on initiation (Kawagoshi et al., 2002).

Exploitation of the estrogen sensitivity of breast cancer cells led to the

development of assays that quantify cell proliferation in the presence of

estrogens or estrogen-like substances. The E-Screen (MCF-7 breast cancer

cells) assay (Soto et al., 1992) provides highly reproducible results when assay

protocols are strictly adhered to and the cell line source is consistent. Payne et

al. (2000) evaluated three MCF-7 cell lines (BUS, UCL, and SOP) for their

sensitivity to E2 and showed EC5o values of 3.6, 3.1, and 2.6 ng/L, respectively.

However, the proliferative effect (growth in excess of control cells) of E2 varied

markedly among the cell lines at 8.9 with BUS, 0.98 with UCL, and 1.45 with

SOP (Payne et al., 2000). The functionality of some estrogen receptors in breast

cancer cell lines may be low, with up to 50 % non-functional (Balmelli-Gallacchi

et al., 1999). In cell proliferation assays, direct counts occur with

hemocytometers or automated Coulter counters.

The addition of reporter genes to cell lines has increased their sensitivity

and allowed for the elucidation of multiple mechanisms of action in a single test








system. Luciferase reporter gene constructs were designed in human breast

cancer cell lines. Addition of the lux reporter gene to MCF-7 and HeLa cells

produced the MVLN and HGELN systems (Balaguer et al., 1999; Gutendorf and

Westendorf, 2001). This adaptation allowed for the identification of substances

whose mode of action was via cell proliferation or ER activation. Katori et al.

(2002) demonstrated that di-butyl phthalate induced cell proliferation, but not

gene transcription.

Additionally, the ER-Chemical Activated Luciferase gene eXpression (ER-

CALUX) assay was developed in T47D human breast cancer cells by

transfection with reporter genes (pEREtata-Luc) (Legler et al., 1999). The steep

E2 dose-response curve with the ER-CALUX assay was nearly 20 times greater

than the reporter gene response in the YES, indicating the higher sensitivity of

the ER-CALUX assay (Legler et al., 2002) (Table 2-11). Other advantages of the

ER-CALUX assay are the small sample volume requirements, which were

roughly 1/10t and 1/100t* the volume required by the YES and ER binding

assays, respectively (Murk et al., 2002).

Cell line assays are not limited to mammalian cells. Monteverdi and

Giulio (1999) combined primary liver hepatocytes from Channel catfish (Ictalurus

punctatus) with an enzyme-linked immunosorbant assay (ELISA) to measure the

induction of Vtg. Petit et al. (1999) developed a test system in yeast that

expressed the Rainbow trout estrogen receptor (rtER).

Competitive binding assays measure the displacement of E2 from the

estrogen receptor. The ERa has a higher sensitivity than the ERI for E2








(Gutendorf and Westendorf, 2001). Generally, these competitive binding assays

have a lower sensitivity than other in vitro assays and are not suited as screening

assays for hormonal activity (Table 2-11). In competitive binding assays the

response to agonistic and antagonistic xenobiotics are measured simultaneously,

which accounts for the lower sensitivity (Murk et al., 2002).

Some novel approaches for detecting hormonally active compounds have

been developed. A biosensor has been constructed, which incorporates the

human estrogen receptor a (ERa) into a lipid bilayer with direct contact to a gold

electrode for quantification (Granek and Rishpon, 2002).

A test battery to determine hormonal activity

An optimum test battery to evaluate aquatic toxicity includes algal,

invertebrate, and bacterial components (Rojickova-Padrtova et al., 1998).

Similarly, a successful investigation of hormonal activity should use a suite of

assays to include a cell proliferation assay, a yeast reporter assay, and a

competitive binding assay (Fang et al., 2000; Coldham et al., 1997). When in

vitro assays are combined in an array, then multiple mechanisms may be studied

simultaneously including the effects of metabolism and/or transport (Baker, 2001;

Vinggaard et al., 1999). The sensitivity of in vitro assays to E2 stands as the

hallmark by which assays for hormonal activity are judged. Gutendorf and

Westendorf (2001) demonstrated an E2 sensitivity that increased in the order of

estrogen receptor binding assays (ERa and ERp) < reporter gene assays < cell

proliferation assays (Table 2-11). The ER-CALUX assay has demonstrated the








overall highest sensitivity to E2, with an EC50 of 1.6 ng/L compared to 27 ng/L

with the YES and 1,360 ng/L with ER binding assays (Murk et al., 2002).

Characterizing Hormonal Activity in MSW Landfill Leachates and Other
Environmental Samples

Until recently, little was known about the hormonal activity of MSW landfill

leachates, despite the threat they posed to the environment (Ejlertsson et al.,

1999). Modern MSW landfills are engineered with barriers to restrict the mobility

of the liquid fraction (leachate) of waste and collection systems; however, this

has not always been the case. In the past, the disposal of waste was largely

unregulated allowing for direct release of toxic substances to ground and surface

waters. Some of these older unregulated landfills continue to release leachates

of unknown strength and chemical composition.

In MSW landfills, biological and chemical processes produce leachates

with high concentrations of organic contaminants (Yasuhara et al., 1999).

Shiraishi et al. (1999) demonstrated the presence of compounds with known

hormonal activity in the leachates of Japanese landfills. Behnisch et al. (2001),

using the E-Screen assay (MCF-7 cells), confirmed the hormonal activity of

waste leachates in a Japanese landfill and tracked reductions after treatment

processes. They reported an estradiol equivalency (EE) of the untreated

leachate at 4.8 ng EE/L and after biological and activated carbon treatment at 2.8

ng EE/L. This was equivalent to a 58 % reduction in hormonal activity (Behnisch

et al., 2001). The composition of waste materials in Japanese domestic landfills

differs significantly from U.S. MSW landfills. Due to space constraints and other

considerations, domestic wastes in Japan are first incinerated to reduce volume








and reactivity, then the ash material is landfilled. The composition of the waste in

the landfill studied by Behnish et al. (2001) was primarily inorganic, with about 70

% incinerator ash. Characterizing some of the organic compounds in the

leachate revealed the presence of known hormonally active compounds,

specifically bisphenol A, nonylphenol, and estradiol at 0.13, 2.8, and 0.005 pg/L,

respectively.

Kawagoshi et al. (2002) used a yeast two-hybrid reporter assay

(Nishikawa et al., 1999) to demonstrate the hormonal activity of waste leachate-

contaminated groundwater at an E2 activity equivalent to 27.2 ng/L. Leachates

that were collected from sites for the disposal of solid municipal wastes and

dredged soils were not hormonally active. In a continuing investigation, the

efficiency of the extraction procedures for the recovery of hormonal activity were

evaluated (Kawagoshi et al., 2003). Their extraction procedures used C-18 SPE

columns and were highly efficient for the recovery of hormonal activity. Together

with the high recovery of activity following elution with polar solvents (acetone),

these results implicated non-polar hydrophobic compounds as causative agents

for the activity (Kawagoshi et al., 2003). Based on the comparison of bioassay

results with one raw leachate and acetone extracts of the same leachate,

Kawagoshi et al. (2003) suggested that anti-estrogenic compounds in the

leachate interfered with the recovery of hormonal activity.

Considering the potential for release of MSW leachate from landfills, the

fate of hormonally active compounds in soils is a concem. The mobility of

hormonally active compounds (HACs) (E2, EE2, nonylphenol, octylphenol, and








Table 2-12. The hormonal activity of selected metal species
MCF-7 luciferasee E-Screen
Sub e reporter assay Assay
Substance (ECo) (EC0)
(EC5o) (EC5o)
(nM) (nM)
E2 0.03 0.14
Bis(tri-n- 1.84 0.55
butyltin)
Antimony 16.4 14.8
chloride
Chromium 34.5 33.3
chloride
Lithium 47.1 49.7
chloride
Cadmium 108 176
chloride
Barium
Barium 743 458
chloride
aChoe et al. (2003).


bisphenol A) in soil (93 to 94 % sand) was investigated in lysimeter experiments,

with bioassays (Dizer et al., 2002). Although the leachates (water extracts)

produced by the lysimeters displayed a low hormonal activity, no attempt was

made to quantify the concentrations of hormonally active compounds in the

leachates. Hence, reduced hormonal activity may have resulted from the

adsorption of the HACs to soil particles (Dizer et al., 2002).

While organic substances are the most widely recognized hormonally active

compounds, some heavy metals are also hormonally active (Stoica et al., 2000).

Although the concentrations of heavy metals in MSW landfill leachates are low,

there is a potential for increased leaching of heavy metals as landfills age

(Bozkurt et al., 2000; Flyhammer, 1997). Stoica et al. (2000) reported cadmium

(as CdCl2) activated the estrogen receptor at low concentrations, but at high








Table 2-13. Reported phthalate concentrations in landfill leachates


Compound Concentration Reference
(ng/L)


Bis(ethylhexyl) 1350a Yasuhara et al.,
phthalate 1999
S61400 Yasuhara et al.,
Bisphenol A 61400 1999
Yasuhara et al.,
Dimethyl phthalate 300a araet1999 al.,
Yasuhara et al.,
Dibutyl phthalate 1800 1999haraet
Yasuhara et al.,
Diethyl phthalate 1600 1999raal
Welander and
Diethyl phthalate 479 Henrysson, 1998
Bis(ethylhexyl) 10,850 Welander and
phthalate Henrysson, 1998
Butyl-benzene 2300 Welander and
sulfonamide Henrysson, 1998
"median concentration

concentrations it blocked the binding of estradiol to the receptor. Choe et al.

(2003) used the E-Screen assay and a cell proliferation/ luciferase reporter gene

assay in MCF-7 cells to determine hormonal activity in twenty species of eight

metals (Table 2-12). The metal species were ranked for their hormonal activity in

the cell proliferation/reporter assay as Bis(tri-n-butyltin) > cadmium chloride >

antimony chloride > barium chloride = chromium chloride. Although the

sensitivity of the E-Screen assay was lower, the ranking was similar with Bis (tri-

n-butyltin) > cadmium chloride > antimony chloride > lithium chloride > barium

chloride (Choe et al., 2003).

As previously discussed, phthalates have demonstrated hormonal activity

in a variety of test systems. Their presence in MSW landfill leachates can be

attributed to the composition of the waste material and the increasing use of


~ --`--








excess packaging in consumer goods. Table 2-13 summarizes the

concentrations of various phthalates identified in MSW landfill leachates.

Hormonal activity has been identified in a variety of other environmental samples,

which include tree debarking mill effluents (Mellanen et al., 1996), wastewater

treatment plant effluents (Shang et al., 1999), industrialized rivers (Lye et al.,

1999) and surface waters (Witters et al., 2001). The sources of hormonal activity

with the widest distribution are the WWTPs. This is attributed to the

concentrations of natural and synthetic hormones in domestic wastewater (Table

2-14). In the absence of WWTPs, septic systems represent a source of

hormonally active compounds (Rudel et al., 1998). The largest sources of

hormonal activity in wastewater treatment plants (WWTPs) are the natural and

synthetic estrogens. They are excreted as inactive glucuronide and sulfonide

conjugates; however, rapid deconjugation occurs in WWTPs (Ternes et al.,

1999a). Deconjugation occurs in the presence of the enzyme glucuronidase,

which is abundantly produced by Escherichia coli (Ternes et al., 1999b). This

enzyme is responsible for the degradation of both the glucuronide and sulfonide

estrogen conjugates (Belfroid et al., 1999), but sulfonide to a lesser degree

(Huang and Sedlak, 2001). Roughly 25 % of excreted estrogens occur as

sulfonide conjugates, and their degradation is closely associated with

arylsulfatase enzymes. Low concentrations of these enzymes in treatment plants

is responsible for the greater persistence of the sulfonide conjugates (D'Ascenzo

et al., 2003). Regardless of the reason for incomplete deconjugation of excreted

estrogens, the underestimation of estrogen loads on treatment facilities may








Table 2-14. Concentrations (ng/L) of natural and synthetic hormones in
wastewater treatment plants(WWTPs)
17p3- Estrone Estriol 177a-ethynyl
Estradiol (EI) (E3) estradio Source
(E2) (ng/L) (ngL) (EE2)
(ng/L) (ngnL)L
WWTP ND ND ND 263 ND
Inf Sole et al.,
TP ND ND ND ND 2000
Eff
WWTP 11.6 51.8b 80.4b 3.0bti .,
inr" Baronti et al.,
WWTP 1.4b 18.4b 3.0b 0.4b 2000
Eff~
WTP <0.5-20 <0.5-75 2-120 <0.5-6 Johnson etal.,
WWTP 2000
Eff <0.5-7 <0.5-52 <0.5-28 <0.5-2.2 2000
T 50.7 NM NM NM Matsuial.,
InO Matsui et al.,
WWTP 7.1 NM NM NM 2000
Eft
WWTP 11b 44b 73b NM
n NM D'Ascenzo et
WWTP 1 23b N al., 2003
Eff' 1.6 17b 2.3b NM
WWTP Rodgers-Gray
Eff' 7-88 15-220 NM NM eta
(winter) et al., 2000
(winter)
VWVTP Rodgers-Gray
Eft 4-8.8 27-56 NM NM etal.2
(summer)
WWTP ND 9b M b Ternes et al.,
EffaN 1999a
WWTP 6b 3b NM 9b Temes et al.,
EfF 1999a
WWTP Belfroid et al.,
Eff. 0.9 4.5 NM <0.3 1999
WWTP Desbrow et
Efr' 2.7-48 1.4-76 NM 0.2-7 al.,1998
WWTP Huang and
E0.2-4.1 NM NM 0.2-2.4 Sedlak,2001
WWTP Snyder et al.,
ElP 1.9-14.6 NM NM <0.05-3.0 ydetal.
Abbreviations: Wastewater treatment plant, WWTP; Inf, influent; Eff, effluent; ND, not detected;
NM, not measured. indicates median value. Samples collected from: d Germany, CCanada,
dUnited Kingdom, USA, 'Netherlands, 'Italy, hSpain, Japan








result (Johnson et al., 2000).

To preface any discussion of reported concentrations of hormonally active

compounds in the environment, it is important to consider the detection limits of

the analytical methods. Often the analytical methods for the identification and

quantification of low-level organic contaminants are ineffective and grossly under

predict environmental burdens (Castillo and Barcelo, 1999). For example, while

some laboratories have reported detection limits for E2, El, E3, and EE2 as low as

0.1-0.6 ng/L for surface waters and 0.1-2.4 ng/L for wastewaters (Belfroid et al.,

1999), others have reported detection limits up to three orders of magnitude

higher for E2(250 ng/L), E1(100 ng/L), E3(50 ng/L) and EE2 (500 ng/L) in WWTP

effluents (Sole et al., 2000).

Alterations in the sexual and developmental characteristics of aquatic

species have been reported worldwide and attributed to the release of

hormonally active micro-organic contaminants (Sheahan et al., 2002b; Desbrow

et al., 1998; Jobling et al., 1993). Although researchers have reported a range of

estrogens in WWTP effluents, concentrations are generally at the low ng/L level.

Chiefly, E2 and EE2 as the hormones with the greatest reported activity have

been identified in effluents at <0.2 to 88 and <0.2 to 9 ng/L, respectively (Table 2-

14). The metabolite El has been identified in effluents at concentrations of 1.4 to

220 ng/L (Desbrow et al., 1998). While E2 is widely recognized as the strongest

estrogen, its metabolites are also potent, with E1 about 1.5 times less active

(Jurgens et al., 2002) and E3, the weakest metabolite, also inducing hormonal

affects albeit orders of magnitude less (Metcalfe et al., 2001). The synthetic








estrogen, EE2, while found at lower concentrations in wastewater (Johnson,

2000) has an activity comparable to that of E2 (Larsson et al., 1999). The lower

activity of E1 is offset by its extensive presence, and gives rise to concerns about

the equivalent E2 activity.

Rodgers-Gray et al. (2000) investigated the influence of seasonal changes

on activated sludge biology and its subsequent effect on the removal of E2 and

E1 from an activated sludge WWTP in England. During the cooler winter months,

concentrations of E2 and E1 ranged from 7 to 88 ng/L and from 15 to 220 ng/L,

respectively. In contrast, there were lower concentrations reported in the

summer months with E2 concentrations ranging from 4 to 8.8 ng/L and E1

concentrations ranging from 27 to 56 nglL. Overall, the concentrations of E1

exceeded those of E2, indicating a greater recalcitrance of E1 to biological

treatment (D' Ascenzo et al., 2003). Few studies have looked at E3, and its

effects may be under estimated.

A limited number of studies have evaluated the concentrations of

endogenous and synthetic estrogens in influents and corresponding effluents of

WWTPs. Despite reported removal rates of up to 90 %, concentrations of

estrogens remain at threshold levels for inducing hormonal effects. Six activated

sludge treatment facilities in Italy (Cobis, Fregene, Ostia, Roma Sud, Roma Est,

and Roma Nord) have been extensively studied by three research teams over a

two-year period. Baronti et al. (2000) reported mean influent concentrations for

the six facilities at 51.8, 11.6, 80.4, and 3.0 ng/L for E1, E2, E3, and EE2,

respectively. Following biological treatment these concentrations were reduced








Table 2-15. Reported concentrations (ng/L) of natural and synthetic estrogens in
surface waters
17p- Estrone Estriol 17a-ethynyl
estradiol (E) (E) estradiol Source
(E2) (ng/L) (ng/L) (EE2)
(ng/L) (nglL)
Surface water <0.5 ND NM <0.5 Tees et al.
1999a
Belfroid et al.,
Surface water <0.3-5.5 NM NM <0.3-4.3 Be et al
1999
Surface water 3.6-5.2 NM NM <0.05-1.4 Snyder et al.,
1999
Surface water <0.05-0.8 NM NM <0.05-0.07 Hang and Sedlak,
2001


to 18.4, 1.4, 3.0, and 0.4 ng/L for E1, E2, E3, and EE2, respectively. These results

are consistent to those reported by Johnson et al. (2000) and D' Ascenzo et al.

(2003), although the later did not measure EE2. Notably, E1 displayed the

greatest range of concentrations (Baronti et al., 2000; Johnson et al., 2000; D'

Ascenzo et al., 2003) and remained the most prevalent estrogen following

treatment. The only other study to evaluate multiple hormone concentrations in

influents and corresponding effluents was conducted with four WWTPs in Spain

(Sole et al., 2000). While analyses were conducted for E1, E2, E3, and EE2, the

only hormone detected was E3 and then only in the influents of 2 WWTPs at

approximately 262 ng/L (Sole et al., 2000). These levels were nearly double the

maximum concentrations detected in the Italian WWTPs (Baronti et al., 2000).

Comparatively, low ambient levels of natural and synthetic hormones have

been detected in surface water samples, however, the potential for extensive

contamination of surface waters exists from animal manure's (Casey et al., 2003)

and WWTP discharges (Kolpin et al., 2002). In surface waters, E2 concentrations





53


ranged from <0.05 to 5.5 ng/L, while those for EE2 were <0.05 to 4.3 ng/L (Table

2-15). As a point of reference, altered sexual characteristics are induced in male

rainbow trout (Oncorhynchus mykiss) at -1.5 ng/L EE2 (Larsson et al., 1999).

Additionally, threshold doses of 1 to 10 ng/L and 25 to 50 ng/L for E2and Ei,

respectively, have been reported for the induction of hormonal effects in O.

mykiss (Metcalfe et al., 2001).













CHAPTER 3
TOXICITY OF LEACHATES FROM FLORIDA MUNICIPAL SOLID WASTE
(MSW) LANDFILLS USING A BATTERY OF TESTS APPROACH

Introduction

The State of Florida currently generates more than 25 million tons of

municipal solid waste (MSW) a year. Fifty-six percent of this waste is disposed in

engineered Class I landfills (FDEP, 2000). The state has sixty-one Class I

landfills (permitted to accept only MSW) that are lined and contain systems for

the collection and transport of waste leachates, that are then subsequently

subjected to biological treatment. Researchers have extensively characterized

the chemical and physical characteristics (Townsend et al., 1996; Booth et al.,

1996; Gettinby et al., 1996) and biological toxicity (Plotkin and Ram, 1984;

Ferrari et al., 1999; Ernst et al., 1994) of waste leachates world-wide. These

waste leachates are a complex mixture of both inorganic (e.g., heavy metals,

ammonia) and organic substances (e.g. pesticides and chlorinated

hydrocarbons). It has been suggested that exposure to MSW landfill leachates

may pose as great a cancer risk as does the exposure to industrial waste

leachates (Brown and Donnelly, 1988) due to their mutagenic properties (Beg

and AI-Muzaini, 1998). The genotoxic potential of MSW landfill leachates was

shown to be higher than that for industrial wastewater, groundwater or drinking

water samples (Helma et al., 1996).








When evaluating the toxicity of complex effluents, the use of a battery-of-

tests approach allows for multiple mechanisms of action to be evaluated

simultaneously (Deanovic et al., 1999; Rutherford et al., 2000). A battery-of-

tests approach with algal, crustacean, and bacterial assays was used to

successfully characterize landfill leachate toxicity (Rojickova-Padrtova et al.,

1998; Clement et al., 1996). No characterization of MSW landfill leachate toxicity

is complete until toxicological assays are combined with analytical procedures for

chemical characterization (Lambolez et al., 1994).

The unique sub-tropical climate in Florida with generally abundant rainfall

and warm temperatures reduces the chemical strength of MSW landfill leachates

(Reinhart and Grosh, 1998). Although researchers have characterized the

composition and site-specific parameters for MSW landfills throughout Florida,

the biological effects of these leachates have not been assessed (Reinhart and

Grosh, 1998). The toxicity of MSW leachates from separate landfills, while

related, may differ due to specific characteristics of the wastes, e.g. pH,

temperature, ammonia levels, presence of recalcitrant organic substances, and

microbiological activity. Little information is currently available conceding the

toxicity of MSW landfill leachates in Florida and such a database of information

could be useful in evaluating leachate treatment options and reuse possibilities

(Ward et al., 2000). The research community now recognizes the importance of

using a tandem approach, with both biological and chemical analyses, when

analyzing environmental samples to achieve a better understanding of the

possible causes of toxic effects.












Site 5

Site 1 Site4

Site 2 and 3
S\ Site 6










Figure 3-1. Locations of the MSW landfills for the collection of leachates in
Florida

The objectives of this research were to 1.). characterize the toxicity of

Florida MSW landfill leachates, 2.). evaluate the use of a battery of algal,

invertebrate and bacterial toxicity assays with Florida leachates, 3.). characterize

the chemical composition of the MSW leachates, and 4.). determine relationships

between selected chemical components and leachate toxicity. Six landfill sites

in north and north-central Florida were sampled monthly over a six-month

sampling period. The sites sampled represented a variety of factors, including

rural and urban areas, some industrial activity, leachate recycle, enhanced

biological treatment in-situ, and those sites currently accepting waste and capped

sites.








Table 3-1. Amount of MSW generated and landfilled at six landfill sites in Florida
Landfill MSW Collected Amoun Waste Landfill
Site (Tons/year)a Land d Type

1 235,662 65 Leachate recycle

2 226,477 69 Rural

3 NAb NA Rural/capped

4 317,694 68 Semi-urban

5 10,197 83 Regional
Urban/enhanced
6 1,890,112 73 biological treatment
'The data presented represents information collected in 1998. "Data were not available (NA) for
site 3 a capped landfill site, no longer permitted to accept MSW.

Materials and Methods

Leachate Collection

Municipal solid waste (MSW) leachates were collected from six sites

located in five landfills in central Florida, USA. The sites were designated as 1

through 6. Site 2, an operating landfill unit, and site 3, a capped landfill unit, are

located at the same landfill (Figure 3-1). Capped landfill units no longer accept

waste materials and are surrounded by a high-density polyethylene (HDPE) liner

to prevent the infiltration of water and the potential for subsequent escape of

leachates. Table 3-1 summarizes the rates of municipal waste disposal at the

sites under study (FDEP, 2000). MSW landfill leachates were collected from

leachate collection wells using a Teflon baler. One sample was collected at each

site, and then apportioned to separate containers for chemical analysis and

toxicity assays. Leachates for chemical analysis were collected in polyethylene

or glass containers and preserved according to U.S. Environmental Protection








Agency (USEPA, 1993b). Samples for toxicity analysis were collected in plastic

cubitainers, transported to the lab on ice and immediately stored at 4C until

sample analysis, within 1 to 2 days.

Chemical and Physical Characterization of Leachates

The chemical/physical characterization of the MSW landfill leachates

began with field measurements that included pH and temperature (Orion, Model

290A), conductivity (HANNA Instruments, Model H19033), dissolved oxygen

(DO) (YSI Inc., Model 55/12 FT), and oxidation/reduction potential (ORP)

(Accumet Co., Model 20). In the laboratory, the MSW landfill leachates were

analyzed for a number of standard chemical and physical parameters, which

included alkalinity, biochemical oxygen demand (BOD), chemical oxygen

demand (COD), ammonia and sulfides, according to methods described by

USEPA (1993b) and APHA (1999). Leachates for metal analysis were digested

and analyzed by Inductively Coupled Plasma (ICP) (Thermo Jarrell Ash, Model

Enviro 36). For ion analysis, a Dionex ion chromatograph (Dionex, Model DX-

500) was used. Total ammonia (NH4* + NHa) and un-ionized ammonia (NHs)

were analyzed by a selective ion probe (Accumet, Model 15).

Maintenance of Test Organisms

Pseudokirchneriella subcapitata

Pseudokirchneriella subcapitata (previously known as Selenastrum

capricomutum) is a freshwater unicellular green algae routinely utilized in both








Table 3-2. Components of the preliminary algal assay procedure (PAAP)
medium

Growth Assay
medium medium
MACRO SALTS
Magnesium sulfate X X
(MgSO4*7H20)
Magnesium chloride X X
(MgCI2*6H20)
Calcium chloride X X
(CaCI2 2H20)
Sodium bicarbonate X X
(NaHCOs)
Sodium nitrate x
(NaNO3)
Potassium phosphate X X
(KH2PO4)
Disodium(Ethylene-
dinitrilo)tetraacetate X
(EDTA)
TRACE METAL SOLUTION
Zinc chloride X X
(ZnCI2)
Cobalt chloride X X
(CoCI2* 6H20)
Sodium molybdate X X
(Na2MoO4 2H20)
Cupric chloride X X
(CuCI2* 2H20)
Boric acid
(H3BO3)
Manganese chloride X X
(MnCI2)
Ferric chloride
(FeCI3*6H20)


FDEP (FDEP, 1997) and USEPA protocols (USEPA, 1978; USEPA, 1994a). The

algae cultures were started from an original algae seed graciously provided by

Hydrosphere Research. Subsequent cultures of algae were maintained in the

laboratories at the University of Florida. The algae growth medium was prepared








according to FDEP (1997) and is referred to as the preliminary algal assay

procedure (PAAP) medium. The components of the PAAP are listed in Table 3-

2. The PAAP was prepared by combining 1 ml of each of the macrosalts with 1

ml of the trace metal solution in a 1-L volumetric flask. The flask was filled with

DDI water and thoroughly mixed. The pH of the PAAP medium was adjusted to

7.5 0.1 with either 0.1 NaOH or 0.1 N HCI. The PAAP medium was then filter-

sterilized (0.45 pm membrane filter) and stored under refrigeration.

The algal cells were grown under a specially designed light unit

(constructed by Martin Dolley). The light unit consisted of a wooden platform (3

feet by 4 feet) supported with legs (4 feet), but with open sides. Three

fluorescent light fixtures (3 1/2 feet long) were suspended from the wooden

platform on adjustable chains. The light intensity inside the unit was regulated by

adjustment of the chain length. Black plastic sheeting surrounded the light unit,

and the interior of the sheeting was lined with aluminum foil to maximize the light

reflection and minimize temperature fluctuations.

When propagating the algal cells, cultures were grown in sterile 1- or 2-L

glass erlenmeyer flasks. The flasks were filled to the three-quarter mark with

sterile PAAP medium and then approximately 100 ml of an algae culture (3 to 5

days old) was added. A 10-ml glass pipette was placed in the flask, which was

then wrapped with parafilm to seal the top of the flask. The flask was swirled

vigorously to mix, placed under the light unit, and then the culture was aerated by

attaching a small hose in series with filters (0.2 im, Acrodisc) to the glass

pipette. A small aquaculture pump supplied air to the algal culture and








continuous gentle mixing of the algae medium was maintained. The algae cells

were cultured for up to one week at 25oC under constant illumination (400 40 ft-

c). After 3-5 days in the light unit, the algae cells were harvested for toxicity

assays, and after 1 week the cells were transferred to start new algae cultures or

were recovered for use as aquatic invertebrate food. The 1-week old algae cells

were transferred to wide-mouth glass containers, covered loosely and placed in

the refrigerator. After settling for approximately one week, the algae cells were

recovered by siphoning off the overlying spent medium. The recovered algae

cells were then resuspended in a minimum volume of DDI. Past experience has

shown that the washing of the algal cells was not required. Algae cultures were

periodically checked for uniformity by transferring small volumes of cells to glass

slides and visually inspecting cell morphology under a phase contrast

microscope. All settled cultures were combined and a final algal cell density was

determined with a hemacytometer. The algae cell density was maintained at 3.5

X 107 cells/ml with DDI before use as aquatic invertebrate food.

Ceriodaphnia dubia and Daphnia pulex

The aquatic invertebrates, Ceriodaphnia dubia and Daphnia pulex are

both members of the family Daphnidae (commonly referred to as daphnids) and

have similar species distributions and life cycles (USEPA, 1993a). Traditionally,

D. pulex (or D. magna) were the invertebrates of choice for determining aquatic

toxicity. Over the past 20 years, assays with C. dubia have increased in

popularity. This is directly related to the greater sensitivity of the C. dubia to

aquatic toxicants (Versteeg et al., 1997). Both invertebrate species are able to








reproduce by parthenogenesis, which ensures a continuous supply of identical

offspring.

Preparation of aquatic invertebrate food

The aquatic invertebrate cultures (C. dubia and D. pulex) were fed a

yeast, cereal leaves, and trout chow (YCT) based food. The YCT was prepared

over a one-week period beginning with the digestion of the trout chow pellets.

The trout chow digestion was performed in a bottomless 3-L inverted plastic

container by combining a 0.5-g portion of trout chow with 1-L of distilled water

(DDI). To ensure adequate mixing, an aquaculture pump was attached to a

glass pipette secured in the inverted cap of the plastic container. After 7 days,

the digestion was complete and the container was covered and placed in the

refrigerator to settle for at least 1 hour. No water was added during the digestion,

despite evaporative losses; however, water was added if needed in the final step

of food preparation to reach the desired solids content. The supernatant from the

trout chow digestion was filtered through a fine mesh, e.g. nylon hose, and then

reserved. Simultaneously, on day 6 of the trout chow digestion a 5-gram portion

of cereal leaves (Sigma) was combined in a blender with 1-L of DDI and mixed

on high speed for 5 minutes. The cereal leaf mixture was covered and reserved

in the refrigerator overnight to settle. Finally, on day 7 of the digestion, a 5-gram

portion of dry yeast (Fleischmann" or equivalent) was combined with 1-L of DDI

water and mixed well on a magnetic stir plate. Equal volumes of the yeast

solution, cereal leaf supematant, and trout chow filtered supernatant were

combined and mixed thoroughly. The YCT food was apportioned into 50-ml








plastic bottles, labeled, and stored in the freezer (-400C) until needed. YCT food

was stored in the refrigerator and unused portions were discarded after 1 week.

The total solids (TS) content of the YCT was maintained between 1.7 and 1.9 g

solids/L by the addition of DDI as needed. The C. dubia and D. pulex cultures

were also fed P. subcapitata algae cells (3.5 X 107 cells/mi), as previously

described.

Maintenance of aquatic invertebrate cultures

Starter cultures of C. dubia and D. pulex were graciously donated by

Hydrosphere Research (Gainesville, FL). The aquatic invertebrates were

cultured in dedicated glassware, which was maintained separately and

thoroughly washed and rinsed between each usage. Daphnids were cultured in

reconstituted moderately hard water (MHW), which was composed of NaHCO3,,

96 mg; CaSO4* 2H20, 60 mg; MgSO4, 60 mg; and KCI, 4 mg per liter of DDI

water. The MHW had the following specifications: pH, 7.4 -7.8; hardness, 80 -

100 mg/L as CaC03; and alkalinity; 60 -70 mg/L (USEPA, 1993a).

Aquatic invertebrate cultures were maintained by adding neonates (< 24-

hour old) of C. dubia or D. pulex to 1-L glass beakers containing MHW. The

daphnid beakers were kept in an environmental chamber (Percival, model E-

30BX) at 20 + 20C and with a light regime of 16 hours of light and 8 hours of

dark. The daphnid cultures were fed 7 ml of YCT and 7 ml of algae cells per liter

of invertebrate culture. Invertebrate cultures were culled daily to remove

neonates and ensure a population with a uniform age distribution. Following the

removal of neonates for toxicity assays, any remaining neonates were used to









Prepare leachate dilutions with
PAAP minus EDTA






Transfer 50 ml of leachate, or its dilution, to
triplicate 125-mi erienmeyer flasks






Spike each flask with 1 ml
P. subcapitata (500,000 cells/ml)







Place erlenmeyers under growth light for
96 hours, shaking daily






Count number algae cells with
hemacytometer and phase/contrast
microscope


Figure 3-2. Flowchart for the P. subcapitata assay








start new cultures or were discarded. Adult females were retained for neonate

production for a period no longer than two weeks.

Toxicity Assays

Pseudokirchneriella subcapitata

The chronic toxicity of the MSW landfill leachates was evaluated

according to the protocols of the 96-hour P. subcapitata assay (USEPA, 1994a).

The leachates were filtered with glass fiber (Whatman, GFIB) and membrane

filters (0.45 pm). The glass fiber pre-filters were used to minimize clogging of the

membrane filter. The dilution media for the algal assays was a PAAP solution

prepared without the disodium (Ethylenedinitrilo) tetra-acetate (EDTA). EDTA

has been shown to form complexes with heavy metals, which confounds assay

results when metal toxicity is suspected. The PAAP growth medium requires the

addition of EDTA, because its presence is crucial for the uptake of many

micronutrients (USEPA, 1994a).

For each algal assay, five dilutions were prepared in a laminar flow hood

with PAAP minus EDTA at a dilution factor of 0.5 (Figure 3-2). A 50-ml aliquot of

the leachate, or its dilution, was added to triplicate 125-ml sterile erlenmeyer

flasks with styrofoam stoppers. The flasks were then inoculated with a 1-ml

aliquot of algae cells (500,000 cells/ml). The inoculum was prepared by

centrifuging a 4 to 5-ml portion of algae stock (3-5 days old) at 4000 rpm for

fifteen minutes. The supernatant was discarded, and the algae cells were

resuspended in PAAP minus EDTA and mixed by vortexing gently. Using a

hemacytometer and a phase contrast microscope, the cell density was

determined. The volume of resuspended algae cells required to prepare an








algae seed with a density of 500,000 cells/ml was determined by the following

equation:

Number test flasks x Vol. test Solution per flask x 10,000 cells per ml
Cell density (cells per ml) in the stock culture


Based on the number of assay flasks and a volume of 1-ml of seed per

flask, the algae cell inoculum was prepared. As an example, if the assay

required 18 flasks containing 50 ml of leachate per flask and the stock algal

culture density was 106 cells/ml, then the required volume of stock culture to

produce 18 ml of algae inoculum is 9 ml. Combining a 9-mi portion of the algae

stock culture (500,000 cells/ml) with a 9-mi portion of PAAP without EDTA

provided the required 18-ml portion of algae inoculum needed for the assay.

The erlenmeyer flasks containing the leachate, or its dilution, and the algal

seed were placed under the light unit (previously described) at 25C. Constant

illumination (400 40 ft-c) was maintained for 96 hours, and the flasks were

rotated and mixed daily by swirling manually. At the conclusion of each assay,

the algae cell density in each flask was measured by algal cell counts using a

hemacytometer and a phase/contrast microscope. Growth inhibition was

determined by comparing the number of algae cells in the leachate containing

flasks to the number in the control flasks. The leachate concentration that

produced a 50 % inhibition (ICso) of algal growth was determined by graphing the

cell density in each flask versus the leachate concentration.









Prepare leachate dilutions with
MHW






Transfer 10 neonates to assay cup; add 20
ml of sample or its dilution






Expose neonates to leachate
for 48 hours







Observe neonates for
death /immobilization


Figure 3-3. Flowchart for the C. dubia assay

Ceiodaphnia dubia and Daphnia pulex

The acute toxicity of the MSW landfill leachates were evaluated with C.

dubia and D. pulex.in the standard 48-hour acute toxicity protocols (USEPA,

1993a; APHA, 1999). Basically, the assay protocols were identical for the two

species of aquatic invertebrates. Before the leachates were evaluated for

toxicity, they were pre-filtered (Whatman, GF/B) to remove large particles. The C.

dubia and D. pulex assays follow similar conditions. Prior to the start of each








aquatic invertebrate assay, the neonates (< 24hrs) were separated from the adult

daphnids and fed a mixture of 7 ml YCT/liter and 7 ml algae/liter. After feeding,

10 neonates were transferred to each test container (30-ml plastic cups) using a

small wide-mouth plastic pipette to minimize the transfer of culture water (Figure

3-3). The leachate dilutions were prepared with MHW at a 0.5 dilution factor, and

the leachate or its dilution was added at 20-ml volumes to triplicate cups

containing the 10 neonates. Containers filled with MHW were used as the

negative controls. The test containers were placed in a water bath at 20 2 C

for 48 hours, with a loose covering to allow light penetration and prevent settling

of air particles. Neonates were exposed to ambient lighting and were not fed

during the assay.

After 48-hours, the invertebrate test containers were placed on a light

table for the determination of viable organisms. The light table was designed to

sit on the laboratory bench (constructed by Martin Dolley). It was composed of a

wooden box with a plexiglass top and three fluorescent lights. The fluorescent

lights were mounted inside the box and below the plexiglass top, so as to

illuminate the work surface. Test containers were swirled gently and neonates

with the power to swim away from the center of the container were counted as

dead/immobilized. Mortality greater than 10% in the controls negated the assay

results.









Add MOAS to leachate for final
concentration of 2% NaCI





Prepare serial dilutions of leachate in with
Microtox diluent





Aliquot 50 g1 of bacterial reagent into
cuvettes; measure initial bioluminescence



JD-

Add 450 i1 of leachate, or its dilution, to
cuvettes containing bacterial reagent;
mix





Measure final bioluminescence after
15-minute exposure


Figure 3-4. Flowchart for the MicrotoxT assay








MicrotoxT

The MicrotoxT toxicity analyzer is a commercially available toxicity system

that measures toxic effects by changes in bacterial (Vibrio fischen)

bioluminescence (Beckman Instruments, 1982). The assay kit includes a diluent,

the freeze-dried Microtox bacterial-reagent, a reconstitution solution, and an

osmotic adjusting solution. Each leachate was assayed in duplicate and each

assay also included a duplicate control (DDI water) (Figure 3-4). A preliminary

investigation indicated that a 15-minute exposure produced the highest

sensitivity, which agrees with the reports of other researchers (Plotkin and Ram,

1984).

The Microtoxm analyzer combines a pre-cooling well for storage of the

reconstituted bacteria reagent during the assay, an incubator well block to cool

the cuvettes containing the sampless, and a turret containing the photomultiplier,

which quantifies the bacterial light output. Before the assay, the analyzer is

brought to thermal equilibrium and the performance calibrated. Clean glass

cuvettes are added to the incubator well block and the pre-cooling well. The

bacterial reagent is rehydrated with 1 ml of reconstitution solution and transferred

to the cuvette in the pre-cooling well. The incubator well block has a grid pattern,

with columns 1 to 5 and rows A to C, row A is designated for the preparation of

the sample dilutions and rows B and C for sample testing in duplicate. Column 1

is dedicated to assay blanks and contains only the bacterial reagent and the

Microtox diluent.








The leachate dilutions are prepared from right to left in columns 5 to 2,

and well A5 corresponded to the highest leachate concentration. The dilution

series is prepared by first adding 1000 pl of the Microtox diluent to the cuvettes in

row A of columns 5 to 2. Then 1000 pl of the leachate is added to columns 4 and

5 of row A and mixed by repeatedly pipetting 500 pl of the mixture and aspirating.

A 1000-pl aliquot from the cuvette in column 4 was transferred to the cuvette in

column 3, mixed, and then a 1000-1 aliquot from column 3 was transferred to

column 2. After column 2 was mixed and aspirated, a 1000-1d aliquot was

discarded. The cuvettes were allowed to cool for approximately 10 minutes.

Following the addition of the reconstituted Microtox bacterial reagent (50 pl) to

wells B1 through B5 and C1 through C5, the cuvettes were allowed to reach

thermal equilibrium, approximately 15 minutes. The luminescence of the

bacterial reagent in each cuvette was determined by placing the cuvette in the

turret and turning the handle to the read position. The luminescence output from

the bacterial reagent was read on the digital panel meter (DPM) at the front of the

analyzer.

The pre-exposure bacterial luminescence was measured beginning with

the cuvette in row B1, then C1, B2, C2, B3, C3, B4, C4, B5, and C5. Next, a

450-pl aliquot of the sample or its dilution was transferred from the cuvettes in

row A to the duplicate cuvettes in rows B and C and mixed, beginning with

column 5. After 15 minutes, the cuvette was placed in the turret and the

luminescence output from the bacteria exposed to the sample or its dilution was

read on the DPM. The loss of bioluminescence is described by gamma (r), which








is measured as the ratio of light lost to light remaining following leachate

exposure.

Data Analysis

The toxicity assay results were expressed as the concentration of leachate

that produced a 50 % effect in the bioassay. The endpoints of the assays were

different and included inhibition of bioluminescence (Microtox as ECso), lethality

or death (C. dubia as LC5o), and growth inhibition (P. subcapitata as IC50). The

results of the 96-hr P. subcapitata assays were determined by graphical

interpolation. The LC5ofor the C. dubia assay was determined using the USEPA

data analysis software (USEPA, 1994b). Bioassay results were presented with

one standard deviation for the C. dubia and P. subcapitata assays. MicrotoxT

test results were determined by least square regression analysis of the natural

log of the sample concentration vs. the natural log of gamma (ratio of light lost to

light remaining). Results were presented as the concentration of leachate

causing 50% inhibition of bioluminescence (gamma). The Microtox assay was

performed in duplicate; therefore, determinations of standard deviations were not

valid. Data was evaluated by least square regression, student's t-test, or the F-

test (Excel, Microsoft 2000), as appropriate. The ECso, ICso, and LC50 results

were transformed to toxicity units (TU); according to the following;

100
TU (unitless) =00
EC, or IC, or LCs,

When appropriate, data were log transformed, for evaluation of linear

relationships.








Table 3-3. Physical and chemical characteristics of MSW landfill leachates at six
sites in Florida
Site Site Site Site Site Site
Parameter 1 2 3 4 5 6
7.5 7.0 7.2 7.5 7.7 7.6
pH (7.3-7.6) (6.5-7.4) (6.8-7.5) (7.3-7.8) (7.5-7.9) (7.3-7.8)
Temperature 35 26 27 26 23 32
(C) (32-38) (25-29) (26-27) (24-28) (19-28) (31-33)
Conductivity 14.1 6.2 5.2 7.6 8.3 9.6
(mS/cm) (13.2-15.2) (3.1-8.4) (2.6-9.5) (6.5-8.6) (3.2-12.1) (1.0-14.2)
Alkalinity 6213 2407 1494 3238 2503 5500
(mg/L as (6075- (1725- (1000- (3125- (1200- (250-8775)
CaCO3) 6625) 2975) 2050) 3350) 4050)
CBODa 140 22 15 66 42 NMc
(mg/L) (89-204) (14-30) (13-21) (55-77) (13-66)
CODb 1850 636 351 1165 857 1245
(mg/L) (107- (522-827) (242-440) (902-1616) (416-1208) 13960)
Sulfide 0.3 20 19.7 47 2.2 3580
(g/IL) (0.08-0.8) (17-23) (15-24) (42-56) (0.1-15.8) 5
Al 0.22 <0.2 0.4 0.2 3.5 10.8
(mg/L) (0.07-0.4) (0.17-0.47) (0.17-0.32) (0.1-4.5) (4.2-23.7)
Cu <0.07 <0.07 <0.07 <0.07 <0.07 <0.07
(mgIL)
Cd 0.01 <0.015
(mCd <0.015 <0.015 <0.015 <0.015 (0.006-
(mg/L) 0.02)
Pb <0.06 <0.06 <0.06 <0.06 0.06 <0.06
(mrgL) (0.04"0.1)
Zn 0.04 <0.03 <0.03 <0.03 0.05 0.45
(mg/L) (0.020.06) (0.03-0.07) (0.050.63)
As 0.16 <0.09 <0.09 <0.09 0.12 0.14
(mg/L) (0.1-0.25) (0.03-0.2) (0.1-0.2)
Cr 0.05 <005 <0.05 <0.05 0.07 0.18
(mg/L) (0.03-0.08) (0.02-0.1) (0.05-0.39)
Ba <0.05 0.05 0.07 0.09 0.1 0.1
(mg/L) (0.03-0.1) (0.03-0.1) (0.08-0.11) (0.03-0.2) (0.08-0.15)
Fe 7.13 12.5 4.7 7.0 7.5 50.4
(mg/L) (5.9-9.5) (9.3-21.4) (2.2-9.2) (3.7-9.9) (3.9-13.6) (3.2-86.5)
Na 1495 735.7 390 1001.5 1039 1663
(mg/L) (1402-
(m1) ( (464-928) (352-445) (989-1014) (554-1890) (76-2670)
K 555 343 202 440 201 644
(mg/L) (520-589) (234-414) (184-220) (431-444) (93-316) (45-1011)
Results are shown as the mean and (range). Abbreviations: "BOD,biological oxygen demand;
bCOD, chemical oxygen demand; CNM, not measured.








Results and Discussion

Chemical Analysis of MSW Leachates

The physical and chemical characteristics of the MSW landfill leachates

are summarized in Table 3-3. Although the mean pH values were near neutral,

with a range from 7.0 to 7.7, lower pH values were measured in some samples.

The toxicity and bioavailability of some leachate toxicants, especially heavy

metals, are pH dependent (Schubauer-Berigan et al., 1993). The alkalinity of the

leachates was highly variable and ranged from 1,494 mg/L as CaC03 at site 3 to

6,213 mg/L as CaCO3 at site 1. These concentrations are typical for landfill

leachates in the early phases of waste stabilization (Kjeldsen et al., 2002).

Conductivity measures ionized molecules in solution, including both

cationic and anionic species. In the MSW landfill leachates, the range of

conductivity values was wide from a low of 5.2 mS/cm at site 3 to a high of 14.1

mS/cm at site 1. Specifically, the mean sulfide concentrations varied widely from

a low of 0.31 ig/L at site 1 to a high of 3,580 pg/L at site 6. Similar mean sulfide

concentrations of 20 pg/L at site 2 and 19.7 pg/L at site 3 were reported. Since

the leachates from sites 2 and 3 were collected from the same landfill and the

waste compositions were comparable, then similar sulfide levels were expected.

This was also true for most of the other chemical characteristics of the sites 2

and 3 leachates, with a few exceptions. The levels of alkalinity in the site 2

leachates ranged from 1725 to 2975 mg/L as CaCO3, while in the site 3

leachates the range was from 1000 to 2050 mg/L as CaCO3. Alkalinity

concentrations generally decrease with increasing landfill age, as the buffering








capacity is consumed by the production of organic acids. Differences in the

BOD/COD ratios were slight, but suggested a higher degree of organic matter

degradation in the leachates from site 3. The BOD/COD ratio was 0.03 in the

leachates from site 2 and 0.04 in the leachates from site 3. The lower

concentration or inorganic cations in the site 3 leachates was due to the "wash-

out" effect typical of older leachates (Kjeldsen et al., 2002). While organic

components in the leachates are degraded by biological activity, inorganic

constituents decrease over time with increased rates of leachate production

(Cameron and Koch, 1980; Chian and DeWalle, 1976). Some variations in the

chemical composition of MSW landfill leachates are expected based on age,

waste degradation and site-specific factors (Ragle et al., 1995).

Generally, the concentrations of heavy metals in the MSW landfill

leachates were below analytical detection limits (Table 3-3). However, some

leachates contained elevated concentrations of heavy metals. Aluminum was

detected in most of the leachates at least once over the 6-month investigation. In

the leachates from site 6, aluminum was detected in each of the leachate

samples collected with a range from 4.2 to 23.7 mg/L. The heavy metals copper

and cadmium were not detected (detection limits of 0.07 mg/L and 0.02 mg/L,

respectively) in any of the leachates analyzed. While lead concentrations were

less than 0.06 mg/L in the leachates from sites 1, 2, 3, 4, and 6, in the leachates

from site 5 lead concentrations ranged from <0.06 to 0.1 mg/L.

The leachates from site 6 contained zinc concentrations that ranged from

0.05 to 0.63 mg/L. Zinc concentrations in the leachates from site 1 exceeded the









2000 50



40
1500

1300



o20 E
10oo





500
10


N N
0 __ i i4 0
Feb March April May June July


Figure 3-5. Concentrations of total (NH4+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 1 over a 6-month
sampling interval. NM indicates sample not measured

detection limit of 0.03 mg/L in five of the six-leachate samples analyzed and

ranged from < 0.03 to 0.06 mg/L. The highest barium levels were measured in

the leachates from sites 2 and 3, although neither exceeded 0.1 mg/L. Similar

concentrations of barium were reported in the leachates from sites 4, 5, and 6,

but barium concentrations were below the detection limit in the leachates from

site 1. In relation to mean arsenic levels in the leachates, 0.16, 0.12, and 0.14

mg/L were identified at sites 1, 5, and 6, respectively. A similar patten was

shown with chromium, but in this case the leachates from site 6 displayed the

highest concentrations with a range from 0.05 to 0.39 mg/L.









500 5



400 4



300 3E
o E-

EC
200 2.5


100 1


0 _0
Feb March April May June July
Figure 3-6. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 2 over a 6-month
sampling interval. NM indicates sample not measured

The concentrations of iron in the MSW landfill leachates were high and

site-specific. The lowest mean iron concentrations were reported for the

leachates from site 3 with a mean of 4.7 mg/L, while with the leachates from site

6 mean concentration of 50.4 mg/L was reported. High iron concentrations in the

MSW landfill leachates directly impact the bioavailability of heavy metals via

influences on the formation of insoluble precipitates. Metallo-sulfide precipitates

reduce metal toxicity; however, at high concentrations iron may out-compete the

toxic metals for binding sites on the sulfide molecule (Bozkurt et al., 2000).

Concentrations of other inorganic cations were also high in the MSW landfill

leachates. Sodium levels ranged from a low mean of 390 mg/L at site 3 to high









250



200 4


0












March April May June July

Figure 3-7. Concentrations of total (NH4+NH3) (bars) and un-ionized ammonia
(NH150 ) (diamonds) in MSW landfill leachates from site 3 over a 6-month








sampling interval. NM indicates sample not measured

mean of 1663 mg/L at site 6. Although the potassium concentrations were lower,

a similar pattern was demonstrated. Reported mean concentrations of

potassium in the MSW landfill leachates from sites 3 and 6 were 202 and 644
E














mg/L,March April May June Julyrespectively.

Figure 3-7. Monthly levelntrations of total (NH4+ NH3) (bars) amnd un-ionized ammonia







leachates fluctuated widely, and the concentrations were dependent on site-

specific conditions. Consistently, the highest overall total ammonia
concentration were identified in the MSW landfill leachates collected from site 3 over a 6-month
sampling interval. NM indicates sample not measured








with a range from 970 to 18601663 mg/L (Figure 3-5)at site 6. Although the potassium concentrations were lower,

total ammonia pattern was demonstrate site 2 and rangeported from 100 to 380 mg/L, theions of
potassium in the MSW landfill leachates from sites 3 and 6 were 202 and 644

mgIL, respectively.

The monthly levels of total (NH4 + NH3) ammonia in the MSW landfill

leachates fluctuated widely, and the concentrations were dependent on site-

specific conditions. Consistently, the highest overall total ammonia

concentrations were identified in the MSW landfill leachates collected from site 1,

with a range from 970 to 1860 mg/L (Figure 3-5). Although the concentrations of

total ammonia were lower at site 2 and ranged from 100 to 380 mg/L, the









400 15



300
10 .0
E














(NH3) (diamonds) in MSW landfill leachates from site 4 over a 6-month
sampling interval. NM indicates sample not measured
100




Feb March April May June

Figure 3-8. Concentrations of total (NH4*+NH3) (bars) and un-ionized ammonia
(N H3) (diamonds) in MSW landfill leachates from site 4 over a 6-month
sampling interval. NM indicates sample not measured

variability remained high (Figure 3-6). The lowest total ammonia concentrations

were displayed by the MSW leachates collected from site 3 (the capped landfill

site), with a range from 82 to 220 mg/L (Figure 3-7). In the MSW landfill

leachates collected from sites 4 and 5, total ammonia concentrations ranged

from 211 to 361 mg/L and 119 to 351.6 mg/L, respectively (Figures 3-8 and 3-9).

This contrasts with total ammonia concentrations measured in the leachates from

site 6, which ranged from 33 mg/L in July 2000 to 1957 mg/L in April of 2000

(Figure 3-10).

Ammonia speciation is dependent on both pH and temperature.

Ammonium is the dominant species at pH values < 9.3, while ammonia









400 15




300
10 .a
0

E 200

o 5

100



NM
0-- 0
Feb March April May June July
Figure 3-9. Concentrations of total (NH4+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 5 over a 6-month
sampling interval. NM indicates not measured

dominates at pH values > 9.3 (Figures 3-5 to 3-10). As previously discussed, the

pH values were similar at the six landfill sites; however, slightly higher

temperatures were reported in the leachates from sites 1 and 6 (Table 3-3). At

the pH of the leachates, the un-ionized ammonia levels were expected to be low,

and this was true for sites 2, 3, 4, and 5 with reported concentrations of less than

6.5 mg/L. Higher total ammonia concentrations were reported in the leachates

from sites 1 and 6 with mean un-ionized ammonia levels of 23.4 and 44.4 mg/L,

respectively. Basically, the un-ionized ammonia concentrations followed the

same pattern as previously described for the total ammonia concentrations.




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EX2AJBWN0_5NPG7X INGEST_TIME 2013-11-16T01:32:13Z PACKAGE AA00014225_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


TOXICITY AND HORMONAL ACTIVITY IN MUNICIPAL SOLID WASTE (MSW)
LEACHATES FROM FLORIDA LANDFILLS
By
MARNIE LYNN WARD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003

Copyright 2003
by
Mamie Ward

This dissertation is dedicated to my family. To my husband, Bill, and my
daughter, Diana Mary, whose love and support fortified me on many long days
and nights. To my parents Nanny and Poppy, my sister Lisa, my brother
Jonathan and my nieces; Deonna, Megan, and Rebecca, for their presence and
guidance in my life.

ACKNOWLEDGMENTS
I wish to thank Dr. Gabriel Bitton for his guidance, patience, humor, and
unwavering steadfastness during the past eight years. He has inspired me to
investigate new ideas, question my theories, and to always ask why. I have been
awed by his wealth of knowledge and experience, while simultaneously humbled
by his understated persona. Dr. Bitton has been my mentor, my teacher, and a
model of all that I could hope to become. He continues to exemplify the strong
values and principles that are the foundation of graduate education.
Special thanks are extended to members of my doctoral committee for their
time and interest in my education and academic growth. Dr. Timothy Townsend
has been a continuous source of support and guidance. Dr. Matthew Booth
provided assistance with analytical questions and performed GC/MS analysis.
Dr. Angela Lindner maintained an "open-door" policy and was always interested
to hear updates on my research. Dr. Nancy Denslow made available
opportunities for further study and has been a reference for many research
questions.
I also extend my gratitude to my fellow students in the Department of
Environmental Engineering Sciences; both past and present. They were always
a source of support and inspiration to me, through many long nights in the lab
and even during times of personal crisis. I specifically wish to recognize the
IV

following students; Kristin Stook, Roi Dagan, Thabet Toylamet, Diana Lee, Libby
Schmidt, Jenna Jambeck, Pradeep, Dubey, Jeff, Jenn, Tracy, and Marissa.
My heartfelt appreciation is extended to my dear friend and personal
mentor, Linda Tyson. Her house became my second home, which was a
welcome respite during my qualifying exams. I first met Linda in the early 1990s
when I was a student at Central Florida Community College and she was an
instructor. It was after listening to her talk about her graduate work that I became
inspired to attend the University of Florida. She has been an unwavering source
of inspiration and wisdom.
I owe a deep appreciation to Peter Meyers and Craig Watts at Hydrosphere
Research. They provided the starter cultures of Ceriodaphnia dubia, Daphnia
pulex, and Pseudokirchneriella subcapitata, which were subcultured to supply in-
house test organisms. Peter Meyers was a reliable and experienced source for
information concerning techniques and methods for culturing aquatic test
organisms and for conducting acute and chronic assays.
I also extend appreciation to the operators of the landfills at which the
leachates were collected. I specifically thank Jim Brunswick for his friendship
and guidance.
v

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ¡V
LIST OF TABLES x
LIST OF FIGURES xiv
ABSTRACT xvill
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 6
MSW Landfills 6
Modern MSW Landfills 6
Characterization of the Chemical and Physical Composition of MSW
Landfill Leachates 9
Bioassays for the Evaluation of Toxicity in the Environment 14
Toxicity of MSW Landfill Leachates 16
Hormonally Active Agents in the Environment 21
Phytoestrogens 23
Phthalates 24
Alkylphenols 26
Natural and Synthetic Estrogens 28
Effects of Hormonally Active Compounds on Humans 31
Bioassays to Identify Hormonal Activity 33
In vivo assays for the determination of hormonal activity 36
In vitro assays for the determination of hormonal activity 37
A test battery to determine hormonal activity 43
Characterizing Hormonal Activity in MSW Landfill Leachates and Other
Environmental Samples 44
3 TOXICITY OF LEACHATES FROM FLORIDA MUNICIPAL SOLID WASTE
(MSW) LANDFILLS USING A BATTERY OF TESTS APPROACH 54
Introduction 54
Materials and Methods 57
Leachate Collection 57
VI

Chemical and Physical Characterization of Leachates 58
Maintenance of Test Organisms 58
Pseudokirchneriella subcapitata 58
Ceriodaphnia dubia and Daphnia pulex 61
Preparation of aquatic invertebrate food 62
Maintenance of aquatic invertebrate cultures 63
Toxicity Assays 65
Pseudokirchneriella subcapitata 65
Ceriodaphnia dubia and Daphnia pulex 67
Microtoxâ„¢ 70
Data Analysis 72
Results and Discussion 74
Chemical Analysis of MSW Leachates 74
Toxicity of MSW Landfill Leachates 81
Regression Analysis 88
Ammonia Toxicity 92
Influence of Site-Specific Factors on Leachate Toxicity 93
4 A SURVEY TO ASSESS THE ACUTE AND CHRONIC TOXICITY OF
LEACHATES FROM MSW LANDFILLS IN FLORIDA: 95
Introduction 95
Materials and Methods 98
Sampling Sites 98
Collection of MSW Landfill Leachates 100
Chemical and Physical Characterization of MSW Leachates 101
Toxicity Assays 101
Data Analysis 103
Results and Discussion 104
Chemical and Physical Characteristics of the MSW Leachates 104
Toxicity of MSW Landfill Leachates 117
Toxicity of MSW leachates to aquatic invertebrates 117
Toxicity of MSW leachates with algae 121
Toxicity of MSW landfill leachates with Microtox 125
Heavy metal toxicity of MSW leachates using MetPLATE 126
NOEC/LOEC vs. EC50 or TU results 126
Monitoring MSW landfill leachate toxicity over time 130
Comparative sensitivity of the bioassays 134
Relationship Between Chemical/Physical Leachate Characteristics and
Leachate Toxicity 138
5 HEAVY METAL BINDING CAPACITY (HMBC) OF MUNICIPAL SOLID
WASTE LANDFILL LEACHATES 140
Introduction 140
Materials and Methods 143
Sample Sites 143
vii

Leachate Collection 143
Chemicals and Reagents 144
Chemical Analysis 144
Determination of Heavy Metal Toxicity 146
Determination of HMBC 146
Influence of Some Leachate Parameters on HMBC 149
Data Analysis 150
Results and Discussion 151
Heavy Metal Toxicity of Landfill Leachates 151
HMBC of MSW Landfill Leachates 157
Leachate toxicity as a function of time 163
Influence of Selected Leachate Parameters on HMBC 164
6 IDENTIFYING TOXICITY IN FLORIDA MSW LANDFILL LEACHATES WITH
A TOXICITY IDENTIFICATION AND EVALUATION (TIE) PROCEDURE . 179
Introduction 179
Material and Methods 182
Sample Collection 182
Chemical Analysis of MSW Landfill Leachates 183
TIE Procedure: Phase 1 185
pH-adjustment of the MSW landfill leachates 185
Filtration of the MSW landfill leachates 186
Solid phase extraction of the MSW landfill leachates 186
Aeration of the MSW landfill leachates 187
Blank preparation 188
Zeolite test 189
T oxicity Assays 189
Initial toxicity assays 189
Baseline toxicity assays 191
Post-manipulation toxicity assays 192
Data Analysis 192
Results and Discussion 194
Chemical/Physical Characterization 194
Determination of Heavy Metal Bioavailability 195
Initial Toxicity 196
Blanks and Controls 197
Baseline Toxicity 197
Effect of Zeolite Treatment 197
Post-manipulation Toxicity 199
Site 7 199
Site 8 202
Site 14 206
viii

7 HORMONAL ACTIVITY OF MUNICIPAL SOLID WASTE (MSW)
LEACHATES FROM FLORIDA LANDFILLS 209
Introduction 209
Materials and Methods 211
Chemicals 211
MSW Landfills and Leachate Collection 212
MSW Landfill Leachate Treatment Facility 212
Solid Phase Extraction (SPE) of MSW Landfill Leachates 214
YES Assay for Determining Hormonal Activity 215
Toxicity of MSW Leachates to Yeast Cells 217
GC/MS Analysis 218
Results 219
Hormonal Activity of MSW Landfill Leachates 219
Effect of Biological Treatment on Hormonal Activity 224
GC/MS Analysis of MSW Landfill Leachates 226
Influence of Concentration Factor on Hormonal Activity of Leachates. 229
E2 Recovery in Spiked Methanol Extracts of Leachates 232
Interpretation of GC/MS Results with the Hormonal Activity of MSW
Landfill Leachates 234
Isolation of Hormonal Activity at LF 12 241
Issues Raised When Analyzing MSW Landfill Leachates for Hormonal
Activity 242
Toxicity of MSW leachates to yeast cells 243
Coliform bacteria 246
CPRG activity issue 248
Assessment of organic solvents 249
8 CONCLUSIONS 250
LIST OF REFERENCES 253
BIOGRAPHICAL SKETCH 287
ix

LIST OF TABLES
Table Eifle
1-1. Frequently used acronyms 2
2-1. Range of selected chemical and physical characteristics reported
in the literature for domestic wastewater and MSW landfill
leachates in Florida and internationally 10
2-2. Toxicity of individual constituents identified in MSW landfill leachates 13
2-3. Reported toxicity in the literature for MSW landfill leachates 17
2-4. Select phthalate compounds and their common usage 24
2-5. Concentrations of nonylphenols in food items in Germany 26
2-6. Rates for the urinary excretion of natural estrogens from men
and women 29
2-7. Advantages and disadvantages associated with the use of in vivo and
in vitro assays for identifying hormonal activity 33
2-8. In vivo assays for the determination of hormonal activity 34
2-9. Threshold dose for the induction of hormonal effects following
exposure of fish to natural and synthetic estrogens 35
2-10. In vitro assays for the determination of hormonal activity 37
2-11. Relative sensitivity of in vitro assays to 17 (1-estradiol (E2) 38
2-12. The hormonal activity of selected metal species 46
2-13. Reported phthalate concentrations in landfill leachates 47
2-14. Concentrations (ng/L) of natural and synthetic hormones in
wastewater treatment plants(WWTPs) 49
2-15. Reported concentrations (ng/L) of natural and synthetic
estrogens in surface waters 52
x

3-1. Amount of MSW generated and landfilled at six landfill
sites in Florida 57
3-2. Components of the preliminary algal assay procedure (PAAP)
medium 59
3-3. Physical and chemical characteristics of MSW landfill leachates
at six sites in Florida 73
3-4. Correlative analysis with the C. dubia, P. subcapitata, and Microtoxâ„¢
assay results versus leachate chemical characteristics 87
4-1. Description of 14 MSW landfill sites where leachates were
collected 99
4-2. Physical and chemical characteristics of MSW leachates collected
from 14 lined landfills in Florida 107
4-3. Distribution of major ions in leachates from 14 lined MSW
landfills in Florida 110
4-4. Mean concentrations (mg/L) of total (NH47NH3) and un-ionized (NH3)
ammonia in leachates from fourteen MSW landfills in Florida 113
4-5. Metal concentrations in leachates from fourteen MSW landfills
in Florida 114
4-6. Toxicity of leachates collected from 14 lined MSW landfills with
C. dubia, D. pulex, and P. subcapitata 120
4-7. Toxicity of the MSW landfill leachates from 14 sites in Florida
using the 15-minute Microtox acute assay 124
4-8. Relationship between the toxic endpoints of IC50 (%),NOEC (%)
and LOEC (%) with the results of the P. subcapitata assay
with leachate from site 1 127
4-9. Coefficients of variation (CV)(%) for the P. subcapitata, C. dubia,
and D. pulex assays 135
4-10. Classification system for ranking the toxicity of MSW landfill
leachates from 16 sites in Florida 137
5-1. ECsofor Cu*2, Zn+2, and Hg*2 determined with the MetPLATE
assay 151
5-2. Toxicity of leachates from 16 lined MSW landfills using
MetPLATE 152
XI

5-3. Physical and chemical characteristics of leachates collected
from 16 lined MSW landfills in Florida 154
5-4. Heavy metal binding capacity (HMBC) (unitless) of leachates
from 16 MSW landfills with copper, zinc, and mercury 158
5-5. MetPLATE and HMBC results with MSW landfill leachates
collected from sites 1, 4, 5, and 8 164
5-6. HMBC of MSW landfill leachates with copper, zinc, and mercury
following fractionation 165
5-7. Changes in physical and chemical characteristics during fractionation
of the MSW landfill leachates from site 1,4, 5, and 8 166
5-8. Coefficients of determination (R2) obtained between MSW landfill
leachate characteristics and the heavy metal binding capacity
(HMBC) for copper, mercury, and zinc 175
6-1. Population served and amount of waste landfilled, as a percent
of total waste generated, at sites 7, 8, and 14 182
6-2. Manipulations to identify suspected toxicants 184
6-3. Chemical and physical characteristics of the MSW landfill leachates
from sites 7, 8, and 14 193
6-4. The initial (day 1) and baseline (day 2) acute and chronic toxicity
of the whole MSW landfill leachates from sites 7, 8, and 14 prior
to fractionation 196
6-5. Ammonia concentrations in MSW landfill leachates before and after
treatment on a Zeolite cation exchange column 198
6-6. Acute toxicity of the whole and post-Zeolite MSW landfill
leachates to C. dubia neonates 199
6-7. Summary of TIE results with MSW landfill leachates from
sites 7, 8, and 14 208
7-1. Hormonal activity of raw MSW landfill leachates and their
methanol extracts 220
7-2. Hormonal activity of raw MSW landfill leachates from LF 8
before (influent) and after (effluent) treatment in a powdered
activated carbon treatment (PACT) facility 224
xii

7-3. Organic compounds tentatively identified in MSW landfill leachates
by GC/MS analysis in full scan mode 226
7-4. Recovery (%) of 17 p-estradiol (E2) from the E2 spiked methanol
extracts of MSW landfill leachates 232
7-5. Categories of hormonal activity in MSW landfill leachates 234
7-6. Presence of hormonal activity in the raw leachates and methanol
extracts of MSW landfill leachates with identified hormonally active
compounds in parenthesis 235
7-7. Effect of extraction procedures on the hormonal activity of leachates
from LF 12 (March 2002) 240
7-8. Total and fecal coliform bacteria determined in MSW landfill
leachates with results expressed as the most probable
number (MPN) of bacteria/100 ml of leachate 247
xiii

LIST OF FIGURES
Figure page
1-1. Pathways for the characterization of the biological effects of MSW
landfill leachates 3
2-1. Representation of the vertebrate endocrine system and the possible
influences of hormonally active compounds on various system
and organs 32
3-1. Locations of the MSW landfills for the collection of leachates
in Florida 56
3-2. Flowchart for the P. subcapitata assay 64
3-3. Flowchart for the C. dubia assay 67
3-4. Flowchart for the Microtoxâ„¢ assay 69
3-5. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 1 over a 6-month
sampling interval 76
3-6. Concentrations of total (NH/+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 2 over a 6-month
sampling interval 77
3-7. Concentrations of total (NH/+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 3 over a 6-month
sampling interval 78
3-8. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 4 over a 6-month
sampling interval 79
3-9. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 5 over a 6-month
sampling interval 80
xiv

3-10. Concentrations of total (NH4*+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 6 over a 6-month
sampling interval 81
3-11. The mean toxicity of MSW leachates collected from six
landfill sites 82
3-12. Toxicity of MSW landfill leachates from site 1 over time 82
3-13. Toxicity of MSW landfill leachates from site 2 over time 83
3-14. Toxicity of MSW landfill leachates from site 3 over time 83
3-15. Toxicity of MSW landfill leachates from site 4 overtime 85
3-16. Toxicity of MSW landfill leachates from site 5 over time 85
3-17. Toxicity of MSW landfill leachates from site 6 overtime 86
3-18. Relationship between the P. subcapitata (EC50) and C. dubia (EC50)
assay results with MSW landfill leachates 89
3-19. Toxicity fluctuations in the leachates collected from the MSW
landfill at site 5 during February 2000 90
4-1. Locations of the MSW landfills for the collection of leachates
in Florida 98
4-2. Acute (48-hr C. dubia) toxicity of MSW landfill leachates collected
from 14 landfill sites in Florida 116
4-3. Correlation between the 48-hour acute toxicity assays using
C. dubia and D. pulex assays with MSW leachates collected
from 14 landfill sites in Florida 118
4-4. Chronic (96-hour P. subcapitata) toxicity of MSW landfill leachates
collected from 14 landfill sites in Florida 121
4-5. Correlation between the results of the standard (125-ml) and
modified (25-ml) P. subcapitata chronic 96-hour assays 122
4-6. Influence of time on A.) conductivity, B.) chemical oxygen demand,
C.) total organic carbon of the MSW landfill leachates from site 1 128
4-7. Influence of time on A.) conductivity, B.) chemical oxygen demand,
C.) total organic carbon of the MSW landfill leachates from site 5 129
xv

4-8. Acute (C. dubia and Microtox) and chronic (P. subcapitata) toxicity
of MSW landfill leachates collected from site 1 between February
2000 and May 2001 132
4-9. Acute (C. dubia and Microtox) and chronic (P. subcapitata) toxicity
of MSW landfill leachates collected from site 5 between February
2000 and March 2001 132
4-10. Relationship between the results of the chronic 96-hour P. subcapitata
(ICso) and the acute 48-hour C. dubia (LC50) assays with MSW
landfill leachates from fourteen sites in Florida 134
4-11. Ranking of sixteen MSW leachates with the results of the
Microtox (MT), P. subcapitata (P. sub), D. pulex (D. p.),
and Ceriodaphnia dubia (C.d.) assays 138
5-1. The MetPLATE assay protocol for determining the heavy metal
toxicity of MSW landfill leachates 145
5-2. The protocol for determining HMBC of MSW landfill leachates 147
5-3. The protocol used for fractionation of HMBC 149
5-4. MetPLATE results for MSW landfill leachates collected from site 1
and site 5 over time 163
5-5. Effect of leachate treatment by filtration (Solids), DEAE resin
(Organics), and Dowex resin (Hardness) on the HMBC 177
6-1. Results of the Phase 1 toxicity fractionation with the 24-hour
C. dubia assay for MSW landfill leachates collected from site 7 200
6-2. Results of the Phase 1 toxicity fractionation with the Microtoxâ„¢
assay for MSW landfill leachates collected from site 7 201
6-3. Results of the Phase 1 toxicity fractionation with the 24-hour
C. dubia assay for MSW landfill leachates collected from site 8 202
6-4. Results of the Phase 1 toxicity fractionation with the Microtoxâ„¢
assay for MSW landfill leachates collected from site 8 204
6-5. Results of the Phase 1 toxicity fractionation with the 24-hour
C. dubia assay for MSW landfill leachates collected from site 14 205
6-6. Results of the Phase 1 toxicity fractionation with the Microtoxâ„¢
assay for MSW landfill leachates collected from site 14 207
XVI

7-1. Procedure for preparing MSW leachates and methanol extracts
of leachates for analysis of hormonal activity 213
7-2. Response of the YES assay to 17-p estradiol (E2) 219
7-3. Total ion chromatogram of MSW leachates from LF 8 (Feb. ’02#1)
in the full scan mode 228
7-4. Dose-response of the LF 1 (Nov. '01) methanol extracts versus
concentration factor 230
7-5. Dose-response of the LF 8 (Dec. ’01) methanol extracts versus
concentration factor 231
7-6. Dose-response of the LF 12 (Nov. ’01) methanol extracts versus
concentration factor 231
7-7. The solid phase extraction protocol used with MSW landfill
leachates 241
7-8. The toxicity of MSW landfill leachates to yeast cells according
to the I NT procedure 244
xvii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
AN INVESTIGATION OF TOXICITY AND HORMONAL ACTIVITY IN
LEACHATES FROM MUNICIPAL SOLID WASTE (MSW) LANDFILLS IN
FLORIDA
By
Marnie Lynn Ward
December, 2003
Chair: Dr. Gabriel Bitton
Major Department: Environmental Engineering Sciences
The purpose of this research was to characterize the chemical composition
and biological effects of leachates from MSW landfills in Florida. Samples were
collected from 16 engineered landfills to encompass a cross-section of leachate
quality and characteristics. The MSW landfill leachates were tested using a suite
of bioassays, which included the chronic Pseudokirchneriella subcapitata and the
acute Ceriodaphnia dubia, D. pulex, and Microtoxâ„¢. Leachates were tested with
MetPLATE, a heavy metal specific assay. Additionally, using a yeast reporter
assay, the leachates were tested for hormonal activity.
Landfill leachates are complex mixtures of organic and inorganic
contaminants with compositions heavily influenced by site-specific parameters
(e.g. waste composition and age). The chemical composition of the Florida
landfill leachates varied widely. In some leachates, high levels of un-ionized
xviii

ammonia, inorganic components, CBOD, and COD were recorded. The
corresponding toxicity at these sites was high. Significant relationships were
shown between the ammonia content of the leachates and toxicity as determined
by the C. dubia (R2 = 0.62) and P. subcapitata (R2 = 0.69). The assays were
ranked for their sensitivities to the MSW landfill leachates as follows: C. dubia *
D. pulex >P. subcapitata (125-ml) ~ P. subcapitata (25-ml) > Microtoxâ„¢.
The heavy metal toxicity/bioavailability and heavy metal binding capacity
(HMBC) of landfill leachates was determined with MetPLATE. The heavy metal
toxicity was low, which was attributed to the presence of complex-forming
ligands. The magnitude of the HMBC was investigated with the metals copper,
zinc, and mercury. The results showed that the HMBC ranged from 3 to 115, 5
to 93 and 4 to 101 for HMBC-Cu+2, HMBC-Zn+2, and HMBC-Hg+2, respectively.
The leachates were chemically/physically treated to reduce or fractionate their
complexity. Fractionation of selected leachates revealed that HMBC was
influenced by the solid, organic, and hardness content of the tested leachates.
Additionally, other unidentified components influenced the HMBC of the landfill
leachates.
The leachates were evaluated for their hormonal activity using a yeast
reporter assay. The hormonal activity of the raw MSW landfill leachates was
highly variable, with an E2 equivalent range from 2.6 to 45.7 ng E2/L. A similar
range from 6.2 to 59.7 ng E2/L was reported for the methanol extracts of the
leachates. The presence of unidentified substances in the leachates reduced the
XIX

recovery of hormonal activity. Treatment processes utilizing powdered activated
carbon (PAC) removed hormonal activity.
xx

CHAPTER 1
INTRODUCTION
Historically, domestic wastes were disposed in open pits or surface piles,
and these sites often posed a significant risk to the surrounding environments
(Reinhart and Townsend, 1998). Concerns with the environmental fate of
discarded materials led to the construction of engineered systems for long-term
storage and management of waste materials and their degradation products.
These engineered systems included lined cells for the disposal of waste and
collection systems for leachate recovery. Government regulations prohibit the
disposal of waste materials except at regulated municipal solid waste (MSW)
disposal facilities. Although MSW landfills may contain small quantities of
hazardous waste materials, they are primarily designed to receive domestic
wastes. These items may include packaging materials, food scraps, furniture,
clothing, and grass clippings (USEPA, 2002).
The acute and chronic toxicity of MSW landfill leachates has been
extensively studied using various bioassays (Rutherford et al., 2000; Clement et
al., 1997; Kauretal., 1996; Plotkin and Ram, 1984); however, little information is
available concerning the biological effects of MSW leachates collected from
landfills In Florida. Reinhart and Grosh (1998) have reported a lower chemical
strength in Florida MSW leachates, resulting from the predominant environmental
conditions, e.g., abundant rainfall and warm temperatures. Therefore, the basis
1

2
Table 1-1. Frequently used acronyms
Acronym
Definition
Acronym
Definition
CBOD
Carbonaceous
biochemical oxygen
demand
HAA
Hormonally active
agent
COD
Chemical oxygen
demand
HAC
Hormonally active
compound
CPRG
Chlorophenol red
galactopyranoside
HMBC
Heavy metal binding
capacity
DOC
Dissolved organic
carbon
IC50
Sample concentration
responsible for 50 %
inhibition in test
organism
DOM
Dissolved organic
matter
LC50
Sample concentration
lethal to 50 % of the
test organisms
Ei
Estrone
MSW
Municipal solid waste
e2
17 p-estradiol
MHW
Moderately hard water
e3
Estriol
ONPG
Ortho-nitrophenyl
galactopyranoside
ee2
17 a-ethinyl estradiol
TDS
Total dissolved solids
EC50
Sample concentration
responsible for a 50 %
effect in test organism
TIE
Toxicity identification
evaluation
ED
Endocrine disrupter
TRE
Toxicity reduction
evaluation
ERa
Estrogen receptor alpha
TS
Total solids
United States
ERp
Estrogen receptor beta
USEPA
Environmental
Protection Agency
ER-CALUX
Estrogen receptor -
chemically activated
luciferase reporter gene
YES
Yeast estrogen
screen
of this investigation was to determine the biological affects associated with
exposure to MSW leachates from Florida landfills.

3
ACUTE
CHRONIC
TOXICITY
TOXICITY
LEACHATE
HORMONE
ACTIVITY
â–¼
METAL
BIOAVAILABILfTY
METAL
BINDING
Figure 1-1. Pathways for the characterization of the biological effects of MSW
landfill leachates
In recent years, the number of operating landfills in the U.S. has
decreased; however, the overall size of the remaining landfills has increased
(USEPA, 2002). Landfills continue to be the most economically feasible and
least environmentally intrusive method for the disposal of discarded waste
materials. In Florida, more than 14 million tons (total 24.8 million tons) of MSW
were landfilled in 1998, the most recent year data were available, with a per
capita generation rate of 9.1 pounds/person/day (FDEP, 2000).
While the chemical and physical composition of MSW landfill leachates in
Florida have been summarized (Reinhart and Grosh, 1998), only one study has
evaluated the toxicity of Florida leachates (Ward et a!., 2000) and then only on a
very limited scale. The scientific community, environmental regulators, landfill
operators, and the operators of facilities treating landfill leachates require

4
comprehensive databases to provide information relative to biological effects and
chemical constituents of Florida MSW landfill leachates.
Numerous acronyms are frequently used in the scientific research
community, and many are commonly recognized; however, some are relatively
new and discipline-specific. Therefore, a table of the acronyms used in the
following chapters has been included to aid the reader as a quick reference
(Table 1-1).
The purpose of this research was to evaluate MSW landfill leachates in
Florida, for their toxicity, hormonal activity, and chemical characteristics. To
date, there have been no reported investigations of MSW landfills of this scope or
magnitude. Most investigations have focussed on leachate from one or several
landfills (Kaur et al., 1996; Wong, 1989), but few evaluated multiple leachates
(Clement et al., 1996, 1997), and none have tracked patterns over time. The
toxicity of leachates from Florida landfills has received little attention. Ward et al.
(2000) evaluated the toxicity of landfill leachates from three Florida landfills;
however, leachates were evaluated with only one acute toxicity assay.
The basis of this research project was to provide a statewide database
for landfill operators, regulators, and other research investigators, that
established a range of biological effects from exposure to MSW landfill leachates.
The overall objectives of this research project were as follows (Figure 1-1):
1. To characterize the toxicity in the MSW landfill leachates using a battery of
toxicity assays including the chronic 96-hr Selenastrum capricomutum, and

5
the acute 48-hour Ceriodaphnia dubia, 48-hour Daphnia pulex, and
Microtoxâ„¢ assay (Chapters 3 and 4).
2. To characterize the chemical and physical composition of MSW landfill
leachates (Chapters 3, 4, 5, 6, and 7).
3. To determine the toxicity of heavy metals in MSW landfill leachates with the
heavy metal specific MetPLATE assay and to quantify the ability of MSW
leachates to reduce the bioavailability of heavy metals with a heavy metal
binding capacity (HMBC) assay (Chapter 5).
4. To conduct a toxicity identification evaluation (TIE) with selected MSW
landfill leachates (Chapter 6).
5. To determine the hormonal activity of MSW landfill leachates with a yeast
estrogen screen (YES) and identify by gas chromatography/mass
spectrometry (GC/MS) organic compounds responsible for hormonal
activity (Chapter 7).
6. To determine the effect of powdered activated carbon treatment on
hormonal activity in landfill leachates (Chapter 7).
7. The results obtained during this research investigation are summarized
(Chapter 8).

CHAPTER 2
LITERATURE REVIEW
Forty years after Rachel Carson first revealed the risks to humans and the
environment posed by a diverse array of man-made substances, the threat from
these anthropogenic chemicals persists (Carson, 1962). Over 100,000 synthetic
chemicals including pesticides, solvents, domestic cleaners, plasticizers, and
flame-retardants are produced yearly for domestic and industrial usage; however,
little is known about the biological effects from long-term exposure to these
compounds (Darnerud et al., 2001; Hale et al., 2001; Lyytikainen et al., 2001). In
the US, the Resource Conservation and Recovery Act (RCRA), Toxic
Substances Control Act (ToSCA), and the Clean Water Act (CWA) and their
amendments have been effective in the control of point source pollution.
However, non-point sources of pollution continue to pose significant threats to
the environment.
MSW Landfills
Modem MSW Landfills
MSW landfills are engineered systems that are designed through the use
of heavy-duty plastic liners and/or low permeability clay barriers to retard the
escape of pollutants to surrounding environments and for the recovery of
leachates. Leachates are formed following rainwater infiltration. Percolation of
this water through the waste materials mobilizes soluble substances (Ross,
1990). These leachates represent the mobile fraction of landfill toxicants, and
6

7
they contain high concentrations of inorganic and organic compounds (Bozkurt et
al., 2000). The majority of MSW landfill leachates are closely regulated;
therefore, their release of leachates from modern landfills is unlikely (Barlaz et
al., 2002). Accidental leachate releases may occur, e g. during periods of high
rainfall when leachates are contaminated by stormwater or from the improper or
faulty installation of leachate collection systems. Additional sources for the
unintentional release of MSW landfill leachates exist. Prior to 1990, federal
regulations did not require landfill liners, therefore, the escape of leachates from
these sites represents a potential adverse environmental impact (Assmuth,
1996). Florida was more proactive and required landfill liners for MSW landfill in
the Solid Waste Act of 1988.
Landfilling remains the predominant management method for municipal
solid waste (MSW), accounting for more than 50% (128.3 million tons) of the
MSW generated in the United States (USEPA, 2002). Under authority granted by
RCRA, subtitle D, the U S. Environmental Protection Agency (USEPA) regulates
the construction, operation, and post-closure of municipal solid waste (MSW)
landfills (CFR 258, 1996). Strict regulations require landfill operators to minimize,
recover, and treat the leachates generated in MSW landfills. Landfill liners are
used to restrict the flow of leachates to ground and surface waters, and they may
be composed of clay, high or low-density polyethylene, or concrete, depending
on the local conditions. In some landfills, leachates are recycled through the
waste to encourage waste stabilization and improve leachate quality (Reinhart
and Townsend, 1998; Nopharatana et al., 1998).

8
The chemical characteristics of MSW leachates are dependent on the type
and amount of waste landfilled, the landfill age and various environmental
conditions, e g. temperature and rainfall. Environmental regulations in the U.S.
greatly limit the disposal of hazardous wastes in municipal landfills, but waste
materials containing toxic chemicals may enter many landfills. Some sources of
these substances include generators of small amounts of hazardous waste, non-
hazardous industrial wastes, and household hazardous wastes (HHW), such as
electrical devices, fluorescent light bulbs, thermometers, batteries, pesticides,
and other chemical products (Boyle and Baetz, 1993). Using a risk-based
assessment, comparable health risks were associated with MSW landfill
leachates and industrial waste leachates (Brown and Donnelly, 1988). Their
assessment was based on the individual leachate constituents, while not
accounting for potential synergistic and/or antagonistic responses (Brown and
Donnelly, 1988).
Landfills are classified based on the composition of the waste materials
landfilled. Class I and II landfills are designated for the disposal of non-
hazardous household wastes and some commercial, industrial and agricultural
wastes, while Class III landfills are designed for yard waste, construction and
demolition debris, carpet, furniture, and similar non-putrescible waste materials.
The basis for the distinction between Class I and Class II landfills is the volume
of MSW landfilled daily. Class I landfills receive 20 or more tons of MSW per
day, while Class II landfills less than 20 tons of MSW per day.

9
Characterization of the Chemical and Physical Composition of MSW
Landfill Leachates
Waste stabilization is based on microbial degradation processes, with the
conversion of organic matter to methane gas occurring predominately under
anaerobic conditions (Barlaz, 1997). Initially, aerobic conditions dominate in
landfills, but oxygen is rapidly depleted with the continuous addition of waste.
The phases of waste degradation are loosely defined by the predominant
chemical characteristics. Early phases are defined by the transition from aerobic
to anoxic and finally strictly anaerobic conditions within the waste.
Concomitantly, microbial degradation converts the large organic molecules to
smaller organic acids, which are further degraded to hydrogen and acetate.
Methanogenesis, the conversion of these small molecules to methane gas by
methanogenic bacteria, is a strictly anaerobic process. Researchers theorize
that landfills may revert to aerobic conditions as oxidized micro-environments
begin to form at the boundaries of the waste. However, no landfill currently
under study has reached this stage of decomposition (Bozkurt et al., 2000;
Kjeldsen et al., 2002). As landfilling Is a continuous and on-going process,
various stages of waste decomposition occur simultaneously, and this may be
reflected in the chemical characteristics of the leachates.
MSW landfill leachates are complex mixtures, with a wide ranging
chemical strength (Table 2-1). In a recent review, Kjeldsen et al. (2002)
summarized the chemical and physical characteristics of MSW landfill leachates.

10
Table 2-1. Range of selected chemical and physical characteristics reported in
the literature for domestic wastewater and MSW landfill leachates in
Florida and internationally
Parameter
Raw Wastewater
Florida MSW
Landfill Leachate
International MSW
Landfill Leachate
BOD5
(mg/L)
110-400c
0.3-4800°
42-10,900°
COD
(mg/L)
250-1,000c
7-50,000°
40-90,000°
Alkalinity
(mg/L)
50-200°
350-8775'
1,350-3,510b
pH
-
6.2-9.7°
3-7.9a
nh3-n
(mg/L)
12-50°
0-4110°
<0.3b-13,000d
Chloride
(mg/L)
20-50°
1.9-2720°
125-2,400b
Sulfide
(mg/L)
-
<0.01-3.8°
<0.02-30b
TDS
(mg/L)
250-850°
1,800-31,700'
2,000-60,0009
Na+
(mg/L)
40-70°
25.6-1963'
128-840b
Ca+2
(mg/L)
-
45-4,400'
10-7.2009
Mg"2
(mg/L)
-
25-122'
30-15,0009
Total phosphorus
(mg/L as POT2)
4-15°
0.1-39.6'
<0.01-2.7°
Conductivity
(mS/cm)
:—.,, • ——
3.4-39.6'
1,200-16,000°
References: "Kadlec and Knight, 1996; “Cameron and Koch, 1980; “Metcalf and Eddy;1991; “Lo,
1996; 'Reinhart and Grosh, 1998; 'this research; aKjeidsen et al., 2002.
They stressed four primary categories of contaminants to consider in discussions
of leachate quality, and these were dissolved organic matter, organic xenobiotics,
inorganic components, and heavy metals (Christensen et al., 1994).
In MSW landfill leachates, dissolved organic matter (DOM) includes the
dissolved and colloidal particles (Gounaris et al., 1993). The molecular structure
and elemental composition of dissolved organic matter in MSW landfill leachates
is strongly influenced by microbial degradative processes. Calace et al. (2001)

11
reported a narrow distribution of organic molecular weight groups in young
landfills (< 5 years old), with primarily low molecular weight constituents (<500
Dalton). This contrasted with their findings in older landfills (>10 years old)
with an increased distribution of molecular weight fractions and high molecular
weight constituents (> 10,000 Dalton) (Calace et al., 2001). These high
molecular weight fractions contain structurally complex humic materials (Croue et
al„ 2003). Kang et al. (2002) reported an increased presence of humic
substances with increasing landfill age and a decrease in the easily degraded
lower molecular weight organic materials. Attributed to the high ammonia
concentrations in the leachate, the humic materials contained a large distribution
of nitrogen functional groups (Kang et al., 2002). This is significant when
considering the strong metal complexes that are formed with organic ligands
containing nitrogen functional groups (Croue et al., 2003; Stumm and Morgan,
1995).
Xenobiotics are frequently detected at low levels in MSW landfill leachates
(Schwarzbauer et al., 2003; Kawagoshi et al., 2002; Yasuhara et al., 1999).
Some xenobiotics of significant environmental concern, relative to reproductive
effects, have been identified in MSW landfill leachates. Wintgens et al. (2003)
reported nonylphenol, a surfactant, and Bisphenol A, a plasticizer, at 60 and 37.5
pg/L, respectively, in MSW landfill leachates. In a Japanese landfill, xenobiotics
identified included Bisphenol A, nonylphenol, octylphenol, and some dioxin-like
substances (Behnisch et al., 2001). The organic contaminants in MSW landfill
leachates have been summarized (Kjeldsen et al., 2002).

12
Inorganic contaminants in MSW landfill leachates include anionic and
cationic species, and some of the most widespread are NH4*, Ca*2, Mg+2, Na+1,
Cl'1, HCO3'1, SO4'2. Typically, the ammonia in MSW landfill leachates occurs as
the ionized ammonium (NH/) species. Ammonia speciation is pH dependent,
with a pKa of 9.3. Therefore, the dominant species is ammonium, rather than the
highly toxic ammonia (NH3) form (McBean et al., 1995). Generally, total
ammonia concentrations are high, with reports of up to 740 mg/L (Kjeldsen et al.,
2002). These high ammonia levels are often attributed to the absence of
degradative pathways for the removal of ammonia from landfills (Burton and
Watson-Craik, 1998). Also found at high concentrations are the hardness
cations. Hardness is a measure of multivalent metallic cations; e.g. Ca *2, Mg*2,
Sr*2, Fe+2, Mn+2, but mainly Ca*2and Mg+2(Stumm and Morgan, 1995).
Hardness has a strong influence on heavy metal bioavailability in landfill
leachates (Heijerick et al., 2003).
Heavy metals are generally reported in the low mg/L range (Reinhart and
Grosh, 1998). However, this represents only a fraction of the total metal
associated with the waste. In some cases, metals that are strongly associated
with waste materials are not easily leached under landfill conditions. Flyhammer
(1995), in a mass balance on cadmium in Swedish landfills, concluded that the
total concentrations associated with the landfilled wastes were up to four orders
of magnitude greater than leached concentrations (Flyhammer, 1995).
Kjeldsen et al. (2002) discussed leachate characteristics in MSW landfills
with a primarily organic composition and the influences of time on these leachate

13
Table 2-2.
Compound
C. dubia
S. capricomutum
Microtoxâ„¢
D. magna
MetPLATEâ„¢
(mg/L)
(mg/L)
(mg/L)
(mg/L)
ÍÜ2SÍÜ
Copper
0.01b
0.04h
7.4'
0.02f
0.22'
5-min EC50
48-hr LC50
Cadmium
0.05c
0.34h
-
-
0.03'
Zinc
-
0.18h
12'
15-min EC50
5.1'
48-hr LC50
0.11'
Total
3607f
ammonia
5-min ECso
Unionized
1.18a
1.7*
3.3e
ammonia
15-min EC»
24-hr EC50
Manganese
14.5d
(MHW)
-
-
-
-
Alkalinity
7819
9219
(HCCV)
Chloride
-
-
-
-
-
Sodium
-
-
-
1995; 'Rhodes, 1992; 'Lassier et al., 2000; 'Clement and Merlin, 1995; 'Qureshi et al., 1982;
“Hoke et al., 1992; hChen et al., 1997; 'Doherty et al., 1999; ’Bitton et al., 1994.
characteristics. In earlier work, Bozkurt et al. (2000) discussed leachates
generated in MSW landfills with either a primarily organic or inorganic waste
composition. The latter case represented ash monofills or co-disposal facilities
for MSW and ash. The focus of their investigation was to predict the long-term
fate of heavy metals using a conceptual model, which included influences from
various organic and inorganic ligands (Bozkurt et al., 2000).
The toxicity of MSW landfill leachates is heavily influenced by chemical
and physical interactions (Table 2-2). In this regard, complexation reactions
have a mitigating influence on toxicity and are frequently underestimated and
poorly understood, due to the sheer number of possible ligands in solution
(Martensson et al., 1999; Stumm and Morgan, 1995). Some of the typical

14
inorganic ligands in leachates include carbonate (Sletten et al., 1995), chloride
(Bolton and Evans, 1991), and sulfide ions (Bozkurt et al., 2000). These
inorganic ligands can form insoluble precipitates with heavy metals (Majone et
al., 1996). Other ligands present are dissolved organic matter (Kaschl et al.,
2002) and colloidal solids (Gounaris et al., 1993). These complexes exert a
strong influence on heavy metal toxicity (Heijerick et al., 2003), with up to 98 % of
metals in some landfills present as organo-metallic complexes (Kang et al., 2002;
Weng et al., 2002). However, there have been some questions concerning the
toxicity of these organo-metallic complexes (Palmer et al., 1998). Fraser et al.
(2000) suggested low-level toxicity associated with complexes between copper
and dissolved organic materials (DOM).
Bioassays for the Evaluation of Toxicity in the Environment
Luoma (1995) described bioassays as tools for investigating the complex
continuum of biochemical, physiological, and reproductive responses that occur
in organisms following exposures to suspect toxicants. Traditionally, bioassays
with various vertebrate and invertebrate organisms were used to monitor and
track environmental perturbations (USEPA, 1993a, 1994a). These bioassays
measured chronic effects in low-level, long-term exposures and acute toxicity in
high-level, short-term exposures. Critically important to all environmental
researchers was how best to reconcile chronic low-level environmental exposure
with laboratory investigations utilizing high dose acute substances (Mowat and
Bundy, 2001; Degen and Bolt, 2000). Some of the most common bioassays
have used algae (Eullaffroy and Vernet, 2003; van der Heever and Grobbelaar,
1998), aquatic plants (Mohan and Hosetti, 1999; Klaine and Lewis, 1995),

15
invertebrates (Heijerick et al., 2003; Kim et al., 2003; Preston and Snell, 2001;
Pereira et al., 1999), or microorganisms (LeBlond et al., 2001; Doherty et al.,
1999; Jung et al., 1997).
While most bioassays have been extensively validated, each has intrinsic
advantages and disadvantages that are specific to the method. For example, in
some algal assays, cell exudates (extracellular organic material) mitigate metal
toxicity by acting as ligands that form complexes with free metal ions. In
microplate assays, these and similar problems are controlled, which may explain
the recent increase in the use of microbiotests. Additionally, these microbiotests
offer an increased affordability, portability, and the availability of results in a short
interval of time (Chial and Persoone, 2002; Bitton et al., 1994). Gabrielson et al.
(2003) recently developed a microplate assay, referred to by the acronym MARA
(microplate assay risk assessment), that utilized 11 lyophilized microbial strains
for determining the toxic fingerprint of a chemical. This assay allows for the
testing of multiple species simultaneously; however, it is not sensitive to any
specific class of toxicants. There are microplate assays (e g., MetPLATE and
MetPAD) that are designed specifically for the detection of heavy metal toxicity
(Bitton et al., 1992b, 1994).
Developments in the field of environmental chemistry have produced
analytical methodologies and techniques that are highly successful at identifying
and quantifying contaminants, even in highly complex matrices (Richardson,
2001). They use a suite of analytical tools that have in common an electrode,
which senses changes based on electronic signals. Some typical electrodes

16
measure dissolved oxygen (DO), conductivity, pH, select ions (e.g. ionized
metals), and oxidative potential. Parallel techniques for application in the field of
environmental toxicology would allow for the rapid identification of toxicity, while
simultaneously reducing the time and cost involved in continuous monitoring
programs (Arikawa et al., 1998). Biosensors are a rapid and convenient
monitoring tool, which incorporate biological tissues in a system highly sensitive
to a broad spectrum of toxic substances (Botre et al., 2000; Buffte and Horvai,
1998; Argese et al., 1996). Although their use is currently limited, biosensors
have been successfully applied to the evaluation of wastewater toxicity (Farre
and Barcello, 2003).
Toxicity of MSW Landfill Leachates
Research investigations that simultaneously combine bioassays with
methods for chemical characterization are highly valued, but the associated
expenses and labor demands constrain their extensive utilization (Ferrari et al.,
1999; Atwater et al., 1983). To date, the most extensive study of waste
leachates was conducted in France. Clement et al. (1996) investigated the
toxicity often domestic landfill leachates and various other hazardous and non-
hazardous waste leachates. Bioassay results with protozoa, bacteria, algae, and
invertebrates demonstrated that the toxicity of the domestic waste leachate was
higher than the industrial or hazardous waste leachates (Clement et al., 1996).
Furthermore, the chemical characterization of the domestic leachates revealed
that ammonia, alkalinity, conductivity, and COD were highly associated with
increased leachate toxicity (Clement et al., 1997). In earlier work, a significant
contribution of ammonia to the acute toxicity of landfill leachate to duckweed

17
Table 2-3. Reported toxicity in the literature for MSW landfill leachates
Leachate Origin
Species
Endpoint
Reference
Solid waste landfill
(unknown composition)
Tilapia
(Sarotherodon
mossambicus)
96-hr LC50 = 1.4 to 12 %
Wong,
1989
Solid waste landfill
(40 % household and
60%
industrial/commercial)
Fathead minnow
(Pimephales
prometas)
96-hrLC5o= 100%
Plotkin and
Ram, 1984
Solid waste landfill
(40 % household and
60%
industrial/commercial)
Daphnia magna
48-hr LC50 = 62 to 66 %
Plotkin and
Ram, 1984
Solid waste landfill
(40 % household and
60%
industrial/commercial)
Selenastrum
capricomutum
13 day EC5o= 1 to 10%
Plotkin and
Ram, 1984
Solid waste landfill
(40 % household and
60%
industrial/commercial)
Microtox
5 min. ECso= 17 %
Plotkin and
Ram, 1984
Solid waste landfill
(unknown composition)
Aquatic plants
ECso= 10%
Devare
and
Bahadir,
1994
Solid waste landfill
(unknown composition)
Microtox
EC5o= 18-35%
Devare
and
Bahadir,
1994
(Lemna sp.) was reported (Clement and Merlin, 1995). Clement et al. (1997)
performed correlative analyses between their bioassay results and various
chemical characteristics and revealed a strong correlation (R2 = 0.92) between
Daphnia magna and combined ammonia and alkalinity concentrations. Similar
relationships were shown between the bioassay results with aquatic plants,
algae, and other crustaceans and ammonia and alkalinity (Clement et al., 1997).
Using the Microtoxâ„¢ assay, the relationship between COD and toxicity was

18
significant (p< 0.01) but weaker (R2 = 0.58). This was the only assay sensitive to
toxicity associated with increasing organic content (Clement et al., 1997).
The toxicity of MSW landfill leachates have been well characterized
around the world (Ernst et al., 1994; Lambolez et al., 1994; Devare and Bahadir,
1994; Cheung et al., 1993; Wong, 1989; Radi et al., 1987; Plotkin and Ram,
1984; Atwater et al., 1983; Millemann and Parkhurst, 1980; Cameron and Koch,
1980), but the toxicity of Florida landfill leachates remains poorly characterized
(Table 2-3). Ward et al. (2002) studied the leachates from six MSW landfill
leachates in Florida and concluded that the leachates were highly toxic. The
toxicity of the MSW landfill leachates varied widely, due to site-specific chemical
characteristics of the leachates. Furthermore, on a monthly basis, fluctuations in
leachate toxicity indicated the heterogenous composition of the waste materials
and local conditions (Ward et al., 2002).
Recently, investigations of waste leachates in countries that do not require
landfill liners, minimization of leachate generation, or leachate collection and
treatment have been reported. These studies offer insight to researchers
concerned with the potential for leachate release to the environment. Magdaleno
and De Rosa (2000) characterized leachates from a waste dump in Argentina
with an algal assay using Selenastrum capricomutum (renamed
Pseudokirchneriella subcapitata), while Sisinno et al. (2000) evaluated waste
leachates in Brazil with the Zebrafish (Brachydanio rerio). The chemical strength
of these leachates were comparable to reports of others (Kjeldsen et al., 2002;
see Table 2-1).

19
The chemical strength of the Argentinian leachates was demonstrated by
COD concentrations from 502 to 4640 mg/L, ammonia from 26.5 to 35 mg/L, and
pH from 7 to 7.3 (Magdaleno and De Rosa, 2000). Similar chemical
characteristics were reported with the leachates from Brazil, with conductivity
values from 3.1 to 6.2 mS/cm, alkalinity from 212 to 372 mg/L as CaC03, and
COD from 5,200 to 11,500 mg/L (Sisinno et al., 2000). Overall, the toxicity of the
Argentinian leachates was low with TU (toxicity unit) values that ranged from 1 to
2.1 (Magdaleno and De Rosa, 2000). Higher toxicity was reported in the
Brazilian leachates and ranged from 17.5 to 45.5 TU (Sisinno et al., 2000). The
leachates from Brazil demonstrated a toxicity similar to that reported with Florida
leachates (Ward et al., 2002). However, the leachates from Argentina displayed
reduced toxicity. The predominance of plastics and other disposable materials in
US MSW landfills may be a factor contributing to their higher toxicity.
The contamination of groundwater by waste leachates is a primary
concern, relative to the escape of leachates into the environment. Baun et al.
(1999) investigated the toxicity of groundwater contaminated by MSW leachates
in Denmark with an algae assay, a crustacean assay, and a bacterial
genotoxicity assay. Using the algae assay, the leachate-contaminated
groundwater sample displayed an EC20 of 17 %; however, the toxicity decreased
by 75 % at twice the distance from the landfill. Similar toxicity was demonstrated
by the crustacean, Daphnia magna, with an EC20 of 18 % at the landfill, but,
further downstream, no toxicity was reported. Further analysis of this
contaminated groundwater revealed that organic contaminants were responsible

20
for the high toxicity, and with increasing distance from the landfill the organic
toxicity decreased, suggesting metabolic degradation or dilution effects (Baun et
al., 2000). Additional bioassays with the organic fraction revealed a low
sensitivity of the D. magna assay to organic toxicants, which contrasted with the
algal and Microtoxâ„¢ assay results (Baun et al., 2000). Other researchers have
reported the higher sensitivity of Microtoxâ„¢ to organic contaminants (Bitton et al.,
1994).
In 1988, reports of comparable carcinogenic risk associated with exposure
to MSW landfill leachates or hazardous waste leachates raised concerns in the
regulatory community (Brown and Donnelly, 1988). Subsequent investigations,
to determine the genotoxic potential of MSW landfill leachates, have revealed
conflicting results. Beg and Al-Muzaini (1998) investigated the genotoxicity of
MSW landfill leachates in Kuwait using a dark mutant strain (nonluminescent) of
Vibrio fisheri, a bioluminescent bacterium. In the presence of a mutagen, the
dark strain reverts to the luminescent state and this response is quantified by
increased light intensity. These results suggested, in some of the Kuwaiti
leachates there was a high degree of genotoxicity, and this was dependent on
the type of waste landfilled and seasonal conditions (Beg and Al-Muzaini, 1998).
Helma et al. (1996) used four bacterial assays to characterize genotoxicity in
landfill leachates, wastewater effluents, pulp and papermill effluents, and
contaminated groundwater. Overall, the highest genotoxicity was displayed by
the MSW landfill leachates with more than 35,000 revertants/L of leachate. This
was comparable to the genotoxicity of the leachates produced by mixed industrial

21
and domestic wastes, which were reported as approximately 40,000 revertants/L
(Helma et al., 1996). Baun et al. (1999) using the umuC strain of Salmonella
typhlmurium showed that leachate-contaminated groundwater was not genotoxic
at concentrations up to 25% by volume. However, bacterial toxicity at higher
concentrations prevented the evaluation of genotoxic effects. After isolation of
the organic fraction of these contaminated groundwaters, a similar mutagenicity
was identified. These results suggested that the organic fraction contained the
mutagens (Baun et al., 2000).
Hormonally Active Agents in the Environment
Some natural and anthropogenic substances may interact or interfere with
the nuclear receptors and chemical messengers of the endocrine system and
have been identified as a threat to the environment and wildlife (McLachlan,
2001). Alterations in both the developmental and reproductive functions of cells
and whole organisms are increasingly documented and attributed to exposure to
these exogenous substances (NRC, 1999). These substances are ubiquitous
environmental contaminants, which are commonly referred to as hormonally
active agents (HAAs) or compounds (HACs), xenoestrogens, estrogen-like
compounds, endocrine disruptors, estrogen-mimics, or estrogen
agonists/antagonists. Substances are labeled based on their interaction with
and/or displacement of an endogenous hormone from its conservative function
(McLachlan, 2001). Recent congressional mandates in the Safe Drinking Water
Act (1996) (Bill No.S.1316) and the Food Quality Protection Act (1996)(Bill
No.P.L. 104-170) have required the USEPA to evaluate the hormonal activity of
all chemicals produced In the U.S.

22
In a recent survey of 139 contaminated U S. surface waters, alkylphenols,
phthalate compounds, and natural and synthetic estrogens were shown to
comprise roughly 75% of the organic contaminant load (Kolpin et al., 2002). Of
critical concern is the exposure to these three classes of compounds, because of
reports of hormone-like effects. Their origin may be due in part to agricultural
non-point source runoffs (Casey et al., 2003), but the majority are discharged
from domestic and industrial wastewater treatment plants (WWTPs) (Sheahan et
al., 2002b; Snyder et al., 2001; Baronti et al., 2000; Rudel et al., 1998).
Environmentally relevant concentrations of these compounds have been linked to
altered sexual characteristics (Jobling et al., 1995) and elevated tissue levels of
hormonally active compounds (Sheahan et al., 2002b) in fish. However, links are
not easily established in some situations (Jacobsen and Guildal, 2000; Fawell et
al., 2001; Sepulveda et al., 2002).
According to the European Union Scientific Committee, hormonally active
agents (HAAs) (referred to as endocrine disruptors) are "exogenous substances
or mixtures that alter function(s) of the endocrine system and consequently
cause adverse health effects in an intact organism, or its progeny, or sub¬
populations" (Baker, 2001). Sweeping in its brevity, this definition fails to address
some concerns (McLachlan, 2001; Ashby, 2000), specifically, altered cellular
functions in relation to the overall health of the organism. Although, the National
Research Council (1999) was even less direct when they defined hormonally
active compounds as any "substance that possesses hormone-like activity,

23
regardless of the mechanism" of action. In light of new research (Wu et al.,
2003), changes to the definition may read, "any substance or influencing factor."
Hormonally active compounds are arranged in three groups; the natural
and synthetic estrogens, anthropogenic chemicals, and phytoestrogens (naturally
produced substances in plants). The effects of these substances may be
agonistic or antagonistic. Agonistic hormonally active compounds act in a
manner similar to an endogenous hormone, while antagonists block the activity
of endogenous substances. Beginning with reports of the estrogen-like effects
following exposure to the insecticide DDT (Burlington and Lindeman, 1950),
researchers continue to study the interaction of non-steroidal compounds with
the estrogen receptor (Miksicek, 1994).
Phytoestrogens
Phytoestrogens are naturally occurring compounds in plants and plant-
derived products (Nilsson, 2000) and include genistein, equol, formononetin,
biochanin A (Latonelle et al., 2000). The hormonal activity of phytoestrogens has
been reviewed, with special emphasis on environmentally relevant dosages
(Nilsson, 2000). One source of phytoestrogens is the urine of vegetarians (Fotsis
and Adlercreutz, 1987). Phytoestrogens impact the reproduction and sexual
health of wildlife (Hughes, 1988), but there is no evidence in humans (Strauss et
al., 1998). In fact, limited evidence suggests phytoestrogens are beneficial in
treating some types of human cancers (DiPaola et al., 1998). Ju et al. (2000)

24
Table 2-4. Select phthalate compounds and their common usage
COMPOUND
USES
Di-ethylhexyl phthalate
(DEHP)
Rain gear, footwear, upholstery materials, I.V.
fluid bags, waterproof gloves
Heat seal coating on metal foils used on portioned
food items.
Butyl benyzl phthalate
(BBP)
Dispersant in insect repellants and perfumes
Component of cellulose plastics
Floor tiles
Di-butyl phthalate
(DBP)
Coatings on cellophane, insect repellants,
Hair spray
Carpet backing
Di-ethyl phthalate
(DEP)
Cellulose acetate plastic films- used as
carton windows to display foods
Molded plastics, i.e. toothbrushes, car
components and children's toys
Di-isononyl phthalate
(DINP)
Vinyl wall coverings, toys, and medical devices
showed low concentrations of some plant substances reduce estrogenic effects,
while at high doses estrogenic effects may be increased. The contamination of
foodstuff by zearalenone, a fungal phytoestrogen, is common and human
consumption is estimated at 3 pg/person/day in North America (McLachlan,
2001).
Phthalates
Phthalates are plasticizers that are commonly used as softeners in the
production of paints, inks, adhesives, and various plastic goods (Table 2-4). In
an extensive study of phthalate compounds, the National Institute for Health
(NIH) concluded that benzyl butyl phthalate (BBP) was both a developmental and
reproductive toxicant (NIH, 2003). Additionally, extensive phthalate
contamination has been reported for over-the-counter beauty products
(Environmental Working Group, 2002). With concern, researchers have shown

25
that body burdens of phthalate compounds in women of child-bearing age (20-40
years) are higher than males and any other age group (Blount et al., 2000), and
the long-term consequences of this are unknown.
Phthalate compounds are ubiquitous contaminants of both terrestrial and
aquatic environments. Freshwater levels of di-ethyl hexyl phthalate (DEHP) and
di-butyl benzyl phthalate (DBP) ranged from 4.6 to 90.5 pg/L and 0.1 to 75.6
pg/L, respectively, while marine concentrations of DEHP and DBP ranged 0.1 to
2306.8 pg/L and 1.0 to 1028.1 pg/L, respectively (Fatoki and Noma, 2002).
Additionally, contamination of raw drinking water by di-ethyl phthalate (DEP) has
been reported (USEPA, 2001). Some phthalate contamination in the
environment may be traced back to WWTP discharges. Fromme et al. (2002)
surveyed 39 German wastewater treatment plants and showed the
concentrations of DEHP and DBP were highly variable in the effluents and
ranged from 1.7 to 182 pg/L and 0.2 to 10.4 pg/L, respectively. Furthermore, the
concentrations of phthalate esters in the WWTP sludge ranged from 27.9 to 154
mg/kg dry weight for DEHP and 0.2 to 1.7 mg/kg dry weight for DBP (Fromme et
al., 2002).
Phthalate esters, including the commonly used di-ethyl phthalate (DEP),
DBP, BBP, and di-isobutyl phthalate (DIBP), are capable of inducing an
estrogenic response in reporter assays, but their potency was one millionth that
of 17 p-estradiol (Jobling etal., 1998; Harris etal., 1997). Legleretal. (2002)
identified the hormonal activity of BBP using an estrogen receptor-chemically
activated luciferase reporter gene (ER-CALUX) construct; however, the

26
Table 2-5.
Food Item
Concentration3
(pg/kg)
Peanut butter
5.2
Marmalade
7.3
Butter
14.4
Tomatoes
18.5
Apples
19.4
Breast milk
0.3
Infant formula
1.6-2.1
’ from Guenther et al. (2002).
responses of other phthalates, e g., DEP and DBP, were weaker. Researchers
continue to investigate the hormonal activity of phthalate compounds; in fact,
there is still a debate surrounding the hormonal activity of the most commonly
used phthalate, DEHP (Metcalfe etal., 2001).
In humans, the main pathway for the conjugation of phthalates prior to
excretion is via glucuronidation (Albro et al., 1982). Evidence for the reduced
hormonal activity of phthalate conjugates comes from rodent assays (Foster et
al., 2000). Although the conjugated phthalates are excreted at concentrations in
the microgram per liter range, they may be rapidly deconjugated in the presence
of the glucuronidase enzymes (Blount et al., 2000). These glucuronidase
enzymes are present in high concentrations in domestic wastewaters.
Alkylphenols
Alkylphenol polyethoxylates (APEs) are one class of non-ionic surfactants,
with numerous industrial and domestic uses (Talmage, 1994). These hydrophilic
APEs are rapidly degraded during biological treatment, e g. in wastewater
treatment plant (WWTP), to hydrophobic and recalcitrant alkylphenols (AP). Due
to the high degree of ethoxylation, APEs are not estrogenic; however, activity has

27
been reported in the degradation products nonylphenol (NP) and octylphenol
(OP) (Routledge and Sumpter, 1996a). Nonylphenols are ubiquitous
contaminants of commercially available food items (Guenther et al., 2002) (Table
2-5).
The affinity of the hydrophobic APs to sediment increases with organic
content (Lye et al., 1999). As a result, the reported half-life of sediment-
associated alkylphenols is roughly 60 years (Shang et al., 1999). In the outfall of
WWTPs, reported concentrations of APs in the sediments range from 2 to 9,050
ng/g dry weight in freshwater environments (Lye et al., 1999) and from 1370 to
1630 ng/g in marine environments (Shang et al., 1999).
Humans excrete AP compounds as glucuronide conjugates (Muller et al.,
1998), and these conjugates are then subjected to biological degradation
processes. Current wastewater treatment technologies are not effective for the
complete removal of APs (Sheahan et al., 2002b); hence, pg/L levels are
discharged to receiving waters and induce hormonal responses in fish (Sheahan
et al., 2002a). In aquatic environments, one of the biomarkers for exposure to
hormonally active compounds is the presence of vitellogenin (Vtg), a fish egg
yolk protein. Jobling and Sumpter (1993) reported a 20- to 90-fold increase in
Vtg production in Rainbow trout (Oncorhynchus mykiss) exposed to various
concentrations of alkylphenols (1 to 100 pM) in a laboratory study. Alkylphenols
bioaccumulate (Sheahan et al., 2002b); in fact, 10-30 ng NP/g of liver (wet
weight) was reported in male flounder (Platichthys flesus) (Lye et al., 1999).
Although WWTP effluent concentrations of the hydrophobic NP and OP have

28
been reported in the mid ng/L range (Snyder et al., 1999), higher concentrations
may be found in the wasted sludge due to partitioning (Ejlertsson et al„ 1999; La
Guardia et al., 2001). While octylphenol and nonylphenol are weakly estrogenic
(Legler et al„ 2002), their conjugated forms are not capable of inducing estrogen¬
like responses (Moffat et al., 2001).
Natural and Synthetic Estrogens
Research indicates that estrogens, both natural and synthetic, represent
the predominant fraction of organic wastewater contaminants and concurrently
induce the highest hormone activity (Metcalfe et al., 2001; Snyder et al., 2001;
Rodgers-Gray et al., 2000; Desbrow et al., 1998). The vertebrate endocrine
system produces chemical messages, called hormones, which regulate body
functions, e g., reproduction, growth, and homeostasis (Figure 2-2). Estrogens
are the hormones produced by the ovaries and they are responsible for the
development and regulation of female secondary sexual characteristics. The
endogenous estrogens, 17 p-estradiol (E2) and estrone (Ei), together with their
degradation product estriol (E3) are rapidly conjugated and excreted from the
body. This made their use in hormone therapies ineffective and led to the
development of synthetic hormones (Bolt, 1979). Although the synthetic
hormones are rapidly absorbed in the bloodstream, they are slowly metabolized
and are; therefore, better suited for drug therapies (Guengerich, 1990). The
most commonly prescribed synthetic hormones are 17a-ethynylestradiol (EE2)
and mestranol, which are both utilized in the production of birth control pills
(BCP) and as inhibitors of ovulation (Ranney, 1977). Although E1 and E3 are the

29
Table 2-6. Rates for the urinary excretion of natural estrogens from men and
women
Women
Menc
(n=2)
Estrogen
Pre-
Menopausal3
(n=114)
Pre¬
menopausal13
(n=25)
Post¬
menopausal3
(n=146)
17p-Estradiol (E2)
(pg/day)
3.5
1.1-2.8
0.7
1.5
Estrone (E1)
(pg/day)
7.0
2.6-78
1.4
3.9
Estriol (E3)
(pg/day)
8.7
4.7-56
r>A„'. .
1.6
1.5
‘Key et al., 1996, reported as geometric mean, “Adlercreutz et al., 1994, reported as range, and
cFotsis and Adlercreutz, 1987 reported as mean. Standard deviations were not reported.
main metabolites of E2, there is also a group of minor metabolites with
inconsequential hormonal activity.
The metabolic pathways for natural and synthetic estrogens have been
extensively reviewed (Bolt, 1979; Guengerich, 1990). Generally, natural and
synthetic hormones in the human body are metabolized to inactive glucuronide or
sulfonide conjugates before excretion (Bolt, 1979). The age distribution and;
hence, the reproductive conditions of women in a population determines the total
concentration of excreted estrogens. Table 2-6 summarizes reported excretion
rates of natural estrogens from both men and women. Keys et al. (1996) showed
that pre-menopausal women excreted 3.5 pg/day of E2, 7.0 pg/day Ei, and 8.7
pg/day E3. In a separate study with pre-menopausal women, similar estrogen
concentrations in urine were reported by Adlercreutz et al. (1994). The slight
variation in estrogen excretion reported by Key et al. (1996) and Adlercreutz et
al. (1994) was probably due to the menstrual phase during urine collection.

