Transformation and fate of dissolved organic matter originating in the Suwannee River watershed

MISSING IMAGE

Material Information

Title:
Transformation and fate of dissolved organic matter originating in the Suwannee River watershed a stable isotope approach
Physical Description:
xii, 158 leaves : ill. ; 29 cm.
Language:
English
Creator:
Hall, Emily R., 1976-
Publication Date:

Subjects

Subjects / Keywords:
Environmental Engineering Sciences thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Environmental Engineering Sciences -- UF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 2004.
Bibliography:
Includes bibliographical references.
Statement of Responsibility:
by Emily R. Hall.
General Note:
Printout.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 024496949
System ID:
AA00014271:00001


This item is only available as the following downloads:


Full Text











TRANSFORMATION AND FATE OF DISSOLVED ORGANIC MATTER
ORIGINATING IN THE SUWANNEE RIVER WATERSHED: A STABLE ISOTOPE
APPROACH














By

EMILY R. HALL


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

UNIVERSITY OF FLORIDA


2004














ACKNOWLEDGMENTS

Financial support for this work was provided in part, by the USDA (Grant # 2001-

35102-09857), NSF (Grant # OCE0240187), the University of Florida Women's Club,

and the University of Florida.

Everyone who I have had contact with in any way throughout my time at the

University of Florida has influenced my life in some way. I thank them all from the

bottom of my heart, even though I know simple thanks are not enough. I must

acknowledge them to the best of my ability on paper, but know that they all mean the

world to me.

First I must start with my family. My parents have been through thick and thin

with me, and I could not have done well without their support, both financially and

emotionally. My sister has been an ear for me to talk to, and she has taught me to live

free. My brother has been a source of energy, and he has taught me to enjoy life while

still working hard.

My mentors have been crucial in my academic journey. Dr. Brian Rood gave me

the first opportunity to pursue a career in environmental science. His enthusiasm and

love for the environment as well as his energy guided me towards this field of science.

Dr. Joseph Delfino, my committee chair for the past five and a half years, took over

where Dr. Rood left off. He guided me through tough times, scientifically and

administratively, and he has had continued faith in my abilities. Dr. Tom Frazer, my co-

chair, willingly provided me with a project without prior knowledge of my capabilities.








He allowed me to learn from my mistakes and he pushed me to become a better scientist.

I also acknowledge my committee members Dr. Jean-Claude Bonzongo and Dr. Mark

Brenner for their mentoring and involvement in my graduate education.

My peers have also had a large influence during my time at the University of

Florida. Unfortunately (but fortunately) this group is the largest, and I cannot mention

everyone individually. First I will start with my research groups in Black Hall and at the

Department of Fisheries and Aquatic Sciences. They provided me with friendships that I

will never forget as well as inspiration to work hard in science and in life. I also thank

the University of Florida Club Soccer program. It gave me an opportunity to continue to

play the sport I love, and to have some of the best friendships. Lastly, I thank Jason

Heggerick for being my strength and support during demanding times. He was a

shoulder to cry on and a friend to laugh with.

"The sea lies all about us. The commerce of all lands must cross it. The very

winds that move over the lands have been cradled on its broad expanse and seek ever to

return to it. The continents themselves dissolve and pass to the sea, in grain after grain of

eroded land. So the rains that rose from it return again in rivers. In its mysterious past it

encompasses all the dim origins of life and receives in the end, after, it may be, many

transmutations, the dead husks of that same life. For all at last returns to the sea-to

Oceanus, the ocean river, like the ever-flowing stream of time, the beginning and the

end" (Rachel Carson, The Sea Around Us). There are no limits for journeys of the mind.














TABLE OF CONTENTS

page

ACKNOW LEDGM ENTS ............................................................ .......................... ii

L IST O F TA B L E S ........................................................................ ............................. vi

LIST O F FIG U R E S ...................................................................... .............................. vii

A B STR A C T ....................................................................... ......................................... x

CHAPTER

S IN TR O D U C TIO N ................................................................. ..............................

B ackground................................... ...................................................................
Suwannee River and Estuary................................................... .........................
Nutrient Loading, Especially Nitrogen........................ ......................... 4
Clam and Oyster Ecology..................................................... ............................6
H um ic Substances.............................. ............................................................... 8
Flocculation .......................................................................................................... 11
Stable Carbon and Nitrogen Isotopes as Tracers of Material Transfer and Energy
Flow ......................... ..... ................................................................... 19
Proposed Research....................... ... .................... ............................. 29

2 STABLE CARBON AND NITROGEN ISOTOPE COMPOSITION OF ORGANIC
AGGREGATES PRODUCED BY SALINITY INDUCED FLOCCULATION OF
DISSOLVED ORGANIC MATTER FROM THE SUWANNEE RIVER................30

Introduction....................... ................................................................... 30
M methods and M materials ....................................................... ............................ 34
Site............................................................... ..............................34
Methods ...................................................................................... 35
DOC and Stable Isotope Analysis........................................ .........................36
Results............... ... ....................................................... 36
Discussion.................. ............................................................................. 46
Summary................ .......................................................................50
Conclusions................ .......................................................................50

3 SPATIAL GRADIENTS IN THE STABLE CARBON AND NITROGEN ISOTOPE
COMPOSITION OF SUSPENDED PARTICLES IN THE SUWANNEE RIVER
ESTUARY DURING DIFFERENT FLOW REGIMES............................................ 51








Introduction......................................................................... ..................................5 1
Methods, Materials, and Site Description..................... ........... ................53
Site D description ........................................ ..............................................53
M methods ......................................................................... ...............................54
R esults............................................................................... ....................................59
Physical Parameters......................... .......................... .........................59
Chemical Parameters..................... .......................................59
Biological Indicator.......................... ..................................65
Stable Isotope Analyses........................... ...... .........................65
Statistics....................................................................... ..................................77
D discussion .......................................................................... ...................................77
Sum m ary ............................................................................. .................................. 94
C onclusions......................................................................... ..................................95

4 STABLE ISOTOPES AS TRACERS IN FOOD WEB INVESTIGATIONS OF THE
SUWANNEE RIVER ESTUARY ................................... ........................96

Introduction.......................................................................... ................................. 96
M methods and M materials ........................................................ ........................... 99
Site D description ........................................................... ............................99
Sampling........................... ............................................100
Stable Isotope Analyses........................... ...... .........................101
R esults..................................................... ........................... ........................ 101
D discussion ......................................................................... ..................................103
Stable Carbon Isotopes..................... ....... .......................103
Stable Nitrogen Isotopes............................................. ............ 112
Multiple Stable Isotopes and Food Source....................................................112
Sum m ary ............................................................................. ................................118
C onclusions........................................................................ .................................118

5 SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS .........................120

Sum m ary ........................................................................... ..................................120
Conclusions............................. ....... ...................................................121
Recommendations for Further Research ........................................................ 122

APPENDIX

A SUPPLEMENTAL STABLE CARBON AND NITROGEN ISOTOPE DATA.....123

B SUPPLEMENTAL SIZE AND WEIGHT DATA OF CLAMS ............................133

C SUPPLEMENTAL SIZE AND WEIGHT DATA OF OYSTERS .......................... 140

LIST OF REFERENCES.................. ............................................................143

BIOGRAPHICAL SKETCH............................................................. ........................158














LIST OF TABLES


Table page

2-1. Analysis of Variance (ANOVA) Results for all of the Floc Studies......................38

3-1. Location and Site Names for Sampling Sites in the Suwannee River and its Estuary.57

3-2. Chemical and Biological Parameters in the Suwannee River and its Estuary for Each
Sam pling Event. .......................................... ...............................................60

3-3. Concentrations of Nitrogen Components (ig/L) in the Suwannee River and its
Estuary for Each Sampling Event. ................................ .........................63

3-4. Concentrations of Phosphorus Components (pg/L) in the Suwannee River and its
Estuary for each Sampling Event.................................... .................................64

3-5. Stable Isotope Data of Suspended Particulate Organic Matter (SPOM) and
Dissolved Inorganic Nitrogen (DIN) for the 1996, 1997, and Two 2003 Sampling
Events. ............................................................. ........... ......................... 68

3-6. ANOVA Tables for the Effects of Salinity on the i'5N and 13C of Suspended
Particulate Organic Matter during Novermber 1996................................... ...80

3-7. ANOVA Tables for the Effects of Salinity on the 5'5N and 13C of Suspended
Particulate Organic Matter during March 1997......................................................81

3-8. ANOVA Tables for the Effects of Salinity on the Physical, Chemical, and
Biological Indicators of Suspended Particulate Organic Matter during December
2003 ....................................................... ............................. ............................82

3-9. ANOVA Tables for the Effects of Salinity on the Physical, Chemical, and
Biological Indicators of Suspended Particulate Organic Matter during May 2003.84

A-1. Stable Carbon and Nitrogen Isotopes of Clams, Oysters, and Suspended Particulate
Organic Matter (SPOM)..........................................................123

B-1. Size (mm) and Weight (g) Data of Clams. ....................... .........................133

C-l. Size (mm) and W eight (g) of Oysters..................................... .......................... 140














LIST OF FIGURES


Figure page

2-1. Salinity versus Dissolved Organic Carbon in the Upper and Lower Suwannee River
Floe Studies................................... .............................. .............. ...............39

2-2. Time versus Dissolved Organic Carbon for Floc Studies I and II...........................40

2-3. Salinity versus 615N for the Upper and Lower Suwannee River Flocculated Organic
A ggregates............................................ ........................................................ 41

2-4. Salinity versus 813C for the Upper and Lower Suwannee River Flocculated Organic
A ggregates................................................ ..........................................................42

2-5. Salinity versus Average 615N Signatures of the Upper and Lower Suwannee River
Floe Studies Including Standard Deviation Error Bars..........................................43

2-6. Salinity versus Average 6SC Signatures of the Upper and Lower Suwannee
River Floe Studies Including Standard Deviation Error Bars................................44

2-7. Salinity versus C:N Ratios of the Flocculated Organic Aggregates from the Upper
and Lower Suwannee River Floe Studies Including Standard Deviation Error
B ars............................................ ........ ................................. ............................... 45

3-1. Map of the Sampling Sites in the Suwannee River and its Estuary........................56

3-2. Monthly Streamflow Data for the Suwannee River near Wilcox, Florida (USGS,
2004).............................................................................. ....................................6 1

