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Detection of Selected Pharmaceutical Compounds and Determination of Their Fate in Modern Lined Landfills

Permanent Link: http://ufdc.ufl.edu/UFE0022013/00001

Material Information

Title: Detection of Selected Pharmaceutical Compounds and Determination of Their Fate in Modern Lined Landfills
Physical Description: 1 online resource (216 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acetaminophen, acetylsalicylic, anaerobic, biodegradation, disposal, ethinylestradiol, ibuprofen, landfill, medication, metoprolol, msw, ppcp, progesterone
Environmental Engineering Sciences -- Dissertations, Academic -- UF
Genre: Environmental Engineering Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Research was performed to characterize discarded household medication disposal in municipal solid waste (MSW) and the fate of selected pharmaceuticals in landfills. A community household pharmaceutical collection program was conducted to examine the quantity and characteristics of pharmaceutical compounds entering MSW and to determine factors affecting the efficiency of special programs for medication waste collection. Collection program data confirmed that the disposal methods for medications most commonly utilized by households were predominantly the sewage system and MSW. Information gathered from participants showed that program efficiency would be optimized by targeting older age groups, developing systems to reduce medication loss due to expiration, and providing a continuous, convenient collection point. The concentration of active pharmaceutical ingredients (APIs) in MSW was determined using a mathematical estimation and directly by performance of a waste composition study. In the mathematical estimation, the variable of greatest uncertainty was the quantity of medications which become unused once dispensed. An estimated range of APIs in MSW of 7.4 mg/kg to 45 mg/kg was calculated. In direct measurement, APIs comprised 8.1 mg/kg of MSW with 22 differing compounds present and another 33 identified as potentially present due to the presence of empty containers. Three laboratory experiments were performed to determine the fate of pharmaceuticals in landfills. Six pharmaceutical compounds were examined: 17alpha-ethinylestradiol, acetaminophen, acetylsalicylic acid, ibuprofen, metoprolol tartrate, and progesterone. Adsorption to solid waste materials and other abiotic mechanisms played a strong role in retention of progesterone and 17alpha-ethinylestradiol in landfills and removal from landfill leachate. Adsorption and biodegradation were both factors in the fate of metoprolol tartrate and acetaminophen. Anaerobic biodegradation of acetylsalicylic acid was significant, while ibuprofen demonstrated a resistance to degradation and adsorption in all experiments. Analysis of actual landfill leachate for the six pharmaceutical compounds from the laboratory experiments plus an additional four compounds (caffeine, cephalexin, hydrochlorothiazide, and prednisone) was conducted to characterize the discharge of pharmaceutical compounds from landfills. Ibuprofen was measured in 9 of 10 landfill leachates and in the leachate of a solid waste transfer station and standing water on the face of a landfill. Other compounds detected included acetaminophen, acetylsalicylic acid (salicylic acid) and caffeine. Landfills which had the greatest number of compounds detected were those of the largest size, serving the largest population. Landfills which currently or previously practiced leachate recirculation, on average had higher concentrations of ibuprofen in their leachate than those which did not. Comparison of acetaminophen, acetylsalicylic acid, and ibuprofen inputs to wastewater treatment plants from landfill leachate treatment versus inputs from the sewage system showed landfills to be a significantly smaller source of active pharmaceutical ingredients.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Townsend, Timothy G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022013:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022013/00001

Material Information

Title: Detection of Selected Pharmaceutical Compounds and Determination of Their Fate in Modern Lined Landfills
Physical Description: 1 online resource (216 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: acetaminophen, acetylsalicylic, anaerobic, biodegradation, disposal, ethinylestradiol, ibuprofen, landfill, medication, metoprolol, msw, ppcp, progesterone
Environmental Engineering Sciences -- Dissertations, Academic -- UF
Genre: Environmental Engineering Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Research was performed to characterize discarded household medication disposal in municipal solid waste (MSW) and the fate of selected pharmaceuticals in landfills. A community household pharmaceutical collection program was conducted to examine the quantity and characteristics of pharmaceutical compounds entering MSW and to determine factors affecting the efficiency of special programs for medication waste collection. Collection program data confirmed that the disposal methods for medications most commonly utilized by households were predominantly the sewage system and MSW. Information gathered from participants showed that program efficiency would be optimized by targeting older age groups, developing systems to reduce medication loss due to expiration, and providing a continuous, convenient collection point. The concentration of active pharmaceutical ingredients (APIs) in MSW was determined using a mathematical estimation and directly by performance of a waste composition study. In the mathematical estimation, the variable of greatest uncertainty was the quantity of medications which become unused once dispensed. An estimated range of APIs in MSW of 7.4 mg/kg to 45 mg/kg was calculated. In direct measurement, APIs comprised 8.1 mg/kg of MSW with 22 differing compounds present and another 33 identified as potentially present due to the presence of empty containers. Three laboratory experiments were performed to determine the fate of pharmaceuticals in landfills. Six pharmaceutical compounds were examined: 17alpha-ethinylestradiol, acetaminophen, acetylsalicylic acid, ibuprofen, metoprolol tartrate, and progesterone. Adsorption to solid waste materials and other abiotic mechanisms played a strong role in retention of progesterone and 17alpha-ethinylestradiol in landfills and removal from landfill leachate. Adsorption and biodegradation were both factors in the fate of metoprolol tartrate and acetaminophen. Anaerobic biodegradation of acetylsalicylic acid was significant, while ibuprofen demonstrated a resistance to degradation and adsorption in all experiments. Analysis of actual landfill leachate for the six pharmaceutical compounds from the laboratory experiments plus an additional four compounds (caffeine, cephalexin, hydrochlorothiazide, and prednisone) was conducted to characterize the discharge of pharmaceutical compounds from landfills. Ibuprofen was measured in 9 of 10 landfill leachates and in the leachate of a solid waste transfer station and standing water on the face of a landfill. Other compounds detected included acetaminophen, acetylsalicylic acid (salicylic acid) and caffeine. Landfills which had the greatest number of compounds detected were those of the largest size, serving the largest population. Landfills which currently or previously practiced leachate recirculation, on average had higher concentrations of ibuprofen in their leachate than those which did not. Comparison of acetaminophen, acetylsalicylic acid, and ibuprofen inputs to wastewater treatment plants from landfill leachate treatment versus inputs from the sewage system showed landfills to be a significantly smaller source of active pharmaceutical ingredients.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Townsend, Timothy G.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022013:00001


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DETECTION OF SELECTED PHAR MACEUTICAL COMPOUNDS AND DETERMINATION OF THEIR FATE IN MODERN LINED LANDFILLS By STEPHEN E. MUSSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Stephen E. Musson 2

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To Grayson, by far my best discovery. 3

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ACKNOWLEDGMENTS I am indebted to my advisor, Dr. Timothy Townsend, for all of the support and guidance he has provided in the more than 12 years I ha ve known him. Dr. Townsend has been a true mentor and friend. He has given me numerous opportunities to learn, teach, and develop as a professional and as a person. I will always be grateful. I wish to express my gratitude to Dr. Thabet Tolaymat for the financial, professional, and personal support that he provided. His time and efforts were invaluable. I would also like to thank my other committee members, Dr. Joseph Delfino, Dr. Angela Lindner, a nd Dr. Hartmut Derendorf for their guidance and willingness to work with me on a project far from the University of Florida. Additionall y, I wish to express my gratitude to Dr. Jean-Claude Bonzongo. I wish to thank the Hinkley Center for So lid and Hazardous Waste Management for their support of household pharmaceutical waste research. I am also thankful to Dr. Makram Suidan for his guidance. I further wish to thank Pablo Campo-Moreno and Ruth Marfil-Vega for their countless hours in the lab. I would also like to thank Dr. Brajesh Dubey and Dr. Hwidong Kim and other members of the Solid and Hazardous Waste Research Group at the University of Florida for assisting in comp letion of this project. I am grateful to my family. I am grateful to my mother, whose life as an educator has extended far beyond her classroom and who gave me the basis to chase my dreams. I am grateful for my Dad who did so many little things that I appreciat ed much too late. I wish to thank Kyle and Kacey whose patience with their fa thers adventures has been endless. Finally, I want to express my gratitude and love to my wife, Grayson, for her love, support, and neverwavering friendship. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES .........................................................................................................................10LIST OF FIGURES .......................................................................................................................13ABSTRACT ...................................................................................................................... .............15CHAPTER 1 INTRODUCTION ................................................................................................................ ..17Background .................................................................................................................... .........17Research Objectives ........................................................................................................... .....19Research Approach .................................................................................................................20Study Overview ......................................................................................................................222 BACKGROUND AND LI TERATURE REVIEW ................................................................23Introduction .................................................................................................................. ...........23Sources of Pharmaceuticals in the Environment ....................................................................23Sewage Treatment Works (STWs) ..................................................................................23Agriculture .......................................................................................................................25Landfills ..................................................................................................................... ......27Effects of PPCPs in the Environment .....................................................................................30Hormones (and Endocrine Disrupting Compounds) .......................................................31Antibiotics .......................................................................................................................32Blood Lipid Regulators ...................................................................................................33Analgesic/Anti-inflammatory Drugs ...............................................................................33Other Drug Classes ..........................................................................................................34Environmental Exposure Factors ....................................................................................35Persistence ................................................................................................................35Synergy .....................................................................................................................36Inconspicuous Change ..............................................................................................36Pharmaceutical Regulation ..................................................................................................... 36European Medicines Agency ...........................................................................................37United States Food and Dr ug Administration (FDA) ......................................................38United States Drug Enforcement Agency (DEA) ...........................................................39United States Environmental Protection Agency (EPA) .................................................40Other Regulation .............................................................................................................41Pharmaceutical Disposal ....................................................................................................... ..42Non-Residential GeneratorsHospitals, Pharmacies, Healthcare Facilities ....................42Household Pharmaceuticals .............................................................................................43Pharmaceuticals in Landfills ...........................................................................................44 5

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Landfills and Landfill Leachate ..............................................................................................4 4Phase I: Initial Adjustment Phase ....................................................................................46Phase II: Transition Phase ...............................................................................................46Phase III: Acid Formation Phase .....................................................................................46Phase IV: Methane Fermentation Phase ..........................................................................47Phase V: Maturation Phase ..............................................................................................47Landfill Leachate .............................................................................................................473 CONTINUOUS COLLECTION SYSTEM FOR HOUSEHOLD PHARMACEUTICAL WASTES: A PILOT PROJECT .............................................................................................55Introduction .................................................................................................................. ...........55Materials and Methods ...........................................................................................................55Results and Discussion ........................................................................................................ ...59Conclusions .............................................................................................................................624 PHARMACEUTICAL COMPOUND CON TENT OF MUNICIPAL SOLID WASTE .......71Introduction .................................................................................................................. ...........71Materials and Methods ...........................................................................................................72Mathematical Estimation .................................................................................................72Direct Measurement ........................................................................................................73Waste Sectors ...........................................................................................................73Sampling and Sorting ...............................................................................................74Results and Discussion ........................................................................................................ ...75Mathematical Estimation .................................................................................................75Direct Measurement ........................................................................................................775 DETERMINATION OF THE ANAEROBIC BIODEGRADATION POTENTIAL OF SELECTED ACTIVE PHARMACE UTICAL INGREDIENTS ...........................................83Introduction .................................................................................................................. ...........83Materials and Methods ...........................................................................................................85Selection of Target Pharmaceutical Compounds ............................................................85Reagents and Test Compounds .......................................................................................85Respirometric Testing .....................................................................................................85Analytical Methods .........................................................................................................88Standards Preparation ......................................................................................................88Sample Preparation ..........................................................................................................88Chromatography and Mass Spectrometry .......................................................................88Anaerobic Biodegradation Model Prediction ..................................................................90Data Analysis ...................................................................................................................90Results and Discussion ........................................................................................................ ...91Biodegradation Gas Production .......................................................................................91Degradation Direct Concentration Measurement ..........................................................92Progesterone and 17 -Ethinylestradiol ....................................................................93Acetaminophen .........................................................................................................94 6

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Acetylsalicylic Acid .................................................................................................94Ibuprofen ..................................................................................................................95Metoprolol Tartrate ..................................................................................................96Anaerobic Biodegradation Model ...................................................................................96Summary ....................................................................................................................... ..........986 EFFECTS OF LANDFILL RETENTION TI ME AND ORGANIC MATTER ON THE FATE OF SELECTED ACTIVE PHAR MACEUTICAL INGREDIENTS ........................105Materials and Methods .........................................................................................................106Selection of Target Pharmaceutical Compounds ..........................................................106Reagents and Test Compounds .....................................................................................107Sample Preparation ........................................................................................................107Respirometric Testing ...................................................................................................110Analytical Method .........................................................................................................110Standards Preparation ....................................................................................................110Sample Preparation ........................................................................................................110Chromatography and Mass Spectrometry .....................................................................111Data Analysis .................................................................................................................112Results and Discussion ........................................................................................................ .112Respirometric Testing ...................................................................................................112Pharmaceutical Compound Degradati on Analytical Measurement ...........................11417 -Ethinylestradiol ...............................................................................................114Acetaminophen .......................................................................................................115Acetylsalicylic Acid. ..............................................................................................117Ibuprofen ................................................................................................................117Metoprolol Tartrate ................................................................................................118Progesterone ...........................................................................................................120Correlation of Results with Prio r Anaerobic Degradation Test .............................120Summary ....................................................................................................................... ........1227 FATE OF SELECTED ACTIVE PHARMACEUTICAL INGREDIENTS PRESENT IN LEACHATE UNDER SIMULATED A NAEROBIC MUNICIPAL SOLID WASTE LANDFILL ENVIRONMENTS ..........................................................................................132Introduction .................................................................................................................. .........132Materials and Methods .........................................................................................................133Solid Waste Fabrication ................................................................................................133Sample Preparation ........................................................................................................134Respirometric Testing ...................................................................................................135Analytical Method .........................................................................................................136Standards Preparation ....................................................................................................136Sample Preparation ........................................................................................................136Solid Sample Extraction ................................................................................................136Chromatography and Mass Spectrometry .....................................................................137Data Analysis .................................................................................................................138Results and Discussion ........................................................................................................ .138 7

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Respirometric Testing ...................................................................................................138Pharmaceutical Compound Degradati on Analytical Measurement ...........................13917 -Ethinylestradiol ...............................................................................................140Acetaminophen .......................................................................................................141Acetylsalicylic Acid ...............................................................................................143Ibuprofen ................................................................................................................144Metoprolol Tartrate ................................................................................................145Progesterone ...........................................................................................................146Correlation of Results with Prio r Experimental Observations ...............................147Summary ....................................................................................................................... ........1488 DETECTION OF SELECTED PH ARMACEUTICAL COMPOUNDS IN MUNICIPAL SOLID WASTE LANDFILL LEACHATE ..................................................158Introduction .................................................................................................................. .........158Methods and Materials .........................................................................................................160Selection of Target Pharmaceutical Compounds ..........................................................160Sample Collection .........................................................................................................160Leachate Chemical Analysis .........................................................................................161Pharmaceutical Compound Analysis .............................................................................161Results and Discussion ........................................................................................................ .162MSW Landfill Characteristics versus Leach ate Pharmaceutical Compound Content ..164MSW Landfill Leachate vs. Wastewater Tr eatment Plant Sewage Influent .................166Correlation of Results with Prio r Experimental Observations ......................................168Summary ....................................................................................................................... ........1699 SUMMARY AND CONCLUSIONS ...................................................................................176Summary ....................................................................................................................... ........176Conclusions ...........................................................................................................................180Future Work ..........................................................................................................................181APPENDIX A METHOD FOR THE SELECTION OF PH ARMACEUTICAL COMPOUNDS FOR EXAMINATION OF LANDFILL ANAE ROBIC BIODEGRADABILITY AND PRESENCE IN MUNICIPAL SOLI D WASTE LANDFI LL LEACHATE ........................185Pharmaceutical Compound Selection ...................................................................................185MS Optimization ..................................................................................................................189B US EPA EPI SUITE BIOWIN ANAEROBIC BIODEGRADATION MODEL RESULTS ....................................................................................................................... ......198C QUALITY ASSURANCE / QUALITY CONTROL ...........................................................201Quality Control .....................................................................................................................201Sample Identification .....................................................................................................201 8

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9 Gas Analysis ..................................................................................................................201Chemical Analysis .........................................................................................................202LIST OF REFERENCES .............................................................................................................204BIOGRAPHICAL SKETCH .......................................................................................................216

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LIST OF TABLES Table page 2-1 Summary of pharmaceuticals detected in sewage treatment plant effluents ....................502-2 Common disposal methods by percentage for household medication disposal .................532-3 Landfill leachate concentration ranges as a function of the degree of landfill stabilization ................................................................................................................. .......533-1 Pharmaceuticals collected for disposal by 2004 Alachua County, Florida collection program ....................................................................................................................... .......654-1 Percent composition of research study re sidential and commercial municipal solid waste vs. United States national averages..........................................................................804-2 Active pharmaceutical ingredients collect ed in measurable quantities within municipal solid waste .........................................................................................................804-3 Active pharmaceutical compounds of empty medication containers collected from municipal solid waste .........................................................................................................815-1 Pharmaceutical compound sample mass equivalent to 50 mg/L organic carbon content in 300 ml sample solution ...................................................................................1005-2 Method detection limit for analysis of tested pharmaceutical compounds in anaerobic sludge media .................................................................................................................. ..1005-3 Comparison of theoretical and experimental methane production ..................................1005-4 Average abiotic removal of selected pharmaceutical compounds in biologically inactive test samples ........................................................................................................1 015-5 Average biodegradation of selected ph armaceutical compounds in anaerobic sludge test samples .................................................................................................................. ....1015-6 Average overall reduction of selected pharmaceutical compounds in anaerobic sludge test samples ........................................................................................................... 1015-7 Anaerobic biodegradation potential for target pharmaceutical compounds predicted by U.S. EPA BIOWIN program model. ..........................................................................1026-1 Anaerobic degradation gas production of pharmaceutical compound samples at concentrations of 50, 250, and 500 g/L with 50 mg/L organic carbon from crystalline cellulose. .........................................................................................................124 10

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6-2 Percentage of methane within anae robic degradation gas of six selected pharmaceutical compounds at 50, 250, and 500 g/L and a corresponding blank sample with 50 mg/L organic carbon from crystalline cellulose. ....................................1246-3 Concentration of pharmaceutical com pounds in biologically active and abiotic samples following 112 days of incubation.......................................................................1256-4 Seven day adsorption to cellulose in u ltrapure water versus autoclaved anaerobic sludge solution of metoprolol tartrate at 50, 250, and 500 g/L initial concentrations. ..1257-1 Composition of fabricated municipal solid waste for pharmaceutical degradation samples ....................................................................................................................... ......1517-2 Method detection limit for analysis of pha rmaceutical compounds in landfill leachate .1517-3 Total gas production and final degradation gas concentrations of pharmaceutical and municipal solid waste mixtures af ter 84 days of degradation. .........................................1527-4 Final Landfill Leachate Concentrations of 500 g/L Selected Pharmaceutical Compound Samples Following 84 days of In cubation with Municipal Solid Waste ......1527-5 Quantity of pharmaceutical compounds rec overed from solid fraction of simulated MSW after 84 days using solid/liquid extraction ............................................................1538-1 Chemical properties of pharmaceutical com pounds selected for analysis in municipal solid waste landfill leachate .............................................................................................1708-2 Operating characteristics of ten Florida MSW landfills examined for leachate pharmaceutical compound content. .................................................................................1718-3 Physical and chemical properties of leachate from ten Florida MSW landfills examined for selected pharmaceutical compounds. .........................................................1728-4 Concentration of selected pharmaceutical compounds in municipal solid waste landfill leachate ................................................................................................................1728-5 Rainfall at Florida landfill sites for 1, 3 and 12 months prior to sampling ......................1738-6 Comparison of selected pharmaceuti cal compound measured landfill leachate concentrations with published maximum wastewater treatment plant influent concentrations ................................................................................................................ ..1738-7 Wastewater treatment plant input of pharmaceutical compounds from MSW landfill leachate versus estimated sewage system influent ...........................................................174A-1 Top 50 prescribed pharmaceuticals by to tal number of prescriptions, 2002-2004. .........191A-2 Top 30 prescribed pharmaceutical s 2002-2004 based on cumulative rank .....................193 11

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12 A-3 Top 20 pharmaceuticals for investigati on based on 2004 total mass prescribed .............194A-4 Twenty-five most commonly taken pres cription and nonprescription medications by U.S. adults in 2004 ...........................................................................................................195A-5 Selective reaction monitoring parameters utilized for target compounds in positive and negative ionization modes .........................................................................................196A-6 Source parameters used for electrospray ionization ........................................................196B-1 Complete anaerobic biodegradati on model results for acetaminophen ...........................198B-2 Complete anaerobic biodegradati on model results for metoprolol ..................................198B-3 Complete anaerobic biodegradation m odel results for acetylsalicylic acid .....................199B-4 Complete anaerobic biodegrada tion model results for ibupofen .....................................199B-5 Complete anaerobic biodeg radation model results for 17 -ethinylestradiol ...................200B-6 Complete anaerobic biodegradati on model results for progesterone ...............................200C-1 Degradation gas analysis ca libration curve data points ...................................................203

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LIST OF FIGURES Figure page 3-1 Collection container used in Alachua County discarded medication collection program ....................................................................................................................... .......683-2 Age of medication users (years) particip ating in pharmaceutical disposal program .........683-3 Histogram of survey responses to advert ising methods recognized by participants .........693-4 Histogram of survey responses to reason for pharmaceutical disposal .............................693-5 Histogram of survey responses pertaining to previously used disposal methods ..............704-1 Procedure for mathematical predicti on of the active pharmaceutical ingredient concentration of United States municipal solid waste. ......................................................825-1 Average cumulative methane production of pharmaceutical and background test samples ....................................................................................................................... ......1035-2 Chemical structure of pharmaceuticals se lected for anaerobic degradation study. .........1046-1 Change in concentration of 17 -ethinylestradiol from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples ......................................................................1266-2 Change in concentration of acetami nophen from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples ......................................................................1276-3 Change in concentration of acetylsalicy lic acid from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples ......................................................................1286-4 Change in concentration of ibuprofen fr om three initial conc entrations following 0, 7, 28, and 112 days of incubation with crys talline cellulose in active anaerobic degradation and abiotic samples ......................................................................................1296-5 Change in concentration of metoprolol tartrate from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples ......................................................................1306-6 Change in concentration of progesterone from three initial concentrations following 0, 7, 28, and 112 days of incubation with cr ystalline cellulose in active anaerobic degradation and abiotic samples ......................................................................................131 13

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14 7-1 Percentage mass of materials discarded in United States municipal solid waste after materials and compost recovery .......................................................................................1537-2 Concentration of 17 -ethinylestradiol in leachate following 0, 28, and 84 days of simulated MSW degradation. ...........................................................................................1547-3 Concentration of Acetaminophen in leachate following 0, 28, and 84 days of simulated MSW degradation. ...........................................................................................1547-4 Concentration of acetylsalicylic acid (as salicylic acid) in le achate following 0, 28, and 84 days of simulated MSW degradation. ..................................................................1557-5 Average weekly degradation gas producti on of triplicate simulated MSW samples containing acetylsalicylic acid. ........................................................................................1557-6 Concentration of ibuprofen in l eachate following 0, 28, and 84 days of MSW degradation ................................................................................................................... ....1567-7 Concentration of metoprolol tartrate in leachate following 0, 28, and 84 days of MSW degradation ............................................................................................................1567-8 Concentration of progester one in leachate following 0, 28, and 84 days of simulated MSW degradation ............................................................................................................1578-1 MSW landfill leachate sampling locations ......................................................................1759-1 Fate of selected pharmaceutical compounds in anaerobic degradab ility testing using the pharmaceutical as the sole source of organic carbon .................................................1839-2 Fate of selected pharmaceutical co mpounds in landfill leachate under simulated anaerobic municipal solid waste landfill conditions. .......................................................184A-1 Selected pharmaceuticals for examina tion in degradation and MSW leachate concentration studies. .......................................................................................................197

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DETECTION OF SELECTED PHAR MACEUTICAL COMPOUNDS AND DETERMINATION OF THEIR FATE IN MODERN LINED LANDFILLS By Stephen E. Musson May 2008 Chair: Timothy G. Townsend Major: Environmental Engineering Sciences Research was performed to characterize di scarded household medi cation disposal in municipal solid waste (MSW) and the fate of selected pharmaceuticals in landfills. A community household pharmaceutical collection pr ogram was conducted to examine the quantity and characteristics of pharmaceutical com pounds entering MSW and to determine factors affecting the efficiency of special programs fo r medication waste collec tion. Collection program data confirmed that the disposal methods for medications most commonly utilized by households were predominantly the sewage system and MSW. Information gathered from participants showed that program efficiency would be op timized by targeting older age groups, developing systems to reduce medication loss due to expi ration, and providing a continuous, convenient collection point. The concentration of active pharmaceutical in gredients (APIs) in MSW was determined using a mathematical estimation and directly by performance of a waste composition study. In the mathematical estimation, the va riable of greatest uncertainty was the quantity of medications which become unused once dispensed. An estimat ed range of APIs in MSW of 7.4 mg/kg to 45 mg/kg was calculated. In direct measuremen t, APIs comprised 8.1 mg/kg of MSW with 22 15

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16 differing compounds present and another 33 identified as potentiall y present due to the presence of empty containers. Three laboratory experiments were performed to determine the fate of pharmaceuticals in landfills. Six pharmaceutical compounds were examined: 17 -ethinylestradio l, acetaminophen, acetylsalicylic acid, ibuprofen, me toprolol tartrate, and progester one. Adsorption to solid waste materials and other abiotic mechanisms played a strong role in reten tion of progesterone and 17 -ethinylestradiol in landfills and removal from landfill leachate. Adsorption and biodegradation were both factors in the fate of metoprolol tartra te and acetaminophen. Anaerobic biodegradation of acetylsalicylic acid wa s significant, while ibuprofen demonstrated a resistance to degradation and adsorption in all experiments. Analysis of actual landfill leachate for the six pharmaceutical compounds from the laboratory experiments plus an additional four compounds (Caffeine, Cephalexin, Hydrochlorothiazide, and Pr ednisone) was conducted to ch aracterize the discharge of pharmaceutical compounds from landfills. Ibupro fen was measured in 9 out of 10 landfill leachates and in the leachate of a solid waste tr ansfer station and standi ng water on the face of a landfill. Other compounds detected included acet aminophen, acetylsalicylic acid (salicylic acid) and caffeine. Landfills which had the greatest nu mber of compounds detected were those of the largest size, serving the largest population. Land fills which currently or previously practiced leachate recirculation, on average had higher concen trations of ibuprofen in their leachate than those which did not. Comparison of acetaminophen, acetylsalicylic acid, and ibuprofen inputs to wastewater treatment plants from landfill leachate treatment versus inputs from the sewage system showed landfills to be a significantly sma ller source of active pharmaceutical ingredients.

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CHAPTER 1 INTRODUCTION Background Each week, 82% of adults in the United Stat es use at least one form of medication and nearly one third are taking five or more (1) In 2003, national health care expenditures in the United States totaled $1.7 trillion, a 7.7% increa se from 2002. From 1995 to 2003, the average annual rate of increase for prescription drug expenditures was 14%, hi gher than any other healthcare expenditure (2) United States prescription drug sp ending is predicted to rise from 200.7 billion dollars in 2005 to 497.5 b illion dollars by 2016, a 148% increase (3) In 2005, over 3.6 billion prescriptions were purch ased at retail pharmacies alone (3) Once prescribed or purchased without a prescription, many medications go unused. The measure of prescription usage by a patient is termed compliance or concordance by the medical community and includes fact ors such as taking the medicati on at the appropriate time or under the appropriate conditions (such as a voiding confounding factors that may reduce its effectiveness) and consuming all of the medi cation prescribed. Estimates on the specific quantities of purchased medications which eventua lly become discarded vary widely with values ranging from as little as 3% up to as large as 65% (4-7) Previously, environmental pollution concer ns and regulations have focused on the traditional point discharges, what emerges from a smokestack or a discharge pipe. Concerns about the disposal of unused or expired medi cation were limited to preventing children or animals from obtaining the medications acci dentally. This resulted in the common recommendation by health care and safety profe ssions to wash pharmaceuticals into the sewage system. However, new studies began to show that many pharmaceuticals were not removed by wastewater treatment systems and enter the aquatic environment from treatment plant discharges 17

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(8-15) In 2002, the United States Geologica l Service (USGS) re leased a national reconnaissance of pharmaceuticals, hormones, and ot her organic pollutants in surface waters that revealed the presence of these compounds in more than 80% of the streams tested (16) As a result of the USGS and other studies new policies on the disposal of unused and expired household medications have emerged. In February 2007, the White House Office of National Drug Policy issued guidance to United States citizens reco mmending disposal of medications in municipal solid waste (MSW) (17) Also within that month the American Pharmacists Association in conj unction with the U.S. Fish and Wildlife Service also issued guidance with the same recomm endation dubbed SMARxT DISPOSAL (18) Many state environmental protection agencies have also developed policies and guidance documents with similar suggestions (19-23) With these new recommendations the amount of medications entering MSW is expected to rise. However, the occurrence and fate of pharmaceuticals in landfills has been largely neglected. Landfill leachate, the liquid which is the result of rainwater and preexisting moisture percolating through municipal solid waste, is captured by collection systems in modern landfills. Once collected, landfill leachate is often treated through wastew ater treatment plants. If the pharmaceutical compounds deposited within the la ndfill become dissolved or entrained in the leachate, then the possibility exists that they wi ll reach the wastewater tr eatment plant; the very process that the new polices were attempti ng to avoid. In a recent publication, Bound and Voulvoulis stated Further studies on the c oncentrations of pharmaceutical compounds within landfill sites and in leachate woul d be informative, and, if necessary, those sites not equipped with the necessary treatment facilities could be upgraded (24) 18

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Research Objectives The purpose of this research was to assess th e role landfills play in the disposal of pharmaceutical compounds and their subsequent emission to the environment. As research continues to identify the impacts of these new pollutants, the development of methods to minimize the release of these compounds is n ecessary. Landfills are characterized by high retention times and varying stages of chemi cal composition and degradation. U.S. landfills receive discarded pharmaceutical s and personal care products (PPCPs) through MSW disposal and the disposal of wastewater treatment plant biosolids. The first objective was to characterize and quantify the active pharmaceutical ingredients entering MSW in the United States. Environmen tal policy makers must weigh the cost and benefits of special collecti on programs for unused medicati ons versus MSW disposal. Determination of the quantity a nd types of medications to be collected is essential to the assessment of these programs. Furthermore, the factors influencing the success of these programs including advertising, target constituents, and regulatory limitations have been largely unexamined. Additionally, the quantity of pharm aceutical compounds entering MSW is of direct importance in determining their potential for emissi on in leachate. While prior research studies have found that households utilize MSW as freque ntly as wastewater treatment systems for the disposal of unwanted medications, none have at tempted to equate thes e levels to the amount entering MSW (24, 25) If the quantities of discarded pharmaceuticals emitted from MSW are determined to be de minimis in comparison to their discard and excr etion into wastewater treatment systems, then diversion of pharmaceu tical disposal from MSW may be unnecessary. A further objective of the research was to de termine the fate of medications once within MSW. Overall, the fates of pharmaceuticals in a landfill incl ude attenuation, degradation or decomposition, mobilization in the leachate, and volatilization and transport with the landfills 19

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gas stream. Examination of the degradation of pharmaceutical compounds has predominantly focused on efficacy and determination of shelflives. Simulating the dry, well-lit, and aerobic conditions of household medication st orage, these studies cannot be co rrelated to the dark, moist, and anaerobic conditions typical of MSW. The final objective was to determine if landf ills are a significant source of pharmaceuticals to the environment. A limited number of studies have evaluated concentrations of pharmaceutical compounds in landfill leachate and its entry into groundwater near the landfills. However, the landfills examined in these studies were unlined landfills of significant age. Thus research addressing the emission of pharmaceuticals from modern, lined landfills is necessary. Research Approach The quantity and characteristics of pha rmaceutical compounds entering MSW were examined by conducting a community household pharmaceutical collection program and a MSW composition study. Collection program data included prior medication disposal methods used by households, demographic information, and length of time from purchase to discard of the medications. Additionally, the reas on for medication disposal, such as expiration or death of the patient was determined. To quantify the amount of pharmaceutical compounds entering MSW, a mathematical estimation based upon pharmaceutical prescripti on rates, disposal practices, and MSW generation was computed. This estimation was th en compared to direct measurement of the concentration of pharmaceutical compounds in MSW by performance of a waste composition study. Waste composition studies are commonly used to assess solid waste management programs, such as measurement of plastics, pape r, glass, and metal objects in MSW to determine capture of these materials by recycling programs. However, specific separation of discarded medications permitted measurement of the concentration of the pharmaceutical active 20

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pharmaceutical ingredients in typical MSW. Additionally, the composition study allowed further identification of medications and medicati on types commonly disposed in MSW. To evaluate the fate of pharmaceuticals in an aerobic landfill environments, a series of laboratory anaerobic degradation studies were performed. While thousands of pharmaceutical compounds and metabolites exist, six pharmaceutical compounds were chosen through examination of prescription quantities, nonprescription usage, and environmental factors. Using standard respirometric tests and direct analytical measurement, the first of the experiments was designed to determine the potential for anaer obic degradation of the compounds without the presence of other substances which may inhibit degradation or cause re moval from solution via other mechanisms. The second experiment was devi sed to examine the degradation of the target compounds at lower concentrations similar to th e expected environmental concentration. These samples also included a readily degradable organi c substrate to create conditions representative of landfills and to include potential cometabolism of the compounds with the organic substrate. A final laboratory experiment utilizing samples comprised of a synthesized municipal solid waste in landfill leachate allowed examination of the combined removal mechanisms as affected by typical MSW components such as food, paper, cardboard, iron, and textiles. To assess the potential of landfills to be a significant source of pharmaceuticals to the environment, analysis of landfill leachate field samples was performed for a total of ten compounds. Leachate samples were obtained fr om ten lined landfills throughout Florida. Additionally, the landfill leachates were analyzed for common phys ical and chemical parameters and landfill operating characterist ics were recorded to determin e possible correlation with any detected concentrations. 21

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22 Study Overview Chapter 2 provides a literature re view of issues pertinent to discarded pharmaceuticals as pollutants in the environment including sources, regulation, disposal methods, and environmental impacts. To address emerging pharmaceutical pollutants, community collection programs have emerged to capture discarded, household pharmaceuticals. Chapter 3 presents the results of a pilot program to characterize discarded medicat ions and issues affec ting collection program efficiency. Chapter 4 presents the results of research to determine the pharmaceutical input to MSW landfills in the United States. The resear ch utilized two approaches: a mathematical prediction of active pharmaceutical concentratio n in MSW and a direct measurement of API concentration in MSW utilizing a waste com position study. Chapters 5 and 6 examine the anaerobic degradation and co-meta bolism of six selected APIs s as a measure of potential for degradation in MSW landfills. The anaerobic de gradation and co-metabolism of the six APIs within landfill leachate and a mixtur e of MSW in differing stages of degradation is presented in Chapter 7. To characterize the current emission of pharmaceuticals from landfills, analysis of 10 target compounds in landfill leachates was conduc ted from sites across Florida. Chapter 8 presents the results of this study. Finally, Chap ter 9 provides a summary and conclusions for all of the completed research. The literature cited as references are include d at the conclusion of this document.