30
Adlercreutz et al. (1994) collected urine samples during the mid-follicular phase
(3-11 days after the onset of the last menstruation), while Key et al. (1996)
analyzed urine collected throughout the entire menstrual cycle.
Overall, pregnant women excrete the highest concentrations of natural
hormones at 600 pg/day, 259 pg/day, and 6000 pg/day for Ep E2, and E3,
respectively (Fotsls et al., 1980). The estrogen concentrations reported for post¬
menopausal women were 0.7 pg/day for E2,1.4 pg/day for Ep and 1.6 pg/day for
E3(Key et al., 1996) and were comparable to male estrogen excretion rates.
Male (n=2) excretion was reported for Ep E2, and E3at 3.9 pg/day, 1.5 pg/day,
and 1.5 pg/day, respectively (Fotsls and Adlercreutz, 1987). Predicting the
excretion rates of synthetic hormones is more difficult and depends on the
number of pre-menopausal females in a population, cultural mores and the brand
of birth control pill used (Johnson et al., 2000). A search of pharmaceutical
information on the Internet showed a range of 30 - 40 pg EE2/ tablet, with a
typical dosing regime of 21-28 days, followed by 7 days of inactive tablets.
Larsson et al. (1999) estimated EE2 excretion rates at 4 pg/day per female
consuming oral contraceptive pills in Sweden. The excretion of endogenous
hormones is predominantly via the urine, while fecal elimination generally
exhibits a minor secondary role; however, for the excretion of synthetic hormones
the fecal route is primary (Ranney, 1977).
Fecal excretion rates of endogenous estrogens from pre-menopausal
women (n=25) were reported as 0.5 ng/day for Ep 0.4 ng/day for E2, and 0.8
ng/day for E3 (Adlercreutz et al., 1994). Daily excretory rates for feces and urine

31
have been reported at 100-400 grams and 1-1.3 kg wet volume/person/day,
respectively. These ranges generally apply to men, with excretion rates for
women generally at the lower limit of this range (Polprasert, 1989 as cited in
Bitton, 1994).
Effects of Hormonally Active Compounds on Humans
Over the past 30 years, what began as anecdotal observations of altered
reproductive and sexual development in humans have coalesced into concern for
long-term species survival (McLachlan, 2001). The early onset of middle-age
vaginal carcinomas and deformed uteri in young women have been linked to the
potent synthetic estrogen diethylstilbesterol, widely prescribed to pregnant
women throughout the 1950's and 60's (Colburn et al., 1996). During the first
trimester of pregnancy, human fetuses are highly sensitive to exposures from
hormonally active compounds.
Industrialized nations, including the U.S., Scandinavia, and Japan, have
reported an increased incidence of hypospadias (displacement of the urethral
opening toward the scrotum) and cryptorchidism (failure of the testicles to
descend into the scrotum) in males (Paulozzi, 1999). Some researchers have
questioned this conclusion and instead cite increased reporting and stricter
definitions as factors artificially inflating the data. Widespread trends are difficult
to establish, but adverse sexual effects from exogenous substances have been
confirmed. The feminization of males has been attributed to work place
exposure to formaldehyde (Finkelstein et al., 1988) and therapeutic treatments
with herbal supplements (DiPaola et al., 1998). Gray (1998a) showed that
sexual differentiation in male rats was altered after exposure to hormonally active

I HORMONALLY ACTIVE COMPOUNDS']
CENTRAL NERVOUS SYSTEM
HYPOTHALAMUS
a
{1
ÍJ.
£
a
THYMUS
| THYROID |
I ADRENAL 1
1 GLANDS 1
PANCREAS
| OVARY
| TESTES |
PINEAL
GLAND
LYMPH TISSUE
MUSCLE,
LIVER
NERVOUS
SYSTEM,
KIDNEYS
LIVER,
MUSCLES
REPRODUCTIVE ORGANS
CIRCADIAN
RYTHYMS
Figure 2-1. Representation of the vertebrate endocrine system and the possible influences of hormonally active
compounds on various systems and organs, (adapted from Mathews and van Holde, 1996)

33
Table 2-7. Advantages and disadvantages associated with the use of in vivo and
in vitro assays for identifying hormonal activity
Advantages
Disadvantages
In Vivo
Metabolic capability
High cost
Multiple nuclear receptors
Established assays
Assay duration (weeks to months)
Non-standardized protocols (dosing
regime, food, endpoint)
Sensitivity to non-hormonal effects
In Vitro
Low cost
Rapid (hours to days)
Simplified culture techniques
No metabolic capability
Predominately measure ER mediated
effects
Lack of pathways to clear hormones
Minimize endocrine system complexity
compounds and pesticides. The human reproductive system is regulated by a
plethora of chemical messengers in a complex relay of signals that control
gametogenesis, ovulation, fertilization and sexual differentiation (Thomas, 1997).
The vertebrate endocrine system produces chemical messages, called
hormones, which regulate body functions, e.g. reproduction, growth, and
homeostasis (Figure 2-1). Numerous reviews have been published that discuss
the effects of endocrine-disrupting compounds on humans (Sultan et at., 2001;
Degen and Bolt, 2000; Paulozzi, 1999; Neubert, 1997).
Bioassays to Identify Hormonal Activity
Pursuant to congressional mandates, the US environmental protection
agency (USEPA, 1998) developed a framework for a tiered screening program

34
Table 2-8. In vivo assays for the determination of hormonal activity
ASSAY
ENDPOINT
Rodent
Measure uterine weight of ovariectomised
rodents
Rodent
Measure vaginal cornification3 of
ovariectomisedb rodents
Hershberger Castrated Rat
Measures androgen sensitive tissue weight of
castrated male rodents
Fish
Gonadosomatic index, Vtgc induction
Turtles
—a ■ ..
Vtg induction
“vaginal lesions, "ovaries removed surgically, “Vtg, vitellogenin (a female egg yolk protein)
that integrated in vivo and in vitro bioassays for the identification and
quantification of hormonally active agents in the environment (Gray, 1998b).
Researchers continue to investigate novel approaches for identifying hormonal
activity, and, although these new methods increase the knowledge base of
hormonal effects, more work is needed in establish the foundation of adverse
hormonal effects. Primarily, increased validation of the more widely utilized
assays, e g., the yeast estrogen screen (Routledge and Sumpter, 1996b) and
rodent assays (Ashby, 2000) and inter-laboratory comparison of these
established assays (Ashby, 2003) are needed. In vitro and in vivo assays each
have their own advantages and disadvantages; therefore, a battery of assays
utilizing both types of assays has the greatest value (Table 2-7).
Endogenous estrogen ligands bind with estrogen receptors at the cellular
level in a well-defined cascade of cellular events (Okamura and Nakahara,
1999). The endogenous ligand enters the cell via active transport mechanisms
or diffusion. Once inside the cell, the ligand enters the nucleus and binds with
the estrogen receptor displacing the heat shock proteins (e.g., Hsp90) associated

35
Table 2-9. Threshold dose for the induction of hormonal effects following
exposure of fish to natural and synthetic estrogens
Species
Hormone3
Cone.
(ng/L)
Response
Source
Oncorhynchus
mykiss
(Rainbow
trout)
m m
ro -»■
25-50
1-10
Vtg induction
Vtg induction
Routledge et al.,
1998
Oncorhynchus
mykiss
(Rainbow
ee2
1.5
Vtg induction
Larsson et al., 1999
trout)
Rutilus
rutilus
(Roach)
e2
1-10
Vtg induction
Routledge et al.,
1998
Oryzias
latipes
(Medaka)
e2
ee2
Ei
e3
4°
0.03b
8b
750b
Alteration in
reproductive
characteristics
Metcalfe etal., 2001
Danio
rerio
(Zebrafish)
ee2
5-10
Vtg induction;
Erratic
spawning
van den Belt et al.,
2001
Ictalurus
punctatus
(Channel
catfish)
e2
2.7
Vtg induction
Monteverdi et al.,
1999
Platichthys
flesus
(Flounder)
ee2
10
Vtg induction
Allen et al., 1999
“Abbreviations: E,, estrone: E2, 17p-estradiol; E3, estriol; EE2, ethynyl estradiol; Vtg, vitellogenin
“lowest observed effect concentration
with the receptor. These proteins maintain the structural conformation of the
estrogen receptor (Fliss et al., 2000). The receptor-ligand complex then binds to
a specific ligand-binding domain on the nuclear DNA (Massaad et al., 1998),
which codes for the transcription of messenger RNA (mRNA). In the cellular

36
machinery, the genomic message on the mRNA is translated into protein. This
suite of events is initiated in response to the estrogenic ligand. Estrogen
receptors are part of a "superfamily" of nuclear receptors and include a large
number of orphan receptors, with no recognized ligands (McLachlan, 2001).
There are two forms of the estrogen receptor, estrogen receptor a (ERa) and
estrogen receptor p (ERp). Although, the tissue distribution of ERa and ERp
differ based on sex (male or female) and organ type, they display similar
sensitivities to the endogenous estrogen 17 p-estradiol (Couse et al., 1997).
While the majority of hormonally active substances exert their influence via
ligand-dependent activation of the estrogen receptor, some substances do not
act via receptor interactions (El-Tanani and Green, 1997).
In vivo assays for the determination of hormonal activity
Traditionally, the potential for hormonal activity was assessed with in vivo
assays (Table 2-8). Typical endpoints measured are increased uterine weight,
altered sex ratios, skewed gonado-somatic index, and induction of vitellogenin
(Vtg) (an egg yolk protein in vertebrates). In vivo assays using various
crustacean species (Andersen et al., 1999; Fingerman et al., 1998), including
Daphnia magna (Shurin and Dodson, 1997; Baldwin et al., 1997; Baldwin et al.,
1995) have been used to evaluate hormonal activity as decreased steroid
metabolism and developmental abnormalities.
Common in vivo assays use rodents (Prinsen and Gouko, 2001), fish
(Sepulveda et al., 2002; Bowman et al., 2000) and some invertebrates (Gagne et
al., 2001; Blaise et al., 1999), but these assays are expensive, labor-intensive

37
Table 2-10. In vitro assays for the determination of hormonal activity
ASSAY
MODE OF ACTION
Cell Proliferation Assays
measures the ability of a substance to
stimulate proliferation in estrogen sensitive
cells
Ex. E-Screen (Soto et al., 1992)
Receptor Binding Assays
measures the affinity between a substance
and the estrogen receptor
Ex. hER a or p (Gutendorf and
Westendorf, 2001)
measures the ability of a substance to induce
Reporter Gene Assays
the reporter gene
Ex. YES (Routledge and Sumpter, 1996b)
Cell Line Assays
measures the induction of a specific proteins
or enzymes by a substance
Ex. Liver cells (Monteverdi et al., 1999)
Cell Proliferation/Reporter Gene
measures the ability of substance to stimulate
cell proliferation and induce reporter gene
transcription
Ex. ER-CALUX (Legler et al., 1999)
and, in some cases, raise ethical concerns. One of the advantages of in vivo
assays is the cellular machinery for the metabolism and/or conjugation
ofhormonally active compounds (HAC). The degradation of the parent HAC may
produce a metabolite with no hormonal activity (Harris et al., 1997). Table 2-9
summarizes literature reports for the threshold dose of natural or synthetic
estrogens required for the induction of a hormonal response in various fish
species.
In vitro assays for the determination of hormonal activity
Generally, the premise on which in vitro assays are based is the defined
mechanism of action between hormonally active ligands and nuclear receptors,
usually the estrogen receptor. Due to the lack of metabolic pathways in the in
vitro assays, the hormonal activity may be over-predicted. In most cases, in vitro

38
Table 2-11. Relative sensitivity of in vitro assays to 17 p-estradiol (E2)
Assay
MDLa
(ng/L)
EC50
(ng/L)
Source
RCBAb
.03
-
Coldham et al., 1997
RCBA
.02
-
Klein et al., 1994
YES
3
-
Routledge and Sumpter, 1996b
YES
2.7
27
Murk et al., 2002
YES
-
19
Beresford et al., 2000
YES
-
27
Tanaka et al., 2001
YES
-
13
Elsby et al., 2001
YES
-
5.4
Vinggaard et al., 2000
YES
-
22.8
Layton et al., 2002
YES
2.7
27
Legler et al., 2002
E-SCREEN
-
1.4
Gutendorf and Westendorf, 2001
E-SCREEN
-
2.7
Soto et al., 1992
E-SCREEN
1.7
Behnisch et al., 2001
Cell line/reporter
(MVLN)
-
1.4
Gutendorf and Westendorf, 2001
Cell line/reporter
(HGELN)
-
10.9
Gutendorf and Westendorf, 2001
Cell line/reporter
(ER-CALUX)
0.1
1.6
Legler et al., 2002
Competitive
binding (ER a)
-
900
Gutendorf and Westendorf, 2001
Competitive
binding (ER p)
-
17700
Gutendorf and Westendorf, 2001
Competitive
binding (ER (?))c
272
1360
Murk et al., 2002
Abbreviations: aMDL, minimum detection limit; “RCBA, recombinant cell bioassay; “estrogen
receptor form not indicated

39
assays are constructed with recombinant molecules or cells from either
mammalian or fish tissues (Zacharewski, 1997; Diel et al., 1999). Most
frequently, yeast cells (Saccharomyces cerevisiae) are used as carriers for the
hormone receptors. Early in vitro assays incorporated an estrogen receptor (ER)
and reported only ER-mediated hormonal activity. Recently, in vitro assays have
been designed with other nuclear receptors, including androgen receptors (AR)
and progesterone receptors (PR) (Nishikawa et al., 1999). Table 2-10
summarizes the general types of in vitro assays currently available.
The hallmark of an in vitro assay is the sensitivity of the assay to the
endogenous estrogen E2. Table 2-11 summarizes the sensitivities of various in
vitro assays. Advantages afforded by in vitro assays include the rapid
identification of hormonal effects, relatively lower cost, and the ability to screen
numerous samples simultaneously. Typical endpoints for in vitro assays include
cell proliferation, enzyme expression (e.g. p-galactosidase), and protein
synthesis. Some of the limitations inherent in in vitro assays are the absence of
metabolic pathways, the over-estimation of binding in a single receptor systems
(Jobling et al., 2002), and reliance on estrogen mediated effects, while ignoring
other receptors (Diel et al., 1999). Despite these concerns in vitro
assayscontinue to be widely used for the identification of hormonal activity and
for the quantification of the contributions from individual chemicals to overall
activity (Degen and Bolt, 2000; Gutendorf and Westendorf, 2001).
The most widely used reporter gene assay Incorporates the human
estrogen receptor (hER) into the genome of the yeast Saccharomyces cerevisiae

40
(Rehmann et al„ 1999; Coldham et al., 1997; Gaido et al„ 1997; Routledge and
Sumpter, 1996b). Yeast cells are stably transfected with the human ER (hER)
and expression plasmids for a reporter gene, usually lac-Z (codes for the enzyme
p- galactosidase). When estrogenic ligands enter the cell, they bind with the ER
to form a ligand-ER complex. This complex then interacts with the estrogen
responsive element (ERE) on the plasmid and initiates transcription of the
reporter gene. The reporter gene product is quantified by the addition of a
suitable substrate. Due to their easy quantification by spectrophotometers,
chromogenic substrates are typically used, e.g., chlorophenol red
galactopyranoside (CPRG) (Routledge and Sumpter, 1996b) or orfbo-nitrophenol
galactopyranoside (ONPG) (Lascombe et al., 2000; Klein et al., 1994). These
estrogen receptor/reporter assays are rapid, reproducible, and have
demonstrated a high degree of sensitivity to hormonally active compounds.
The YES (Routledge and Sumpter, 1996b) has been widely used to
identify hormonal activity in pure compounds (Beresford et al., 2000), wastewater
treatment plant influents and effluents (Holbrook et al., 2002), flue gases
(Muthumbi et al., 2002) and recycled materials (Vinggaard et al., 2000). This
assay has also been adapted to include the androgen receptor and thus quantify
androgenic effects (Beresford et al., 2000).
Other yeast-based assays have been developed, and some are gaining
wide acceptance due to their use of multiple nuclear receptors. A novel ligand-
receptor binding assay was constructed in a yeast (Y190) two-hybrid assay with
expression plasmids (pGBT9 and pGAD424) to determine the interaction

41
between selected hormone receptors (ER, AR, PR, MR, TR) and co-activators
(TIF2, SRC1.TIF1, RIP140) in the presence ofHACs (Nishikawa et al„ 1999).
This assay is highly sensitive to phytoestrogens and nonylphenol (Nakano et al.,
2002) and environmentally relevant concentrations of HACs (Kawagoshi et al.,
2003). The role of the co-activator is poorly understood, but, after binding of the
ligand to the nuclear receptor, it appears to influence processes that initiate gene
transcription (Nishikawa et al., 1999). The TIF 2 co-activator had the greatest
influence on initiation (Kawagoshi et al., 2002).
Exploitation of the estrogen sensitivity of breast cancer cells led to the
development of assays that quantify cell proliferation in the presence of
estrogens or estrogen-like substances. The E-Screen (MCF-7 breast cancer
cells) assay (Soto et al., 1992) provides highly reproducible results when assay
protocols are strictly adhered to and the cell line source is consistent. Payne et
al. (2000) evaluated three MCF-7 cell lines (BUS, UCL, and SOP) for their
sensitivity to E2 and showed EC50 values of 3.6, 3.1, and 2.6 ng/L, respectively.
However, the proliferative effect (growth in excess of control cells) of E2 varied
markedly among the cell lines at 8.9 with BUS, 0.98 with UCL, and 1.45 with
SOP (Payne et al., 2000). The functionality of some estrogen receptors in breast
cancer cell lines may be low, with up to 50 % non-functional (Balmelli-Gallacchi
et al., 1999). In cell proliferation assays, direct counts occur with
hemocytometers or automated Coulter counters.
The addition of reporter genes to cell lines has Increased their sensitivity
and allowed for the elucidation of multiple mechanisms of action in a single test

42
system. Luciferase reporter gene constructs were designed in human breast
cancer cell lines. Addition of the lux reporter gene to MCF-7 and HeLa cells
produced the MVLN and HGELN systems (Balaguer et al., 1999; Gutendorf and
Westendorf, 2001). This adaptation allowed for the identification of substances
whose mode of action was via cell proliferation or ER activation. Katori et al.
(2002) demonstrated that di-butyl phthalate induced cell proliferation, but not
gene transcription.
Additionally, the ER-Chemical Activated Luciferase gene expression (ER-
CALUX) assay was developed in T47D human breast cancer cells by
transfection with reporter genes (pEREtata-Luc) (Legler et al., 1999). The steep
E2 dose-response curve with the ER-CALUX assay was nearly 20 times greater
than the reporter gene response in the YES, indicating the higher sensitivity of
the ER-CALUX assay (Legler et al., 2002) (Table 2-11). Other advantages of the
ER-CALUX assay are the small sample volume requirements, which were
roughly 1 /10th and 1/100lh the volume required by the YES and ER binding
assays, respectively (Murk et al., 2002).
Cell line assays are not limited to mammalian cells. Monteverdi and
Giulio (1999) combined primary liver hepatocytes from Channel catfish (Ictalurus
punctatus) with an enzyme-linked immunosorbant assay (ELISA) to measure the
induction of Vtg. Petit et al. (1999) developed a test system in yeast that
expressed the Rainbow trout estrogen receptor (rtER).
Competitive binding assays measure the displacement of E2 from the
estrogen receptor. The ERa has a higher sensitivity than the ERp for E2

43
(Gutendorf and Westendorf, 2001). Generally, these competitive binding assays
have a lower sensitivity than other in vitro assays and are not suited as screening
assays for hormonal activity (Table 2-11). In competitive binding assays the
response to agonistic and antagonistic xenobiotics are measured simultaneously,
which accounts for the lower sensitivity (Murk et al., 2002).
Some novel approaches for detecting hormonally active compounds have
been developed. A biosensor has been constructed, which incorporates the
human estrogen receptor a (ERa) into a lipid bilayer with direct contact to a gold
electrode for quantification (Granek and Rishpon, 2002).
A test battery to determine hormonal activity
An optimum test battery to evaluate aquatic toxicity includes algal,
invertebrate, and bacterial components (Rojickova-Padrtova et al., 1998).
Similarly, a successful investigation of hormonal activity should use a suite of
assays to include a cell proliferation assay, a yeast reporter assay, and a
competitive binding assay (Fang et al., 2000; Coldham et al., 1997). When in
vitro assays are combined in an array, then multiple mechanisms may be studied
simultaneously including the effects of metabolism and/or transport (Baker, 2001;
Vinggaard et al., 1999). The sensitivity of in vitro assays to E2 stands as the
hallmark by which assays for hormonal activity are judged. Gutendorf and
Westendorf (2001) demonstrated an E2 sensitivity that increased in the order of
estrogen receptor binding assays (ERa and ERp) < reporter gene assays < cell
proliferation assays (Table 2-11). The ER-CALUX assay has demonstrated the

44
overall highest sensitivity to E2, with an EC50 of 1.6 ng/L compared to 27 ng/L
with the YES and 1,360 ng/L with ER binding assays (Murk et al., 2002).
Characterizing Hormonal Activity in MSW Landfill Leachates and Other
Environmental Samples
Until recently, little was known about the hormonal activity of MSW landfill
leachates, despite the threat they posed to the environment (Ejlertsson et al.,
1999). Modern MSW landfills are engineered with barriers to restrict the mobility
of the liquid fraction (leachate) of waste and collection systems; however, this
has not always been the case. In the past, the disposal of waste was largely
unregulated allowing for direct release of toxic substances to ground and surface
waters. Some of these older unregulated landfills continue to release leachates
of unknown strength and chemical composition.
In MSW landfills, biological and chemical processes produce leachates
with high concentrations of organic contaminants (Yasuhara et al., 1999).
Shiraishi et al. (1999) demonstrated the presence of compounds with known
hormonal activity in the leachates of Japanese landfills. Behnisch et al. (2001),
using the E-Screen assay (MCF-7 cells), confirmed the hormonal activity of
waste leachates in a Japanese landfill and tracked reductions after treatment
processes. They reported an estradiol equivalency (EE) of the untreated
leachate at 4.8 ng EE/L and after biological and activated carbon treatment at 2.8
ng EE/L. This was equivalent to a 58 % reduction in hormonal activity (Behnisch
et al., 2001). The composition of waste materials in Japanese domestic landfills
differs significantly from U.S. MSW landfills. Due to space constraints and other
considerations, domestic wastes in Japan are first incinerated to reduce volume

45
and reactivity, then the ash material is landfilled. The composition of the waste in
the landfill studied by Behnish et al. (2001) was primarily inorganic, with about 70
% incinerator ash. Characterizing some of the organic compounds in the
leachate revealed the presence of known hormonally active compounds,
specifically bisphenol A, nonylphenol, and estradiol at 0.13, 2.8, and 0.005 pg/L,
respectively.
Kawagoshi et al. (2002) used a yeast two-hybrid reporter assay
(Nishikawa et al., 1999) to demonstrate the hormonal activity of waste leachate-
contaminated groundwater at an E2 activity equivalent to 27.2 ng/L. Leachates
that were collected from sites for the disposal of solid municipal wastes and
dredged soils were not hormonally active. In a continuing investigation, the
efficiency of the extraction procedures for the recovery of hormonal activity were
evaluated (Kawagoshi et al., 2003). Their extraction procedures used C-18 SPE
columns and were highly efficient for the recovery of hormonal activity. Together
with the high recovery of activity following elution with polar solvents (acetone),
these results implicated non-polar hydrophobic compounds as causative agents
for the activity (Kawagoshi et al„ 2003). Based on the comparison of bioassay
results with one raw leachate and acetone extracts of the same leachate,
Kawagoshi et al. (2003) suggested that anti-estrogenic compounds in the
leachate interfered with the recovery of hormonal activity.
Considering the potential for release of MSW leachate from landfills, the
fate of hormonally active compounds in soils is a concern. The mobility of
hormonally active compounds (HACs) (E2, EE2, nonylphenol, octylphenol, and

46
MCF-7 /luciferase
E-Screen
Substance
reporter assay
(ECso)
(nM)
Assay
(ECso)
(nM)
e2
0.03
0.14
Bis(tri-n-
butyltin)
1.84
0.55
Antimony
chloride
16.4
14.8
Chromium
chloride
34.5
33.3
Lithium
chloride
47.1
49.7
Cadmium
chloride
108
176
Barium
chloride
743
458
aChoe et al. (2003).
bisphenol A) in soil (93 to 94 % sand) was investigated in lysimeter experiments,
with bioassays (Dizer et al., 2002). Although the leachates (water extracts)
produced by the lysimeters displayed a low hormonal activity, no attempt was
made to quantify the concentrations of hormonally active compounds in the
leachates. Hence, reduced hormonal activity may have resulted from the
adsorption of the HACs to soil particles (Dizer et al., 2002).
While organic substances are the most widely recognized hormonally active
compounds, some heavy metals are also hormonally active (Stoica et al., 2000).
Although the concentrations of heavy metals in MSW landfill leachates are low,
there is a potential for increased leaching of heavy metals as landfills age
(Bozkurt et al., 2000; Flyhammer, 1997). Stoica et al. (2000) reported cadmium
(as CdCI2) activated the estrogen receptor at low concentrations, but at high

47
Compound
Concentration
(ng/L)
Reference
Bis(ethylhexyl)
phthalate
1350a
Yasuhara et al.,
1999
Bisphenol A
61400a
Yasuhara et al.,
1999
Dimethyl phthalate
300a
Yasuhara et al.,
1999
Dibutyl phthalate
1800a
Yasuhara et al.,
1999
Diethyl phthalate
1600a
Yasuhara et al.,
1999
Diethyl phthalate
479
Welander and
Henrysson, 1998
Bis(ethylhexyl)
phthalate
10,850
Welander and
Henrysson, 1998
Butyl-benzene
sulfonamide
2300
Welander and
Henrysson, 1998
median concentration
concentrations it blocked the binding of estradiol to the receptor. Choe et al.
(2003) used the E-Screen assay and a cell proliferation/ luciferase reporter gene
assay in MCF-7 cells to determine hormonal activity in twenty species of eight
metals (Table 2-12). The metal species were ranked for their hormonal activity in
the cell proliferation/reporter assay as Bis(tri-n-butyltin) > cadmium chloride >
antimony chloride > barium chloride = chromium chloride. Although the
sensitivity of the E-Screen assay was lower, the ranking was similar with Bis (tri-
n-butyltin) > cadmium chloride > antimony chloride > lithium chloride > barium
chloride (Choe et al., 2003).
As previously discussed, phthalates have demonstrated hormonal activity
in a variety of test systems. Their presence in MSW landfill leachates can be
attributed to the composition of the waste material and the increasing use of

48
excess packaging in consumer goods. Table 2-13 summarizes the
concentrations of various phthalates identified in MSW landfill leachates.
Hormonal activity has been identified in a variety of other environmental samples,
which include tree debarking mill effluents (Mellanen et al., 1996), wastewater
treatment plant effluents (Shang et al., 1999), industrialized rivers (Lye et al.,
1999) and surface waters (Witters etal., 2001). The sources of hormonal activity
with the widest distribution are the WWTPs. This is attributed to the
concentrations of natural and synthetic hormones in domestic wastewater (Table
2-14). In the absence of WWTPs, septic systems represent a source of
hormonally active compounds (Rudel et al., 1998). The largest sources of
hormonal activity in wastewater treatment plants (WWTPs) are the natural and
synthetic estrogens. They are excreted as inactive glucuronide and sulfonide
conjugates; however, rapid deconjugation occurs in WWTPs (Ternes et al.,
1999a). Deconjugation occurs in the presence of the enzyme glucuronidase,
which is abundantly produced by Escherichia coli (Ternes et al., 1999b). This
enzyme is responsible for the degradation of both the glucuronide and sulfonide
estrogen conjugates (Belfroid et al., 1999), but sulfonide to a lesser degree
(Huang and Sedlak, 2001). Roughly 25 % of excreted estrogens occur as
sulfonide conjugates, and their degradation is closely associated with
arylsulfatase enzymes. Low concentrations of these enzymes in treatment plants
Is responsible for the greater persistence of the sulfonide conjugates (D'Ascenzo
et al., 2003). Regardless of the reason for incomplete deconjugation of excreted
estrogens, the underestimation of estrogen loads on treatment facilities may

49
Table 2-14. Concentrations (ng/L) of natural and synthetic hormones in
wastewater treatment plants(WWTPs)
1715-
Estradiol
(E2)
(ng/L)
Estrone
(Et)
(ng/L)
Estriol
(Ej)
(ng/L)
17a-ethynyl
estradiol
(EE2)
(ng/L)
Source
WWTP
Inf
ND
ND
ND - 263
ND
Sole et al.,
WWTP
Eff
ND
ND
ND
ND
2000
WWTP
Inf"
11.6b
51.8b
80.4b
3.0b
Baronti et al.,
WWTP
Eff"
1.4b
18.4b
3.0b
0.4b
2000
WWTP
Inf*1
<0.5-20
<0.5-75
2-120
<0.5-6
Johnson et al.,
WWTP
Eff11
<0.5-7
<0.5-52
<0.5-28
<0.5-22
2000
WWTP
Inf
50.7
NM
NM
NM
Matsui et al.,
WWTP
Eff
7.1
NM
NM
NM
2000
WWTP
Inf1
11b
44b
73b
NM
D'Ascenzo et
WWTP
Eff1
1 6b
17b
2.3b
NM
al., 2003
WWTP
Eff"
(winter)
7-88
15-220
NM
NM
Rodgers-Gray
et al., 2000
WWTP
Eff1
(summer)
4-8.8
27-56
NM
NM
Rodgers-Gray
et al., 2000
WWTP
Eff
ND
9b
NM
1b
Ternes et al.,
1999a
WWTP
Eff
6b
3b
NM
9b
Ternes et al.,
1999a
WWTP
Eff"
0.9
4.5
NM
<0.3
Belfroid et al.,
1999
WWTP
Eff"
2.7-48
1.4-76
NM
0.2-7
Desbrow et
al., 1998
WWTP
Eff"
0.2-4.1
NM
NM
0.2-2.4
Huang and
Sedlak, 2001
WWTP
Eff
1.9-14.6
NM
NM
<0.05-3.0
Snyder et al.,
1999
Abbreviations: Wastewater treatment plant, WWTP; Inf, influent; Eff, effluent; ND, not detected;
NM, not measured.b indicates median value. Samples collected from:a Germany, cCanada,
dUnited Kingdom, e USA, 'Netherlands, 8ltaly, "Spain, 'Japan

50
result (Johnson et al., 2000).
To preface any discussion of reported concentrations of hormonally active
compounds in the environment, it is important to consider the detection limits of
the analytical methods. Often the analytical methods for the identification and
quantification of low-level organic contaminants are ineffective and grossly under
predict environmental burdens (Castillo and Barcelo, 1999). For example, while
some laboratories have reported detection limits for E2, E1, E3, and EE2 as low as
0.1-0.6 ng/L for surface waters and 0.1-2.4 ng/L for wastewaters (Belfroid et al.,
1999), others have reported detection limits up to three orders of magnitude
higher for E2(250 ng/L), E,(100 ng/L), E3(50 ng/L) and EE2 (500 ng/L) in WWTP
effluents (Sole et al., 2000).
Alterations in the sexual and developmental characteristics of aquatic
species have been reported worldwide and attributed to the release of
hormonally active micro-organic contaminants (Sheahan et al., 2002b; Desbrow
et al., 1998; Jobling et al., 1993). Although researchers have reported a range of
estrogens in WWTP effluents, concentrations are generally at the low ng/L level.
Chiefly, E2 and EE2 as the hormones with the greatest reported activity have
been identified in effluents at <0.2 to 88 and <0.2 to 9 ng/L, respectively (Table 2-
14). The metabolite E1 has been identified in effluents at concentrations of 1.4 to
220 ng/L (Desbrow et al., 1998). While E2 is widely recognized as the strongest
estrogen, its metabolites are also potent, with E1 about 1.5 times less active
(Jurgens et al., 2002) and E3, the weakest metabolite, also inducing hormonal
affects albeit orders of magnitude less (Metcalfe et al., 2001). The synthetic

51
estrogen, EE2, while found at lower concentrations in wastewater (Johnson,
2000) has an activity comparable to that of E2 (Larsson et al., 1999). The lower
activity of Ei is offset by its extensive presence, and gives rise to concerns about
the equivalent E2 activity.
Rodgers-Gray etal. (2000) investigated the influence of seasonal changes
on activated sludge biology and its subsequent effect on the removal of E2 and
Ei from an activated sludge WWTP in England. During the cooler winter months,
concentrations of E2 and Ei ranged from 7 to 88 ng/L and from 15 to 220 ng/L,
respectively. In contrast, there were lower concentrations reported in the
summer months with E2 concentrations ranging from 4 to 8.8 ng/L and Ei
concentrations ranging from 27 to 56 ng/L. Overall, the concentrations of Ei
exceeded those of E2, indicating a greater recalcitrance of Ei to biological
treatment (D' Ascenzo et al., 2003). Few studies have looked at E3, and its
effects may be under estimated.
A limited number of studies have evaluated the concentrations of
endogenous and synthetic estrogens in influents and corresponding effluents of
WWTPs. Despite reported removal rates of up to 90 %, concentrations of
estrogens remain at threshold levels for inducing hormonal effects. Six activated
sludge treatment facilities in Italy (Cobis, Fregene, Ostia, Roma Sud, Roma Est,
and Roma Nord) have been extensively studied by three research teams over a
two-year period. Baronti et al. (2000) reported mean influent concentrations for
the six facilities at 51.8, 11.6, 80.4, and 3.0 ng/L for Ei, E2, E3, and EE2,
respectively. Following biological treatment these concentrations were reduced

52
Table 2-15. Reported concentrations (ng/L) of natural and synthetic estrogens in
surface waters
1713-
estradiol
(E2)
(ng/L)
Estrone
(Ei)
(ng/L)
Estriol
(E3)
(ng/L)
17a-ethynyl
estradiol
(EE2)
(ng/L)
Source
Surface water
<0.5
ND
NM
<0.5
Ternes et al.,
1999a
Surface water
<03-5.5
NM
NM
<0.3-4.3
Belfroid etal.,
1999
Surface water
36-5.2
NM
NM
<0.05-1.4
Snyder et al.,
1999
Surface water
<0.05-0.8
NM
NM
<0.05-0.07
Huang and Sedlak,
2001
to 18.4, 1.4, 3.0, and 0.4 ng/L for Ei, E2, E3, and EE2, respectively. These results
are consistent to those reported by Johnson et al. (2000) and D' Ascenzo et al.
(2003), although the later did not measure EE2. Notably, E1 displayed the
greatest range of concentrations (Baronti et al., 2000; Johnson et al., 2000; D'
Ascenzo et al., 2003) and remained the most prevalent estrogen following
treatment. The only other study to evaluate multiple hormone concentrations in
influents and corresponding effluents was conducted with four WWTPs in Spain
(Sole et al., 2000). While analyses were conducted for Ei, E2, E3, and EE2, the
only hormone detected was E3 and then only in the influents of 2 WWTPs at
approximately 262 ng/L (Sole et al., 2000). These levels were nearly double the
maximum concentrations detected in the Italian WWTPs (Baronti et al., 2000).
Comparatively, low ambient levels of natural and synthetic hormones have
been detected in surface water samples, however, the potential for extensive
contamination of surface waters exists from animal manure's (Casey et al., 2003)
and WWTP discharges (Kolpin et al., 2002). In surface waters, E2 concentrations

53
ranged from <0.05 to 5.5 ng/L, while those for EE2 were <0.05 to 4.3 ng/L (Table
2-15). As a point of reference, altered sexual characteristics are induced in male
rainbow trout (Oncortiynchus mykiss) at ~1.5 ng/L EE2 (Larsson et al., 1999).
Additionally, threshold doses of 1 to 10 ng/L and 25 to 50 ng/L for E2and E1,
respectively, have been reported for the induction of hormonal effects in O.
mykiss (Metcalfe et al., 2001).

CHAPTER 3
TOXICITY OF LEACHATES FROM FLORIDA MUNICIPAL SOLID WASTE
(MSW) LANDFILLS USING A BATTERY OF TESTS APPROACH
Introduction
The State of Florida currently generates more than 25 million tons of
municipal solid waste (MSW) a year. Fifty-six percent of this waste is disposed in
engineered Class I landfills (FDEP, 2000). The state has sixty-one Class I
landfills (permitted to accept only MSW) that are lined and contain systems for
the collection and transport of waste leachates, that are then subsequently
subjected to biological treatment. Researchers have extensively characterized
the chemical and physical characteristics (Townsend etal., 1996; Booth etal.,
1996; Gettinby et al., 1996) and biological toxicity (Plotkin and Ram, 1984;
Ferrari et al., 1999; Ernst et al., 1994) of waste leachates world-wide. These
waste leachates are a complex mixture of both inorganic (e.g., heavy metals,
ammonia) and organic substances (e g. pesticides and chlorinated
hydrocarbons). It has been suggested that exposure to MSW landfill leachates
may pose as great a cancer risk as does the exposure to industrial waste
leachates (Brown and Donnelly, 1988) due to their mutagenic properties (Beg
and Al-Muzaini, 1998). The genotoxic potential of MSW landfill leachates was
shown to be higher than that for industrial wastewater, groundwater or drinking
water samples (Helma et al., 1996).
54

55
When evaluating the toxicity of complex effluents, the use of a battery-of-
tests approach allows for multiple mechanisms of action to be evaluated
simultaneously (Deanovic et al., 1999; Rutherford et al., 2000). A battery-of -
tests approach with algal, crustacean, and bacterial assays was used to
successfully characterize landfill leachate toxicity (Rojickova-Padrtova et al.,
1998; Clement et al., 1996). No characterization of MSW landfill leachate toxicity
is complete until toxicological assays are combined with analytical procedures for
chemical characterization (Lambolez et al., 1994).
The unique sub-tropical climate in Florida with generally abundant rainfall
and warm temperatures reduces the chemical strength of MSW landfill leachates
(Reinhart and Grosh, 1998). Although researchers have characterized the
composition and site-specific parameters for MSW landfills throughout Florida,
the biological effects of these leachates have not been assessed (Reinhart and
Grosh, 1998). The toxicity of MSW leachates from separate landfills, while
related, may differ due to specific characteristics of the wastes, e.g. pH,
temperature, ammonia levels, presence of recalcitrant organic substances, and
microbiological activity. Little information is currently available concerning the
toxicity of MSW landfill leachates in Florida and such a database of information
could be useful in evaluating leachate treatment options and reuse possibilities
(Ward et al., 2000). The research community now recognizes the importance of
using a tandem approach, with both biological and chemical analyses, when
analyzing environmental samples to achieve a better understanding of the
possible causes of toxic effects.

56
Site 5
Site 4
Site 6
Figure 3-1. Locations of the MSW landfills for the collection of leachates In
Florida
The objectives of this research were to 1.). characterize the toxicity of
Florida MSW landfill leachates, 2.). evaluate the use of a battery of algal,
invertebrate and bacterial toxicity assays with Florida leachates, 3.). characterize
the chemical composition of the MSW leachates, and 4.). determine relationships
between selected chemical components and leachate toxicity. Six landfill sites
in north and north-central Florida were sampled monthly over a six-month
sampling period. The sites sampled represented a variety of factors, including
rural and urban areas, some industrial activity, leachate recycle, enhanced
biological treatment in-situ, and those sites currently accepting waste and capped
sites.

57
Table 3-1. Amount of MSW generated and landfilled at six landfill sites in Florida
Landfill
Site
MSW Collected
(Tons/year)3
Amount of Waste
Landfilled
(%)
Landfill
Type
1
235,662
65
Leachate recycle
2
226,477
69
Rural
3
NAb
NA
Rural/capped
4
317,694
68
Semi-urban
5
10,197
83
Regional
6
1,890,112
73
Urban/enhanced
biological treatment
®The data presented represents information collected in 1998. “Data were not available (NA) for
site 3 a capped landfill site, no longer permitted to accept MSW,
Materials and Methods
Leachate Collection
Municipal solid waste (MSW) leachates were collected from six sites
located in five landfills in central Florida, USA. The sites were designated as 1
through 6. Site 2, an operating landfill unit, and site 3, a capped landfill unit, are
located at the same landfill (Figure 3-1). Capped landfill units no longer accept
waste materials and are surrounded by a high-density polyethylene (HDPE) liner
to prevent the infiltration of water and the potential for subsequent escape of
leachates. Table 3-1 summarizes the rates of municipal waste disposal at the
sites under study (FDEP, 2000). MSW landfill leachates were collected from
leachate collection wells using a Teflon baler. One sample was collected at each
site, and then apportioned to separate containers for chemical analysis and
toxicity assays. Leachates for chemical analysis were collected in polyethylene
or glass containers and preserved according to U S. Environmental Protection

58
Agency (USEPA, 1993b). Samples for toxicity analysis were collected in plastic
cubitainers, transported to the lab on ice and immediately stored at4°C until
sample analysis, within 1 to 2 days.
Chemical and Physical Characterization of Leachates
The chemical/physical characterization of the MSW landfill leachates
began with field measurements that included pH and temperature (Orion, Model
290A), conductivity (HANNA Instruments, Model H19033), dissolved oxygen
(DO) (YSI Inc., Model 55/12 FT), and oxidation/reduction potential (ORP)
(Accumet Co., Model 20). In the laboratory, the MSW landfill leachates were
analyzed for a number of standard chemical and physical parameters, which
included alkalinity, biochemical oxygen demand (BOD), chemical oxygen
demand (COD), ammonia and sulfides, according to methods described by
USEPA (1993b) and APHA (1999). Leachates for metal analysis were digested
and analyzed by Inductively Coupled Plasma (ICP) (Thermo Jarrell Ash, Model
Enviro 36). For ion analysis, a Dionex ion chromatograph (Dionex, Model DX-
500) was used. Total ammonia (NH4* + NH3) and un-ionized ammonia (NH3)
were analyzed by a selective ion probe (Accumet, Model 15).
Maintenance of Test Organisms
Pseudokirchneriella subcapitata
Pseudokirchneriella subcapitata (previously known as Selenastrum
capricomutum) is a freshwater unicellular green algae routinely utilized in both

59
Table 3-2. Components of the preliminary algal assay procedure (PAAP)
medium
Growth
Assay
medium
medium
MACRO SALTS
Magnesium sulfate
x
X
(MgS04*7H20)
Magnesium chloride
x
x
(MgCI2 * 6H20)
Calcium chloride
x
x
(CaCI2 * 2H20)
Sodium bicarbonate
Y
x
(NaHC03)
Sodium nitrate
x
x
(NaN03)
Potassium phosphate
x
x
(KH2P04)
Disodium(Ethylene-
dinitrilo)tetraacetate
X
(EDTA)
TRACE METAL SOLUTION
Zinc chloride
X
x
(ZnCI2)
Cobalt chloride
x
x
(CoCI2* 6H20)
Sodium molybdate
x
x
(Na2Mo04 * 2H20)
Cupric chloride
x
x
(CuCI2 * 2H20)
Boric acid
x
(H3BO3)
Manganese chloride
x
x
(MnCI2)
Ferric chloride
(FeCI3*6H20)
FDEP (FDEP, 1997) and USEPA protocols (USEPA, 1978; USEPA, 1994a). The
algae cultures were started from an original algae seed graciously provided by
Hydrosphere Research. Subsequent cultures of algae were maintained in the
laboratories at the University of Florida. The algae growth medium was prepared

60
according to FDEP (1997) and is referred to as the preliminary algal assay
procedure (PAAP) medium. The components of the PAAP are listed in Table 3-
2. The PAAP was prepared by combining 1 ml of each of the macrosalts with 1
ml of the trace metal solution in a 1-L volumetric flask. The flask was filled with
DDI water and thoroughly mixed. The pH of the PAAP medium was adjusted to
7.5 ± 0.1 with either 0.1 NaOH or 0.1 N HCI. The PAAP medium was then filter-
sterilized (0.45 pm membrane filter) and stored under refrigeration.
The algal cells were grown under a specially designed light unit
(constructed by Martin Dolley). The light unit consisted of a wooden platform (3
feet by 4 feet) supported with legs (4 feet), but with open sides. Three
fluorescent light fixtures (3 1/2 feet long) were suspended from the wooden
platform on adjustable chains. The light intensity inside the unit was regulated by
adjustment of the chain length. Black plastic sheeting surrounded the light unit,
and the interior of the sheeting was lined with aluminum foil to maximize the light
reflection and minimize temperature fluctuations.
When propagating the algal cells, cultures were grown in sterile 1- or 2-L
glass erlenmeyer flasks. The flasks were filled to the three-quarter mark with
sterile PAAP medium and then approximately 100 ml of an algae culture (3 to 5
days old) was added. A 10-ml glass pipette was placed in the flask, which was
then wrapped with parafilm to seal the top of the flask. The flask was swirled
vigorously to mix, placed under the light unit, and then the culture was aerated by
attaching a small hose in series with filters (0.2 pm, Acrodisc) to the glass
pipette. A small aquaculture pump supplied air to the algal culture and

61
continuous gentle mixing of the algae medium was maintained. The algae cells
were cultured for up to one week at 25°C under constant illumination (400 + 40 ft-
c). After 3-5 days in the light unit, the algae cells were harvested for toxicity
assays, and after 1 week the cells were transferred to start new algae cultures or
were recovered for use as aquatic invertebrate food. The 1-week old algae cells
were transferred to wide-mouth glass containers, covered loosely and placed in
the refrigerator. After settling for approximately one week, the algae cells were
recovered by siphoning off the overlying spent medium. The recovered algae
cells were then resuspended in a minimum volume of DDL Past experience has
shown that the washing of the algal cells was not required. Algae cultures were
periodically checked for uniformity by transferring small volumes of cells to glass
slides and visually inspecting cell morphology under a phase contrast
microscope. All settled cultures were combined and a final algal cell density was
determined with a hemacytometer. The algae cell density was maintained at 3.5
X 107 cells/ml with DDI before use as aquatic invertebrate food.
Ceriodaphnia dubia and Daphnia pulex
The aquatic invertebrates, Ceriodaphnia dubia and Daphnia pulex are
both members of the family Daphnidae (commonly referred to as daphnids) and
have similar species distributions and life cycles (USEPA, 1993a). Traditionally,
D. pulex (or D. magna) were the invertebrates of choice for determining aquatic
toxicity. Over the past 20 years, assays with C. dubia have increased in
popularity. This is directly related to the greater sensitivity of the C. dubia to
aquatic toxicants (Versteeg et al., 1997). Both invertebrate species are able to

62
reproduce by parthenogenesis, which ensures a continuous supply of identical
offspring.
Preparation of aquatic invertebrate food
The aquatic invertebrate cultures (C. dubia and D. pulex) were fed a
yeast, cereal leaves, and trout chow (YCT) based food. The YCT was prepared
over a one-week period beginning with the digestion of the trout chow pellets.
The trout chow digestion was performed in a bottomless 3-L inverted plastic
container by combining a 0.5-g portion of trout chow with 1-L of distilled water
(DDI). To ensure adequate mixing, an aquaculture pump was attached to a
glass pipette secured in the inverted cap of the plastic container. After 7 days,
the digestion was complete and the container was covered and placed in the
refrigerator to settle for at least 1 hour. No water was added during the digestion,
despite evaporative losses; however, water was added if needed in the final step
of food preparation to reach the desired solids content. The supernatant from the
trout chow digestion was filtered through a fine mesh, e g. nylon hose, and then
reserved. Simultaneously, on day 6 of the trout chow digestion a 5-gram portion
of cereal leaves (Sigma) was combined in a blender with 1-L of DDI and mixed
on high speed for 5 minutes. The cereal leaf mixture was covered and reserved
in the refrigerator overnight to settle. Finally, on day 7 of the digestion, a 5-gram
portion of dry yeast (Fleischmann® or equivalent) was combined with 1-L of DDI
water and mixed well on a magnetic stir plate. Equal volumes of the yeast
solution, cereal leaf supernatant, and trout chow filtered supernatant were
combined and mixed thoroughly. The YCT food was apportioned into 50-ml

63
plastic bottles, labeled, and stored in the freezer (~40°C) until needed. YCT food
was stored in the refrigerator and unused portions were discarded after 1 week.
The total solids (TS) content of the YCT was maintained between 1.7 and 1.9 g
solids/L by the addition of DDI as needed. The C. dubia and D. pulex cultures
were also fed P. subcapitata algae cells (3.5 X 107 cells/ml), as previously
described.
Maintenance of aquatic invertebrate cultures
Starter cultures of C. dubia and D. pulex were graciously donated by
Hydrosphere Research (Gainesville, FL). The aquatic invertebrates were
cultured in dedicated glassware, which was maintained separately and
thoroughly washed and rinsed between each usage. Oaphnids were cultured in
reconstituted moderately hard water (MHW), which was composed of NaHC03,,
96 mg; CaSOí» 2H2O, 60 mg; MgSCXi, 60 mg; and KCI, 4 mg per liter of DDI
water. The MHW had the following specifications: pH, 7.4 -7.8; hardness, 80 -
100 mg/L as CaC03; and alkalinity; 60 -70 mg/L (USEPA, 1993a).
Aquatic invertebrate cultures were maintained by adding neonates (< 24-
hour old) of C. dubia or D. pulex to 1-L glass beakers containing MHW. The
daphnid beakers were kept in an environmental chamber (Percival, model E-
30BX) at 20 ± 2°C and with a light regime of 16 hours of light and 8 hours of
dark. The daphnid cultures were fed 7 ml of YCT and 7 ml of algae cells per liter
of invertebrate culture. Invertebrate cultures were culled daily to remove
neonates and ensure a population with a uniform age distribution. Following the
removal of neonates for toxicity assays, any remaining neonates were used to

64
Prepare leachate dilutions with
PAAP minus EDTA
Transfer 50 ml of leachate, or its dilution, to
triplicate 125-ml erlenmeyer flasks
ja
Spike each flask with 1 ml
P. subcapitata (500,000 cells/ml)
JH
Place erlenmeyers under growth light for
96 hours, shaking daily
Count number algae cells with
hemacytometer and phase/contrast
microscope
Figure 3-2. Flowchart for the P. subcapitata assay

65
start new cultures or were discarded. Adult females were retained for neonate
production for a period no longer than two weeks.
Toxicity Assays
Pseudokirchneriella subcapitata
The chronic toxicity of the MSW landfill leachates was evaluated
according to the protocols of the 96-hour P. subcapitata assay (USEPA, 1994a).
The leachates were filtered with glass fiber (Whatman, GF/B) and membrane
filters (0.45 pm). The glass fiber pre-filters were used to minimize clogging of the
membrane filter. The dilution media for the algal assays was a PAAP solution
prepared without the disodium (Ethylenedinitrilo) tetra-acetate (EDTA). EDTA
has been shown to form complexes with heavy metals, which confounds assay
results when metal toxicity is suspected. The PAAP growth medium requires the
addition of EDTA, because its presence is crucial for the uptake of many
micronutrients (USEPA, 1994a).
For each algal assay, five dilutions were prepared in a laminar flow hood
with PAAP minus EDTA at a dilution factor of 0.5 (Figure 3-2). A 50-ml aliquot of
the leachate, or its dilution, was added to triplicate 125-ml sterile erienmeyer
flasks with styrofoam stoppers. The flasks were then inoculated with a 1-ml
aliquot of algae cells (500,000 cells/ml). The inoculum was prepared by
centrifuging a 4 to 5-ml portion of algae stock (3-5 days old) at 4000 rpm for
fifteen minutes. The supernatant was discarded, and the algae cells were
resuspended in PAAP minus EDTA and mixed by vortexing gently. Using a
hemacytometer and a phase contrast microscope, the cell density was
determined. The volume of resuspended algae cells required to prepare an

66
algae seed with a density of 500,000 cells/ml was determined by the following
equation:
Number test flasks x Vol. test Solution per flask x 10,000 cells per ml
Cell density (cells per ml) in the stock culture
Based on the number of assay flasks and a volume of 1-ml of seed per
flask, the algae cell inoculum was prepared. As an example, if the assay
required 18 flasks containing 50 ml of leachate per flask and the stock algal
culture density was 106 cells/ml, then the required volume of stock culture to
produce 18 ml of algae inoculum is 9 ml. Combining a 9-ml portion of the algae
stock culture (500,000 cells/ml) with a 9-ml portion of PAAP without EDTA
provided the required 18-ml portion of algae inoculum needed for the assay.
The erlenmeyer flasks containing the leachate, or its dilution, and the algal
seed were placed under the light unit (previously described) at 25°C. Constant
illumination (400 ± 40 ft-c) was maintained for 96 hours, and the flasks were
rotated and mixed daily by swirling manually. At the conclusion of each assay,
the algae cell density in each flask was measured by algal cell counts using a
hemacytometer and a phase/contrast microscope. Growth inhibition was
determined by comparing the number of algae cells in the leachate containing
flasks to the number in the control flasks. The leachate concentration that
produced a 50 % inhibition (IC50) of algal growth was determined by graphing the
cell density in each flask versus the leachate concentration.

67
Prepare leachate dilutions with
MHW
33
Transfer 10 neonates to assay cup; add 20
ml of sample or its dilution
43
Expose neonates to leachate
for 48 hours
34
Observe neonates for
death /immobilization
Figure 3-3. Flowchart for the C. dubia assay
Ceriodaphnia dubia and Daphnia pulex
The acute toxicity of the MSW landfill leachates were evaluated with C.
dubia and D. pulex.in the standard 48-hour acute toxicity protocols (USEPA,
1993a; APHA, 1999). Basically, the assay protocols were identical for the two
species of aquatic invertebrates. Before the leachates were evaluated for
toxicity, they were pre-filtered (Whatman, GF/B) to remove large particles. The C.
dubia and D. pulex assays follow similar conditions. Prior to the start of each

68
aquatic invertebrate assay, the neonates (< 24hrs) were separated from the adult
daphnids and fed a mixture of 7 ml YCT/liter and 7 ml algae/liter. After feeding,
10 neonates were transferred to each test container (30-ml plastic cups) using a
small wide-mouth plastic pipette to minimize the transfer of culture water (Figure
3-3). The leachate dilutions were prepared with MHW at a 0.5 dilution factor, and
the leachate or its dilution was added at 20-ml volumes to triplicate cups
containing the 10 neonates. Containers filled with MHW were used as the
negative controls. The test containers were placed in a water bath at 20 ± 2° C
for 48 hours, with a loose covering to allow light penetration and prevent settling
of air particles. Neonates were exposed to ambient lighting and were not fed
during the assay.
After 48-hours, the invertebrate test containers were placed on a light
table for the determination of viable organisms. The light table was designed to
sit on the laboratory bench (constructed by Martin Dolley). It was composed of a
wooden box with a plexiglass top and three fluorescent lights. The fluorescent
lights were mounted inside the box and below the plexiglass top, so as to
illuminate the work surface. Test containers were swirled gently and neonates
with the power to swim away from the center of the container were counted as
dead/immobilized. Mortality greater than 10% in the controls negated the assay
results.

69
J3-
Add 450 nl of leachate, or its dilution, to
cuvettes containing bacterial reagent;
mix
Measure final bioluminescence after
15-minute exposure
Figure 3-4. Flowchart for the Microtoxâ„¢ assay

70
Microtoxâ„¢
The Microtoxâ„¢ toxicity analyzer is a commercially available toxicity system
that measures toxic effects by changes in bacterial (Vibrio fischeri)
bioluminescence (Beckman Instruments, 1982). The assay kit includes a diluent,
the freeze-dried Microtox bacterial-reagent, a reconstitution solution, and an
osmotic adjusting solution. Each leachate was assayed in duplicate and each
assay also included a duplicate control (DDI water) (Figure 3-4). A preliminary
investigation indicated that a 15-minute exposure produced the highest
sensitivity, which agrees with the reports of other researchers (Plotkin and Ram,
1984).
The Microtoxâ„¢ analyzer combines a pre-cooling well for storage of the
reconstituted bacteria reagent during the assay, an incubator well block to cool
the cuvettes containing the sample(s), and a turret containing the photomultiplier,
which quantifies the bacterial light output. Before the assay, the analyzer is
brought to thermal equilibrium and the performance calibrated. Clean glass
cuvettes are added to the incubator well block and the pre-cooling well. The
bacterial reagent is rehydrated with 1 ml of reconstitution solution and transferred
to the cuvette in the pre-cooling well. The incubator well block has a grid pattern,
with columns 1 to 5 and rows A to C, row A is designated for the preparation of
the sample dilutions and rows B and C for sample testing in duplicate. Column 1
is dedicated to assay blanks and contains only the bacterial reagent and the
Microtox diluent.

71
The leachate dilutions are prepared from right to left in columns 5 to 2,
and well A5 corresponded to the highest leachate concentration. The dilution
series is prepared by first adding 1000 pi of the Microtox diluent to the cuvettes in
row A of columns 5 to 2. Then 1000 pi of the leachate is added to columns 4 and
5 of row A and mixed by repeatedly pipetting 500 pi of the mixture and aspirating.
A 1000-pl aliquot from the cuvette in column 4 was transferred to the cuvette in
column 3, mixed, and then a 1000-pl aliquot from column 3 was transferred to
column 2. After column 2 was mixed and aspirated, a 1000-pl aliquot was
discarded. The cuvettes were allowed to cool for approximately 10 minutes.
Following the addition of the reconstituted Microtox bacterial reagent (50 pi) to
wells B1 through B5 and C1 through C5, the cuvettes were allowed to reach
thermal equilibrium, approximately 15 minutes. The luminescence of the
bacterial reagent in each cuvette was determined by placing the cuvette in the
turret and turning the handle to the read position. The luminescence output from
the bacterial reagent was read on the digital panel meter (DPM) at the front of the
analyzer.
The pre-exposure bacterial luminescence was measured beginning with
the cuvette in row B1, then C1, B2, C2, B3, C3, B4, C4, B5, and C5. Next, a
450-pl aliquot of the sample or its dilution was transferred from the cuvettes in
row A to the duplicate cuvettes in rows B and C and mixed, beginning with
column 5. After 15 minutes, the cuvette was placed in the turret and the
luminescence output from the bacteria exposed to the sample or its dilution was
read on the DPM. The loss of bioluminescence is described by gamma (r), which

72
is measured as the ratio of light lost to light remaining following leachate
exposure.
Data Analysis
The toxicity assay results were expressed as the concentration of leachate
that produced a 50 % effect in the bioassay. The endpoints of the assays were
different and included inhibition of bioluminescence (Microtox as EC50), lethality
or death (C. dubia as LC50), and growth inhibition (P. subcapitata as IC50). The
results of the 96-hr P. subcapitata assays were determined by graphical
Interpolation. The LCsofor the C. dubia assay was determined using the USEPA
data analysis software (USEPA, 1994b). Bioassay results were presented with
one standard deviation for the C. dubia and P. subcapitata assays. Microtoxâ„¢
test results were determined by least square regression analysis of the natural
log of the sample concentration vs. the natural log of gamma (ratio of light lost to
light remaining). Results were presented as the concentration of leachate
causing 50% inhibition of bioluminescence (gamma). The Microtox assay was
performed in duplicate; therefore, determinations of standard deviations were not
valid. Data was evaluated by least square regression, student's t-test, or the F-
test (Excel, Microsoft 2000), as appropriate. The EC50, IC50, and LC50 results
were transformed to toxicity units (TU); according to the following;
TU (unitless)
100
ECxor ICM or LCS0
When appropriate, data were log transformed, for evaluation of linear
relationships.

73
Table 3-3. Physical and chemical characteristics of MSW landfill leachates at six
sites in Florida
Parameter
Site
1
Site
2
Site
3
Site
4
Site
5
Site
6
pH
Temperature
(°C)
Conductivity
(mS/cm)
Alkalinity
(mg/L as
CaC03)
CBODa
(mg/L)
CODb
(mg/L)
7.5'
(7.3-76)
35
(32-38)
14.1
(13.2-15.2)
6213
(6075-
6625)
140
(89-204)
1850
(1070-
2584)
7.0
(6.5-7.4)
26
(25-29)
6.2
(3.1-8.4)
2407
(1725-
2975)
22
(14-30)
636
(522-827)
7.2
(6.8-75)
27
(26-27)
5.2
(2.6-95)
1494
(1000-
2050)
15
(13-21)
351
(242-440)
7.5
(7.3-78)
26
(24-28)
7.6
(6.5-8.6)
3238
(3125-
3350)
66
(55-77)
1165
(902-1616)
7.7
(7.5-79)
23
(19-28)
8.3
(3.2-12.1)
2503
(1200-
4050)
42
(13-66)
857
(416-1208)
7.6
(7.3-78)
32
(31-33)
9.6
(1.0-14.2)
5500
(250-8775)
NMC
12245
(10530-
13960)
Sulfide
0.3
20
19.7
47
2.2
3580
(pg/L)
(0.08-0.8)
(17-23)
(15-24)
(42-56)
(0.1-15.8)
(1060-
5460)
Al
0.22
<0.2
0.4
0.2
3.5
10.8
(mg/L)
(0.07-0.4)
(0.17-0.47)
(0.17-0.32)
(0.1-4.5)
(4.2-23.7)
Cu
<0.07
<0.07
<0.07
<0.07
<0.07
<0.07
Cd
(mg/L)
<0.015
<0.015
<0.015
<0.015
0.01
(0.006-
0.02)
<0.015
Pb
(mg/L)
<0.06
<0.06
<0.06
<0.06
0.06
(0.04-0.1)
<0.06
Zn
0.04
<0.03
<0.03
<0.03
0.05
0.45
(mg/L)
(0.02-0.06)
(0.03-0.07)
(0.05-0.63)
As
0.16
<0.09
<0.09
<0.09
0.12
0.14
(mg/L)
(0.1-0.25)
(0.03-0.2)
(0.1-0.2)
Cr
0.05
<0.05
<0.05
<0.05
0.07
0.18
(mg/L)
(0.03-0.08)
(0.02-0.1)
(0.05-0.39)
Ba
<0.05
0.05
0.07
0.09
0.1
0.1
(mg/L)
(0.03-0.1)
(0.03-0.1)
(0.08-0.11)
(0.03-0.2)
(0.08-0.15)
Fe
7.13
12.5
4.7
7.0
7.5
50.4
(mg/L)
(5.9-9.5)
(9.3-21.4)
(2.2-9.2)
(3.7-99)
(3.9-13.6)
(3.2-86.5)
Na
1495
735.7
390
1001.5
1039
1663
(mg/L)
(1402-
1600)
(464-928)
(352-445)
(989-1014)
(554-1890)
(76-2670)
K
555
343
202
440
201
644
(mg/L)
(520-589)
(234-414)
(184-220)
(431-444)
(93-316)
(45-1011)
Results are shown as the mean and (range). Abbreviations: “BOD,biological oxygen demand;
bCOD, chemical oxygen demand; CNM, not measured.

74
Results and Discussion
Chemical Analysis of MSW Leachates
The physical and chemical characteristics of the MSW landfill leachates
are summarized in Table 3-3. Although the mean pH values were near neutral,
with a range from 7.0 to 7.7, lower pH values were measured in some samples.
The toxicity and bioavailability of some leachate toxicants, especially heavy
metals, are pH dependent (Schubauer-Berigan et al., 1993). The alkalinity of the
leachates was highly variable and ranged from 1,494 mg/L as CaCÜ3 at site 3 to
6,213 mg/L as CaCC>3 at site 1. These concentrations are typical for landfill
leachates in the early phases of waste stabilization (Kjeldsen et al., 2002).
Conductivity measures ionized molecules in solution, including both
cationic and anionic species. In the MSW landfill leachates, the range of
conductivity values was wide from a low of 5.2 mS/cm at site 3 to a high of 14.1
mS/cm at site 1. Specifically, the mean sulfide concentrations varied widely from
a low of 0.31 pg/L at site 1 to a high of 3,580 pg/L at site 6. Similar mean sulfide
concentrations of 20 pg/L at site 2 and 19.7 pg/L at site 3 were reported. Since
the leachates from sites 2 and 3 were collected from the same landfill and the
waste compositions were comparable, then similar sulfide levels were expected.
This was also true for most of the other chemical characteristics of the sites 2
and 3 leachates, with a few exceptions. The levels of alkalinity in the site 2
leachates ranged from 1725 to 2975 mg/L as CaCC>3, while in the site 3
leachates the range was from 1000 to 2050 mg/L as CaC03. Alkalinity
concentrations generally decrease with increasing landfill age, as the buffering

75
capacity is consumed by the production of organic acids. Differences in the
BOD/COD ratios were slight, but suggested a higher degree of organic matter
degradation in the leachates from site 3. The BOD/COD ratio was 0.03 in the
leachates from site 2 and 0.04 in the leachates from site 3. The lower
concentration or inorganic cations in the site 3 leachates was due to the "wash¬
out" effect typical of older leachates (Kjeldsen et al., 2002). While organic
components in the leachates are degraded by biological activity, inorganic
constituents decrease over time with increased rates of leachate production
(Cameron and Koch, 1980; Chian and DeWalle, 1976). Some variations in the
chemical composition of MSW landfill leachates are expected based on age,
waste degradation and site-specific factors (Ragle et al., 1995).
Generally, the concentrations of heavy metals in the MSW landfill
leachates were below analytical detection limits (Table 3-3). However, some
leachates contained elevated concentrations of heavy metals. Aluminum was
detected in most of the leachates at least once over the 6-month investigation. In
the leachates from site 6, aluminum was detected in each of the leachate
samples collected with a range from 4.2 to 23.7 mg/L. The heavy metals copper
and cadmium were not detected (detection limits of 0.07 mg/L and 0.02 mg/L,
respectively) in any of the leachates analyzed. While lead concentrations were
less than 0.06 mg/L in the leachates from sites 1,2,3, 4, and 6, in the leachates
from site 5 lead concentrations ranged from <0.06 to 0.1 mg/L.
The leachates from site 6 contained zinc concentrations that ranged from
0.05 to 0.63 mg/L. Zinc concentrations in the leachates from site 1 exceeded the

76
Figure 3-5. Concentrations of total (NhV+NHs) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 1 over a 6-month
sampling interval. NM indicates sample not measured
detection limit of 0.03 mg/L in five of the six-leachate samples analyzed and
ranged from < 0.03 to 0.06 mg/L. The highest barium levels were measured in
the leachates from sites 2 and 3, although neither exceeded 0.1 mg/L. Similar
concentrations of barium were reported in the leachates from sites 4, 5, and 6,
but barium concentrations were below the detection limit in the leachates from
site 1. In relation to mean arsenic levels in the leachates, 0.16, 0.12, and 0.14
mg/L were identified at sites 1,5, and 6, respectively. A similar pattern was
shown with chromium, but in this case the leachates from site 6 displayed the
highest concentrations with a range from 0.05 to 0.39 mg/L.
Un-ionized ammonia
(mg/L)

77
500
400
1 300
200
100
Feb
March April May June July
Figure 3-6. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 2 over a 6-month
sampling interval. NM indicates sample not measured
The concentrations of iron in the MSW landfill leachates were high and
site-specific. The lowest mean iron concentrations were reported for the
leachates from site 3 with a mean of 4.7 mg/L, while with the leachates from site
6 mean concentration of 50.4 mg/L was reported. High iron concentrations in the
MSW landfill leachates directly impact the bioavailability of heavy metals via
influences on the formation of insoluble precipitates. Metallo-sulfide precipitates
reduce metal toxicity; however, at high concentrations iron may out-compete the
toxic metals for binding sites on the sulfide molecule (Bozkurt et al., 2000).
Concentrations of other inorganic cations were also high in the MSW landfill
leachates. Sodium levels ranged from a low mean of 390 mg/L at site 3 to high
Un-lonized ammonia
(mg/L)

78
250 y
200
1 150
o _
E 5
E oi
® £
<0
o 100
50
0
March April May June July
Figure 3-7. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 3 over a 6-month
sampling interval. NM indicates sample not measured
mean of 1663 mg/L at site 6. Although the potassium concentrations were lower,
a similar pattern was demonstrated. Reported mean concentrations of
potassium in the MSW landfill leachates from sites 3 and 6 were 202 and 644
mg/L, respectively.
The monthly levels of total (NH/+ NH3) ammonia in the MSW landfill
leachates fluctuated widely, and the concentrations were dependent on site-
specific conditions. Consistently, the highest overall total ammonia
concentrations were identified in the MSW landfill leachates collected from site 1,
with a range from 970 to 1860 mg/L (Figure 3-5). Although the concentrations of
total ammonia were lower at site 2 and ranged from 100 to 380 mg/L, the
I
Un-ionized ammonia
(mg/L)

79
400 y T 15
Feb March April May June
Figure 3-8. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 4 over a 6-month
sampling interval. NM indicates sample not measured
variability remained high (Figure 3-6). The lowest total ammonia concentrations
were displayed by the MSW leachates collected from site 3 (the capped landfill
site), with a range from 82 to 220 mg/L (Figure 3-7). In the MSW landfill
leachates collected from sites 4 and 5, total ammonia concentrations ranged
from 211 to 361 mg/L and 119 to 351.6 mg/L, respectively (Figures 3-8 and 3-9).
This contrasts with total ammonia concentrations measured in the leachates from
site 6, which ranged from 33 mg/L in July 2000 to 1957 mg/L in April of 2000
(Figure 3-10).
Ammonia speciation is dependent on both pH and temperature.
Ammonium is the dominant species at pH values < 9.3, while ammonia

80
Figure 3-9. Concentrations of total (NH4*+NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 5 over a 6-month
sampling interval. NM indicates not measured
dominates at pH values > 9.3 (Figures 3-5 to 3-10). As previously discussed, the
pH values were similar at the six landfill sites; however, slightly higher
temperatures were reported in the leachates from sites 1 and 6 (Table 3-3). At
the pH of the leachates, the un-ionized ammonia levels were expected to be low,
and this was true for sites 2, 3, 4, and 5 with reported concentrations of less than
6.5 mg/L. Higher total ammonia concentrations were reported in the leachates
from sites 1 and 6 with mean un-ionized ammonia levels of 23.4 and 44.4 mg/L,
respectively. Basically, the un-ionized ammonia concentrations followed the
same pattern as previously described for the total ammonia concentrations.
Un-ionized ammonia
(mg/L)

81
2500
150
2000 -
100
50
0
March
April
May
June
July
Figure 3-10. Concentrations of total (NH4++NH3) (bars) and un-ionized ammonia
(NH3) (diamonds) in MSW landfill leachates from site 6 over a 6-month
sampling interval. NM indicates sample not measured
According to the total ammonia concentrations, the relative strength of the
MSW landfill leachates were ranked as site 1 > site 6 > site 4 > site 5 > site 2 >
site 3. When the un-ionized ammonia concentrations were evaluated, a similar
pattern was identified with one exception. The leachates from site 6 had higher
levels of un-ionized ammonia compared to the leachates from site 1.
Toxicity of MSW Landfill Leachates
The MSW landfill leachates from the six sites in Florida were found to be
toxic in both the 48-hour C.dubia and 96-hour P. subcapitata assays; however,
this contrasted with the results of the Microtox assay (Figure 3-11). According to
the results of the 48-hour C. dubia assay, the MSW landfill leachates collected
Un-ionized ammonia
(mg/L)

82
Landfill Site
Figure 3-11. The mean toxicity of MSW leachates collected from six landfill sites.
Results are presented as the mean + one standard deviation
100 -i
ES C. dubia 0 P. subcapitata B Microtox
75
25
Figure 3-12. Toxicity of MSW landfill leachates from site 1 over time. Results are
presented as the mean ± one standard deviation

TU (unitless)
83
100
C. dubia HD P. subcapitata S Microtox
75
50
25
0
Figure 3-13. Toxicity of MSW landfill leachates from site 2 over time. Results are
presented as the mean ± one standard deviation
50 -
C. dubia im P. subcapitata B Microtox
Figure 3-14. Toxicity of MSW landfill leachates from site 3 over time. Results are
presented as the mean + one standard deviation

84
from sites 1 and 6 displayed the greatest toxic effects, and the leachates
collected from site 3 were the least toxic. Figure 3-12 shows the pattern of toxic
effects measured monthly. The MSW landfill leachates collected from site 1
displayed TU values that ranged from 45.4 to 72.1 (Figure 3-12). Although, on
average, the toxicity of the site 2 leachates was low with a mean TU of 29.7, the
range of toxicity was variable. The TU response of the C. dubia to the leachate
from site 2 in May 2000 was nearly double the mean toxicity of this leachate over
the six-month investigation (Figure 3-13). Generally, the MSW leachates from
site 3 displayed lowest toxicity. The TU values reported with the leachates from
site 3 ranged from 6 to 21.1, with a mean TU of 12.9 (Figure 3-14). The toxicity
of the site 4 leachates displayed a mean TU response of 24.2 ± 5.8 with the
greatest TU reported in April 2000 at 33.8 (Figure 3-15). The toxicity of the site 5
leachates varied from a low of 9.5 in March to a high of 39.1 in April (Figure 3-
16). The response of the 48-hour C. dubia assay to the leachates collected from
site

TU (unitless)
85
50
3 C. dubia ID P. subcapitata B Microtox
Figure 3-15. Toxicity of MSW landfill leachates from site 4 over time. Results are
presented as the mean ± one standard deviation
50
SC. dubia IDP subcapitata BMicrotox
Figure 3-16. Toxicity of MSW landfill leachates from site 5 over time. Results are
presented as the mean ± one standard deviation

86
Figure 3-17. Toxicity of MSW landfill leachates from site 6 over time. Results are
presented as the mean ± one standard deviation
6 displayed the widest variability in toxicity. The site 6 leachates displayed TU
values in March, April, and May 2000 with a range from 56 to 58 (Figure 3-17).
The toxicity of the leachates from site 6 dropped dramatically in July of 2000
when a large rain event immediately preceded sample collection. The collection
well was inundated with rainwater, and this resulted in a nearly 100% reduction in
toxicity. A similar reduction in leachate chemical strength was reported for this
sample (Table 3-3).
A similar pattern of toxic response was demonstrated by the results of the
96-hour P. subcapitata assay (Figure 3-11). According to the algal assay, the
leachates from site 1 displayed the greatest toxicity compared to those from sites
2, 3, 4, and 5 (Figure 3-11). When comparing leachates from sites 2 and 3,

87
Table 3-4. Correlative analysis with the C. dubia, P. subcapitata, and Microtoxâ„¢
assay results versus leachate chemical characteristics
Parameters
C. dubia
(48-hour)
P. subcapitata
(96-hour)
Microtoxâ„¢
(15-minute)
Total Ammonia
(NKf + NH3)
R2= 0.62
(p<0.01)
R^ 0.69
(p<0.01)
R2= 0.19
(p<0.05)
Un-ionized Ammonia
(NH3)
f *
o II
o o
—' CO
00
o M
o o
^ cn
R2= 0.24
(p<0.01)
Alkalinity
Conductivity
TDSa
CBODb
CODc
R2= 0.56
(p<0.01)
R2= 0.47
R2= 0.18
(p<0.01)
R2= 0.26
(p<0.05)
R^ 0.47
(p<0.01)
R2= 0.71
(p<0.01)
R^ 0.48
(p<0.01)
R2= 0.39
(p<0.01)
R^ 0.19
(p<0.07)
0.59
(p<0.01)
R^ 0.21
(P=0.01)
NRd
NR
NR
R^ 0.21
(p<0.05)
The coefficient of determination and (significance of the relationships) are reported.
Abbreviations: “TDS , total dissolved solids; bCBOD, carbonaceous oxygen demand; cCOD,
chemical oxygen demand; |1NR, no relationship.
the leachates from the capped site (site 3) were less toxic. The range of toxicity
in the leachates from site 2 was from 9.3 to 24, while the site 3 leachates ranged
from 4.4 to 18 (Figures 3-13 and 3-14). With the leachates from site 4, there
were peaks in the TU responses during May at 29.2 and July at 32.2. This
pattern of toxicity was not consistent with the results of the C. dubia assay. The
toxicity of the leachates from site 5 ranged from TUs of 5.6 to 28. Therefore, the
toxic response of the MSW landfill leachates from site 5 was comparable to that
reported with the leachates from site 2 (Figure 3-11). The same was true for the
algal assay results with the leachates from sites 1 and 6, which were roughly
equivalent and displayed mean TU values of 44 and 64, respectively (Figure 3-
11).

88
The Microtox assay was not sensitive to the toxicity of the MSW landfill
leachates (Figure 3-11). The Microtox assays were all conducted in duplicate,
and; therefore, results are expressed only as mean values. According to the
results of the Microtox assay, the mean toxicity of the leachates from sites 1 and
6 were similar with TU values of 7.7 and 7.5, respectively. A similar toxicity was
also identified at sites 2 and 4 with TU values of 2.8 and 2.1, respectively. The
lowest toxicity was measured in the leachates from site 3 at a TU of 1.6.
Regression Analysis
The MSW landfill leachates at all 6 sites demonstrated both acute and
chronic toxicity over the 6-month sampling period. There was significant
variability in the response of the C. dubia assay to the leachate toxicity both
between each of the sites sampled and within each landfill site over the 6-month
sampling period. A simple regression analysis of the toxicity results versus
various chemical characteristics was performed (Table 3-4). Significant
relationships were found between both the C. dubia (r2 = 0.62, p<0.01) and P.
subcapitata (r2 = 0.69, p<0.01) toxicity results and the total ammonia content of
the leachates. For the un-ionized ammonia (NH3) content of the leachates the
relationship was not as strong with either the C. dubia (r2 = 0.38, p<0.01) or the
P. subcapitata (r2 = 0.50, p<0.01) toxicity data. Weaker relationships were found
between the Microtox™ results and total ammonia (r2 = 0.19, p<0.05) or un¬
ionized ammonia (r2 = 0.24, p<0.01). A simple regression analysis of the
leachate toxicity values with C. dubia (EC50) versus those with P. subcapitata
(EC50) revealed a high degree of data correlation (R2 = 0.64, p<0.01) (Figure 3-

89
C. dubia ECk(%)
Figure 3-18. Relationship between the P. subcapitata (EC50) and C. dubia (EC50)
assay results with MSW landfill leachates
18). The Microtoxâ„¢ assay was less sensitive and showed no relationship with
either the C. dubia or P. subcapitata assay results.
The toxicity results for the leachates collected from site 2 (active site)
(Figure 3-13) versus site 3 (capped site) (Figure 3-14) were compared.
Leachates collected from the active unit demonstrated a significantly higher
toxicity in both the P. subcapitata and C. dubia assays (p<0.05) except for the
month of April when leachate toxicity was similar. The results of the Microtox
assay were also similar for the site 2 and site 3 leachate samples.
The results of the 6-month investigation indicated that the acute and
chronic toxicity of the MSW landfill leachates was highly variable. In order to
assess the toxicity variations within a shorter time frame, the acute/chronic

90
Feb. 2 Feb. 15 Feb. 29
Figure 3-19. Toxicity fluctuations in the leachates collected from the MSW landfill
at site 5 during February 2000. Results are presented as the mean ±
one standard deviation. Standard deviations are not shown for the
Microtoxâ„¢ data. Significant differences * p < 0.05 are indicated
toxicity of leachates collected from site 5 were monitored on three occasions over
a period of one month to determine temporal variation in leachate strength. The
TU results of the P. subcapitata assay were 4.3 ± 0.04, 5.6 ± 0.02, and 5.6 ± 0
with the leachates collected on 2nd, 15th, and 29th of February 2000, respectively.
There was no significant (p<0.05) difference in the toxicity of the leachates
collected during February from site 5, as measured with the algae assay. In
contrast, according to the results of the C. dubia assay the toxicity was
significantly (p<0.05) increased during February. On the 2nd, 15th, and 29th of
February 2000, the TU values in the C. dubia assay were 11.3 ± 0, 27.1 ± 1.5,
and 19.5 ± 1.3, respectively. With the Microtox™ assay results there was a

91
toxicity decrease (p<0.05) between February 2nd and 15th from 4.9 to 2.0, but no
change between the 15th and 29th (Figure 3-19).
The assays were ranked according to their sensitivity to the MSW
leachates: C. dubia > P. subcapitata > Microtox. The mean TU values with the C.
dubia assay ranged from 13 to 52, while a similar range from 9.5 to 64 was
reported in the P. subcapitata assays. The comparable sensitivities of these two
widely used toxicity assays verifies reports in the literature (Suedel et al., 1996;
Rojickova-Padrtova et al., 1998; Ferrari et al., 1999; Clement et al., 1996). In a
study of 9 domestic waste leachates in France, the TU values with C. dubia
ranged from 15.2 to 111 with 8 of the 9 leachates evaluated. The authors also
evaluated leachates from non-hazardous and hazardous industrial solid wastes
and found a lower TU range from 1.9 to 17.9 with C. dubia, leading to the
conclusion that leachates from domestic solid wastes should be considered as
dangerous as those from industrial wastes (Clement et al., 1996). Multiple
bioassays were also used in a study of leachates from a landfill receiving mixed
(domestic and some industrial) wastes. The researchers found P. subcapitata
(chlorophyll a content) was highly sensitive with TU values ranging 10 to 100
(Plotkin and Ram, 1984). They also showed 48-hour TUs for Daphnia magna
from 1.5 to 1.6 and a Microtoxâ„¢ TU of 5.9. Others (Devare and Bahadir, 1994)
showed variability in toxicity between separate landfill sites and reported a higher
sensitivity with plant (Lemna minor) assays over Microtoxâ„¢. In a European
study, leachates from domestic wastes were found more toxic than those of
industrial origin, and there was a high variability in toxicity of landfill leachates of

92
similar origin (Clement et al., 1996). These results support our finding of the
toxic nature of the Florida MSW leachates, demonstrating that the toxicity is still
of concern despite the dilution effect suggested by Reinhart and Grosh (1998).
Ammonia Toxicity
The ammonia content of MSW landfill leachates in Florida has previously
been shown to explain significant amounts of toxicity (Ward et al., 2000). The un¬
ionized ammonia concentrations of the individual leachates under investigation
ranged from 0.6 to 101 mg/L, while the total ammonia concentrations ranged
from 99.6 to 1,957 mg/L. Ammonia toxicity is primarily associated with the un¬
ionized form of ammonia, which is dependent on the pH, temperature, and the
concentrations of total ammonia in the leachates (Clement and Merlin, 1995;
Rutherford et al., 2000).
The MSW landfill leachates evaluated from sites 1 to 6 contained high
levels of un-ionized ammonia at concentrations that were sufficient to cause a
significant portion of the leachate toxicity. Anderson and Buckley (1998) reported
that 1.2 mg/L of un-ionized ammonia was lethal to 50 % of C. dubia in a 48-hour
acute assay. This level was lower than the concentrations actually found in the
MSW landfill leachates studied in Florida. The Microtox results with the MSW
landfill leachates suggested that this assay was not suitable for the evaluation of
leachate toxicity, probably due to the low sensitivity of this assay to ammonia
(Stronkhurst et al„ 2003). Qureshi et al. (1982) reported an EC» of 3,607 mg/L
for ionized ammonia using the Microtox test system. In contrast to the results
reported here, Doherty et al. (1999), using the Microtox assay, reported an un¬
ionized ammonia EC50 of 1.7 mg/L (15-minute). The low sensitivity of the

93
Microtoxâ„¢ assay to un-ionized ammonia, as shown here, may lead to a
conclusion of its suitability for detecting the presence of other toxicants in the
leachate; however, this was not the case with the MSW landfill leachates.
The results reported demonstrate that the concentrations of ammonia in
the Florida leachates explained a significant amount of the toxicity in both the C.
dubia and P. subcapitata assays. In contrast, the Microtoxâ„¢ assay showed a
small relationship between the ammonia content of MSW leachates and their
toxicity. Previously, researchers have correlated Microtoxâ„¢ test results with
ammonia concentrations in sediment elutriates (Cheung et al., 1997) and mine
effluents (LeBlond and Duffy, 2001).
Influence of Site-Specific Factors on Leachate Toxicity
Previously, the physico-chemical characteristics of domestic, industrial
and mixed waste leachates were compared to their measured toxicity with a
battery of tests that included C. dubia and Microtox (Clement et al., 1997). Their
results showed a relationship between COD concentrations and the Microtox (5-
minute EC50) (^=0.58, p<0.0001) results. A weaker relationship (^=0.21,
p<0.05). was demonstrated with a 15-minute exposure. The Florida leachates
were strictly of domestic origin, while the waste leachates investigated by
Clement et al. (1997) consisted of 13 domestic waste leachates and 9 industrial
or mixed industrial/ domestic waste leachates. The results with the Florida
leachates suggest that the Microtox bioluminescence assay is more sensitive for
evaluating the toxicity of samples with predominantly organic toxicants (Bitton et

94
al., 1996), rather than waste leachates with high inorganic contents, e.g.
ammonia or alkalinity (Clement et al., 1997).
The alkalinity of the MSW leachates assayed was high. The predominant
role of alkalinity in leachate toxicity seems to be the buffering of pH and its
influence on ammonia speciation (Clement and Merlin, 1995; Hoke et al., 1992).
Alkalinity concentrations were correlated with P. subcapitata EC50 results
(1^=0.71, p<0.01) and with C. dubia EC50 results (^=0.56, p<0.01), but the
relationship with Microtoxâ„¢ (^=0.21, p=0.01) was weaker. The relationship
showed for C. dubia and alkalinity was higher than that found by Clement et al.
(1997) with mixed waste leachates (^=0.38) but agrees with their report for un¬
ionized ammonia (^=0.34).

CHAPTER 4
A SURVEY TO ASSESS THE ACUTE AND CHRONIC TOXICITY OF
LEACHATES FROM MSW LANDFILLS IN FLORIDA:
Introduction
Rubbish, garbage, trash, unwanted materials, and discarded wastes are
some of the phrases commonly used to describe the by-products of dally life.
The regulatory and scientific communities refer to these by-products as municipal
solid waste (MSW) a term that includes packaging materials, grass clippings,
furniture, clothing, bottles, food scraps and other discarded materials (USEPA,
2002). The per capita generation of municipal solid waste (MSW) in the United
States remains relatively constant at approximately 4.5 lbs. MSW/person/day
(USEPA, 2002). Although the percentage of waste landfilled has decreased in
recent years, roughly 55.3 % of MSW continues to be disposed in landfills and
this continues to be the method of choice for waste disposal.
Municipal solid wastes are composed of a variety of natural and synthetic
materials, including packaging items, furniture, plastics, appliances, batteries,
and organic materials, e.g. food and animal feces. In recent years,
manufacturers have enhanced some consumer products with environmentally
friendly properties including biodegradability or photodegradability; however, the
storage conditions in modern landfills do not optimize these characteristics
(Hamilton et al., 1995). Modern landfills are engineered systems, which are
typically designed to minimize the infiltration of water, maintain anaerobic
95

96
conditions and ensure the recovery of leachates. In some areas, landfill designs
are being modified to encourage chemical and biological degradation processes
by increasing moisture levels and oxidized conditions (Reinhart and Townsend,
1998; Nopharatana et al., 1998).
MSW landfill leachates are produced when rainwater infiltrates into the
waste and exceeds the field capacity, or the ability of the waste to hold water. As
leachates pass through the landfilled waste materials, soluble constituents are
mobilized. Leachates pose a potential threat to the surrounding environment
from groundwater contamination or direct escape of leachate to surface waters
(Noaksson et al., 2003; Kawagoshi et al., 2002; Al-Muzaini et al., 1995). The
pollutants of concern in MSW landfill leachates are primarily dissolved organic
matter, inorganic macrocomponents, heavy metals, and xenobiotic organic
compounds (Kjeldsen et al., 2002).
Landfill operators are required to treat landfill leachates and treatment
typically occurs at domestic wastewater treatment plants, although, small on-site
treatment facilities are utilized at some landfills (CFR, 1996). Landfill leachates
in Florida are collected and treated as necessary to meet surface and
groundwater standards Chapter 62-3 and 62-302 F.A.C. (F.A.C., 1994a; F.A.C.,
1994b). The chemical and physical characteristics of the MSW leachates vary
widely and can significantly alter the chemical composition of domestic
wastewater. Consequently, when these leachates are combined with domestic
wastewater the microflora of the treatment facility can be enhanced, as
evidenced by a doubling of biosolids (Booth et al., 1996). Furthermore, the

97
treatment of MSW leachates at domestic wastewater treatment plants (WWTPs)
may inhibit biological processes due to the high concentrations of contaminants,
especially ammonia. Hence, the treatability of MSW landfill leachates is heavily
dependent on the leachate strength and the presence of toxic substances
(Alkalay et at., 1998).
In previous research (Ward et al., 2002), the toxicity of MSW leachates from
six landfill sites in Florida was investigated to identify toxicants of concern and to
establish toxicity methods for use with the leachates (see Chapter 3). Surveys of
MSW landfill leachate quality and toxicity provide a framework for long-term
leachate management strategies (Barlaz et al., 2002). In a continuation of the
initial survey, additional leachate samples were collected from MSW landfills
across the state of Florida. These leachates were selected to represent a cross-
section of leachate quality and strength; therefore, sampling sites included the
smallest and one of the largest landfills in the state. The scope of the continued
investigation included municipal solid waste (MSW) leachates collected from
fourteen landfill sites in Florida.
The objectives of this investigation were; 1). to increase the acute and
chronic toxicity database for Florida MSW landfill leachates, 2). to characterize
the chemical composition of these leachates, 3). to evaluate relationships
between leachate toxicity and chemical characteristics, 4). to determine the
influence of time on MSW landfill leachate toxicity.

98
Site 5
Figure 4-1. Locations of the MSW landfills for the collection of leachates in
Florida
Materials and Methods
Sampling Sites
MSW landfill leachates were collected from fourteen engineered landfills
throughout the state of Florida (Figure 4-1). Some of these landfill sites (1,4, 5,
and 6) were previously investigated as part of a larger research project (see
Chapter 3). All the MSW landfills were lined and contained leachate collection
systems. Descriptions of the MSW landfills are provided (Table 4-1). Additional
background information is available for sites 1, 5, and 6 (see Chapter 3) and sites
7, 8, and 14 (see Chapter 5). A unique situation was provided at site 11, which
contained three individual landfill cells with separate leachate collection systems.
The site 11 landfills Included an operating Class I (site 11a), a capped Class I
(site 11c), and a lined Class III landfill (site 11b).

99
Site
Landfill Class
Population
Tons/year
Landfilled
1
I
(capped)
209,000
-
2
I
113,000
158,000
3
I
(capped)
-
-
4
I
243,000
216,000
5
I
Data not
available
6
I
825,000
1,400,000
7
1
466,000
379,000
8
1
9
II
10,000
6,400
10
1
197,000
136,000
11a
1
1,030,000
624,000
11b
III
11c
1
(capped)
12
1
466,000
670,000
13
1
72,000
59,000
14
1
430,000
525,000
Class I landfills are designated for the disposal of non-hazardous household
wastes and some commercial, industrial and agricultural wastes, while Class III
landfills are designed for yard wastes, construction and demolition debris, carpet,
furniture, and similar non-putrescible waste materials. Depending on their age,

100
capped (closed) landfills are generally In the methanogenic or humic phase of
waste stabilization (Bozkurt et al., 2000). Low concentrations of easily
biodegraded materials and higher concentrations of recalcitrant molecules
characterize these phases. The only Class II landfill in Florida is located at site
9. The distinction between Class I and Class II landfills is solely based on the
amount of MSW received, with Class I landfills receiving 20 or more tons of
waste per day and Class II landfills less than 20 tons of MSW per day. The
Class I landfill located at site 1 was capped in 1998 and no longer receives
waste. Instead, the waste from site 1 is currently shipped to site 5, a regional
landfill.
Collection of MSW Landfill Leachates
The MSW landfill leachates were collected from the leachate collection
sumps of the lined landfills using a Teflon baler. However, at site 14 a collection
sump was not accessible, so the leachate was collected from a discharge pipe
that transported the leachate to an evaporation pond. By holding the individual
sampling containers under the pipe, only leachate was collected and not
rainwater that collected in the evaporation pond. The leachates were split for
chemical analysis and toxicity assays. For chemical analysis, the leachates were
collected in polyethylene or glass bottles and preserved according to U.S.
Environmental Protection Agency (USEPA) methods (USEPA, 1993b). Plastic
containers were used to store the leachates for toxicity analysis. All leachates
were transported to the lab on ice and immediately stored at 4°C until analysis.

101
Chemical and Physical Characterization of MSW Leachates
Field measurements included pH and temperature (Orion, Model 290A),
conductivity (HANNA Instruments, Model H19033), dissolved oxygen (DO) (YSI
Inc. Model 55/12 FT), and oxidation/reduction potential (ORP) (Accumet Co.
Model 20). In the laboratory, alkalinity, carbonaceous biochemical oxygen
demand (CBOD), chemical oxygen demand (COD), total organic carbon (TOC),
and sulfides were measured according to methods described by USEPA (1993b)
and APHA (1999). Hardness was measured by colorimetric analysis, HACH
method 8030 (HACH, Loveland, CO). Samples for total metal content were
digested (USEPA Method SW-846 3010A) (USEPA, 1996) and analyzed by
atomic emission spectroscopy (AES) (Thermo Jarrell Ash, Model Enviro 36). For
major ion analysis, a Dionex ion chromatograph (Dionex, Model DX-500) was
used. Total ammonia (NH4+ + NH3) and un-ionized ammonia (NH3) were
analyzed by a selective ion probe (Accumet). Some of the un-ionized ammonia
concentrations were calculated according to Emerson et al. (1975):
% un-ionized ammonia (NH~) = —__
3' (i + io(p*»-p»>)
where pKa = 0.09018 + 2729.92/T and T = temperature (°K)
Toxicity Assays
The acute toxicity of the MSW leachates were assayed with the static 48-
hour Ceriodaphnia dubia and Daphnia pulex assays (USEPA, 1993a) and the 15-
minute Microtoxâ„¢ test (Beckman Instruments, 1982). The green alga
Pseudokirchneriella subcapitata (formerly Selenastrum capricomutum) was
utilized in a 96-hr chronic algal assay (USEPA, 1994a). This suite of four acute

102
and chronic assays provided a comprehensive measure of MSW landfill leachate
toxicity with effects quantifiable by various endpoints. These endpoints included
lethality/immobilization, inhibition of bioluminescence, and growth inhibition for C.
dubia and D.pulex, Microtoxâ„¢, and P. subcapitata, respectively.
The toxicity assay protocols were previously described in detail (Chapter 3).
Briefly, for the C. dubia assays each MSW landfill leachate was assayed using 5
dilutions in triplicate. All dilutions and controls were prepared with synthetic
moderately hard water (MHW). C. dubia neonates (<24-hr old) were used in all
tests. Ten neonates were exposed to each dilution in triplicate with appropriate
controls. Neonates were exposed for 48-hrs at 24 ± 2°C under conditions of
ambient light (16hrs light/ 8hrs dark).
A second aquatic invertebrate assay utilizing Daphnia pulex was utilized to
determine the toxicity of MSW landfill leachates. The assay procedure was
similar to that previously described for the C. dubia. D. pulex neonates (<24
hours old) were exposed for 48-hours to the leachates or their dilutions in
triplicate at 24 ± 2°C under conditions of ambient light (16hrs light/ 8hrs dark).
For Microtoxâ„¢ analysis, color correction by absorbance at 490 nm was
required due to the reddish or amber color of the leachates. Leachate salinity
was adjusted to 2 % with NaCI, while neglecting the actual leachate salinity.
Four leachate dilutions were prepared, in duplicate, using the Microtoxâ„¢ diluent.
The inhibition of Vibrio fisheri bioluminescence was determined following a 15-
minute exposure to each leachate sample (Doherty et al., 1999). Due to
constraints of the Microtoxâ„¢ method, the maximum leachate concentration tested

103
was 90 %. This was attributed to dilution of the leachate after the bacterial
suspension was added.
For the P. subcapitata assays, leachates were filter-sterilized (0.45 pm
membrane filter). Five dilutions of each MSW landfill leachate, in triplicate, were
prepared in algal assay medium. Stock cultures of P. subcapitata were
maintained to provide a continuous supply of 3-5 day old cells for testing. Algal
cell counts were performed using a hemacytometer following a 96-hour exposure
at 25 ± 1 °C under conditions of continuous lighting (86 ± 8.6 pE/m2/s).
Data Analysis
Results for the toxicity assays were expressed as the 50 % effective
concentration (EC50), 50 % inhibitory concentration (IC50), or 50 % lethal
concentration (LC») for the 15-minute Microtox™, 96-hr P. subcapitata and 48-hr
C. dubia or D. pulex assays, respectively. All bioassay endpoints (EC50, IC50, or
LC50) were expressed as a percent (%) of the leachate sample. The EC50 and
IC50 results were determined by graphical interpolation for Microtoxâ„¢ and the 96-
hr P. subcapitata assays, respectively. The LC50 values were determined using
the USEPA data analysis software (USEPA, 1994b). Error bars included with the
bioassay results represent one standard deviation. Correlations within the data
were determined by least square regression, a student's t-test, or the F-test
(Excel, Microsoft 2000), as appropriate. The coefficient of variation (CV)(%) for
each assay was expressed as the percent of toxic response (EC50, IC50, or LC50)
represented by the standard deviation. All EC50, IC50, and LC50 endpoints were
transformed (100/ECso) to toxicity units (TU) and were unitless. When the assay

104
results are provided as TU values, a direct relation is expressed between
increased toxicity and higher TU values, which contrasts with the inverse
relationship between ECso(%) values and increased toxicity.
Results and Discussion
Chemical and Physical Characteristics of the MSW Leachates
Leachates from fourteen MSW landfill sites in Florida were selected to
demonstrate the varied and complex organic and inorganic contaminants
typically found in MSW landfill leachates (Kjeldsen et al., 2002; Chen, 1996;
Bolton and Evans, 1991). The compositions of the MSW landfill leachates are
influenced by site-specific factors including temperature, rainfall, waste
composition, and landfill age (Vadillo et al., 1999; Chian and DeWalle, 1976).
While the chemical strength of MSW landfill leachates may vary widely, general
trends in these parameters are often used as indicators of overall quality.
Table 4-2 summarizes the range of selected chemical/physical
characteristics of the fourteen MSW landfill leachates in Florida. For comparative
purposes, a typical range of MSW landfill leachate chemical characteristics is
provided in Table 2-1. The mean pH values measured in the leachates were
around neutral with mean values from 6.5 to 8.1 pH units. Generally, young
leachates display pH values that are less than neutral due to the predominance
of organic acids in the initial stages of waste decomposition (Kang et al., 2002).
More neutral pH values are expected in leachates that have already undergone
some stabilization, while mature landfills display pH values greater than 7 (Chian
and DeWalle, 1976). The leachate collected from site 10 displayed the lowest
pH value at 6.5, and this was attributed to the low buffering capacity of this

105
leachate as demonstrated by the low alkalinity. Mean pH values of 8.1 were
reported in the leachates collected from sites 6 and 8. The pH of leachates
affects the mobility of contaminants (Roy and Dzombak, 1997; Clevenger and
Rao, 1996) by its influence on the stability of humic complexes (Masion et al„
2000) and toxicant bioavailability (Meyer, 2002). Sletten et al. (1995) reported
that, above a pH of 8, soluble heavy metals in MSW landfill leachates decreased
to below the analytical detection limit of 0.1 mg/L for Zn, Ni, Cr, Cu, Pb, and Cd.
Overall, the alkalinity measurements were high in all of the MSW landfill
leachates with one exception. The alkalinity measured in the leachate from site
10 was 350 mg/L as CaC03. In the other leachates, alkalinity concentrations
ranged from 1175 to 6850 mg/L as CaC03. The leachates collected from site 6
displayed the highest alkalinity values. This was attributed to the practice at this
landfill of co-disposing wastewater treatment plant (WWTP) sludge with the MSW
as a means to increase waste stabilization. Alkalinity is often described for its
acid-neutralizing properties. Therefore, the low pH measured in the leachate
from site 10 may be linked to the low alkalinity of the leachate.
Conductivity is a bulk parameter that indicates the magnitude of ionized
species in solution. Although the mean conductivity values ranged from a low of
3.4 mS/cm at sites 11 b and 11 c to a high of 39.6 mS/cm at site 10, the majority
of the leachates displayed conductivity values of <14 mS/cm. The high
conductivity in the leachate from site 10 can be attributed to the co-disposal of
incinerated MSW ash at site 10, which produced a leachate with a high inorganic

106
content (Table 4-2). Similar results were shown in the leachates from site 11a,
which also practiced co-disposal and displayed a conductivity of 26.9 mS/cm.
The bulk parameter of total dissolved solids (TDS), when combined with
measured conductivity values, can indicate the ionic strength of a solution by the
Langelier approximation (Langelier, 1936). While in this research, TDS was
determined by analytical methods, the approximation remains valid. Therefore, it
follows that the leachates with the highest conductivity also display the highest
values for TDS. This was true for the site 10 leachates with a mean TDS at 31.7
g/L, distinguishing the high ionic strength of this leachate. In all other cases, the
TDS values were <17 g/L, with the lowest value of 2.4 g/L reported at site 8.

Table 4-2. Physical and chemical characteristics of MSW leachates collected from 14 lined landfills in Florida
Site
pH
Conductivity
(mS/cm)
Alkalinity
(mg/L as
CaCOa)
TDSa
(g/L)
Sulfides
(Wl/L)
DOCb
(mg/L)
CBOD°
(mg/L)
CODd
(mg/L)
CBOD/
COD
1
7.7
(7.3-8.1)
13.9
(8.1-18.0)
4948
(3615-7100)
5.7
(4.1-7.4)
0.13
(0.1-0.2)
675
(443-1126)
190
(159-231)
2355
(1718-3116)
0.08
4
7.8
(7-7-7.9)
9.7
(6.1-13.3)
3900
(2500-5300)
4.4
(3.3-5.5)
NMe
NM
NM
NM
5
7.8
(7.6-8.1)
10.6
(7.4-12.7)
3285
(875-4700)
6.3
(4.6-7.8)
25.2
(2-86)
467.6
(313-738.8)
163
(97-214)
2161
(1320-4100)
0.08
6
8.1
20.1
6850
9.6
3170
NM
NM
9528
-
7
6.6
(6.5-6.7)
4.7
(4.6-48)
1175
(1100-1250)
2.75
(2.6-2.9)
10
NM
73d
792
0.09
8
8.1
(8-8.4)
4.7
(4.5-52)
1200
2.4
(2.2-21)
31.9
(20-43)
85.4
(22-149)
NM
444
(360-500)
-
9
7.4
3.9
2800
3.9
48
NM
62
992
0.06
10
6.5
39.6
350
31.7
52
NM
189
2272
0.08
11(a)
6.8
26.9
1400
16.8
67
NM
182
2648
0.07
11(b)
7.3
3.4
1800
2.5
31
NM
BDL
2232
-
11(c)
7.1
3.4
2900
3.6
66
NM
110
852
0.13
12
NM
NM
NM
NM
NM
NM
NM
NM
-
13
7.5
10.1
3450
3.8
42
NM
NM
1024
-
14
7.9
(7.5-8.2)
4.7
(38-5.6)
2000
(1550-2450)
2.9
(2.0-3.9)
3838
(700-6975)
NM
182
1260
0.14
Mean
7.6
10.7
3137
5.6
1272
480
144
2213
Min
6.5
3.4
350
2.4
< 1
85.4
62
444
0.06
Max
8.1
39.6
X a-rr^
6850
31.7
j Di
3838
675
189
9528
0.14
biochemical oxygen demand, dCOD = chemical oxygen demand, ®NM= not measured. Results 'mean and (range) for
multiple sampling events and 9 mean ± one standard deviation for one sampling event
107

108
The presence of organic substances in the MSW leachates were
measured as chemical oxygen demand (COD) and carbonaceous biochemical
oxygen demand (CBOD) procedures (Table 4-3). Both of these parameters
measure the concentration of oxidizable organic matter, including both the
biologically assimilable and the recalcitrant compounds. When determining only
the fraction of organic matter that is subject to biological degradation
(assimilable), a CBOD test is performed. Therefore, in a given leachate the COD
concentrations are higher than the CBOD concentrations. For comparative
purposes, the ratio of CBOD to COD is a rough indicator of the degree of waste
degradation.
The mean CBOD concentrations in the MSW landfill leachates ranged
from 62 mg/L at site 9 to 190 mg/L at site 14. In most cases, the magnitude of
COD demand ranged from 444 mg/L at site 8 to 2648 mg/L at site 11a. By
comparison, the COD concentration of > 9,500 mg/L reported in the leachates
from site 6 was excessively high but within the range reported by others
(Kjeldsen et al., 2002).
When considering the CBOD/COD, the range was from 0.06 at site 9 to
0.14 at site 14, indicating a similar degree of waste stabilization among the
different landfills. The CBOD/COD for the leachates collected from site 6 was
not determined, but based on past experience with leachates from this site, a
similar range of CBOD/COD is expected. Several attempts to determine the
CBOD of the site 6 leachates were unsuccessful due to the high organic matter
content and an insufficient dilution factor. Based on the limited data that was
108

109
obtained at this site, the CBOD range for the site 6 leachate was estimated to be
at least 500 mg/L.
Little is known about the dissolved organic carbon (DOC) concentrations
in the leachates, because of the limited number of samples analyzed for this
parameter (Table 4-3). The mean DOC concentrations reported for this research
were 85.4, 467.6, and 675 mg/L for sites 8, 5, and 1, respectively. Previous
research showed that the DOC concentrations in the leachates from site 6 were
quite high with a mean of 4956 mg/L. This may have been caused by the co¬
disposal of WWTP sludges with the MSW.
Chapter 5 discusses the heavy metal binding capacity of MSW landfill leachates,
and as part of that investigation the DOC was determined in leachates from sites
1,4, 5, and 8 at concentrations of 1126, 368, 377, and 149, respectively. These
DOC levels are comparable to previous reports and ranged from a low of 190
mg/L to a high of 18,362 mg/L (Marttinen et al., 2002; Kang et al., 2002).
Additionally, DOC measurements are a quasi-surrogate for humic materials,
which influences the complexation of heavy metal and some organic ligands
(Calace et al., 2001; Martensson et al., 1999).

110
Table 4-3. Distribution of major ions in leachates from 14 lined MSW landfills in
Florida. Results are presented as the mean and (range)
Site
Sodium
(mq/L)
Potassium
(mg/L)
Calcium
(mg/L)
Magnesium
(mg/L)
Chloride
(mg/L)
Sulfate
(mg'1-)
1
4
5
1309a
(1103-
1496)
1395
1329
(946-1850)
486
(418-561)
604
351
(276-426)
250
138
(67-210)
210
90
104.5
(49-160)
160
1380
(1104-
1608)
400
987
(699-1380)
12.5
(0-57)
32
243
(226-348)
6
1920
813
NM
NM
2145
100.5
566
24.3
290
67.5
589
69.4
(532-600)
(23-25)
(275-304)
(65-70)
(300-879)
(36-103)
8
NMa
NM
123
(46-200)
51.7
(33-70)
NM
NM
9
790
424
175
44.9
920
9
10
3812
2228
4406
75
19294
234
11a
3410
1456
1191
122
9164
375
11b
273
256
278
66
406
234
11c
724
340
203
41
875
26
12
NM
NM
NM
NM
NM
NM
13
1010
365
158
54
1069
10.9
14
479
162
224
50
1022
131
(367-591)
(123-201)
(174-275)
(36-65)
(544-1500)
(126-135)
Abbreviations: aNM, not measured,
The concentrations of select major ions in the landfill leachates are
summarized (Table 4-3). These inorganic ions are not subject to biological
degradative processes, hence, their concentrations depend solely on the volume
of leachate generated (Chian and DeWalle, 1976). While reviewing the results
for the major ion analysis, it became apparent that the high conductivity reported
for the leachates from site 10 was a good indicator of the major ion distribution.
As previously discussed, the landfills associated with the leachates from sites 10

111
and 11a practice the co-disposal of MSW incinerator ash, and this resulted in the
production of leachates with high ionic strength. Overall, the site 10 leachates
exhibited the highest cation concentrations for sodium, potassium, and calcium at
3812, 2228, and 4406 mg/L, respectively. A similar pattern was demonstrated in
the leachates from site 11a with sodium, potassium, and calcium concentrations
at 3410,1456, and 1191 mg/L, respectively. The magnesium concentration at
site 11a was 122 mg/L or nearly double the 75 mg/L at site 10. Across all
leachates, the highest magnesium concentration was reported as 160 mg/L at
site 5, and the lowest was 45 mg/L at site 11c. In all cases, the most abundant
cation in the leachates was the sodium ion, which ranged from a low of 273 mg/L
at site 11 b to a high of 3812 at site 10.
Referring to the anions, the magnitude of the concentrations was similar to
those measured for the cations. An excessively high level of chloride ions was
reported in the site 10 leachate at a concentration > 15,000 mg/L. This high
concentration was a recurring problem in the leachate, and was limiting available
leachate treatment options (Gary Debo, landfill operator, personal
communication). In all of the other leachates, the chloride levels were < 1500
mg/L, with the lowest chloride concentrations identified in the leachates from site
4 at 400 mg/L. Chloride complexes with metals in solution have been shown to
modify metal speciation and bioavailability (Bolton and Evans, 1991).
Under typical landfill conditions the sulfate molecule is reduced to sulfide,
and this reduced ion forms complexes with heavy metals (Bozkurt et al., 2000).
By comparison, the concentrations of sulfate ions in the MSW leachates were

112
much lower than the reported chloride concentrations. In the MSW landfill
leachates the mean levels of sulfate ions ranged from 12.5 to 375 mg/L. The
highest sulfate concentrations were reported in the leachate from site 11a, while
the lowest were in the site 1 leachates. Decreasing sulfate ion concentrations
can be used as an indicator of the degree of reducing conditions in an anaerobic
environment. In the case of the site 1 leachates, the low mean sulfide ion
concentrations (< 1 pg/L) corresponded with mean sulfate level of 12.5 mg/L. In
all other leachates, the mean sulfide concentrations were generally low (< 70
pg/L), with two notable exceptions at 3170 and 3838 pg/L at sites 6 and 14,
respectively. Since, the majority of the landfill leachates were not monitored over
time, it was not possible to identify any trends toward decreasing sulfate and
increasing sulfide concentrations.
Hardness is a measure of divalent metallic cations in solution, although
the major contributors are the calcium and magnesium ions. The hardness of the
leachates from each landfill site was determined as the sum of the calcium and
magnesium ions, measured as calcium carbonate (CaC03>. As predicted by the
high conductivity and TDS values, the leachate from site 10 contained the
greatest hardness at over 4,000 mg/L. According to classical water quality
indices, the hardness of the MSW landfill leachates ranged from very to
extremely hard (Sawyer et al., 1994). The high degree of hardness in the MSW
leachates contributes to their ability to reduce heavy metal bioavailability (see
Chapter 5; George et al., 1995).

113
Table 4-4. Mean concentrations (mg/L) of total (NH4VNH3) and un-ionized (NH3)
ammonia in leachates from fourteen MSW landfills in Florida
Site
Total Ammonia
(NH//NH3)
(mg/L)
Ammonia
(NH3)
(mg/L)
pH
1
1063
32
8.1
4
832.8
39.7
7.9
5
479
32
8.1
6
1492.3
100.1
8.1
7
20
0.5
6.5
8
258
15
8
9
140.4
NMa
7.4
10
67.3
<0.5
6.5
11a
436.6
1.9
6.8
11b
96.3
1.3
7.3
11c
333.6
4.5
7.1
12
NM
NM
NM
13
614
18.9
7.5
14
127.8
STTT. x "A '
7.1
8.2
Abbreviation: aNM, not measured.
In landfills the nitrogen cycle is inhibited by anaerobic conditions, as
nitrification requires the presence of oxygen for the conversion of ammonia to
nitrate (Burton and Watson-Craik, 1998). Therefore, in the absence of oxygen
microbial degradation of organic matter in landfills results in the accumulation of
ammonia (Cecen and Gursoy, 2000). Table 4-4 summarizes the mean total
ammonia concentrations in the MSW landfill leachates, which ranged widely from

114
Table 4-5. Metal concentrations in leachates from fourteen MSW landfills in
Florida
Site
Al
(mg/L)
Cu°
(mg/L)
Cd°
(mg/L)
Fe
(mg/L)
Pb°
(mg/L)
Zne
(mg/L)
1
0.42
(<0.1-0.89)
BDL
BDL
12.4
(5.7-30.7)
BDL
0.1
(0.1-0.11)
4
NMa
BDL
BDL
BDL
BDL
BDL
5
2.5
BDL
BDL
8.5
0.13
0.99
(0.9 - 5.8)
(4.2-16.2)
6
NM
NM
NM
NM
NM
NM
7
0.11
BDL
BDL
15.1
BDL
0.3
8
0.17
BDL
BDL
5.0
BDL
BDL
(<0.1-0.27)
(3.0-10.6)
9
0.4
BDL
BDL
7.2
BDL
BDL
10
1.61
0.16
BDL
70.0
BDL
BDL
11(a)
0.54
0.12
BDL
26.4
BDL
BDL
11(b)
0.23
BDL
BDL
7.4
BDL
BDL
11(c)
0.29
BDL
BDL
3.6
BDL
BDL
12
0.6
BDL
BDL
14.6
BDL
BDL
(0.1-1.1)
(8.5-20.6)
13
1.44
BDL
BDL
21.2
BDL
BDL
14
... 7"T
4.21
BDL
BDL
1.9
BDL
BDL
“NM, not measured. Metal concentrations below the detection limit (BDL) of each analytica
method were determined based on the minimum detection limits for each heavy metal, “Cu,
0.07mg/L; cCd, 0.01 mg/L; dPb, 0.07 mg/L; eZn, 0.01 mg/L..
67.3 mg/L at site 10 to nearly 1500 mg/L at site 6. Ammonia levels in landfills
have been shown to increase with age with three times higher ammonia
concentrations reported in mature landfills (Marttinen etal., 2002). Ammonia
speciation is pH dependent with equivalent concentrations of the ionized (NH4+)
or un-ionized (NH3) forms at the pKa of 9.3. Based on the measured pH values,
the concentrations of un-ionized ammonia in the MSW leachates were expected

115
to be low, and this was generally true. Compared to the ionized ammonia
concentrations, the un-ionized ammonia (NH3) ranged from < 0.5 mg/L at site 10
to 100 mg/L at site 6 (Table 4-4). The high total ammonia concentrations
corresponded with elevated un-ionized ammonia levels in some leachates.
The concentrations of heavy metals in the fourteen MSW landfill leachates
were summarized in Table 4-5. High and variable iron concentrations were
reported in the MSW landfill leachates with a range from 1.9 to 70 mg/L. The
leachates from site 1 demonstrated the lowest iron concentrations, while the
leachates from site 10 had the highest concentrations. Iron is routinely found at
high concentrations in MSW landfill leachates, and the range reported for Florida
leachates is comparable to the range reported in Danish landfills from < 1 to 218
mg/L (Baun et al., 1999). The abundance of iron in the leachates from site 10
may be attributed to the poor recovery of ferrous compounds by the magnet at
the incinerator facility (Gary Debo, personal communication). Aluminum was a
low-level, but ubiquitous, contaminant of all the MSW leachates with
concentrations that ranged from 0.1 to 4.2 mg/L. The presence, speciation, and
bioavailability of aluminum in the environment has been reviewed (Gensemer
and Playle, 1999).
The analytical detection limits for the heavy metals were 0.07, 0.01, 0.07,
and 0.01, for copper, cadmium, lead, and zinc, respectively. Generally, the
concentrations of toxic heavy metals in the leachates were below the analytical
detection limits. Notable exceptions were shown in the leachates from sites 10
and 11a, as they contained 0.16 and 0.12 mg/L of copper, respectively. The only

116
Landfill
Figure 4-2. Acute (48-hr C. dubia) toxicity of MSW landfill leachates collected
from 14 landfill sites in Florida with results expressed as TU (IOO/EC50)
and one standard deviation
leachate that contained lead above the detection limit was collected from site 5,
with a level of 0.13 mg/L. Zinc was the most prevalent heavy metal with 0.10,
0.99, and 0.30 mg/L identified in leachates from sites 1, 5, and 7, respectively.
Despite reports of higher concentrations of cationic contaminants including some
heavy metals, researchers do not agree about the risks to the environment from
this facilities (Wiles, 1996). Some researchers have suggested that landfills
practicing the co-disposal of MSW with incinerator ash are no more
likely to leach heavy metals than are MSW only landfills (Chichester and
Landsberger, 1996; Bozkurt et al., 2000).

117
Toxicity of MSW Landfill Leachates
Toxicity of MSW leachates to aquatic invertebrates
The acute toxicity of the MSW leachates collected at fourteen separate
landfill sites was evaluated with the 48-hour C. dubia assay (Figure 4-2). Control
survival for all results shown was greater than 90 % (USEPA, 1993a). The
endpoint utilized for the aquatic Invertebrate assays was death or Immobilization,
and this was quantified as the concentration of leachate lethal to 50 % of the test
organisms (LCso). All LC50 results were transformed (IOO/LC50) and expressed
as toxicity units (TU). The results show that the C. dubia assay was sensitive to
MSW landfill leachate toxicity. Although the range of toxicity in the MSW landfill
leachates was broad, it was consistent with the heterogeneous chemical
composition of the leachates.
The C. dubia response to the leachates from the 14 landfill sites was
variable with mean TU values ranging from 2.9 (site 7) to 51.7(site 6). Earlier
work, also demonstrated the toxic nature of the leachates from site 6 (Ward et
al., 2002). The acute toxicity of the leachates from sites 1 and 8 are represented
by TU values of 39.9 and 16.2, respectively. MSW landfill leachates from sites 1
and 8 were previously assayed for their acute toxicity (Ward et al., 2000). When
compared to the C. dubia assay responses in 1997, the toxicity of the leachates
from site 1 remained consistent; however, the leachates from site 8 increased in
toxicity (Ward et al., 2000). In 1997, some leachate samples from site 8 were
collected from a leachate storage tank, and degradation of toxic leachate
components may have occurred (Ward, 1997).

118
Figure 4-3. Correlation between the 48-hour acute toxicity assays using C. dubia
and D. pulex assays with MSW leachates collected from 14 landfill
sites in Florida
The TU responses of the C. dubia to leachates from sites 11a, 11b, and
11c were 26.3, 36.1, and 28.3, respectively. As previously described, site 11a
was an active Class I landfill, site 11b a Class III landfill, and site 11c was a
capped Class I landfill. There were no statistical differences (f-test) in the toxicity
of the leachates from 11a and 11c, even though the chemical composition of the
leachate from 11c was consistently lower for nearly all parameters except
ammonia. The ammonia concentration in the leachate from site 11c was 4.5
mg/L, which was double the 1.9 mg/L measured in the leachate from site 11c and
could explain the higher toxicity in this leachate. The toxicity of the leachates
produced at site 9 (TU = 28.9 ± 2.8), the only Class II landfill studied, were not

119
statistically different (f-test) from the toxicity of the leachates from site 11 a (TU =
26.3 ±2.4).
It should be noted that the site 9 and site 11a landfills are both permitted
for the disposal of domestic municipal waste; however, the two sites are located
in vastly different areas of the state and receive different volumes of waste
material. Site 9 is located in a sparsely populated rural area, and utilizes the only
operating Class II landfill, in Florida. On the other hand, the site 11a landfill is
located in a heavily populated area, and relative to other areas with lower
population densities, there is an increased potential for domestic hazardous
waste disposal.
While some researchers have reported a strong influence of high salt
concentrations on toxicity, the results of this research do not support this
conclusion (Wundram and Bahadir, 1999). The ionic strength of the leachates
from sites 10 and 11a were high, but there were no corresponding elevated toxic
responses. In contrast, others have demonstrated the strong influence of
inorganic macro-ions on leachate toxicity (Baun et al., 1999). Hoke et al. (1992)
reported an LC50 for sodium ranging from 781 to 825 mg/L for C. dubia.
Considering the prevalence of sodium in the MSW leachates, this may represent
a significant source of toxicity.
Additionally, some of the MSW landfill leachates were assayed for acute
toxicity with the 48-hour Daphnia pulex assay. Researchers have shown that the
sensitivity of C.dubia is comparable to or slightly greater than that of D. pulex
(Suedel et al., 1996). To date, the sensitivity of the C.dubia and D. pulex assays

120
Table 4-6. Toxicity of leachates collected from 14 lined MSW landfills with C.
dubia, D. pulex, and P. subcapitata with results expressed as the mean
toxicity unit (TU)(unitless) and one standard deviation (SD)
C. dubia
(48-hour)
D. pulex
(48-hour)
P. subcapitata
(96-hour)
1
4
39.9 ±12.8
(24.6-69.4)
28.6 ±4.4
Mean toxicity (TU) ± 1 SD
(range)
52.9 ±8.9
(44.4-62.6)
NMa
38.2 ±12
(25.2 - 60.6)
24.1 ±0.5
23.1 ±4.9
21.5 ±3.3
20.6 ± 5.2
(14.8-30.9)
(18.9-25.2)
(13.5-29.4)
6
51.7 ±5.1
NM
42.2 ±1.1
7
2.9 ±1.4
(1.9-3.9)
NM
1
8
16.2 ±3.5
20.5 ±6.9
10.9 ±3.0
(1.1-18.4)
(14.3-27.9)
(8.7-15.1)
9
28.9 ±2.8
14.7 ± 3.1b
17.9 ± 0.9C
10
47.6 ±1.9
44.4 ± 1.8b
6.6 ±0.5°
11(a)
26.3 ±2.4
27 ±1.1
11.1 ±0.7°
(b)
36.1 ±1.4
11.6±0.9b
8.5 ± 0.5C
(c)
28.3 ±3.7
25.5 ±1.7
19.6 ±0.4°
12
21 ±18.5
25.9 ±115.1
23.3 ±13.9
(6.1-44.2)
(5.1-39.9)
(5.2-38.5)
13
37.4 ±7.9
43.6 ±4.6
14.2 ± 1.9C
14
... . a.
8.3 ±4.3
11.3
12.6 ±7.4
(5.2-11.3)
(7.4-17.8)
Abbreviation: iNM, not measured “ D. pulex results statistically different from C. dubia results at p
< 0.01c P. subcapitata results statistically different from C. dubia results at p < 0.01.
have not been compared using MSW landfill leachates. The results shown here
support the comparable sensitivity of both daphnid species to MSW landfill
leachate toxicity (Figure 4-3). The TU values with the D. pulex assay ranged
from 1.6 to 50, while the response of the C. dubia assay ranged from 1.5 to 54
(Table 4-6). There was no statistically significant difference (f-test) in the
response of the C. dubia and D. pulex assays. The simple linear regression

121
E
I
100
75
50
25
0
â–¡ 1 â–  4 05 ID6 0 7 08 CJ9 H10 â–  11a 011b 511c 012 013 Q14
Landfill Site
Figure 4-4. Chronic (96-hour P. subcapitata) toxicity of MSW landfill leachates
collected from 14 landfill sites in Florida with results expressed as TU
(IOO/EC50) and one standard deviation
analysis of the invertebrate data show a significant (R2 = 0.94, p < 0.01)
correlation between the D. pulex and C. dubia results.
Toxicity of MSW leachates with algae
The chronic toxicity of the MSW landfill leachates was assayed by the 96-hour P.
subcapitata assay. The response of the algae to the leachates was similar to the
response of the aquatic invertebrates with algal mean TU values that ranged
from 1 to 42.2 (Figure 4-4). This agrees with earlier reports of the sensitivity of
algal assays (Marttinen et al., 2002). Species from lower trophic levels, like
algae and bacteria, are more likely to be adversely affected by MSW landfill
leachates (Plotkin and Ram, 1984).. According to the algal assay results, site 7
leachate was the least toxic with a TU of 1. This was similar to the low toxicity

122
O 4- t 1
O 1 2
P. subcapitata (ICso(%) 125-ml)
Figure 4-5. Correlation between the results of the standard (125-ml) and
modified (25-ml) P. subcapitata chronic 96-hour assays
reported for the site 7 leachate in the invertebrate assays. At the other extreme,
the leachates from site 6 produced a mean TU of 42.2. A similar toxicity was
identified in the leachates from site 6 over a six-month period in 2000 (Ward et
al., 2002). The leachates from sites 11a (an operating Class I landfill) and 11b
(an operating Class III landfill) demonstrated comparable toxicity with TU values
of 11.1 and 8.5, respectively. In contrast to the C. dubia assay results, the
leachates from site 11c were significantly more toxic (TU = 19.6) than either the
11a or 11b leachates. This was surprising considering that the composition of
the waste at sites 11a and 11c were similar. Both were designed for MSW,
which contrasted with the primarily non-putrescible waste composition at site
11b. The toxicity of the leachates from sites 9 and 14 were equivalent and

123
displayed TU values of 17.9 and 17.8, respectively. The site 9 leachates
represented the only Class II landfill studied, and comparatively the volume of
waste disposed at site 14 was nearly 5 times greater (Table 4-1). These results
indicate that the amount of waste landfilled does not influence the toxicity of the
waste leachates.
The chronic toxicity of the MSW landfill leachates was also evaluated in a
modified 96-hour P. subcapitata assay. This assay attempted to miniaturize the
protocol and minimize space requirements. Selected leachate samples were
tested with both the standard 125-ml (50-ml portion of leachate) protocol and the
modified 25-ml (10-ml portion of leachate) protocol. The TU values for the
leachates in the standard P. subcapitata protocol ranged from 1 to 31, while in
the modified protocol they ranged from 1.2 to 45.5. Generally, the magnitude of
the toxic response was consistent in both the standard and the modified
protocols. A least squares regression was performed with the results and
showed a significant correlation (r2 = 0.76, p < 0.01) between the standard and
modified P. subcapitata assays (Figure 4-5). Examination of the data revealed
that, while most of the leachates induced comparable toxicity responses in both
protocols, some did not. Overall, the results with the modified protocol
suggested a higher sensitivity to leachate toxicity, and this was especially true for
the leachates from sites 10,13, and 14. In fact, when the regression analysis
was performed without the toxicity data from sites 10, 13, and 14, the correlation
between the standard and modified protocols was stronger (R2 = 0.93, p < 0.01).
The low number of leachates evaluated precluded any strong conclusions

124
Table 4-7. Toxicity of the MSW landfill leachates from 14 sites in Florida using
the 15-minute Microtox acute assay with results expressed as toxicity
units (TU) and mean values of duplicate measures
Landfill Site
TU
(mean)
1
8.4
4
-
5
1.6
6
-
7
1.1
8
1.5
9
1.1
10
1.1
11a
1.1
11b
1.1
11c
1.1
12
4.7
13
1.1
14
1.1
concerning the use of the modified protocol, although the higher sensitivity
requires further investigation.
It may be that the color tone of the leachates reduced light penetration,
and by decreasing the volume of leachate the light penetration increased. The
color of the MSW landfill leachates ranged from a light golden to a deep reddish-
brown; however, the influence on algal growth rates from decreased light
intensity in growth chambers is poorly understood. Cleuvers and Ratte (2002)
found no relationship between colored samples and algal growth rates, but they
and others concluded decreasing sample volume increased algae sensitivity
when evaluating colored samples (Geis et al., 2000).
In the algal assays, observations indicated that below a certain threshold
(~3%), leachates from some sites produced an overgrowth of algal cells.

125
Observations made throughout the research investigation suggested that at low
concentrations the leachates were stimulatory to algal cell growth, which may be
attributed to the additional nutrients in the leachates (Cheung et al., 1993). This
suggested a stimulatory effect due to substances in the leachates, which was
often well above the cell growth observed in the negative controls (minus
leachate). Some researchers have suggested that moderate treatment of MSW
leachates could produce an effluent suitable for the irrigation of agricultural crops
and tree farms (Revel et al., 1999).
Toxicity of MSW landfill leachates with Microtox
The acute toxicity of the MSW landfill leachates was evaluated with the
Microtox assay in a 15-minute exposure period. Overall, the Microtox assay was
insensitive to the leachate toxicity with mean TU values that ranged from 1.1 to
8.4 (Table 4-7). The majority of the leachates displayed TU values less than 2
with TU responses of 1.1 common. As a point of reference, a TU value of 1.1 is
equivalent to an EC50 of ~ 90 % or very low toxicity. The Microtox assay
identified low toxicity in the leachates from sites 1 and 12 with TU values of 8.4
and 4.7, respectively. Due to the low sensitivity of the Microtox assay, there
were some instances when an EC50 could not be determined even at the highest
possible concentration of leachate (90 %). When a definitive EC50 was not
determined, then the EC50 was reported as greater than the highest
concentration assayed. For comparative purposes, when the EC50 values were
converted to toxicity units (TU) the greater than sign was omitted.

126
Heavy metal toxicity of MSW leachates using MetPLATE
MSW landfill leachates were evaluated for heavy metal toxicity with the
MetPLATE assay, which is specifically sensitive to the presence of heavy metal
toxicants (Bitton et al., 1994). The detailed MetPLATE results with the MSW
landfill leachates are presented in Chapter 5. The results demonstrated that in
the MetPLATE assay the MSW leachates were slightly inhibitory, but the mean
inhibition was generally quite low. To briefly summarize, roughly 95 % of the
leachates assayed produced inhibitory responses less than 50 %.
NOEC/LOEC vs. EC50 or TU results
Toxicity assays are conducted to evaluate the concentration of a
substance responsible for an adverse biological response (e.g. death or
reproductive failure) in a specified period of time. Traditionally, these assays
have been conducted at 24, 48, or 96 hours, although some rapid bioassays
produce results in a matter of hours (Nelson and Roline, 1998; Jung and Bitton,
1997). By graphical or statistical procedures, the response of a toxicity test
organism can be interpolated to yield a point estimate of the concentration
producing the toxic effect. These point estimates are determined as the
concentration producing the desired effect in the test population. Generally,
responses are reported as the concentration that produces a 50 % effect (EC50),
death (lethality) (LC50) or inhibition (IC50) in a test population (Rand, 1995).
During hypothesis testing, the no observed effect concentration (NOEC)
and the lowest observed effect concentration (LOEC) are determined statistically.
Unlike the point estimate, the NOEC/LOEC results depend on the leachate
dilution series, and; therefore, are not independent of the test concentrations.

127
Table 4-8. Relationship between the toxic endpoints of IC50 (%),NOEC (%) and
LOEC (%) with the results of the P. subcapitata assay with leachate
from site 1
DATE
IC50
NOECa
LOECD
(%)
(%)
m
Feb '00
3.7
3.12
6.25
March '00
2.4
<3.12
3.12
April '00
3.5
<3.12
3.12
May '00
2.0
< 1.56
1.56
June '00
2.1
< 1.56
1.56
July '00
1.5
< 1.56
1.56
Oct '00
2.6
< 1.56
1.56
Nov '00
3.6
1.56
3.12
Dec '00
2.5
1.56
3.12
Jan '01
1.7
< 1.56
1.56
Feb '01
2.3
1.56
3.12
March '01
3.2
1.56
3.12
May '01
3.9
3.12
6.25
... x:
Abbreviations: “LC50, concentration lethal to 50 % of the test population; °NOEC, no
observed effect concentration; cLOEC, lowest observed effect concentration.
For example, consider the case with two toxicity assays conducted
simultaneously. The first assay is prepared with a dilution factor of 0.5 (e g. 100,
50, 25 %) and the second at a dilution factor of 0.1 (100,10,1 %). The
NOEC/LOEC results at a 0.5 dilution factor represent a tighter margin of
response than the 0.1 dilution factor. NOEC/LOEC responses are used to
establish threshold doses of adverse effects, which aid in the establishment of
ranges for the purpose of comparing test organism responses to samples with
similar matrices. Bierkens et al. (1998) evaluated twenty bioassays for their
relative sensitivities and by using NOEC/LOEC values established a risk-based
prediction of toxicity. To demonstrate the use of NOEC/LOEC values, the P.
subcapitata IC50, and NOEC/LOEC results are compared for the MSW landfill
leachates collected from site 1 (Table 4-8). Generally, the IC50 results were

128
CONDUCTIVITY
A. (mS/cm)
0
Feb March April June July Sept Oct Nov Dec Jan Feb March
•00 '00 '00 '00 '00 ’00 '00 '00 '00 *01 '01 ’01
TOTAL ORGANIC CARBON
(mg/L)
Figure 4-6. Influence of time on A.) conductivity, B.) chemical oxygen demand
C.) total organic carbon of the MSW landfill leachates from site 1

129
A. CONDUCTIVITY
Figure 4-7. Influence of time on A.) conductivity, B.) chemical oxygen demand,
C.) total organic carbon of the MSW landfill leachates from site 5

130
within the range of the NOEC/LOEC. Exceptions were noted when the NOEC
could not be determined, due to insufficient leachate dilution. In many cases, the
margin of safety implied by the NOEC/LOEC values was < 20 %. These results
reinforced the artificial nature of the NOEC/LOEC measurements (Crane and
Newman, 2000).
Monitoring MSW landfill leachate toxicity over time
Trends in the chemical characteristics and toxicity were monitored in the
leachates collected from sites 1 and 5 between February 2000 and May
2001 .These leachates were tracked over time to determine temporal variability or
spatial trends in assay results. When considering the distribution of various
chemical characteristics in the leachates from site 1, it becomes apparent that
the time frame of the investigation was insufficient to establish stable trends in
the data (Figure 4-6). The conductivity values remained relatively consistent
over time (Figure 4-6A). The landfill at site 1 no longer receives MSW; therefore,
the total concentrations of inorganic constituents in the waste are now finite.
Since, the concentrations of inorganic contaminants are sensitive to 'washout'
effects it can be expected that with time these concentrations may decline.
In contrast, the data indicated a trend for increasing COD concentrations
with time (Figure 4-6B). With time, changes in the physical structure of the waste
material may result in increased leaching of organic compounds. Generally,
COD concentrations are expected to decrease as the biologically degradable
organic materials in the landfill are consumed, although the fraction of COD
representing the recalcitrant organic material remains constant. When the TOC
concentrations in the leachates from site 1 were considered a wide fluctuation

131
was identified (Figure 4-6C). Further interpretation of the chemical composition
of the leachates from site 1 revealed that the COD/TOC ratio fluctuated between
1.4 to 4.7. This ratio is used as an indicator of highly recalcitrant organic
materials, and generally increases with landfill age (Chian and DeWalle, 1976).
The chemical characteristics of the leachates from site 5 were more varied
over time (Figure 4-7). The level of conductivity in the leachates from site 5
ranged from 3.2 to 12.7 mS/cm (Figure 4-7A). During this investigation, there
was a large overall fluctuation in the conductivity levels; however, the initial
conductivity concentration of 6.1 mS/cm measured in February 2000 was very
similar to the final concentration of 7.4 mS/cm in March 2001. When interpreting
leachate characteristics over time, it is critical to recognize the limitation of the
data set presented. For example, the leachate samples were collected from only
one area of the landfill and did not represent a complete mix of leachate
characteristics. Furthermore, leachate generation is a continuous process, and
so the portion collected at one point in time may not necessarily represent a
single time period. The COD concentrations in the leachates from site 5 ranged
one order of magnitude, from 416 to 4100 mg/L (Figure 4-7B). When considering
these COD concentrations, a general trend for increasing COD with time was
identified. There was no pattern or trend to the TOC concentrations in the
leachates from site 5 (Figure 4-7C). While the concentrations ranged from 210 to
831 mg/L, variations over time remained close to the median value of 467 mg/L.
The COD/TOC ranged from 1 to 7.4, reflecting the dramatic fluctuations in the
COD and TOC concentrations.

132
0 C.dubia â–¡ P. subcapiata 0 Microtox
Figure 4-8. Acute (C. dubia and Microtox) and chronic (P. subcapitata) toxicity of
MSW landfill leachates collected from site 1 between February 2000
and May 2001. Results as TU (100/ECso) with one standard deviation
for C. dubia and P. subcapitata, Microtox mean of two replicates
BC.dubia mP subcapitata B Microtox
Figure 4-9. Acute (C. dubia and Microtox) and chronic (P. subcapitata) toxicity of
MSW landfill leachates collected from site 5 between February 2000
and March 2001. Results as TU (IOO/EC50) with one standard
deviation for C. dubia and P. subcapitata, Microtox mean of 2
replicates

133
Based on the continued high strength of the MSW landfill leachates from
sites 1 and 5, alterations in leachate toxicity were unlikely. This conclusion was
supported by the results of the toxicity assays, which are summarized for the
MSW landfill leachates from site 1 (Figure 4-8) and site 5 (Figure 4-9). In the
C.dubia assay, the toxicity of the MSW leachates from site 1 displayed a TU
range from 24.6 to 72.1. Neglecting the extreme responses, the range of TU
values was from 41 to 69, indicating that the acute toxicity of these leachates
remained relatively consistent relative to the time frame evaluated.
Comparable C. dubia toxicity was identified in the MSW landfill leachates
from site 5 with TU values that ranged from 9.5 to 30.1. When considering the
response of the P. subcapitata assays, the TU ranges were 25.2 to 60.6 % and
4.3 to 29.4 % for the MSW landfill leachates from sites 1 and 5, respectively. In
most cases, the Microtox assay was insensitive to the toxicity in the leachates
from site 1 with most TU values less than 8. The site 1 leachate collected in May
2001 produced a TU value of 26.7 with the Microtox assay, which was lower than
the TU of 48.5 measured with the C. dubia assay, but equivalent to the 25.2 with
the algal assay. The response of the Microtox assay to the leachates from site 5
was inadequate, with measured TU values generally < 9, although one leachate
sample collected in March 2000 displayed a TU of 16.3.
Overall, the results indicated that the toxicity of the leachates from site 1
was greater than that in the site 5 leachates. Although there were no
relationships identified between the concentration of various chemical
parameters and time, the fluctuations were large. Additionally, the acute and

134
Figure 4-10. Relationship between the results of the chronic 96-hour P.
subcapitata (IC50) and the acute 48-hour C. dubia (LC50) assays with
MSW landfill leachates from fourteen sites in Florida
chronic toxicity of the MSW landfill leachates remained consistent. It is important
to recognize that a relatively short time course was evaluated here compared
with the entire life of a MSW landfill; therefore, these trends may not be true over
the entire life of a landfill.
Comparative sensitivity of the bioassays
A least squares regression analysis of the acute C. dubia (LC50) and the
chronic P. subcapitata (IC50) assays indicated a similar sensitivity (R2 = 0.53, p <
0.01) (Figure 4-10). There were no relationships between the C. dubia, D. pulex,
or P. subcapitata assay results when compared to the parallel Microtox results.