3-3. 615N Signatures of Surface Suspended Particulate Organic Matter (SPOM) for the
1996 and 1997 Sampling Events................................ ................................66

3-4. S&5N Signatures of Surface Suspended Particulate Organic Matter (SPOM) for the
Two 2003 Sampling Events. ........................ .......... ..........................67

3-5. 815N Signatures of Surface Dissolved Inorganic Nitrogen (DIN) for the March 1997
Sam pling E vent. ..................................................................... .................................70

3-6. S15N Signatures of Surface Dissolved Inorganic Nitrogen (DIN-Nitrate) for the 2003
Sam pling Events................................................................................................. 71








3-7. '15N Signatures of Zooplankton Collected during the May 2003 (High Flow)
Sampling Event (Cope = Calanoid Copepods, Chaet = Chaetognaths, Cten =
Ctenophores). .....................................................................................................72

3-8. 6'5N Signatures of Zooplankton Collected during the December 2003 (Low Flow)
Sampling Event (Cope = Calanoid Copepods, Chaet = Chaetognaths, Cten =
Ctenophores). ............................................................... ..................................73

3-9. Stable Carbon and Nitrogen Isotopes of Oysters Collected During the December
2003 Sam pling Event. .................................................. .............................74

3-10. 813C of the Suspended Particulate Organic Matter (SPOM) for the 1996 and 1997
Sam pling Events.................................................... ..................................... 75

3-11. S63C of the Suspended Particulate Organic Matter (SPOM) for the Two 2003
Sam pling Events........................................ ... .......................................... 76

3-12. 63C of Zooplankton Collected During the May 2003 Sampling Event (Cope =
Calanoid Copepods, Chaet = Chaetognaths, Cten = Ctenophores)........................78

3-13. 86"C of Zooplankton Collected During the December 2003 Sampling Event (Cope
= Calanoid Copepods, Chaet = Chaetognaths, Cten = Ctenophores). .....................79

3-14. Model of Spatial Gradients in the 1'5N Signatures of a Hypothetical Closed System
(redrawn from Montoya and McCarthy, 1995).......................................................89

3-15. Theoretical Curves Illustrating Spatial Variation in the f15N of Phytoplankton
along an Onshore/Offshore Gradient. Losses in the Bottom Panel Occur as a
Consequence of Grazing and Sedimentation Processes.........................................90

3-16. 615N vs. 613C of Oysters, Zooplankton, and SPOM for the December 2003
Sam pling Event.............................................................. .............................91

3-17. ~'N vs. 613C of Oysters, Zooplankton, and SPOM for the May 2003
Sampling Event..................... ...............................................................................92

4-1. Map of Sampling Sites for Clams, Oysters, and Suspended Particulate Organic
Matter (SPOM) in the Suwannee River Estuary ...............................................102

4-2. 61C of Suspended Particulate Organic Matter (SPOM) from Two Suwannee River
Estuary Sites......................... ........................ .........................104

4-3. 615N of Suspended Particulate Organic Matter (SPOM) from Two Suwannee River
Estuary Sites...................................... ...... .............. ..........................105

4-4. 6"C of Clams from Two Suwannee River Estuary Sites......................................106








4-5. 13C of Oysters from Two Suwannee River Estuary Sites....................................107

4-6. 615N of Clams from Two Suwannee River Estuary Sites......................................109

4-7. 615N of Oysters from Two Suwannee River Estuary Sites.................................... 10

4-8. 613C vs. Dry Body Weight (g) of Clams from Two Suwannee River Estuary
Sites.................................................................................. ................................. 113

4-9. 13C vs. Dry Body Weight (g) of Oysters from Two Suwannee River Estuary
Sites...................... ............... ......................... .............. ............. ............ .. 114

4-10. 815N vs. Dry Body Weight (g) of Clams from Two Suwannee River Estuary
Sites................................................................................. ................................. 115

4-11. s65N vs. Dry Body Weight (g) of Oysters from Two Suwannee River Estuary
Sites............................................ .............................. ................................... 16

4-12. The Suwannee River Estuary Food Web Structure Using 5"C and 65N............. 117













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
TRANSFORMATION AND FATE OF DISSOLVED ORGANIC MATTER
ORIGINATING IN THE SUWANNEE RIVER WATERSHED: A STABLE ISOTOPE
APPROACH

By

Emily R. Hall

December 2004

Chair: Joseph J. Delfino
Cochair: Thomas K. Frazer
Major Department: Environmental Engineering Sciences

Anthropogenic activities in a watershed can have significant impacts on the

ecology of downstream coastal environments. Surface runoffand groundwater inputs

from the Suwannee River watershed contribute high concentrations of nutrients (nitrates

>1 mg/L) to this blackwater river before it discharges into the Gulf of Mexico. High

concentrations of nutrients have the potential to affect the sustainability, productivity, and

growth of the oyster and clam aquaculture industries in the estuary. In determining

detrimental effects, it is important to establish a link between dissolved carbon and

nitrogen, suspended particulate organic matter (SPOM), and production of clam and

oyster biomass in the Suwannee River estuary.

Organic aggregates (a possible food source for the bivalves in the Suwannee River

estuary) were produced in the laboratory by adding salts to unfiltered fresh organic-

enriched Suwannee River surface water. They were analyzed for stable carbon and

nitrogen isotopes and the results indicate that the 615N signatures can be used as a tracer








of organic aggregate development. More importantly, however, the results suggest that

3"C signatures of organic aggregates can be used to quantify their role as a food source

for estuarine fauna.

Spatial gradients in nutrient concentrations, particularly nitrate, are characteristic of

estuarine systems. These gradients occur as a result of mixing processes and uptake and

assimilation by phytoplankton. Isotopic fractionation associated with this uptake and

assimilation can generate strong spatial gradients in the stable nitrogen isotope

composition of particulate nitrogen. In essence, stable nitrogen isotopes serve as in situ

tracers of the processing of nitrogen as it moves through an estuarine system.

Clams and oysters were seeded in the Suwannee River estuary near the river

mouth. Clams, oysters, and SPOM were sampled monthly for approximately one year

and subsequently analyzed for stable carbon and nitrogen isotopes. The results indicate

that the clams consumed the same source of organic material throughout the year (marine

SPOM), while the oysters' food source changed consistently with hydrologic patterns in

the river (e.g., more terrestrial origin during high flow and more marine origin during low

flow). Neither clams nor oysters consumed flocculated organic aggregates as a dominant

food source.













CHAPTER 1
INTRODUCTION

Background

Anthropogenic activities in a watershed can have significant impacts on the

ecology of downstream coastal environments. The Suwannee River drains 28,000 km2 of

southern Georgia and north central Florida (Wolfe and Wolfe, 1985). Surface runoff and

groundwater inputs contribute high levels of nutrients to this blackwater river before it

discharges into the region of the Gulf of Mexico known as the Big Bend. This region of

Florida has a relatively low population density; however, there have been significant

increases in a variety of agricultural activities in the river basin. Nitrogen-enriched run-

off from these agricultural areas filters into the groundwater because of the porous nature

of the karst sediments characteristic of the region. Ultimately, these increases in

groundwater nitrogen are manifested as higher nitrate concentrations in the river, due to

numerous spring inflows. Nitrate levels within the Suwannee River are increasing and

concentrations near the mouth and in the upper reaches of the estuary now regularly

exceed 1 mg/L (Phlips and Bledsoe, 1997). This has brought on widespread concern

about the potential consequences of cultural eutrophication of the region on the

ecological health of the river and the coastal waters of the Big Bend.

Suwannee River and Estuary

The Suwannee River is a blackwater, tidally influenced river with its origin in the

Okefenokee Swamp in Georgia. The river is approximately 394 km in length, flows

southwest through Florida, and discharges into the Gulf of Mexico just north of Cedar








Key. Tributaries include the Alapaha, Withlacoochee, and Santa Fe Rivers. At the

mouth of the river there is a delta region which is characterized by a network of intertidal

and subtidal oyster reefs (Wolfe and Wolfe, 1985).

The geographical features of the Suwannee River and the Okefenokee Swamp

include flat terrace, the Trail Ridge sand barrier, and the Hawthorne Formation (Malcom

et al., 1989a). There are also shallow clay lenses near the headwaters of the river. Much

of the river and its watershed are underlain by karst limestone, allowing nutrients to leach

into the aquifer and ultimately enter the Suwannee River through the groundwater springs

along its banks (Crandall et al., 1999; Bledsoe and Phlips, 2000). Water is mixed

between the river and aquifer primarily through springs and diffuse groundwater seepage

(Pittman et al., 1997).

The water quality of the upper Suwannee River is indicated by low pH, low

concentration of dissolved inorganic solids, and high concentration of dissolved organic

substances, especially fulvic acids (Malcolm et al., 1989a). The lower Suwannee River

maintains a higher pH (6 to 9) (Wolfe and Wolfe, 1985). The source of the humic

substances in the water is predominantly fresh litter, leaf, and root exudates, and leaf

leachates. Very little of the colored dissolved organic matter (CDOM) is due to older and

more slowly decaying peat (Malcolm et al., 1989a). The concentration of these humic

substances has been shown to be an order of magnitude greater than that in most natural

stream waters. The dissolved organic carbon concentrations are in excess of 25 mg/L

(Malcolm et al., 1989b). High nutrient concentrations are also present in the river due to

the large amounts of fertilizer entering the system through groundwater and overland

flow (Suwannee River Water Management District, 2003).








The climate along the river is characterized by long, warm summers and short, mild

winters (Malcolm et al., 1989a; Crandall et al., 1999; Katz et al., 2001). Peak mean daily

discharge in the river usually occurs in March or April, and September due to continental

frontal systems (Crandall et al., 1999); however, discharge rates are generally fairly

uniform throughout much of the year (Malcolm et al., 1989b). A large source of water

for the Okefenokee Swamp, and hence the Suwannee River, is rainfall. Overland flow

and groundwater (via numerous springs) also contribute to the amount of water flowing

through this system (Wolfe and Wolfe, 1985; Crane, 1986). During the months of heavy

rains, the river flows more rapidly, and there is generally a higher organic content as a

result of drainage from the swamp and watershed to the river. When precipitation is low,

water is contributed to the river through springs and diffuse seepage fed by the Floridan

Aquifer (Crane, 1986).