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CHAPTER 2 BACKGROUND AND LITERATURE REVIEW Introduction This chapter discusses background information and pertinent literature. Current and potential sources of pharmaceuticals to the envi ronment, the effects of pharmaceuticals in the environment, and factors influenc ing both of these topics are reviewed. Additionally, regulations concerning pharmaceutical disposal and househol d and municipal pharmaceutical disposal practices are examined. The review is comp leted by a discussion of municipal solid waste landfill design and characteristics of operation. Sources of Pharmaceuticals in the Environment Sewage Treatment Works (STWs) Pharmaceuticals and personal care products (PPCPs) enter the environment through a myriad of pathways. Their widespread use in hospitals, homes, manufacturing, and animal facilities provides ample opportuni ty to enter the water and land ecosystems. In the 1970s, the first reports of pharmaceuticals entering the environment were reported (26) These reports found pharmaceutical compounds in the influent and effluent of wastewater treatment plants. Largely unnoticed by the public, the fate of drugs outside of their medical use remained unexplored. Further hindering discovery, the analytical abilities to de tect most pharmaceutical compounds at the low concentrations at which th ey exist in the environment were not possible until recently. The most commonly identified source of PPC Ps into waterways is domestic sewage (27) For many years, health professionals recomm ended the disposal of expired or unwanted medications by flushing or washing them into th e sewage system. The intent was to prevent children from obtaining them or to prevent the poisoning of animals who scavenged them from 23

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the trash in the local neighborhood or at the landf ill. Despite the evidence of pharmaceutical pollution from sewage systems, this practice continues widely today. Another source to the waste water stream is the excretion of pharmaceutical active ingredients and their metabolites by the patients using the medications. Unlike other pollutants, PPCPs can be found in almost all areas people ex ist since medications whether prescription or over-the-counter (OTC) are commonly used. Th ese medications once taken are metabolized by the body. The body may excrete via urine and feces a portion of either the original medication or its metabolites. As an example, the blocker nadolol is unmetabolized in the body and 80 to 90% of the antibiotic amoxici llin is excreted unchanged (27) Even those that are metabolized may return to their original form once excreted into the environment. Research has shown that the metabolites of chloramphenical (antibiotic) and sulphadimidine (antibiotic, animal growth promoter) return to their parent compounds when excreted (28) These compounds become part of the domestic sewage system. Once in the domestic sewage, it is the responsibility of the wa stewater treatment plant to remove the pollutants. Wastewater treatm ent plants (WWTPs), however, were designed primarily for the removal and treatment of natural human excrements and not the various anthropogenic substances such as PPCPs that beco me part of the waste water. An extensive study of German WWTPs for 14 common drugs indicated a range of removal efficiency of 7% to 96% depending on the medication and method of treatment. Th e average for all 14 drugs was approximately 60% (14) Other reports indicate removal efficiencies of 38% to 80% (29) Although some tertiary treatment methods ar e used in WWTPs, the primary methods of pollutant removal and water treatment are microbial degradation and removal through filtration of materials adsorbed to the sludge. The low removal rates of pharmaceuticals by these methods 24

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may be due to their low concentrations and thei r chemistry. At low concentrations, sufficient levels of the PPCP may not be present to support the microbial fauna necessary for its decomposition. Additionally, PPCPs are larg ely water soluble making them less likely to adsorb to the particulate ma tter and be removed in the WWTP sludge. Even for those compounds with high removal rates, upset of the equilibrium of the water treatment plant such as in wet weather runoff surge or hi gh influent flow, can result in dramatic decreases in removal efficiency (14) Table 2-1 summarizes studies of pharmaceu tical compounds detected in sewage treatment plant effluents. Agriculture Farming and animal rearing have been iden tified as affecting water quality primarily through pesticides and animal excrement (30, 31) Recently, the large qu antities of antibiotics and hormones used by the industry have also come under scrutiny. Uses include antibiotics in feed additives for disease protection, hormones for growth enhancement, and parasiticides for animal health. Research in Denmark indicate d that the annual applic ation of antibiotics (200 tons) in farming practices outstripped the use of insecticides (185 tons) (27). Runoff and sewage treatment facilities from ag ricultural operations can contain any and all of these compounds. In a study of Canadian stre ams and rivers with primarily agricultural inputs, Lissemore et al. (32) measured detectable levels of 14 of 28 pharmaceutical compounds analyzed. Twelve of these pharmaceuticals had agricultural purposes, with the test area predominantly supporting cattle, swine, and poultry production. Soto et al. (33) attempted to make comparisons of the effluents from cattle fe edlots that used hormone therapies with those that did not. However, the rese archers were unable locate a feed lot in the study region that did not use hormone therapy and research results sh owed the effluent waters to have endocrine 25

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disrupting properties. In a related study, Orlando et al. (34) found significant effects of feedlot effluent upon fathead minnows collected from th e discharge waters of the effluent. Land application of wastewater treatment sludge as fertilizer may also play a role in pharmaceutical compounds entering the environment. Lissemore et al. (32) detected carbamazepine, a solely human pharmaceutical, in 3 of 7 streams with primarily agricultural input. Record review indicated that sewage treatment bi osolids had been applied to lands surrounding the 3 streams with m easured carbamazepine within the last four years. Those streams without measurable quantities had not r eceived any biosolids. In a study of German agricultural fields fertilized with liquid ma nure from animal rearing operations, researchers discovered tetracycline and chlortetracycline in the soil. The researchers further discovered no significant degradation of these compounds afte r six months and signi ficant increases in concentrations following a second applica tion of the liquid manure one year later (35) In addition to WWTP solids applied as fertiliz er, other animal wastes may be applied to land. These wastes are the result of animal dippi ng, animal excreta, and ot her animal treatment. Once on land, land-feeding animals may ingest these compounds as plant uptake has been observed in laboratory studies with pharmaceutical agents (36) The effects may occur directly in these animals or result in exposure further upwards in the food chain. As an example, a concentration of 0.5 g per kg of ivermectin (Anti-parasi tic, heartworm treatment) in cow dung yields aberrations in the wings of the yellow dung fly (37) In perhaps the most direct adverse effect of pharmaceutical use upon w ildlife noted to date, the use of diclofenac for the treatment of cattl e in Pakistan and India has resulted in the devastating reduction of Asian vulture populations in those regions. Residua l diclofenac in cattle carcasses consumed by the vulture s has resulted in re nal failure and gout-l ike deposits in the 26

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joints and internal organs for three vulture spec ies. The resulting deaths have lead to a 95% population decrease in one species and the listing of all three speci es as critically endangered by Birdlife International (38) Landfills Meager research has been completed concer ning the occurrence and fate of PPCPs in landfills. The method of disposal for PPCPs most commonly recommended in previous years has been the sewage system. Nonetheless, this has not prevented the use of municipal refuse and landfills for their disposal and landfills are increasingly becoming the recommended disposal practice for households (19, 21, 23, 39) Holm et al. (40) examined an unlined landfill near Grinsted, Germany for pharmaceutical compounds. The landfill was loca ted directly on the surface of a flat landscape and had no leachate collection system. Landfilling of household waste took place from 1930 until 1977 when the landfill was closed. From 1962-1975, the landfill was also used for the disposal of an estimated 85,000 tons of solid and liquid waste from pharmaceutical production. The pharmaceutical waste was comprised primarily of activated carbon and filter aid used in the purification of sulfonamides, barbiturates, and some water-solubl e vitamins, but also included calcium sulfate, sodium chloride, and some pha rmaceutical compounds and distillation residues. Groundwater samples at depths from 5.5 to 10 m were taken from 23 sampling points at nine distances (0-260 m) from the landfill. Si x sulfonamides (sulfanilic acid, sulfanilamide, sulfaguanidine, sulfadiazine, sulfadimidine, sulfamethizol) and thr ee byproducts of their production (aniline, o-chloroaniline, p-chloroaniline ), one barbiturate (5,5-diallylbarbituric acid), an analgesic (propyphenazone), an intermediate in the production of meprobamate (2-methyl-2n-propyl-1,3-propanediol), and an anti-foaming agent used in pharmaceutical production (tri-(2methylpropyl)-phosphate) were found in the groundwat er. Concentrations ranged from less than 27

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1 g/L to 18 mg/L across the compounds, sampli ng locations, and depths. The pharmaceutical compounds accounted for approximately 5% of the NVOC (nonvolatile organic carbon) measured in the groundwater within the firs t 50 m downgradient of the landfill. Large attenuation of the pharmaceuticals was observed w ithin the first 50 m from the landfill and the authors concluded this was caused by the me thanogenic, sulfate-reducing, and iron reducing conditions. In 1993, Eckel et al. reported groundwater contamination by pentobarbital 300m away from and 21 years after the closing of an unlined landfill in Jacksonville, Florida. The landfill, active from 1968 to 1969 received waste from two large naval bases and is believed to have received waste from a large hospital located at one of th e bases. Re-analyzing gas chromatography/mass spectrometry data from a samp le collected in 1984 as part of an earlier study, the researchers were able to identify the presence of the sedatives pentobarbital and meprobamate and the anticonvulsant phensuximide in the groundwater. To confirm the analysis, the investigators drilled a new well adjacent to the 1984 sampling location and analyzed the groundwater for pentobarbital. The com pound was found at a concentration of 1 g/L (41) In a 1998 study by Schwarzbauer et al., the researchers used gas chromatography/mass spectroscopy to analyze groundwater containing seepage from a sanitary landfill in Germany. Additionally, direct sampling of the seepage water was possible due to leakage and failure into a former mining shaft below the landfill. Propyphenazone, a wide ly used analgesic and antipyretic, was detected with concentrations of 110 mg /1. Ibuprofen was detected in leach ate and leakage water samples. A third pharmaceutical compound, clofibric acid, a metabolite of a blood lipid regulator was also identified (42) In a follow-up study, an investigation into the persistence of or ganic chemicals in the groundwater surrounding the leaking landfill was conducted. Propyphenazone and clofibric acid were detected in the groundwater and in th e leakage water collecte d from the exit of the 28

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mine shaft running below the landfill (approximately 2 km from the landfill). Propyphenazone was present at concentrations up to 1.4 g/L in the groundwater adjacent to the landfill and up to 100 ng/L in the leakage water. Clofibric ac id was found at concentrations up to 1.1 g/L in the groundwater and up to 55 ng/L in the leakage water. These concentrations were 100-1000 times less than the concentrations measur ed in the leachate of the prior study (43) Two research studies of an active, unlined landfill in Croatia also detected pharmaceuticals in several differing areas. In the initial study, researchers examined solid waste and underlying soil (1 m) from three different locations. The analge sic propyphenazone was found at concentrations up to 0.1 mg/kg in the soil and 10 mg/kg in th e solid waste. Isop ropylidene carbohydrate derivatives from the manufacture of Vitamin C were also found at concentrations in excess of 10 mg/kg in the solid waste and up to 1 mg/kg in the underlying soil. The authors linked these concentrations to disposal of pharmaceutical pr oduction waste, rather th an from disposal of municipal refuse (44) In a following study, the researchers also exam ined the concentrations of pharmaceutical compounds in the landfill leacha te and groundwater underlying th e landfill. Three phenazone analgesics (propyphenazone, aminopyrine, and antip yrine) were detected in the solid waste, leachate, underlying soil, and groundwater. Propy phenazone was found at concentrations up to approximately 50 g/L in the leachate and in the groundwat er. Aminopyrine was also present in the leachate at concentrations up to 16 g/L and the groundwater at up to 36 g/L. Antipyrine was found in the leachate near detection lim its at concentrations less than 50 ng/L (45) In the United States, an investigation or orga nic wastewater contaminants from an unlined landfill in Norman, Oklahoma tested for 76 contam inants in groundwater wells down gradient of the landfill. The landfill was ope rated from 1920 to 1985 and received residential, commercial and some hazardous waste. It was then closed, capped with clay and vegetated. Although not 29

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detected in all samples, the researchers did de tect the antibiotic lincomycin and continine, a metabolite of a human non-prescription drug. Li ncomycin was found at concentrations ranging from < 0.05 to 0.1 g/L and cotinine was found at concen trations ranging from < 0.05 to 0.13 g/L (46) In 2004, a literature review by Metzger describe d two studies of drugs in landfill leachates in Germany. The first of the studies measur ed the concentrations of several pharmaceutical compounds in two municipal landfills in German y. The drugs quantified included clofibric acid, diclofenac, ibuprofen, indomethacin, pentoxyfylline and prim idone. The concentrations measured ranged from 1 to 20 g/L. The second study investigated the presence of several drugs in the leachate from five active municipal la ndfills in Germany. Clofibric acid, ibuprofen, carbamazepine, and phenacetin were found (47) Beyond the direct disposal of unwanted pha rmaceuticals or manufacturing wastes in landfills, wastewater treatment bi osolids sent to landfills is a po tential source of PPCPs. In 2004, approximately 2 million tons of the biosolids generated in the United States were disposed of in municipal landfills (48) The release of pharmaceuticals from biosolids has been detected in research studies (32, 49) Thus the pharmaceutical compounds contained in the sludge may become released within the landfill environmen t and enter the landfill s leachate. Conversely, pharmaceuticals in the leachate of the landfill may then be sent to wastewater treatment plants, resulting in pharmaceuticals be ing released in the effluent or the biosolids. Effects of PPCPs in the Environment One of the daunting tasks now facing scien tists and environmental regulators is the determination of the ultimate effects on plants and animals from exposure to the environmental levels of PPCPs. These concentrations are very low, often below the therapeutic value of the substance, commonly at parts per billion and parts per trillion (50) Within the environment the 30

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pharmaceutical compounds can be pseudo-persistent, i.e. they remain in the environment for long periods of time due to their continual low-level introduction in to the environment. While research of pharmaceuticals often measur es the acute physiological effects of a drug in the target patient, little knowledge is ob tained concerning the chronic, low dosage effects upon health, reproduction, and other functions. Drugs designed for specific modes of action in one organism, humans for example, may elicit an entirely different response in a non-target organism such as a fish in an affected river. Compounding the task of identifying potential effects in the environment is the large number of differing chemical fo rmulations utilized in pharmaceuticals. The measurement of pharmaceuticals has focu sed on the aquatic environment. Although initial research was focused in Europe, studi es have found pharmaceuticals in surface and ground waters in Austria, Brazil, Canada, Croatia, Germany, Greece, Italy, Spain, Switzerland, the UK, and the United States (29) The existence of PPCPs in the air and terrestrial habitats has been either largely ignored or undetectable. Few PPCPs are volatile enough to be significantly detected in the air (32, 50) However, recent research shows that pharmaceutical compounds which may be associated with particulate transport such as caffe ine, nicotine, and cocaine, may be detected in the atmosphere near areas of concentrated usage (51) Sources of pharmaceutical compounds to the land environment are primarily through the application of WWTP biosolids to the land and from animal wastes applied to the land. The following paragraphs briefly discuss the types of pharmaceuticals commonly found in the environment. Hormones (and Endocrine Disrupting Compounds) The endocrine system is an organisms signal transmitting system. The hormones produced by the endocrine system are lipid (steroids) and amino acid based, transmitting information that can regulate multiple body functions, including reproduction, growth, and 31

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coordination of homeostasis. Some of the first physiologic com pounds reported in WWTP effluent were steroids, specifi cally estrogenic compounds. Estr ogenic drugs are utilized in estrogen-replacement therapy, oral contraceptives, and in veterinary medicine. These estrogen compounds, including 17 -ethinylestradiol which is used as an oral contraceptive, have been suspected in combination with other endocri ne disrupting compounds such as pesticides to cause the feminization of ma le fish. This is expressed by the production of vitellogenin, an egg yolk precursor protein, normally only found in females (34, 50, 52) Other reports indicate irregularities in reproductiv e and sexual development in fish such as egg development in males, disruption of gonad development, and reduced sperm production and motility. Estrogens, however, are not the sole endoc rine disrupting compounds (EDCs). Other compounds have been identified as hormonally active substances, mimicking the action of hormones, blocking hormone production, or stimulating natural hormone production. These compounds are found in a wide range of usage such as plastics production The interaction of these compounds with pharmaceuticals that may or may not be steroid based is undetermined. Antibiotics Antibiotics in the environmen t have received a great amount of attention. The primary concern for antibiotics in the environment is the pot ential to result in resistant microbial strains, reducing the effectiveness of the antibiotics for h ealthcare use. The widespread use of antibiotics in animal farming and human healthcare provides ample opportunity for th eir introduction to the environment. Additionally, their long term eff ects upon bacteria, causing a shift in the microbial roles and structures in nature, may have an impact on the upper food chain (53) Costanzo et al. tested bacteria isolated in a WWTP with antibiotics detected in the influent, effluent, and up to 500m downstream of the WWTP discharge. Four strains of bacteria 32

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from the bioreactor each showed resistance to at least 2 out of 6 antibiotics applied. Further, tests showed that two bacteria isolated from the downstream water ha d resistance either to erythromycin or ampicillin (54) In a study of antibiotics used in Vietnamese shrimp farming, Le et al. discovered a high inciden ce of bacterial resist ance, although the relati on of resistance to antibiotic residues detected in the farmi ng environment could not be correlated (55) Blood Lipid Regulators Blood lipid regulators are drugs designed to re duce excess fatty substances in the blood stream, such as cholesterol, resu lting in reduction of diseases su ch as atherosclerosis and heart disease. Related to the herbicide mecoprop, clof ibric acid is an active metabolite of many blood lipid regulators. It was one of the first prescription drugs repo rted in sewage effluent and continues to be one of the most widely reported PPCPs in monitoring studie s. Clofibric acid has been reported in Berlin tap water, Sw iss surface waters, and Brazilian WWTPs (50) Despite their widespread use, the mechan ism of the fibrates therapeutic action is not fully understood and therefore the implications for thei r environmental effects are uncertain. Analgesic/Anti-inflammatory Drugs Common analgesic and anti-infla mmatory drugs have been meas ured in sewage and river waters throughout the world. Ibuprofen, acetylsa licylic acid, naproxen, ketoprofen, and other drugs commonly referred to as nonsteroidal an ti-inflammatory drugs (NSAIDs) have been measured in g/L concentrations throughout the world (14, 56) These studies have found measurable concentrations in surface waters, gr ound waters, and WWTP in fluents and effluents. In humans, NSAIDs inhibit the cyclooxygena ses, the key enzymes responsible for the production of prostaglandins. Pr ostaglandin creation by cells serv es several functions including the promotion of inflammation, pain, fever, supporting the function of platelets, and the protection of the stomach lining from stomach acid. Thus by inhibiting the production of 33

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prostaglandins, NSAIDs create their anti-inflammatory and anal gesic effects, although their effects on platelets and stomach lin ing can lead to detrimental side effects such as internal bleeding and stomach ulcers. Prostaglandins do occur in other nonmammalia n vertebrates such as fish, amphibians, and birds, as well as in invertebrates such as corals, sponges, coelenterates, mollusks, crustaceans, insects, and in marine algae and higher plants. These prostaglandins carry out different functions in these organisms. In a st udy of the toxicity of several of these compounds, researchers found the lowest no observable e ffect concentration to be 10,000 to 100,000 times the measured environmental concentrations (57) However, long-term chronic effects were not examined. Examination of environmental concen trations of ibuprofen upon the growth of the cyanobaceterium Synechocystis and L. minor showed ibuprofen to promote the growth of Synechocystis while inhibiting the growth of L. minor (58) Other Drug Classes Many other common drugs have demonstrated biological effects on organisms outside of their intended use. A popular diet drug, fenfluramine, removed from use by the EPA in 1998 has proven to trigger reproductiv e effects in fiddler crabs (59) and crayfish (60) Antidepressants that affect serotonin have long been used to promote the spawning of bivalves, with commercial farmers adding serotonin to stimulate spawni ng. Widely common in both vertebrate and invertebrate nervous systems, the potential for en vironmental effects is a subject of extensive research (50) Antiepileptics, antineoplastics, retinoids, tra nquilizers, and impotence drugs are all widely used and while needing further investigati on could result in environmental effects (50) New drugs such as Viagra, have distinct modes of action and may have unexpected consequences in nontarget organisms. W ith recent work in human genomes, dr ugs developed to address specific 34

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genomic actions will be developed with uncertain consequences for the environment. Recognizing the potential environmental impact, more stringent environmental assessments have been required by the United States Food a nd Drug Administration a nd the European Union, although the need for modifying the assessmen t requirements has been questioned as new research results are published (6) Environmental Exposure Factors Several factors can determine the environmen tal exposure of organisms to pharmaceuticals in the environment. Typicall y, testing involves measurement of the acute effects a pollutant has on organisms. However, other factors must be considered in determining the exposure of organisms to pharmaceutical compounds and the potential of these compounds to induce change. Ecotoxilogical data are available for less than 1% of human pharmaceuticals, despite the fact that 10-15% of high volume pharmaceuticals found in surface waters are intrinsically toxic (61) Persistence In studies of the effects of chemicals, whether primary pollutants, PPCPs, or other compounds, a significant factor in determining th e potential to harm the environment is its environmental half life. This is a measure of the time for a pollutant to be reduced to one half of its original concentration. PPC Ps with long half lives include blood lipid regulators and musks (50) Normal calculations measure factors which remo ve the pollutant from the environment. PPCPs, however, demonstrate pseudo persistence in the environment due to their continuous introduction. PPCPs although low in concentr ation are continually replenished in the environment due to their continuous use and eventu al discharge via wastewat er treatment plants. Thus compounds which would normally decrease in concentration rapidly due to short half lives remain at steady or increasing values. 35

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Synergy When chemicals or drugs are used togeth er, they may show synergy. Synergy occurs when the sum of the effects of ch emicals acting together is greater than the additive effects of the individual chemicals. This has been comm only demonstrated in human medication use. Therefore, even with testing showing no de leterious effects from a pharmaceutical at concentrations found in the environment, the combination of two or more pharmaceuticals may be capable of inducing change. While antagonism may also occur (less effect than the two individually), the potentia l for synergy of PPCPs requi res further investigation. Inconspicuous Change Due to the low concentrations of PPCPs in the environment and their continuous introduction to the environment, exposure to organisms is typically chronic instead of acute. Research regarding the long term effects of PPCPs in the environment is a newly developing field. The risk created by extended exposure at low levels is the possibilit y that changes in the organisms will take several generations to express themselves and will be unnoticed. Furthermore, the ability to take action to prevent such changes will have long since passed. One published study has highlighted the subtle effects that PPCPs may have in the environment. In a study of fathead minnows, Mar tinovic et al. noted no effects from exposure to estradiol in a concentration of parts per trillion. However, wh en the treated group was removed from isolation and placed with the untreated gr oup, disparities in the treated groups spawning competitiveness were shown (62) Thus, simple toxicity tests may not be enough to demonstrate the subtle effects of PPCPs in the environment (53) Pharmaceutical Regulation Approximately 50 new drugs enter th e United States market every year (63) With the introduction of each new medication, a potential new waste for disposal is also introduced. Past 36

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regulation of pharmaceuticals has focused on their efficacy and safety for use. Awareness of their environmental impacts has only recently been realized. In the United States, the Federal Interagency Task Group on Pharmaceuticals a nd Personal Care Products was formed in September 2004. In Canada, the Environmental Impact Initiative was formed in 2001 and in Japan the process of formulating a plan for e nvironmental risk assessment of pharmaceuticals with sales exceeding one ton per year is underway (63) However, these are emerging efforts and regulations concerning final disp osal of pharmaceuticals are meager. European Medicines Agency The European Medicines Agency (EMEA) is the body of the European Union responsible for the protection and promotion of public a nd animal health, thr ough the evaluation and supervision of medicines for human and veteri nary use. Beginning in 1995, pharmaceutical manufacturers were allowed to submit a single ma rketing authorization a pplication to the EMEA for approval for the whole European Union. Th e EMEA does this through the Committee for Medicinal Products for Human Use (CHMP) or Committee for Medicinal Products for Veterinary Use (CVMP). If the relevant co mmittee concludes that the quality, safety, and efficacy of the medicinal product are sufficiently proven, it adopts a positive opinion. This is sent to a commission to be authorized fo r the whole of the European Union. In 1999, in response to rising evidence of pha rmaceuticals in the environment and their impacts, the EMEA began drafting environmental risk assessment procedures to accompany new pharmaceutical applications in Europe. The fina l draft was closed for public comment in August 2005 and received final approval in 2006. The Eur opean guidance is the first to include longterm ecotoxicity testing as well as to consider the environmental effects from extremely low concentrations of bioactive substan ces, such as endocrine disruptors. 37

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The guidance outlines a risk assessment procedure for new active pharmaceutical substances, their metabolites, and their excipients (non-active ingredients). It does not apply to drugs already on the market. The EMEA risk assessm ent process is divided into two phases. The first phase involves the calculati on of the aquatic predicted envi ronmental concentration (PEC) of the new drug. A substances PEC is deemed too low to be of concern to environmental health when below 0.01 g/L in surface waters and is ru led out for further assessment. However, the guidance does note that certain substances th at are likely to cause effects at very low concentrations, such as endocrine disruptors, may need to be addressed regardless of the quantity released into the environment. If the PEC is above the 0.01 g/L limit, th en Phase II assessment is required. Phase II requires gradually increased levels of testing including degradability, potential to bioaccumulate, adsorption on sewage sludge, and toxicity to sewa ge microbial populations. Also included is the long-term testing of fish, Daphnia (water fleas) and algae to assess th e predicted no effect concentration (PNEC) of the new drug for each of these species. After completion of the increasing levels of testing, if the PEC is deemed to be higher than the determined PNEC, then pharmaceutical companies must propose recommendations to limit the drugs impact on the environment such as labeling to educate people about how best to dispose of expired or unused medicines. United States Food and Drug Administration (FDA) The Food and Drug Administration (FDA) is the federal agency responsible for the regulation of pharmaceutical development and us age in the United States. The Federal Food, Drug, and Cosmetic Act requires the FDA to determine whether new drugs developed by pharmaceutical companies are safe and effective. This largely involves the testing of the drugs for stability and their ability to produce the desired effects safely. 38

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However, the National Environmental Polic y Act of 1969 (NEPA) requires all federal agencies to assess the environmental impacts of thei r actions and to ensure that the interested and affected public is informed of environmental analyses. The FDA is required under NEPA to consider the environmental impacts of approvi ng drug applications. FDA's regulations in 21 CFR part 25 specify that environmental assessments (EAs) must be submitted as part of certain applications, unless the application qualifies fo r an exclusion. EAs were required by the FDA for all drugs as a part of new drug applications but in 1998 the FDA revised the requirements. EAs are now only required when the expected in troduction concentration (EIC) of the active ingredient of the drug in the aquatic environment (EIC-aquatic) exceeds 1 g/L (e.g. the WWTP effluent concentration). If a dilution factor of 10 is assumed as the average dilution for WWTP effluent into a stream or river, then the resulting 0.10 g/L concen tration is ten times greater than the proposed European standard of 0.01 g/L and mu ch greater than the results of new research examining the affects of pharmaceuticals in the e nvironment at parts per trillion concentrations. United States Drug Enforcement Agency (DEA) Many pharmaceuticals are narcotics, depre ssants and stimulants manufactured for legitimate medical purposes but w ith the potential for abuse. To prevent their abuse these pharmaceuticals have been designated as controll ed substances and brought under legal control through the Drug Enforcement Agency (DEA) and the Controlled Substances Act. The goal of these controls is to ensure that these "controll ed substances" are readily available for medical use, while preventing their distribu tion for illicit sale and abuse. Five schedules have been established which classify controlled substances according to their potential for abuse. Drugs are placed into categories according to how dangerous they are, how great their potential for abuse, and whether they have any legitimate medical value. The schedule a drug falls under determines the cont rols required by the DEA, including differing 39

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documentation to ensure proper final destruction of the waste pharmaceutical. The schedules, as well as other information concerning controlled substances, may be found in 21 CFR Part 13001316. Prior to 1995, the DEA received and perfor med the destruction of all controlled substances. Due to the large cost and time necessa ry for the destruction of controlled substances, the DEA privatized the destruction of contro lled substances in 1995 through a program of certifying commercial waste companies as disposing agencies. These agencies primarily use incineration for the destruction of the medications; however, sewer disposal has been and may be used in the future with DEA approval. The DE A does not allow pharmacies reverse distributors, or disposal companies to accept controlled subs tances once given to the prescription user, resulting in unused household medica tions to be either flushed into the sewage system or thrown in the trash with a measurable introduction to the environment. The number one prescribed pharmaceutical for each year from 2002 to 2004 was the controlled substance hydrocodone (64) Hydrocodone has been measured in surface waters (65) United States Environmental Protection Agency (EPA) The Environmental Protection Agency (EPA) is the primary agency responsible for regulation of the disposal of solid wastes in the United States, including unused and discarded pharmaceuticals. Specific EPA regulations pertaini ng to discarded pharmaceuticals do not exist. Thus under EPA regulations pharmaceuticals are treated as any other solid waste. As a solid waste, pharmaceuticals may be clas sified as a hazardous waste. Pharmacies, hospitals, and other pharmaceutical waste gene rators must be able to identify those pharmaceuticals that are hazardous wastes as defi ned in 40 CFR Part 261. Antineoplastic agents are the most common pharmaceuticals which are sp ecifically listed as hazardous wastes in 40 CFR Part 261 Subpart D (66) Many pharmaceuticals also fall under the definition of a 40

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hazardous waste by meeting one of the characteris tics of hazardous waste set forth in 40 CFR Part 261 Subpart C. In an effort to promote the collection of unused medications instead of flushing to wastewater systems, several states have cla ssified hazardous waste pharmaceuticals as universal wastes (67, 68) Universal wastes are a special subset of hazardous wastes which are subject to less stringent administrative practi ces to promote their collection from a wide variety of sources. The United States federal government is consid ering a similar proposed change in federal regulations as well. However, these changes will not affect the capture of discard of pharmaceuticals from households. Under RCRA, househol d hazardous waste is exempted from regulation, and thus household generated pharmaceutical wastes cannot be regulated as hazardous wastes and fall under general solid waste regulations. States have the ability to enact more stringent regulations as in California which does not allow a household exemption to the definition of hazardous waste. Therefore, with the exception of the few medications designated as a hazardous waste, pharmaceutical disposal into landfills is allowed under EPA regulations. Other Regulation The eventual fate of pharmaceuticals can be affected by multiple other regulations and regulatory agencies. Some pharmaceuticals may be considered or managed as medical waste. Medical waste management is generally define d under state regulati ons and differs amongst various states. In each state a Board of Phar macy or its equivalent regulates pharmacists, pharmacies, and prescription drugs and devices to protect consumer health and safety. Once dispensed, the improper storage, de terioration due to age, or tamp ering could alter the quality of medications. As a result the return of pharm aceuticals for reuse is often prohibited and many states forbid the possession of a prescription pharmaceutical by anyone other than the original 41

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prescribed patient. This limits the role pharmacists can play within the United States in receiving unwanted household medications for disposal. Pharmaceutical Disposal The growing pharmaceutical waste stream is indicated by the ever expanding use of prescription medications and ove r-the-counter preparations. In 2003 national health care expenditures in the United States totaled $1.7 trillion, a 7.7% increase from 2002. From 1995 to 2003 the average annual rate of increase for pr escription drug expenditures was 14%, higher than any other healthcare expenditure. (2) Between 1988 and 1999 the percentage of Americans who reported using any prescription drug during the past month increased from 39% to 44%. During the same period the percentage of persons who reported using three or more drugs in the past month increased from 12% to more than 17% (69) Non-Residential GeneratorsHospitals, Pharmacies, Healthcare Facilities Typical generators of unused pharmaceutical wastes outside of consumer use are hospitals, medical and dental offices, commercial pharmacies and other healthcare facilities. In a survey of community and hospital pharmacies, Kuspis and Krenzelok found that only 3% of the pharmacies surveyed did not have a specific unuse d medication disposal plan. The method most often used was return to the producer. Howeve r, for nonreturnable medications, the pharmacies cited incineration (15%), hazardous waste dispos al contractor (17%), or conventional waste disposal (trash or sewer flushing, 68%) (25) Reverse distributors manage unwanted pharmaceuticals from pharmacies, hospitals, and clinics. Typically a complicated process, re verse distributors ease the burden on individual pharmacies in sorting unwanted pharmaceuticals for the proper return or disposal method. Some collected pharmaceuticals are returned to manuf acturers who often provide credits for the returned products. Those unable to be returned to the manufacturer are sent for off-site disposal 42

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by the reverse distributor. A revi ew of the literature did not rev eal any information pertaining to the off-site disposal methods util ized by reverse di stributors. Since the reverse distributors make the de termination if an unwanted pharmaceutical can be returned to the manufacturer or needs altern ate disposal, the reverse distributor and not the pharmacy is considered the generator of waste for those pharmaceuticals designated for disposal. Prior to this determination, the pharmaceutical ha s a potential value as a manufacturer return and thus is a product and not a waste. As such, h azardous or medical waste management or transfer station permits are not required since they are not generators under the la w. However, reverse distributors cannot accept pharmaceuticals that have been prescribed and given to patients. Once outside the control of the pharmacy, concerns for a pharmaceuticals purity and safety prevent its value as a potential product and therefore the me dication is a waste before entering the reverse distribution system. This prevents reverse distributors from accepting collected household pharmaceutical wastes. Household Pharmaceuticals The disposal options utilized by consumers c onsist primarily of flushing to the sewage system, throwing into the trash, and return to a pharmaceutical collection program. Table 2-2 summarizes the results of several studies of co mmon disposal methods used by households. As education of the potential dangers of sewer disposal has increa sed, growing efforts have been undertaken to divert discarded pharmaceuticals to environmentally friendly disposal methods, primarily through community collection progr ams. Increased capture of discarded pharmaceuticals is often achieved through pharmacy return programs as highlighted for Canada and the United Kingdom in Table 2-2. Preval ent outside of the United States, drop-off of unwanted pharmaceuticals at pharmacies exists in British Columbia (Canada), Prince Edward Island (Canada), Australia, and eleven European Union nations (70) In Australia, through its 43