135
Table 4-9. Coefficients of variation (CV)(%) for the P. subcapitata, C. dubia, and
0. pulex assays
Coefficient of Variation (CV)
96-hour
48-hour
48-hour
Site
P. subcapitata
C. dubia
D. pulex
(%)
(%)
i%]
i*
31.3
25.6
52.9
2°
41.6
52.3
3d
54.4
46.3
-
4°
38.5
22
-
5®
47.9
36.7
15.2
6D
74
24.9
-
7
NDa
48.7
ND
8
27.4
21.4
33.7
9
5.0
9.7
21.1
10
7.6
4.0
4.1
11a
6.3
9.1
4.1
11b
5.9
3.9
7.8
11c
2.0
13.1
6.7
12
59.6
88
72.8
13
13.4
21
10.6
14
58.4
52.3
tttt—■—7r~tr,—rrrrrrr.
ND
“The CV not determined (ND) when standard deviation was 0. ° Included data presented in
Chapter 3.
This contrasted with reports that showed a relationship (R2 = 0.36) between the
48-hour C. dubia and 15-minute Microtox acute toxicity assays (Jung and Bitton,
1997). Although contrary to the results reported here, the samples evaluated by
Jung and Bitton (1997) were industrial effluents of limited complexity.
While the leachate from only one Class III landfill (site 11b) was assayed,
the bioassay results suggest that these leachates displayed toxicity that was
comparable with the toxicity of the Class I and II MSW landfill leachates. The site
11b leachates displayed a TU of 36.1 in the C. dubia assay, which was within the
range of 8.3 to 51.7 reported for the MSW landfill leachates. The toxicity of the

136
leachates from site 11 b was lower In the D. pulex and P. subcapitata assays with
TU values of 11.6 and 8.5, respectively.
The coefficient of variation (CV) is a measure of the reproducibility of
biological responses (Ferrari et al., 1999). As predicted by the highly variable
chemical composition of the MSW landfill leachates, the CVs for the C. dubia
assays with the fourteen MSW landfill leachates ranged from 3.9 to 88 % (Table
4-9). The leachate samples collected from site 12 displayed the highest variation
(CV = 88 %) with the C. dubia assay (Table 4-2). The low reproducibility of
thetoxic responses in the leachates from site 12 may be attributed to a change in
sampling location during the investigation. After the first sampling point at site 12
became inaccessible, it was necessary to collect the leachates from a second
point. Low variability was reported in the responses of the C.dubia toxicity
assays to MSW landfill leachates from sites 10 and 11b, with CVs of 4.3 and 3.6
%.
From a broad perspective, the degree of inter-replicate variability with the
results of the P. subcapitata toxicity assays was greater than described for the C.
dubia assays. This may be a function of the method used to quantify the algal
response. During this investigation, algal cells were counted using a
hemacytometer and a microscope. Manual counts of algal cells are labor-
intensive and require an experienced technician. Geis et al. (2000) observed
replicate variability ranging from 2 to 35 % in flasks counted manually.
Combining the intrinsic variability of the quantification method and the extremely

137
Table 4-10. Classification system for ranking the toxicity of MSW landfill
leachates from 16 sites in Florida (adapted from Clement et al.,1996;
Bulich, 1982)
Class
TU
(unitless)
1
< 1
2
1 to 3
3
3 to 10
4
10 to 30
5
30 to 100
6
> 100
variable MSW landfill leachate composition, then a possible source of the high
CVs becomes apparent.
The sensitivity of the P. subcapitata, C. dubia, D. pulex, and Microtoxâ„¢
assays to MSW landfill leachates were summarized (Clement et al., 1996; Bulich,
1982). Table 4-10 describes the classification system used to rank the
sensitivities of the assays. When ranking the sensitivity of the various toxicity
assays additional acute and chronic toxicity results, obtained during the initial
phase of MSW landfill leachate characterization, were included (Chapter 3).
According to the classification system, the toxicity was distributed in
classes 2 through 5 (Figure 4-11). Disproportionately, the majority of the toxicity
associated with the MSW landfill leachates was distributed in class 4. This was
true for the results of the C.dubia (50 %), D. pulex (72.7 %), and P. subcapitata
(62.5 %) assays, but not the results of the Microtox assay. Instead, the Microtox
assay results showed that 75 % of the MSW landfill leachates were ranked in
class 2, while the remaining 25 % were in class 3. For the C.dubia, D. pulex, and
P. subcapitata assays the proportionality of MSW landfill leachate toxicity was

138
■1 ¡ -M
SE MI
D.P-
Figure 4-11. Ranking of sixteen MSW leachates with the results of the Microtox
(MT), P. subcapitata (P. sub), D. pulex (D. p.), and Ceriodaphnia dubia
(C.d.) assays using the classification in Table 4-10. The leachates
were ranked least toxic (class 1) to most toxic (class 6)
12.5 %, 0 %, and 18.8 % in class 3 and 6.3 %, 0 %, and 6.3 % in class 2,
respectively. Additionally, a significant portion of the MSW landfill leachate
toxicity was in class 5 at 31.3, 27.3, and 12.5 % for the C.dubia, D. pulex, and P.
subcapitata assays, respectively.
Relationship Between Chemical/Physical Leachate Characteristics and
Leachate Toxicity
Using simple linear regression analysis, the toxicity of the MSW landfill
leachates and various leachate chemical parameters were compared. When the
C. dubia assay results were compared with the alkalinity concentrations in the
leachates, this relationship explained < 15 % of the toxic response (R2 = 0.14, p

139
= 0.01). This relationship improved when the conductivity values were compared
to the C. dubia toxicity results (R2 = 0.36, p < 0.05). There was a stronger
relationship between the total ammonia concentrations (R2 = 0.49, p < 0.01) and
the un-ionized ammonia concentrations (R2 = 0.57, p < 0.01). Heijerick et al.
(2003) showed that physicochemical factors in MSW landfill leachates (e.g.
hardness, alkalinity, and conductivity) exert a strong influence on toxicity.
Regression analysis was also performed with the results of the algal
toxicity assays. The alkalinity concentrations in the MSW landfill leachates had
no influence on the chronic toxicity (R2 = 0.09). When the P. subcapitata toxicity
results were compared to the conductivity concentrations a relationship was
identified (R2 = 0.21, p <0.01). A significant relationship was found between the
algae toxicity results and the total ammonia concentrations in the MSW landfill
leachates (R2 = 0.85, p < 0.01). Similarly, Marttinen et al. (2002) reported a
correlation between algal toxicity and total ammonia concentrations (R2 = 0.53).
When the un-ionized ammonia concentrations were compared to the P.
subcapitata assay results the correlation was lower (R2 = 0.61, p < 0.01).
The low sensitivity of the Microtox assay to the MSW landfill leachates
precluded any comparisons with chemical parameters. Although the Microtox
assay is a widely utilized toxicity assay, it is insensitive to ammonia toxicity
(Stronkhurst et al., 2003). Considering that ammonia is a major toxicant in MSW
landfill leachates, it is apparent that the usefulness of the Microtox assay is
seriously limited during toxicity investigations in these types of samples.

CHAPTER 5
HEAVY METAL BINDING CAPACITY (HMBC) OF MUNICIPAL SOLID WASTE
LANDFILL LEACHATES
Introduction
Researchers have shown that heavy metal toxicity and bioavailability are
strongly influenced by the activity of ionized metal species in solution, but not by
the total metal concentrations (Grabowski et al., 2001; Mowat and Bundy, 2001).
In 1983, Morel (1983) summarized the results of research to date and proposed
the free ion activity model (FIAM), recognizing that heavy metals are most toxic
in their ionized state (Campbell, 1995). MSW leachates may contain metals due
to the composition of waste materials, and degradative processes in the landfill.
While researchers agree that the total concentration of heavy metals in MSW
landfill leachates cannot predict toxicity or bioavailability (Morgan and Stumm,
1991; Luoma, 1995), there is no consensus within the research community for
identifying or quantifying the various metal species. Nimmo et al. (1995) used
bioassays with invertebrates and plants to investigate the toxicity of landfill
leachates in Virginia, and determined that metals in the landfill leachates were
not bioavailable, although the complexation potential of the leachate was not
quantified.
The most widely used analytical method for determining metal speciation
was developed by Tessier et al. (1979). With a sequential extraction procedure,
they proposed five distinct metal fractions; exchangeable, bound to carbonate,
140

141
bound to iron and manganese oxides, bound to organic matter, and residual
heavy metals. Other methods for metal speciation include dialysis and ion
exchange. While the mobility and extractability of metals in landfills has been
studied (Clevenger and Rao, 1996), no data are available on the quantitative
determination of the bioavailability and the ability of landfill leachates to bind
metals, using biological assays. Reliance on metal speciation models, derived
from sequential extraction procedures, may underestimate bioavailable metal
concentrations (Scheifler et al., 2003). The bioavailability of metals in
contaminated soils was investigated with a microbial respiration assay and a
microbial nitrification assay. Although nitrification was sensitive to bioavailable
metals, respiration was highly sensitive (Ge et al., 2002). Other researchers
have shown that microbial respiration provides a good measure of both general
toxicity (Bitton and Koopman, 1982) and heavy metal toxicity (Bitton, et al.,
1984).
The ability of MSW leachates to reduce metal toxicity has been
recognized, but no work has been published to quantify this ability or to attempt
to elucidate the mechanisms responsible. Huang et al. (1999) measured the
heavy metal binding capacity (HMBC) of surface waters as the ratio of the EC50
of a selected metal in site water to the EC50 of the same metal in control water.
This methodology is similar to that previously described for the water effect ratio
(WER) proposed by the USEPA (1984). Basically, both incorporate the influence
of site-specific parameters on the quantity and bioavailability of metals in
environmental matrices, but the HMBC test, based on MetPLATE, is rapid, cost-

142
effective and can easily be used in field assessments. Recently, technical issues
with the WER have been raised, in relation to test species (acclimation to test
conditions and interspecies variability) and chemical issues (alkalinity and
calciunrmagnesium ratios in control waters and pH variability in site waters)
(Welsh et al„ 2000).
The utility of MetPLATE, a microbial enzyme assay specifically sensitive to
heavy metal toxicity, was investigated with MSW landfill leachates in Florida.
Earlier work showed that MSW landfill leachates are both acutely and chronically
toxic in bioassays using aquatic invertebrates and algae (refer to Chapters 3 and
4). By adaptation of the MetPLATEâ„¢ assay, the heavy metal binding capacity
(HMBC) of the MSW landfill leachates could be evaluated. A review of current
literature revealed a lack of research concerned with the quantitative
determination of metal toxicity, bioavailability and metal binding capacity in
landfill leachates using toxicity assays. The majority of the degradative
processes in landfills are due the activity of microbial populations (Barlaz, 1997).
Therefore, the use of a microbial assay to evaluate the toxicity or bioavailability of
landfill leachates is appropriate (Suflita et al., 1992). The MetPLATE assay
quantifies changes in the activity of the p-galactosidase enzyme. The enzyme
activity is dependent on conformational changes and competition with cationic
heavy metals, for functional binding sites, decreases enzyme activity (Juers et
al., 2001). During earlier investigations with MSW landfill leachates, results
suggested that inorganic compounds, but not heavy metals, were primarily
responsible for the toxic effects (Ward et al., 2002).

143
The objectives of the present work were; 1.) to evaluate the heavy metal
toxicity of MSW leachates, 2.) to quantify the heavy metal binding capacity
(HMBC) of the MSW landfill leachates, and 3.) to tentatively identify chemical
characteristics of MSW landfill leachates responsible for the binding capacity.
This investigation was designed to provide information concerning heavy metal
bioavailability, and heavy metal interactions and reactions within MSW landfill
leachates.
Materials and Methods
Sample Sites
MSW leachates were collected over a two-year period from 16 lined
landfills in Florida. Sites were selected to provide a range of landfill properties
(landfill size, quantity and type of waste and age of landfill) and leachate
characteristics (chemical strength, inorganic or organic content). Further
descriptions of the sites at which the MSW landfill leachates were collected are
available (Chapters 3, 4, and 6). Additional baseline water samples were
collected from two local lakes (Lake Alice in Gainesville, FL and Lake Beverly in
Beverly Hills, FL) and from a municipal wastewater treatment plant (WWTP), in
Gainesville, FL.
Leachate Collection
MSW landfill leachates were collected from the leachate collection sumps
of the lined landfills using a Teflon baler. Samples for chemical analysis were
collected in polyethylene or glass containers and preserved according to the U.S.
Environmental Protection Agency (USEPA) methods (USEPA, 1993b). The
leachates for toxicity analysis were collected in plastic containers, transported to

144
the lab on ice and immediately stored at 4°C until sample analysis, within 1 or 2
days. The lake water and WWTP effluent samples were collected in plastic
containers for toxicity analysis; however, samples were not collected for chemical
analysis.
Chemicals and Reagents
Metal stocks, Cu+2 (CuS04), Hg+2(HgCI2) and Zn*2 (ZnS04), were
prepared in Milli-Q water at a concentration of 500 mg/L. The exchange resins,
diethylaminoethyl cellulose (DEAE) capacity 0.99 meq/g, and Dowex-50W
(Styrene DVB, gel) dry mesh 50-100 were purchased from Sigma-Aldrich
(Milwaukee, Wl). Columns were prepared in 10-ml borosilicate glass pipettes
and resins were conditioned according to manufacturer specifications with
reagent grade materials.
Chemical Analysis
Field measurements included; pH and temperature (Orion, Model 290A),
electrical conductivity (HANNA Instruments, Model H19033), dissolved oxygen
(DO) (YSI Inc. Model 55/12 FT), and oxidation/reduction potential (ORP)
(Accumet Co. Model 20). Alkalinity, biochemical oxygen demand (BOD),
chemical oxygen demand (COD), dissolved organic carbon (DOC), ammonia and
sulfides were measured according to methods described by USEPA (1993) and
APHA (1999). Hardness was measured by colorimetric analysis, HACH method
8030 (HACH, Loveland, CO). Samples for total metal content were digested

145
Prepare sample dilutions
Aliquot 900-pl of sample, or its dilution, into dean test
tubes
Aliquot 100-pl of bacterial reagent into each of the
test tubes; vortex
Incubate for 1 1/2 hoirs at 35°C
Aliquot 200-pl from each test tube into separate wells
of a 96-well microplate and add 100pi of
chromogenic substrate
Incubate at 35° C for color development in the
negative control
Figure 5-1. The MetPLATE assay protocol for determining the heavy metal
toxicity of MSW landfill leachates (adapted from Jung, 1995)

146
(USEPA Method SW-846 301OA) (USEPA, 1996) and analyzed by atomic
emission spectroscopy (AES) (Thermo Jarrell Ash, Model Enviro 36).
Determination of Heavy Metal Toxicity
The MetPLATEâ„¢ test kit was developed at the University of Florida for
determining heavy metal toxicity, and the kit contains a bacterial reagent (an
E.coli strain), buffer, chlorophenol red galactopyranoside (CPRG), which serves
as the substrate for p-galactosidase, and moderately hard water (MHW) as a
diluent. The toxicity test was previously described (Bitton et al., 1994,
http://www.ees.ufl.edu/homepp/bitton) (Figure 5-1). Briefly, the bacterial reagent
was rehydrated with 5-ml of diluent and thoroughly mixed by vortexing. A 900-pl
aliquot of the leachate or its dilution was added to a test tube to which was added
100-pl of bacterial reagent. Test tubes were vortexed, and then incubated for 1.5
hours at 35°C. A 200-pl aliquot of the suspension (leachate + bacteria) was
transferred to a 96-well microplate to which was added 100-pl of CPRG, the
enzyme substrate, followed by shaking. The microplate was then incubated at
35°C for color development. The response was quantified at 570 nm using a
Multiskan microplate reader.
Determination of HMBC
The ability of MSW landfill leachates to reduce the bioavailability of heavy
metals was assessed using the MetPLATEâ„¢ test kit to determine the HMBC of
the leachates following a methodology previously developed (Huang et al.,
1999). The heavy metals copper, zinc and mercury were selected for testing
based on their increased concentrations in the natural environment and their

147
Metal Solution
J
J-
J
â–¡
CONTROL WATER
Spike moderately hard water
(MHW) with heavy metal
MSW LANDFILL LEACHATE
Spike with heavy metal
J
J
â–¡
L
Incubate at 25°C for 60
minutes while shaking
Incubate at 25°C for 60
minutes while shaking
J
â–¡
L
J
â–¡
L
Prepare dilutions with MHW
and incubate for 60 minutes at
25°C;shake
Prepare dilutions with
leachate and incubate for 60
minutes at 25°C; shake
J
â–¡
L
r
L
Add 0.1 ml test bacteria to 0.9
ml solution (triplicate tubes)
incubate 90 min. at 35°C
Add 0.1 ml test bacteria to 0.9
ml solution (triplicate tubes)
incubate 90 min. at 35°C
J
â–¡
L
r
L
METPLATE ASSAY
Add 0.2ml from each tube to a microplate well. Add 0.1ml
of buffered substrate to each well. Incubate at 35°C until
color development in negative control. Read color at
570nm. Determine EC» for control water and leachate.
HMBC
EC50 MSW LANDFILL LEACHATE
ec50 control water
Figure 5-2. The protocol for determining HMBC of MSW landfill leachates
(adapted from Huang et al., 1999)

148
significant threat to environmental health resulting from interactions with
biologically important functional groups, e.g. sulfur and nitrogen groups (Morgan
and Stumm, 1991). The HMBC method has been used with water samples
obtained from rivers and wetlands, but never before with MSW landfill leachates.
Using the MetPLATE assay the concentration of each heavy metal
prepared in moderately hard water (MHW), which produced a 50 % inhibition in
enzyme activity (EC50), was determined. Simultaneously, the MSW landfill
leachates were spiked with the same metal and the concentration of the metal in
the leachate responsible for a 50 % inhibition in enzyme activity (EC50) was
determined. The HMBC was expressed as the ratio of the ECsoof a given metal
in leachate to the ECsoof a given metal in MHW (Figure 5-2). The EC50 values
were determined by the following procedure. A 10-ml aliquot of each MSW
landfill leachate or MHW was spiked with an appropriate volume of metal stock
solution (Cu+2, Zn+2 or Hg+2) and shaken (160 rpm) for 60 minutes at room
temperature. Generally 10 to 50 pi of the metal stock was added, but this was
dependent on the response of the MetPLATE assay. After the addition of the
metal spike, the leachates and/or MHW were serially diluted at a dilution factor of
0.5. The spiked MSW landfill leachates were serially diluted in additional MSW
landfill leachate and the spiked MHW was serially diluted with MHW. The spiked
MSW leachates and MHW were covered and shaken (160 rpm) for an additional
60 minutes at 25°C. The spiked leachates and the spiked MHW were then
assayed in triplicate according to the MetPLATE assay, as previously described.

149
Figure 5-3. The protocol used for fractionation of HMBC
Influence of Some Leachate Parameters on HMBC
A fractionation scheme was proposed to identify the influence of some
chemical parameters of the MSW landfill leachates on HMBC (Figure 5-3).
Leachate samples were collected from sites 1,4,5 and 8 and assayed for their
HMBC with copper (Cu+2), zinc (Zn+2) and mercury (Hg+2). A 2-L volume of each

150
leachate sample was filtered (0.45 um membrane filter, GN-6 Metricel), passed
through diethylamlnoethyl cellulose (DEAE, 3 g resin/column) a weakly basic
anion exchange resin, and finally passed through a strong acidic cation (in the
hydrogen-saturated form) exchange resin (Dowex-50W, 3 g resin/column). Ion
exchange resins were chosen based on their selectivity for the ions of interest.
The anion exchange resin, DEAE, has a high affinity for humic and fulvic acids
(Yamada et al., 2000) as well as phosphates, carbonates and chlorides. Kim et
al. (1999) reported > 99 % removal of hardness cations by exchange resins.
Each of the MSW landfill leachate fractions generated was assayed
according to the HMBC protocol. The chemical parameters of dissolved organic
carbon (DOC), alkalinity, pH and conductivity were monitored following each
fractionation procedure. For purposes of this research the binding capacity of the
leachates were divided into the solids-associated fraction (removed by filtration),
organic-associated fraction (removed by anion exchange) and hardness-
associated fraction (removed by cation exchange).
Data Analysis
The results of the MetPLATE assays were expressed as the leachate
concentration that produced a 50% decrease in enzyme activity (ECso). When
the inhibition of enzyme activity was less than 50 %, then the results were
expressed as the percent (%) inhibition caused by the MSW landfill leachate at a
concentration of 100 %. The EC50 (%) or inhibition (%) results were determined
by graphical interpolation. All MetPLATE assays were performed in triplicate and
results are presented as the mean ± one standard deviation. For the

151
Table 5-1. ECwfor Cu*2, Zn+2, and Hg*2 determined with the MetPLATE assay.
Metal
EC50
(mg/L)
Cu+2 (as CuSO„)
0.13 ±0.05
Zn+2(as ZnCI2)
0.33 ±0.16
Hg+2 (as HgCI2)
0.15 ±0.06
determination of HMBC, the individual EC50 values were obtained by graphical
interpolation for the metal spiked MSW leachates and the metal spiked
moderately hard water (MHW). The HMBC of each individual MSW landfill
leachate was determined from triplicate MetPLATE assays; therefore, results are
presented as the mean ± one standard deviation.
Results and Discussion
Heavy Metal Toxicity of Landfill Leachates
The prevalence of heavy metals as constituents of landfill leachates has
been widely reported, but little is known about their bioavailability and toxicity.
This research investigated the bioavailability of heavy metals In the leachates
from sixteen lined MSW landfills in Florida with MetPLATE, an assay specific for
heavy metal toxicity. The sensitivity of the MetPLATE assay to heavy metals has
been previously demonstrated (Bitton et al., 1994) and was validated for the
three metals (copper (as CuS04), zinc (as ZnCI2), and mercury (as HgCI2)),
utilized in the current study. The metal EC50S obtained were 0.13 ± 0.05 mg/L,
0.38 ±0.16 mg/L and 0.15 ± 0.06 mg/L for Cu*2, Zn*2, and Hg*2, respectively
(Table 5-1). These EC50 values were comparable to those previously reported
(Jung, 1995).

152
% Inhibition
Mean
Range
1
14.8
0 - 36.8
2
5.8
0-46.4
3
8.3
0-26.1
4
8.6
0-53
5
14.9
0-61.7
6
28.5
12.2-37.0
7
20.9
1.6-31.1
8
2.5
0-11.9
9
0
-
10
65 ±2.4
a
11(a)
0
11(b)
0
-
11(c)
0
12
22.4
0 - 56.7
13
0
-
14
22.3
6.6 - 37.9
Lake Alice
0
-
Lake Beverly
0
-
WWTP
Effluent
0
-
aN= number of sampling events

153
Table 5-2 summarizes the results of the MetPLATE assay for the sixteen
MSW landfill leachates from Florida. A range of MetPLATE inhibition (%) was
reported with the leachates. There was no toxicity (inhibition = 0) in the MSW
landfill leachates from sites 9,11a, 11b, 11c, and 13. The leachates collected
from site 10 produced a 65 % inhibition of enzyme activity in the MetPLATE
assay. However, the mean toxicity was generally low, with 95% of the samples
displaying less than a 50% inhibition. For comparison, samples were collected
from two local lakes and a WWTP and these samples showed no toxicity in the
MetPLATE assay.
Higher heavy metal concentrations have been reported in the leachates
from landfills co-disposing MSW and MSW incinerator ash (Bozkurt et at., 2000),
so these types of leachates are expected to elicit a greater inhibitory response in
the MetPLATE assay. Landfills at sites 10 and 11a practice the co-disposal of
MSW and MSW incinerator ash. The MetPLATE results with these leachates
were vastly different. The leachate from site 10 inhibited the response of the
MetPLATE assay by 65 %, which contrasts with the absence of toxicity in the
leachate from site 11a. Surprisingly, metal analysis of the leachates revealed
similar copper concentrations of 0.16 mg/L and 0.12 mg/L at site 10 and site 11a,
respectively. These concentrations were roughly equivalent to the EC» of 0.13
mg/L reported for copper in the MetPLATE assay (Table 5-1). Therefore, the
inhibition caused by the leachate from site 10 was attributable to the measured
copper concentration. Based on the metal analysis, the toxicity of the leachate
from site 11a was expected to be high; however, this was not the case. Instead,

154
Table 5-3. Physical and chemical characteristics of leachates collected from 16
lined MSW landfills in Florida. Results are presented as mean and
(range) or mean ± one standard deviation for sites sampled on one
occasion
Site
pH
Conductivity
(mS/cm)
Alkalinity
(mgIL as
CaC03)
TDSa
(g/U
Sulfides
(pg'L)
CODb
(mg/L)
DOC
(mgiL)
1
2
7.6
(7.3-8.1)
7.0
(6.5-7.4)
14.0
(8.1-18.0)
6.2
(4.3-8.4)
5580
(3200
7100)
2406
(1725-
2975)
6.0
(4.1-7.4)
2.74
(2.03.5)
0.18
(0.1-0.8)
18.3
(13-23)
2144
(1070
3116)
636
(522-827)
670
(483-1126)
204
(60.2-
287.6)
3
7.2
(6.8-7.5)
5.2
(2.6-95)
1493
(1000-
2050)
1.8
(1.2-2.5)
19.7
(15-24)
351.3
(242-490)
136.9
(67.8-
262.3)
4
7.6
(7.3-7.9)
8.5
(6.5-13.3)
3650
(3125-
5300)
4.2
(3.8-5.5)
57.6
(42-100)
1411.8
(902-2152)
289
(188-427)
5
7.7
(7.5-8.1)
9.5
(3.2-13.5)
2803
(875-4700)
5.3
(2.3-7.8)
12.9
(0.1-
27.4)
1465
(416-2348)
373.7
(114.7-
831.4)
6
7.7
(7.3-8.1)
12.3
(1-20.1)
5770
(2508775)
10.4
(7.2-
14.2)
3498
(1060
5460)
11339
(9528-
13960)
4956
(3747-
6165)
7
6.6
(65-6.7)
4.7
(4.6-48)
1175
(1100
1250)
2.75
(2.6-29)
10
792
NM
8
8.1
4.7
1200
2.4
31.9
444
85.4
(8-8.4)
(45-5.2)
(2.2-27)
(20-43)
(360-500)
(21.8-149)
9
7.4
3.9
2800
3.9
48
992
NM
10
6.5
39.6
350
31.7
52
2272
NM
11(a)
6.8
26.9
1400
16.8
67
2648
NM
11(b)
7.3
3.4
1800
2.5
31
2232
NM
11(c)
7.1
3.4
2900
3.6
66
852
NM
12
NMC
NM
NM
NM
NM
NM
NM
13
7.5
10.1
3450
3.8
42
1024
NM
14
7.9
(7.5-8.2)
4.7
(3.8-5.6)
2000
(1550
2450)
2.9
(2.03.9)
3838
(700
6975)
1260
Trm—:—
NM
Abbreviations: "TDS. total dissolved solids, “COD, chemical oxygen demand;BNM, not
measured.a one sample collected.

155
a low toxicity was shown which suggested that the metals were not present in a
bioavailable form.
Some physical and chemical characteristics of the sixteen MSW landfill
leachates are summarized in Table 5-3. The range of concentrations was typical
for MSW landfill leachates in Florida (Ward et al„ 2002). The pH values were
approximately neutral with mean values that ranged from 6.6 to 8.0. Alkalinity
concentrations varied by more than an order of magnitude, from 350 mg/L as
CaC03 at site 10 to 5770 mg/L as CaC03at site 6. COD, a surrogate parameter
for organic matter, ranged from 792 to >10,000 mg/L. The sulfide concentrations
in the leachates ranged from a mean of < 1 to 3838 pg/L. Leachate
characteristics are dependent on the waste composition, age, and environmental
conditions, and this was typified at site 11. There were three separate landfills
located at site 11; including an operating Class I MSW landfill (11a), a Class III
landfill, for non-putrescible wastes (11b) and a capped Class I MSW landfill
(11c). A similar chemical composition was reported for the leachates from sites
11b and 11c. The characteristics of the leachate from site 11 c were more closely
similar to 11b than to 11a.
Taken together, the leachates from sites 2, 3 and 8 represented different
phases of degradation in municipal waste materials from one landfill facility. The
leachates from site 2 represent an active landfill cell currently accepting waste,
the leachates from site 3 were from a capped MSW landfill cell, and finally site 8
represents a mixture of the leachates from sites 2 and 3, but predominantly the
leachates from site 2. The chemical characteristics of the leachates were very

156
similar, with a few exceptions. The sulfide concentrations in leachates from sites
2 and 3 were 18.3 and 19.7 pg/L, respectively, but at site 8 the concentration was
31.9 pg/L. While unexplained, this finding agrees with reports of low sulfate
concentrations in the leachates from site 8. Other leachate parameters for site 8
were within the range reported for the leachates from sites 2 and 3. Although the
toxicity of the leachates from sites 2 and 3 was low at less than 10 % inhibition,
the leachates from site 8 were even less toxic (mean inhibition = 2.5 %),
The greater toxicity and bioavailability of heavy metals in the leachates
from site 10 may be attributed to site-specific characteristics of the leachates
(Table 5-3). The quality of a landfill leachate directly impacts metal
bioavailability, and; hence, toxicity due to the modifying influences from alkalinity,
COD, TDS, and pH (Welsh et al., 1996). Reinhart and Grosh (1998) reported
that landfill leachates generated by the co-disposal of MSW ash and MSW are
typically characterized by low alkalinity, high TDS and high conductivity, which
agrees with the leachate characteristics at sites 10 and 11a. Reports of low
concentrations of organic materials in landfills containing a primarily ash-based
waste, translates into a greater influence of pH, buffering capacity and inorganic
ligands, e.g., sulfide and chloride, on heavy metal bioavailability. Insoluble metal
sulfides reduce the bioavailability of metals (Bozkurt et al., 2000).
Unique differences were demonstrated between the chemical
characteristics of the leachates from sites 10 and 11a (Table 5-3). The leachates
from site 10 displayed alkalinity, TDS, and conductivity concentrations of 350
mg/L as CaC03, 31.7 g/L, and 39.6 mS/cm, respectively. Underscoring the

157
influence of site specific parameters, alkalinity, TDS, and conductivity were 1400
mg/L as CaC03, 16.8 g/L, and 26.9 mS/cm, respectively, in the leachate from site
11a. The four times greater alkalinity at site 11a may explain the decreased
metal toxicity and bioavailability in this leachate when compared with the
leachate from site 10. The results of the chemical characterization suggested
decreased metal bioavailability with increasing leachate strength. One leachate
sample collected from site 6 displayed low toxicity, despite zinc concentrations of
0.44 mg/L in the leachate. This exceeded the reported MetPLATE EC50 of 0.38
mg/L. The chemical strength of the leachates from site 6 was high and
corresponded with a mean MetPLATE inhibition of 28.5 %. Comparatively, the
leachates from site 1 also displayed high concentrations of ions (as conductivity,
14 mS/cm), alkalinity (5,580 mg/L as CaC03) and TDS (6,000 mg/L), with a
comparable range of toxicity. Due to the lack of toxic response in nearly 25 % of
the MetPLATE assays, relationships between the MetPLATE toxicity and MSW
leachate parameters could not be determined.
HMBC of MSW Landfill Leachates
The bioavailability and toxicity of heavy metals in MSW leachates was
further characterized by determining the heavy metal binding capacity (HMBC) of
each leachate. HMBC quantifies the metal bioavailability/toxicity in aquatic
environments and is dependent on physicochemical parameters such as pH,
alkalinity, hardness, and the presence of complexing ligands (Huang et al.,
1999). The scope of the HMBC concept is similar to the water effect ratio (WER)
proposed by USEPA (USEPA, 1984). The USEPA recognized the relationship
between site-specific water quality parameters and metal bioavailability when

158
Table 5-4. Heavy metal binding capacity (HMBC) (unitless) of leachates from 16
MSW landfills with copper, zinc, and mercury. Results are presented
as the mean and (range) or the mean ± one standard deviation for
Site
Cu+2(as CuSO-t)
HMBCa
Zn*2 (as ZnCI2)
Hq+2 (as HgCI2)
Mean
Mean
Mean
1
54.5
25.7
30 5 + 2 4
(1.6-162.9)
(8.8-42.7)
2
15.8
32.2
NMC
(3.7-29.8)
(10.6-83.1)
3
12.3
27.9
NM
(0.5-24.1)
(3.1-64.3)
34.9
48.1
21.7
(7.2-56.2)
(6.2-115.7)
(16.7-26.7)
114.9
32.3
86.8
(7.9-327.9)
(8.8-101.1)
(85.2-88.3)
6
NM
93
NM
(0.9-218.1)
2 9
10.9
7
(2.7-3.1)
(10.7-11.1)
3.6 ±0.2
27.5
6.1
12.9
(1.1-79.7)
(1.4-10.7)
(6.8-19.1)
9
24.0 ±0.8
46.0 ±4.2
NMb
10
NM
NM
NM
11(a)
3.6 ±0.2
NM
NM
(b)
7.5 ±1.2
NM
NM
(c)
21.4 ±0.4
10.7 ±0.3
NM
12
59.7 ±3.6
45.2 ±9.9
100.8 ±8.4
13
70.7 ±2.3
12.9 ±0.5
NM
14
26.7
4.9
13 3+1 1
(9.9-43.5)
(38-5.9)
Lake Alice
1.1 ±0.1
1.8 ±0.3
2.8 ±0.1
Lake Beverly
<1
<1
<1
WWTP Effluent
" ai\ x: . á, it inr-
2.4 ±0.1
2.6 ±0.3
t7r—vr7r.—- * - ■ r
2.3 ±0.1
evaluating aquatic toxicity (USEPA, 1977). The WER protocol utilizes indigenous
species and incorporates site-specific water quality parameters, e.g., pH,

159
hardness, alkalinity, to derive acceptable maximum allowable total metal
concentrations (Welsh et al., 2000). Since the WER protocol relies on bioassays
with whole organisms, the tests are labor-intensive, expensive, and not easily
adapted to in-field testing.
The HMBC assay allows for the rapid determination of metal bioavailability
and toxicity (Huang et al., 1999). The protocol incorporates the microbial assay
MetPLATE to quantify the influence of site-specific water quality parameters on
metal bioavailability. The HMBC assay has been validated with surface waters,
and direct relationships were identified between water quality and the range of
HMBC.
Theoretically, the potential for complexation between heavy metals and
MSW landfill leachates is high, especially, when the chemical characteristics of
the MSW leachates are considered. Table 5-4 summarizes the HMBC of
leachates from sixteen MSW landfills using the heavy metals copper, zinc and
mercury. The HMBCs with the MSW landfill leachates were high and ranged
from 3 to 115, 5 to 93, and 4 to 101 for HMBC-Cu+2, HMBC-Zn+2, and HMBC-
Hg+2, respectively. The capacity for metal binding at each landfill varied widely for
the three metals. The MSW landfill leachate from site 5 displayed the highest
HMBC-Cu+2 (114.9), while leachates from sites 6 and 12 had the highest HMBC-
Zn+2 (93), and HMBC-Hg+2 (100.8), respectively. When multiple leachate
samples were collected from individual landfill sites, the HMBC results displayed
high variability. This was demonstrated by the leachates from site 5, which
displayed HMBC-Cu+2 values that ranged from 7.9 to 327.9. Although the

160
magnitude of the response was lower, the variability in the HMBC-Cu+2 values
with the leachates from site 1 was similar and ranging from 1.6 to 162.9. The
leachates from site 10 were not assayed for their HMBC due to the formation of
precipitates during preliminary testing. Based on the results of the MetPLATE
assay the HMBC of the leachate collected from site 10 was predicted to be low.
By comparison, the HMBC of the samples collected from Lake Alice and
Lake Beverly were low, with all values< 3. The HMBC-Cu+2 for the two lake
samples ranged from <1 to 1.1, while the HMBC-Zn*2 ranged from <1 to 1.8, and
the HMBC-Hg+2 ranged from <1 to 2.8. These HMBC-Cu+2 values were within
the range (<1 to 4.2) previously reported for surface waters (Huang et al., 1999).
Slightly higher HMBC values were measured in the WWTP effluent at 2.4, 2.6,
and 2.3 for HMBC-Cu+2, HMBC-Zn+2, and HMBC-Hg+2, respectively.
The HMBC results indicated a high capacity for binding to copper, zinc,
and mercury by the MSW landfill leachates, which was attributed to the chemical
characteristics of the leachates. Relationships between individual physico¬
chemical parameters and the capacity of the leachates to form complexes with
copper, zinc and mercury were not discernible. This was attributed to the
heterogeneous leachate composition and the wide range of chemical
characteristics in the MSW landfill leachates. The results suggested that HMBC
was heavily influenced by site-specific factors and that the physico-chemical
parameters relevant to binding at one site were insignificant at others.
Correlative analysis of the HMBCs with various chemical characteristics
revealed no significant relationships or general trends. Relationships were

161
investigated between HMBC and pH, alkalinity, conductivity, total dissolved
solids, sulfide, chemical oxygen demand, and hardness concentrations.
Comparisons revealed no relationships and the coefficient of determination (R2)
were all < 0.2. There was a trend between increasing chloride concentrations
and the increased binding of zinc by MSW landfill leachates (R2 = 0.7); however,
chloride had no influence on the binding of copper or mercury. From a broad
perspective, the large number of landfills investigated together with the frequency
of leachate collection and various site-specific conditions (e.g., rainfall, waste
composition, age and temperature) contributed to the failure to identify predictive
characteristics for toxicity in the leachates.
While there were no clear patterns between chemical characteristics and
HMBC, generally the leachates with the greatest strength displayed the highest
HMBC. Elevated concentrations of complexing agents, e g., organic and
inorganic ligands, contribute to the HMBC (Sletten et al., 1995). This was the
case with the leachates from site 6, which displayed high concentrations of
alkalinity, sulfide, and chloride. The high strength of the leachates from site 6
corresponded with the greatest mean HMBC for zinc at 218.1. Based on
knowledge of the leachates from site 6, the predicted binding capacity for copper
and mercury was high. However, these metals were not assayed with the
leachates from site 6. Instead, as discussed earlier, the highest HMBC-Cu+2
(114.9) and HMBC-Hg+2 (100.8) were measured at sites 5 and 12, respectively.
The chemical strength of the MSW landfill leachates from site 5 was on average

162
greater than other leachates, but the leachates from site 12 were not
characterized.
Interpreting relationships within the HMBC results was simplified when the
landfill sites were considered individually. Examining the HMBC results for the
leachates collected from site 6, revealed the strong influence of environmental
factors on the range of HMBC-Zn+2 values. The mean HMBC-Zn*2 with the
leachates from site 6 was 93; however, this represented a range from 0.88 to
218.1. Preceding leachate collection in July 2000, a large rain event caused a
dramatic dilution in leachate strength. A similar dilution effect was noted when
evaluating the acute and chronic toxicity of the leachates from site 6 (Chapter 3).
These results underscore the importance of site-specific parameters on the
overall magnitude of HMBC for each MSW landfill leachate. The chemical
strength of MSW leachates in Florida has been described as more dilute than
that reported for other MSW leachates, which reduces the overall potential for
heavy metal binding (Reinhart and Grosh, 1998).

163
a Site 1 â–  Site 5
75 i
'00 '00 '00 '00 '00 '00 '00 '00 '00 '00 '00 '01 '01 '01 '01 '01
•indicates sample inhibition = 0; N = sample not tested
Figure 5-4. MetPLATE results for MSW landfill leachates collected from site 1
and site 5 over time with results presented as the mean ± one standard
deviation and dashed line representing the 50% inhibition level
Leachate toxicity as a function of time
Temporal influences on heavy metal toxicity in leachates from sites 1 and
5 were evaluated, and the results are presented in Figure 5-4. According to the
results of the MetPLATE assay, the toxicity of the leachates collected from site 1
remained low throughout the year with a range of inhibition from 0 to 36.8 %.
Similar results were shown for the MSW leachates from site 5 with one
exception. The leachate collected from site 5 in January 2001 caused an
inhibition of 61.7 % in the MetPLATE assay. There were no trends in the
MetPLATE results for heavy metal toxicity versus time with the leachates from

164
Table 5-5. MetPLATE and HMBC results with MSW landfill leachates collected
from sites 1,4,5, and 8. Results are presented as the mean ± one
standard deviation
Site
MetPLATE
(% Inhibition)
HMBCa
Cu**
(as CUSO4)
Zn¿
(as ZnCh)
Hg~
(as HgCI2)
1
0.9 ±3.8
162.9 ± 13.0
17.1 ±0.7
30.5 ±2.4
4
7.3 ±5.7
41.4 ±2.1
6.2 ±0.4
26.7 ±1.5
5
0
300.2 ± 35.8
22.3 ±0.2
88.3 ±6.3
8
0
79.7 ±6.9
6.1 ±0.2
19.1 ±0.5
aHMBC, heavy metal binding capacity (unitless).
sites 1 and 5, which agreed with the results of the acute and chronic assays with
these leachates (Chapter 4).
Influence of Selected Leachate Parameters on HMBC
The HMBC results obtained for the leachates collected from the sixteen
lined MSW landfills raised questions related to the influence of physico-chemical
parameters on HMBC. Table 5-3 shows strong variability in the physicochemical
parameters of the MSW landfill leachates collected from the sixteen Florida sites.
As previously discussed, this variability made it difficult to observe relationships
between HMBC and individual parameters. Therefore, additional leachate
samples collected from sites 1,4,5, and 8 were subjected to a partial
fractionation. The effect of some leachate parameters (solids, organics and
hardness) on the binding capacity of these MSW leachates for copper, zinc and
mercury were Investigated.
Table 5-5 presents the MetPLATE and HMBC results obtained with these
four additional leachate samples. The MSW landfill leachates form sites 1, 5,
and 8 were not toxic (inhibition <1 %), but the leachate from site 4 was slightly

165
Table 5-6. HMBC of MSW landfill leachates with copper, zinc, and mercury
following fractionation with results expressed as the mean ± one
standard deviation
Treatment
Site 1
Site 4
Site 5
Site 8
HMBC-Cu*2
Whole
162.9 ±13.0
41.4 ±2.1
300.2 ± 35.8
79.7 ±6.9
Post-filtration
104.9 ±4.4"
51.1± 3.6aa
140.4 ±8.9”
29.2 ±1.9"
Post-DEAE
65.1 ±8.7"
28.4 ± 2.2"
211.8 ±15.4aa
24.3 ± 3.6
Post-Dowex
51 ± 8.4
97.1 ±16.9 33
104.0 ± 15.2”
6.8 ±0.1”
HMBC-Hg*2
Whole
30.5 ±2.4
26.7 ± 1.5
88.3 ±6.3
19.1 ±0.5
Post-filtration
17.2 ±0.7"
33.7 ±0.9“
26.1 ±2.5"
6.8 ± 0.2”
Post-DEAE
12.4 ±2.4"
43.7 ± 1.2a3
76.0 ±1.033
7.8 ± 0.633
Post-Dowex
11.1 ±0.1
38.3 ± 0.9"
60.0 ±1.0"
9.0 ±1.0
HMBC-Zn+2
Whole
17.1 ±0.7
6.2 ±0.4
22.3 ±0.2
6.1 ±0.2
Post-filtration
11.9 ± 0.1”
9.3 ± 0.333
14 ±0.7"
5.9 ± 0.4
Post-DEAE
10.2 ±0.2"
7.3 ±0.1"
18.6 ± 0.433
3.5 ± 0.2"
Post-Dowex
8.4 ± 0.5"
26.9 ±1.6a3
15.2 ±0.6”
2.7±0.1”
Relative changes in HMBC for each metal were designated by for significant reductions at p <
0.05 and 38 for significant increases at p < 0.05, based on comparison to the HMBC result of the
previous treatment
toxic with 7.3 % inhibition. The magnitude of the binding capacity for each metal
with the four leachates differed markedly. HMBC values ranged from 41.4 to
300.2,19.1 to 88.3, and 6.1 to 22.3, for copper, mercury and zinc, respectively.
The heavy metal binding capacity of the four leachates followed the order of
HMBC-Cu+2 > HMBC-Hg+2>HMBC-Zn*2. This trend resulted from the high
affinity of organic substances for free copper ions. Unlike mercury and zinc ions,
the copper ions form strong complexes with both humic and fulvic acids (Weng et
al., 2002; Calace et al., 2001). Therefore, more copper relative to zinc and
mercury is complexed. Overall, the MSW landfill leachates from site 5

166
Table 5-7. Changes in physical and chemical characteristics during fractionation
of the MSW landfill leachates from site 1,4,5, and 8
Site 1
Site 4
Site 5
Site 8
Whole Leachate
pH
8.0
7.7
7.8
7.9
Conductivity
8.1
6.1
10.8
5.2
(mS/cm)
Alkalinity
3200
2500
4700
1300
(mg/L as CaC03)
Hardness
340
370
360
270
(mg/L as CaCQj)
DOC
1126
368.0
332.1
149.1
Total Solids
(mg/L)
4100
-
7800
2850
Post-Filtration
pH
8.1
7.8
8.0
8.0
Conductivity
8.2
7.2
11.1
5.2
(mS/cm)
Alkalinity
3200
2700
4700
1700
(mg/L as CaC03)
Hardness
210
360
340
190
(mg/L as CaC03)
DOC
1130
304.9
377.2
131.4
Total dissolved solids
(mg/L)
4070
-
7750
2540
Post-DEAE
pH
8.4
8.3
8.5
8.3
Conductivity
8.0
7.7
11.5
5.5
(mS/cm)
Alkalinity
3100
1300
4300
900
(mg/L as CaC03)
Hardness
140
300
320
190
(mg/L as CaC03)
DOC
(mg/L)
299.4
193
466.9
81.5
Post-Dowex
pH
7.1
6.3
7.9
6.9
Conductivity
6.5
5.5
11.5
4.1
(mS/cm)
Alkalinity
625
500
2625
250
(mg/L as CaC03)
Hardness
67
61
110
30
(mg/L as CaC03)
DOC
(mg/L)
330.5
450
686.1
107

167
displayed the highest capacity for binding with heavy metals, and this was
followed by leachates from sites 1, 8, and 4.
The fractionation of the MSW landfill leachates collected from sites 1,4,5,
and 8 began with membrane filtration (0.45 pm), continued with passage through
an anion exchange column (DEAE cellulose) and then passage through a cation
(Dowex) exchange column. The treatments produced statistically significant
reductions (p <0.05) in the binding capacity of the leachates for the metals, Cu+2,
Zn+2 and Hg+2 (Table 5-6). Researchers have shown the effectiveness of the
DEAE cellulose resins for the recovery of humic substances (Yamada et al.,
2000).
The filtration of the MSW landfill leachates removed coarse (> 0.45 pm),
but not the colloidal particles (< 0.1pm). In some cases, 40 to 90% of free metal
ions are associated with colloidal solids (Guo et al., 2002), and this increases the
mobility of heavy metals (Roy and Dzombak, 1997). Table 5-7 summarizes
chemical characteristics of the MSW landfill leachates, and the changes in these
characteristics following each of the fractionation treatments. Filtration reduced
the solids content of the leachates by approximately 11 % at site 8, and 5 % at
site 4, but by < 1 % at sites 1 and 5. This agrees with reports of low
concentrations of suspended solids (< 20 %) in MSW landfill leachates (Jensen
and Christensen, 1999; Chian and DeWalle, 1976). Following the 11 %
reduction in the solids content of the leachate collected at site 8, there were large
decreases in both HMBC-Cu+2and HMBC-Zn+2, which were roughly 60 %. The
same leachate showed a HMBC-Hg+2 decrease of less than 3 %. This

168
suggested that in the leachate from site 8 the characteristics important to the
binding of copper and zinc were associated with solids, while mercury binding
was dependent on other factors.
Overall, filtration of the MSW landfill leachates yielded the greatest
reductions for HMBC with copper, zinc, and mercury (Table 5-7). Despite low
solids removal (< 1 %) in the MSW landfill leachates from sites 1 and 5, the
HMBC for copper, zinc, and mercury were significantly reduced (p < 0.05). In
fact, in the leachates from sites 1 and 5 the HMBC-Cu+2, HMBC-Zn+2, and
HMBC-Hg*2 were reduced by 36 and 53 %, 44 and 70 %, and 30 and 37 %,
respectively. Although filtration reduced the solids content of the leachates from
site 4 by 5 %, there were no concurrent reductions in the HMBC for copper, zinc,
or mercury. Instead, the HMBC for each of the metals increased after filtration.
With the site 4 leachate the HMBC increased (24 to 50 %) for each of the metals,
which suggested that during filtration of the leachate the composition of the was
altered. Basically, the results reported for the effect of filtration on HMBC are
consistent with reports of high associations between particulate materials and
metal ions (Guo et al., 2002; Jensen and Christensen, 1999; Gounaris et al.,
1993).
In both terrestrial (Parat et al., 2002; Ge et al., 2002) and aquatic (Voelker
and Kogut, 2001; Benedetti et al., 2002) environments, the reduced
bioavailability of heavy metals in the presence of organic matter has been
reported. Roughly 40 to 70 % of organic matter is humic in nature (Weng et al.,

169
2002; Masion et al., 2000), and the size of these humic ligands has a strong
influence on metal mobility (Kang et al., 2002). Schroth and Sposito (1998)
suggested that in the presence of clay solids, humic substances display an
increased affinity for heavy metals. Clay is an integral part of many landfill liner
systems; therefore, clay materials may become associated with the waste
materials.
Fractionation showed that the organic matter content of landfill leachates
was an important determinate of HMBC. High concentrations of dissolved
organic matter have been reported in MSW leachates (Calace et al., 2001), and
metal complexation by this organic fraction has been extensively reported
(Kaschl et al., 2002). These organo-metallic complexes are highly resistant to
biodegradation (Parat et al., 2002) resulting in a slower breakdown of organic
matter in landfills. The formation of complexes with organic materials increases
the mobility of metals (Barlaz et al., 2002). In unlined landfills, these complexes
facilitated the transport of metals to groundwater. Castagnoli et al. (1990)
reported low concentrations of organic matter in leachate-contaminated soils
beneath unlined landfills, and this was attributed to the rapid degradation of
organo-metallic complexes. Organo-metallic complexes are rapidly degraded in
carbon-poor environments, which results in the release of metals (Bozkurt et al.,
2000).
Passage of the MSW landfill leachates from sites 1,4,5, and 8 through
the DEAE resin columns reduced DOC concentrations by 73, 48, 2.7, and 45 %
respectively (Table 5-7). Visual observations and the organic characteristics of

170
the MSW landfill leachates confirmed the presence of humic substances.
Dissolved organic carbon (DOC) concentrations are used to describe humic
substances in MSW landfill leachates (Smith and Weber, 1990). The leachate
collected from site 1 showed the highest association between the presence of
organic substances and HMBC for copper, zinc, and mercury. When the DOC
was reduced by 73 %, there were corresponding reductions of 24, 16 and 10 %
for HMBC-Cu*2, HMBC-Hg*2 and HMBC-Zn*2, respectively (Table 5-6). The
leachate from site 1 represents a closed and capped landfill unit and the
concentrations of humic substances in closed landfills are expected to increase
with age and waste degradation (Castagnoli et al., 1990). The association
between copper binding and organics removal was probably due to the affinity of
copper for dissolved organic matter (Calace et al., 2001), specifically humic
materials (Weng et al., 2002). The formation of organo-copper complexes in
MSW landfill leachates reduces copper toxicity (Fraser et al., 2000; Palmer et al.,
1998). This same property has been widely reported in aquatic systems, albeit at
greatly reduced DOC concentrations (30.3 mg/L)(Winch et al., 2002).
After the MSW landfill leachate from site 5 was passed through the DEAE
resin, the HMBCs for copper, zinc, and mercury did not decrease. Instead, the
HMBC-Cu+2 and HMBC-Hg*2 nearly doubled and the HMBC-Zn*2 increased by
roughly 25 %. This increased binding was unique to this sample and the DEAE
treatment, but the low removal of DOC (2.7 %) was probably an influential factor.
Based on these results, there was no association between the removal of anionic
species by the DEAE resin and the HMBC for the three heavy metals.

171
Competition between the organic material and other anionic species, e.g.,
chloride and carbonate ions, for binding sites on the DEAE resin may have been
responsible for the low DOC removal. The organic content of the leachate from
site 8 was reduced by 45 % after DEAE treatment, and there was a
corresponding 41 % reduction of the leachates HMBC for zinc. The HMBC-Cu+2
decreased, but not significantly, and the HMBC-Hg*2 increased by 15 %.
Metal toxicity is strongly influenced by the presence of dissolved organic
matter (DOM) and its ability to complex heavy metals (Heijerick et al., 2003;
Richards et al., 2001; Kim et al., 1999). In MSW landfill leachates, organic
substances occur predominantly in two distinct molecular weight regions, one of
low molecular weight (< 3000 Dalton) and a second of high molecular weight (>
10,000 Dalton). Surprisingly, in both municipal and industrial waste leachates
the distribution of the organic matter in these regions is similar (Smith and
Weber, 1990). Overall, the composition of humic substances in the landfill
depends on various environmental factors like rainfall and temperature, but also
on the type of waste materials and more importantly landfill age (Kang et al.,
2002). The molecular weight of organic compounds in leachates increases with
time (Calace et al., 2001). From its influence on humic structure, pH regulates
metal mobility (Martensson et al., 1999). As the pH increases, the deprotonation
of humic substances causes conformational changes, and lowers the affinity of
humic substances for metal ions (Masion et al., 2000).
Humic substances are complex organic molecules, and due to this
complexity, the structure of these molecules is poorly characterized. The

172
functional group on the humic material determines the relative binding affinity for
metal ions. Generally, stronger metal binding is associated with the functional
groups containing nitrogen and sulfur, rather than those containing phenolic and
carboxylic functional groups (Calace et al., 2001; Stumm and Morgan, 1995).
This property has been demonstrated for humic substances in MSW landfill
leachates (Parat et al., 2002; Croue et al., 2003). The high nitrogen content of
the leachates contributes to stronger binding (Burton and Watson-Craik, 1998).
For comparison, the nitrogen content of MSW landfill leachates is often
measured at concentrations five times greater than in the aquatic environments
(Kang et al., 2002).
The MSW landfill leachates at sites 1,4 and 5 contained similar hardness
concentrations at 340, 370 and 360 mg/L, respectively, while at site 8 hardness
was slightly lower at 270 mg/L (Table 5-7). Hardness is a measure of the cations
in solution, and the most prevalent hardness cations, in both the environment
and MSW landfill leachates, are calcium and magnesium. In the leachates from
sites 1,4,5, and 8, the calcium concentrations were generally greater than or
equivalent to the magnesium concentrations, which is relevant since the calcium
ions are stronger competitors than magnesium for metal binding sites. The
cation exchange resin (Dowex) reduced hardness concentrations by 52, 80, 66,
and 84 % in the MSW landfill leachates from sites 1,4,5, and 8, respectively
(Table 5-7).
Unique differences were demonstrated for the role of hardness on metal
toxicity in the MSW landfill leachates. While a large fraction of the total hardness

173
was reduced, the results of the metal toxicity were highly variable. The strongest
relationship between the hardness concentrations in the leachates and HMBC
was demonstrated for the leachate from site 8. In this leachate, an 84 %
reduction in hardness coincided with a 22 % reduction in the capacity of the
leachate from site 8 to bind with copper. Under the same conditions, the
leachate from site 8 displayed no association between hardness concentrations
and the binding of mercury, but there was a 12 % decrease in the ability of the
leachate to bind zinc (Table 5-6).
Considering the MSW landfill leachates from sites 1 and 5, hardness was
an important factor to the binding of copper, mercury, and zinc. In the leachates
from site 1, the removal of 52 % of the hardness cations reduced the HMBC of
the leachates by 9, 4, and 10 %, respectively. An in-depth analysis of the
leachates from site 5 revealed that 10 to 20 % of the metal binding capacity was
explained by the removal of 66 % of the hardness cations. According to the
HMBC assay, the capacity of the site 5 leachates to bind with copper, mercury,
and zinc was reduced by 12,18, and 15 %, respectively.
The results with the leachate from site 4 are more difficult to interpret.
Despite an 80 % reduction of hardness cations, the leachates from site 4
exhibited significant increases for binding capacity (decreased toxicity) for both
copper and zinc. Specifically, the HMBC of the leachate for copper and zinc ions
increased from 28.4 to 97.1 and from 7.3 to 26.9, respectively. There was a
small, but significant, decrease in the binding of the site 4 leachate for mercury
from 43.7 to 38.3.

174
Knox and Jones (1979) demonstrated that the formation oforgano-
metallic complexes was not influenced by the presence of hardness cations. In
contrast, other researchers have reported strong competition between metal
cations and the predominant hardness cations (calcium and magnesium)
(Morgan and Stumm, 1991). Bozkurt et al. (2000) showed that MSW landfills
contained sufficient binding capacity for the removal of typical concentrations of
heavy metals, but they acknowledged that high concentrations of hardness
decreases the metal binding capacity, as calcium and magnesium compete with
metal ions. Since hardness cations may be strongly associated with colloidal
solids, the filtration of the leachates probably removed a large portion of this
binding capacity (Park et al., 1999). This was demonstrated by a roughly 30%
decrease in total hardness concentrations for site 1 and 8 after filtration, but for
sites 4 and 5 only a 3 to 5 % decrease was reported. Although to differing
degrees, the DOC concentrations of the MSW landfill leachates increased
following cation exchange treatment. This increase was unexpected and
probably an artifact of the resin, caused by insufficient washing of the Dowexâ„¢
material.
Other landfill characteristics influence metal speciation and bioavailability.
For example, the aerobic/anaerobic profile in a landfill directly regulates metal
mobility. Landfills are operated to maximize anaerobic conditions for the
stabilization of waste materials during acidic, methanogenic and humic phases of
degradation. Bioreactor landfills, that maximize aerobic conditions for waste

175
Table 5-8. Coefficients of determination (R2) obtained between MSW landfill
leachate characteristics and the heavy metal binding capacity (HMBC)
for copper, mercury, and zinc with strong relationships in bold
HMBC-Cu*2
HMBC-Hg*2
HMBC-Zn*2
Pre-filtration
TSa
0.89
0.99
0.78
Alkalinity
0.78
0.82
0.84
DOCb
0.02
0.01
0.17
Hardness
0.09
0.23
0.18
Post-filtration
TDSC
0.84
0.14
0.77
Alkalinity
0.92
0.27
0.93
DOC
0.24
0
0.26
Hardness
0.1
0.89
0.21
Post-DEAE
Alkalinity
0.82
0.38
0.91
DOC
0.67
0.43
0.88
Hardness
0.26
0.82
0.25
Post-Dowex
Alkalinity
0.42
0.72
0.03
DOC
0.89
0.88
0.38
Hardness
0.67
0.71
0.14
Abbreviations: °TS, total solids; “DOC, dissolved organic carbon; CTDS, total dissolved solids.
Total solids were determined after drying at 105°C and total dissolved solids after filtration and
drying at 105°C.
stabilization, are a relatively new concept (Reinhart and Townsend, 1998). Some
researchers have hypothesized that traditional anaerobic landfill conditions
transition to aerobic during the late humic phase of waste stabilization (Bozkurt et
al., 2000). Under these aerobic conditions metal mobility and bioavailability
increase (Martensson et al., 1999; Van Ryssen et al., 1998).
General tendencies in the direction of the relationships between physico¬
chemical parameters in the MSW landfill leachates from sites 1,4, 5, and 8 and
the HMBC for copper, zinc, and mercury were evaluated by pooling the data from
the four sites. Table 5-8 summarizes the coefficients of determination that were

176
obtained. In the whole (before filtration) MSW landfill leachates the total solids
content made the highest contribution to the metal binding capacity, with R2
values of 0.89, 0.99, and 0.78 for HMBC-Cu*2, HMBC-Hg*2, and HMBC-Zn*2,
respectively. The removal of the suspended solids from the MSW landfill
leachates by filtration (0.45 pm) did not impact the contribution of solids to the
HMBC for copper (R2 = 0.84) and zinc (R2 = 0.77). In contrast, the removal of
suspended solids decreased the relationship (R2 = 0.14). Strong associations
were identified between anionic species, e.g. alkalinity and DOC, and HMBC-
Cu+2 and HMBC-Zn+2, but were lower for HMBC-Hg*2. There were direct
relationships between the removal of alkalinity from the MSW landfill leachates
and decreased HMBC-Cu*2 (R2 = 0.82) and HMBC-Zn*2 (R2 = 0.91). A lower
relationship was demonstrated between the removal of alkalinity and HMBC-Hg+2
(R2 = 0.38). With the reduction of DOC in the MSW landfill leachates, the
association with HMBC-Zn*2 (R2 = 0.88) was high. The relationships with HMBC-
Cu+2 (R2 = 0.67) and DOC was lower than with the other anionic parameter,
alkalinity. The association between DOC and HMBC-Hg*2 (R2 = 0.43) was low
and comparable to that previously shown with alkalinity. The relationships
between hardness and the heavy metal binding capacity with copper, zinc, and
mercury diverge from those previously stated with the other leachate parameters.
While the HMBC-Cu*2 (R2 = 0.67) remains strong, there was no relationship with
HMBC-Zn+2 (R2 = 0.14) and hardness removal. Instead, the HMBC-Hg*2 (R2 =
0.71) displayed the highest association with hardness.

A.
D.
G.
Solids
63%
B.
Solids
44%
Other
36%
Hardness Organics
4% 16%
E.
H.
Solids
58%
Other
42%
c.
Hardness 65%
11%
Solids
Hardness
12%
Organics
41%
Figure 5-5. Effect of leachate treatment by filtration (Solids), DEAE resin (Organics), and Dowex resin (Hardness) on the
HMBC. A. Site 1 with Cut2, B. Site 1 with Hg+2, C. Site 1 with Zn+2; D. Site 5 with Cu*2, E. Site 5 with Hg+2, F.
Site 5 with Zn+2; G. Site 8 with Cu+2, H. Site 8 with Hgt2,1. Site 8 with Zn+2
177

178
The binding affinity for the MSW landfill leachates with heavy metals
followed the order of copper > mercury >zinc. This agrees with the results of the
correlative analysis, which demonstrated that the binding of copper with the
MSW landfill leachates was strongly associated with total solids, total dissolved
solids, DOC, alkalinity, and hardness. Although there was a strong association
between the binding of the MSW landfill leachates for mercury and total solids
(R2 = 0.99), the removal of the suspended solids reduced this relationship (R2 =
0.14). Furthermore, there were only weaker relationships between DOC (R2 =
0.43) and alkalinity (R2 =0.38) with the binding capacity for mercury. However,
the hardness (R2 = 0.71) levels in the MSW landfill leachates were correlated
with HMBC-Hg+2. Comparatively, the association between zinc binding and total
solids content of the MSW landfill leachates was the lowest for the three heavy
metals. Zinc binding was strongly associated with alkalinity (R2 = 0.91) and DOC
(R2 = 0.88) in the MSW landfill leachates, but not with hardness (R2 = 0.14). In
summary, the HMBC of the MSW landfill leachates was high, attributable to
various chemical characteristics of the leachates, and was not completely
explained by the investigated characteristics (Figure 5-5).

CHAPTER 6
IDENTIFYING TOXICITY IN FLORIDA MSW LANDFILL LEACHATES WITH A
TOXICITY IDENTIFICATION AND EVALUATION (TIE) PROCEDURE
Introduction
In 1991, the United States Environmental Protection Agency
(USEPA) published a series of methods for the determination of aquatic
toxicity. These methods combined chemical and physical fractionation
procedures with biological assays (USEPA, 1991a). Initially, these methods
were developed as a tool for wastewater treatment plant operators, and
were used in the identification of pre-treatment strategies prior to effluent
discharge. The goal of these methods was to minimize the potential for
adverse impacts to aquatic species in receiving waters.
When investigating aquatic toxicity, effluent dischargers are required
to conduct a three-phase toxicity reduction evaluation (TRE) to explore
options for the reduction or removal of target compounds. The initial or
Phase I segment of the investigation requires the physical/chemical
characterization of the suspect sample, while during Phase II options are
explored for the removal of the toxic substance or the identification of it's
source. Finally, Phase III monitors toxicity using chemical analysis and
biological assays.
179

180
The first phase (Phase I) of the TRE describes the protocols for a toxicity
identification and evaluation (TIE), which has three distinct components. The TIE
Phase I includes methods for the characterization of the suspected toxic
substance(s) (USEPA, 1991a). Phase II describes methods for the identification
of toxic substance(s), and is limited to non-polar organics, ammonia, and heavy
metals (USEPA, 1991b). Phase III confirms the results obtained during the
Phase I and Phase II procedures (USEPA, 1991c). Phase I investigations
combine the chemical and physical characterization of a sample with the results
of biological assays to identify the sample fractions with altered toxicity profiles.
These fractionation procedures include pH adjustment, aeration, filtration, and
solid phase extraction (SPE).
The TIE is a powerful tool for characterizing and identifying toxicants as it
combines the chemical and physical manipulation of samples with the evaluation
of biological effects. Therefore, the bioavailability of a toxicant can be used as an
indicator of the form and species of a substance present. The various
manipulations separate the classes of toxicants into distinct biologically available
forms. While the results of a TIE provide an insight into the types of toxic
substances present, it is a complex procedure that requires a large investment of
time and resources.
The selection of toxicity test organism for use in a TIE may be a crucial
factor in the evaluation procedure. Several factors must be considered when
selecting the test organism including volume of sample required, cost
effectiveness, easily quantified endpoint, and the availability of large numbers of

181
test organisms at the initiation of the assay (Coombe et al., 1999). The aquatic
invertebrate, Ceriodaphnia dubia, is uniquely suited for use in TIE investigations
(Norberg-King et al., 1991) due to its sensitivity, population structure (asexual
reproduction), and ease for culturing (USEPA, 1993a). The TIE procedure has
been applied to the monitoring of ambient water quality (Stronkhorst et al., 2003),
and for assessing the effectiveness of remediation technologies (Deanovic et al.,
1999). Recently, the TIE has been expanded for use as a tool for identifying
substances capable of inducing hormone-like effects in domestic wastewaters
(Desbrowet al., 1998) and surface waters (Thomas et al., 2001).
Municipal solid waste (MSW) landfill leachates are a byproduct of
waste stabilization processes, and their composition is complex with high
concentrations of both organic and inorganic substances (Kjeldsen et al.,
2002). These leachates are generated as rainfall infiltrates the landfill and
percolates through the waste materials. Modern landfills are lined and
contain systems for the collection of leachates, which are then treated at on¬
site facilities or more frequently at off-site domestic wastewater treatment
plants (WWTP). Therefore, toxic substances in leachates have the potential
to disrupt biological treatment processes, and may impact the quality of
WWTP effluents. This makes the MSW landfill leachates a prime candidate
for a TIE investigation; however, the heterogeneous chemical composition
and variable toxicity must be taken into consideration when designing the
fractionation protocol (Ward et al., 2002).

182
Table 6-1. Population served and amount of waste landfilled, as a percent of
total waste generated, at sites 7, 8, and 14
Site
Population
Landfilled Waste
(tons) (% of total)
7
470,000
380,000 (57 %)
8
115,000
160,000 (69 %)
14
421,000
525,000 (70 %)
The objective of the present work was to tentatively identify the class or
classes of toxic substances in MSW landfill leachates collected from three sites
in Florida. The toxicity of the whole MSW leachates was evaluated with both
acute and chronic assays. The results of these assays served as a basis for the
subsequent chemical/physical fractionation. During the TIE fractionation, the 24-
hr Ceriodaphnia dubia and 15-minute Microtoxâ„¢ bioassays were utilized to
measure the relative changes in leachate toxicity.
Material and Methods
Sample Collection
As part of a larger research investigation, MSW landfill leachates were
collected from three engineered landfill sites located in Florida, USA. The
selected leachates represent a typical cross-section of leachate quality in Florida.
The leachates were collected at various MSW landfills across the state of Florida.
Specifically, the leachates from site 7 were collected from a MSW landfill in
southern Florida, while the leachates from sites 8 and 14 were from MSW
landfills in western and eastern Florida (Table 6-1). A 3-L sample of MSW landfill
leachate was collected from the leachate collection sump at sites 7 and 8 using a
Teflon baler; however, at site 14 a collection sump was not accessible.

183
Therefore, the leachate from site 14 was collected from a discharge pipe that
transported the leachate to an evaporation pond. By holding the individual
sampling containers under the pipe, only leachate was collected and not
rainwater that had mixed with the leachate in the evaporation pond. Samples for
chemical analysis were collected in polyethylene or glass containers and
preserved according to U.S. Environmental Protection Agency (USEPA)(USEPA,
1993b) protocols, while samples for toxicity analysis were collected in plastic
containers and were not preserved. All samples were transported to the lab on
ice and immediately stored at 4°C. All biological assays were initiated upon
arrival of the sample in the laboratory.
Chemical Analysis of MSW Landfill Leachates
The three MSW landfill leachates were characterized for their
chemical and physical composition. Field measurements included pH and
temperature (Orion, Model 290A), conductivity (Hanna Instruments, Model
H19033), dissolved oxygen (DO)(YSI Inc., Model 55/12 FT), and
oxidation/reduction potential (ORP) (Accumet Co., Model 20). In the
laboratory samples were analyzed for alkalinity, carbonaceous biochemical
oxygen demand (CBOD), total dissolved solids (TDS), and sulfides,
according to methods described by USEPA (1993b) and APHA (1999).
Ammonia was measured with an ion selective electrode (ISE, Orion)
according to the manufacturer's instructions. The ISE measures total
ammonia (NH4VNH3) concentrations following the adjustment of leachate

184
Phase I Manipulation
Suspected Toxicant(s)
pH adjustment
lonizable compounds
Filtration
Particle-associated
compounds
Solid phase extraction
(SPE)
Non-polar organic
compounds
Aeration
Volatile or oxidizable
compounds
MetPLATE
Heavy metals
pH to 11 with 5N NaOH, which converts any ionized ammonia species
(NH4+) to the un-ionized (NH3) form. The ISE was calibrated before each
use with a five- point calibration curve. According to the manufacturer, the
un-ionized (NH3) ammonia concentration could be determined with the ISE
prior to the pH adjustment to 11 (Orion technical support, personal
communication). Samples for total metal content were digested (USEPA
Method SW-846 3010A)(USEPA, 1996) and analyzed by atomic emission
spectroscopy (AES) (Thermo Jarrell Ash, Model Enviro 36). Dissolved
oxygen (DO, YSI Inc., Model 55), pH and temperature (Accumet, Fisher
Scientific, Model 15), and electrical conductivity (Hanna Instruments, Model
H19033) were monitored in the P. subcapitata and C. dubia assays.

185
TIE Procedure: Phase I
The initial phase (Phase I) of the TIE protocol required the fractionation of
the MSW landfill leachates by chemical and physical manipulations for the
characterization of toxic substances (Table 6-2). The leachate fractions were
generated by sample manipulations. The manipulations included: pH adjustment
to 3 (pH 3/ADJ) and 11 (pH 11/ADJ), pH adjustment and filtration (pH 3/FILT and
pH 11/FILT), pH adjustment and aeration (pH 3/AER and pH 11/AER), and solid
phase extraction (SPE) (pH 3/SPE and pH 9/SPE) (USEPA, 1991b). The integrity
of the SPE columns may be compromised at pH values greater than 10;
therefore, the pH 11 leachates were adjusted to a pH value of 9 (USEPA,
1991b). Simultaneously, a third portion of each MSW landfill leachate was also
manipulated as described for the pH 3 and pH 11 fractions. However, the pH of
this portion was maintained at the pH value in the leachate upon collection and
was subsequently referred to as the initial pH (pH¡) of the leachates. The MSW
landfill leachates from sites 7, 8, and 14 were assayed for their initial toxicity
upon arrival in the laboratory and an 80-ml aliquot of each MSW landfill leachate
was set aside for baseline toxicity testing on day 2.
pH-adjustment of the MSW landfill leachates
The MSW landfill leachates from sites 7, 8, and 14 were subdivided
into three aliquots for the pH adjustment manipulation. The initial pH values
for the leachates were determined as 6.9, 7.6, and 7.4 for sites 7, 8, and 14,
respectively. A 305-ml aliquot of leachate from each of the three sites was
raised to pH 11 with 1N NaOH, a 605-ml aliquot was held at the initial pH

186
(pHi), and a third aliquot of 305-ml was lowered to pH 3 with 1N HCI. A 35-
ml aliquot of the pH 11-adjusted leachate and the pH 3-adjusted leachate
were set aside for the post-pH adjustment toxicity test. The remaining pH
11 and pH 3 leachates from sites 7, 8, and 14 were divided into a 35-ml
aliquot for the pH-adjusted aeration manipulation and 235-ml aliquots for the
pH-adjusted filtration step. The MSW landfill leachates at pH, were
separated into two aliquots of 35-ml for the aeration and 570-ml for filtration
manipulation.
Filtration of the MSW landfill leachates
Solids-associated toxicity may be removed by filtration of the MSW
landfill leachates. The leachate filtration utilized membrane filters (0.45um,
Gelman) pre-conditioned with pH 11, pH¡, or pH 3-adjusted DDI (Nanopure,
Barnstead). The pH-adjusted (pH 11, pH initial, and pH 3) leachates from
the three sites were filtered under vacuum (2 ml/min). Following filtration,
the pH 1 land pH 3 leachates were divided into 35-ml aliquots for the post¬
filtration toxicity test and 200-ml aliquots for passage through the SPE
columns. The pH, leachates were also separated for the post-filtration
toxicity test (35-ml), a 200-ml aliquot for SPE treatment, and a 335-ml
aliquot for Zeolite treatment.
Solid phase extraction of the MSW landfill leachates
Solid phase extraction techniques were employed for the removal of
non-polar organic toxicants from the leachates. The SPE columns (C-18,
500mg, EXTRA-SEP, Lida Manufacturing, Bensenville, CO) were placed on
the SPE reservoir (Visiprep 12 port, Supelco, Bellefonte, NY). The SPE

187
reservoir was attached to a vacuum with a flow rate of 0.5ml/min. The solid
phase extraction columns were conditioned with ~ 6 ml of methanol. The
methanol was loaded onto the column and following conditioning the
columns remained saturated. The integrity of the SPE column is sensitive
to pH values greater than 10; therefore, the pH 11 sample was adjusted to a
pH value of 9 (USEPA, 1991b). After conditioning, each of the SPE
columns was washed with pH-adjusted (pH 9, pH¡, or pH 3) DDI water, as
indicated. The pH 9, pH¡, and pH 3-adjusted leachates were loaded onto
their respective columns. A 25-ml aliquot of each pH-adjusted or pHi
leachate was passed through the column and then the next 30-ml portion
that passed through the column was collected for testing. Additionally, a 30-
ml portion was again collected after a total of 150-ml of the pH-adjusted
leachates or pH¡ leachate had passed through the column. All fractions
were held for the post-SPE toxicity tests.
Aeration of the MSW landfill leachates
Aeration removes or reduces volatile toxicants. The 35-ml aliquots of
pH 3, pH 11, or pH¡ leachates previously set aside, during the pH adjustment
manipulation, were utilized for this step. The pH-adjusted and pH¡ leachates
were transferred to individual 100-ml glass graduated cylinders, to which
was added a small air stone. The pH of each leachate was checked prior to
the initiation of the aeration procedure and four times during the one-hour
treatment. Adjustment with 1N, 0.1N, 0.01 N NaOH or HCI maintained
sample pH within 0.1 pH units. The leachate fractions were held for the
post-aeration toxicity test.

188
Blank preparation
Fractionation blanks were prepared with moderately hard water
(MHW) according to USEPA (1991a) (see Chapter 4). Briefly, the water had
the following specifications: pH 7.4 -7.8; hardness 80 -100 mg/L as CaCC>3;
and alkalinity 60 -70 mg/L (USEPA, 1993a).
The MHW fractionation blanks were generated parallel to the
leachate fractions with a 105-ml aliquot at pH 11, a 105-ml aliquot at pH¡,
and a 105-ml aliquot at pH 3. A 20-ml aliquot of both the pH 11 and pH 3
adjusted MHW were set aside for the pH-adjustment toxicity testing, as the
pH adjustment blanks. The pH 11, pH¡, and pH 3-adjusted MHW was then
passed through their respective 0.45 pm filters (GN-6 Metricel), prior to
passage of the pH-adjusted leachate fractions. The filtered pH 11, pH¡, and
pH 3-adjusted MHW was divided into 20-ml aliquots for the post-filtration
toxicity test and 30-ml aliquots for passage through the SPE columns. The
30-ml aliquots of pH adjusted/filtered MHW were passed through their
respective columns, with the final 10-ml portion collected for the post-SPE
toxicity test. Then the 35-ml aliquots of pH 11, pH¡, and pH 3-adjusted
MHW, set aside previously, were placed in 100-ml glass graduated
cylinders with a small air stone. The portions of pH 3 and 11-adjusted and
the pH¡ MHW were aerated for 1 hour with pH maintained within 0.1 pH
units from the starting pH of 3,11, or initial.
At the end of day 1, all of the pH 3 and pH 11-adjusted leachate
samples were returned to the initial pH, by addition of 1N, 0.1 N, 0.01 N

189
NaOH or HCI. The pH readjusted fractions for the three leachates were
stored under refrigeration at 4°C overnight for post-manipulation toxicity
assays on day 2.
Zeolite test
When identifying ammonia as a possible toxicant during the TIE phase II a
Zeolite treatment is included as a part of the investigation. A 335-ml aliquot of
MSW landfill leachate from sites 7, 8, and 14 was passed through 3 individual
cation exchange columns (USEPA, 1991c). The cation exchange columns were
prepared by combining 30 grams of a zeolite resin with 60 ml of DDI (Nanopure,
Barnstead) to form a slurry. This slurry was then transferred to a PVC tube fitted
with a mesh retainer to prevent loss of resin particles. Columns were washed
with three bed volumes of DDI and the final 10-ml from each column was
retained for use as the cation-exchange column toxicity blank. The pH-adjusted
and pHi MSW landfill leachates were passed through their respective columns
and the post-Zeolite effluents were collected. The post-Zeolite effluents were
assayed with the other post-manipulation leachate fractions. The concentrations
of ionized and un-ionized ammonia in the MSW landfill leachates were monitored
before and after the Zeolite treatment.
Toxicity Assays
Initial toxicity assays
The initial toxicity assays were initiated immediately following arrival
of the leachates in the laboratory, which was approximately five hours after
collection. The results of the initial toxicity assays were used to establish

190
the dilution series on day 2 of the fractionation procedure. The acute
toxicity of each leachate was determined with the aquatic invertebrate
Ceriodaphnia dubia with exposures of 24 and 48 hours (USEPA, 1993a).
The procedures for the culturing of C. dubia and the protocols for the acute
C. dubia toxicity assays are described in Chapter 3. Slight modifications to
the C. dubia toxicity assay protocol were required for the TIE and are
described. The initial dilution series utilized in the C. dubia assays were
from 0.78 to 12.5 % with sites 8 and 14 and from 3.12 to 50% with site 7,
with each concentration tested in duplicate. MHW was used for the dilution
water and the control water. At the start of each assay, five C. dubia
neonates (<24-hours old) were transferred to 30-ml plastic cups, and then a
10-ml aliquot of each leachate dilution was added to the cup. Each assay
included a duplicate control with 5 neonates in 10 ml of MHW. The assay
cups were placed in a water bath at 20 + 2° C under ambient lighting.
Neonates were fed prior to use in the assay but not during the testing.
The initial MSW leachate toxicity was also evaluated by the
Microtoxâ„¢ acute toxicity assay using the bacteria Vibrio fisheri (Beckman
Instruments, 1982). A fifteen-minute exposure interval was selected, based
on earlier research (Ward et al., 2002). In the Microtox assay, the MSW
landfill leachate toxicity was assayed without dilution, due to the large
number of leachate fractions. However, when accounting for the dilution of
the MSW landfill leachates by the bacterial reagent, the highest
concentration assayed was 90 %.

191
Additionally, for the determination of the initial toxicity of the three
whole MSW landfill leachates a chronic and a heavy metal specific assay
were included. The chronic toxicity was measured with the green alga,
Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), in a
96-hour assay (USEPA, 1994a). Toxicity due to the presence of heavy
metals was determined with MetPLATE, an assay specific for heavy metal
toxicity (Bitton et al„ 1994). The protocols for the P. subcapitata and
Microtoxâ„¢ assays have been described previously (Chapter 3). All toxicity
assays met the requirements for test acceptability according to USEPA.
Baseline toxicity assays
The baseline toxicity assays using the MSW landfill leachates
collected from sites 7, 8, and 14 were started on day 2 of the TIE using the
80-ml portions of leachate, which had been set aside on day 1. The pH of
each whole MSW landfill leachate was checked and adjusted to within 0.1
pH units of the initial pH of the leachates, which were 6.9, 7.6, and 7.4 for
sites 7, 8, and 14, respectively. These baseline assays were included to
monitor changes in leachate toxicity during the holding period that extended
from the start of the initial toxicity assays to the start of the baseline toxicity
assays (USEPA, 1991a). In this research, the holding time for the MSW
landfill leachates was approximately 18 hours. The baseline assays were
performed with the 24-hour C. dubia and the 15-minute Microtoxâ„¢ toxicity
assays, as previously described for the initial toxicity assays. The baseline
toxicity assays with C. dubia utilized the same dilution series as reported for

192
the initial toxicity assays and there were no dilutions assayed with the
Microtoxâ„¢ test.
Post-manipulation toxicity assays
At the start of day 2, the pH of the MSW landfill leachate fractions from
sites 7, 8, and 14 were checked and adjusted to within 0.1 pH units of the initial
pH of the leachate. The toxicity of the MSW landfill leachate fractions was
determined with the 24-hour C. dubia and 15-minute Microtoxâ„¢ acute assays, as
previously described. The C. dubia assays utilized four leachate dilutions from
3.1 to 25% for the leachates from sites 8 and 14. The leachate from site 7 was
assayed in dilutions from 6.2 to 50 %. The C. dubia assays were conducted
without replication and with MHW as the dilution water and control. There were
no changes to the Microtox method.
Data Analysis
The toxicity assay results were expressed as the leachate concentration
that produced a 50 % effect (EC50), e g. growth inhibition, death, or decreased
enzyme activity. In the case of the MetPLATE assay, the results were expressed
as the inhibition (%) to enzyme activity from the whole leachate; therefore, EC50
values were not determined. Graphical interpolation methods were used to
generate the EC50 values for the 96-hour P. subcapitata and the 15-minute
Microtoxâ„¢ assays, by the relationship between the leachate concentration and
the assay endpoint. Data analysis software was used to determine the ECsofor
the C. dubia assay (USEPA, 1994). All EC50 results were transformed
(IOO/EC50) and expressed as toxicity units (TU), a unitless measure. When

193
Table 6-3. Chemical and physical characteristics of the MSW landfill leachates
from sites 7, 8, and 14
Parameter
Site 7
Site 8
Site 14
pH
6.7
7.2
7.5
Conductivity
(mS/cm)
4.8
6.8
5.6
Alkalinity
(mg/L as CaC03)
1100
2400
2450
TDSa
(g/L)
2.9
3.0
3.9
Hardness
(mg/L)
374
116
339
CBODD
(mg/L)
73
180
182
Total ammonia
(mg/L)
21
428.9
335.9
nh3
(mg/L)
BDL
18.9
5.2
Sodium
(mg/L)
600
728
591
Chloride
(mg/L)
879
854
544
Sulfate
(mg/L)
103
<1
135
Copperc
(mg/L)
BDL
BDL
BDL
Zinc6
(mg/L)
0.3
BDL
BDL
Nickel6
(mg/L)
BDL
BDL
0.1
Detection limits for metal analysis °0.07 mg/L, a0.1 mg/L and “0.07 mg/L
results are expressed as TU values, and an increase in the TU value
corresponds to an increase in toxicity. In contrast, the EC50 values decrease with
increasing toxicity, as the concentration of leachate required to produce a 50 %
effect decreases. Data correlation was determined with the Student's f-test and
by least-squares regression in the Excel program (Microsoft, version 2000).

194
Results and Discussion
Chemical/Physical Characterization
Selected chemical and physical characteristics of the MSW leachates
from sites 7, 8, and 14 are summarized in Table 6-3. Generally the
chemical strength of the site 7 leachate was lower than that reported for
sites 8 or 14. The pH values were circum-neutral, which was typical for the
landfills tested, and ranged from 6.7 at site 7 to 7.5 at site 14, while a pH of
7.2 was reported at site 8. When determining toxicity, pH has a strong
influence on ionizable substances. For example, ammonia is highly toxic
and predominates at pH values greater than 9.3, whereas heavy metals are
generally more toxic or bioavailable at pH values below 7.
Conductivity values were reported as 4.8, 6.8, and 5.6 mS/cm for site
7, 8, and 14, respectively. The leachate carbonaceous oxygen demand
(CBOD) was highest for sites 8 and 14, at 180 and 182 mg/L, respectively,
while for site 7 the CBOD was 73 mg/L. The hardness concentrations, as
calcium and magnesium, were similar at sites 7 and 14 at 374 and 339
mg/L, respectively; however, at site 8 the hardness concentration was 116
mg/L. A striking difference was noted In the ammonia concentrations in the
three MSW landfill leachates. The total ammonia concentration in the site 7
leachate was 21 mg/L. This was less than 10% of the 428.9 and 335.9
mg/L of total ammonia reported at sites 8 and 14, respectively. A similar
pattern was shown for un-ionized ammonia concentrations with site 7
concentrations below the detection limit of the assay (0.5 mg/L), while 18.9
and 5.2 mg/L were reported for sites 8 and 14, respectively. Similar sodium

195
concentrations were reported for the 3 leachates with a range from 591 to
600 mg/L. The chloride levels for sites 7 and 8 were 879 and 854 mg/L,
respectively, while the site 14 levels were lower at 544 mg/L. Copper
concentrations were below the detection limit (0.07 mg/L) of the analytical
method with all leachates, but zinc was detected at site 7 and nickel at site
14 at 0.3 and 0.1 mg/L, respectively.
Determination of Heavy Metal Bioavailability
The TIE describes the addition of the metal chelator, ethylenediamine
tetraacetic acid (EDTA) for the identification of metal toxicity (USEPA, 1991a). It
should be recognized that the use of EDTA introduces toxicity, which may result
in false positive results (Kong et al., 1995). In some cases, EDTA is not effective
in the removal of metal toxicity. Mount and Hockett (2000) identified heavy metal
toxicants in an industrial effluent despite earlier TIE results that indicated no
reduction of toxicity following EDTA treatment. As an alternative in this research,
the heavy metal toxicity of the MSW landfill leachates was determined with
MetPLATE, an assay specific to heavy metal toxicity. Although, low
concentrations of heavy metals have been reported in MSW leachates (Ward et
al., 2002), the potential for metal leaching from the waste remains high
(Flyhammer, 1997). MSW leachates are by definition a heterogeneous mixture
of site-specific inorganic and organic contaminants, which can influence metal
bioavailability via complexation, precipitation, and adsorption reactions. Previous
research has shown that these leachate characteristics contribute to the low
toxicity of MSW landfill leachates using MetPLATE as the toxicity test (see

196
Table 6-4. The Initial (day 1) and baseline (day 2) acute and chronic toxicity of
the whole MSW landfill leachates from sites 7, 8, and 14 prior to
fractionation with results expressed as toxicity units (100/ECso)
Site 7
Site 8
Site 14
Initial
Baseline
Initial Baseline
Initial
Baseline
C dubia
(48-hr)
1.9
2.2
20 26.3
11.4
22.7
P. subcapitata
(96-hr)
<1
-
15.1
17.8
-
Microtoxâ„¢
(15-min)
< 1
1.3
< 1
Chapter 3 and 4). The heavy metal toxicity of the leachates was investigated with
MetPLATEâ„¢, an assay specific for heavy metal toxicity. The MetPLATEâ„¢ results
showed that the leachate toxicity was not due to the presence of heavy metals
with inhibitions of less than 10 % for each leachate (see Chapter 5).
Initial Toxicity
The results for the initial toxicity assays utilizing invertebrates, algae,
and Microtox have been summarized (Table 6-4). Generally, the leachate
samples were highly toxic in the invertebrate and algal assays, while low or
no toxicity was displayed in the Microtox assay. Ward et al. (2002) has
shown that the overall toxicity of MSW landfill leachates is best evaluated in
a suite of assays. The toxicity endpoints for the site 8 and 14 leachates
displayed similar profiles with algal TUs of 15.1 and 17.8 and invertebrate
TUs of 20 and 11.3, respectively. In contrast, the site 7 leachate displayed

197
a low toxicity in the algal and invertebrate assays with TUs of < land 1.9,
respectively.
Blanks and Controls
The pH values were monitored during each sample manipulation and
adjusted with 0.01 or 0.1 N HCI or NaOH as needed. Throughout the
fractionation procedures, slight pH adjustments were required to maintain
the pH of the pH 3 and 11-adjusted and pH, leachate samples.
Baseline Toxicity
The baseline toxicity of the MSW leachates from sites 7, 8, and 14
were evaluated with the 24-hr C. dubia and 15-min Microtox assays (Table
6-4). The toxicity of the three MSW landfill leachates increased with
increased holding time. During the 24-hour interval between the sample
collection and the start of the post-manipulation toxicity assays, the toxicity
associated with the leachate from site 8 increased from 20 to 26.3 TU.
Furthermore, the toxicity of the site 14 leachate doubled from 11.4 TU in the
initial toxicity assay to 22.7 TU during the baseline assay. In all cases, the
Microtox assay displayed a low sensitivity to the leachate toxicity from sites
7, 8, and 14 with TUs of < 1, 1.3, and 1.1, respectively.
Effect of Zeolite Treatment
The positive identification of ammonia as a toxicant in the MSW leachates
required an additional leachate treatment that included the passage of the three
raw MSW landfill leachates through a cation exchange column (Zeolite).
Hedstrom (2001) reported a highly efficient exchange of ammonium ions by

198
Table 6-5. Ammonia concentrations in MSW landfill leachates before and after
treatment on a Zeolite cation exchange column
Site
Total ammonia (NH4VNH3)
(mg/L)
Pre-Zeolite
Post-Zeolite
7
21
BDL
8
426.9
11.2
14
335.9
4.9
Zeolite columns. Ammonia toxicity in aquatic systems has been widely reported
(Adamsson et al., 1998; Game et al., 1996). While ammonia is found as both
ammonium (NH«+) and ammonia (NH3) forms, toxicity is primarily associated with
ammonia (Anderson and Buckley, 1998; Clement and Merlin, 1995); therefore,
pH is a critical factor when evaluating toxicity. The high ammonia concentrations
in Florida MSW leachates contribute to the overall toxicity (Ward, 1997; Ward et
al., 2002).
The ammonia concentrations in the three MSW landfill leachates
before and after passage through the Zeolite column are presented (Table
6-5). When the leachates from sites 7, 8, and 14 were passed through the
cation exchange columns the ammonia concentrations were reduced by
more than 95 %. The ammonia concentration in the un-treated whole site 7
leachate was 21 mg/L, but after the Zeolite treatment the concentration was
reduced to below the detection limit of the ISE (0.5 mg/L). The
concentrations of ammonia at sites 8 and 14 were reduced from 426.9 to
11.2 mg/L and from 335.9 to 4.9 mg/L, respectively. When the toxicity of
the post-Zeolite column leachates were assayed with the C. dubia toxicity
test the results were mixed (Table 6-6). After passage of sites 7 and 14

199
Table 6-6. Acute toxicity of the whole and post-Zeolite MSW landfill leachates to
C. dubia neonates with the ratio of live to total neonates presented at
the concentration of MSW landfill leachate assayed
Site
Neonate survival
Raw
Leachate
Post-Zeolite
Leachate
Control
7
3/5 (50 %)
5/5 (50 %)
5/5
8
4/5 (3.12 %)
4/5 (3.12 %)
5/5
14
4/5 (12.5 %)
3/5 (12.5 %)
5/5
leachates through the Zeolite columns, there were toxicity reductions of 100
% and roughly 50 %, respectively. In contrast, the site 8 leachate showed
no change in toxicity. The Zeolite resin exchanges ammonium ions (NH/)
for sodium (Na*) and due to the high concentrations of ammonium ions in
the raw leachates from sites 8 and 14 the added sodium ions may have
decreased test organism viability (Van Sprang and Janssen, 1997). When
the toxicity of the post-Zeolite MSW leachates were assayed by Microtox
the leachates from site 7 exhibited a large toxicity increase. The post-
Zeolite leachates from sites 8 and 14 displayed small toxicity reductions.
Post-manipulation Toxicity
Site 7
The whole leachate from site 7 displayed a low toxicity in the initial
survey of acute and chronic toxicity (Table 6-4). The results for the TIE
were summarized for the 24-hour C.dubia (Figure 6-1). It is worth noting
that the fractionation protocols caused an increased toxicity in the leachates

200
Figure 6-1. Results of the Phase 1 toxicity fractionation with the 24-hour C. dubia
assay for MSW landfill leachates collected from site 7 with results
shown as the percent (%) difference from the baseline toxicity
collected from site 7, following the pH adjustment, aeration, and solid phase
extraction procedures. Fractionation protocols are known to introduce
artifactual effects especially in samples with low initial toxicity (Jop and
Askew, 1994; Qureshi et al., 1982). This effect has also been reported
when leachates are modified during biological treatment processes
(Marttinen et al., 2002). The TIE results suggested that filtration reduced
the toxicity of the site 7 leachate. General conclusions were made
concerning characteristics of the toxicants in the site 7 leachate. Namely,
the toxicants were non-volatile and not hydrophobic, although they may
have been associated with leachate solids. Furthermore, the Zeolite resin
treatment of the site 7 leachate reduced the total ammonia concentrations

201
Figure 6-2. Results of the Phase 1 toxicity fractionation with the Microtoxâ„¢ assay
for MSW landfill leachates collected from site 7 with results shown as
the percent (%) difference from the baseline toxicity
from 21 to < 0.5 mg/L, which resulted in the removal of all acute toxicity
from the leachate. Although not implicated during Phase I fractionation, the
Zeolite treatment suggested that the toxicity may have been due to
ammonia or another cationic species.
The low sensitivity of the Microtox assay with a TU of < 1, did not
accurately reflect the baseline toxicity of the leachate (Table 6-4). The
effects of the fractionation procedures on site 7 leachate toxicity using
the15-minute Microtoxâ„¢ were summarized (Figure 6-2). Toxicity was
reduced with all treatments, except for the Zeolite treatment as previously
described.

202
Figure 6-3. Results of the Phase 1 toxicity fractionation with the 24-hour C. dubia
assay for MSW landfill leachates collected from site 8 with results
shown as the percent (%) difference from the baseline toxicity
Site 8
The TIE results with the site 8 leachate are summarized for the 24-
hour C. dubia assay in Figure 6-3. In most cases, the toxicity associated
with the leachate from site 8 was pH-dependent. When the pH was
reduced to 3, a foamy-white precipitate was formed, and there were
corresponding toxicity reductions in each pH 3-adjusted leachate fraction
(FILT, AER, and SPE). The toxicity of the SPE fractions (pH3 or pH9) was
decreased after treatment, suggesting the presence of a hydrophobic non¬
polar toxicant (Stronkhorst et al., 2003).
The strong influence of pH reduction on toxicity suggests an
ionizable toxicant. At pH 3, ammonia species are exclusively in the ionized

203
(and less toxic) ammonium form. On the other extreme, when the leachate
was adjusted to pH 11 the toxicity increased, probably due to the
conversion of ionized ammonium to un-ionized and highly toxic ammonia
ammonia. Marttinen et al. (2002) reported an increase in leachate toxicity
to Daphnia magna after ammonia stripping at pH 11. Furthermore, aeration
of the pH 3-adjusted leachate decreased toxicity by roughly 90 % and the
aeration of the pH 11-adjusted leachate reduced toxicity by 30 %,
suggesting a volatile component to the toxicant (Brack and Frank, 1997).
The toxicity of ammonia is pH-dependent, and with a pKa of 9.25 the
ionized form predominates under the generally neutral pH conditions found
in MSW leachates. Researchers have shown that the fraction of ammonia
present may be biologically relevant when concentrations of total ammonia
(NH4+/NH3) are high. C. dubia is sensitive to the presence of ammonia with
an EC50 of 1.2 mg/L (Andersen and Buckley, 1998). The Phase I TIE
results strongly suggest that ammonia was responsible for leachate toxicity;
however, the Phase II procedure did not positively identify ammonia toxicity.
Although the Zeolite treatment reduced total ammonia concentrations from
426 to 11 mg/L, the corresponding toxicity assays showed only a slight
decline in toxicity of approximately 5 %.
Sodium ions enhance toxicity when present in high concentrations
(Van Sprang and Janssen, 1997). The concentration of sodium in the site 8
leachate was 728 mg/L, much higher than the 600 mg/L and 591 mg/L at
site 7 and 14, respectively. Additionally, the high concentrations of anionic

204
Figure 6-4. Results of the Phase 1 toxicity fractionation with the Microtoxâ„¢ assay
for MSW landfill leachates collected from site 8 with results shown as
the percent (%) difference from the baseline toxicity
and cationic species in the MSW leachates may have contributed to the
overall toxicity (Baun et al., 1999). As evidence, the conductivity of the
three leachates were 4.8, 6.8, and 5.6 mS/cm for sites 7, 8, and 14,
respectively.
Conductivity is used as an indicator of total dissolved solids, and the
C. dubia LC50 for conductivity is 3 mS/cm (USEPA, 1991a). Cooman et al.
(2003) concluded conductivity levels that were 50 % of the LC50 influenced
the toxicity of a tannery wastewater. The control water, MHW, has a
conductivity of ~0.28 mS/cm, an order of magnitude lower than the reported
LC50.

205
Figure 6-5. Results of the Phase 1 toxicity fractionation with the 24-hour C. dubia
assay for MSW landfill leachates collected from site 14 with results
shown as the percent (%) difference from the baseline toxicity
The toxicity assay results for site 8 leachates using the 15-minute
Microtoxâ„¢ assay are summarized in Figure 6-4. From a broad perspective,
the pH adjustment, aeration, and filtration procedures produced comparable
reductions in leachate toxicity, although the pH3-adjusted leachate
displayed a very slight decline. The assay showed that the greatest toxicity
reduction was following the SPE treatments, suggesting the presence of a
non-polar organic toxicant. As discussed previously, the failure of the
Microtox assay to implicate ammonia toxicity was not surprising considering
the insensitivity of the assay relative to ammonia toxicity (Stronkhorst et al.,
2003; Ankley et al., 1990). The suggestion of a non-polar organic

206
compound agrees with the sensitivity of the Microtox ” assay for this
category of contaminants (Stronkhorst et al., 2003).
Site 14
The TIE results with the MSW landfill leachate collected from site 14
are more difficult to interpret, because of the uniform toxicity reduction
following each fractionation step. Figure 6-5 summarizes the 24-hour C.
dubia TIE results. When an aliquot of site 14 leachate was lowered to pH 3
the color of the leachate lightened. Similar effects were observed when the
pH of a second aliquot was increased to pH 11. The color of the pH 11
aliquot lightened, and a heavy precipitate was formed. This precipitate was
removed by filtration (0.45 pm). The Phase I results showed that the toxicity
declined following each manipulation regardless of pH; however, the largest
toxicity decline (75 %) was reported following the SPE treatment at pH 3.
The SPE results suggest that a non-polar organic toxicant in the
leachate contributed to the toxicity. Overall, the magnitude of toxicity
reduction was consistent for each TIE step indicating a pH insensitive
component contributing to the toxicity. This contrasts with the results of the
Zeolite column treatment, which showed a reduction of total ammonia
concentrations from 335 to 4.9 mg/L, and an overall toxicity reduction of 68
%. Toxicity reductions following exchange on a Zeolite_column suggest the
presence of an inorganic cation as the toxicant (Cooman et al., 2003).
According to the results of the TIE, ammonia was identified as a responsible
toxicant in the leachate from site14.

207
â–¡ Zeolite
HpH11/SPE
B pHi/SPE
BpH3/SPE
BpH11/aer
S pHi/aer
0 pH3/aer
in pm 1/fii
(DpHi/fil
CD pH3/fil
SpH11/adj
S pHi/adj
BpH3/adj
-100 -50 0 50 100
Toxidty (%) relative to baseline
Figure 6-6. Results of the Phase 1 toxicity fractionation with the Microtoxâ„¢ assay
for MSW landfill leachates collected from site 14 with results shown as
the percent (%) difference from the baseline toxicity
The results of the 15-minute Microtoxâ„¢ assay are summarized in
Figure 6-6. With the Microtox assay there were toxicity increases following
the aeration and filtration of the pH 3 and pH 11-adjusted site 14 leachate.
A similar phenomenon was reported by Stronkhorst et al. (2003) following
the filtration of sediment extracts. In the Microtoxâ„¢ assay, the SPE of the
site 14 leachate suggested the presence of a non-polar toxicant. The
leachate toxicity was reduced irrespective of the pH of the sample.
The results of the TIE investigations were summarized (Table 6-7).
The bioassays indicated various species were responsible for the leachate
toxicity. The toxicants in the leachates from site 7 were characterized as
non-volatile, not hydrophobic, and possibly associated with the leachate
solids. The results with the site 8 leachates indicated ionized substances

208
Table 6-7. Summary of TIE results with MSW landfill leachates from sites 7, 8,
and 14
Site
Toxicant Characteristic(s)
7
Non-volatile
Not hydrophobic
(?) Solids associated
8
Ionized substance
Ammonia
Cations
14
Hydrophobic compound
Ammonia
and cations were potentially responsible for the toxicity. Ammonia was
positively identified as a toxicant in the site 8 leachate. Similarly, ammonia
was identified as a toxicant in the site 14 leachates. The TIE results
indicated that hydrophobic compounds were associated with the toxicity of
the site 14 leachates.

CHAPTER 7
HORMONAL ACTIVITY OF MUNICIPAL SOLID WASTE (MSW) LEACHATES
FROM FLORIDA LANDFILLS
Introduction
Adverse reproductive and developmental effects have been documented
worldwide (Jobling et al., 1995; Routledge et al., 1998) following exposure to
substances that can alter chemical messengers in living systems (McLachlan,
2001). These hormonally active compounds (NRC, 1998) are defined not by
their chemical structures, but by their biological effects (Lopez de Alda and
Barcelo, 2001). There are multiple sources of hormonally active compounds in
the environment, including industrial effluents (Sheahan et al., 2002b; Lye et al.,
1999; Mellanen et al., 1996), and wastewater treatment plant (WWTP) effluents
(Holbrook et al., 2002; Jobling et al., 2002; Angus et al., 2002).
MSW landfills contain a multitude of discarded consumer products and the
leachates generated include numerous organic compounds. A wide variety of
organic compounds are disposed in landfills, and additional compounds are
produced as a result of waste decomposition processes (Kjeldsen et al., 2002).
While some of these compounds are hormonally active, such as the phthalates
(Vinggaard et al., 2000; van Wezel et al., 2000; Harris et al., 1997), surfactants
(Routledge and Sumpter, 1996a), and flame-retardants (Meerts et al., 2001), little
is known about the majority of the contaminants. MSW landfills may also contain
small quantities of industrial and hazardous wastes of unknown composition.
209

210
Phthalates are extensively used in the production of consumer goods, including
paints, inks, adhesives and various plastic goods to impart flexibility. Although
recycling programs in the U.S. encourage the recovery of plastics, only about 7%
of all manufactured plastics are recycled (USEPA, 2002). As a direct
consequence, MSW landfill leachates may contain large quantities of phthalate
compounds (Bauer and Herrmann, 1997). Generally, parts per billion levels of
phthalate contamination have been reported in MSW leachates; however, the
concentrations may range over an order of magnitude (Kjeldsen et al., 2002).
Contrary to previous work in this lab, which had shown extensive phthalate
contamination of MSW leachates (Ward and Townsend, unpublished data), the
phthalate contamination in the surveyed leachates was limited. In addition,
phytoestrogens found in plants and plant products (Nilsson, 2000) could also
occur in MSW leachates when yard wastes are not separated from household
wastes
While the acute and chronic toxicity of MSW landfill leachates has been
characterized (Ward et al., 2002; Ferrari et al., 1999; Bernard et al., 1996), less
has been published to address concerns about their potential hormonal activity.
Behnisch et al. (2001) using the E-screen cell proliferation assay (with MCF-7
cells) reported hormonal activity equivalent to 4.8 ng 17 p-estradiol (E2)/L in the
leachate from one solid waste landfill. Additionally, hormonal activity equivalent
to 28.4 ng/L E2 was reported in a groundwater sample contaminated with waste
leachate (Kawagoshi et al., 2002). Chronic exposure to waste leachates
produced alterations in the reproductive characteristics of fish in a Swedish lake

211
with decreased gonadal size and hormone levels (Noaksson, et al., 2003).
However, the hormonal activity of MSW landfill leachates in the United States
has received little attention.
The objectives of this research were to: 1.) determine if MSW landfill
leachates were hormonally active with a yeast reporter assay, 2.) tentatively
identify compounds in the leachates responsible for this activity, 3.) monitor the
effect of biological treatment on hormonal activity at one particular MSW landfill
leachate treatment plant, and 4.) provide monitoring data for researchers and
environmental regulators concerned with the hormonal activity of MSW landfill
leachates.
Materials and Methods
Chemicals
Unless otherwise specified, all chemicals were obtained from Fisher
Scientific at the highest possible purity. The hormone 17p-estradiol (E2) was
purchased from Sigma (St. Louis, MO, USA). Reference standards were
purchased from Ultra Scientific (North Kingston, Rl): dimethyl phthalate (DMP),
benzyl butyl phthalate (BBP), dibutyl phthalate (DBP), diethyl phthalate (DEP),
Bisphenol A, Tetrabromobisphenol A, and 3-tert-butyl-4-hydroxyanisole. The
phenolic compounds, nonylphenol (NP), octylphenol (OP) and tert-butyl phenol
(tBP), were obtained from ChemServe (Westchester, PA). Stock solutions of E2
and the analytical standards were prepared with HPLC grade methanol in
volumetric flasks sealed with Teflon stoppers. All stock solutions were stored
under refrigeration at 4°C with minimum light exposure, until needed.

212
MSW Landfills and Leachate Collection
The MSW landfill leachates were collected at eight engineered landfill
sites, in the state of Florida, USA. These leachates were selected based on
previous investigations. Additional information concerning the chemical/physical
characteristics and toxicity of these leachates is available (see Chapter 4). LF 1
is a capped landfill that stopped accepting waste in late 1999 and LF 5 is a
regional landfill currently accepting waste from three surrounding counties. LF 4,
LF 6, LF 12, and LF 14 are located throughout central Florida, while LF 7 is
located in southeastern Florida. LF 8 has an on-site leachate treatment facility
and samples were collected before and after treatment.
For comparative purposes, water samples were collected from the influent
(INF) and effluent (EFF) of a wastewater treatment plant (WWTP) in Gainesville,
FL and from Lake Alice in Gainesville, FL and Lake Beverly in Beverly Hills, FL.
A first flush stormwater sample was collected from a drainage basin in Beverly
Hills, FL.
MSW Landfill Leachate Treatment Facility
The treatment facility at LF3 is a two-stage powdered activated carbon
(PAC) system (Zimpro/Vivendi) that treats approximately 6000 gallons of MSW
leachate per day. The facility operates with a hydraulic retention time (HRT) of 25
hours and a sludge retention time (SRT) of 30 days. The mixed liquor
suspended solids (MLSS) is maintained at roughly 6000-8000 mg/L and sludge is

213
1-L
SAMPLE
PARTICULATES
YEAST
ASSAY
MéOH
FILTER
DISSOLVED PHASE
SOLID PHASE YEAST
EXTRACTION ASSAY
YEAST
ASSAY
Figure 7-1. Procedure for preparing MSW leachates and methanol extracts of
leachates for analysis of hormonal activity
wasted three times weekly from the aerobic and daily from the anoxic stages.
Influent strength varies, but generally the carbonaceous biological oxygen
demand (CBOD) ranges from 100 to 150 mg/L and the total ammonia
concentrations are approximately 450 mg/L. Batch plants based on
nitrification/denitrification are effective in the removal of ammonia from MSW
landfill leachate (Harper et al., 1996). The treated effluents are discharged to
percolation ponds in accordance with Florida standards for groundwater.
The dedicated glassware utilized throughout this investigation was acid-
washed, Dl-rinsed, dried, and methanol-rinsed prior to each use. MSW landfill
leachates were collected from the leachate collection sumps of each lined landfill

214
using a methanol-rinsed Teflon baler. Leachates were collected for the YES
assay in 4-L amber glass jugs with Teflon-lined septa. For the chemical and
physical characterization of the MSW leachates, samples were also collected in
glass or polyethylene bottles and preserved according to the U.S. Environmental
Protection Agency (USEPA, 1993). Field measurements were pH and
temperature (Orion, Model 290A), electrical conductivity (HANNA Instruments,
Model H19033), dissolved oxygen (DO) (YSI Inc. Model 55/12 FT), and
oxidation/reduction potential (ORP) (Accumet Co. Model 20). Samples were
transported on ice to the laboratory where they were stored at 4°C until analysis.
Solid Phase Extraction (SPE) of MSW Landfill Leachates
The MSW landfill leachates were concentrated by solid phase extraction
(SPE) to identify low concentrations of hormonally active compounds (Figure 7-
1). The leachates were pre-filtered with methanol-rinsed glass fiber (GF) (~1pm,
GF/B glass, Whatman) filters and membrane filters (MF) (0.45 pm, Fisher
Scientific), following delivery to the laboratory. The pre-filtration was used for the
removal of solids or bacterial contaminants, which might interfere with the assay
procedure. Two 1-L volumes of each pre-filtered raw MSW leachate were then
extracted in parallel on methanol conditioned C-18 extraction disks (47mm,
Empore, 3M Corp., St. Paul, MN) with vacuum filtration according to USEPA
method 3535 (SW-846). Due to the high organic content in the MSW leachates,
multiple extraction disks were utilized to prevent disk saturation or breakthrough.
Each C-18 extraction disk was eluted with two 15-ml aliquots of methanol. The
eluents from the individual disks were combined and further concentrated under

215
a stream of nitrogen (N2) (Turbovap, Zymark Corp., Hopkinton, MA) to a final
volume of 10 ml and a final concentration factor of 100X. The methanol extracts
were then transferred to a 40-ml glass VOC vial with Teflon septum, and stored
under refrigeration at 4°C until required. An aliquot of each methanol extract of
the leachates was exchanged with hexane, evaporated to 1 ml under a
continuous N2 stream, and analyzed by gas chromatography/mass spectrometry
(GC/MS). Method controls using distilled de-ionized water (DDI) (Nanopure,
Barnstead) and E2 spiked DDI were extracted following the same methodology
as described for the leachates.
A MSW landfill leachate collected from LF 12 in March 2002 was subject
to additional extractions and analysis. To remove compounds of the widest
polarity range, the C-18 extraction disks were sequentially eluted with solvents of
decreasing polarity, methanol (MeOH), acetonitrile (ACN), dichloromethane
(DCM), and hexane (HEX). Each solvent extract (MeOH, ACN, DCM, and HEX)
was further concentrated under nitrogen (Turbovap, Zymark Corp., Hopkinton,
MA) to a final volume of 30 ml, transferred to a 40 ml glass VOC vial with a
Teflon septa, and stored under refrigeration at4°C, until required.
YES Assay for Determining Hormonal Activity
The recombinant yeast assay (YES) has been extensively utilized for
identifying hormone activity in environmental samples (Sheahan et al., 2002a;
Witters et al., 2001; Desbrow et al., 1998). The recombinant yeast cells,
Saccharomyces cerevisiae, used for the assay were generously provided by Dr.
Sumpter of Brunei University (Middlesex, UK). The development of the yeast

216
cells for the assay has been previously described (Routledge and Sumpter,
1996b). The hormonal activity of individual samples and controls was quantified
by measuring the absorbance increases (570nm) with a microplate
spectrophotometer (Multiskan Plus MK II, ICN Biomedicals, Huntsville, AL).
To date, the YES has been used to identify hormonal activity in solvent
extracts of environmental samples (Holbrook et al„ 2002; Witters et al., 2001) or
with pure compounds (Miller et al., 2001). Therefore, when determining the
hormonal activity of raw MSW landfill leachates modifications to the YES assay
were required. The standard assay media was prepared for tests with the
solvent extracts of the MSW landfill leachates and with the E2 (Routledge and
Sumpter, 1996b). A three times concentrated assay media (3X) was prepared
for use with the raw leachates, so that when raw leachates were combined with
the 3X assay media the raw leachate was diluted by one-third.
Prior to the assay, 10-ml aliquots of the raw leachates were filter-sterilized
(0.2 pm, Acrodisc), and then a 100-pl aliquots of each filtered raw leachate was
combined with a 50-pl aliquot of the 3X assay media in a 96-well microplate and
mixed. The microplate also included 10- to 50-pl aliquots of the solvent extract of
the leachates and E2-spiked (E2 final concentration 120 to 160 ng/L) solvent
extracts. The solvent extracts were evaporated to dryness and mixed with 200 pi
of standard assay media.
In a separate 96-well microplate, a dose-response curve with the positive
control E2 was prepared with 10-pl aliquots of the eleven E2 dilutions, which
ranged from 7.3x1 O'12 (2 ng/well) to 5x10'9(1246 ng/well). The raw leachate

217
samples, solvent extracts of the MSW landfill leachates, E2- spiked solvent
extracts, positive (E2) and negative (methanol) controls were each assayed in six
replicates. To account for any p-galactosidase degradative capacity in the raw
leachate, a blank was included for each of the raw leachates. The leachate
blanks combined the raw leachates and the 3X assay media that had been
prepared without yeast cells. An increase in the absorbance of the leachate
blank wells was an indicator of the presence of exogenous enzyme degradative
capacity in the leachate. The GF and MF filters and their associated solids were
extracted with methanol and assayed for hormonal activity.
For the E2 positive controls, scatter plots were constructed with
absorbance measurements versus the E2 concentration (ng/L). The E2
equivalents for the raw leachates, leachate extracts and environmental samples
were determined by extrapolation from the linear region of the E2 positive control
curve (Sheahan et al., 2002b; Flolbrook et al, 2002). Following the assay, a
student's t-test was used to determine differences between the control
absorbance and sample absorbance.
Toxicity of MSW Leachates to Yeast Cells
The complex nature of the MSW leachates raised concerns about the
potential for toxicity to yeast cells. Cell growth was monitored visually as
increased turbidity (Routledge and Sumpter, 1996b), but alternative protocols
were needed (Beresford et al., 2000). To meet this concern, yeast cells were
assayed for viability with the oxido-reduction dye, 2-[4-iodo-phenyl]-3-[4-
nitrophenyl]-5- phenyl tetrazolium chloride (INT) (Sigma) (Bitton and Koopman,

218
1982). In the presence of viable yeast cells, INT (a clear liquid) is reduced to red
INT-formazan crystals. Concurrently with the YES assay, the viability of the yeast
cells was evaluated with aliquots of each raw leachate, leachate concentrate, or
water sample. After the YES incubation period, an aliquot of INT (final
concentration 0.02 %/well) was added to designated microplate wells for the
viability assay. Designated wells for the toxicity assays contained 100-pl aliquots
of the raw leachate sample, which was combined with 50-pl aliquots of the 3X
assay media prepared without the chromogenic substrate (CPRG). The
microplate was then incubated for an additional 30-45 minutes. Color
development in the microplate wells was quantified with a microplate reader
(Multiskan Plus MK II, ICN Biomedicals, Huntsville, AL) at 490nm.
GC/MS Analysis
The leachates were analyzed in triplicate using a Finnigan Trace 2000 gas
chromatography/mass spectrometry (GC/MS) instrument. The GC column was a
DB-5MS (J&W Scientific), 30-m long, 0.25-mm i.d., coated with 0.5-pm film
thickness. The injection port temperature was 250°C. The temperature of the
interface transfer line to the mass spectrometer was maintained at 290°C. Initial
oven temperature was 40°C and was held for 5 minutes. The oven was then
heated to 290°C at a heating rate of 20°C/minute and was held at that
temperature for 8 minutes. The GC/MS was operated in full scan mode (m/z 35-
500). Compounds were tentatively identified by library searches using a
NIST/EPA/NIH mass spectral library of 107,886 compounds, version 1.6D 1998.
The reference standards NP, OP, tBP, DMP, BBP, DBP, Bisphenol A,

219
O 50 100 150 200 250 300 350
17Beta-estrad¡ol (ng/L)
Figure 7-2. Response of the YES assay to 17-p estradiol (E2), (n=34) with error
bars representing the 95% confidence interval and the solid line the
solvent blank
Tetrabromobisphenol A and 3-tert-butyl~4-hydroxyanisole were analyzed using a
single standard of 100 mg/ml.
Results
Hormonal Activity of MSW Landfill Leachates
The hormonal activity of each leachate was expressed as the E2
equivalent response. The minimum detection limit (MDL) for the YES assay was
2.5 ng E2/L, which was determined as the mean absorbance of the solvent
control plus two times the standard deviation. There was a significant (p<0.01)
difference between the lowest concentration of E2 assayed and the solvent blank
(Figure 7-2).

220
Table 7-1. Hormonal activity of raw MSW landfill leachates and their methanol
extracts, as equivalent 17 p-estradiol concentrations with other water
samples included for comparison
Hormonal Activity
Site
(ng E2/L)
Raw
Methanol
Leachate3
Extract11
LF 1
Sept. 2001
NHAC
NT
Nov. 2001
NHA
59.7"
LF 4
Feb. 2002
NHA
NHA
Sept. 2001
NHA
NT
LF 5
Nov. 2001
NHA
NHA
March 2002
2.6"
NHA
LF 6
Feb. 2002
NHA
NHA
LF 7
Feb. 2002
3.4 (p=0.07)
NHA
Sept. 2001
NHA
NT
Nov. 2001
45.7"
29.6”
LF 8
Dec. 2001
NHA
25.4"
Jan. 2002
4.1"
13.7"
Feb. 2002#1
7.9 "
NHA
Feb. 2002#2
3*
NHA
LF 12
Nov. 2001
28.4
6.2
March. 2002
11.8"
NHA
LF 14
Feb. 2002
NHA
NHA
Lake Alice
Jan. 2001
6.8"
NT
Lake Beverly
Jan. 2001
NHA
NT
WWTP Inf
Sept. 2001
35.1
NT
WWTP Eff
Sept. 2001
NHA
NT
SW
Sept. 2001
NHA
NT
Blank
NHA
NHA
“Assayed at 1/3 dilution,
“Assayed at 5.5 times raw leachate,
“detection limit 2.5 ng/L.
Abbreviations: NUA = not hormonally active, NT = not tested, WWTP INF = wastewater treatment
plant influent, WWTP EFF = wastewater treatment plant effluent, SW = stormwater
Statistical difference from control: p £ 0.05,
p < 0.01
Twenty leachate samples were collected from eight-lined MSW landfills in
Florida, USA and tested for their hormonal activity with a recombinant yeast
estrogen screen (YES) assay. The surveyed samples represent typical MSW
landfill leachates from Florida (Reinhart and Grosh, 1998; Ward et al„ 2002).

221
Table 7-1 summarizes the hormonal activity of the raw MSW landfill leachates.
The results represent the activity in the raw leachates after a one-third dilution by
addition of the assay medium. The hormonal activity of the raw landfill leachates
was variable. Hormonal activity was identified in the raw MSW landfill leachates
from LF 5 (March '02) at 2.6 ng E2/L, but not in leachates collected in September
and November of 2001. The raw leachate collected from LF 8 in November 2001
displayed the highest hormonal activity at 45.7 ng E2/L. Comparatively,
leachates with low hormonal activity were collected from LF 8 in January 2002,
February 2002 #1, and February 2002 #2, with concentrations of at 4.1, 7.9, and
3 ng E2/L, respectively. The raw leachates collected from LF 12 displayed
hormonal activity equivalent to 28.4 ng E2/L in November 2001 and 11.8 ng E2/L
in March 2002. All of the hormonal responses associated with the raw leachates
were significantly different from the controls at either p < 0.05 or p < 0.01, with
one exception. The hormonal activity shown by the leachates collected from LF
7 were equivalent to 3.4 ng E2/L; however, the significance of this activity was at
the p = 0.07 level.
The hormonal activity in the raw MSW landfill leachates was variable and
fluctuated over time. This indicated the influence of site-specific parameters,
e.g., heterogeneous waste composition, degree of waste decomposition and
environmental conditions, like rainfall and temperature, on leachate production
and quality (Kjeldsen et al., 2002). Over a five-month period, the YES assay
showed that the hormonal activity at LF 8 ranged from no activity to 45.7 ng E2/L.
Similar fluctuations were previously reported with wastewater treatment plant

222
effluents and were attributed to environmental factors, like temperature and
rainfall (Murk et al., 2002). Hormonally active compounds were not associated
with solids in the Florida landfill leachates. Desbrow et al. (1998) found that
hormonal activity was not associated with solids in WWTP effluents; however,
suspended particulate matter in river water samples collected in the Netherlands
were extensively contaminated by hormonally active compounds (Murk et al.,
2002).
For comparison, the hormonal activity of various environmental samples
was investigated. The hormonal activity of the water sample collected from Lake
Alice was equivalent to 6.8 ng E2/L, while the Lake Beverly sample was not
active. Although beyond the scope of this research, the low hormonal activity
reported in the Lake Alice sample warrants further investigation. Since the lake
does not receive any direct discharges, then runoff or other non-point sources
may be contributing factors (Snyder et al., 1999). In Flemish surface waters,
hormonal activity ranging from 10.6 to 81.4 ng E2/L was reported using the same
YES assay (Witters et al., 2001).
The domestic WWTP influent displayed significant hormonal activity (p <
0.01) equivalent to 35.1 ng E2/L. This was higher than the hormonal activity of
WWTP influents assayed in Virginia, which ranged from 17.7 to 23.9 ng E2/L
(Holbrook et al., 2002). When compared to reports of hormonal activity in the
influents of WWTPs, this activity was low. In Japan, the hormonal activity of
WWTP influents has been reported at 150 ng E2/L (Matsui et al., 2000). In this
investigation, tertiary biological treatment reduced the hormonal activity to below

223
the detection limit of the assay (2.5 ng/L). Earlier research has shown hormonal
activity in WWTP effluents that ranged from 6.3 ng E2/L (Thomas et al., 2001) to
90 ng E2/L (Desbrow et al., 1998). Finally, the sample collected from a
stormwater basin was not hormonally active. To date, there have been no
reports concerning the identification of hormonal activity in stormwater.
To investigate trace substances, the MSW landfill leachates were treated
by solid phase extraction with subsequent methanol elution. The complex nature
of MSW leachates coupled with the limited information concerning hormonal
activity in MSW leachates led to the selection of a C-18 solid phase for an initial
gross separation of chemical classes (Snyder et al., 1999). Solid phase
extraction disks have a large surface area and low susceptibility to clogging;
therefore, they are a highly efficient choice for use with MSW landfill leachates
(Lopez and Barcelo, 2001).
The methanol extracts of the leachates were assayed for hormonal
activity, and their responses were lower than the raw leachates. With the
methanol extracts, five of the seventeen samples were hormonally activity (Table
7-1). The methanol extracts of the leachates from LF 1 (Nov. 2001) displayed
the highest hormonal activity at 59.7 ng E2/L, while the lowest activity was
determined in extracts from LF 12 (Nov. 2001) at 6.2 ng E2/L. Hormonal activity
was identified in the methanol extracts of leachates collected from LF 8 in
November and December 2001 and January 2002 at 29.6, 25.4, and 13.7 ng
E2/L, respectively. There was no hormonal activity in the methanol extracts of
the leachates collected from LF 7, LF 5, LF 4, LF 6, and LF 14. Methanol

224
Table 7-2. Hormonal activity of raw MSW landfill leachates from LF 8 before
(influent) and after (effluent) treatment in a powdered activated carbon
Sample
Hormonal Activity3
(ng E2/L)
Influent 1
7.9 ±1.0
Effluent 1
3.2 ±0.7
Influent 2
3.0 ±1.5
Effluent 2
NHA
“Results presented as mean of six replicates ± one standard deviation.
"Statistically significant at p< 0.01, NHA = not hormonally active, detection limit of the
assay was 2.5 ng E^L
extracts of the environmental samples were not assayed for hormonal activity.
The response of the methanol extracts was lower in some of the assays, and this
may be attributed to matrix interference or the presence of non-extractable
compounds (Murk et al., 2002; Holbrook et al., 2002; Baun et a!., 1999).
Recently, researchers in Japan have reported on the hormonal activity of
solid waste leachates. The hormonal activity of Japanese waste leachates
ranged from 4.8 to 28.4 ng E2/L (Behnisch et al., 2001; Kawagoshi et al., 2002).
This was similar to the range reported here for Florida MSW landfill leachates.
While the MSW landfill leachates assayed in this research were produced in
landfills receiving only MSW, the waste composition in the Japanese landfills was
primarily incinerator ash and incombustible wastes (Behnish et al., 2001;
Kawagoshi et al., 2002).
Effect of Biological Treatment on Hormonal Activity
Powdered activated carbon (PAC) treatment of MSW landfill leachates
was investigated for its effect on hormonal activity at LF 8 in February 2002

225
during two separate treatment cycles (Table 7-2). The MSW landfill leachates
entering the treatment facility (influents) displayed hormonal activity equivalent to
7.9 and 3.0 ng E2/L for treatment cycles 1 and 2, respectively. In the
corresponding treated effluents, the hormonal activity was reduced to below the
detection limit of the assay (2.5 ng/L) during cycle 2, but not in cycle 1. Instead,
during cycle 1 a 59.6 % reduction of hormonal activity was reported. It should be
noted, that the methanol extracts of the treatment plant influents and effluents
were assayed, but displayed no hormonal activity.
State and federal environmental regulations require the treatment of MSW
landfill leachates at either an on-site treatment facility or at a domestic
wastewater treatment plant (F.A.C. 62-701). Treatment processes can
metabolize some xenoestrogens to more active products (Sole et al., 1998).
Processes based on activated carbon remove organic compounds, including
those with hormonal activity (USEPA, 2001). Coors et al. (2003) recently
reported highly efficient removal of hormonal activity from MSW landfill leachates
after biological and reverse osmosis processes. Similarly, Wintgens et al. (2003)
found strong removal with reverse osmosis, but ultrafiltration processes had the
highest removal rates with an 80 % reduction in hormonally active substances.
Based on mass balance investigations of hormonal activity in domestic
wastewater treatment facilities, the activity was transferred to the sludge
(Holbrook et al., 2002; Takigami et al., 2000).