There is much less land development and point source pollution in this river and its

plume than in most other coastal regions in Florida (Bass and Cox, 1988). Major land

uses in the watershed include forestry and agriculture (row crops, dairy, poultry, swine

farming operations, and pasture) (Katz et al., 2001). In 1995, wetlands comprised about

2% to 36% of the land use in Suwannee and Lafayette Counties (Katz et al., 2001).

Asbury and Oaksford (1997) characterized the watershed in the Suwannee basin as

50.4% forested, 15.9% wetland, 31.2% agriculture, and 0.8% residential. The land

immediately surrounding the river is predominantly used for pine forestry operations.

Fertilizer from pine forestry operations and waste material from dairy practices are

thought to be a significant source of non-point source nutrient loading to the river

(Asbury and Oaksford, 1997). The Suwannee River could potentially be highly








influenced by these large amounts of nitrogen coming into the system. The clam beds

and oyster reefs at the mouth of the river and in the estuary are harvested periodically

throughout the year. Increased nutrient loads may negatively affect these and other

species, although elevated nutrient concentrations can lead to high production of

phytoplankton. This production can lead to low dissolved oxygen concentrations, and, in

turn affect production and survivorship.

Nutrient Loading, Especially Nitrogen

River discharge provides a significant source of nutrients to estuaries; however this

can be considered contamination as well as a pathway for increased productivity. The

potential impact of a contaminant such as nitrogen in the environment depends not only

on the concentration and mobility of the species, but also on its bioavailability (Suffet

and MacCarthy, 1989). Increased nutrient loads can lead to the eutrophication of surface

waters, and high nitrate concentrations (>10 mg/L) especially pose a possible health risk

in drinking water (Asbury and Oaksford, 1997). Other possible effects include harmful

cause algal blooms and low dissolved oxygen concentrations, which can have detrimental

effects on the entire ecosystem.

Sources of nutrients in the Suwannee River include atmospheric deposition,

fertilizer, animal waste, and septic discharge, with nitrogen from fertilizer and animal

wastes predominating (Asbury and Oaksford, 1997). Asbury and Oaksford (1997)

estimated the nitrogen stream load per year in the Suwannee River as 9,700,000 kg/yr and

476 (kg/yr)/km2, and found that organic nitrogen was the most abundant nitrogen

compound. Nitrogen exists in the river largely in the form of NO3; however, nitrogen is

also a trace constituent in aquatic humic substances. According to Thurman and

Malcolm (1989), the nitrogen content of fulvic acids varies from 0.5 to 1.5 percent;








however aquatic humic acid has twice as much nitrogen as fulvic acid. In the Suwannee

River, the concentration of nitrogen in fulvic acid is 0.56 percent, and in humic acid it is

1.08 percent (Thurman and Malcolm, 1989). Frazer et al. (2002) found that nitrate

discharged from the lower Suwannee River was quickly diluted and/or assimilated as it

entered the Gulf of Mexico.

Agricultural activities and other land uses have impaired the quality of spring

waters by contributing large quantities of nutrients to groundwater recharge in many parts

of the world (Katz et al., 2001). During the past 50 years, nitrate-N concentrations in

groundwater discharging from several springs in the middle Suwannee River Basin have

increased from near background concentrations (less than 0.1 mg/L) in groundwater to

more than 5 mg/L (Katz et al., 2001). However, nitrate concentrations in the water from

four springs in Suwannee County generally have remained constant or have decreased

slightly in the last few decades following a decrease in the use of fertilizers (Katz et al.,

2001).

Many different factors in an ecosystem such as the Suwannee River impact the

nutrients once they enter the system, and nutrient concentrations can change with time.

Rates of change are a function of mixing and transport, emission rate, sinks, and

reactivity (Suffet and MacCarthy, 1989). Chemical influences on nutrients include

physiochemical reactions of the compounds (e.g., sorption, coagulation, acid-base

interactions, and complexation reactions), and chemical changes (e.g., oxidation,

reduction, hydrolysis, and photochemical reactions). Physical influences include

transport and dispersal. Biological influences include uptake and mineralization by

organisms and metabolic changes associated with receptor organisms and food web








interactions (Suffet and MacCarthy, 1989; Horrigan et al., 1990). Nutrients from

fertilizers will be taken up and used by organisms, such as algae, which will, in turn, be

consumed by higher trophic levels in river and estuarine systems. Nutrient flows through

these types of ecosystems have been studied in great detail (e.g., Kaul and Froelich, 1984;

Horrigan et al., 1990; Wada and Hattori, 1991; Schelske and Hodell, 1995).

Nitrate and ammonium are taken up by microbes, algae, and macrophytes, but

organic particles are generally consumed by organisms such as clams and oysters in

estuarine environments. As previously stated, there is a small amount of nitrogen present

in humic substances. Clams and oysters can readily consume phytoplankton (a known

source of good quality food), but organic nitrogen aggregates that are formed from

dissolved compounds have also been found to be an important source of nitrogen for

these consumers (Alber and Valiela, 1996).

Clam and Oyster Ecology

At the mouth of the Suwannee River and in the estuary oyster reefs are common.

In addition, there are a number of lease sites where hard clams are allowed to grow for

harvest. The predominant species are Crassostrea virginica and Mercenaria mercenaria.

The oysters, Crassostrea virginica, are well suited to estuarine conditions. They can

tolerate wide ranges of temperatures, salinity, turbidity, and dissolved oxygen. They can

also generate their own substrate by producing their own shells to which they attach (Day

et al., 1989; Ruppert and Barnes, 1994). These organisms are typically limited by space

(Day et al., 1989). Oysters are suspension feeders (pump and filter a large quantity of

water to obtain sufficient food of the proper quality), and it is important that the quality

(nutrient content) of the food be very high to achieve maximal growth. Adult oysters

actively pump (through ciliary action) several liters of water per hour across their gills








(Day et al., 1989; Ruppert and Barnes, 1994). Therefore, dense populations of

suspension feeders can become limited by food in areas with a lack of circulation (Day et

al., 1989). Ammonia and phosphorus are regenerated by these organisms as a

consequence of excretion and fecal production (as subsequent remineralization). Oyster

harvesting is an important industry for Florida's economy, and oysters also provide a

source of food for birds and several aquatic organisms (e.g., sea anemone, crabs,

flatworms, etc.) (Dunning and Adams, 1995; Colson and Stunner, 2000; Newell et al.,

2000).

The clam, Mercenaria mercenaria, is also an important food source for humans,

some birds, and aquatic organisms. It is one of the most important commercial bivalve

species on the eastern coast of the United States, and the Suwannee River estuary is the

most economical area to grow these clams in the natural environment (Pfeiffer et al.,

1999; Colson and Sturmer, 2000). They feed in a similar manner to the oysters

(suspension feeders), and as clams grow and population numbers increase, food

requirements grow exponentially (Ruppert and Barnes, 1994; Pfeiffer et al, 1999).

There are three trophic pathways by which clams and oysters might ingest and

subsequently assimilate detritus: (1) via direct uptake of dissolved organic matter (which

is released by both live and decaying producers), (2) ingestion of particulate detritus, and

(3) ingestion of organic aggregates (Alber and Valiela, 1994a). Alber and Valiela

(1994a) have also found that bivalves are capable of taking up bacteria. In a study by

Crosby et al. (1990), the oyster, Crassostrea virginica, was found to assimilate refractory

Spartina alterniflora-derived detritus with an efficiency of 2.7 percent, whereas material








that was colonized by bacteria was assimilated with an efficiency of 10.3 percent. These

findings suggest that bacteria can actually enhance the quality of food for the consumers.

Suspension feeders encounter food supplies of varying nutrient quality, quantity,

and size. Many bivalves are able to alter their capture rates and strategies to account for

lower quality food. Ward et al. (1997), for example, showed that oysters can select living

particles from non-living detritus on the gills. Ward et al. (1998), in a subsequent study,

determined that in the oyster, Crassostrea virginica, the ctenidia are responsible for

particle sorting while the labial palps have an accessory role in particle selection. There

have been several studies with bivalves demonstrating the ability to sort particles based

on size (e.g., Vahl, 1972; Stenton-Dozey and Brown, 1992; Defossez and Hawkins, 1997;

Raby et al., 1997; Brillant and MacDonald, 2000).

Humic Substances

Organics within a river system can be allochthonous (produced outside of the

system) or autochthonous (produced within the system). Allochthonous organic

materials include humic substances. The dissolved organic matter (DOM) in an average

river is typically composed of about 50% humic substances; however DOM is highly

variable depending on source of organic matter, temperature, ionic strength, pH, major

cation composition of the water, sorption processes, and microbial degradation processes

(Leenheer and Crou6, 2003; Schwarzenbach et al., 2003). In the case of the Suwannee

River, a blackwater river, there are high concentrations of dissolved humic substances in

the water, ranging up to 60 mg/L at its source. The water in the Suwannee River is dark

and acidic due to the high amounts of humic substances from decomposition of swamp

vegetation (Malcolm et al., 1989a). Much of this humic material originates in the








Okeefenokee Swamp. Along the river channel there are many pine forests which also

contribute to the high concentration of dissolved humic acids.

Humic substances are ubiquitous organic materials in the environment that result

from the decomposition of plant and animal residues (Suffet and MacCarthy, 1989;

MacCarthy, 2001). They can be expressed as dissolved organic carbon (aqueous

materials that pass through a 0.45-am filter); however, some of these materials may be in

colloidal form rather than in solution. Humic substances can assist with the transport of

metals through complexation and ion-exchange mechanisms, depending on the redox

state of the metal ions, leading to either solubilization or immobilization of the metals.

Humic substances also interact with nonionic compounds (pesticides and other river

contaminants) via sorption, causing the immobilization of such compounds in

sedimentary organic matter (Schwarzenbach et al., 2003). Humic substances in solution

consist of amino acids, sugars, various aliphatic and aromatic acids, and a variety of more

complex organic compounds. They can be somewhat polar in that they contain numerous

oxygen-containing functional groups including carboxy-, phenoxy-, hydroxy-, and

carbonyl- substituents (Stumm and Morgan, 1996; MacCarthy, 2001; Schwarzenbach et

al., 2003). This allows them to interact with water-soluble compounds. However, they

also consist of nonpolar constituents which allow them to interact with water-insoluble

compounds (Calace et al., 1999). In soils, sediments, and suspensions, humic substances

are often bound with inorganic materials (i.e., clays and metals) (Suffet and MacCarthy,

1989; Meier et al., 1999; Schwarzenbach et al., 2003).