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Return Unwanted Medicines (RUM) program, an average of 31,103 kg per month were collected at pharmacies throughout the coun try from July 2006 to June 2007 (71) Other potential collection locations may include nursing hom es, retirement communities, and household hazardous waste collection centers. Collection programs vary among one day collection events, continuous collection, or periodic gathering. Within the United States, overall collection has been limited by the regulatory structure previously described, but efforts have been ma de. In Clark County, Washington a program with some restrictions allows the re turn of pharmaceuticals to nearly 80% of the pharmacies in the county (70) Other one day collections events conti nue to be conducted th roughout the country. In Maine, Public Law 2003, Chapter 679 created the Unused Pharmaceutical Disposal Program, an unwanted pharmaceutical mail-back program for collection (72) Pharmaceuticals in Landfills The potential for PPCPs in a landfill increases with specific demographic characteristics of the surrounding area. In many cases of groundw ater pollution by pharmaceuticals in landfills, specific pharmaceutical contributions were made to the landfills (40, 41) Thus pharmaceutical manufacture and hospitals within the community can be expected contributing factors. Additional factors include, ag e of the population, urban vs. rural community, and community access to healthcare. A Literature review did not locate information concerning the effects of these parameters on landfill pharmaceutical content. Landfills and Landfill Leachate A landfill is an engineered site for the dis posal of solid waste on land. In the past, a landfill represented nothing more than a common area where refuse was indiscriminately placed. However, the hazards associated with these dumps such as rodents, flies, fires, and odors led to the development of sanitary landfills. Sanitary landfills use engineering controls such as daily 44

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cover soil (soil used to cover the waste at th e end of each day) and compaction. Despite these controls, less obvious threats to human health arose from leachate (liquid percolating from deposited waste) and gas produced by these landfills. To address these concerns, modern landfills have become highly engineered facilities under strict regu lations for design and operation. In the United States, municipal so lid waste (MSW) landfills are regulated under subtitle D of the Resource Conservation and Recovery Act (RCRA) of 1976. Specific landfill design and operational regulati ons are located in 40 CFR part 258. The design of a typical modern sanitary landfill is shown in Figure 2-1. Approximately 4.6 pounds of MSW is generate d per person per day in the US, and over half of the waste generated is disposed in landfills (73) Approximately 50 to 70% of MSW is biodegradable materials compos ed mainly of proteins, lipi ds, carbohydrates (c ellulose and hemicellulose), and lignins (74) Once deposited in a landfill, microorganisms degrade this material. The microorganisms present are diverse and vary due to the heterogeneous nature of waste and landfill operating characteristics. Additionally, landfill degradation and microorganisms are strongly influenced by enviro nmental conditions, such as temperature, pH, toxins, moisture content, and the oxidation-reduction potential. Throughout the lifetime of a landfill a complex se ries of chemical and biological reactions occur. Landfill studies have suggested that the stabilization of waste proceeds in five sequential and distinct phases (74-76) As shown in Figure 2-2, the pr ocesses taking place inside the landfill, the active bacteria popula tions present, leachate character istics, and gas production vary from one phase to another. A description of th e phases of refuse decomposition is presented in the following section. This is followed by info rmation on the composition of leachate with a focus on xenobiotic organic compou nds such as pharmaceuticals. 45

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Phase I: Initial Adjustment Phase This phase is associated with initial placement of solid wast e and accumulation of moisture within landfills. Aerobic conditions occur for the in itial few days and oxygen is quickly depleted following isolation of the waste by cover soil. Initially the waste is below field capacity and an acclimation period (or initial lag time) is observe d until sufficient moisture develops to support an active microbial community. Preliminary ch anges in environmental components occur in order to create favorable conditions for biochemical decomposition. Phase II: Transition Phase In the transition phase, the field capacity is reached and exceeded, and a transformation from an aerobic to an anaerobic environment occurs. The anaerobic conditions result in fermentation reactions and decomposition carrie d out by three groups of bacteria: (1) the hydrolytic and fermentative bacteria that hydrolyze polymers and fe rment the resulting monosaccharides to carboxylic acid s and alcohols; (2) the acetogeni c bacteria that convert these acids and alcohols to acetate, hydrogen, and carbon dioxide; and (3) the methanogens that convert the end products of the acetogenic reactions to methane and carbon dioxide (75). During Phase II these bacteria begin their growth and measurable concentrations of chemical oxygen demand (COD) and volatile organic acids (VOA) can be dete cted in the leachate. Phase III: Acid Formation Phase In Phase III the hydrolytic, fermentative, and ac etogenic bacteria dominate, resulting in an accumulation of intermediate volatile organic acids at high concentrations throughout this phase. An accompanying decrease in leachate pH and rapid consumption of substrate and nutrients are the predominant features. 46

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Phase IV: Methane Fermentation Phase The onset of Phase IV occurs when measurab le quantities of methane are produced as the pH of the refuse becomes sufficiently neutralized to permit growth of methanogenic bacteria. During this phase the acids that accumulated in Phase III are converted to methane and carbon dioxide by methanogenic bacteria, and the meth ane production rate will increase. COD and BOD concentrations begin to decrease and th e pH increases as acids are consumed. The methane production rate reaches a maximum, and decreases thereafter as the pool of soluble substrate (carboxylic acids) decreases. Phase V: Maturation Phase During the final state of landf ill stabilization, nutrients and available substrate become limiting, as the BOD:COD ratio generally will fall below 0.1 in this phase because carboxylic acids are consumed as rapidly as they are produced (75) Gas production drops dramatically and the reappearance of oxygen and oxidized species may slowly be observed. Leachate strengths remain constant at much lower concentrations than in previous phases. Landfill Leachate Leachate is generated as water contained in the deposited waste and precipitation such as rain and snow percolates through the landfill. As the water travels through the landfill, contaminants are released or leached from the solid waste. Mechanisms of contaminant removal include leaching of inherently so luble materials, leaching of so luble biodegradation products of complex organic molecules, leaching of soluble products of chemical reactions, and washout of fines and colloids. The characteristics of the l eachate produced are highly variable, depending on the composition of the solid waste, precipitati on rate, site hydrology, co mpaction, cover design, waste age, sampling procedures, interaction of the leachate with the environment, and landfill 47

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design and operation (74) Typical characteristics of landfill leachate during landfill stabilization reproduced from Reinhart and Townsend are shown in Table 2-3 (74) Pollutants in MSW landfill leachate can be divided into four major groups: (75) Dissolved organic matter, quantified as Chemical Oxygen Demand (COD) or Total Organic Carbon (TOC), volatile fatty acids, and more refractory compounds such as fulviclike and humic-like compounds. Inorganic macrocomponents: calcium (Ca2 +), magnesium (Mg2+), sodium (Na+), potassium (K+), ammonium (N H4+), iron (Fe2+), manganese (Mn2+), chloride (Cl-), sulfate ( SO42-) and hydr ogen carbonate (HCO3-). Heavy metals: arsenic (As3+, As 5+), cadmi um (Cd2+), chromium (Cr3+), copper (Cu2+), lead (Pb2+), nickel (Ni 2+) and zinc (Zn2+). Xenobiotic organic compounds (XOCs) origina ting from household or industrial chemicals and present in relatively low concentrations (usually less than 1 mg/l of individual compounds). These compounds include among othe rs a variety of aromatic hydrocarbons, phenols, chlorinated al iphatics, pesticides, and plasticizers. These pollutants vary significantly among landf ills depending on waste composition, waste age, and landfilling technology. Additionally, the quan tity of each of these pollutants in leachate varies throughout the lifetime of an individual landfill. XOCs in landfill leachate are the result of the disposal of manmade chemical compounds in landfills. In the past these compounds were pr imarily the result of th e disposal of industrial wastes within MSW landfills. However, the amount of these wastes has decreased as hazardous waste regulations have decreased the amount of chemical wastes entering landfills, although older landfills may continue to contain waste fr om a time period with fewer restrictions on the disposal of hazardous wa ste in MSW landfills. The most frequently found XOCs are the monoaromatic hydrocarbons (benzene, toluene, ethylbenzene, and xylenes) and halogenated hydrocarbons such as tetrac hloroethylene and trichloroethylene (74, 75) These pollutants are also the ones found in the highest concentrations. These pollutants are well documente d due to their negative effects in the aquatic 48

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environment and tracking of several of these as priority pollutants under the Clean Water Act of 1972. In addition, these organic compounds are relativ ely easy to detect and analyze, in spite of the very complicated matrix of leachates from landfills (75) Data on other pollutants is scarcer. Pesticides and phthalate plasti cizers are two other more commonl y reported components found in leachates. In total, studies have shown over 150 to 200 different identifiable organic compounds present in landfill leachate (74, 75) Once leachate is collected at a landfill, it must be disposed of in an environmentally and legally responsible manner. This can involve a tr eatment process at the landfill or at an offsite facility, or some combination of both. Howe ver, regardless of the treatment method used, regulatory limits for wastewater must be me t prior to discharge to the environment. Methods of treating leachate vary and new and innovative tec hnologies continue to emerge such as leachate recirculati on, evaporation, and reverse osmosis. However, the most common and often simplest approach is the treatment of the leachate by a loca l wastewater treatment plant. This can occur either by directly piping the leachate collection system to the treatment plant or by the more common method of transp orting the leachate to the treatment site. Pharmaceuticals contained within the leachate may th en enter the wastewater process and as with compounds excreted by medication users, become a part of the effluent and biosolids emitted from the treatment plant. 49

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Table 2-1. Summary of pharmaceuticals detected in sewage treatment plant effluents Pharmaceutical compound or metabolite Location of study Effluent concentration (g/l)* Reference Antibiotics Acetylsulfamethoxazole United Kingdom
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Table 2-1. Continued Pharmaceutical compound or metabolite Location of study Effluent concentration (g/l)* Reference Antiepileptic Carbamazepine France, Greece, Italy, Sweden 0.87 (1.2) (8) France, Greece, Italy, Sweden, Denmark 0.44 (1.2) (8) Germany 2.1 (78) Beta Blockers Acebutorol France, Greece, Italy, Sweden 0.06 (0.13) (8) Atenolol France, Greece, Italy, Sweden, Denmark 0.19 (0.73) (8) Germany 0.36 (78) Celiprolol Germany 0.28 (78) Metoprolol France, Greece, Italy, Sweden 0.08 (0.39) (8) France, Greece, Italy, Sweden, Denmark 0.08 (0.39) (8) Germany 1.7 (78) Oxeprenolol France, Greece, Italy, Sweden 0.02 (0.05) (8) Propanolol France, Greece, Italy, Sweden 0.01 (0.09) (8) France, Greece, Italy, Sweden 0.06 (0.13) (8) Germany 0.18 (78) Sotalol Germany 1.32 (78) Hormones 17 -ethinylestradiol Canada 0.009 (0.042) (79) Germany 0.001 (0.005) (79) Italy
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Table 2-1. Continued Pharmaceutical compound or metabolite Location of study Effluent concentration (g/l)* Reference Norway
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Table 2-2. Common disposal methods by percentage for household medication disposal Method of Disposal Pittsburgh, PA(25) Alberta, Canada(80) United Kingdom( 24) Toilet 35.4% 46% 11.5% Trash 54.0% 31% 63.2% Pharmacy 1.4% 17% 21.8% Other 9.2% 6% Table 2-3. Landfill leachate concentration range s as a function of the degree of landfill stabilization Parameter Phase II: transition Phase III: acid formation Phase IV: methane formation Phase V: final maturation BOD, mg/L 100-10,000 1,000-57,000 600-3,400 4-120 COD, mg/L 480-18,000 1,500-71,000 580-9,760 31-900 Total volatile acids as acetic acid mg/L 100-3000 3000-18,800 250-4,000 0 BOD/COD 0.23-0.87 0.4-0.8 0.17-0.64 0.02-0.13 Ammonia mg/lN 120-125 2-1030 6-430 6-430 pH 6.7 4.7-7.7 6.3-8.8 7.1-8.8 Conductivity mhos/cm 2450-3310 1600-17,200 2900-7700 1400-4500 (Source: Reinhart, D. R.; Townsend, T. G ., Landfill Bioreactor Design and Operation. CRC Press: 1998.) 53

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54 Figure 2-1. Typical design of a modern sanitary landfill (Source: Reinhart, D. R.; Townsend, T. G., Landfill Bioreactor Design and Operation CRC Press: 1998.) Figure 2-2. Phases of landfill stabilization (Source: Pohland, F. G.; Harper, S. R. Critical Review and Summary of Leachate and Gas Production from Landfills; EPA/600/2-86/073; United States Environmental Protection Ag ency: Washington, DC, 1986.)

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CHAPTER 3 CONTINUOUS COLLECTION SYSTEM FOR HOUSEHOLD PHARMACEUTICAL WASTES: A PILOT PROJECT Introduction As pharmaceuticals have emerged as trace e nvironmental contaminants, the development of an effective method for diverting medications from sewage treatment systems has become of greater importance. Pharmaceutical compounds en ter the sewage system primarily through the discard of unwanted pharmaceuticals and the ex cretion of pharmaceuticals and their metabolites by the patients using them. Unable to reduce the excretion of pharmaceuticals, environmental professionals must focus efforts on the redirection of discarded medications from sewage treatment to more environmentally protective m easures. Additional information concerning the technical and logistical issues of collection and proper disposal of unwanted medications from the general public is needed. A pilot project to collect and properly di spose of old and unwanted prescription and nonprescription medications from residents of Alachua County, Florida was implemented in conjunction with the Alachua C ounty Environmental Protection De partment. The program was conducted free of charge to the participants. An objective of the collection program was to determine parameters to maximize the effectiven ess of a continuous coll ection program for the disposal of unwanted pharmaceuticals. Thes e factors include: age of medication, age of medication user, reason for disposal, and advertising methods. The second objective was to characterize the type and quantity of discarded household pharmaceuticals. Materials and Methods Unwanted pharmaceuticals were collected at twelve locations throughout Alachua County, Florida with an estimated population of 222,568 (81) To reach the largest number of residents, the twelve locations were chosen to include the smaller communities within the county while 55

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concentrating the largest number of collection points in the city of Gainesville, FL, the countys largest city with a 2003 estimated population of 109,146 (81) The participation of major drug store chains and pharmacies was sought. However, these businesses declined participation in the program. Reasons cited for not particip ating included the following concerns: The potential public perception that the pharmaci es would reuse the returned medications to fill new prescriptions; The legal implications of accepting drugs dispensed by another pharmacy; Concerns that patient privacy would be comp romised by return of pr escription containers; Security of controlled subs tances would be problematic; Lease provisions for the stores prevente d the acceptance and st orage of returned merchandise. The final locations chosen were three locally owned pharmacies, the pharmacy of a national chain retail store, six county health department offices, one fire department location, and the Alachua County Household Hazardous Waste Collection Center. The collection program was designed to allo w participants to drop-off unwanted medications at the collection si te. To obtain the largest amount of data from the collection process, locations were to 1) accept the pharmaceutic al waste from the participant, 2) inventory the medication, and 3) store the pharmaceutical until collection by Alachua County Household Hazardous Waste staff. This pr ocedure would have allowed the mo st accurate collection of data on pharmaceutical identity and quantity relinquish ed and allowed optimum characterization of the waste to include type of medication, hazardous waste characterization, and controlled substance status. Pharmaceutical waste can be a hazardous waste if it is a listed hazardous waste (U and P coded material) or meets the criteria of a characteristic ha zardous waste (D coded material) due to ignitable, corrosive, reactive, or toxic properties (82) 56

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However, based upon discussions with the Florida Board of Pharmacy, the Florida Department of Environmental Protection (FDEP), and the Florida Health Department, it was determined that it would be illegal for anyone ot her than the originally prescribed patient to possess the medication. Only the customer could handle and dispose of the material. Thus a method was necessary that allowed the participant to dispose of the material directly. Additional concerns included violations of patient privacy and the Health Information and Patient Privacy Act (HIPPA), controlled substa nce tracking, and possible theft of the collected pharmaceuticals. The final collection system was designed as a customer self-serve system in which the participants deposited the medications directly in to a collection container that contained either tap water or an aqueous acidic solution. It was desired that the medications became no longer recognizable and unusable when de posited into the container. It was determined that a mild hydrochloric acid solution (1 ml of 20 Baume hydrochloric acid to 12L of water) with a pH of 2 was capable of dissolving a majority of pha rmaceuticals and rendering them unusable. This acidic solution was used in collection containers at all locations except one. Due to concerns expressed by store management at the major chain store pharmacy about having an acidic solution in the store, 100% water was used in the collection containe r for that location. Three gallons of solution were placed into 20L steel, epoxy-lined, clos ed-head pails with a 2 inch bung. A steel mini-drum funnel with s ealable lid was screwed into the bung and each drum labeled with a corrosive liquid label and a pharmaceutical waste label as shown in Figure 3-1. The collection container was weighed prio r to placement at the collection point and upon its retrieval from the collection point. The amount of pharmaceuticals collected was determined by the difference between the initial and final we ights. The average in itial weight of each collection pail with solution was approximately 10 kilograms. 57

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Participants were asked to ope n their containers and pour the medications directly into the funnel. To prevent the steel funnels from re taining medications, a small plastic funnel was inserted within the steel funnels. Following dispos al of their medications, patrons were asked to fill out an informational survey to obtain da ta concerning the medication identity and other pertinent information. To ensure patient conf identiality, customers were asked to take their empty containers with them. When full, contai ners were collected by Alachua County staff and taken to the Alachua County Household Hazardous Waste Center where they were weighed and emptied into a 55 ga llon collection drum. To maximize participation in the pharmaceu tical collection program, a comprehensive advertising campaign was implemented. The goals of the advertising campaign were to educate and raise the awareness of the public about the environmental concerns associated with the disposal of unwanted pharmaceuticals and to make them aware of the pilot collection program. The media campaign included: Press releases to local media outlets; Public service announcements; Advertising in the major local newspaper; Advertising in a newspaper targeted to older adults; Information included in the Weather Channel crawl on the local cable TV system; Information posted on the Alachua County web page; Information posted on the Earth 911 web page. A news story highlighting the project published in the local newspaper and within an insert in the newspaper. 58

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Results and Discussion The collection period spanned from January 6, 2004 until May 28, 2004. A total weight of 305 pounds of discarded pharmaceuticals was collected during the pilot program period. The waste pharmaceutical solutions collected filled a to tal of two 55 gallon drums and had a final pH of 6. The solids content of the collected so lutions was estimated as 10% by volume based upon visual observation of the drum column by a thie f sampler. Although the vast majority of the pharmaceuticals were disintegrated by the collec tion solution, some partially decomposed medications were observed. Additionally, during emptying of the collection containers, it was observed that several medications remained in their tamper-proof packaging and had not been removed by the participants during their disposal. The number of residents who participated in the project could not be accurately determined. Official records were not kept by the participating pharm acies and only 51 surveys were completed by collection program participants However some general information could be obtained that allowed an estimat e of participation in the progr am. The national chain stores pharmacy reported the highest partic ipation rate of approximately 2-3 participants per day. The local household hazardous waste collection cente r averaged approximately 5 participants per week. From these limited observations it is esti mated that a minimum of 400 and no greater than 520 residents participated in the program. If it is assumed that each participant was from a single household, then using U.S. census data for Alach ua County, 520 participants would represent a total of 0.55% of the total households in Alac hua County. However, a lower percentage of participation is possible if households pa rticipated more th an once in the study. Based on participation within Alachua Count y, a national estimate of the quantity of pharmaceutical waste can be made. Using U.S. Census data, there were approximately 110,750,000 homes in the United States in 2004. E quating the 305 lbs collected from Alachua 59

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Countys 94,480 households over 143 days would resu lt in the collection of 456 tons of waste medications annually in the United States. Howeve r, this number assume s participation of only 0.55% of households. If the partic ipation rate were to increase from 0.55% to only 5% of the households, an estimated 4148 tons/year would be ge nerated nationally. If th e participation rate were to increase to 25%, th en an appreciable 20,740 tons/year would be collected. For comparison, the Australian unwanted medica tion collection program currently collects approximately 411 tons/year of medications with 7,968,400 households (71) Equivalent participation in the United States would equal 5,751 tons/year. Measurement of the effectiveness of the tec hniques used in the collection program was necessary to determine changes and goals for future pharmacy collection programs. A six question survey for patron completion upon disposal of their medications was created. However, only a total of 51 surveys were collected, far less than the estimated number of participants. Several factors contributed to th e low survey completion rate. Wh en interviewed, participants of the program cited privacy concerns and lack of time as two reasons for not completing the surveys. Due to other duties, collection site employees ofte n could not assist patrons in completing the survey or ensure a survey was completed. Thus surveys were simply placed near the collection containers for patrons to comple te, resulting in a small number of completed surveys or incomplete surveys. While the survey requested the names of th e medications patrons were disposing, it was not possible to fully report the types and amounts of medications discarded due to the lack of survey completion. Table 3-1 provides a list of those medications cited by patrons on the survey forms. The medications cover a wide variety of uses and include both prescription and over-thecounter formulations. 60

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Additionally, the surveys do provi de useful data concerning pa rticipant demographics and program effectiveness. Although 51 surveys were completed, subsequent figures may show a total greater than 51 responses. This was due to survey participants providing multiple responses for some questions such as no longer use a nd expired as reasons for disposal. These multiple responses were counted in each category. According to the National Center for Health Statistics for the year 2000, over 80% of people in the US over the age 65 had taken a prescr iption within the last month in comparison to less than 40% of pe ople age 18 to 44 (69) This corresponds with th e collection program data concerning the age of the medication users. Th e average age of the medication users reported by program participants was 63 years and 7 months, w ith greater than 65% of reported users over 60 years in age and 85% being great er than 50 years in age. Figure 3-2 shows the number of responses in each age group. Focusing limited progr am resources to this demographic would be expected to yield th e greatest benefit. With limited budgets, unwanted pharmaceutical collection programs must determine the best possible methods to make patrons aware of their services. A wide variety of media were utilized to inform the public of the pharmaceutic al collection program. Survey responses show that a majority of participants learned of th e collection program through newspaper articles and ads. Other methods not specifically listed included referral by lo cal officials and posted flyers. Figure 3-4 shows the survey re sults for advertising methods. An important factor in reducing the quanti ty of pharmaceutical waste generated is the medication users reason for discard. With this information future initiatives may target not only the collection of unused medications, but also prevent or reduce pharmaceutical waste generation. As shown in figure 3-4, expiration of the medicati on was the primary reason given 61

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for disposal by the survey participants. The resu lts suggest that the quan tity of pharmaceutical waste could be reduced by conducting research into the extension of expiration dates, manufacturing pharmaceuticals with greater effic acy, or reduction of the quantities sold per package or prescription. It was not possibl e to determine those pharmaceuticals which had expired after the patient had failed to complete th e prescribed medicine as directed but survey results indicated the average age of th e medications was 2 years and 11 months. Figure 3-5 shows the methods of medication di sposal utilized by the survey respondents prior to the collection program. A total of 49% of the participants cited the sewer system, 45% the trash, and 7.8% cited return to the pharmacy or other methods. Previous studies found 31% to 63.2% of households utilized trash disposal while 11.5% to 46% utilized the sewer system (5, 24, 25) Conclusions The creation of a pharmaceutical waste collectio n program faces several challenges in its development, but appears to be an issue of concern for the public. As with other household hazardous wastes, residents have to be motivated to participate and spend the time and resources to drop off products that may be hazardous to th e environment. An advantage of the Alachua County collection program was the ease with wh ich participants coul d dispose of their pharmaceuticals. In general, the location of co llection containers at local pharmacies allowed participants to drop off their pharmaceutical wastes at a place that was convenient. This conclusion is supported by the majority of waste being collected at the major chain store pharmacy. Had this store been unwilling to part icipate, the success of the program would have been greatly reduced. Future collection programs should endeavor to include convenient, public locations to increase participa tion. This may include mail-in programs with envelopes for return 62

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of unused medications to manufact urers or collection cen ters such as is the goal of the Maine return program (72) Continuous collection over seve ral months eliminated the restrictions of a one-day collection program. Interference with the coll ection program by special events, weather, and logistical restrictions were removed by having the collection program over a large period of time. Furthermore, the long term collection combined w ith the visibility of the containers at the collection sites (particularly pharmacies) allowed re sidents to learn of the program and return at a later time with unwanted pharmaceuticals. The c ontinuous inflow of participants throughout the program indicates that participation was not limited to a few, eager resident s, but that a sustained interest in the program was maintained. Despite the apparent success, the system had many restrictions. The inability to directly collect the medications severely reduced data collection. Collection site staffs, as voluntary participants, were primarily focused on assisting customers and their daily routines. Combined with the fact that many participants were unwil ling to complete the program survey, accurate participation numbers and additi onal beneficial information was limited in comparison to direct collection. The legal regulations concerning prescripti on and other pharmaceutical handling provided several obstacles to the effec tive, continuous collection of pharmaceutical waste. While the innovative collection technique employed in this study overcame many of th ese obstacles, future changes in the regulations may be necessary. Temporary changes such as limited exemptions issued to pilot programs for the direct collect ion of pharmaceuticals by pharmacies would allow for more effective data collection. With the data collected in this and future collection programs, legislatures and regulators could make permanent changes that are protectiv e of the public health 63

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64 and the environment by allowing for the efficient diversion of pharmaceutical waste to environmentally sound disposal options.

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Table 3-1. Pharmaceuticals collected for disposal by 2004 Alachua County, Florida collection program Brand name Generic name Drug class Activella Estradiol/Norethindrone acetate Hormone therapy Adequan Polysulfated gycosaminoglycan Veterinary Afrin Oxymetazoline HCl Vasoconstrictor Aleve Naproxen sodium Nonsteroid al antiinflammatory agents Allegra Fexofenadine Antihistamine Altace Ramipril Hypotensive agent Aquatab Guaifenesin/Dextromethorphan Decongestant Aspirin Acetylsalicylic acid Nonste roidal antiinflammatory agents Atrovent Ipratropium bromide Antimuscarinics/antispasmodics Augmenten Amoxicillin/Clavulanate potassium Antibiotic Azithromycin Azithromycin Antibiotic Azmacort Triamcinolone acetonide Antiinflammatory agent Benadryl Diphenhydramine HCl Antihistamine Bumetanide Bumetanide Diuretic Butalbital Butalbital Analgesic/antipyretic Cardura Doxazosin mesylate Hypotensive agent Cipro Ciprofloxacin Antibiotic Colace Docusate sodium Carthatics And laxatives Cyclophosphamide Cyclophosphamide Antineoplastic agents Detrol Tolterodine tartrate Urinary antispasmodic Dexamethasone Dexamethasone Antiinflammatory agent Digoxin Digoxin Cardiac Diltiaz Er Diltiazem HCl Cardiac Diovan Hct Valsartan/Hydrochlorothiazine Hypotensive agent Dyazide Hydrochlorothiazid e/Triamterene Diuretic Echinacea Echinacea Herbal Effexor Xr Venlafaxine HCl Antidepressant E-Mycin Erythromycin Antibiotic Enalpril Maleate Enalpril maleate Antihypertensive Excedrin Migraine Acetaminophen/acety lsalicylic acid/caffein e Nonsteroidal antiinflammatory agents Flagyl Metronidazole Antiinfective 65

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Table 3-1. Continued Brand name Generic name Drug class Flexeril Cyclobenzaprine hcl Skeletal Muscle Relaxant Flonaise Fluticasone propionate Antiinfective Flumadine Rimantadine hcl Antiviral Furosemide Furosemide Diuretic Glucovance Glyburide And metformin hcl tablets Antidiabetic Ibuprofen Ibuprofen Nonsteroidal antiinflammatory agents Immodium Loperamide hcl Antidiarrhea Keflex Cephalexin Antibiotic Konsyl Psyllium Carthatics And laxatives Lexapro Escitalopram oxalate Antidepressant Lisinopril Lisinopril Hypotensive agent Meclizine Meclizine hcl Antiemetic Megestrol Megrestrol acetat e Antineoplastic agents Moban Molindone hcl Tranquilizer Modicon Norethindrone/Ethinyl estradiol/mestranol Contraceptive Nasacort Triamcinolone acetonide Antiinflammatory agent Neomycin Neomycin Sulfate Antibiotic Nexium Esomeprazole magnesium GI Proton pump inhibitor Nitrostat Nitroglycerine Vasodilator Norpace Disopyramide phosphate Cardiac Nortriptylin Nortriptyline hcl Antidepressant Paxil Paroxetine hcl Antidepressant Pepcid Famotidine GI H2 antagonist Pravachol Pravastatin sodium Antilipemic agent Premarin Estrogens conjugated Hormone therapy Prevacid Lansoprazole GI antacid Progesterone Progesterone Hormone Prograf Tacrolimus Antirejection Promethazine Promethazine hcl Antihistamine Relafen Nabumetone Nonsteroidal antiinflammatory agents Robitussen Guaifenesin Expectorant 66

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67Table 3-1. Continued Brand name Generic name Drug class Rythmol Propafenone HCl Antiarrythmics Skelaxin Metaxalone Skeletal Muscle Relaxant Sudafed Pseudophendrine HCl Sympatho mimetic (Adrenergic) Agents Sulfameth Sulfamethoxazole Antiinfective Synthroid Levothyroxine Sodium Hypothyroidism Tagamet Hb Cimetidine GI Antacid Tetracycline Tetracycline HCl Antibiotic Thyrotab Thyroid Ho rmone Hyporthyroidism Toprol Xl Metoprol ol Hypotensive Agent Tylenol Acetaminophen Nonsteroidal Antiinflammatory Agents Tylenol Cold/Sinus Acetaminophen/Diphenhydramin e Nonsteroidal Antiinflammatory Agents Tylenol Pm Acetaminophen/Diphenhydramine N onsteroidal Antiinflammatory Agents Ursodiol Ursodiol Chol elitholytic Agents Valtrex Valacyclovir HCl Antiviral Warfarin Warfarin Anticoagulant Zantac Ramitidine HCl GI H2 Antagonist Zantaflex Tizanidine HCl Skeletal Muscle Relaxant Zofran Ondansetron HCl Antiemetic Zoloft Sertraline Antidepressant

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Figure 3-1. Collection container used in Al achua County discarded medication collection program 0 2 4 6 8 10 12 14 16 18 20-2930-3940-4950-5960-6970-7980-8990-99 Age of Medication UserNumber of Survey Responses Figure 3-2. Age of medication users (years) participating in pharmaceutical disposal program 68

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0 5 10 15 20 25 30 Radio TV Newspaper Friend Other Advertising MethodNumber of Survey Responses Figure 3-3. Histogram of survey responses to advertising methods reco gnized by participants 0 5 10 15 20 25 30 Expired No Longer Used Death Reason Cited for Medication DisposalNumber of Survey Responses Figure 3-4. Histogram of survey responses to reason for pharmaceutical disposal 69

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70 0 5 10 15 20 25 30 Sewer Trash Pharmacist Other Method Of Pharmaceutical Disposal Previously Used by Collection Program ParticipantsNumber of Survey Responses Figure 3-5. Histogram of survey responses pert aining to previously used disposal methods

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CHAPTER 4 PHARMACEUTICAL COMPOUND CONTEN T OF MUNICIPAL SOLID WASTE Introduction Previously, the recommended disposal method for unused pharmaceuticals was the sewage system. This method was chosen as a means to pr otect children from accide ntal poisoning in the home, prevent animal poisoning from scavenging either at the home or the landfill, and more recently to prevent scavenging of prescription medicat ions for illicit use. However, despite this recommendation the use of municipal refuse a nd landfills for pharmaceuti cal disposal was not uncommon. Prior research has shown that nearly half of all people have used their household trash as a disposal method, just slightly le ss than those who had used sewer disposal (83) Results of other studies showed that from 31 to 63% of respondents used municipal waste for disposal of medications.(24, 25, 80) Only a limited number of studies have investigated the presence of pharmaceutical compounds in landfills. These studies have focused on the analysis of landfill leachate (42, 45, 47) or leachate contaminated groundwater (40, 42, 43, 45, 46) Even fewer studies have examined the potential pharmaceutical waste composition of MSW (84, 85) Few specific regulations a pply to the management of discarded pharmaceuticals, particularly household medications and a myriad of pathways for disposal may be followed (66) Many U.S. states have begun to recommend refuse disposal instead of sewage disposal to their residents (19-21, 23, 39) In February of 2007, the United States White House Office of National Drug Control Policy released guidance on th e proper disposal of unused or unwanted prescription drugs (17) The guidance directs consumers to dispose unused drugs in household trash or to take advantage of drug take-back programs, rather than flushing the drugs down the drain. These new policies will undoubtedly re sult in increased amounts of pharmaceuticals entering MSW landfills. 71

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While studies have indicated landfills as potential sources of active pharmaceutical ingredients (APIs) to the envir onment, research could not be lo cated to quantify the input of APIs to landfills and how the quantity of APIs entering MSW affects their fate and potential leachate concentrations. The objectives of the research described in this chapter were to determine the quantity of active pharmaceutical i ngredients entering MSW of the United States and to identify pharmaceutical compounds common to municipal solid waste disposal for future research purposes. Materials and Methods Mathematical Estimation Information concerning the distributed mass of a pharmaceutical compound is not widely published. Instead, pharmaceutical data is more ofte n expressed in terms of the total dollar value sold. However, in Australia, the Drug U tilization Sub-Committee (DUSC), formed in 1988, annually assembles data on prescription drug us age. The DUSC publishes for each prescription medication the number of World Health Organi zations Defined Daily Dose (DDD) taken each day per 1000 people. In a previ ous study, Fisher and Borland used this report to estimate the pharmaceutical burden to the environment in Sydney (29) Employing a similar technique, it was possible to estimate the input of pharmaceu ticals to landfills in the United States. Figure 4-1 illustrates the met hod and statistical values util ized. The United States and Australia are similar in health care de velopment and medication availability (86) Therefore, the per capita medication usage of Australia may be c onverted to a corresponding per capita usage of the United States. This was accomplished utilizing a ratio of the prescriptions per person in the United States versus Australia (87-89) Using the U.S. population, this value was then equated to the total annual activ e pharmaceutical ingredient (API) usag e in the United States. The annual total API usage was then corrected for othe r factors based upon published research and 72