226
Table 7-3. Organic compounds tentatively identified in MSW landfill leachates by
GC/MS analysis in full scan mode
Compound3
Eucalyptol
a,a,-dimethyl-benzenemethanol
L-Fenchone
Triethyl phosphate
Terpineol
T ricyclo[5.2.10(2,6)dec-3-en-8-one
Benzenamine, 2-chloro-4-methyl-
2,4,7,9-Tetramethyl-5-decyn-4,7-diol
Diethyltoluamide (DEET)
Dibutyl phthalate (DBP)
Tris(3-chloropropyl) phosphate
Benzenesulfonamide, N-butyl-
3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid
Hexadecanoic acid, butyl ester
Octadecanoic acid, 2-hydroxy-1,3-propanediyl ester
3-propyl-phenol
1 -methylyethyl-phenol
Di-octylphthalate
Benzenepropanoic acid
“Only representative compounds listed.
GC/MS Analysis of MSW Landfill Leachates
One objective of this research was to identify substances responsible for
hormonal activity. Efforts were centered on the segment of organic compounds,
which were specifically retained by C-18 and subsequently eluted with methanol.
Table 7-3 summarizes selected compounds tentatively identified in MSW landfill
leachates by GC/MS. Comprehensive lists of organic contaminants in waste
leachates have been published, but considering the scope of this research they
are overly broad (Yasuhara et al., 1999). Compound classes identified included
the phthalates, hydrocarbons, fatty acid methyl esters (FAME), phenols, and
phosphates. All identifications were tentative, as analytical standards were

227
available for only a few of the compounds. However, the identified compounds
were all present in concentrations of 100 pg/L or higher (Figure 7-3). The only
phthalate compound specifically identified was dimethyl phthalate (DMP) in the
leachate from LF 12 (Nov. '01), other non-specific phthalates were also identified
in this leachate. Although the analytical standards (BBP, DBP, Bisphenol A,
Tetrabromobisphenol A and 3-tert-butyl-4-hydroxyanisole) were not identified, a
wide variety of other organic compounds were present in the leachates. These
organic compounds were not evaluated for hormonal activity. Therefore, no
direct or inferred relationships were made between the identified compounds and
hormonal activity.
While no clear pattern was observed between hormonal activity and the
presence of a specific class of organic compounds, the recurrence of some
compounds is notable (Schwarzbauer et al., 2002). Diethyl-toluamide (DEET), a
commonly used insect repellant, was identified in 54 % of the leachate samples
analyzed by GC/MS. Its presence was also detected in all four of the samples
collected at LF 8, but was removed following leachate treatment. This insecticide
has also been identified in 74 % of polluted surface (Kolpin et al., 2002) and 80
% of marine (Weigel et al., 2002) waters, but no data are available related to the
effects of long-term exposure. Additionally, a plasticizer, n-butyl-benzene
sulfonamide, used in the production of polycarbonates, and triethyl phosphate
(TEP) (Carlsson et al., 2000), a flame-retardant compound, were identified in
multiple leachate extracts. While many organic compounds in MSW leachates
are degraded or undergo transformations, others like DEET and benzene

228
A.
RT: 8.02 - 39.75
Figure 7-3. Total ion chromatogram of MSW leachates from LF 8 (Feb. ’02#1) in
the full scan mode A.) before treatment B.) after treatment in the PACT
facility

229
has also been identified in 74 % of surface (Kolpin et al., 2002) and 80 % of
sulfonamide persist (Schwarzbauer et al., 2002). The most frequently detected
compound was 2,4,7,9-tetramethyl-5-decyn-4, 7-diol, a surfactant used as an
adhesive component in the furniture industry. Various fatty acids, which included
those in animal fat, milk fat, and vegetable material were identified in all
leachates.
Other hormonally active compounds associated with waste materials are
the polybrominated diphenyl ethers (PBDEs) (Darnerud et al., 2001; Rahman et
al., 2001), which are widely used as flame retardants in consumer goods
including circuit boards, furniture, clothing, power cables, and casings for
televisions, computers and audio equipment. Although PBDEs were not
identified in the MSW leachates surveyed in this investigation, the flame
retardant triethyl phosphate (TEP) was found. TEP is used in vinyl polymers and
polyesters.
Influence of Concentration Factor on Hormonal Activity of Leachates
Early in this investigation, questions were raised concerning the variability
of YES results between raw leachates and methanol extracts. The hypothesis
was advanced that the extraction procedure altered the hormonal activity,
possibly via antagonistic actions. By combining E2 with the methanol extracts,
the influence of substances in the extracts were measured via decreases in the
activity of E2. The solid phase extraction procedure produced methanol extracts,
which were 100 times more concentrated than the raw leachates. These 100X
methanol extracts of selected leachates were assayed at concentration factors of

230
Figure 7-4. Dose-response of the LF 1 (Nov. '01) methanol extracts versus
concentration factor. A factor of 1 indicates the concentration of
substances in the extract is equivalent to the concentration in the raw
leachate with the solid line representing the solvent (MeOH) blank
0.66, 2.2, 5.5, and 11. A factor of 0.66 indicates that the concentration of
substances in the extracts were equivalent to their concentrations in the assayed
raw leachates. Graphical interpolations of the absorbance response of these
extracts versus the concentration factors were performed. The results revealed
that, in general, the maximum absorbance was achieved at a concentration
factor of 5.5.
In the methanol extracts of leachates collected at LF 1 in November 2001,
a dose-dependent increase in absorbance values was noted (Figure 7-4).
However, at a concentration factor of 11 the absorbance decreased by 50 %.
Similar results were shown with the methanol extracts of leachates collected from
LF 8 (Dec. '01). There was a dose-dependent increase in the absorbance of the

231
O 5 10 15
Concentration factor
Figure 7-5. Dose-response of the LF 8 (Dec. ’01) methanol extracts versus
concentration factor. A factor of 1 indicates the concentration of
substances in the extract is equivalent to the concentration in the raw
leachate with the solid line representing the solvent (MeOH) blank
Concentration factor
Figure 7-6. Dose-response of the LF 12 (Nov. ’01) methanol extracts versus
concentration factor. A factor of 1 Indicates the concentration of
substances in the extract is equivalent to the concentration in the raw
leachate with the solid line representing the solvent (MeOH) blank

232
Table 7-4. Recovery (%) of 17 (3-estradiol (E2) from the E2 spiked methanol
extracts of MSW landfill leachates
Methanol Extract
RECOVERY3
(%)
LF 1
(Nov. '01)
19.9 ±2.2
LF 5
(Nov. '01)
79.5 ±8.4
LF 8
(Dec. '01)
86.5 ±5.8
LF 8
(Jan. '02)
31.1 ± 5.1
LF 12
(Nov. '01)
52.4 ±4.3
DDI
94.5 ±7.3
a Recovery reported as mean ± 1 standard deviation.
methanol extracts from 0.66 to 5.5 (Figure 7-5). When assayed at a
concentration factor of 11, there was a 10 % decrease in absorbance. A different
pattern was observed with the methanol extract of the MSW landfill leachates
from LF 12 (Nov. 2001) with the highest absorbance at a concentration factor of
0.66. Although the absorbance values decreased by 38 % between the
concentration factors of 0.66 and 2.2, there was a slight increase between 2.2
and 11 (Figure 7-6). Overall, the hormonal activity of the MSW landfill leachates
was altered by the extraction procedure. However, more investigation is required
before assertions can be made concerning the cause of the reduced E2 activity.
E2 Recovery in Spiked Methanol Extracts of Leachates
Taken together, the results of the YES assays with the raw leachates and
the methanol extracts of raw leachates suggested two possibilities for the pattern
of hormonal activity observed. First that substances co-eluted during the
extraction procedure were masking the response of the assay or second that

233
hormonally active compounds were not retained by the solid phase extraction
disk. It was also possible that substances retained by the solid phase extraction
disk were not eluted by the methanol; however, this was not investigated. The
first possibility was investigated by the addition of E2 to the methanol extracts of
the leachates, followed by a YES assay to quantify the activity of the E2. Table
7-4 summarizes the variable recovery of E2 activity from the spiked methanol
extracts. In the methanol extract of the DDI, nearly 95 % of the E2 activity was
recovered. After spiking, the recovery of E2 activity from the methanol extracts of
the MSW landfill leachates was lower. In the methanol extract of the leachate
collected from LF 8 (Dec. '01) 86.5 % of the E2 activity was recovered, but in the
January (’02) sample only 31.1 % of the activity was recovered. The poorest
recovery for E2 activity ( < 20 %) was in the methanol extract of the leachate from
LF 1 (Nov. '01). The addition of E2 to the methanol extract of LF 12 (Nov. '01)
resulted in the loss of roughly 50 % of the E2 activity. The low recovery suggests
the presence of compounds that mask or interfere with E2 activity.
The erratic recovery of E2 from methanol extracts suggested an inhibitory
effect was causing the reduced response of E2 in the assay. It is difficult to
assign a cause for this behavior, although some possibilities are evident. The
raw MSW leachates contained an elevated organic content, and many were
highly colored (see Chapter 5). Passage of the leachates on the C-18 disks
reduced their color, but the solvent elution may have caused some break-through
of organic material. Complexation of organic material with hormonally active
substances may have reduced interactions with the estrogen receptor. Similarly,

234
Category
Comment
1
NHADa
2
Hormonal activity due to
nonpolar or polar molecules
with large alkyl tails
3
Concentration effect
4
Unknown agent masks
presence of hormonal activity
NHAD = No hormonal activity detected
Kawagoshi et al. (2002) reported a loss of activity following the combination of E2
with leachate extracts. They attributed the loss of E2 activity to co-extraction and
co-elution of anti-estrogenic substances (Kawagoshi et al., 2002). Based on their
results, this explanation was premature and overstated (Ashby, 2000).
Kawagoshi et al. (2002) do not discuss any evaluation of leachate toxicity to
yeast cells, despite concerns of others (Eguchi et al., 2003). The primarily ash
composition of solid waste landfills in Japan means that inorganic species are
dominant and may interfere with E2 recovery.
Interpretation of GC/MS Results with the Hormonal Activity of MSW Landfill
Leachates
The hormonal activities of the raw and methanol extracts of the MSW
landfill leachates as determined by the YES assay were compared with the
analytical results obtained by GC/MS techniques. There were four cases

235
Table 7-6. Presence of hormonal activity in the raw leachates and methanol
extracts of MSW landfill leachates with identified hormonally active
Hormonal Activity?
Category
bite
Raw
Methanol Extract3
LF 1 (Nov. '01)
No
Yes
3b
LF 4 (Feb. ’02)
No
No
1
LF 5 (Nov. ’01)
No
No
1
LF 5 (March '02)
Yes
No
4
(nonylphenol)
LF 7 (Feb. '02)
Yes
No
4b
LF 8 (Nov. '01)
Yes
Yes
2b
LF 8 (Dec. '01)
No
Yes
3b
LF 8 (Jan. '02)
Yes
Yes
2b
LF 8 (Feb. '02) #1
Yes
No
4b
LF 8 (Feb. '02) #2
Yes
No
4b
LF 12 (Nov. '01)
Yes
Yes
2
(t-butyl phenol)
LF 12 (March ’02)
Yes
No
4
(phthalate compounds)
LF 14 (Feb. ’02)
a .. T—T TTTTT
No
No
1
specific hormonally active compounds were not identified
observed when attempting to analyze the results of the YES and the analytical
data (Table 7-5). The first category describes MSW landfill leachates with no
hormonal activity detected (NHAD) in either the raw leachate or the methanol
extract from the C-18 disk. The second category represents samples with
hormonal activity in both the raw leachate and the methanol extract from the C-
18 disk. The third category illustrates the case of raw leachates with hormonal

236
activity below the detection limit of the YES assay. This category is different from
the first one because hormonal activity was subsequently detected in the
methanol extract from the C-18 disk. In this category, the trace contaminants
were concentrated to detectable levels following passage through the C-18 disks.
In the fourth category, the raw landfill leachates display hormonal activity, but the
methanol extracts from the C-18 disk did not display hormonal activity.
Table 7-6 describes the classification of the MSW landfill leachates
according to the four categories listed in Table 7-5. The first category was
illustrated by MSW landfill leachates collected from LF 4 and LF 14 in February
2002 and from LF 5 in November 2001 with no hormonal activity in the raw
leachates or methanol extracts. The absence of hormonal activity was confirmed
by GC/MS analysis, which failed to identify any known hormonally active
compounds. For the second category, hormonal activity was identified in both
the raw leachate and the methanol extracts. This was exemplified by MSW
landfill leachates from LF 8 (Nov. '01 and Jan. '02) and LF 12 (Nov. '01). The
hormonal activity of the LF 8 (Nov. '01) raw leachate was equivalent to 45.7 ng
E2/L. After the solid phase extraction, the hormonal activity of the methanol
extract was 29.6 ng E2/L. These results indicated that the raw MSW leachate
contained both polar and non-polar hormonally active compounds. The reduced
hormonal activity of the extract was probably due to the failure of the non-polar
C-18 disk to retain the smaller polar compounds. This suggests that the bulk of
the hormonal activity (65 %) was due to the presence of non-polar compounds.
With the raw leachates from LF 8 (Jan. ’02), the hormonal activity was equivalent

237
to 4.1 ng E2/L, but the activity increased to 34.3 ng E2/L after solid phase
extraction. Since the methanol extract was 5.5 times more concentrated than the
raw leachate, the majority of the increased hormonal activity can be explained by
a simple concentration effect. Therefore, the hormonally active compounds
implicated in this leachate sample are non-polar compounds or large polar
compounds with a significant alkyl portion to the molecule. The LF 12 (Nov. '01)
raw leachate induced a hormonal response equivalent to 28.4 ng E2/L, but the
activity was reduced to 6.2 ng E2/L after solid phase extraction. While this raw
leachate sample was not analyzed by GC/MS, an analysis of the methanol
extract showed the presence of t-butyl phenol. Although the large alkyl tail on
the t-butylphenol molecule allowed it to be retained on the non-polar C-18 solid
phase extraction disk, other smaller polar compounds were not retained. Thus,
for this MSW landfill leachate the results suggest that a large portion of the
hormonal activity in the raw leachate was due to polar compounds, which were
not retained during the extraction procedure.
The third category is illustrated by raw leachates collected from LF 1 (Nov.
'01) and LF 8 (Dec. ’01), which were not hormonally activity. Hormonal activity
was detected when these leachates were concentrated by solid phase extraction.
The 100X methanol extracts were diluted to 5.5X following addition of the assay
medium. The hormonal activity in the methanol extract from LF 1 (Nov. '01) was
equivalent to 59.7 ng E2/L, while the raw leachate was not active. Considering
only the concentration factor and assuming 100 % concentration efficiency, then
the raw leachate activity should have been equivalent to 10.8 ng E2/L, but this

238
was not the case. Instead, there was no hormonal activity detected in the raw
leachate. Further analysis revealed that when this extract was diluted to 2.2X,
the hormonal activity was equivalent to 12.9 ng E2/L or roughly half the predicted
activity of 23.8 ng E2/L. Taking this into account, the expected raw leachate
activity should have been roughly 4-5 ng E2/L. While this value was
approximately double the detection limit of the assay, it may be that other factors
in addition to concentration were influencing the detection of hormonal activity. A
similar effect was shown with the leachate from LF 8 (Dec. '01). The raw
leachate was not hormonally active, but the 5.5X methanol extract displayed
activity equivalent to 25.4 ng E2/L. Additionally, a 2.2X extract displayed
hormonal activity equivalent to 7.1 ng E2/L. Based on these results, the
predicted activity in the raw leachate was probably just below the detection limit
of the assay (2.5 ng E2/L).
The fourth category was illustrated by MSW landfill leachates from LF 7
(Feb. ’02), LF 8 (Feb. '02)#1, and LF 8 (Feb. '02)#2, with hormonal activity
identified in the raw but not the extracted leachates. The equivalent hormonal
activity was 3.4, 7.9, and 3 ng E2/L in the raw leachates from LF 7 (Feb. '02), LF
8 (Feb. '02)#1, and LF 8 (Feb. '02) #2, respectively. According to the results of
the GC/MS analysis, no specific hormonally active compounds were identified in
these leachates. Since the hormonal activity was not recovered on the C-18
disks, polar compounds were probably causing the hormonal activity. Overall,
these results provide support for the conclusion that polar compounds were
responsible for the hormonal activity in these three landfill leachates.

239
Interpreting the results obtained with the LF 5 (March '02) and LF 12
(March '02) leachates, was more difficult. Although the LF 5 (March '02) raw
leachate displayed hormonal activity equivalent to 2.6 ng E2/L, there was no
hormonal activity in the methanol extract. This initially suggested that the
hormonally active compounds were of a polar nature and; therefore, were not
captured by the C-18 extraction disk. However, subsequent GC/MS analysis
revealed the presence of nonylphenol, a known xenoestrogen, in both the raw
and methanol extracts. While nonylphenol is a polar compound, it does have
certain non-polar characteristics. Large polar compounds like nonylphenol
contain a significant non-polar alkyl tail, readily retained by the C-18 extraction
disk (Simpson and Van Home, 1993). Based on the identification of
nonylphenol, there was an expectation of hormonal activity in the extracts. The
LF 12 (March '02) raw leachate was hormonally active at 11.8 ng E2/L, but the
methanol extract did not display hormonal activity. The GC/MS analysis of this
leachate revealed the presence of some non-polar phthalate compounds, which
are recognized for their ability to induce hormonal responses (Picard et al., 2001;
Jobling and Sumpter, 1993). In contrast, some researchers have suggested that
some phthalates display anti-estrogenic activity (Kawagoshi et al., 2002)
including diethyl-hexyl phthalate (DEHP) (Metcalfe et al., 2001; Hams et al.,
1997), as well as other phthalates (Foster et al., 2000). Based on these results,
the lack of hormonal activity in the LF 5 (March ’02) and LF 12 (March ’02)
methanol extracts required further investigation.

240
Table 7-7. Effect of extraction procedures on the hormonal activity of leachates
from LF 12 (March 2002)
Sample
Hormonal Activity
(nq E2/L)
Raw leachate
11.8"
Filtrate
2.7"
Methanol
extract
NHAa
Acetonitrile
extract
NHA
Dichloromethane
extract
NHA
Hexane
extract
NHA
aNHA=not hormonally active
Caution should be exercised when describing relationships between
hormonal activity and organic contaminants of MSW landfill leachates. MSW
landfill leachates are a complex matrix and some organic compounds can not be
identified by GC/MS analysis, e g. non-volatile or high molecular weight
compounds (Reineke et al., 2002). However, the GC/MS technique employed
has been successfully used to determine similar compounds (Croley et al., 2000)
and here served to produce a list of organic contaminants from which to start an
investigation. The MSW leachates contained a wide variety of organic
contaminants, which was expected based on the stages (early to middle) of
waste decomposition represented by the selected landfills (Bozkurt et al., 2000).
However, the identified compounds may account for only 10-24 % of the total
organic carbon content of the leachates (Yasuhara et al., 1999), which agrees
with reports that 90 % of organic carbon may be attributed to unidentified organic

241
Figure 7-7. The solid phase extraction protocol used with MSW landfill leachates
chemicals (Castillo and Barcelo, 1999). As landfills age the organic materials
become more recalcitrant and the influence of this on estrogenic leachate
properties has yet to be investigated. Based on the results of the GC/MS
analysis and the YES assay with the methanol extracts, a conclusion was drawn
that the matrix of the MSW landfill leachates interfered with the determination of
hormonal activity.
Isolation of Hormonal Activity at LF 12
One leachate sample from LF 12 collected in March 2002 was subject to
additional analysis by GC/MS and YES assays. The hormonal activity of the raw
MSW leachate was equivalent to 11.8 ng E2/L (Table 7-7). Additionally, after
passage on the C-18 disk the filtrate was reserved and assayed for hormonal
activity (Figure 7-7). The hormonal activity of the filtrate was equivalent to 2.7 ng
E2/L, indicating that some of the hormonal activity was due to the presence of
polar compounds, which were not retained by the C-18 sorbent. To remove

242
compounds of the widest polarity range from the C-18 disks, they were
sequentially eluted with solvents of decreasing polarity, beginning with methanol
(MeOH), then acetonitrile (ACN), and dichloromethane (DCM), and finally hexane
(HEX). Accounting for the 11.8 ng E2/L in the raw leachate and the 2.7 ng E2/L in
the filtrate, the extraction disk retained 77 % of the hormonal activity.
The in-depth analysis of the MSW leachate collected from LF 12 revealed
general characteristics of the hormonally active compounds. The majority of the
hormonal activity was associated with non-polar compounds, as established by
the retention of 77 % of the raw hormonal activity on the C-18 disk. Desbrow and
workers (1998) identified a similar pattern when non-polar compounds were
responsible for hormonal activity. Unexpectedly, sequential elution of the C-18
disks with solvents of decreasing polarity (MeOH, ACN, DCM, and HEX) and
subsequent testing in the YES assay revealed that the substances responsible
for the hormonal activity were not eluted from the disk. These results support the
hypothesis that the recovery of hormonal activity may be influenced by more than
the polarity of the solid phase and the solvents.
Issues Raised When Analyzing MSW Landfill Leachates for Hormonal
Activity
Early in this investigation, issues were encountered concerning the use of
the YES assay with MSW landfill leachates (Routledge and Sumpter, 1996b).
These leachates contain a multitude of organic and inorganic contaminants,
which may be highly toxic (Chapters 3, 4, 5, and 6). Previously, researchers
have demonstrated the applicability of the YES assay with a variety of sample
matrices, which utilized domestic wastewater (Holbrook et al„ 2002), flue gases

243
(Muthumbi et al., 2002), recycled materials (Vinggaard etal., 2000) and surface
waters (Witters et al., 2001). Comparatively, MSW landfill leachates display a
greater chemical complexity (Table 2-1). This increased the potential for false
negative responses, due to toxic influences of the leachates on the yeast cells.
When investigating potential hormonal activity, other factors including the
solvent utilized during extraction procedures, and the presence of microbial
populations in the MSW landfill leachates, may influence the response of the
yeast cells. While the research presented here was not concerned with the
development of a yeast estrogen screen assay or improving an established
assay, these issues were addressed relative to concerns with MSW leachates
analysis.
Toxicity of MSW leachates to yeast cells
Issues related to the viability and overall health of yeast cells have been
raised, especially related to concerns of false negative results (Dizer et al., 2002;
Picard et al., 2001). For the purposes of this research, the viability of the yeast
cells was assessed with the oxido-reduction dye, 4-[iodo-phenyl]-3-[4-
nitrophenyl]-5- phenyl tetrazolium chloride (INT) (Bitton and Dutka, 1986). Yeast
cells were exposed to the MSW landfill leachates and were then assayed for their
dehydrogenase activity. The results of the YES assays showed that the raw
MSW landfill leachates, and methanol extracts were not toxic to the yeast cells.
In some cases, dehydrogenase activity was higher in the leachate wells when
compared to the control wells, based on the absorbance values (Figure 7-8).
These results suggested a higher density of yeast cells in the microplate wells

244
Dec. '01
-50
Figure 7-8. The toxicity of MSW landfill leachates to yeast cells according to the
INT procedure
containing leachates. Therefore, it was hypothesized that there was an increased
proliferation of yeast cells in the nutrient rich leachate. A slight inhibition (< 20 %)
of yeast respiration was noted following exposure to leachates from site LF5
(Nov. '01) and site LF8 (Sept. '01 and Dec. ’01).
Although the viability assay demonstrated that the yeast cells were
actively multiplying and respiring, it did not establish the absence of inhibitory
influences from the leachates. Yeast cells (Saccharomyces cerevisiae) are
sensitive to environmental changes, e g., temperature, pH, osmotic stress
(Gasch et al., 2000; Palkova et al., 2002), but may readily adapt to new
environments (Causton et al., 2001). One primary toxicant identified in the MSW
landfill leachates was ammonia (see Chapters 3, 4, and 6). In this research,

245
ammonia did not decrease yeast respiration, which may be due to the design of
the YES assay. The small volume of leachate utilized in combination with
frequent mixing may have reduced the ammonia concentrations in the microplate
wells to below toxic levels. The leachates also contain a variety of other
inorganic and organic toxicants with the potential for adversely affecting overall
yeast cell health. The influence of other substances on biochemical pathways in
yeast cells was not investigated, although potentially they may reduce the ability
of yeast cells to correctly identify hormonal activity.
Nakano et al. (2002) reported that decreased yeast cell proliferation was
associated with nearly 25 % of the substances that exhibited weakly estrogenic
effects. This suggests that in some cases weakly estrogenic responses may be
attributed to toxic effects. Additionally, leachate toxicity can be a confounding
factor when determining hormonal activity by preventing the transcription and
translation required for quantification of an interaction with the hER (Layton et al.,
2002).
Eguchi et al. (2003) evaluated the recovery of E2 activity by standard
addition experiments. A known volume of the positive control, E2, was added to
the test solution, and the solution was then assayed for hormonal activity. The
reduced activity of E2 in spiked test solutions, indicated an inhibitory influence on
the yeast cells (Eguchi et al., 2003). As previously discussed, the methanol
extracts of the MSW landfill leachates, in this work, were spiked with E2 to
quantify the recovery of hormonal activity. Beresford et al. (2000) addressed
concerns for yeast cell viability by tracking metabolic activity with the non-

246
fluorescent stain, carboxyfluorescein (cFDA). Esterase activity resulted in the
metabolism of cFDA to the fluorescent compound carboxyfluorescein (cF). The
fluorescent compound is retained in viable yeast cells, but escapes from cells
that have been compromised, and with this assay viability of yeast cells was
demonstrated (Beresford et al., 2000).
Coliform bacteria
MSW landfill leachates contain large amounts of coliform bacteria
resulting from the composition of the waste materials and deposition conditions.
As previously discussed (Chapter 2), these bacteria produce enzymes that are
responsible for the microbial degradation of conjugated estrogens in domestic
wastewater treatment plants (Ternes et al., 1999). The specificity of microbial
enzymes is exploited for the identification and quantification of total and fecal
coliform bacteria. Total coliform bacteria are identified by the production of p-
galactosidase, while fecal bacteria are identified by p-glucuronidase (APHA,
1999; Bitton et al., 1995). In the YES assay, the presence of hormonally active
compounds is indicated by the production of the p-galactosidase enzyme.
Therefore, the presence of total conforms in the MSW landfill leachates provides
another source for the p-galactosidase enzyme, which may interfere with the
YES assay results.
To quantify the presence of total and fecal coliform in the MSW landfill
leachates a rapid enzymatic assay, ColiPAD, was used (Bitton etal., 1995). The
ColiPAD assay contains a pad saturated with the enzyme substrates
chlorophenol red galactopyranoside (CPRG) and 4-methylumbelliferone

247
Table 7-8. Total and fecal conform bacteria determined in MSW landfill leachates
with results expressed as the most probable number (MPN) of
bacteria/100 ml of leachate
Coliform Bacteria
Site
Date
(MPN/100 ml)a
Total
£. coli
LF 1
Sept. 2001
Nov. 2001
ND6
11
ND
0
LF 4
Feb. 2002
500
500
Sept. 2001
2800
60
LF 5
Nov. 2001
900
0
March 2002
ND
ND
LF 6
Feb. 2002
22,000
5,000
LF 7
Feb. 2002
400
40
Sept. 2001
900
300
Nov. 2001
2,400
4
LF 8
Dec. 2001
1700
70
Jan. 2002
270
20
Feb. 2002#1
500
400
Feb. 2002#2
ND
ND
LF 12
Nov. 2001
50
4
March. 2002
ND
ND
LF 14
Feb. 2002
28,000
3,000
Lake Alice
Jan. 2001
ND
ND
Lake Beverly
Jan. 2001
ND
ND
WWTP Inf
Sept. 2001
TNTC
230,000
WWTP Eff
Sept. 2001
80,000
500
SW
Sept. 2001
70
0
treatment plant influent; |1WWTP Eft, wastewater treatment plant effluent; 'SW, stormwater.
glucuronide (MUG). The assay is conducted by preparing five dilutions of the
leachate in 0.9 % NaCI, transferring 1-ml aliquots of leachate to tubes with 1 ml
of double strength lauryl tryptose broth-MUG (LTB-MUG, Difco), and incubating
for 22 hours at 35 °C. Enzyme activity was determined by adding 10-pl aliquots
from each tube to the substrate soaked pad and then incubating for two hours at
44.5 ° C. Purple spots on the pad indicated the presence of p-galactosidase and,

248
thus, total coliform bacteria. Following the addition of an alkaline buffer, 2-amino-
2-methyl-1-propanol (Sigma) to the purple spots, the purple spots were observed
for fluorescence with a long-wave ultraviolet lamp. Fluorescence indicated the
presence of p-glucuronidase enzyme and, thus, the presence of E. coli.
The results of the ColiPAD assay with MSW landfill leachates revealed a
varied distribution of total and E.coli bacteria (Table 7-8). By comparison, the
leachates from sites 6 and 14 contained the highest density of total coliform
bacteria. In the case of the leachates from site 6, the high number of conforms
was attributable to the co-disposal of domestic wastewater sludge with the MSW.
The leachates from site 14 were collected from a leachate storage pond, and the
large population of various bird species in and around the area may have
contributed to the increased microflora.
CPRG activity issue
Vanderperren et al. (2001) reported that with an increased incubation
period there was a decrease in sensitivity for the YES assay, which they
attributed to estrogenic activity caused by exposure to the chromogenic substrate
CPRG. In contrast, Beresford et al. (2000) reported an increased sensitivity with
increased assay duration and no toxicity. DeBoever et al. (2001) described a
modification to the Routledge and Sumpter (1996b) method to eliminate issues
related to possible hormonal effects from CPRG. In their modification, DeBoever
et al. (2001) described the addition of CPRG following the incubation interval in
conjunction with cycloheximide, a protein inhibitor. The cycloheximide inhibited
production of the enzyme, which allowed for the quantification of p-galactosidase

249
produced in the absence of CPRG. Subsequent to their original (Routledge and
Sumpter, 1996b) publication, a decrease of the incubation temperature for the
final day of the assay has been indicated (Elsby et al., 2001). However, the
modification suggested by DeBoever et al. (2001) has not been incorporated into
the widely utilized YES protocol, and there have been no further reports of
estrogen-like effects from CPRG or any other enzyme substrate.
Assessment of organic solvents
There have been reports of toxic effects in yeast cells exposed to solvent
extracts of some samples (Vinggaard et al., 2000) indicating the need for
constant monitoring of the overall health of yeast cells during YES assays. For
the determination of hormonal activity, various solvents have been evaluated for
the preparation of reference compounds and suspect chemicals. While some
researchers have encouraged the use of the solvent dimethyl sulfoxide (DMSO)
(Soto et al, 1992), others have shown a direct adverse influence of this solvent
on the outcome of the assay (Beresford et al., 2000).
As with any assay for hormonal activity, the purity of each reagent is
critical. In some cases, trace contaminants have been responsible for the
erroneous labeling of substances as hormonally active (Miller et al., 2001). In the
YES assay, stock solutions of E2 were prepared in high purity solvents. Initial
investigations with the YES assay, revealed the importance of solvent purity.

CHAPTER 8
CONCLUSIONS
The purpose of this research was to characterize the chemical composition
and evaluate the biological effects of leachates from MSW landfills in Florida. To
meet the goals of this research, MSW landfill leachates were tested in acute and
chronic exposures with a suite of bioassays. Additionally, using a yeast reporter
assay, the hormonal activity of the MSW landfill leachates was evaluated. Based
on the results of this investigation the following conclusions were drawn:
• According to a suite of bioassays (C. dubia, P. subcapitata, D. pulex, and
Microtox), Florida MSW landfill leachates were toxic. The range of mean
TU values reported were from 2.9 to 50.2 with C. dubia and from 1 to 54.9
with P. subcapitata. The toxicity of the Florida leachates was similar to that
reported for landfill leachates world-wide.
• The bioassay results established the relative sensitivity of each assay to the
MSW landfill leachates. The sensitivity of the two invertebrate and the algal
assay were comparable, while the Microtox assay was insensitive to the
leachate toxicants.
• The results of the bioassays (C. dubia, P. subcapitata, D. pulex, and
Microtox) with the MSW landfill leachates established a range of biological
effects, which provide a statewide database for landfill operators,
regulators, and other research investigators.
250

251
• The chemical/physical characteristics of the MSW landfill leachates are site-
specific and were influenced by waste composition, landfill age, and rainfall.
• The acute and chronic toxicity of MSW landfill leachates from a closed
landfill (Site 3) were significantly lower than leachates collected from a
younger landfill (Site 2).
• MSW landfill leachates are toxic and contain a complex mixture of organic
and inorganic contaminants. Although the bioassay results were correlated
with several chemical characteristics of the leachate, the wide variation in
these characteristics made identifying relationships difficult.
• Ammonia concentrations were correlated with the results of the P.
subcapitata (R2 = 0.69) and C.dubia (R2 = 0.62) bioassays, during the initial
investigation of leachate toxicity (Chapter 3). When additional MSW landfill
leachates were evaluated, the relationship with ammonia increased for P.
subcapitata (R2= 0.85) and decreased for C. dubia (R2 = 0.49) (Chapter 4).
• A TIE investigation with MSW landfill leachates implicated ammonia as a
probable toxicant (Chapter 6). Furthermore, other properties of leachate
toxicants were characterized as non-volatile, ionized, and hydrophobic.
• MSW landfill leachates displayed low toxicity in the heavy metal specific
assay, MetPLATE.

252
MSW landfill leachates were characterized by a high heavy metal binding
capacity (HMBC). The binding affinity for the MSW landfill leachates with
heavy metals followed the order of copper > mercury >zinc.
HMBC fractionation scheme indicated that solids, organics, and hardness
were factors that contributed to metal binding by leachates; however, other
unidentified factors contributed to the overall HMBC.
Raw MSW landfill leachates and their methanol extracts were shown to be
hormonally active in a yeast reporter assay, although the responses of the
leachates were variable. The hormonal activity of the raw leachates ranged
from below the detection limit of the assay (2.5 ng E2/L) to 45.7 ng E2/L.
Results suggested that unidentified substances in MSW landfill leachates
masked the recovery of hormonal activity in some leachate concentrates.
This was confirmed by the variable recovery of 17 (i-estradiol (E2) activity
from the leachate extracts.
Leachate treatment reduced the hormonal activity of the MSW landfill
leachates.

LIST OF REFERENCES
Adamsson, M„ G. Dave, L. Forsberg, and B. Guterstam. 1998. Toxicity
identification evaluation of ammonia, nitrite and heavy metals at the
Stensund wastewater aquaculture plant, Sweden. Water Science and
Technology 38(3): 151-157.
Adlercreutz, H., S.L. Gorbach, B.R. Goldin, M.N. Woods, J.Y. Dwyer and E.
Hamalainen. 1994. Estrogen metabolism and excretion in Oriental and
Caucasian women. Journal of the National Cancer Institute 86:1076-
1082.
Albro, P.W., C.J.T. Corbett, J.L. Shroeder, S. Jordan, H.B. Matthews. 1982.
Pharmacokinetics, interactions with macromolecules and species
differences in metabolism of DEHP. Environmental Health Perspectives
45: 19-25.
Alkalay, D., L. Guerrero, J.M. Lema, R. Mendez, and R. Chamy. 1998. Anaerobic
treatment of municipal sanitary landfill leachates: the problem of refractory
and toxic compounds. World Journal of Microbiology and Biotechnology
14: 309-320.
Allen, Y., A.P. Scott, R. Matthiessen, S. Haworth, J.E. Thain, and S. Feist. 1999.
Survey of estrogenic activity in United Kingdom estuarine and coastal
waters and its effects on gonadal development of the flounder Platichthys
flesus. Environmental Toxicology and Chemistry 18(8): 1791-1800.
Al-Muzaini, S., M. U. Beg, and K. Musimani. 1995. Characterization of landfill
leachates at a waste disposal site in Kuwait. Environment International
21(4): 399-405.
Andersen, H.R., B. Halling-Sorensen, and K.O. Kusk. 1999. A parameter for
detecting estrogenic exposure in the copepod Acartia tonsa.
Ecotoxlcology and Environmental Safety 44: 56-61.
Anderson, H.B. and J.A. Buckley. 1998. Acute toxicity of ammonia to
Ceriodaphnia dubia and a procedure to improve control survival. Bulletin
of Environmental Contamination and Toxicology 61: 116-122.
253

254
Angus, R.A., S.A. Weaver, J.M. Grizzle, and R.D. Watson. 2002. Reproductive
characteristics of male mosquitofish (Gambusia affinis) inhabiting a small
southeastern U.S. river receiving treated domestic sewage effluent.
Environmental Toxicology and Chemistry 21(7): 1404-1409.
Ankley, G.T., A. Katko, and J. Arthur. 1990. Identification of ammonia as an
important sediment-associated toxicant in the lower Fox river and Green
Bay, Wisconsin. Environmental Toxicology and Chemistry 9: 312-322.
APHA.1999. Standard Methods for the Examination of Water and Wastewater.
Author, Washington, DC.
Argese, E., C. Bettiol, P. Miaña, L. luzzolino, and G. Giurin. 1996.
Submitochondrial particles as in vitro biosensors of heavy metal toxicity.
Journal fo Aquatic Ecosystem Health 5:125-134.
Arikawa, Y., K. Ikebukuro and I. Karube. 1998. Microbial biosensors based on
respiratory inhibition, pp. 225-235. In Enzyme and Microbial Biosensors:
Techniques and Protocols. A. Mulchandani and K.R. Rogers (eds.).
Humana Press, Totowa.
Ashby, J. 2003. The leading role and responsibility of the international scientific
community in test development. Toxicology Letters 140-141: 37-42.
Ashby, J. 2000. Getting the problem of endocrine disruption into focus: the need
for a pause for thought. APMIS 108: 805-813.
Assmuth, T.W. 1996. Comparative risk analysis of waste site toxicants by indices
based on concentration distributions, fluxes, environmental fate and
critical effects. Journal of Hazardous Materials 48:121-135.
Atwater, J.W., S. Jasper, D.S. Mavinic, and F.A. Koch. 1983. Experiments using
daphnia to measure landfill leachate toxicity. Water Research 17(12):
1855-1861.
Baker, V.A. 2001. Endocrine disruptors - testing strategies to assess human
hazard. Toxicology in Vitro 15: 413-419.
Balaguer, P., F. Francois, F. Comunale, H. Fenet, A-M. Boussioux, M. Pons, J-C.
Nicolas, and C. Casellas. 1999. Reporter cell lines to study the estrogenic
effects of xenoestrogens. Science of the Total Environment 233(1-3):
47-56.
Baldwin, W.S., S.E. Graham, D. Shea, and G.A. LeBlanc. 1997. Metabolic
androgenization of female Daphnia magna by the xenoestrogen 4-
nonylphenol. Environmental Toxicology and Chemistry 16(9): 1905-1911.

255
Baldwin, W.S., D.L. Milam, and G.A. LeBlanc. 1995. Physiological and
biochemical perturbations in Daphnia magna following exposure to the
model environmental estrogen diethylstilbestrol. Environmental Toxicology
and Chemistry 14(6): 945-952.
Balmelli-Gallacchi, P., F. Schoumacher, J. W. Liu, U. Eppenberger, H. Mueller,
and D. Picard. 1999. A yeast-based bioassay for the determination of
functional and non-functional estrogen receptors. Nucleic Acids Research
27(8): 1875-1881.
Barlaz, M.A. 1997. Microbial studies of landfills and anaerobic refuse
decomposition, pp 541-557. In Manual of Environmental Microbiology.
C.J. Hurst, G.R. Knudsen, M.J. Mclnerney, L.D. Stetzenbach, and
M.V. Walter (eds.). American Society for Microbiology, Washington, DC.
894 pp.
Barlaz, M.A., A.P. Rooker, P. Kjeldsen, M.A. Gabr, and R.C. Borden. 2002.
Critical evaluation of factors required to terminate the postclosure
monitoring period at solid waste landfills. Environmental Science and
Technology 36(16): 3457-3464.
Baronti, C., R. Curini, G. D Ascenzo, A. Di Corda, A. Gentili, and R. Samperi.
2000. Monitoring natural and synthetic estrogens at activated sludge
sewage treatment plants and in a receiving river water. Environmental
Science and Technology 34(24): 5059-5066.
Bauer, M.J. and R. Herrmann. 1997. Estimation of the environmental
contamination by phthalic add esters leaching from household wastes.
The Science of the Total Environment 208: 49-57.
Baun, A., S.D. Jensen, P.L. Bjerg, T.H. Christensen, and N. Nyholm. 2000.
Toxicity of organic chemical pollution in groundwater downgradient of a
landfill (Grindsted, Denmark). Environmental Science and Technology 34:
1647-1652.
Baun, A., L. Kloft, P.L. Bjerg, and N. Nyholm. 1999. Toxidty testing of organic
chemicals in groundwater polluted with landfill leachate. Environmental
Toxicology and Chemistry 18(9): 2046-2053.
Beckman Instruments. 1982. Microtox System Operating Manual. No. 015-555-
879. Author, Carlsbad, CA.
Beg, M.U. and S. Al-Muzaini. 1998. Genotoxicity assay of landfill leachates.
Environmental Toxicology and Water Quality 13: 127-131.

256
Behnisch, P.A., K. Fujii, K. Shiozaki, I. Sawakami and S-l. Sakai. 2001.
Estrogenic and dioxin-like potency in each step of a controlled landfill
leachate treatment plant in Japan. Chemosphere 43: 977-984.
Belfroid, A.C., A. Van der Horst, A.D. Vethaak, A.J. Schafer, G.B.J. Rijs, J.
Wegener,and W.P. Cofino. 1999. Analysis and occurrence of estrogenic
hormones and their glucuronides in surface water and waste water in the
Netherlands. The Science of the Total Environment 225: 101-108.
Benedetti, M., J.F. Ranville, M. Ponthieu and J.P. Pinheiro. 2002. Field-flow
fractionation characterization and binding properties of particulate and
colloidal organic matter from the Rio Amazon and Rio Negro. Organic
Chemistry 33: 269-279.
Beresford, N., E.J. Routledge, C.A. Harris and J.P. Sumpter. 2000. Issues arising
when interpreting results from an in vitro assay for estrogenic activity.
Toxicology and Applied Pharmacology 162: 22-33.
Bierkens, J., G. Klein, P. Corbisier, R. Van Den Heuvel, L. Verschaeve, R.
Weltens, and G. Schoeters. 1998. Comparative sensitivity of 20 bioassays
for soil quality. Chemosphere 37(14-15): 2935-2947.
Bitton, G. and B.J. Dutka. 1986. Toxicity Testing Using Microorganisms, Vol. 1.
CRC Press, Boca Raton, FL.
Bitton, G., B. Koopman and H.D. Wang. 1984. Baker's yeast assay for testing
heavy metal toxicity. Bulletin of Environmental Contamination and
Toxicology 32: 80.
Bitton, G. and B. Koopman. 1982. Tetrazolium reduction-malachite green method
for assessing the viability of filamentous bacteria in activated sludge.
Applied and Environmental Microbiology 43: 964-966.
Bitton, G., K. Rhodes, and B. Koopman. 1996. CerioFASTâ„¢: an acute toxicity
test based on Ceriodaphnia dubia feeding behavior. Environmental
Toxicology and Chemistry 15: 123-125.
Bitton, G., B. Koopman, and K. Jung. 1995. An assay for the enumeration of total
conforms and Escherichia coli in water and wastewater. Water
Environment Research 67: 906-909.
Bitton, G., K. Jung, and B. Koopman. 1994. Evaluation of a microplate assay
specific for heavy metal toxicity. Archives of Environmental Contamination
and Toxicology 27: 25-28.

257
Blaise, C„ F. Gagne, J. Pellerin, P.D. Hansen. 1999. Determination of
vitellogenin-like properties in Mya arenaria hemolymph (Saguenay Fjord,
Canada): a potential biomarker for endocrine disruption. Environmental
Toxicology 14: 455-465.
Blount, B.C., M.J. Silva, S.P. Caudill, L.L. Needham, J.L. Pirkle, E.J. Sampson,
G.W. Lucier, R.J. Jackson and J.W. Brock. 2000. Levels of seven urinary
phthalate metabolites in a human reference population. Environmental
Health Perspectives 108(10): 979-982.
Bolt, H.M. 1979. Metabolism of estrogens - natural and synthetic. Pharmacology
and Therapeutics 4: 155-181.
Bolton, K.A. and L.J. Evans. 1991. Elemental composition and speciation of
some landfill leachates with particular reference to cadmium. Water, Air
and Soil Pollution 60: 43-53.
Booth, S.D.J., Urfer, D., Rereira, G., Cober, K.J. 1996. Assessing the impact of a
landfill leachate on a Canadian wastewater treatment plant. Water
Environment Research 68:1179-1186.
Botre, C. F. Botre, F. Mazzei, and E. Podesta. 2000. Inhibition-based biosensors
for the detection of environmental contaminants: determination of 2,4-
dichlorophenoxyacetic acid. Environmental Toxicology and Chemistry
19(12); 2876-2881.
Bowman, C.J., K.J. Kroll, M.J. Hemmer, L.C. Folmar, and N.D. Denslow. 2000.
Estrogen-induced vitellogenin mRNA and protein in Sheepshead minnow
(Cyprinodon variegatus). General and Comparative Endocrinology 120:
300-313.
Boyle, C. and B. Baetz. 1993. Household hazardous wastes: options for
management. Canadian Journal of Civil Engineering 20: 543-549.
Bozkurt, S., L. Moreno and I. Neretnieks. 2000. Long-term processes in waste
deposits. Science of the Total Environment 250: 101-121.
Brack, W. and H. Frank. 1997. A bioassay-directed method for the separation of
volatile compounds from landfill leachates. Chemosphere 34(4): 849-854.
Brown, K.W. and K.C. Donnelly. 1988. An estimation of the risk associated with
the organic constituents of hazardous and municipal waste landfill
leachates. Hazardous Waste & Hazardous Materials 5(1): 1-30.

258
Buffle, J. and G. Horvai. 1998. In situ Monitoring of Aquatic Systems:
Chemical Analysis and Speciation. IUPAC Series on Analytical and
Physical Chemistry of Environmental Systems. Volume 6. John Wiley &
Sons, New York.
Bulich, A.A. 1982. A practival and reliable method for monitoring the toxicity of
aquatic samples. Process Biochemistry: 45-47.
Burlington, H., and V.F. Lindeman. 1950. Effect of DDT on testes and secondary
sex characteristics of white leghorn cockerels. Proceedings of the Society
for Experimental Biology and Medicine 74: 48-51.
Burton, S.A.Q. and I .A. Watson-Craik. 1998. Ammonia and nitrogen fluxes in
landfill sites: applicability to sustainable landfilling. Waste Management
Research 16(1): 41-53.
Calace, N., A. Liberatori, B.M. Petronio, and M. Pietroletti. 2001. Characteristics
of different molecular weight fractions of organic matter in landfill leachate
and their role in soil sorption of heavy metals. Environmental Pollution
113: 331-339.
Cameron, R.D. and F.A. Koch. 1980. Toxicity of landfill leachates. Journal of the
Water Pollution Control Federation 52(4): 760-769.
Campbell, P.G.C. 1995. Interactions between trace metals and aquatic
organisms: a critique of the free-ion activity model, pp. 45-102. In Metal
Speciation and Bioavailability in Aquatic Systems. Tessier A, Turner DR
(eds). Wiley. New York, New York.
Carlsson, H., U. Nlsson, and C. Ostman. 2000. Video display units: an emission
source of the contact allergenic flame retardant triphenyl phosphate in the
indoor environment. Environmental Science and Technology 34(18):
3885-3889.
Carson, R. 1962. Silent Spring. Houghton Mifflin, New York.
Casey, F.X.M., G.L. Larsen, H. Hakk, and J. Simunek. 2003. Fate and transport
of 17[i-Estradiol in soil-water systems. Environmental Science and
Technology 37(11): 2400-2409.
Castagnoli, O., L. Musmeci, E. Zavattiero and M. Chirico. 1990. Humic
substances and humification rate in a municipal refuse disposed of in a
landfill. Water, Air, and Soil Pollution 53: 1-12.

259
Castillo, M. and D. Barcelo. 1999. Identification of polar toxicants in industrial
wastewaters using toxicity-based fractionation with liquid
chromatography/mass spectrometry. Analytical Chemistry 71: 3769-3776.
Causton, H.C., B. Ren, S.S. Koh, C.T. Harbison, E. Kanin, E.G. Jennings, T.l.
Lee, H.L. True, E.S. Lander, and R.A. Young. 2001. Remodeling of yeast
genome expression in response to environmental changes. Molecular
Biology of the Cell 12: 323-337.
Cecen, F. and G. Gursoy. 2000. Characterization of landfill leachates and studies
on heavy metal removal. Journal of Environmental Monitoring 2: 436-442.
CFR. 1996. Protection of the Environment. Title 40 Code of Federal Regulations
Pt. 258.
Chen, C-Y., K-C. Lin, and D-T. Yang. 1997. Comparison of the relative toxicity
relationships based on batch and continuous algal toxicity tests.
Chemosphere 35(9): 1959-1965.
Chen, P.H. 1996. Assessment of leachates from sanitary landfills: impact of age,
rainfall, and treatment. Environment International 22(2): 225-237.
Cheung, K.C., L.M. Chu and M.H. Wong. 1997. Ammonia stripping as a
pretreatment for landfill leachate. Water, Air, and Soil Pollution 94:
209-221.
Cheung, K.C., L.M. Chu, and M.H. Wong. 1993. Toxic effects of landfill leachate
on microalgae. Water, Air and Soil Pollution 69: 337-349.
Chial, B. and G. Persoone. 2002. Cyst-based toxicity tests XlV-application of the
ostracod solid-phase microbiotest for toxicity monitoring of river sediments
in Flanders (Belgium). Environmental Toxicology 17: 533-537.
Chian, E., and F.B. DeWalle. 1976. Sanitary landfill leachates and their
treatment. Journal of the Environmental Engineering Division 4:411-431.
Chichester, D.L. and S. Landsberger. 1996. Determination of the leaching
dynamics of metals from municipal solid waste incinerator fly ash using a
column test. Journal of Air and Waste Management 46: 643-649.
Choe, S.Y., S.J. Kim, H.G. Kim, J.H. Lee, Y. Choi, H. Lee, Y. Kim. 2003.
Evaluation of estrogenicity of major heavy metals. Science of the
Total Environment 312: 15-21.

260
Christensen, J.B., J.J. Botma, and T.H. Christensen. 1999. Complexation of Cu
and Pb by DOC in polluted groundwater: a comparison of experimental
data and predictions by computer speciation models (WHAM and
MINTEQA2). Water Research 33(15): 3231-3238.
Christensen, T.H., R. Kjeldsen, H.J. Albrechtsen, G. Heron, P.H. Nielsen, P.L.
Bjerg, and P.E. Holm. 1994. Attenuation of landfill leachate pollutants in
aquifers. Critical Reviews in Environmental Science and Technology 24:
119
Ciccotelli, M., S. Crippa, and A. Colombo. 1998. Bioindicators for toxicity
assessment of effluents from a wastewater treatment plant. Chemosphere
37(14-15): 2823-2832.
Clement, B., Colin, J.R., Le Du-Delepierre, A. 1997. Estimation of the hazard of
landfills through toxicity testing of leachates 2. Comparison of physico¬
chemical characteristics of landfill leachates with their toxicity determined
with a battery of tests. Chemosphere 35:2783-2796.
Clement, B. and G. Merlin. 1995. The contribution of ammonia and alkalinity to
landfill leachate toxicity to duckweed. Science of the Total Environment
170: 71-79.
Clement, B.; Persoone, G.; Janssen, C.; Le Du-Delepierre, A. 1996. Estimation
of the hazard of landfills through toxicity testing of leachates.
Chemosphere 33: 2303-2320.
Cleuvers, M. and H.T. Ratte. 2002. The importance of light intensity in algal tests
with coloured substances. Water Research 36: 2173-2178.
Clevenger, T.E. and D. Rao. 1996. Mobility of lead in mine tailings due to landfill
leachate. Water, Air, and Soil Pollution 91: 197-207.
Colburn, T., D. Dumanoski, and J.P. Myers. 1996. Our Stolen Future. Penguin
Books, Inc., New York.
Coldham, N.G., M. Dave, S. Sivapathasundaram, D.P. McDonnell, C. Connor,
and M.J. Sauer. 1997. Evaluation of a recombinant yeast cell estrogen
screening assay. Environmental Health Perspectives 105(7): 734-742.
Cooman, K., M. Gajardo, J. Nieto, C. Bornhardt, and G. Vidal. 2003. Tannery
wastewater characterization and toxicity effects on Daphnia spp.
Environmental Toxicology 18: 45-51.

261
Coombe, V.T., K.W. Moore, and M.J. Hutchings. 1999. TIE and TRE: an
abbreviated guide to dealing with toxicity. Water Science and Technology
39(10-11): 91-97.
Coors, A., P.D. Jones, J.P. Giesy, and H.T. Ratte. 2003. Removal of estrogenic
activity from municipal waste landfill leachate assessed with a bioassay
based on reporter gene expression. Environmental Science and
Technology 37 (15): 3430-3434.
Couse, J.F., J. Lindzey, K. Grandien, J-A. Gustafsson, and K.S. Korach. 1997.
Tissue distribution and quantitative analysis of estrogen receptor-a(ERa)
and estrogen receptor-p (ERp) messenger ribonucleic acid in the wild-type
and ERa-knockout mouse. Endocrinology 138(11): 4613-4621.
Crane, M. and M.C. Newman. 2000. What level of effect is a no observed effect?
Environmental Toxicology and Chemistry 19(2): 516-519.
Croley, T.R., R.J. Hughes, B.G. Koenig, C.D Metcalfe, and R.E. March. 2000.
Mass spectrometry applied to the analysis of estrogens in the
environment. Rapid Communications in Mass Spectrometry 14:
1087-1093.
Croue, J.P., M.F. Benedetti, D. Violleau and J.A. Leenheer. 2003.
Characterization and copper binding of humic and nonhumic organic
matter isolated from the South Platte River: evidence for the presence of
nitrogenous binding site. Environmental Science and Technology 37:
328-336.
Darnerud, P.O., G.S. Eriksen, T. Johannesson, P.B. Larsen, and M. Viluksela.
2001. Polybrominated diphenyl ethers: occurrence, dietary exposure, and
toxicology. Environmental Health Perspectives 109 (suppl. 1): 49-68.
D'Ascenzo, G., A. Di Corda, A. Gentili, R. Mancini, R. Mastropasqua, M. Nazzari
and R. Samperi. 2003. Fate of natural estrogen conjugates in municipal
sewage transport and treatment facilities. Science of the Total
Environment 302: 199-209.
Davies, C.M., S.C. Apte, S.M. Peterson, and J.L. Stauber. 1994. Plant and algal
interference in bacterial p-D-galactosidase and p-D-glucuronidase assays.
Applied and Environmental Microbiology 60(11): 3959-3964.
Deanovic, L., V.M. Connor, A.W. Knight and K.J. Maier. 1999. The use of
bioassays and toxicity identification evaluation (TIE) procedures to assess
recovery and effectiveness of remedial activities in a mine drainage-
impacted stream system. Archives of Environmental Contamination and
Toxicology 36: 21-27.

262
Degen, G.H. and H.M. Bolt. 2000. Endocrine disruptors: update on
xenoestrogens. International Archives of Occupational and Environmental
Health 73: 433-441.
Desbrow, C., E.J. Routledge, G.C. Brighty, J.P. Sumpter and M. Waldock. 1998.
Identification of estrogenic chemicals in STW effluent. 1. chemical
fractionation and in vitro biological screening. Environmental Science and
Technology 32(11):1549-1558.
Devare, M. and M. Bahadir. 1994. Biological monitoring of landfill leachate using
plants and luminescent bacteria. Chemosphere 28(2): 261-271.
Diel, P., K. Smolnikar, and H. Michna. 1999. In vitro test systems for the
evaluation of the estrogenic activity of natural products. Planta Medica 65:
197-203.
DiPaola, R.S., H. Zhang, G.H. Lambert, R. Meeker, E. Licitra, M.M. Rafi, B.T.
Zhu, H. Spaulding, S. Goodin, M.B. Toledano, W.N. Hait and M.A. Gallo.
1998. Clinical and biologic activity of an estrogenic herbal combination
(PC-SPES) in prostate cancer. The New England Journal of Medicine
339(12): 785-791.
Dizer, H., B. Fischer, I. Sepulveda, E. Loffredo, N. Senesi, F. Santana, P.D.
Hansen. 2002. Estrogenic effect of leachates and soil extracts from
lysimeters spiked with sewage sludge and reference endocrine disrupters.
Environmental Toxicology 17:105-112.
Doherty, F. G.; Qureshi, A. A.; Razza, J. B. 1999. Comparison of the
Ceriodaphnia dubia and Microtoxâ„¢ inhibition tests for toxicity assessment
of industrial and municipal wastewaters. Environmental Toxicology 14:
375-382.
Dyer, S.D., J.R. Lauth, S.W. Morrall, R.R. Herzog, and D.S. Cherry. 1997.
Development of a chronic toxicity structure-activity relationship for alkyl
sulfates. Environmental Toxicology and Water Quality 12(4): 295-303.
Eguchi, K., M. Ozawa, Y.S. Endoh, J. Nishikawa, T. Nishihara, K. Goto, and H.
Yoshimura. 2003. Validity test for a yeast two-hybrid assay to screen for
estrogenic activity, and its application to insecticides and disinfectants for
veterinary use. Bulletin of Environmental Contamination and Toxicology
70: 226-232.
Ejlertsson, J., M L. Nilsson, H. Kylin, A. Bergman, L. Karlson, M. Oquist and B.H.
Svensson. 1999. Anaerobic degradation of nonylphenol mono- and
diethoxylates in digestor sludge, landfilled municipal solid waste, and
landfilled sludge. Environmental Science and Technology 33: 301-306.

263
Elsby, R., J. Ashby, J.P. Sumpter, A.N. Brooks, W.D. Pennie, J.L. Maggs, P.A.
Lefevre, J. Odum, N. Beresford, D. Patón, and B.K. Park. 2000. Obstacles
to the prediction of estrogenicity from chemical structure: assay-mediated
metabolic transformation and the apparent promiscuous nature of the
estrogen receptor. Biochemical Pharmacology 60:1519-1530.
Elsby, R., J.L. Maggs, J. Ashby, D. Patón, J.P. Sumpter, and B.K. Park. 2001.
Assessment of the effects of metabolism on the estrogenic activity of
xenoestrogens: a two-stage approach coupling human liver microsomes
and a yeast estrogenicity assay. Journal of Pharmacology and
Experimental Therapeutics 296(2): 329-337.
El-Tanani, M.K.K. and C.D. Green. 1997. Two separate mechanisms for ligand-
independent activation of the estrogen receptor. Molecular Endocrinology
11: 928-937.
Emerson, K., R.C. Russo, R.E. Lund, and R.V. Thurston. 1975. Aqueous
ammonia equilibrium calculations: effect of pH and temperature. Journal of
Fisheries Research Board of Canada 32: 2379-2383.
Environmental Working Group. 2002. Not too pretty; phthalates, beauty products
and the FDA.
http://www.ewg.org/reports/nottoopretty/NotTooPretty_final.pdf (retrieved
October 2003).
Ernst, W. R.; Hennigar, P.; Doe, K.; Wade, S.; Julien, G. 1994. Characterization
of the chemical constituents and toxicity to aquatic organisms of a
municipal landfill leachate. Water Pollution Research Journal of Canada
29: 89-101.
Eullaffroy, P. and G. Vernet. 2003. The F684/F735 chlorophyll fluorescence ratio:
a potential tool for rapid detection and determination of herbicide
phytotoxicity in algae. Water Research 37: 1983-1990.
FAC. 1994a. Surface Water Quality Standards. Chapter 62. Florida
Administrative Code. Section 62-302.
FAC. 1994b. Division of Waste Management: Description of Organization.
Chapter 62. Florida Administrative Code. Sect. 62-300.
Fang, H., W. Tong, R. Perkins, A.M. Soto, N.V. Prechtl, and D M. Sheehan.
2000. Quantitative comparisons of in vitro assays for estrogenic activities.
Environmental Health Perspectives 108(8): 723-729.

264
Farre, M. and D. Barcelo. 2003. Toxicity testing of wastewater and sewage
sludge by biosensors, bioassays, and chemical analysis. Trends in
Analytical Chemistry 22(5): 299-310.
Fatoki, O.S. and A. Noma. 2002. Solid phase extraction methods for selective
determination of phthalate esters in the aquatic environment. Water, Air,
and Soil Pollution 140: 85-98.
Fawell, J.K. and J.K. Chipman. 2001. Potential endocrine disrupting substances
from materials in contact with drinking water. Journal of the Chartered
Institution of Water and Environmental Management 15(2): 92-96.
FDEP. 2000. Solid Waste Management Annual Report; Florida Department of
Environmental Protection, Tallahassee, FL.
FDEP. 1997. Selenastrum capricomutum Chronic Toxicity Testing Methods.
SOP#TA-07.10. Florida Department of Environmental Protection,
Tallahassee, FL.
Ferrari, B„ C.M. Radetski, A-M. Veber, and J-F. Ferard. 1999. Ecotoxicological
assessment of solid wastes: a combined liquid- and solid-phase testing
approach using a battery of bioassays and biomarkers. Environmental
Toxicology and Chemistry 18(6): 1195-1202.
Fingerman, M., N.C. Jackson, and R. Nagabhushanam. 1998. Hormonally-
regulated functions in crustaceans as biomarkers of environmental
pollution. Comparative Biochemistry and Physiology Part C 120: 343-350.
Finkelstein, J.S., W.F. McCully, D.T. MacLaughlin, J.E. Godine, and W.F.
Crowley Jr. 1988. The morticians's mystery. Gynecomastia and reversible
hypogonadotropic hypogonadism in an embalmer. New England Journal
of Medicine 318: 961-965.
Fliss, A.E., S. Benzano, J. Rao, and A.J. Captan. 2000. Control of estrogen
receptor ligand binding by Hsp90. Journal of Steroid Biochemistry and
Molecular Biology 72: 223-230.
Flyhammer, P. 1997. Estimation of heavy metal transformations in municipal
solid waste. Science of the Total Environment 198; 123-133.
Foster, P.M., M.W. Cook, L.V. Thomas, D.G. Walters, and S.D. Gangoli. 2000.
Differences in urinary metabolic profile from di-n-butyl phthalate-treated
rats and hamsters. A possible explanation for species differences in
susceptibility to testicular atrophy. Drug Metabolism and Disposition 11:
59-61.

265
Fotsis, T. and H. Adlercreutz. 1987. The multicomponent analysis of estrogens in
urine by ion exchange chromatography and GCMS-1. Quantification of
estrogens after initial hydrolysis of conjugates. Journal of Steroid
Biochemistry 28: 203-213.
Fotsis, T., P. Jarvenpaa, H. Adlercreutz. 1980. Purification of urine for
quantification of the complete estrogen profile. Journal of Steroid
Biochemistry 12: 503-508.
Fraser, J.K., C.A. Butler, M.H. Timperley, and C.W. Evans. 2000. Formation of
copper complexes in landfill leachate and their toxicity to zebrafish
embryos. Environmental Toxicology and Chemistry 19(5): 1397-1402.
Fromme, H., T. Kuchler, T. Otto, K. Pilz, J. Muller and A. Wenzel. 2002.
Occurrence of phthalates and bisphenol a and f in the environment. Water
Research 36: 1429-1438.
Gabrielson, J., I. Kuhn, P. Colque-Navarro, M. Hart, A. Iversen, D. McKenzie,
and R. Mollby. 2003. Microplate-based microbial assay for risk
assessment and (eco)toxic fingerprinting of chemicals. Analytics Chimica
Acta 85:121-130.
Gagne, F., D.J. Marcogliese, C. Blaise, A.D. Gendron. 2001. Occurrence of
compounds estrogenic to freshwater mussels in surface waters in an
urban area. Environmental Toxicology 16: 260-268.
Gaido, K.W., L.S. Leonard, S.Lovell, J.C. Gould, D. Babal, C.J. Porter, and D.P.
McDonnell. 1997. Evaluation of chemicals with endocrine modulating
activity in a yeast-based steroid hormone receptor gene transcription
assay. Toxicology and Applied Pharmacology 143: 205-212.
Game, J., B. Vollat, D.K. Nguyen, M. Bray, B. Migeon, and A. Kosmala. 1996.
Ecotoxicological and chemical characterization of municipal wastewater
treatment plant effluents. Water Science and Technology 33(6): 83-91.
Gasch, A.P., P.T. Spellman, C.M. Kao, O. Carmel-Harmel, M B. Eisen, G. Storz,
D. Botstein, and P.O. Brown. 2000. Genomic expression programs in the
response of yeast cells to environmental changes. Molecular Biology of
the Cell 11:4241-4257.
Ge, Y., P. Murray, S. Sauve and W. Hendershot. 2002. Low metal bioavailability
in a contaminated urban site. Environmental Toxicology and Chemistry
21(5): 954-961.
Geis, S.W., K.L. Fleming, E.T. Korthals, G. Searie, L. Reynolds, and D.A. Karner.
2000. Modifications to the algal growth inhibition test for use as a
regulatory assay. Environmental Toxicology and Chemistry 19(1): 36-41.

266
Gensemer, R.W. and R.C. Playle. 1999. The bioavailability and toxicity of
aluminum in aquatic environments. Critical Reviews in Environmental
Science and Technology 29(4): 315-450.
George, D.B., S.G. Berk, V.D. Adams, R.S. Ting, R.O. Roberts, L.H. Parks, and
R.C. Lott. 1995. Toxicity of alum sludge extracts to a freshwater alga,
protozoan, fish, and marine bacterium. Archives of Environmental
Contamination and Toxicology 29: 149-158.
Gettinby, J. H.; Sarsby, R. W.; Nedwell, J. C. 1996. The composition of leachate
from landfilled refuse. Proceeding of the Institue of Civil Engineer-
Municipal Engineer 115: 47-59.
Gounaris, V., P.R. Anderson and T.M. Holsen. 1993. Characteristics and
environmental significance of colloids in landfill leachate. Environmental
Science and Technology 27(7): 1381-1387.
Grabowski, L.A., J.L.J. Houpis, W.l. Woods, and K.A. Johnson. 2001. Seasonal
bioavailability of sediment-associated heavy metals along the Mississippi
river floodplain. Chemosphere 45: 643-651.
Granek, V. and J. Rishpon. 2002. Detecting endocrine-disrupting compounds by
fast impedence measurements. Environmental Science and Technology
36(7): 1574-1578.
Gray, L.E.Jr. 1998a. Xenoendocrine disrupters: laboratory studies on male
reproductive effects. Toxicology Letters 102-103: 331-335.
Gray, L.E.Jr. 1998b. Tiered screening and testing strategy for xenoestrogens and
antiandrogens. Toxicology Letters 102-103: 677-680.
Guengerich, F.P. 1990. Metabolism of 17a - ethynylestradiol in humans. Life
Sciences 47: 1981-1988.
Guenther, K., V. Heinke, B. Thiele, E. Kleist, H. Prast, and T. Raecker. 2002.
Endocrine disrupting nonylphenols are ubiquitous in food. Environmental
Science and Technology (8): 1676-1680.
Guo, L., P.H. Santschi, and S.M. Ray. 2002. Metal partitioning between colloidal
and dissolved phases and its relation with bioavailability to American
oysters. Marine Environmental Research 54: 49-64.
Gutendorf, B. and J. Westendorf. 2001. Comparison of an array of in vitro assays
for the assessment of the estrogenic potential of natural and synthetic
estrogens, phytoestrogens, and xenoestrogens. Toxicology 166: 79-89.

267
Hale, R.C., M.J. La Guardia, E.P. Harvey, T.M. Mainor, W.H. Duff, and M.O.
Gaylor. 2001. Polybrominated diphenyl ether flame retardants in Virginia
freshwater fishes (USA). Environmental Science and Technology 35(23):
4585-4591.
Hamilton, J.D., K.H. Reinert, J.V. Hagan, and W.V. Lord. 1995. Polymers as solid
waste in municipal landfills. Journal of the Air and Waste Management
Association 45: 247-251.
Harper, S.C., R. Manoharan, D.S. Mavinic, C.W. Randall. 1996. Chromium and
nickel toxicity during the biotreatment of high ammonia landfill leachate.
Water Environment Research 68(1): 19-24.
Harris, C.A., P. Henttu, M.G. Parker and J.P. Sumpter. 1997. The estrogenic
activity of phthalate esters in vitro. Environmental Health Perspectives
105(8): 802-811.
Hedstrom, A. 2001. Ion exchange of ammonium in zeolites: a literature review.
Journal of Environmental Engineering 127(8): 673-681.
Heijerick, D.G., C.R. Janssen, and W.M. DeCoen. 2003. The combined effects of
hardness, pH, and dissolved organic carbon on the chronic toxicity of Zn
to D. magna: development of a surface response model. Archives of
Environmental Contamination and Toxicology 44: 210-217.
Helma, C., V. Mersch-Sundermann, V.S. Houk, U. Glasbrenner, C. Klein, L.
Wenquing, F. Kassie, R. Schulte-Hermann, and S. Knasmuller. 1996.
Comparative evaluation of four bacterial assays for the detection of
genotoxic effects in the dissolved water phases of aqueous matrices.
Environmental Science and Technology 30(3): 897-907.
Hoke, R.A., W.R. Gala, J.B. Drake, and J.P. Giesy. 1992. Bicarbonate as a
potential confounding factor in cladoceran toxicity assessments of pore
water from contaminated sediments. Canadian Journal of Fisheries and
Aquatic Science 49: 1633-1640.
Holbrook, R.D., J.T. Novak, T.J. Grizzard and N.G. Love. 2002. Estrogen
receptor agonist fate during wastewater and biosolids treatment
processes: a mass balance analysis. Environmental Science and
Technology 36 (21): 4533-4539.
Holm, P.E., S. Andersen and T.H. Christensen. 1995. Speciation of dissolved
cadmium: interpretation of dialysis, ion exchange and computer
(GEOCHEM) methods. Water Research 29(3): 803-809.

268
Huang, C-H., and D. Sedlak. 2001. Analysis of estrogenic hormones in municipal
wastewater effluent and surface water using enzyme-linked
immunosorbent assay and gas chromatography/tandem mass
spectrometry. Environmental Toxicology and Chemistry 20(1); 133-139.
Huang, F., G. Bitton and l-C. Kong. 1999. Determination of the heavy metal
binding capacity of aquatic samples using MetPLATEâ„¢: a preliminary
study. The Science of the Total Environment 234: 139-145.
Hughes Jr., C.L.1988. Phytochemical mimicry of reproductive hormones and
modulation of herbivore fertility by phytoestrogens. Environmental Health
Perspectives 78: 171-175.
Jacobsen, B.N. and T. Guildal. 2000. Novel aspects for management of
xenobiotic compounds in wastewater treatment plants - linking theory, field
studies, regulation, engineering, and experience. Water Science and
Technology 42(7-8): 315-322.
Jensen, D.L. and T.H. Christensen. 1999. Colloidal and dissolved metals in
leachates from four Danish landfills. Water Research 33(9): 2139-2147.
Jobling, S. N. Beresford, M. Nolan, T. Rodgers-Gray, G.C. Brighty, J.P. Sumpter
and C.R. Tyler. 2002. Altered sexual maturation and gamete production in
wild roach (Rutilus rutilus) living in rivers that receive treated sewage
effluents. Biology of Reproduction 66: 272-281.
Jobling, S., T. Reynolds, R. White, M.G. Parker and J.P. Sumpter. 1995. A
variety of environmentally persistent chemicals, including some phthalate
plasticizers, are weakly estrogenic. Environmental Health Perspectives
103(6): 582-587.
Jobling, S. and J.P. Sumpter. 1993. Detergent components in sewage effluent
are weakly oestrogenic to fish: an in vitro study using rainbow trout
(Oncorhynchus mykiss) hepatocytes. Aquatic Toxicology 27: 361-372.
Johnson, A.C., A. Belfroid, A. Di Corcia. 2000. Estimating steroid oestrogen
inputs into activated sludge treatment works and observations on their
removal from the effluent. Science of the Total Environment 256:
163-173.
Jop, K.M. and A.M. Askew. 1994. Toxicity identification evaluation using a short¬
term chronic test with Ceriodaphnia dubia. Bulletin of Environmental
Contamination and Toxicology 53: 91-97.

269
Jop, K.M., T.Z. Kendall, A M. Askew, and R.B. Foster. 1991. Use of fractionation
procedures and extensive chemical analysis for toxicity identification of a
chemical plant effluent. Environmental Toxicology and Chemistry 10:
981-990.
Ju, Y.H., K.E. Carlson, J. Sun, D. Pathak, B.S. Katzenellenbogen, J.A.
Katzenellenbogen, and W.G. Helferich. 2000. Estrogenic effects of
extracts from cabbage, fermented cabbage, and acidified brussels sprouts
on growth and gene expression of estrogen-dependent human breast
cancer (MCF-7) cells. Journal of Agricultural and Food Chemistry 48:
4628-4634.
Juers, D.H., T.D. Fleightman, A. Vasella, J.D. McCarter, L. Mackenzie, S.G.
Withers, and B. W. Matthews. 2001. A structural view of the action of
Escherichia coli (lacZ) p-galactosidase. Biochemistry 40:14781-14794.
Jung, K. and G. Bitton. 1997. Use of CerioFASTâ„¢ for monitoring the toxicity of
industrial effluents: comparison with the 48-h acute Ceriodaphnia toxicity
test and Microtox®. Environmental Toxicology and Chemistry 16(11):
2264-2267.
Jung, K. 1995. Development of short-term bioassays for toxicity testing in aquatic
environments. Doctoral dissertation. University of Florida, Gainesville.
Kadlec, R.H., and R.L. Knight. 1996. Treatment Wetlands. Lewis Publishers,
New York.
Kang, K-H., FI S. Shin and H. Park. 2002. Characterization of humic substances
present in landfill leachates with different landfill ages and its implications.
Water Research 36: 4023-4032.
Kaschl, A., V. Romheld and Y. Chen. 2002. The influence of soluble organic
matter from municipal solid waste compost on trace metal leaching in
calcareous soils. Science of the Total Environment 291: 45-57.
Katori, Y., Y. Ksu, and H. Utsumi. 2002. Estrogen-like effect and cytotoxicity of
chemical compounds. Water Science and Technology 46(11-12): 363-366.
Kaur, R., B. Buckley, S.S. Park, Y.K. Kim, and K.R. Cooper. 1996. Toxicity test of
Nanji Island landfill (Seoul, Korea) leachate using Japanese Medaka
(Oryzias latipes) embryo larval assay. Bulletin of Environmental
Contamination and Toxicology 57: 84-90.

270
Kawagoshi, Y., Y. Fujita, I. Kishi and I. Fukunaga. 2003. Estrogenic chemicals
and estrogenic activity in leachate from municipal waste landfill
determined by yeast two-hybrid assay. Journal of Environmental
Monitoring 5(2): 269-274.
Kawagoshi, K., Y. Tsukagoshi and I. Fukunaga. 2002. Determination of
estrogenic activity in landfill leachate by simplified yeast two-hybrid assay.
Journal of Environmental Monitoring 4(6): 1040-1046.
Key, T.J.A., M.C. Pike, J.B. Brown, C. Flermon, D.S. Allen, and D.Y. Wang. 1996.
Cigarette smoking and urinary oestrogen excretion in premenopausal and
post-menopausal women. British Journal of Cancer 74: 1313-1316.
Kim, J.H., B.D. Yoon, and H.M. Oh. 2003. Rapid bioassay for microcystin toxicity
based on feeding activity of Daphnia. Bulletin of Environmental
Contamination and Toxicology 70: 861-867.
Kim, S.D., H. Ma, H.E. Allen and D.K. Cha. 1999. Influence of dissolved organic
matter on the toxicity of copper to Ceriodaphnia dubia: effect of
complexation kinetics. Environmental Toxicology and Chemistry 18(11):
2433-2437.
Kjeldsen, P., M.A. Barlaz, A.P. Rooker, A.Baun, A. Ledin, and T.H. Christensen.
2002. Present and long-term composition of MSW landfill leachate: a
review. Critical Reviews in Environmental Science and Technology 32(4):
297-336.
Klaine, S.J. and M.A. Lewis. 1995. Algal and plant toxicity testing. In Handbook
of Ecotoxicology. pp 163-184. D.J. Hoffman, B.A. Rattner, G.A. Burton, Jr.
and J. Cairns Jr.(eds) Lewis, CRC Press, Boca Raton, FL.
Klein, K.O., J. Baron, M.J. Colli, D.P. McDonnell, and G.B. Cutler, Jr. 1994.
Estrogen levels in childhood determined by an ultrasensitive recombinant
cell bioassay. Journal of Clinical Investigation 9: 2475-2480.
Knox, K. and P.H. Jones. 1979. Complexation characteristics of sanitary landfill
leachates. Water Research 13: 839-846.
Kolpin, D.W., E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber
and H.T. Buxton. 2002. Pharmaceuticals, Hormones, and other organic
wastewater contaminants in U.S. streams, 1999-2000: a national
reconnaissance. Environmental Science and Technology 36(6):
1202-1211.

271
Kong, l-C., G. Bitton, B. Koopman, and K-H. Jung. 1995. Heavy metal toxicity
testing in environmental samples. Reviews of Environmental
Contamination and Toxicology 142: 119-147.
La Guardia, M.J., R.C. Hale, E. Harvey and T.M. Mainor. 2001. Alkylphenol
ethoxylate degradation in land-applied sewage sludge (biosolids).
Environmental Science and Technology 35(24): 4798-4804.
Lambolez, L.; Vasser, P.; Ferard, J. F.; Gisbert, T. 1994. The environmental risks
of industrial waste disposal: an experimental approach including acute and
chronic toxicity studies. Ecotoxicology and Environmental Safety 28:
317-328.
Langelier, W.F. 1936. The analytical control of anti-corrosion water treatment.
Journal of the American Water Works Association 28:1500.
Larsson, D.G.J., M. Adolfsson-Erici, J. Parkkonen, M. Pettersson, A.H. Berg,
P.E. Olsson, and L. Forlin. 1999. Ethinyloestradiol - an undesired fish
contraceptive? Aquatic Toxicology 45: 91-97.
Lascombe, I., D. Beffa, U. Ruegg, J. Tarradellas, and W. Wahli. 2000. Estrogenic
activity assessment of environmental chemicals using in vitro assays:
identification of two new estrogenic compounds. Environmental Health
Perspectives 108(7): 621-629.
Lassier, P.J., P.V. Winger, and K.J. Bogenrider. 2000. Toxicity of manganese to
Ceriodaphnia dubia and Hyalella azteca. Archives of Environmental
Contamination and Toxicology 38: 298-304.
Latonnelle, K., F. Le Menn, and C. Bennetau-Pelissero. 2000. In vitro estrogenic
effects of phytoestrogens in Rainbow Trout and Siberian Sturgeon.
Ecotoxicology 9: 115-125.
Layton, A.C., J. Sanseverino, B.W. Gregory, J.P. Easter, G.S. Sayler, and T.W.
Schultz. 2002. In vitro estrogen receptor binding of PCBs: measured
activity and detection of hydroxylated metabolites in a recombinant yeast
assay. Toxicology and Applied Pharmacology 180:157-163.
LeBlond, J. B.; Duffy, L. K. 2001. Toxicity assessment of total dissolved solids in
effluent of Alaskan mines using 22-h Microtoxâ„¢ and Selenastrum
capricomutum assays. Science of the Total Environment 271: 49-59.
Legler, J., M. Dennekamp, A.D. Vethaak, A. Brouwer, J.H. Koeman, B. van der
Burg, and A.J. Murk. 2002. Detection of estrogenic activity in sediment-
associated compounds using in vitro reporter gene assays. Science
of the Total Environment 293: 69-83.

272
Legler, J., C.E. van den Brink, A. Brouwer, A.J. Murk, P.T. van der Saag, A.D.
Vethaak, and B. van der Burg. 1999. Development of a stably transfected
estrogen receptor-mediated luciferase reporter gene assay in the human
T47D breast cancer cell line. Toxicological Sciences 48: 55-66.
Lo, I.M.C. 1996. Characteristics and treatment of leachates from domestic
landfills. Environment International 22(4): 433-442.
Lopez de Alda, M.J. and D. Barcelo. 2001. Review of analytical methods for the
determination of estrogens and progestogens in waste waters. Fresenius
Journal of Analytical Chemistry 371: 437-447.
Luoma, S.N. 1995. Prediction of metal toxicity in nature from bioassays:
limitations and research needs, pp. 609-659. In Metal Speciation and
Bioavailability in Aquatic Systems. Eds. A. Tessier and D.R. Turner (eds.)
John Wiley and Sons, New York.
Lye, C.M., C.L.J. Frid, M.E. Gill, D.W. Cooperand D M. Jones. 1999. Estrogenic
alkylphenols in fish tissues, sediments, and waters from the U.K. Tyne and
Tees estuaries. Environmental Science and Technology 33:1009-1014.
Lyytikainen, M., A. Sormunen, S. Peraniemi, and J.V.K. Kukkonen. 2001.
Environmental fate and bioavailability of wood preservatives in freshwater
sediments near an old sawmill site. Chemosphere 44: 341-350.
Magdaleno, A. and E. De Rosa. 2000. Chemical composition and toxicity of
waste dump leachates using Selenastrum capricomutum Printz
(chlorococcales, chlorophyta). Environmental Toxicology 15: 76-80.
Majone, M., M.P. Papine and E. Rolle. 1996. Heavy metal speciation in landfill
leachates by exchange on chelex-100 resin. Environmental Technology
17: 587-595.
Martensson, A M., C. Aulin, O. Wahlberg, and S. Agren. 1999. Effect of humic
substances on the mobility of toxic metals in a mature landfill. Waste
Management Research 17: 296-304.
Marttinen, S.K., R.H. Kettunen, K.M. Sormunen, R.M. Soimasuo, and J.A.
Rintala. 2002. Screening of physical-chemical methods for removal of
organic material, nitrogen and toxicity from low strength landfill leachates.
Chemosphere 46: 851-858.
Masion, A., A.V. Ritter, J. Rose, W.E.E. Stone, B.J. Teppen, D. Rybacki and J-Y.
Bottero. 2000. Coagulation-flocculation of natural organic matter with A!
salts: speciation and structure of the aggregates. Environmental Science
and Technology 34(15): 3242-3246.

273
Massaad, C., X. Coumoul, M. Sabbah, M. Garlatti, G. Redeuilh, and R. Barouki.
1998. Properties of overlapping EREs: synergistic activation of
transcription and cooperative binding of ER. Biochemistry 37: 6023-6032.
Matsui, S., H. Takigami, T. Matsuda, N. Taniguchi, J. Adachi, H. Kawami and Y.
Shimizu. 2000. Estrogen and estrogen mimics contamination in water and
the role of sewage treatment. Water Science and Technology 42(12):
173-179.
Mathews, C.K. and K.E. van Holde. 1996. Biochemistry. 2nd Ed.
Benjamin/Cummings, Inc., New York.
McBean, E.A., F.A. Rovers, and G.J. Farquhar. 1995. Solid Waste Landfill
Engineering and Design. Prentice-Hall, Englewood Cliffs, NJ.
McLachlan, J.A. 2001. Environmental signaling: what embryos and evolution
teach us about endocrine disrupting chemicals. Endocrine Reviews 22(3):
319-341.
Meerts, I.A.T.M., R.J. Letcher, S. Hoving, G. Marsh, A. Bergman, J.G. Lemmen,
B. van der Burg, and A. Brouwer. 2001. In vitro estrogenicity of
polybrominated diphenyl ethers, hydroxylated PBDEs, and polybrominated
bisphenol a compounds. Environmental Health Perspectives 109(4):
399-407.
Mellanen, P., T. Patanen, J. Lehtimaki, S. Makela, G. Bylund, B. Holmbom, E.
Mannila, A. Oikari and R. Santti. 1996. Wood-derived estrogens: studies in
vitro with breast cancer cell lines and in vivo in trout. Toxicology and
Applied Pharmacology 136: 381-388.
Metcalfe, C.D., T.L. Metcalfe, Y. Kiparissis, B.G. Koenig, C. Khan, R.J. Hughes,
T.R. Croley, R.E. March and T. Potter. 2001. Estrogenic potency of
chemicals detected in sewage treatment plant effluents as determined by
in vivo assays with Japanese Medaka (Oryzias latipes). Environmental
Toxicology and Chemistry 20(2): 297-308.
Meyer, J.S. 2002. The utility of the terms "bioavailability" and "bioavailable
fraction" for metals. Marine Environmental Research 53: 417-423.
Miksicek, R.J. 1994. Interaction of naturally occurring nonsteroidal estrogens with
expressed recombinant human estrogen receptor. Journal of Steroidal
Biochemistry and Molecular Biology 49(2/3): 153160.
Millemann, R.E. and B.R. Parkhurst. 1980. Comparative toxicity of solid waste
leachates to Daphnia magna. Environment International 4: 255-260.

274
Miller, D„ B.B. Wheals, N. Beresford, and J.P. Sumpter. 2001. Estrogenic activity
of phenolic additives determined by an in vitro yeast bioassay.
Environmental Health Perspectives 109(2): 133-138.
Moffat, G.J., A. Burns, J. Van Miller, R. Joiner and J. Ashby. 2001.
Glucuronidation of nonylphenol and octylphenol eliminates their ability to
activate transcription via the estrogen receptor. Regulatory Toxicology and
Pharmacology 34: 182-187.
Mohan, B.S. and B.B. Hosetti. 1999. Aquatic plants for toxicity assessment.
Environmental Research Section A 81: 259-274.
Monteverdi, G.H. and R.T. Giulio. 1999. An enzyme-linked immunosorbent assay
for estrogenicity using primary hepatocyte cultures from the Channel
Catfish (Ictalurus punctatus). Archives of Environmental Contamination
and Toxicology 37: 62-69.
Morel, F.M.M.1983. Principles of Aquatic Chemistry. Wiley-lnterscience, New
York.
Morgan, J.J. and W. Stumm. 1991. Chemical processes in the environment,
relevance of chemical speciation. pp. 67-103. In Metals and Their
Compounds in the Environment Occurrence, Analysis and Biological
Relevance. E. Merian (ed ). VCH, New York.
Mount, D.R. and J.R. Hockett. 2000. Use of toxicity identification evaluation
methods to characterize, identify, and confirm hexavalent chromium
toxicity in an industrial effluent. Water Research 34: 1379-1385.
Mowat, F.S. and K.J. Bundy. 2001. Correlation of field-measured toxicity with
chemical concentration and pollutant availability. Environment
International 27: 479-489.
Muller, S., P. Schmid and C. Schlatter. 1998. Pharmacokinetic behaviour of 4-
nonylphenol in humans. Environmental Toxicology and Pharmacology 5:
257-265.
Murk, A.J., J. Legler, M.M.H. van Lipzig, J.H.N. Meerman, A.C. Belfroid, A.
Spenkelink, B. van der Burg, G.B.J. Rijs, and D. Vethaak. 2002. Detection
of estrogenic potency in wastewater and surface water with three in vitro
bioassays. Environmental Toxicology and Chemistry 21(1): 16-23.

275
Muthumbi, W., P. DeBoever, I. D'Haese, W. D'Hooge, E.M.Top, J.G. Pieters, F.
Comhaire and W. Verstraete. 2002. Assessment of the estrogenic activity
of flue gases from burning processes by means of the yeast based human
estrogen receptor (hER) bioassay. Environmental Technology 23:
287-291.
Nakano, S., Y. Nagao, T. Kobayashi, M. Tanaka, S. Hirano, Y. Nobuhara, and T.
Yamada. 2002. Problems with methods used to screen estrogenic
chemicals by yeast two-hybrid assays. Journal of Health Science 48(1):
83-88.
Nelson, S.M. and R.A. Roline. 1998. Evaluation of the sensitivity of rapid toxicity
tests relative to daphnid acute lethality tests. Bulletin of Environmental
Contamination and Toxicology 60: 292-299.
Neubert, D. 1997. Vulnerability of the endocrine system to xenobiotic influence.
Regulatory Toxicology and Pharmacology 26: 9-29.
NIH. 2003. NTP-CERHR Monograph on the potential human reproductive and
developmental effects of butyl benzyl phthalate (BBP). Publication No. 03-
4487.National Institutes of Health, Bethesda, MD
Nilsson, R. 2000. Endocrine modulators in the food chain and environment.
Toxicologic Pathology 28(3): 420-431.
Nimmo, D.W.R., M.J. Willox, J.F. Karish, J.D. Tessari, T.L. Craig, E.G. Gasser
and J.R. Self. 1995. Non-availability of metals from an urban landfill in
Virginia. Chemical Speciation and Bioavailability 7(2):65-72.
Nishikawa, J-l., K. Saito, j. Goto, F. Dakeyama, M. Matsuo, and T. Nishihara.
1999. New screening methods for chemicals with hormonal activities using
interaction of nuclear hormone receptor with coactivator. Toxicology and
Applied Pharmacology 154: 76-83.
Noaksson, E., M. Linderoth, A.T.C. Bosveld, L. Norrgren, Y. Zebuhr, and L. Balk.
2003. Endocrine disruption in brook trout (Salvelinus fontinalis) exposed to
leachate from a public refuse dump. Science of the Total Environment
305: 87-103.
Nopharatana, A., W.P. Clarke, P C. Pullammanappallil, P. Silvey, and D.P.
Chynoweth. 1998. Evaluation of methanogenic activities during anaerobic
digestion of municipal solid waste. Bioresource Technology 64: 169-174.

276
Norberg-King, T.J., E.J. Durhan, and G.T. Ankley. 1991. Application of toxicity
identification evaluation procedures to the ambient waters of the Colusa
basin drain, California. Environmental Toxicology and Chemistry 10:
891-900.
NRC. 1999. Hormonally Active Agents in the Environment. National Research
Council, National Academy of Sciences. Washington, DC.
Okamura, E. and M. Nakahara. 1999. NMR study directly determining drug
delivery sites in phospholipid bilayer membranes. Journal of Physical
Chemistry B 103: 3505-3509.
Palkova, Z., F. Devaux, M. Ricicova, L. Minarikova, S. Le Crom, and C. Jacq.
2002. Ammonia pulses and metabolic oscillations guide yeast colony
development. Molecular Biology of the Cell 13: 3901-3914.
Palmer, F.B., C.A. Butler, M.H. Timperley, and C.W. Evans. 1998. Toxicity to
embryo and adult Zebrafish of copper complexes with two malonic acids
as models for dissolved organic matter. Environmental Toxicology and
Chemistry 17(8): 1538-1545.
Parat, C., R. Chaussod, J. Leveque, S. Dousset and F. Andreux. 2002. The
relationship between copper accumulated in vineyard calcareous soils and
soil organic matter and iron. European Journal of Soil Science 53:
663-669.
Park, S., K.S. Joe, S.H. Han, T.Y. Eom and H.S. Kim. 1999. Characteristics and
distribution of metallic elements in landfill leachates. Environmental
Technology 20(4): 443-448.
Paulozzi, L.J. 1999. International trends in rates of hypospadias and
cryptorchidism. Environmental Health Perspectives 107(4): 297-302.
Payne, J., C. Jones, S. Lakhani, and A. Kortenkamp. 2000. Improving the
reproducibility of the MCF-7 cell proliferation assay for the detection of
xenoestrogens. Science of the Total Environment 248: 51-62.
Pereira, A.M.M., A.M. Velho da Maia Soares, F. Goncalves, and R. Ribeiro.
1999. Test chambers and test procedures for in situ toxicity testing with
zooplankton. Environmental Toxicology and Chemistry 18(9): 1956-1964.
Petit, F., P. Le Goff, J-P. Cravedi, O. Kah, Y. Valotaire, and F. Pakdel. 1999.
Trout oestrogen receptor sensitivity to xenobiotics as tested by different
bioassays. Aquaculture 177: 353-365.

277
Picard, K., J-C. Lhuguenot, M-C. Lavier-Canivene, and M-C. Chagnon. 2001.
Estrogenic activity and metabolism of n-butyl benzyl phthalate in vitro:
identification of the active molecule(s). Toxicology and Applied
Pharmacology 172: 108-118.
Plotkin, S. and N.M. Ram. 1984. Multiple bioassays to assess the toxicity of a
sanitary landfill leachate. Archives of Environmental Contamination and
Toxicology 13: 197-206.
Preston, B.L. and T.W. Snell. 2001. Full life-cycle toxicity assessment using
rotifer resting egg production: implications for ecological risk assessment.
Environmental Pollution 114: 399-406.
Prinsen, M.K. and N. Gouko. 2001. Determination of the oestrogenic
(uterotrophic) activity of extracts of'general purpose polystyrene (GPPS)'
using immature female rats. Journal of Applied Toxicology 21: 235-239.
Qureshi, A.A., K.W. Flood, S.R. Thompson, and S.M. Janhurst. 1982.
Comparison of a luminescent bacterial test with other bioassays for
determining toxicity of pure compounds and complex effluents, pp. 179-
195.In Aquatic Toxicology and Hazard Assessment: Fifth conference.
ASTM STP 766. J.G. Pearson, R.B. Foster, and W.E. Bishop, (eds.).
ASTM, Philadelphia.
Radi, L.M., D.J. Kuntz, G. Padmanabhan, I.E. Berg, and A.K. Chaturvedi. 1987.
Toxicological evaluation of the leachate from a closed urban landfill.
Bulletin of Environmental Contamination and Toxicology 38: 337-344.
Ragle, N., J. Kissel, J.E. Ongerth, and F.B. DeWalle. 1995. Composition and
variability of leachate from recent and aged areas within a municipal
landfill. Water Environment Research 67(2): 238-243.
Rand, G.M., P.G. Wells, and L.S. McCarty. 1995. Introduction to aquatic
toxicology, pp. 3-70. In Fundamentals of Aquatic Toxicology: Effects,
Environmental Fate, and Risk Assessment. G.M. Rand (ed.), Taylor and
Francis, Washington, DC.
Ranney, R E. 1977. Comparative metabolism of 17a-ethynyl steroids used in oral
contraceptives. Journal of Toxicology and Environmental Health 3:
139-166.
Rahman, F., K.H. Langford, M.D. Scrimshaw, and J.N. Lester. 2001.
Polybrominated diphenyl ether (PBDE) flame retardants. Science of
the Total Environment 275: 1-17.

278
Rehmann, K., K-W. Schramm, and A.A. Kettrup. 1999. Applicability of a yeast
oestrogen screen for the detection of oestrogen-like activities in
environmental samples. Chemosphere 38(14): 3303-3312.
Reineke, N., K. Bester, H. Huhnerfuss, B. Jastorff, and S. Weigel. 2002.
Bioassay-directed chemical analysis of River Elbe surface water including
large volume extractions and high performance fractionation.
Chemosphere 47: 717-723.
Reinhart, D.R. and C.J. Grosh. 1998. Analysis of Florida MSW landfill leachate
quality data. Submitted to Florida Center for Solid and Hazardous Waste
Management, Gainesville, FL.
Reinhart, D.R. and T.G. Townsend. 1998. Landfill Bioreactor Design and
Operation. Lewis, New York.
Revel, J. C.; Morard, R.; Labbe, J. R.; Berthout, C.; Kaemmerer, M. J. 1999.
Plants use of leachate derived from municipal solid waste. Environmental
Quality 28: 1083-1089.
Rhodes, K. 1992. A rapid acute toxicity test based on daphnid feeding behavior.
M.S. Thesis, University of Florida, Gainesville.
Richards, J.G., P.J. Curtis, B.K. Burnison, and R.C. Playle. 2001. Effects of
natural organic matter source on reducing metal toxicity to Rainbow trout
(Oncortiynchus mykiss) and on metal binding to their gills. Environmental
Toxicology and Chemistry 20(6): 1159-1166.
Richardson, S.D. 2001. Mass spectrometry in environmental sciences. Chemical
Reviews 101: 211-254.
Rodgers-Gray, T.P., S. Jobling, S. Morris, C. Kelly, S. Kirby, A. Janbakhsh, J. E.
Harries, M.J. Waldock, J.P. Sumpter and C.R. Tyler. 2000. Long-term
temporal changes in the estrogenic composition of treated sewage effluent
and its biological effects on fish. Environmental Science and Technology
34(8): 1524-1528.
Rojickova-Padrtova, R., B. Marsalek, and I. Holoubek. 1998. Evaluation of
alternative and standard toxcicity assays for screening of environmental
samples: selection of an optimal test battery. Chemosphere 37(3):
495-507.
Ross, W.R. 1990. Factors Influencing the Chemical Characteristics of Landfill
Leachates. 1sl Methane from Landfill Summer School, Rhodes University,
Grahamstown.

279
Routledge, E.J., D. Sheahan, C. Desbrow, G.C. Brighty, M. Waldock, and J.P.
Sumpter. 1998. Identification of estrogenic chemicals in STW effluent. 2.
In vivo responses in Trout and Roach. Environmental Science and
Technology 32(11): 1559-1565.
Routledge, E.J. and J.P. Sumpter. 1996a. Structural features of alkylphenolic
chemicals associated with estrogenic activity. Journal of Biological
Chemistry 272(6): 3280-3288.
Routledge, E.J. and J.P. Sumpter. 1996b. Estrogenic activity of surfactants and
some of their degradation products assessed using a recombinant yeast
screen. Environmental Toxicology and Chemistry 15(3): 241-248.
Roy, S.B. and D.A. Dzombak. 1997. Chemical factors influencing colloid-
facilitated transport of contaminants in porous media. Environmental
Science and Technology 31(3): 656-664.
Rudel, R.A., S.J. Melly, P.W. Geno, G. Sun, and J.G. Brody. 1998. Identification
of alkylphenols and other estrogenic phenolic compounds in wastewater,
septage, and groundwater on Cape Cod, Massachusetts. Environmental
Science and Technology 32(7): 861-869.
Rutherford, L. A.; Matthews, S. L; Doe, K. G.; Julien, G. R. J. 2000. Aquatic
toxicity and environmental impact of leachate discharges from a municipal
landfill. Water Quality Research Journal of Canada. 35: 39-57.
Sawyer, C.N., P.L. McCarty, and G.F. Parkin. 1994. Chemistry for Environmental
Engineering. 4th Ed. McGraw-Hill, Inc. New York.
Scheifler, R., C. Schwartz, G. Echevarria, A. De Vaufleury, P-M. Badot and J.L.
Morel. 2003. "Nonavailable" soil cadmium is bioavailable to snails:
evidence from isotopic dilution experiments. Environmental Science and
Technology 37(1): 81-86.
Schmieder, P.K., G. Ankley, O. Mekenyan, J.D. Walker, and S. Bradbury. 2003.
Quantitative structure-activity relationship models for prediction of
estrogen receptor binding affinity of structurally diverse chemicals.
Environmental Toxicology and Chemistry 22(8): 1844-1854.
Schroth, B.K. and G. Sposito. 1998. Effect of landfill leachate organic acids on
trace metal adsorption by kaolinite. Environmental Science and
Technology 32(10): 1404-1408.

280
Schubauer-Berigan, M.K., J.R. Dierkes, P.D. Monson, and G.T. Ankley. 1993.
pH-dependent toxicity of Cd, Cu, Ni, Pb, and Zn to Ceriodaphnia dubia,
Pimephales prometas, Hyalella azteca and Lumbriculus variegatus.
Environmental Toxicology and Chemistry 12: 1261-1266.
Schultz, T.W., J.R. Seward, and G.D. Sinks. 2000. Estrogenicity of
benzophenones evaluated with a recombinant yeast assay: comparison of
experimental and rules-based predicted activity. Environmental Toxicology
and Chemistry 19(2): 301-304.
Schwarzbauer, J., S. Heim, S. Brinker, and R. Littke. 2002. Occurrence and
alteration of organic contaminants in seepage and leakage water from a
waste deposit landfill. Water Research 36: 2275-2287.
Sepulveda, M.S., W.E. Johnson, J.C. Higman, N.D. Denslow, T.R. Schoeb and
T.S. Gross. 2002. An evaluation of biomarkers of reproductive function
and potential contaminant effects in Florida largemouth bass (Microptems
salmoides floridanus) sampled from the St. Johns river. Science of
the Total Environment 289: 133-144.
Shang, D.Y., R.W. Macdonald and M.G. Ikonomou. 1999. Persistence of
nonylphenol ethoxylate surfactants and their primary degradatin products
in sediments from near a municipal outfall in the Strait of Georgia, British
Columbia, Canada. Environmental Science and Technology 33(9):
1366-1372.
Sheahan, D.A., G.C. Brightly, M. Daniel, S.J. Kirby, M.R. Hurst, J. Kennedy, S.
Morris, E.J. Routledge, J.P. Sumpter, and M.J. Waldock. 2002a.
Estrogenic activity measured in a sewage treatment works treating
industrial inputs containing high concentrations of alkylphenolic
ompounds - a case study. Environmental Toxicology and Chemistry
21(3): 507-514.
Sheahan, D.A., G.C. Brighty, M. Daniel, S. Jobling, J.E. Harries, M R. Hurst, J.
Kennedy, S.J. Kirby, S. Morris, E.J. Routledge, J.P. Sumpter, and M.J.
Waldock. 2002b. Reduction in the estrogenic activity of a treated sewage
effluent discharge to an English river as a result of a decrease in the
concentration of industrially derived surfactants. Environmental Toxicology
and Chemistry 21(3): 515-519.
Shiraishi, H., O. Nakasugi, S. Hashimoto, T. Yamamoto, A. Yasuhara, and K.
Yasuda. 1999. Endocrine disrupters in the leachate from waste disposal
sites. Waste Management Research 10: 33-45.

281
Shurin, J.B. and S I. Dodson. 1997. Sublethal toxic effects of cyanobacteria and
nonylphenol on environmental sex determination and development in
Daphnia. Environmental Toxicology and Chemistry 16(6): 1269-1276.
Sisinno, C.L.S., E.C. Oliveira-Filho, M.C. Dufrayer, J.C. Moreira, and F.J.R.
Paumgartten. 2000. Toxicity evaluation of a municipal dump leachate
using Zebrafish acute test. Bulletin of Environmental Contamination and
Toxicology 64: 107-113.
Sletten, R.S., M.M. Benjamin, J.J. Horng, and J.F. Ferguson. 1995. Physical-
chemical treatment of landfill leachate for metals removal. Water
Research 29(10): 2376-2386.
Smith, E.H. and W.J. Weber. 1990. Comparative assessment of the chemical
and adsorptive characteristics of leachates from a municipal and an
industrial landfill. Water, Air, and Soil Pollution 53: 279-295.
Snyder, S.A., D.L. Villeneuve, E M. Snyder and J.P. Giesy. 2001. Identification
and quantification of estrogen receptor agonists in wastewater effluents.
Environmental Science and Technology 35: 3620-3625.
Sole, M„ M.J. Lopez de Alda, M. Castillo, C. Porte, K. Ladegaard-Pedersen, and
D. Barcelo. 2000. Estrogenicity determination in sewage treatment plants
and surface waters from Catalonian area (NE Spain). Environmental
Science and Technology 34: 5076-5083.
Soto, A.M., T-M. Lin, H. Justicia, R.M. Silvia and C. Sonnenschein. 1992. An "in
culture" bioassay to assess the estrogenicity of xenobiotics (E-screen).
Journal of Clean Technology, Environmental Toxicology, and
Occupational Medicine 7(3): 331-343.
Stewart, A.J. 1996. Ambient bioassays for assessing water-quality conditions in
receiving streams. Ecotoxicology 5: 377-393.
Stoica, A., B.S. Katzenellenbogen, and M.B. Martin. 2000. Activation of estrogen
receptor-a by the heavy metal cadmium. Molecular Endocrinology 14(4):
545-553.
Strauss, L., R. Santti, N. Saarinen, T. Streng, S. Joshi and S. Makela. 1998.
Dietary phytoestrogens and their role in hormonally dependent disease.
Toxicology Letters 102-103: 349-354.
Stronkhorst, J., M.E. Schot, M.C. Dubbeldam, and K.T. Ho. 2003. A toxicity
identification evaluation of silty marine harbor sediments to characterize
persistant and non-persistent constituents. Marine Pollution Bulletin 46:
56-64.

282
Stumm, W. and J. Morgan. 1995. Aquatic Chemistry: Chemical Equilibria and
Rates in Natural Waters, 3rd Ed. Wiley. New York.
Suedel, B.C., E. Deaver, J.H. Rodgers, Jr. 1996. Experimental factors that may
affect toxicity of aqueous and sediment-bound copper to freshwater
organisms. Archives of Environmental Contamination and Toxicology 30:
40-46.
Suflita, J.M., C.P. Gerba, R.K. Ham, A.C. Palmisano, W.L. Rathje and J.A.
Robinson. 1992. The world's largest landfill. Environmental Science and
Technology 26(8): 1486-1495.
Sultan, C„ P. Balaguer, B. Terouanne, V. Georget, F. Paris, C. Jeandel, S.
Lumbroso, and J-C. Nicolas. 2001. Environmental xenoestrogens,
antiandrogens and disorders of male sexual differentiation. Molecular and
Cellular Endocrinology 178: 99-105.
Takigami, N., N. Taniguchi, T. Matsuda, M. Yamada, Y. Shimizu and S. Matsui.
2000. The fate and behaviour of human estrogens in a night soil treatment
process. Water Science and Technology 42(7-8): 45-51.
Tanaka, H„ Y. Yakou, A. Takahashi, T. Higashitani and K. Komori. 2001.
Comparison between estrogenicities estimated from DNA recombinant
yeast assay and from chemical analyses of endocrine disruptors during
sewage treatment. Water Science and Technology 43(2): 125-132.
Ternes, T.A., M. Stumpf, J. Mueller, K. Haberer, R.D. Wilken, and M. Servos.
1999a. Behavior and occurrence of estrogens in municipal sewage
treatment plants-1, investigations in Germany, Canada and Brazil. The
Science of the Total Environment 225; 81-90.
Ternes, T.A., R. Kreckel, and J. Mueller. 1999b. Behaviour and occurrence of
estrogens in municipal sewage treatment plants - II. aerobic batch
experiments with activated sludge. Science of the Total Environment
225: 91-99.
Tessier, A., P.G.C. Campbell and M. Bisson. 1979. Sequential extraction
procedure for the speciation of particulate trace metals. Analytical
Chemistry 51: 844-851.
Thomas, K.V., M.R. Hurst, P. Matthiessen, and M.J. Waldock. 2001.
Characterization of estrogenic compounds in water samples collected
from United Kingdom estuaries. Environmental Toxicology and Chemistry
20(10): 2165-2170.

283
Townsend, T., W. Miller, H. Lee, and J. Earie.1996. Acceleration of landfill
stabilization using leachate recycle. Journal of Environmental Engineering
ASCE 122: 263-268.
USEPA. 2002. Municipal solid waste in the United States: 2000 facts and figures
executive summary. Office of Solid Waste and Emergency Response.
EPA-530-5-02-001; Washington, DC.
USEPA, 2001. Removal of endocrine disruptor chemicals using drinking water
treatment processes. Office of Research and Development. EPA/625/R-
00/015; Washington, DC.
USEPA. 1996. Test methods for evaluating solid waste. SW-846, 3rd Ed. Office
of Solid Waste, Cincinnati, OH.
USEPA. 1994a. Short-term methods for estimating the chronic toxicity of
effluents and receiving water to freshwater organisms. 3rd Ed. EPA-600-4-
91-002. Office of Research and Development, Cincinnati, OH.
USEPA. 1994b. USEPA toxicity data analysis software; EPA: Cincinnati, OH.
USEPA. 1993a. Methods for measuring the acute toxicity of effluents and
receiving waters to freshwater and marine organisms. 4th Ed. EPA/600/4-
90/027F. Office of Research and Development, Cincinnati, OH.
USEPA. 1991a. Methods for aquatic toxicity identification evaluation: phase I
toxicity characterization procedures. EPA/600/6-91/003. Office of
Research and Development. Cincinnati, OH.
USEPA. 1991b. Methods for aquatic toxicity identification evaluation: phase II
toxicity identification procedures. EPA/600/3-88/035. Office of Research
and Development, Cincinnati, OH.
USEPA. 1991c. Methods for aquatic toxicity identification evaluation: phase III
toxicity confirmation procedures. EPA/600/3-88/036. Office of Research
and Development, Cincinnati, OH.
USEPA. 1984. Guidelines for deriving numerical aquatic site-specific water
quality criteria by modifying national criteria. EPA-600/3-84-099. Office of
Research and Development, Cincinnati, OH.
USEPA. 1978. The Selenastrum capricomutum printz algal assay bottle test.
EPA-600/9-78-018. Office of Research and Development, Cincinnati, OH.
USEPA. 1977. Recent advances in fish toxicology: a symposium. EPA-600/3-77-
085. Ecological Research Series, Cincinnati, OH.

284
Vadillo, I., F. Carrasco, B. Andreo, A. Garcia de Torres, and C. Bosch. 1999.
Chemical composition of landfill leachate in a karst area with a
Mediterranean climate (Marbella, southern Spain). Environmental Geology
37(4): 326-332.
Van den Belt, K., R. Verheyen, and H. Witters. 2001. Reproductive effects of
ethynylestradiol and t-octylphenol on the Zebrafish (Danio rerio). Archives
of Environmental Contamination and Toxicology 41: 458-467.
van der Heever, J.A. and J.U. Grobbelaar. 1998. In vivo chlorophyll a
fluorescence of Selenastrum capricomutum as a screening bioassay in
toxicity studies. Archives of Environmental Contamination and Toxicology
35: 281-286.
Vanderperren, E., W. Demare, R. Blust, K. Cooreman, and P. Bossier. 2001.
Oestrogenic activity of CPRG (chlorophenol red-p-/d-galactopyranoside),
a p-galactosidase substrate commonly used in recombinant yeast
oestrogenic assays. Biomarkers 6(5): 375-380.
Van Ryssen, R., M. Alam, L. Goeyens, and W. Baeyens. 1998. The use of fiux-
corer experiments in the determination of heavy metal re-distribution in
and of potential leaching from the sediments. Water Science and
Technology 37(6-7): 283-290.
Van Sprang, P.A. and C.R. Janssen. 1997. Identification and confirmation of
ammonia toxicity in contaminated sediments using a modified toxicity
identification evaluation approach. Environmental Toxicology and
Chemistry 16(12): 2501-2507.
van Wezel, A.P., P. van Vlaardingen, R. Posthumus, G.H. Crommentuijn and
D.T.H.M. Sijm. 2000. Environmental risk limits for two phthalates, with
special emphasis on endocrine disruptive properties. Ecotoxicology and
Environmental Safety 46: 305-321.
Versteeg, D.J., M. Stalmans, S.D. Dyer, and C. Janssen. 1997. Ceriodaphnia
and Daphnia: a comparison of their sensitivity to xenobiotics and utility as
a test species. Chemosphere 34(4): 869-892.
Vinggaard, A.M., W. Korner, K.H. Lund, U. Bolz, and J.H. Petersen. 2000.
Identification and quantification of estrogenic compounds in recycled and
virgin paper for household use as determined by an in vitro yeast estrogen
screen and chemical analysis. Chemical Research and Toxicology 13:
1214-1222.

285
Voelker, B.M. and M B. Kogut. 2001. Interpretation of metal speciation data in
coastal waters: the effects of humic substances on copper binding as a
test case. Marine Chemistry 74: 303-318.
Ward, M. 1997. Toxicological aspects of municipal solid waste landfill leachates.
Master's Thesis, University of Florida, Gainesville, FL.
Ward, M., G. Bitton, and T. Townsend. 2000. Toxicity testing of municipal solid
waste leachates with CerioFAST. Bulletin of Environmental Contamination
and Toxicology 64: 100-106.
Ward, M., G. Bitton, T. Townsend and M. Booth. 2002. Determining toxicity of
leachates from Florida municipal solid waste landfills using a battery-of-
tests approach. Environmental Toxicology 17: 258-266.
Weigel, S., J. Kuhlmann, and h. Huhnerfuss. 2002. Drugs and personal care
products as ubiquitous pollutanats: occurrence and distribution of clofibric
acid, caffeine, and DEET in the North Sea. Science of the Total
Environment 295: 131-141.
Welander, U. and T. Henrysson. 1998. Degradation of organic compounds in a
municipal landfill leachate treated in a suspended-carrier biofilm process.
Water Environment Research 70(7): 1236-1241.
Welsh, P.G., J. Upton and G.A. Chapman. 2000. Evaluation of water-effect ratio
methodology for establishing site-specific water quality criteria.
Environmental Toxicology and Chemistry 19(6): 1616-1623.
Weng, L, E.J.M. Temmlnghoff, S. Lofts, E. Tipping and W.H. Van Rlemsdijk.
2002. Complexation with dissolved organic matter and solubility control of
heavy metals in a sandy soil. Environmental Science and Technology
36(22): 4804-4810.
Wiles, C.C. 1996. Municipal solid waste combustion ash: state-of-the-knowledge.
Journal of Hazardous Materials 47: 325-344.
Winch, S„ J. Ridal and D. Lean. 2002. Increased metal bioavailability following
alteration of freshwater dissolved organic carbon by ultraviolet B radiation
exposure. Environmental Toxicology 17: 267-274.
Wintgens, T., M. Gallenkemper, and T. Melin. 2003. Occurrence and removal of
endocrine disrupters in landfill leachate treatment plants. Water Science
and Technology 48(3): 127-134.

286
Witters, H.E., C. Vangenechten, and P. Berckmans. 2001. Detection of
estrogenic activity in Flemish surface waters using an in vitro recombinant
assay with yeast cells. Water Science and Technology 43 (2): 117-123.
Wong, M.H. 1989. Toxicity test of landfill leachate using Sarotherodon
mossambicus (freshwater fish). Ecotoxicology and Environmental Safety
17: 149-156.
Wu, R.S.S., B.S. Zhou, D.J. Randall, N.Y.S. Woo, and P.K.S. Lam. 2003. Aquatic
hypoxia is an endocrine disruptor and impairs fish reproduction.
Environmental Science and Technology 37(6): 1137-1141.
Wundram, M. and M. Bahadir. 1999. Ecotoxicological test systems for prediction
of environmental behaviour of toxic compounds in underground disposals.
Fresenius Environmental Bulletin 8: 280-287.
Yamada, E., K. Doi, K. Okano and Y. Fuse. 2000. Simultaneous determinations
of the concentration and molecular weight of humic substances in
environmental water by gel chromatography with a fluorescence detector.
Analytical Sciences 16: 125-129.
Yasuhara, A., H. Shiraishi, M. Nishikawa, T. Yamamoto, O. Nakasugi, T.
Okumura, K. Kenmotsu, H. Fukui, M. Nagase, and Y. Kawagoshi. 1999.
Organic components in leachates from hazardous waste disposal sites.
Waste Management Research 17: 186-197.
Zacharewski, T. 1997. In vitro bioassays for assessing estrogenic substances.
Environmental Science and Technology 31(3): 613-623.

BIOGRAPHICAL SKETCH
Marnie Ward was born on April 23, 1967, in Sidney, New York. She was
raised in upstate New York and attended Mt. Upton Central Elementary and High
Schools. In 1986, Marnie and her family relocated to Beverly Hills, Florida.
There she met and married her husband, Bill Ward. They have one daughter,
Diana Mary and she was born on June 20, 2002.
Marnie earned her associate of arts degree at Central Florida Community
College, in Ocala, Florida. She received her Bachelor of Science degree from
the Department of Zoology at the University of Florida. While an undergraduate
student, Marnie worked at the University of Florida Museum of Natural History.
She was responsible for the identification and cataloging of vertebrate fossils
from the Aucilla River, Taylor County, Florida. Marnie also conducted an
independent research project with the Southwest Florida Water Management
District, Brooksville office. The project investigated the diversity of aquatic
invertebrate fauna in stormwater retention basins.
Marnie was accepted into the Department of Environmental Engineering
Sciences in 1995. There she earned a Master of Science degree specializing in
environmental toxicology, Dr. Gabriel Bitton directed her work. Upon graduation
in 1997, Marnie taught as an adjunct instructor at the Central Florida Community
College and worked for the city of Ocala in their water quality lab. Marnie
returned to the University of Florida in 1998 and began working towards a
287

288
doctorate of philosophy. She worked as a graduate research assistant for Dr.
Gabriel Bitton and assisted other professors in teaching duties. Mamie's
research interests include MSW landfills, specifically the generation, treatment,
and long-term fate of MSW landfill leachates. She is also concerned with the use
of bioassays to determine site-specific influences on toxicity. In future research,
Marnie would like to investigate the presence of hormonally active compounds in
the wastewater treatment plants and other aquatic environments, relative to the
influence of these contaminants on "in-situ" populations. Marnie is a current
member of the Society of Environmental Toxicology and Chemistry.