Humic substances are typically fractionated into the following groups: humic acid -

the fraction of humic substances that is not soluble in water under acidic conditions








(pH<2) but is soluble at higher pH values; fulvic acid the fraction of humic substances

that is soluble in water under all pH conditions; and humin the fraction of humic

substances that is not soluble in water at any pH value. There are different concentrations

of each of these groups in different river systems. Studies show that typically 90% of

dissolved humic substances is fulvic acid and 10% is humic acid (Suffet and MacCarthy,

1989; Hautala et al., 1998). In the Suwannee River, fulvic acids are most dominant

(Malcolm et al., 1989a).

Humic substances can be a food source for organisms in river and estuarine

environments. Detritus humicc substances) is generally a year-round source of food and

may be more abundant than phytoplankton at times (Alber and Valiela, 1996). However,

it is also found to be very recalcitrant to biological and chemical degradation (Wang et

al., 2000). Therefore, humic substances are often poor quality food (Alber and Valiela,

1994a). Before many humic substances can be eaten, some detritivores depend on

microbes to decompose and alter the quality of the detritus by increasing both its

available nitrogen content (see below) and availability to consumers (Alber and Valiela,

1994a). The dissolved organic matter is, therefore, a growth substrate for bacteria (Sun et

al., 1997). It has been shown that bacteria use dissolved organic matter at specific size

ranges (small>medium>large); however higher molecular weight dissolved organic

matter (as humic substances) is generally most available (Engelhaupt et al., 2002). Sun et

al. (1997) found that bacterial growth was positively correlated with H:C and N:C ratios,

and negatively correlated with O:C ratios. Colonization by bacteria can enhance the

quality of the food for consumers through metabolism of larger nitrogen containing

molecules to smaller, more available forms.








The dissolved organic carbon concentration in colored surface waters is extremely

variable and typically ranges from about five to more than 50 mg of carbon/L (Suffet and

MacCarthy, 1989). The fraction of the total dissolved organic carbon that is in the form

of humic substances varies considerably and can be up to 80% (Suffet and MacCarthy,

1989). According to Wang et al. (2000), the nitrogen contained in humic substances can

be released as low molecular weight species through photochemical processes or

reactions, and therefore becomes more readily available for biological use. They found

that solar irradiation of dissolved organic matter results in conversion of recalcitrant

dissolved organic nitrogen to ammonium, which is more readily assimilated by microbes.

These photochemical reactions may play a significant role in the cycling of the nitrogen

through the ecosystem.

Flocculation

The terms flocculant and aggregate, often used interchangeably refer to "fragile,

amorphous, macroscopic or microscopic particles consisting of organic and inorganic

material" (Zimmerman-Timm, 2002; p. 197). Aggregates are ubiquitous in moving

surface water (rivers, tidally-influenced rivers, and estuaries); however, they have been

found to be more concentrated in an estuarine environment where fresh water meets salt

water, and where there is wave influence and turbulence. The main characteristics that

affect organic aggregates in moving water have been presented by Zimmerman-Timm

(2002) as hydrological regime, waves, shipping, channel deepening, precipitation,

stagnation, transport, aggregate composition, organic content, size, sedimentation,

saltwater interaction, and resuspension.

The hydrological regime affecting aggregates includes current velocity, horizontal

transport, wave action, and discharge rate. According to Berger et al. (1996) and








Zimmerman (1997), increasing current velocity and freshwater discharge rate result in a

large amount of aggregates in the water column. With a higher density of aggregates in

the water column, however, the probability of particle contact is more likely, and an

increase in the number of aggregates is unlikely. Horizontal transport and water

residence times are other hydrological factors that can affect aggregates in most running

surface water systems. The water residence times in estuaries are influenced by

interactions between tides and water flow, and circulation patterns (Day et al., 1989;

Zimmerman-Timm, 2002). Wave action also influences the generation of aggregates, as

waves can stir up the bottom sediment, causing particles to interact with each other and

aggregate. In areas of stagnation, however, low amounts of aggregates are generally

present (Zimmerman-Timm, 2002). In these locations, particles settle to the bottom as

there are few physical disturbances to bring them back into the water column.

The composition of aggregates can be made up of organic and inorganic,

allochthonous and autochthonous materials. According to Zimmerman-Timm (2002), the

composition of aggregates may include non-living geologic components (clays, mica,

quartz, etc.), anthropogenic components (organic and inorganic contaminants), non-living

biogenic material (silica, calcite, etc.), attached organisms (algae, bacteria, fungi, etc.),

macrophytes, detritus (dead and dying organic material), and fecal pellets. Conners and

Naiman (1984) have shown that 85-97 percent of the particulate organic carbon in large

rivers is autochthonous in nature. Suffet and MacCarthy (1989) found that, in most

cases, higher humic substance concentrations correspond to higher concentration of

aggregates. The aggregates in the Suwannee River consist predominantly of humic

substances brought into the system from the Okefenokee Swamp. However, clays and








bacteria, as well as salinity, might have a strong influence on the formation of the

aggregates (Meier et al., 1999). Algae and bacteria hold the aggregates together with

sticky mucous substances. Kepkay and Johnson (1988) found that after 15 minutes of

bubbling coastal seawater, DOC was reduced by 43% and the production of organic

particles increased after surface coagulation initiated a rapid bacterial response. Alber

and Valiela (1994b) showed that aggregates from dissolved organic matter released by

macrophytes are biotic in nature and contain large numbers of bacteria. Alber and

Valiela (1994b), in studying the carbohydrate, protein, lipid, carbon, and nitrogen

composition of aggregates, macrophytes, and bacteria, showed also that the biochemical

composition of the aggregates is closer to that of bacteria than the macrophytes.

Flocculated organic aggregates have different forms and sizes, all of which are

greater than or equal to 0.45 Im. Size is important when determining transport properties

(settling and movement through a system) (Jackson and Burd, 1998). Three size classes

of these flocculants have been determined by Alldredge and Silver (1988) and Perret et

al. (1994) and include macroscopic aggregates (>150 pm), microaggregates (<150 pm),

and submicron particles (<1 ljm). Typical sizes of aggregates are 5,000 aim and smaller

(Zimmerman-Timm, 2002). Size of the aggregates influences how quickly they will

settle out. Aggregates in the 2,400 to greater than 5,000 Am size range settle at an

average of 25 times more rapidly than single algal cells (Alldredge and Gotschalk, 1988).

Larger size aggregates can be easily broken down by mechanical disruption (waves,

wind, mixing, etc.), consumer interaction, and microbial decomposition. Physical motion

breaks down aggregates near the surface in rivers (Argaman and Kaufman, 1970; Parker

et al., 1972). In the Elbe, the River Rhine, and the Zaire River Estuary, a decrease in








aggregate size has been found to occur with an increase in salinity due to decreased

colonization by microbes and bacteria (Zimmerman-Timm, 2002). Larger size

aggregates are also more accessible to larger consumers that are less efficient at

consuming small particles (Alber and Valiela, 1994).

The mechanisms underlying the formation of aggregates are often debated.

Although flocculation of dissolved organic matter has been shown to occur in many

estuarine ecosystems, it has not been proven if the cause of this flocculation is solely salt

induced, bacteria induced, physically induced, or a mixture of these. Many argue that it

is a complex process controlled by several variables (O'Melia, 1987; Eisma, 1993;

Stumm and Morgan, 1996) including particle concentration, stickiness of the particle

surface, velocities, and probability of attachment. The higher the concentration of

particles, the more aggregates will be formed. If the concentration is too low,

flocculation will be less likely to occur because the collision frequency will be too low

(Zimmerman-Timm, 2002).

Physical and chemical processes that have been found to produce aggregates

include Brownian motion, shear forces, differential settling, filtration, bubbling surface

coagulation, precipitation, adsorption, attractive and repulsive properties, bridging with

divalent cations, and spontaneous coagulation of dissolved organic matter. Brownian

motion is the movement and collision of particles smaller than 8 im under the influence

of heat energy induced motion of water molecules (Eisma, 1986; Zimmerman-Timm,

2002; Schwarzenbach, 2003). Shear forces refers to the strength of laminar and turbulent

shear which determines the frequency and the push of particles (Jackson and Burd, 1998;

Zimmerman-Timm, 2002). Differential settling is a mechanism that is dependent on








aggregate composition, size, and abundance. Larger particles are formed as the larger

and more rapidly sinking particles interact with the smaller particles during vertical

transport (Zimmerman-Timm, 2002). Gas bubbles can be released from sediments or

formed by waves breaking at the surface. They will rise through the water column until

they burst or collapse. Particles can concentrate along the border of the bubble, and can

then be transported as aggregates (Johnson, 1976; Kepkay and Johnson, 1988).

Attractive and repulsive surface properties refer to the charge on particles and depend on

the size, composition, and chemical coating on the particle. Most particles are negatively

charged at their surface because the organic material has many carboxyl or hydroxyl

groups associated with it (especially humic substances) (Suffet and MacCarthy, 1989;

Zimmerman-Timm, 2002). Positive cations (such as those found in saline waters) can act

as chemical bridging agents, enhancing the attraction properties of electronegatively

charged particles. This is why salt-induced flocculation is one explanation for the

aggregates found in rivers where fresh water enters salt water.

Schwarzenbach et al. (2003) presented a discussion on how the presence of the

predominant inorganic ionic species found in natural waters generally decrease the

aqueous solubility, or increase the aqueous activity coefficient, of nonpolar or weakly

polar organic compounds. The magnitude of this effect is termed "salting-out". An

empirical formula established by Setschenow (1889) (as cited by Schwarzenbach et al.,

2003) is presented to support this idea. This formula relates organic compound

solubilities in saline aqueous solutions to those in pure water by relating the effectiveness

of a particular salt or combination of salts to the change in solubility of a given








compound (in terms of a salting constant). It shows that the "salting-out" effect increases

exponentially with increasing salt concentration (see below).

Csaiw,sa = Csaiw 10-Ki[salt]to (1-1)

In the above equation, Csaiw,sat is the organic compound solubility in saline aqueous

solutions, C"ai is the organic compound solubility in pure water, [salt]otal is the total

molar salt concentration, and Ksi is the Setschenow, or salting constant (Schwarzenbach

et al., 2003). This can also be presented in terms of activity coefficients:

_w, at = w* 1+Ksi[salt]tot (1-2)

This equation shows that -, ,,lt increases exponentially with increasing salt

concentration. Essentially, predominant ionic species, such as salts from sea water,

generally decreases the solubility of nonpolar or weakly polar, such as natural dissolved

organic matter.