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governmental statistics. These factors include d: 1) Over-the-Counter (OTC) pharmaceutical usage, 2) The percentage of medications which go unused, an d 3) Percentage of unused medications discarded to MSW. Based upon these values and the total annual MSW disposal rate of the United States, the concentration of API within United States MSW was predicted. Direct Measurement A waste composition study of MSW was conduc ted to measure the percentage by mass and types of pharmaceuticals in the MSW of Or ange County, FL. The study was conducted in December 2006 at the McLeod Road Municipal Solid Waste Transfer Station, Orlando, FL. The choice of a transfer station afforded four primary advantages: A covered location to reduce the impact of inclement weather Reduced potential error introduced from la ndfill soil becoming intermingled with selected trash Ease of obtaining samples due to the immedi ate access to equipment at the facility, and A concrete partition within the facility to provide additional safe ty for personnel from operating machinery and waste vehicles. Waste Sectors The composition of waste from residential and commercial sector s was independently determined during the study. Residential wastes were comprised of MS W from single-family and multi-family residential dwellings. Commercial sectors included commercial and institutional businesses of any type, including offices, restaurants, retail establishments, warehouses, hotels, schools, and government buildi ngs. Samples were not taken from segregated loads of bulky items, recovered materials, or solely construction and demolition debris. Commercial waste from the medical industry, such as doc tor offices, nursing homes, and hospitals was not targeted nor ex cluded from the waste examined. 73

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Sampling and Sorting The commercial or residential origin of each MSW sample was verified by the transfer facility staff through interview with the collection vehicle driv er. Once identified, a grab sample of MSW, visually estimated to weigh from 200 to 400 pounds, was obtained from the MSW on the tipping floor of the facility. I ndustry standard practice specifies a minimum average sample size of 200 pounds (90, 91) However, variation in load materials and their density can result in some samples weighing less or more than the 200-pound target. Due to this, efforts were made to ensure that samples were of significant size to achieve well greater than 200 lbs on average. To minimize bias, a vertical slice was taken from the waste stack and the sample was compared to the visible characteristics of the full waste load for any obviously nonrepresentative material prior to sorting. The municipal solid waste was segregated into twenty-three indi vidual categories and grouped into ten major groups. This permitted comparison to US national waste composition data to ensure the waste selected was typical of the national average. Each sample was manually loaded onto a sorting table with bagged waste carried to the tabl e and loose waste transferred via plastic containers. Large or bulky items, such as tires, were separated a nd weighed directly. The sorting table was covered by 1/ 2-inch (1.2 cm) screening and particles small enough to fall through the screen were characte rized as organic fines. Rema ining waste was manually sorted into bins labeled for each of the twenty-thr ee categories. Bagged and boxed materials were opened and all waste sorted. Upon completion of sorting each sample load, each category was weighed to determine its individual weight. Ph armaceutical wastes were then further processed to determine the quantity (numbe r of tablets or volume of liquid), active pharmaceutical ingredient, and medication brand information. 74

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Results and Discussion Mathematical Estimation In Australia, approximately 233.4 million prescriptions were filled in 2004 (88) With a 2004 population of 20.1 million people, a total of 11.6 prescriptions per person were filled. In 2004, the number of retail prescr iptions filled in the United States was 3.27 billion and the number of outpatient prescriptions filled by ve terinary facilities, hospitals, and other medical clinics was estimated to be 229.9 million, for a total of 3.50 billion prescriptions (87, 89) Dividing this by the United States populat ion for 2004 (293,638,158), the average number of prescriptions per person in the U.S. was 11.9 or a factor of 1.03 greater than in Australia. The average consumption of prescription pharm aceuticals per thousand people in Australia was calculated to be 178.21 g of active pharmaceu tical ingredients (APIs) per day in 2004 (88) Thus, as shown in Figure 4-1, multiplying the Australian consumption by the 1.03 factor yielded a total of 183 g/day per thousand people in the Unite d States. This is equivalent to a total of 2.16x104 tons/year of active ingredie nts purchased via prescription. To determine the amount of the dispensed AP Is entering MSW, the percentage of the prescribed drugs which become unused is required. The measure of pres cription fulfillment by a patient is termed compliance or concordance by the medical community and includes factors such as consuming all medication prescribed, ta king the medication at th e appropriate time and under the appropriate conditions (such as a voiding confounding factors that may reduce its effectiveness). Studies of pres cription compliance can be found exte nsively in the literature, but specific details on the remaining unused medicati on quantity are seldom mentioned. In a study conducted in Alberta, Canada, it was estimated that 60% of the original drugs dispensed in a prescription were returned to the lo cal pharmaceutical collection program (5) In another German study, it was determined that 65% of the original medication was returned (4) Other 75

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studies have measured only 3% or estimated 4 to 15% (6, 7) In light of the wide range of potential values, three percentages were used to estimate the amount of medication which becomes unused 10%, 30%, and 60%. To this point, only prescription medications have been included in the estimation. Nonprescription or Over-the-Coun ter (OTC) medications may also be discarded and must be included in the estimation. Garey et al. report ed 65% of medications collected in a Houston, Texas program were prescription drugs, while 27% were over-the-counter (OTC) and 8% were pharmaceutical sales samples (92) A Swedish collection progra m found that only 7% of the unused medications were OTC drugs (7) An average of these two studies, 17%, was assumed as the percentage of unused medications attributable to OTC medications. The disposal options for unused medications practiced by consumers consist primarily of flushing to the sewage system, household tras h, and return to a pharmaceutical collection program. The results of several studies on pharmaceutical disposal methods have been previously published. The percentage of people utilizing MSW disposal averaged 48.3% over four studies and the total APIs for dispos al were multiplied by this percentage (5, 24, 25, 83) As seen in Figure 4-1, the total APIs entering US landfills in 2004 are estimated to be 1.26x103tons (10% unused), 3.78x103 tons (30%), and 7.56x103 tons (60%). These computations account for only the active ingredients of the medications and do not account for the salts and other materials included in the formulations which comprise a much larger percentage of the medicine. Dividing th e total APIs landfilled by the total of 169.6 million tons of MSW landfilled in 2004 (73) the average concentration of pharmaceutical compounds deposited to US landfills that year was 7.4 mg/kg (10%), 22 mg/ kg (30%), or 45 mg/kg (60%). 76

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These concentrations encompass all pharmaceu ticals and the concentration of any single pharmaceutical can be expected to be much lower. Direct Measurement A total of 6,204 lbs (2820 kg) of MSW was sorted in 22 samples comprised of an equal number of residential and comme rcial loads. Samples ranged in weight from 163.9 to 437.9 lbs, with an average of 282.0.9 lbs. The final compos ition is presented in Table 4-1. As seen in the table, the waste collected during the composition study was comparable to the United States national average composition. The pharmaceutical compounds collected are shown in Table 4-2. A total of 22 differing APIs were collected co mprising a total of 22,910 m g. This resulted in a final concentration of 8.1 mg/kg w ithin the municipal solid waste, a value within the range of the prior calculations. Of note, liquid medications we re collected as frequently as solid medications. Of the MSW received from commercial so urces, only 1 of 11 samples contained a measureable API, ciprofloxacin. The remaining APIs collected during th e study were received from 9 of the 11 residential MSW samples studied. However, in both sources, a large number of empty pharmaceutical containers were located. It was not possible to determine if these compounds were discarded as empty containers or became ruptured during collection and transport and their contents lost, dissolved, or crushed within the MSW. The 33 APIs listed on the 45 empty collected containe rs is given in Table 4-3. In a recent pharmaceutical indus try report on the potential rel eases of medications in MSW landfill leachate, calculations were based upon the assumption of 5%, 10%, and 15% of all medications being disposed via landfills (85) In the statistical estimation employed in this research, it was estimated that 10 to 60% of medicati ons become unused and that 48.3% of those unused medications are disposed via MSW. This corresponds to approxim ately 5 to 31% of all dispensed medications becoming unused a nd is comparable to the industry study. 77

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The large range in mathematically estimated API concentration in MSW, 7.4 to 45 mg/kg, is due to the large uncertainty in the quantity of medications wh ich become unused once given to the patient/consumer. The direct measurement re sult of 8.1 mg/kg is equivalent to a calculated value of 11% of all medications becoming unuse d. However, the percentage of medications which become unused may be lower as the prior calculations were unable to account for internet prescription sales, an increasingl y popular source of prescription medications. This would result in a higher initial API input. Additionally, an increase in number of prescriptions between the calculated year (2004) a nd the measurement year (2006) may result in an increase in the measured API concentration over the predicted concentration. The pharmaceuticals comprising the greates t API concentration in MSW included antibiotics (Ciprofloxacin, Amoxicillin) and nonsteroidal anti-inflammatory drugs (acetaminophen, ibuprofen). The Alzheimers treat ment, ampicillin, which was collected from a single residential sample, also appe ared in significant quantity but may be skewed due to this one time disposal. The large quantity of cold medicat ions including antihista mines, decongestants, cough suppressants, and fever reducers may be du e to the time of year in which the study was conducted and other medications may be expected to be prevalent during ot her seasons, such as allergy medications in spring. The concentration determined in this st udy may underestimate the final disposal of pharmaceutical compounds within a landfill. Beyond the direct dis posal of unwanted pharmaceuticals, pharmaceutical manufacturing wast es in landfills and waste water treatment biosolids sent to landfills are also a potenti al source of pharmaceuticals. Pharmaceuticals adsorbed to the biosolids may become released within the landfill environment and enter the landfills leachate (11, 49, 93) Furthermore, new government policies to direct unused 78

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medications to MSW disposal may result in a significant increase, possibly doubling the measured concentration if the roughly 50% of di scarded medications previously flushed to the sewage system become discarded within MSW. 79

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Table 4-1. Percent composition of research stud y residential and commercial municipal solid waste vs. United States national averages Residential Commercial Total United States average Paper & paper board 33.5 44.9 39.2 25.2 Plastic 19.8 16.8 18.3 16.4 Glass 3.8 3.1 3.4 6.0 Metals 5.4 6.2 5.8 7.1 Yard waste 8.8 1.2 5.0 7.3 Wood 0.9 4.0 2.5 7.6 Food waste 9.8 8.9 9.3 17.1 Textiles 4.7 1.3 3.0 5.6 Other wastes 8.7 4.3 6.5 5.5 Miscellaneous inorganic 4.7 9.4 7.1 2.2 Table 4-2. Active pharmaceutical ingredients collected in measurable quantities within municipal solid waste Active pharmaceutical ingredient (API) Pharmaceutical category Form of delivery (liquid/solid) Number of medications Total quantity (mg) Ciprofloxacin hcl Antibiotic Solid 1 6500 Acetaminophen NSAID Liquid/Solid 3/1 5380 Amoxicillin Antibiotic Liquid 1 4000 Ampicillin Antibiotic Solid 1 3000 Donpezil hcl Alzheimer's disease Solid 1 1050 Ibuprofen NSAID Liquid 1 900 Pseudoephedrine hcl Decongestant Liquid/Solid 3/1 737 Metoprolol succinate Antihypertensive/Beta blocker Solid 1 475 Bismuth subsalicylate Gastro intestinal Liquid 1 349 Dextromethorphan hbr Cough suppre ssant Liquid/Solid 3/1 306 Minoxidil Hair loss Liquid 1 100 Albuterol sulfate Bronchodila tors Liquid 3 26.4 Tegaserod maleate Irritable bowel syndrome Solid 1 24 Cetirizine hcl Antihistamine Solid 1 20 Polymyxin B sulfate Antibiotic Liquid 1 16.7 Trimethoprim Antibiotic Liquid 10 Clobetasol propionate Corticoste roid Liquid 1 5.7 Phenylephrine Decongestant Solid 1 5 Brompheniramine maleate Antihistamine Solid 1 2 Nicotine Smoking treatment Solid 1 2 Levothyroxine sodium Hypothyroidism Solid 1 0.75 Clotrimazole Antifungal Solid 1 0.3 Note: Some medications, such as cold me dications, contained more than one API. 80

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Table 4-3. Active pharmaceutical compounds of empty medication containers collected from municipal solid waste Active pharmaceutical ingredient (API) Pharmaceutical category Acetaminophen NSAID Amlodipine besylate Hypertensive Aripiprazole Schi zophrenia/Mania Atorvastatin calcium Cholesterol Azithromycin Antibiotic Caffeine Stimulant Celebrex NSAID Chlorpheniramine Antihistamine Dexamethasone Corticosteroid Dextromethorphan Cough suppressant Diazepam Antianxiety Diclofenac NSAID Digoxin Heart Failure Dutasteride Prostate Enlargement Esomeprazole Gastrointestinal Ethinylestradiol Oral Contraceptive Genotropin Growth Hormone Glycerine Laxative Ibuprofen NSAID Losartan potassium Antihypertensive Methscopolamine Anticholinergic Metoprolol succinate Anti hypertensive/Beta blocker Montelukast Asthma/Allergy Neomycin Antibiotic Norethindrone Oral contraceptive Pamabrom Diuretic Pseudoephedrine Decongestant Pyrilamine maleate Antihistamine Quinapril ACE inhibitor Simethicone Gastrointestinal Thiabendazole Antifungal Tussin ex Expectorant Ziprasidone HCI Schizophrenia/Mania 81

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82 2004 Australian Prescription API Usage 178.21 g/1000 people/day 2004 Prescriptions US 3.5 billion/293 million people = 11.9/person AUS 233.4 million/20.1 million people = 11.6/person Ratio = 1.03 2004 U.S. Prescription API Usage 183.13 g/1000 people/day = 21638 Tons Unused Medications Discarded to MSW 48.3% 10% Unused 2164 tons 30% Unused 6491 tons 60% Unused 12983 tons 2607 tons 7821 tons 15642 tons 1259 tons 3778 tons 7555 tons 7.4 m g /L 22 m g /L 45 m g /L 2004 169.6 million Tons of MSW for Disposal Over-the-Counter Medications 17% of Unused Figure 4-1. Procedure for mathematical pred iction of the active pharmaceutical ingredient concentration of United Stat es municipal solid waste.

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CHAPTER 5 DETERMINATION OF THE ANAEROBIC BIODEGRADATION POTENTIAL OF SELECTED ACTIVE PHARMA CEUTICAL INGREDIENTS Introduction Throughout the lifetime of a landfill a complex se ries of chemical and biological reactions occur. Studies have suggested that the stab ilization of landfilled wa ste proceeds in five sequential and distinct phases (74-76) The chemical and biological processes, active bacterial populations, leachate characteristics, and gas emissi ons vary from one phase to another; however anaerobic conditions comprise the largest portio n of the lifespan of a modern landfill. As demonstrated by the unused medication colle ction program and waste composition study presented in chapters 3 and 4, pharmaceutical co mpounds are routinely discarded in municipal solid waste (MSW) and will predominantly be subj ect to anaerobic conditions once placed in MSW landfills. Overall, the fates of pharmaceuticals in a landfill include attenuation, degradation or decomposition, mobilization in the leachate, and volatilization and transport with the landfills gas stream. Examination of the degradation of pharmaceutical compounds has predominantly focused on efficacy and determination of shelflives. Simulating the dry, well-lit, and aerobic conditions of household medication st orage, these studies cannot be co rrelated to the dark, moist, and anaerobic conditions typical of MSW. Therefore, the study of th e biodegradability of discarded medications under anae robic conditions is necessary. In a study by Joss et al., researchers determin ed that extending the hydraulic retention time of wastewater treatment plants may play a key in the breakdown and removal of estrogens, common birth control medication compounds (94) Wastewater treatment anaerobic digesters based on attached growth typically have hydrauli c retention times between 1 and 10 days while mesophilic and thermophilic digesters range between 25 and 35 days, but may be lower (95) 83

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Landfills potentially provide si gnificantly longer retention ti mes due to the low hydraulic conductivity of MSW and high organic matter content for potential adsorption of the pharmaceutical compound. A landfill with waste at thirty meters in height and a hydraulic conductivity of 1x10-4 cm/sec (96) would result in leachate requiring 347 days to pass through the landfill. Respirometric techniques that measure ga s production (primarily methane and carbon dioxide) under anaerobic conditi ons have been used successf ully for assessing anaerobic biodegradability (97, 98) As a result, standard test me thods have been developed including ASTM E1192-92 (Standard Test Method for Determining the Anaerobic Biodegradation Potential of Organic Chemicals), USEPA OP PTS Method 835.3400 (Anaerobic Biodegradability of Organic Chemicals), and OECD Method 311 (Anaerobic Biodegrad ability of Organic Compounds in Digested Sludge: By Measurement of Gas Production.) (99-101) However, less than 0.01% of known organic compounds ha ve been tested for biodegradation (102) Numerous efforts to predict the biodegradab ility of specific compounds have been made, with most derived from rules based on structure (102) The fundamental units of biodegradation are taken to be organic functi onal groups, with each group subj ect to multiple but specific enzymatic transformations (102) Therefore, the anaerobic degradation of pharmaceutical compounds can be expected to have some basis in the structure and functional groups of each compound. The objective of the research described in this chapter was to determine the potential for anaerobic degradation of six pharmaceutical com pounds. Using a respirometric test procedure based on standard test methods and direct analy tical measurement, the anaerobic degradation of the compounds was examined without the pres ence of other substances which may inhibit 84

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degradation or cause removal from solution via other mechanisms. The pharmaceutical compound was the primary source of organic car bon. These anaerobic degr adation results were then compared to the predicted degradabil ity of the compounds by a US Environmental Protection Agency anaerobi c degradation model. Materials and Methods Selection of Target Pharmaceutical Compounds Various methods have been used to identif y pharmaceutical compounds with the potential for release to the environment and previous rese archers have commonly utilized prescription rate data (103-105) Using a similar method, six pharmaceuti cal compounds were selected for study based upon the number of prescriptions from 2002 to 2004, the defined daily dose of the medications, and over-the-counter usage and environmental detection of medications. A detailed description of the compound selection method is given in Appendix A. The six pharmaceutical compounds selected for degradation study were acetaminophen, acetylsalicylic acid (measured as salicylic acid), 17 -ethinylestradiol, ibuprofen, metoprol ol tartrate, and progesterone. Reagents and Test Compounds All the pharmaceutical standards used were of high purity grade (> 98%). 17 ehtinylestradiol, acetylsalicylic acid, ibuprofen, metoprolol ta rtrate, and progesterone were purchased form Sigma (WI, USA). Acetami nophen was obtained from Sigma-Aldrich (MO, USA). Ammonium formate and HPLC water were from Fisher (PA, USA). HPLC methanol was from Tedia (OH, USA). Respirometric Testing Respirometric testing for anaerobic degradation of the pharmaceutical compounds was performed using a modified procedure based upon US EPA test method OPPTS 835.3400 (101) Samples of each compound were prepared in trip licate in 500 ml glass sample bottles (Wheaton 85

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Lab 45 Graduated Safety Coated Bottles Cat # 06-451-29). Each compound was weighed into a sample bottle in the quantity shown in Table 51 to obtain 50 mg/L of organic carbon in each sample. Two milliliters of ultr apure water were added to each bottle to prevent loss of the powdered compound, and continuous sparging was commenced using a nitrogen (N2, 80%) and carbon dioxide (CO2, 20%) gas mixture through a glass tube inserted into the bottle. A nutrient solution was prep ared per method OPPTS 835.3400 (101) The solution was heated to a boil with conti nuous stirring and sparging with oxygen-free nitrogen. Upon boiling, the solution was placed in an ice bath and th e sparging gas was changed to a mixture of N2(80%)/CO2 (20%). When the medium cooled to 37C, the flask was removed from the ice bath and remaining nutrients, 10.56 g of sodium bicarbonate, and 400 ml of anaerobic digester sludge were added to reach a final volume of approximately 4L. The anaerobic sludge was obtained from a laboratory digester with a retention time of approxi mately 20 days. The digester was maintained on a hormone and additive free dog food for three months prior to the experiment (Pet Promise dry adult dog food). E ach sample bottle was then filled with 300 ml of inoculated media and a butyl rubber two-le g lyophilization stopper (Wheaton #224100-507) was inserted while the glass tube used to deliv er the sparging gas was removed. The sample headspace was then flushed for an additional five minutes through a supply and vent needle inserted through the bottle septum. To measure background gas production of the te st solution, triplicate samples without any pharmaceutical addition were prepared in the sa me manner. To verify sufficient bacterial activity of the solution, triplicat e samples containing cellulose as the carbon source (50 mg/L carbon) were also prepared. The cellulose sa mples were required to exhibit anaerobic biodegradation as measured by gas production of gr eater than 50% of the theoretical maximum. 86

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Otherwise the biological activity would be rega rded as inadequate a nd the test repeated. Additionally, these samples were used as quality control blank samples during chemical analysis. To measure potential adsorption of the pharmaceutical compounds to the sample bottles and the solution solids, triplicate samples of each compound were prepared using autoclaved inoculation media. Prior to addition to each sa mple bottle, the inoculation media was autoclaved for 17 minutes at 250C, held at room temperat ure for 24 hours, and then autoclaved a second time. Upon final removal from the autoclave, the solution was cooled in an ice bath to 37C and added to the sample bottles as previously described. All sample bottles were wrapped in alumin um foil to prevent po ssible photodegradation and stored in an incubator at 37C over a peri od of 56 days. This time frame was chosen to match the time allotted by standard anaerobic degradation test methods (99-101) Approximately 3 to 5 hours after filling, the sample bottles were vented by inserting a gastight glass syringe through the septum. This permitted the increased pressure formed due to the heating of the flushing gases to equilibrate with atmospheric pressure through expansion within the syringe. Measurements of gas volume produced and me thane content were performed at 3, 7, 14, 21, 28, 42, and 56 days following sample creation. Gas volume was measured with a gas-tight syringe (Popper Micro-mate 5, 10, and 20cc) with a 20 gage needle inserted through the septum and held in a horizontal position, allowing the sy ringe plunger to move freely. Methane content was determined via analysis of a 100 L headspace sample using an Agilent 6980 Gas Chromatograph with a thermal conductivity dete ctor (GC/TCD). At 56 days, the final gas composition was determined for all samples and the samples processed for chemical analysis. 87

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Analytical Methods Standards Preparation Stock solutions were prepared containing each of the pharmaceuticals of interest at a concentration of 1000 mg/L in HPLC methanol using Class A volumetric glassware. These stock solutions were stored at -20 C. Calib ration standards were prepared from the stock solutions by a series of 10 and 100-fold diluti ons with the sample matrix. These working standards were stored at 4 C. Sample Preparation Following final headspace gas analysis, samp les were vacuum filtered through a 1.2 um glass filter paper (Fisher Scientif ic, Fisherbrand G4 Glass Fiber Filt er Circles). An aliquot of 30 to 50 ml of filtrate was placed into a silanized bo rosilicate glass vial with a teflon coated cap. The pH of each sample was adjusted to 5 usi ng concentrated hydrochlor ic acid and the samples were stored at 4C until analys is. Immediately prior to analys is, a 2ml aliquot of each sample was placed into a 2ml sampling vi al with filtration through a 0.2u m syringe filter (Supelco IsoDisc Filters PTFE-25-2). Chromatography and Mass Spectrometry LC analysis was performed with an Agilent 1200 Series rapid re solution LC system coupled to an Agilent 6410 Triple Quad MS /MS (Palo Alto CA, USA) equipped with an orthogonal ESI interface. For analys is of samples containing ibuprofen and acetylsalicylic acid, compounds were separated at 40 C by means of a Zorbax extended C18, RRHT column (2.1 mm 100 mm, 1.8 m) from Agilent. The injec tion volume was 1 L with a flow rate maintained at 0.4 ml/min. The mobile phase was comprised of a mixture of (A) 5mM ammonium formate in water and (B) 5mM ammonium format e in methanol. The mobile phase solvent gradient started with 5% of solvent B and was increased to 90% solvent B evenly over 12.0 88

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minutes. The solvent mixture was returned to th e initial proportions following completion of sample analysis at 12.1 minutes and a post-run tim e of 2 minutes was requ ired to re-equilibrate the column. The analysis of samples c ontaining acetaminophen, metoprolol tartrate, 17 ethinylestradiol, and progesterone was complete d without chromatography. A volume of 1L of each sample was directly injected (bypassing the LC column) with a flow rate of 0.4 ml/min in a mobile phase solvent mixture comprised of 50% of solvent B and 50% of solvent A. Calibration was monitored through the use of calibration verification samples every six samples. The calibration check sample was required to be within % of the initial sample. Instrument blanks to monitor po tential carryover between injections were analyzed prior to each calibration check. For samples in which carryove r was indicated, one to three system washes were placed between analytical samples and the samples reanalyzed. Acquisition, peak identification and integration, and final quantiz ation were performed with Agilent MassHunter Workstation Software B.01.00 9(B48). The method detection limit (MDL) was determined according to US Environmental Protection Agency guidelines (106) The MDL is defined as the minimum concentration of a substance that can be measured and reporte d with a 99% confidence that the compound concentration is greater than zer o, and is determined from at l east seven replicate analyses of samples containing the compounds of interest. Seven samples containing each pharmaceutical compound at 10 g/L in the anaerobic test solution were analyzed. The MDL for each compound was determined from the standard devi ation of the concentration for the replicate measurements, which is multiplied by the Students t-value for (n 1) degrees of freedom. The resulting MDL for each of the six tested compounds is listed in Table 5-2. 89

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Anaerobic Biodegradation Model Prediction The potential for anaerobic biodegradation of the six selected pharmaceutical compounds was analyzed using the degradation predicti on model BIOWIN. The BIOWIN model is a component of EPI (Estimation Programs Inte rface) Suite, a group of physical/chemical property and environmental fate estimation models developed by the EPAs Office of Pollution Prevention & Toxics and Syracuse Research. It provides screening level estimations of physical/chemical properties and environmental fate properties of organic compounds. The BIOWIN model estimates aerobic and anaerobic biodegradability of organic chemicals using 7 different models including a seventh and newest model which estimates anaerobic biodegradation potential (107) Data Analysis The theoretical methane yields for each of the compounds were calculated using Equation 5-1 (108) Using the compounds molecular formul a, Equation 5-1 perm its prediction of methane generation from the anaerobic decompos ition of the compounds. Experimental methane yields were divided by the theore tical methane yield to calculate the percent yield of methane. Statistical comparison of the total methane prod uced by each compounds triplicate samples at 56 days with background media samples and cellulose samples was conducted using ttest analysis ( =0.05, P < .05). Ttest statistical comparis on of compound concentrations determined by direct analytical measurem ent was also performed. 2 4 2324324 324 CO dcba CH dcba OH dcba NOHCdcba 8 8 4 (5-1) 90

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Results and Discussion Biodegradation Gas Production Due to the use of a sparging gas comprised of 20% carbon dioxide during sample preparation, degradation gas production was de termined by methane gas production of the samples. Table 5-3 shows the experimental and theoretical methane yield for each of the tested compounds under anaerobic decomposition. 17 -ethinylestradiol yielde d an average of 0.4 ml less methane than that produced solely by the in oculated media as background. Thus, inhibition of the degradation organisms by 17 -ethinylestradiol is indica ted. Ibuprofen (-1.3 ml) and progesterone (-1.8 ml) also produced meth ane at levels less than background. To be considered anaerobically degradable, standard test methods require gas production equivalent to a minimum of 75% of the theoretical value (100, 101) None of the selected pharmaceutical compounds exceeded this threshold, with the largest percentage of the theoretical total methane coming from acet ylsalicylic acid with an average of 6.4%. However the deficiency in gas production was not due to poor biological activity of th e inoculation media as the reference substance, cellulo se, yielded 80% of the theoretically predicted methane. One reason for the measured gas production of the te st compounds being less than the theoretical value may be the incomplete conversion of th e organic carbon of each sample into methane (CH4) and carbon dioxide (CO2). This may be due to abiotic chemical reactions which do not result in methane production or incomplete metabolism of the compounds. Figures 5-1 shows the average cumulative methane production for each compound and the background gas samples over the 56 day test period. T-test analysis (p<0.05) showed that at 56 days, the difference between background methane generation and that of acetylsalicylic acid, acetaminophen, ibuprofen, and progesterone were st atistically significant. During the first 7 days, inhibition of methanogenic bacteria is indicated for acety lsalicylic acid, ibuprofen, 17 91

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ethinylestradiol, and progesterone as their methane production lags that of background samples. Of these four compounds, only the acetylsalicy lic acid samples recovered from the initial inhibition to levels greater th an background. This may be du e to the rapid hydrolysis of acetylsalicylic acid to salicylic acid. This hydr olysis generates acetic acid, which may be more readily degraded. Furthermore, acclimation of the bacteria to the compound may have occurred. Following the in itial inhibition, 17 -ethinylestradiol appeared to maintain near background levels indicating a pro cess which eliminated its interf erence. This may include its adsorption out of solution or acclimation of the bacteria. Ibuprofen and progesterone demonstrated continued inhibition with the margin between their methane production and background increasing over the experiment. Ba sed upon respirometric te sting, acetylsalicylic acid showed the greatest degr adation over the test period. Degradation Direct Concentration Measurement Table 5-4 shows the average pharmaceutical concentration for each compound after 56 days in the abiotic samples (inactivated by auto claving). These samples measured the potential of the pharmaceutical compound to adsorb to th e test vessel and the medium. They also measured potential losses due to other abiotic mechanisms. The biodegradation of each compound was calculated by subtracting the aver age concentration of each compound following 56 days in the biologically activ e samples from those in the abio tic samples as shown in Table 55. This is a conservative estimation of the degradation potential as compounds may have desorbed and reentered the solution upon their degradation in solution or may have been degraded at their adsorption sites. Table 5-6 shows the overall reduction of each compound at the end of the 56 day period, accounting for both biode gradation and abiotic losses. A review of literature concerning biodegradation and chemical structure reveals several general rules which 92

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are discussed for each individual compound ba sed on its structure shown in Figure 5-2 (12, 109, 110) Progesterone and 17 -Ethinylestradiol In the abiotic samples, 17 -ethinylestradiol and progest erone showed significant reductions of greater than 90% from their initia l added target concentr ations of 61.75 mg/L and 62.39 mg/L. This indicates that adsorption is a strong mechan ism for their removal from solution. Due to adsorption, these compounds show ed the largest reductions of all compounds in the biologically active samples with the final 17 -ethinylestradiol concentrations resulting in a 99% decrease to less than 1 mg/L and progester one to less than the method detection limit. Minor biodegradation of 17 -ethinylestradiol was indicated wi th a difference of 4mg/L between the abiotic and biologically active samples. This is equivalent to 6% of th e initial content of the sample. Progesterone displayed only a 0.4 mg/L difference between the samples and therefore had only 0.6% of the initial content potentia lly removed by biodegradation. However, the aqueous solubility of these compounds (17 -ethinylestradiol = 11.3 mg/L, progesterone = 8.81 mg/L at 25C) is less than the targeted concentr ation and may have played a role in restricting their concentration. 17 -ethinylestradiol and progest erone are large polycyclic compounds. Cyclic compounds are generally less biodegradable than alipha tic compounds and polycyclic less than cyclic compounds. It is also hypothesized that a molecules larger size decreases the probability of its passage through the cell membrane for metabolis m. Additionally, compounds of smaller size and straighter carbon chains are spatially favored to allow the a pproach of metabolizing enzymes to the active biodegradation sites of the molecu le. Thus these compounds may be predicted to have the least degradation potential of the six compounds tested. 93

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Acetaminophen Acetaminophen with a final average concentrat ion of 79 mg/L in the abiotic samples showed no reduction in concentration over the 56 day period, indicating adsorption and other abiotic mechanisms were not significant. In the biologically activ e samples, acetaminophen concentrations decreased to 70 mg/L or by 11% and statistical analysis showed this decrease in concentration to be significant. Acetaminophen contains an aromatic ri ng with substitutions. Substitution upon an aromatic ring affects biodegradation in two possible manners. First, the substituent may facilitate or hinder biodegradat ion. Hydroxylation of adjoining carbon atoms in the ring is the primary mechanism for ring cleavage. Constituen ts such as COOH and OH which can promote hydroxylation increase biodegradabili ty while those that cannot, such as halogens, decrease biodegradability. Furthermore, the greater the number of substitutions, the fewer the potential sites for direct hydroxylation on the aromatic ring, causing reduced biodegradability. Additionally, chances of spatia lly interfering with the prom oting enzyme are increased. Acetaminophen contains a short, branched chai n comprised of an amino group, both of which indicate a resistan ce to biodegradation. Acetylsalicylic Acid Acetylsalicylic acid was measured in solution as its primary hydrolysis product, salicylic acid. The carboxylic acid group contained in acetylsali cylic acid reacts with water to form acetic acid and salicylic acid. Salicylic acid demonstrated the greatest amount of biodegradation with a reduction of 18 mg/L over 56 days. This is equi valent to a 21% reduction. In the abiotic samples, acetylsalicylic acid concentration av eraged 88 mg/L and showed no reduction in concentration over the 56 day period. This indicates that adsorp tion and other abiotic mechanisms were not significant removal mechan isms for salicylic acid. The reduction in 94

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salicylic acid concentration was expected as it was the top methane producer of the compounds tested. As with acetaminophen, salicyli c acid contains an aromatic ring with substitutions. However, the functional groups of salicylic acid, COOH and OH, can promote hydroxylation and increase biodegradability. Furthermore, the lo cation of these groups in the ortho configuration opens the potential sites for direct hydroxylation on the aromatic ring at the opposite side of the ring and reduces the chances of spatially interfering with the promoting enzyme. The carboxylic acid group contained in each of th ese compounds may be the most likely point of degradation. This group increases the biode gradability by providing a s ite for decarboxylation or -oxidation, resulting in the release of carbon dioxide or acetic acid. Ibuprofen In the abiotic samples, ibuprofen decreased from the initial concentrat ion of 66 mg/L to an average concentration of 53 mg/L. This is an average decrease of 19% and indicates adsorption to be an appreciable mechanism for its removal from solution. Over the 56 day test period, the biologically active samples showed a d ecrease to an average concentration of 49 mg/L. This was not statistically different from the abiotic sample concentrations and therefore biodegradation of ibuprofen was not indicated. This was also expected as ibuprofen methane production lagged behind background methane produc tion indicating a lack of biodegradation. As with acetaminophen and salicylic acid, ibup rofen contains an aromatic ring with substitutions which affect its biodegradation. Ib uprofens functional group s, due to their highly branched structure, are resistant to biodegrad ation themselves. Additionally, their presence on opposite ends of the aromatic ring blocks hydr oxylation of adjoini ng carbon atoms in the aromatic ring and increases the chance of spatially interfering with promoting enzymes. 95