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctpr of Philosg^y.
Gabriel Bitfon, Chairman
TOfessor of Environmental Engineering
Sciences
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a thesis for the degree of Docl
nsend
Professor of Environmental
Engineering Sciences
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a thesis for the degree of
Assistant Professor of Environmental
Engineering Sciences
Doctor of Philosophy
Angela eindner
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a thesis for the degree of Doctor of Philosophy.
Matthew Booth
Assistant in Environmental Engineering
Sciences
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a thesis for the degree of Doctor of Philosophy.
'y .
Nancy Denslow
Scientist in Biochemistry and Molecular
Biology

This dissertation was submitted to the Graduate Faculty of the College of
Engineering and to the Graduate School and was accepted as partial fulfillment
of the requirements for the degree of Doctor of Philosophy.
December, 2003 )
Pramod P. Khargonekar
Dean, College of Engineering
Winfred M. Phillips
Dean, Graduate School

UN'yERS'TY OF FLORIDA
3 1262 08556 6247




PAGE 1

72;,&,7< $1' +25021$/ $&7,9,7< ,1 081,&,3$/ 62/,' :$67( 06:f /($&+$7(6 )520 )/25,'$ /$1'),//6 %\ 0$51,( /<11 :$5' $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

&RS\ULJKW E\ 0DPLH :DUG

PAGE 3

7KLV GLVVHUWDWLRQ LV GHGLFDWHG WR P\ IDPLO\ 7R P\ KXVEDQG %LOO DQG P\ GDXJKWHU 'LDQD 0DU\ ZKRVH ORYH DQG VXSSRUW IRUWLILHG PH RQ PDQ\ ORQJ GD\V DQG QLJKWV 7R P\ SDUHQWV 1DQQ\ DQG 3RSS\ P\ VLVWHU /LVD P\ EURWKHU -RQDWKDQ DQG P\ QLHFHV 'HRQQD 0HJDQ DQG 5HEHFFD IRU WKHLU SUHVHQFH DQG JXLGDQFH LQ P\ OLIH

PAGE 4

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

PAGE 5

IROORZLQJ VWXGHQWV .ULVWLQ 6WRRN 5RL 'DJDQ 7KDEHW 7R\ODPHW 'LDQD /HH /LEE\ 6FKPLGW -HQQD -DPEHFN 3UDGHHS 'XEH\ -HII -HQQ 7UDF\ DQG 0DULVVD 0\ KHDUWIHOW DSSUHFLDWLRQ LV H[WHQGHG WR P\ GHDU IULHQG DQG SHUVRQDO PHQWRU /LQGD 7\VRQ +HU KRXVH EHFDPH P\ VHFRQG KRPH ZKLFK ZDV D ZHOFRPH UHVSLWH GXULQJ P\ TXDOLI\LQJ H[DPV ILUVW PHW /LQGD LQ WKH HDUO\ V ZKHQ ZDV D VWXGHQW DW &HQWUDO )ORULGD &RPPXQLW\ &ROOHJH DQG VKH ZDV DQ LQVWUXFWRU ,W ZDV DIWHU OLVWHQLQJ WR KHU WDON DERXW KHU JUDGXDWH ZRUN WKDW EHFDPH LQVSLUHG WR DWWHQG WKH 8QLYHUVLW\ RI )ORULGD 6KH KDV EHHQ DQ XQZDYHULQJ VRXUFH RI LQVSLUDWLRQ DQG ZLVGRP RZH D GHHS DSSUHFLDWLRQ WR 3HWHU 0H\HUV DQG &UDLJ :DWWV DW +\GURVSKHUH 5HVHDUFK 7KH\ SURYLGHG WKH VWDUWHU FXOWXUHV RI &HULRGDSKQLD GXELD 'DSKQLD SXOH[ DQG 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD ZKLFK ZHUH VXEFXOWXUHG WR VXSSO\ LQ KRXVH WHVW RUJDQLVPV 3HWHU 0H\HUV ZDV D UHOLDEOH DQG H[SHULHQFHG VRXUFH IRU LQIRUPDWLRQ FRQFHUQLQJ WHFKQLTXHV DQG PHWKRGV IRU FXOWXULQJ DTXDWLF WHVW RUJDQLVPV DQG IRU FRQGXFWLQJ DFXWH DQG FKURQLF DVVD\V DOVR H[WHQG DSSUHFLDWLRQ WR WKH RSHUDWRUV RI WKH ODQGILOOV DW ZKLFK WKH OHDFKDWHV ZHUH FROOHFWHG VSHFLILFDOO\ WKDQN -LP %UXQVZLFN IRU KLV IULHQGVKLS DQG JXLGDQFH Y

PAGE 6

7$%/( 2) &217(176 3DJH $&.12:/('*0(176 c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f /$1'),//6 86,1* $ %$77(5< 2) 7(676 $3352$&+ ,QWURGXFWLRQ 0DWHULDOV DQG 0HWKRGV /HDFKDWH &ROOHFWLRQ 9,

PAGE 7

&KHPLFDO DQG 3K\VLFDO &KDUDFWHUL]DWLRQ RI /HDFKDWHV 0DLQWHQDQFH RI 7HVW 2UJDQLVPV 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD &HULRGDSKQLD GXELD DQG 'DSKQLD SXOH[ 3UHSDUDWLRQ RI DTXDWLF LQYHUWHEUDWH IRRG 0DLQWHQDQFH RI DTXDWLF LQYHUWHEUDWH FXOWXUHV 7R[LFLW\ $VVD\V 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD &HULRGDSKQLD GXELD DQG 'DSKQLD SXOH[ 0LFURWR[r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f 2) 081,&,3$/ 62/,' :$67( /$1'),// /($&+$7(6 ,QWURGXFWLRQ 0DWHULDOV DQG 0HWKRGV 6DPSOH 6LWHV YLL

PAGE 8

/HDFKDWH &ROOHFWLRQ &KHPLFDOV DQG 5HDJHQWV &KHPLFDO $QDO\VLV 'HWHUPLQDWLRQ RI +HDY\ 0HWDO 7R[LFLW\ 'HWHUPLQDWLRQ RI +0%& ,QIOXHQFH RI 6RPH /HDFKDWH 3DUDPHWHUV RQ +0%& 'DWD $QDO\VLV 5HVXOWV DQG 'LVFXVVLRQ +HDY\ 0HWDO 7R[LFLW\ RI /DQGILOO /HDFKDWHV +0%& RI 06: /DQGILOO /HDFKDWHV /HDFKDWH WR[LFLW\ DV D IXQFWLRQ RI WLPH ,QIOXHQFH RI 6HOHFWHG /HDFKDWH 3DUDPHWHUV RQ +0%& ,'(17,)<,1* 72;,&,7< ,1 )/25,'$ 06: /$1'),// /($&+$7(6 :,7+ $ 72;,&,7< ,'(17,),&$7,21 $1' (9$/8$7,21 7,(f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

PAGE 9

+25021$/ $&7,9,7< 2) 081,&,3$/ 62/,' :$67( 06:f /($&+$7(6 )520 )/25,'$ /$1'),//6 ,QWURGXFWLRQ 0DWHULDOV DQG 0HWKRGV &KHPLFDOV 06: /DQGILOOV DQG /HDFKDWH &ROOHFWLRQ 06: /DQGILOO /HDFKDWH 7UHDWPHQW )DFLOLW\ 6ROLG 3KDVH ([WUDFWLRQ 63(f RI 06: /DQGILOO /HDFKDWHV <(6 $VVD\ IRU 'HWHUPLQLQJ +RUPRQDO $FWLYLW\ 7R[LFLW\ RI 06: /HDFKDWHV WR
PAGE 10

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f 7KH KRUPRQDO DFWLYLW\ RI VHOHFWHG PHWDO VSHFLHV 5HSRUWHG SKWKDODWH FRQFHQWUDWLRQV LQ ODQGILOO OHDFKDWHV &RQFHQWUDWLRQV QJ/f RI QDWXUDO DQG V\QWKHWLF KRUPRQHV LQ ZDVWHZDWHU WUHDWPHQW SODQWV::73Vf 5HSRUWHG FRQFHQWUDWLRQV QJ/f RI QDWXUDO DQG V\QWKHWLF HVWURJHQV LQ VXUIDFH ZDWHUV [

PAGE 11

$PRXQW RI 06: JHQHUDWHG DQG ODQGILOOHG DW VL[ ODQGILOO VLWHV LQ )ORULGD &RPSRQHQWV RI WKH SUHOLPLQDU\ DOJDO DVVD\ SURFHGXUH 3$$3f PHGLXP 3K\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI 06: ODQGILOO OHDFKDWHV DW VL[ VLWHV LQ )ORULGD &RUUHODWLYH DQDO\VLV ZLWK WKH & GXELD 3 VXEFDSLWDWD DQG 0LFURWR[r1 DVVD\ UHVXOWV YHUVXV OHDFKDWH FKHPLFDO FKDUDFWHULVWLFV 'HVFULSWLRQ RI 06: ODQGILOO VLWHV ZKHUH OHDFKDWHV ZHUH FROOHFWHG 3K\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI 06: OHDFKDWHV FROOHFWHG IURP OLQHG ODQGILOOV LQ )ORULGD 'LVWULEXWLRQ RI PDMRU LRQV LQ OHDFKDWHV IURP OLQHG 06: ODQGILOOV LQ )ORULGD 0HDQ FRQFHQWUDWLRQV PJ/f RI WRWDO 1+1+f DQG XQLRQL]HG 1+f DPPRQLD LQ OHDFKDWHV IURP IRXUWHHQ 06: ODQGILOOV LQ )ORULGD 0HWDO FRQFHQWUDWLRQV LQ OHDFKDWHV IURP IRXUWHHQ 06: ODQGILOOV LQ )ORULGD 7R[LFLW\ RI OHDFKDWHV FROOHFWHG IURP OLQHG 06: ODQGILOOV ZLWK & GXELD SXOH[ DQG 3 VXEFDSLWDWD 7R[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV LQ )ORULGD XVLQJ WKH PLQXWH 0LFURWR[ DFXWH DVVD\ 5HODWLRQVKLS EHWZHHQ WKH WR[LF HQGSRLQWV RI ,& bf12(& bf DQG /2(& bf ZLWK WKH UHVXOWV RI WKH 3 VXEFDSLWDWD DVVD\ ZLWK OHDFKDWH IURP VLWH &RHIILFLHQWV RI YDULDWLRQ &9fbf IRU WKH 3 VXEFDSLWDWD & GXELD DQG SXOH[ DVVD\V &ODVVLILFDWLRQ V\VWHP IRU UDQNLQJ WKH WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWHV LQ )ORULGD (&VRIRU &Xr =Q DQG +Jr GHWHUPLQHG ZLWK WKH 0HW3/$7( DVVD\ 7R[LFLW\ RI OHDFKDWHV IURP OLQHG 06: ODQGILOOV XVLQJ 0HW3/$7( ;,

PAGE 12

3K\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI OHDFKDWHV FROOHFWHG IURP OLQHG 06: ODQGILOOV LQ )ORULGD +HDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f XQLWOHVVf RI OHDFKDWHV IURP 06: ODQGILOOV ZLWK FRSSHU ]LQF DQG PHUFXU\ 0HW3/$7( DQG +0%& UHVXOWV ZLWK 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWHV DQG +0%& RI 06: ODQGILOO OHDFKDWHV ZLWK FRSSHU ]LQF DQG PHUFXU\ IROORZLQJ IUDFWLRQDWLRQ &KDQJHV LQ SK\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV GXULQJ IUDFWLRQDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH DQG &RHIILFLHQWV RI GHWHUPLQDWLRQ 5f REWDLQHG EHWZHHQ 06: ODQGILOO OHDFKDWH FKDUDFWHULVWLFV DQG WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f IRU FRSSHU PHUFXU\ DQG ]LQF 3RSXODWLRQ VHUYHG DQG DPRXQW RI ZDVWH ODQGILOOHG DV D SHUFHQW RI WRWDO ZDVWH JHQHUDWHG DW VLWHV DQG 0DQLSXODWLRQV WR LGHQWLI\ VXVSHFWHG WR[LFDQWV &KHPLFDO DQG SK\VLFDO FKDUDFWHULVWLFV RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG 7KH LQLWLDO GD\ f DQG EDVHOLQH GD\ f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f DQG DIWHU HIIOXHQWf WUHDWPHQW LQ D SRZGHUHG DFWLYDWHG FDUERQ WUHDWPHQW 3$&7f IDFLOLW\ [LL

PAGE 13

2UJDQLF FRPSRXQGV WHQWDWLYHO\ LGHQWLILHG LQ 06: ODQGILOO OHDFKDWHV E\ *&06 DQDO\VLV LQ IXOO VFDQ PRGH 5HFRYHU\ bf RI SHVWUDGLRO (f IURP WKH ( VSLNHG PHWKDQRO H[WUDFWV RI 06: ODQGILOO OHDFKDWHV &DWHJRULHV RI KRUPRQDO DFWLYLW\ LQ 06: ODQGILOO OHDFKDWHV 3UHVHQFH RI KRUPRQDO DFWLYLW\ LQ WKH UDZ OHDFKDWHV DQG PHWKDQRO H[WUDFWV RI 06: ODQGILOO OHDFKDWHV ZLWK LGHQWLILHG KRUPRQDOO\ DFWLYH FRPSRXQGV LQ SDUHQWKHVLV (IIHFW RI H[WUDFWLRQ SURFHGXUHV RQ WKH KRUPRQDO DFWLYLW\ RI OHDFKDWHV IURP /) 0DUFK f 7RWDO DQG IHFDO FROLIRUP EDFWHULD GHWHUPLQHG LQ 06: ODQGILOO OHDFKDWHV ZLWK UHVXOWV H[SUHVVHG DV WKH PRVW SUREDEOH QXPEHU 031f RI EDFWHULD PO RI OHDFKDWH [LLL

PAGE 14

/,67 2) ),*85(6 )LJXUH SDJH 3DWKZD\V IRU WKH FKDUDFWHUL]DWLRQ RI WKH ELRORJLFDO HIIHFWV RI 06: ODQGILOO OHDFKDWHV 5HSUHVHQWDWLRQ RI WKH YHUWHEUDWH HQGRFULQH V\VWHP DQG WKH SRVVLEOH LQIOXHQFHV RI KRUPRQDOO\ DFWLYH FRPSRXQGV RQ YDULRXV V\VWHP DQG RUJDQV /RFDWLRQV RI WKH 06: ODQGILOOV IRU WKH FROOHFWLRQ RI OHDFKDWHV LQ )ORULGD )ORZFKDUW IRU WKH 3 VXEFDSLWDWD DVVD\ )ORZFKDUW IRU WKH & GXELD DVVD\ )ORZFKDUW IRU WKH 0LFURWR[r1 DVVD\ &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO [LY

PAGE 15

&RQFHQWUDWLRQV RI WRWDO 1+r1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO 7KH PHDQ WR[LFLW\ RI 06: OHDFKDWHV FROOHFWHG IURP VL[ ODQGILOO VLWHV 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHUWLPH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHUWLPH 5HODWLRQVKLS EHWZHHQ WKH 3 VXEFDSLWDWD (&f DQG & GXELD (&f DVVD\ UHVXOWV ZLWK 06: ODQGILOO OHDFKDWHV 7R[LFLW\ IOXFWXDWLRQV LQ WKH OHDFKDWHV FROOHFWHG IURP WKH 06: ODQGILOO DW VLWH GXULQJ )HEUXDU\ /RFDWLRQV RI WKH 06: ODQGILOOV IRU WKH FROOHFWLRQ RI OHDFKDWHV LQ )ORULGD $FXWH KU & GXELDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP ODQGILOO VLWHV LQ )ORULGD &RUUHODWLRQ EHWZHHQ WKH KRXU DFXWH WR[LFLW\ DVVD\V XVLQJ & GXELD DQG SXOH[ DVVD\V ZLWK 06: OHDFKDWHV FROOHFWHG IURP ODQGILOO VLWHV LQ )ORULGD &KURQLF KRXU 3 VXEFDSLWDWDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP ODQGILOO VLWHV LQ )ORULGD &RUUHODWLRQ EHWZHHQ WKH UHVXOWV RI WKH VWDQGDUG POf DQG PRGLILHG POf 3 VXEFDSLWDWD FKURQLF KRXU DVVD\V ,QIOXHQFH RI WLPH RQ $f FRQGXFWLYLW\ %f FKHPLFDO R[\JHQ GHPDQG &f WRWDO RUJDQLF FDUERQ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH ,QIOXHQFH RI WLPH RQ $f FRQGXFWLYLW\ %f FKHPLFDO R[\JHQ GHPDQG &f WRWDO RUJDQLF FDUERQ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH [Y

PAGE 16

$FXWH & GXELD DQG 0LFURWR[f DQG FKURQLF 3 VXEFDSLWDWDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH EHWZHHQ )HEUXDU\ DQG 0D\ $FXWH & GXELD DQG 0LFURWR[f DQG FKURQLF 3 VXEFDSLWDWDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH EHWZHHQ )HEUXDU\ DQG 0DUFK 5HODWLRQVKLS EHWZHHQ WKH UHVXOWV RI WKH FKURQLF KRXU 3 VXEFDSLWDWD ,&VRf DQG WKH DFXWH KRXU & GXELD /&f DVVD\V ZLWK 06: ODQGILOO OHDFKDWHV IURP IRXUWHHQ VLWHV LQ )ORULGD 5DQNLQJ RI VL[WHHQ 06: OHDFKDWHV ZLWK WKH UHVXOWV RI WKH 0LFURWR[ 07f 3 VXEFDSLWDWD 3 VXEf SXOH[ Sf DQG &HULRGDSKQLD GXELD &Gf DVVD\V 7KH 0HW3/$7( DVVD\ SURWRFRO IRU GHWHUPLQLQJ WKH KHDY\ PHWDO WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV 7KH SURWRFRO IRU GHWHUPLQLQJ +0%& RI 06: ODQGILOO OHDFKDWHV 7KH SURWRFRO XVHG IRU IUDFWLRQDWLRQ RI +0%& 0HW3/$7( UHVXOWV IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH DQG VLWH RYHU WLPH (IIHFW RI OHDFKDWH WUHDWPHQW E\ ILOWUDWLRQ 6ROLGVf '($( UHVLQ 2UJDQLFVf DQG 'RZH[ UHVLQ +DUGQHVVf RQ WKH +0%& 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH KRXU & GXELD DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH 0LFURWR[r1 DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH KRXU & GXELD DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH 0LFURWR[r1 DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH KRXU & GXELD DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH 0LFURWR[r1 DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ;9,

PAGE 17

3URFHGXUH IRU SUHSDULQJ 06: OHDFKDWHV DQG PHWKDQRO H[WUDFWV RI OHDFKDWHV IRU DQDO\VLV RI KRUPRQDO DFWLYLW\ 5HVSRQVH RI WKH <(6 DVVD\ WR S HVWUDGLRO (f 7RWDO LRQ FKURPDWRJUDP RI 06: OHDFKDWHV IURP /) )HE ff LQ WKH IXOO VFDQ PRGH 'RVHUHVSRQVH RI WKH /) 1RY nf PHWKDQRO H[WUDFWV YHUVXV FRQFHQWUDWLRQ IDFWRU 'RVHUHVSRQVH RI WKH /) 'HF ff PHWKDQRO H[WUDFWV YHUVXV FRQFHQWUDWLRQ IDFWRU 'RVHUHVSRQVH RI WKH /) 1RY ff PHWKDQRO H[WUDFWV YHUVXV FRQFHQWUDWLRQ IDFWRU 7KH VROLG SKDVH H[WUDFWLRQ SURWRFRO XVHG ZLWK 06: ODQGILOO OHDFKDWHV 7KH WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV WR \HDVW FHOOV DFFRUGLQJ WR WKH 17 SURFHGXUH [YLL

PAGE 18

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH 6FKRRO RI WKH 8QLYHUVLW\ RI )ORULGD LQ 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ $1 ,19(67,*$7,21 2) 72;,&,7< $1' +25021$/ $&7,9,7< ,1 /($&+$7(6 )520 081,&,3$/ 62/,' :$67( 06:f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r1 /HDFKDWHV ZHUH WHVWHG ZLWK 0HW3/$7( D KHDY\ PHWDO VSHFLILF DVVD\ $GGLWLRQDOO\ XVLQJ D \HDVW UHSRUWHU DVVD\ WKH OHDFKDWHV ZHUH WHVWHG IRU KRUPRQDO DFWLYLW\ /DQGILOO OHDFKDWHV DUH FRPSOH[ PL[WXUHV RI RUJDQLF DQG LQRUJDQLF FRQWDPLQDQWV ZLWK FRPSRVLWLRQV KHDYLO\ LQIOXHQFHG E\ VLWHVSHFLILF SDUDPHWHUV HJ ZDVWH FRPSRVLWLRQ DQG DJHf 7KH FKHPLFDO FRPSRVLWLRQ RI WKH )ORULGD ODQGILOO OHDFKDWHV YDULHG ZLGHO\ ,Q VRPH OHDFKDWHV KLJK OHYHOV RI XQLRQL]HG [YLLL

PAGE 19

DPPRQLD LQRUJDQLF FRPSRQHQWV &%2' DQG &2' ZHUH UHFRUGHG 7KH FRUUHVSRQGLQJ WR[LFLW\ DW WKHVH VLWHV ZDV KLJK 6LJQLILFDQW UHODWLRQVKLSV ZHUH VKRZQ EHWZHHQ WKH DPPRQLD FRQWHQW RI WKH OHDFKDWHV DQG WR[LFLW\ DV GHWHUPLQHG E\ WKH & GXELD 5 f DQG 3 VXEFDSLWDWD 5 f 7KH DVVD\V ZHUH UDQNHG IRU WKHLU VHQVLWLYLWLHV WR WKH 06: ODQGILOO OHDFKDWHV DV IROORZV & GXELD r SXOH[ !3 VXEFDSLWDWD POf a 3 VXEFDSLWDWD POf 0LFURWR[r1 7KH KHDY\ PHWDO WR[LFLW\ELRDYDLODELOLW\ DQG KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f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

PAGE 20

UHFRYHU\ RI KRUPRQDO DFWLYLW\ 7UHDWPHQW SURFHVVHV XWLOL]LQJ SRZGHUHG DFWLYDWHG FDUERQ 3$&f UHPRYHG KRUPRQDO DFWLYLW\ [[

PAGE 21

&+$37(5 ,1752'8&7,21 +LVWRULFDOO\ GRPHVWLF ZDVWHV ZHUH GLVSRVHG LQ RSHQ SLWV RU VXUIDFH SLOHV DQG WKHVH VLWHV RIWHQ SRVHG D VLJQLILFDQW ULVN WR WKH VXUURXQGLQJ HQYLURQPHQWV 5HLQKDUW DQG 7RZQVHQG f &RQFHUQV ZLWK WKH HQYLURQPHQWDO IDWH RI GLVFDUGHG PDWHULDOV OHG WR WKH FRQVWUXFWLRQ RI HQJLQHHUHG V\VWHPV IRU ORQJWHUP VWRUDJH DQG PDQDJHPHQW RI ZDVWH PDWHULDOV DQG WKHLU GHJUDGDWLRQ SURGXFWV 7KHVH HQJLQHHUHG V\VWHPV LQFOXGHG OLQHG FHOOV IRU WKH GLVSRVDO RI ZDVWH DQG FROOHFWLRQ V\VWHPV IRU OHDFKDWH UHFRYHU\ *RYHUQPHQW UHJXODWLRQV SURKLELW WKH GLVSRVDO RI ZDVWH PDWHULDOV H[FHSW DW UHJXODWHG PXQLFLSDO VROLG ZDVWH 06:f GLVSRVDO IDFLOLWLHV $OWKRXJK 06: ODQGILOOV PD\ FRQWDLQ VPDOO TXDQWLWLHV RI KD]DUGRXV ZDVWH PDWHULDOV WKH\ DUH SULPDULO\ GHVLJQHG WR UHFHLYH GRPHVWLF ZDVWHV 7KHVH LWHPV PD\ LQFOXGH SDFNDJLQJ PDWHULDOV IRRG VFUDSV IXUQLWXUH FORWKLQJ DQG JUDVV FOLSSLQJV 86(3$ f 7KH DFXWH DQG FKURQLF WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV KDV EHHQ H[WHQVLYHO\ VWXGLHG XVLQJ YDULRXV ELRDVVD\V 5XWKHUIRUG HW DO &OHPHQW HW DO .DXUHWDO 3ORWNLQ DQG 5DP f KRZHYHU OLWWOH LQIRUPDWLRQ LV DYDLODEOH FRQFHUQLQJ WKH ELRORJLFDO HIIHFWV RI 06: OHDFKDWHV FROOHFWHG IURP ODQGILOOV LQ )ORULGD 5HLQKDUW DQG *URVK f KDYH UHSRUWHG D ORZHU FKHPLFDO VWUHQJWK LQ )ORULGD 06: OHDFKDWHV UHVXOWLQJ IURP WKH SUHGRPLQDQW HQYLURQPHQWDO FRQGLWLRQV HJ DEXQGDQW UDLQIDOO DQG ZDUP WHPSHUDWXUHV 7KHUHIRUH WKH EDVLV

PAGE 22

7DEOH )UHTXHQWO\ XVHG DFURQ\PV $FURQ\P 'HILQLWLRQ $FURQ\P 'HILQLWLRQ &%2' &DUERQDFHRXV ELRFKHPLFDO R[\JHQ GHPDQG +$$ +RUPRQDOO\ DFWLYH DJHQW &2' &KHPLFDO R[\JHQ GHPDQG +$& +RUPRQDOO\ DFWLYH FRPSRXQG &35* &KORURSKHQRO UHG JDODFWRS\UDQRVLGH +0%& +HDY\ PHWDO ELQGLQJ FDSDFLW\ '2& 'LVVROYHG RUJDQLF FDUERQ ,& 6DPSOH FRQFHQWUDWLRQ UHVSRQVLEOH IRU b LQKLELWLRQ LQ WHVW RUJDQLVP '20 'LVVROYHG RUJDQLF PDWWHU /& 6DPSOH FRQFHQWUDWLRQ OHWKDO WR b RI WKH WHVW RUJDQLVPV (L (VWURQH 06: 0XQLFLSDO VROLG ZDVWH H HVWUDGLRO 0+: 0RGHUDWHO\ KDUG ZDWHU H (VWULRO 213* 2UWKRQLWURSKHQ\O JDODFWRS\UDQRVLGH HH DHWKLQ\O HVWUDGLRO 7'6 7RWDO GLVVROYHG VROLGV (& 6DPSOH FRQFHQWUDWLRQ UHVSRQVLEOH IRU D b HIIHFW LQ WHVW RUJDQLVP 7,( 7R[LFLW\ LGHQWLILFDWLRQ HYDOXDWLRQ (' (QGRFULQH GLVUXSWHU 75( 7R[LFLW\ UHGXFWLRQ HYDOXDWLRQ (5D (VWURJHQ UHFHSWRU DOSKD 76 7RWDO VROLGV 8QLWHG 6WDWHV (5S (VWURJHQ UHFHSWRU EHWD 86(3$ (QYLURQPHQWDO 3URWHFWLRQ $JHQF\ (5&$/8; (VWURJHQ UHFHSWRU FKHPLFDOO\ DFWLYDWHG OXFLIHUDVH UHSRUWHU JHQH <(6
PAGE 23

$&87( &+521,& 72;,&,7< 72;,&,7< /($&+$7( +25021( $&7,9,7< 0(7$/ %,2$9$,/$%,/I7< 0(7$/ %,1',1* )LJXUH 3DWKZD\V IRU WKH FKDUDFWHUL]DWLRQ RI WKH ELRORJLFDO HIIHFWV RI 06: ODQGILOO OHDFKDWHV ,Q UHFHQW \HDUV WKH QXPEHU RI RSHUDWLQJ ODQGILOOV LQ WKH 86 KDV GHFUHDVHG KRZHYHU WKH RYHUDOO VL]H RI WKH UHPDLQLQJ ODQGILOOV KDV LQFUHDVHG 86(3$ f /DQGILOOV FRQWLQXH WR EH WKH PRVW HFRQRPLFDOO\ IHDVLEOH DQG OHDVW HQYLURQPHQWDOO\ LQWUXVLYH PHWKRG IRU WKH GLVSRVDO RI GLVFDUGHG ZDVWH PDWHULDOV ,Q )ORULGD PRUH WKDQ PLOOLRQ WRQV WRWDO PLOOLRQ WRQVf RI 06: ZHUH ODQGILOOHG LQ WKH PRVW UHFHQW \HDU GDWD ZHUH DYDLODEOH ZLWK D SHU FDSLWD JHQHUDWLRQ UDWH RI SRXQGVSHUVRQGD\ )'(3 f :KLOH WKH FKHPLFDO DQG SK\VLFDO FRPSRVLWLRQ RI 06: ODQGILOO OHDFKDWHV LQ )ORULGD KDYH EHHQ VXPPDUL]HG 5HLQKDUW DQG *URVK f RQO\ RQH VWXG\ KDV HYDOXDWHG WKH WR[LFLW\ RI )ORULGD OHDFKDWHV :DUG HW DO f DQG WKHQ RQO\ RQ D YHU\ OLPLWHG VFDOH 7KH VFLHQWLILF FRPPXQLW\ HQYLURQPHQWDO UHJXODWRUV ODQGILOO RSHUDWRUV DQG WKH RSHUDWRUV RI IDFLOLWLHV WUHDWLQJ ODQGILOO OHDFKDWHV UHTXLUH

PAGE 24

FRPSUHKHQVLYH GDWDEDVHV WR SURYLGH LQIRUPDWLRQ UHODWLYH WR ELRORJLFDO HIIHFWV DQG FKHPLFDO FRQVWLWXHQWV RI )ORULGD 06: ODQGILOO OHDFKDWHV 1XPHURXV DFURQ\PV DUH IUHTXHQWO\ XVHG LQ WKH VFLHQWLILF UHVHDUFK FRPPXQLW\ DQG PDQ\ DUH FRPPRQO\ UHFRJQL]HG KRZHYHU VRPH DUH UHODWLYHO\ QHZ DQG GLVFLSOLQHVSHFLILF 7KHUHIRUH D WDEOH RI WKH DFURQ\PV XVHG LQ WKH IROORZLQJ FKDSWHUV KDV EHHQ LQFOXGHG WR DLG WKH UHDGHU DV D TXLFN UHIHUHQFH 7DEOH f 7KH SXUSRVH RI WKLV UHVHDUFK ZDV WR HYDOXDWH 06: ODQGILOO OHDFKDWHV LQ )ORULGD IRU WKHLU WR[LFLW\ KRUPRQDO DFWLYLW\ DQG FKHPLFDO FKDUDFWHULVWLFV 7R GDWH WKHUH KDYH EHHQ QR UHSRUWHG LQYHVWLJDWLRQV RI 06: ODQGILOOV RI WKLV VFRSH RU PDJQLWXGH 0RVW LQYHVWLJDWLRQV KDYH IRFXVVHG RQ OHDFKDWH IURP RQH RU VHYHUDO ODQGILOOV .DXU HW DO :RQJ f EXW IHZ HYDOXDWHG PXOWLSOH OHDFKDWHV &OHPHQW HW DO f DQG QRQH KDYH WUDFNHG SDWWHUQV RYHU WLPH 7KH WR[LFLW\ RI OHDFKDWHV IURP )ORULGD ODQGILOOV KDV UHFHLYHG OLWWOH DWWHQWLRQ :DUG HW DO f HYDOXDWHG WKH WR[LFLW\ RI ODQGILOO OHDFKDWHV IURP WKUHH )ORULGD ODQGILOOV KRZHYHU OHDFKDWHV ZHUH HYDOXDWHG ZLWK RQO\ RQH DFXWH WR[LFLW\ DVVD\ 7KH EDVLV RI WKLV UHVHDUFK SURMHFW ZDV WR SURYLGH D VWDWHZLGH GDWDEDVH IRU ODQGILOO RSHUDWRUV UHJXODWRUV DQG RWKHU UHVHDUFK LQYHVWLJDWRUV WKDW HVWDEOLVKHG D UDQJH RI ELRORJLFDO HIIHFWV IURP H[SRVXUH WR 06: ODQGILOO OHDFKDWHV 7KH RYHUDOO REMHFWLYHV RI WKLV UHVHDUFK SURMHFW ZHUH DV IROORZV )LJXUH f 7R FKDUDFWHUL]H WKH WR[LFLW\ LQ WKH 06: ODQGILOO OHDFKDWHV XVLQJ D EDWWHU\ RI WR[LFLW\ DVVD\V LQFOXGLQJ WKH FKURQLF KU 6HOHQDVWUXP FDSULFRPXWXP DQG

PAGE 25

WKH DFXWH KRXU &HULRGDSKQLD GXELD KRXU 'DSKQLD SXOH[ DQG 0LFURWR[r1 DVVD\ &KDSWHUV DQG f 7R FKDUDFWHUL]H WKH FKHPLFDO DQG SK\VLFDO FRPSRVLWLRQ RI 06: ODQGILOO OHDFKDWHV &KDSWHUV DQG f 7R GHWHUPLQH WKH WR[LFLW\ RI KHDY\ PHWDOV LQ 06: ODQGILOO OHDFKDWHV ZLWK WKH KHDY\ PHWDO VSHFLILF 0HW3/$7( DVVD\ DQG WR TXDQWLI\ WKH DELOLW\ RI 06: OHDFKDWHV WR UHGXFH WKH ELRDYDLODELOLW\ RI KHDY\ PHWDOV ZLWK D KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f DVVD\ &KDSWHU f 7R FRQGXFW D WR[LFLW\ LGHQWLILFDWLRQ HYDOXDWLRQ 7,(f ZLWK VHOHFWHG 06: ODQGILOO OHDFKDWHV &KDSWHU f 7R GHWHUPLQH WKH KRUPRQDO DFWLYLW\ RI 06: ODQGILOO OHDFKDWHV ZLWK D \HDVW HVWURJHQ VFUHHQ <(6f DQG LGHQWLI\ E\ JDV FKURPDWRJUDSK\PDVV VSHFWURPHWU\ *&06f RUJDQLF FRPSRXQGV UHVSRQVLEOH IRU KRUPRQDO DFWLYLW\ &KDSWHU f 7R GHWHUPLQH WKH HIIHFW RI SRZGHUHG DFWLYDWHG FDUERQ WUHDWPHQW RQ KRUPRQDO DFWLYLW\ LQ ODQGILOO OHDFKDWHV &KDSWHU f 7KH UHVXOWV REWDLQHG GXULQJ WKLV UHVHDUFK LQYHVWLJDWLRQ DUH VXPPDUL]HG &KDSWHU f

PAGE 26

&+$37(5 /,7(5$785( 5(9,(: )RUW\ \HDUV DIWHU 5DFKHO &DUVRQ ILUVW UHYHDOHG WKH ULVNV WR KXPDQV DQG WKH HQYLURQPHQW SRVHG E\ D GLYHUVH DUUD\ RI PDQPDGH VXEVWDQFHV WKH WKUHDW IURP WKHVH DQWKURSRJHQLF FKHPLFDOV SHUVLVWV &DUVRQ f 2YHU V\QWKHWLF FKHPLFDOV LQFOXGLQJ SHVWLFLGHV VROYHQWV GRPHVWLF FOHDQHUV SODVWLFL]HUV DQG IODPHUHWDUGDQWV DUH SURGXFHG \HDUO\ IRU GRPHVWLF DQG LQGXVWULDO XVDJH KRZHYHU OLWWOH LV NQRZQ DERXW WKH ELRORJLFDO HIIHFWV IURP ORQJWHUP H[SRVXUH WR WKHVH FRPSRXQGV 'DUQHUXG HW DO +DOH HW DO /\\WLNDLQHQ HW DO f ,Q WKH 86 WKH 5HVRXUFH &RQVHUYDWLRQ DQG 5HFRYHU\ $FW 5&5$f 7R[LF 6XEVWDQFHV &RQWURO $FW 7R6&$f DQG WKH &OHDQ :DWHU $FW &:$f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f 7KHVH OHDFKDWHV UHSUHVHQW WKH PRELOH IUDFWLRQ RI ODQGILOO WR[LFDQWV DQG

PAGE 27

WKH\ FRQWDLQ KLJK FRQFHQWUDWLRQV RI LQRUJDQLF DQG RUJDQLF FRPSRXQGV %R]NXUW HW DO f 7KH PDMRULW\ RI 06: ODQGILOO OHDFKDWHV DUH FORVHO\ UHJXODWHG WKHUHIRUH WKHLU UHOHDVH RI OHDFKDWHV IURP PRGHUQ ODQGILOOV LV XQOLNHO\ %DUOD] HW DO f $FFLGHQWDO OHDFKDWH UHOHDVHV PD\ RFFXU H J GXULQJ SHULRGV RI KLJK UDLQIDOO ZKHQ OHDFKDWHV DUH FRQWDPLQDWHG E\ VWRUPZDWHU RU IURP WKH LPSURSHU RU IDXOW\ LQVWDOODWLRQ RI OHDFKDWH FROOHFWLRQ V\VWHPV $GGLWLRQDO VRXUFHV IRU WKH XQLQWHQWLRQDO UHOHDVH RI 06: ODQGILOO OHDFKDWHV H[LVW 3ULRU WR IHGHUDO UHJXODWLRQV GLG QRW UHTXLUH ODQGILOO OLQHUV WKHUHIRUH WKH HVFDSH RI OHDFKDWHV IURP WKHVH VLWHV UHSUHVHQWV D SRWHQWLDO DGYHUVH HQYLURQPHQWDO LPSDFW $VVPXWK f )ORULGD ZDV PRUH SURDFWLYH DQG UHTXLUHG ODQGILOO OLQHUV IRU 06: ODQGILOO LQ WKH 6ROLG :DVWH $FW RI /DQGILOOLQJ UHPDLQV WKH SUHGRPLQDQW PDQDJHPHQW PHWKRG IRU PXQLFLSDO VROLG ZDVWH 06:f DFFRXQWLQJ IRU PRUH WKDQ b PLOOLRQ WRQVf RI WKH 06: JHQHUDWHG LQ WKH 8QLWHG 6WDWHV 86(3$ f 8QGHU DXWKRULW\ JUDQWHG E\ 5&5$ VXEWLWOH WKH 8 6 (QYLURQPHQWDO 3URWHFWLRQ $JHQF\ 86(3$f UHJXODWHV WKH FRQVWUXFWLRQ RSHUDWLRQ DQG SRVWFORVXUH RI PXQLFLSDO VROLG ZDVWH 06:f ODQGILOOV &)5 f 6WULFW UHJXODWLRQV UHTXLUH ODQGILOO RSHUDWRUV WR PLQLPL]H UHFRYHU DQG WUHDW WKH OHDFKDWHV JHQHUDWHG LQ 06: ODQGILOOV /DQGILOO OLQHUV DUH XVHG WR UHVWULFW WKH IORZ RI OHDFKDWHV WR JURXQG DQG VXUIDFH ZDWHUV DQG WKH\ PD\ EH FRPSRVHG RI FOD\ KLJK RU ORZGHQVLW\ SRO\HWK\OHQH RU FRQFUHWH GHSHQGLQJ RQ WKH ORFDO FRQGLWLRQV ,Q VRPH ODQGILOOV OHDFKDWHV DUH UHF\FOHG WKURXJK WKH ZDVWH WR HQFRXUDJH ZDVWH VWDELOL]DWLRQ DQG LPSURYH OHDFKDWH TXDOLW\ 5HLQKDUW DQG 7RZQVHQG 1RSKDUDWDQD HW DO f

PAGE 28

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f VXFK DV HOHFWULFDO GHYLFHV IOXRUHVFHQW OLJKW EXOEV WKHUPRPHWHUV EDWWHULHV SHVWLFLGHV DQG RWKHU FKHPLFDO SURGXFWV %R\OH DQG %DHW] f 8VLQJ D ULVNEDVHG DVVHVVPHQW FRPSDUDEOH KHDOWK ULVNV ZHUH DVVRFLDWHG ZLWK 06: ODQGILOO OHDFKDWHV DQG LQGXVWULDO ZDVWH OHDFKDWHV %URZQ DQG 'RQQHOO\ f 7KHLU DVVHVVPHQW ZDV EDVHG RQ WKH LQGLYLGXDO OHDFKDWH FRQVWLWXHQWV ZKLOH QRW DFFRXQWLQJ IRU SRWHQWLDO V\QHUJLVWLF DQGRU DQWDJRQLVWLF UHVSRQVHV %URZQ DQG 'RQQHOO\ f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

PAGE 29

&KDUDFWHUL]DWLRQ RI WKH &KHPLFDO DQG 3K\VLFDO &RPSRVLWLRQ RI 06: /DQGILOO /HDFKDWHV :DVWH VWDELOL]DWLRQ LV EDVHG RQ PLFURELDO GHJUDGDWLRQ SURFHVVHV ZLWK WKH FRQYHUVLRQ RI RUJDQLF PDWWHU WR PHWKDQH JDV RFFXUULQJ SUHGRPLQDWHO\ XQGHU DQDHURELF FRQGLWLRQV %DUOD] f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f $V ODQGILOOLQJ ,V D FRQWLQXRXV DQG RQJRLQJ SURFHVV YDULRXV VWDJHV RI ZDVWH GHFRPSRVLWLRQ RFFXU VLPXOWDQHRXVO\ DQG WKLV PD\ EH UHIOHFWHG LQ WKH FKHPLFDO FKDUDFWHULVWLFV RI WKH OHDFKDWHV 06: ODQGILOO OHDFKDWHV DUH FRPSOH[ PL[WXUHV ZLWK D ZLGH UDQJLQJ FKHPLFDO VWUHQJWK 7DEOH f ,Q D UHFHQW UHYLHZ .MHOGVHQ HW DO f VXPPDUL]HG WKH FKHPLFDO DQG SK\VLFDO FKDUDFWHULVWLFV RI 06: ODQGILOO OHDFKDWHV

PAGE 30

7DEOH 5DQJH RI VHOHFWHG FKHPLFDO DQG SK\VLFDO FKDUDFWHULVWLFV UHSRUWHG LQ WKH OLWHUDWXUH IRU GRPHVWLF ZDVWHZDWHU DQG 06: ODQGILOO OHDFKDWHV LQ )ORULGD DQG LQWHUQDWLRQDOO\ 3DUDPHWHU 5DZ :DVWHZDWHU )ORULGD 06: /DQGILOO /HDFKDWH ,QWHUQDWLRQDO 06: /DQGILOO /HDFKDWH %2' PJ/f F r r &2' PJ/f F r r $ONDOLQLW\ PJ/f r n E S+ r D QKQ PJ/f r r EG &KORULGH PJ/f r r E 6XOILGH PJ/f r E 7'6 PJ/f r n 1D PJ/f r n E &D PJ/f n 0J PJ/f n 7RWDO SKRVSKRUXV PJ/ DV 327f r n r &RQGXFWLYLW\ P6FPf f§ U!af§Q n r 5HIHUHQFHV .DGOHF DQG .QLJKW f&DPHURQ DQG .RFK f0HWFDOI DQG (GG\ f/R n5HLQKDUW DQG *URVK nWKLV UHVHDUFK D.MHLGVHQ HW DO 7KH\ VWUHVVHG IRXU SULPDU\ FDWHJRULHV RI FRQWDPLQDQWV WR FRQVLGHU LQ GLVFXVVLRQ" RI OHDFKDWH TXDOLW\ DQG WKHVH ZHUH GLVVROYHG RUJDQLF PDWWHU RUJDQLF [HQRELRWLFV LQRUJDQLF FRPSRQHQWV DQG KHDY\ PHWDOV &KULVWHQVHQ HW DO f ,Q 06: ODQGILOO OHDFKDWHV GLVVROYHG RUJDQLF PDWWHU '20f LQFOXGHV WKH GLVVROYHG DQG FROORLGDO SDUWLFOHV *RXQDULV HW DO f 7KH PROHFXODU VWUXFWXUH DQG HOHPHQWDO FRPSRVLWLRQ RI GLVVROYHG RUJDQLF PDWWHU LQ 06: ODQGILOO OHDFKDWHV LV VWURQJO\ LQIOXHQFHG E\ PLFURELDO GHJUDGDWLYH SURFHVVHV &DODFH HW DO f

PAGE 31

UHSRUWHG D QDUURZ GLVWULEXWLRQ RI RUJDQLF PROHFXODU ZHLJKW JURXSV LQ \RXQJ ODQGILOOV \HDUV ROGf ZLWK SULPDULO\ ORZ PROHFXODU ZHLJKW FRQVWLWXHQWV 'DOWRQf 7KLV FRQWUDVWHG ZLWK WKHLU ILQGLQJV LQ ROGHU ODQGILOOV \HDUV ROGf ZLWK DQ LQFUHDVHG GLVWULEXWLRQ RI PROHFXODU ZHLJKW IUDFWLRQV DQG KLJK PROHFXODU ZHLJKW FRQVWLWXHQWV 'DOWRQf &DODFH HW DO f 7KHVH KLJK PROHFXODU ZHLJKW IUDFWLRQV FRQWDLQ VWUXFWXUDOO\ FRPSOH[ KXPLF PDWHULDOV &URXH HW DOf f .DQJ HW DO f UHSRUWHG DQ LQFUHDVHG SUHVHQFH RI KXPLF VXEVWDQFHV ZLWK LQFUHDVLQJ ODQGILOO DJH DQG D GHFUHDVH LQ WKH HDVLO\ GHJUDGHG ORZHU PROHFXODU ZHLJKW RUJDQLF PDWHULDOV $WWULEXWHG WR WKH KLJK DPPRQLD FRQFHQWUDWLRQV LQ WKH OHDFKDWH WKH KXPLF PDWHULDOV FRQWDLQHG D ODUJH GLVWULEXWLRQ RI QLWURJHQ IXQFWLRQDO JURXSV .DQJ HW DO f 7KLV LV VLJQLILFDQW ZKHQ FRQVLGHULQJ WKH VWURQJ PHWDO FRPSOH[HV WKDW DUH IRUPHG ZLWK RUJDQLF OLJDQGV FRQWDLQLQJ QLWURJHQ IXQFWLRQDO JURXSV &URXH HW DO 6WXPP DQG 0RUJDQ f ;HQRELRWLFV DUH IUHTXHQWO\ GHWHFWHG DW ORZ OHYHOV LQ 06: ODQGILOO OHDFKDWHV 6FKZDU]EDXHU HW DO .DZDJRVKL HW DO
PAGE 32

,QRUJDQLF FRQWDPLQDQWV LQ 06: ODQGILOO OHDFKDWHV LQFOXGH DQLRQLF DQG FDWLRQLF VSHFLHV DQG VRPH RI WKH PRVW ZLGHVSUHDG DUH 1+r &Dr 0J 1D &On +&2n 62n 7\SLFDOO\ WKH DPPRQLD LQ 06: ODQGILOO OHDFKDWHV RFFXUV DV WKH LRQL]HG DPPRQLXP 1+f VSHFLHV $PPRQLD VSHFLDWLRQ LV S+ GHSHQGHQW ZLWK D S.D RI 7KHUHIRUH WKH GRPLQDQW VSHFLHV LV DPPRQLXP UDWKHU WKDQ WKH KLJKO\ WR[LF DPPRQLD 1+f IRUP 0F%HDQ HW DO f *HQHUDOO\ WRWDO DPPRQLD FRQFHQWUDWLRQV DUH KLJK ZLWK UHSRUWV RI XS WR PJ/ .MHOGVHQ HW DO f 7KHVH KLJK DPPRQLD OHYHOV DUH RIWHQ DWWULEXWHG WR WKH DEVHQFH RI GHJUDGDWLYH SDWKZD\V IRU WKH UHPRYDO RI DPPRQLD IURP ODQGILOOV %XUWRQ DQG :DWVRQ&UDLN f $OVR IRXQG DW KLJK FRQFHQWUDWLRQV DUH WKH KDUGQHVV FDWLRQV +DUGQHVV LV D PHDVXUH RI PXOWLYDOHQW PHWDOOLF FDWLRQV HJ &D r 0Jr 6Ur )H 0Q EXW PDLQO\ &DrDQG 0J6WXPP DQG 0RUJDQ f +DUGQHVV KDV D VWURQJ LQIOXHQFH RQ KHDY\ PHWDO ELRDYDLODELOLW\ LQ ODQGILOO OHDFKDWHV +HLMHULFN HW DO f +HDY\ PHWDOV DUH JHQHUDOO\ UHSRUWHG LQ WKH ORZ PJ/ UDQJH 5HLQKDUW DQG *URVK f +RZHYHU WKLV UHSUHVHQWV RQO\ D IUDFWLRQ RI WKH WRWDO PHWDO DVVRFLDWHG ZLWK WKH ZDVWH ,Q VRPH FDVHV PHWDOV WKDW DUH VWURQJO\ DVVRFLDWHG ZLWK ZDVWH PDWHULDOV DUH QRW HDVLO\ OHDFKHG XQGHU ODQGILOO FRQGLWLRQV )O\KDPPHU f LQ D PDVV EDODQFH RQ FDGPLXP LQ 6ZHGLVK ODQGILOOV FRQFOXGHG WKDW WKH WRWDO FRQFHQWUDWLRQV DVVRFLDWHG ZLWK WKH ODQGILOOHG ZDVWHV ZHUH XS WR IRXU RUGHUV RI PDJQLWXGH JUHDWHU WKDQ OHDFKHG FRQFHQWUDWLRQV )O\KDPPHU f .MHOGVHQ HW DO f GLVFXVVHG OHDFKDWH FKDUDFWHULVWLFV LQ 06: ODQGILOOV ZLWK D SULPDULO\ RUJDQLF FRPSRVLWLRQ DQG WKH LQIOXHQFHV RI WLPH RQ WKHVH OHDFKDWH

PAGE 33

7DEOH &RPSRXQG & GXELD 6 FDSULFRPXWXP 0LFURWR[r1 PDJQD 0HW3/$7(r1 PJ/f PJ/f PJ/f PJ/f PJ/f &RSSHU E K n I n PLQ (& KU /& &DGPLXP F K n =LQF K n PLQ (& n KU /& n 7RWDO I DPPRQLD PLQ (&VR 8QLRQL]HG D r H DPPRQLD PLQ (&} KU (& 0DQJDQHVH G 0+:f $ONDOLQLW\ +&&9f &KORULGH 6RGLXP n5KRGHV n/DVVLHU HW DOf n&OHPHQW DQG 0HUOLQ n4XUHVKL HW DO f+RNH HW DO K&KHQ HW DO n'RKHUW\ HW DO f%LWWRQ HW DO FKDUDFWHULVWLFV ,Q HDUOLHU ZRUN %R]NXUW HW DO f GLVFXVVHG OHDFKDWHV JHQHUDWHG LQ 06: ODQGILOOV ZLWK HLWKHU D SULPDULO\ RUJDQLF RU LQRUJDQLF ZDVWH FRPSRVLWLRQ 7KH ODWWHU FDVH UHSUHVHQWHG DVK PRQRILOOV RU FRGLVSRVDO IDFLOLWLHV IRU 06: DQG DVK 7KH IRFXV RI WKHLU LQYHVWLJDWLRQ ZDV WR SUHGLFW WKH ORQJWHUP IDWH RI KHDY\ PHWDOV XVLQJ D FRQFHSWXDO PRGHO ZKLFK LQFOXGHG LQIOXHQFHV IURP YDULRXV RUJDQLF DQG LQRUJDQLF OLJDQGV %R]NXUW HW DO f 7KH WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV LV KHDYLO\ LQIOXHQFHG E\ FKHPLFDO DQG SK\VLFDO LQWHUDFWLRQV 7DEOH f ,Q WKLV UHJDUG FRPSOH[DWLRQ UHDFWLRQV KDYH D PLWLJDWLQJ LQIOXHQFH RQ WR[LFLW\ DQG DUH IUHTXHQWO\ XQGHUHVWLPDWHG DQG SRRUO\ XQGHUVWRRG GXH WR WKH VKHHU QXPEHU RI SRVVLEOH OLJDQGV LQ VROXWLRQ 0DUWHQVVRQ HW DO 6WXPP DQG 0RUJDQ f 6RPH RI WKH W\SLFDO

PAGE 34

LQRUJDQLF OLJDQGV LQ OHDFKDWHV LQFOXGH FDUERQDWH 6OHWWHQ HW DO f FKORULGH %ROWRQ DQG (YDQV f DQG VXOILGH LRQV %R]NXUW HW DO f 7KHVH LQRUJDQLF OLJDQGV FDQ IRUP LQVROXEOH SUHFLSLWDWHV ZLWK KHDY\ PHWDOV 0DMRQH HW DO f 2WKHU OLJDQGV SUHVHQW DUH GLVVROYHG RUJDQLF PDWWHU .DVFKO HW DO f DQG FROORLGDO VROLGV *RXQDULV HW DO f 7KHVH FRPSOH[HV H[HUW D VWURQJ LQIOXHQFH RQ KHDY\ PHWDO WR[LFLW\ +HLMHULFN HW DO f ZLWK XS WR b RI PHWDOV LQ VRPH ODQGILOOV SUHVHQW DV RUJDQRPHWDOOLF FRPSOH[HV .DQJ HW DO :HQJ HW DO f +RZHYHU WKHUH KDYH EHHQ VRPH TXHVWLRQV FRQFHUQLQJ WKH WR[LFLW\ RI WKHVH RUJDQRPHWDOOLF FRPSOH[HV 3DOPHU HW DO f )UDVHU HW DO f VXJJHVWHG ORZOHYHO WR[LFLW\ DVVRFLDWHG ZLWK FRPSOH[HV EHWZHHQ FRSSHU DQG GLVVROYHG RUJDQLF PDWHULDOV '20f %LRDVVD\V IRU WKH (YDOXDWLRQ RI 7R[LFLW\ LQ WKH (QYLURQPHQW /XRPD f GHVFULEHG ELRDVVD\V DV WRROV IRU LQYHVWLJDWLQJ WKH FRPSOH[ FRQWLQXXP RI ELRFKHPLFDO SK\VLRORJLFDO DQG UHSURGXFWLYH UHVSRQVHV WKDW RFFXU LQ RUJDQLVPV IROORZLQJ H[SRVXUHV WR VXVSHFW WR[LFDQWV 7UDGLWLRQDOO\ ELRDVVD\V ZLWK YDULRXV YHUWHEUDWH DQG LQYHUWHEUDWH RUJDQLVPV ZHUH XVHG WR PRQLWRU DQG WUDFN HQYLURQPHQWDO SHUWXUEDWLRQV 86(3$ D Df 7KHVH ELRDVVD\V PHDVXUHG FKURQLF HIIHFWV LQ ORZOHYHO ORQJWHUP H[SRVXUHV DQG DFXWH WR[LFLW\ LQ KLJKOHYHO VKRUWWHUP H[SRVXUHV &ULWLFDOO\ LPSRUWDQW WR DOO HQYLURQPHQWDO UHVHDUFKHUV ZDV KRZ EHVW WR UHFRQFLOH FKURQLF ORZOHYHO HQYLURQPHQWDO H[SRVXUH ZLWK ODERUDWRU\ LQYHVWLJDWLRQV XWLOL]LQJ KLJK GRVH DFXWH VXEVWDQFHV 0RZDW DQG %XQG\ 'HJHQ DQG %ROW f 6RPH RI WKH PRVW FRPPRQ ELRDVVD\V KDYH XVHG DOJDH (XOODIIUR\ DQG 9HUQHW YDQ GHU +HHYHU DQG *UREEHODDU f DTXDWLF SODQWV 0RKDQ DQG +RVHWWL .ODLQH DQG /HZLV f

PAGE 35

LQYHUWHEUDWHV +HLMHULFN HW DO .LP HW DO 3UHVWRQ DQG 6QHOO 3HUHLUD HW DO f RU PLFURRUJDQLVPV /H%ORQG HW DO 'RKHUW\ HW DO -XQJ HW DO f :KLOH PRVW ELRDVVD\V KDYH EHHQ H[WHQVLYHO\ YDOLGDWHG HDFK KDV LQWULQVLF DGYDQWDJHV DQG GLVDGYDQWDJHV WKDW DUH VSHFLILF WR WKH PHWKRG )RU H[DPSOH LQ VRPH DOJDO DVVD\V FHOO H[XGDWHV H[WUDFHOOXODU RUJDQLF PDWHULDOf PLWLJDWH PHWDO WR[LFLW\ E\ DFWLQJ DV OLJDQGV WKDW IRUP FRPSOH[HV ZLWK IUHH PHWDO LRQV ,Q PLFURSODWH DVVD\V WKHVH DQG VLPLODU SUREOHPV DUH FRQWUROOHG ZKLFK PD\ H[SODLQ WKH UHFHQW LQFUHDVH LQ WKH XVH RI PLFURELRWHVWV $GGLWLRQDOO\ WKHVH PLFURELRWHVWV RIIHU DQ LQFUHDVHG DIIRUGDELOLW\ SRUWDELOLW\ DQG WKH DYDLODELOLW\ RI UHVXOWV LQ D VKRUW LQWHUYDO RI WLPH &KLDO DQG 3HUVRRQH %LWWRQ HW DO f *DEULHOVRQ HW DO f UHFHQWO\ GHYHORSHG D PLFURSODWH DVVD\ UHIHUUHG WR E\ WKH DFURQ\P 0$5$ PLFURSODWH DVVD\ ULVN DVVHVVPHQWf WKDW XWLOL]HG O\RSKLOL]HG PLFURELDO VWUDLQV IRU GHWHUPLQLQJ WKH WR[LF ILQJHUSULQW RI D FKHPLFDO 7KLV DVVD\ DOORZV IRU WKH WHVWLQJ RI PXOWLSOH VSHFLHV VLPXOWDQHRXVO\ KRZHYHU LW LV QRW VHQVLWLYH WR DQ\ VSHFLILF FODVV RI WR[LFDQWV 7KHUH DUH PLFURSODWH DVVD\V H J 0HW3/$7( DQG 0HW3$'f WKDW DUH GHVLJQHG VSHFLILFDOO\ IRU WKH GHWHFWLRQ RI KHDY\ PHWDO WR[LFLW\ %LWWRQ HW DO E f 'HYHORSPHQWV LQ WKH ILHOG RI HQYLURQPHQWDO FKHPLVWU\ KDYH SURGXFHG DQDO\WLFDO PHWKRGRORJLHV DQG WHFKQLTXHV WKDW DUH KLJKO\ VXFFHVVIXO DW LGHQWLI\LQJ DQG TXDQWLI\LQJ FRQWDPLQDQWV HYHQ LQ KLJKO\ FRPSOH[ PDWULFHV 5LFKDUGVRQ f 7KH\ XVH D VXLWH RI DQDO\WLFDO WRROV WKDW KDYH LQ FRPPRQ DQ HOHFWURGH ZKLFK VHQVHV FKDQJHV EDVHG RQ HOHFWURQLF VLJQDOV 6RPH W\SLFDO HOHFWURGHV

PAGE 36

PHDVXUH GLVVROYHG R[\JHQ '2f FRQGXFWLYLW\ S+ VHOHFW LRQV HJ LRQL]HG PHWDOVf DQG R[LGDWLYH SRWHQWLDO 3DUDOOHO WHFKQLTXHV IRU DSSOLFDWLRQ LQ WKH ILHOG RI HQYLURQPHQWDO WR[LFRORJ\ ZRXOG DOORZ IRU WKH UDSLG LGHQWLILFDWLRQ RI WR[LFLW\ ZKLOH VLPXOWDQHRXVO\ UHGXFLQJ WKH WLPH DQG FRVW LQYROYHG LQ FRQWLQXRXV PRQLWRULQJ SURJUDPV $ULNDZD HW DO f %LRVHQVRUV DUH D UDSLG DQG FRQYHQLHQW PRQLWRULQJ WRRO ZKLFK LQFRUSRUDWH ELRORJLFDO WLVVXHV LQ D V\VWHP KLJKO\ VHQVLWLYH WR D EURDG VSHFWUXP RI WR[LF VXEVWDQFHV %RWUH HW DO %XIIWH DQG +RUYDL $UJHVH HW DO f $OWKRXJK WKHLU XVH LV FXUUHQWO\ OLPLWHG ELRVHQVRUV KDYH EHHQ VXFFHVVIXOO\ DSSOLHG WR WKH HYDOXDWLRQ RI ZDVWHZDWHU WR[LFLW\ )DUUH DQG %DUFHOOR f 7R[LFLW\ RI 06: /DQGILOO /HDFKDWHV 5HVHDUFK LQYHVWLJDWLRQV WKDW VLPXOWDQHRXVO\ FRPELQH ELRDVVD\V ZLWK PHWKRGV IRU FKHPLFDO FKDUDFWHUL]DWLRQ DUH KLJKO\ YDOXHG EXW WKH DVVRFLDWHG H[SHQVHV DQG ODERU GHPDQGV FRQVWUDLQ WKHLU H[WHQVLYH XWLOL]DWLRQ )HUUDUL HW DO $WZDWHU HW DO f 7R GDWH WKH PRVW H[WHQVLYH VWXG\ RI ZDVWH OHDFKDWHV ZDV FRQGXFWHG LQ )UDQFH &OHPHQW HW DO f LQYHVWLJDWHG WKH WR[LFLW\ RIWHQ GRPHVWLF ODQGILOO OHDFKDWHV DQG YDULRXV RWKHU KD]DUGRXV DQG QRQ KD]DUGRXV ZDVWH OHDFKDWHV %LRDVVD\ UHVXOWV ZLWK SURWR]RD EDFWHULD DOJDH DQG LQYHUWHEUDWHV GHPRQVWUDWHG WKDW WKH WR[LFLW\ RI WKH GRPHVWLF ZDVWH OHDFKDWH ZDV KLJKHU WKDQ WKH LQGXVWULDO RU KD]DUGRXV ZDVWH OHDFKDWHV &OHPHQW HW DO f )XUWKHUPRUH WKH FKHPLFDO FKDUDFWHUL]DWLRQ RI WKH GRPHVWLF OHDFKDWHV UHYHDOHG WKDW DPPRQLD DONDOLQLW\ FRQGXFWLYLW\ DQG &2' ZHUH KLJKO\ DVVRFLDWHG ZLWK LQFUHDVHG OHDFKDWH WR[LFLW\ &OHPHQW HW DO f ,Q HDUOLHU ZRUN D VLJQLILFDQW FRQWULEXWLRQ RI DPPRQLD WR WKH DFXWH WR[LFLW\ RI ODQGILOO OHDFKDWH WR GXFNZHHG

PAGE 37

7DEOH 5HSRUWHG WR[LFLW\ LQ WKH OLWHUDWXUH IRU 06: ODQGILOO OHDFKDWHV /HDFKDWH 2ULJLQ 6SHFLHV (QGSRLQW 5HIHUHQFH 6ROLG ZDVWH ODQGILOO XQNQRZQ FRPSRVLWLRQf 7LODSLD 6DURWKHURGRQ PRVVDPELFXVf KU /& WR b :RQJ 6ROLG ZDVWH ODQGILOO b KRXVHKROG DQG b LQGXVWULDOFRPPHUFLDOf )DWKHDG PLQQRZ 3LPHSKDOHV SURPHWDVf KU/&R b 3ORWNLQ DQG 5DP 6ROLG ZDVWH ODQGILOO b KRXVHKROG DQG b LQGXVWULDOFRPPHUFLDOf 'DSKQLD PDJQD KU /&VR WR b 3ORWNLQ DQG 5DP 6ROLG ZDVWH ODQGILOO b KRXVHKROG DQG b LQGXVWULDOFRPPHUFLDOf 6HOHQDVWUXP FDSULFRPXWXP GD\ (&R WR b 3ORWNLQ DQG 5DP 6ROLG ZDVWH ODQGILOO b KRXVHKROG DQG b LQGXVWULDOFRPPHUFLDOf 0LFURWR[ PLQ (&VR b 3ORWNLQ DQG 5DP 6ROLG ZDVWH ODQGILOO XQNQRZQ FRPSRVLWLRQf $TXDWLF SODQWV (&VR b 'HYDUH DQG %DKDGLU 6ROLG ZDVWH ODQGILOO XQNQRZQ FRPSRVLWLRQf 0LFURWR[ (&R b 'HYDUH DQG %DKDGLU /HPQD VSf ZDV UHSRUWHG &OHPHQW DQG 0HUOLQ f &OHPHQW HW DO f SHUIRUPHG FRUUHODWLYH DQDO\VHV EHWZHHQ WKHLU ELRDVVD\ UHVXOWV DQG YDULRXV FKHPLFDO FKDUDFWHULVWLFV DQG UHYHDOHG D VWURQJ FRUUHODWLRQ 5 f EHWZHHQ 'DSKQLD PDJQD DQG FRPELQHG DPPRQLD DQG DONDOLQLW\ FRQFHQWUDWLRQV 6LPLODU UHODWLRQVKLSV ZHUH VKRZQ EHWZHHQ WKH ELRDVVD\ UHVXOWV ZLWK DTXDWLF SODQWV DOJDH DQG RWKHU FUXVWDFHDQV DQG DPPRQLD DQG DONDOLQLW\ &OHPHQW HW DO f 8VLQJ WKH 0LFURWR[r1 DVVD\ WKH UHODWLRQVKLS EHWZHHQ &2' DQG WR[LFLW\ ZDV

PAGE 38

VLJQLILFDQW S f EXW ZHDNHU 5 f 7KLV ZDV WKH RQO\ DVVD\ VHQVLWLYH WR WR[LFLW\ DVVRFLDWHG ZLWK LQFUHDVLQJ RUJDQLF FRQWHQW &OHPHQW HW DO f 7KH WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV KDYH EHHQ ZHOO FKDUDFWHUL]HG DURXQG WKH ZRUOG (UQVW HW DO /DPEROH] HW DO 'HYDUH DQG %DKDGLU &KHXQJ HW DO :RQJ 5DGL HW DO 3ORWNLQ DQG 5DP $WZDWHU HW DO 0LOOHPDQQ DQG 3DUNKXUVW &DPHURQ DQG .RFK f EXW WKH WR[LFLW\ RI )ORULGD ODQGILOO OHDFKDWHV UHPDLQV SRRUO\ FKDUDFWHUL]HG 7DEOH f :DUG HW DO f VWXGLHG WKH OHDFKDWHV IURP VL[ 06: ODQGILOO OHDFKDWHV LQ )ORULGD DQG FRQFOXGHG WKDW WKH OHDFKDWHV ZHUH KLJKO\ WR[LF 7KH WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV YDULHG ZLGHO\ GXH WR VLWHVSHFLILF FKHPLFDO FKDUDFWHULVWLFV RI WKH OHDFKDWHV )XUWKHUPRUH RQ D PRQWKO\ EDVLV IOXFWXDWLRQV LQ OHDFKDWH WR[LFLW\ LQGLFDWHG WKH KHWHURJHQRXV FRPSRVLWLRQ RI WKH ZDVWH PDWHULDOV DQG ORFDO FRQGLWLRQV :DUG HW DO f 5HFHQWO\ LQYHVWLJDWLRQV RI ZDVWH OHDFKDWHV LQ FRXQWULHV WKDW GR QRW UHTXLUH ODQGILOO OLQHUV PLQLPL]DWLRQ RI OHDFKDWH JHQHUDWLRQ RU OHDFKDWH FROOHFWLRQ DQG WUHDWPHQW KDYH EHHQ UHSRUWHG 7KHVH VWXGLHV RIIHU LQVLJKW WR UHVHDUFKHUV FRQFHUQHG ZLWK WKH SRWHQWLDO IRU OHDFKDWH UHOHDVH WR WKH HQYLURQPHQW 0DJGDOHQR DQG 'H 5RVD f FKDUDFWHUL]HG OHDFKDWHV IURP D ZDVWH GXPS LQ $UJHQWLQD ZLWK DQ DOJDO DVVD\ XVLQJ 6HOHQDVWUXP FDSULFRPXWXP UHQDPHG 3VHXGRNLUFKQHULHOOD VXEFDSLWDWDf ZKLOH 6LVLQQR HW DO f HYDOXDWHG ZDVWH OHDFKDWHV LQ %UD]LO ZLWK WKH =HEUDILVK %UDFK\GDQLR UHULRf 7KH FKHPLFDO VWUHQJWK RI WKHVH OHDFKDWHV ZHUH FRPSDUDEOH WR UHSRUWV RI RWKHUV .MHOGVHQ HW DO VHH 7DEOH f

PAGE 39

7KH FKHPLFDO VWUHQJWK RI WKH $UJHQWLQLDQ OHDFKDWHV ZDV GHPRQVWUDWHG E\ &2' FRQFHQWUDWLRQV IURP WR PJ/ DPPRQLD IURP WR PJ/ DQG S+ IURP WR 0DJGDOHQR DQG 'H 5RVD f 6LPLODU FKHPLFDO FKDUDFWHULVWLFV ZHUH UHSRUWHG ZLWK WKH OHDFKDWHV IURP %UD]LO ZLWK FRQGXFWLYLW\ YDOXHV IURP WR P6FP DONDOLQLW\ IURP WR PJ/ DV &D& DQG &2' IURP WR PJ/ 6LVLQQR HW DO f 2YHUDOO WKH WR[LFLW\ RI WKH $UJHQWLQLDQ OHDFKDWHV ZDV ORZ ZLWK 78 WR[LFLW\ XQLWf YDOXHV WKDW UDQJHG IURP WR 0DJGDOHQR DQG 'H 5RVD f +LJKHU WR[LFLW\ ZDV UHSRUWHG LQ WKH %UD]LOLDQ OHDFKDWHV DQG UDQJHG IURP WR 78 6LVLQQR HW DO f 7KH OHDFKDWHV IURP %UD]LO GHPRQVWUDWHG D WR[LFLW\ VLPLODU WR WKDW UHSRUWHG ZLWK )ORULGD OHDFKDWHV :DUG HW DO f +RZHYHU WKH OHDFKDWHV IURP $UJHQWLQD GLVSOD\HG UHGXFHG WR[LFLW\ 7KH SUHGRPLQDQFH RI SODVWLFV DQG RWKHU GLVSRVDEOH PDWHULDOV LQ 86 06: ODQGILOOV PD\ EH D IDFWRU FRQWULEXWLQJ WR WKHLU KLJKHU WR[LFLW\ 7KH FRQWDPLQDWLRQ RI JURXQGZDWHU E\ ZDVWH OHDFKDWHV LV D SULPDU\ FRQFHUQ UHODWLYH WR WKH HVFDSH RI OHDFKDWHV LQWR WKH HQYLURQPHQW %DXQ HW DO f LQYHVWLJDWHG WKH WR[LFLW\ RI JURXQGZDWHU FRQWDPLQDWHG E\ 06: OHDFKDWHV LQ 'HQPDUN ZLWK DQ DOJDH DVVD\ D FUXVWDFHDQ DVVD\ DQG D EDFWHULDO JHQRWR[LFLW\ DVVD\ 8VLQJ WKH DOJDH DVVD\ WKH OHDFKDWHFRQWDPLQDWHG JURXQGZDWHU VDPSOH GLVSOD\HG DQ (& RI b KRZHYHU WKH WR[LFLW\ GHFUHDVHG E\ b DW WZLFH WKH GLVWDQFH IURP WKH ODQGILOO 6LPLODU WR[LFLW\ ZDV GHPRQVWUDWHG E\ WKH FUXVWDFHDQ 'DSKQLD PDJQD ZLWK DQ (& RI b DW WKH ODQGILOO EXW IXUWKHU GRZQVWUHDP QR WR[LFLW\ ZDV UHSRUWHG )XUWKHU DQDO\VLV RI WKLV FRQWDPLQDWHG JURXQGZDWHU UHYHDOHG WKDW RUJDQLF FRQWDPLQDQWV ZHUH UHVSRQVLEOH

PAGE 40

IRU WKH KLJK WR[LFLW\ DQG ZLWK LQFUHDVLQJ GLVWDQFH IURP WKH ODQGILOO WKH RUJDQLF WR[LFLW\ GHFUHDVHG VXJJHVWLQJ PHWDEROLF GHJUDGDWLRQ RU GLOXWLRQ HIIHFWV %DXQ HW DO f $GGLWLRQDO ELRDVVD\V ZLWK WKH RUJDQLF IUDFWLRQ UHYHDOHG D ORZ VHQVLWLYLW\ RI WKH PDJQD DVVD\ WR RUJDQLF WR[LFDQWV ZKLFK FRQWUDVWHG ZLWK WKH DOJDO DQG 0LFURWR[r1 DVVD\ UHVXOWV %DXQ HW DO f 2WKHU UHVHDUFKHUV KDYH UHSRUWHG WKH KLJKHU VHQVLWLYLW\ RI 0LFURWR[r1 WR RUJDQLF FRQWDPLQDQWV %LWWRQ HW DO f ,Q UHSRUWV RI FRPSDUDEOH FDUFLQRJHQLF ULVN DVVRFLDWHG ZLWK H[SRVXUH WR 06: ODQGILOO OHDFKDWHV RU KD]DUGRXV ZDVWH OHDFKDWHV UDLVHG FRQFHUQV LQ WKH UHJXODWRU\ FRPPXQLW\ %URZQ DQG 'RQQHOO\ f 6XEVHTXHQW LQYHVWLJDWLRQV WR GHWHUPLQH WKH JHQRWR[LF SRWHQWLDO RI 06: ODQGILOO OHDFKDWHV KDYH UHYHDOHG FRQIOLFWLQJ UHVXOWV %HJ DQG $O0X]DLQL f LQYHVWLJDWHG WKH JHQRWR[LFLW\ RI 06: ODQGILOO OHDFKDWHV LQ .XZDLW XVLQJ D GDUN PXWDQW VWUDLQ QRQOXPLQHVFHQWf RI 9LEULR ILVKHUL D ELROXPLQHVFHQW EDFWHULXP ,Q WKH SUHVHQFH RI D PXWDJHQ WKH GDUN VWUDLQ UHYHUWV WR WKH OXPLQHVFHQW VWDWH DQG WKLV UHVSRQVH LV TXDQWLILHG E\ LQFUHDVHG OLJKW LQWHQVLW\ 7KHVH UHVXOWV VXJJHVWHG LQ VRPH RI WKH .XZDLWL OHDFKDWHV WKHUH ZDV D KLJK GHJUHH RI JHQRWR[LFLW\ DQG WKLV ZDV GHSHQGHQW RQ WKH W\SH RI ZDVWH ODQGILOOHG DQG VHDVRQDO FRQGLWLRQV %HJ DQG $O0X]DLQL f +HOPD HW DO f XVHG IRXU EDFWHULDO DVVD\V WR FKDUDFWHUL]H JHQRWR[LFLW\ LQ ODQGILOO OHDFKDWHV ZDVWHZDWHU HIIOXHQWV SXOS DQG SDSHUPLOO HIIOXHQWV DQG FRQWDPLQDWHG JURXQGZDWHU 2YHUDOO WKH KLJKHVW JHQRWR[LFLW\ ZDV GLVSOD\HG E\ WKH 06: ODQGILOO OHDFKDWHV ZLWK PRUH WKDQ UHYHUWDQWV/ RI OHDFKDWH 7KLV ZDV FRPSDUDEOH WR WKH JHQRWR[LFLW\ RI WKH OHDFKDWHV SURGXFHG E\ PL[HG LQGXVWULDO

PAGE 41

DQG GRPHVWLF ZDVWHV ZKLFK ZHUH UHSRUWHG DV DSSUR[LPDWHO\ UHYHUWDQWV/ +HOPD HW DO f %DXQ HW DO f XVLQJ WKH XPX& VWUDLQ RI 6DOPRQHOOD W\SKLPXUOXP VKRZHG WKDW OHDFKDWHFRQWDPLQDWHG JURXQGZDWHU ZDV QRW JHQRWR[LF DW FRQFHQWUDWLRQV XS WR b E\ YROXPH +RZHYHU EDFWHULDO WR[LFLW\ DW KLJKHU FRQFHQWUDWLRQV SUHYHQWHG WKH HYDOXDWLRQ RI JHQRWR[LF HIIHFWV $IWHU LVRODWLRQ RI WKH RUJDQLF IUDFWLRQ RI WKHVH FRQWDPLQDWHG JURXQGZDWHUV D VLPLODU PXWDJHQLFLW\ ZDV LGHQWLILHG 7KHVH UHVXOWV VXJJHVWHG WKDW WKH RUJDQLF IUDFWLRQ FRQWDLQHG WKH PXWDJHQV %DXQ HW DO f +RUPRQDOO\ $FWLYH $JHQWV LQ WKH (QYLURQPHQW 6RPH QDWXUDO DQG DQWKURSRJHQLF VXEVWDQFHV PD\ LQWHUDFW RU LQWHUIHUH ZLWK WKH QXFOHDU UHFHSWRUV DQG FKHPLFDO PHVVHQJHUV RI WKH HQGRFULQH V\VWHP DQG KDYH EHHQ LGHQWLILHG DV D WKUHDW WR WKH HQYLURQPHQW DQG ZLOGOLIH 0F/DFKODQ f $OWHUDWLRQV LQ ERWK WKH GHYHORSPHQWDO DQG UHSURGXFWLYH IXQFWLRQV RI FHOOV DQG ZKROH RUJDQLVPV DUH LQFUHDVLQJO\ GRFXPHQWHG DQG DWWULEXWHG WR H[SRVXUH WR WKHVH H[RJHQRXV VXEVWDQFHV 15& f 7KHVH VXEVWDQFHV DUH XELTXLWRXV HQYLURQPHQWDO FRQWDPLQDQWV ZKLFK DUH FRPPRQO\ UHIHUUHG WR DV KRUPRQDOO\ DFWLYH DJHQWV +$$Vf RU FRPSRXQGV +$&Vf [HQRHVWURJHQV HVWURJHQOLNH FRPSRXQGV HQGRFULQH GLVUXSWRUV HVWURJHQPLPLFV RU HVWURJHQ DJRQLVWVDQWDJRQLVWV 6XEVWDQFHV DUH ODEHOHG EDVHG RQ WKHLU LQWHUDFWLRQ ZLWK DQGRU GLVSODFHPHQW RI DQ HQGRJHQRXV KRUPRQH IURP LWV FRQVHUYDWLYH IXQFWLRQ 0F/DFKODQ f 5HFHQW FRQJUHVVLRQDO PDQGDWHV LQ WKH 6DIH 'ULQNLQJ :DWHU $FW f %LOO 1R6f DQG WKH )RRG 4XDOLW\ 3URWHFWLRQ $FW f%LOO 1R3/ f KDYH UHTXLUHG WKH 86(3$ WR HYDOXDWH WKH KRUPRQDO DFWLYLW\ RI DOO FKHPLFDOV SURGXFHG ,Q WKH 86

PAGE 42

,Q D UHFHQW VXUYH\ RI FRQWDPLQDWHG 8 6 VXUIDFH ZDWHUV DON\OSKHQROV SKWKDODWH FRPSRXQGV DQG QDWXUDO DQG V\QWKHWLF HVWURJHQV ZHUH VKRZQ WR FRPSULVH URXJKO\ b RI WKH RUJDQLF FRQWDPLQDQW ORDG .ROSLQ HW DO f 2I FULWLFDO FRQFHUQ LV WKH H[SRVXUH WR WKHVH WKUHH FODVVHV RI FRPSRXQGV EHFDXVH RI UHSRUWV RI KRUPRQHOLNH HIIHFWV 7KHLU RULJLQ PD\ EH GXH LQ SDUW WR DJULFXOWXUDO QRQSRLQW VRXUFH UXQRIIV &DVH\ HW DO f EXW WKH PDMRULW\ DUH GLVFKDUJHG IURP GRPHVWLF DQG LQGXVWULDO ZDVWHZDWHU WUHDWPHQW SODQWV ::73Vf 6KHDKDQ HW DO E 6Q\GHU HW DO %DURQWL HW DO 5XGHO HW DO f (QYLURQPHQWDOO\ UHOHYDQW FRQFHQWUDWLRQV RI WKHVH FRPSRXQGV KDYH EHHQ OLQNHG WR DOWHUHG VH[XDO FKDUDFWHULVWLFV -REOLQJ HW DO f DQG HOHYDWHG WLVVXH OHYHOV RI KRUPRQDOO\ DFWLYH FRPSRXQGV 6KHDKDQ HW DO Ef LQ ILVK +RZHYHU OLQNV DUH QRW HDVLO\ HVWDEOLVKHG LQ VRPH VLWXDWLRQV -DFREVHQ DQG *XLOGDO )DZHOO HW DO 6HSXOYHGD HW DO f $FFRUGLQJ WR WKH (XURSHDQ 8QLRQ 6FLHQWLILF &RPPLWWHH KRUPRQDOO\ DFWLYH DJHQWV +$$Vf UHIHUUHG WR DV HQGRFULQH GLVUXSWRUVf DUH H[RJHQRXV VXEVWDQFHV RU PL[WXUHV WKDW DOWHU IXQFWLRQVf RI WKH HQGRFULQH V\VWHP DQG FRQVHTXHQWO\ FDXVH DGYHUVH KHDOWK HIIHFWV LQ DQ LQWDFW RUJDQLVP RU LWV SURJHQ\ RU VXEn SRSXODWLRQV %DNHU f 6ZHHSLQJ LQ LWV EUHYLW\ WKLV GHILQLWLRQ IDLOV WR DGGUHVV VRPH FRQFHUQV 0F/DFKODQ $VKE\ f VSHFLILFDOO\ DOWHUHG FHOOXODU IXQFWLRQV LQ UHODWLRQ WR WKH RYHUDOO KHDOWK RI WKH RUJDQLVP $OWKRXJK WKH 1DWLRQDO 5HVHDUFK &RXQFLO f ZDV HYHQ OHVV GLUHFW ZKHQ WKH\ GHILQHG KRUPRQDOO\ DFWLYH FRPSRXQGV DV DQ\ VXEVWDQFH WKDW SRVVHVVHV KRUPRQHOLNH DFWLYLW\

PAGE 43

UHJDUGOHVV RI WKH PHFKDQLVP RI DFWLRQ ,Q OLJKW RI QHZ UHVHDUFK :X HW DO f FKDQJHV WR WKH GHILQLWLRQ PD\ UHDG DQ\ VXEVWDQFH RU LQIOXHQFLQJ IDFWRU +RUPRQDOO\ DFWLYH FRPSRXQGV DUH DUUDQJHG LQ WKUHH JURXSV WKH QDWXUDO DQG V\QWKHWLF HVWURJHQV DQWKURSRJHQLF FKHPLFDOV DQG SK\WRHVWURJHQV QDWXUDOO\ SURGXFHG VXEVWDQFHV LQ SODQWVf 7KH HIIHFWV RI WKHVH VXEVWDQFHV PD\ EH DJRQLVWLF RU DQWDJRQLVWLF $JRQLVWLF KRUPRQDOO\ DFWLYH FRPSRXQGV DFW LQ D PDQQHU VLPLODU WR DQ HQGRJHQRXV KRUPRQH ZKLOH DQWDJRQLVWV EORFN WKH DFWLYLW\ RI HQGRJHQRXV VXEVWDQFHV %HJLQQLQJ ZLWK UHSRUWV RI WKH HVWURJHQOLNH HIIHFWV IROORZLQJ H[SRVXUH WR WKH LQVHFWLFLGH ''7 %XUOLQJWRQ DQG /LQGHPDQ f UHVHDUFKHUV FRQWLQXH WR VWXG\ WKH LQWHUDFWLRQ RI QRQVWHURLGDO FRPSRXQGV ZLWK WKH HVWURJHQ UHFHSWRU 0LNVLFHN f 3K\WRHVWURJHQV 3K\WRHVWURJHQV DUH QDWXUDOO\ RFFXUULQJ FRPSRXQGV LQ SODQWV DQG SODQW GHULYHG SURGXFWV 1LOVVRQ f DQG LQFOXGH JHQLVWHLQ HTXRO IRUPRQRQHWLQ ELRFKDQLQ $ /DWRQHOOH HW DO f 7KH KRUPRQDO DFWLYLW\ RI SK\WRHVWURJHQV KDV EHHQ UHYLHZHG ZLWK VSHFLDO HPSKDVLV RQ HQYLURQPHQWDOO\ UHOHYDQW GRVDJHV 1LOVVRQ f 2QH VRXUFH RI SK\WRHVWURJHQV LV WKH XULQH RI YHJHWDULDQV )RWVLV DQG $GOHUFUHXW] f 3K\WRHVWURJHQV LPSDFW WKH UHSURGXFWLRQ DQG VH[XDO KHDOWK RI ZLOGOLIH +XJKHV f EXW WKHUH LV QR HYLGHQFH LQ KXPDQV 6WUDXVV HW DO f ,Q IDFW OLPLWHG HYLGHQFH VXJJHVWV SK\WRHVWURJHQV DUH EHQHILFLDO LQ WUHDWLQJ VRPH W\SHV RI KXPDQ FDQFHUV 'L3DROD HW DO f -X HW DO f

PAGE 44

7DEOH 6HOHFW SKWKDODWH FRPSRXQGV DQG WKHLU FRPPRQ XVDJH &203281' 86(6 'LHWK\OKH[\O SKWKDODWH '(+3f 5DLQ JHDU IRRWZHDU XSKROVWHU\ PDWHULDOV ,9 IOXLG EDJV ZDWHUSURRI JORYHV +HDW VHDO FRDWLQJ RQ PHWDO IRLOV XVHG RQ SRUWLRQHG IRRG LWHPV %XW\O EHQ\]O SKWKDODWH %%3f 'LVSHUVDQW LQ LQVHFW UHSHOODQWV DQG SHUIXPHV &RPSRQHQW RI FHOOXORVH SODVWLFV )ORRU WLOHV 'LEXW\O SKWKDODWH '%3f &RDWLQJV RQ FHOORSKDQH LQVHFW UHSHOODQWV +DLU VSUD\ &DUSHW EDFNLQJ 'LHWK\O SKWKDODWH '(3f &HOOXORVH DFHWDWH SODVWLF ILOPV XVHG DV FDUWRQ ZLQGRZV WR GLVSOD\ IRRGV 0ROGHG SODVWLFV LH WRRWKEUXVKHV FDU FRPSRQHQWV DQG FKLOGUHQnV WR\V 'LLVRQRQ\O SKWKDODWH ',13f 9LQ\O ZDOO FRYHULQJV WR\V DQG PHGLFDO GHYLFHV VKRZHG ORZ FRQFHQWUDWLRQV RI VRPH SODQW VXEVWDQFHV UHGXFH HVWURJHQLF HIIHFWV ZKLOH DW KLJK GRVHV HVWURJHQLF HIIHFWV PD\ EH LQFUHDVHG 7KH FRQWDPLQDWLRQ RI IRRGVWXII E\ ]HDUDOHQRQH D IXQJDO SK\WRHVWURJHQ LV FRPPRQ DQG KXPDQ FRQVXPSWLRQ LV HVWLPDWHG DW SJSHUVRQGD\ LQ 1RUWK $PHULFD 0F/DFKODQ f 3KWKDODWHV 3KWKDODWHV DUH SODVWLFL]HUV WKDW DUH FRPPRQO\ XVHG DV VRIWHQHUV LQ WKH SURGXFWLRQ RI SDLQWV LQNV DGKHVLYHV DQG YDULRXV SODVWLF JRRGV 7DEOH f ,Q DQ H[WHQVLYH VWXG\ RI SKWKDODWH FRPSRXQGV WKH 1DWLRQDO ,QVWLWXWH IRU +HDOWK 1,+f FRQFOXGHG WKDW EHQ]\O EXW\O SKWKDODWH %%3f ZDV ERWK D GHYHORSPHQWDO DQG UHSURGXFWLYH WR[LFDQW 1,+ f $GGLWLRQDOO\ H[WHQVLYH SKWKDODWH FRQWDPLQDWLRQ KDV EHHQ UHSRUWHG IRU RYHUWKHFRXQWHU EHDXW\ SURGXFWV (QYLURQPHQWDO :RUNLQJ *URXS f :LWK FRQFHUQ UHVHDUFKHUV KDYH VKRZQ

PAGE 45

WKDW ERG\ EXUGHQV RI SKWKDODWH FRPSRXQGV LQ ZRPHQ RI FKLOGEHDULQJ DJH \HDUVf DUH KLJKHU WKDQ PDOHV DQG DQ\ RWKHU DJH JURXS %ORXQW HW DO f DQG WKH ORQJWHUP FRQVHTXHQFHV RI WKLV DUH XQNQRZQ 3KWKDODWH FRPSRXQGV DUH XELTXLWRXV FRQWDPLQDQWV RI ERWK WHUUHVWULDO DQG DTXDWLF HQYLURQPHQWV )UHVKZDWHU OHYHOV RI GLHWK\O KH[\O SKWKDODWH '(+3f DQG GLEXW\O EHQ]\O SKWKDODWH '%3f UDQJHG IURP WR SJ/ DQG WR SJ/ UHVSHFWLYHO\ ZKLOH PDULQH FRQFHQWUDWLRQV RI '(+3 DQG '%3 UDQJHG WR SJ/ DQG WR SJ/ UHVSHFWLYHO\ )DWRNL DQG 1RPD f $GGLWLRQDOO\ FRQWDPLQDWLRQ RI UDZ GULQNLQJ ZDWHU E\ GLHWK\O SKWKDODWH '(3f KDV EHHQ UHSRUWHG 86(3$ f 6RPH SKWKDODWH FRQWDPLQDWLRQ LQ WKH HQYLURQPHQW PD\ EH WUDFHG EDFN WR ::73 GLVFKDUJHV )URPPH HW DO f VXUYH\HG *HUPDQ ZDVWHZDWHU WUHDWPHQW SODQWV DQG VKRZHG WKH FRQFHQWUDWLRQV RI '(+3 DQG '%3 ZHUH KLJKO\ YDULDEOH LQ WKH HIIOXHQWV DQG UDQJHG IURP WR SJ/ DQG WR SJ/ UHVSHFWLYHO\ )XUWKHUPRUH WKH FRQFHQWUDWLRQV RI SKWKDODWH HVWHUV LQ WKH ::73 VOXGJH UDQJHG IURP WR PJNJ GU\ ZHLJKW IRU '(+3 DQG WR PJNJ GU\ ZHLJKW IRU '%3 )URPPH HW DO f 3KWKDODWH HVWHUV LQFOXGLQJ WKH FRPPRQO\ XVHG GLHWK\O SKWKDODWH '(3f '%3 %%3 DQG GLLVREXW\O SKWKDODWH ',%3f DUH FDSDEOH RI LQGXFLQJ DQ HVWURJHQLF UHVSRQVH LQ UHSRUWHU DVVD\V EXW WKHLU SRWHQF\ ZDV RQH PLOOLRQWK WKDW RI SHVWUDGLRO -REOLQJ HWDO +DUULV HW DO f /HJOHUHWDO f LGHQWLILHG WKH KRUPRQDO DFWLYLW\ RI %%3 XVLQJ DQ HVWURJHQ UHFHSWRUFKHPLFDOO\ DFWLYDWHG OXFLIHUDVH UHSRUWHU JHQH (5&$/8;f FRQVWUXFW KRZHYHU WKH

PAGE 46

7DEOH )RRG ,WHP &RQFHQWUDWLRQ SJNJf 3HDQXW EXWWHU 0DUPDODGH %XWWHU 7RPDWRHV $SSOHV %UHDVW PLON ,QIDQW IRUPXOD r IURP *XHQWKHU HW DO f UHVSRQVHV RI RWKHU SKWKDODWHV H J '(3 DQG '%3 ZHUH ZHDNHU 5HVHDUFKHUV FRQWLQXH WR LQYHVWLJDWH WKH KRUPRQDO DFWLYLW\ RI SKWKDODWH FRPSRXQGV LQ IDFW WKHUH LV VWLOO D GHEDWH VXUURXQGLQJ WKH KRUPRQDO DFWLYLW\ RI WKH PRVW FRPPRQO\ XVHG SKWKDODWH '(+3 0HWFDOIH HWDO f ,Q KXPDQV WKH PDLQ SDWKZD\ IRU WKH FRQMXJDWLRQ RI SKWKDODWHV SULRU WR H[FUHWLRQ LV YLD JOXFXURQLGDWLRQ $OEUR HW DO f (YLGHQFH IRU WKH UHGXFHG KRUPRQDO DFWLYLW\ RI SKWKDODWH FRQMXJDWHV FRPHV IURP URGHQW DVVD\V )RVWHU HW DO f $OWKRXJK WKH FRQMXJDWHG SKWKDODWHV DUH H[FUHWHG DW FRQFHQWUDWLRQV LQ WKH PLFURJUDP SHU OLWHU UDQJH WKH\ PD\ EH UDSLGO\ GHFRQMXJDWHG LQ WKH SUHVHQFH RI WKH JOXFXURQLGDVH HQ]\PHV %ORXQW HW DO f 7KHVH JOXFXURQLGDVH HQ]\PHV DUH SUHVHQW LQ KLJK FRQFHQWUDWLRQV LQ GRPHVWLF ZDVWHZDWHUV $ON\OSKHQROV $ON\OSKHQRO SRO\HWKR[\ODWHV $3(Vf DUH RQH FODVV RI QRQLRQLF VXUIDFWDQWV ZLWK QXPHURXV LQGXVWULDO DQG GRPHVWLF XVHV 7DOPDJH f 7KHVH K\GURSKLOLF $3(V DUH UDSLGO\ GHJUDGHG GXULQJ ELRORJLFDO WUHDWPHQW H J LQ ZDVWHZDWHU WUHDWPHQW SODQW ::73f WR K\GURSKRELF DQG UHFDOFLWUDQW DON\OSKHQROV $3f 'XH WR WKH KLJK GHJUHH RI HWKR[\ODWLRQ $3(V DUH QRW HVWURJHQLF KRZHYHU DFWLYLW\ KDV

PAGE 47

EHHQ UHSRUWHG LQ WKH GHJUDGDWLRQ SURGXFWV QRQ\OSKHQRO 13f DQG RFW\OSKHQRO 23f 5RXWOHGJH DQG 6XPSWHU Df 1RQ\OSKHQROV DUH XELTXLWRXV FRQWDPLQDQWV RI FRPPHUFLDOO\ DYDLODEOH IRRG LWHPV *XHQWKHU HW DO f 7DEOH f 7KH DIILQLW\ RI WKH K\GURSKRELF $3V WR VHGLPHQW LQFUHDVHV ZLWK RUJDQLF FRQWHQW /\H HW DO f $V D UHVXOW WKH UHSRUWHG KDOIOLIH RI VHGLPHQW DVVRFLDWHG DON\OSKHQROV LV URXJKO\ \HDUV 6KDQJ HW DO f ,Q WKH RXWIDOO RI ::73V UHSRUWHG FRQFHQWUDWLRQV RI $3V LQ WKH VHGLPHQWV UDQJH IURP WR QJJ GU\ ZHLJKW LQ IUHVKZDWHU HQYLURQPHQWV /\H HW DO f DQG IURP WR QJJ LQ PDULQH HQYLURQPHQWV 6KDQJ HW DO f +XPDQV H[FUHWH $3 FRPSRXQGV DV JOXFXURQLGH FRQMXJDWHV 0XOOHU HW DO f DQG WKHVH FRQMXJDWHV DUH WKHQ VXEMHFWHG WR ELRORJLFDO GHJUDGDWLRQ SURFHVVHV &XUUHQW ZDVWHZDWHU WUHDWPHQW WHFKQRORJLHV DUH QRW HIIHFWLYH IRU WKH FRPSOHWH UHPRYDO RI $3V 6KHDKDQ HW DO Ef KHQFH SJ/ OHYHOV DUH GLVFKDUJHG WR UHFHLYLQJ ZDWHUV DQG LQGXFH KRUPRQDO UHVSRQVHV LQ ILVK 6KHDKDQ HW DO Df ,Q DTXDWLF HQYLURQPHQWV RQH RI WKH ELRPDUNHUV IRU H[SRVXUH WR KRUPRQDOO\ DFWLYH FRPSRXQGV LV WKH SUHVHQFH RI YLWHOORJHQLQ 9WJf D ILVK HJJ \RON SURWHLQ -REOLQJ DQG 6XPSWHU f UHSRUWHG D WR IROG LQFUHDVH LQ 9WJ SURGXFWLRQ LQ 5DLQERZ WURXW 2QFRUK\QFKXV P\NLVVf H[SRVHG WR YDULRXV FRQFHQWUDWLRQV RI DON\OSKHQROV WR S0f LQ D ODERUDWRU\ VWXG\ $ON\OSKHQROV ELRDFFXPXODWH 6KHDKDQ HW DO Ef LQ IDFW QJ 13J RI OLYHU ZHW ZHLJKWf ZDV UHSRUWHG LQ PDOH IORXQGHU 3ODWLFKWK\V IOHVXVf /\H HW DO f $OWKRXJK ::73 HIIOXHQW FRQFHQWUDWLRQV RI WKH K\GURSKRELF 13 DQG 23 KDYH

PAGE 48

EHHQ UHSRUWHG LQ WKH PLG QJ/ UDQJH 6Q\GHU HW DO f KLJKHU FRQFHQWUDWLRQV PD\ EH IRXQG LQ WKH ZDVWHG VOXGJH GXH WR SDUWLWLRQLQJ (MOHUWVVRQ HW DOf /D *XDUGLD HW DO f :KLOH RFW\OSKHQRO DQG QRQ\OSKHQRO DUH ZHDNO\ HVWURJHQLF /HJOHU HW DOf f WKHLU FRQMXJDWHG IRUPV DUH QRW FDSDEOH RI LQGXFLQJ HVWURJHQn OLNH UHVSRQVHV 0RIIDW HW DO f 1DWXUDO DQG 6\QWKHWLF (VWURJHQV 5HVHDUFK LQGLFDWHV WKDW HVWURJHQV ERWK QDWXUDO DQG V\QWKHWLF UHSUHVHQW WKH SUHGRPLQDQW IUDFWLRQ RI RUJDQLF ZDVWHZDWHU FRQWDPLQDQWV DQG FRQFXUUHQWO\ LQGXFH WKH KLJKHVW KRUPRQH DFWLYLW\ 0HWFDOIH HW DO 6Q\GHU HW DO 5RGJHUV*UD\ HW DO 'HVEURZ HW DO f 7KH YHUWHEUDWH HQGRFULQH V\VWHP SURGXFHV FKHPLFDO PHVVDJHV FDOOHG KRUPRQHV ZKLFK UHJXODWH ERG\ IXQFWLRQV H J UHSURGXFWLRQ JURZWK DQG KRPHRVWDVLV )LJXUH f (VWURJHQV DUH WKH KRUPRQHV SURGXFHG E\ WKH RYDULHV DQG WKH\ DUH UHVSRQVLEOH IRU WKH GHYHORSPHQW DQG UHJXODWLRQ RI IHPDOH VHFRQGDU\ VH[XDO FKDUDFWHULVWLFV 7KH HQGRJHQRXV HVWURJHQV SHVWUDGLRO (f DQG HVWURQH (Lf WRJHWKHU ZLWK WKHLU GHJUDGDWLRQ SURGXFW HVWULRO (f DUH UDSLGO\ FRQMXJDWHG DQG H[FUHWHG IURP WKH ERG\ 7KLV PDGH WKHLU XVH LQ KRUPRQH WKHUDSLHV LQHIIHFWLYH DQG OHG WR WKH GHYHORSPHQW RI V\QWKHWLF KRUPRQHV %ROW f $OWKRXJK WKH V\QWKHWLF KRUPRQHV DUH UDSLGO\ DEVRUEHG LQ WKH EORRGVWUHDP WKH\ DUH VORZO\ PHWDEROL]HG DQG DUH WKHUHIRUH EHWWHU VXLWHG IRU GUXJ WKHUDSLHV *XHQJHULFK f 7KH PRVW FRPPRQO\ SUHVFULEHG V\QWKHWLF KRUPRQHV DUH DHWK\Q\OHVWUDGLRO ((f DQG PHVWUDQRO ZKLFK DUH ERWK XWLOL]HG LQ WKH SURGXFWLRQ RI ELUWK FRQWURO SLOOV %&3f DQG DV LQKLELWRUV RI RYXODWLRQ 5DQQH\ f $OWKRXJK ( DQG ( DUH WKH

PAGE 49

7DEOH 5DWHV IRU WKH XULQDU\ H[FUHWLRQ RI QDWXUDO HVWURJHQV IURP PHQ DQG ZRPHQ :RPHQ 0HQF Q f (VWURJHQ 3UH 0HQRSDXVDO Q f 3UHn PHQRSDXVDO Q f 3RVWn PHQRSDXVDO Q f S(VWUDGLRO (f SJGD\f (VWURQH (f SJGD\f (VWULRO (f SJGD\f U!$fn f.H\ HW DO UHSRUWHG DV JHRPHWULF PHDQ f$GOHUFUHXW] HW DO UHSRUWHG DV UDQJH DQG F)RWVLV DQG $GOHUFUHXW] UHSRUWHG DV PHDQ 6WDQGDUG GHYLDWLRQV ZHUH QRW UHSRUWHG PDLQ PHWDEROLWHV RI ( WKHUH LV DOVR D JURXS RI PLQRU PHWDEROLWHV ZLWK LQFRQVHTXHQWLDO KRUPRQDO DFWLYLW\ 7KH PHWDEROLF SDWKZD\V IRU QDWXUDO DQG V\QWKHWLF HVWURJHQV KDYH EHHQ H[WHQVLYHO\ UHYLHZHG %ROW *XHQJHULFK f *HQHUDOO\ QDWXUDO DQG V\QWKHWLF KRUPRQHV LQ WKH KXPDQ ERG\ DUH PHWDEROL]HG WR LQDFWLYH JOXFXURQLGH RU VXOIRQLGH FRQMXJDWHV EHIRUH H[FUHWLRQ %ROW f 7KH DJH GLVWULEXWLRQ DQG KHQFH WKH UHSURGXFWLYH FRQGLWLRQV RI ZRPHQ LQ D SRSXODWLRQ GHWHUPLQHV WKH WRWDO FRQFHQWUDWLRQ RI H[FUHWHG HVWURJHQV 7DEOH VXPPDUL]HV UHSRUWHG H[FUHWLRQ UDWHV RI QDWXUDO HVWURJHQV IURP ERWK PHQ DQG ZRPHQ .H\V HW DO f VKRZHG WKDW SUHPHQRSDXVDO ZRPHQ H[FUHWHG SJGD\ RI ( SJGD\ (L DQG SJGD\ ( ,Q D VHSDUDWH VWXG\ ZLWK SUHPHQRSDXVDO ZRPHQ VLPLODU HVWURJHQ FRQFHQWUDWLRQV LQ XULQH ZHUH UHSRUWHG E\ $GOHUFUHXW] HW DO f 7KH VOLJKW YDULDWLRQ LQ HVWURJHQ H[FUHWLRQ UHSRUWHG E\ .H\ HW DO f DQG $GOHUFUHXW] HW DO f ZDV SUREDEO\ GXH WR WKH PHQVWUXDO SKDVH GXULQJ XULQH FROOHFWLRQ

PAGE 50

$GOHUFUHXW] HW DO f FROOHFWHG XULQH VDPSOHV GXULQJ WKH PLGIROOLFXODU SKDVH GD\V DIWHU WKH RQVHW RI WKH ODVW PHQVWUXDWLRQf ZKLOH .H\ HW DO f DQDO\]HG XULQH FROOHFWHG WKURXJKRXW WKH HQWLUH PHQVWUXDO F\FOH 2YHUDOO SUHJQDQW ZRPHQ H[FUHWH WKH KLJKHVW FRQFHQWUDWLRQV RI QDWXUDO KRUPRQHV DW SJGD\ SJGD\ DQG SJGD\ IRU (S ( DQG ( UHVSHFWLYHO\ )RWVOV HW DO f 7KH HVWURJHQ FRQFHQWUDWLRQV UHSRUWHG IRU SRVWn PHQRSDXVDO ZRPHQ ZHUH SJGD\ IRU ( SJGD\ IRU (S DQG SJGD\ IRU (.H\ HW DO f DQG ZHUH FRPSDUDEOH WR PDOH HVWURJHQ H[FUHWLRQ UDWHV 0DOH Q f H[FUHWLRQ ZDV UHSRUWHG IRU (S ( DQG (DW SJGD\ SJGD\ DQG SJGD\ UHVSHFWLYHO\ )RWVOV DQG $GOHUFUHXW] f 3UHGLFWLQJ WKH H[FUHWLRQ UDWHV RI V\QWKHWLF KRUPRQHV LV PRUH GLIILFXOW DQG GHSHQGV RQ WKH QXPEHU RI SUHPHQRSDXVDO IHPDOHV LQ D SRSXODWLRQ FXOWXUDO PRUHV DQG WKH EUDQG RI ELUWK FRQWURO SLOO XVHG -RKQVRQ HW DO f $ VHDUFK RI SKDUPDFHXWLFDO LQIRUPDWLRQ RQ WKH ,QWHUQHW VKRZHG D UDQJH RI SJ (( WDEOHW ZLWK D W\SLFDO GRVLQJ UHJLPH RI GD\V IROORZHG E\ GD\V RI LQDFWLYH WDEOHWV /DUVVRQ HW DO f HVWLPDWHG (( H[FUHWLRQ UDWHV DW SJGD\ SHU IHPDOH FRQVXPLQJ RUDO FRQWUDFHSWLYH SLOOV LQ 6ZHGHQ 7KH H[FUHWLRQ RI HQGRJHQRXV KRUPRQHV LV SUHGRPLQDQWO\ YLD WKH XULQH ZKLOH IHFDO HOLPLQDWLRQ JHQHUDOO\ H[KLELWV D PLQRU VHFRQGDU\ UROH KRZHYHU IRU WKH H[FUHWLRQ RI V\QWKHWLF KRUPRQHV WKH IHFDO URXWH LV SULPDU\ 5DQQH\ f )HFDO H[FUHWLRQ UDWHV RI HQGRJHQRXV HVWURJHQV IURP SUHPHQRSDXVDO ZRPHQ Q f ZHUH UHSRUWHG DV QJGD\ IRU (S QJGD\ IRU ( DQG QJGD\ IRU ( $GOHUFUHXW] HW DO f 'DLO\ H[FUHWRU\ UDWHV IRU IHFHV DQG XULQH

PAGE 51

KDYH EHHQ UHSRUWHG DW JUDPV DQG NJ ZHW YROXPHSHUVRQGD\ UHVSHFWLYHO\ 7KHVH UDQJHV JHQHUDOO\ DSSO\ WR PHQ ZLWK H[FUHWLRQ UDWHV IRU ZRPHQ JHQHUDOO\ DW WKH ORZHU OLPLW RI WKLV UDQJH 3ROSUDVHUW DV FLWHG LQ %LWWRQ f (IIHFWV RI +RUPRQDOO\ $FWLYH &RPSRXQGV RQ +XPDQV 2YHU WKH SDVW \HDUV ZKDW EHJDQ DV DQHFGRWDO REVHUYDWLRQV RI DOWHUHG UHSURGXFWLYH DQG VH[XDO GHYHORSPHQW LQ KXPDQV KDYH FRDOHVFHG LQWR FRQFHUQ IRU ORQJWHUP VSHFLHV VXUYLYDO 0F/DFKODQ f 7KH HDUO\ RQVHW RI PLGGOHDJH YDJLQDO FDUFLQRPDV DQG GHIRUPHG XWHUL LQ \RXQJ ZRPHQ KDYH EHHQ OLQNHG WR WKH SRWHQW V\QWKHWLF HVWURJHQ GLHWK\OVWLOEHVWHURO ZLGHO\ SUHVFULEHG WR SUHJQDQW ZRPHQ WKURXJKRXW WKH nV DQG nV &ROEXUQ HW DO f 'XULQJ WKH ILUVW WULPHVWHU RI SUHJQDQF\ KXPDQ IHWXVHV DUH KLJKO\ VHQVLWLYH WR H[SRVXUHV IURP KRUPRQDOO\ DFWLYH FRPSRXQGV ,QGXVWULDOL]HG QDWLRQV LQFOXGLQJ WKH 86 6FDQGLQDYLD DQG -DSDQ KDYH UHSRUWHG DQ LQFUHDVHG LQFLGHQFH RI K\SRVSDGLDV GLVSODFHPHQW RI WKH XUHWKUDO RSHQLQJ WRZDUG WKH VFURWXPf DQG FU\SWRUFKLGLVP IDLOXUH RI WKH WHVWLFOHV WR GHVFHQG LQWR WKH VFURWXPf LQ PDOHV 3DXOR]]L f 6RPH UHVHDUFKHUV KDYH TXHVWLRQHG WKLV FRQFOXVLRQ DQG LQVWHDG FLWH LQFUHDVHG UHSRUWLQJ DQG VWULFWHU GHILQLWLRQV DV IDFWRUV DUWLILFLDOO\ LQIODWLQJ WKH GDWD :LGHVSUHDG WUHQGV DUH GLIILFXOW WR HVWDEOLVK EXW DGYHUVH VH[XDO HIIHFWV IURP H[RJHQRXV VXEVWDQFHV KDYH EHHQ FRQILUPHG 7KH IHPLQL]DWLRQ RI PDOHV KDV EHHQ DWWULEXWHG WR ZRUN SODFH H[SRVXUH WR IRUPDOGHK\GH )LQNHOVWHLQ HW DO f DQG WKHUDSHXWLF WUHDWPHQWV ZLWK KHUEDO VXSSOHPHQWV 'L3DROD HW DO f *UD\ Df VKRZHG WKDW VH[XDO GLIIHUHQWLDWLRQ LQ PDOH UDWV ZDV DOWHUHG DIWHU H[SRVXUH WR KRUPRQDOO\ DFWLYH

PAGE 52

, +25021$//< $&7,9( &203281'6n@ &(175$/ 1(59286 6<67(0 +<327+$/$086 D ^ D e D 7+<086 7+<52,' $'5(1$/ */$1'6 3$1&5($6 29$5< 7(67(6 3,1($/ */$1' /<03+ 7,668( 086&/( /,9(5 1(59286 6<67(0 .,'1(<6 /,9(5 086&/(6 5(352'8&7,9( 25*$16 &,5&$',$1 5<7+<06 )LJXUH 5HSUHVHQWDWLRQ RI WKH YHUWHEUDWH HQGRFULQH V\VWHP DQG WKH SRVVLEOH LQIOXHQFHV RI KRUPRQDOO\ DFWLYH FRPSRXQGV RQ YDULRXV V\VWHPV DQG RUJDQV DGDSWHG IURP 0DWKHZV DQG YDQ +ROGH f

PAGE 53

7DEOH $GYDQWDJHV DQG GLVDGYDQWDJHV DVVRFLDWHG ZLWK WKH XVH RI LQ YLYR DQG LQ YLWUR DVVD\V IRU LGHQWLI\LQJ KRUPRQDO DFWLYLW\ $GYDQWDJHV 'LVDGYDQWDJHV ,Q 9LYR 0HWDEROLF FDSDELOLW\ +LJK FRVW 0XOWLSOH QXFOHDU UHFHSWRUV (VWDEOLVKHG DVVD\V $VVD\ GXUDWLRQ ZHHNV WR PRQWKVf 1RQVWDQGDUGL]HG SURWRFROV GRVLQJ UHJLPH IRRG HQGSRLQWf 6HQVLWLYLW\ WR QRQKRUPRQDO HIIHFWV ,Q 9LWUR /RZ FRVW 5DSLG KRXUV WR GD\Vf 6LPSOLILHG FXOWXUH WHFKQLTXHV 1R PHWDEROLF FDSDELOLW\ 3UHGRPLQDWHO\ PHDVXUH (5 PHGLDWHG HIIHFWV /DFN RI SDWKZD\V WR FOHDU KRUPRQHV 0LQLPL]H HQGRFULQH V\VWHP FRPSOH[LW\ FRPSRXQGV DQG SHVWLFLGHV 7KH KXPDQ UHSURGXFWLYH V\VWHP LV UHJXODWHG E\ D SOHWKRUD RI FKHPLFDO PHVVHQJHUV LQ D FRPSOH[ UHOD\ RI VLJQDOV WKDW FRQWURO JDPHWRJHQHVLV RYXODWLRQ IHUWLOL]DWLRQ DQG VH[XDO GLIIHUHQWLDWLRQ 7KRPDV f 7KH YHUWHEUDWH HQGRFULQH V\VWHP SURGXFHV FKHPLFDO PHVVDJHV FDOOHG KRUPRQHV ZKLFK UHJXODWH ERG\ IXQFWLRQV HJ UHSURGXFWLRQ JURZWK DQG KRPHRVWDVLV )LJXUH f 1XPHURXV UHYLHZV KDYH EHHQ SXEOLVKHG WKDW GLVFXVV WKH HIIHFWV RI HQGRFULQHGLVUXSWLQJ FRPSRXQGV RQ KXPDQV 6XOWDQ HW DW 'HJHQ DQG %ROW 3DXOR]]L 1HXEHUW f %LRDVVD\V WR ,GHQWLI\ +RUPRQDO $FWLYLW\ 3XUVXDQW WR FRQJUHVVLRQDO PDQGDWHV WKH 86 HQYLURQPHQWDO SURWHFWLRQ DJHQF\ 86(3$ f GHYHORSHG D IUDPHZRUN IRU D WLHUHG VFUHHQLQJ SURJUDP

PAGE 54

7DEOH ,Q YLYR DVVD\V IRU WKH GHWHUPLQDWLRQ RI KRUPRQDO DFWLYLW\ $66$< (1'32,17 5RGHQW 0HDVXUH XWHULQH ZHLJKW RI RYDULHFWRPLVHG URGHQWV 5RGHQW 0HDVXUH YDJLQDO FRUQLILFDWLRQ RI RYDULHFWRPLVHGE URGHQWV +HUVKEHUJHU &DVWUDWHG 5DW 0HDVXUHV DQGURJHQ VHQVLWLYH WLVVXH ZHLJKW RI FDVWUDWHG PDOH URGHQWV )LVK *RQDGRVRPDWLF LQGH[ 9WJF LQGXFWLRQ 7XUWOHV f§D ‘ 9WJ LQGXFWLRQ fYDJLQDO OHVLRQV RYDULHV UHPRYHG VXUJLFDOO\ n9WJ YLWHOORJHQLQ D IHPDOH HJJ \RON SURWHLQf WKDW LQWHJUDWHG LQ YLYR DQG LQ YLWUR ELRDVVD\V IRU WKH LGHQWLILFDWLRQ DQG TXDQWLILFDWLRQ RI KRUPRQDOO\ DFWLYH DJHQWV LQ WKH HQYLURQPHQW *UD\ Ef 5HVHDUFKHUV FRQWLQXH WR LQYHVWLJDWH QRYHO DSSURDFKHV IRU LGHQWLI\LQJ KRUPRQDO DFWLYLW\ DQG DOWKRXJK WKHVH QHZ PHWKRGV LQFUHDVH WKH NQRZOHGJH EDVH RI KRUPRQDO HIIHFWV PRUH ZRUN LV QHHGHG LQ HVWDEOLVK WKH IRXQGDWLRQ RI DGYHUVH KRUPRQDO HIIHFWV 3ULPDULO\ LQFUHDVHG YDOLGDWLRQ RI WKH PRUH ZLGHO\ XWLOL]HG DVVD\V H J WKH \HDVW HVWURJHQ VFUHHQ 5RXWOHGJH DQG 6XPSWHU Ef DQG URGHQW DVVD\V $VKE\ f DQG LQWHUODERUDWRU\ FRPSDULVRQ RI WKHVH HVWDEOLVKHG DVVD\V $VKE\ f DUH QHHGHG ,Q YLWUR DQG LQ YLYR DVVD\V HDFK KDYH WKHLU RZQ DGYDQWDJHV DQG GLVDGYDQWDJHV WKHUHIRUH D EDWWHU\ RI DVVD\V XWLOL]LQJ ERWK W\SHV RI DVVD\V KDV WKH JUHDWHVW YDOXH 7DEOH f (QGRJHQRXV HVWURJHQ OLJDQGV ELQG ZLWK HVWURJHQ UHFHSWRUV DW WKH FHOOXODU OHYHO LQ D ZHOOGHILQHG FDVFDGH RI FHOOXODU HYHQWV 2NDPXUD DQG 1DNDKDUD f 7KH HQGRJHQRXV OLJDQG HQWHUV WKH FHOO YLD DFWLYH WUDQVSRUW PHFKDQLVPV RU GLIIXVLRQ 2QFH LQVLGH WKH FHOO WKH OLJDQG HQWHUV WKH QXFOHXV DQG ELQGV ZLWK WKH HVWURJHQ UHFHSWRU GLVSODFLQJ WKH KHDW VKRFN SURWHLQV HJ +VSf DVVRFLDWHG

PAGE 55

7DEOH 7KUHVKROG GRVH IRU WKH LQGXFWLRQ RI KRUPRQDO HIIHFWV IROORZLQJ H[SRVXUH RI ILVK WR QDWXUDO DQG V\QWKHWLF HVWURJHQV 6SHFLHV +RUPRQH &RQH QJ/f 5HVSRQVH 6RXUFH 2QFRUK\QFKXV P\NLVV 5DLQERZ WURXWf P P UR }‘ 9WJ LQGXFWLRQ 9WJ LQGXFWLRQ 5RXWOHGJH HW DO 2QFRUK\QFKXV P\NLVV 5DLQERZ &0 /8 /8 9WJ LQGXFWLRQ /DUVVRQ HW DO WURXWf 5XWLOXV UXWLOXV 5RDFKf H 9WJ LQGXFWLRQ 5RXWOHGJH HW DO 2U\]LDV ODWLSHV 0HGDNDf H HH (U H r E E E $OWHUDWLRQ LQ UHSURGXFWLYH FKDUDFWHULVWLFV 0HWFDOIH HWDO 'DQLR UHULR =HEUDILVKf HH 9WJ LQGXFWLRQ (UUDWLF VSDZQLQJ YDQ GHQ %HOW HW DO ,FWDOXUXV SXQFWDWXV &KDQQHO FDWILVKf H 9WJ LQGXFWLRQ 0RQWHYHUGL HW DO 3ODWLFKWK\V IOHVXV )ORXQGHUf HH 9WJ LQGXFWLRQ $OOHQ HW DO f$EEUHYLDWLRQV ( HVWURQH ( SHVWUDGLRO ( HVWULRO (( HWK\Q\O HVWUDGLRO 9WJ YLWHOORJHQLQ fORZHVW REVHUYHG HIIHFW FRQFHQWUDWLRQ ZLWK WKH UHFHSWRU 7KHVH SURWHLQV PDLQWDLQ WKH VWUXFWXUDO FRQIRUPDWLRQ RI WKH HVWURJHQ UHFHSWRU )OLVV HW DO f 7KH UHFHSWRUOLJDQG FRPSOH[ WKHQ ELQGV WR D VSHFLILF OLJDQGELQGLQJ GRPDLQ RQ WKH QXFOHDU '1$ 0DVVDDG HW DO f ZKLFK FRGHV IRU WKH WUDQVFULSWLRQ RI PHVVHQJHU 51$ P51$f ,Q WKH FHOOXODU

PAGE 56

PDFKLQHU\ WKH JHQRPLF PHVVDJH RQ WKH P51$ LV WUDQVODWHG LQWR SURWHLQ 7KLV VXLWH RI HYHQWV LV LQLWLDWHG LQ UHVSRQVH WR WKH HVWURJHQLF OLJDQG (VWURJHQ UHFHSWRUV DUH SDUW RI D VXSHUIDPLO\ RI QXFOHDU UHFHSWRUV DQG LQFOXGH D ODUJH QXPEHU RI RUSKDQ UHFHSWRUV ZLWK QR UHFRJQL]HG OLJDQGV 0F/DFKODQ f 7KHUH DUH WZR IRUPV RI WKH HVWURJHQ UHFHSWRU HVWURJHQ UHFHSWRU D (5Df DQG HVWURJHQ UHFHSWRU S (5Sf $OWKRXJK WKH WLVVXH GLVWULEXWLRQ RI (5D DQG (5S GLIIHU EDVHG RQ VH[ PDOH RU IHPDOHf DQG RUJDQ W\SH WKH\ GLVSOD\ VLPLODU VHQVLWLYLWLHV WR WKH HQGRJHQRXV HVWURJHQ SHVWUDGLRO &RXVH HW DO f :KLOH WKH PDMRULW\ RI KRUPRQDOO\ DFWLYH VXEVWDQFHV H[HUW WKHLU LQIOXHQFH YLD OLJDQGGHSHQGHQW DFWLYDWLRQ RI WKH HVWURJHQ UHFHSWRU VRPH VXEVWDQFHV GR QRW DFW YLD UHFHSWRU LQWHUDFWLRQV (O7DQDQL DQG *UHHQ f ,Q YLYR DVVD\V IRU WKH GHWHUPLQDWLRQ RI KRUPRQDO DFWLYLW\ 7UDGLWLRQDOO\ WKH SRWHQWLDO IRU KRUPRQDO DFWLYLW\ ZDV DVVHVVHG ZLWK LQ YLYR DVVD\V 7DEOH f 7\SLFDO HQGSRLQWV PHDVXUHG DUH LQFUHDVHG XWHULQH ZHLJKW DOWHUHG VH[ UDWLRV VNHZHG JRQDGRVRPDWLF LQGH[ DQG LQGXFWLRQ RI YLWHOORJHQLQ 9WJf DQ HJJ \RON SURWHLQ LQ YHUWHEUDWHVf ,Q YLYR DVVD\V XVLQJ YDULRXV FUXVWDFHDQ VSHFLHV $QGHUVHQ HW DO )LQJHUPDQ HW DO f LQFOXGLQJ 'DSKQLD PDJQD 6KXULQ DQG 'RGVRQ %DOGZLQ HW DO %DOGZLQ HW DO f KDYH EHHQ XVHG WR HYDOXDWH KRUPRQDO DFWLYLW\ DV GHFUHDVHG VWHURLG PHWDEROLVP DQG GHYHORSPHQWDO DEQRUPDOLWLHV &RPPRQ LQ YLYR DVVD\V XVH URGHQWV 3ULQVHQ DQG *RXNR f ILVK 6HSXOYHGD HW DO %RZPDQ HW DO f DQG VRPH LQYHUWHEUDWHV *DJQH HW DO %ODLVH HW DO f EXW WKHVH DVVD\V DUH H[SHQVLYH ODERULQWHQVLYH

PAGE 57

7DEOH ,Q YLWUR DVVD\V IRU WKH GHWHUPLQDWLRQ RI KRUPRQDO DFWLYLW\ $66$< 02'( 2) $&7,21 &HOO 3UROLIHUDWLRQ $VVD\V PHDVXUHV WKH DELOLW\ RI D VXEVWDQFH WR VWLPXODWH SUROLIHUDWLRQ LQ HVWURJHQ VHQVLWLYH FHOOV ([ (6FUHHQ 6RWR HW DO f 5HFHSWRU %LQGLQJ $VVD\V PHDVXUHV WKH DIILQLW\ EHWZHHQ D VXEVWDQFH DQG WKH HVWURJHQ UHFHSWRU ([ K(5 D RU S *XWHQGRUI DQG :HVWHQGRUI f PHDVXUHV WKH DELOLW\ RI D VXEVWDQFH WR LQGXFH 5HSRUWHU *HQH $VVD\V WKH UHSRUWHU JHQH ([ <(6 5RXWOHGJH DQG 6XPSWHU Ef &HOO /LQH $VVD\V PHDVXUHV WKH LQGXFWLRQ RI D VSHFLILF SURWHLQV RU HQ]\PHV E\ D VXEVWDQFH ([ /LYHU FHOOV 0RQWHYHUGL HW DO f &HOO 3UROLIHUDWLRQ5HSRUWHU *HQH PHDVXUHV WKH DELOLW\ RI VXEVWDQFH WR VWLPXODWH FHOO SUROLIHUDWLRQ DQG LQGXFH UHSRUWHU JHQH WUDQVFULSWLRQ ([ (5&$/8; /HJOHU HW DO f DQG LQ VRPH FDVHV UDLVH HWKLFDO FRQFHUQV 2QH RI WKH DGYDQWDJHV RI LQ YLYR DVVD\V LV WKH FHOOXODU PDFKLQHU\ IRU WKH PHWDEROLVP DQGRU FRQMXJDWLRQ RIKRUPRQDOO\ DFWLYH FRPSRXQGV +$&f 7KH GHJUDGDWLRQ RI WKH SDUHQW +$& PD\ SURGXFH D PHWDEROLWH ZLWK QR KRUPRQDO DFWLYLW\ +DUULV HW DO f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

PAGE 58

7DEOH 5HODWLYH VHQVLWLYLW\ RI LQ YLWUR DVVD\V WR SHVWUDGLRO (f $VVD\ 0'/D QJ/f HF QJ/f 6RXUFH 5&%$E &ROGKDP HW DO 5&%$ .OHLQ HW DO <(6 5RXWOHGJH DQG 6XPSWHU E <(6 0XUN HW DO <(6 %HUHVIRUG HW DO <(6 7DQDND HW DO <(6 (OVE\ HW DO <(6 9LQJJDDUG HW DO <(6 /D\WRQ HW DO <(6 /HJOHU HW DO (6&5((1 *XWHQGRUI DQG :HVWHQGRUI (6&5((1 6RWR HW DO (6&5((1 %HKQLVFK HW DO &HOO OLQHUHSRUWHU 09/1f *XWHQGRUI DQG :HVWHQGRUI &HOO OLQHUHSRUWHU +*(/1f *XWHQGRUI DQG :HVWHQGRUI &HOO OLQHUHSRUWHU (5&$/8;f /HJOHU HW DO &RPSHWLWLYH ELQGLQJ (5 Df *XWHQGRUI DQG :HVWHQGRUI &RPSHWLWLYH ELQGLQJ (5 Sf *XWHQGRUI DQG :HVWHQGRUI &RPSHWLWLYH ELQGLQJ (5 "ffF 0XUN HW DO $EEUHYLDWLRQV D0'/ PLQLPXP GHWHFWLRQ OLPLW f5&%$ UHFRPELQDQW FHOO ELRDVVD\ fHVWURJHQ UHFHSWRU IRUP QRW LQGLFDWHG

PAGE 59

DVVD\V DUH FRQVWUXFWHG ZLWK UHFRPELQDQW PROHFXOHV RU FHOOV IURP HLWKHU PDPPDOLDQ RU ILVK WLVVXHV =DFKDUHZVNL 'LHO HWDO f 0RVW IUHTXHQWO\ \HDVW FHOOV 6DFFKDURP\FHV FHUHYLVLDHf DUH XVHG DV FDUULHUV IRU WKH KRUPRQH UHFHSWRUV (DUO\ LQ YLWUR DVVD\V LQFRUSRUDWHG DQ HVWURJHQ UHFHSWRU (5f DQG UHSRUWHG RQO\ (5PHGLDWHG KRUPRQDO DFWLYLW\ 5HFHQWO\ LQ YLWUR DVVD\V KDYH EHHQ GHVLJQHG ZLWK RWKHU QXFOHDU UHFHSWRUV LQFOXGLQJ DQGURJHQ UHFHSWRUV $5f DQG SURJHVWHURQH UHFHSWRUV 35f 1LVKLNDZD HW DO f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f DQG SURWHLQ V\QWKHVLV 6RPH RI WKH OLPLWDWLRQV LQKHUHQW LQ LQ YLWUR DVVD\V DUH WKH DEVHQFH RI PHWDEROLF SDWKZD\V WKH RYHUHVWLPDWLRQ RI ELQGLQJ LQ D VLQJOH UHFHSWRU V\VWHPV -REOLQJ HW DO f DQG UHOLDQFH RQ HVWURJHQ PHGLDWHG HIIHFWV ZKLOH LJQRULQJ RWKHU UHFHSWRUV 'LHO HW DO f 'HVSLWH WKHVH FRQFHUQV LQ YLWUR DVVD\VFRQWLQXH WR EH ZLGHO\ XVHG IRU WKH LGHQWLILFDWLRQ RI KRUPRQDO DFWLYLW\ DQG IRU WKH TXDQWLILFDWLRQ RI WKH FRQWULEXWLRQV IURP LQGLYLGXDO FKHPLFDOV WR RYHUDOO DFWLYLW\ 'HJHQ DQG %ROW *XWHQGRUI DQG :HVWHQGRUI f 7KH PRVW ZLGHO\ XVHG UHSRUWHU JHQH DVVD\ ,QFRUSRUDWHV WKH KXPDQ HVWURJHQ UHFHSWRU K(5f LQWR WKH JHQRPH RI WKH \HDVW 6DFFKDURP\FHV FHUHYLVLDH

PAGE 60

5HKPDQQ HW DOf &ROGKDP HW DO *DLGR HW DOf 5RXWOHGJH DQG 6XPSWHU Ef
PAGE 61

EHWZHHQ VHOHFWHG KRUPRQH UHFHSWRUV (5 $5 35 05 75f DQG FRDFWLYDWRUV 7,) 65&7,) 5,3f LQ WKH SUHVHQFH RI+$&V 1LVKLNDZD HW DOf f 7KLV DVVD\ LV KLJKO\ VHQVLWLYH WR SK\WRHVWURJHQV DQG QRQ\OSKHQRO 1DNDQR HW DO f DQG HQYLURQPHQWDOO\ UHOHYDQW FRQFHQWUDWLRQV RI +$&V .DZDJRVKL HW DO f 7KH UROH RI WKH FRDFWLYDWRU LV SRRUO\ XQGHUVWRRG EXW DIWHU ELQGLQJ RI WKH OLJDQG WR WKH QXFOHDU UHFHSWRU LW DSSHDUV WR LQIOXHQFH SURFHVVHV WKDW LQLWLDWH JHQH WUDQVFULSWLRQ 1LVKLNDZD HW DO f 7KH 7,) FRDFWLYDWRU KDG WKH JUHDWHVW LQIOXHQFH RQ LQLWLDWLRQ .DZDJRVKL HW DO f ([SORLWDWLRQ RI WKH HVWURJHQ VHQVLWLYLW\ RI EUHDVW FDQFHU FHOOV OHG WR WKH GHYHORSPHQW RI DVVD\V WKDW TXDQWLI\ FHOO SUROLIHUDWLRQ LQ WKH SUHVHQFH RI HVWURJHQV RU HVWURJHQOLNH VXEVWDQFHV 7KH (6FUHHQ 0&) EUHDVW FDQFHU FHOOVf DVVD\ 6RWR HW DO f SURYLGHV KLJKO\ UHSURGXFLEOH UHVXOWV ZKHQ DVVD\ SURWRFROV DUH VWULFWO\ DGKHUHG WR DQG WKH FHOO OLQH VRXUFH LV FRQVLVWHQW 3D\QH HW DO f HYDOXDWHG WKUHH 0&) FHOO OLQHV %86 8&/ DQG 623f IRU WKHLU VHQVLWLYLW\ WR ( DQG VKRZHG (& YDOXHV RI DQG QJ/ UHVSHFWLYHO\ +RZHYHU WKH SUROLIHUDWLYH HIIHFW JURZWK LQ H[FHVV RI FRQWURO FHOOVf RI ( YDULHG PDUNHGO\ DPRQJ WKH FHOO OLQHV DW ZLWK %86 ZLWK 8&/ DQG ZLWK 623 3D\QH HW DO f 7KH IXQFWLRQDOLW\ RI VRPH HVWURJHQ UHFHSWRUV LQ EUHDVW FDQFHU FHOO OLQHV PD\ EH ORZ ZLWK XS WR b QRQIXQFWLRQDO %DOPHOOL*DOODFFKL HW DO f ,Q FHOO SUROLIHUDWLRQ DVVD\V GLUHFW FRXQWV RFFXU ZLWK KHPRF\WRPHWHUV RU DXWRPDWHG &RXOWHU FRXQWHUV 7KH DGGLWLRQ RI UHSRUWHU JHQHV WR FHOO OLQHV KDV ,QFUHDVHG WKHLU VHQVLWLYLW\ DQG DOORZHG IRU WKH HOXFLGDWLRQ RI PXOWLSOH PHFKDQLVPV RI DFWLRQ LQ D VLQJOH WHVW

PAGE 62

V\VWHP /XFLIHUDVH UHSRUWHU JHQH FRQVWUXFWV ZHUH GHVLJQHG LQ KXPDQ EUHDVW FDQFHU FHOO OLQHV $GGLWLRQ RI WKH OX[ UHSRUWHU JHQH WR 0&) DQG +H/D FHOOV SURGXFHG WKH 09/1 DQG +*(/1 V\VWHPV %DODJXHU HW DO *XWHQGRUI DQG :HVWHQGRUI f 7KLV DGDSWDWLRQ DOORZHG IRU WKH LGHQWLILFDWLRQ RI VXEVWDQFHV ZKRVH PRGH RI DFWLRQ ZDV YLD FHOO SUROLIHUDWLRQ RU (5 DFWLYDWLRQ .DWRUL HW DO f GHPRQVWUDWHG WKDW GLEXW\O SKWKDODWH LQGXFHG FHOO SUROLIHUDWLRQ EXW QRW JHQH WUDQVFULSWLRQ $GGLWLRQDOO\ WKH (5&KHPLFDO $FWLYDWHG /XFLIHUDVH JHQH H[SUHVVLRQ (5 &$/8;f DVVD\ ZDV GHYHORSHG LQ 7' KXPDQ EUHDVW FDQFHU FHOOV E\ WUDQVIHFWLRQ ZLWK UHSRUWHU JHQHV S(5(WDWD/XFf /HJOHU HW DO f 7KH VWHHS ( GRVHUHVSRQVH FXUYH ZLWK WKH (5&$/8; DVVD\ ZDV QHDUO\ WLPHV JUHDWHU WKDQ WKH UHSRUWHU JHQH UHVSRQVH LQ WKH <(6 LQGLFDWLQJ WKH KLJKHU VHQVLWLYLW\ RI WKH (5&$/8; DVVD\ /HJOHU HW DO f 7DEOH f 2WKHU DGYDQWDJHV RI WKH (5&$/8; DVVD\ DUH WKH VPDOO VDPSOH YROXPH UHTXLUHPHQWV ZKLFK ZHUH URXJKO\ WK DQG OK WKH YROXPH UHTXLUHG E\ WKH <(6 DQG (5 ELQGLQJ DVVD\V UHVSHFWLYHO\ 0XUN HW DO f &HOO OLQH DVVD\V DUH QRW OLPLWHG WR PDPPDOLDQ FHOOV 0RQWHYHUGL DQG *LXOLR f FRPELQHG SULPDU\ OLYHU KHSDWRF\WHV IURP &KDQQHO FDWILVK ,FWDOXUXV SXQFWDWXVf ZLWK DQ HQ]\PHOLQNHG LPPXQRVRUEDQW DVVD\ (/,6$f WR PHDVXUH WKH LQGXFWLRQ RI 9WJ 3HWLW HW DO f GHYHORSHG D WHVW V\VWHP LQ \HDVW WKDW H[SUHVVHG WKH 5DLQERZ WURXW HVWURJHQ UHFHSWRU UW(5f &RPSHWLWLYH ELQGLQJ DVVD\V PHDVXUH WKH GLVSODFHPHQW RI ( IURP WKH HVWURJHQ UHFHSWRU 7KH (5D KDV D KLJKHU VHQVLWLYLW\ WKDQ WKH (5S IRU (

PAGE 63

*XWHQGRUI DQG :HVWHQGRUI f *HQHUDOO\ WKHVH FRPSHWLWLYH ELQGLQJ DVVD\V KDYH D ORZHU VHQVLWLYLW\ WKDQ RWKHU LQ YLWUR DVVD\V DQG DUH QRW VXLWHG DV VFUHHQLQJ DVVD\V IRU KRUPRQDO DFWLYLW\ 7DEOH f ,Q FRPSHWLWLYH ELQGLQJ DVVD\V WKH UHVSRQVH WR DJRQLVWLF DQG DQWDJRQLVWLF [HQRELRWLFV DUH PHDVXUHG VLPXOWDQHRXVO\ ZKLFK DFFRXQWV IRU WKH ORZHU VHQVLWLYLW\ 0XUN HW DO f 6RPH QRYHO DSSURDFKHV IRU GHWHFWLQJ KRUPRQDOO\ DFWLYH FRPSRXQGV KDYH EHHQ GHYHORSHG $ ELRVHQVRU KDV EHHQ FRQVWUXFWHG ZKLFK LQFRUSRUDWHV WKH KXPDQ HVWURJHQ UHFHSWRU D (5Df LQWR D OLSLG ELOD\HU ZLWK GLUHFW FRQWDFW WR D JROG HOHFWURGH IRU TXDQWLILFDWLRQ *UDQHN DQG 5LVKSRQ f $ WHVW EDWWHU\ WR GHWHUPLQH KRUPRQDO DFWLYLW\ $Q RSWLPXP WHVW EDWWHU\ WR HYDOXDWH DTXDWLF WR[LFLW\ LQFOXGHV DOJDO LQYHUWHEUDWH DQG EDFWHULDO FRPSRQHQWV 5RMLFNRYD3DGUWRYD HW DO f 6LPLODUO\ D VXFFHVVIXO LQYHVWLJDWLRQ RI KRUPRQDO DFWLYLW\ VKRXOG XVH D VXLWH RI DVVD\V WR LQFOXGH D FHOO SUROLIHUDWLRQ DVVD\ D \HDVW UHSRUWHU DVVD\ DQG D FRPSHWLWLYH ELQGLQJ DVVD\ )DQJ HW DO &ROGKDP HW DO f :KHQ LQ YLWUR DVVD\V DUH FRPELQHG LQ DQ DUUD\ WKHQ PXOWLSOH PHFKDQLVPV PD\ EH VWXGLHG VLPXOWDQHRXVO\ LQFOXGLQJ WKH HIIHFWV RI PHWDEROLVP DQGRU WUDQVSRUW %DNHU 9LQJJDDUG HW DO f 7KH VHQVLWLYLW\ RI LQ YLWUR DVVD\V WR ( VWDQGV DV WKH KDOOPDUN E\ ZKLFK DVVD\V IRU KRUPRQDO DFWLYLW\ DUH MXGJHG *XWHQGRUI DQG :HVWHQGRUI f GHPRQVWUDWHG DQ ( VHQVLWLYLW\ WKDW LQFUHDVHG LQ WKH RUGHU RI HVWURJHQ UHFHSWRU ELQGLQJ DVVD\V (5D DQG (5Sf UHSRUWHU JHQH DVVD\V FHOO SUROLIHUDWLRQ DVVD\V 7DEOH f 7KH (5&$/8; DVVD\ KDV GHPRQVWUDWHG WKH

PAGE 64

RYHUDOO KLJKHVW VHQVLWLYLW\ WR ( ZLWK DQ (& RI QJ/ FRPSDUHG WR QJ/ ZLWK WKH <(6 DQG QJ/ ZLWK (5 ELQGLQJ DVVD\V 0XUN HW DO f &KDUDFWHUL]LQJ +RUPRQDO $FWLYLW\ LQ 06: /DQGILOO /HDFKDWHV DQG 2WKHU (QYLURQPHQWDO 6DPSOHV 8QWLO UHFHQWO\ OLWWOH ZDV NQRZQ DERXW WKH KRUPRQDO DFWLYLW\ RI 06: ODQGILOO OHDFKDWHV GHVSLWH WKH WKUHDW WKH\ SRVHG WR WKH HQYLURQPHQW (MOHUWVVRQ HW DO f 0RGHUQ 06: ODQGILOOV DUH HQJLQHHUHG ZLWK EDUULHUV WR UHVWULFW WKH PRELOLW\ RI WKH OLTXLG IUDFWLRQ OHDFKDWHf RI ZDVWH DQG FROOHFWLRQ V\VWHPV KRZHYHU WKLV KDV QRW DOZD\V EHHQ WKH FDVH ,Q WKH SDVW WKH GLVSRVDO RI ZDVWH ZDV ODUJHO\ XQUHJXODWHG DOORZLQJ IRU GLUHFW UHOHDVH RI WR[LF VXEVWDQFHV WR JURXQG DQG VXUIDFH ZDWHUV 6RPH RI WKHVH ROGHU XQUHJXODWHG ODQGILOOV FRQWLQXH WR UHOHDVH OHDFKDWHV RI XQNQRZQ VWUHQJWK DQG FKHPLFDO FRPSRVLWLRQ ,Q 06: ODQGILOOV ELRORJLFDO DQG FKHPLFDO SURFHVVHV SURGXFH OHDFKDWHV ZLWK KLJK FRQFHQWUDWLRQV RI RUJDQLF FRQWDPLQDQWV
PAGE 65