Some experiments have been performed to produce aggregates in a laboratory

setting. Sholkovitz (1976), in a classic study, produced aggregates at varying salinities

from 0%o (parts per thousand) to 30%o by mixing filtered river water and sea water. He

found that no more changes were noticeable in color or concentration of flocculants after

about 159%o to 20%o, naming this the salinity of maximum removal of dissolved organic

carbon. Other elements were present in his river water sample that could have had an

effect on the flocculants, including aluminum and calcium. Fox (1983) attempted to

repeat (with minor differences) the study by Sholkovitz. In two of three estuaries studied,

he found that salt-induced removal of dissolved humic acid was insignificant. Eisma

(1986) also determined that salt induced flocculation plays only a minor role, and that

flocs are formed more by a combination of forces including viscous flow, gravity, and








Brownian motion. Alber and Valiela (1994) produced aggregates in the laboratory using

dissolved organic matter from different macrophyte species. They bubbled the samples

and the formed aggregates ranged in size from a few micrometers to 500 jim across.

Esteves et al. (1999) concluded that mixing freshwater and seawater in a laboratory

setting removes 5% to 10% of dissolved organic matter as particulate matter. Thill et al.

(2001), in studying water from Rhone River, France, found that particle aggregation and

adsorption are not an instantaneous process. The Rhone river particulate matter had a

poor average reactivity regarding salt induced flocculation. Reaction kinetics depend on

the electrostatic repulsion between particles, which is dependent on the salinity.

However, when shear collision dominates a system, the kinetics are proportional to the

particle volume fraction. They also stated that higher particle concentrations favor the

faster formation of aggregates (Thill et al., 2001).

Field studies have also shown the production of aggregates. A study by Del

Castillo et al. (2000) found that flocculation of dissolved organic matter in the Peace

River, Florida occurred at the mixing interface of salt and fresh water. Del Castillo and

colleagues hypothesized that the importance of the salt induced flocculation might be

overshadowed by large concentrations of CDOM in the mouth of the river. To test this

hypothesis, the possible effects of flocculation were modeled, and it was found that 11%

of the dissolved organic matter was lost and 20% of the color was removed during

flocculation as reported by Sholkovitz et al. (1976). Milligan et al. (2001) found that in

the ACE Basin, South Carolina, suspended material was dominated by high

concentrations of flocculated sediment. They stated that this was probably due to a

suppression of turbulence and salinity gradients. In the Rhone River, France, salt induced








flocculation was found to be a minor factor unless mixing conditions were adequate for

the formation of floes (Thill et al., 2001).

The use of organic aggregates as a food resource by consumers has been

hypothesized by several investigators (e.g., Alber and Valiela, 1994; Zimmerman-Timm,

2002). However, this hypothesis has not been adequately addressed. Alber and Valiela

(1996) found that in the aggregate detritus pathway, dissolved organic matter released by

primary producers is transformed into organic aggregates, which are then ingested by

consumers (in this case, bivalves). They also found that the aggregates could meet a

substantial portion of the nutritional requirements of bay scallops (Argopecten irradians)

in the field. Other experiments have also shown that benthic organisms are able to ingest

aggregates as a food source. The food quality of the aggregates can be characterized by

their composition. Alber and Valiela (1994) found that aggregates have lower C:N

values and are higher in protein than the macrophyte-derived detritus in their study,

explaining why aggregates are more readily consumed. The ingestion of aggregates as a

food source forms a link between the microbial food web and the higher trophic levels.

As bivalves consume the aggregates, they are also consuming the bacteria associated with

the aggregates, thus repackaging the smaller microbial particles.

Collection of aggregates can be troublesome. Filtration and centrifugation have

been shown to destroy the typical structure of the fragile aggregates, producing an

amorphous mass of organic and inorganic material (Thill et al., 2001; Zimmerman-Timm,

2002). It can be difficult to analyze aggregates as well. Laboratory produced aggregates

are often different from field aggregates because the conditions of their formation are

somewhat different. In the natural environment, there are higher numbers of organisms








and larger size classes than those produced in the lab, and the physical parameters are not

completely representative. However, it is worthwhile to construct laboratory experiments

to mimic field conditions to get an understanding of different mechanisms that may cause

the formation of aggregates.

Stable Carbon and Nitrogen Isotopes as Tracers of Material Transfer and Energy
Flow

Anthropogenic perturbations can alter the pathways of material transfer and energy

flow through an ecosystem. For example, excess fertilization in a watershed can alter the

abundance and flow of elements such as nitrogen in a stream within that watershed.

Stable carbon and nitrogen isotope ratios (5sN) can be used to follow the changes caused

by excessive fertilization and show which processes or components are altered (i.e.

foodweb, trophic changes, etc.), as well as determine the source of carbon and nitrogen

fueling production of consumers (Mariotti et al., 1984; Owens, 1985; Peterson and Fry,

1987; Fry, 1988; D'Avanzo et al., 1991; Wada et al., 1991; Holmes et al., 1996; Riera,

1998; Fry, 1999; Hamilton et al., 2001; Lehmann et al., 2001; McKinney et al., 2002).

Owens (1987) collected data available on the distribution of '5N for various

environments. In estuarine systems, the average s15N value of all collected materials was

4.6 2.0 for 199 observations. The average stable nitrogen isotope signature for all

materials in freshwater was 4.3 2.7 for 64 observations, and the terrestrial average was

3.9 + 3.1. Differences in ecosystems provide a possible reason for using "SN as a tracer.

The use of stable isotopes to address issues related to energy flow is increasing because

stable isotope data can contribute both source-sink data (tracer) and process information

(Peterson and Fry, 1987; Fry, 1988; Montoya et al., 1990; D'Avanzo et al., 1991; Holmes








et al., 1996; Riera, 1998; Hamilton et al., 2001; Lehmann et al., 2001; McKinney et al.,

2002).

The stable isotopic signature of inorganic and organic nitrogen provides an

integrative tool for studying the sources of nitrogen supporting production in an

ecosystem (Valiela et al., 1997). The stable carbon isotope signature provides a tool for

determining the source of organic matter in these ecosystems. Most systems have several

inputs of organic carbon as a food source, e.g. C3 terrestrial plant material (8'3C = -23 to

-30%o), seagrasses (-3 to -15%o), macroalgae (-8 to -27%o), C3 marsh plants (-23 to -

26%o), C4 marsh plants (-12 to -14%o), benthic algae (-10 to -20%o), and marine

phytoplankton (-18 to -24%o) (Fry and Sherr, 1984; Michener and Schell, 1994). The

major source of terrestrial-derived DOM is vascular plants, which are confined

essentially to land and characteristically contain high concentrations of recalcitrant,

nitrogen-free biomacromolecules such as lignin and tannin (Hedges et al., 1997). These

plants have distinct stable carbon isotope compositions and can be used as a biomarker of

terrestrial origin (Fry and Sherr, 1984; Hedges et al., 1997). There can be, however,

some overlap of stable isotope signatures (e.g., C3 terrestrial plants, C3 marsh plants, and

marine phytoplankton). Detailed investigations of individual ecosystems should be

studied to differentiate sources of organic carbon.

The implications for the use of stable nitrogen and carbon isotopes in food web

investigations are also significant (DeNiro and Epstein, 1981; Fry, 1988; Wada et al.,

1991; Canuel et al., 1995; Cabana and Rasmussen, 1996; Vander Zanden and Rasmussen,

1999; Jepsen and Winemiller, 2002). DeNiro and Epstein (1981) found that the 6'5N of

organisms reflects the 1'5N signature of the diet; however, the animal is generally








enriched in 5N, and isN values are approximately 3 to 5%o greater than the diet. This is

mainly due to excretion of isotopically light nitrogen in urine (Minagawa and Wada,

1984; Owens, 1987; Peterson and Fry, 1987; Fry, 1988; Wada et al., 1991; Lajtha and

Michener, 1994; Cabana and Rasmussen, 1996; Gue et al., 1996). Stable carbon isotopes

of animals reflect those of the diet within approximately 1%o (DeNiro and Epstein, 1978;

Peterson and Fry, 1987; Michener and Schell, 1994; O'Donnell et al., 2003). Animals

may be isotopically enriched relative to their diet as a consequence of the preferential loss

of 12CO2 during respiration, the preferential uptake of 13C compounds during digestion, or

metabolic fractionation during synthesis of different tissue types (DeNiro and Epstein,

1978; Rau et al, 1983; Tieszen et al., 1983; Fry et al., 1984). A determination of an

organisms trophic status can be facilitated by the simultaneous measure of 6'N and "3C

signatures (Peterson et al., 1985). Stable carbon and nitrogen isotopes are a useful

method for following the flow of energy in a system such as the Suwannee River and its

estuary.

Many chemical and physical processes are accompanied by significant isotopic

fractionation which results in an enrichment or depletion of the heavy isotope (Peterson

and Fry, 1987; Wada et al., 1991; Lajtha and Michener, 1994). Fractionation can occur

during kinetic and equilibrium processes. Equilibrium isotope effects are common where

chemical exchange occurs between two molecules, whereas kinetic isotope effects are

involved with more complex reactions (Peterson and Fry, 1987). In biochemical

processes involving nutrient uptake, the substrate is enriched and the product is depleted

in heavy isotopes when the substrate pool is infinite (Montoya et al., 1990). This is due

to the fact that autotrophs, e.g. phytoplankton, preferentially assimilate lighter nitrogen.








In the process of nitrogen fixation, however, the 1'SN values are near 0%o. In the

processes of nitrification and denitrification, residual ammonium-N and nitrate-N,

respectively, are enriched with heavy nitrogen. According to Owens (1987), there is a

large fractionation effect associated with denitrification. The residual nitrate substrate

exhibits isotopic enrichment in situ, however, a large pool of dissolved N2 can preclude

any significant depletion of the accumulated product (Owens, 1987). With regard to

animals, preferential excretion of 5N-depleted nitrogen has been found, generally in the

form of urea and ammonia, resulting in and isotopic enrichment of the animal tissue

(Minagawa and Wada, 1984).