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Metoprolol Tartrate Metoprolol experienced the thir d largest overall reduction of 42%. However, its reduction was appreciably larger than may be attributed to biodegradation alone and mechanisms such as adsorption and other abiotic reactio ns play a larger role in its reduction. Examination of the abiotic sample concentrations shows a decrea se from 71.49 mg/L to 54 mg/L over the 56 day period. Thus, abiotic mechanisms accounted fo r 24% out of the 42% reduction. However, statistical analysis confirmed that the reducti on in concentration of the biologically active samples to 41 mg/L was significantly different and biodegradation of metoprolol was shown. Metoprolol, while also containing a substitu ted aromatic ring, contains several other significant functional groups affect ing its biodegradability. Thes e groups include two ethers, an alcohol, and an amine group. The ethers and amine groups are pr edicted to be less biodegradable, but the long chain length of the molecule and the alcohol group may increase its biodegradability. Anaerobic Biodegradation Model In developing BIOWIN, the anaerobic degrad ation of 169 compounds wa s initially used in the preparation of the model. Each compound was scored with a "Pass" if the compound yielded 60% or greater of the theoretical gas production within 60 days. It was scored as "Fail" if it did not. Model output includes a de gradation rating factor for each structural fragment of the compound if contained within the model library and an overall anaerobic linear biodegradation probability factor. A factor greater than 0.5 indicates that the compound will degrade quickly, i.e. pass the test, while a factor less than 0.5 indicates that it will not. The Anaerobic Linear Biodegradation Probabi lity Factor for each of the six tested pharmaceutical compounds is presented in Table 5-7 and the complete BIOWIN Anaerobic model results are given in Appendix B. Of the six compounds, only acetylsalicylic acid with a 96

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factor of 0.8 was predicted to degrade quickly ov er the test span. Furthermore, with negative factors, acetaminophen, 17 -ethinylestradiol, and progesterone were predicted to be the least biodegradable of the six compounds. Based upon these results, the predicted amount of anaerobic degradation of the compou nds is in the order: acetylsalic ylic acid > metoprolol tartrate > ibuprofen > acetaminophen > 17 -ethinylestradiol > progesterone. This corresponds well with the experimental findings which found ace tylsalicylic acid > metoprolol tartrate > acetami nophen > ibuprofen. Due to adso rption to less than the method detection limit, it was not possible to ra nk the experimental biodegradation of 17 ethinylestradiol and progesterone. Although expe rimentally acetylsalicylic acid did not produce sufficient degradation gas to be considered biod egradable as predicted by the model, the high concentration of the test compound may have been inhibitory to the bact eria and biodegradation may occur more readily at a lower concentration. The actual biodegradability of any organic compound while dependent on the chemical structure of the molecule is also highly depe ndent on other factors. The presence of an appropriate microbial communit y, sufficient acclimation time, pH, temperature, presence of oxygen, and numerous other factors influence a compounds degradation. In assessing the biodegradation of any compound, tests of the compound under the expected environmental conditions are the most accurate means available. Acclimation of microorganisms to an orga nic carbon source plays a vital role and the biodegradation rate of many compounds increases af ter an initial lag time. The lag time required for these compounds may have been greater than the 56 days examined in this research. Additionally, if the chemical di splays ready degradation at th e high concentration of test chemical in this procedure, it is assumed that it would also do so at low concentrations. 97

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However, this is not inversely tr ue and degradation at lower concentrations, below an inhibitory level, may be possible. Pharmaceutical compounds are typically detected at concentrations of parts per billion and lower in the environment. Thus the potential inhibitory effects observed in the results may not occur at the lower environmental levels or at levels within landfill leachate as discarded pharmaceuticals become dissolved and entrained. Further resear ch of the degradation and removal of these compounds at lower concentrations is necessary. Summary The potential for the anaerobic degradation of six pharmaceutical compounds within municipal solid waste landfills was assessed using an anaerobic degradation model of the US EPA (BIOWIN), direct sample analysis, and th rough respirometric testing using a modified version of OPPTS Method 835.3400. Respirometric testing measured the total volume of the anaerobic degradation gas, methane, produced by samples containing an anaerobic sludge medium and each pharmaceutical compound at a con centration equivalent to 50 mg/L organic carbon. The samples were then directly analy zed for change in pharmaceutical concentration following 56 days of incubation at 37C using liquid chromatography tandem mass spectrometry. The degradation model predicted that of the tested compounds, acetylsalicylic acid would be readily degradable while acetami nophen, ibuprofen, metopr olol tartrate, 17 -ethinylestradiol and progesterone would not. Experimental te sting resulted in none of the six compounds producing 75% of the potential degradation gas to be classified as readily degradable per the test method. However, as predicted by the model, acet ylsalicylic acid (21%) a nd metoprolol tartrate (18%) demonstrated the greatest degree of biodegradation based on direct analytical measurement. 17 -ethinylestradiol (>94%) and progesteron e (>99%) showed si gnificant abiotic 98

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removal from solution while metoprolol tartrate (24%) and ibuprofen ( 20%) also demonstrated appreciable abiotic losses but to a much lesser extent. 99

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100 Table 5-1. Pharmaceutical compound sample mass equivalent to 50 mg/L organic carbon content in 300 ml sample solution Compound Formula Formula weight Sample mass (mg) Acetaminophen C8H9O2N1 151.2 23.62 Acetylsalicylic acid C9H9O4 180.2 25.02 Ibuprofen C13H18O2 206.3 19.84 Metoprolol tartrate C34H56O12N2 684.8 25.18 17 -ethinylestradiol C20H24O2 296.4 18.53 Progesterone C21H30O2 314.5 18.72 Sample Volume = 300 ml Table 5-2. Method detection limit for analysis of tested pharmaceutical compounds in anaerobic sludge media Compound Method detection limit (MDL) (g/L) Acetaminophen 1 Acetylsalicylic acid 5 17 -ethinylestradiol 6 Ibuprofen 10 Metoprolol 1 Progesterone 8 Table 5-3. Comparison of theoretical and experimental methane production Compound Theoretical methane yield (ml) Experimental average methane yield (ml) Methane (%) Acetaminophen 16.9 1.0 6.0 Acetylsalicylic acid 15.9 1.0 6.4 Ibuprofen 20.2 1.3 6.4 Metoprolol tartrate 18.9 0.7 3.5 17 -ethinylestradiol 19.9 0.4 2.1 Progesterone 20.8 1.8 8.5 Cellulose 15.9 12.9 80.8 Experimental values reported as average of tr iplicate samples and are corrected for average background solution methane production.

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Table 5-4. Average abiotic removal of sel ected pharmaceutical compounds in biologically inactive test samples Compound Initial concentration (mg/L) Abiotic samples (mg/L) Abiotic removal (mg/L) % Acetaminophen 78.73 79 0 Acetylsalicylic acid 83.41 88 0 17 -ethinylestradiol 61.75 4 58 94 Ibuprofen 66.12 53 13 20 Metoprolol 71.49 54 17 24 Progesterone 62.39 0.4.3 62 99 Average standard deviation of triplicate samples Table 5-5. Average biodegradation of selected pharmaceutical compounds in anaerobic sludge test samples Compound Adsorption samples (mg/L) Biodegradation samples (mg/L) Theoretical loss to biodegradation (mg/L) Acetaminophen 79 70 9 Acetylsalicylic acid 88 70 18 17 -ethinylestradiol 4 0.1 4 Ibuprofen 53 49 5 Metoprolol tartrate 54 41 13 Progesterone 0.4.3 99 Ibuprofen 66.12 49 18 27 Metoprolol tartrate 71.49 41 30 42 Progesterone 62.39 62 >99 101

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Table 5-7. Anaerobic biodegradation potential for target pharmaceutical compounds predicted by U.S. EPA BIOWIN program model. Compound Anaerobic linear biodegradation factor Predicted rapid biodegradation Acetaminophen 0.1124 No Acetylsalicylic acid 0.8122 Yes 17 -ethinylestradiol -0.8418 No Ibuprofen 0.0334 No Metoprolol 0.0709 No Progesterone 1.7307 No Biodegradation factor > 0.5 = fast biodegradation 102

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Figure 5-1. Average cumulative methane produ ction of pharmaceutical and background test samples 0 1 2 3 4 5 6 010203040506mlDays 0 Acetylsalicylic acid Acetaminophen Metoprolol Background Ethinylestradiol Ibuprofen Progesterone 103

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104 AB C D E F Figure 5-2. Chemical structure of pharmaceuticals selected for anaerobic degradation study. A) Acetaminophen B) Acetylsalicylic Acid (Asp irin) C) Ibuprofen D) Metoprolol E) 17 -Ethinylestradiol F) Progesterone

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CHAPTER 6 EFFECTS OF LANDFILL RETENTION TIME AND ORGANIC MATTER ON THE FATE OF SELECTED ACTIVE PHARMACEUTICAL INGREDIENTS The anaerobic degradation research conducte d in the previous ch apter showed that significant degradation of the six tested pharmaceutical compounds did not occur over a period of 56 days. However, the test method employed in that and other standa rd respirometric test methods examine the degradation of the organic compound at a high concentration (> 50 mg/L) when compared to the concentration typical of trace environmenta l pollutants such as pharmaceuticals and personal care products (PPCPs). These compounds are typically detected at the g/L or ng/L level (50, 100, 101) The high test concentrati ons may inhibit biological activity which would otherwise occur and thus result in inaccurate results and erroneous conclusions of nondegradability for these compounds. The potential for inhibition was demonstrated in the previous experiment by three out of the six compounds producing methane, the measured degradation product gas, in quant ities less than measured background quantities. Consideration must also be gi ven to the impact of bacteria l acclimation upon degradation. Studies of pharmaceutical degradation in wastew ater treatment plants have theorized that acclimation of the sludge to pharmaceutical co mpounds and treatment methods which promote more diverse biological popul ations result in lower effluent concentrations (11, 111) Typical anaerobic degradation test methods permit a test period of 56 days (100, 101) This period may be inadequate to permit the growth and acclimation of bacteria capable of degradation of the test compounds and may not adequately simulate envir onmental conditions. In a landfill setting with a hydraulic conductivity of 1x10-4 cm/sec (96) leachate generated from waste at a height of 30 meters would take 347 days to reach the bottom of the landfill. To address this concern, the degradation time permitted in this study was doubled from the standard methods to a period of 112 days. 105

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Finally the standard test met hods utilize the test compounds as the sole source of organic carbon within the system. As such, these methods fail to account for the potential degradation of the test compound through the process of cometabo lism. Degradation research of chlorinated organic compounds and the active pharmaceutical i ngredient diclofenac have demonstrated that bacterial subsistence on a differing substrate perm itted either sufficient time for acclimation or resulted in the incidental metabolism of the contaminant (112-115) The potential for cometabolism of pharmaceutical compounds within a municipal solid waste (MSW) landfill is greater than in many other envi ronmental compartments due to the initially large amount of degradable organic carbon and the vast variety of organic compounds present. The objective of this research was to determin e the potential for anaerobic degradation of six selected pharmaceutical compounds in the presence of a readily degradable organic substrate. To reduce the possibility of toxic/inhibitory effects, three lower concentrations than prescribed by the standard test methods were examined. Th ese concentrations permitted reliable analytical measurement while being similar to expected environmental and landf ill concentrations. Anaerobic degradation and abiotic removal mech anisms of the pharmaceutical compounds were determined using respirometric and di rect analytical test methods. Materials and Methods Selection of Target Pharmaceutical Compounds Various methods have been used to identif y pharmaceutical compounds with the potential for release to the environment and previous rese archers have commonly utilized prescription rate data (103-105) Using a similar method, six pharmaceuti cal compounds were selected for study based upon the number of prescriptions from 2002 to 2004, the defined daily dose of the medications, and over-the-counter usage and environmental detection of medications. A detailed description of the compound selection method is given in Appendix A. The six pharmaceutical 106

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compounds selected for degradation study were acetaminophen, acetylsalicylic acid (measured as salicylic acid), 17 -ethinylestradiol, ibuprofen, metoprol ol tartrate, and progesterone. Reagents and Test Compounds All the pharmaceutical standards used were of high purity grade (> 98%). 17 ehtinylestradiol, acetylsalicylic acid, ibuprofen, metoprolol ta rtrate, and progesterone were purchased form Sigma (WI, USA). Acetami nophen was obtained from Sigma-Aldrich (MO, USA). Ammonium formate and HPLC water were from Fisher (PA, USA). HPLC methanol was from Tedia (OH, USA). Sample Preparation Samples of each compound were prepared at th ree initial concentrations in 500 ml glass sample bottles (Wheaton Lab 45 Graduated Safety Coated Bottles Cat # 06-451-29): 50 g/L, 250 g/L, and 500 g/L. For each concentration, tr iplicate samples were created for each of four differing incubation times: 0 days to determin e immediate changes in concentration, 7 and 28 days to detect changes during the period expected to have the most ac tive degradation, and 112 days to permit sufficient time for biodegradation a nd allow for acclimation of the bacteria. Thus a total of 36 active degradation samples were prepared for each compound. Aqueous stock solutions of 75 mg/L were pr epared for acetaminophen, acetylsalicylic acid, and metoprolol tartrate. Due to their low aque ous solubility, 75 mg/L stock solutions of 17 ethinylestradiol and progesterone were prepared in ethanol and ibuprofen was prepared as an aqueous solution at 20 mg/L. Stock solution wa s pipetted into each empty sample bottle to achieve the desired final concentr ation and the ethanol of the 17 -ethinylestradiol and progesterone samples was allowed to evaporate to dryness to prevent any impact on available organic carbon or bacterial function. Continuous sparging of each sample bottle through a glass tube inserted into the bottle was commenced us ing oxygen-free nitrogen vice the nitrogen/carbon 107

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dioxide mixture required by the standard met hods. This permitted the measurement of carbon dioxide generated from anaerobic degradation of the samples. A sample nutrient solution was prepared per OPPTS method 835.3400 (101) The nutrient solution was heated to a boil with continuous st irring and sparging with oxygen-free nitrogen. Upon boiling, the solution was placed in an ice bath and sparging with nitrogen continued. When the solution cooled to 37C, the flask was removed from the ice bath and stirring continued. Remaining nutrients, 5.28 g of sodium bicarbonate, and 400 ml of anaerobic digester sludge were added to reach a final volume of a pproximately 4 L. The sludge was obtained from a laboratory anaerobic digester wi th a retention time of approxima tely 20 days. The digester had been maintained on a hormone and additive free dog food (Pet Promise) for three months prior to use and thus was not previously exposed to the test compounds to pr event prior acclimation. Following addition of the sludge to the solu tion, 450 milligrams of crystalline cellulose were added to provide 50mg/L of organic carbon. Each sample bottle was then filled with 300 ml of the inoculation media and a butyl r ubber two-leg lyophili zation stopper (Wheaton #224100-507) was inserted while the glass tube used to deliver the sparging gas was removed. The headspace of all samples was then flushed with nitrogen for an additional five minutes through a supply and vent needle in serted through the bottle septum. A total of three, 4L batches of inoculated media were required to create the number of samples necessary for each compound. Due to li mited anaerobic digester volume and the need to maintain the desired retention time of the dige ster only three 4L batc hes were possible every three days. Therefore, the cr eation of samples for each of th e compounds was staggered. This resulted in all metoprolol tartrate samples being created on day 1 of sample preparation, followed by all acetaminophen samples on day 4, and so on to completion of all 6 compounds. 108

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To determine the background gas production of the media, a background sample was created during sample preparation of each compound. On the last 4L batch created for each compound, 300 ml of solution were collected prior to cellulose addition. 416.5mg of cellulose were then added to the flask vice the prior 450 mg to achieve the desired 50 mg/L of organic carbon in the solution. To verify sufficient bacterial activity of the media, to provide a reference for fluctuations in bacterial activity over the 3 weeks of sample creation, and to act as quality control blank samples for later analytical meas urements, one sample containing cellulose, but without any pharmaceutical compound was prepared with each compounds samples. Thus the samples of each compound had a corresponding refe rence sample created on the same day, for a total of six reference samples. To measure the adsorption and other abiotic losses of each compound, five samples of each compound were prepared using autoclaved media fo r each of the initial test concentrations (15 total samples = 5 samples x 3 concentrations). One sample was incubated for 7 days before analysis, one sample was incubated for 28 days before analysis, and triplicate samples were incubated for 112 days before concentration analys is. The solution was prepared as previously described, however, prior to addition of cellulose to the media, the solution was autoclaved for 17 minutes at 250C, held at room temperature for 24 hours, and then autoclaved a second time for 17 minutes at 250C. Upon final removal from the autoclave, the solution was cooled in an ice bath to 37C, 450 mg of cellulose were a dded, and 300 ml of solution placed into each sample bottle. The bottles were sealed and fl ushed with nitrogen as previously described. All sample bottles were wrapped in alumin um foil to prevent po ssible photodegradation and stored in an incubator at 37C for either 7, 28, or 112 days. Three to five hours after filling, the sample bottles were ve nted by inserting a gas-tight glass syringe through the septum. 109

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This permitted the increased pressure formed due to the heating of the headspace flushing gas to equilibrate with atmospheric pressure through expansion within the syringe. Respirometric Testing Measurements of degradation gas volume and methane and carbon dioxide content were performed at 3, 7, 14, 21, 28, 42, 56, 70, 84, and 112 days following sample creation. Gas volume was measured with a gas-tight syringe (Popper Micro-mate 5, 10, and 20cc) with a 20 gage needle inserted through the septum and he ld in a horizontal posit ion, allowing the syringe plunger to move freely. Methane and carbon dioxide content was determined via analysis of a 100 uL headspace sample manually injected into th e packed inlet (temperature = 200C) of an Agilent 6980 Gas Chromatograph with a 9m Porapack N packed column and a 9m Molesieve packed column with a constant flow (19.3 ml/min of argon carrier gas) and a thermal conductivity detector (t emperature = 250C). Analytical Method Standards Preparation Stock solutions were prepared containing each of the pharmaceuticals of interest at a concentration of 1000 mg/L in HPLC grade meth anol using Class A volumetric glassware. These stock solutions were stored at -20 C. Ca libration standards were prepared from the stock solutions by a series of 10 and 100-fold diluti ons with the sample matrix. These working standards were stored at 4 C. Sample Preparation Triplicate samples were collected at initial sample preparation and after 7 days, 28 days, and 112 days of incubation. Following obtaining a headspace gas sample, samples were vacuum filtered through a 1.2 m glass filte r paper (Fisher Scientific, Fisherbrand G4 Glass Fiber Filter Circles). An aliquot of 30 to 50 ml of filtrate wa s placed in a silanized borosilicate glass vial 110

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with a teflon coated cap. The pH of each sample was adjusted to 5 using concentrated hydrochloric acid and the samples were stored at 4C until analysis. Immediately prior to analysis, a 2ml aliquot of each sample was placed into a 2ml sampling vial via filtration through a 0.2um syringe filter (Supelco Is o-Disc Filters PTFE-25-2). Chromatography and Mass Spectrometry LC analysis was performed with an Agilent 1200 Series rapid re solution LC system coupled to an Agilent 6410 Triple Quad MS /MS (Palo Alto CA, USA) equipped with an orthogonal ESI interface. For analys is of samples containing ibuprofen and acetylsalicylic acid, compounds were separated, at 40 C, by means of a Zorbax extended C18, RRHT column (2.1 mm 100 mm, 1.8 m) from Agilent. The injec tion volume was 1 L with a flow rate maintained at 0.4 ml/min. The mobile phase was comprised of a mixture of (A) 5mM ammonium formate in water and (B) 5mM ammonium format e in methanol. The mobile phase solvent gradient started with 5% of solvent B and was increased to 90% solvent B evenly over 12.0 minutes. The solvent mixture was returned to th e initial proportions following completion of sample analysis at 12.1 minutes and a post-run tim e of 2 minutes was requ ired to re-equilibrate the column. The analysis of samples c ontaining acetaminophen, metoprolol tartrate, 17 ethinylestradiol, and progesterone was complete d without chromatography. A volume of 1L of each sample was directly injected (bypassing the LC column) with a flow rate of 0.4 ml/min in a mobile phase solvent mixture comprised of 50% of solvent B and 50% of solvent A. Calibration was monitored through the use of calibration verification samples every six samples required to be within % of the initial sample. Instrument blanks to monitor potential carryover between injections were analyzed pr ior to each calibration check. For samples in which carryover was indicated, one to three system washes were placed between analytical 111

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samples and the samples reanalyzed. Acquisition peak identification a nd integration, and final quantification were performed with Agilent MassHunter Workstation Software B.01.00 9(B48). Data Analysis For respirometric testing, the average total gas production and total methane production for each compounds triplicate samples were comput ed. In each case, the amount of gas was corrected for background production by the solution by subtracti ng the gas production of the corresponding gas blank sample. This blank samp le was created on the same day as the given compounds samples and therefore was representative of the background or ganic content and the bacterial activity of those samples. The total gas production quantity was computed as the sum of the total production of me thane and carbon dioxide. Statistical ttest analysis ( =0.05, P < .05) was performed to determine if the difference in gas production of each compound in comparison to background levels was significant. The aver age concentration and st andard deviation was calculated for the triplicate samples of each pharmaceutical compound at the four differing sampling time periods. Statistical analysis using ttest( =0.05, P < .05) comparison of compound concentrations between time periods was performe d to determine the statistical significance of concentration changes over time. Results and Discussion Respirometric Testing Anaerobic degradation within th e test samples relied upon cellulose as the primary source of organic carbon with the pharmaceutical comp ound contributing less than one percent of the total organic carbon in any sample. Therefore, degradation gas producti on was not a direct indicator of pharmaceutical biod egradation. However, degradation gas production may be a reliable indicator of either the inhibition or enhancement of cellulose degradation by the 112

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pharmaceutical compound. Furthermore, headspace gas monitoring ensured oxygen levels remained low and anaerobic conditions were maintained. The average total gas production, corrected for background, of each compound after 112 days is given in Table 6-1. Total gas quantities varied from as little as 13 ml for 500 g/L ibuprofen samples to a maximum of 26 ml for 50 g/L progesterone samples. However, gas quantities were directly affected by the bacterial activity of the inoculating solution at the time of sample creation. Thus, direct comparison of ga s production between compounds is not possible. To correct this, the table also shows an aver age percentage yield of gas for each set of samples. This percentage was computed by dividing the average gas production of each compound by the gas production of the corresponding cellulose-only control sample created with that compound. Thus, the control sample had equiva lent bacterial activity and this percentage permits detection of impacts of the pharmaceutic al on cellulose degradation. The percentage also normalizes the individual compound result s and permits comparison of the compounds despite potentially differing ini tial bacterial activities. Overall gas production percenta ges varied including amongst the individual compounds. As an example, the lowest relative total gas yi eld of 74% was produced by the 250 g/L progesterone samples while the greatest relative yield of 141% also came from progesterone samples at 50 g/L. However, no pharmaceutical compound resulted in gas production below 50% of its corresponding control sample. Therefore it is concluded that none of the pharmaceuticals resulted in a detrimental effect on biological degradation activity. However, it should be noted that for ibuprofen, ttest analysis of the total gas production showed the decrease in total gas production at 500 g/L to be statistica lly significant when compared to 50 g/L and 113

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250 g/L. Furthermore, although not st atistically significant, acetaminophen, 17 ethinylestradiol, and ac etylsalicylic acid displayed similar trends. Table 6-2 shows the percentage of methan e produced for each compound and for their corresponding cellulose-only blanks. Carbon dioxide, not show n in this table, comprised the remainder of the degradation gas. The methane concentration achieved by the pharmaceutical samples ranged from 54.3% to 64.3% while the control sample concen trations ranged from 55.6% to 64.8%. The greatest difference occurre d with a difference of 6.0% between the 250 g/L progesterone sample and its matching contro l sample. However, this difference was less than two standard deviations of the 250 g/L progesterone samples. Therefore, the difference in methane concentrations was negligible and supports the conclusion that the pharmaceutical compounds did not affect anaerobic bi odegradation of the cellulose. Pharmaceutical Compound Degradation Analytical Measurement Table 6-3 presents the final concentra tions of each pharmaceutical compound in the biologically active and the abiotic samples following 112 days of incubation. Compounds which were not detected in any of the final samples are indicated by the abbr eviation ND, while those samples which were detected below the method de tection limit of quantif ication are indicated by the abbreviation
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samples showed corresponding decreases in con centration. The decrease in concentration continued and at 28 days decreased to below detection limits for both active and abiotic samples with the exception of the abiotic 500g/L samp les which had an average concentration of 13 g/L. All samples then showed an increase in concentration at 112 days of incubation. The active samples obtained concentratio ns even higher than at time zero, with concentrations of 20 g/L, 106 g/L, and 254 g/L for the 50 g/L, 250 g/L and 500 g/L samples respectively. The 250 g/L and 500 g/L abiotic samples also show ed smaller increases in concentration. Due to the decrease in concentration for bot h the abiotic and active biological samples, adsorption to the cellulose a nd other sample components and other abiotic mechanisms are theorized to be the predominant removal mechanisms for 17 -ethinylestradiol. Adsorption continued until the concentrati ons reached undetectable levels at 28 days. However, as biological activity consumed the cellulose, deso rption of the compound occurred, resulting in an increase in concentrations to the levels seen at 112 days. The small increase in the concentrations of the abiotic samples is believed to be due to abiotic mechanisms resulting in desorption of 17 -ethinylestradiol from the cellulose, such as limited hydrolysis of the cellulose. Anaerobic biodegradation was not indicated for 17 -ethinylestradiol. Acetaminophen Evaluation of the concentrations for acetam inophen following initial sample preparation and at 7 days indicates that the target concen tration of the samples was exceeded. This is theorized to be due to a high stock solution concen tration in preparing the samples as each of the sample concentrations are proporti onately greater than their inte nded concentration. However, it was not possible to confirm this suspicion due to degradation of the stoc k solution upon exposure to oxygen and light after sample preparation resulting in the st ock solution turning an opaque 115

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brown color. Despite the higher concentra tions, the examination of the behavior of acetaminophen was still possible. Inspection of the abiotic samples s hows a gradual decrease in acetaminophen concentration over time with each of the three test concentrations. The low concentration samples decreased from 62 g/L at 7 days to 47 g /L at 28 days then con tinued to decrease to 34 g/L at 112 days. The mid concentration sample s decreased from 308 g/L to 189 g/L and the high concentration samples decreased from 621 g/L to 363 g/L over the test period. This loss resulted in a total average decrease of approxi mately 45%, 39%, and 41% at 112 days for the 50 g/L, 250 g/L, and 500 g/L samples respectivel y. The lack of biological activity for these samples was confirmed by zero methane and carb on dioxide production over the test period and zero total gas generated. Based on the abiotic samples and corresponding decreases in th e biologically active samples, the loss of acetaminophen is believed to be due to adsorption and other abiotic mechanisms. Furthermore, examination of the concentration results of the biologically active samples provides evidence that as with 17 -ethinylestradiol, the primary adsorption losses observed in the samples were due to adsorption to the organic matter/cellul ose of the samples. Initial adsorption of the acetaminophen to the organic substrate resulted in an initial decrease in concentration during the first 1 to 20 days of incubation. Howe ver, as biological activity degraded the organic substrate, the acetaminophen returned to solution and concentrations increased to approximately their initial solution concentrations. As an example, the 250 g/L samples decreased from 594 g/L following initial sample preparation to 517 g/L after 28 days of incubation. However, the concentration then rose to 644 g/L by 112 days of incubation. It should be noted that the large e rror shown for the biologically active 50 g/L samples is due to 116

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one of the triplicate samples diffe ring significantly from the others It is believed this is an analytical outlier but was included due to the small number of samples in the study. Acetylsalicylic Acid. As shown in Figure 6-3, the abiotic samples of acetylsalicylic acid s howed no statistically significant change throughout the experiment. Following 112 days of incubation, abiotic samples averaged 472 g/L, 210 g/L, and 50 g/L for the initial concentrations of 500 g/L, 250 g/L, and 50 g/L respectively. The biol ogically samples, however, showed a rapid decrease in concentration, with the average sa mple concentrations be ing 10 g/L, 10 g/L, and less than the method detection limit at 28 days for the 50 g/L, 250 g/L, and 500 g/L samples respectively. All samples were below th e method detection limit by 112 days. Due to the constant concentrations observed within the abiotic samples, unlike the prior pharmaceutical compounds, adsorption and other abio tic losses were not indicated as significant removal mechanisms. This observation, coupled w ith the rapid decrease in acetylsalicylic acid concentration of the biologically active samples indicates significant degradation of the compound. At 28 days, acetylsalicylic acid concen trations were reduced by greater than 99%, 95%, and 90% for the 500 g/L, 250 g/L, and 50 g/L samples respectiv ely. This is in agreement with biodegradability mode l prediction of ready degradation (107) Ibuprofen Table 6-1 shows that following 112 days of incubation, ibuprofen concentrations of both the abiotic and biologically active samples showed minor decreases. The biologically active 50 g/L samples decreased to an average concen tration of 44 g/L the 250 g/L samples decreased to 194 g/L while the 500 g/L samples decreased to an average of 411 g/L Statistical comparison of abiotic and biologically active samples conc entration at 112 days showed no statistical difference. The parallel between the abiotic and biologically active 117

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samples is also supported by review of Figure 64 which shows near equal values for the two sample types at 7 and 28 days as well. The bi ologically active samples taken following sample preparation appear lower than thos e taken at 7 days. However, th e difference is not statistically significant and may be due to variations in analytical recovery or instrument response during analysis, or undetermined mechanisms. Adsorption of ibuprofen to the organic substr ate appears to be minor throughout the course of the experiment, with some minor adsorption possible initially in both the abiotic and active samples. However, unlike acetaminophen which showed continual adsorption in the abiotic samples and a return to solution over time in th e biologically active samples, the minor loss of ibuprofen appears to remain constant. Theref ore, any loss to adsorption may not be due to adsorption to the cellulose, but may be due to ad sorption to other sample surfaces, such as the sample bottle surface. The final concentrations show that anaerobic degradation of ibuprofen is not expected at low concentrations, nor is adsorption to the orga nic substrate. While, minor decreases in gas production with increasing concen tration were noted, inhibition is not apparent at the low concentrations of this experiment and the lack of degradation app ears to be due to resistance to degradation of ibuprofen. Metoprolol Tartrate In prior research at high compound concentratio ns, metoprolol tartra te demonstrated the largest potential for loss due to adsorption of the six compounds tested with a reported 24% removed from solution by adsorption. These tests also demonstrated a potential for minor biodegradation as an average biodegradation of 17.9% of the compound occurred. Review of Figure 6-5 shows metoprol ol tartrate concentrations decrea sed rapidly to below the limit of detection within the biologically active samples for each of the th ree initial concentrations. 118

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The rapid decrease in con centration was unexpected based upon the prior studys indication of its lack of adsorption or degradation. It was theorized that the inclusion of cellulose in the samples resulted in significant adsorptive re moval of the metoprolol from solution. To test this theory, a further experiment was conducted by creating triplicate anaerobic aqueous samples of metoprolol at 50, 250, and 500 g/L with cellulose (50mg/L organic carbon) using only ultrapure water. These samples were incubated fo r 7 days at 37C and analyzed. The results are shown in Table 6-4. The water-cellulose samples did exhibit decreases in metoprolol concentration with the 50 g/L samples decr easing to an average of 38 g/L, the 250 g/L samples decreasing to 185 g/L, and the 500 g/L samples decreasing to 390 g/L. However, these values differ greatly from the reduction to less than the detection limit experienced in the sludge matrix samples. Furthermore, the large difference between the abiotic samples and the pure water samples indicates that adsorption lo sses may be affected by other parameters introduced by the sludge media. These factors may include adsorption to the sludge bacteria, adsorption to other background organic matter, ch emical reactions betwee n the sludge media and metoprolol, or the pH of the test solution ( 7.0-7.2 for sludge solution, 5.8 for ultrapure water). Returning to Figure 6-5, the differences be tween the abiotic and biologically active samples may have several potentia l explanations. The first possi ble explanation is the rapid biodegradation of metoprolol by the active bacteria resulting in a decrease in concentration greater than by adsorption alone (abiotic samples) The second potential e xplanation is alteration of adsorption properties of the medium between the abiotic and biologically active samples. Autoclaving the sludge solution may have resulted in changes in the ability of the metoprolol to adsorb to the bacteria or other organic matter within the samples. 119

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Finally, the most surprising observation of Figure 6-5 is the increase in metoprolol concentration over time. In acetaminophen, theo rized adsorption of the acetaminophen to the cellulose resulted in decreasing concentrations ov er time for abiotic samples while biologically active samples saw an increase over time due to desorption from the biologically degraded cellulose. However, Figure 6-5 shows increasing values for not only biologically active samples over time, but abiotic as well. Therefore, desorpti on of metoprolol is theo rized not to be due to biological degradation, but a physic al or chemical mechanism. Performance of a literature search did not reveal potential mechanisms and fu rther research is necessary to isolate this phenomenon. Progesterone Similar to 17 -ethynylestradiol, the hormone progesterone showed rapid removal from solution to undetectable levels. Plot of the change in concen tration was not possible as all samples (abiotic and active) were below the method detection limit or not detected at all. It was not possible to determine if any anaerobic biodegr adation of the compound occurred or if the removal was simply due to adsorption and ot her abiotic mechanisms, however as with 17 ethynylestradiol, adsorption and abiotic mechanisms are theorized to be the predominant removal mechanisms for progesterone. Correlation of Results with Prior Anaerobic Degradation Test The anaerobic degradation research conducte d in the previous ch apter examined the pharmaceutical compounds at a high concentration that may have inhibited biological activity. Therefore, in the current study, the pharmaceutic al test compound concentrations were reduced to less than one percent of the value used in the prior tests. Additionally, the experiments of this chapter included an organic substrate, cellulo se, to support bacterial metabolism and permit potential cometabolism of the pharmaceutical com pounds. To permit a greater time for bacterial 120

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acclimation to the test compounds, the 56 day test period of the prior studies were extended to 112 days in the current study. The primary process for removal from solution for progesterone and 17 -ethinylestradiol was adsorption. In each experiment, progesteron e concentrations rapidly decreased to and remained at less than the method detecti on limit. In the second experiment, 17 -ethinylestradiol demonstrated the potential for de sorption and return to solution as the organic substrate to which it adsorbed is depleted by biological activit y. Unlike the high concentration tests which displayed no adsorption of acetaminophen, the in clusion of the organic substrate permitted limited adsorption. However, as with 17 -ethinylestradiol, this adsorption was reversed as the organic substrate was consumed by biological activity and only minor degradation of acetaminophen was possible. Under the high test concentrations of the pr ior experiments acetylsalicylic acid was not readily degradable. With the lower test conc entrations employed in the current experiment, acetylsalicylic acid did demonstrate rapid degrad ation to below the method detection limit at each concentration. Thus inhibition of bacteria l activity by the high test concentration of the previous experiment was confirmed. In both expe riments, adsorption of acetylsalicylic acid was not detected. In the initial experiments, metoprolol tartra te demonstrated a mixture of adsorptive and degradation losses resulting in the greatest re duction in concentration of the nonhormone compounds. This trend continued in the current experiment with significant adsorption to the organic substrate indicated with some return to solution following substrate consumption. Unlike prior experiments, ibuprofen demonstr ated only minor decreases in concentration 121