DQG UHDFWLYLW\ WKHQ WKH DVK PDWHULDO LV ODQGILOOHG 7KH FRPSRVLWLRQ RI WKH ZDVWH LQ WKH ODQGILOO VWXGLHG E\ %HKQLVK HW DO f ZDV SULPDULO\ LQRUJDQLF ZLWK DERXW b LQFLQHUDWRU DVK &KDUDFWHUL]LQJ VRPH RI WKH RUJDQLF FRPSRXQGV LQ WKH OHDFKDWH UHYHDOHG WKH SUHVHQFH RI NQRZQ KRUPRQDOO\ DFWLYH FRPSRXQGV VSHFLILFDOO\ ELVSKHQRO $ QRQ\OSKHQRO DQG HVWUDGLRO DW DQG SJ/ UHVSHFWLYHO\ .DZDJRVKL HW DO f XVHG D \HDVW WZRK\EULG UHSRUWHU DVVD\ 1LVKLNDZD HW DO f WR GHPRQVWUDWH WKH KRUPRQDO DFWLYLW\ RI ZDVWH OHDFKDWH FRQWDPLQDWHG JURXQGZDWHU DW DQ ( DFWLYLW\ HTXLYDOHQW WR QJ/ /HDFKDWHV WKDW ZHUH FROOHFWHG IURP VLWHV IRU WKH GLVSRVDO RI VROLG PXQLFLSDO ZDVWHV DQG GUHGJHG VRLOV ZHUH QRW KRUPRQDOO\ DFWLYH ,Q D FRQWLQXLQJ LQYHVWLJDWLRQ WKH HIILFLHQF\ RI WKH H[WUDFWLRQ SURFHGXUHV IRU WKH UHFRYHU\ RI KRUPRQDO DFWLYLW\ ZHUH HYDOXDWHG .DZDJRVKL HW DO f 7KHLU H[WUDFWLRQ SURFHGXUHV XVHG & 63( FROXPQV DQG ZHUH KLJKO\ HIILFLHQW IRU WKH UHFRYHU\ RI KRUPRQDO DFWLYLW\ 7RJHWKHU ZLWK WKH KLJK UHFRYHU\ RI DFWLYLW\ IROORZLQJ HOXWLRQ ZLWK SRODU VROYHQWV DFHWRQHf WKHVH UHVXOWV LPSOLFDWHG QRQSRODU K\GURSKRELF FRPSRXQGV DV FDXVDWLYH DJHQWV IRU WKH DFWLYLW\ .DZDJRVKL HW DOf f %DVHG RQ WKH FRPSDULVRQ RI ELRDVVD\ UHVXOWV ZLWK RQH UDZ OHDFKDWH DQG DFHWRQH H[WUDFWV RI WKH VDPH OHDFKDWH .DZDJRVKL HW DO f VXJJHVWHG WKDW DQWLHVWURJHQLF FRPSRXQGV LQ WKH OHDFKDWH LQWHUIHUHG ZLWK WKH UHFRYHU\ RI KRUPRQDO DFWLYLW\ &RQVLGHULQJ WKH SRWHQWLDO IRU UHOHDVH RI 06: OHDFKDWH IURP ODQGILOOV WKH IDWH RI KRUPRQDOO\ DFWLYH FRPSRXQGV LQ VRLOV LV D FRQFHUQ 7KH PRELOLW\ RI KRUPRQDOO\ DFWLYH FRPSRXQGV +$&Vf ( (( QRQ\OSKHQRO RFW\OSKHQRO DQG

PAGE 66

0&) OXFLIHUDVH (6FUHHQ 6XEVWDQFH UHSRUWHU DVVD\ (&VRf Q0f $VVD\ (&VRf Q0f H %LVWULQ EXW\OWLQf $QWLPRQ\ FKORULGH &KURPLXP FKORULGH /LWKLXP FKORULGH &DGPLXP FKORULGH %DULXP FKORULGH D&KRH HW DO f ELVSKHQRO $f LQ VRLO WR b VDQGf ZDV LQYHVWLJDWHG LQ O\VLPHWHU H[SHULPHQWV ZLWK ELRDVVD\V 'L]HU HW DO f $OWKRXJK WKH OHDFKDWHV ZDWHU H[WUDFWVf SURGXFHG E\ WKH O\VLPHWHUV GLVSOD\HG D ORZ KRUPRQDO DFWLYLW\ QR DWWHPSW ZDV PDGH WR TXDQWLI\ WKH FRQFHQWUDWLRQV RI KRUPRQDOO\ DFWLYH FRPSRXQGV LQ WKH OHDFKDWHV +HQFH UHGXFHG KRUPRQDO DFWLYLW\ PD\ KDYH UHVXOWHG IURP WKH DGVRUSWLRQ RI WKH +$&V WR VRLO SDUWLFOHV 'L]HU HW DO f :KLOH RUJDQLF VXEVWDQFHV DUH WKH PRVW ZLGHO\ UHFRJQL]HG KRUPRQDOO\ DFWLYH FRPSRXQGV VRPH KHDY\ PHWDOV DUH DOVR KRUPRQDOO\ DFWLYH 6WRLFD HW DO f $OWKRXJK WKH FRQFHQWUDWLRQV RI KHDY\ PHWDOV LQ 06: ODQGILOO OHDFKDWHV DUH ORZ WKHUH LV D SRWHQWLDO IRU LQFUHDVHG OHDFKLQJ RI KHDY\ PHWDOV DV ODQGILOOV DJH %R]NXUW HW DO )O\KDPPHU f 6WRLFD HW DO f UHSRUWHG FDGPLXP DV &G&,f DFWLYDWHG WKH HVWURJHQ UHFHSWRU DW ORZ FRQFHQWUDWLRQV EXW DW KLJK

PAGE 67

&RPSRXQG &RQFHQWUDWLRQ QJ/f 5HIHUHQFH %LVHWK\OKH[\Of SKWKDODWH D
PAGE 68

H[FHVV SDFNDJLQJ LQ FRQVXPHU JRRGV 7DEOH VXPPDUL]HV WKH FRQFHQWUDWLRQV RI YDULRXV SKWKDODWHV LGHQWLILHG LQ 06: ODQGILOO OHDFKDWHV +RUPRQDO DFWLYLW\ KDV EHHQ LGHQWLILHG LQ D YDULHW\ RI RWKHU HQYLURQPHQWDO VDPSOHV ZKLFK LQFOXGH WUHH GHEDUNLQJ PLOO HIIOXHQWV 0HOODQHQ HW DO f ZDVWHZDWHU WUHDWPHQW SODQW HIIOXHQWV 6KDQJ HW DO f LQGXVWULDOL]HG ULYHUV /\H HW DO f DQG VXUIDFH ZDWHUV :LWWHUV HW DO f 7KH VRXUFHV RI KRUPRQDO DFWLYLW\ ZLWK WKH ZLGHVW GLVWULEXWLRQ DUH WKH ::73V 7KLV LV DWWULEXWHG WR WKH FRQFHQWUDWLRQV RI QDWXUDO DQG V\QWKHWLF KRUPRQHV LQ GRPHVWLF ZDVWHZDWHU 7DEOH f ,Q WKH DEVHQFH RI ::73V VHSWLF V\VWHPV UHSUHVHQW D VRXUFH RI KRUPRQDOO\ DFWLYH FRPSRXQGV 5XGHO HW DO f 7KH ODUJHVW VRXUFHV RI KRUPRQDO DFWLYLW\ LQ ZDVWHZDWHU WUHDWPHQW SODQWV ::73Vf DUH WKH QDWXUDO DQG V\QWKHWLF HVWURJHQV 7KH\ DUH H[FUHWHG DV LQDFWLYH JOXFXURQLGH DQG VXOIRQLGH FRQMXJDWHV KRZHYHU UDSLG GHFRQMXJDWLRQ RFFXUV LQ ::73V 7HUQHV HW DO Df 'HFRQMXJDWLRQ RFFXUV LQ WKH SUHVHQFH RI WKH HQ]\PH JOXFXURQLGDVH ZKLFK LV DEXQGDQWO\ SURGXFHG E\ (VFKHULFKLD FROL 7HUQHV HW DO Ef 7KLV HQ]\PH LV UHVSRQVLEOH IRU WKH GHJUDGDWLRQ RI ERWK WKH JOXFXURQLGH DQG VXOIRQLGH HVWURJHQ FRQMXJDWHV %HOIURLG HW DO f EXW VXOIRQLGH WR D OHVVHU GHJUHH +XDQJ DQG 6HGODN f 5RXJKO\ b RI H[FUHWHG HVWURJHQV RFFXU DV VXOIRQLGH FRQMXJDWHV DQG WKHLU GHJUDGDWLRQ LV FORVHO\ DVVRFLDWHG ZLWK DU\OVXOIDWDVH HQ]\PHV /RZ FRQFHQWUDWLRQV RI WKHVH HQ]\PHV LQ WUHDWPHQW SODQWV ,V UHVSRQVLEOH IRU WKH JUHDWHU SHUVLVWHQFH RI WKH VXOIRQLGH FRQMXJDWHV 'n$VFHQ]R HW DO f 5HJDUGOHVV RI WKH UHDVRQ IRU LQFRPSOHWH GHFRQMXJDWLRQ RI H[FUHWHG HVWURJHQV WKH XQGHUHVWLPDWLRQ RI HVWURJHQ ORDGV RQ WUHDWPHQW IDFLOLWLHV PD\

PAGE 69

7DEOH &RQFHQWUDWLRQV QJ/f RI QDWXUDO DQG V\QWKHWLF KRUPRQHV LQ ZDVWHZDWHU WUHDWPHQW SODQWV::73Vf (VWUDGLRO (f QJ/f (VWURQH (Wf QJ/f (VWULRO (Mf QJ/f DHWK\Q\O HVWUDGLRO ((f QJ/f 6RXUFH ::73 ,QI 1' 1' 1' 1' 6ROH HW DO ::73 (II 1' 1' 1' 1' ::73 ,QI E E E E %DURQWO HW DO ::73 (II E E E E ::73 ,QIr -RKQVRQ HW DO ::73 (II ::73 ,QIr 10 10 10 0DWVXL HW DO ::73 (II 10 10 10 ::73 ,QIr E E E 10 'n$VFHQ]R HW ::73 (IIr E E E 10 DO ::73 (II ZLQWHUf 10 10 5RGJHUV*UD\ HW DO ::73 (II VXPPHUf 10 10 5RGJHUV*UD\ HW DO ::73 (II 1' E 10 E 7HUQHV HW DO D ::73 (II E E 10 E 7HUQHV HW DO D ::73 (II 10 %HOIUROG HW DO ::73 (II 10 'HVEURZ HW DO ::73 (II 10 10 +XDQJ DQG 6HGODN ::73 (II 10 10 6Q\GHU HW DO $EEUHYLDWLRQV :DVWHZDWHU WUHDWPHQW SODQW ::73 ,QI LQIOXHQW (II HIIOXHQW 1' QRW GHWHFWHG 10 QRW PHDVXUHGE LQGLFDWHV PHGLDQ YDOXH 6DPSOHV FROOHFWHG IURPD *HUPDQ\ F&DQDGD G8QLWHG .LQJGRP H 86$ n1HWKHUODQGV OWDO\ 6SDLQ n-DSDQ

PAGE 70

UHVXOW -RKQVRQ HW DO f 7R SUHIDFH DQ\ GLVFXVVLRQ RI UHSRUWHG FRQFHQWUDWLRQV RI KRUPRQDOO\ DFWLYH FRPSRXQGV LQ WKH HQYLURQPHQW LW LV LPSRUWDQW WR FRQVLGHU WKH GHWHFWLRQ OLPLWV RI WKH DQDO\WLFDO PHWKRGV 2IWHQ WKH DQDO\WLFDO PHWKRGV IRU WKH LGHQWLILFDWLRQ DQG TXDQWLILFDWLRQ RI ORZOHYHO RUJDQLF FRQWDPLQDQWV DUH LQHIIHFWLYH DQG JURVVO\ XQGHU SUHGLFW HQYLURQPHQWDO EXUGHQV &DVWLOOR DQG %DUFHOR f )RU H[DPSOH ZKLOH VRPH ODERUDWRULHV KDYH UHSRUWHG GHWHFWLRQ OLPLWV IRU ( ( ( DQG (( DV ORZ DV QJ/ IRU VXUIDFH ZDWHUV DQG QJ/ IRU ZDVWHZDWHUV %HOIURLG HW DO f RWKHUV KDYH UHSRUWHG GHWHFWLRQ OLPLWV XS WR WKUHH RUGHUV RI PDJQLWXGH KLJKHU IRU ( QJ/f ( QJ/f ( QJ/f DQG (( QJ/f LQ ::73 HIIOXHQWV 6ROH HW DO f $OWHUDWLRQV LQ WKH VH[XDO DQG GHYHORSPHQWDO FKDUDFWHULVWLFV RI DTXDWLF VSHFLHV KDYH EHHQ UHSRUWHG ZRUOGZLGH DQG DWWULEXWHG WR WKH UHOHDVH RI KRUPRQDOO\ DFWLYH PLFURRUJDQLF FRQWDPLQDQWV 6KHDKDQ HW DO E 'HVEURZ HW DO -REOLQJ HW DO f $OWKRXJK UHVHDUFKHUV KDYH UHSRUWHG D UDQJH RI HVWURJHQV LQ ::73 HIIOXHQWV FRQFHQWUDWLRQV DUH JHQHUDOO\ DW WKH ORZ QJ/ OHYHO &KLHIO\ ( DQG (( DV WKH KRUPRQHV ZLWK WKH JUHDWHVW UHSRUWHG DFWLYLW\ KDYH EHHQ LGHQWLILHG LQ HIIOXHQWV DW WR DQG WR QJ/ UHVSHFWLYHO\ 7DEOH f 7KH PHWDEROLWH ( KDV EHHQ LGHQWLILHG LQ HIIOXHQWV DW FRQFHQWUDWLRQV RI WR QJ/ 'HVEURZ HW DO f :KLOH ( LV ZLGHO\ UHFRJQL]HG DV WKH VWURQJHVW HVWURJHQ LWV PHWDEROLWHV DUH DOVR SRWHQW ZLWK ( DERXW WLPHV OHVV DFWLYH -XUJHQV HW DO f DQG ( WKH ZHDNHVW PHWDEROLWH DOVR LQGXFLQJ KRUPRQDO DIIHFWV DOEHLW RUGHUV RI PDJQLWXGH OHVV 0HWFDOIH HW DO f 7KH V\QWKHWLF

PAGE 71

HVWURJHQ (( ZKLOH IRXQG DW ORZHU FRQFHQWUDWLRQV LQ ZDVWHZDWHU -RKQVRQ f KDV DQ DFWLYLW\ FRPSDUDEOH WR WKDW RI ( /DUVVRQ HW DO f 7KH ORZHU DFWLYLW\ RI (L LV RIIVHW E\ LWV H[WHQVLYH SUHVHQFH DQG JLYHV ULVH WR FRQFHUQV DERXW WKH HTXLYDOHQW ( DFWLYLW\ 5RGJHUV*UD\ HWDO f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n $VFHQ]R HW DO f )HZ VWXGLHV KDYH ORRNHG DW ( DQG LWV HIIHFWV PD\ EH XQGHU HVWLPDWHG $ OLPLWHG QXPEHU RI VWXGLHV KDYH HYDOXDWHG WKH FRQFHQWUDWLRQV RI HQGRJHQRXV DQG V\QWKHWLF HVWURJHQV LQ LQIOXHQWV DQG FRUUHVSRQGLQJ HIIOXHQWV RI ::73V 'HVSLWH UHSRUWHG UHPRYDO UDWHV RI XS WR b FRQFHQWUDWLRQV RI HVWURJHQV UHPDLQ DW WKUHVKROG OHYHOV IRU LQGXFLQJ KRUPRQDO HIIHFWV 6L[ DFWLYDWHG VOXGJH WUHDWPHQW IDFLOLWLHV LQ ,WDO\ &RELV )UHJHQH 2VWLD 5RPD 6XG 5RPD (VW DQG 5RPD 1RUGf KDYH EHHQ H[WHQVLYHO\ VWXGLHG E\ WKUHH UHVHDUFK WHDPV RYHU D WZR\HDU SHULRG %DURQWL HW DO f UHSRUWHG PHDQ LQIOXHQW FRQFHQWUDWLRQV IRU WKH VL[ IDFLOLWLHV DW DQG QJ/ IRU (L ( ( DQG (( UHVSHFWLYHO\ )ROORZLQJ ELRORJLFDO WUHDWPHQW WKHVH FRQFHQWUDWLRQV ZHUH UHGXFHG

PAGE 72

7DEOH 5HSRUWHG FRQFHQWUDWLRQV QJ/f RI QDWXUDO DQG V\QWKHWLF HVWURJHQV LQ VXUIDFH ZDWHUV HVWUDGLRO (f QJ/f (VWURQH (Lf QJ/f (VWULRO (f QJ/f DHWK\Q\O HVWUDGLRO ((f QJ/f 6RXUFH 6XUIDFH ZDWHU 1' 10 7HUQHV HW DO D 6XUIDFH ZDWHU 10 10 %HOIURLG HWDO 6XUIDFH ZDWHU 10 10 6Q\GHU HW DO 6XUIDFH ZDWHU 10 10 +XDQJ DQG 6HGODN WR DQG QJ/ IRU (L ( ( DQG (( UHVSHFWLYHO\ 7KHVH UHVXOWV DUH FRQVLVWHQW WR WKRVH UHSRUWHG E\ -RKQVRQ HW DO f DQG 'n $VFHQ]R HW DO f DOWKRXJK WKH ODWHU GLG QRW PHDVXUH (( 1RWDEO\ ( GLVSOD\HG WKH JUHDWHVW UDQJH RI FRQFHQWUDWLRQV %DURQWL HW DO -RKQVRQ HW DO 'n $VFHQ]R HW DO f DQG UHPDLQHG WKH PRVW SUHYDOHQW HVWURJHQ IROORZLQJ WUHDWPHQW 7KH RQO\ RWKHU VWXG\ WR HYDOXDWH PXOWLSOH KRUPRQH FRQFHQWUDWLRQV LQ LQIOXHQWV DQG FRUUHVSRQGLQJ HIIOXHQWV ZDV FRQGXFWHG ZLWK IRXU ::73V LQ 6SDLQ 6ROH HW DO f :KLOH DQDO\VHV ZHUH FRQGXFWHG IRU (L ( ( DQG (( WKH RQO\ KRUPRQH GHWHFWHG ZDV ( DQG WKHQ RQO\ LQ WKH LQIOXHQWV RI ::73V DW DSSUR[LPDWHO\ QJ/ 6ROH HW DO f 7KHVH OHYHOV ZHUH QHDUO\ GRXEOH WKH PD[LPXP FRQFHQWUDWLRQV GHWHFWHG LQ WKH ,WDOLDQ ::73V %DURQWL HW DO f &RPSDUDWLYHO\ ORZ DPELHQW OHYHOV RI QDWXUDO DQG V\QWKHWLF KRUPRQHV KDYH EHHQ GHWHFWHG LQ VXUIDFH ZDWHU VDPSOHV KRZHYHU WKH SRWHQWLDO IRU H[WHQVLYH FRQWDPLQDWLRQ RI VXUIDFH ZDWHUV H[LVWV IURP DQLPDO PDQXUHnV &DVH\ HW DO f DQG ::73 GLVFKDUJHV .ROSLQ HW DO f ,Q VXUIDFH ZDWHUV ( FRQFHQWUDWLRQV

PAGE 73

UDQJHG IURP WR QJ/ ZKLOH WKRVH IRU (( ZHUH WR QJ/ 7DEOH f $V D SRLQW RI UHIHUHQFH DOWHUHG VH[XDO FKDUDFWHULVWLFV DUH LQGXFHG LQ PDOH UDLQERZ WURXW 2QFRUWL\QFKXV P\NLVVf DW a QJ/ (( /DUVVRQ HW DO f $GGLWLRQDOO\ WKUHVKROG GRVHV RI WR QJ/ DQG WR QJ/ IRU (DQG ( UHVSHFWLYHO\ KDYH EHHQ UHSRUWHG IRU WKH LQGXFWLRQ RI KRUPRQDO HIIHFWV LQ 2 P\NLVV 0HWFDOIH HW DO f

PAGE 74

&+$37(5 72;,&,7< 2) /($&+$7(6 )520 )/25,'$ 081,&,3$/ 62/,' :$67( 06:f /$1'),//6 86,1* $ %$77(5< 2) 7(676 $3352$&+ ,QWURGXFWLRQ 7KH 6WDWH RI )ORULGD FXUUHQWO\ JHQHUDWHV PRUH WKDQ PLOOLRQ WRQV RI PXQLFLSDO VROLG ZDVWH 06:f D \HDU )LIW\VL[ SHUFHQW RI WKLV ZDVWH LV GLVSRVHG LQ HQJLQHHUHG &ODVV ODQGILOOV )'(3 f 7KH VWDWH KDV VL[W\RQH &ODVV ODQGILOOV SHUPLWWHG WR DFFHSW RQO\ 06:f WKDW DUH OLQHG DQG FRQWDLQ V\VWHPV IRU WKH FROOHFWLRQ DQG WUDQVSRUW RI ZDVWH OHDFKDWHV WKDW DUH WKHQ VXEVHTXHQWO\ VXEMHFWHG WR ELRORJLFDO WUHDWPHQW 5HVHDUFKHUV KDYH H[WHQVLYHO\ FKDUDFWHUL]HG WKH FKHPLFDO DQG SK\VLFDO FKDUDFWHULVWLFV 7RZQVHQG HWDO %RRWK HWDO *HWWLQE\ HW DO f DQG ELRORJLFDO WR[LFLW\ 3ORWNLQ DQG 5DP )HUUDUL HW DO (UQVW HW DO f RI ZDVWH OHDFKDWHV ZRUOGZLGH 7KHVH ZDVWH OHDFKDWHV DUH D FRPSOH[ PL[WXUH RI ERWK LQRUJDQLF HJ KHDY\ PHWDOV DPPRQLDf DQG RUJDQLF VXEVWDQFHV H J SHVWLFLGHV DQG FKORULQDWHG K\GURFDUERQVf ,W KDV EHHQ VXJJHVWHG WKDW H[SRVXUH WR 06: ODQGILOO OHDFKDWHV PD\ SRVH DV JUHDW D FDQFHU ULVN DV GRHV WKH H[SRVXUH WR LQGXVWULDO ZDVWH OHDFKDWHV %URZQ DQG 'RQQHOO\ f GXH WR WKHLU PXWDJHQLF SURSHUWLHV %HJ DQG $O0X]DLQL f 7KH JHQRWR[LF SRWHQWLDO RI 06: ODQGILOO OHDFKDWHV ZDV VKRZQ WR EH KLJKHU WKDQ WKDW IRU LQGXVWULDO ZDVWHZDWHU JURXQGZDWHU RU GULQNLQJ ZDWHU VDPSOHV +HOPD HW DO f

PAGE 75

:KHQ HYDOXDWLQJ WKH WR[LFLW\ RI FRPSOH[ HIIOXHQWV WKH XVH RI D EDWWHU\RI WHVWV DSSURDFK DOORZV IRU PXOWLSOH PHFKDQLVPV RI DFWLRQ WR EH HYDOXDWHG VLPXOWDQHRXVO\ 'HDQRYLF HW DO 5XWKHUIRUG HW DO f $ EDWWHU\RI WHVWV DSSURDFK ZLWK DOJDO FUXVWDFHDQ DQG EDFWHULDO DVVD\V ZDV XVHG WR VXFFHVVIXOO\ FKDUDFWHUL]H ODQGILOO OHDFKDWH WR[LFLW\ 5RMLFNRYD3DGUWRYD HW DO &OHPHQW HW DO f 1R FKDUDFWHUL]DWLRQ RI 06: ODQGILOO OHDFKDWH WR[LFLW\ LV FRPSOHWH XQWLO WR[LFRORJLFDO DVVD\V DUH FRPELQHG ZLWK DQDO\WLFDO SURFHGXUHV IRU FKHPLFDO FKDUDFWHUL]DWLRQ /DPEROH] HW DO f 7KH XQLTXH VXEWURSLFDO FOLPDWH LQ )ORULGD ZLWK JHQHUDOO\ DEXQGDQW UDLQIDOO DQG ZDUP WHPSHUDWXUHV UHGXFHV WKH FKHPLFDO VWUHQJWK RI 06: ODQGILOO OHDFKDWHV 5HLQKDUW DQG *URVK f $OWKRXJK UHVHDUFKHUV KDYH FKDUDFWHUL]HG WKH FRPSRVLWLRQ DQG VLWHVSHFLILF SDUDPHWHUV IRU 06: ODQGILOOV WKURXJKRXW )ORULGD WKH ELRORJLFDO HIIHFWV RI WKHVH OHDFKDWHV KDYH QRW EHHQ DVVHVVHG 5HLQKDUW DQG *URVK f 7KH WR[LFLW\ RI 06: OHDFKDWHV IURP VHSDUDWH ODQGILOOV ZKLOH UHODWHG PD\ GLIIHU GXH WR VSHFLILF FKDUDFWHULVWLFV RI WKH ZDVWHV HJ S+ WHPSHUDWXUH DPPRQLD OHYHOV SUHVHQFH RI UHFDOFLWUDQW RUJDQLF VXEVWDQFHV DQG PLFURELRORJLFDO DFWLYLW\ /LWWOH LQIRUPDWLRQ LV FXUUHQWO\ DYDLODEOH FRQFHUQLQJ WKH WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV LQ )ORULGD DQG VXFK D GDWDEDVH RI LQIRUPDWLRQ FRXOG EH XVHIXO LQ HYDOXDWLQJ OHDFKDWH WUHDWPHQW RSWLRQV DQG UHXVH SRVVLELOLWLHV :DUG HW DO f 7KH UHVHDUFK FRPPXQLW\ QRZ UHFRJQL]HV WKH LPSRUWDQFH RI XVLQJ D WDQGHP DSSURDFK ZLWK ERWK ELRORJLFDO DQG FKHPLFDO DQDO\VHV ZKHQ DQDO\]LQJ HQYLURQPHQWDO VDPSOHV WR DFKLHYH D EHWWHU XQGHUVWDQGLQJ RI WKH SRVVLEOH FDXVHV RI WR[LF HIIHFWV

PAGE 76

6LWH 6LWH 6LWH )LJXUH /RFDWLRQV RI WKH 06: ODQGILOOV IRU WKH FROOHFWLRQ RI OHDFKDWHV ,Q )ORULGD 7KH REMHFWLYHV RI WKLV UHVHDUFK ZHUH WR f FKDUDFWHUL]H WKH WR[LFLW\ RI )ORULGD 06: ODQGILOO OHDFKDWHV f HYDOXDWH WKH XVH RI D EDWWHU\ RI DOJDO LQYHUWHEUDWH DQG EDFWHULDO WR[LFLW\ DVVD\V ZLWK )ORULGD OHDFKDWHV f FKDUDFWHUL]H WKH FKHPLFDO FRPSRVLWLRQ RI WKH 06: OHDFKDWHV DQG f GHWHUPLQH UHODWLRQVKLSV EHWZHHQ VHOHFWHG FKHPLFDO FRPSRQHQWV DQG OHDFKDWH WR[LFLW\ 6L[ ODQGILOO VLWHV LQ QRUWK DQG QRUWKFHQWUDO )ORULGD ZHUH VDPSOHG PRQWKO\ RYHU D VL[PRQWK VDPSOLQJ SHULRG 7KH VLWHV VDPSOHG UHSUHVHQWHG D YDULHW\ RI IDFWRUV LQFOXGLQJ UXUDO DQG XUEDQ DUHDV VRPH LQGXVWULDO DFWLYLW\ OHDFKDWH UHF\FOH HQKDQFHG ELRORJLFDO WUHDWPHQW LQVLWX DQG WKRVH VLWHV FXUUHQWO\ DFFHSWLQJ ZDVWH DQG FDSSHG VLWHV

PAGE 77

7DEOH $PRXQW RI 06: JHQHUDWHG DQG ODQGILOOHG DW VL[ ODQGILOO VLWHV LQ )ORULGD /DQGILOO 6LWH 06: &ROOHFWHG 7RQV\HDUf $PRXQW RI :DVWH /DQGILOOHG bf /DQGILOO 7\SH /HDFKDWH UHF\FOH 5XUDO 1$E 1$ 5XUDOFDSSHG 6HPLXUEDQ 5HJLRQDO 8UEDQHQKDQFHG ELRORJLFDO WUHDWPHQW p7KH GDWD SUHVHQWHG UHSUHVHQWV LQIRUPDWLRQ FROOHFWHG LQ f'DWD ZHUH QRW DYDLODEOH 1$f IRU VLWH D FDSSHG ODQGILOO VLWH QR ORQJHU SHUPLWWHG WR DFFHSW 06: 0DWHULDOV DQG 0HWKRGV /HDFKDWH &ROOHFWLRQ 0XQLFLSDO VROLG ZDVWH 06:f OHDFKDWHV ZHUH FROOHFWHG IURP VL[ VLWHV ORFDWHG LQ ILYH ODQGILOOV LQ FHQWUDO )ORULGD 86$ 7KH VLWHV ZHUH GHVLJQDWHG DV WKURXJK 6LWH DQ RSHUDWLQJ ODQGILOO XQLW DQG VLWH D FDSSHG ODQGILOO XQLW DUH ORFDWHG DW WKH VDPH ODQGILOO )LJXUH f &DSSHG ODQGILOO XQLWV QR ORQJHU DFFHSW ZDVWH PDWHULDOV DQG DUH VXUURXQGHG E\ D KLJKGHQVLW\ SRO\HWK\OHQH +'3(f OLQHU WR SUHYHQW WKH LQILOWUDWLRQ RI ZDWHU DQG WKH SRWHQWLDO IRU VXEVHTXHQW HVFDSH RI OHDFKDWHV 7DEOH VXPPDUL]HV WKH UDWHV RI PXQLFLSDO ZDVWH GLVSRVDO DW WKH VLWHV XQGHU VWXG\ )'(3 f 06: ODQGILOO OHDFKDWHV ZHUH FROOHFWHG IURP OHDFKDWH FROOHFWLRQ ZHOOV XVLQJ D 7HIORQ EDOHU 2QH VDPSOH ZDV FROOHFWHG DW HDFK VLWH DQG WKHQ DSSRUWLRQHG WR VHSDUDWH FRQWDLQHUV IRU FKHPLFDO DQDO\VLV DQG WR[LFLW\ DVVD\V /HDFKDWHV IRU FKHPLFDO DQDO\VLV ZHUH FROOHFWHG LQ SRO\HWK\OHQH RU JODVV FRQWDLQHUV DQG SUHVHUYHG DFFRUGLQJ WR 8 6 (QYLURQPHQWDO 3URWHFWLRQ

PAGE 78

$JHQF\ 86(3$ Ef 6DPSOHV IRU WR[LFLW\ DQDO\VLV ZHUH FROOHFWHG LQ SODVWLF FXELWDLQHUV WUDQVSRUWHG WR WKH ODE RQ LFH DQG LPPHGLDWHO\ VWRUHG DWr& XQWLO VDPSOH DQDO\VLV ZLWKLQ WR GD\V &KHPLFDO DQG 3K\VLFDO &KDUDFWHUL]DWLRQ RI /HDFKDWHV 7KH FKHPLFDOSK\VLFDO FKDUDFWHUL]DWLRQ RI WKH 06: ODQGILOO OHDFKDWHV EHJDQ ZLWK ILHOG PHDVXUHPHQWV WKDW LQFOXGHG S+ DQG WHPSHUDWXUH 2ULRQ 0RGHO $f FRQGXFWLYLW\ +$11$ ,QVWUXPHQWV 0RGHO +f GLVVROYHG R[\JHQ '2f <6, ,QF 0RGHO )7f DQG R[LGDWLRQUHGXFWLRQ SRWHQWLDO 253f $FFXPHW &R 0RGHO f ,Q WKH ODERUDWRU\ WKH 06: ODQGILOO OHDFKDWHV ZHUH DQDO\]HG IRU D QXPEHU RI VWDQGDUG FKHPLFDO DQG SK\VLFDO SDUDPHWHUV ZKLFK LQFOXGHG DONDOLQLW\ ELRFKHPLFDO R[\JHQ GHPDQG %2'f FKHPLFDO R[\JHQ GHPDQG &2'f DPPRQLD DQG VXOILGHV DFFRUGLQJ WR PHWKRGV GHVFULEHG E\ 86(3$ Ef DQG $3+$ f /HDFKDWHV IRU PHWDO DQDO\VLV ZHUH GLJHVWHG DQG DQDO\]HG E\ ,QGXFWLYHO\ &RXSOHG 3ODVPD ,&3f 7KHUPR -DUUHOO $VK 0RGHO (QYLUR f )RU LRQ DQDO\VLV D 'LRQH[ LRQ FKURPDWRJUDSK 'LRQH[ 0RGHO '; f ZDV XVHG 7RWDO DPPRQLD 1+r 1+f DQG XQLRQL]HG DPPRQLD 1+f ZHUH DQDO\]HG E\ D VHOHFWLYH LRQ SUREH $FFXPHW 0RGHO f 0DLQWHQDQFH RI 7HVW 2UJDQLVPV 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD SUHYLRXVO\ NQRZQ DV 6HOHQDVWUXP FDSULFRPXWXPf LV D IUHVKZDWHU XQLFHOOXODU JUHHQ DOJDH URXWLQHO\ XWLOL]HG LQ ERWK

PAGE 79

7DEOH &RPSRQHQWV RI WKH SUHOLPLQDU\ DOJDO DVVD\ SURFHGXUH 3$$3f PHGLXP *URZWK $VVD\ PHGLXP PHGLXP 0$&52 6$/76 0DJQHVLXP VXOIDWH [ ; 0J6r+f 0DJQHVLXP FKORULGH [ [ 0J&, r +f &DOFLXP FKORULGH [ [ &D&, r +f 6RGLXP ELFDUERQDWH < [ 1D+&f 6RGLXP QLWUDWH [ [ 1D1f 3RWDVVLXP SKRVSKDWH [ [ .+3f 'LVRGLXP(WK\OHQH GLQLWULORfWHWUDDFHWDWH ; ('7$f 75$&( 0(7$/ 62/87,21 =LQF FKORULGH ; [ =Q&,f &REDOW FKORULGH [ [ &R&,r +f 6RGLXP PRO\EGDWH [ [ 1D0R r +f &XSULF FKORULGH [ [ &X&, r +f %RULF DFLG [ +%2f 0DQJDQHVH FKORULGH [ [ 0Q&,f )HUULF FKORULGH )H&,r+f )'(3 )'(3 f DQG 86(3$ SURWRFROV 86(3$ 86(3$ Df 7KH DOJDH FXOWXUHV ZHUH VWDUWHG IURP DQ RULJLQDO DOJDH VHHG JUDFLRXVO\ SURYLGHG E\ +\GURVSKHUH 5HVHDUFK 6XEVHTXHQW FXOWXUHV RI DOJDH ZHUH PDLQWDLQHG LQ WKH ODERUDWRULHV DW WKH 8QLYHUVLW\ RI )ORULGD 7KH DOJDH JURZWK PHGLXP ZDV SUHSDUHG

PAGE 80

DFFRUGLQJ WR )'(3 f DQG LV UHIHUUHG WR DV WKH SUHOLPLQDU\ DOJDO DVVD\ SURFHGXUH 3$$3f PHGLXP 7KH FRPSRQHQWV RI WKH 3$$3 DUH OLVWHG LQ 7DEOH 7KH 3$$3 ZDV SUHSDUHG E\ FRPELQLQJ PO RI HDFK RI WKH PDFURVDOWV ZLWK PO RI WKH WUDFH PHWDO VROXWLRQ LQ D / YROXPHWULF IODVN 7KH IODVN ZDV ILOOHG ZLWK '', ZDWHU DQG WKRURXJKO\ PL[HG 7KH S+ RI WKH 3$$3 PHGLXP ZDV DGMXVWHG WR s ZLWK HLWKHU 1D2+ RU 1 +&, 7KH 3$$3 PHGLXP ZDV WKHQ ILOWHU VWHULOL]HG SP PHPEUDQH ILOWHUf DQG VWRUHG XQGHU UHIULJHUDWLRQ 7KH DOJDO FHOOV ZHUH JURZQ XQGHU D VSHFLDOO\ GHVLJQHG OLJKW XQLW FRQVWUXFWHG E\ 0DUWLQ 'ROOH\f 7KH OLJKW XQLW FRQVLVWHG RI D ZRRGHQ SODWIRUP IHHW E\ IHHWf VXSSRUWHG ZLWK OHJV IHHWf EXW ZLWK RSHQ VLGHV 7KUHH IOXRUHVFHQW OLJKW IL[WXUHV IHHW ORQJf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f ZDV DGGHG $ PO JODVV SLSHWWH ZDV SODFHG LQ WKH IODVN ZKLFK ZDV WKHQ ZUDSSHG ZLWK SDUDILOP WR VHDO WKH WRS RI WKH IODVN 7KH IODVN ZDV VZLUOHG YLJRURXVO\ WR PL[ SODFHG XQGHU WKH OLJKW XQLW DQG WKHQ WKH FXOWXUH ZDV DHUDWHG E\ DWWDFKLQJ D VPDOO KRVH LQ VHULHV ZLWK ILOWHUV SP $FURGLVFf WR WKH JODVV SLSHWWH $ VPDOO DTXDFXOWXUH SXPS VXSSOLHG DLU WR WKH DOJDO FXOWXUH DQG

PAGE 81

FRQWLQXRXV JHQWOH PL[LQJ RI WKH DOJDH PHGLXP ZDV PDLQWDLQHG 7KH DOJDH FHOOV ZHUH FXOWXUHG IRU XS WR RQH ZHHN DW r& XQGHU FRQVWDQW LOOXPLQDWLRQ IW Ff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f DQG KDYH VLPLODU VSHFLHV GLVWULEXWLRQV DQG OLIH F\FOHV 86(3$ Df 7UDGLWLRQDOO\ SXOH[ RU PDJQDf ZHUH WKH LQYHUWHEUDWHV RI FKRLFH IRU GHWHUPLQLQJ DTXDWLF WR[LFLW\ 2YHU WKH SDVW \HDUV DVVD\V ZLWK & GXELD KDYH LQFUHDVHG LQ SRSXODULW\ 7KLV LV GLUHFWO\ UHODWHG WR WKH JUHDWHU VHQVLWLYLW\ RI WKH & GXELD WR DTXDWLF WR[LFDQWV 9HUVWHHJ HW DO f %RWK LQYHUWHEUDWH VSHFLHV DUH DEOH WR

PAGE 82

UHSURGXFH E\ SDUWKHQRJHQHVLV ZKLFK HQVXUHV D FRQWLQXRXV VXSSO\ RI LGHQWLFDO RIIVSULQJ 3UHSDUDWLRQ RI DTXDWLF LQYHUWHEUDWH IRRG 7KH DTXDWLF LQYHUWHEUDWH FXOWXUHV & GXELD DQG SXOH[f ZHUH IHG D \HDVW FHUHDO OHDYHV DQG WURXW FKRZ <&7f EDVHG IRRG 7KH <&7 ZDV SUHSDUHG RYHU D RQHZHHN SHULRG EHJLQQLQJ ZLWK WKH GLJHVWLRQ RI WKH WURXW FKRZ SHOOHWV 7KH WURXW FKRZ GLJHVWLRQ ZDV SHUIRUPHG LQ D ERWWRPOHVV / LQYHUWHG SODVWLF FRQWDLQHU E\ FRPELQLQJ D J SRUWLRQ RI WURXW FKRZ ZLWK / RI GLVWLOOHG ZDWHU '',f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f ZDV FRPELQHG LQ D EOHQGHU ZLWK / RI '', DQG PL[HG RQ KLJK VSHHG IRU PLQXWHV 7KH FHUHDO OHDI PL[WXUH ZDV FRYHUHG DQG UHVHUYHG LQ WKH UHIULJHUDWRU RYHUQLJKW WR VHWWOH )LQDOO\ RQ GD\ RI WKH GLJHVWLRQ D JUDP SRUWLRQ RI GU\ \HDVW )OHLVFKPDQQp RU HTXLYDOHQWf ZDV FRPELQHG ZLWK / RI '', ZDWHU DQG PL[HG ZHOO RQ D PDJQHWLF VWLU SODWH (TXDO YROXPHV RI WKH \HDVW VROXWLRQ FHUHDO OHDI VXSHUQDWDQW DQG WURXW FKRZ ILOWHUHG VXSHUQDWDQW ZHUH FRPELQHG DQG PL[HG WKRURXJKO\ 7KH <&7 IRRG ZDV DSSRUWLRQHG LQWR PO

PAGE 83

SODVWLF ERWWOHV ODEHOHG DQG VWRUHG LQ WKH IUHH]HU ar&f XQWLO QHHGHG <&7 IRRG ZDV VWRUHG LQ WKH UHIULJHUDWRU DQG XQXVHG SRUWLRQV ZHUH GLVFDUGHG DIWHU ZHHN 7KH WRWDO VROLGV 76f FRQWHQW RI WKH <&7 ZDV PDLQWDLQHG EHWZHHQ DQG J VROLGV/ E\ WKH DGGLWLRQ RI '', DV QHHGHG 7KH & GXELD DQG SXOH[ FXOWXUHV ZHUH DOVR IHG 3 VXEFDSLWDWD DOJDH FHOOV ; FHOOVPOf DV SUHYLRXVO\ GHVFULEHG 0DLQWHQDQFH RI DTXDWLF LQYHUWHEUDWH FXOWXUHV 6WDUWHU FXOWXUHV RI & GXELD DQG SXOH[ ZHUH JUDFLRXVO\ GRQDWHG E\ +\GURVSKHUH 5HVHDUFK *DLQHVYLOOH )/f 7KH DTXDWLF LQYHUWHEUDWHV ZHUH FXOWXUHG LQ GHGLFDWHG JODVVZDUH ZKLFK ZDV PDLQWDLQHG VHSDUDWHO\ DQG WKRURXJKO\ ZDVKHG DQG ULQVHG EHWZHHQ HDFK XVDJH 2DSKQLGV ZHUH FXOWXUHG LQ UHFRQVWLWXWHG PRGHUDWHO\ KDUG ZDWHU 0+:f ZKLFK ZDV FRPSRVHG RI 1D+& PJ &D62} +2 PJ 0J6&;L PJ DQG .&, PJ SHU OLWHU RI '', ZDWHU 7KH 0+: KDG WKH IROORZLQJ VSHFLILFDWLRQV S+ KDUGQHVV PJ/ DV &D& DQG DONDOLQLW\ PJ/ 86(3$ Df $TXDWLF LQYHUWHEUDWH FXOWXUHV ZHUH PDLQWDLQHG E\ DGGLQJ QHRQDWHV KRXU ROGf RI & GXELD RU SXOH[ WR / JODVV EHDNHUV FRQWDLQLQJ 0+: 7KH GDSKQLG EHDNHUV ZHUH NHSW LQ DQ HQYLURQPHQWDO FKDPEHU 3HUFLYDO PRGHO ( %;f DW s r& DQG ZLWK D OLJKW UHJLPH RI KRXUV RI OLJKW DQG KRXUV RI GDUN 7KH GDSKQLG FXOWXUHV ZHUH IHG PO RI <&7 DQG PO RI DOJDH FHOOV SHU OLWHU RI LQYHUWHEUDWH FXOWXUH ,QYHUWHEUDWH FXOWXUHV ZHUH FXOOHG GDLO\ WR UHPRYH QHRQDWHV DQG HQVXUH D SRSXODWLRQ ZLWK D XQLIRUP DJH GLVWULEXWLRQ )ROORZLQJ WKH UHPRYDO RI QHRQDWHV IRU WR[LFLW\ DVVD\V DQ\ UHPDLQLQJ QHRQDWHV ZHUH XVHG WR

PAGE 84

3UHSDUH OHDFKDWH GLOXWLRQV ZLWK 3$$3 PLQXV ('7$ 7UDQVIHU PO RI OHDFKDWH RU LWV GLOXWLRQ WR WULSOLFDWH PO HUOHQPH\HU IODVNV MD 6SLNH HDFK IODVN ZLWK PO 3 VXEFDSLWDWD FHOOVPOf -+ 3ODFH HUOHQPH\HUV XQGHU JURZWK OLJKW IRU KRXUV VKDNLQJ GDLO\ &RXQW QXPEHU DOJDH FHOOV ZLWK KHPDF\WRPHWHU DQG SKDVHFRQWUDVW PLFURVFRSH )LJXUH )ORZFKDUW IRU WKH 3 VXEFDSLWDWD DVVD\

PAGE 85

VWDUW QHZ FXOWXUHV RU ZHUH GLVFDUGHG $GXOW IHPDOHV ZHUH UHWDLQHG IRU QHRQDWH SURGXFWLRQ IRU D SHULRG QR ORQJHU WKDQ WZR ZHHNV 7R[LFLW\ $VVD\V 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD 7KH FKURQLF WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV ZDV HYDOXDWHG DFFRUGLQJ WR WKH SURWRFROV RI WKH KRXU 3 VXEFDSLWDWD DVVD\ 86(3$ Df 7KH OHDFKDWHV ZHUH ILOWHUHG ZLWK JODVV ILEHU :KDWPDQ *)%f DQG PHPEUDQH ILOWHUV SPf 7KH JODVV ILEHU SUHILOWHUV ZHUH XVHG WR PLQLPL]H FORJJLQJ RI WKH PHPEUDQH ILOWHU 7KH GLOXWLRQ PHGLD IRU WKH DOJDO DVVD\V ZDV D 3$$3 VROXWLRQ SUHSDUHG ZLWKRXW WKH GLVRGLXP (WK\OHQHGLQLWULORf WHWUDDFHWDWH ('7$f ('7$ KDV EHHQ VKRZQ WR IRUP FRPSOH[HV ZLWK KHDY\ PHWDOV ZKLFK FRQIRXQGV DVVD\ UHVXOWV ZKHQ PHWDO WR[LFLW\ LV VXVSHFWHG 7KH 3$$3 JURZWK PHGLXP UHTXLUHV WKH DGGLWLRQ RI ('7$ EHFDXVH LWV SUHVHQFH LV FUXFLDO IRU WKH XSWDNH RI PDQ\ PLFURQXWULHQWV 86(3$ Df )RU HDFK DOJDO DVVD\ ILYH GLOXWLRQV ZHUH SUHSDUHG LQ D ODPLQDU IORZ KRRG ZLWK 3$$3 PLQXV ('7$ DW D GLOXWLRQ IDFWRU RI )LJXUH f $ PO DOLTXRW RI WKH OHDFKDWH RU LWV GLOXWLRQ ZDV DGGHG WR WULSOLFDWH PO VWHULOH HULHQPH\HU IODVNV ZLWK VW\URIRDP VWRSSHUV 7KH IODVNV ZHUH WKHQ LQRFXODWHG ZLWK D PO DOLTXRW RI DOJDH FHOOV FHOOVPOf 7KH LQRFXOXP ZDV SUHSDUHG E\ FHQWULIXJLQJ D WR PO SRUWLRQ RI DOJDH VWRFN GD\V ROGf DW USP IRU ILIWHHQ PLQXWHV 7KH VXSHUQDWDQW ZDV GLVFDUGHG DQG WKH DOJDH FHOOV ZHUH UHVXVSHQGHG LQ 3$$3 PLQXV ('7$ DQG PL[HG E\ YRUWH[LQJ JHQWO\ 8VLQJ D KHPDF\WRPHWHU DQG D SKDVH FRQWUDVW PLFURVFRSH WKH FHOO GHQVLW\ ZDV GHWHUPLQHG 7KH YROXPH RI UHVXVSHQGHG DOJDH FHOOV UHTXLUHG WR SUHSDUH DQ

PAGE 86

DOJDH VHHG ZLWK D GHQVLW\ RI FHOOVPO ZDV GHWHUPLQHG E\ WKH IROORZLQJ HTXDWLRQ 1XPEHU WHVW IODVNV [ 9RO WHVW 6ROXWLRQ SHU IODVN [ FHOOV SHU PO &HOO GHQVLW\ FHOOV SHU POf LQ WKH VWRFN FXOWXUH %DVHG RQ WKH QXPEHU RI DVVD\ IODVNV DQG D YROXPH RI PO RI VHHG SHU IODVN WKH DOJDH FHOO LQRFXOXP ZDV SUHSDUHG $V DQ H[DPSOH LI WKH DVVD\ UHTXLUHG IODVNV FRQWDLQLQJ PO RI OHDFKDWH SHU IODVN DQG WKH VWRFN DOJDO FXOWXUH GHQVLW\ ZDV FHOOVPO WKHQ WKH UHTXLUHG YROXPH RI VWRFN FXOWXUH WR SURGXFH PO RI DOJDH LQRFXOXP LV PO &RPELQLQJ D PO SRUWLRQ RI WKH DOJDH VWRFN FXOWXUH FHOOVPOf ZLWK D PO SRUWLRQ RI 3$$3 ZLWKRXW ('7$ SURYLGHG WKH UHTXLUHG PO SRUWLRQ RI DOJDH LQRFXOXP QHHGHG IRU WKH DVVD\ 7KH HUOHQPH\HU IODVNV FRQWDLQLQJ WKH OHDFKDWH RU LWV GLOXWLRQ DQG WKH DOJDO VHHG ZHUH SODFHG XQGHU WKH OLJKW XQLW SUHYLRXVO\ GHVFULEHGf DW r& &RQVWDQW LOOXPLQDWLRQ s IWFf ZDV PDLQWDLQHG IRU KRXUV DQG WKH IODVNV ZHUH URWDWHG DQG PL[HG GDLO\ E\ VZLUOLQJ PDQXDOO\ $W WKH FRQFOXVLRQ RI HDFK DVVD\ WKH DOJDH FHOO GHQVLW\ LQ HDFK IODVN ZDV PHDVXUHG E\ DOJDO FHOO FRXQWV XVLQJ D KHPDF\WRPHWHU DQG D SKDVHFRQWUDVW PLFURVFRSH *URZWK LQKLELWLRQ ZDV GHWHUPLQHG E\ FRPSDULQJ WKH QXPEHU RI DOJDH FHOOV LQ WKH OHDFKDWH FRQWDLQLQJ IODVNV WR WKH QXPEHU LQ WKH FRQWURO IODVNV 7KH OHDFKDWH FRQFHQWUDWLRQ WKDW SURGXFHG D b LQKLELWLRQ ,&f RI DOJDO JURZWK ZDV GHWHUPLQHG E\ JUDSKLQJ WKH FHOO GHQVLW\ LQ HDFK IODVN YHUVXV WKH OHDFKDWH FRQFHQWUDWLRQ

PAGE 87

3UHSDUH OHDFKDWH GLOXWLRQV ZLWK 0+: 7UDQVIHU QHRQDWHV WR DVVD\ FXS DGG PO RI VDPSOH RU LWV GLOXWLRQ ([SRVH QHRQDWHV WR OHDFKDWH IRU KRXUV 2EVHUYH QHRQDWHV IRU GHDWK LPPRELOL]DWLRQ )LJXUH )ORZFKDUW IRU WKH & GXELD DVVD\ &HULRGDSKQLD GXELD DQG 'DSKQLD SXOH[ 7KH DFXWH WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV ZHUH HYDOXDWHG ZLWK & GXELD DQG SXOH[LQ WKH VWDQGDUG KRXU DFXWH WR[LFLW\ SURWRFROV 86(3$ D $3+$ f %DVLFDOO\ WKH DVVD\ SURWRFROV ZHUH LGHQWLFDO IRU WKH WZR VSHFLHV RI DTXDWLF LQYHUWHEUDWHV %HIRUH WKH OHDFKDWHV ZHUH HYDOXDWHG IRU WR[LFLW\ WKH\ ZHUH SUHILOWHUHG :KDWPDQ *)%f WR UHPRYH ODUJH SDUWLFOHV 7KH & GXELD DQG SXOH[ DVVD\V IROORZ VLPLODU FRQGLWLRQV 3ULRU WR WKH VWDUW RI HDFK

PAGE 88

DTXDWLF LQYHUWHEUDWH DVVD\ WKH QHRQDWHV KUVf ZHUH VHSDUDWHG IURP WKH DGXOW GDSKQLGV DQG IHG D PL[WXUH RI PO <&7OLWHU DQG PO DOJDHOLWHU $IWHU IHHGLQJ QHRQDWHV ZHUH WUDQVIHUUHG WR HDFK WHVW FRQWDLQHU PO SODVWLF FXSVf XVLQJ D VPDOO ZLGHPRXWK SODVWLF SLSHWWH WR PLQLPL]H WKH WUDQVIHU RI FXOWXUH ZDWHU )LJXUH f 7KH OHDFKDWH GLOXWLRQV ZHUH SUHSDUHG ZLWK 0+: DW D GLOXWLRQ IDFWRU DQG WKH OHDFKDWH RU LWV GLOXWLRQ ZDV DGGHG DW PO YROXPHV WR WULSOLFDWH FXSV FRQWDLQLQJ WKH QHRQDWHV &RQWDLQHUV ILOOHG ZLWK 0+: ZHUH XVHG DV WKH QHJDWLYH FRQWUROV 7KH WHVW FRQWDLQHUV ZHUH SODFHG LQ D ZDWHU EDWK DW s r & IRU KRXUV ZLWK D ORRVH FRYHULQJ WR DOORZ OLJKW SHQHWUDWLRQ DQG SUHYHQW VHWWOLQJ RI DLU SDUWLFOHV 1HRQDWHV ZHUH H[SRVHG WR DPELHQW OLJKWLQJ DQG ZHUH QRW IHG GXULQJ WKH DVVD\ $IWHU KRXUV WKH LQYHUWHEUDWH WHVW FRQWDLQHUV ZHUH SODFHG RQ D OLJKW WDEOH IRU WKH GHWHUPLQDWLRQ RI YLDEOH RUJDQLVPV 7KH OLJKW WDEOH ZDV GHVLJQHG WR VLW RQ WKH ODERUDWRU\ EHQFK FRQVWUXFWHG E\ 0DUWLQ 'ROOH\f ,W ZDV FRPSRVHG RI D ZRRGHQ ER[ ZLWK D SOH[LJODVV WRS DQG WKUHH IOXRUHVFHQW OLJKWV 7KH IOXRUHVFHQW OLJKWV ZHUH PRXQWHG LQVLGH WKH ER[ DQG EHORZ WKH SOH[LJODVV WRS VR DV WR LOOXPLQDWH WKH ZRUN VXUIDFH 7HVW FRQWDLQHUV ZHUH VZLUOHG JHQWO\ DQG QHRQDWHV ZLWK WKH SRZHU WR VZLP DZD\ IURP WKH FHQWHU RI WKH FRQWDLQHU ZHUH FRXQWHG DV GHDGLPPRELOL]HG 0RUWDOLW\ JUHDWHU WKDQ b LQ WKH FRQWUROV QHJDWHG WKH DVVD\ UHVXOWV

PAGE 89

$GG QO RI OHDFKDWH RU LWV GLOXWLRQ WR FXYHWWHV FRQWDLQLQJ EDFWHULDO UHDJHQW PL[ 0HDVXUH ILQDO ELROXPLQHVFHQFH DIWHU PLQXWH H[SRVXUH )LJXUH )ORZFKDUW IRU WKH 0LFURWR[r1 DVVD\

PAGE 90

0LFURWR[r1 7KH 0LFURWR[r1 WR[LFLW\ DQDO\]HU LV D FRPPHUFLDOO\ DYDLODEOH WR[LFLW\ V\VWHP WKDW PHDVXUHV WR[LF HIIHFWV E\ FKDQJHV LQ EDFWHULDO 9LEULR ILVFKHULf ELROXPLQHVFHQFH %HFNPDQ ,QVWUXPHQWV f 7KH DVVD\ NLW LQFOXGHV D GLOXHQW WKH IUHH]HGULHG 0LFURWR[ EDFWHULDOUHDJHQW D UHFRQVWLWXWLRQ VROXWLRQ DQG DQ RVPRWLF DGMXVWLQJ VROXWLRQ (DFK OHDFKDWH ZDV DVVD\HG LQ GXSOLFDWH DQG HDFK DVVD\ DOVR LQFOXGHG D GXSOLFDWH FRQWURO '', ZDWHUf )LJXUH f $ SUHOLPLQDU\ LQYHVWLJDWLRQ LQGLFDWHG WKDW D PLQXWH H[SRVXUH SURGXFHG WKH KLJKHVW VHQVLWLYLW\ ZKLFK DJUHHV ZLWK WKH UHSRUWV RI RWKHU UHVHDUFKHUV 3ORWNLQ DQG 5DP f 7KH 0LFURWR[r1 DQDO\]HU FRPELQHV D SUHFRROLQJ ZHOO IRU VWRUDJH RI WKH UHFRQVWLWXWHG EDFWHULD UHDJHQW GXULQJ WKH DVVD\ DQ LQFXEDWRU ZHOO EORFN WR FRRO WKH FXYHWWHV FRQWDLQLQJ WKH VDPSOHVf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

PAGE 91

7KH OHDFKDWH GLOXWLRQV DUH SUHSDUHG IURP ULJKW WR OHIW LQ FROXPQV WR DQG ZHOO $ FRUUHVSRQGHG WR WKH KLJKHVW OHDFKDWH FRQFHQWUDWLRQ 7KH GLOXWLRQ VHULHV LV SUHSDUHG E\ ILUVW DGGLQJ SL RI WKH 0LFURWR[ GLOXHQW WR WKH FXYHWWHV LQ URZ $ RI FROXPQV WR 7KHQ SL RI WKH OHDFKDWH LV DGGHG WR FROXPQV DQG RI URZ $ DQG PL[HG E\ UHSHDWHGO\ SLSHWWLQJ SL RI WKH PL[WXUH DQG DVSLUDWLQJ $ SO DOLTXRW IURP WKH FXYHWWH LQ FROXPQ ZDV WUDQVIHUUHG WR WKH FXYHWWH LQ FROXPQ PL[HG DQG WKHQ D SO DOLTXRW IURP FROXPQ ZDV WUDQVIHUUHG WR FROXPQ $IWHU FROXPQ ZDV PL[HG DQG DVSLUDWHG D SO DOLTXRW ZDV GLVFDUGHG 7KH FXYHWWHV ZHUH DOORZHG WR FRRO IRU DSSUR[LPDWHO\ PLQXWHV )ROORZLQJ WKH DGGLWLRQ RI WKH UHFRQVWLWXWHG 0LFURWR[ EDFWHULDO UHDJHQW SLf WR ZHOOV % WKURXJK % DQG & WKURXJK & WKH FXYHWWHV ZHUH DOORZHG WR UHDFK WKHUPDO HTXLOLEULXP DSSUR[LPDWHO\ PLQXWHV 7KH OXPLQHVFHQFH RI WKH EDFWHULDO UHDJHQW LQ HDFK FXYHWWH ZDV GHWHUPLQHG E\ SODFLQJ WKH FXYHWWH LQ WKH WXUUHW DQG WXUQLQJ WKH KDQGOH WR WKH UHDG SRVLWLRQ 7KH OXPLQHVFHQFH RXWSXW IURP WKH EDFWHULDO UHDJHQW ZDV UHDG RQ WKH GLJLWDO SDQHO PHWHU '30f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f ZKLFK

PAGE 92

LV PHDVXUHG DV WKH UDWLR RI OLJKW ORVW WR OLJKW UHPDLQLQJ IROORZLQJ OHDFKDWH H[SRVXUH 'DWD $QDO\VLV 7KH WR[LFLW\ DVVD\ UHVXOWV ZHUH H[SUHVVHG DV WKH FRQFHQWUDWLRQ RI OHDFKDWH WKDW SURGXFHG D b HIIHFW LQ WKH ELRDVVD\ 7KH HQGSRLQWV RI WKH DVVD\V ZHUH GLIIHUHQW DQG LQFOXGHG LQKLELWLRQ RI ELROXPLQHVFHQFH 0LFURWR[ DV (&f OHWKDOLW\ RU GHDWK & GXELD DV /&f DQG JURZWK LQKLELWLRQ 3 VXEFDSLWDWD DV ,&f 7KH UHVXOWV RI WKH KU 3 VXEFDSLWDWD DVVD\V ZHUH GHWHUPLQHG E\ JUDSKLFDO ,QWHUSRODWLRQ 7KH /&VRIRU WKH & GXELD DVVD\ ZDV GHWHUPLQHG XVLQJ WKH 86(3$ GDWD DQDO\VLV VRIWZDUH 86(3$ Ef %LRDVVD\ UHVXOWV ZHUH SUHVHQWHG ZLWK RQH VWDQGDUG GHYLDWLRQ IRU WKH & GXELD DQG 3 VXEFDSLWDWD DVVD\V 0LFURWR[r1 WHVW UHVXOWV ZHUH GHWHUPLQHG E\ OHDVW VTXDUH UHJUHVVLRQ DQDO\VLV RI WKH QDWXUDO ORJ RI WKH VDPSOH FRQFHQWUDWLRQ YV WKH QDWXUDO ORJ RI JDPPD UDWLR RI OLJKW ORVW WR OLJKW UHPDLQLQJf 5HVXOWV ZHUH SUHVHQWHG DV WKH FRQFHQWUDWLRQ RI OHDFKDWH FDXVLQJ b LQKLELWLRQ RI ELROXPLQHVFHQFH JDPPDf 7KH 0LFURWR[ DVVD\ ZDV SHUIRUPHG LQ GXSOLFDWH WKHUHIRUH GHWHUPLQDWLRQV RI VWDQGDUG GHYLDWLRQV ZHUH QRW YDOLG 'DWD ZDV HYDOXDWHG E\ OHDVW VTXDUH UHJUHVVLRQ VWXGHQWnV WWHVW RU WKH ) WHVW ([FHO 0LFURVRIW f DV DSSURSULDWH 7KH (& ,& DQG /& UHVXOWV ZHUH WUDQVIRUPHG WR WR[LFLW\ XQLWV 78f DFFRUGLQJ WR WKH IROORZLQJ 78 XQLWOHVVf (&[RU ,&0 RU /&6 :KHQ DSSURSULDWH GDWD ZHUH ORJ WUDQVIRUPHG IRU HYDOXDWLRQ RI OLQHDU UHODWLRQVKLSV

PAGE 93

7DEOH 3K\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI 06: ODQGILOO OHDFKDWHV DW VL[ VLWHV LQ )ORULGD 3DUDPHWHU 6LWH 6LWH 6LWH 6LWH 6LWH 6LWH S+ 7HPSHUDWXUH r&f &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ/ DV &D&f &%2'D PJ/f &2'E PJ/f n f f f f f f f f f f f f f f f f f f f f f f f f f f f f mf f f f f f 10& f 6XOILGH SJ/f f f f f f f $O PJ/f f f f f f &X &G PJ/f f 3E PJ/f f =Q PJ/f f f f $V PJ/f f f f &U PJ/f f f f %D PJ/f f f f f f )H PJ/f f f f f f f 1D PJ/f f f f f f f PJ/f f f f f f f 5HVXOWV DUH VKRZQ DV WKH PHDQ DQG UDQJHf $EEUHYLDWLRQV f%2'ELRORJLFDO R[\JHQ GHPDQG E&2' FKHPLFDO R[\JHQ GHPDQG &10 QRW PHDVXUHG

PAGE 94

5HVXOWV DQG 'LVFXVVLRQ &KHPLFDO $QDO\VLV RI 06: /HDFKDWHV 7KH SK\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI WKH 06: ODQGILOO OHDFKDWHV DUH VXPPDUL]HG LQ 7DEOH $OWKRXJK WKH PHDQ S+ YDOXHV ZHUH QHDU QHXWUDO ZLWK D UDQJH IURP WR ORZHU S+ YDOXHV ZHUH PHDVXUHG LQ VRPH VDPSOHV 7KH WR[LFLW\ DQG ELRDYDLODELOLW\ RI VRPH OHDFKDWH WR[LFDQWV HVSHFLDOO\ KHDY\ PHWDOV DUH S+ GHSHQGHQW 6FKXEDXHU%HULJDQ HW DO f 7KH DONDOLQLW\ RI WKH OHDFKDWHV ZDV KLJKO\ YDULDEOH DQG UDQJHG IURP PJ/ DV &D& DW VLWH WR PJ/ DV &D&&! DW VLWH 7KHVH FRQFHQWUDWLRQV DUH W\SLFDO IRU ODQGILOO OHDFKDWHV LQ WKH HDUO\ SKDVHV RI ZDVWH VWDELOL]DWLRQ .MHOGVHQ HW DO f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

PAGE 95

FDSDFLW\ LV FRQVXPHG E\ WKH SURGXFWLRQ RI RUJDQLF DFLGV 'LIIHUHQFHV LQ WKH %2'&2' UDWLRV ZHUH VOLJKW EXW VXJJHVWHG D KLJKHU GHJUHH RI RUJDQLF PDWWHU GHJUDGDWLRQ LQ WKH OHDFKDWHV IURP VLWH 7KH %2'&2' UDWLR ZDV LQ WKH OHDFKDWHV IURP VLWH DQG LQ WKH OHDFKDWHV IURP VLWH 7KH ORZHU FRQFHQWUDWLRQ RU LQRUJDQLF FDWLRQV LQ WKH VLWH OHDFKDWHV ZDV GXH WR WKH ZDVKn RXW HIIHFW W\SLFDO RI ROGHU OHDFKDWHV .MHOGVHQ HW DO f :KLOH RUJDQLF FRPSRQHQWV LQ WKH OHDFKDWHV DUH GHJUDGHG E\ ELRORJLFDO DFWLYLW\ LQRUJDQLF FRQVWLWXHQWV GHFUHDVH RYHU WLPH ZLWK LQFUHDVHG UDWHV RI OHDFKDWH SURGXFWLRQ &DPHURQ DQG .RFK &KLDQ DQG 'H:DOOH f 6RPH YDULDWLRQV LQ WKH FKHPLFDO FRPSRVLWLRQ RI 06: ODQGILOO OHDFKDWHV DUH H[SHFWHG EDVHG RQ DJH ZDVWH GHJUDGDWLRQ DQG VLWHVSHFLILF IDFWRUV 5DJOH HW DO f *HQHUDOO\ WKH FRQFHQWUDWLRQV RI KHDY\ PHWDOV LQ WKH 06: ODQGILOO OHDFKDWHV ZHUH EHORZ DQDO\WLFDO GHWHFWLRQ OLPLWV 7DEOH f +RZHYHU VRPH OHDFKDWHV FRQWDLQHG HOHYDWHG FRQFHQWUDWLRQV RI KHDY\ PHWDOV $OXPLQXP ZDV GHWHFWHG LQ PRVW RI WKH OHDFKDWHV DW OHDVW RQFH RYHU WKH PRQWK LQYHVWLJDWLRQ ,Q WKH OHDFKDWHV IURP VLWH DOXPLQXP ZDV GHWHFWHG LQ HDFK RI WKH OHDFKDWH VDPSOHV FROOHFWHG ZLWK D UDQJH IURP WR PJ/ 7KH KHDY\ PHWDOV FRSSHU DQG FDGPLXP ZHUH QRW GHWHFWHG GHWHFWLRQ OLPLWV RI PJ/ DQG PJ/ UHVSHFWLYHO\f LQ DQ\ RI WKH OHDFKDWHV DQDO\]HG :KLOH OHDG FRQFHQWUDWLRQV ZHUH OHVV WKDQ PJ/ LQ WKH OHDFKDWHV IURP VLWHV DQG LQ WKH OHDFKDWHV IURP VLWH OHDG FRQFHQWUDWLRQV UDQJHG IURP WR PJ/ 7KH OHDFKDWHV IURP VLWH FRQWDLQHG ]LQF FRQFHQWUDWLRQV WKDW UDQJHG IURP WR PJ/ =LQF FRQFHQWUDWLRQV LQ WKH OHDFKDWHV IURP VLWH H[FHHGHG WKH

PAGE 96

)LJXUH &RQFHQWUDWLRQV RI WRWDO 1K91+Vf EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf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f

PAGE 97

)HE 0DUFK $SULO 0D\ -XQH -XO\ )LJXUH &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf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f &RQFHQWUDWLRQV RI RWKHU LQRUJDQLF FDWLRQV ZHUH DOVR KLJK LQ WKH 06: ODQGILOO OHDFKDWHV 6RGLXP OHYHOV UDQJHG IURP D ORZ PHDQ RI PJ/ DW VLWH WR KLJK 8QORQL]HG DPPRQLD PJ/f

PAGE 98

\ 7 R B ( ( R p ( R 0DUFK $SULO 0D\ -XQH -XO\ )LJXUH &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO 10 LQGLFDWHV VDPSOH QRW PHDVXUHG PHDQ RI PJ/ DW VLWH $OWKRXJK WKH SRWDVVLXP FRQFHQWUDWLRQV ZHUH ORZHU D VLPLODU SDWWHUQ ZDV GHPRQVWUDWHG 5HSRUWHG PHDQ FRQFHQWUDWLRQV RI SRWDVVLXP LQ WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG ZHUH DQG PJ/ UHVSHFWLYHO\ 7KH PRQWKO\ OHYHOV RI WRWDO 1+ 1+f DPPRQLD LQ WKH 06: ODQGILOO OHDFKDWHV IOXFWXDWHG ZLGHO\ DQG WKH FRQFHQWUDWLRQV ZHUH GHSHQGHQW RQ VLWH VSHFLILF FRQGLWLRQV &RQVLVWHQWO\ WKH KLJKHVW RYHUDOO WRWDO DPPRQLD FRQFHQWUDWLRQV ZHUH LGHQWLILHG LQ WKH 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ZLWK D UDQJH IURP WR PJ/ )LJXUH f $OWKRXJK WKH FRQFHQWUDWLRQV RI WRWDO DPPRQLD ZHUH ORZHU DW VLWH DQG UDQJHG IURP WR PJ/ WKH ff§ 8QLRQL]HG DPPRQLD PJ/f

PAGE 99

\ 7 )HE 0DUFK $SULO 0D\ -XQH )LJXUH &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO 10 LQGLFDWHV VDPSOH QRW PHDVXUHG YDULDELOLW\ UHPDLQHG KLJK )LJXUH f 7KH ORZHVW WRWDO DPPRQLD FRQFHQWUDWLRQV ZHUH GLVSOD\HG E\ WKH 06: OHDFKDWHV FROOHFWHG IURP VLWH WKH FDSSHG ODQGILOO VLWHf ZLWK D UDQJH IURP WR PJ/ )LJXUH f ,Q WKH 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWHV DQG WRWDO DPPRQLD FRQFHQWUDWLRQV UDQJHG IURP WR PJ/ DQG WR PJ/ UHVSHFWLYHO\ )LJXUHV DQG f 7KLV FRQWUDVWV ZLWK WRWDO DPPRQLD FRQFHQWUDWLRQV PHDVXUHG LQ WKH OHDFKDWHV IURP VLWH ZKLFK UDQJHG IURP PJ/ LQ -XO\ WR PJ/ LQ $SULO RI )LJXUH f $PPRQLD VSHFLDWLRQ LV GHSHQGHQW RQ ERWK S+ DQG WHPSHUDWXUH $PPRQLXP LV WKH GRPLQDQW VSHFLHV DW S+ YDOXHV ZKLOH DPPRQLD

PAGE 100

)LJXUH &RQFHQWUDWLRQV RI WRWDO 1+r1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf LQ 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU D PRQWK VDPSOLQJ LQWHUYDO 10 LQGLFDWHV QRW PHDVXUHG GRPLQDWHV DW S+ YDOXHV )LJXUHV WR f $V SUHYLRXVO\ GLVFXVVHG WKH S+ YDOXHV ZHUH VLPLODU DW WKH VL[ ODQGILOO VLWHV KRZHYHU VOLJKWO\ KLJKHU WHPSHUDWXUHV ZHUH UHSRUWHG LQ WKH OHDFKDWHV IURP VLWHV DQG 7DEOH f $W WKH S+ RI WKH OHDFKDWHV WKH XQLRQL]HG DPPRQLD OHYHOV ZHUH H[SHFWHG WR EH ORZ DQG WKLV ZDV WUXH IRU VLWHV DQG ZLWK UHSRUWHG FRQFHQWUDWLRQV RI OHVV WKDQ PJ/ +LJKHU WRWDO DPPRQLD FRQFHQWUDWLRQV ZHUH UHSRUWHG LQ WKH OHDFKDWHV IURP VLWHV DQG ZLWK PHDQ XQLRQL]HG DPPRQLD OHYHOV RI DQG PJ/ UHVSHFWLYHO\ %DVLFDOO\ WKH XQLRQL]HG DPPRQLD FRQFHQWUDWLRQV IROORZHG WKH VDPH SDWWHUQ DV SUHYLRXVO\ GHVFULEHG IRU WKH WRWDO DPPRQLD FRQFHQWUDWLRQV 8QLRQL]HG DPPRQLD PJ/f

PAGE 101

0DUFK $SULO 0D\ -XQH -XO\ )LJXUH &RQFHQWUDWLRQV RI WRWDO 1+1+f EDUVf DQG XQLRQL]HG DPPRQLD 1+f GLDPRQGVf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f $FFRUGLQJ WR WKH UHVXOWV RI WKH KRXU & GXELD DVVD\ WKH 06: ODQGILOO OHDFKDWHV FROOHFWHG 8QLRQL]HG DPPRQLD PJ/f

PAGE 102

)LJXUH 7KH PHDQ WR[LFLW\ RI 06: OHDFKDWHV FROOHFWHG IURP VL[ ODQGILOO VLWHV 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ RQH VWDQGDUG GHYLDWLRQ )LJXUH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ

PAGE 103

78 XQLWOHVVf t & GXELD IIO 3 VXEFDSLWDWD i 0LFURWR[ )HE 0DUFK $SULO 0D\ -XQH -XO\ )LJXUH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ L 6 & GXELD PL 3 VXEFDSLWDWD % 0LFURWR[ )LJXUH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ RQH VWDQGDUG GHYLDWLRQ

PAGE 104

IURP VLWHV DQG GLVSOD\HG WKH JUHDWHVW WR[LF HIIHFWV DQG WKH OHDFKDWHV FROOHFWHG IURP VLWH ZHUH WKH OHDVW WR[LF )LJXUH VKRZV WKH SDWWHUQ RI WR[LF HIIHFWV PHDVXUHG PRQWKO\ 7KH 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH GLVSOD\HG 78 YDOXHV WKDW UDQJHG IURP WR )LJXUH f $OWKRXJK RQ DYHUDJH WKH WR[LFLW\ RI WKH VLWH OHDFKDWHV ZDV ORZ ZLWK D PHDQ 78 RI WKH UDQJH RI WR[LFLW\ ZDV YDULDEOH 7KH 78 UHVSRQVH RI WKH & GXELD WR WKH OHDFKDWH IURP VLWH LQ 0D\ ZDV QHDUO\ GRXEOH WKH PHDQ WR[LFLW\ RI WKLV OHDFKDWH RYHU WKH VL[PRQWK LQYHVWLJDWLRQ )LJXUH f *HQHUDOO\ WKH 06: OHDFKDWHV IURP VLWH GLVSOD\HG ORZHVW WR[LFLW\ 7KH 78 YDOXHV UHSRUWHG ZLWK WKH OHDFKDWHV IURP VLWH UDQJHG IURP WR ZLWK D PHDQ 78 RI )LJXUH f 7KH WR[LFLW\ RI WKH VLWH OHDFKDWHV GLVSOD\HG D PHDQ 78 UHVSRQVH RI s ZLWK WKH JUHDWHVW 78 UHSRUWHG LQ $SULO DW )LJXUH f 7KH WR[LFLW\ RI WKH VLWH OHDFKDWHV YDULHG IURP D ORZ RI LQ 0DUFK WR D KLJK RI LQ $SULO )LJXUH f 7KH UHVSRQVH RI WKH KRXU & GXELD DVVD\ WR WKH OHDFKDWHV FROOHFWHG IURP VLWH

PAGE 105

78 XQLWOHVVf & GXELD ,' 3 VXEFDSWDWD % 0LFURWR[ )LJXUH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ 6& GXELD ,'3 VXEFDSWDWD %0LFURWR[ )LJXUH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ

PAGE 106

& GXELD ', 3 VXEFDSLWDWD % 0LFURWR[ )LJXUH 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWH RYHU WLPH 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ GLVSOD\HG WKH ZLGHVW YDULDELOLW\ LQ WR[LFLW\ 7KH VLWH OHDFKDWHV GLVSOD\HG 78 YDOXHV LQ 0DUFK $SULO DQG 0D\ ZLWK D UDQJH IURP WR )LJXUH f 7KH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWH GURSSHG GUDPDWLFDOO\ LQ -XO\ RI ZKHQ D ODUJH UDLQ HYHQW LPPHGLDWHO\ SUHFHGHG VDPSOH FROOHFWLRQ 7KH FROOHFWLRQ ZHOO ZDV LQXQGDWHG ZLWK UDLQZDWHU DQG WKLV UHVXOWHG LQ D QHDUO\ b UHGXFWLRQ LQ WR[LFLW\ $ VLPLODU UHGXFWLRQ LQ OHDFKDWH FKHPLFDO VWUHQJWK ZDV UHSRUWHG IRU WKLV VDPSOH 7DEOH f $ VLPLODU SDWWHUQ RI WR[LF UHVSRQVH ZDV GHPRQVWUDWHG E\ WKH UHVXOWV RI WKH KRXU 3 VXEFDSLWDWD DVVD\ )LJXUH f $FFRUGLQJ WR WKH DOJDO DVVD\ WKH OHDFKDWHV IURP VLWH GLVSOD\HG WKH JUHDWHVW WR[LFLW\ FRPSDUHG WR WKRVH IURP VLWHV DQG )LJXUH f :KHQ FRPSDULQJ OHDFKDWHV IURP VLWHV DQG

PAGE 107

7DEOH &RUUHODWLYH DQDO\VLV ZLWK WKH & GXELD 3 VXEFDSLWDWD DQG 0LFURWR[r1 DVVD\ UHVXOWV YHUVXV OHDFKDWH FKHPLFDO FKDUDFWHULVWLFV 3DUDPHWHUV & GXELD KRXUf 3 VXEFDSLWDWD KRXUf 0LFURWR[r1 PLQXWHf 7RWDO $PPRQLD 1.I 1+f 5 Sf 5A Sf 5 Sf 8QLRQL]HG $PPRQLD 1+f I r R ,, R R &2 I r R 0 R R A FQ 5 Sf $ONDOLQLW\ &RQGXFWLYLW\ 7'6D &%2'E &2'F 5 Sf 5 Sf 5 Sf 5 Sf 5A Sf 5 Sf U Sf 5 Sf 5A Sf Sf 5A 3 f 15G 15 15 5A Sf 7KH FRHIILFLHQW RI GHWHUPLQDWLRQ DQG VLJQLILFDQFH RI WKH UHODWLRQVKLSVf DUH UHSRUWHG $EEUHYLDWLRQV f7'6 WRWDO GLVVROYHG VROLGV E&%2' FDUERQDFHRXV R[\JHQ GHPDQG F&2' FKHPLFDO R[\JHQ GHPDQG _15 QR UHODWLRQVKLS WKH OHDFKDWHV IURP WKH FDSSHG VLWH VLWH f ZHUH OHVV WR[LF 7KH UDQJH RI WR[LFLW\ LQ WKH OHDFKDWHV IURP VLWH ZDV IURP WR ZKLOH WKH VLWH OHDFKDWHV UDQJHG IURP WR )LJXUHV DQG f :LWK WKH OHDFKDWHV IURP VLWH WKHUH ZHUH SHDNV LQ WKH 78 UHVSRQVHV GXULQJ 0D\ DW DQG -XO\ DW 7KLV SDWWHUQ RI WR[LFLW\ ZDV QRW FRQVLVWHQW ZLWK WKH UHVXOWV RI WKH & GXELD DVVD\ 7KH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWH UDQJHG IURP 78V RI WR 7KHUHIRUH WKH WR[LF UHVSRQVH RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH ZDV FRPSDUDEOH WR WKDW UHSRUWHG ZLWK WKH OHDFKDWHV IURP VLWH )LJXUH f 7KH VDPH ZDV WUXH IRU WKH DOJDO DVVD\ UHVXOWV ZLWK WKH OHDFKDWHV IURP VLWHV DQG ZKLFK ZHUH URXJKO\ HTXLYDOHQW DQG GLVSOD\HG PHDQ 78 YDOXHV RI DQG UHVSHFWLYHO\ )LJXUH f

PAGE 108

7KH 0LFURWR[ DVVD\ ZDV QRW VHQVLWLYH WR WKH WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV )LJXUH f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f 6LJQLILFDQW UHODWLRQVKLSV ZHUH IRXQG EHWZHHQ ERWK WKH & GXELD U Sf DQG 3 VXEFDSLWDWD U Sf WR[LFLW\ UHVXOWV DQG WKH WRWDO DPPRQLD FRQWHQW RI WKH OHDFKDWHV )RU WKH XQLRQL]HG DPPRQLD 1+f FRQWHQW RI WKH OHDFKDWHV WKH UHODWLRQVKLS ZDV QRW DV VWURQJ ZLWK HLWKHU WKH & GXELD U Sf RU WKH 3 VXEFDSLWDWD U Sf WR[LFLW\ GDWD :HDNHU UHODWLRQVKLSV ZHUH IRXQG EHWZHHQ WKH 0LFURWR[r1 UHVXOWV DQG WRWDO DPPRQLD U Sf RU XQn LRQL]HG DPPRQLD U Sf $ VLPSOH UHJUHVVLRQ DQDO\VLV RI WKH OHDFKDWH WR[LFLW\ YDOXHV ZLWK & GXELD (&f YHUVXV WKRVH ZLWK 3 VXEFDSLWDWD (&f UHYHDOHG D KLJK GHJUHH RI GDWD FRUUHODWLRQ 5 Sf )LJXUH

PAGE 109

& GXELD (&Nbf )LJXUH 5HODWLRQVKLS EHWZHHQ WKH 3 VXEFDSLWDWD (&f DQG & GXELD (&f DVVD\ UHVXOWV ZLWK 06: ODQGILOO OHDFKDWHV f 7KH 0LFURWR[r1 DVVD\ ZDV OHVV VHQVLWLYH DQG VKRZHG QR UHODWLRQVKLS ZLWK HLWKHU WKH & GXELD RU 3 VXEFDSLWDWD DVVD\ UHVXOWV 7KH WR[LFLW\ UHVXOWV IRU WKH OHDFKDWHV FROOHFWHG IURP VLWH DFWLYH VLWHf )LJXUH f YHUVXV VLWH FDSSHG VLWHf )LJXUH f ZHUH FRPSDUHG /HDFKDWHV FROOHFWHG IURP WKH DFWLYH XQLW GHPRQVWUDWHG D VLJQLILFDQWO\ KLJKHU WR[LFLW\ LQ ERWK WKH 3 VXEFDSLWDWD DQG & GXELD DVVD\V Sf H[FHSW IRU WKH PRQWK RI $SULO ZKHQ OHDFKDWH WR[LFLW\ ZDV VLPLODU 7KH UHVXOWV RI WKH 0LFURWR[ DVVD\ ZHUH DOVR VLPLODU IRU WKH VLWH DQG VLWH OHDFKDWH VDPSOHV 7KH UHVXOWV RI WKH PRQWK LQYHVWLJDWLRQ LQGLFDWHG WKDW WKH DFXWH DQG FKURQLF WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV ZDV KLJKO\ YDULDEOH ,Q RUGHU WR DVVHVV WKH WR[LFLW\ YDULDWLRQV ZLWKLQ D VKRUWHU WLPH IUDPH WKH DFXWHFKURQLF

PAGE 110

& GXELD ,% 3 VXEFDSEDWD % 0LFURWR[ V )LJXUH 7R[LFLW\ IOXFWXDWLRQV LQ WKH OHDFKDWHV FROOHFWHG IURP WKH 06: ODQGILOO DW VLWH GXULQJ )HEUXDU\ 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ 6WDQGDUG GHYLDWLRQV DUH QRW VKRZQ IRU WKH 0LFURWR[r1 GDWD 6LJQLILFDQW GLIIHUHQFHV r S DUH LQGLFDWHG WR[LFLW\ RI OHDFKDWHV FROOHFWHG IURP VLWH ZHUH PRQLWRUHG RQ WKUHH RFFDVLRQV RYHU D SHULRG RI RQH PRQWK WR GHWHUPLQH WHPSRUDO YDULDWLRQ LQ OHDFKDWH VWUHQJWK 7KH 78 UHVXOWV RI WKH 3 VXEFDSLWDWD DVVD\ ZHUH s s DQG s ZLWK WKH OHDFKDWHV FROOHFWHG RQ QG WK DQG WK RI )HEUXDU\ UHVSHFWLYHO\ 7KHUH ZDV QR VLJQLILFDQW Sf GLIIHUHQFH LQ WKH WR[LFLW\ RI WKH OHDFKDWHV FROOHFWHG GXULQJ )HEUXDU\ IURP VLWH DV PHDVXUHG ZLWK WKH DOJDH DVVD\ ,Q FRQWUDVW DFFRUGLQJ WR WKH UHVXOWV RI WKH & GXELD DVVD\ WKH WR[LFLW\ ZDV VLJQLILFDQWO\ Sf LQFUHDVHG GXULQJ )HEUXDU\ 2Q WKH QG WK DQG WK RI )HEUXDU\ WKH 78 YDOXHV LQ WKH & GXELD DVVD\ ZHUH s s DQG s UHVSHFWLYHO\ :LWK WKH 0LFURWR[r1 DVVD\ UHVXOWV WKHUH ZDV D

PAGE 111

WR[LFLW\ GHFUHDVH Sf EHWZHHQ )HEUXDU\ QG DQG WK IURP WR EXW QR FKDQJH EHWZHHQ WKH WK DQG WK )LJXUH f 7KH DVVD\V ZHUH UDQNHG DFFRUGLQJ WR WKHLU VHQVLWLYLW\ WR WKH 06: OHDFKDWHV & GXELD 3 VXEFDSLWDWD 0LFURWR[ 7KH PHDQ 78 YDOXHV ZLWK WKH & GXELD DVVD\ UDQJHG IURP WR ZKLOH D VLPLODU UDQJH IURP WR ZDV UHSRUWHG LQ WKH 3 VXEFDSLWDWD DVVD\V 7KH FRPSDUDEOH VHQVLWLYLWLHV RI WKHVH WZR ZLGHO\ XVHG WR[LFLW\ DVVD\V YHULILHV UHSRUWV LQ WKH OLWHUDWXUH 6XHGHO HW DO 5RMLFNRYD3DGUWRYD HW DO )HUUDUL HW DO &OHPHQW HW DO f ,Q D VWXG\ RI GRPHVWLF ZDVWH OHDFKDWHV LQ )UDQFH WKH 78 YDOXHV ZLWK & GXELD UDQJHG IURP WR ZLWK RI WKH OHDFKDWHV HYDOXDWHG 7KH DXWKRUV DOVR HYDOXDWHG OHDFKDWHV IURP QRQKD]DUGRXV DQG KD]DUGRXV LQGXVWULDO VROLG ZDVWHV DQG IRXQG D ORZHU 78 UDQJH IURP WR ZLWK & GXELD OHDGLQJ WR WKH FRQFOXVLRQ WKDW OHDFKDWHV IURP GRPHVWLF VROLG ZDVWHV VKRXOG EH FRQVLGHUHG DV GDQJHURXV DV WKRVH IURP LQGXVWULDO ZDVWHV &OHPHQW HW DO f 0XOWLSOH ELRDVVD\V ZHUH DOVR XVHG LQ D VWXG\ RI OHDFKDWHV IURP D ODQGILOO UHFHLYLQJ PL[HG GRPHVWLF DQG VRPH LQGXVWULDOf ZDVWHV 7KH UHVHDUFKHUV IRXQG 3 VXEFDSLWDWD FKORURSK\OO D FRQWHQWf ZDV KLJKO\ VHQVLWLYH ZLWK 78 YDOXHV UDQJLQJ WR 3ORWNLQ DQG 5DP f 7KH\ DOVR VKRZHG KRXU 78V IRU 'DSKQLD PDJQD IURP WR DQG D 0LFURWR[r1 78 RI 2WKHUV 'HYDUH DQG %DKDGLU f VKRZHG YDULDELOLW\ LQ WR[LFLW\ EHWZHHQ VHSDUDWH ODQGILOO VLWHV DQG UHSRUWHG D KLJKHU VHQVLWLYLW\ ZLWK SODQW /HPQD PLQRUf DVVD\V RYHU 0LFURWR[r1 ,Q D (XURSHDQ VWXG\ OHDFKDWHV IURP GRPHVWLF ZDVWHV ZHUH IRXQG PRUH WR[LF WKDQ WKRVH RI LQGXVWULDO RULJLQ DQG WKHUH ZDV D KLJK YDULDELOLW\ LQ WR[LFLW\ RI ODQGILOO OHDFKDWHV RI

PAGE 112

VLPLODU RULJLQ &OHPHQW HW DO f 7KHVH UHVXOWV VXSSRUW RXU ILQGLQJ RI WKH WR[LF QDWXUH RI WKH )ORULGD 06: OHDFKDWHV GHPRQVWUDWLQJ WKDW WKH WR[LFLW\ LV VWLOO RI FRQFHUQ GHVSLWH WKH GLOXWLRQ HIIHFW VXJJHVWHG E\ 5HLQKDUW DQG *URVK f $PPRQLD 7R[LFLW\ 7KH DPPRQLD FRQWHQW RI 06: ODQGILOO OHDFKDWHV LQ )ORULGD KDV SUHYLRXVO\ EHHQ VKRZQ WR H[SODLQ VLJQLILFDQW DPRXQWV RI WR[LFLW\ :DUG HW DO f 7KH XQn LRQL]HG DPPRQLD FRQFHQWUDWLRQV RI WKH LQGLYLGXDO OHDFKDWHV XQGHU LQYHVWLJDWLRQ UDQJHG IURP WR PJ/ ZKLOH WKH WRWDO DPPRQLD FRQFHQWUDWLRQV UDQJHG IURP WR PJ/ $PPRQLD WR[LFLW\ LV SULPDULO\ DVVRFLDWHG ZLWK WKH XQn LRQL]HG IRUP RI DPPRQLD ZKLFK LV GHSHQGHQW RQ WKH S+ WHPSHUDWXUH DQG WKH FRQFHQWUDWLRQV RI WRWDO DPPRQLD LQ WKH OHDFKDWHV &OHPHQW DQG 0HUOLQ 5XWKHUIRUG HW DO f 7KH 06: ODQGILOO OHDFKDWHV HYDOXDWHG IURP VLWHV WR FRQWDLQHG KLJK OHYHOV RI XQLRQL]HG DPPRQLD DW FRQFHQWUDWLRQV WKDW ZHUH VXIILFLHQW WR FDXVH D VLJQLILFDQW SRUWLRQ RI WKH OHDFKDWH WR[LFLW\ $QGHUVRQ DQG %XFNOH\ f UHSRUWHG WKDW PJ/ RI XQLRQL]HG DPPRQLD ZDV OHWKDO WR b RI & GXELD LQ D KRXU DFXWH DVVD\ 7KLV OHYHO ZDV ORZHU WKDQ WKH FRQFHQWUDWLRQV DFWXDOO\ IRXQG LQ WKH 06: ODQGILOO OHDFKDWHV VWXGLHG LQ )ORULGD 7KH 0LFURWR[ UHVXOWV ZLWK WKH 06: ODQGILOO OHDFKDWHV VXJJHVWHG WKDW WKLV DVVD\ ZDV QRW VXLWDEOH IRU WKH HYDOXDWLRQ RI OHDFKDWH WR[LFLW\ SUREDEO\ GXH WR WKH ORZ VHQVLWLYLW\ RI WKLV DVVD\ WR DPPRQLD 6WURQNKXUVW HW DOf f 4XUHVKL HW DO f UHSRUWHG DQ (&} RI PJ/ IRU LRQL]HG DPPRQLD XVLQJ WKH 0LFURWR[ WHVW V\VWHP ,Q FRQWUDVW WR WKH UHVXOWV UHSRUWHG KHUH 'RKHUW\ HW DO f XVLQJ WKH 0LFURWR[ DVVD\ UHSRUWHG DQ XQn LRQL]HG DPPRQLD (& RI PJ/ PLQXWHf 7KH ORZ VHQVLWLYLW\ RI WKH

PAGE 113

0LFURWR[r1 DVVD\ WR XQLRQL]HG DPPRQLD DV VKRZQ KHUH PD\ OHDG WR D FRQFOXVLRQ RI LWV VXLWDELOLW\ IRU GHWHFWLQJ WKH SUHVHQFH RI RWKHU WR[LFDQWV LQ WKH OHDFKDWH KRZHYHU WKLV ZDV QRW WKH FDVH ZLWK WKH 06: ODQGILOO OHDFKDWHV 7KH UHVXOWV UHSRUWHG GHPRQVWUDWH WKDW WKH FRQFHQWUDWLRQV RI DPPRQLD LQ WKH )ORULGD OHDFKDWHV H[SODLQHG D VLJQLILFDQW DPRXQW RI WKH WR[LFLW\ LQ ERWK WKH & GXELD DQG 3 VXEFDSLWDWD DVVD\V ,Q FRQWUDVW WKH 0LFURWR[r1 DVVD\ VKRZHG D VPDOO UHODWLRQVKLS EHWZHHQ WKH DPPRQLD FRQWHQW RI 06: OHDFKDWHV DQG WKHLU WR[LFLW\ 3UHYLRXVO\ UHVHDUFKHUV KDYH FRUUHODWHG 0LFURWR[r1 WHVW UHVXOWV ZLWK DPPRQLD FRQFHQWUDWLRQV LQ VHGLPHQW HOXWULDWHV &KHXQJ HW DO f DQG PLQH HIIOXHQWV /H%ORQG DQG 'XII\ f ,QIOXHQFH RI 6LWH6SHFLILF )DFWRUV RQ /HDFKDWH 7R[LFLW\ 3UHYLRXVO\ WKH SK\VLFRFKHPLFDO FKDUDFWHULVWLFV RI GRPHVWLF LQGXVWULDO DQG PL[HG ZDVWH OHDFKDWHV ZHUH FRPSDUHG WR WKHLU PHDVXUHG WR[LFLW\ ZLWK D EDWWHU\ RI WHVWV WKDW LQFOXGHG & GXELD DQG 0LFURWR[ &OHPHQW HW DO f 7KHLU UHVXOWV VKRZHG D UHODWLRQVKLS EHWZHHQ &2' FRQFHQWUDWLRQV DQG WKH 0LFURWR[ PLQXWH (&f A Sf UHVXOWV $ ZHDNHU UHODWLRQVKLS A Sf ZDV GHPRQVWUDWHG ZLWK D PLQXWH H[SRVXUH 7KH )ORULGD OHDFKDWHV ZHUH VWULFWO\ RI GRPHVWLF RULJLQ ZKLOH WKH ZDVWH OHDFKDWHV LQYHVWLJDWHG E\ &OHPHQW HW DO f FRQVLVWHG RI GRPHVWLF ZDVWH OHDFKDWHV DQG LQGXVWULDO RU PL[HG LQGXVWULDO GRPHVWLF ZDVWH OHDFKDWHV 7KH UHVXOWV ZLWK WKH )ORULGD OHDFKDWHV VXJJHVW WKDW WKH 0LFURWR[ ELROXPLQHVFHQFH DVVD\ LV PRUH VHQVLWLYH IRU HYDOXDWLQJ WKH WR[LFLW\ RI VDPSOHV ZLWK SUHGRPLQDQWO\ RUJDQLF WR[LFDQWV %LWWRQ HW

PAGE 114

DO f UDWKHU WKDQ ZDVWH OHDFKDWHV ZLWK KLJK LQRUJDQLF FRQWHQWV HJ DPPRQLD RU DONDOLQLW\ &OHPHQW HW DO f 7KH DONDOLQLW\ RI WKH 06: OHDFKDWHV DVVD\HG ZDV KLJK 7KH SUHGRPLQDQW UROH RI DONDOLQLW\ LQ OHDFKDWH WR[LFLW\ VHHPV WR EH WKH EXIIHULQJ RI S+ DQG LWV LQIOXHQFH RQ DPPRQLD VSHFLDWLRQ &OHPHQW DQG 0HUOLQ +RNH HW DO f $ONDOLQLW\ FRQFHQWUDWLRQV ZHUH FRUUHODWHG ZLWK 3 VXEFDSLWDWD (& UHVXOWV UA2 Sf DQG ZLWK & GXELD (& UHVXOWV A Sf EXW WKH UHODWLRQVKLS ZLWK 0LFURWR[r1 A S f ZDV ZHDNHU 7KH UHODWLRQVKLS VKRZHG IRU & GXELD DQG DONDOLQLW\ ZDV KLJKHU WKDQ WKDW IRXQG E\ &OHPHQW HW DO f ZLWK PL[HG ZDVWH OHDFKDWHV A f EXW DJUHHV ZLWK WKHLU UHSRUW IRU XQn LRQL]HG DPPRQLD A f

PAGE 115

&+$37(5 $ 6859(< 72 $66(66 7+( $&87( $1' &+521,& 72;,&,7< 2) /($&+$7(6 )520 06: /$1'),//6 ,1 )/25,'$ ,QWURGXFWLRQ 5XEELVK JDUEDJH WUDVK XQZDQWHG PDWHULDOV DQG GLVFDUGHG ZDVWHV DUH VRPH RI WKH SKUDVHV FRPPRQO\ XVHG WR GHVFULEH WKH E\SURGXFWV RI GDOO\ OLIH 7KH UHJXODWRU\ DQG VFLHQWLILF FRPPXQLWLHV UHIHU WR WKHVH E\SURGXFWV DV PXQLFLSDO VROLG ZDVWH 06:f D WHUP WKDW LQFOXGHV SDFNDJLQJ PDWHULDOV JUDVV FOLSSLQJV IXUQLWXUH FORWKLQJ ERWWOHV IRRG VFUDSV DQG RWKHU GLVFDUGHG PDWHULDOV 86(3$ f 7KH SHU FDSLWD JHQHUDWLRQ RI PXQLFLSDO VROLG ZDVWH 06:f LQ WKH 8QLWHG 6WDWHV UHPDLQV UHODWLYHO\ FRQVWDQW DW DSSUR[LPDWHO\ OEV 06:SHUVRQGD\ 86(3$ f $OWKRXJK WKH SHUFHQWDJH RI ZDVWH ODQGILOOHG KDV GHFUHDVHG LQ UHFHQW \HDUV URXJKO\ b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f 0RGHUQ ODQGILOOV DUH HQJLQHHUHG V\VWHPV ZKLFK DUH W\SLFDOO\ GHVLJQHG WR PLQLPL]H WKH LQILOWUDWLRQ RI ZDWHU PDLQWDLQ DQDHURELF

PAGE 116

FRQGLWLRQV DQG HQVXUH WKH UHFRYHU\ RI OHDFKDWHV ,Q VRPH DUHDV ODQGILOO GHVLJQV DUH EHLQJ PRGLILHG WR HQFRXUDJH FKHPLFDO DQG ELRORJLFDO GHJUDGDWLRQ SURFHVVHV E\ LQFUHDVLQJ PRLVWXUH OHYHOV DQG R[LGL]HG FRQGLWLRQV 5HLQKDUW DQG 7RZQVHQG 1RSKDUDWDQD HW DO f 06: ODQGILOO OHDFKDWHV DUH SURGXFHG ZKHQ UDLQZDWHU LQILOWUDWHV LQWR WKH ZDVWH DQG H[FHHGV WKH ILHOG FDSDFLW\ RU WKH DELOLW\ RI WKH ZDVWH WR KROG ZDWHU $V OHDFKDWHV SDVV WKURXJK WKH ODQGILOOHG ZDVWH PDWHULDOV VROXEOH FRQVWLWXHQWV DUH PRELOL]HG /HDFKDWHV SRVH D SRWHQWLDO WKUHDW WR WKH VXUURXQGLQJ HQYLURQPHQW IURP JURXQGZDWHU FRQWDPLQDWLRQ RU GLUHFW HVFDSH RI OHDFKDWH WR VXUIDFH ZDWHUV 1RDNVVRQ HW DO .DZDJRVKL HW DO $O0X]DLQL HW DO f 7KH SROOXWDQWV RI FRQFHUQ LQ 06: ODQGILOO OHDFKDWHV DUH SULPDULO\ GLVVROYHG RUJDQLF PDWWHU LQRUJDQLF PDFURFRPSRQHQWV KHDY\ PHWDOV DQG [HQRELRWLF RUJDQLF FRPSRXQGV .MHOGVHQ HW DO f /DQGILOO RSHUDWRUV DUH UHTXLUHG WR WUHDW ODQGILOO OHDFKDWHV DQG WUHDWPHQW W\SLFDOO\ RFFXUV DW GRPHVWLF ZDVWHZDWHU WUHDWPHQW SODQWV DOWKRXJK VPDOO RQVLWH WUHDWPHQW IDFLOLWLHV DUH XWLOL]HG DW VRPH ODQGILOOV &)5 f /DQGILOO OHDFKDWHV LQ )ORULGD DUH FROOHFWHG DQG WUHDWHG DV QHFHVVDU\ WR PHHW VXUIDFH DQG JURXQGZDWHU VWDQGDUGV &KDSWHU DQG )$& )$& D )$& Ef 7KH FKHPLFDO DQG SK\VLFDO FKDUDFWHULVWLFV RI WKH 06: OHDFKDWHV YDU\ ZLGHO\ DQG FDQ VLJQLILFDQWO\ DOWHU WKH FKHPLFDO FRPSRVLWLRQ RI GRPHVWLF ZDVWHZDWHU &RQVHTXHQWO\ ZKHQ WKHVH OHDFKDWHV DUH FRPELQHG ZLWK GRPHVWLF ZDVWHZDWHU WKH PLFURIORUD RI WKH WUHDWPHQW IDFLOLW\ FDQ EH HQKDQFHG DV HYLGHQFHG E\ D GRXEOLQJ RI ELRVROLGV %RRWK HW DO f )XUWKHUPRUH WKH

PAGE 117

WUHDWPHQW RI 06: OHDFKDWHV DW GRPHVWLF ZDVWHZDWHU WUHDWPHQW SODQWV ::73Vf PD\ LQKLELW ELRORJLFDO SURFHVVHV GXH WR WKH KLJK FRQFHQWUDWLRQV RI FRQWDPLQDQWV HVSHFLDOO\ DPPRQLD +HQFH WKH WUHDWDELOLW\ RI 06: ODQGILOO OHDFKDWHV LV KHDYLO\ GHSHQGHQW RQ WKH OHDFKDWH VWUHQJWK DQG WKH SUHVHQFH RI WR[LF VXEVWDQFHV $ONDOD\ HW DW f ,Q SUHYLRXV UHVHDUFK :DUG HW DO f WKH WR[LFLW\ RI 06: OHDFKDWHV IURP VL[ ODQGILOO VLWHV LQ )ORULGD ZDV LQYHVWLJDWHG WR LGHQWLI\ WR[LFDQWV RI FRQFHUQ DQG WR HVWDEOLVK WR[LFLW\ PHWKRGV IRU XVH ZLWK WKH OHDFKDWHV VHH &KDSWHU f 6XUYH\V RI 06: ODQGILOO OHDFKDWH TXDOLW\ DQG WR[LFLW\ SURYLGH D IUDPHZRUN IRU ORQJWHUP OHDFKDWH PDQDJHPHQW VWUDWHJLHV %DUOD] HW DO f ,Q D FRQWLQXDWLRQ RI WKH LQLWLDO VXUYH\ DGGLWLRQDO OHDFKDWH VDPSOHV ZHUH FROOHFWHG IURP 06: ODQGILOOV DFURVV WKH VWDWH RI )ORULGD 7KHVH OHDFKDWHV ZHUH VHOHFWHG WR UHSUHVHQW D FURVV VHFWLRQ RI OHDFKDWH TXDOLW\ DQG VWUHQJWK WKHUHIRUH VDPSOLQJ VLWHV LQFOXGHG WKH VPDOOHVW DQG RQH RI WKH ODUJHVW ODQGILOOV LQ WKH VWDWH 7KH VFRSH RI WKH FRQWLQXHG LQYHVWLJDWLRQ LQFOXGHG PXQLFLSDO VROLG ZDVWH 06:f OHDFKDWHV FROOHFWHG IURP IRXUWHHQ ODQGILOO VLWHV LQ )ORULGD 7KH REMHFWLYHV RI WKLV LQYHVWLJDWLRQ ZHUH f WR LQFUHDVH WKH DFXWH DQG FKURQLF WR[LFLW\ GDWDEDVH IRU )ORULGD 06: ODQGILOO OHDFKDWHV f WR FKDUDFWHUL]H WKH FKHPLFDO FRPSRVLWLRQ RI WKHVH OHDFKDWHV f WR HYDOXDWH UHODWLRQVKLSV EHWZHHQ OHDFKDWH WR[LFLW\ DQG FKHPLFDO FKDUDFWHULVWLFV f WR GHWHUPLQH WKH LQIOXHQFH RI WLPH RQ 06: ODQGILOO OHDFKDWH WR[LFLW\

PAGE 118

6LWH )LJXUH /RFDWLRQV RI WKH 06: ODQGILOOV IRU WKH FROOHFWLRQ RI OHDFKDWHV LQ )ORULGD 0DWHULDOV DQG 0HWKRGV 6DPSOLQJ 6LWHV 06: ODQGILOO OHDFKDWHV ZHUH FROOHFWHG IURP IRXUWHHQ HQJLQHHUHG ODQGILOOV WKURXJKRXW WKH VWDWH RI )ORULGD )LJXUH f 6RPH RI WKHVH ODQGILOO VLWHV DQG f ZHUH SUHYLRXVO\ LQYHVWLJDWHG DV SDUW RI D ODUJHU UHVHDUFK SURMHFW VHH &KDSWHU f $OO WKH 06: ODQGILOOV ZHUH OLQHG DQG FRQWDLQHG OHDFKDWH FROOHFWLRQ V\VWHPV 'HVFULSWLRQV RI WKH 06: ODQGILOOV DUH SURYLGHG 7DEOH f $GGLWLRQDO EDFNJURXQG LQIRUPDWLRQ LV DYDLODEOH IRU VLWHV DQG VHH &KDSWHU f DQG VLWHV DQG VHH &KDSWHU f $ XQLTXH VLWXDWLRQ ZDV SURYLGHG DW VLWH ZKLFK FRQWDLQHG WKUHH LQGLYLGXDO ODQGILOO FHOOV ZLWK VHSDUDWH OHDFKDWH FROOHFWLRQ V\VWHPV 7KH VLWH ODQGILOOV ,QFOXGHG DQ RSHUDWLQJ &ODVV VLWH Df D FDSSHG &ODVV VLWH Ff DQG D OLQHG &ODVV ,,, ODQGILOO VLWH Ef

PAGE 119

6LWH /DQGILOO &ODVV 3RSXODWLRQ 7RQV\HDU /DQGILOOHG FDSSHGf , FDSSHGf , 'DWD QRW DYDLODEOH ,, D E ,,, F FDSSHGf &ODVV ODQGILOOV DUH GHVLJQDWHG IRU WKH GLVSRVDO RI QRQKD]DUGRXV KRXVHKROG ZDVWHV DQG VRPH FRPPHUFLDO LQGXVWULDO DQG DJULFXOWXUDO ZDVWHV ZKLOH &ODVV ,,, ODQGILOOV DUH GHVLJQHG IRU \DUG ZDVWHV FRQVWUXFWLRQ DQG GHPROLWLRQ GHEULV FDUSHW IXUQLWXUH DQG VLPLODU QRQSXWUHVFLEOH ZDVWH PDWHULDOV 'HSHQGLQJ RQ WKHLU DJH

PAGE 120

FDSSHG FORVHGf ODQGILOOV DUH JHQHUDOO\ ,Q WKH PHWKDQRJHQLF RU KXPLF SKDVH RI ZDVWH VWDELOL]DWLRQ %R]NXUW HW DO f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f PHWKRGV 86(3$ Ef 3ODVWLF FRQWDLQHUV ZHUH XVHG WR VWRUH WKH OHDFKDWHV IRU WR[LFLW\ DQDO\VLV $OO OHDFKDWHV ZHUH WUDQVSRUWHG WR WKH ODE RQ LFH DQG LPPHGLDWHO\ VWRUHG DW r& XQWLO DQDO\VLV

PAGE 121

&KHPLFDO DQG 3K\VLFDO &KDUDFWHUL]DWLRQ RI 06: /HDFKDWHV )LHOG PHDVXUHPHQWV LQFOXGHG S+ DQG WHPSHUDWXUH 2ULRQ 0RGHO $f FRQGXFWLYLW\ +$11$ ,QVWUXPHQWV 0RGHO +f GLVVROYHG R[\JHQ '2f <6, ,QF 0RGHO )7f DQG R[LGDWLRQUHGXFWLRQ SRWHQWLDO 253f $FFXPHW &R 0RGHO f ,Q WKH ODERUDWRU\ DONDOLQLW\ FDUERQDFHRXV ELRFKHPLFDO R[\JHQ GHPDQG &%2'f FKHPLFDO R[\JHQ GHPDQG &2'f WRWDO RUJDQLF FDUERQ 72&f DQG VXOILGHV ZHUH PHDVXUHG DFFRUGLQJ WR PHWKRGV GHVFULEHG E\ 86(3$ Ef DQG $3+$ f +DUGQHVV ZDV PHDVXUHG E\ FRORULPHWULF DQDO\VLV +$&+ PHWKRG +$&+ /RYHODQG &2f 6DPSOHV IRU WRWDO PHWDO FRQWHQW ZHUH GLJHVWHG 86(3$ 0HWKRG 6: $f 86(3$ f DQG DQDO\]HG E\ DWRPLF HPLVVLRQ VSHFWURVFRS\ $(6f 7KHUPR -DUUHOO $VK 0RGHO (QYLUR f )RU PDMRU LRQ DQDO\VLV D 'LRQH[ LRQ FKURPDWRJUDSK 'LRQH[ 0RGHO ';f ZDV XVHG 7RWDO DPPRQLD 1+ 1+f DQG XQLRQL]HG DPPRQLD 1+f ZHUH DQDO\]HG E\ D VHOHFWLYH LRQ SUREH $FFXPHWf 6RPH RI WKH XQLRQL]HG DPPRQLD FRQFHQWUDWLRQV ZHUH FDOFXODWHG DFFRUGLQJ WR (PHUVRQ HW DO f b XQLRQL]HG DPPRQLD 1+af f§BB n nrfS+ff ZKHUH S.D 7 DQG 7 WHPSHUDWXUH r.f 7R[LFLW\ $VVD\V 7KH DFXWH WR[LFLW\ RI WKH 06: OHDFKDWHV ZHUH DVVD\HG ZLWK WKH VWDWLF KRXU &HULRGDSKQLD GXELD DQG 'DSKQLD SXOH[ DVVD\V 86(3$ Df DQG WKH PLQXWH 0LFURWR[r1 WHVW %HFNPDQ ,QVWUXPHQWV f 7KH JUHHQ DOJD 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD IRUPHUO\ 6HOHQDVWUXP FDSULFRPXWXPf ZDV XWLOL]HG LQ D KU FKURQLF DOJDO DVVD\ 86(3$ Df 7KLV VXLWH RI IRXU DFXWH

PAGE 122

DQG FKURQLF DVVD\V SURYLGHG D FRPSUHKHQVLYH PHDVXUH RI 06: ODQGILOO OHDFKDWH WR[LFLW\ ZLWK HIIHFWV TXDQWLILDEOH E\ YDULRXV HQGSRLQWV 7KHVH HQGSRLQWV LQFOXGHG OHWKDOLW\LPPRELOL]DWLRQ LQKLELWLRQ RI ELROXPLQHVFHQFH DQG JURZWK LQKLELWLRQ IRU & GXELD DQG 'SXOH[ 0LFURWR[r1 DQG 3 VXEFDSLWDWD UHVSHFWLYHO\ 7KH WR[LFLW\ DVVD\ SURWRFROV ZHUH SUHYLRXVO\ GHVFULEHG LQ GHWDLO &KDSWHU f %ULHIO\ IRU WKH & GXELD DVVD\V HDFK 06: ODQGILOO OHDFKDWH ZDV DVVD\HG XVLQJ GLOXWLRQV LQ WULSOLFDWH $OO GLOXWLRQV DQG FRQWUROV ZHUH SUHSDUHG ZLWK V\QWKHWLF PRGHUDWHO\ KDUG ZDWHU 0+:f & GXELD QHRQDWHV KU ROGf ZHUH XVHG LQ DOO WHVWV 7HQ QHRQDWHV ZHUH H[SRVHG WR HDFK GLOXWLRQ LQ WULSOLFDWH ZLWK DSSURSULDWH FRQWUROV 1HRQDWHV ZHUH H[SRVHG IRU KUV DW s r& XQGHU FRQGLWLRQV RI DPELHQW OLJKW KUV OLJKW KUV GDUNf $ VHFRQG DTXDWLF LQYHUWHEUDWH DVVD\ XWLOL]LQJ 'DSKQLD SXOH[ ZDV XWLOL]HG WR GHWHUPLQH WKH WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV 7KH DVVD\ SURFHGXUH ZDV VLPLODU WR WKDW SUHYLRXVO\ GHVFULEHG IRU WKH & GXELD SXOH[ QHRQDWHV KRXUV ROGf ZHUH H[SRVHG IRU KRXUV WR WKH OHDFKDWHV RU WKHLU GLOXWLRQV LQ WULSOLFDWH DW s r& XQGHU FRQGLWLRQV RI DPELHQW OLJKW KUV OLJKW KUV GDUNf )RU 0LFURWR[r1 DQDO\VLV FRORU FRUUHFWLRQ E\ DEVRUEDQFH DW QP ZDV UHTXLUHG GXH WR WKH UHGGLVK RU DPEHU FRORU RI WKH OHDFKDWHV /HDFKDWH VDOLQLW\ ZDV DGMXVWHG WR b ZLWK 1D&, ZKLOH QHJOHFWLQJ WKH DFWXDO OHDFKDWH VDOLQLW\ )RXU OHDFKDWH GLOXWLRQV ZHUH SUHSDUHG LQ GXSOLFDWH XVLQJ WKH 0LFURWR[r1 GLOXHQW 7KH LQKLELWLRQ RI 9LEULR ILVKHUL ELROXPLQHVFHQFH ZDV GHWHUPLQHG IROORZLQJ D PLQXWH H[SRVXUH WR HDFK OHDFKDWH VDPSOH 'RKHUW\ HW DO f 'XH WR FRQVWUDLQWV RI WKH 0LFURWR[r1 PHWKRG WKH PD[LPXP OHDFKDWH FRQFHQWUDWLRQ WHVWHG

PAGE 123

ZDV b 7KLV ZDV DWWULEXWHG WR GLOXWLRQ RI WKH OHDFKDWH DIWHU WKH EDFWHULDO VXVSHQVLRQ ZDV DGGHG )RU WKH 3 VXEFDSLWDWD DVVD\V OHDFKDWHV ZHUH ILOWHUVWHULOL]HG SP PHPEUDQH ILOWHUf )LYH GLOXWLRQV RI HDFK 06: ODQGILOO OHDFKDWH LQ WULSOLFDWH ZHUH SUHSDUHG LQ DOJDO DVVD\ PHGLXP 6WRFN FXOWXUHV RI 3 VXEFDSLWDWD ZHUH PDLQWDLQHG WR SURYLGH D FRQWLQXRXV VXSSO\ RI GD\ ROG FHOOV IRU WHVWLQJ $OJDO FHOO FRXQWV ZHUH SHUIRUPHG XVLQJ D KHPDF\WRPHWHU IROORZLQJ D KRXU H[SRVXUH DW s r& XQGHU FRQGLWLRQV RI FRQWLQXRXV OLJKWLQJ s S(PVf 'DWD $QDO\VLV 5HVXOWV IRU WKH WR[LFLW\ DVVD\V ZHUH H[SUHVVHG DV WKH b HIIHFWLYH FRQFHQWUDWLRQ (&f b LQKLELWRU\ FRQFHQWUDWLRQ ,&f RU b OHWKDO FRQFHQWUDWLRQ /&}f IRU WKH PLQXWH 0LFURWR[r1 KU 3 VXEFDSLWDWD DQG KU & GXELD RU SXOH[ DVVD\V UHVSHFWLYHO\ $OO ELRDVVD\ HQGSRLQWV (& ,& RU /&f ZHUH H[SUHVVHG DV D SHUFHQW bf RI WKH OHDFKDWH VDPSOH 7KH (& DQG ,& UHVXOWV ZHUH GHWHUPLQHG E\ JUDSKLFDO LQWHUSRODWLRQ IRU 0LFURWR[r1 DQG WKH KU 3 VXEFDSLWDWD DVVD\V UHVSHFWLYHO\ 7KH /& YDOXHV ZHUH GHWHUPLQHG XVLQJ WKH 86(3$ GDWD DQDO\VLV VRIWZDUH 86(3$ Ef (UURU EDUV LQFOXGHG ZLWK WKH ELRDVVD\ UHVXOWV UHSUHVHQW RQH VWDQGDUG GHYLDWLRQ &RUUHODWLRQV ZLWKLQ WKH GDWD ZHUH GHWHUPLQHG E\ OHDVW VTXDUH UHJUHVVLRQ D VWXGHQWnV WWHVW RU WKH )WHVW ([FHO 0LFURVRIW f DV DSSURSULDWH 7KH FRHIILFLHQW RI YDULDWLRQ &9fbf IRU HDFK DVVD\ ZDV H[SUHVVHG DV WKH SHUFHQW RI WR[LF UHVSRQVH (& ,& RU /&f UHSUHVHQWHG E\ WKH VWDQGDUG GHYLDWLRQ $OO (& ,& DQG /& HQGSRLQWV ZHUH WUDQVIRUPHG (&VRf WR WR[LFLW\ XQLWV 78f DQG ZHUH XQLWOHVV :KHQ WKH DVVD\

PAGE 124

UHVXOWV DUH SURYLGHG DV 78 YDOXHV D GLUHFW UHODWLRQ LV H[SUHVVHG EHWZHHQ LQFUHDVHG WR[LFLW\ DQG KLJKHU 78 YDOXHV ZKLFK FRQWUDVWV ZLWK WKH LQYHUVH UHODWLRQVKLS EHWZHHQ (&VRbf YDOXHV DQG LQFUHDVHG WR[LFLW\ 5HVXOWV DQG 'LVFXVVLRQ &KHPLFDO DQG 3K\VLFDO &KDUDFWHULVWLFV RI WKH 06: /HDFKDWHV /HDFKDWHV IURP IRXUWHHQ 06: ODQGILOO VLWHV LQ )ORULGD ZHUH VHOHFWHG WR GHPRQVWUDWH WKH YDULHG DQG FRPSOH[ RUJDQLF DQG LQRUJDQLF FRQWDPLQDQWV W\SLFDOO\ IRXQG LQ 06: ODQGILOO OHDFKDWHV .MHOGVHQ HW DO &KHQ %ROWRQ DQG (YDQV f 7KH FRPSRVLWLRQV RI WKH 06: ODQGILOO OHDFKDWHV DUH LQIOXHQFHG E\ VLWHVSHFLILF IDFWRUV LQFOXGLQJ WHPSHUDWXUH UDLQIDOO ZDVWH FRPSRVLWLRQ DQG ODQGILOO DJH 9DGLOOR HW DO &KLDQ DQG 'H:DOOH f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f 0RUH QHXWUDO S+ YDOXHV DUH H[SHFWHG LQ OHDFKDWHV WKDW KDYH DOUHDG\ XQGHUJRQH VRPH VWDELOL]DWLRQ ZKLOH PDWXUH ODQGILOOV GLVSOD\ S+ YDOXHV JUHDWHU WKDQ &KLDQ DQG 'H:DOOH f 7KH OHDFKDWH FROOHFWHG IURP VLWH GLVSOD\HG WKH ORZHVW S+ YDOXH DW DQG WKLV ZDV DWWULEXWHG WR WKH ORZ EXIIHULQJ FDSDFLW\ RI WKLV

PAGE 125

OHDFKDWH DV GHPRQVWUDWHG E\ WKH ORZ DONDOLQLW\ 0HDQ S+ YDOXHV RI ZHUH UHSRUWHG LQ WKH OHDFKDWHV FROOHFWHG IURP VLWHV DQG 7KH S+ RI OHDFKDWHV DIIHFWV WKH PRELOLW\ RI FRQWDPLQDQWV 5R\ DQG ']RPEDN &OHYHQJHU DQG 5DR f E\ LWV LQIOXHQFH RQ WKH VWDELOLW\ RI KXPLF FRPSOH[HV 0DVLRQ HW DOf f DQG WR[LFDQW ELRDYDLODELOLW\ 0H\HU f 6OHWWHQ HW DO f UHSRUWHG WKDW DERYH D S+ RI VROXEOH KHDY\ PHWDOV LQ 06: ODQGILOO OHDFKDWHV GHFUHDVHG WR EHORZ WKH DQDO\WLFDO GHWHFWLRQ OLPLW RI PJ/ IRU =Q 1L &U &X 3E DQG &G 2YHUDOO WKH DONDOLQLW\ PHDVXUHPHQWV ZHUH KLJK LQ DOO RI WKH 06: ODQGILOO OHDFKDWHV ZLWK RQH H[FHSWLRQ 7KH DONDOLQLW\ PHDVXUHG LQ WKH OHDFKDWH IURP VLWH ZDV PJ/ DV &D& ,Q WKH RWKHU OHDFKDWHV DONDOLQLW\ FRQFHQWUDWLRQV UDQJHG IURP WR PJ/ DV &D& 7KH OHDFKDWHV FROOHFWHG IURP VLWH GLVSOD\HG WKH KLJKHVW DONDOLQLW\ YDOXHV 7KLV ZDV DWWULEXWHG WR WKH SUDFWLFH DW WKLV ODQGILOO RI FRGLVSRVLQJ ZDVWHZDWHU WUHDWPHQW SODQW ::73f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

PAGE 126

FRQWHQW 7DEOH f 6LPLODU UHVXOWV ZHUH VKRZQ LQ WKH OHDFKDWHV IURP VLWH D ZKLFK DOVR SUDFWLFHG FRGLVSRVDO DQG GLVSOD\HG D FRQGXFWLYLW\ RI P6FP 7KH EXON SDUDPHWHU RI WRWDO GLVVROYHG VROLGV 7'6f ZKHQ FRPELQHG ZLWK PHDVXUHG FRQGXFWLYLW\ YDOXHV FDQ LQGLFDWH WKH LRQLF VWUHQJWK RI D VROXWLRQ E\ WKH /DQJHOLHU DSSUR[LPDWLRQ /DQJHOLHU f :KLOH LQ WKLV UHVHDUFK 7'6 ZDV GHWHUPLQHG E\ DQDO\WLFDO PHWKRGV WKH DSSUR[LPDWLRQ UHPDLQV YDOLG 7KHUHIRUH LW IROORZV WKDW WKH OHDFKDWHV ZLWK WKH KLJKHVW FRQGXFWLYLW\ DOVR GLVSOD\ WKH KLJKHVW YDOXHV IRU 7'6 7KLV ZDV WUXH IRU WKH VLWH OHDFKDWHV ZLWK D PHDQ 7'6 DW J/ GLVWLQJXLVKLQJ WKH KLJK LRQLF VWUHQJWK RI WKLV OHDFKDWH ,Q DOO RWKHU FDVHV WKH 7'6 YDOXHV ZHUH J/ ZLWK WKH ORZHVW YDOXH RI J/ UHSRUWHG DW VLWH

PAGE 127

7DEOH 3K\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI 06: OHDFKDWHV FROOHFWHG IURP OLQHG ODQGILOOV LQ )ORULGD 6LWH S+ &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ/ DV &D&2Df 7'6D J/f 6XOILGHV :O/f '2&E PJ/f &%2'r PJ/f &2'G PJ/f &%2' &2' f f f f f f f f f f f f 10H 10 10 10 f f f f f f f f 10 10 f f f f 10 G f f f f f 10 f 10 10 Df 10 Ef 10 %'/ Ff 10 10 10 10 10 10 10 10 10 10 10 f f f f f 10 0HDQ 0LQ 0D[ ; DUAR M 'L ELRFKHPLFDO R[\JHQ GHPDQG G&2' FKHPLFDO R[\JHQ GHPDQG p10 QRW PHDVXUHG 5HVXOWV nPHDQ DQG UDQJHf IRU PXOWLSOH VDPSOLQJ HYHQWV DQG PHDQ s RQH VWDQGDUG GHYLDWLRQ IRU RQH VDPSOLQJ HYHQW

PAGE 128

7KH SUHVHQFH RI RUJDQLF VXEVWDQFHV LQ WKH 06: OHDFKDWHV ZHUH PHDVXUHG DV FKHPLFDO R[\JHQ GHPDQG &2'f DQG FDUERQDFHRXV ELRFKHPLFDO R[\JHQ GHPDQG &%2'f SURFHGXUHV 7DEOH f %RWK RI WKHVH SDUDPHWHUV PHDVXUH WKH FRQFHQWUDWLRQ RI R[LGL]DEOH RUJDQLF PDWWHU LQFOXGLQJ ERWK WKH ELRORJLFDOO\ DVVLPLODEOH DQG WKH UHFDOFLWUDQW FRPSRXQGV :KHQ GHWHUPLQLQJ RQO\ WKH IUDFWLRQ RI RUJDQLF PDWWHU WKDW LV VXEMHFW WR ELRORJLFDO GHJUDGDWLRQ DVVLPLODEOHf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f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

PAGE 129

REWDLQHG DW WKLV VLWH WKH &%2' UDQJH IRU WKH VLWH OHDFKDWH ZDV HVWLPDWHG WR EH DW OHDVW PJ/ /LWWOH LV NQRZQ DERXW WKH GLVVROYHG RUJDQLF FDUERQ '2&f FRQFHQWUDWLRQV LQ WKH OHDFKDWHV EHFDXVH RI WKH OLPLWHG QXPEHU RI VDPSOHV DQDO\]HG IRU WKLV SDUDPHWHU 7DEOH f 7KH PHDQ '2& FRQFHQWUDWLRQV UHSRUWHG IRU WKLV UHVHDUFK ZHUH DQG PJ/ IRU VLWHV DQG UHVSHFWLYHO\ 3UHYLRXV UHVHDUFK VKRZHG WKDW WKH '2& FRQFHQWUDWLRQV LQ WKH OHDFKDWHV IURP VLWH ZHUH TXLWH KLJK ZLWK D PHDQ RI PJ/ 7KLV PD\ KDYH EHHQ FDXVHG E\ WKH FRn GLVSRVDO RI ::73 VOXGJHV ZLWK WKH 06: &KDSWHU GLVFXVVHV WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ RI 06: ODQGILOO OHDFKDWHV DQG DV SDUW RI WKDW LQYHVWLJDWLRQ WKH '2& ZDV GHWHUPLQHG LQ OHDFKDWHV IURP VLWHV DQG DW FRQFHQWUDWLRQV RI DQG UHVSHFWLYHO\ 7KHVH '2& OHYHOV DUH FRPSDUDEOH WR SUHYLRXV UHSRUWV DQG UDQJHG IURP D ORZ RI PJ/ WR D KLJK RI PJ/ 0DUWWLQHQ HW DO .DQJ HW DO f $GGLWLRQDOO\ '2& PHDVXUHPHQWV DUH D TXDVLVXUURJDWH IRU KXPLF PDWHULDOV ZKLFK LQIOXHQFHV WKH FRPSOH[DWLRQ RI KHDY\ PHWDO DQG VRPH RUJDQLF OLJDQGV &DODFH HW DO 0DUWHQVVRQ HW DO f

PAGE 130

7DEOH 'LVWULEXWLRQ RI PDMRU LRQV LQ OHDFKDWHV IURP OLQHG 06: ODQGILOOV LQ )ORULGD 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ DQG UDQJHf 6LWH 6RGLXP PT/f 3RWDVVLXP PJ/f &DOFLXP PJ/f 0DJQHVLXP PJ/f &KORULGH PJ/f 6XOIDWH :f D f f f f f f f f f f 10 10 f f f f f f 10D 10 f f 10 10 D E F 10 10 10 10 10 10 f f f f f f $EEUHYLDWLRQV D10 QRW PHDVXUHG 7KH FRQFHQWUDWLRQV RI VHOHFW PDMRU LRQV LQ WKH ODQGILOO OHDFKDWHV DUH VXPPDUL]HG 7DEOH f 7KHVH LQRUJDQLF LRQV DUH QRW VXEMHFW WR ELRORJLFDO GHJUDGDWLYH SURFHVVHV KHQFH WKHLU FRQFHQWUDWLRQV GHSHQG VROHO\ RQ WKH YROXPH RI OHDFKDWH JHQHUDWHG &KLDQ DQG 'H:DOOH f :KLOH UHYLHZLQJ WKH UHVXOWV IRU WKH PDMRU LRQ DQDO\VLV LW EHFDPH DSSDUHQW WKDW WKH KLJK FRQGXFWLYLW\ UHSRUWHG IRU WKH OHDFKDWHV IURP VLWH ZDV D JRRG LQGLFDWRU RI WKH PDMRU LRQ GLVWULEXWLRQ $V SUHYLRXVO\ GLVFXVVHG WKH ODQGILOOV DVVRFLDWHG ZLWK WKH OHDFKDWHV IURP VLWHV