Most nitrogen is present as N2 gas in the atmosphere with an isotopic composition

essentially constant at 0%o (Peterson and Fry, 1987). Nitrogen in the rest of the biosphere

has a typical isotopic composition around 0%o as well (ranging from -10 to 10%o). The

nitrogen cycle, however, involves processes with notable fractionation effects that affect

i'5N signatures of nitrogen pools. For example, there is a cumulative faster loss of '4N

than 15N during particulate nitrogen decomposition in soil and water, resulting in 85N

increases of 5 to 10%o with increasing depth (Peterson and Fry, 1987). Nitrification and

denitrification in the oceans involve substantial isotope effects. As animals are on

average 3 to 5%0 heavier than dietary nitrogen (Minagawa and Wada, 1984; Owens,

1987; Peterson and Fry, 1987; Fry, 1988; Wada et al., 1991; Lajtha and Michener, 1994;

Cabana and Rasmussen, 1996; Gu et al., 1996), nitrogen isotopic values may increase by

10 to 15%o in many food webs, due to the presence of three to five successive trophic

transfers (Peterson and Fry, 1987). Volatization of nitrogen also causes fractionation

effects because the lighter isotope (14N) is more volatile than the heavier isotope (15N).








For example, Flipse and Bonner (1985) found that the average 615N values of fertilizers

was 0.2%0 (potato farm) and -5.9%0 (golf course), and the average 3 5N values of the

groundwater nitrate in these systems were 6.2%o and 6.5%0, respectively. It was

determined that higher 5"N values of groundwater nitrate were caused by isotopic

fractionation during the volatile loss of ammonia from nitrogen applied in reduced forms.

The elements carbon, nitrogen, sulfur, hydrogen, and oxygen all have more than

one stable isotope and their relative abundances can be measured with a mass

spectrometer (Peterson and Fry, 1987). The isotopic compositions of different materials

will change in predictable ways as elements cycle through the biosphere and also as a

consequence of isotopic fractionation (Owens, 1987; Peterson and Fry, 1987; Lajtha and

Michener, 1994). According to Owens (1987), however, these changes aren't always

apparent due to a degree of overlap between different types of samples. Therefore, a

combination of elements (e.g. carbon, nitrogen, and sulfur) and their isotopes are often

used. For example, Peters et al. (1978) found a high correlation between tSN and 63C

values in sedimentary organic matter. Peterson et al. (1985) found that the use of a

combination of the stable isotopes of sulfur, carbon, and nitrogen helped to delineate food

sources and characterize the trophic structure in an estuary where there was more than

one source of food for the ribbed mussel (Geukensia demissa). Gu et al. (1996) used

stable carbon and nitrogen isotopes to identify food sources and describe trophic transfer

pathways within a lake ecosystem. Vander Zanden and Rasmussen (1999) also used

stable carbon and nitrogen isotopes as indicators of trophic structure within a freshwater

lake system, and determined that 5N values of higher consumers could not be used

alone because of the high variability of 65N values of primary consumers. Carbon and








nitrogen element ratios might also assist in understanding 5N values. For example, Guo

and Santschi (1997) studied the elements carbon, nitrogen, and sulfur, as well as isotopic

composition of colloidal organic matter to investigate sources and turnover rates of

dissolved organic matter in Chesapeake Bay and Galveston Bay, USA.

A number of studies have been done on nitrogen isotopes in freshwater, estuarine,

and marine ecosystems. Wada et al. (1981) studied highly enriched algal felt in saline

and non-saline lakes in the Antarctic. The epibenthic algae in this system were depleted

in '5N as a result of isotopic fractionation associated with slow growth rates in low light

and a high nitrate environment. The source of nitrogen for the algae was avian excreted

ammonium, which had a large fractionation effect during volatization of ammonium.

Therefore the nitrogen available to the algae was likely to be highly enriched (Wada et

al., 1981). Owens (1987) presented data from numerous studies and found that the 65'N

signature ofphytoplankton ranges from 3%o to 12%o. It was predicted that phytoplankton

in the field would tend to exhibit a lower 15N content because of fractionation effects

(Owens, 1987). In the turbidity maximum (area where material is periodically

resuspended from the surface sediments) of the Tamar River estuary, Britain, Owens

(1985) showed a distinction between at least two different populations of particles using

'"N analyses. In the marine end of the estuary (salinity 33.8%0), the 6'iN values were

4.96 1.03%o, in the freshwater end, the 8'5N values were 2.29 2.23%o, and

intermediate 6i5N values ranged from 1.79 1.63%o to 14.73 0.7%0. The highest values

were found in the turbidity maximum, due to the longer residence time (and thus,

increased microbial decomposition) of the particles at this site (Owens, 1985). The low

815N values (close to 0%o) were similar to values typical of terrestrial material, which








reflects the dominance of atmospheric nitrogen (Owens, 1985). Wada et al. (1991) also

found this to be the case for particulate organic matter in Otsuchi River and its receiving

waters. Low 8'SN values (0.2 to 0.7%o) were found in the particulate matter of the upper

reaches of the river, while high 615N values (6.4 1.8%o) were found in the bay (Wada et

al., 1991). Sigleo and Macko (1985) also reported similar findings for suspended

particulate material in the Patuxent Estuary, Maryland.

Mariotti et al. (1984) found substantial spatial differences in the 15N content of

ammonium and nitrate in particulate material in the Scheldt Estuary, France. The

upstream zone was depleted in dissolved oxygen and was a site of denitrification (low

concentration of nitrate and high concentration of ammonium). In this zone, the 615N of

ammonium was 10%o and the 'SN of nitrate was 18%o. However, in the lower estuary,

where dissolved oxygen was high and nitrification was active, the 68"N values were

reversed (nitrate 2 to 5%o and ammonium 29%o) (Mariotti et al., 1984). This indicates

that the nitrogen cycle is very important when interpreting '5N data. Katz et al. (2001)

studied nitrate-N flowing out of springs into the Suwannee River using 8'SN as well as

other tracers. Water discharging from springs in Suwannee County had lower S15N

values (2.7-6.2 %o) than Lafayette County (4.5-9.1%o). The average concentration of

nitrate in Suwannee County springs was higher (1.5-37 mg/L) than the average

concentration of nitrate in Lafayette County springs (1.7-5.5 mg/L). These differences

were attributed to local differences in land use and nitrate source. The springs with the

highest concentration of nitrate (and lowest 15N values) were near cropland farming

areas that were intensively fertilized and irrigated, and the springs with the lowest

concentration of nitrate (and highest i 5N values) were near recharge areas with








concentrated animal feeding operations (animal waste sources of nitrate) (Katz et al.,

2001).

A number of studies have been done with 15N being used as a food web tracer as

well. DeNiro and Epstein (1980) found through studying animals raised in the laboratory

on diets of known and constant 85N values, that the isotopic composition of nitrogen in

an animal reflects the nitrogen isotopic composition of its diet. In general, the 85N value

of an organism is greater than the 515N of its diet (DeNiro and Epstein, 1980; Owens,

1987, Wada et al., 1991). According to Peterson and Fry (1987), stable nitrogen isotopes

in animals average 3-5 %o heavier than dietary nitrogen. Alber and Valiela (1994) found

that two different species of mussels incorporated 15N-labelled nitrogen when fed

aggregates derived from dissolved organic matter released by labeled macrophytes,

demonstrating that nitrogen can be transferred from decomposing macrophytes to

suspension-feeding consumers through the aggregate detrital pathway. They also showed

that both species of mussels incorporated significantly more nitrogen when they were fed

aggregates than when they were fed either dissolved organic matter or particulate detritus

(Alber and Valiela, 1994; Alber and Valiela, 1996). However, the nitrogen incorporation

rate of scallops that were fed phytoplankton was significantly greater than that of scallops

fed aggregates (Alber and Valiela, 1996). Fry (1999) studied the clam, Potamocorbula

amurensis, in San Francisco Bay and found that nitrogen isotopic compositions of clams

were representative of watershed nutrient loading. Higher b 5N values were found in the

South Bay, where there was a stronger input of anthropogenic nitrogen than in the North

Bay. In the freshwater end of the North Bay, however, a discrepancy was noted between

the 515N values of the filter-feeders and seston (seston 5%o and large zooplankton 9 to








14%o), and three possible explanations were presented: (1) there was an intermediate

trophic linkage in the microbial component of the food web leading to higher 61"N values

in filter-feeders, (2) detrital river material colonized by bacteria immobilize enriched 15N

from the water column, and filter-feeders consuming these bacteria have higher 15N

values, and (3) a large quantity of riverine seston (which is largely terrestrial and

refractory to food web use) masked a relatively minor "1N-enriched river/estuarine

phytoplankton component that was selectively used by filter-feeders (Fry, 1999).

s15N values have also been used to provide insight into the source of nitrogen

supporting autotrophic production in a system. For example, Frazer et al. (2002) found

that the 61sN values of submerged aquatic vegetation were low in the Chassahowitzka

River. This was consistent with the idea that fertilizers have contributed significantly to

nitrate pollution of the system. Fry et al. (2001) determined that nitrogen isotope assays

were useful for detecting watershed nitrogen loading in four National Estuarine Research

Reserve (NERR) sites on the west coast of the United States. Isotope values of

ammonium and nitrate (ranging from -0.3 to 19.6%o) showed a trend towards higher

values in those estuaries associated with higher watershed nitrogen loading (Fry et al.,

2001). Some studies have even broken the S15N values of each organism down to

individual tissues and excretory products because isotopic variation can exist among

different tissues and metabolites of different animals (DeNiro and Epstein, 1981;

Peterson and Fry, 1987). For example, Alber and Valiela (1994) found that whatever '5N

was present in the guts of two species of mussels was released after 48 hours. Excretory

products are consistently depleted in 5N relative to the diets, which also explains why

'5SN values are higher in the organism compared to its diet (DeNiro and Epstein, 1981;








Owens, 1987; Peterson and Fry, 1987; Alber and Valiela, 1994). Another problem to

consider is that there could be variations in the 815N values of food sources over time.

For example, a study by Zieman et al. (1984) showed that while the 15N content of sea

grasses did not change as they degraded over time, the 1SN content of mangrove leaves

varied by up to 10%o.

Studies have also been done using 15N values as indicators of trophic level.