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attributable to abiotic mechanisms and biodegrad ation was not indicated. This may be due to bacterial preference for the more readily degradable cellulose in solution. Summary The experiments of this chapter examined the effects of landfills extended hydraulic retention periods and the presence of other organic matter on the fate of 17 -ethynylestradiol, acetaminophen, acetylsalicylic acid, ibuprofen, met oprolol tartrate, and progesterone. The anaerobic degradation of each compound was meas ured over 112 days at three differing initial concentrations: 500 g/L, 250 g/L, and 50 g/L. Each sample contained an anaerobic sludge medium and cellulose at a concentration equi valent to 50 mg/L organic carbon to support biological activity. The samples were then dire ctly analyzed for change in pharmaceutical concentration following 7 days, 28 days, and 112 days of incubation at 37C using liquid chromatography tandem mass spectrometry. The results of the experiments show that the potential for an aerobic degradation and other removal processes vary widely between pha rmaceutical compounds. Of the six compounds tested at low concentrations, acetylsalicylic ac id was the only compound to display a reduction in concentration which may be linked to signi ficant degradation or cometabolism of the pharmaceutical. Metoprolol tartrate, 17 -ethinylestradiol, and proge sterone displayed significant removal from solution from due to abiotic mechanisms including adsorption to the cellulose within solution. For acetaminophen, the degradation of the solutio ns organic matter (cellulose) over time resulted in desorption of the compound a nd return to solution with time. Additionally, through mechanisms which could not be determined by the test methods, desorption of metoprolol also occurred following its initial ad sorption. The results for metoprolol tartrate, however, are sufficient to indicate that in a landfill environment, metoprolol at low 122

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concentrations will be removed from solution, but return to the leachate may occur as the chemical and physical properties of the waste ch ange and/or the waste is degraded. Finally, ibuprofen demonstrated neither appreciable adsorption nor anaer obic biodegradation, remaining near initial concentr ations throughout the experimental period. 123

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Table 6-1. Anaerobic degradation gas producti on of pharmaceutical compound samples at concentrations of 50, 250, and 500 g/L with 50 mg/L organic carbon from crystalline cellulose. Compound Total Gas (ml) Percentage of Total Gas Compared to Positive Control Sample 50 g/L 250 g/L 500 g/L 50 g/L 250 g/L 500 g/L 17 -Ethinylestradiol 21 19 18 124 110 104 Acetaminophen 17 16 16 86 80 79 Acetylsalicylic acid 19 15 16 130 99 106 Ibuprofen 19 15 13 127 103 91 Metoprolol tartrate 14 14 2 16 94 96 109 Progesterone 26.4 14 19 141 74 103 Average standard deviation of triplicate samples Table 6-2. Percentage of methane within anaerobic degradation gas of six selected pharmaceutical compounds at 50, 250, and 500 g/L and a corresponding blank sample with 50 mg/L organic ca rbon from crystalline cellulose. Compound 50 g/L (%) 250 g/L (%) 500 g/L (%) Corresponding cellulose blank (%) 17 -Ethynylestradiol 62.0.9 62.6.1 58.3.2 58.1 Acetaminophen 63.0.6 61.7.6 62.9.0 64.8 Acetylsalicylic acid 61.7.9 61.7.2 61.8.3 58.8 Ibuprofen 64.3.7 62.9.9 63.4.4 63.4 Metoprolol tartrate 55.0.0 54.3.2 54.5.4 55.6 Progesterone 60.9.8 57.6.7 60.9.4 63.6 Average standard deviation of triplicate samples 124

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Table 6-3. Concentration of pharmaceutical compounds in biologically active and abiotic samples following 112 days of incubation Biologically active degradation Abiotic biologically inactivated Initial Concentration 50 g/L 250 g/L 500 g/L 50 g/L 250 g/L 500 g/L 17 -Ethynylestradiol 20 106 254 < MDL 28 82 Acetaminophen 87 302 644 34 189 363 Acetylsalicylic acid
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0 10 20 30 40 50 0728112g/LDays 50 g/L 0 50 100 150 200 250 300 0728112g/LDays 250 g/L Active Abiotic 0 100 200 300 400 500 0728112g/LDays 500 g/LFigure 6-1. Change in concentration of 17 -ethinylestradiol from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples 126

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0 20 40 60 80 100 120 140 0728112g/LDays 50 g/L 0 50 100 150 200 250 300 350 0728112g/LDays 250 g/L Active Abiotic 0 100 200 300 400 500 600 700 0728112g/LDays 500 g/LFigure 6-2. Change in concentration of acet aminophen from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples 127

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0 20 40 60 80 100 120 140 0728112g/LDays 50 g/L 0 50 100 150 200 250 300 0728112g/LDays 250 g/L Active Abiotic 0 100 200 300 400 500 600 0728112g/LDays 500 g/LFigure 6-3. Change in concentration of acetylsa licylic acid from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples 128

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0 10 20 30 40 50 60 0728112g/LDays 50 g/L 0 50 100 150 200 250 300 0728112g/LDays 250 g/L Active Abiotic 0 100 200 300 400 500 600 0728112g/LDays 500 g/LFigure 6-4. Change in concentration of ibuprofen from three initial c oncentrations following 0, 7, 28, and 112 days of incubation with crys talline cellulose in active anaerobic degradation and abiotic samples 129

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0 10 20 30 40 50 0728112g/LDays 50 g/L 0 50 100 150 200 250 300 0728112g/LDays 250 g/L Active Abiotic 0 100 200 300 400 500 600 0728112g/LDays 500 g/LFigure 6-5. Change in concentration of metoprol ol tartrate from three initial concentrations following 0, 7, 28, and 112 days of incubation with crystalline cel lulose in active anaerobic degradation and abiotic samples 130

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131 0 10 20 30 40 50 0728112g/LDays 50 g/L 0 50 100 150 200 250 300 0728112g/LDays250 g/L Active Abiotic 0 100 200 300 400 500 600 0728112g/LDays500 g/LFigure 6-6. Change in concentration of progester one from three initial concentrations following 0, 7, 28, and 112 days of incubation with cr ystalline cellulose in active anaerobic degradation and abiotic samples

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CHAPTER 7 FATE OF SELECTED ACTIVE PHARMACE UTICAL INGREDIENTS PRESENT IN LEACHATE UNDER SIMULATED ANAEROBI C MUNICIPAL SOLI D WASTE LANDFILL ENVIRONMENTS Introduction In the preceding experiments, the anaerobic de gradation and adsorption of the selected pharmaceutical compounds was examined. Initial experiments utilized the pharmaceutical compounds as the sole source of organic carbon. Latter expe riments expanded the potential removal mechanisms to include cometabolism and adsorption to an organic substrate through the introduction of a common landfill organic substance, cellulose. However, the experiments were limited in their ability to characterize the fate of the pharmaceuticals in a landfill environment. The organic carbon content of landfills is comp rised of a diverse group of compounds including lignin, cellulose, and plastic polymers. The com ponents of municipal solid waste (MSW) affect the anaerobic fate of pharmaceuticals within la ndfills by altering the potential for cometabolism or providing differing sites for adsorption. Furt hermore, the diverse bacterial population of a landfill may also affect the fate of the pharmaceu tical compounds. Prior studies have examined the fate of pharmaceuticals in sewage treatment pl ants, but not studies concerning their fate in the varied environment of MSW landfills are known (116-119) The objective of this research was to measure the change in landfill leachate pharmaceutical concentration over time during th e degradation of municipal solid waste materials. Samples of landfill leachate were spiked with six pharmaceutical compounds. The landfill leachate was then placed in sample bottle s with solid waste material in two differing stages of degradation to simulate landfills of differing ages and samples were analyzed immediately following sample preparation, followi ng 28 days of degradation, and finally at 84 days of degradation. The results provide info rmation which permits general predictions on the 132

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fate of the selected compounds upon disposal in landfills and the poten tial emission of the compounds through landfill leachate. Materials and Methods Solid Waste Fabrication The MSW composition of the United Stat es in 2005 is given in Figure 7-1 (73) From this data, a fabricated municipal solid waste was created. Among various MSW components, several non-biodegradable materials listed as misce llaneous inorganics were excluded from the fabricated waste stream. The mass of these materials was compensated for by an equivalent increase in the mass of glass. The mass corres ponding to yard waste was replaced by a matching increase in paper and paperboard mass. To de termine the type and quantities of paper products to include, EPA data concerning paper compos ition was referenced for nondurable papers, containers, and packaging. Nondurable papers of less than 1% in overall content were excluded in the fabricated waste and the percentage of office paper was increased to compensate (73) Detailed waste components and their sources for th e fabricated waste are pr esented in Table 7-1. Two sets of waste were created with a total weight of 3 kg each. One set was chosen to simulate newly landfilled waste and was not altere d prior to sample creation. The second set was aerobically composted to simulate older, landfilled waste. The se t to be degraded was placed in a closed, plastic container out doors. 2.0 L of ultrapure wate r were added. The waste was aerobically composted in the outdoor container for a period of 90 days with temperatures ranging from 25C to 40C. The waste was manually mixe d twice a week and an additional 500 ml of ultrapure water were added with each mixing. At the end of 90 days, approximately 600 ml of free standing water was drained fr om the container and the waste was transferred to a laboratory tray and allowed to dry in a fume hood at room temperature for 90 days. The final weight of the dried waste was 1959.2g, resulting in a tota l mass loss of 1040.8g, or 34.7%. This is 133

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approximately equivalent to the weight of the paper and paperboard products (33.8%) which were no longer visibly recognizable in the composted mixture. Volatile solids analysis per method 2540E was performed on the composted and new waste streams for comparison of initial organic content (120) Sample Preparation Landfill leachate was obtained from New River Regional Landfill in Raiford, Florida. In a 4L Erlenmeyer flask, 3.6 L of leachate were heated to 37C with continuous stirring and sparging with oxygen-free nitrogen. Upon reachi ng the desired temperature, 1.8 mg of the desired pharmaceutical compound was added to the flask to obtain a minimum initial concentration of 500 g/L. The flask was th en stirred for an additional 30 minutes. For each of the six pharmaceutical compounds, triplicate samples of each waste type (composted and new) were prepar ed by weighing 15g of either wa ste into 500ml glass sample bottles (Wheaton Lab 45 Graduated Safety Coated Bottles Cat # 06-451-29). After waste addition and beginning five minutes prior to the addition of the spiked leachate, sparging with oxygen-free nitrogen was commenced for each sa mple bottle through a glass tube inserted through the bottle mouth. Each sample bottle was th en filled with 300 ml of the spiked leachate and a butyl rubber two-leg lyophilization stopper (Wheaton #224100-507) was inserted while the glass tube used to deliver the sparging gas was removed. The sample headspace was flushed with nitrogen for an additional five minutes th rough a supply and vent needle inserted through the bottle septum. To detect potential inhibitory or toxic effects of the pharm aceutical compounds and verify bacterial activity of th e leachate, duplicate samples were pr epared for each of the waste types containing solely the waste and l eachate. These samples were also used as quality assurance blanks for later analytical analysis. 134

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To measure the adsorption of the compound to the sample bottles, the fabricated waste, and other solution constituents, triplicate sample s of each compound were also prepared for each waste type using autoclaved leachate. For these samples, leachate was autoclaved for 20 minutes at 250C, held at room temperature for 24 hours, and then autoclaved for a second time for 20 minutes at 250C. Upon final removal from th e autoclave, the solution was allowed to cool to room temperature and the samples prep ared as previously described. All sample bottles were wrapped in alumin um foil to prevent po ssible photodegradation and stored in an incubator at 37C for the re mainder of the experiment. Three to five hours after filling, the sample bottles were vented by inserting a gas-tight gl ass syringe through the septum. This permitted the increased pressure formed due to the heating of the flushing gas to equilibrate with atmospheric pressure through expansion within the syringe. Respirometric Testing For the new waste, measurements of gas volume produced and methane and carbon dioxide content were performed at 3 and 7 da ys following sample creation and every 7 days thereafter up to 84 days. Due to the redu ced gas production of the composted waste, gas measurements were suspended at 56 days and th en measured again at 84 days. Gas volume was measured with one of two gas-tight syringes (Popper Micro-mate 20cc or Hamilton 500cc) with a 20 gage needle inserted thr ough the septum and held in a ho rizontal position, allowing the syringe plunger to move freely. Methane a nd carbon dioxide content was determined via analysis of a 100 uL headspace sample manually in jected into the packed inlet (temperature = 200C) of an Agilent 6890 Gas Chromatograph with a 9m Porapack N packed column and a 9m Molesieve packed column with a constant flow (19.3 ml/min of argon carri er gas) and a thermal conductivity detector (t emperature = 250C). 135

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Analytical Method Standards Preparation Stock solutions were prepared containing each of the six pharmaceuticals of interest at a concentration of 1000 mg/L in HPLC methanol using Class A volumetric glassware. These stock solutions were stored at -20 C. Calib ration standards were prepared from the stock solutions by a series of 10 and 100-fold dilutions with the sample matrix of interest. These working standards were stored at 4 C. Sample Preparation Following 84 days of sample incubation and final headspace gas analysis, samples were vacuum filtered through a 1.2 um glass filter paper (Fisher Scie ntific, Fisherbrand G4 Glass Fiber Filter Circles). An aliquot of 30 to 50 ml of filtrate was placed into a silanized borosilicate glass vial with a teflon coated cap. The pH of e ach aliquot was adjusted to 5 using concentrated hydrochloric acid and the samples were stored at 4C until analysis. Immediately prior to analysis, a 2ml aliquot of each sample was placed into a 2ml sampling vial with filtration through a 0.2um syringe filter (Supelc o Iso-Disc Filters PTFE-25-2). Solid Sample Extraction Due to failure of the previously discussed autoclaved samples to cease biological activity, a solid/liquid extraction was performed on the biol ogically active samples as a measurement of compound adsorption. Following initial sample filtra tion of the biologically active samples, the filter paper and filtered solids were returned to the sample bottle. To each bottle, 200 ml of a methanol and acetone mixture (1:1) were added a nd the bottles sealed with the original stopper and cap. The samples were then maintained at 37C and shaken at 100 rpm for 36 hours. Following the extraction period, each extraction sa mple was vacuum filtered and processed in the same manner as the previous leachate samples. 136

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Chromatography and Mass Spectrometry LC analysis was performed with an Agilent 1200 Series rapid re solution LC System coupled to an Agilent 6410 Triple Quad LC/M S/MS (Palo Alto CA, USA) equipped with an orthogonal ESI interface. All compounds were separated at 40 C by means of a Zorbax Eclipse XDB-C18 column (2.1 mm 50 mm, 3.5 m) from Agilent. The injection volume was 1 L with a flow rate maintained at 0.4 ml/min. The mobile phase was comprised of a mixture of (A) 5mM ammonium formate in water and (B) 5mM a mmonium formate in methanol. The mobile phase solvent gradient started with 5% of solv ent B and was increased to 90% solvent B evenly over 12.0 minutes. The solvent mixture was retu rned to the initial proportions following completion of sample analysis at 12.1 minutes and a post-run time of 2 minutes was required in to re-equilibrate the column. Calibration was monitored through the use of calibration verification samples every six samples. The calibration check sample was required to be within % of the initial sample. Instrument blanks to monitor po tential carryover between injections were analyzed prior to each calibration check. For samples in which carryove r was indicated, one to three blank system washes were placed between analytical samples and the samples reanalyzed. Acquisition, peak identification and integration, and final quantiz ation were performed with Agilent MassHunter Workstation Software B.01.00 9 (B48). The method detection limit (MDL) was determined according to US Environmental Protection Agency guidelines (106) The MDL is defined as the minimum concentration of a substance that can be measured and reporte d with a 99% confidence that the compound concentration is greater than zer o, and is determined from at l east seven replicate analyses of samples containing the compounds of interest. Seven samples of each pharmaceutical compound at 10 or 50 g/L were analyzed. The MDL for each compound was determined from the 137

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standard deviation of the concentration for the replicate measurements, which is multiplied by the Students t-value for (n 1) degrees of freedom. The resulting MDL for each of the six tested compounds is listed in Table 7-2. Data Analysis For respirometric testing, the average tota l gas production, total methane production, and total carbon dioxide production for each compounds triplicate samples were computed. The total gas production quantity was computed as the sum of the total production of methane and carbon dioxide. To determine any inhibition of biological activity by the pharmaceutical compound, statistical ttest analysis ( =0.05, P < .05) was performed to determine if the difference in average gas production of each co mpound in comparison to positive controls was statistically significant. The average concentra tion and standard deviation was calculated for the triplicate measurements of each pharmaceutical compound at each of the three sampling periods. Statistical analysis using ttest( =0.05, P < .05) comparison of compound concentrations between the time periods was performed to determine st atistical significance of concentration changes over time. Results and Discussion Respirometric Testing Respirometric testing was conducted as a means to detect the potential inhibition of waste degradation by the selected pharmaceutical co mpounds. Table 7-3 lists the average gas production and average final methane and carbon dioxide composition of each pharmaceutical compound sample for both the new and composted waste bottles. The total gas produced ranged from 3275 ml to 4663 ml for new waste samples and from 51 to 132 ml for composted waste samples. Average carbon dioxide concentra tions ranged from 39% to 45% for new waste 138

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samples and from 9% to 18% for composted waste while average methane concentrations ranged from 55% to 60% and 6% to 17% respectively. The lower content of degradable material within the composted waste samples was demonstrated by their reduced total gas producti on and degradation gas concentrations. The composted waste samples produced gas in quant ities of only 1.4% to 5.5% of the corresponding new waste samples. Additionally, methane and ca rbon dioxide concentrations of the composted waste averaged less than one third of the levels obtained in the new waste samples. Statistical analysis (ttest, =0.05) of the average total gas pr oduction shows that for the new waste samples the only compound to produce st atistically less gas than the control samples was acetylsalicylic acid, while for the compos ted waste samples acetaminophen, acetylsalicylic acid, ibuprofen, and progesterone were statistically less than the controls. However, these results are believed to be due to the hete rogeneity of the waste mixture vice any inhibitory effect. This is supported by the large standard deviation of the sample averages. Furthermore, while each bottle received 15g of waste, it wa s not possible to ensure each bot tle received equal quantities of biodegradable materials. For example, it was observed during sample preparation that some samples received greater amounts of dog food due to being samples being randomly drawn from the entire simulated waste mixt ure versus individually created. Pharmaceutical Compound Degradation Analytical Measurement Table 7-4 presents the final concentrati ons of each pharmaceutical compound in the new and composted municipal solid waste samples following 84 days of incubation. Compounds which were below the method detection limit ar e indicated by the abbreviation
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solids fraction remaining at the completion of the te st period. Table 7-5 shows the quantity of each pharmaceutical recovered in the extract solution. In each case, a total of 150 g of the test compound were placed in the bottle. The quantity recovered in the extract varied from less than the method detection limit for progesterone samples to 115 g for 17 -ethinylestradiol samples. The specific results of each compound are discusse d in the following sections. The final pH of the composted waste samples ranged from 7.44 to 7.99. The final pH of the new waste samples ranged from 7.67 to 8.26 with the exception of a si ngle metoprolol tartrate sample which had a final pH of 5.14. 17 -Ethinylestradiol Figure 7-2 shows the initial, 28 day incubati on, and 84 day incubation concentrations of 17 -ethinylestradiol for the new and composted wa ste samples. An appreciable decrease from the initial concentration of 500 g/L occurred immediately as seen by the low concentration of 20 g/L measured following initial sample preparation for the new waste samples and 26 g/L measured in the composted waste samples. These values showed little difference at 28 days and demonstrated a slight increase at 84 da ys with an average new waste concentration of 67 g/L and composted waste average concentration of 29 g/L. The rapid decrease in concentration was the result of adsorption of 17 -ethinylestradiol to the solid fraction of the waste. This is substantiated by the large quantity recovered in the extract of the solid materials. As shown in Table 7-5, an average of 99 g out of an original 150 g placed in the new waste samples was recovered in the methanol/acetone extract. This is an average of 66% of the initial compound and result ed in an average of 75% of the original compound remaining in the samples at the end of the experiment. An even greater 115 g was recovered in the extract from the composted wast e samples resulting in a final average recovery 140

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of 82% of the compound. The small increase in concentration observed at 84 days is thought to be due to desorption of the compound as de gradation of the solid materials proceeded over time. Acetaminophen In the previous experiment examining acetaminophen removal in the presence of cellulose, it was determined that the reduction in acetaminophen concentration was primarily due to adsorption to the cellulose and other organic components. This was demonstrated by an initial decrease in concentration in both the abiotic a nd biologically active sample s. This was followed by a subsequent increase in concen tration in the biologi cally active samples as the substrate was consumed by the microflora. Figure 7-2 shows the change in concentration of acetaminophen with measurements of the concentration following sample preparation, at 28 days, and at 84 days for both the new and composted waste samples. The new waste demonstrated an initial decrease to an average concentration of 449 g/L following sample prep aration and decreased further to an average final concentration of 376 g/L. This is a 25% reduction in concen tration over the test period. The solid/liquid extraction yielded only an average of 6 g of acetaminophen accounting for only 4% of the original acetaminophen and fa r less than the 25% removed from solution. The composted waste showed a similar starting concentration of 406 g/L. However, unlike the fresh waste, the concentra tion of acetaminophen was below the method detection limit at 28 and 84 days of incubation. Furthermore, this loss was not recovered during the solid/liquid extraction performed at the end of the experiment as the analysis of the composted waste extract was less than the method detection limit. The low quantity of acetaminophen recovered in the solid/liquid extract indicates the possible biodegradation of acetaminophen within th e samples. However, biological activity was 141

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significantly greater in the new waste samples versus the composted waste samples with the new waste yielding an average of 4662 ml of degradat ion gases compared to only an average of 132 ml for the composted waste. Thus, if bi odegradation of acetami nophen was the source of removal for the composted waste samples, a ma tching or greater reduction would have been expected for the new waste samples. Therefor e, biodegradation is believed to be unlikely although a difference in microbial species betw een the new and composted waste causing the difference in removal cannot be eliminated. As in the prior experiments, the primary mechanism for acetaminophen removal is thought to be adsorption to the waste materials. The difference in removal capacity between the two types of waste material may be explained by a change in the composition of the composted waste during composting. As shown in Table 7-1, the simulated waste used in the experiment contained nearly 38% by weight of paper and wood products. These products, comprised of natural fibers, contain varying amounts of ligni n, cellulose, and hemicelluloses materials. Typically, lignin is considered to be the most resistant to degradati on of these materials, especially in an anaerobic environment. Howe ver, the aerobic composting conducted for sample preparation of the composted wa ste can result in the degradation of lignin by certain fungi and bacteria to form humic substances (121, 122) During composting, the presence of fungi was visually observed and the composted waste demons trated the dark, spongy appearance typical of humic mixtures. The biodegradation of the comp osted waste is indicated by volatile solids (VS) measurement with the composted waste having an average VS content of 52%, while the new waste had an average of 58%. The formation of the humic substances pe rmitted the greater adsorption of acetaminophen from solution in the composted waste samples. Humic substances possess a greater number of 142

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active adsorption sites than the initial lignin primarily through the formation of available carboxylic acid functional groups. These active site s are especially active in the adsorption of micronutrients such as metal cations and polar organic compounds such as acetaminophen (123) The lack of humic substances in the new wast e samples reduced the adsorption capacity of the waste materials resulting in a smaller reducti on in solution concentra tion. This adsorption, however, could not be verified by recovery using solid/liquid extr action and further investigation is warranted. Acetylsalicylic Acid The concentrations of acetyl salicylic acid (salic ylic acid) measured following sample preparation, after 28 days of incubation, and after 84 days of incubation are shown in Figure 7-5. Concentrations measured after sample prepara tion were below the spik ed concentration of 500 g/L with the new waste samples having an av erage concentration of 334 g/L and the composted waste exhibiting a concentration of 414 g/L. The lower concentrations may be the result of adsorption or initia l biodegradation of the samples. Salicylic acid concentrations then increased at 28 days to 606 g/L a nd 505 g/L for the new waste and composted waste samples respectively. The new waste samples then showed a dramatic decrease to 23 g/L while the composted waste samples remained near initial levels at 472 g/L. In the prior experiments utiliz ing cellulose and anaerobic slu dge media, acetylsalicylic acid showed a dramatic decrease in concentration wi thin 28 days. However, this decrease did not occur within the leachate/ MSW test samples and in contrast sh owed an increase in concentration. This increase may be explained by a potential source of salicy lic acid and its hydroxybenzoic acid isomers within the municipal solid waste mi xture. Potential sour ces are the dry dog food used as the food component of the MSW mixture or other humic substances. The ingredients of the dog food included rice, oats, and barley products. In agricultura l research, these plants have 143

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been shown to contain and emit upon degrada tion salicylic acid and hydroxybenzoic acids (124, 125) Other research has shown the production of hydroxybenzoic acid degradation products from humic substances (126) Therefore, during the first 28 da ys, degradation of these products may have resulted in the additional release of sa licylic acid into the leachate and a corresponding increase in concentration. Figure 7-5 show s the degradation gas production of the new and composted waste samples containing acetaminophen. As seen in this figure, the peak weekly gas production for the new waste samples occurred in the week prior to the 28 day measurement and thus the release from these sources may have peaked at this point. At some time after the 28 day measurement, th e rate of introduction of salicylic acid from these sources became less than the rate of rem oval by degradation and l eachate concentrations decreased in both waste types. The larger microbial activity of the new waste samples resulted in a significantly larger reducti on to less than the method detecti on limit. Solid/liquid extraction of the new waste samples resulted in no quantities greater than the detection limit. Extraction of the solid fraction of the composted waste samples resulted in the recovery of an average of 11 g/L of salicylic acid. Thus the average recovery of acetylsalicylic acid composted waste samples was 102%. However, this number ma y have been heightened by the potential input from the waste materials. In comparison, the reco very of salicylic acid in the new waste samples was 6%. Ibuprofen Examination of Figure 7-6 shows that the c oncentration of ibuprofen in the new and composted waste samples following sample creation averaged 733 g/L and 659 g/L respectively. These concentrations exceeded th e target ibuprofen concentration of 500 g/L by 47% and 32% respectively. However, this was not unexpected. Pr ior analytical method development utilized the same landfill leachate used to create the MSW/leachate samples. 144

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During this work, analysis indicated that the landfill leachate contained a background concentration of ibuprofen. Therefore, measurem ent of ibuprofen concentrations for each of the samples was determined by the method of standard additions. The concentration of ibuprofen in the samples of each waste type showed a slow continued decrease in concentration at both 28 days and 84 days of degr adation. The new waste samples contained an average leachate concentrati on of 348 g/L at 84 days and solid/liquid extraction recovered an average of 40 g of ibuprofen from the solid fraction. The composted waste samples had a final concentrati on of 464 g/L with an average of 22 g recovered by solid/liquid extraction. This corres ponds to an average total recovery of 96% of the ibuprofen from the new waste samples and 107% for the composted waste samples. The lack degradation of ibupr ofen under the test conditions is indicated by the almost complete recovery of the compound from the new waste and composted waste samples. Thus, a lack of degradation within landf ills may be expected. The new waste samples, while showing a larger reduction in leachate concentration, also exhibited a greater fraction of the ibuprofen partitioned to the solid material. This may be due to the preferential adsorption of ibuprofen by a constituent present in the new waste samples which had been either eliminated or reduced in the composted waste samples. Metoprolol Tartrate Following an initial decline, the concentrati on of metoprolol tart rate did not change considerably over the test period. As displayed in figure 7-7, the concentra tion of metoprolol in the new waste samples declined from the spiked concentration of 500 g/L to an average of 244 g/L following sample preparation, then ro se to a concentrati on of 310 g/L at 28 days of incubation, and remained near that va lue, finishing at 297 g/L at 84 days. The 145

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composted waste samples showed a similar trend with concentrations of 186 g/L following sample preparation, 133 g/L at 28 days, and 154 g/L at 84 days. The greater reduction in met oprolol concentration observed in the composted waste samples indicates the possibility of greater adsorption of metoprol ol to the more well composted waste or degradation products than the new waste components. As seen in Table 7-5, 39 g of metoprolol was recovered from the solid fraction of the composted waste, while less than half this amount, 18 g, was recovered from the ne w waste sample solids. Thus, adsorption to older waste material, possibly th e humic substances formed from waste degradation is favored by metoprolol. Despite the larger recovery of metoprolol from the solids fr action of the composted waste samples, the samples showed a lower overall reco very of metoprolol, 57 % versus 71%, than the new waste samples. Due to the lower degradation activity within the composted waste samples, this difference recovery is not believed to be due to degradation of the metoprolol. Although a shift in the bacterial composition of the composted waste leading to greater degradation cannot be excluded, it is believed that a difference in the extraction efficiency between the two wastes types is more probable. Regardless, minor biodegr adation of metoprolol cannot be discounted. Progesterone As shown in Figure 7-8, progesterone showed rapid removal from solution and decreased to less than the limit of detection within 28 days in both waste types. Furthermore, as shown in Table 7-5, no measureable quantity of progester one was recovered from the solids fraction of either waste type. While it is not possible to eliminate anaerobic biodegradation as a mechanism for removal, the similarity between the two wast e types of differing biological activity and the complex structure of progesterone leads to the conclusion of adsorption and abiotic mechanisms 146

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as the probable removal mechanisms. Future re search utilizing radiolabeled progesterone may permit differentiation between mineralization and adsorptive losses of progesterone. Correlation of Results with Prior Experimental Observations In the experiments of the preceding chapters, the anaerobic degradation and adsorption of the selected pharmaceutical compounds was exam ined. Initial experiments utilized the pharmaceutical compounds as the sole source of organic carbon. Latter experiments expanded the potential removal mechanisms to include cometabolism and adsorption to an organic substrate through the introduction of a common landfill organic subs tance, cellulose. The final experiment presented in this ch apter substituted actual landfill leachate for the nutrient solution of the prior experiments and components typical of municipal solid waste as the substrate. This created conditions more typical of actual MSW landfill environments. The strong adsorption losses observe d in prior experiments for 17 -ethinylestradiol and progesterone were confirmed in the MSW experi ments. As in the prior experiments, the potential for 17 -ethinylestradiol desorption followi ng organic substrate consumption was demonstrated by a slight increase in concentr ation after initial losse s and by the large amount extracted from the solids fraction fo llowing experimental completion. Unlike the prior experiments, acetaminophe n displayed a significant reduction in concentration within the compos ted waste samples to less than the method detection limit. While the degree of reduction differs between th e experiments, the mechanism for this reduction continues to be theorized as adsorption. The reduction in concentration observed in this experiment is believed to be the result of strong adsorption of acetaminophen to components formed in the more highly degraded waste during its aerobic composting preparation. 147

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Acetylsalicylic acid (salicylic acid) as in the second experiment, s howed a large reduction in concentration attribut able to biodegradation. However, in this last experiment rising concentrations indicate that natu rally occurring sources of salicy lic acid within municipal solid waste may exist. Metoprolol tartrate demonstr ated a minor initial reduction in concentration attributable to adsorption, although minor bi odegradation of the compound could not be discounted. This is consistent with prior e xperimental findings which showed a mixture of adsorption as primary mechanism for removal of metoprolol from test solutions and biodegradation as a secondary pathway. Finally, as in the previous experiments, i buprofen continued to show a resistance to degradation under anaerobic landf ill conditions. While values were affected by differing concentrations and matrices, resu lts of the initial experiment s howed that 73% of the ibuprofen remained in solution after 56 days the second experiment showed that 82% remained in solution of the 500 g/L biologically activ e samples after 112 days, and the final experiment showed that 70% to 93% remained in the leachate after 84 da ys, depending on the waste type. Ibuprofen was the only compound not exhibit a re duction in concentration to less than the method detection limit in any of the experimental samples. Summary The experiments of this chapter examined th e fate of six target pharmaceutical compounds in laboratory experiments designed to simulate municipal solid waste landfill conditions. The anaerobic degradation of each compound was measured over 84 days from an initial concentration of 500 g/L. Each sample contai ned 300ml of landfill leachate and 15g of one of two differing synthesized municipal solid waste mixtures. The first mixture was comprised of newly generated, undegraded waste to simulate municipal solid waste following initial waste placement and the acid phase of landfill degradation. The second mixture was comprised of the 148

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same synthesized waste mixture which had b een aerobically composted for 90 days. This mixture was intended to simulate degraded wast es present in the methanogenic phase of landfill stabilization. Each sample was directly anal yzed for change in pharmaceutical concentration following 28 and 84 days of incubation at 37C using liquid chromatography tandem mass spectrometry. The results of the experiments show that the potential for an aerobic degradation and other removal processes vary widely not only between the pharmaceutical compounds, but under differing waste composition conditions. Acetyl salicylic acid (measured as salicylic acid) displayed ready biodegradation removal from landfill leachate in the presence of the high biological activity occurring with new, undegraded waste (94%), while biodegradation was not significant in the presence of pr eviously degraded waste with 102% of the original content remaining at the end of the test. Acetaminophen displayed the opposite trend, sh owing a significant removal from leachate in the presence of degraded waste (>99%) but le ss during the active degradation of new waste (25%). This was believed to be due to differen ce in the composition of the two waste types and their capabilities for ace taminophen adsorption. 17 -ethinylestradiol displayed significant removal from solution due to adsorption in th e new waste samples (87%) and composted waste samples (94%). Similarly, progesterone displaye d greater than 99% removal from solution for both waste types possibly due to adsorpti on although other abiotic mechanisms and biodegradation could not be eliminated as possible mechanisms. Metoprolol tartrate demonstrated minor reduction in leachate conc entration in the new waste (41%) and composted waste (69%) samples due to a mixture of ad sorption and potential biodegradation. Finally, ibuprofen also displayed a small reduction in concen tration attributable to adsorption to the solid 149

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material, but continued to show resistance to biodegradation with 96 to 107% of the initial ibuprofen recovered at the end of th e experiment for both waste types. 150