PAGE 131

DQG D SUDFWLFH WKH FRGLVSRVDO RI 06: LQFLQHUDWRU DVK DQG WKLV UHVXOWHG LQ WKH SURGXFWLRQ RI OHDFKDWHV ZLWK KLJK LRQLF VWUHQJWK 2YHUDOO WKH VLWH OHDFKDWHV H[KLELWHG WKH KLJKHVW FDWLRQ FRQFHQWUDWLRQV IRU VRGLXP SRWDVVLXP DQG FDOFLXP DW DQG PJ/ UHVSHFWLYHO\ $ VLPLODU SDWWHUQ ZDV GHPRQVWUDWHG LQ WKH OHDFKDWHV IURP VLWH D ZLWK VRGLXP SRWDVVLXP DQG FDOFLXP FRQFHQWUDWLRQV DW DQG PJ/ UHVSHFWLYHO\ 7KH PDJQHVLXP FRQFHQWUDWLRQ DW VLWH D ZDV PJ/ RU QHDUO\ GRXEOH WKH PJ/ DW VLWH $FURVV DOO OHDFKDWHV WKH KLJKHVW PDJQHVLXP FRQFHQWUDWLRQ ZDV UHSRUWHG DV PJ/ DW VLWH DQG WKH ORZHVW ZDV PJ/ DW VLWH F ,Q DOO FDVHV WKH PRVW DEXQGDQW FDWLRQ LQ WKH OHDFKDWHV ZDV WKH VRGLXP LRQ ZKLFK UDQJHG IURP D ORZ RI PJ/ DW VLWH E WR D KLJK RI DW VLWH 5HIHUULQJ WR WKH DQLRQV WKH PDJQLWXGH RI WKH FRQFHQWUDWLRQV ZDV VLPLODU WR WKRVH PHDVXUHG IRU WKH FDWLRQV $Q H[FHVVLYHO\ KLJK OHYHO RI FKORULGH LRQV ZDV UHSRUWHG LQ WKH VLWH OHDFKDWH DW D FRQFHQWUDWLRQ PJ/ 7KLV KLJK FRQFHQWUDWLRQ ZDV D UHFXUULQJ SUREOHP LQ WKH OHDFKDWH DQG ZDV OLPLWLQJ DYDLODEOH OHDFKDWH WUHDWPHQW RSWLRQV *DU\ 'HER ODQGILOO RSHUDWRU SHUVRQDO FRPPXQLFDWLRQf ,Q DOO RI WKH RWKHU OHDFKDWHV WKH FKORULGH OHYHOV ZHUH PJ/ ZLWK WKH ORZHVW FKORULGH FRQFHQWUDWLRQV LGHQWLILHG LQ WKH OHDFKDWHV IURP VLWH DW PJ/ &KORULGH FRPSOH[HV ZLWK PHWDOV LQ VROXWLRQ KDYH EHHQ VKRZQ WR PRGLI\ PHWDO VSHFLDWLRQ DQG ELRDYDLODELOLW\ %ROWRQ DQG (YDQV f 8QGHU W\SLFDO ODQGILOO FRQGLWLRQV WKH VXOIDWH PROHFXOH LV UHGXFHG WR VXOILGH DQG WKLV UHGXFHG LRQ IRUPV FRPSOH[HV ZLWK KHDY\ PHWDOV %R]NXUW HW DO f %\ FRPSDULVRQ WKH FRQFHQWUDWLRQV RI VXOIDWH LRQV LQ WKH 06: OHDFKDWHV ZHUH

PAGE 132

PXFK ORZHU WKDQ WKH UHSRUWHG FKORULGH FRQFHQWUDWLRQV ,Q WKH 06: ODQGILOO OHDFKDWHV WKH PHDQ OHYHOV RI VXOIDWH LRQV UDQJHG IURP WR PJ/ 7KH KLJKHVW VXOIDWH FRQFHQWUDWLRQV ZHUH UHSRUWHG LQ WKH OHDFKDWH IURP VLWH D ZKLOH WKH ORZHVW ZHUH LQ WKH VLWH OHDFKDWHV 'HFUHDVLQJ VXOIDWH LRQ FRQFHQWUDWLRQV FDQ EH XVHG DV DQ LQGLFDWRU RI WKH GHJUHH RI UHGXFLQJ FRQGLWLRQV LQ DQ DQDHURELF HQYLURQPHQW ,Q WKH FDVH RI WKH VLWH OHDFKDWHV WKH ORZ PHDQ VXOILGH LRQ FRQFHQWUDWLRQV SJ/f FRUUHVSRQGHG ZLWK PHDQ VXOIDWH OHYHO RI PJ/ ,Q DOO RWKHU OHDFKDWHV WKH PHDQ VXOILGH FRQFHQWUDWLRQV ZHUH JHQHUDOO\ ORZ SJ/f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f 7KH KLJK GHJUHH RI KDUGQHVV LQ WKH 06: OHDFKDWHV FRQWULEXWHV WR WKHLU DELOLW\ WR UHGXFH KHDY\ PHWDO ELRDYDLODELOLW\ VHH &KDSWHU *HRUJH HW DO f

PAGE 133

7DEOH 0HDQ FRQFHQWUDWLRQV PJ/f RI WRWDO 1+91+f DQG XQLRQL]HG 1+f DPPRQLD LQ OHDFKDWHV IURP IRXUWHHQ 06: ODQGILOOV LQ )ORULGD 6LWH 7RWDO $PPRQLD 1,91+f PJ/f $PPRQLD 1+f PJ/f S+ 10D D E F 10 10 10 VUUU r $EEUHYLDWLRQ D10 QRW PHDVXUHG ,Q ODQGILOOV WKH QLWURJHQ F\FOH LV LQKLELWHG E\ DQDHURELF FRQGLWLRQV DV QLWULILFDWLRQ UHTXLUHV WKH SUHVHQFH RI R[\JHQ IRU WKH FRQYHUVLRQ RI DPPRQLD WR QLWUDWH %XUWRQ DQG :DWVRQ&UDLN f 7KHUHIRUH LQ WKH DEVHQFH RI R[\JHQ PLFURELDO GHJUDGDWLRQ RI RUJDQLF PDWWHU LQ ODQGILOOV UHVXOWV LQ WKH DFFXPXODWLRQ RI DPPRQLD &HFHQ DQG *XUVR\ f 7DEOH VXPPDUL]HV WKH PHDQ WRWDO DPPRQLD FRQFHQWUDWLRQV LQ WKH 06: ODQGILOO OHDFKDWHV ZKLFK UDQJHG ZLGHO\ IURP

PAGE 134

7DEOH 0HWDO FRQFHQWUDWLRQV LQ OHDFKDWHV IURP IRXUWHHQ 06: ODQGILOOV LQ )ORULGD 6LWH $O PJ/f &Xr PJ/f &Gr PJ/f )H PJ/f 3Er PJ/f =QH PJ/f f %'/ %'/ f %'/ f 10D %'/ %'/ %'/ %'/ %'/ %'/ %'/ f f 10 10 10 10 10 10 %'/ %'/ %'/ %'/ %'/ %'/ %'/ f f %'/ %'/ %'/ %'/ %'/ %'/ %'/ Df %'/ %'/ %'/ Ef %'/ %'/ %'/ %'/ Ff %'/ %'/ %'/ %'/ %'/ %'/ %'/ %'/ f f %'/ %'/ %'/ %'/ 7 %'/ %'/ %'/ %'/ D10 QRW PHDVXUHG 0HWDO FRQFHQWUDWLRQV EHORZ WKH GHWHFWLRQ OLPLW %'/f RI HDFK DQDO\WLFV PHWKRG ZHUH GHWHUPLQHG EDVHG RQ WKH PLQLPXP GHWHFWLRQ OLPLWV IRU HDFK KHDY\ PHWDO f&X PJ/ F&G PJ/ G3E PJ/ H=Q PJ/ PJ/ DW VLWH WR QHDUO\ PJ/ DW VLWH $PPRQLD OHYHOV LQ ODQGILOOV KDYH EHHQ VKRZQ WR LQFUHDVH ZLWK DJH ZLWK WKUHH WLPHV KLJKHU DPPRQLD FRQFHQWUDWLRQV UHSRUWHG LQ PDWXUH ODQGILOOV 0DUWWLQHQ HWDO f $PPRQLD VSHFLDWLRQ LV S+ GHSHQGHQW ZLWK HTXLYDOHQW FRQFHQWUDWLRQV RI WKH LRQL]HG 1+f RU XQLRQL]HG 1+f IRUPV DW WKH S.D RI %DVHG RQ WKH PHDVXUHG S+ YDOXHV WKH FRQFHQWUDWLRQV RI XQLRQL]HG DPPRQLD LQ WKH 06: OHDFKDWHV ZHUH H[SHFWHG

PAGE 135

WR EH ORZ DQG WKLV ZDV JHQHUDOO\ WUXH &RPSDUHG WR WKH LRQL]HG DPPRQLD FRQFHQWUDWLRQV WKH XQLRQL]HG DPPRQLD 1+f UDQJHG IURP PJ/ DW VLWH WR PJ/ DW VLWH 7DEOH f 7KH KLJK WRWDO DPPRQLD FRQFHQWUDWLRQV FRUUHVSRQGHG ZLWK HOHYDWHG XQLRQL]HG DPPRQLD OHYHOV LQ VRPH OHDFKDWHV 7KH FRQFHQWUDWLRQV RI KHDY\ PHWDOV LQ WKH IRXUWHHQ 06: ODQGILOO OHDFKDWHV ZHUH VXPPDUL]HG LQ 7DEOH +LJK DQG YDULDEOH LURQ FRQFHQWUDWLRQV ZHUH UHSRUWHG LQ WKH 06: ODQGILOO OHDFKDWHV ZLWK D UDQJH IURP WR PJ/ 7KH OHDFKDWHV IURP VLWH GHPRQVWUDWHG WKH ORZHVW LURQ FRQFHQWUDWLRQV ZKLOH WKH OHDFKDWHV IURP VLWH KDG WKH KLJKHVW FRQFHQWUDWLRQV ,URQ LV URXWLQHO\ IRXQG DW KLJK FRQFHQWUDWLRQV LQ 06: ODQGILOO OHDFKDWHV DQG WKH UDQJH UHSRUWHG IRU )ORULGD OHDFKDWHV LV FRPSDUDEOH WR WKH UDQJH UHSRUWHG LQ 'DQLVK ODQGILOOV IURP WR PJ/ %DXQ HW DO f 7KH DEXQGDQFH RI LURQ LQ WKH OHDFKDWHV IURP VLWH PD\ EH DWWULEXWHG WR WKH SRRU UHFRYHU\ RI IHUURXV FRPSRXQGV E\ WKH PDJQHW DW WKH LQFLQHUDWRU IDFLOLW\ *DU\ 'HER SHUVRQDO FRPPXQLFDWLRQf $OXPLQXP ZDV D ORZOHYHO EXW XELTXLWRXV FRQWDPLQDQW RI DOO WKH 06: OHDFKDWHV ZLWK FRQFHQWUDWLRQV WKDW UDQJHG IURP WR PJ/ 7KH SUHVHQFH VSHFLDWLRQ DQG ELRDYDLODELOLW\ RI DOXPLQXP LQ WKH HQYLURQPHQW KDV EHHQ UHYLHZHG *HQVHPHU DQG 3OD\OH f 7KH DQDO\WLFDO GHWHFWLRQ OLPLWV IRU WKH KHDY\ PHWDOV ZHUH DQG IRU FRSSHU FDGPLXP OHDG DQG ]LQF UHVSHFWLYHO\ *HQHUDOO\ WKH FRQFHQWUDWLRQV RI WR[LF KHDY\ PHWDOV LQ WKH OHDFKDWHV ZHUH EHORZ WKH DQDO\WLFDO GHWHFWLRQ OLPLWV 1RWDEOH H[FHSWLRQV ZHUH VKRZQ LQ WKH OHDFKDWHV IURP VLWHV DQG D DV WKH\ FRQWDLQHG DQG PJ/ RI FRSSHU UHVSHFWLYHO\ 7KH RQO\

PAGE 136

/DQGILOO )LJXUH $FXWH KU & GXELDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP ODQGILOO VLWHV LQ )ORULGD ZLWK UHVXOWV H[SUHVVHG DV 78 ,22(&f DQG RQH VWDQGDUG GHYLDWLRQ OHDFKDWH WKDW FRQWDLQHG OHDG DERYH WKH GHWHFWLRQ OLPLW ZDV FROOHFWHG IURP VLWH ZLWK D OHYHO RI PJ/ =LQF ZDV WKH PRVW SUHYDOHQW KHDY\ PHWDO ZLWK DQG PJ/ LGHQWLILHG LQ OHDFKDWHV IURP VLWHV DQG UHVSHFWLYHO\ 'HVSLWH UHSRUWV RI KLJKHU FRQFHQWUDWLRQV RI FDWLRQLF FRQWDPLQDQWV LQFOXGLQJ VRPH KHDY\ PHWDOV UHVHDUFKHUV GR QRW DJUHH DERXW WKH ULVNV WR WKH HQYLURQPHQW IURP WKLV IDFLOLWLHV :LOHV f 6RPH UHVHDUFKHUV KDYH VXJJHVWHG WKDW ODQGILOOV SUDFWLFLQJ WKH FRGLVSRVDO RI 06: ZLWK LQFLQHUDWRU DVK DUH QR PRUH OLNHO\ WR OHDFK KHDY\ PHWDOV WKDQ DUH 06: RQO\ ODQGILOOV &KLFKHVWHU DQG /DQGVEHUJHU %R]NXUW HW DO f

PAGE 137

7R[LFLW\ RI 06: /DQGILOO /HDFKDWHV 7R[LFLW\ RI 06: OHDFKDWHV WR DTXDWLF LQYHUWHEUDWHV 7KH DFXWH WR[LFLW\ RI WKH 06: OHDFKDWHV FROOHFWHG DW IRXUWHHQ VHSDUDWH ODQGILOO VLWHV ZDV HYDOXDWHG ZLWK WKH KRXU & GXELD DVVD\ )LJXUH f &RQWURO VXUYLYDO IRU DOO UHVXOWV VKRZQ ZDV JUHDWHU WKDQ b 86(3$ Df 7KH HQGSRLQW XWLOL]HG IRU WKH DTXDWLF ,QYHUWHEUDWH DVVD\V ZDV GHDWK RU ,PPRELOL]DWLRQ DQG WKLV ZDV TXDQWLILHG DV WKH FRQFHQWUDWLRQ RI OHDFKDWH OHWKDO WR b RI WKH WHVW RUJDQLVPV /&VRf $OO /& UHVXOWV ZHUH WUDQVIRUPHG ,22/&f DQG H[SUHVVHG DV WR[LFLW\ XQLWV 78f 7KH UHVXOWV VKRZ WKDW WKH & GXELD DVVD\ ZDV VHQVLWLYH WR 06: ODQGILOO OHDFKDWH WR[LFLW\ $OWKRXJK WKH UDQJH RI WR[LFLW\ LQ WKH 06: ODQGILOO OHDFKDWHV ZDV EURDG LW ZDV FRQVLVWHQW ZLWK WKH KHWHURJHQHRXV FKHPLFDO FRPSRVLWLRQ RI WKH OHDFKDWHV 7KH & GXELD UHVSRQVH WR WKH OHDFKDWHV IURP WKH ODQGILOO VLWHV ZDV YDULDEOH ZLWK PHDQ 78 YDOXHV UDQJLQJ IURP VLWH f WR VLWH f (DUOLHU ZRUN DOVR GHPRQVWUDWHG WKH WR[LF QDWXUH RI WKH OHDFKDWHV IURP VLWH :DUG HW DO f 7KH DFXWH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWHV DQG DUH UHSUHVHQWHG E\ 78 YDOXHV RI DQG UHVSHFWLYHO\ 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG ZHUH SUHYLRXVO\ DVVD\HG IRU WKHLU DFXWH WR[LFLW\ :DUG HW DO f :KHQ FRPSDUHG WR WKH & GXELD DVVD\ UHVSRQVHV LQ WKH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWH UHPDLQHG FRQVLVWHQW KRZHYHU WKH OHDFKDWHV IURP VLWH LQFUHDVHG LQ WR[LFLW\ :DUG HW DO f ,Q VRPH OHDFKDWH VDPSOHV IURP VLWH ZHUH FROOHFWHG IURP D OHDFKDWH VWRUDJH WDQN DQG GHJUDGDWLRQ RI WR[LF OHDFKDWH FRPSRQHQWV PD\ KDYH RFFXUUHG :DUG f

PAGE 138

)LJXUH &RUUHODWLRQ EHWZHHQ WKH KRXU DFXWH WR[LFLW\ DVVD\V XVLQJ & GXELD DQG SXOH[ DVVD\V ZLWK 06: OHDFKDWHV FROOHFWHG IURP ODQGILOO VLWHV LQ )ORULGD 7KH 78 UHVSRQVHV RI WKH & GXELD WR OHDFKDWHV IURP VLWHV D E DQG F ZHUH DQG UHVSHFWLYHO\ $V SUHYLRXVO\ GHVFULEHG VLWH D ZDV DQ DFWLYH &ODVV ODQGILOO VLWH E D &ODVV ,,, ODQGILOO DQG VLWH F ZDV D FDSSHG &ODVV ODQGILOO 7KHUH ZHUH QR VWDWLVWLFDO GLIIHUHQFHV IWHVWf LQ WKH WR[LFLW\ RI WKH OHDFKDWHV IURP D DQG F HYHQ WKRXJK WKH FKHPLFDO FRPSRVLWLRQ RI WKH OHDFKDWH IURP F ZDV FRQVLVWHQWO\ ORZHU IRU QHDUO\ DOO SDUDPHWHUV H[FHSW DPPRQLD 7KH DPPRQLD FRQFHQWUDWLRQ LQ WKH OHDFKDWH IURP VLWH F ZDV PJ/ ZKLFK ZDV GRXEOH WKH PJ/ PHDVXUHG LQ WKH OHDFKDWH IURP VLWH F DQG FRXOG H[SODLQ WKH KLJKHU WR[LFLW\ LQ WKLV OHDFKDWH 7KH WR[LFLW\ RI WKH OHDFKDWHV SURGXFHG DW VLWH 78 s f WKH RQO\ &ODVV ,, ODQGILOO VWXGLHG ZHUH QRW

PAGE 139

VWDWLVWLFDOO\ GLIIHUHQW IWHVWf IURP WKH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWH D 78 sf ,W VKRXOG EH QRWHG WKDW WKH VLWH DQG VLWH D ODQGILOOV DUH ERWK SHUPLWWHG IRU WKH GLVSRVDO RI GRPHVWLF PXQLFLSDO ZDVWH KRZHYHU WKH WZR VLWHV DUH ORFDWHG LQ YDVWO\ GLIIHUHQW DUHDV RI WKH VWDWH DQG UHFHLYH GLIIHUHQW YROXPHV RI ZDVWH PDWHULDO 6LWH LV ORFDWHG LQ D VSDUVHO\ SRSXODWHG UXUDO DUHD DQG XWLOL]HV WKH RQO\ RSHUDWLQJ &ODVV ,, ODQGILOO LQ )ORULGD 2Q WKH RWKHU KDQG WKH VLWH D ODQGILOO LV ORFDWHG LQ D KHDYLO\ SRSXODWHG DUHD DQG UHODWLYH WR RWKHU DUHDV ZLWK ORZHU SRSXODWLRQ GHQVLWLHV WKHUH LV DQ LQFUHDVHG SRWHQWLDO IRU GRPHVWLF KD]DUGRXV ZDVWH GLVSRVDO :KLOH VRPH UHVHDUFKHUV KDYH UHSRUWHG D VWURQJ LQIOXHQFH RI KLJK VDOW FRQFHQWUDWLRQV RQ WR[LFLW\ WKH UHVXOWV RI WKLV UHVHDUFK GR QRW VXSSRUW WKLV FRQFOXVLRQ :XQGUDP DQG %DKDGLU f 7KH LRQLF VWUHQJWK RI WKH OHDFKDWHV IURP VLWHV DQG D ZHUH KLJK EXW WKHUH ZHUH QR FRUUHVSRQGLQJ HOHYDWHG WR[LF UHVSRQVHV ,Q FRQWUDVW RWKHUV KDYH GHPRQVWUDWHG WKH VWURQJ LQIOXHQFH RI LQRUJDQLF PDFURLRQV RQ OHDFKDWH WR[LFLW\ %DXQ HW DO f +RNH HW DO f UHSRUWHG DQ /& IRU VRGLXP UDQJLQJ IURP WR PJ/ IRU & GXELD &RQVLGHULQJ WKH SUHYDOHQFH RI VRGLXP LQ WKH 06: OHDFKDWHV WKLV PD\ UHSUHVHQW D VLJQLILFDQW VRXUFH RI WR[LFLW\ $GGLWLRQDOO\ VRPH RI WKH 06: ODQGILOO OHDFKDWHV ZHUH DVVD\HG IRU DFXWH WR[LFLW\ ZLWK WKH KRXU 'DSKQLD SXOH[ DVVD\ 5HVHDUFKHUV KDYH VKRZQ WKDW WKH VHQVLWLYLW\ RI &GXELD LV FRPSDUDEOH WR RU VOLJKWO\ JUHDWHU WKDQ WKDW RI SXOH[ 6XHGHO HW DO f 7R GDWH WKH VHQVLWLYLW\ RI WKH &GXELD DQG SXOH[ DVVD\V

PAGE 140

7DEOH 7R[LFLW\ RI OHDFKDWHV FROOHFWHG IURP OLQHG 06: ODQGILOOV ZLWK & GXELD SXOH[ DQG 3 VXEFDSLWDWD ZLWK UHVXOWV H[SUHVVHG DV WKH PHDQ WR[LFLW\ XQLW 78fXQLWOHVVf DQG RQH VWDQGDUG GHYLDWLRQ 6'f & GXELD KRXUf SXOH[ KRXUf 3 VXEFDSLWDWD KRXUf s f s 0HDQ WR[LFLW\ 78f s 6' UDQJHf s f 10D s f s s s s f f f s 10 s s f 10 s s s f f f s s E s & s s E s & Df s s sr Ef s sE s & Ff s s sr s s s f f f s s s & D s s f f $EEUHYLDWLRQ L10 QRW PHDVXUHG f SXOH[ UHVXOWV VWDWLVWLFDOO\ GLIIHUHQW IURP & GXELD UHVXOWV DW S F 3 VXEFDSLWDWD UHVXOWV VWDWLVWLFDOO\ GLIIHUHQW IURP & GXELD UHVXOWV DW S KDYH QRW EHHQ FRPSDUHG XVLQJ 06: ODQGILOO OHDFKDWHV 7KH UHVXOWV VKRZQ KHUH VXSSRUW WKH FRPSDUDEOH VHQVLWLYLW\ RI ERWK GDSKQLG VSHFLHV WR 06: ODQGILOO OHDFKDWH WR[LFLW\ )LJXUH f 7KH 78 YDOXHV ZLWK WKH SXOH[ DVVD\ UDQJHG IURP WR ZKLOH WKH UHVSRQVH RI WKH & GXELD DVVD\ UDQJHG IURP WR 7DEOH f 7KHUH ZDV QR VWDWLVWLFDOO\ VLJQLILFDQW GLIIHUHQFH IWHVWf LQ WKH UHVSRQVH RI WKH & GXELD DQG SXOH[ DVVD\V 7KH VLPSOH OLQHDU UHJUHVVLRQ

PAGE 141

( + ‘ D E F /DQGILOO 6LWH )LJXUH &KURQLF KRXU 3 VXEFDSLWDWDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP ODQGILOO VLWHV LQ )ORULGD ZLWK UHVXOWV H[SUHVVHG DV 78 ,22(&f DQG RQH VWDQGDUG GHYLDWLRQ DQDO\VLV RI WKH LQYHUWHEUDWH GDWD VKRZ D VLJQLILFDQW 5 S f FRUUHODWLRQ EHWZHHQ WKH SXOH[ DQG & GXELD UHVXOWV 7R[LFLW\ RI 06: OHDFKDWHV ZLWK DOJDH 7KH FKURQLF WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV ZDV DVVD\HG E\ WKH KRXU 3 VXEFDSLWDWD DVVD\ 7KH UHVSRQVH RI WKH DOJDH WR WKH OHDFKDWHV ZDV VLPLODU WR WKH UHVSRQVH RI WKH DTXDWLF LQYHUWHEUDWHV ZLWK DOJDO PHDQ 78 YDOXHV WKDW UDQJHG IURP WR )LJXUH f 7KLV DJUHHV ZLWK HDUOLHU UHSRUWV RI WKH VHQVLWLYLW\ RI DOJDO DVVD\V 0DUWWLQHQ HW DO f 6SHFLHV IURP ORZHU WURSKLF OHYHOV OLNH DOJDH DQG EDFWHULD DUH PRUH OLNHO\ WR EH DGYHUVHO\ DIIHFWHG E\ 06: ODQGILOO OHDFKDWHV 3ORWNLQ DQG 5DP f $FFRUGLQJ WR WKH DOJDO DVVD\ UHVXOWV VLWH OHDFKDWH ZDV WKH OHDVW WR[LF ZLWK D 78 RI 7KLV ZDV VLPLODU WR WKH ORZ WR[LFLW\

PAGE 142

2 L 7 n 2 3 VXEFDSLWDWD ,&VRbf POf )LJXUH &RUUHODWLRQ EHWZHHQ WKH UHVXOWV RI WKH VWDQGDUG POf DQG PRGLILHG POf 3 VXEFDSLWDWD FKURQLF KRXU DVVD\V UHSRUWHG IRU WKH VLWH OHDFKDWH LQ WKH LQYHUWHEUDWH DVVD\V $W WKH RWKHU H[WUHPH WKH OHDFKDWHV IURP VLWH SURGXFHG D PHDQ 78 RI $ VLPLODU WR[LFLW\ ZDV LGHQWLILHG LQ WKH OHDFKDWHV IURP VLWH RYHU D VL[PRQWK SHULRG LQ :DUG HW DO f 7KH OHDFKDWHV IURP VLWHV D DQ RSHUDWLQJ &ODVV ODQGILOOf DQG E DQ RSHUDWLQJ &ODVV ,,, ODQGILOOf GHPRQVWUDWHG FRPSDUDEOH WR[LFLW\ ZLWK 78 YDOXHV RI DQG UHVSHFWLYHO\ ,Q FRQWUDVW WR WKH & GXELD DVVD\ UHVXOWV WKH OHDFKDWHV IURP VLWH F ZHUH VLJQLILFDQWO\ PRUH WR[LF 78 f WKDQ HLWKHU WKH D RU E OHDFKDWHV 7KLV ZDV VXUSULVLQJ FRQVLGHULQJ WKDW WKH FRPSRVLWLRQ RI WKH ZDVWH DW VLWHV D DQG F ZHUH VLPLODU %RWK ZHUH GHVLJQHG IRU 06: ZKLFK FRQWUDVWHG ZLWK WKH SULPDULO\ QRQSXWUHVFLEOH ZDVWH FRPSRVLWLRQ DW VLWH E 7KH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWHV DQG ZHUH HTXLYDOHQW DQG

PAGE 143

GLVSOD\HG 78 YDOXHV RI DQG UHVSHFWLYHO\ 7KH VLWH OHDFKDWHV UHSUHVHQWHG WKH RQO\ &ODVV ,, ODQGILOO VWXGLHG DQG FRPSDUDWLYHO\ WKH YROXPH RI ZDVWH GLVSRVHG DW VLWH ZDV QHDUO\ WLPHV JUHDWHU 7DEOH f 7KHVH UHVXOWV LQGLFDWH WKDW WKH DPRXQW RI ZDVWH ODQGILOOHG GRHV QRW LQIOXHQFH WKH WR[LFLW\ RI WKH ZDVWH OHDFKDWHV 7KH FKURQLF WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV ZDV DOVR HYDOXDWHG LQ D PRGLILHG KRXU 3 VXEFDSLWDWD DVVD\ 7KLV DVVD\ DWWHPSWHG WR PLQLDWXUL]H WKH SURWRFRO DQG PLQLPL]H VSDFH UHTXLUHPHQWV 6HOHFWHG OHDFKDWH VDPSOHV ZHUH WHVWHG ZLWK ERWK WKH VWDQGDUG PO PO SRUWLRQ RI OHDFKDWHf SURWRFRO DQG WKH PRGLILHG PO PO SRUWLRQ RI OHDFKDWHf SURWRFRO 7KH 78 YDOXHV IRU WKH OHDFKDWHV LQ WKH VWDQGDUG 3 VXEFDSLWDWD SURWRFRO UDQJHG IURP WR ZKLOH LQ WKH PRGLILHG SURWRFRO WKH\ UDQJHG IURP WR *HQHUDOO\ WKH PDJQLWXGH RI WKH WR[LF UHVSRQVH ZDV FRQVLVWHQW LQ ERWK WKH VWDQGDUG DQG WKH PRGLILHG SURWRFROV $ OHDVW VTXDUHV UHJUHVVLRQ ZDV SHUIRUPHG ZLWK WKH UHVXOWV DQG VKRZHG D VLJQLILFDQW FRUUHODWLRQ U S f EHWZHHQ WKH VWDQGDUG DQG PRGLILHG 3 VXEFDSLWDWD DVVD\V )LJXUH f ([DPLQDWLRQ RI WKH GDWD UHYHDOHG WKDW ZKLOH PRVW RI WKH OHDFKDWHV LQGXFHG FRPSDUDEOH WR[LFLW\ UHVSRQVHV LQ ERWK SURWRFROV VRPH GLG QRW 2YHUDOO WKH UHVXOWV ZLWK WKH PRGLILHG SURWRFRO VXJJHVWHG D KLJKHU VHQVLWLYLW\ WR OHDFKDWH WR[LFLW\ DQG WKLV ZDV HVSHFLDOO\ WUXH IRU WKH OHDFKDWHV IURP VLWHV DQG ,Q IDFW ZKHQ WKH UHJUHVVLRQ DQDO\VLV ZDV SHUIRUPHG ZLWKRXW WKH WR[LFLW\ GDWD IURP VLWHV DQG WKH FRUUHODWLRQ EHWZHHQ WKH VWDQGDUG DQG PRGLILHG SURWRFROV ZDV VWURQJHU 5 S f 7KH ORZ QXPEHU RI OHDFKDWHV HYDOXDWHG SUHFOXGHG DQ\ VWURQJ FRQFOXVLRQV

PAGE 144

7DEOH 7R[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV LQ )ORULGD XVLQJ WKH PLQXWH 0LFURWR[ DFXWH DVVD\ ZLWK UHVXOWV H[SUHVVHG DV WR[LFLW\ XQLWV 78f DQG PHDQ YDOXHV RI GXSOLFDWH PHDVXUHV /DQGILOO 6LWH 78 PHDQf D E F FRQFHUQLQJ WKH XVH RI WKH PRGLILHG SURWRFRO DOWKRXJK WKH KLJKHU VHQVLWLYLW\ UHTXLUHV IXUWKHU LQYHVWLJDWLRQ ,W PD\ EH WKDW WKH FRORU WRQH RI WKH OHDFKDWHV UHGXFHG OLJKW SHQHWUDWLRQ DQG E\ GHFUHDVLQJ WKH YROXPH RI OHDFKDWH WKH OLJKW SHQHWUDWLRQ LQFUHDVHG 7KH FRORU RI WKH 06: ODQGILOO OHDFKDWHV UDQJHG IURP D OLJKW JROGHQ WR D GHHS UHGGLVK EURZQ KRZHYHU WKH LQIOXHQFH RQ DOJDO JURZWK UDWHV IURP GHFUHDVHG OLJKW LQWHQVLW\ LQ JURZWK FKDPEHUV LV SRRUO\ XQGHUVWRRG &OHXYHUV DQG 5DWWH f IRXQG QR UHODWLRQVKLS EHWZHHQ FRORUHG VDPSOHV DQG DOJDO JURZWK UDWHV EXW WKH\ DQG RWKHUV FRQFOXGHG GHFUHDVLQJ VDPSOH YROXPH LQFUHDVHG DOJDH VHQVLWLYLW\ ZKHQ HYDOXDWLQJ FRORUHG VDPSOHV *HLV HW DO f ,Q WKH DOJDO DVVD\V REVHUYDWLRQV LQGLFDWHG WKDW EHORZ D FHUWDLQ WKUHVKROG abf OHDFKDWHV IURP VRPH VLWHV SURGXFHG DQ RYHUJURZWK RI DOJDO FHOOV

PAGE 145

2EVHUYDWLRQV PDGH WKURXJKRXW WKH UHVHDUFK LQYHVWLJDWLRQ VXJJHVWHG WKDW DW ORZ FRQFHQWUDWLRQV WKH OHDFKDWHV ZHUH VWLPXODWRU\ WR DOJDO FHOO JURZWK ZKLFK PD\ EH DWWULEXWHG WR WKH DGGLWLRQDO QXWULHQWV LQ WKH OHDFKDWHV &KHXQJ HW DO f 7KLV VXJJHVWHG D VWLPXODWRU\ HIIHFW GXH WR VXEVWDQFHV LQ WKH OHDFKDWHV ZKLFK ZDV RIWHQ ZHOO DERYH WKH FHOO JURZWK REVHUYHG LQ WKH QHJDWLYH FRQWUROV PLQXV OHDFKDWHf 6RPH UHVHDUFKHUV KDYH VXJJHVWHG WKDW PRGHUDWH WUHDWPHQW RI 06: OHDFKDWHV FRXOG SURGXFH DQ HIIOXHQW VXLWDEOH IRU WKH LUULJDWLRQ RI DJULFXOWXUDO FURSV DQG WUHH IDUPV 5HYHO HW DO f 7R[LFLW\ RI 06: ODQGILOO OHDFKDWHV ZLWK 0LFURWR[ 7KH DFXWH WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV ZDV HYDOXDWHG ZLWK WKH 0LFURWR[ DVVD\ LQ D PLQXWH H[SRVXUH SHULRG 2YHUDOO WKH 0LFURWR[ DVVD\ ZDV LQVHQVLWLYH WR WKH OHDFKDWH WR[LFLW\ ZLWK PHDQ 78 YDOXHV WKDW UDQJHG IURP WR 7DEOH f 7KH PDMRULW\ RI WKH OHDFKDWHV GLVSOD\HG 78 YDOXHV OHVV WKDQ ZLWK 78 UHVSRQVHV RI FRPPRQ $V D SRLQW RI UHIHUHQFH D 78 YDOXH RI LV HTXLYDOHQW WR DQ (& RI a b RU YHU\ ORZ WR[LFLW\ 7KH 0LFURWR[ DVVD\ LGHQWLILHG ORZ WR[LFLW\ LQ WKH OHDFKDWHV IURP VLWHV DQG ZLWK 78 YDOXHV RI DQG UHVSHFWLYHO\ 'XH WR WKH ORZ VHQVLWLYLW\ RI WKH 0LFURWR[ DVVD\ WKHUH ZHUH VRPH LQVWDQFHV ZKHQ DQ (& FRXOG QRW EH GHWHUPLQHG HYHQ DW WKH KLJKHVW SRVVLEOH FRQFHQWUDWLRQ RI OHDFKDWH bf :KHQ D GHILQLWLYH (& ZDV QRW GHWHUPLQHG WKHQ WKH (& ZDV UHSRUWHG DV JUHDWHU WKDQ WKH KLJKHVW FRQFHQWUDWLRQ DVVD\HG )RU FRPSDUDWLYH SXUSRVHV ZKHQ WKH (& YDOXHV ZHUH FRQYHUWHG WR WR[LFLW\ XQLWV 78f WKH JUHDWHU WKDQ VLJQ ZDV RPLWWHG

PAGE 146

+HDY\ PHWDO WR[LFLW\ RI 06: OHDFKDWHV XVLQJ 0HW3/$7( 06: ODQGILOO OHDFKDWHV ZHUH HYDOXDWHG IRU KHDY\ PHWDO WR[LFLW\ ZLWK WKH 0HW3/$7( DVVD\ ZKLFK LV VSHFLILFDOO\ VHQVLWLYH WR WKH SUHVHQFH RI KHDY\ PHWDO WR[LFDQWV %LWWRQ HW DO f 7KH GHWDLOHG 0HW3/$7( UHVXOWV ZLWK WKH 06: ODQGILOO OHDFKDWHV DUH SUHVHQWHG LQ &KDSWHU 7KH UHVXOWV GHPRQVWUDWHG WKDW LQ WKH 0HW3/$7( DVVD\ WKH 06: OHDFKDWHV ZHUH VOLJKWO\ LQKLELWRU\ EXW WKH PHDQ LQKLELWLRQ ZDV JHQHUDOO\ TXLWH ORZ 7R EULHIO\ VXPPDUL]H URXJKO\ b RI WKH OHDFKDWHV DVVD\HG SURGXFHG LQKLELWRU\ UHVSRQVHV OHVV WKDQ b 12(&/2(& YV (& RU 78 UHVXOWV 7R[LFLW\ DVVD\V DUH FRQGXFWHG WR HYDOXDWH WKH FRQFHQWUDWLRQ RI D VXEVWDQFH UHVSRQVLEOH IRU DQ DGYHUVH ELRORJLFDO UHVSRQVH HJ GHDWK RU UHSURGXFWLYH IDLOXUHf LQ D VSHFLILHG SHULRG RI WLPH 7UDGLWLRQDOO\ WKHVH DVVD\V KDYH EHHQ FRQGXFWHG DW RU KRXUV DOWKRXJK VRPH UDSLG ELRDVVD\V SURGXFH UHVXOWV LQ D PDWWHU RI KRXUV 1HOVRQ DQG 5ROLQH -XQJ DQG %LWWRQ f %\ JUDSKLFDO RU VWDWLVWLFDO SURFHGXUHV WKH UHVSRQVH RI D WR[LFLW\ WHVW RUJDQLVP FDQ EH LQWHUSRODWHG WR \LHOG D SRLQW HVWLPDWH RI WKH FRQFHQWUDWLRQ SURGXFLQJ WKH WR[LF HIIHFW 7KHVH SRLQW HVWLPDWHV DUH GHWHUPLQHG DV WKH FRQFHQWUDWLRQ SURGXFLQJ WKH GHVLUHG HIIHFW LQ WKH WHVW SRSXODWLRQ *HQHUDOO\ UHVSRQVHV DUH UHSRUWHG DV WKH FRQFHQWUDWLRQ WKDW SURGXFHV D b HIIHFW (&f GHDWK OHWKDOLW\f /&f RU LQKLELWLRQ ,&f LQ D WHVW SRSXODWLRQ 5DQG f 'XULQJ K\SRWKHVLV WHVWLQJ WKH QR REVHUYHG HIIHFW FRQFHQWUDWLRQ 12(&f DQG WKH ORZHVW REVHUYHG HIIHFW FRQFHQWUDWLRQ /2(&f DUH GHWHUPLQHG VWDWLVWLFDOO\ 8QOLNH WKH SRLQW HVWLPDWH WKH 12(&/2(& UHVXOWV GHSHQG RQ WKH OHDFKDWH GLOXWLRQ VHULHV DQG WKHUHIRUH DUH QRW LQGHSHQGHQW RI WKH WHVW FRQFHQWUDWLRQV

PAGE 147

7DEOH 5HODWLRQVKLS EHWZHHQ WKH WR[LF HQGSRLQWV RI ,& bf12(& bf DQG /2(& bf ZLWK WKH UHVXOWV RI WKH 3 VXEFDSLWDWD DVVD\ ZLWK OHDFKDWH IURP VLWH '$7( ,& 12(&D /2(&' bf bf P )HE n 0DUFK n $SULO n 0D\ n -XQH n -XO\ n 2FW n 1RY n 'HF n -DQ n )HE n 0DUFK n 0D\ n $EEUHYLDWLRQV f/& FRQFHQWUDWLRQ OHWKDO WR b RI WKH WHVW SRSXODWLRQ r12(& QR REVHUYHG HIIHFW FRQFHQWUDWLRQ F/2(& ORZHVW REVHUYHG HIIHFW FRQFHQWUDWLRQ )RU H[DPSOH FRQVLGHU WKH FDVH ZLWK WZR WR[LFLW\ DVVD\V FRQGXFWHG VLPXOWDQHRXVO\ 7KH ILUVW DVVD\ LV SUHSDUHG ZLWK D GLOXWLRQ IDFWRU RI H J bf DQG WKH VHFRQG DW D GLOXWLRQ IDFWRU RI bf 7KH 12(&/2(& UHVXOWV DW D GLOXWLRQ IDFWRU UHSUHVHQW D WLJKWHU PDUJLQ RI UHVSRQVH WKDQ WKH GLOXWLRQ IDFWRU 12(&/2(& UHVSRQVHV DUH XVHG WR HVWDEOLVK WKUHVKROG GRVHV RI DGYHUVH HIIHFWV ZKLFK DLG LQ WKH HVWDEOLVKPHQW RI UDQJHV IRU WKH SXUSRVH RI FRPSDULQJ WHVW RUJDQLVP UHVSRQVHV WR VDPSOHV ZLWK VLPLODU PDWULFHV %LHUNHQV HW DO f HYDOXDWHG WZHQW\ ELRDVVD\V IRU WKHLU UHODWLYH VHQVLWLYLWLHV DQG E\ XVLQJ 12(&/2(& YDOXHV HVWDEOLVKHG D ULVNEDVHG SUHGLFWLRQ RI WR[LFLW\ 7R GHPRQVWUDWH WKH XVH RI 12(&/2(& YDOXHV WKH 3 VXEFDSLWDWD ,& DQG 12(&/2(& UHVXOWV DUH FRPSDUHG IRU WKH 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH 7DEOH f *HQHUDOO\ WKH ,& UHVXOWV ZHUH

PAGE 148

&21'8&7,9,7< $ P6FPf )HE 0DUFK $SULO -XQH -XO\ 6HSW 2FW 1RY 'HF -DQ )HE 0DUFK f n n n n f n n n r n f 727$/ 25*$1,& &$5%21 PJ/f )LJXUH ,QIOXHQFH RI WLPH RQ $f FRQGXFWLYLW\ %f FKHPLFDO R[\JHQ GHPDQG &f WRWDO RUJDQLF FDUERQ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH

PAGE 149

$ &21'8&7,9,7< )LJXUH ,QIOXHQFH RI WLPH RQ $f FRQGXFWLYLW\ %f FKHPLFDO R[\JHQ GHPDQG &f WRWDO RUJDQLF FDUERQ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH

PAGE 150

ZLWKLQ WKH UDQJH RI WKH 12(&/2(& ([FHSWLRQV ZHUH QRWHG ZKHQ WKH 12(& FRXOG QRW EH GHWHUPLQHG GXH WR LQVXIILFLHQW OHDFKDWH GLOXWLRQ ,Q PDQ\ FDVHV WKH PDUJLQ RI VDIHW\ LPSOLHG E\ WKH 12(&/2(& YDOXHV ZDV b 7KHVH UHVXOWV UHLQIRUFHG WKH DUWLILFLDO QDWXUH RI WKH 12(&/2(& PHDVXUHPHQWV &UDQH DQG 1HZPDQ f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f 7KH FRQGXFWLYLW\ YDOXHV UHPDLQHG UHODWLYHO\ FRQVLVWHQW RYHU WLPH )LJXUH $f 7KH ODQGILOO DW VLWH QR ORQJHU UHFHLYHV 06: WKHUHIRUH WKH WRWDO FRQFHQWUDWLRQV RI LQRUJDQLF FRQVWLWXHQWV LQ WKH ZDVWH DUH QRZ ILQLWH 6LQFH WKH FRQFHQWUDWLRQV RI LQRUJDQLF FRQWDPLQDQWV DUH VHQVLWLYH WR nZDVKRXWn HIIHFWV LW FDQ EH H[SHFWHG WKDW ZLWK WLPH WKHVH FRQFHQWUDWLRQV PD\ GHFOLQH ,Q FRQWUDVW WKH GDWD LQGLFDWHG D WUHQG IRU LQFUHDVLQJ &2' FRQFHQWUDWLRQV ZLWK WLPH )LJXUH %f :LWK WLPH FKDQJHV LQ WKH SK\VLFDO VWUXFWXUH RI WKH ZDVWH PDWHULDO PD\ UHVXOW LQ LQFUHDVHG OHDFKLQJ RI RUJDQLF FRPSRXQGV *HQHUDOO\ &2' FRQFHQWUDWLRQV DUH H[SHFWHG WR GHFUHDVH DV WKH ELRORJLFDOO\ GHJUDGDEOH RUJDQLF PDWHULDOV LQ WKH ODQGILOO DUH FRQVXPHG DOWKRXJK WKH IUDFWLRQ RI &2' UHSUHVHQWLQJ WKH UHFDOFLWUDQW RUJDQLF PDWHULDO UHPDLQV FRQVWDQW :KHQ WKH 72& FRQFHQWUDWLRQV LQ WKH OHDFKDWHV IURP VLWH ZHUH FRQVLGHUHG D ZLGH IOXFWXDWLRQ

PAGE 151

ZDV LGHQWLILHG )LJXUH &f )XUWKHU LQWHUSUHWDWLRQ RI WKH FKHPLFDO FRPSRVLWLRQ RI WKH OHDFKDWHV IURP VLWH UHYHDOHG WKDW WKH &2'72& UDWLR IOXFWXDWHG EHWZHHQ WR 7KLV UDWLR LV XVHG DV DQ LQGLFDWRU RI KLJKO\ UHFDOFLWUDQW RUJDQLF PDWHULDOV DQG JHQHUDOO\ LQFUHDVHV ZLWK ODQGILOO DJH &KLDQ DQG 'H:DOOH f 7KH FKHPLFDO FKDUDFWHULVWLFV RI WKH OHDFKDWHV IURP VLWH ZHUH PRUH YDULHG RYHU WLPH )LJXUH f 7KH OHYHO RI FRQGXFWLYLW\ LQ WKH OHDFKDWHV IURP VLWH UDQJHG IURP WR P6FP )LJXUH $f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f :KHQ FRQVLGHULQJ WKHVH &2' FRQFHQWUDWLRQV D JHQHUDO WUHQG IRU LQFUHDVLQJ &2' ZLWK WLPH ZDV LGHQWLILHG 7KHUH ZDV QR SDWWHUQ RU WUHQG WR WKH 72& FRQFHQWUDWLRQV LQ WKH OHDFKDWHV IURP VLWH )LJXUH &f :KLOH WKH FRQFHQWUDWLRQV UDQJHG IURP WR PJ/ YDULDWLRQV RYHU WLPH UHPDLQHG FORVH WR WKH PHGLDQ YDOXH RI PJ/ 7KH &2'72& UDQJHG IURP WR UHIOHFWLQJ WKH GUDPDWLF IOXFWXDWLRQV LQ WKH &2' DQG 72& FRQFHQWUDWLRQV

PAGE 152

% &GXEWD 3 VXEFDSLWDWD 0LFURWR[ )LJXUH $FXWH & GXELD DQG 0LFURWR[f DQG FKURQLF 3 VXEFDSLWDWDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH EHWZHHQ )HEUXDU\ DQG 0D\ 5HVXOWV DV 78 (&VRf ZLWK RQH VWDQGDUG GHYLDWLRQ IRU & GXELD DQG 3 VXEFDSLWDWD 0LFURWR[ PHDQ RI WZR UHSOLFDWHV )LJXUH $FXWH & GXELD DQG 0LFURWR[f DQG FKURQLF 3 VXEFDSLWDWDf WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH EHWZHHQ )HEUXDU\ DQG 0DUFK 5HVXOWV DV 78 ,22(&f ZLWK RQH VWDQGDUG GHYLDWLRQ IRU & GXELD DQG 3 VXEFDSLWDWD 0LFURWR[ PHDQ RI UHSOLFDWHV

PAGE 153

%DVHG RQ WKH FRQWLQXHG KLJK VWUHQJWK RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG DOWHUDWLRQV LQ OHDFKDWH WR[LFLW\ ZHUH XQOLNHO\ 7KLV FRQFOXVLRQ ZDV VXSSRUWHG E\ WKH UHVXOWV RI WKH WR[LFLW\ DVVD\V ZKLFK DUH VXPPDUL]HG IRU WKH 06: ODQGILOO OHDFKDWHV IURP VLWH )LJXUH f DQG VLWH )LJXUH f ,Q WKH &GXELD DVVD\ WKH WR[LFLW\ RI WKH 06: OHDFKDWHV IURP VLWH GLVSOD\HG D 78 UDQJH IURP WR 1HJOHFWLQJ WKH H[WUHPH UHVSRQVHV WKH UDQJH RI 78 YDOXHV ZDV IURP WR LQGLFDWLQJ WKDW WKH DFXWH WR[LFLW\ RI WKHVH OHDFKDWHV UHPDLQHG UHODWLYHO\ FRQVLVWHQW UHODWLYH WR WKH WLPH IUDPH HYDOXDWHG &RPSDUDEOH & GXELD WR[LFLW\ ZDV LGHQWLILHG LQ WKH 06: ODQGILOO OHDFKDWHV IURP VLWH ZLWK 78 YDOXHV WKDW UDQJHG IURP WR :KHQ FRQVLGHULQJ WKH UHVSRQVH RI WKH 3 VXEFDSLWDWD DVVD\V WKH 78 UDQJHV ZHUH WR b DQG WR b IRU WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG UHVSHFWLYHO\ ,Q PRVW FDVHV WKH 0LFURWR[ DVVD\ ZDV LQVHQVLWLYH WR WKH WR[LFLW\ LQ WKH OHDFKDWHV IURP VLWH ZLWK PRVW 78 YDOXHV OHVV WKDQ 7KH VLWH OHDFKDWH FROOHFWHG LQ 0D\ SURGXFHG D 78 YDOXH RI ZLWK WKH 0LFURWR[ DVVD\ ZKLFK ZDV ORZHU WKDQ WKH 78 RI PHDVXUHG ZLWK WKH & GXELD DVVD\ EXW HTXLYDOHQW WR WKH ZLWK WKH DOJDO DVVD\ 7KH UHVSRQVH RI WKH 0LFURWR[ DVVD\ WR WKH OHDFKDWHV IURP VLWH ZDV LQDGHTXDWH ZLWK PHDVXUHG 78 YDOXHV JHQHUDOO\ DOWKRXJK RQH OHDFKDWH VDPSOH FROOHFWHG LQ 0DUFK GLVSOD\HG D 78 RI 2YHUDOO WKH UHVXOWV LQGLFDWHG WKDW WKH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWH ZDV JUHDWHU WKDQ WKDW LQ WKH VLWH OHDFKDWHV $OWKRXJK WKHUH ZHUH QR UHODWLRQVKLSV LGHQWLILHG EHWZHHQ WKH FRQFHQWUDWLRQ RI YDULRXV FKHPLFDO SDUDPHWHUV DQG WLPH WKH IOXFWXDWLRQV ZHUH ODUJH $GGLWLRQDOO\ WKH DFXWH DQG

PAGE 154

)LJXUH 5HODWLRQVKLS EHWZHHQ WKH UHVXOWV RI WKH FKURQLF KRXU 3 VXEFDSLWDWD ,&f DQG WKH DFXWH KRXU & GXELD /&f DVVD\V ZLWK 06: ODQGILOO OHDFKDWHV IURP IRXUWHHQ VLWHV LQ )ORULGD FKURQLF WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV UHPDLQHG FRQVLVWHQW ,W LV LPSRUWDQW WR UHFRJQL]H WKDW D UHODWLYHO\ VKRUW WLPH FRXUVH ZDV HYDOXDWHG KHUH FRPSDUHG ZLWK WKH HQWLUH OLIH RI D 06: ODQGILOO WKHUHIRUH WKHVH WUHQGV PD\ QRW EH WUXH RYHU WKH HQWLUH OLIH RI D ODQGILOO &RPSDUDWLYH VHQVLWLYLW\ RI WKH ELRDVVD\V $ OHDVW VTXDUHV UHJUHVVLRQ DQDO\VLV RI WKH DFXWH & GXELD /&f DQG WKH FKURQLF 3 VXEFDSLWDWD ,&f DVVD\V LQGLFDWHG D VLPLODU VHQVLWLYLW\ 5 S f )LJXUH f 7KHUH ZHUH QR UHODWLRQVKLSV EHWZHHQ WKH & GXELD SXOH[ RU 3 VXEFDSLWDWD DVVD\ UHVXOWV ZKHQ FRPSDUHG WR WKH SDUDOOHO 0LFURWR[ UHVXOWV

PAGE 155

7DEOH &RHIILFLHQWV RI YDULDWLRQ &9fbf IRU WKH 3 VXEFDSLWDWD & GXELD DQG SXOH[ DVVD\V &RHIILFLHQW RI 9DULDWLRQ &9f KRXU KRXU KRXU 6LWH 3 VXEFDSLWDWD & GXELD SXOH[ bf bf -b@ Lr r G r p 1'D 1' D E F WWWWf§‘f§UaQf§UUUUUUU 1' f7KH &9 QRW GHWHUPLQHG 1'f ZKHQ VWDQGDUG GHYLDWLRQ ZDV r ,QFOXGHG GDWD SUHVHQWHG LQ &KDSWHU 7KLV FRQWUDVWHG ZLWK UHSRUWV WKDW VKRZHG D UHODWLRQVKLS 5 f EHWZHHQ WKH KRXU & GXELD DQG PLQXWH 0LFURWR[ DFXWH WR[LFLW\ DVVD\V -XQJ DQG %LWWRQ f $OWKRXJK FRQWUDU\ WR WKH UHVXOWV UHSRUWHG KHUH WKH VDPSOHV HYDOXDWHG E\ -XQJ DQG %LWWRQ f ZHUH LQGXVWULDO HIIOXHQWV RI OLPLWHG FRPSOH[LW\ :KLOH WKH OHDFKDWH IURP RQO\ RQH &ODVV ,,, ODQGILOO VLWH Ef ZDV DVVD\HG WKH ELRDVVD\ UHVXOWV VXJJHVW WKDW WKHVH OHDFKDWHV GLVSOD\HG WR[LFLW\ WKDW ZDV FRPSDUDEOH ZLWK WKH WR[LFLW\ RI WKH &ODVV DQG ,, 06: ODQGILOO OHDFKDWHV 7KH VLWH E OHDFKDWHV GLVSOD\HG D 78 RI LQ WKH & GXELD DVVD\ ZKLFK ZDV ZLWKLQ WKH UDQJH RI WR UHSRUWHG IRU WKH 06: ODQGILOO OHDFKDWHV 7KH WR[LFLW\ RI WKH

PAGE 156

OHDFKDWHV IURP VLWH E ZDV ORZHU ,Q WKH SXOH[ DQG 3 VXEFDSLWDWD DVVD\V ZLWK 78 YDOXHV RI DQG UHVSHFWLYHO\ 7KH FRHIILFLHQW RI YDULDWLRQ &9f LV D PHDVXUH RI WKH UHSURGXFLELOLW\ RI ELRORJLFDO UHVSRQVHV )HUUDUL HW DO f $V SUHGLFWHG E\ WKH KLJKO\ YDULDEOH FKHPLFDO FRPSRVLWLRQ RI WKH 06: ODQGILOO OHDFKDWHV WKH &9V IRU WKH & GXELD DVVD\V ZLWK WKH IRXUWHHQ 06: ODQGILOO OHDFKDWHV UDQJHG IURP WR b 7DEOH f 7KH OHDFKDWH VDPSOHV FROOHFWHG IURP VLWH GLVSOD\HG WKH KLJKHVW YDULDWLRQ &9 bf ZLWK WKH & GXELD DVVD\ 7DEOH f 7KH ORZ UHSURGXFLELOLW\ RI WKHWR[LF UHVSRQVHV LQ WKH OHDFKDWHV IURP VLWH PD\ EH DWWULEXWHG WR D FKDQJH LQ VDPSOLQJ ORFDWLRQ GXULQJ WKH LQYHVWLJDWLRQ $IWHU WKH ILUVW VDPSOLQJ SRLQW DW VLWH EHFDPH LQDFFHVVLEOH LW ZDV QHFHVVDU\ WR FROOHFW WKH OHDFKDWHV IURP D VHFRQG SRLQW /RZ YDULDELOLW\ ZDV UHSRUWHG LQ WKH UHVSRQVHV RI WKH &GXELD WR[LFLW\ DVVD\V WR 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG E ZLWK &9V RI DQG b )URP D EURDG SHUVSHFWLYH WKH GHJUHH RI LQWHUUHSOLFDWH YDULDELOLW\ ZLWK WKH UHVXOWV RI WKH 3 VXEFDSLWDWD WR[LFLW\ DVVD\V ZDV JUHDWHU WKDQ GHVFULEHG IRU WKH & GXELD DVVD\V 7KLV PD\ EH D IXQFWLRQ RI WKH PHWKRG XVHG WR TXDQWLI\ WKH DOJDO UHVSRQVH 'XULQJ WKLV LQYHVWLJDWLRQ DOJDO FHOOV ZHUH FRXQWHG XVLQJ D KHPDF\WRPHWHU DQG D PLFURVFRSH 0DQXDO FRXQWV RI DOJDO FHOOV DUH ODERU LQWHQVLYH DQG UHTXLUH DQ H[SHULHQFHG WHFKQLFLDQ *HLV HW DO f REVHUYHG UHSOLFDWH YDULDELOLW\ UDQJLQJ IURP WR b LQ IODVNV FRXQWHG PDQXDOO\ &RPELQLQJ WKH LQWULQVLF YDULDELOLW\ RI WKH TXDQWLILFDWLRQ PHWKRG DQG WKH H[WUHPHO\

PAGE 157

7DEOH &ODVVLILFDWLRQ V\VWHP IRU UDQNLQJ WKH WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV IURP VLWHV LQ )ORULGD DGDSWHG IURP &OHPHQW HW DO %XOLFK f &ODVV 78 XQLWOHVVf WR WR WR WR YDULDEOH 06: ODQGILOO OHDFKDWH FRPSRVLWLRQ WKHQ D SRVVLEOH VRXUFH RI WKH KLJK &9V EHFRPHV DSSDUHQW 7KH VHQVLWLYLW\ RI WKH 3 VXEFDSLWDWD & GXELD SXOH[ DQG 0LFURWR[r1 DVVD\V WR 06: ODQGILOO OHDFKDWHV ZHUH VXPPDUL]HG &OHPHQW HW DO %XOLFK f 7DEOH GHVFULEHV WKH FODVVLILFDWLRQ V\VWHP XVHG WR UDQN WKH VHQVLWLYLWLHV RI WKH DVVD\V :KHQ UDQNLQJ WKH VHQVLWLYLW\ RI WKH YDULRXV WR[LFLW\ DVVD\V DGGLWLRQDO DFXWH DQG FKURQLF WR[LFLW\ UHVXOWV REWDLQHG GXULQJ WKH LQLWLDO SKDVH RI 06: ODQGILOO OHDFKDWH FKDUDFWHUL]DWLRQ ZHUH LQFOXGHG &KDSWHU f $FFRUGLQJ WR WKH FODVVLILFDWLRQ V\VWHP WKH WR[LFLW\ ZDV GLVWULEXWHG LQ FODVVHV WKURXJK )LJXUH f 'LVSURSRUWLRQDWHO\ WKH PDMRULW\ RI WKH WR[LFLW\ DVVRFLDWHG ZLWK WKH 06: ODQGILOO OHDFKDWHV ZDV GLVWULEXWHG LQ FODVV 7KLV ZDV WUXH IRU WKH UHVXOWV RI WKH &GXELD bf SXOH[ bf DQG 3 VXEFDSLWDWD bf DVVD\V EXW QRW WKH UHVXOWV RI WKH 0LFURWR[ DVVD\ ,QVWHDG WKH 0LFURWR[ DVVD\ UHVXOWV VKRZHG WKDW b RI WKH 06: ODQGILOO OHDFKDWHV ZHUH UDQNHG LQ FODVV ZKLOH WKH UHPDLQLQJ b ZHUH LQ FODVV )RU WKH &GXELD SXOH[ DQG 3 VXEFDSLWDWD DVVD\V WKH SURSRUWLRQDOLW\ RI 06: ODQGILOO OHDFKDWH WR[LFLW\ ZDV

PAGE 158

I P M£\IOOOf§ ‘ PPP0 ? X L Q 'S )LJXUH 5DQNLQJ RI VL[WHHQ 06: OHDFKDWHV ZLWK WKH UHVXOWV RI WKH 0LFURWR[ 07f 3 VXEFDSLWDWD 3 VXEf SXOH[ Sf DQG &HULRGDSKQLD GXELD &Gf DVVD\V XVLQJ WKH FODVVLILFDWLRQ LQ 7DEOH 7KH OHDFKDWHV ZHUH UDQNHG OHDVW WR[LF FODVV f WR PRVW WR[LF FODVV f b b DQG b LQ FODVV DQG b b DQG b LQ FODVV UHVSHFWLYHO\ $GGLWLRQDOO\ D VLJQLILFDQW SRUWLRQ RI WKH 06: ODQGILOO OHDFKDWH WR[LFLW\ ZDV LQ FODVV DW DQG b IRU WKH &GXELD SXOH[ DQG 3 VXEFDSLWDWD DVVD\V UHVSHFWLYHO\ 5HODWLRQVKLS %HWZHHQ &KHPLFDO3K\VLFDO /HDFKDWH &KDUDFWHULVWLFV DQG /HDFKDWH 7R[LFLW\ 8VLQJ VLPSOH OLQHDU UHJUHVVLRQ DQDO\VLV WKH WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV DQG YDULRXV OHDFKDWH FKHPLFDO SDUDPHWHUV ZHUH FRPSDUHG :KHQ WKH & GXELD DVVD\ UHVXOWV ZHUH FRPSDUHG ZLWK WKH DONDOLQLW\ FRQFHQWUDWLRQV LQ WKH OHDFKDWHV WKLV UHODWLRQVKLS H[SODLQHG b RI WKH WR[LF UHVSRQVH 5 S

PAGE 159

f 7KLV UHODWLRQVKLS LPSURYHG ZKHQ WKH FRQGXFWLYLW\ YDOXHV ZHUH FRPSDUHG WR WKH & GXELD WR[LFLW\ UHVXOWV 5 S f 7KHUH ZDV D VWURQJHU UHODWLRQVKLS EHWZHHQ WKH WRWDO DPPRQLD FRQFHQWUDWLRQV 5 S f DQG WKH XQLRQL]HG DPPRQLD FRQFHQWUDWLRQV 5 S f +HLMHULFN HW DO f VKRZHG WKDW SK\VLFRFKHPLFDO IDFWRUV LQ 06: ODQGILOO OHDFKDWHV HJ KDUGQHVV DONDOLQLW\ DQG FRQGXFWLYLW\f H[HUW D VWURQJ LQIOXHQFH RQ WR[LFLW\ 5HJUHVVLRQ DQDO\VLV ZDV DOVR SHUIRUPHG ZLWK WKH UHVXOWV RI WKH DOJDO WR[LFLW\ DVVD\V 7KH DONDOLQLW\ FRQFHQWUDWLRQV LQ WKH 06: ODQGILOO OHDFKDWHV KDG QR LQIOXHQFH RQ WKH FKURQLF WR[LFLW\ 5 f :KHQ WKH 3 VXEFDSLWDWD WR[LFLW\ UHVXOWV ZHUH FRPSDUHG WR WKH FRQGXFWLYLW\ FRQFHQWUDWLRQV D UHODWLRQVKLS ZDV LGHQWLILHG 5 S f $ VLJQLILFDQW UHODWLRQVKLS ZDV IRXQG EHWZHHQ WKH DOJDH WR[LFLW\ UHVXOWV DQG WKH WRWDO DPPRQLD FRQFHQWUDWLRQV LQ WKH 06: ODQGILOO OHDFKDWHV 5 S f 6LPLODUO\ 0DUWWLQHQ HW DO f UHSRUWHG D FRUUHODWLRQ EHWZHHQ DOJDO WR[LFLW\ DQG WRWDO DPPRQLD FRQFHQWUDWLRQV 5 f :KHQ WKH XQLRQL]HG DPPRQLD FRQFHQWUDWLRQV ZHUH FRPSDUHG WR WKH 3 VXEFDSLWDWD DVVD\ UHVXOWV WKH FRUUHODWLRQ ZDV ORZHU 5 S f 7KH ORZ VHQVLWLYLW\ RI WKH 0LFURWR[ DVVD\ WR WKH 06: ODQGILOO OHDFKDWHV SUHFOXGHG DQ\ FRPSDULVRQV ZLWK FKHPLFDO SDUDPHWHUV $OWKRXJK WKH 0LFURWR[ DVVD\ LV D ZLGHO\ XWLOL]HG WR[LFLW\ DVVD\ LW LV LQVHQVLWLYH WR DPPRQLD WR[LFLW\ 6WURQNKXUVW HW DO f &RQVLGHULQJ WKDW DPPRQLD LV D PDMRU WR[LFDQW LQ 06: ODQGILOO OHDFKDWHV LW LV DSSDUHQW WKDW WKH XVHIXOQHVV RI WKH 0LFURWR[ DVVD\ LV VHULRXVO\ OLPLWHG GXULQJ WR[LFLW\ LQYHVWLJDWLRQV LQ WKHVH W\SHV RI VDPSOHV

PAGE 160

&+$37(5 +($9< 0(7$/ %,1',1* &$3$&,7< +0%&f 2) 081,&,3$/ 62/,' :$67( /$1'),// /($&+$7(6 ,QWURGXFWLRQ 5HVHDUFKHUV KDYH VKRZQ WKDW KHDY\ PHWDO WR[LFLW\ DQG ELRDYDLODELOLW\ DUH VWURQJO\ LQIOXHQFHG E\ WKH DFWLYLW\ RI LRQL]HG PHWDO VSHFLHV LQ VROXWLRQ EXW QRW E\ WKH WRWDO PHWDO FRQFHQWUDWLRQV *UDERZVNL HW DO 0RZDW DQG %XQG\ f ,Q 0RUHO f VXPPDUL]HG WKH UHVXOWV RI UHVHDUFK WR GDWH DQG SURSRVHG WKH IUHH LRQ DFWLYLW\ PRGHO ),$0f UHFRJQL]LQJ WKDW KHDY\ PHWDOV DUH PRVW WR[LF LQ WKHLU LRQL]HG VWDWH &DPSEHOO f 06: OHDFKDWHV PD\ FRQWDLQ PHWDOV GXH WR WKH FRPSRVLWLRQ RI ZDVWH PDWHULDOV DQG GHJUDGDWLYH SURFHVVHV LQ WKH ODQGILOO :KLOH UHVHDUFKHUV DJUHH WKDW WKH WRWDO FRQFHQWUDWLRQ RI KHDY\ PHWDOV LQ 06: ODQGILOO OHDFKDWHV FDQQRW SUHGLFW WR[LFLW\ RU ELRDYDLODELOLW\ 0RUJDQ DQG 6WXPP /XRPD f WKHUH LV QR FRQVHQVXV ZLWKLQ WKH UHVHDUFK FRPPXQLW\ IRU LGHQWLI\LQJ RU TXDQWLI\LQJ WKH YDULRXV PHWDO VSHFLHV 1LPPR HW DO f XVHG ELRDVVD\V ZLWK LQYHUWHEUDWHV DQG SODQWV WR LQYHVWLJDWH WKH WR[LFLW\ RI ODQGILOO OHDFKDWHV LQ 9LUJLQLD DQG GHWHUPLQHG WKDW PHWDOV LQ WKH ODQGILOO OHDFKDWHV ZHUH QRW ELRDYDLODEOH DOWKRXJK WKH FRPSOH[DWLRQ SRWHQWLDO RI WKH OHDFKDWH ZDV QRW TXDQWLILHG 7KH PRVW ZLGHO\ XVHG DQDO\WLFDO PHWKRG IRU GHWHUPLQLQJ PHWDO VSHFLDWLRQ ZDV GHYHORSHG E\ 7HVVLHU HW DO f :LWK D VHTXHQWLDO H[WUDFWLRQ SURFHGXUH WKH\ SURSRVHG ILYH GLVWLQFW PHWDO IUDFWLRQV H[FKDQJHDEOH ERXQG WR FDUERQDWH

PAGE 161

ERXQG WR LURQ DQG PDQJDQHVH R[LGHV ERXQG WR RUJDQLF PDWWHU DQG UHVLGXDO KHDY\ PHWDOV 2WKHU PHWKRGV IRU PHWDO VSHFLDWLRQ LQFOXGH GLDO\VLV DQG LRQ H[FKDQJH :KLOH WKH PRELOLW\ DQG H[WUDFWDELOLW\ RI PHWDOV LQ ODQGILOOV KDV EHHQ VWXGLHG &OHYHQJHU DQG 5DR f QR GDWD DUH DYDLODEOH RQ WKH TXDQWLWDWLYH GHWHUPLQDWLRQ RI WKH ELRDYDLODELOLW\ DQG WKH DELOLW\ RI ODQGILOO OHDFKDWHV WR ELQG PHWDOV XVLQJ ELRORJLFDO DVVD\V 5HOLDQFH RQ PHWDO VSHFLDWLRQ PRGHOV GHULYHG IURP VHTXHQWLDO H[WUDFWLRQ SURFHGXUHV PD\ XQGHUHVWLPDWH ELRDYDLODEOH PHWDO FRQFHQWUDWLRQV 6FKHLIOHU HW DO f 7KH ELRDYDLODELOLW\ RI PHWDOV LQ FRQWDPLQDWHG VRLOV ZDV LQYHVWLJDWHG ZLWK D PLFURELDO UHVSLUDWLRQ DVVD\ DQG D PLFURELDO QLWULILFDWLRQ DVVD\ $OWKRXJK QLWULILFDWLRQ ZDV VHQVLWLYH WR ELRDYDLODEOH PHWDOV UHVSLUDWLRQ ZDV KLJKO\ VHQVLWLYH *H HW DO f 2WKHU UHVHDUFKHUV KDYH VKRZQ WKDW PLFURELDO UHVSLUDWLRQ SURYLGHV D JRRG PHDVXUH RI ERWK JHQHUDO WR[LFLW\ %LWWRQ DQG .RRSPDQ f DQG KHDY\ PHWDO WR[LFLW\ %LWWRQ HW DO f 7KH DELOLW\ RI 06: OHDFKDWHV WR UHGXFH PHWDO WR[LFLW\ KDV EHHQ UHFRJQL]HG EXW QR ZRUN KDV EHHQ SXEOLVKHG WR TXDQWLI\ WKLV DELOLW\ RU WR DWWHPSW WR HOXFLGDWH WKH PHFKDQLVPV UHVSRQVLEOH +XDQJ HW DO f PHDVXUHG WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f RI VXUIDFH ZDWHUV DV WKH UDWLR RI WKH (& RI D VHOHFWHG PHWDO LQ VLWH ZDWHU WR WKH (& RI WKH VDPH PHWDO LQ FRQWURO ZDWHU 7KLV PHWKRGRORJ\ LV VLPLODU WR WKDW SUHYLRXVO\ GHVFULEHG IRU WKH ZDWHU HIIHFW UDWLR :(5f SURSRVHG E\ WKH 86(3$ f %DVLFDOO\ ERWK LQFRUSRUDWH WKH LQIOXHQFH RI VLWHVSHFLILF SDUDPHWHUV RQ WKH TXDQWLW\ DQG ELRDYDLODELOLW\ RI PHWDOV LQ HQYLURQPHQWDO PDWULFHV EXW WKH +0%& WHVW EDVHG RQ 0HW3/$7( LV UDSLG FRVW

PAGE 162

HIIHFWLYH DQG FDQ HDVLO\ EH XVHG LQ ILHOG DVVHVVPHQWV 5HFHQWO\ WHFKQLFDO LVVXHV ZLWK WKH :(5 KDYH EHHQ UDLVHG LQ UHODWLRQ WR WHVW VSHFLHV DFFOLPDWLRQ WR WHVW FRQGLWLRQV DQG LQWHUVSHFLHV YDULDELOLW\f DQG FKHPLFDO LVVXHV DONDOLQLW\ DQG FDOFLXQUPDJQHVLXP UDWLRV LQ FRQWURO ZDWHUV DQG S+ YDULDELOLW\ LQ VLWH ZDWHUVf :HOVK HW DOf f 7KH XWLOLW\ RI 0HW3/$7( D PLFURELDO HQ]\PH DVVD\ VSHFLILFDOO\ VHQVLWLYH WR KHDY\ PHWDO WR[LFLW\ ZDV LQYHVWLJDWHG ZLWK 06: ODQGILOO OHDFKDWHV LQ )ORULGD (DUOLHU ZRUN VKRZHG WKDW 06: ODQGILOO OHDFKDWHV DUH ERWK DFXWHO\ DQG FKURQLFDOO\ WR[LF LQ ELRDVVD\V XVLQJ DTXDWLF LQYHUWHEUDWHV DQG DOJDH UHIHU WR &KDSWHUV DQG f %\ DGDSWDWLRQ RI WKH 0HW3/$7(r1 DVVD\ WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f RI WKH 06: ODQGILOO OHDFKDWHV FRXOG EH HYDOXDWHG $ UHYLHZ RI FXUUHQW OLWHUDWXUH UHYHDOHG D ODFN RI UHVHDUFK FRQFHUQHG ZLWK WKH TXDQWLWDWLYH GHWHUPLQDWLRQ RI PHWDO WR[LFLW\ ELRDYDLODELOLW\ DQG PHWDO ELQGLQJ FDSDFLW\ LQ ODQGILOO OHDFKDWHV XVLQJ WR[LFLW\ DVVD\V 7KH PDMRULW\ RI WKH GHJUDGDWLYH SURFHVVHV LQ ODQGILOOV DUH GXH WKH DFWLYLW\ RI PLFURELDO SRSXODWLRQV %DUOD] f 7KHUHIRUH WKH XVH RI D PLFURELDO DVVD\ WR HYDOXDWH WKH WR[LFLW\ RU ELRDYDLODELOLW\ RI ODQGILOO OHDFKDWHV LV DSSURSULDWH 6XIOLWD HW DO f 7KH 0HW3/$7( DVVD\ TXDQWLILHV FKDQJHV LQ WKH DFWLYLW\ RI WKH JDODFWRVLGDVH HQ]\PH 7KH HQ]\PH DFWLYLW\ LV GHSHQGHQW RQ FRQIRUPDWLRQDO FKDQJHV DQG FRPSHWLWLRQ ZLWK FDWLRQLF KHDY\ PHWDOV IRU IXQFWLRQDO ELQGLQJ VLWHV GHFUHDVHV HQ]\PH DFWLYLW\ -XHUV HW DO f 'XULQJ HDUOLHU LQYHVWLJDWLRQV ZLWK 06: ODQGILOO OHDFKDWHV UHVXOWV VXJJHVWHG WKDW LQRUJDQLF FRPSRXQGV EXW QRW KHDY\ PHWDOV ZHUH SULPDULO\ UHVSRQVLEOH IRU WKH WR[LF HIIHFWV :DUG HW DO f

PAGE 163

7KH REMHFWLYHV RI WKH SUHVHQW ZRUN ZHUH f WR HYDOXDWH WKH KHDY\ PHWDO WR[LFLW\ RI 06: OHDFKDWHV f WR TXDQWLI\ WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f RI WKH 06: ODQGILOO OHDFKDWHV DQG f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f DQG OHDFKDWH FKDUDFWHULVWLFV FKHPLFDO VWUHQJWK LQRUJDQLF RU RUJDQLF FRQWHQWf )XUWKHU GHVFULSWLRQV RI WKH VLWHV DW ZKLFK WKH 06: ODQGILOO OHDFKDWHV ZHUH FROOHFWHG DUH DYDLODEOH &KDSWHUV DQG f $GGLWLRQDO EDVHOLQH ZDWHU VDPSOHV ZHUH FROOHFWHG IURP WZR ORFDO ODNHV /DNH $OLFH LQ *DLQHVYLOOH )/ DQG /DNH %HYHUO\ LQ %HYHUO\ +LOOV )/f DQG IURP D PXQLFLSDO ZDVWHZDWHU WUHDWPHQW SODQW ::73f LQ *DLQHVYLOOH )/ /HDFKDWH &ROOHFWLRQ 06: ODQGILOO OHDFKDWHV ZHUH FROOHFWHG IURP WKH OHDFKDWH FROOHFWLRQ VXPSV RI WKH OLQHG ODQGILOOV XVLQJ D 7HIORQ EDOHU 6DPSOHV IRU FKHPLFDO DQDO\VLV ZHUH FROOHFWHG LQ SRO\HWK\OHQH RU JODVV FRQWDLQHUV DQG SUHVHUYHG DFFRUGLQJ WR WKH 86 (QYLURQPHQWDO 3URWHFWLRQ $JHQF\ 86(3$f PHWKRGV 86(3$ Ef 7KH OHDFKDWHV IRU WR[LFLW\ DQDO\VLV ZHUH FROOHFWHG LQ SODVWLF FRQWDLQHUV WUDQVSRUWHG WR

PAGE 164

WKH ODE RQ LFH DQG LPPHGLDWHO\ VWRUHG DW r& XQWLO VDPSOH DQDO\VLV ZLWKLQ RU GD\V 7KH ODNH ZDWHU DQG ::73 HIIOXHQW VDPSOHV ZHUH FROOHFWHG LQ SODVWLF FRQWDLQHUV IRU WR[LFLW\ DQDO\VLV KRZHYHU VDPSOHV ZHUH QRW FROOHFWHG IRU FKHPLFDO DQDO\VLV &KHPLFDOV DQG 5HDJHQWV 0HWDO VWRFNV &X &X6f +J+J&,f DQG =Qr =Q6f ZHUH SUHSDUHG LQ 0LOOL4 ZDWHU DW D FRQFHQWUDWLRQ RI PJ/ 7KH H[FKDQJH UHVLQV GLHWK\ODPLQRHWK\O FHOOXORVH '($(f FDSDFLW\ PHTJ DQG 'RZH[: 6W\UHQH '9% JHOf GU\ PHVK ZHUH SXUFKDVHG IURP 6LJPD$OGULFK 0LOZDXNHH :Of &ROXPQV ZHUH SUHSDUHG LQ PO ERURVLOLFDWH JODVV SLSHWWHV DQG UHVLQV ZHUH FRQGLWLRQHG DFFRUGLQJ WR PDQXIDFWXUHU VSHFLILFDWLRQV ZLWK UHDJHQW JUDGH PDWHULDOV &KHPLFDO $QDO\VLV )LHOG PHDVXUHPHQWV LQFOXGHG S+ DQG WHPSHUDWXUH 2ULRQ 0RGHO $f HOHFWULFDO FRQGXFWLYLW\ +$11$ ,QVWUXPHQWV 0RGHO +f GLVVROYHG R[\JHQ '2f <6, ,QF 0RGHO )7f DQG R[LGDWLRQUHGXFWLRQ SRWHQWLDO 253f $FFXPHW &R 0RGHO f $ONDOLQLW\ ELRFKHPLFDO R[\JHQ GHPDQG %2'f FKHPLFDO R[\JHQ GHPDQG &2'f GLVVROYHG RUJDQLF FDUERQ '2&f DPPRQLD DQG VXOILGHV ZHUH PHDVXUHG DFFRUGLQJ WR PHWKRGV GHVFULEHG E\ 86(3$ f DQG $3+$ f +DUGQHVV ZDV PHDVXUHG E\ FRORULPHWULF DQDO\VLV +$&+ PHWKRG +$&+ /RYHODQG &2f 6DPSOHV IRU WRWDO PHWDO FRQWHQW ZHUH GLJHVWHG

PAGE 165

3UHSDUH VDPSOH GLOXWLRQV $OLTXRW SO RI VDPSOH RU LWV GLOXWLRQ LQWR GHDQ WHVW WXEHV $OLTXRW SO RI EDFWHULDO UHDJHQW LQWR HDFK RI WKH WHVW WXEHV YRUWH[ ,QFXEDWH IRU KRLUV DW r& $OLTXRW SO IURP HDFK WHVW WXEH LQWR VHSDUDWH ZHOOV RI D ZHOO PLFURSODWH DQG DGG SL RI FKURPRJHQLF VXEVWUDWH ,QFXEDWH DW r & IRU FRORU GHYHORSPHQW LQ WKH QHJDWLYH FRQWURO )LJXUH 7KH 0HW3/$7( DVVD\ SURWRFRO IRU GHWHUPLQLQJ WKH KHDY\ PHWDO WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV DGDSWHG IURP -XQJ f

PAGE 166

86(3$ 0HWKRG 6: 2$f 86(3$ f DQG DQDO\]HG E\ DWRPLF HPLVVLRQ VSHFWURVFRS\ $(6f 7KHUPR -DUUHOO $VK 0RGHO (QYLUR f 'HWHUPLQDWLRQ RI +HDY\ 0HWDO 7R[LFLW\ 7KH 0HW3/$7(r1 WHVW NLW ZDV GHYHORSHG DW WKH 8QLYHUVLW\ RI )ORULGD IRU GHWHUPLQLQJ KHDY\ PHWDO WR[LFLW\ DQG WKH NLW FRQWDLQV D EDFWHULDO UHDJHQW DQ (FROL VWUDLQf EXIIHU FKORURSKHQRO UHG JDODFWRS\UDQRVLGH &35*f ZKLFK VHUYHV DV WKH VXEVWUDWH IRU SJDODFWRVLGDVH DQG PRGHUDWHO\ KDUG ZDWHU 0+:f DV D GLOXHQW 7KH WR[LFLW\ WHVW ZDV SUHYLRXVO\ GHVFULEHG %LWWRQ HW DO KWWSZZZHHVXIOHGXKRPHSSELWWRQf )LJXUH f %ULHIO\ WKH EDFWHULDO UHDJHQW ZDV UHK\GUDWHG ZLWK PO RI GLOXHQW DQG WKRURXJKO\ PL[HG E\ YRUWH[LQJ $ SO DOLTXRW RI WKH OHDFKDWH RU LWV GLOXWLRQ ZDV DGGHG WR D WHVW WXEH WR ZKLFK ZDV DGGHG SO RI EDFWHULDO UHDJHQW 7HVW WXEHV ZHUH YRUWH[HG DQG WKHQ LQFXEDWHG IRU KRXUV DW r& $ SO DOLTXRW RI WKH VXVSHQVLRQ OHDFKDWH EDFWHULDf ZDV WUDQVIHUUHG WR D ZHOO PLFURSODWH WR ZKLFK ZDV DGGHG SO RI &35* WKH HQ]\PH VXEVWUDWH IROORZHG E\ VKDNLQJ 7KH PLFURSODWH ZDV WKHQ LQFXEDWHG DW r& IRU FRORU GHYHORSPHQW 7KH UHVSRQVH ZDV TXDQWLILHG DW QP XVLQJ D 0XOWLVNDQ PLFURSODWH UHDGHU 'HWHUPLQDWLRQ RI +0%& 7KH DELOLW\ RI 06: ODQGILOO OHDFKDWHV WR UHGXFH WKH ELRDYDLODELOLW\ RI KHDY\ PHWDOV ZDV DVVHVVHG XVLQJ WKH 0HW3/$7(r1 WHVW NLW WR GHWHUPLQH WKH +0%& RI WKH OHDFKDWHV IROORZLQJ D PHWKRGRORJ\ SUHYLRXVO\ GHYHORSHG +XDQJ HW DO f 7KH KHDY\ PHWDOV FRSSHU ]LQF DQG PHUFXU\ ZHUH VHOHFWHG IRU WHVWLQJ EDVHG RQ WKHLU LQFUHDVHG FRQFHQWUDWLRQV LQ WKH QDWXUDO HQYLURQPHQW DQG WKHLU

PAGE 167

0HWDO 6ROXWLRQ &21752/ :$7(5 6SLNH PRGHUDWHO\ KDUG ZDWHU 0+:f ZLWK KHDY\ PHWDO 06: /$1'),// /($&+$7( 6SLNH ZLWK KHDY\ PHWDO Â’ Â’ / ,QFXEDWH DW r& IRU PLQXWHV ZKLOH VKDNLQJ ,QFXEDWH DW r& IRU PLQXWHV ZKLOH VKDNLQJ Â’ / Â’ / 3UHSDUH GLOXWLRQV ZLWK 0+: DQG LQFXEDWH IRU PLQXWHV DW r&VKDNH 3UHSDUH GLOXWLRQV ZLWK OHDFKDWH DQG LQFXEDWH IRU PLQXWHV DW r& VKDNH Â’ / U / $GG PO WHVW EDFWHULD WR PO VROXWLRQ WULSOLFDWH WXEHVf LQFXEDWH PLQ DW r& $GG PO WHVW EDFWHULD WR PO VROXWLRQ WULSOLFDWH WXEHVf LQFXEDWH PLQ DW r& Â’ / U / 0(73/$7( $66$< $GG PO IURP HDFK WXEH WR D PLFURSODWH ZHOO $GG PO RI EXIIHUHG VXEVWUDWH WR HDFK ZHOO ,QFXEDWH DW r& XQWLO FRORU GHYHORSPHQW LQ QHJDWLYH FRQWURO 5HDG FRORU DW QP 'HWHUPLQH (&} IRU FRQWURO ZDWHU DQG OHDFKDWH +0%& (& 06: /$1'),// /($&+$7( HF FRQWURO ZDWHU )LJXUH 7KH SURWRFRO IRU GHWHUPLQLQJ +0%& RI 06: ODQGILOO OHDFKDWHV DGDSWHG IURP +XDQJ HW DO f

PAGE 168

VLJQLILFDQW WKUHDW WR HQYLURQPHQWDO KHDOWK UHVXOWLQJ IURP LQWHUDFWLRQV ZLWK ELRORJLFDOO\ LPSRUWDQW IXQFWLRQDO JURXSV HJ VXOIXU DQG QLWURJHQ JURXSV 0RUJDQ DQG 6WXPP f 7KH +0%& PHWKRG KDV EHHQ XVHG ZLWK ZDWHU VDPSOHV REWDLQHG IURP ULYHUV DQG ZHWODQGV EXW QHYHU EHIRUH ZLWK 06: ODQGILOO OHDFKDWHV 8VLQJ WKH 0HW3/$7( DVVD\ WKH FRQFHQWUDWLRQ RI HDFK KHDY\ PHWDO SUHSDUHG LQ PRGHUDWHO\ KDUG ZDWHU 0+:f ZKLFK SURGXFHG D b LQKLELWLRQ LQ HQ]\PH DFWLYLW\ (&f ZDV GHWHUPLQHG 6LPXOWDQHRXVO\ WKH 06: ODQGILOO OHDFKDWHV ZHUH VSLNHG ZLWK WKH VDPH PHWDO DQG WKH FRQFHQWUDWLRQ RI WKH PHWDO LQ WKH OHDFKDWH UHVSRQVLEOH IRU D b LQKLELWLRQ LQ HQ]\PH DFWLYLW\ (&f ZDV GHWHUPLQHG 7KH +0%& ZDV H[SUHVVHG DV WKH UDWLR RI WKH (&VRRI D JLYHQ PHWDO LQ OHDFKDWH WR WKH (&VRRI D JLYHQ PHWDO LQ 0+: )LJXUH f 7KH (& YDOXHV ZHUH GHWHUPLQHG E\ WKH IROORZLQJ SURFHGXUH $ PO DOLTXRW RI HDFK 06: ODQGILOO OHDFKDWH RU 0+: ZDV VSLNHG ZLWK DQ DSSURSULDWH YROXPH RI PHWDO VWRFN VROXWLRQ &X =Q RU +Jf DQG VKDNHQ USPf IRU PLQXWHV DW URRP WHPSHUDWXUH *HQHUDOO\ WR SL RI WKH PHWDO VWRFN ZDV DGGHG EXW WKLV ZDV GHSHQGHQW RQ WKH UHVSRQVH RI WKH 0HW3/$7( DVVD\ $IWHU WKH DGGLWLRQ RI WKH PHWDO VSLNH WKH OHDFKDWHV DQGRU 0+: ZHUH VHULDOO\ GLOXWHG DW D GLOXWLRQ IDFWRU RI 7KH VSLNHG 06: ODQGILOO OHDFKDWHV ZHUH VHULDOO\ GLOXWHG LQ DGGLWLRQDO 06: ODQGILOO OHDFKDWH DQG WKH VSLNHG 0+: ZDV VHULDOO\ GLOXWHG ZLWK 0+: 7KH VSLNHG 06: OHDFKDWHV DQG 0+: ZHUH FRYHUHG DQG VKDNHQ USPf IRU DQ DGGLWLRQDO PLQXWHV DW r& 7KH VSLNHG OHDFKDWHV DQG WKH VSLNHG 0+: ZHUH WKHQ DVVD\HG LQ WULSOLFDWH DFFRUGLQJ WR WKH 0HW3/$7( DVVD\ DV SUHYLRXVO\ GHVFULEHG

PAGE 169

)LJXUH 7KH SURWRFRO XVHG IRU IUDFWLRQDWLRQ RI +0%& ,QIOXHQFH RI 6RPH /HDFKDWH 3DUDPHWHUV RQ +0%& $ IUDFWLRQDWLRQ VFKHPH ZDV SURSRVHG WR LGHQWLI\ WKH LQIOXHQFH RI VRPH FKHPLFDO SDUDPHWHUV RI WKH 06: ODQGILOO OHDFKDWHV RQ +0%& )LJXUH f /HDFKDWH VDPSOHV ZHUH FROOHFWHG IURP VLWHV DQG DQG DVVD\HG IRU WKHLU +0%& ZLWK FRSSHU &Xf ]LQF =Qf DQG PHUFXU\ +Jf $ / YROXPH RI HDFK

PAGE 170

OHDFKDWH VDPSOH ZDV ILOWHUHG XP PHPEUDQH ILOWHU *1 0HWULFHOf SDVVHG WKURXJK GLHWK\ODPOQRHWK\O FHOOXORVH '($( J UHVLQFROXPQf D ZHDNO\ EDVLF DQLRQ H[FKDQJH UHVLQ DQG ILQDOO\ SDVVHG WKURXJK D VWURQJ DFLGLF FDWLRQ LQ WKH K\GURJHQVDWXUDWHG IRUPf H[FKDQJH UHVLQ 'RZH[: J UHVLQFROXPQf ,RQ H[FKDQJH UHVLQV ZHUH FKRVHQ EDVHG RQ WKHLU VHOHFWLYLW\ IRU WKH LRQV RI LQWHUHVW 7KH DQLRQ H[FKDQJH UHVLQ '($( KDV D KLJK DIILQLW\ IRU KXPLF DQG IXOYLF DFLGV
PAGE 171

7DEOH (&ZIRU &Xr =Q DQG +Jr GHWHUPLQHG ZLWK WKH 0HW3/$7( DVVD\ 0HWDO (& PJ/f &X DV &X62ff s =QDV =Q&,f s +J DV +J&,f s GHWHUPLQDWLRQ RI +0%& WKH LQGLYLGXDO (& YDOXHV ZHUH REWDLQHG E\ JUDSKLFDO LQWHUSRODWLRQ IRU WKH PHWDO VSLNHG 06: OHDFKDWHV DQG WKH PHWDO VSLNHG PRGHUDWHO\ KDUG ZDWHU 0+:f 7KH +0%& RI HDFK LQGLYLGXDO 06: ODQGILOO OHDFKDWH ZDV GHWHUPLQHG IURP WULSOLFDWH 0HW3/$7( DVVD\V WKHUHIRUH UHVXOWV DUH SUHVHQWHG DV WKH PHDQ s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f DQG ZDV YDOLGDWHG IRU WKH WKUHH PHWDOV FRSSHU DV &X6f ]LQF DV =Q&,f DQG PHUFXU\ DV +J&,ff XWLOL]HG LQ WKH FXUUHQW VWXG\ 7KH PHWDO (&6 REWDLQHG ZHUH s PJ/ s PJ/ DQG s PJ/ IRU &X =Qr DQG +Jr UHVSHFWLYHO\ 7DEOH f 7KHVH (& YDOXHV ZHUH FRPSDUDEOH WR WKRVH SUHYLRXVO\ UHSRUWHG -XQJ f

PAGE 172

b ,QKLELWLRQ 0HDQ 5DQJH s D Df Ef Ff /DNH $OLFH /DNH %HYHUO\ ::73 (IIOXHQW D1 QXPEHU RI VDPSOLQJ HYHQWV

PAGE 173

7DEOH VXPPDUL]HV WKH UHVXOWV RI WKH 0HW3/$7( DVVD\ IRU WKH VL[WHHQ 06: ODQGILOO OHDFKDWHV IURP )ORULGD $ UDQJH RI 0HW3/$7( LQKLELWLRQ bf ZDV UHSRUWHG ZLWK WKH OHDFKDWHV 7KHUH ZDV QR WR[LFLW\ LQKLELWLRQ f LQ WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV D E F DQG 7KH OHDFKDWHV FROOHFWHG IURP VLWH SURGXFHG D b LQKLELWLRQ RI HQ]\PH DFWLYLW\ LQ WKH 0HW3/$7( DVVD\ +RZHYHU WKH PHDQ WR[LFLW\ ZDV JHQHUDOO\ ORZ ZLWK b RI WKH VDPSOHV GLVSOD\LQJ OHVV WKDQ D b LQKLELWLRQ )RU FRPSDULVRQ VDPSOHV ZHUH FROOHFWHG IURP WZR ORFDO ODNHV DQG D ::73 DQG WKHVH VDPSOHV VKRZHG QR WR[LFLW\ LQ WKH 0HW3/$7( DVVD\ +LJKHU KHDY\ PHWDO FRQFHQWUDWLRQV KDYH EHHQ UHSRUWHG LQ WKH OHDFKDWHV IURP ODQGILOOV FRGLVSRVLQJ 06: DQG 06: LQFLQHUDWRU DVK %R]NXUW HW DO f VR WKHVH W\SHV RI OHDFKDWHV DUH H[SHFWHG WR HOLFLW D JUHDWHU LQKLELWRU\ UHVSRQVH LQ WKH 0HW3/$7( DVVD\ /DQGILOOV DW VLWHV DQG D SUDFWLFH WKH FRGLVSRVDO RI 06: DQG 06: LQFLQHUDWRU DVK 7KH 0HW3/$7( UHVXOWV ZLWK WKHVH OHDFKDWHV ZHUH YDVWO\ GLIIHUHQW 7KH OHDFKDWH IURP VLWH LQKLELWHG WKH UHVSRQVH RI WKH 0HW3/$7( DVVD\ E\ b ZKLFK FRQWUDVWV ZLWK WKH DEVHQFH RI WR[LFLW\ LQ WKH OHDFKDWH IURP VLWH D 6XUSULVLQJO\ PHWDO DQDO\VLV RI WKH OHDFKDWHV UHYHDOHG VLPLODU FRSSHU FRQFHQWUDWLRQV RI PJ/ DQG PJ/ DW VLWH DQG VLWH D UHVSHFWLYHO\ 7KHVH FRQFHQWUDWLRQV ZHUH URXJKO\ HTXLYDOHQW WR WKH (&} RI PJ/ UHSRUWHG IRU FRSSHU LQ WKH 0HW3/$7( DVVD\ 7DEOH f 7KHUHIRUH WKH LQKLELWLRQ FDXVHG E\ WKH OHDFKDWH IURP VLWH ZDV DWWULEXWDEOH WR WKH PHDVXUHG FRSSHU FRQFHQWUDWLRQ %DVHG RQ WKH PHWDO DQDO\VLV WKH WR[LFLW\ RI WKH OHDFKDWH IURP VLWH D ZDV H[SHFWHG WR EH KLJK KRZHYHU WKLV ZDV QRW WKH FDVH ,QVWHDG

PAGE 174

7DEOH 3K\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI OHDFKDWHV FROOHFWHG IURP OLQHG 06: ODQGILOOV LQ )ORULGD 5HVXOWV DUH SUHVHQWHG DV PHDQ DQG UDQJHf RU PHDQ s RQH VWDQGDUG GHYLDWLRQ IRU VLWHV VDPSOHG RQ RQH RFFDVLRQ 6LWH S+ &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ,/ DV &D&f 7'6D J8 6XOILGHV SJ/! &2'E PJ/f '2& PJIOf f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f 10 f f f f f f 10 10 Df 10 Ef 10 Ff 10 10& 10 10 10 10 10 10 10 f f f f f 7nHf§f§ 10 $EEUHYLDWLRQV 7'6 WRWDO GLVVROYHG VROLGV f&2' FKHPLFDO R[\JHQ GHPDQG f 10 QRW PHDVXUHGD RQH VDPSOH FROOHFWHG

PAGE 175

D ORZ WR[LFLW\ ZDV VKRZQ ZKLFK VXJJHVWHG WKDW WKH PHWDOV ZHUH QRW SUHVHQW LQ D ELRDYDLODEOH IRUP 6RPH SK\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI WKH VL[WHHQ 06: ODQGILOO OHDFKDWHV DUH VXPPDUL]HG LQ 7DEOH 7KH UDQJH RI FRQFHQWUDWLRQV ZDV W\SLFDO IRU 06: ODQGILOO OHDFKDWHV LQ )ORULGD :DUG HW DOf f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f D &ODVV ,,, ODQGILOO IRU QRQSXWUHVFLEOH ZDVWHV Ef DQG D FDSSHG &ODVV 06: ODQGILOO Ff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

PAGE 176

VLPLODU ZLWK D IHZ H[FHSWLRQV 7KH VXOILGH FRQFHQWUDWLRQV LQ OHDFKDWHV IURP VLWHV DQG ZHUH DQG SJ/ UHVSHFWLYHO\ EXW DW VLWH WKH FRQFHQWUDWLRQ ZDV SJ/ :KLOH XQH[SODLQHG WKLV ILQGLQJ DJUHHV ZLWK UHSRUWV RI ORZ VXOIDWH FRQFHQWUDWLRQV LQ WKH OHDFKDWHV IURP VLWH 2WKHU OHDFKDWH SDUDPHWHUV IRU VLWH ZHUH ZLWKLQ WKH UDQJH UHSRUWHG IRU WKH OHDFKDWHV IURP VLWHV DQG $OWKRXJK WKH WR[LFLW\ RI WKH OHDFKDWHV IURP VLWHV DQG ZDV ORZ DW OHVV WKDQ b LQKLELWLRQ WKH OHDFKDWHV IURP VLWH ZHUH HYHQ OHVV WR[LF PHDQ LQKLELWLRQ bf 7KH JUHDWHU WR[LFLW\ DQG ELRDYDLODELOLW\ RI KHDY\ PHWDOV LQ WKH OHDFKDWHV IURP VLWH PD\ EH DWWULEXWHG WR VLWHVSHFLILF FKDUDFWHULVWLFV RI WKH OHDFKDWHV 7DEOH f 7KH TXDOLW\ RI D ODQGILOO OHDFKDWH GLUHFWO\ LPSDFWV PHWDO ELRDYDLODELOLW\ DQG KHQFH WR[LFLW\ GXH WR WKH PRGLI\LQJ LQIOXHQFHV IURP DONDOLQLW\ &2' 7'6 DQG S+ :HOVK HW DO f 5HLQKDUW DQG *URVK f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f 8QLTXH GLIIHUHQFHV ZHUH GHPRQVWUDWHG EHWZHHQ WKH FKHPLFDO FKDUDFWHULVWLFV RI WKH OHDFKDWHV IURP VLWHV DQG D 7DEOH f 7KH OHDFKDWHV IURP VLWH GLVSOD\HG DONDOLQLW\ 7'6 DQG FRQGXFWLYLW\ FRQFHQWUDWLRQV RI PJ/ DV &D& J/ DQG P6FP UHVSHFWLYHO\ 8QGHUVFRULQJ WKH

PAGE 177

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b &RPSDUDWLYHO\ WKH OHDFKDWHV IURP VLWH DOVR GLVSOD\HG KLJK FRQFHQWUDWLRQV RI LRQV DV FRQGXFWLYLW\ P6FPf DONDOLQLW\ PJ/ DV &D&f DQG 7'6 PJ/f ZLWK D FRPSDUDEOH UDQJH RI WR[LFLW\ 'XH WR WKH ODFN RI WR[LF UHVSRQVH LQ QHDUO\ b RI WKH 0HW3/$7( DVVD\V UHODWLRQVKLSV EHWZHHQ WKH 0HW3/$7( WR[LFLW\ DQG 06: OHDFKDWH SDUDPHWHUV FRXOG QRW EH GHWHUPLQHG +0%& RI 06: /DQGILOO /HDFKDWHV 7KH ELRDYDLODELOLW\ DQG WR[LFLW\ RI KHDY\ PHWDOV LQ 06: OHDFKDWHV ZDV IXUWKHU FKDUDFWHUL]HG E\ GHWHUPLQLQJ WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f RI HDFK OHDFKDWH +0%& TXDQWLILHV WKH PHWDO ELRDYDLODELOLW\WR[LFLW\ LQ DTXDWLF HQYLURQPHQWV DQG LV GHSHQGHQW RQ SK\VLFRFKHPLFDO SDUDPHWHUV VXFK DV S+ DONDOLQLW\ KDUGQHVV DQG WKH SUHVHQFH RI FRPSOH[LQJ OLJDQGV +XDQJ HW DO f 7KH VFRSH RI WKH +0%& FRQFHSW LV VLPLODU WR WKH ZDWHU HIIHFW UDWLR :(5f SURSRVHG E\ 86(3$ 86(3$ f 7KH 86(3$ UHFRJQL]HG WKH UHODWLRQVKLS EHWZHHQ VLWHVSHFLILF ZDWHU TXDOLW\ SDUDPHWHUV DQG PHWDO ELRDYDLODELOLW\ ZKHQ

PAGE 178

7DEOH +HDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f XQLWOHVVf RI OHDFKDWHV IURP 06: ODQGILOOV ZLWK FRSSHU ]LQF DQG PHUFXU\ 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ DQG UDQJHf RU WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ IRU 6LWH &XDV &X62Wf +0%&D =Qr DV =Q&,f +J DV +J&,f 0HDQ 0HDQ 0HDQ f f 10& f f 10 f f f f f f f f 10 10 f f f s f f f s s 10E 10 10 10 Df s 10 10 Ef s 10 10 Ff s s 10 s s s s s 10 f f /DNH $OLFH s s s /DNH %HYHUO\ ::73 (IIOXHQW n DL? [ £ LW LQU s s WUf§YUUf§W ‘ U s HYDOXDWLQJ DTXDWLF WR[LFLW\ 86(3$ f 7KH :(5 SURWRFRO XWLOL]HV LQGLJHQRXV VSHFLHV DQG LQFRUSRUDWHV VLWHVSHFLILF ZDWHU TXDOLW\ SDUDPHWHUV HJ S+

PAGE 179

KDUGQHVV DONDOLQLW\ WR GHULYH DFFHSWDEOH PD[LPXP DOORZDEOH WRWDO PHWDO FRQFHQWUDWLRQV :HOVK HW DO f 6LQFH WKH :(5 SURWRFRO UHOLHV RQ ELRDVVD\V ZLWK ZKROH RUJDQLVPV WKH WHVWV DUH ODERULQWHQVLYH H[SHQVLYH DQG QRW HDVLO\ DGDSWHG WR LQILHOG WHVWLQJ 7KH +0%& DVVD\ DOORZV IRU WKH UDSLG GHWHUPLQDWLRQ RI PHWDO ELRDYDLODELOLW\ DQG WR[LFLW\ +XDQJ HW DO f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f ZKLOH OHDFKDWHV IURP VLWHV DQG KDG WKH KLJKHVW +0%& =Q f DQG +0%&+J f UHVSHFWLYHO\ :KHQ PXOWLSOH OHDFKDWH VDPSOHV ZHUH FROOHFWHG IURP LQGLYLGXDO ODQGILOO VLWHV WKH +0%& UHVXOWV GLVSOD\HG KLJK YDULDELOLW\ 7KLV ZDV GHPRQVWUDWHG E\ WKH OHDFKDWHV IURP VLWH ZKLFK GLVSOD\HG +0%&&X YDOXHV WKDW UDQJHG IURP WR $OWKRXJK WKH

PAGE 180

PDJQLWXGH RI WKH UHVSRQVH ZDV ORZHU WKH YDULDELOLW\ LQ WKH +0%&&X YDOXHV ZLWK WKH OHDFKDWHV IURP VLWH ZDV VLPLODU DQG UDQJLQJ IURP WR 7KH OHDFKDWHV IURP VLWH ZHUH QRW DVVD\HG IRU WKHLU +0%& GXH WR WKH IRUPDWLRQ RI SUHFLSLWDWHV GXULQJ SUHOLPLQDU\ WHVWLQJ %DVHG RQ WKH UHVXOWV RI WKH 0HW3/$7( DVVD\ WKH +0%& RI WKH OHDFKDWH FROOHFWHG IURP VLWH ZDV SUHGLFWHG WR EH ORZ %\ FRPSDULVRQ WKH +0%& RI WKH VDPSOHV FROOHFWHG IURP /DNH $OLFH DQG /DNH %HYHUO\ ZHUH ORZ ZLWK DOO YDOXHV 7KH +0%&&X IRU WKH WZR ODNH VDPSOHV UDQJHG IURP WR ZKLOH WKH +0%&=Qr UDQJHG IURP WR DQG WKH +0%&+J UDQJHG IURP WR 7KHVH +0%&&X YDOXHV ZHUH ZLWKLQ WKH UDQJH WR f SUHYLRXVO\ UHSRUWHG IRU VXUIDFH ZDWHUV +XDQJ HW DO f 6OLJKWO\ KLJKHU +0%& YDOXHV ZHUH PHDVXUHG LQ WKH ::73 HIIOXHQW DW DQG IRU +0%&&X +0%&=Q DQG +0%&+J UHVSHFWLYHO\ 7KH +0%& UHVXOWV LQGLFDWHG D KLJK FDSDFLW\ IRU ELQGLQJ WR FRSSHU ]LQF DQG PHUFXU\ E\ WKH 06: ODQGILOO OHDFKDWHV ZKLFK ZDV DWWULEXWHG WR WKH FKHPLFDO FKDUDFWHULVWLFV RI WKH OHDFKDWHV 5HODWLRQVKLSV EHWZHHQ LQGLYLGXDO SK\VLFRn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

PAGE 181

LQYHVWLJDWHG EHWZHHQ +0%& DQG S+ DONDOLQLW\ FRQGXFWLYLW\ WRWDO GLVVROYHG VROLGV VXOILGH FKHPLFDO R[\JHQ GHPDQG DQG KDUGQHVV FRQFHQWUDWLRQV &RPSDULVRQV UHYHDOHG QR UHODWLRQVKLSV DQG WKH FRHIILFLHQW RI GHWHUPLQDWLRQ 5f ZHUH DOO 7KHUH ZDV D WUHQG EHWZHHQ LQFUHDVLQJ FKORULGH FRQFHQWUDWLRQV DQG WKH LQFUHDVHG ELQGLQJ RI ]LQF E\ 06: ODQGILOO OHDFKDWHV 5 f KRZHYHU FKORULGH KDG QR LQIOXHQFH RQ WKH ELQGLQJ RI FRSSHU RU PHUFXU\ )URP D EURDG SHUVSHFWLYH WKH ODUJH QXPEHU RI ODQGILOOV LQYHVWLJDWHG WRJHWKHU ZLWK WKH IUHTXHQF\ RI OHDFKDWH FROOHFWLRQ DQG YDULRXV VLWHVSHFLILF FRQGLWLRQV HJ UDLQIDOO ZDVWH FRPSRVLWLRQ DJH DQG WHPSHUDWXUHf FRQWULEXWHG WR WKH IDLOXUH WR LGHQWLI\ SUHGLFWLYH FKDUDFWHULVWLFV IRU WR[LFLW\ LQ WKH OHDFKDWHV :KLOH WKHUH ZHUH QR FOHDU SDWWHUQV EHWZHHQ FKHPLFDO FKDUDFWHULVWLFV DQG +0%& JHQHUDOO\ WKH OHDFKDWHV ZLWK WKH JUHDWHVW VWUHQJWK GLVSOD\HG WKH KLJKHVW +0%& (OHYDWHG FRQFHQWUDWLRQV RI FRPSOH[LQJ DJHQWV H J RUJDQLF DQG LQRUJDQLF OLJDQGV FRQWULEXWH WR WKH +0%& 6OHWWHQ HW DO f 7KLV ZDV WKH FDVH ZLWK WKH OHDFKDWHV IURP VLWH ZKLFK GLVSOD\HG KLJK FRQFHQWUDWLRQV RI DONDOLQLW\ VXOILGH DQG FKORULGH 7KH KLJK VWUHQJWK RI WKH OHDFKDWHV IURP VLWH FRUUHVSRQGHG ZLWK WKH JUHDWHVW PHDQ +0%& IRU ]LQF DW %DVHG RQ NQRZOHGJH RI WKH OHDFKDWHV IURP VLWH WKH SUHGLFWHG ELQGLQJ FDSDFLW\ IRU FRSSHU DQG PHUFXU\ ZDV KLJK +RZHYHU WKHVH PHWDOV ZHUH QRW DVVD\HG ZLWK WKH OHDFKDWHV IURP VLWH ,QVWHDG DV GLVFXVVHG HDUOLHU WKH KLJKHVW +0%&&X f DQG +0%&+J f ZHUH PHDVXUHG DW VLWHV DQG UHVSHFWLYHO\ 7KH FKHPLFDO VWUHQJWK RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH ZDV RQ DYHUDJH

PAGE 182

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f 7KHVH UHVXOWV XQGHUVFRUH WKH LPSRUWDQFH RI VLWHVSHFLILF SDUDPHWHUV RQ WKH RYHUDOO PDJQLWXGH RI +0%& IRU HDFK 06: ODQGILOO OHDFKDWH 7KH FKHPLFDO VWUHQJWK RI 06: OHDFKDWHV LQ )ORULGD KDV EHHQ GHVFULEHG DV PRUH GLOXWH WKDQ WKDW UHSRUWHG IRU RWKHU 06: OHDFKDWHV ZKLFK UHGXFHV WKH RYHUDOO SRWHQWLDO IRU KHDY\ PHWDO ELQGLQJ 5HLQKDUW DQG *URVK f

PAGE 183

* 6LWH ‘ 6LWH n n n n n n n n n n n n n n n n fLQGLFDWHV VDPSOH LQKLELWLRQ 1 VDPSOH QRW WHVWHG )LJXUH 0HW3/$7( UHVXOWV IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH DQG VLWH RYHU WLPH ZLWK UHVXOWV SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ DQG GDVKHG OLQH UHSUHVHQWLQJ WKH b LQKLELWLRQ OHYHO /HDFKDWH WR[LFLW\ DV D IXQFWLRQ RI WLPH 7HPSRUDO LQIOXHQFHV RQ KHDY\ PHWDO WR[LFLW\ LQ OHDFKDWHV IURP VLWHV DQG ZHUH HYDOXDWHG DQG WKH UHVXOWV DUH SUHVHQWHG LQ )LJXUH $FFRUGLQJ WR WKH UHVXOWV RI WKH 0HW3/$7( DVVD\ WKH WR[LFLW\ RI WKH OHDFKDWHV FROOHFWHG IURP VLWH UHPDLQHG ORZ WKURXJKRXW WKH \HDU ZLWK D UDQJH RI LQKLELWLRQ IURP WR b 6LPLODU UHVXOWV ZHUH VKRZQ IRU WKH 06: OHDFKDWHV IURP VLWH ZLWK RQH H[FHSWLRQ 7KH OHDFKDWH FROOHFWHG IURP VLWH LQ -DQXDU\ FDXVHG DQ LQKLELWLRQ RI b LQ WKH 0HW3/$7( DVVD\ 7KHUH ZHUH QR WUHQGV LQ WKH 0HW3/$7( UHVXOWV IRU KHDY\ PHWDO WR[LFLW\ YHUVXV WLPH ZLWK WKH OHDFKDWHV IURP

PAGE 184

7DEOH 0HW3/$7( DQG +0%& UHVXOWV ZLWK 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWHV DQG 5HVXOWV DUH SUHVHQWHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ 6LWH 0HW3/$7( b ,QKLELWLRQf +0%&D &Xrr DV &862f =Q DV =Q&Ef +Ja DV +J&,f s s s s s s s s s s s s s s D+0%& KHDY\ PHWDO ELQGLQJ FDSDFLW\ XQLWOHVVf VLWHV DQG ZKLFK DJUHHG ZLWK WKH UHVXOWV RI WKH DFXWH DQG FKURQLF DVVD\V ZLWK WKHVH OHDFKDWHV &KDSWHU f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f RQ WKH ELQGLQJ FDSDFLW\ RI WKHVH 06: OHDFKDWHV IRU FRSSHU ]LQF DQG PHUFXU\ ZHUH LQYHVWLJDWHG 7DEOH SUHVHQWV WKH 0HW3/$7( DQG +0%& UHVXOWV REWDLQHG ZLWK WKHVH IRXU DGGLWLRQDO OHDFKDWH VDPSOHV 7KH 06: ODQGILOO OHDFKDWHV IRUP VLWHV DQG ZHUH QRW WR[LF LQKLELWLRQ bf EXW WKH OHDFKDWH IURP VLWH ZDV VOLJKWO\

PAGE 185

7DEOH +0%& RI 06: ODQGILOO OHDFKDWHV ZLWK FRSSHU ]LQF DQG PHUFXU\ IROORZLQJ IUDFWLRQDWLRQ ZLWK UHVXOWV H[SUHVVHG DV WKH PHDQ s RQH VWDQGDUG GHYLDWLRQ 7UHDWPHQW 6LWH 6LWH 6LWH 6LWH +0%&&Xr :KROH s s s s 3RVWILOWUDWLRQ s s DD s s 3RVW'($( s s f sDD s 3RVW'RZH[ s s s sf +0%&+Jr :KROH s s s s 3RVWILOWUDWLRQ s sf s s 3RVW'($( s s D s s 3RVW'RZH[ s s s s +0%&=Q :KROH s s s s 3RVWILOWUDWLRQ s f s s s 3RVW'($( s s s s 3RVW'RZH[ s sD sf sf 5HODWLYH FKDQJHV LQ +0%& IRU HDFK PHWDO ZHUH GHVLJQDWHG E\ IRU VLJQLILFDQW UHGXFWLRQV DW S DQG IRU VLJQLILFDQW LQFUHDVHV DW S EDVHG RQ FRPSDULVRQ WR WKH +0%& UHVXOW RI WKH SUHYLRXV WUHDWPHQW WR[LF ZLWK b LQKLELWLRQ 7KH PDJQLWXGH RI WKH ELQGLQJ FDSDFLW\ IRU HDFK PHWDO ZLWK WKH IRXU OHDFKDWHV GLIIHUHG PDUNHGO\ +0%& YDOXHV UDQJHG IURP WR WR DQG WR IRU FRSSHU PHUFXU\ DQG ]LQF UHVSHFWLYHO\ 7KH KHDY\ PHWDO ELQGLQJ FDSDFLW\ RI WKH IRXU OHDFKDWHV IROORZHG WKH RUGHU RI +0%&&X +0%&+J!+0%&=Qr 7KLV WUHQG UHVXOWHG IURP WKH KLJK DIILQLW\ RI RUJDQLF VXEVWDQFHV IRU IUHH FRSSHU LRQV 8QOLNH PHUFXU\ DQG ]LQF LRQV WKH FRSSHU LRQV IRUP VWURQJ FRPSOH[HV ZLWK ERWK KXPLF DQG IXOYLF DFLGV :HQJ HW DO &DODFH HW DO f 7KHUHIRUH PRUH FRSSHU UHODWLYH WR ]LQF DQG PHUFXU\ LV FRPSOH[HG 2YHUDOO WKH 06: ODQGILOO OHDFKDWHV IURP VLWH

PAGE 186

7DEOH &KDQJHV LQ SK\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV GXULQJ IUDFWLRQDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWH DQG 6LWH 6LWH 6LWH 6LWH :KROH /HDFKDWH S+ &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ/ DV &D&f +DUGQHVV PJ/ DV &D&4Mf '2& 7RWDO 6ROLGV PJ/f 3RVW)LOWUDWLRQ S+ &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ/ DV &D&f +DUGQHVV PJ/ DV &D&f '2& 7RWDO GLVVROYHG VROLGV PJ/f 3RVW'($( S+ &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ/ DV &D&f +DUGQHVV PJ/ DV &D&f '2& PJ/f 3RVW'RZH[ S+ &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ/ DV &D&f +DUGQHVV PJ/ DV &D&f '2& PJ/f

PAGE 187

GLVSOD\HG WKH KLJKHVW FDSDFLW\ IRU ELQGLQJ ZLWK KHDY\ PHWDOV DQG WKLV ZDV IROORZHG E\ OHDFKDWHV IURP VLWHV DQG 7KH IUDFWLRQDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWHV DQG EHJDQ ZLWK PHPEUDQH ILOWUDWLRQ SPf FRQWLQXHG ZLWK SDVVDJH WKURXJK DQ DQLRQ H[FKDQJH FROXPQ '($( FHOOXORVHf DQG WKHQ SDVVDJH WKURXJK D FDWLRQ 'RZH[f H[FKDQJH FROXPQ 7KH WUHDWPHQWV SURGXFHG VWDWLVWLFDOO\ VLJQLILFDQW UHGXFWLRQV S f LQ WKH ELQGLQJ FDSDFLW\ RI WKH OHDFKDWHV IRU WKH PHWDOV &X =Q DQG +J 7DEOH f 5HVHDUFKHUV KDYH VKRZQ WKH HIIHFWLYHQHVV RI WKH '($( FHOOXORVH UHVLQV IRU WKH UHFRYHU\ RI KXPLF VXEVWDQFHV
PAGE 188

VXJJHVWHG WKDW LQ WKH OHDFKDWH IURP VLWH WKH FKDUDFWHULVWLFV LPSRUWDQW WR WKH ELQGLQJ RI FRSSHU DQG ]LQF ZHUH DVVRFLDWHG ZLWK VROLGV ZKLOH PHUFXU\ ELQGLQJ ZDV GHSHQGHQW RQ RWKHU IDFWRUV 2YHUDOO ILOWUDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV \LHOGHG WKH JUHDWHVW UHGXFWLRQV IRU +0%& ZLWK FRSSHU ]LQF DQG PHUFXU\ 7DEOH f 'HVSLWH ORZ VROLGV UHPRYDO bf LQ WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG WKH +0%& IRU FRSSHU ]LQF DQG PHUFXU\ ZHUH VLJQLILFDQWO\ UHGXFHG S f ,Q IDFW LQ WKH OHDFKDWHV IURP VLWHV DQG WKH +0%&&X +0%&=Q DQG +0%&+Jr ZHUH UHGXFHG E\ DQG b DQG b DQG DQG b UHVSHFWLYHO\ $OWKRXJK ILOWUDWLRQ UHGXFHG WKH VROLGV FRQWHQW RI WKH OHDFKDWHV IURP VLWH E\ b WKHUH ZHUH QR FRQFXUUHQW UHGXFWLRQV LQ WKH +0%& IRU FRSSHU ]LQF RU PHUFXU\ ,QVWHDG WKH +0%& IRU HDFK RI WKH PHWDOV LQFUHDVHG DIWHU ILOWUDWLRQ :LWK WKH VLWH OHDFKDWH WKH +0%& LQFUHDVHG WR bf IRU HDFK RI WKH PHWDOV ZKLFK VXJJHVWHG WKDW GXULQJ ILOWUDWLRQ RI WKH OHDFKDWH WKH FRPSRVLWLRQ RI WKH ZDV DOWHUHG %DVLFDOO\ WKH UHVXOWV UHSRUWHG IRU WKH HIIHFW RI ILOWUDWLRQ RQ +0%& DUH FRQVLVWHQW ZLWK UHSRUWV RI KLJK DVVRFLDWLRQV EHWZHHQ SDUWLFXODWH PDWHULDOV DQG PHWDO LRQV *XR HW DO -HQVHQ DQG &KULVWHQVHQ *RXQDULV HW DO f ,Q ERWK WHUUHVWULDO 3DUDW HW DO *H HW DO f DQG DTXDWLF 9RHONHU DQG .RJXW %HQHGHWWL HW DO f HQYLURQPHQWV WKH UHGXFHG ELRDYDLODELOLW\ RI KHDY\ PHWDOV LQ WKH SUHVHQFH RI RUJDQLF PDWWHU KDV EHHQ UHSRUWHG 5RXJKO\ WR b RI RUJDQLF PDWWHU LV KXPLF LQ QDWXUH :HQJ HW DO

PAGE 189

0DVLRQ HW DOf f DQG WKH VL]H RI WKHVH KXPLF OLJDQGV KDV D VWURQJ LQIOXHQFH RQ PHWDO PRELOLW\ .DQJ HW DO f 6FKURWK DQG 6SRVLWR f VXJJHVWHG WKDW LQ WKH SUHVHQFH RI FOD\ VROLGV KXPLF VXEVWDQFHV GLVSOD\ DQ LQFUHDVHG DIILQLW\ IRU KHDY\ PHWDOV &OD\ LV DQ LQWHJUDO SDUW RI PDQ\ ODQGILOO OLQHU V\VWHPV WKHUHIRUH FOD\ PDWHULDOV PD\ EHFRPH DVVRFLDWHG ZLWK WKH ZDVWH PDWHULDOV )UDFWLRQDWLRQ VKRZHG WKDW WKH RUJDQLF PDWWHU FRQWHQW RI ODQGILOO OHDFKDWHV ZDV DQ LPSRUWDQW GHWHUPLQDWH RI +0%& +LJK FRQFHQWUDWLRQV RI GLVVROYHG RUJDQLF PDWWHU KDYH EHHQ UHSRUWHG LQ 06: OHDFKDWHV &DODFH HW DO f DQG PHWDO FRPSOH[DWLRQ E\ WKLV RUJDQLF IUDFWLRQ KDV EHHQ H[WHQVLYHO\ UHSRUWHG .DVFKO HW DO f 7KHVH RUJDQRPHWDOOLF FRPSOH[HV DUH KLJKO\ UHVLVWDQW WR ELRGHJUDGDWLRQ 3DUDW HW DO f UHVXOWLQJ LQ D VORZHU EUHDNGRZQ RI RUJDQLF PDWWHU LQ ODQGILOOV 7KH IRUPDWLRQ RI FRPSOH[HV ZLWK RUJDQLF PDWHULDOV LQFUHDVHV WKH PRELOLW\ RI PHWDOV %DUOD] HW DO f ,Q XQOLQHG ODQGILOOV WKHVH FRPSOH[HV IDFLOLWDWHG WKH WUDQVSRUW RI PHWDOV WR JURXQGZDWHU &DVWDJQROL HW DO f UHSRUWHG ORZ FRQFHQWUDWLRQV RI RUJDQLF PDWWHU LQ OHDFKDWHFRQWDPLQDWHG VRLOV EHQHDWK XQOLQHG ODQGILOOV DQG WKLV ZDV DWWULEXWHG WR WKH UDSLG GHJUDGDWLRQ RI RUJDQRPHWDOOLF FRPSOH[HV 2UJDQRPHWDOOLF FRPSOH[HV DUH UDSLGO\ GHJUDGHG LQ FDUERQSRRU HQYLURQPHQWV ZKLFK UHVXOWV LQ WKH UHOHDVH RI PHWDOV %R]NXUW HW DO f 3DVVDJH RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG WKURXJK WKH '($( UHVLQ FROXPQV UHGXFHG '2& FRQFHQWUDWLRQV E\ DQG b UHVSHFWLYHO\ 7DEOH f 9LVXDO REVHUYDWLRQV DQG WKH RUJDQLF FKDUDFWHULVWLFV RI

PAGE 190

WKH 06: ODQGILOO OHDFKDWHV FRQILUPHG WKH SUHVHQFH RI KXPLF VXEVWDQFHV 'LVVROYHG RUJDQLF FDUERQ '2&f FRQFHQWUDWLRQV DUH XVHG WR GHVFULEH KXPLF VXEVWDQFHV LQ 06: ODQGILOO OHDFKDWHV 6PLWK DQG :HEHU f 7KH OHDFKDWH FROOHFWHG IURP VLWH VKRZHG WKH KLJKHVW DVVRFLDWLRQ EHWZHHQ WKH SUHVHQFH RI RUJDQLF VXEVWDQFHV DQG +0%& IRU FRSSHU ]LQF DQG PHUFXU\ :KHQ WKH '2& ZDV UHGXFHG E\ b WKHUH ZHUH FRUUHVSRQGLQJ UHGXFWLRQV RI DQG b IRU +0%&&Xr +0%&+Jr DQG +0%&=Qr UHVSHFWLYHO\ 7DEOH f 7KH OHDFKDWH IURP VLWH UHSUHVHQWV D FORVHG DQG FDSSHG ODQGILOO XQLW DQG WKH FRQFHQWUDWLRQV RI KXPLF VXEVWDQFHV LQ FORVHG ODQGILOOV DUH H[SHFWHG WR LQFUHDVH ZLWK DJH DQG ZDVWH GHJUDGDWLRQ &DVWDJQROL HW DO f 7KH DVVRFLDWLRQ EHWZHHQ FRSSHU ELQGLQJ DQG RUJDQLFV UHPRYDO ZDV SUREDEO\ GXH WR WKH DIILQLW\ RI FRSSHU IRU GLVVROYHG RUJDQLF PDWWHU &DODFH HW DO f VSHFLILFDOO\ KXPLF PDWHULDOV :HQJ HW DO f 7KH IRUPDWLRQ RI RUJDQRFRSSHU FRPSOH[HV LQ 06: ODQGILOO OHDFKDWHV UHGXFHV FRSSHU WR[LFLW\ )UDVHU HW DO 3DOPHU HW DO f 7KLV VDPH SURSHUW\ KDV EHHQ ZLGHO\ UHSRUWHG LQ DTXDWLF V\VWHPV DOEHLW DW JUHDWO\ UHGXFHG '2& FRQFHQWUDWLRQV PJ/f:LQFK HW DO f $IWHU WKH 06: ODQGILOO OHDFKDWH IURP VLWH ZDV SDVVHG WKURXJK WKH '($( UHVLQ WKH +0%&V IRU FRSSHU ]LQF DQG PHUFXU\ GLG QRW GHFUHDVH ,QVWHDG WKH +0%&&X DQG +0%&+Jr QHDUO\ GRXEOHG DQG WKH +0%&=Qr LQFUHDVHG E\ URXJKO\ b 7KLV LQFUHDVHG ELQGLQJ ZDV XQLTXH WR WKLV VDPSOH DQG WKH '($( WUHDWPHQW EXW WKH ORZ UHPRYDO RI '2& bf ZDV SUREDEO\ DQ LQIOXHQWLDO IDFWRU %DVHG RQ WKHVH UHVXOWV WKHUH ZDV QR DVVRFLDWLRQ EHWZHHQ WKH UHPRYDO RI DQLRQLF VSHFLHV E\ WKH '($( UHVLQ DQG WKH +0%& IRU WKH WKUHH KHDY\ PHWDOV