Nitrogen isotopes are assumed to increase progressively with each trophic transfer (Wada

et al., 1981). Minagawa and Wada (1984) found, in a study on marine and freshwater

animals from the East China Sea, the Bering Sea, Lake Ahinoko, and the Usujiri

intertidal zone, that all consumers, zooplankton, fish, and birds exhibited stepwise

enrichment of 15N with increasing trophic level. Fry (1988) found a general trend of

increasing s35N and 813C with increasing trophic levels; however, he also found that 8'3C

and 8'5N were not correlated. "N increases were more consistent indicating that LSN is a

more reliable trophic indicator than a13C (Fry, 1988). Gu et al. (1996) found that

enriched 81N values in medium and large fish in Lake Apopka, Florida resulted from

diets consisting of other fishes occupying high trophic levels. Riera (1998) found

through analysis of 81SN of Crassostrea gigas in Marennes-Oleron Bay, France, that the

oysters exhibited direct utilization of benthic diatoms as a food source near intertidal

mudflats. However, in the upper estuarine reaches, they were also found to utilize

terrestrial detritus as a food source. It was suggested that 6'5N can be a useful tool in

characterizing trophic levels in an estuarine food web as long as different feeding habitats

are considered (Riera, 1998).






29

Proposed Research

The overall goal of this research is to study the transformation and fate of dissolved

organic matter (DOM) originating in the Suwannee River using stable carbon and

nitrogen isotopes. This includes three main objectives: (1) determining if flocculated

organic aggregates could be produced in the laboratory and if they maintained a

terrestrial isotopic signature, (2) characterizing the spatial pattern of stable isotope

signatures in particulate organic matter in the Suwannee River and its estuary during two

different flow regimes, and (3) using stable isotope ratios to investigate aspects of the

food web in the Suwannee River estuary.













CHAPTER 2
STABLE CARBON AND NITROGEN ISOTOPE COMPOSITION OF ORGANIC
AGGREGATES PRODUCED BY SALINITY INDUCED FLOCCULATION OF
DISSOLVED ORGANIC MATTER FROM THE SUWANNEE RIVER

Introduction

Terrestrial dissolved organic matter (DOM) discharged by rivers into estuarine and

marine systems represents a significant source of carbon and other elements (e.g.,

McCarthy et al., 1996; Opsahl and Benner, 1997; Dagg et al., 2004) and is a potentially

important source of energy for consumers in coastal ecosystems. Much of the DOM

originating in terrestrial systems is degraded by a broad suite of physical, chemical, and

biological processes as it is transported to and mixed with ocean waters (Gardner et al.,

1994; Amon and Benner, 1996; Hedges et al., 1997; Twardowski and Donaghay, 2001;

Heres and Benner, 2002; Zimmerman-Timm, 2002; Dagg et al., 2004; Oberosterer and

Benner, 2004). Examples include photochemical processes and microbial degradation.

However, flocculation of DOM has been shown to occur in estuarine systems where fresh

water meets salt water (Scholkovitz, 1976; Fox, 1983; Mannino and Harvey, 2000;

Sondergaard et al., 2003) and may result in the formation of organic aggregates of a

suitable size and quality for filter- and suspension-feeding organisms, such as clams and

oysters. The DOM, from which flocs are formed in the Suwannee River estuary has a

high nitrogen component as a result of fertilizers and animal wastes present in the system

(Asbury and Oaksford, 1997; Katz et al., 2001).

Laboratory produced organic aggregates have historically been difficult to produce,

even though flocculation of DOM has been shown to occur in many estuarine systems








(Fox, 1983; Eisma, 1986; de Souza Sierra et al., 1997; Del Castillo et al., 2000; Thill et

al., 2001; Sondergaard et al., 2003). Sholkovitz et al. (1976) presented a classic study

where organic aggregates were produced at various salinities by mixing filtered river

water (Glen Burn, Water of Luce, River Cree, and River Stinchar in Scotland) with

filtered seawater (collected off the coast of southeast Scotland). The amounts of

flocculated material increased as salinity increased from 0 to -15%o. Above 15%o, little

removal of the dissolved organic matter occurred. Studies following Sholkovitz's

methods have been less successful in creating flocs (Fox, 1983; de Souza Sierra et al.,

1997; Thill et al., 2001).

The mechanisms underlying the formation of organic aggregates have often been

debated, and it has not been proven that flocculation is solely induced by salt or bacteria,

physically induced, or a combination of these processes. Fox (1983) repeated, with

minor differences, the Sholkovitz et al. (1976) study with unfiltered river water and

filtered seawater from the Delaware Bay and its tributaries. After analysis of the

dissolved humic acid carbon, it was determined that salt-induced removal of dissolved

humic acid was insignificant. Humic substances from different environments are

compositionally different, and therefore, their response to changes in ionic strength and

removal mechanisms may be different (Fox, 1983). De Souza Sierra et al. (1997) used

filtered river water from the Scheldt estuary (Holland) and artificial seawater in an

attempt to produce organic aggregates in the laboratory. No flocculation was noticed and

it was ascertained that under natural conditions, the removal of humic material may

proceed slowly by aggregation through various chemical, physical, and biological

interactions or by adsorption onto suspended matter which was not present in their








samples due to filtering. Thill et al. (2001) included analysis of particle size distribution

along the mixing zone of the Rhone river estuary, France, during high and low flow

regimes to determine if salt induced flocculation played an important role. No obvious

instantaneous change in particle size of the natural river particle size occurred and it was

determined that salt induced aggregation did not play an important role concerning

increased particle size.

Eisma (1986), in a review of observations of flocculated material in European

coastal waters and along the US east coast found that salt-induced mechanisms play only

a minor role in organic aggregate formation, and that organic aggregates are formed more

by a combination of forces including viscous flow, gravity, and Brownian motion.

Bacteria have also been shown to be a major factor in the production of flocculated

material, either by direct colonization or through excretions of mucous which enhances

the stickiness of particle surfaces (Zimmermann-Timm, 2002). Rivers supply inorganic

and organic nutrients that can be used by bacteria as determined in the Hudson River

(USA) by Findlay et al. (1992). Gardner et al. (1994) found that most bacterial

production was supported by dissolved organic matter rather than by particulate materials

during microcosm experiments with filtered and unfiltered water from the northern Gulf

of Mexico near the main delta of the Mississippi River. Also in the Mississippi River

Delta, it was determined that the bioreactivity of DOM increased with increasing size of

the organic matter (Amon and Benner, 1996). Womer et al. (2000) found that bacteria

colonized laboratory-produced aggregates, using the aggregates as a haven in the water

column, over a period of 14 days using water from the Elbe Estuary (Germany). The

aggregates were largest after 9 days because large omnivorous and carnivorous ciliates








colonized the aggregates (W6rner et al., 2000). The combination of physical and

bacterial influences can combine to produce flocculated materials from DOM.

Del Castillo et al. (2000) were able to produce organic aggregates in a laboratory

experiment using Peace River water (a colored river with high concentrations of

dissolved organic matter; west coast of Florida, USA). The Suwannee River (west coast

of Florida, USA) similarly has large concentrations of colored dissolved organic matter

(CDOM), and organic aggregates have been observed in the water column at the salt and

freshwater interface (personal observation). Regardless of exactly how organic

aggregates are formed, it is important to understand what happens to the organic

aggregates once they are produced. It has been hypothesized that organic aggregates are

used as a food source by estuarine organisms (Alber and Valiela, 1994; Zimmerman-

Timm, 2002). Alber and Valiela (1994b) determined that macrophyte-derived aggregates

are more readily consumed than detritus by bay scallops. The DOM released by primary

producers in an aggregate detrital pathway is transformed into organic aggregates, and

then ingested by bivalves, e.g., bay scallops. The aggregates were shown to meet a

substantial portion of the nutritional requirements of the bay scallops because they had

lower C:N ratios and higher protein content than detritus (Alber and Valiela, 1994b). In a

review of river-born aggregates, Zimmerman-Timm (2002) suggests that aggregates

support growth and production of riverine protozoa and metazoa as a long term food

supply.

The major source of terrestrial-derived DOM is vascular plants, which are confined

largely to land and characteristically contain high concentrations of recalcitrant, nitrogen-

free biomacromolecules such as lignin and tannin (Hedges et al., 1997). These plants








have distinct stable carbon isotope ( 3C) compositions which can be used as a biomarker

indicating a terrestrial origin (Fry and Sherr, 1984; Hedges et al., 1997). Most terrestrial

plants have 8'3C values of -28 to -25%0, versus -22 to -19%0 for temperate marine

phytoplankton, whereas sea grasses have heavier "3C values near -12%o (Fry and Sherr,

1984). The f15N values of vascular land plants and marine plankton can also be

sufficiently different (Hedges et al., 1997). Terrestrial plants typically have average 815N

values of 3%, whereas phytoplankton have heavier average 515N values ranging from 6

to 10%o (Owens, 1987). There is a prevalence of nitrogen fixation on land, and therefore

the light organic '5N compositions have been suggested as being typical of terrestrial

origin (Peters et al., 1987), however blue green algae also present light '5N values

because of the capability to fix nitrogen.

Here, it is proposed that DOM in the fresh water of the Suwannee River flocculates

when mixed with salt water from the Gulf of Mexico, and that the organic aggregates

may be consumed by clams and oysters in the estuary. A laboratory study was performed

to produce organic aggregates under varying salinity conditions and to determine if

natural abundances of stable nitrogen and carbon isotopes might be used as markers to

characterize organic aggregates and identify their potential role as a food source for fauna

in the Suwannee River estuary. Further, the study sought to determine if organic

aggregates maintain their stable isotope signature along the salinity gradient utilized in

the laboratory to simulate such gradients in the estuary.

Methods and Materials

Site

The Suwannee River is a blackwater, tidally influenced river with its principal

origin in the Okefenokee Swamp in Georgia. It flows southwest through Florida and








discharges into the Gulf of Mexico just north of Cedar Key. There is a delta region at the

mouth of the river characterized by a network of intertidal and subtidal oyster reefs

(Wolfe and Wolfe, 1985). The water quality of the upper Suwannee River is

characterized by low pH (around 4), low concentrations of dissolved inorganic solids, and

high concentrations of dissolved organic substances, especially fulvic acids (Malcolm et

al., 1989a). The lower Suwannee River maintains a higher pH (6 to 9) (Wolfe and Wolfe,

1985). The source of the humic substances in the water is predominantly litter, leaf and

root exudates, and leaf leachates. The concentration of these humic substances has been

shown to be an order of magnitude greater than that in most natural stream waters. The

dissolved organic carbon concentrations in the Suwannee River near the Okefenokee

Swamp are in excess of 25 mg/L (Malcolm et al., 1989b).

Methods

Laboratory experiments were designed to flocculate the dissolved organic matter

(predominantly humic substances) from the Suwannee River across a range of salinities.