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Table 7-1. Composition of fabricated municipal solid waste for pharmaceutical degradation samples Waste components Source Processing for size reduction Particle size/length (cm) Percentage Of waste (mass %) Office paper Used office paper Paper shredder 0.5cm x 0.5cm 15.8% Cardboard Mixed corrugated boxes Scissors 2cm x 2cm 13.6% Newspaper Local newspaper Paper shredder 0.5cm x 0.5cm 1.8% Tissue paper/towels Purchased at local store Pa per shredder 0.5cm x 0.5cm 2.6% Plastics PET/HDPE bottles Scissors 1cm x 1cm 17.5% Food waste Commercial dog food Mortar and pestle < 0.25 cm 16.3% Wood non treated Home improvement store Chipped 3cm x 1cm x 1cm 7.5% Ferrous metal Steel Wool Pads Scissors 0.25 cm x 2 cm 5.4% Aluminum Aluminum cans Scissors 1cm x 1cm 1.6% Other metal Copper wire Scissors 1 cm 0.3% Glass Mixed cullet Crush with a hammer 1cm x 1cm 8.6% Textiles Cloth rags Scisso rs-cut strips 3cm x 1cm 5.5% Rubber and textiles Old shoes Scissors & shears 2cm x 2cm 3.5% Table 7-2. Method detection limit for analysis of pharmaceutical compounds in landfill leachate Compound Method detection limit (MDL) (g/L) Acetaminophen 5 Acetylsalicylic acid 4 17 -ethinylestradiol 6 Ibuprofen Metoprolol 2 Progesterone 3 MDL for ibuprofen not determined concentrations determined by method of standard additions 151

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Table 7-3. Total gas production and final degrada tion gas concentrations of pharmaceutical and municipal solid waste mixtures after 84 days of degradation. Compound Waste type Average total gas production (ml) Average carbon dioxide concentration (%) Average methane concentration (%) Acetaminophen New 4663 39 60 Composted 132 18 17 Acetylsalicylic acid New 3668 40.8 59.6 Composted 70 15 9 17 -ethinylestradiol New 4493 45 55 Composted 249 16 37 Ibuprofen New 3293 44 55 Composted 51 9 6 Metoprolol tartrate New 3275 44 55 Composted 131 11 20 Progesterone New 4615 41.7 59.7 Composted 64 10 8 Controls New 5053 43 57 Composted 324 22 39 Average standard deviation of triplicate samples Table 7-4. Final Landfill Leachate Concentrat ions of 500 g/L Selected Pharmaceutical Compound Samples Following 84 days of In cubation with Municipal Solid Waste N ew waste (ug/L) Composted waste (ug/L) Acetaminophen 376
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Table 7-5. Quantity (g) of pharmaceutical compounds recovered from solid fraction of simulated MSW after 84 days using solid/liquid extraction N ew waste (g) Composted waste (g) Acetaminophen 6
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Figure 7-2. Concentration of 17 -ethinylestradiol in leachate following 0, 28, and 84 days of simulated MSW degradation. 0 100 200 300 400 500 600 02 88 4g/LDays New Waste Degraded Waste Initial Concentration Figure 7-3. Concentration of Acetaminophen in leachate following 0, 28, and 84 days of simulated MSW degradation. 0 100 200 300 400 500 600 02 88 4g/LDays New Waste Degraded Waste Initial Concentration 154

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155 Figure 7-4. Concentration of acetylsalicylic acid (as salicylic acid) in leachate following 0, 28, and 84 days of simulated MSW degradation. 0 100 200 300 400 500 600 700 02 88 4g/LDays New Waste Degraded Waste Initial Concentration Figure 7-5. Average weekly degradation gas production of triplicate simulated MSW samples containing acetylsalicylic acid. 0 200 400 600 800 1000 1200 1400 020406080100Degradation Gas Production (ml)Days New Waste Degraded Waste

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Figure 7-6. Concentration of ibuprofen in leachate following 0, 28, and 84 days of MSW degradation 0 100 200 300 400 500 600 700 800 02 88 4g/LDays New Waste Degraded Waste Figure 7-7. Concentration of metoprolol tartrate in leacha te following 0, 28, and 84 days of MSW degradation 0 100 200 300 400 500 600 02 88 4g/LDays New Waste Degraded Waste Initial Concentration 156

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157 Figure 7-8. Concentration of progesterone in leachate followi ng 0, 28, and 84 days of simulated MSW degradation

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CHAPTER 8 DETECTION OF SELECTED PHARMACEUTIC AL COMPOUNDS IN MUNICIPAL SOLID WASTE LANDFILL LEACHATE Introduction Meager research has been completed concerning the occurrence and fate of pharmaceuticals and personal care products (PPCPs) in landfills. The method of disposal for PPCPs most commonly recommended in previo us years has been the sewage system. Nonetheless, this has not prevented the use of municipal solid waste (MSW) and landfills for their disposal and MSW landfills are increasingly becoming the recommended disposal practice for household medications (19, 21, 23, 39) Indirect evidence of the presence of pharmaceu ticals in landfill leachate has been presented in studies of groundwater contamination from MSW landfills. Holm et al. examined an unlined landfill near Grinsted, Germany for pharmaceutic al compounds and found six sulfonamides, three byproducts of their production, one barbiturate, an analgesic, an intermediate in the production of meprobamate, and an anti-foaming agent used in pharmaceutical production in groundwater (40) In 1993, Eckel et al. reported gr oundwater contamination by pentobarbital from a closed landfill in Florida (41) Schwarzbauer et al. analyzed groundwater containing seepage from a sanitary landfill and directly sampled the seep age below the landfill. Propyphenazone, a widely used analgesic and antipyretic was detected as well as ibuprofen and clofibric acid (42) In the United States, an investigation of orga nic wastewater contaminants from an unlined landfill in Norman, Oklahoma detected the antibiotic lincomycin and continine in the downgradient groundwater (46) Few studies have sampled and measured th e pharmaceutical compound concentration of MSW landfills and landfill leachate directly. Research studies of an active, unlined landfill in Croatia detected pharmaceuticals in solid waste, leachate and underlying soil (1 m). However the authors 158

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attributed these compounds to the disposal of pharmaceutical production waste rather than from disposal of municipal refuse (44, 45) In 2004, a literature review by Metzger described two studies of drugs in landfill leach ates in Germany. The medications quantified included clofibric acid, diclofenac, ibuprofen, indomethacin, pentoxyfylline and primidone. The second study investigated the presence of se veral drugs in the leachate from five active municipal landfills in Germany including clofibric acid, ibupr ofen, carbamazepine, and phenacetin (47) However, the potential sources of these compounds were not examined. No studies of the pharmaceutical content of landfill leachate in the United States have been performed to date. Beyond the direct disposal of unwanted pharm aceuticals or manufacturing wastes in MSW landfills, wastewater treatment biosolids sent to MSW landfills is a potential source of PPCPs. In 2004, approximately 2 million tons of the biosolids generated in the United States annually were disposed of in municipal landfills (48) The release of pharmaceuticals from wastewater biosolids has been detected (32, 49) Thus the pharmaceutical compounds contained in the biosolids may become released within the landfill envir onment and enter the leachate. Conversely, pharmaceuticals in the leachate may then be sent to wastewater treatment plants, resulting in pharmaceuticals being released in th e effluent or the biosolids. Prior research examined the fate of selected active pharmaceutical ingredients under anaerobic landfill conditions. The objective of th is research was to determine if the compounds predicted to persist by that research resulted in emission in the leachate of actual MSW landfills. A survey of landfill leachate from the state of Florida was performed to determine the concentrations of ten active pharmaceutical ingred ients. The leachate from a total of ten MSW landfills was analyzed to measure pharmaceutical concentrations and physical and chemical parameters such as pH, COD, and ammonia comm only used to characteri ze landfill leachate. 159

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Demographic and operational data were also co llected to examine for potential trends for correlation with pharmaceuti cal concentrations. Methods and Materials Selection of Target Pharmaceutical Compounds Various methods have been used to identif y pharmaceutical compounds with the greatest potential for release to the environment and previous researchers have commonly utilized prescription rate data (103-105) Using a similar method, ten pharmaceutical compounds were selected for study based upon the number of pr escriptions from 2002 to 2004, the defined daily dose of the medications, and/or the over-the-coun ter usage and environmen tal detection of the medication. A detailed description of the comp ound selection method is given in Appendix A. The pharmaceutical compounds and their pertinent physical and chemical properties are listed in Table 8-1. Sample Collection The leachate from a total of ten MSW la ndfills, a MSW transfer station, and water collected at the face of a landfill were collected fro m ten sites in the state of Florida in January 2008. The location of the sites is shown in Figur e 8-1. Table 8-2 presen ts a summary of the characteristics of the landfills, including age/status of operation, leachate generation rates, waste disposal rates, and population serv ed by the landfill. The leachates were collected at the point closest to immediate discharge from the landfill as possible. Samples were typically taken at leachate collection wells at the base of the landf ill, with the exception of landfills E and G which were sampled from leachate collection tanks and landfill B which was sampled from an aerated leachate holding pond. Samples were collected w ith a teflon baler and were placed into polyethylene and glass containers for chemical analysis. Samples for pharmaceutical analysis were placed in silanized 1L am ber bottles. Samples for chemi cal analysis were preserved as 160

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required by the appropriate EPA method. Once colle cted samples were transported to the lab on ice and immediately stored at 4C until analysis. Leachate Chemical Analysis Samples for chemical analysis were collected from each landfill site and replicate measurements were made as required in the individual methods. Field measurements included pH, conductivity, and oxidation/redu ction potential (ORP; Orion 5 St ar Portable Multimeter). The leachates were analyzed for biochemi cal oxygen demand (BOD), chemical oxygen demand (COD), total organic carbon, ammonia, and total di ssolved solids according to standard methods described by the US EPA and the American Public Health Association.(120, 127) Total ammonia (NH4 and NH3) was analyzed by a selective ion probe (Accumet). Pharmaceutical Compound Analysis Leachate samples were vac uum filtered through a 1.2 m glass filter paper (Fisher Scientific, Fisherbrand G4 Glass Fi ber Filter Circles). An aliquot of 30 to 50 ml of filtrate was placed in a silanized borosilicate gl ass vial with a teflon coated cap. The pH of each sample was adjusted to 5 using concentrated hydrochloric acid and the samples were stored at 4C until analysis. Immediately prior to analysis, a 2ml aliquot of each sample was placed into a 2ml sampling vial via filtration through a 0.2um syringe f ilter (Supelco Iso-Disc Filters PTFE-25-2). LC analysis was performed with an Agilent 1200 Series rapid re solution LC System coupled to an Agilent 6410 Triple Quad LC/M S/MS (Palo Alto CA, USA) equipped with an orthogonal ESI interface. All compounds were separated at 40 C by means of a Zorbax Eclipse XDB-C18 column (2.1 mm 50 mm, 3.5 m) from Agilent. The injection volume was 10 L with a flow rate maintained at 0.4 ml/min. The mobile phase was comprised of a mixture of (A) 5mM ammonium formate in water and (B) 5mM a mmonium formate in methanol. The mobile phase solvent gradient started with 5% of solv ent B and was increased to 90% solvent B evenly 161

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over 12.0 minutes. The solvent mixture was retu rned to the initial proportions following completion of sample analysis at 12.1 minutes and a post-run time of 2 minutes was required in to re-equilibrate the column. An initial scan for the presence of the sele cted pharmaceutical compounds was performed on each sample. Samples for which a pharmaceutical compound was detected were further analyzed by the method of standard additions to determine the concentration within the leachate. A stock solution was prepared containing each of the pharmaceuticals of interest at a concentration of 20 mg/L in HPLC grade methanol using Cl ass A volumetric glassware. Standards were prepared from the stock solution at concentration additions of 10 g/L 20 g/L 100 g/L 200 g/L 1000 g/L and 2000 g/L Calibration was monitored through the use of calibration verification samples every six samples required to be within % of the initial sample. Instrument blanks to monitor potential carryover between injections were analyzed pr ior to each calibration check. For samples in which carryover was indicated, one to three system washes were placed between analytical samples and the samples reanalyzed. Acquisition peak identification a nd integration, and final quantification were performed with Agilent MassHunter Workstation Software B.01.00 9(B48). Results and Discussion The results of analysis for leachate pharm aceutical concentration are shown in table 8-4. 17 -ethinyestradiol, cephalexin, hydrochlorothia zide, metoprolol tart rate, prednisone and progesterone were not detected in any of the leachate samples. This was not unexpected however. The method detection of each of these compounds was greater than 1 g/L and each compound is normally detected in concentrations less than this limit in environmental samples. 162

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Acetaminophen was detected in two of the te n samples at concentrations of 64 g/L and 3 g/L. Notably, acetaminophen was also detected in the leachate from the MSW transfer station sample and from the leachate pond on the work ing face of landfill B at 18 g/L and 13 g/L respectively. Salicylic acid, a degradation product of acetylsalicylic acid, was detected in 4 out of 10 landfill samples. Concentrations ranged from 2 g/L to 1006 g/L. As with acetaminophen, salicylic acid was also present in the MSW transfer station sample at 44 g/L and in the landfill face leachate pond at 10 g/L. Although vi ewed more commonly as a cola beverage component, caffeine is also included in many headache medication formulations and was included as a compound of interest. Caffeine was identified in two of the leachate samples at concentrations of 4 g/L and 54 g/L. Ibuprofen was the compound detected most fre quently, appearing in 9 of the 10 landfill samples and in the waste transfer station leacha te and landfill face pond sa mple. Concentrations detected ranged from 23 g/L up to 256 g/L. The leachate sample for which ibuprofen was not detected was obtained from an aerated leachate collection pond. The lack of ibuprofen within this sample is believed to be due either th e aerobic degradation of the compound within the pond, the photodegradation of the compound, or a combination of these mechanisms. Table 8-1 shows chemical properties of the selected pharmaceutical compounds pertinent to their potential landfill leachate concentra tions. Due to limited compound detection, the determination of a discernable trend in leachat e concentrations versus Kow or pKa was not possible. As an aqueous solution, the wate r solubility of the compounds would have a significant effect on potential leachate concentrations with greater solubility expected to result in higher leachate concentrations. However, the pharmaceutical compound detected most often in MSW landfill leachate was ibuprofen, ranking as th e third least soluble of the ten compounds. 163

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This is believed to be due to ibuprofens resist ance to degradation and its common use resulting in larger quantities of disposal Compounds such as acetaminophen or acetylsalicylic acid which have much higher solubility and relatively equal usage rates may be more readily degraded or simply flushed quickly from the waste and not observ ed in later leachate samples. Other highly soluble compounds such as metoprolol tartrate may simply not have been present in quantities sufficient to be observed above the method detection limits. Ibuprofen, as the only compound seen in the ma jority of samples, was used to compare leachate chemical properties and local rainfa ll quantities (Table 85) with pharmaceutical compound concentration. No pattern was observed in comparing rainfall over 1 month, 3 months and 12 months prior to sampling with ib uprofen concentrations. Therefore, sample dilution effects are not expected to be a factor in the results. Additionally, no discernable trend was seen in comparison of ibuprofen concentra tions with landfill pH, ORP, conductivity, or any of the other leachate parameters shown in Table 8-3. MSW Landfill Characteristics versus Leachate Pharmaceutical Compound Content The landfill with the greatest number of co mpounds detected was landfill E, with acetaminophen, salicylic acid, ibuprofen, and caffe ine. With measurable quantities of acetaminophen, salicylic acid, and ibuprofen, land fill I had the second greatest number of detections. Review of Table 8-2 shows that these two landfills have the greatest waste deposition rate and serve the largest populations of all the landfills. Additionally, these landfills provided service to urban areas. Thus, landfills receiving greater amounts of waste from larger cities and densely populated areas may have a greater probability of pharmaceutical compound emission. A total of six of the surveyed MSW landfills were actively accepting waste. Notably, the only landfills to have a measureable quantity of a pharmaceutical compound other than ibuprofen 164

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were four of these currently active landfills. Thus, the detection of salicylic acid and acetaminophen may be dependent on the age of the la ndfill. As noted previously, acetylsalicylic acid degraded readily under MSW landfill conditions and acetaminophen showed significant reductions in concentration in the composted waste test samples. The presence of these two compounds in the MSW transfer station leacha te and the leachate pond from the landfill face provides further evidence for this trend. Leachate recirculation back to the landfill had been or was currently being practiced by 5 out of 10 of the landfills surveyed. Examination of the ibuprofen c oncentrations of each of these landfills shows that they contain 5 of the 6 hi ghest concentrations, with landfill E, an active major metropolitan landfill, being the only landfil l with a similar concentration that did not recirculate leachate. Notably, la ndfill H, had the highest ibuprofe n concentration of all landfills despite being closed for many years. Additiona lly, despite being one of the smaller landfills, landfill H possessed a large leachate generation ra te due to leachate re circulation. Landfill operators reported that the leachate injection rate and generation rate had become equal, indicating a constant recircul ation of the leachat e through the landfill. Thus, leachate recirculation may have led to elevated ibuprofen concentrations in this and the other landfills. Other MSW landfill operation factors examined included receipt of any unusual wastes or the acceptance of wastewater treatment biosolid s. No association between the acceptance of biosolids and the number of pharm aceuticals detected or their measured concentration could be identified. Examination of special waste accep tance provides only circumstantial evidence as landfill E, the only landfill to report having receive d specific lots of discarded medications, had the largest number of pharmaceu tical compound detections 4. Further research is necessary before any conclusion can be reached. 165

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MSW Landfill Leachate vs. Wastewater Treatment Plant Sewage Influent A comparison of the maximum and average m easured landfill leachate pharmaceutical concentrations with published wastewater treatm ent plant influent maximum concentrations is shown in Table 8-6. For prednisone and progest erone, a comparison cannot be made due to a lack of any reported concentrations of either in wastewater treatment plant influent and the lack of detection in any of the tested landfill leach ates. For the remaining compounds, 6 out of 8 compounds had maximum measured concentratio ns in the landfill le achate less than the maximum published levels in WWTP influe nt. The two compounds which had leachate maximums greater than WWTP influent were ib uprofen and acetylsalicylic acid. In the prior anaerobic degradation experiment s, ibuprofen displayed the grea test resistance reduction in solution concentration. Additiona lly, salicylic acid, the actual molecular structure used to measure acetylsalicylic acid concentration in the landfill leachate, was hypothesized as a degradation product of municipal solid waste by th e tests of Chapter 7 and thus may be a source of the higher measured concentrations. However, when considering the overall i nput of pharmaceutical compounds from other sources of WWTP influent versus wastewater tr eatment of landfill leachate, the volume of liquid to be treated must also be considered. Table 8-7 allows this and presents the WWTP input of three of the pharmaceuticals detect ed in landfill leachate expressed as mg/person-year. This number was calculated by multiplying the measur ed leachate concentration by the quantity of leachate generated per year and then dividing by the number of people served by the landfill. If this leachate is assumed to be wholly treated by the local wastewater treatment plant, then this permits comparison on a per person basis the inpu t of the MSW landfills with the input from medication user excretion and sewage disposal of unwanted medications. The expected input from sewage disposal of unused medications wa s computed by multiplying the quantity of each 166

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medication sold in 2005 by a conservative estima te of 10% becoming unused and 50% of those unused medications being disposed in the sewage system (85, 128) The input due to patient excretion was calculated by multiplying the quantity sold by the 90% of the medication assumed to be used, then multiplying by the fraction excreted by the patient. Each of these figures was then divided by 296 million, the population of the Un ited States in 2005 to equate their input to a per person basis. Acetaminophen with an average input to wast ewater treatment plan ts from landfills of 0.3 mg/person-year was significantly less than the predicted input of 961 mg/person-year from unused medication sewage disposal and 865 mg/year-person from excretion. Acetylsalicylic acid showed similar trends with 4 mg/person-year from landfills versus 1843 mg/person-year and 3317 mg/person-year from sewage sources. The input of ibuprofen from landfills is the closest of the three compounds to the sewage input rate s; however, an average of 11 mg/person-year remains much less than the 175 mg/person-year estimated for unused medication sewage disposal and 315 mg/person-year from excreti on. Thus the contribution of landfills to wastewater treatment plant effluent discharge of pharmaceuticals appears to not be appreciable. However, as new policies divert medication dispos al from sewage systems to landfills this may change. Of unusual interest in table 8-6, the estimat ed wastewater treatment plant input of acetaminophen from unused drug sewage disposal was greater than the expected excretion input. The unused medication input for acetylsalicylic acid and ibuprofen were over 30% of the total input. Thus the high potential input of these compounds from unus ed drug disposal may provide evidence of the need for diversi on to landfills or special collecti on programs. Howe ver, these are only three of thousands of potential compounds and the inputs of any individual compound 167

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would vary with usage, degradability, and disp osal habits. Additionally, the sewage system inputs to waste water treatment plants predicted in table 8-6 may be reduced due to degradation and abiotic reactions of the compounds dur ing transit to the treatment facility. Correlation of Results with Prior Experimental Observations In the experiments of the preceding chapters, the anaerobic degradation and adsorption of the selected pharmaceutical compounds was examined. In each experiment, 17 -ethinylestradiol and progesterone demonstrated rapid reduction in concentration due to adsorption. These compounds are typically taken in low dosages (Defined Daily Dose for 17 -ethinylestradiol =25 g, progesterone= 5 to 300 mg) (129) With these combined fact ors, the lack of detection within the MSW leachate samples was not unexpected. Acetaminophen displayed mixed results in prior experiments di splaying a significant reduction in concentration within the composted waste samples of the last experiment showing only minor loss in the first two experiments. The detection of acetaminophen, was therefore, not unexpected due to the majority of experiments sh owing a slow reduction in concentration and as seen in Appendix A, acetaminophen ranking as th e top medication used in the United States measured both in mass prescribed and percenta ge of persons using the medication each week. Acetylsalicylic acid (salicylic acid), show ed the greatest biodegr adation of all the compounds tested. Therefore, its presence in MSW landfill leachate may not be expected. However, as shown in the last experiment, natu rally occurring sources of salicylic acid from municipal solid waste may exist providing suppleme ntal sources to landfill leachate. Metoprolol tartrate demonstrated measurable degradation an d/or adsorption in each experiment and in the second experiment displayed adsorptive reductio n to less than the method detection limit. 168

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169 However, no conclusion may be reached on the role any of the potential mechanisms played in the lack of metoprolol detection in municipal solid waste leachates. Finally in the previous experiments, i buprofen was the only compound not exhibit a reduction in concentration to less than the method detection limit in any of the experimental samples and ibuprofen demonstrated a resistance to degradation. Furthermore, as shown in Appendix A, ibuprofen lagged only acetaminophen in total mass prescribed annually and was the third most commonly used drugs by Americans each week. Ibuprofen was also collected in both the pharmaceutical waste collecti on program and in the municipal solid waste composition study. Thus the disposal of ibuprofen by medications us ers is common and the presence of ibuprofen 11 out of the 12 leachate samples and the prior expe rimental results confirm ibuprofens resistance to degradation in a landfill environment. Summary A survey of MSW landfill leachates from the st ate of Florida was performed to determine the concentrations of ten active pharmaceutical ingredients. Of the ten compounds, ibuprofen was the most frequently detected, found in 9 out of the ten MSW landfills. This was expected due to the resistance to degradation displaye d by ibuprofen in the prior experiments. Calculations of mass input of APIs to wastewater treatment plants from MSW landfills versus patient excretion and sewage disposal show that treatment of MSW landfill leachate by wastewater treatment systems ma y not be a significant source of APIs to the environment. Results also indicated that leachate of MSW landfills with high daily loading rates and serving large population centers may contain a greate r number of pharmaceutical compounds. While leachate recirculation may permit further time for the degradation of pharmaceutical compounds, compounds resistant to degradation may accumulate and reach higher leachate concentrations.

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Table 8-1. Chemical properties of pharmaceu tical compounds selected for analysis in municipal solid waste landfill leachate pKa Water solubility at 25C ( mg/L) Log Kow Henry's law constant 17 -ethinylestradiol 10.4 11.3 (27C) 3.67 2.67E-09 Acetaminophen 9.5 1.4x104 0.46 6.42E-13 Acetylsalicylic acid 3.5 4600 1.19 2.52E-05 Caffeine 10.4 2.16x104 -0.07 7.33E-09 Cephalexin 1790 0.65 3.24E-15 Hydrochlorothiazide 7.9 722 -0.07 1.32E-09 Ibuprofen 4.9 21 3.97 1.86E-04 Metoprolol (tartrate) 9.6 1.69x104 1.88 2.88E-07 Prednisone 312 1.46 5.09E-13 Progesterone 8.81 3.87 1.30E-06 (Source: Syracuse Research Corporation, P HYSPROP The Physical Properties Database. http://www.syrres.com/esc/physdemo.htm (accessed February 19, 2008)) 170

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Table 8-2. Operating characteristics of ten Florida MSW landfills examined for leachate pharmaceutical compound content. Landfill Population served Size (acres) Sample point Year opened Year closed Waste placement rate (tons/day) Leachate recirculation Leachate generation rate (gal/month) Biosolids accepted Special waste received Notes A 124,131 (1995) 7.3 Sump 1991 1995 300 No 138,343 No None B 295,426 74 Holding Pond 1990 1100 No 1,404,000 Yes Fly ash, auto fluff Leachate generation high due to interception trench for unlined cells C 68,693 10.5 Sump 1992 210 No 159,794 No None D 294,181 (2000) 9.6 Sump 1997 2000 450 Yes 210,000 No None E 1,043,500 105 Storage Tank 1991 3900 No 835,600 Yes Coal ash, pharmaceuticals, orange juice, beer, industrial waste F 561,606 5.1 Sump 2000 2008 1250 Yes 45,000 Pharmaceutical waste, incinerator ash G 316,183 43 Storage Tank 1988 550 Yes 75,000 No None Leachate Recirculated 2000-2003 H 217,955 (2000) 27 Sump 1988 1998 300 Yes 933,600 No None I 1,274,013 330 Sump 1989 1944 No 2,633,750 Yes Waste to energy ash approximately 50% of waste J 97,987 35 Sump 2003 400 Yes 314,507 No None 40,000 gal recirculated 171

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Table 8-3. Physical and chemical propertie s of leachate from ten Florida MSW landfills examined for selected pharmaceutical compounds. Landfill pH Conductivity (us/cm) ORP (RmV) COD (mg/L) BOD (mg/L) TDS (mg/L) TOC (mg/L) Ammonia (mg/L) A 7.16 9480 289.8 2154 20.4 5.15 526.9 707.1 B 8.49 6060 6.4 1158 3.1 3.92 385.7 564.3 C 7.03 7330 33.5 780 17.0 3.00 212.0 662.9 D 6.82 11560 102.5 3120 153.6 6.71 659.7 647.1 E 7.67 12130 499.2 5016 1399.8 10.48 1265.7 1070.0 F 7.15 11850 89.5 2008 63.3 9.11 509.1 575.7 G 7.72 9560 182.2 2112 31.4 4.20 358.1 780.0 H 7.47 14540 10.5 3081 49.1 6.34 341.0 705.7 I 6.84 25470 150.8 2436 168.4 14.76 338.5 562.9 J 7.57 12890 73.2 3185 122.2 6.57 662.4 108.6 Transfer station 6.90 2245 95.5 Landfill face 6.73 1761 12.8 172Table 8-4. Concentration (g/L) of selected pharmaceutical compounds in municipal solid waste landfill leachate Landfill A B C D E F G H I J Transfer station Landfill face Acetaminophen 64 3 18 13 Acetylsalicylic acid 5 1006 55 2 44 10 17 -ethinylestradiol Ibuprofen 79 23 227 123 119 118 256 92 191 15 8 Metoprolol tartrate Progesterone Caffeine 4 54 Hydrochlorothiazide Cephalexin Prednisone

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Table 8-5. Rainfall at Florida landfill site s for 1, 3 and 12 months prior to sampling Landfill A B C D E F G H I J 1 month 2.35 4.47 4.61 2.75 5.30 3.27 1.85 1.79 0.14 1.40 3 months 3.83 10.44 8.98 4.68 7.68 5.74 2.51 3.72 3.04 2.90 12 months 31.36 49.52 46.80 27.95 55.02 46.18 14.12 21.73 31.04 46.73 Table 8-6. Comparison of selected pharm aceutical compound measured landfill leachate concentrations with published maximum wastewater treatment plant influent concentrations Compound Average measured landfill leachate concentration ( g/L) Maximum measured landfill leachate concentration ( g/L) Wastewater treatment plant influent maximum cited concentration ( g/L) Source 17 -ethinylestradiol ND ND 0.005 (94) Acetaminophen 7 64 246 (130) Acetylsalicylic Acid (salicylic acid) 107 1006 38.5 (131) Caffeine 6 54 230 (132) Cephalexin ND ND 5.6 (133) Hydrochlorothiazide ND ND 6.4 (134) Ibuprofen 123 256 168 (130) Metoprolol ND ND 7.2 (135) Prednisone ND ND N/A Progesterone ND ND N/A 173 ND = None Detected

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174Table 8-7. Wastewater treatment plant input of pharmaceutical compounds from MSW landfill leachate versus estimated sewage system influent (mg/person-year) Landfill A B C D E F G H I J Average Sewage system input due to unused disposal Sewage system input due to excretion Acetaminophen 2 0.3 0.3 961 865 Acetylsalicylic acid 37 5 0.3 4 1843 3317 Ibuprofen 4 2 7 4 0.4 1 50 8 28 11 175 315 Acetaminophen excretion=10%, ace tylsalicylic acid =10%, a nd ibuprofen = 5% (Sources: Physicians' Desk Reference 2005 Medical Economics: 2005; Vol. 59.0); Critchley, J.; Cr itchley, L. A. H.; Anderson, P. J.; Tom linson, B., Differences in the single-oral -dose pharmacokinetics and urinary excretion of paracetamol and its conjugates between Hong Kong Chinese and Caucasian subjects. Journal of Clinical Pharmacy and Therapeutics 2005, 30, (2), 179-184.)