PAGE 191

&RPSHWLWLRQ EHWZHHQ WKH RUJDQLF PDWHULDO DQG RWKHU DQLRQLF VSHFLHV HJ FKORULGH DQG FDUERQDWH LRQV IRU ELQGLQJ VLWHV RQ WKH '($( UHVLQ PD\ KDYH EHHQ UHVSRQVLEOH IRU WKH ORZ '2& UHPRYDO 7KH RUJDQLF FRQWHQW RI WKH OHDFKDWH IURP VLWH ZDV UHGXFHG E\ b DIWHU '($( WUHDWPHQW DQG WKHUH ZDV D FRUUHVSRQGLQJ b UHGXFWLRQ RI WKH OHDFKDWHV +0%& IRU ]LQF 7KH +0%&&X GHFUHDVHG EXW QRW VLJQLILFDQWO\ DQG WKH +0%&+Jr LQFUHDVHG E\ b 0HWDO WR[LFLW\ LV VWURQJO\ LQIOXHQFHG E\ WKH SUHVHQFH RI GLVVROYHG RUJDQLF PDWWHU '20f DQG LWV DELOLW\ WR FRPSOH[ KHDY\ PHWDOV +HLMHULFN HW DO 5LFKDUGV HW DO .LP HW DO f ,Q 06: ODQGILOO OHDFKDWHV RUJDQLF VXEVWDQFHV RFFXU SUHGRPLQDQWO\ LQ WZR GLVWLQFW PROHFXODU ZHLJKW UHJLRQV RQH RI ORZ PROHFXODU ZHLJKW 'DOWRQf DQG D VHFRQG RI KLJK PROHFXODU ZHLJKW 'DOWRQf 6XUSULVLQJO\ LQ ERWK PXQLFLSDO DQG LQGXVWULDO ZDVWH OHDFKDWHV WKH GLVWULEXWLRQ RI WKH RUJDQLF PDWWHU LQ WKHVH UHJLRQV LV VLPLODU 6PLWK DQG :HEHU f 2YHUDOO WKH FRPSRVLWLRQ RI KXPLF VXEVWDQFHV LQ WKH ODQGILOO GHSHQGV RQ YDULRXV HQYLURQPHQWDO IDFWRUV OLNH UDLQIDOO DQG WHPSHUDWXUH EXW DOVR RQ WKH W\SH RI ZDVWH PDWHULDOV DQG PRUH LPSRUWDQWO\ ODQGILOO DJH .DQJ HW DO f 7KH PROHFXODU ZHLJKW RI RUJDQLF FRPSRXQGV LQ OHDFKDWHV LQFUHDVHV ZLWK WLPH &DODFH HW DO f )URP LWV LQIOXHQFH RQ KXPLF VWUXFWXUH S+ UHJXODWHV PHWDO PRELOLW\ 0DUWHQVVRQ HW DO f $V WKH S+ LQFUHDVHV WKH GHSURWRQDWLRQ RI KXPLF VXEVWDQFHV FDXVHV FRQIRUPDWLRQDO FKDQJHV DQG ORZHUV WKH DIILQLW\ RI KXPLF VXEVWDQFHV IRU PHWDO LRQV 0DVLRQ HW DO f +XPLF VXEVWDQFHV DUH FRPSOH[ RUJDQLF PROHFXOHV DQG GXH WR WKLV FRPSOH[LW\ WKH VWUXFWXUH RI WKHVH PROHFXOHV LV SRRUO\ FKDUDFWHUL]HG 7KH

PAGE 192

IXQFWLRQDO JURXS RQ WKH KXPLF PDWHULDO GHWHUPLQHV WKH UHODWLYH ELQGLQJ DIILQLW\ IRU PHWDO LRQV *HQHUDOO\ VWURQJHU PHWDO ELQGLQJ LV DVVRFLDWHG ZLWK WKH IXQFWLRQDO JURXSV FRQWDLQLQJ QLWURJHQ DQG VXOIXU UDWKHU WKDQ WKRVH FRQWDLQLQJ SKHQROLF DQG FDUER[\OLF IXQFWLRQDO JURXSV &DODFH HW DO 6WXPP DQG 0RUJDQ f 7KLV SURSHUW\ KDV EHHQ GHPRQVWUDWHG IRU KXPLF VXEVWDQFHV LQ 06: ODQGILOO OHDFKDWHV 3DUDW HW DO &URXH HW DO f 7KH KLJK QLWURJHQ FRQWHQW RI WKH OHDFKDWHV FRQWULEXWHV WR VWURQJHU ELQGLQJ %XUWRQ DQG :DWVRQ&UDLN f )RU FRPSDULVRQ WKH QLWURJHQ FRQWHQW RI 06: ODQGILOO OHDFKDWHV LV RIWHQ PHDVXUHG DW FRQFHQWUDWLRQV ILYH WLPHV JUHDWHU WKDQ LQ WKH DTXDWLF HQYLURQPHQWV .DQJ HW DO f 7KH 06: ODQGILOO OHDFKDWHV DW VLWHV DQG FRQWDLQHG VLPLODU KDUGQHVV FRQFHQWUDWLRQV DW DQG PJ/ UHVSHFWLYHO\ ZKLOH DW VLWH KDUGQHVV ZDV VOLJKWO\ ORZHU DW PJ/ 7DEOH f +DUGQHVV LV D PHDVXUH RI WKH FDWLRQV LQ VROXWLRQ DQG WKH PRVW SUHYDOHQW KDUGQHVV FDWLRQV LQ ERWK WKH HQYLURQPHQW DQG 06: ODQGILOO OHDFKDWHV DUH FDOFLXP DQG PDJQHVLXP ,Q WKH OHDFKDWHV IURP VLWHV DQG WKH FDOFLXP FRQFHQWUDWLRQV ZHUH JHQHUDOO\ JUHDWHU WKDQ RU HTXLYDOHQW WR WKH PDJQHVLXP FRQFHQWUDWLRQV ZKLFK LV UHOHYDQW VLQFH WKH FDOFLXP LRQV DUH VWURQJHU FRPSHWLWRUV WKDQ PDJQHVLXP IRU PHWDO ELQGLQJ VLWHV 7KH FDWLRQ H[FKDQJH UHVLQ 'RZH[f UHGXFHG KDUGQHVV FRQFHQWUDWLRQV E\ DQG b LQ WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG UHVSHFWLYHO\ 7DEOH f 8QLTXH GLIIHUHQFHV ZHUH GHPRQVWUDWHG IRU WKH UROH RI KDUGQHVV RQ PHWDO WR[LFLW\ LQ WKH 06: ODQGILOO OHDFKDWHV :KLOH D ODUJH IUDFWLRQ RI WKH WRWDO KDUGQHVV

PAGE 193

ZDV UHGXFHG WKH UHVXOWV RI WKH PHWDO WR[LFLW\ ZHUH KLJKO\ YDULDEOH 7KH VWURQJHVW UHODWLRQVKLS EHWZHHQ WKH KDUGQHVV FRQFHQWUDWLRQV LQ WKH OHDFKDWHV DQG +0%& ZDV GHPRQVWUDWHG IRU WKH OHDFKDWH IURP VLWH ,Q WKLV OHDFKDWH DQ b UHGXFWLRQ LQ KDUGQHVV FRLQFLGHG ZLWK D b UHGXFWLRQ LQ WKH FDSDFLW\ RI WKH OHDFKDWH IURP VLWH WR ELQG ZLWK FRSSHU 8QGHU WKH VDPH FRQGLWLRQV WKH OHDFKDWH IURP VLWH GLVSOD\HG QR DVVRFLDWLRQ EHWZHHQ KDUGQHVV FRQFHQWUDWLRQV DQG WKH ELQGLQJ RI PHUFXU\ EXW WKHUH ZDV D b GHFUHDVH LQ WKH DELOLW\ RI WKH OHDFKDWH WR ELQG ]LQF 7DEOH f &RQVLGHULQJ WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG KDUGQHVV ZDV DQ LPSRUWDQW IDFWRU WR WKH ELQGLQJ RI FRSSHU PHUFXU\ DQG ]LQF ,Q WKH OHDFKDWHV IURP VLWH WKH UHPRYDO RI b RI WKH KDUGQHVV FDWLRQV UHGXFHG WKH +0%& RI WKH OHDFKDWHV E\ DQG b UHVSHFWLYHO\ $Q LQGHSWK DQDO\VLV RI WKH OHDFKDWHV IURP VLWH UHYHDOHG WKDW WR b RI WKH PHWDO ELQGLQJ FDSDFLW\ ZDV H[SODLQHG E\ WKH UHPRYDO RI b RI WKH KDUGQHVV FDWLRQV $FFRUGLQJ WR WKH +0%& DVVD\ WKH FDSDFLW\ RI WKH VLWH OHDFKDWHV WR ELQG ZLWK FRSSHU PHUFXU\ DQG ]LQF ZDV UHGXFHG E\ DQG b UHVSHFWLYHO\ 7KH UHVXOWV ZLWK WKH OHDFKDWH IURP VLWH DUH PRUH GLIILFXOW WR LQWHUSUHW 'HVSLWH DQ b UHGXFWLRQ RI KDUGQHVV FDWLRQV WKH OHDFKDWHV IURP VLWH H[KLELWHG VLJQLILFDQW LQFUHDVHV IRU ELQGLQJ FDSDFLW\ GHFUHDVHG WR[LFLW\f IRU ERWK FRSSHU DQG ]LQF 6SHFLILFDOO\ WKH +0%& RI WKH OHDFKDWH IRU FRSSHU DQG ]LQF LRQV LQFUHDVHG IURP WR DQG IURP WR UHVSHFWLYHO\ 7KHUH ZDV D VPDOO EXW VLJQLILFDQW GHFUHDVH LQ WKH ELQGLQJ RI WKH VLWH OHDFKDWH IRU PHUFXU\ IURP WR

PAGE 194

.QR[ DQG -RQHV f GHPRQVWUDWHG WKDW WKH IRUPDWLRQ RIRUJDQR PHWDOOLF FRPSOH[HV ZDV QRW LQIOXHQFHG E\ WKH SUHVHQFH RI KDUGQHVV FDWLRQV ,Q FRQWUDVW RWKHU UHVHDUFKHUV KDYH UHSRUWHG VWURQJ FRPSHWLWLRQ EHWZHHQ PHWDO FDWLRQV DQG WKH SUHGRPLQDQW KDUGQHVV FDWLRQV FDOFLXP DQG PDJQHVLXPf 0RUJDQ DQG 6WXPP f %R]NXUW HW DO f VKRZHG WKDW 06: ODQGILOOV FRQWDLQHG VXIILFLHQW ELQGLQJ FDSDFLW\ IRU WKH UHPRYDO RI W\SLFDO FRQFHQWUDWLRQV RI KHDY\ PHWDOV EXW WKH\ DFNQRZOHGJHG WKDW KLJK FRQFHQWUDWLRQV RI KDUGQHVV GHFUHDVHV WKH PHWDO ELQGLQJ FDSDFLW\ DV FDOFLXP DQG PDJQHVLXP FRPSHWH ZLWK PHWDO LRQV 6LQFH KDUGQHVV FDWLRQV PD\ EH VWURQJO\ DVVRFLDWHG ZLWK FROORLGDO VROLGV WKH ILOWUDWLRQ RI WKH OHDFKDWHV SUREDEO\ UHPRYHG D ODUJH SRUWLRQ RI WKLV ELQGLQJ FDSDFLW\ 3DUN HW DO f 7KLV ZDV GHPRQVWUDWHG E\ D URXJKO\ b GHFUHDVH LQ WRWDO KDUGQHVV FRQFHQWUDWLRQV IRU VLWH DQG DIWHU ILOWUDWLRQ EXW IRU VLWHV DQG RQO\ D WR b GHFUHDVH ZDV UHSRUWHG $OWKRXJK WR GLIIHULQJ GHJUHHV WKH '2& FRQFHQWUDWLRQV RI WKH 06: ODQGILOO OHDFKDWHV LQFUHDVHG IROORZLQJ FDWLRQ H[FKDQJH WUHDWPHQW 7KLV LQFUHDVH ZDV XQH[SHFWHG DQG SUREDEO\ DQ DUWLIDFW RI WKH UHVLQ FDXVHG E\ LQVXIILFLHQW ZDVKLQJ RI WKH 'RZH[r1 PDWHULDO 2WKHU ODQGILOO FKDUDFWHULVWLFV LQIOXHQFH PHWDO VSHFLDWLRQ DQG ELRDYDLODELOLW\ )RU H[DPSOH WKH DHURELFDQDHURELF SURILOH LQ D ODQGILOO GLUHFWO\ UHJXODWHV PHWDO PRELOLW\ /DQGILOOV DUH RSHUDWHG WR PD[LPL]H DQDHURELF FRQGLWLRQV IRU WKH VWDELOL]DWLRQ RI ZDVWH PDWHULDOV GXULQJ DFLGLF PHWKDQRJHQLF DQG KXPLF SKDVHV RI GHJUDGDWLRQ %LRUHDFWRU ODQGILOOV WKDW PD[LPL]H DHURELF FRQGLWLRQV IRU ZDVWH

PAGE 195

7DEOH &RHIILFLHQWV RI GHWHUPLQDWLRQ 5f REWDLQHG EHWZHHQ 06: ODQGILOO OHDFKDWH FKDUDFWHULVWLFV DQG WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ +0%&f IRU FRSSHU PHUFXU\ DQG ]LQF ZLWK VWURQJ UHODWLRQVKLSV LQ EROG +0%&&Xr +0%&+Jr +0%&=Qr 3UHILOWUDWLRQ 76D $ONDOLQLW\ '2&E +DUGQHVV 3RVWILOWUDWLRQ 7'6& $ONDOLQLW\ '2& +DUGQHVV 3RVW'($( $ONDOLQLW\ '2& +DUGQHVV 3RVW'RZH[ $ONDOLQLW\ '2& +DUGQHVV $EEUHYLDWLRQV r76 WRWDO VROLGV f'2& GLVVROYHG RUJDQLF FDUERQ &7'6 WRWDO GLVVROYHG VROLGV 7RWDO VROLGV ZHUH GHWHUPLQHG DIWHU GU\LQJ DW r& DQG WRWDO GLVVROYHG VROLGV DIWHU ILOWUDWLRQ DQG GU\LQJ DW r& VWDELOL]DWLRQ DUH D UHODWLYHO\ QHZ FRQFHSW 5HLQKDUW DQG 7RZQVHQG f 6RPH UHVHDUFKHUV KDYH K\SRWKHVL]HG WKDW WUDGLWLRQDO DQDHURELF ODQGILOO FRQGLWLRQV WUDQVLWLRQ WR DHURELF GXULQJ WKH ODWH KXPLF SKDVH RI ZDVWH VWDELOL]DWLRQ %R]NXUW HW DO f 8QGHU WKHVH DHURELF FRQGLWLRQV PHWDO PRELOLW\ DQG ELRDYDLODELOLW\ LQFUHDVH 0DUWHQVVRQ HW DO 9DQ 5\VVHQ HW DO f *HQHUDO WHQGHQFLHV LQ WKH GLUHFWLRQ RI WKH UHODWLRQVKLSV EHWZHHQ SK\VLFRn FKHPLFDO SDUDPHWHUV LQ WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG DQG WKH +0%& IRU FRSSHU ]LQF DQG PHUFXU\ ZHUH HYDOXDWHG E\ SRROLQJ WKH GDWD IURP WKH IRXU VLWHV 7DEOH VXPPDUL]HV WKH FRHIILFLHQWV RI GHWHUPLQDWLRQ WKDW ZHUH

PAGE 196

REWDLQHG ,Q WKH ZKROH EHIRUH ILOWUDWLRQf 06: ODQGILOO OHDFKDWHV WKH WRWDO VROLGV FRQWHQW PDGH WKH KLJKHVW FRQWULEXWLRQ WR WKH PHWDO ELQGLQJ FDSDFLW\ ZLWK 5 YDOXHV RI DQG IRU +0%&&Xr +0%&+Jr DQG +0%&=Qr UHVSHFWLYHO\ 7KH UHPRYDO RI WKH VXVSHQGHG VROLGV IURP WKH 06: ODQGILOO OHDFKDWHV E\ ILOWUDWLRQ SPf GLG QRW LPSDFW WKH FRQWULEXWLRQ RI VROLGV WR WKH +0%& IRU FRSSHU 5 f DQG ]LQF 5 f ,Q FRQWUDVW WKH UHPRYDO RI VXVSHQGHG VROLGV GHFUHDVHG WKH UHODWLRQVKLS 5 f 6WURQJ DVVRFLDWLRQV ZHUH LGHQWLILHG EHWZHHQ DQLRQLF VSHFLHV HJ DONDOLQLW\ DQG '2& DQG +0%& &X DQG +0%&=Q EXW ZHUH ORZHU IRU +0%&+Jr 7KHUH ZHUH GLUHFW UHODWLRQVKLSV EHWZHHQ WKH UHPRYDO RI DONDOLQLW\ IURP WKH 06: ODQGILOO OHDFKDWHV DQG GHFUHDVHG +0%&&Xr 5 f DQG +0%&=Qr 5 f $ ORZHU UHODWLRQVKLS ZDV GHPRQVWUDWHG EHWZHHQ WKH UHPRYDO RI DONDOLQLW\ DQG +0%&+J 5 f :LWK WKH UHGXFWLRQ RI '2& LQ WKH 06: ODQGILOO OHDFKDWHV WKH DVVRFLDWLRQ ZLWK +0%&=Qr 5 f ZDV KLJK 7KH UHODWLRQVKLSV ZLWK +0%& &X 5 f DQG '2& ZDV ORZHU WKDQ ZLWK WKH RWKHU DQLRQLF SDUDPHWHU DONDOLQLW\ 7KH DVVRFLDWLRQ EHWZHHQ '2& DQG +0%&+Jr 5 f ZDV ORZ DQG FRPSDUDEOH WR WKDW SUHYLRXVO\ VKRZQ ZLWK DONDOLQLW\ 7KH UHODWLRQVKLSV EHWZHHQ KDUGQHVV DQG WKH KHDY\ PHWDO ELQGLQJ FDSDFLW\ ZLWK FRSSHU ]LQF DQG PHUFXU\ GLYHUJH IURP WKRVH SUHYLRXVO\ VWDWHG ZLWK WKH RWKHU OHDFKDWH SDUDPHWHUV :KLOH WKH +0%&&Xr 5 f UHPDLQV VWURQJ WKHUH ZDV QR UHODWLRQVKLS ZLWK +0%&=Q 5 f DQG KDUGQHVV UHPRYDO ,QVWHDG WKH +0%&+Jr 5 f GLVSOD\HG WKH KLJKHVW DVVRFLDWLRQ ZLWK KDUGQHVV

PAGE 197

$ 6ROLGV b % 6ROLGV b 2WKHU b +DUGQHVV 2UJDQLFV b b ( + 6ROLGV b 2WKHU b F +DUGQHVV b b 6ROLGV +DUGQHVV b 2UJDQLFV b )LJXUH (IIHFW RI OHDFKDWH WUHDWPHQW E\ ILOWUDWLRQ 6ROLGVf '($( UHVLQ 2UJDQLFVf DQG 'RZH[ UHVLQ +DUGQHVVf RQ WKH +0%& $ 6LWH ZLWK &XW % 6LWH ZLWK +J & 6LWH ZLWK =Q 6LWH ZLWK &Xr ( 6LWH ZLWK +Jr ) 6LWH ZLWK =Q 6LWH ZLWK &X + 6LWH ZLWK +JW 6LWH ZLWK =Q

PAGE 198

7KH ELQGLQJ DIILQLW\ IRU WKH 06: ODQGILOO OHDFKDWHV ZLWK KHDY\ PHWDOV IROORZHG WKH RUGHU RI FRSSHU PHUFXU\ !]LQF 7KLV DJUHHV ZLWK WKH UHVXOWV RI WKH FRUUHODWLYH DQDO\VLV ZKLFK GHPRQVWUDWHG WKDW WKH ELQGLQJ RI FRSSHU ZLWK WKH 06: ODQGILOO OHDFKDWHV ZDV VWURQJO\ DVVRFLDWHG ZLWK WRWDO VROLGV WRWDO GLVVROYHG VROLGV '2& DONDOLQLW\ DQG KDUGQHVV $OWKRXJK WKHUH ZDV D VWURQJ DVVRFLDWLRQ EHWZHHQ WKH ELQGLQJ RI WKH 06: ODQGILOO OHDFKDWHV IRU PHUFXU\ DQG WRWDO VROLGV 5 f WKH UHPRYDO RI WKH VXVSHQGHG VROLGV UHGXFHG WKLV UHODWLRQVKLS 5 f )XUWKHUPRUH WKHUH ZHUH RQO\ ZHDNHU UHODWLRQVKLSV EHWZHHQ '2& 5 f DQG DONDOLQLW\ 5 f ZLWK WKH ELQGLQJ FDSDFLW\ IRU PHUFXU\ +RZHYHU WKH KDUGQHVV 5 f OHYHOV LQ WKH 06: ODQGILOO OHDFKDWHV ZHUH FRUUHODWHG ZLWK +0%&+J &RPSDUDWLYHO\ WKH DVVRFLDWLRQ EHWZHHQ ]LQF ELQGLQJ DQG WRWDO VROLGV FRQWHQW RI WKH 06: ODQGILOO OHDFKDWHV ZDV WKH ORZHVW IRU WKH WKUHH KHDY\ PHWDOV =LQF ELQGLQJ ZDV VWURQJO\ DVVRFLDWHG ZLWK DONDOLQLW\ 5 f DQG '2& 5 f LQ WKH 06: ODQGILOO OHDFKDWHV EXW QRW ZLWK KDUGQHVV 5 f ,Q VXPPDU\ WKH +0%& RI WKH 06: ODQGILOO OHDFKDWHV ZDV KLJK DWWULEXWDEOH WR YDULRXV FKHPLFDO FKDUDFWHULVWLFV RI WKH OHDFKDWHV DQG ZDV QRW FRPSOHWHO\ H[SODLQHG E\ WKH LQYHVWLJDWHG FKDUDFWHULVWLFV )LJXUH f

PAGE 199

&+$37(5 ,'(17,)<,1* 72;,&,7< ,1 )/25,'$ 06: /$1'),// /($&+$7(6 :,7+ $ 72;,&,7< ,'(17,),&$7,21 $1' (9$/8$7,21 7,(f 352&('85( ,QWURGXFWLRQ ,Q WKH 8QLWHG 6WDWHV (QYLURQPHQWDO 3URWHFWLRQ $JHQF\ 86(3$f SXEOLVKHG D VHULHV RI PHWKRGV IRU WKH GHWHUPLQDWLRQ RI DTXDWLF WR[LFLW\ 7KHVH PHWKRGV FRPELQHG FKHPLFDO DQG SK\VLFDO IUDFWLRQDWLRQ SURFHGXUHV ZLWK ELRORJLFDO DVVD\V 86(3$ Df ,QLWLDOO\ WKHVH PHWKRGV ZHUH GHYHORSHG DV D WRRO IRU ZDVWHZDWHU WUHDWPHQW SODQW RSHUDWRUV DQG ZHUH XVHG LQ WKH LGHQWLILFDWLRQ RI SUHWUHDWPHQW VWUDWHJLHV SULRU WR HIIOXHQW GLVFKDUJH 7KH JRDO RI WKHVH PHWKRGV ZDV WR PLQLPL]H WKH SRWHQWLDO IRU DGYHUVH LPSDFWV WR DTXDWLF VSHFLHV LQ UHFHLYLQJ ZDWHUV :KHQ LQYHVWLJDWLQJ DTXDWLF WR[LFLW\ HIIOXHQW GLVFKDUJHUV DUH UHTXLUHG WR FRQGXFW D WKUHHSKDVH WR[LFLW\ UHGXFWLRQ HYDOXDWLRQ 75(f WR H[SORUH RSWLRQV IRU WKH UHGXFWLRQ RU UHPRYDO RI WDUJHW FRPSRXQGV 7KH LQLWLDO RU 3KDVH VHJPHQW RI WKH LQYHVWLJDWLRQ UHTXLUHV WKH SK\VLFDOFKHPLFDO FKDUDFWHUL]DWLRQ RI WKH VXVSHFW VDPSOH ZKLOH GXULQJ 3KDVH ,, RSWLRQV DUH H[SORUHG IRU WKH UHPRYDO RI WKH WR[LF VXEVWDQFH RU WKH LGHQWLILFDWLRQ RI LWnV VRXUFH )LQDOO\ 3KDVH ,,, PRQLWRUV WR[LFLW\ XVLQJ FKHPLFDO DQDO\VLV DQG ELRORJLFDO DVVD\V

PAGE 200

7KH ILUVW SKDVH 3KDVH ,f RI WKH 75( GHVFULEHV WKH SURWRFROV IRU D WR[LFLW\ LGHQWLILFDWLRQ DQG HYDOXDWLRQ 7,(f ZKLFK KDV WKUHH GLVWLQFW FRPSRQHQWV 7KH 7,( 3KDVH LQFOXGHV PHWKRGV IRU WKH FKDUDFWHUL]DWLRQ RI WKH VXVSHFWHG WR[LF VXEVWDQFHVf 86(3$ Df 3KDVH ,, GHVFULEHV PHWKRGV IRU WKH LGHQWLILFDWLRQ RI WR[LF VXEVWDQFHVf DQG LV OLPLWHG WR QRQSRODU RUJDQLFV DPPRQLD DQG KHDY\ PHWDOV 86(3$ Ef 3KDVH ,,, FRQILUPV WKH UHVXOWV REWDLQHG GXULQJ WKH 3KDVH DQG 3KDVH ,, SURFHGXUHV 86(3$ Ff 3KDVH LQYHVWLJDWLRQV FRPELQH WKH FKHPLFDO DQG SK\VLFDO FKDUDFWHUL]DWLRQ RI D VDPSOH ZLWK WKH UHVXOWV RI ELRORJLFDO DVVD\V WR LGHQWLI\ WKH VDPSOH IUDFWLRQV ZLWK DOWHUHG WR[LFLW\ SURILOHV 7KHVH IUDFWLRQDWLRQ SURFHGXUHV LQFOXGH S+ DGMXVWPHQW DHUDWLRQ ILOWUDWLRQ DQG VROLG SKDVH H[WUDFWLRQ 63(f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

PAGE 201

WHVW RUJDQLVPV DW WKH LQLWLDWLRQ RI WKH DVVD\ &RRPEH HW DO f 7KH DTXDWLF LQYHUWHEUDWH &HULRGDSKQLD GXELD LV XQLTXHO\ VXLWHG IRU XVH LQ 7,( LQYHVWLJDWLRQV 1RUEHUJ.LQJ HW DO f GXH WR LWV VHQVLWLYLW\ SRSXODWLRQ VWUXFWXUH DVH[XDO UHSURGXFWLRQf DQG HDVH IRU FXOWXULQJ 86(3$ Df 7KH 7,( SURFHGXUH KDV EHHQ DSSOLHG WR WKH PRQLWRULQJ RI DPELHQW ZDWHU TXDOLW\ 6WURQNKRUVW HW DO f DQG IRU DVVHVVLQJ WKH HIIHFWLYHQHVV RI UHPHGLDWLRQ WHFKQRORJLHV 'HDQRYLF HW DO f 5HFHQWO\ WKH 7,( KDV EHHQ H[SDQGHG IRU XVH DV D WRRO IRU LGHQWLI\LQJ VXEVWDQFHV FDSDEOH RI LQGXFLQJ KRUPRQHOLNH HIIHFWV LQ GRPHVWLF ZDVWHZDWHUV 'HVEURZHW DO f DQG VXUIDFH ZDWHUV 7KRPDV HW DO f 0XQLFLSDO VROLG ZDVWH 06:f ODQGILOO OHDFKDWHV DUH D E\SURGXFW RI ZDVWH VWDELOL]DWLRQ SURFHVVHV DQG WKHLU FRPSRVLWLRQ LV FRPSOH[ ZLWK KLJK FRQFHQWUDWLRQV RI ERWK RUJDQLF DQG LQRUJDQLF VXEVWDQFHV .MHOGVHQ HW DO f 7KHVH OHDFKDWHV DUH JHQHUDWHG DV UDLQIDOO LQILOWUDWHV WKH ODQGILOO DQG SHUFRODWHV WKURXJK WKH ZDVWH PDWHULDOV 0RGHUQ ODQGILOOV DUH OLQHG DQG FRQWDLQ V\VWHPV IRU WKH FROOHFWLRQ RI OHDFKDWHV ZKLFK DUH WKHQ WUHDWHG DW RQn VLWH IDFLOLWLHV RU PRUH IUHTXHQWO\ DW RIIVLWH GRPHVWLF ZDVWHZDWHU WUHDWPHQW SODQWV ::73f 7KHUHIRUH WR[LF VXEVWDQFHV LQ OHDFKDWHV KDYH WKH SRWHQWLDO WR GLVUXSW ELRORJLFDO WUHDWPHQW SURFHVVHV DQG PD\ LPSDFW WKH TXDOLW\ RI ::73 HIIOXHQWV 7KLV PDNHV WKH 06: ODQGILOO OHDFKDWHV D SULPH FDQGLGDWH IRU D 7,( LQYHVWLJDWLRQ KRZHYHU WKH KHWHURJHQHRXV FKHPLFDO FRPSRVLWLRQ DQG YDULDEOH WR[LFLW\ PXVW EH WDNHQ LQWR FRQVLGHUDWLRQ ZKHQ GHVLJQLQJ WKH IUDFWLRQDWLRQ SURWRFRO :DUG HW DO f

PAGE 202

7DEOH 3RSXODWLRQ VHUYHG DQG DPRXQW RI ZDVWH ODQGILOOHG DV D SHUFHQW RI WRWDO ZDVWH JHQHUDWHG DW VLWHV DQG 6LWH 3RSXODWLRQ /DQGILOOHG :DVWH WRQVf b RI WRWDOf bf bf bf 7KH REMHFWLYH RI WKH SUHVHQW ZRUN ZDV WR WHQWDWLYHO\ LGHQWLI\ WKH FODVV RU FODVVHV RI WR[LF VXEVWDQFHV LQ 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP WKUHH VLWHV LQ )ORULGD 7KH WR[LFLW\ RI WKH ZKROH 06: OHDFKDWHV ZDV HYDOXDWHG ZLWK ERWK DFXWH DQG FKURQLF DVVD\V 7KH UHVXOWV RI WKHVH DVVD\V VHUYHG DV D EDVLV IRU WKH VXEVHTXHQW FKHPLFDOSK\VLFDO IUDFWLRQDWLRQ 'XULQJ WKH 7,( IUDFWLRQDWLRQ WKH KU &HULRGDSKQLD GXELD DQG PLQXWH 0LFURWR[r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f $ / VDPSOH RI 06: ODQGILOO OHDFKDWH ZDV FROOHFWHG IURP WKH OHDFKDWH FROOHFWLRQ VXPS DW VLWHV DQG XVLQJ D 7HIORQ EDOHU KRZHYHU DW VLWH D FROOHFWLRQ VXPS ZDV QRW DFFHVVLEOH

PAGE 203

7KHUHIRUH WKH OHDFKDWH IURP VLWH ZDV FROOHFWHG IURP D GLVFKDUJH SLSH WKDW WUDQVSRUWHG WKH OHDFKDWH WR DQ HYDSRUDWLRQ SRQG %\ KROGLQJ WKH LQGLYLGXDO VDPSOLQJ FRQWDLQHUV XQGHU WKH SLSH RQO\ OHDFKDWH ZDV FROOHFWHG DQG QRW UDLQZDWHU WKDW KDG PL[HG ZLWK WKH OHDFKDWH LQ WKH HYDSRUDWLRQ SRQG 6DPSOHV IRU FKHPLFDO DQDO\VLV ZHUH FROOHFWHG LQ SRO\HWK\OHQH RU JODVV FRQWDLQHUV DQG SUHVHUYHG DFFRUGLQJ WR 86 (QYLURQPHQWDO 3URWHFWLRQ $JHQF\ 86(3$f86(3$ Ef SURWRFROV ZKLOH VDPSOHV IRU WR[LFLW\ DQDO\VLV ZHUH FROOHFWHG LQ SODVWLF FRQWDLQHUV DQG ZHUH QRW SUHVHUYHG $OO VDPSOHV ZHUH WUDQVSRUWHG WR WKH ODE RQ LFH DQG LPPHGLDWHO\ VWRUHG DW r& $OO ELRORJLFDO DVVD\V ZHUH LQLWLDWHG XSRQ DUULYDO RI WKH VDPSOH LQ WKH ODERUDWRU\ &KHPLFDO $QDO\VLV RI 06: /DQGILOO /HDFKDWHV 7KH WKUHH 06: ODQGILOO OHDFKDWHV ZHUH FKDUDFWHUL]HG IRU WKHLU FKHPLFDO DQG SK\VLFDO FRPSRVLWLRQ )LHOG PHDVXUHPHQWV LQFOXGHG S+ DQG WHPSHUDWXUH 2ULRQ 0RGHO $f FRQGXFWLYLW\ +DQQD ,QVWUXPHQWV 0RGHO +f GLVVROYHG R[\JHQ '2f<6, ,QF 0RGHO )7f DQG R[LGDWLRQUHGXFWLRQ SRWHQWLDO 253f $FFXPHW &R 0RGHO f ,Q WKH ODERUDWRU\ VDPSOHV ZHUH DQDO\]HG IRU DONDOLQLW\ FDUERQDFHRXV ELRFKHPLFDO R[\JHQ GHPDQG &%2'f WRWDO GLVVROYHG VROLGV 7'6f DQG VXOILGHV DFFRUGLQJ WR PHWKRGV GHVFULEHG E\ 86(3$ Ef DQG $3+$ f $PPRQLD ZDV PHDVXUHG ZLWK DQ LRQ VHOHFWLYH HOHFWURGH ,6( 2ULRQf DFFRUGLQJ WR WKH PDQXIDFWXUHUnV LQVWUXFWLRQV 7KH ,6( PHDVXUHV WRWDO DPPRQLD 1+91+f FRQFHQWUDWLRQV IROORZLQJ WKH DGMXVWPHQW RI OHDFKDWH

PAGE 204

3KDVH 0DQLSXODWLRQ 6XVSHFWHG 7R[LFDQWVf S+ DGMXVWPHQW ORQL]DEOH FRPSRXQGV )LOWUDWLRQ 3DUWLFOHDVVRFLDWHG FRPSRXQGV 6ROLG SKDVH H[WUDFWLRQ 63(f 1RQSRODU RUJDQLF FRPSRXQGV $HUDWLRQ 9RODWLOH RU R[LGL]DEOH FRPSRXQGV 0HW3/$7( +HDY\ PHWDOV S+ WR ZLWK 1 1D2+ ZKLFK FRQYHUWV DQ\ LRQL]HG DPPRQLD VSHFLHV 1+f WR WKH XQLRQL]HG 1+f IRUP 7KH ,6( ZDV FDOLEUDWHG EHIRUH HDFK XVH ZLWK D ILYH SRLQW FDOLEUDWLRQ FXUYH $FFRUGLQJ WR WKH PDQXIDFWXUHU WKH XQLRQL]HG 1+f DPPRQLD FRQFHQWUDWLRQ FRXOG EH GHWHUPLQHG ZLWK WKH ,6( SULRU WR WKH S+ DGMXVWPHQW WR 2ULRQ WHFKQLFDO VXSSRUW SHUVRQDO FRPPXQLFDWLRQf 6DPSOHV IRU WRWDO PHWDO FRQWHQW ZHUH GLJHVWHG 86(3$ 0HWKRG 6: $f86(3$ f DQG DQDO\]HG E\ DWRPLF HPLVVLRQ VSHFWURVFRS\ $(6f 7KHUPR -DUUHOO $VK 0RGHO (QYLUR f 'LVVROYHG R[\JHQ '2 <6, ,QF 0RGHO f S+ DQG WHPSHUDWXUH $FFXPHW )LVKHU 6FLHQWLILF 0RGHO f DQG HOHFWULFDO FRQGXFWLYLW\ +DQQD ,QVWUXPHQWV 0RGHO +f ZHUH PRQLWRUHG LQ WKH 3 VXEFDSLWDWD DQG & GXELD DVVD\V

PAGE 205

7,( 3URFHGXUH 3KDVH 7KH LQLWLDO SKDVH 3KDVH ,f RI WKH 7,( SURWRFRO UHTXLUHG WKH IUDFWLRQDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV E\ FKHPLFDO DQG SK\VLFDO PDQLSXODWLRQV IRU WKH FKDUDFWHUL]DWLRQ RI WR[LF VXEVWDQFHV 7DEOH f 7KH OHDFKDWH IUDFWLRQV ZHUH JHQHUDWHG E\ VDPSOH PDQLSXODWLRQV 7KH PDQLSXODWLRQV LQFOXGHG S+ DGMXVWPHQW WR S+ $'-f DQG S+ $'-f S+ DGMXVWPHQW DQG ILOWUDWLRQ S+ ),/7 DQG S+ ),/7f S+ DGMXVWPHQW DQG DHUDWLRQ S+ $(5 DQG S+ $(5f DQG VROLG SKDVH H[WUDFWLRQ 63(f S+ 63( DQG S+ 63(f 86(3$ Ef 7KH LQWHJULW\ RI WKH 63( FROXPQV PD\ EH FRPSURPLVHG DW S+ YDOXHV JUHDWHU WKDQ WKHUHIRUH WKH S+ OHDFKDWHV ZHUH DGMXVWHG WR D S+ YDOXH RI 86(3$ Ef 6LPXOWDQHRXVO\ D WKLUG SRUWLRQ RI HDFK 06: ODQGILOO OHDFKDWH ZDV DOVR PDQLSXODWHG DV GHVFULEHG IRU WKH S+ DQG S+ IUDFWLRQV +RZHYHU WKH S+ RI WKLV SRUWLRQ ZDV PDLQWDLQHG DW WKH S+ YDOXH LQ WKH OHDFKDWH XSRQ FROOHFWLRQ DQG ZDV VXEVHTXHQWO\ UHIHUUHG WR DV WKH LQLWLDO S+ S+cf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

PAGE 206

S+Lf DQG D WKLUG DOLTXRW RI PO ZDV ORZHUHG WR S+ ZLWK 1 +&, $ PO DOLTXRW RI WKH S+ DGMXVWHG OHDFKDWH DQG WKH S+ DGMXVWHG OHDFKDWH ZHUH VHW DVLGH IRU WKH SRVWS+ DGMXVWPHQW WR[LFLW\ WHVW 7KH UHPDLQLQJ S+ DQG S+ OHDFKDWHV IURP VLWHV DQG ZHUH GLYLGHG LQWR D PO DOLTXRW IRU WKH S+DGMXVWHG DHUDWLRQ PDQLSXODWLRQ DQG PO DOLTXRWV IRU WKH S+DGMXVWHG ILOWUDWLRQ VWHS 7KH 06: ODQGILOO OHDFKDWHV DW S+ ZHUH VHSDUDWHG LQWR WZR DOLTXRWV RI PO IRU WKH DHUDWLRQ DQG PO IRU ILOWUDWLRQ PDQLSXODWLRQ )LOWUDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV 6ROLGVDVVRFLDWHG WR[LFLW\ PD\ EH UHPRYHG E\ ILOWUDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV 7KH OHDFKDWH ILOWUDWLRQ XWLOL]HG PHPEUDQH ILOWHUV XP *HOPDQf SUHFRQGLWLRQHG ZLWK S+ S+c RU S+ DGMXVWHG '', 1DQRSXUH %DUQVWHDGf 7KH S+DGMXVWHG S+ S+ LQLWLDO DQG S+ f OHDFKDWHV IURP WKH WKUHH VLWHV ZHUH ILOWHUHG XQGHU YDFXXP POPLQf )ROORZLQJ ILOWUDWLRQ WKH S+ ODQG S+ OHDFKDWHV ZHUH GLYLGHG LQWR PO DOLTXRWV IRU WKH SRVWn ILOWUDWLRQ WR[LFLW\ WHVW DQG PO DOLTXRWV IRU SDVVDJH WKURXJK WKH 63( FROXPQV 7KH S+ OHDFKDWHV ZHUH DOVR VHSDUDWHG IRU WKH SRVWILOWUDWLRQ WR[LFLW\ WHVW POf D PO DOLTXRW IRU 63( WUHDWPHQW DQG D PO DOLTXRW IRU =HROLWH WUHDWPHQW 6ROLG SKDVH H[WUDFWLRQ RI WKH 06: ODQGILOO OHDFKDWHV 6ROLG SKDVH H[WUDFWLRQ WHFKQLTXHV ZHUH HPSOR\HG IRU WKH UHPRYDO RI QRQSRODU RUJDQLF WR[LFDQWV IURP WKH OHDFKDWHV 7KH 63( FROXPQV & PJ (;75$6(3 /LGD 0DQXIDFWXULQJ %HQVHQYLOOH &2f ZHUH SODFHG RQ WKH 63( UHVHUYRLU 9LVLSUHS SRUW 6XSHOFR %HOOHIRQWH 1
PAGE 207

UHVHUYRLU ZDV DWWDFKHG WR D YDFXXP ZLWK D IORZ UDWH RI POPLQ 7KH VROLG SKDVH H[WUDFWLRQ FROXPQV ZHUH FRQGLWLRQHG ZLWK a PO RI PHWKDQRO 7KH PHWKDQRO ZDV ORDGHG RQWR WKH FROXPQ DQG IROORZLQJ FRQGLWLRQLQJ WKH FROXPQV UHPDLQHG VDWXUDWHG 7KH LQWHJULW\ RI WKH 63( FROXPQ LV VHQVLWLYH WR S+ YDOXHV JUHDWHU WKDQ WKHUHIRUH WKH S+ VDPSOH ZDV DGMXVWHG WR D S+ YDOXH RI 86(3$ Ef $IWHU FRQGLWLRQLQJ HDFK RI WKH 63( FROXPQV ZDV ZDVKHG ZLWK S+DGMXVWHG S+ S+c RU S+ f '', ZDWHU DV LQGLFDWHG 7KH S+ S+c DQG S+ DGMXVWHG OHDFKDWHV ZHUH ORDGHG RQWR WKHLU UHVSHFWLYH FROXPQV $ PO DOLTXRW RI HDFK S+DGMXVWHG RU S+L OHDFKDWH ZDV SDVVHG WKURXJK WKH FROXPQ DQG WKHQ WKH QH[W PO SRUWLRQ WKDW SDVVHG WKURXJK WKH FROXPQ ZDV FROOHFWHG IRU WHVWLQJ $GGLWLRQDOO\ D PO SRUWLRQ ZDV DJDLQ FROOHFWHG DIWHU D WRWDO RI PO RI WKH S+DGMXVWHG OHDFKDWHV RU S+c OHDFKDWH KDG SDVVHG WKURXJK WKH FROXPQ $OO IUDFWLRQV ZHUH KHOG IRU WKH SRVW63( WR[LFLW\ WHVWV $HUDWLRQ RI WKH 06: ODQGILOO OHDFKDWHV $HUDWLRQ UHPRYHV RU UHGXFHV YRODWLOH WR[LFDQWV 7KH PO DOLTXRWV RI S+ S+ RU S+c OHDFKDWHV SUHYLRXVO\ VHW DVLGH GXULQJ WKH S+ DGMXVWPHQW PDQLSXODWLRQ ZHUH XWLOL]HG IRU WKLV VWHS 7KH S+DGMXVWHG DQG S+c OHDFKDWHV ZHUH WUDQVIHUUHG WR LQGLYLGXDO PO JODVV JUDGXDWHG F\OLQGHUV WR ZKLFK ZDV DGGHG D VPDOO DLU VWRQH 7KH S+ RI HDFK OHDFKDWH ZDV FKHFNHG SULRU WR WKH LQLWLDWLRQ RI WKH DHUDWLRQ SURFHGXUH DQG IRXU WLPHV GXULQJ WKH RQHKRXU WUHDWPHQW $GMXVWPHQW ZLWK 1 1 1 1D2+ RU +&, PDLQWDLQHG VDPSOH S+ ZLWKLQ S+ XQLWV 7KH OHDFKDWH IUDFWLRQV ZHUH KHOG IRU WKH SRVWDHUDWLRQ WR[LFLW\ WHVW

PAGE 208

%ODQN SUHSDUDWLRQ )UDFWLRQDWLRQ EODQNV ZHUH SUHSDUHG ZLWK PRGHUDWHO\ KDUG ZDWHU 0+:f DFFRUGLQJ WR 86(3$ Df VHH &KDSWHU f %ULHIO\ WKH ZDWHU KDG WKH IROORZLQJ VSHFLILFDWLRQV S+ KDUGQHVV PJ/ DV &D&&! DQG DONDOLQLW\ PJ/ 86(3$ Df 7KH 0+: IUDFWLRQDWLRQ EODQNV ZHUH JHQHUDWHG SDUDOOHO WR WKH OHDFKDWH IUDFWLRQV ZLWK D PO DOLTXRW DW S+ D PO DOLTXRW DW S+c DQG D PO DOLTXRW DW S+ $ PO DOLTXRW RI ERWK WKH S+ DQG S+ DGMXVWHG 0+: ZHUH VHW DVLGH IRU WKH S+DGMXVWPHQW WR[LFLW\ WHVWLQJ DV WKH S+ DGMXVWPHQW EODQNV 7KH S+ S+c DQG S+ DGMXVWHG 0+: ZDV WKHQ SDVVHG WKURXJK WKHLU UHVSHFWLYH SP ILOWHUV *1 0HWULFHOf SULRU WR SDVVDJH RI WKH S+DGMXVWHG OHDFKDWH IUDFWLRQV 7KH ILOWHUHG S+ S+c DQG S+ DGMXVWHG 0+: ZDV GLYLGHG LQWR PO DOLTXRWV IRU WKH SRVWILOWUDWLRQ WR[LFLW\ WHVW DQG PO DOLTXRWV IRU SDVVDJH WKURXJK WKH 63( FROXPQV 7KH PO DOLTXRWV RI S+ DGMXVWHGILOWHUHG 0+: ZHUH SDVVHG WKURXJK WKHLU UHVSHFWLYH FROXPQV ZLWK WKH ILQDO PO SRUWLRQ FROOHFWHG IRU WKH SRVW63( WR[LFLW\ WHVW 7KHQ WKH PO DOLTXRWV RI S+ S+c DQG S+ DGMXVWHG 0+: VHW DVLGH SUHYLRXVO\ ZHUH SODFHG LQ PO JODVV JUDGXDWHG F\OLQGHUV ZLWK D VPDOO DLU VWRQH 7KH SRUWLRQV RI S+ DQG DGMXVWHG DQG WKH S+c 0+: ZHUH DHUDWHG IRU KRXU ZLWK S+ PDLQWDLQHG ZLWKLQ S+ XQLWV IURP WKH VWDUWLQJ S+ RI RU LQLWLDO $W WKH HQG RI GD\ DOO RI WKH S+ DQG S+ DGMXVWHG OHDFKDWH VDPSOHV ZHUH UHWXUQHG WR WKH LQLWLDO S+ E\ DGGLWLRQ RI 1 1 1

PAGE 209

1D2+ RU +&, 7KH S+ UHDGMXVWHG IUDFWLRQV IRU WKH WKUHH OHDFKDWHV ZHUH VWRUHG XQGHU UHIULJHUDWLRQ DW r& RYHUQLJKW IRU SRVWPDQLSXODWLRQ WR[LFLW\ DVVD\V RQ GD\ =HROLWH WHVW :KHQ LGHQWLI\LQJ DPPRQLD DV D SRVVLEOH WR[LFDQW GXULQJ WKH 7,( SKDVH ,, D =HROLWH WUHDWPHQW LV LQFOXGHG DV D SDUW RI WKH LQYHVWLJDWLRQ $ PO DOLTXRW RI 06: ODQGILOO OHDFKDWH IURP VLWHV DQG ZDV SDVVHG WKURXJK LQGLYLGXDO FDWLRQ H[FKDQJH FROXPQV 86(3$ Ff 7KH FDWLRQ H[FKDQJH FROXPQV ZHUH SUHSDUHG E\ FRPELQLQJ JUDPV RI D ]HROLWH UHVLQ ZLWK PO RI '', 1DQRSXUH %DUQVWHDGf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

PAGE 210

WKH GLOXWLRQ VHULHV RQ GD\ RI WKH IUDFWLRQDWLRQ SURFHGXUH 7KH DFXWH WR[LFLW\ RI HDFK OHDFKDWH ZDV GHWHUPLQHG ZLWK WKH DTXDWLF LQYHUWHEUDWH &HULRGDSKQLD GXELD ZLWK H[SRVXUHV RI DQG KRXUV 86(3$ Df 7KH SURFHGXUHV IRU WKH FXOWXULQJ RI & GXELD DQG WKH SURWRFROV IRU WKH DFXWH & GXELD WR[LFLW\ DVVD\V DUH GHVFULEHG LQ &KDSWHU 6OLJKW PRGLILFDWLRQV WR WKH & GXELD WR[LFLW\ DVVD\ SURWRFRO ZHUH UHTXLUHG IRU WKH 7,( DQG DUH GHVFULEHG 7KH LQLWLDO GLOXWLRQ VHULHV XWLOL]HG LQ WKH & GXELD DVVD\V ZHUH IURP WR b ZLWK VLWHV DQG DQG IURP WR b ZLWK VLWH ZLWK HDFK FRQFHQWUDWLRQ WHVWHG LQ GXSOLFDWH 0+: ZDV XVHG IRU WKH GLOXWLRQ ZDWHU DQG WKH FRQWURO ZDWHU $W WKH VWDUW RI HDFK DVVD\ ILYH & GXELD QHRQDWHV KRXUV ROGf ZHUH WUDQVIHUUHG WR PO SODVWLF FXSV DQG WKHQ D PO DOLTXRW RI HDFK OHDFKDWH GLOXWLRQ ZDV DGGHG WR WKH FXS (DFK DVVD\ LQFOXGHG D GXSOLFDWH FRQWURO ZLWK QHRQDWHV LQ PO RI 0+: 7KH DVVD\ FXSV ZHUH SODFHG LQ D ZDWHU EDWK DW r & XQGHU DPELHQW OLJKWLQJ 1HRQDWHV ZHUH IHG SULRU WR XVH LQ WKH DVVD\ EXW QRW GXULQJ WKH WHVWLQJ 7KH LQLWLDO 06: OHDFKDWH WR[LFLW\ ZDV DOVR HYDOXDWHG E\ WKH 0LFURWR[r1 DFXWH WR[LFLW\ DVVD\ XVLQJ WKH EDFWHULD 9LEULR ILVKHUL %HFNPDQ ,QVWUXPHQWV f $ ILIWHHQPLQXWH H[SRVXUH LQWHUYDO ZDV VHOHFWHG EDVHG RQ HDUOLHU UHVHDUFK :DUG HW DO f ,Q WKH 0LFURWR[ DVVD\ WKH 06: ODQGILOO OHDFKDWH WR[LFLW\ ZDV DVVD\HG ZLWKRXW GLOXWLRQ GXH WR WKH ODUJH QXPEHU RI OHDFKDWH IUDFWLRQV +RZHYHU ZKHQ DFFRXQWLQJ IRU WKH GLOXWLRQ RI WKH 06: ODQGILOO OHDFKDWHV E\ WKH EDFWHULDO UHDJHQW WKH KLJKHVW FRQFHQWUDWLRQ DVVD\HG ZDV b

PAGE 211

$GGLWLRQDOO\ IRU WKH GHWHUPLQDWLRQ RI WKH LQLWLDO WR[LFLW\ RI WKH WKUHH ZKROH 06: ODQGILOO OHDFKDWHV D FKURQLF DQG D KHDY\ PHWDO VSHFLILF DVVD\ ZHUH LQFOXGHG 7KH FKURQLF WR[LFLW\ ZDV PHDVXUHG ZLWK WKH JUHHQ DOJD 3VHXGRNLUFKQHULHOOD VXEFDSLWDWD IRUPHUO\ 6HOHQDVWUXP FDSULFRUQXWXPf LQ D KRXU DVVD\ 86(3$ Df 7R[LFLW\ GXH WR WKH SUHVHQFH RI KHDY\ PHWDOV ZDV GHWHUPLQHG ZLWK 0HW3/$7( DQ DVVD\ VSHFLILF IRU KHDY\ PHWDO WR[LFLW\ %LWWRQ HW DOf f 7KH SURWRFROV IRU WKH 3 VXEFDSLWDWD DQG 0LFURWR[r1 DVVD\V KDYH EHHQ GHVFULEHG SUHYLRXVO\ &KDSWHU f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f ,Q WKLV UHVHDUFK WKH KROGLQJ WLPH IRU WKH 06: ODQGILOO OHDFKDWHV ZDV DSSUR[LPDWHO\ KRXUV 7KH EDVHOLQH DVVD\V ZHUH SHUIRUPHG ZLWK WKH KRXU & GXELD DQG WKH PLQXWH 0LFURWR[r1 WR[LFLW\ DVVD\V DV SUHYLRXVO\ GHVFULEHG IRU WKH LQLWLDO WR[LFLW\ DVVD\V 7KH EDVHOLQH WR[LFLW\ DVVD\V ZLWK & GXELD XWLOL]HG WKH VDPH GLOXWLRQ VHULHV DV UHSRUWHG IRU

PAGE 212

WKH LQLWLDO WR[LFLW\ DVVD\V DQG WKHUH ZHUH QR GLOXWLRQV DVVD\HG ZLWK WKH 0LFURWR[r1 WHVW 3RVWPDQLSXODWLRQ WR[LFLW\ DVVD\V $W WKH VWDUW RI GD\ WKH S+ RI WKH 06: ODQGILOO OHDFKDWH IUDFWLRQV IURP VLWHV DQG ZHUH FKHFNHG DQG DGMXVWHG WR ZLWKLQ S+ XQLWV RI WKH LQLWLDO S+ RI WKH OHDFKDWH 7KH WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWH IUDFWLRQV ZDV GHWHUPLQHG ZLWK WKH KRXU & GXELD DQG PLQXWH 0LFURWR[r1 DFXWH DVVD\V DV SUHYLRXVO\ GHVFULEHG 7KH & GXELD DVVD\V XWLOL]HG IRXU OHDFKDWH GLOXWLRQV IURP WR b IRU WKH OHDFKDWHV IURP VLWHV DQG 7KH OHDFKDWH IURP VLWH ZDV DVVD\HG LQ GLOXWLRQV IURP WR b 7KH & GXELD DVVD\V ZHUH FRQGXFWHG ZLWKRXW UHSOLFDWLRQ DQG ZLWK 0+: DV WKH GLOXWLRQ ZDWHU DQG FRQWURO 7KHUH ZHUH QR FKDQJHV WR WKH 0LFURWR[ PHWKRG 'DWD $QDO\VLV 7KH WR[LFLW\ DVVD\ UHVXOWV ZHUH H[SUHVVHG DV WKH OHDFKDWH FRQFHQWUDWLRQ WKDW SURGXFHG D b HIIHFW (&f H J JURZWK LQKLELWLRQ GHDWK RU GHFUHDVHG HQ]\PH DFWLYLW\ ,Q WKH FDVH RI WKH 0HW3/$7( DVVD\ WKH UHVXOWV ZHUH H[SUHVVHG DV WKH LQKLELWLRQ bf WR HQ]\PH DFWLYLW\ IURP WKH ZKROH OHDFKDWH WKHUHIRUH (& YDOXHV ZHUH QRW GHWHUPLQHG *UDSKLFDO LQWHUSRODWLRQ PHWKRGV ZHUH XVHG WR JHQHUDWH WKH (& YDOXHV IRU WKH KRXU 3 VXEFDSLWDWD DQG WKH PLQXWH 0LFURWR[r1 DVVD\V E\ WKH UHODWLRQVKLS EHWZHHQ WKH OHDFKDWH FRQFHQWUDWLRQ DQG WKH DVVD\ HQGSRLQW 'DWD DQDO\VLV VRIWZDUH ZDV XVHG WR GHWHUPLQH WKH (&} IRU WKH & GXELD DVVD\ 86(3$ f $OO (&} UHVXOWV ZHUH WUDQVIRUPHG O22(&}f DQG H[SUHVVHG DV WR[LFLW\ XQLWV 78f D XQLWOHVV PHDVXUH :KHQ

PAGE 213

7DEOH &KHPLFDO DQG SK\VLFDO FKDUDFWHULVWLFV RI WKH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG 3DUDPHWHU 6LWH 6LWH 6LWH S+ &RQGXFWLYLW\ P6FPf $ONDOLQLW\ PJ/ DV &D&f 7'6D J/f +DUGQHVV PJ/f &%2'' PJ/f 7RWDO DPPRQLD PJ/f QK PJ/f %'/ 6RGLXP PJ/f &KORULGH PJ/f 6XOIDWH PJ/f &RSSHUF PJ/f %'/ %'/ %'/ =LQF PJ/f %'/ %'/ 1LFNHO PJ/f %'/ %'/ 'HWHFWLRQ OLPLWV IRU PHWDO DQDO\VLV r PJ/ D PJ/ DQG H PJ/ UHVXOWV DUH H[SUHVVHG DV 78 YDOXHV DQG DQ LQFUHDVH LQ WKH 78 YDOXH FRUUHVSRQGV WR DQ LQFUHDVH LQ WR[LFLW\ ,Q FRQWUDVW WKH (& YDOXHV GHFUHDVH ZLWK LQFUHDVLQJ WR[LFLW\ DV WKH FRQFHQWUDWLRQ RI OHDFKDWH UHTXLUHG WR SURGXFH D b HIIHFW GHFUHDVHV 'DWD FRUUHODWLRQ ZDV GHWHUPLQHG ZLWK WKH 6WXGHQWnV IWHVW DQG E\ OHDVWVTXDUHV UHJUHVVLRQ LQ WKH ([FHO SURJUDP 0LFURVRIW YHUVLRQ f

PAGE 214

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f ZDV KLJKHVW IRU VLWHV DQG DW DQG PJ/ UHVSHFWLYHO\ ZKLOH IRU VLWH WKH &%2' ZDV PJ/ 7KH KDUGQHVV FRQFHQWUDWLRQV DV FDOFLXP DQG PDJQHVLXP ZHUH VLPLODU DW VLWHV DQG DW DQG PJ/ UHVSHFWLYHO\ KRZHYHU DW VLWH WKH KDUGQHVV FRQFHQWUDWLRQ ZDV PJ/ $ VWULNLQJ GLIIHUHQFH ZDV QRWHG ,Q WKH DPPRQLD FRQFHQWUDWLRQV LQ WKH WKUHH 06: ODQGILOO OHDFKDWHV 7KH WRWDO DPPRQLD FRQFHQWUDWLRQ LQ WKH VLWH OHDFKDWH ZDV PJ/ 7KLV ZDV OHVV WKDQ b RI WKH DQG PJ/ RI WRWDO DPPRQLD UHSRUWHG DW VLWHV DQG UHVSHFWLYHO\ $ VLPLODU SDWWHUQ ZDV VKRZQ IRU XQLRQL]HG DPPRQLD FRQFHQWUDWLRQV ZLWK VLWH FRQFHQWUDWLRQV EHORZ WKH GHWHFWLRQ OLPLW RI WKH DVVD\ PJ/f ZKLOH DQG PJ/ ZHUH UHSRUWHG IRU VLWHV DQG UHVSHFWLYHO\ 6LPLODU VRGLXP

PAGE 215

FRQFHQWUDWLRQV ZHUH UHSRUWHG IRU WKH OHDFKDWHV ZLWK D UDQJH IURP WR PJ/ 7KH FKORULGH OHYHOV IRU VLWHV DQG ZHUH DQG PJ/ UHVSHFWLYHO\ ZKLOH WKH VLWH OHYHOV ZHUH ORZHU DW PJ/ &RSSHU FRQFHQWUDWLRQV ZHUH EHORZ WKH GHWHFWLRQ OLPLW PJ/f RI WKH DQDO\WLFDO PHWKRG ZLWK DOO OHDFKDWHV EXW ]LQF ZDV GHWHFWHG DW VLWH DQG QLFNHO DW VLWH DW DQG PJ/ UHVSHFWLYHO\ 'HWHUPLQDWLRQ RI +HDY\ 0HWDO %LRDYDLODELOLW\ 7KH 7,( GHVFULEHV WKH DGGLWLRQ RI WKH PHWDO FKHODWRU HWK\OHQHGLDPLQH WHWUDDFHWLF DFLG ('7$f IRU WKH LGHQWLILFDWLRQ RI PHWDO WR[LFLW\ 86(3$ Df ,W VKRXOG EH UHFRJQL]HG WKDW WKH XVH RI ('7$ LQWURGXFHV WR[LFLW\ ZKLFK PD\ UHVXOW LQ IDOVH SRVLWLYH UHVXOWV .RQJ HW DO f ,Q VRPH FDVHV ('7$ LV QRW HIIHFWLYH LQ WKH UHPRYDO RI PHWDO WR[LFLW\ 0RXQW DQG +RFNHWW f LGHQWLILHG KHDY\ PHWDO WR[LFDQWV LQ DQ LQGXVWULDO HIIOXHQW GHVSLWH HDUOLHU 7,( UHVXOWV WKDW LQGLFDWHG QR UHGXFWLRQ RI WR[LFLW\ IROORZLQJ ('7$ WUHDWPHQW $V DQ DOWHUQDWLYH LQ WKLV UHVHDUFK WKH KHDY\ PHWDO WR[LFLW\ RI WKH 06: ODQGILOO OHDFKDWHV ZDV GHWHUPLQHG ZLWK 0HW3/$7( DQ DVVD\ VSHFLILF WR KHDY\ PHWDO WR[LFLW\ $OWKRXJK ORZ FRQFHQWUDWLRQV RI KHDY\ PHWDOV KDYH EHHQ UHSRUWHG LQ 06: OHDFKDWHV :DUG HW DO f WKH SRWHQWLDO IRU PHWDO OHDFKLQJ IURP WKH ZDVWH UHPDLQV KLJK )O\KDPPHU f 06: OHDFKDWHV DUH E\ GHILQLWLRQ D KHWHURJHQHRXV PL[WXUH RI VLWHVSHFLILF LQRUJDQLF DQG RUJDQLF FRQWDPLQDQWV ZKLFK FDQ LQIOXHQFH PHWDO ELRDYDLODELOLW\ YLD FRPSOH[DWLRQ SUHFLSLWDWLRQ DQG DGVRUSWLRQ UHDFWLRQV 3UHYLRXV UHVHDUFK KDV VKRZQ WKDW WKHVH OHDFKDWH FKDUDFWHULVWLFV FRQWULEXWH WR WKH ORZ WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV XVLQJ 0HW3/$7( DV WKH WR[LFLW\ WHVW VHH

PAGE 216

7DEOH 7KH ,QLWLDO GD\ f DQG EDVHOLQH GD\ f DFXWH DQG FKURQLF WR[LFLW\ RI WKH ZKROH 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG SULRU WR IUDFWLRQDWLRQ ZLWK UHVXOWV H[SUHVVHG DV WR[LFLW\ XQLWV (&VRf 6LWH 6LWH 6LWH ,QLWLDO %DVHOLQH ,QLWLDO %DVHOLQH ,QLWLDO %DVHOLQH & GXELD KUf 3 VXEFDSLWDWD KUf 0LFURWR[r1 PLQf &KDSWHU DQG f 7KH KHDY\ PHWDO WR[LFLW\ RI WKH OHDFKDWHV ZDV LQYHVWLJDWHG ZLWK 0HW3/$7(r1 DQ DVVD\ VSHFLILF IRU KHDY\ PHWDO WR[LFLW\ 7KH 0HW3/$7(r1 UHVXOWV VKRZHG WKDW WKH OHDFKDWH WR[LFLW\ ZDV QRW GXH WR WKH SUHVHQFH RI KHDY\ PHWDOV ZLWK LQKLELWLRQV RI OHVV WKDQ b IRU HDFK OHDFKDWH VHH &KDSWHU f ,QLWLDO 7R[LFLW\ 7KH UHVXOWV IRU WKH LQLWLDO WR[LFLW\ DVVD\V XWLOL]LQJ LQYHUWHEUDWHV DOJDH DQG 0LFURWR[ KDYH EHHQ VXPPDUL]HG 7DEOH f *HQHUDOO\ WKH OHDFKDWH VDPSOHV ZHUH KLJKO\ WR[LF LQ WKH LQYHUWHEUDWH DQG DOJDO DVVD\V ZKLOH ORZ RU QR WR[LFLW\ ZDV GLVSOD\HG LQ WKH 0LFURWR[ DVVD\ :DUG HW DO f KDV VKRZQ WKDW WKH RYHUDOO WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV LV EHVW HYDOXDWHG LQ D VXLWH RI DVVD\V 7KH WR[LFLW\ HQGSRLQWV IRU WKH VLWH DQG OHDFKDWHV GLVSOD\HG VLPLODU SURILOHV ZLWK DOJDO 78V RI DQG DQG LQYHUWHEUDWH 78V RI DQG UHVSHFWLYHO\ ,Q FRQWUDVW WKH VLWH OHDFKDWH GLVSOD\HG

PAGE 217

D ORZ WR[LFLW\ LQ WKH DOJDO DQG LQYHUWHEUDWH DVVD\V ZLWK 78V RI ODQG UHVSHFWLYHO\ %ODQNV DQG &RQWUROV 7KH S+ YDOXHV ZHUH PRQLWRUHG GXULQJ HDFK VDPSOH PDQLSXODWLRQ DQG DGMXVWHG ZLWK RU 1 +&, RU 1D2+ DV QHHGHG 7KURXJKRXW WKH IUDFWLRQDWLRQ SURFHGXUHV VOLJKW S+ DGMXVWPHQWV ZHUH UHTXLUHG WR PDLQWDLQ WKH S+ RI WKH S+ DQG DGMXVWHG DQG S+ OHDFKDWH VDPSOHV %DVHOLQH 7R[LFLW\ 7KH EDVHOLQH WR[LFLW\ RI WKH 06: OHDFKDWHV IURP VLWHV DQG ZHUH HYDOXDWHG ZLWK WKH KU & GXELD DQG PLQ 0LFURWR[ DVVD\V 7DEOH f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f +HGVWURP f UHSRUWHG D KLJKO\ HIILFLHQW H[FKDQJH RI DPPRQLXP LRQV E\

PAGE 218

7DEOH $PPRQLD FRQFHQWUDWLRQV LQ 06: ODQGILOO OHDFKDWHV EHIRUH DQG DIWHU WUHDWPHQW RQ D =HROLWH FDWLRQ H[FKDQJH FROXPQ 6LWH 7RWDO DPPRQLD 1+91+f PJ/f 3UH=HROLWH 3RVW=HROLWH %'/ =HROLWH FROXPQV $PPRQLD WR[LFLW\ LQ DTXDWLF V\VWHPV KDV EHHQ ZLGHO\ UHSRUWHG $GDPVVRQ HW D *DPH HW DO f :KLOH DPPRQLD LV IRXQG DV ERWK DPPRQLXP 1+mf DQG DPPRQLD 1+f IRUPV WR[LFLW\ LV SULPDULO\ DVVRFLDWHG ZLWK DPPRQLD $QGHUVRQ DQG %XFNOH\ &OHPHQW DQG 0HUOLQ f WKHUHIRUH S+ LV D FULWLFDO IDFWRU ZKHQ HYDOXDWLQJ WR[LFLW\ 7KH KLJK DPPRQLD FRQFHQWUDWLRQV LQ )ORULGD 06: OHDFKDWHV FRQWULEXWH WR WKH RYHUDOO WR[LFLW\ :DUG :DUG HW DO f 7KH DPPRQLD FRQFHQWUDWLRQV LQ WKH WKUHH 06: ODQGILOO OHDFKDWHV EHIRUH DQG DIWHU SDVVDJH WKURXJK WKH =HROLWH FROXPQ DUH SUHVHQWHG 7DEOH f :KHQ WKH OHDFKDWHV IURP VLWHV DQG ZHUH SDVVHG WKURXJK WKH FDWLRQ H[FKDQJH FROXPQV WKH DPPRQLD FRQFHQWUDWLRQV ZHUH UHGXFHG E\ PRUH WKDQ b 7KH DPPRQLD FRQFHQWUDWLRQ LQ WKH XQWUHDWHG ZKROH VLWH OHDFKDWH ZDV PJ/ EXW DIWHU WKH =HROLWH WUHDWPHQW WKH FRQFHQWUDWLRQ ZDV UHGXFHG WR EHORZ WKH GHWHFWLRQ OLPLW RI WKH ,6( PJ/f 7KH FRQFHQWUDWLRQV RI DPPRQLD DW VLWHV DQG ZHUH UHGXFHG IURP WR PJ/ DQG IURP WR PJ/ UHVSHFWLYHO\ :KHQ WKH WR[LFLW\ RI WKH SRVW=HROLWH FROXPQ OHDFKDWHV ZHUH DVVD\HG ZLWK WKH & GXELD WR[LFLW\ WHVW WKH UHVXOWV ZHUH PL[HG 7DEOH f $IWHU SDVVDJH RI VLWHV DQG

PAGE 219

7DEOH $FXWH WR[LFLW\ RI WKH ZKROH DQG SRVW=HROLWH 06: ODQGILOO OHDFKDWHV WR & GXELD QHRQDWHV ZLWK WKH UDWLR RI OLYH WR WRWDO QHRQDWHV SUHVHQWHG DW WKH FRQFHQWUDWLRQ RI 06: ODQGILOO OHDFKDWH DVVD\HG 6LWH 1HRQDWH VXUYLYDO 5DZ /HDFKDWH 3RVW=HROLWH /HDFKDWH &RQWURO bf bf bf bf bf bf OHDFKDWHV WKURXJK WKH =HROLWH FROXPQV WKHUH ZHUH WR[LFLW\ UHGXFWLRQV RI b DQG URXJKO\ b UHVSHFWLYHO\ ,Q FRQWUDVW WKH VLWH OHDFKDWH VKRZHG QR FKDQJH LQ WR[LFLW\ 7KH =HROLWH UHVLQ H[FKDQJHV DPPRQLXP LRQV 1+f IRU VRGLXP 1Drf DQG GXH WR WKH KLJK FRQFHQWUDWLRQV RI DPPRQLXP LRQV LQ WKH UDZ OHDFKDWHV IURP VLWHV DQG WKH DGGHG VRGLXP LRQV PD\ KDYH GHFUHDVHG WHVW RUJDQLVP YLDELOLW\ 9DQ 6SUDQJ DQG -DQVVHQ f :KHQ WKH WR[LFLW\ RI WKH SRVW=HROLWH 06: OHDFKDWHV ZHUH DVVD\HG E\ 0LFURWR[ WKH OHDFKDWHV IURP VLWH H[KLELWHG D ODUJH WR[LFLW\ LQFUHDVH 7KH SRVW =HROLWH OHDFKDWHV IURP VLWHV DQG GLVSOD\HG VPDOO WR[LFLW\ UHGXFWLRQV 3RVWPDQLSXODWLRQ 7R[LFLW\ 6LWH 7KH ZKROH OHDFKDWH IURP VLWH GLVSOD\HG D ORZ WR[LFLW\ LQ WKH LQLWLDO VXUYH\ RI DFXWH DQG FKURQLF WR[LFLW\ 7DEOH f 7KH UHVXOWV IRU WKH 7,( ZHUH VXPPDUL]HG IRU WKH KRXU &GXELD )LJXUH f ,W LV ZRUWK QRWLQJ WKDW WKH IUDFWLRQDWLRQ SURWRFROV FDXVHG DQ LQFUHDVHG WR[LFLW\ LQ WKH OHDFKDWHV

PAGE 220

)LJXUH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH KRXU & GXELD DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ZLWK UHVXOWV VKRZQ DV WKH SHUFHQW bf GLIIHUHQFH IURP WKH EDVHOLQH WR[LFLW\ FROOHFWHG IURP VLWH IROORZLQJ WKH S+ DGMXVWPHQW DHUDWLRQ DQG VROLG SKDVH H[WUDFWLRQ SURFHGXUHV )UDFWLRQDWLRQ SURWRFROV DUH NQRZQ WR LQWURGXFH DUWLIDFWXDO HIIHFWV HVSHFLDOO\ LQ VDPSOHV ZLWK ORZ LQLWLDO WR[LFLW\ -RS DQG $VNHZ 4XUHVKL HW DO f 7KLV HIIHFW KDV DOVR EHHQ UHSRUWHG ZKHQ OHDFKDWHV DUH PRGLILHG GXULQJ ELRORJLFDO WUHDWPHQW SURFHVVHV 0DUWWLQHQ HW DO f 7KH 7,( UHVXOWV VXJJHVWHG WKDW ILOWUDWLRQ UHGXFHG WKH WR[LFLW\ RI WKH VLWH OHDFKDWH *HQHUDO FRQFOXVLRQV ZHUH PDGH FRQFHUQLQJ FKDUDFWHULVWLFV RI WKH WR[LFDQWV LQ WKH VLWH OHDFKDWH 1DPHO\ WKH WR[LFDQWV ZHUH QRQYRODWLOH DQG QRW K\GURSKRELF DOWKRXJK WKH\ PD\ KDYH EHHQ DVVRFLDWHG ZLWK OHDFKDWH VROLGV )XUWKHUPRUH WKH =HROLWH UHVLQ WUHDWPHQW RI WKH VLWH OHDFKDWH UHGXFHG WKH WRWDO DPPRQLD FRQFHQWUDWLRQV

PAGE 221

)LJXUH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH 0LFURWR[r1 DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ZLWK UHVXOWV VKRZQ DV WKH SHUFHQW bf GLIIHUHQFH IURP WKH EDVHOLQH WR[LFLW\ IURP WR PJ/ ZKLFK UHVXOWHG LQ WKH UHPRYDO RI DOO DFXWH WR[LFLW\ IURP WKH OHDFKDWH $OWKRXJK QRW LPSOLFDWHG GXULQJ 3KDVH IUDFWLRQDWLRQ WKH =HROLWH WUHDWPHQW VXJJHVWHG WKDW WKH WR[LFLW\ PD\ KDYH EHHQ GXH WR DPPRQLD RU DQRWKHU FDWLRQLF VSHFLHV 7KH ORZ VHQVLWLYLW\ RI WKH 0LFURWR[ DVVD\ ZLWK D 78 RI GLG QRW DFFXUDWHO\ UHIOHFW WKH EDVHOLQH WR[LFLW\ RI WKH OHDFKDWH 7DEOH f 7KH HIIHFWV RI WKH IUDFWLRQDWLRQ SURFHGXUHV RQ VLWH OHDFKDWH WR[LFLW\ XVLQJ WKHPLQXWH 0LFURWR[r1 ZHUH VXPPDUL]HG )LJXUH f 7R[LFLW\ ZDV UHGXFHG ZLWK DOO WUHDWPHQWV H[FHSW IRU WKH =HROLWH WUHDWPHQW DV SUHYLRXVO\ GHVFULEHG

PAGE 222

)LJXUH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH KRXU & GXELD DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ZLWK UHVXOWV VKRZQ DV WKH SHUFHQW bf GLIIHUHQFH IURP WKH EDVHOLQH WR[LFLW\ 6LWH 7KH 7,( UHVXOWV ZLWK WKH VLWH OHDFKDWH DUH VXPPDUL]HG IRU WKH KRXU & GXELD DVVD\ LQ )LJXUH ,Q PRVW FDVHV WKH WR[LFLW\ DVVRFLDWHG ZLWK WKH OHDFKDWH IURP VLWH ZDV S+GHSHQGHQW :KHQ WKH S+ ZDV UHGXFHG WR D IRDP\ZKLWH SUHFLSLWDWH ZDV IRUPHG DQG WKHUH ZHUH FRUUHVSRQGLQJ WR[LFLW\ UHGXFWLRQV LQ HDFK S+ DGMXVWHG OHDFKDWH IUDFWLRQ ),/7 $(5 DQG 63(f 7KH WR[LFLW\ RI WKH 63( IUDFWLRQV S+ RU S+f ZDV GHFUHDVHG DIWHU WUHDWPHQW VXJJHVWLQJ WKH SUHVHQFH RI D K\GURSKRELF QRQn SRODU WR[LFDQW 6WURQNKRUVW HW DO f 7KH VWURQJ LQIOXHQFH RI S+ UHGXFWLRQ RQ WR[LFLW\ VXJJHVWV DQ LRQL]DEOH WR[LFDQW $W S+ DPPRQLD VSHFLHV DUH H[FOXVLYHO\ LQ WKH LRQL]HG

PAGE 223

DQG OHVV WR[LFf DPPRQLXP IRUP 2Q WKH RWKHU H[WUHPH ZKHQ WKH OHDFKDWH ZDV DGMXVWHG WR S+ WKH WR[LFLW\ LQFUHDVHG SUREDEO\ GXH WR WKH FRQYHUVLRQ RI LRQL]HG DPPRQLXP WR XQLRQL]HG DQG KLJKO\ WR[LF DPPRQLD DPPRQLD 0DUWWLQHQ HW DO f UHSRUWHG DQ LQFUHDVH LQ OHDFKDWH WR[LFLW\ WR 'DSKQLD PDJQD DIWHU DPPRQLD VWULSSLQJ DW S+ )XUWKHUPRUH DHUDWLRQ RI WKH S+ DGMXVWHG OHDFKDWH GHFUHDVHG WR[LFLW\ E\ URXJKO\ b DQG WKH DHUDWLRQ RI WKH S+ DGMXVWHG OHDFKDWH UHGXFHG WR[LFLW\ E\ b VXJJHVWLQJ D YRODWLOH FRPSRQHQW WR WKH WR[LFDQW %UDFN DQG )UDQN f 7KH WR[LFLW\ RI DPPRQLD LV S+GHSHQGHQW DQG ZLWK D S.D RI WKH LRQL]HG IRUP SUHGRPLQDWHV XQGHU WKH JHQHUDOO\ QHXWUDO S+ FRQGLWLRQV IRXQG LQ 06: OHDFKDWHV 5HVHDUFKHUV KDYH VKRZQ WKDW WKH IUDFWLRQ RI DPPRQLD SUHVHQW PD\ EH ELRORJLFDOO\ UHOHYDQW ZKHQ FRQFHQWUDWLRQV RI WRWDO DPPRQLD 1+1+f DUH KLJK & GXELD LV VHQVLWLYH WR WKH SUHVHQFH RI DPPRQLD ZLWK DQ (& RI PJ/ $QGHUVHQ DQG %XFNOH\ f 7KH 3KDVH 7,( UHVXOWV VWURQJO\ VXJJHVW WKDW DPPRQLD ZDV UHVSRQVLEOH IRU OHDFKDWH WR[LFLW\ KRZHYHU WKH 3KDVH ,, SURFHGXUH GLG QRW SRVLWLYHO\ LGHQWLI\ DPPRQLD WR[LFLW\ $OWKRXJK WKH =HROLWH WUHDWPHQW UHGXFHG WRWDO DPPRQLD FRQFHQWUDWLRQV IURP WR PJ/ WKH FRUUHVSRQGLQJ WR[LFLW\ DVVD\V VKRZHG RQO\ D VOLJKW GHFOLQH LQ WR[LFLW\ RI DSSUR[LPDWHO\ b 6RGLXP LRQV HQKDQFH WR[LFLW\ ZKHQ SUHVHQW LQ KLJK FRQFHQWUDWLRQV 9DQ 6SUDQJ DQG -DQVVHQ f 7KH FRQFHQWUDWLRQ RI VRGLXP LQ WKH VLWH OHDFKDWH ZDV PJ/ PXFK KLJKHU WKDQ WKH PJ/ DQG PJ/ DW VLWH DQG UHVSHFWLYHO\ $GGLWLRQDOO\ WKH KLJK FRQFHQWUDWLRQV RI DQLRQLF

PAGE 224

)LJXUH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH 0LFURWR[r1 DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ZLWK UHVXOWV VKRZQ DV WKH SHUFHQW bf GLIIHUHQFH IURP WKH EDVHOLQH WR[LFLW\ DQG FDWLRQLF VSHFLHV LQ WKH 06: OHDFKDWHV PD\ KDYH FRQWULEXWHG WR WKH RYHUDOO WR[LFLW\ %DXQ HW DO f $V HYLGHQFH WKH FRQGXFWLYLW\ RI WKH WKUHH OHDFKDWHV ZHUH DQG P6FP IRU VLWHV DQG UHVSHFWLYHO\ &RQGXFWLYLW\ LV XVHG DV DQ LQGLFDWRU RI WRWDO GLVVROYHG VROLGV DQG WKH & GXELD /& IRU FRQGXFWLYLW\ LV P6FP 86(3$ Df &RRPDQ HW DO f FRQFOXGHG FRQGXFWLYLW\ OHYHOV WKDW ZHUH b RI WKH /& LQIOXHQFHG WKH WR[LFLW\ RI D WDQQHU\ ZDVWHZDWHU 7KH FRQWURO ZDWHU 0+: KDV D FRQGXFWLYLW\ RI a P6FP DQ RUGHU RI PDJQLWXGH ORZHU WKDQ WKH UHSRUWHG /&

PAGE 225

)LJXUH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH KRXU & GXELD DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ZLWK UHVXOWV VKRZQ DV WKH SHUFHQW bf GLIIHUHQFH IURP WKH EDVHOLQH WR[LFLW\ 7KH WR[LFLW\ DVVD\ UHVXOWV IRU VLWH OHDFKDWHV XVLQJ WKH PLQXWH 0LFURWR[r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f 7KH VXJJHVWLRQ RI D QRQSRODU RUJDQLF

PAGE 226

FRPSRXQG DJUHHV ZLWK WKH VHQVLWLYLW\ RI WKH 0LFURWR[ f DVVD\ IRU WKLV FDWHJRU\ RI FRQWDPLQDQWV 6WURQNKRUVW HW DO f 6LWH 7KH 7,( UHVXOWV ZLWK WKH 06: ODQGILOO OHDFKDWH FROOHFWHG IURP VLWH DUH PRUH GLIILFXOW WR LQWHUSUHW EHFDXVH RI WKH XQLIRUP WR[LFLW\ UHGXFWLRQ IROORZLQJ HDFK IUDFWLRQDWLRQ VWHS )LJXUH VXPPDUL]HV WKH KRXU & GXELD 7,( UHVXOWV :KHQ DQ DOLTXRW RI VLWH OHDFKDWH ZDV ORZHUHG WR S+ WKH FRORU RI WKH OHDFKDWH OLJKWHQHG 6LPLODU HIIHFWV ZHUH REVHUYHG ZKHQ WKH S+ RI D VHFRQG DOLTXRW ZDV LQFUHDVHG WR S+ 7KH FRORU RI WKH S+ DOLTXRW OLJKWHQHG DQG D KHDY\ SUHFLSLWDWH ZDV IRUPHG 7KLV SUHFLSLWDWH ZDV UHPRYHG E\ ILOWUDWLRQ SPf 7KH 3KDVH UHVXOWV VKRZHG WKDW WKH WR[LFLW\ GHFOLQHG IROORZLQJ HDFK PDQLSXODWLRQ UHJDUGOHVV RI S+ KRZHYHU WKH ODUJHVW WR[LFLW\ GHFOLQH bf ZDV UHSRUWHG IROORZLQJ WKH 63( WUHDWPHQW DW S+ 7KH 63( UHVXOWV VXJJHVW WKDW D QRQSRODU RUJDQLF WR[LFDQW LQ WKH OHDFKDWH FRQWULEXWHG WR WKH WR[LFLW\ 2YHUDOO WKH PDJQLWXGH RI WR[LFLW\ UHGXFWLRQ ZDV FRQVLVWHQW IRU HDFK 7,( VWHS LQGLFDWLQJ D S+ LQVHQVLWLYH FRPSRQHQW FRQWULEXWLQJ WR WKH WR[LFLW\ 7KLV FRQWUDVWV ZLWK WKH UHVXOWV RI WKH =HROLWH FROXPQ WUHDWPHQW ZKLFK VKRZHG D UHGXFWLRQ RI WRWDO DPPRQLD FRQFHQWUDWLRQV IURP WR PJ/ DQG DQ RYHUDOO WR[LFLW\ UHGXFWLRQ RI b 7R[LFLW\ UHGXFWLRQV IROORZLQJ H[FKDQJH RQ D =HROLWHBFROXPQ VXJJHVW WKH SUHVHQFH RI DQ LQRUJDQLF FDWLRQ DV WKH WR[LFDQW &RRPDQ HW DO f $FFRUGLQJ WR WKH UHVXOWV RI WKH 7,( DPPRQLD ZDV LGHQWLILHG DV D UHVSRQVLEOH WR[LFDQW LQ WKH OHDFKDWH IURP VLWH

PAGE 227

Â’ =HROLWH +S+63( S+L63( %S+63( %S+DHU S+LDHU 6 S+DHU LQ SP ILL 'S+LILO &' S+ILO +S+DGM % S+LDGM %S+DGM 7R[LFLW\ bf UHODWLYH WR EDVHOLQH )LJXUH 5HVXOWV RI WKH 3KDVH WR[LFLW\ IUDFWLRQDWLRQ ZLWK WKH 0LFURWR[r1 DVVD\ IRU 06: ODQGILOO OHDFKDWHV FROOHFWHG IURP VLWH ZLWK UHVXOWV VKRZQ DV WKH SHUFHQW bf GLIIHUHQFH IURP WKH EDVHOLQH WR[LFLW\ 7KH UHVXOWV RI WKH PLQXWH 0LFURWR[r1 DVVD\ DUH VXPPDUL]HG LQ )LJXUH :LWK WKH 0LFURWR[ DVVD\ WKHUH ZHUH WR[LFLW\ LQFUHDVHV IROORZLQJ WKH DHUDWLRQ DQG ILOWUDWLRQ RI WKH S+ DQG S+ DGMXVWHG VLWH OHDFKDWH $ VLPLODU SKHQRPHQRQ ZDV UHSRUWHG E\ 6WURQNKRUVW HW DO f IROORZLQJ WKH ILOWUDWLRQ RI VHGLPHQW H[WUDFWV ,Q WKH 0LFURWR[r1 DVVD\ WKH 63( RI WKH VLWH OHDFKDWH VXJJHVWHG WKH SUHVHQFH RI D QRQSRODU WR[LFDQW 7KH OHDFKDWH WR[LFLW\ ZDV UHGXFHG LUUHVSHFWLYH RI WKH S+ RI WKH VDPSOH 7KH UHVXOWV RI WKH 7,( LQYHVWLJDWLRQV ZHUH VXPPDUL]HG 7DEOH f 7KH ELRDVVD\V LQGLFDWHG YDULRXV VSHFLHV ZHUH UHVSRQVLEOH IRU WKH OHDFKDWH WR[LFLW\ 7KH WR[LFDQWV LQ WKH OHDFKDWHV IURP VLWH ZHUH FKDUDFWHUL]HG DV QRQYRODWLOH QRW K\GURSKRELF DQG SRVVLEO\ DVVRFLDWHG ZLWK WKH OHDFKDWH VROLGV 7KH UHVXOWV ZLWK WKH VLWH OHDFKDWHV LQGLFDWHG LRQL]HG VXEVWDQFHV

PAGE 228

7DEOH 6XPPDU\ RI 7,( UHVXOWV ZLWK 06: ODQGILOO OHDFKDWHV IURP VLWHV DQG 6LWH 7R[LFDQW &KDUDFWHULVWLFVf 1RQYRODWLOH 1RW K\GURSKRELF "f 6ROLGV DVVRFLDWHG ,RQL]HG VXEVWDQFH $PPRQLD &DWLRQV +\GURSKRELF FRPSRXQG $PPRQLD DQG FDWLRQV ZHUH SRWHQWLDOO\ UHVSRQVLEOH IRU WKH WR[LFLW\ $PPRQLD ZDV SRVLWLYHO\ LGHQWLILHG DV D WR[LFDQW LQ WKH VLWH OHDFKDWH 6LPLODUO\ DPPRQLD ZDV LGHQWLILHG DV D WR[LFDQW LQ WKH VLWH OHDFKDWHV 7KH 7,( UHVXOWV LQGLFDWHG WKDW K\GURSKRELF FRPSRXQGV ZHUH DVVRFLDWHG ZLWK WKH WR[LFLW\ RI WKH VLWH OHDFKDWHV

PAGE 229

&+$37(5 +25021$/ $&7,9,7< 2) 081,&,3$/ 62/,' :$67( 06:f /($&+$7(6 )520 )/25,'$ /$1'),//6 ,QWURGXFWLRQ $GYHUVH UHSURGXFWLYH DQG GHYHORSPHQWDO HIIHFWV KDYH EHHQ GRFXPHQWHG ZRUOGZLGH -REOLQJ HW DO 5RXWOHGJH HW DO f IROORZLQJ H[SRVXUH WR VXEVWDQFHV WKDW FDQ DOWHU FKHPLFDO PHVVHQJHUV LQ OLYLQJ V\VWHPV 0F/DFKODQ f 7KHVH KRUPRQDOO\ DFWLYH FRPSRXQGV 15& f DUH GHILQHG QRW E\ WKHLU FKHPLFDO VWUXFWXUHV EXW E\ WKHLU ELRORJLFDO HIIHFWV /RSH] GH $OGD DQG %DUFHOR f 7KHUH DUH PXOWLSOH VRXUFHV RI KRUPRQDOO\ DFWLYH FRPSRXQGV LQ WKH HQYLURQPHQW LQFOXGLQJ LQGXVWULDO HIIOXHQWV 6KHDKDQ HW DO E /\H HW DO 0HOODQHQ HW DO f DQG ZDVWHZDWHU WUHDWPHQW SODQW ::73f HIIOXHQWV +ROEURRN HW DO -REOLQJ HW DO $QJXV HW DO f 06: ODQGILOOV FRQWDLQ D PXOWLWXGH RI GLVFDUGHG FRQVXPHU SURGXFWV DQG WKH OHDFKDWHV JHQHUDWHG LQFOXGH QXPHURXV RUJDQLF FRPSRXQGV $ ZLGH YDULHW\ RI RUJDQLF FRPSRXQGV DUH GLVSRVHG LQ ODQGILOOV DQG DGGLWLRQDO FRPSRXQGV DUH SURGXFHG DV D UHVXOW RI ZDVWH GHFRPSRVLWLRQ SURFHVVHV .MHOGVHQ HW DO f :KLOH VRPH RI WKHVH FRPSRXQGV DUH KRUPRQDOO\ DFWLYH VXFK DV WKH SKWKDODWHV 9LQJJDDUG HW DO YDQ :H]HO HW DO +DUULV HW DO f VXUIDFWDQWV 5RXWOHGJH DQG 6XPSWHU Df DQG IODPHUHWDUGDQWV 0HHUWV HW DO f OLWWOH LV NQRZQ DERXW WKH PDMRULW\ RI WKH FRQWDPLQDQWV 06: ODQGILOOV PD\ DOVR FRQWDLQ VPDOO TXDQWLWLHV RI LQGXVWULDO DQG KD]DUGRXV ZDVWHV RI XQNQRZQ FRPSRVLWLRQ

PAGE 230

3KWKDODWHV DUH H[WHQVLYHO\ XVHG LQ WKH SURGXFWLRQ RI FRQVXPHU JRRGV LQFOXGLQJ SDLQWV LQNV DGKHVLYHV DQG YDULRXV SODVWLF JRRGV WR LPSDUW IOH[LELOLW\ $OWKRXJK UHF\FOLQJ SURJUDPV LQ WKH 86 HQFRXUDJH WKH UHFRYHU\ RI SODVWLFV RQO\ DERXW b RI DOO PDQXIDFWXUHG SODVWLFV DUH UHF\FOHG 86(3$ f $V D GLUHFW FRQVHTXHQFH 06: ODQGILOO OHDFKDWHV PD\ FRQWDLQ ODUJH TXDQWLWLHV RI SKWKDODWH FRPSRXQGV %DXHU DQG +HUUPDQQ f *HQHUDOO\ SDUWV SHU ELOOLRQ OHYHOV RI SKWKDODWH FRQWDPLQDWLRQ KDYH EHHQ UHSRUWHG LQ 06: OHDFKDWHV KRZHYHU WKH FRQFHQWUDWLRQV PD\ UDQJH RYHU DQ RUGHU RI PDJQLWXGH .MHOGVHQ HW DO f &RQWUDU\ WR SUHYLRXV ZRUN LQ WKLV ODE ZKLFK KDG VKRZQ H[WHQVLYH SKWKDODWH FRQWDPLQDWLRQ RI 06: OHDFKDWHV :DUG DQG 7RZQVHQG XQSXEOLVKHG GDWDf WKH SKWKDODWH FRQWDPLQDWLRQ LQ WKH VXUYH\HG OHDFKDWHV ZDV OLPLWHG ,Q DGGLWLRQ SK\WRHVWURJHQV IRXQG LQ SODQWV DQG SODQW SURGXFWV 1LOVVRQ f FRXOG DOVR RFFXU LQ 06: OHDFKDWHV ZKHQ \DUG ZDVWHV DUH QRW VHSDUDWHG IURP KRXVHKROG ZDVWHV :KLOH WKH DFXWH DQG FKURQLF WR[LFLW\ RI 06: ODQGILOO OHDFKDWHV KDV EHHQ FKDUDFWHUL]HG :DUG HW DO )HUUDUL HW DO %HUQDUG HW DO f OHVV KDV EHHQ SXEOLVKHG WR DGGUHVV FRQFHUQV DERXW WKHLU SRWHQWLDO KRUPRQDO DFWLYLW\ %HKQLVFK HW DO f XVLQJ WKH (VFUHHQ FHOO SUROLIHUDWLRQ DVVD\ ZLWK 0&) FHOOVf UHSRUWHG KRUPRQDO DFWLYLW\ HTXLYDOHQW WR QJ SHVWUDGLRO (f/ LQ WKH OHDFKDWH IURP RQH VROLG ZDVWH ODQGILOO $GGLWLRQDOO\ KRUPRQDO DFWLYLW\ HTXLYDOHQW WR QJ/ ( ZDV UHSRUWHG LQ D JURXQGZDWHU VDPSOH FRQWDPLQDWHG ZLWK ZDVWH OHDFKDWH .DZDJRVKL HW DO f &KURQLF H[SRVXUH WR ZDVWH OHDFKDWHV SURGXFHG DOWHUDWLRQV LQ WKH UHSURGXFWLYH FKDUDFWHULVWLFV RI ILVK LQ D 6ZHGLVK ODNH

PAGE 231

ZLWK GHFUHDVHG JRQDGDO VL]H DQG KRUPRQH OHYHOV 1RDNVVRQ HW DO f +RZHYHU WKH KRUPRQDO DFWLYLW\ RI 06: ODQGILOO OHDFKDWHV LQ WKH 8QLWHG 6WDWHV KDV UHFHLYHG OLWWOH DWWHQWLRQ 7KH REMHFWLYHV RI WKLV UHVHDUFK ZHUH WR f GHWHUPLQH LI 06: ODQGILOO OHDFKDWHV ZHUH KRUPRQDOO\ DFWLYH ZLWK D \HDVW UHSRUWHU DVVD\ f WHQWDWLYHO\ LGHQWLI\ FRPSRXQGV LQ WKH OHDFKDWHV UHVSRQVLEOH IRU WKLV DFWLYLW\ f PRQLWRU WKH HIIHFW RI ELRORJLFDO WUHDWPHQW RQ KRUPRQDO DFWLYLW\ DW RQH SDUWLFXODU 06: ODQGILOO OHDFKDWH WUHDWPHQW SODQW DQG f SURYLGH PRQLWRULQJ GDWD IRU UHVHDUFKHUV DQG HQYLURQPHQWDO UHJXODWRUV FRQFHUQHG ZLWK WKH KRUPRQDO DFWLYLW\ RI 06: ODQGILOO OHDFKDWHV 0DWHULDOV DQG 0HWKRGV &KHPLFDOV 8QOHVV RWKHUZLVH VSHFLILHG DOO FKHPLFDOV ZHUH REWDLQHG IURP )LVKHU 6FLHQWLILF DW WKH KLJKHVW SRVVLEOH SXULW\ 7KH KRUPRQH SHVWUDGLRO (f ZDV SXUFKDVHG IURP 6LJPD 6W /RXLV 02 86$f 5HIHUHQFH VWDQGDUGV ZHUH SXUFKDVHG IURP 8OWUD 6FLHQWLILF 1RUWK .LQJVWRQ 5Of GLPHWK\O SKWKDODWH '03f EHQ]\O EXW\O SKWKDODWH %%3f GLEXW\O SKWKDODWH '%3f GLHWK\O SKWKDODWH '(3f %LVSKHQRO $ 7HWUDEURPRELVSKHQRO $ DQG WHUWEXW\OK\GUR[\DQLVROH 7KH SKHQROLF FRPSRXQGV QRQ\OSKHQRO 13f RFW\OSKHQRO 23f DQG WHUWEXW\O SKHQRO W%3f ZHUH REWDLQHG IURP &KHP6HUYH :HVWFKHVWHU 3$f 6WRFN VROXWLRQV RI ( DQG WKH DQDO\WLFDO VWDQGDUGV ZHUH SUHSDUHG ZLWK +3/& JUDGH PHWKDQRO LQ YROXPHWULF IODVNV VHDOHG ZLWK 7HIORQ VWRSSHUV $OO VWRFN VROXWLRQV ZHUH VWRUHG XQGHU UHIULJHUDWLRQ DW r& ZLWK PLQLPXP OLJKW H[SRVXUH XQWLO QHHGHG

PAGE 232

06: /DQGILOOV DQG /HDFKDWH &ROOHFWLRQ 7KH 06: ODQGILOO OHDFKDWHV ZHUH FROOHFWHG DW HLJKW HQJLQHHUHG ODQGILOO VLWHV LQ WKH VWDWH RI )ORULGD 86$ 7KHVH OHDFKDWHV ZHUH VHOHFWHG EDVHG RQ SUHYLRXV LQYHVWLJDWLRQV $GGLWLRQDO LQIRUPDWLRQ FRQFHUQLQJ WKH FKHPLFDOSK\VLFDO FKDUDFWHULVWLFV DQG WR[LFLW\ RI WKHVH OHDFKDWHV LV DYDLODEOH VHH &KDSWHU f /) LV D FDSSHG ODQGILOO WKDW VWRSSHG DFFHSWLQJ ZDVWH LQ ODWH DQG /) LV D UHJLRQDO ODQGILOO FXUUHQWO\ DFFHSWLQJ ZDVWH IURP WKUHH VXUURXQGLQJ FRXQWLHV /) /) /) DQG /) DUH ORFDWHG WKURXJKRXW FHQWUDO )ORULGD ZKLOH /) LV ORFDWHG LQ VRXWKHDVWHUQ )ORULGD /) KDV DQ RQVLWH OHDFKDWH WUHDWPHQW IDFLOLW\ DQG VDPSOHV ZHUH FROOHFWHG EHIRUH DQG DIWHU WUHDWPHQW )RU FRPSDUDWLYH SXUSRVHV ZDWHU VDPSOHV ZHUH FROOHFWHG IURP WKH LQIOXHQW ,1)f DQG HIIOXHQW ())f RI D ZDVWHZDWHU WUHDWPHQW SODQW ::73f LQ *DLQHVYLOOH )/ DQG IURP /DNH $OLFH LQ *DLQHVYLOOH )/ DQG /DNH %HYHUO\ LQ %HYHUO\ +LOOV )/ $ ILUVW IOXVK VWRUPZDWHU VDPSOH ZDV FROOHFWHG IURP D GUDLQDJH EDVLQ LQ %HYHUO\ +LOOV )/ 06: /DQGILOO /HDFKDWH 7UHDWPHQW )DFLOLW\ 7KH WUHDWPHQW IDFLOLW\ DW /) LV D WZRVWDJH SRZGHUHG DFWLYDWHG FDUERQ 3$&f V\VWHP =LPSUR9LYHQGLf WKDW WUHDWV DSSUR[LPDWHO\ JDOORQV RI 06: OHDFKDWH SHU GD\ 7KH IDFLOLW\ RSHUDWHV ZLWK D K\GUDXOLF UHWHQWLRQ WLPH +57f RI KRXUV DQG D VOXGJH UHWHQWLRQ WLPH 657f RI GD\V 7KH PL[HG OLTXRU VXVSHQGHG VROLGV 0/66f LV PDLQWDLQHG DW URXJKO\ PJ/ DQG VOXGJH LV

PAGE 233

/ 6$03/( 3$57,&8/$7(6 <($67 $66$< 02+ ),/7(5 ',662/9(' 3+$6( 62/,' 3+$6( <($67 (;75$&7,21 $66$< <($67 $66$< )LJXUH 3URFHGXUH IRU SUHSDULQJ 06: OHDFKDWHV DQG PHWKDQRO H[WUDFWV RI OHDFKDWHV IRU DQDO\VLV RI KRUPRQDO DFWLYLW\ ZDVWHG WKUHH WLPHV ZHHNO\ IURP WKH DHURELF DQG GDLO\ IURP WKH DQR[LF VWDJHV ,QIOXHQW VWUHQJWK YDULHV EXW JHQHUDOO\ WKH FDUERQDFHRXV ELRORJLFDO R[\JHQ GHPDQG &%2'f UDQJHV IURP WR PJ/ DQG WKH WRWDO DPPRQLD FRQFHQWUDWLRQV DUH DSSUR[LPDWHO\ PJ/ %DWFK SODQWV EDVHG RQ QLWULILFDWLRQGHQLWULILFDWLRQ DUH HIIHFWLYH LQ WKH UHPRYDO RI DPPRQLD IURP 06: ODQGILOO OHDFKDWH +DUSHU HW DO f 7KH WUHDWHG HIIOXHQWV DUH GLVFKDUJHG WR SHUFRODWLRQ SRQGV LQ DFFRUGDQFH ZLWK )ORULGD VWDQGDUGV IRU JURXQGZDWHU 7KH GHGLFDWHG JODVVZDUH XWLOL]HG WKURXJKRXW WKLV LQYHVWLJDWLRQ ZDV DFLG ZDVKHG 'OULQVHG GULHG DQG PHWKDQROULQVHG SULRU WR HDFK XVH 06: ODQGILOO OHDFKDWHV ZHUH FROOHFWHG IURP WKH OHDFKDWH FROOHFWLRQ VXPSV RI HDFK OLQHG ODQGILOO

PAGE 234

XVLQJ D PHWKDQROULQVHG 7HIORQ EDOHU /HDFKDWHV ZHUH FROOHFWHG IRU WKH <(6 DVVD\ LQ / DPEHU JODVV MXJV ZLWK 7HIORQOLQHG VHSWD )RU WKH FKHPLFDO DQG SK\VLFDO FKDUDFWHUL]DWLRQ RI WKH 06: OHDFKDWHV VDPSOHV ZHUH DOVR FROOHFWHG LQ JODVV RU SRO\HWK\OHQH ERWWOHV DQG SUHVHUYHG DFFRUGLQJ WR WKH 86 (QYLURQPHQWDO 3URWHFWLRQ $JHQF\ 86(3$ f )LHOG PHDVXUHPHQWV ZHUH S+ DQG WHPSHUDWXUH 2ULRQ 0RGHO $f HOHFWULFDO FRQGXFWLYLW\ +$11$ ,QVWUXPHQWV 0RGHO +f GLVVROYHG R[\JHQ '2f <6, ,QF 0RGHO )7f DQG R[LGDWLRQUHGXFWLRQ SRWHQWLDO 253f $FFXPHW &R 0RGHO f 6DPSOHV ZHUH WUDQVSRUWHG RQ LFH WR WKH ODERUDWRU\ ZKHUH WKH\ ZHUH VWRUHG DW r& XQWLO DQDO\VLV 6ROLG 3KDVH ([WUDFWLRQ 63(f RI 06: /DQGILOO /HDFKDWHV 7KH 06: ODQGILOO OHDFKDWHV ZHUH FRQFHQWUDWHG E\ VROLG SKDVH H[WUDFWLRQ 63(f WR LGHQWLI\ ORZ FRQFHQWUDWLRQV RI KRUPRQDOO\ DFWLYH FRPSRXQGV )LJXUH f 7KH OHDFKDWHV ZHUH SUHILOWHUHG ZLWK PHWKDQROULQVHG JODVV ILEHU *)f aSP *)% JODVV :KDWPDQf ILOWHUV DQG PHPEUDQH ILOWHUV 0)f SP )LVKHU 6FLHQWLILFf IROORZLQJ GHOLYHU\ WR WKH ODERUDWRU\ 7KH SUHILOWUDWLRQ ZDV XVHG IRU WKH UHPRYDO RI VROLGV RU EDFWHULDO FRQWDPLQDQWV ZKLFK PLJKW LQWHUIHUH ZLWK WKH DVVD\ SURFHGXUH 7ZR / YROXPHV RI HDFK SUHILOWHUHG UDZ 06: OHDFKDWH ZHUH WKHQ H[WUDFWHG LQ SDUDOOHO RQ PHWKDQRO FRQGLWLRQHG & H[WUDFWLRQ GLVNV PP (PSRUH 0 &RUS 6W 3DXO 01f ZLWK YDFXXP ILOWUDWLRQ DFFRUGLQJ WR 86(3$ PHWKRG 6:f 'XH WR WKH KLJK RUJDQLF FRQWHQW LQ WKH 06: OHDFKDWHV PXOWLSOH H[WUDFWLRQ GLVNV ZHUH XWLOL]HG WR SUHYHQW GLVN VDWXUDWLRQ RU EUHDNWKURXJK (DFK & H[WUDFWLRQ GLVN ZDV HOXWHG ZLWK WZR PO DOLTXRWV RI PHWKDQRO 7KH HOXHQWV IURP WKH LQGLYLGXDO GLVNV ZHUH FRPELQHG DQG IXUWKHU FRQFHQWUDWHG XQGHU

PAGE 235

D VWUHDP RI QLWURJHQ 1f 7XUERYDS =\PDUN &RUS +RSNLQWRQ 0$f WR D ILQDO YROXPH RI PO DQG D ILQDO FRQFHQWUDWLRQ IDFWRU RI ; 7KH PHWKDQRO H[WUDFWV ZHUH WKHQ WUDQVIHUUHG WR D PO JODVV 92& YLDO ZLWK 7HIORQ VHSWXP DQG VWRUHG XQGHU UHIULJHUDWLRQ DW r& XQWLO UHTXLUHG $Q DOLTXRW RI HDFK PHWKDQRO H[WUDFW RI WKH OHDFKDWHV ZDV H[FKDQJHG ZLWK KH[DQH HYDSRUDWHG WR PO XQGHU D FRQWLQXRXV 1 VWUHDP DQG DQDO\]HG E\ JDV FKURPDWRJUDSK\PDVV VSHFWURPHWU\ *&06f 0HWKRG FRQWUROV XVLQJ GLVWLOOHG GHLRQL]HG ZDWHU '',f 1DQRSXUH %DUQVWHDGf DQG ( VSLNHG '', ZHUH H[WUDFWHG IROORZLQJ WKH VDPH PHWKRGRORJ\ DV GHVFULEHG IRU WKH OHDFKDWHV $ 06: ODQGILOO OHDFKDWH FROOHFWHG IURP /) LQ 0DUFK ZDV VXEMHFW WR DGGLWLRQDO H[WUDFWLRQV DQG DQDO\VLV 7R UHPRYH FRPSRXQGV RI WKH ZLGHVW SRODULW\ UDQJH WKH & H[WUDFWLRQ GLVNV ZHUH VHTXHQWLDOO\ HOXWHG ZLWK VROYHQWV RI GHFUHDVLQJ SRODULW\ PHWKDQRO 0H2+f DFHWRQLWULOH $&1f GLFKORURPHWKDQH '&0f DQG KH[DQH +(;f (DFK VROYHQW H[WUDFW 0H2+ $&1 '&0 DQG +(;f ZDV IXUWKHU FRQFHQWUDWHG XQGHU QLWURJHQ 7XUERYDS =\PDUN &RUS +RSNLQWRQ 0$f WR D ILQDO YROXPH RI PO WUDQVIHUUHG WR D PO JODVV 92& YLDO ZLWK D 7HIORQ VHSWD DQG VWRUHG XQGHU UHIULJHUDWLRQ DWr& XQWLO UHTXLUHG <(6 $VVD\ IRU 'HWHUPLQLQJ +RUPRQDO $FWLYLW\ 7KH UHFRPELQDQW \HDVW DVVD\ <(6f KDV EHHQ H[WHQVLYHO\ XWLOL]HG IRU LGHQWLI\LQJ KRUPRQH DFWLYLW\ LQ HQYLURQPHQWDO VDPSOHV 6KHDKDQ HW DO D :LWWHUV HW DO 'HVEURZ HW DO f 7KH UHFRPELQDQW \HDVW FHOOV 6DFFKDURP\FHV FHUHYLVLDH XVHG IRU WKH DVVD\ ZHUH JHQHURXVO\ SURYLGHG E\ 'U 6XPSWHU RI %UXQHL 8QLYHUVLW\ 0LGGOHVH[ 8.f 7KH GHYHORSPHQW RI WKH \HDVW

PAGE 236

FHOOV IRU WKH DVVD\ KDV EHHQ SUHYLRXVO\ GHVFULEHG 5RXWOHGJH DQG 6XPSWHU Ef 7KH KRUPRQDO DFWLYLW\ RI LQGLYLGXDO VDPSOHV DQG FRQWUROV ZDV TXDQWLILHG E\ PHDVXULQJ WKH DEVRUEDQFH LQFUHDVHV QPf ZLWK D PLFURSODWH VSHFWURSKRWRPHWHU 0XOWLVNDQ 3OXV 0. ,, ,&1 %LRPHGLFDOV +XQWVYLOOH $/f 7R GDWH WKH <(6 KDV EHHQ XVHG WR LGHQWLI\ KRUPRQDO DFWLYLW\ LQ VROYHQW H[WUDFWV RI HQYLURQPHQWDO VDPSOHV +ROEURRN HW DOf :LWWHUV HW DO f RU ZLWK SXUH FRPSRXQGV 0LOOHU HW DO f 7KHUHIRUH ZKHQ GHWHUPLQLQJ WKH KRUPRQDO DFWLYLW\ RI UDZ 06: ODQGILOO OHDFKDWHV PRGLILFDWLRQV WR WKH <(6 DVVD\ ZHUH UHTXLUHG 7KH VWDQGDUG DVVD\ PHGLD ZDV SUHSDUHG IRU WHVWV ZLWK WKH VROYHQW H[WUDFWV RI WKH 06: ODQGILOO OHDFKDWHV DQG ZLWK WKH ( 5RXWOHGJH DQG 6XPSWHU Ef $ WKUHH WLPHV FRQFHQWUDWHG DVVD\ PHGLD ;f ZDV SUHSDUHG IRU XVH ZLWK WKH UDZ OHDFKDWHV VR WKDW ZKHQ UDZ OHDFKDWHV ZHUH FRPELQHG ZLWK WKH ; DVVD\ PHGLD WKH UDZ OHDFKDWH ZDV GLOXWHG E\ RQHWKLUG 3ULRU WR WKH DVVD\ PO DOLTXRWV RI WKH UDZ OHDFKDWHV ZHUH ILOWHUVWHULOL]HG SP $FURGLVFf DQG WKHQ D SO DOLTXRWV RI HDFK ILOWHUHG UDZ OHDFKDWH ZDV FRPELQHG ZLWK D SO DOLTXRW RI WKH ; DVVD\ PHGLD LQ D ZHOO PLFURSODWH DQG PL[HG 7KH PLFURSODWH DOVR LQFOXGHG WR SO DOLTXRWV RI WKH VROYHQW H[WUDFW RI WKH OHDFKDWHV DQG (VSLNHG ( ILQDO FRQFHQWUDWLRQ WR QJ/f VROYHQW H[WUDFWV 7KH VROYHQW H[WUDFWV ZHUH HYDSRUDWHG WR GU\QHVV DQG PL[HG ZLWK SL RI VWDQGDUG DVVD\ PHGLD ,Q D VHSDUDWH ZHOO PLFURSODWH D GRVHUHVSRQVH FXUYH ZLWK WKH SRVLWLYH FRQWURO ( ZDV SUHSDUHG ZLWK SO DOLTXRWV RI WKH HOHYHQ ( GLOXWLRQV ZKLFK UDQJHG IURP [ 2n QJZHOOf WR [n QJZHOOf 7KH UDZ OHDFKDWH

PAGE 237

VDPSOHV VROYHQW H[WUDFWV RI WKH 06: ODQGILOO OHDFKDWHV ( VSLNHG VROYHQW H[WUDFWV SRVLWLYH (f DQG QHJDWLYH PHWKDQROf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f 7KH ( HTXLYDOHQWV IRU WKH UDZ OHDFKDWHV OHDFKDWH H[WUDFWV DQG HQYLURQPHQWDO VDPSOHV ZHUH GHWHUPLQHG E\ H[WUDSRODWLRQ IURP WKH OLQHDU UHJLRQ RI WKH ( SRVLWLYH FRQWURO FXUYH 6KHDKDQ HW DO E )OROEURRN HW DO f )ROORZLQJ WKH DVVD\ D VWXGHQWnV WWHVW ZDV XVHG WR GHWHUPLQH GLIIHUHQFHV EHWZHHQ WKH FRQWURO DEVRUEDQFH DQG VDPSOH DEVRUEDQFH 7R[LFLW\ RI 06: /HDFKDWHV WR LRGRSKHQ\O@> QLWURSKHQ\O@ SKHQ\O WHWUD]ROLXP FKORULGH ,17f 6LJPDf %LWWRQ DQG .RRSPDQ

PAGE 238

f ,Q WKH SUHVHQFH RI YLDEOH \HDVW FHOOV ,17 D FOHDU OLTXLGf LV UHGXFHG WR UHG ,17IRUPD]DQ FU\VWDOV &RQFXUUHQWO\ ZLWK WKH <(6 DVVD\ WKH YLDELOLW\ RI WKH \HDVW FHOOV ZDV HYDOXDWHG ZLWK DOLTXRWV RI HDFK UDZ OHDFKDWH OHDFKDWH FRQFHQWUDWH RU ZDWHU VDPSOH $IWHU WKH <(6 LQFXEDWLRQ SHULRG DQ DOLTXRW RI ,17 ILQDO FRQFHQWUDWLRQ bZHOOf ZDV DGGHG WR GHVLJQDWHG PLFURSODWH ZHOOV IRU WKH YLDELOLW\ DVVD\ 'HVLJQDWHG ZHOOV IRU WKH WR[LFLW\ DVVD\V FRQWDLQHG SO DOLTXRWV RI WKH UDZ OHDFKDWH VDPSOH ZKLFK ZDV FRPELQHG ZLWK SO DOLTXRWV RI WKH ; DVVD\ PHGLD SUHSDUHG ZLWKRXW WKH FKURPRJHQLF VXEVWUDWH &35*f 7KH PLFURSODWH ZDV WKHQ LQFXEDWHG IRU DQ DGGLWLRQDO PLQXWHV &RORU GHYHORSPHQW LQ WKH PLFURSODWH ZHOOV ZDV TXDQWLILHG ZLWK D PLFURSODWH UHDGHU 0XOWLVNDQ 3OXV 0. ,, ,&1 %LRPHGLFDOV +XQWVYLOOH $/f DW QP *&06 $QDO\VLV 7KH OHDFKDWHV ZHUH DQDO\]HG LQ WULSOLFDWH XVLQJ D )LQQLJDQ 7UDFH JDV FKURPDWRJUDSK\PDVV VSHFWURPHWU\ *&06f LQVWUXPHQW 7KH *& FROXPQ ZDV D '%06 -t: 6FLHQWLILFf P ORQJ PP LG FRDWHG ZLWK SP ILOP WKLFNQHVV 7KH LQMHFWLRQ SRUW WHPSHUDWXUH ZDV r& 7KH WHPSHUDWXUH RI WKH LQWHUIDFH WUDQVIHU OLQH WR WKH PDVV VSHFWURPHWHU ZDV PDLQWDLQHG DW r& ,QLWLDO RYHQ WHPSHUDWXUH ZDV r& DQG ZDV KHOG IRU PLQXWHV 7KH RYHQ ZDV WKHQ KHDWHG WR r& DW D KHDWLQJ UDWH RI r&PLQXWH DQG ZDV KHOG DW WKDW WHPSHUDWXUH IRU PLQXWHV 7KH *&06 ZDV RSHUDWHG LQ IXOO VFDQ PRGH P] f &RPSRXQGV ZHUH WHQWDWLYHO\ LGHQWLILHG E\ OLEUDU\ VHDUFKHV XVLQJ D 1,67(3$1,+ PDVV VSHFWUDO OLEUDU\ RI FRPSRXQGV YHUVLRQ 7KH UHIHUHQFH VWDQGDUGV 13 23 W%3 '03 %%3 '%3 %LVSKHQRO $

PAGE 239

2 %HWDHVWUDGLRO QJ/f )LJXUH 5HVSRQVH RI WKH <(6 DVVD\ WR S HVWUDGLRO (f Q f ZLWK HUURU EDUV UHSUHVHQWLQJ WKH b FRQILGHQFH LQWHUYDO DQG WKH VROLG OLQH WKH VROYHQW EODQN 7HWUDEURPRELVSKHQRO $ DQG WHUWEXW\OaK\GUR[\DQLVROH ZHUH DQDO\]HG XVLQJ D VLQJOH VWDQGDUG RI PJPO 5HVXOWV +RUPRQDO $FWLYLW\ RI 06: /DQGILOO /HDFKDWHV 7KH KRUPRQDO DFWLYLW\ RI HDFK OHDFKDWH ZDV H[SUHVVHG DV WKH ( HTXLYDOHQW UHVSRQVH 7KH PLQLPXP GHWHFWLRQ OLPLW 0'/f IRU WKH <(6 DVVD\ ZDV QJ (/ ZKLFK ZDV GHWHUPLQHG DV WKH PHDQ DEVRUEDQFH RI WKH VROYHQW FRQWURO SOXV WZR WLPHV WKH VWDQGDUG GHYLDWLRQ 7KHUH ZDV D VLJQLILFDQW Sf GLIIHUHQFH EHWZHHQ WKH ORZHVW FRQFHQWUDWLRQ RI ( DVVD\HG DQG WKH VROYHQW EODQN )LJXUH f

PAGE 240

7DEOH +RUPRQDO DFWLYLW\ RI UDZ 06: ODQGILOO OHDFKDWHV DQG WKHLU PHWKDQRO H[WUDFWV DV HTXLYDOHQW SHVWUDGLRO FRQFHQWUDWLRQV ZLWK RWKHU ZDWHU VDPSOHV LQFOXGHG IRU FRPSDULVRQ +RUPRQDO $FWLYLW\ 6LWH QJ (/f 5DZ 0HWKDQRO /HDFKDWH ([WUDFW /) 6HSW 1+$& 17 1RY 1+$ /) )HE 1+$ 1+$ 6HSW 1+$ 17 /) 1RY 1+$ 1+$ 0DUFK 1+$ /) )HE 1+$ 1+$ /) )HE S f 1+$ 6HSW 1+$ 17 1RY f /) 'HF 1+$ -DQ )HE 1+$ )HE r 1+$ /) 1RY 0DUFK 1+$ /) )HE 1+$ 1+$ /DNH $OLFH -DQ 17 /DNH %HYHUO\ -DQ 1+$ 17 ::73 ,QI 6HSW 17 ::73 (II 6HSW 1+$ 17 6: 6HSW 1+$ 17 %ODQN 1+$ 1+$ f$VVD\HG DW GLOXWLRQ f$VVD\HG DW WLPHV UDZ OHDFKDWH fGHWHFWLRQ OLPLW QJ/ $EEUHYLDWLRQV 18$ QRW KRUPRQDOO\ DFWLYH 17 QRW WHVWHG ::73 ,1) ZDVWHZDWHU WUHDWPHQW SODQW LQIOXHQW ::73 ()) ZDVWHZDWHU WUHDWPHQW SODQW HIIOXHQW 6: VWRUPZDWHU 6WDWLVWLFDO GLIIHUHQFH IURP FRQWURO S e S 7ZHQW\ OHDFKDWH VDPSOHV ZHUH FROOHFWHG IURP HLJKWOLQHG 06: ODQGILOOV LQ )ORULGD 86$ DQG WHVWHG IRU WKHLU KRUPRQDO DFWLYLW\ ZLWK D UHFRPELQDQW \HDVW HVWURJHQ VFUHHQ <(6f DVVD\ 7KH VXUYH\HG VDPSOHV UHSUHVHQW W\SLFDO 06: ODQGILOO OHDFKDWHV IURP )ORULGD 5HLQKDUW DQG *URVK :DUG HW DOf f

PAGE 241

7DEOH VXPPDUL]HV WKH KRUPRQDO DFWLYLW\ RI WKH UDZ 06: ODQGILOO OHDFKDWHV 7KH UHVXOWV UHSUHVHQW WKH DFWLYLW\ LQ WKH UDZ OHDFKDWHV DIWHU D RQHWKLUG GLOXWLRQ E\ DGGLWLRQ RI WKH DVVD\ PHGLXP 7KH KRUPRQDO DFWLYLW\ RI WKH UDZ ODQGILOO OHDFKDWHV ZDV YDULDEOH +RUPRQDO DFWLYLW\ ZDV LGHQWLILHG LQ WKH UDZ 06: ODQGILOO OHDFKDWHV IURP /) 0DUFK nf DW QJ (/ EXW QRW LQ OHDFKDWHV FROOHFWHG LQ 6HSWHPEHU DQG 1RYHPEHU RI 7KH UDZ OHDFKDWH FROOHFWHG IURP /) LQ 1RYHPEHU GLVSOD\HG WKH KLJKHVW KRUPRQDO DFWLYLW\ DW QJ (/ &RPSDUDWLYHO\ OHDFKDWHV ZLWK ORZ KRUPRQDO DFWLYLW\ ZHUH FROOHFWHG IURP /) LQ -DQXDU\ )HEUXDU\ DQG )HEUXDU\ ZLWK FRQFHQWUDWLRQV RI DW DQG QJ (/ UHVSHFWLYHO\ 7KH UDZ OHDFKDWHV FROOHFWHG IURP /) GLVSOD\HG KRUPRQDO DFWLYLW\ HTXLYDOHQW WR QJ (/ LQ 1RYHPEHU DQG QJ (/ LQ 0DUFK $OO RI WKH KRUPRQDO UHVSRQVHV DVVRFLDWHG ZLWK WKH UDZ OHDFKDWHV ZHUH VLJQLILFDQWO\ GLIIHUHQW IURP WKH FRQWUROV DW HLWKHU S RU S ZLWK RQH H[FHSWLRQ 7KH KRUPRQDO DFWLYLW\ VKRZQ E\ WKH OHDFKDWHV FROOHFWHG IURP /) ZHUH HTXLYDOHQW WR QJ (/ KRZHYHU WKH VLJQLILFDQFH RI WKLV DFWLYLW\ ZDV DW WKH S OHYHO 7KH KRUPRQDO DFWLYLW\ LQ WKH UDZ 06: ODQGILOO OHDFKDWHV ZDV YDULDEOH DQG IOXFWXDWHG RYHU WLPH 7KLV LQGLFDWHG WKH LQIOXHQFH RI VLWHVSHFLILF SDUDPHWHUV HJ KHWHURJHQHRXV ZDVWH FRPSRVLWLRQ GHJUHH RI ZDVWH GHFRPSRVLWLRQ DQG HQYLURQPHQWDO FRQGLWLRQV OLNH UDLQIDOO DQG WHPSHUDWXUH RQ OHDFKDWH SURGXFWLRQ DQG TXDOLW\ .MHOGVHQ HW DO f 2YHU D ILYHPRQWK SHULRG WKH <(6 DVVD\ VKRZHG WKDW WKH KRUPRQDO DFWLYLW\ DW /) UDQJHG IURP QR DFWLYLW\ WR QJ (/ 6LPLODU IOXFWXDWLRQV ZHUH SUHYLRXVO\ UHSRUWHG ZLWK ZDVWHZDWHU WUHDWPHQW SODQW

PAGE 242

HIIOXHQWV DQG ZHUH DWWULEXWHG WR HQYLURQPHQWDO IDFWRUV OLNH WHPSHUDWXUH DQG UDLQIDOO 0XUN HW DO f +RUPRQDOO\ DFWLYH FRPSRXQGV ZHUH QRW DVVRFLDWHG ZLWK VROLGV LQ WKH )ORULGD ODQGILOO OHDFKDWHV 'HVEURZ HW DO f IRXQG WKDW KRUPRQDO DFWLYLW\ ZDV QRW DVVRFLDWHG ZLWK VROLGV LQ ::73 HIIOXHQWV KRZHYHU VXVSHQGHG SDUWLFXODWH PDWWHU LQ ULYHU ZDWHU VDPSOHV FROOHFWHG LQ WKH 1HWKHUODQGV ZHUH H[WHQVLYHO\ FRQWDPLQDWHG E\ KRUPRQDOO\ DFWLYH FRPSRXQGV 0XUN HW DO f )RU FRPSDULVRQ WKH KRUPRQDO DFWLYLW\ RI YDULRXV HQYLURQPHQWDO VDPSOHV ZDV LQYHVWLJDWHG 7KH KRUPRQDO DFWLYLW\ RI WKH ZDWHU VDPSOH FROOHFWHG IURP /DNH $OLFH ZDV HTXLYDOHQW WR QJ (/ ZKLOH WKH /DNH %HYHUO\ VDPSOH ZDV QRW DFWLYH $OWKRXJK EH\RQG WKH VFRSH RI WKLV UHVHDUFK WKH ORZ KRUPRQDO DFWLYLW\ UHSRUWHG LQ WKH /DNH $OLFH VDPSOH ZDUUDQWV IXUWKHU LQYHVWLJDWLRQ 6LQFH WKH ODNH GRHV QRW UHFHLYH DQ\ GLUHFW GLVFKDUJHV WKHQ UXQRII RU RWKHU QRQSRLQW VRXUFHV PD\ EH FRQWULEXWLQJ IDFWRUV 6Q\GHU HW DO f ,Q )OHPLVK VXUIDFH ZDWHUV KRUPRQDO DFWLYLW\ UDQJLQJ IURP WR QJ (/ ZDV UHSRUWHG XVLQJ WKH VDPH <(6 DVVD\ :LWWHUV HW DO f 7KH GRPHVWLF ::73 LQIOXHQW GLVSOD\HG VLJQLILFDQW KRUPRQDO DFWLYLW\ S f HTXLYDOHQW WR QJ (/ 7KLV ZDV KLJKHU WKDQ WKH KRUPRQDO DFWLYLW\ RI ::73 LQIOXHQWV DVVD\HG LQ 9LUJLQLD ZKLFK UDQJHG IURP WR QJ (/ +ROEURRN HW DO f :KHQ FRPSDUHG WR UHSRUWV RI KRUPRQDO DFWLYLW\ LQ WKH LQIOXHQWV RI ::73V WKLV DFWLYLW\ ZDV ORZ ,Q -DSDQ WKH KRUPRQDO DFWLYLW\ RI ::73 LQIOXHQWV KDV EHHQ UHSRUWHG DW QJ (/ 0DWVXL HW DO f ,Q WKLV LQYHVWLJDWLRQ WHUWLDU\ ELRORJLFDO WUHDWPHQW UHGXFHG WKH KRUPRQDO DFWLYLW\ WR EHORZ

PAGE 243

WKH GHWHFWLRQ OLPLW RI WKH DVVD\ QJ/f (DUOLHU UHVHDUFK KDV VKRZQ KRUPRQDO DFWLYLW\ LQ ::73 HIIOXHQWV WKDW UDQJHG IURP QJ (/ 7KRPDV HW DO f WR QJ (/ 'HVEURZ HW DO f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f 6ROLG SKDVH H[WUDFWLRQ GLVNV KDYH D ODUJH VXUIDFH DUHD DQG ORZ VXVFHSWLELOLW\ WR FORJJLQJ WKHUHIRUH WKH\ DUH D KLJKO\ HIILFLHQW FKRLFH IRU XVH ZLWK 06: ODQGILOO OHDFKDWHV /RSH] DQG %DUFHOR f 7KH PHWKDQRO H[WUDFWV RI WKH OHDFKDWHV ZHUH DVVD\HG IRU KRUPRQDO DFWLYLW\ DQG WKHLU UHVSRQVHV ZHUH ORZHU WKDQ WKH UDZ OHDFKDWHV :LWK WKH PHWKDQRO H[WUDFWV ILYH RI WKH VHYHQWHHQ VDPSOHV ZHUH KRUPRQDOO\ DFWLYLW\ 7DEOH f 7KH PHWKDQRO H[WUDFWV RI WKH OHDFKDWHV IURP /) 1RY f GLVSOD\HG WKH KLJKHVW KRUPRQDO DFWLYLW\ DW QJ (/ ZKLOH WKH ORZHVW DFWLYLW\ ZDV GHWHUPLQHG LQ H[WUDFWV IURP /) 1RY f DW QJ (/ +RUPRQDO DFWLYLW\ ZDV LGHQWLILHG LQ WKH PHWKDQRO H[WUDFWV RI OHDFKDWHV FROOHFWHG IURP /) LQ 1RYHPEHU DQG 'HFHPEHU DQG -DQXDU\ DW DQG QJ (/ UHVSHFWLYHO\ 7KHUH ZDV QR KRUPRQDO DFWLYLW\ LQ WKH PHWKDQRO H[WUDFWV RI WKH OHDFKDWHV FROOHFWHG IURP /) /) /) /) DQG /) 0HWKDQRO

PAGE 244

7DEOH +RUPRQDO DFWLYLW\ RI UDZ 06: ODQGILOO OHDFKDWHV IURP /) EHIRUH LQIOXHQWf DQG DIWHU HIIOXHQWf WUHDWPHQW LQ D SRZGHUHG DFWLYDWHG FDUERQ 6DPSOH +RUPRQDO $FWLYLW\ QJ (/f ,QIOXHQW s