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Figure 8-1. MSW landfill leachate sampling locations 175

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CHAPTER 9 SUMMARY AND CONCLUSIONS Summary In previous years, the common recommendation by health care and safe ty professionals for the disposal of unused medications was to wash the pharmaceuticals into the sewage system. However, new studies have shown that ma ny pharmaceuticals are not removed by sewage treatment systems and enter the aquatic environm ent from sewage treatment plant discharges. Further studies have also shown the potential en vironmental impact of these compounds, such as the feminization of male fish and other species. As a result, new po licies and guidance on the disposal of unused and expired household medications have emerged nationally and recommending disposal of unused medi cations in munici pal solid waste. With these new recommendations the amount of medications entering the municipal solid waste stream is expected to rise. However, the occurrence and fate of PPCPs in landfills has been largely neglected. Landfill leachate, the liquid which is the result of rain water and preexisting moisture percolat ing through municipal solid wa ste, is captured by designed collection systems in modern landfills. Once co llected, landfill leachate is often treated through waste water treatment plants. If the pharmaceutical compounds deposited within the landfill become dissolved or entrained in the leachate, th en the possibility exists that they will reach the waste water treatment plant; the very process that the new polices were attempting to avoid. A total of six research studies were perf ormed to characterize discarded household medication disposal in municipal so lid waste and the fate of selected pharmaceuticals in landfills. The first study examined the quantity and characteristics of pharmaceutical compounds entering the municipal solid waste stream by conduc tion of a community household pharmaceutical collection program. Collection program data confirmed that the disposal methods for 176

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medications most commonly utiliz ed by households were predominantly the sewage system and municipal solid waste. Examination of the collection program partic ipants showed that collection program efficiency would be best ac hieved by targeting older age groups, developing systems to capture medications at the death of a patient, and that providing a continuous collection system was favorable. The second study employed a mathematical me thod to estimate the quantity of active pharmaceutical ingredients within municipal solid waste and then compared this estimation with a direct measurement by performance of a wa ste composition study. In the mathematical estimation, the variable of greatest uncertainty was the quantity of medications which become unused once dispensed. Using literature values ranging from 10% to 60% of medications becoming unused and with current household dis posal characteristics, it was estimated that active pharmaceutical ingredients comprised from 7.4 mg/kg to 45 mg/kg of municipal solid waste. In direct measurement, active pharmaceuticals comprised 8.1 mg/kg of the greater than 3 tons of municipal solid waste sorted. Additionally, 22 differing active pharmaceutical ingredients were identified with another 33 APIs identified as pot entially distributed into the solid waste through collection of empty containers. The remaining studies, three laboratory experiments and a survey of landfill leachate pharmaceutical compound concentration were designe d to determine the fate of pharmaceuticals in landfills and the potential of landfills to be a significant source of pharmaceuticals to the environment. Six pharmaceutical compounds were selected for laborat ory studies of their anaerobic degradation. In the initial experiment using only the pharmaceutical compound as a carbon source, both respirometric and direct analytical measurements showed only acetylsalicylic acid and metoprolol tartrate to have measureable degradation. The fate of each 177

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compound express as a percentage of the original sample content is give in Figure 9-1. Ibuprofen showed minor biodegr adation (7.10%) with a greate r reduction due to abiotic mechanisms (19.48%). Acetaminophen also showed minor degradation (11.10%) with no demonstrated losses through abioti c means while progesterone and 17 -ethinylestradiol demonstrated significant reduction due to adsorption or other abiotic mechanisms. In the second experiment, the degradation of the six the target compounds at lower concentrations with a common landfill organic substrate, cellulose, was examined. Inhibition of biological activity was demonstrated by none of the pharmaceuticals at the 500 g/L, 250 g/L or 50g/L test concentration. Acetylsalicy lic acid and progesterone showed significant reductions in concentration. The reduction in pr ogesterone was attributable to adsorption and other abiotic mechanisms while acetylsalicylic acid demonstrated considerable degradation. 17 -ethinylestradiol and metoprolol tartrate also displayed si gnificant abiotic reductions in concentration, however, these compounds returned to solution over time indicating desorption from the organic substrate over time and resistance to degrada tion. Ibuprofen and acetaminophen also showed minor initial losses and an increasing concentration over time but the overall reduction in concentration was much less than the prior compounds and were concluded to be undegraded. The final laboratory experiment utilizing sa mples comprised of a synthesized municipal solid waste in landfill leachate a llowed the best examination of the fate of the compounds in a MSW landfill due to inclusion of typical MSW co mponents. As in the prior experiment, the pharmaceutical compounds did not inhibit biological degradation of the MSW components. As can be seen in Figure 9-2, adsorption was confirmed as the predominant removal mechanism for 17 -ethinylestradiol due to the large percentage which was extracted from the solids fraction at 178

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the completion of the experiment. Progest erone on the other hand demonstrated a large percentage removal from solution. However, de termination of the exact mechanism for removal was not possible, but it is hypot hesized to be adsorption and other abiotic mechanisms. Ibuprofen demonstrated a decrease in con centration that based upon recover from the solids fraction is attributable to adsorption. Degradation of ib uprofen was not indicated as a possible mechanism due to near complete recove ry of the initially spiked ibuprofen at the completion of the experiment. Acetaminophen, while experiencing a small reduction in concentration in actively degrading waste, experi enced a dramatic reduction in waste previously degraded. Acetylsalicylic acid continued to demonstrate degradation in samples with active biological activity but had reduced removal rates in waste which had already passed the period of active degradation. Metoprolol tartrate demonstrated a potentia l mixture of degradation and adsorption in both waste types. The final study examined the landfill leachate concentrations of the six pharmaceutical compounds from the laboratory experiments plus an additional four compounds of interest. Ibuprofen, as expected due to the large quantity manufactured and dispensed in the United States and its resistance to biodegradation in laborator y studies, was detected most often. Ibuprofen was measured in 9 out of 10 landfill leachates and in the leachate of a solid waste transfer station and from a leachate pond on the face of a la ndfill. Other compounds detected included acetaminophen, acetylsalicylic acid (salicylic acid) and caffeine. A correlation between these compounds and measured chemical and physical properties of the leachate was not found. However, the landfills which had the greatest number of compounds detect ed were those of the largest size, serving the largest population. 179

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Conclusions This doctoral research examined the quant ity, identity, and fate of pharmaceutical compounds in municipal solid waste. While researchers have increa singly examined the fate and emission of pharmaceutical compounds at wastew ater treatment plants, only a few have examined the emission of these compounds from municipal solid waste landfills. Furthermore, no prior research has examined the fate of the targeted compounds w ithin MSW landfills. The following specific conclusions were reached: The national retail store pharmacy provided the greatest number of participants and medication quantity to the household medication waste collection program. Therefore, the participation of large national re tailers and drug store chains to allow greater convenience for consumers may result in greater particip ation in future co llection programs. Medications collected in the medication wast e collection program were primarily used by patients 50 years of age and above. Medicati on collection programs w ith limited resources may increase collection efficiency if targ eted to people 50 years of age and above. Newspaper advertisement was the most effective means of soliciting participation in the household medication waste collection pilot program. Survey of household medication waste pilot progra m participants showed that the percentage of unused medications disposed vi a municipal solid waste is equi valent to that disposed via the sewage system. The percentage of dispos ed via municipal solid waste is expected to increase with new management policies. The concentration of active pharmaceutical ingred ients in municipal solid waste is estimated to be between 7.4 and 45 mg/kg and is depende nt on the quantity of medications dispensed which become unused. Based on direct measuremen t, the quantity is expected to be near 8.1 mg/kg and the quantity of medicatio ns which become unused are 11% On a mass basis, nonsteroidal anti-inflammato ry medications and antibiotic medications comprise the largest percentage of active ph armaceutical ingredients in municipal solid waste. In a landfill, 17 -ethinylestadiol will be retained during initial waste degradation within the waste due to adsorption to the waste materials. Acetylsalicylic acid and its degradation product salicylic acid readily degrade under anaerobic municipal solid waste landfill conditions. 180

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Progesterone under anaerobic municipal solid wa ste disposal conditi ons displayed rapid removal from solution due to adsorption and other abiotic mechanisms. Metoprolol tartrate a nd acetaminophen demonstrated a mixture of biodegradation and abiotic removal mechanisms under anaerobic muni cipal solid waste landfill conditions. Due to resistance to anaerobi c degradation, the large mass di spensed and discarded, and low adsorption to solid waste materials, ibuprofen is expected to be a common contaminant of landfill leachate. The greatest numbers of pharmaceutical compounds were detected in the landfills serving the largest population. Based upon measured leachate ibuprofen concentr ations, landfill leachat e recirculation may result in higher concentrations of degradation resistant pharmaceutical compounds in landfill leachate due to accumulation or greater mobilization within the landfill. Landfill leachate treatment by wastewater treatment plants is not expected to be significant source in comparison to patient excretion a nd sewage disposal for the ten pharmaceutical compounds tested. Future Work The research conducted will provide informa tion pertinent to solid waste management regulators and industry professiona ls in determining measuring the effectiveness of current policies on the disposal of unused household medi cations. Several of the compounds such as progesterone and 17 -ethinylestradiol exhibited adsorption with the potential for later desorption following degradation of the organic substrate. However, the experiments did not permit sufficient time to measure this possibility. Ther efore, to determine the lifetime release potential of pharmaceutical compounds from landfills, long te rm municipal solid waste lysimeter studies would be of benefit. These would allow leach ate monitoring and releas e of the pharmaceutical compounds throughout each of the st ages of landfill degradation. In addition, an interesting follow-up research would include adsorption isot herms of the differing compounds to individual components of MSW. 181

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182 In terms of MSW pharmaceutical content, the re search was limited to a single time of year and a single location. Waste composition studies occurring during differing times of the year and at differing locations throughout the United States would allow a more comprehensive characterization to be completed. In addition, examination of specific sources such as MSW from hospitals and doctors offices and retirement communities would also provide practical information for collection programs and solid waste managers. In terms of landfill leachate c oncentrations, a national survey of landfill leachates should be conducted. The landfills surveyed in the current research were entirely within the state of Florida. Leachate production a nd characteristics are known to vary geographically across the United States and internationally with differing cl imates and waste generation characteristics. Analysis of these differing leachates would permit examination of the effects of these differences. Furthermore, research to lower the detection limits for pharmaceutical compounds in landfill leachates to parts per trillion concen trations is necessary to further research and detection of pharmaceuticals in landfill leachate and environments.

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Figure 9-1. Fate of selected pharmaceu tical compounds in anaerobic degradability testing using the pharmaceutical as the sole source of organic carbon 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% In Solution Biodegradation Removal Abiotic Removal183

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184 Figure 9-2. Fate of selected pharma ceutical compounds in landfill leachate under simulated anaerobic municipal solid waste landfill conditions. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%New Degraded New Degraded New Degraded New Degraded New Degraded New DegradedAcetaminophenAcetylsalicylic Acid Metoprolol Tartrate 17 ethinylestradiol ProgesteroneIbuprofen Sample Loss Extracted from Solids Leachate

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APPENDIX A METHOD FOR THE SELECTION OF PH ARMACEUTICAL COMPOUNDS FOR EXAMINATION OF LANDFILL ANAEROBIC BI ODEGRADABILITY AND PRESENCE IN MUNICIPAL SOLID WASTE LANDFILL LEACHATE Pharmaceutical Compound Selection Various methods have been used to identify pharmaceutical compounds with the greatest potential for release to the environment and previous researchers have commonly utilized prescription rate data (103-105) However, data c oncerning annual usage of medications is not easily translated into environmen tal input. Annual reports for drugs are often reported in units of total monetary sales. These are not intercha ngeable with compound mass as dosages and price per dose differ amongst medications. Other f actors include medication dispensed through internet sales and sales of medi cations under multiple trade names. Finally, while information on the sale and usage of prescrip tion medication is reasonably atta inable, determination of the distribution of nonprescription or over-the-counter (OTC ) medication quantities is problematic due to the multiple outlets for thei r sale, lack of scient ific literature, and cost of industry trade publications. To determine the six target pharmaceutical compounds for this research, a method similar to the prior studies was employed. The top 200 pr escribed pharmaceuticals in the United States for the years 2002 through 2004 were obtained from the industry trade website www.rxlist.com (64) These rankings were derived from the data of NDC Health, now Per-Se technologies, a leading health information technology provi der and were based upon the total number of prescriptions filled at retail pharmacies. Th e top 50 prescribed pharmaceuticals for 2002, 2003, and 2004 are shown in Table A-1. These years we re selected based upon an average age of discarded pharmaceuticals of 2 years and 11 m onths determined in the collection program 185

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described by Musson et al. (83) Thus, it was expected that pharmaceuticals prescribed from 2002 to 2004 would be commonly disposed during the research period. Each medication was assigned a cumulative sc ore based upon the sum of its individual rankings from each year. As an example, Lipitor (atorvastatin) was ranked #2 in each year and therefore would have a cumulative score of 6. The medications were then ranked based upon their cumulative score. The top 30 medications based upon their cumulative score are shown in Table A-2. Of these medications, hydrocodone, ambien, and alprazolam were removed from consideration due to their regul ation as controlled substances by the United States Drug Enforcement Agency, causing increased regul atory requirements for use in research. To determine the relative mass dispensed of th e thirty pharmaceuticals, the World Health Organization (WHO) defined daily dose (DDD) was used. The DDD was established as part of a classification system and unit of measure to al low international comparison in drug utilization studies. The DDD is the assumed average maintenance dose per day for a drug used for its main indication in adults (129) It should be emphasized that th e defined daily dose is a unit of measurement and does not necessarily reflect the recommended or prescribed daily dose. Doses for individual patients and patient groups often differ from the DDD based on individual characteristics. However, DDDs do provide a fixed unit of meas urement independent of price and formulation. With the number of prescriptions per pharm aceutical and the defined daily dose of each prescription, the total mass prescribed (TMP) was computed using equation A-1: TMP = N DDD DPP (A-1) TMP = total mass prescribed N = number of prescriptions DDD = Defined Daily Dose DPP = days per prescription. 186

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The days per prescription (DPP) were based upon the pharmaceutical type as previously utilized by Sedlak et al (86). Medications given on a one-tim e basis (e.g., an tibiotics) were assumed to include a sufficient number of doses to treat the ailment, a period of 10 days. For drugs administered on a continuing basis (e.g., beta -blockers, birth control pills) it was assumed that each prescription was renewed monthly with a DPP equal to 30 days. Using these values and the number of prescriptions for 2004, the top 20 of the prior 30 pharmaceuticals was determined based upon total mass prescribed and is shown in Table A-3. However, the 20 candidate medications do no account for nonprescription (Over-theCounter, OTC) sales of medica tions and it was not possible to identify a source of data concerning the total quantity of nonprescription me dications dispensed. Therefore, to include potential nonprescription medi cations, qualitative data for 2 004 from the annual report on medication use of the Sloan Epidemiology Center at Boston University was examined. The 2004 report estimated that nonprescription analge sics (acetaminophen, acetylsalicylic acid, and ibuprofen) are the most frequently used drugs in the United States, taken by 16-20% of U.S. adults (136) Table A-4 reproduces the findings of the report for twenty-five of the most commonly used prescription and OTC drugs taken by U.S. adults in 2004. Five of the top ten medications listed in the table are available OTC. Therefore OTC medications cannot be neglected. From tables A-3 and A-4, several compounds were selected for analysis. These compounds include ibuprofen, acetaminophen, and aspirin (acetylsalicylic acid), the three most common analgesics. These compounds have been detected in sewage treatment effluent and environmental samples (9, 14-16, 26) Due to the inclusion of these more common analgesic non steroidal anti-inflammatory drugs (NSAIDs) Celcoxib, another NSAID was not selected. 187

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The next three compounds selected for exam ination were the antib iotics amoxicillin, azithromycin, and cephalexin. Despite the absen ce of antibiotics from the Slone Epidemiology Report, these compounds comprised three of th e top six medications by prescribed mass. Furthermore, each was collected during pr evious pharmaceutical collection programs (83, 137) Development of antibiotic resist ance due to environmental releas e of pharmaceuticals has been the subject of several research studies (55, 138) Therefore, the inclusion of antibiotics is important in characterizing pharmaceuticals in landfills. The fifth and seventh most prescribed medi cines by relative mass dispensed include the beta-blockers metoprolol and atenolol. These compounds have also been detected in sewage treatment plant effluents and environmental samples (132, 139) Additionally, these compounds are commonly used and found in pharmaceutical collection programs (83, 136, 137) Therefore, metoprolol was selected as the target compound fo r this class of medicatio ns due to its greater estimated mass. The diuretics triamterene, furosemide, and hydrochlorothiazide were the eighth, eleventh, and thirteenth most prescribed medications by mass. Furosimide and hydrochlorothiazide have been widely observed in sewage and envi ronmental samples and are stable compounds (140) Additionally, each of these compounds were present during discarded pharmaceutical collection programs (83, 137) However, despite the greater prescribed mass of triamt erene, literature citing its environmental detecti on was not located and it was not among the top medications cited in the Slone Epidemiology Center report. Theref ore, hydrochlorothiazide, detected in several research studies and reported as commonly used was chosen as th e target compound of this class of pharmaceuticals. 188

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Finally, despite their low prescribed ma ss, prescription horm ones have received appreciable attention du e to their newly discovered effects on wildlife (141-143) With contraceptives now applied as dermal patches an d vaginal inserts disposed via household trash after use, these compounds become of even grea ter research interest. Therefore, the common contraceptive synthetic hormones, 17 -ethinylestradiol and progesteron e were selected as target compounds due to their environmen tal relevance and common usage (136) Additionally, due to its routine prescription and previous capture in a pharmaceutical collection program, the steroid prednisone, was included to complete the selection of targeted compounds (64, 137) Figure A-1 shows the chemical structur e of the selected compounds. MS Optimization Following selection of the target phar maceutical compounds, analytical method development using HPLC/MS/MS analysis wa s performed. Based upon the method detection limits of the compounds, six were selected for anaerobic degradation i nvestigation. The six pharmaceutical compounds selected for degrad ation study were acetaminophen, acetylsalicylic acid (measured as salicylic acid), 17 -ethinylestradiol, ibuprofen, me toprolol (tartrate salt), and progesterone. To determine the proper ionization mode fo r each target compound, individual standards of each compound were created at a concentration of 20 mg/L in water-methanol (1:1). 1 uL of each standard was directly injected (i.e., bypassi ng the LC column) with a flow rate of 0.4 ml/min in a solvent mixture comprised of a equal parts (50/50) 5mM ammonium formate in water and 5mM ammonium formate in methanol The MS was operated in full scan mode. Precursor ions with the largest response were id entified for each compound in each mode. Upon identification of the precursor ions, product ion s cans were conducted to identify the product ions of each compound and the parameters were optimized to achieve the maximum sensitivity for 189

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each compound when the instrument was operated in selected reaction monitoring. Table A-5 lists the SRM parameters determined for each compound. Of the target compounds, poor response was obtained for amoxicillin and azithromycin. As a result, azithromycin and amoxicillin were removed from consideration. In the positive ionization mode, all precu rsor ions were selected as [M+H]+. In the negative ionization mode, acetylsali cylic acid was detected as [M-C2H3O](i.e. salicylic acid) and prednisone formed an adduct with formate [M+CH2O2 H]-. The remaining compounds were detected as [M-H]-. In addition to the SRM conditions the source-dependent parameters were optimized for each ionization mode These are listed in Table A-6. 190

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Table A-1. Top 50 prescribed pharmaceuticals by total number of prescriptions, 2002-2004. Rank 2004 2003 2002 1 Hydrocodone w/Acetaminophen Hydrocodone w/Acetaminphen Hydrocodone w/Acetaminophen 2 Lipitor Lipitor Lipitor 3 Lisinopril Synthroid Atenolol 4 Atenolol Atenolol Synthroid 5 Synthroid Zithromax Premarin 6 Amoxicillin Amoxicillin Zithromax 7 Hydrochlorothiazide Furosemide Furosemide 8 Zithromax Hydrochlorothiazide Amoxicillin 9 Furosemide Norvasc Norvasc 10 Norvasc Lisinopril Hydrochlorothiazide 11 Toprol Xl Alprazolam Alprazolam 12 Alprazolam Zoloft Albuterol Aerosol 13 Albuterol Albuterol Aerosol Zoloft 14 Zoloft Toprol-XL Paxil 15 Zocor Zocor Zocor 16 Metformin Hcl Premarin Prevacid 17 Ibuprofen Prevacid Lbuprofen 18 Triamterene W/Hctz Zyrtec Triamterene/HCTZ 19 Ambien lbuprofen Toprol-XL 20 Cephalexin Levoxyl Cephalexin 21 Nexium Propoxyphene N/APAP Celebrex 22 Prevacid Triamterene/HCTZ Zyrtec 23 Lexapro Celebrex Levoxyl 24 Prednisone Ambien Allegra 25 Zyrtec Allegra Ortho Tri-Cyclen 26 Singulair Cephalexin Celexa 27 Celebrex Nexium Prednisone 28 Fluoxetine Hcl Fosamax Prilosec 29 Fosamax Vioxx Vioxx 30 Metoprolol Tartrate Singulair Claritin 31 Premarin Ortho Tri-Cyclen Fluoxetine 32 Levoxyl Prednisone Acetaminophen/Codeine 33 Lorazepam Metoprolol Tartrate Ambien 34 Allegra Fluoxetine Metoprolol Tartrate 35 Plavix Effexor XR Lorazepam 36 Effexor Xr Neurontin Fosamax 37 Potassium Chloride Lorazepam Propoxyphene N/APAP 38 Protonix Clonazepam Metformin 39 Propoxyphene Nap W/Apap Celexa Ranitidine HCl 40 Advair Diskus Viagra Amitriptyline 41 Warfarin Sodium Wellbutrin SR Viagra 42 Acetaminophen W/Codeine Paxil Prempro 191

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Table A-1. Continued Rank 2004 2003 2002 43 Clonazepam Pravachol Trimox 44 Neurontin Plavix Neurontin 45 Flonase Trimox Wellbutrin SR 46 Amitriptyline Hcl Potassium Chloride Pravachol 47 Ranitidine Hcl Protonix Augmentin 48 Trazodone Hcl Advair Diskus Nexium 49 Naproxen Flonase Accupril 50 Amox Tr-Potassium Clavulanate Metformin Lisinopril Source: WebMD Inc., Top 200 Drugs. http://www.rxlist.com/script/main/hp.asp (accessed Feburary 15, 2006) 192

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Table A-2. Top 30 prescribed pharmaceu ticals 2002-2004 based on cumulative rank Cumulative Score Compound N umber of Prescriptions in 2004 (83) Defined Daily Dose (DDD) (129) (mg) 3 Hydrocodone w/Acetaminophen 92,719,975 15/3000 6 Lipitor 69,766,431 10 11 Atenolol 44,162,229 75 12 Synthroid 44,056,176 0.15 19 Zithromax 37,171,754 300 20 Amoxicillin 41,393,538 1000 23 Furosemide 36,508,251 40 25 Hydrochlorothiazide 41,345,733 25 28 N orvasc 34,729,004 5 34 Alprazolam 32,404,743 -38 Albuterol 31,219,862 10 39 Zoloft 29,877,707 50 44 Toprol Xl (Metoprolol) 32,794,562 150 45 Zocor 27,234,005 15 52 Premarin 20,324,619 0.625 53 Ibuprofen 25,188,051 1200 55 Prevacid 23,628,587 30 58 Triamterene W/Hydrochlorothiazide24,616,014 100/25 65 Zyrtec 22,382,823 10 66 Cephalexin 23,665,172 2000 71 Celebrex 21,916,220 200 75 Levoxyl 19,760,520 0.15 76 Ambien 24,494,669 -83 Allegra 18,772,070 120 83 Prednisone 22,506,888 10 93 Fosamax 20,972,548 10 97 Metoprolol Tartrate 20,840,044 150 108 Singulair 22,020,478 10 114 Vioxx 13,226,546 35 122 Effexor Xr 18,574,507 100 193

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Table A-3. Top 20 pharmaceuticals for investig ation based on 2004 total mass prescribed Pharmaceutical Common Name Active Pharmaceutical Ingredient Pharmaceutical Category Estimated Total Mass Prescribed, 2004 (tons) Acetaminophen Acetaminophen NSAID 3066.19 Ibuprofen Ibuprofen NSAID 997.45 Cephalexin Cephalexin Antibiotic 520.63 Amoxicillin Amoxicillin Antibiotic 455.33 Metoprolol Metoprolol Beta Blocker 265.49 Celebrex Celcoxib NSAID 144.65 Zithromax Azithromycin Antibiotic 122.67 Atenolol Atenolol Beta Blocker 109.30 Triamterene Triamterene Diuretic 81.23 Allegra Fexofenadine Antihistamine 74.34 Effexor Xr Venlafaxine Antidepressant 61.30 Hydrochlorothiazide Hydrochlor thiazide Diuretic 54.42 Zoloft Sertraline Antidepressant 49.30 Furosemide Furosemide Diuretic 48.19 Prevacid Lansoprazole Proton Pump Inhibitor 23.39 Lipitor Atorvastatin Calcium Blood lipid 23.02 Vioxx Rofecoxib NSAID 15.28 Zocor Simvastatin Blood Lipid 13.48 Albuterol Salbutamol Bronchodilator 10.30 Prednisone Prednisone Corticosteroid 7.43 Note: Acetaminophen mass based on prescrip tions with hydrocodone. Hydrochlorothiazide total prescribed mass based upon sum of mass from prescrip tions in combination with triamterene and hydrochlorothiazide solely. 194

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Table A-4. Twenty-five most commonly taken prescription and nonprescription medications by U.S. adults in 2004 Medication Percentage of Survey Respondents Taking Medication during Previous Week Acetaminophen 20 Aspirin 19 Ibuprofen 16 Levothyroxine 6.2 Atorvastatin 6.1 Hydrochlorothiazide 5.8 Naproxen 5 Pseudoephedrine 4.8 Lisinopril 4.6 Metoprolol 4 Simvastatin 3.7 Atenolol 3.7 Metformin 3.5 Diphenhydramine 3.1 Amlodipine 3.1 Fluticasone 2.9 Albuterol 2.7 Furosemide 2.6 Fexofenadine 2.5 Alendronate 2.2 Conjugated estrogens 2.1 Hydrocodone 2.1 Celecoxib 2 Lansoprazole 1.8 Omeprazole 1.8 Source: Patterns of Medication Use in the United States 2004, A Report from the Slone Survey ; Slone Epidemiology Center at Boston University: Boston, MA, 2004. 195

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Table A-5. Selective reaction monitoring paramete rs utilized for target compounds in positive and negative ionization modes Compound Precursor Ion (m/z) Fragment Ion (m/z) Fragmentor Voltage (V) Collision Energy (V) Negative Ionization Acetylsalicylic acid 137.1 93.1 80 15 Hydrochlorothiazide 296.0 268.9 160 10 Prednisone 403.2 327.2 100 10 Ibuprofen 205.2 161.1 60 0 Positive Ionization Acetaminophen 152.1 110.2 100 17 Cephalexin 348.2 106.1 100 20 Metoprolol 268.0 116.2 140 15 EE2 297.2 107.1 120 23 Progesterone 315.3 97.2 80 18 Table A-6. Source parameters us ed for electrospray ionization Parameter Positive Ionization Negative Ionization Gas Temperature (C) 350 350 Gas Flos (L/min) 8 13 Nebulizer pressure (psi) 60 50 Capillary (V) 3500 3500 196

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AB C D E G H I J K F Figure A-1. Selected pharmaceuticals for exam ination in degradation and MSW leachate concentration studies. A) Acetaminophen B) Acetylsalicylic Acid (Aspirin) C) Amoxicillin D) Azithromycin E) Ce phalexin F) Ibuprofen G) 17 -ethinyl estradiol H) Hydrochlorothiazide I) Prednisone J) Progesterone K) Metoprolol (Tartrate) 197

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APPENDIX B US EPA EPI SUITE BIOWIN ANAERO BIC BIODEGRADATION MODEL RESULTS Table B-1. Complete anaerobic biodeg radation model results for acetaminophen ------+-----+------------------------------------------+---------+--------TYPE | NUM | Biowin7 FRAGMENT DES CRIPTION | COEFF | VALUE ------+-----+------------------------------------------+---------+--------Frag | 1 | Aromatic alcohol [-OH] | 0.0807 | 0.0807 Frag | 1 | Amide [-C(=O)-N or -C(=S)-N] | -0.5679 | -0.5679 Frag | 4 | Aromatic-H | -0.0954 | -0.3817 Frag | 1 | Methyl [-CH3] | -0.0796 | -0.0796 Const| | Equation Constant | | 0.8361 ============+============ ==================== ============+===== RESULT | Biowin7 (Anaerobic Linear Biodeg Prob) | | -0.1124 ============+============ ==================== ============+===== Table B-2. Complete anaerobic biodeg radation model results for metoprolol ------+-----+------------------------------------------+---------+--------TYPE | NUM | Biowin7 FRAGMENT DES CRIPTION | COEFF | VALUE ------+-----+------------------------------------------+---------+--------Frag | 1 | Aliphatic alcohol [-OH] | 0.1328 | 0.1328 Frag | 1 | Aliphatic amine [-NH2 or -NH-] | 0.1773 | 0.1773 Frag | 1 | Aromatic ether [-Oaromatic carbon] | 0.1780 | 0.1780 Frag | 1 | Aliphatic ether [C -O-C] | -0.2573 | -0.2573 Frag | 1 | Alkyl substitue nt on aromatic ring | -0.1145 | -0.1145 Frag | 1 | Aromatic-CH2 | -0.0073 | -0.0073 Frag | 4 | Aromatic-H | -0.0954 | -0.3817 Frag | 3 | Methyl [-CH3] | -0.0796 | -0.2387 Frag | 3 | -CH2[linear] | 0.0260 | 0.0780 Frag | 2 | -CH[linear] | -0.1659 | -0.3317 Const| | Equation Constant | | 0.8361 ============+============ ==================== ============+===== RESULT | Biowin7 (Anaerobic Linear Biodeg Prob) | | 0.0709 ============+============ ==================== ============+===== 198

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Table B-3. Complete anaerobic biodegrada tion model results for acetylsalicylic acid ------+-----+------------------------------------------+---------+--------TYPE | NUM | Biowin7 FRAGMENT DES CRIPTION | COEFF | VALUE ------+-----+------------------------------------------+---------+--------Frag | 1 | Aromatic acid [-C(=O)-OH] | 0.2656 | 0.2656 Frag | 1 | Ester [-C(=O)-O-C] | 0.1719 | 0.1719 Frag | 4 | Aromatic-H | -0.0954 | -0.3817 Frag | 1 | Methyl [-CH3] | -0.0796 | -0.0796 Const| | Equation Constant | | 0.8361 ============+============ ==================== ============+===== RESULT | Biowin7 (Anaerobi c Linear Biodeg Prob) | | 0.8122 ============+============ ==================== ============+===== Table B-4. Complete anaerobic biod egradation model results for ibupofen ------+-----+------------------------------------------+---------+--------TYPE | NUM | Biowin7 FRAGMENT DES CRIPTION | COEFF | VALUE ------+-----+------------------------------------------+---------+--------Frag | 1 | Aliphatic acid [-C(=O)OH] | 0.1868 | 0.1868 Frag | 2 | Alkyl substitue nt on aromatic ring | -0.1145 | -0.2289 Frag | 1 | Aromatic-CH2 | -0.0073 | -0.0073 Frag | 1 | Aromatic-CH | 0.0331 | 0.0331 Frag | 4 | Aromatic-H | -0.0954 | -0.3817 Frag | 3 | Methyl [-CH3] | -0.0796 | -0.2387 Frag | 1 | -CH[linear] | -0.1659 | -0.1659 Const| | Equation Constant | | 0.8361 ============+============ ==================== ============+===== RESULT | Biowin7 (Anaerobic Linear Biodeg Prob) | | 0.0334 ============+============ ==================== ============+===== 199

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200 Table B-5. Complete anaerobic bi odegradation model results for 17 -ethinylestradiol ------+-----+------------------------------------------+---------+--------TYPE | NUM | Biowin7 FRAGMENT DES CRIPTION | COEFF | VALUE ------+-----+------------------------------------------+---------+--------Frag | 1 | Aromatic alcohol [-OH] | 0.0807 | 0.0807 Frag | 2 | Carbon with 4 singl e bonds & no hydrogens | -0.3342 | -0.6685 Frag | 2 | Alkyl substituent on aromatic ring | -0.1145 | -0.2289 Frag | 1 | Aromatic-CH2 | -0.0073 | -0.0073 Frag | 1 | Aromatic-CH | 0.0331 | 0.0331 Frag | 3 | Aromatic-H | -0.0954 | -0.2863 Frag | 1 | Methyl [-CH3] | -0.0796 | -0.0796 Frag | 5 | -CH2[cyclic] | -0.1200 | -0.6001 Frag | 2 | -CH [cyclic] | 0.0395 | 0.0789 Const| | Equation Constant | | 0.8361 ============+============ ==================== ============+===== RESULT | Biowin7 (Anaerobic Linear Biodeg Prob) | | -0.8418 ============+============ ==================== ============+===== Table B-6. Complete anaerobic biodeg radation model results for progesterone ------+-----+------------------------------------------+---------+--------TYPE | NUM | Biowin7 FRAGMENT DES CRIPTION | COEFF | VALUE ------+-----+------------------------------------------+---------+--------Frag | 2 | Carbon with 4 si ngle bonds & no hydrogens | -0.3342 | -0.6685 Frag | 2 | Ketone [-C-C(=O)-C-] | -0.3919 | -0.7838 Frag | 3 | Methyl [-CH3] | -0.0796 | -0.2387 Frag | 8 | -CH2[cyclic] | -0.1200 | -0.9601 Frag | 4 | -CH [cyclic] | 0.0395 | 0.1578 Frag | 1 | -C=CH [alkenyl hydrogen] | -0.0735 | -0.0735 Const| | Equation Constant | | 0.8361 ============+============ ==================== ============+===== RESULT | Biowin7 (Anaerobic Linear Biodeg Prob) | | -1.7307 ============+============ ==================== ============+===== A Probability Greater Than or Equal to 0.5 indicates --> Biodegrades Fast A Probability Less Than 0.5 indi cates --> Does NOT Biodegrade

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APPENDIX C QUALITY ASSURANCE / QUALITY CONTROL Quality Control Sample Identification For each sample a unique identification numbe r was given. The identification number was comprised of a letter equivalent to the experiment al test method (A,B, or C), a single number for the time period (in weeks) the sample was to be collected, a three letter abbreviation for the PPCP of the sample (ex. IBU = Ibuprofen), and a unique number identifier for the sample. As an example, a set of triplicate samples of Ibuprof en samples to be collected at 4 weeks of experiment B may be labeled B4-Ibu-1, B4-Ibu-2, and B4-Ibu-3. Gas Analysis Gas concentrations were measured on a gas chromatograph TCD/FID instrument (Agilent 6980N). Gas concentration calibrations were obtained using calibration gases of 99.99% methane (Linde), 99.999% hydrogen (Linde) a nd 99.996% carbon dioxide (Linde) diluted by laboratory air. Seven calibration points were ut ilized and the individu al gas compositions are shown in Table C-1. Linear regression analysis of the calibration curves was required to be a minimum of 0.99. Laboratory experience has show n that the calibration curve can remains valid for several days when the GC is not completely shut down. Thus it was possible to use the same calibration curve over several days. Whenever d eemed necessary, for example in the event of a power failure or a dramatic decrease in the carr ier gas pressure, a new calibration was created but no curve was used for more than 72 hours of sampling. Calibration was monitored through the use of continuing calibrati on verification (CCV) samples performed once for every 12 samples. The calibration verificatio n was required to be 201

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within 10 % of the initial calibration value for me thane and carbon dioxide. Additionally, replicate gas sample analysis was performed once for every 12 samples. Chemical Analysis LC analysis was performed with an Agilent 1200 Series rapid re solution LC system coupled to an Agilent 6410 Triple Quad MS /MS (Palo Alto CA, USA) equipped with an orthogonal ESI interface. For analys is of samples containing ibuprofen and acetylsalicylic acid, compounds were separated at 40 C by means of a Zorbax extended C18, RRHT column (2.1 mm 100 mm, 1.8 m) from Agilent. The injec tion volume was 1 L with a flow rate maintained at 0.4 ml/min. The mobile phase was comprised of a mixture of (A) 5mM ammonium formate in water and (B) 5mM ammonium format e in methanol. The mobile phase solvent gradient started with 5% of solvent B and was increased to 90% solvent B evenly over 12.0 minutes. The solvent mixture was returned to th e initial proportions following completion of sample analysis at 12.1 minutes and a post-run tim e of 2 minutes was requ ired to re-equilibrate the column. The analysis of samples c ontaining acetaminophen, metoprolol tartrate, 17 ethinylestradiol, and progesterone was complete d without chromatography. A volume of 1L of each sample was directly injected (bypassing the LC column) with a flow rate of 0.4 ml/min in a mobile phase solvent mixture comprised of 50% of solvent B and 50% of solvent A. In experiment A (Chapter 5) and B (Chapter 6), calibration standard s were created using anaerobic digester sludge nutrient solution, the test sample matrix. In experiment C (Chapter 7), calibration standards were created using landfill leachate, the test sample matrix with the exception of ibuprofen which was present in back ground concentrations within the leachate. For ibuprofen samples of experiment C and the landf ill leachate analysis of Chapter 8, the method of standard additions was utilized to quantify compound concentrations. 202

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203 A new calibration curve was created for each an alytical batch. A minimum of a 5 point calibration curve was created for each compound with concentrations ranging from 10 to 5000 ppb. Samples with concentrations greater than the maximum calibration point were diluted with sample matrix as necessary. Calibration was monitored through th e use of calibration verification samples every six samples. The calibration check sample was required to be within % of the initial sample value. Instrument blanks to monitor potential carryover between injections were analyzed prior to each calibrati on check. For samples in which carryover was indicated, one to three system washes were plac ed between analytical samples and the samples reanalyzed. Table C-1. Degradation gas analysis calibration curve data points Calibration point Methane (%) Carbon dioxide (%) Hydrogen (%) Air (%) 1 42.8 42.8 11.4 3.00 2 15.0 15.0 4.00 66.0 3 5.00 5.00 1.33 88.7 4 1.67 1.67 0.444 96.2 5 0.556 0.556 0.148 98.7 6 0.278 0.278 0.074 99.4 7 0.139 0.139 0.037 99.7

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BIOGRAPHICAL SKETCH Stephen Musson received his Bachelor of Scie nce degree in chemistry from the University of Florida in 1991 and his Master of Science degr ee in environmental engi neering sciences from the University of Florida in 2000. From 1991 to 1996 he served as a nucl ear power and surface warfare officer in the United States Navy. He initially worked as an assistant to the Environmental Engineer at the Naval Training Center, Orland o, FL before completing his training and proceeding to his duties onboard the USS Mississippi. His assignments included supervision of personnel in the operation and maintenance of two shipboard nuclear reactors and steam propulsion plants, training personnel in nuclear plant oper ations and shipboard damage control, and helicopter flight operations officer. From 1996 to 2005, Stephen worked for the University of Florida as the Hazardous Waste Coor dinator, supervising the handling and disposal of hazardous materials for the universitys labora tories and statewide research centers. Stephen became a Certified Hazardous Materials Manage r (CHMM) in 2001 and a Certified Industrial Hygienist (CIH) in 2002. 216