Whole water samples were collected from one site in the upper Suwannee River fresh

water (August 2002 and September 2003) and one in the lower Suwannee River fresh

water (May 2003 and August 2003). When returned to the laboratory, samples were

shaken and 1-L aliquots were distributed to 6 beakers. Samples were not filtered because

it had been noted that filtering samples would remove suspended material that could be

involved in the flocculation and adsorption process (De Souza Sierra et al., 1997).

Instant Ocean Synthetic Sea SaltM (Aquarium Systems, Mentor, OH) was added as a

solid and mixed with the 1-L samples until dissolved and the desired salinity was

reached. The salinity varied from 0 to 30 ppt in increments of 0, 5, 10, 15, 20, and 30 ppt

and was determined with a YSI Conductivity and Salinity field monitor.








The samples were stirred (Phipps and Bird jar tester) for 6 to 10 consecutive days at

approximately 14 rpm. Samples were taken for DOC analyses daily for experiments I

and II, and initially for experiments III and IV (see below). At the end of each

experiment, organic aggregates were allowed to settle and the residual water was

decanted and the organic aggregates were collected onto a combusted (450* C, 4 hours)

GF/F filter. Samples were packed and frozen prior to stable nitrogen and carbon isotope

analysis.

DOC and Stable Isotope Analysis

Immediately after the organic aggregates were collected from each beaker, the

decanted water was filtered through a Millipore HNWP 0.45 um filter (pre-washed with

E-Pure water). The filtrate was analyzed with a Tekmar Dohrman Apollo 9000 HS

Combustion TOC Analyzer with an STS 8000 Autosampler. The reproducibility of the

DOC measurements was generally within 0.05%. 13C and 8'5N isotope ratios of the

organic aggregates on filters were analyzed with a Finnigan Delta-C continuous flow

mass spectrometer. Results are reported in standard 8 notation where:

i'5N (%o)= [('sN/14N)sampl(e5N/14N)sdard)-l] x 1000 (2-la)

and

13C (%) = [(13C/12C)smpd(13C/12C)standard)-l] x 1000 (2-1b)

Atmospheric nitrogen and carbon from the Pee Dee Belemnite served as reference

standards. The reproducibility of the stable 13C and 'SN isotopes was within 0.3%o.

Results

Organic aggregates were formed almost immediately after Instant OceanT was

added to the reaction beaker and stirring was initiated. The organic aggregate densities

increased, as observed visually, with increasing salinity.








Comparisons of DOC versus salinity are presented in Figure 2-1. All four of the

experiments showed that increasing the salinity from 0 to 30 ppt had undetectable or

minor effects on DOC concentrations. Only one experiment from the upper Suwannee

River showed a significant relationship (p < 0.0001); DOC decreased with increasing

salinity. There was some association for two of the other three experiments (p = 0.0396,

0.0188), and one showed no significant relationship (p = 0.3687). Continuous stirring for

longer than 24 hours showed no measurable changes in DOC concentrations for organic

Floe studies I and II (Figure 2-2).

The relationships between '5N and 6"3C signatures and salinity are presented in

Figures 2-3 and 2-4, respectively. Average stable isotope values for all four studies are

presented in Figures 2-5 and 2-6, respectively. The 615N of the laboratory produced

organic aggregates increased by approximately 4%o with increasing salinity using pooled

data from all four experiments. The upper Suwannee River organic aggregate

experiments showed significant relationships with a p-value < 0.0001 for both

experiments. One of the lower Suwannee River organic aggregate experiments also

showed significant relationship with a p-value = 0.0046 (Floe II). The second of the

lower Suwannee River organic aggregate studies did not show a significant relationship

(Floe IV, p = 0.3245). The 13C signature of aggregates decreased significantly in two of

the four experiments (Floe II, p = 0.0062 for the lower Suwannee and Floe IV, p = 0.0035

for the upper Suwannee) (Figure 2-4). The 13C of organic aggregates formed during one

experiment using upper Suwannee River water increased very slightly with increasing

salinity (Floe I, p = 0.0151). The average '15N and 6'3C for all the four of the floe studies

combined are given in Figures 2-5 and 2-6. Salinity had a significant effect on s15N and






38


Table 2-1. Analysis of Variance (ANOVA) Results for all of the Floc Studies.

Floc I Floe II Floc III Floc IV Average
Source of Variation Salinity Salinity Salinity Salinity Salinity Expriment
'15N F Ratio 10.59 23.46 8.30 1.2596 3.62 33.61
P <0.0001 0.0046 0.0450 0.3245 0.0240 <0.0001
613C F Ratio 2.41 10.52 0.30 38.29 0.97 24.58
P 0.0729 0.0062 0.6158 0.0035 0.4669 <0.0001
C:N F Ratio 14.91 7.34 n.d. n.d. 4.35 5.80
P <0.0001 0.0154 n.d. n.d. 0.0120 0.0077
DOC F Ratio 108.14 4.88 6.76 1.32 n.d. n.d.
P <0.0001 0.0396 0.0188 0.3687 n.d. n.d.

*P Values at the 95% confidence level. Analyzed with JMP. (n.d. = not determined)











Floc Study I (Upper Suwannee)


0 5 10 15 20 25 30 35
Salinity


Floc Study IV (Upper Suwannee)

90
80o
70 "- "-
60


Floe Study II (Lower Suwannee)

16.8

16.2
16.21 .o 1,1
ax 16.0
4 15.816 c
15.6
o 15.4
15.2
15.0
14.8


0 5 10 15 20 25 30 35
Salinity


Floe Study III (Lower Suwannee)


13.0
2 12.5
CL
- 12.0
g 11.5
11.0
Insn


0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Salinity Salinity


Figure 2-1. Salinity versus Dissolved Organic Carbon in the Upper and Lower Suwannee River Floe Studies.


S


.









Floc Study I


0 1 2 3 4 5 6 7 8 9 10
Time (Days)


Floc Study II


19

18 ......::
';K ....--------
17 -

16 IK

S15

14

13
12
0 1 2 3 4
Time (Days)


-- 0 Salinity
- 15 Salinity
-- 30 Salinity
--x .-- 5 Salinity
--*x--- 10 Salinity
- 0-.--- 20 Salinity


5 6 7


Figure 2-2. Time versus Dissolved Organic Carbon for Floc Studies I and II.


70
60
E 50
, 40
o 30
0
0 20
10
0


~z z2 Z2


-*- 0 Saliniy
-a- 5 Saliniry
- 10 Salinlry
- 15 Saliniry










Upper Suwannee


Floc Study I



Floc Study IV




S 5 10 15 20 25 30 35
Salinity


Lower Suwannee


0 5 10 15 20 25 30 35
Salinity


Figure 2-3. Salinity versus 85N for the Upper and Lower Suwannee River Flocculated
Organic Aggregates.


7
6
5
4
3
z 2
S1
S0
-1
-2
-3
-4
-5


8

6

z 4
2

0


Floc Study II



Floc Study III










Upper Suwannee

-20

-22

-24

-26 Floc Study I

-28
-30 'Floc Study IV
0 5 10 15 20 25 30 35
Salinity




Lower Suwannee

-20

-22

o-24
b -26
S. Floc Study III
-28
Floc Study II
-30
0 5 10 15 20 25 30 35
Salinity


Figure 2-4. Salinity versus t13C for the Upper and Lower Suwannee River Flocculated
Organic Aggregates.

















5



4



3



2



1



0



-1
-5 0 5 10 15 20 25 30 35
Salinity

Figure 2-5. Salinity versus Average 615N Signatures of the Upper and Lower Suwannee River Floc Studies Including Standard
Error Bars.











-27.3


-27.4


-27.5


-27.6


-27.7


-27.8


-27.9


-28.0


-28.1


-28.2
-5 0 5 10 15 20 25 30 35
Salinity

Figure 2-6. Salinity versus Average 6t3C Signatures of the Upper and Lower Suwannee River Floc Studies Including Standard
Error Bars.











18



16



14



S12


z
10



8-



6-


10 15
Salinity


20 25 30 35


Figure 2-7. Salinity versus Average C:N Ratios of the Flocculated Organic Aggregates from the Upper and Lower Suwannee
River Floc Studies Including Standard Error Bars.


I ( I








no significant effect on 13C (Table 2-1).

The C:N ratios of the filtered organic aggregates are presented in Figure 2-7. C:N

ratios of the organic aggregates averaged between 10 and 12 and were highest at the

highest salinities (Figure 2-7). Salinity had a significant effect on the C:N ratios (p <

0.05).

Discussion

It was necessary to determine if organic aggregates could be formed by mixing

Suwannee River water with salt (Instant OceanTM) so that organic aggregates could be

analyzed for stable nitrogen and carbon isotopes. Salt-induced organic aggregate

formation has been shown to be optimal for salinities between 1 and 7 ppt (Thill et al.,

2001), however, in our experiments, organic aggregates formed almost instantly (within

the first half hour of mixing) in each salinity treatment (0 to 30 ppt). The initial idea for

determining organic aggregate formation was to measure DOC throughout the duration of

a 10-day experiment, given the hypothesis that DOC would decrease each day as the

organic aggregates formed. The results showed, however, that DOC did not change

significantly throughout the 10-day experiment (Figure 2-2). During this study, the

Suwannee River exhibited very high levels of DOC in the upper and lower portions of the

river (ranging from 13 to greater than 50 ppm). Because there was such a high

concentration of DOC in this system, it is likely that only a small percentage of the DOC

was transformed into an aggregate form, and as a result, no substantial loss of DOC was

observed. Sondergaard et al. (2003) also found that fast aggregation of DOC was not a

major removal process of DOC in highly colored rivers. Therefore, a different approach

had to be taken to be able to quantitatively model the behavior of the organic aggregates.








Stable nitrogen and carbon isotope ratios have been used to model theoretically the

behavior of particulate organic material (Montoya et al., 1991; Kendall et al., 2001; De

Brabandere et al., 2002), but not organic aggregates, per se. In each of the four

experiments, the change in the S15N signatures of organic aggregates was consistent from

0 to 30 ppt (Figure 2-3), indicating that i'5N could be used as markers of organic

aggregates as they are formed and move throughout the Suwannee River and estuary.

The 6i5N of the organic aggregates followed a pattern showing a transition from a

terrestrial signature (i.e., near 0) to a higher, more marine signature as the salinity

increased. It is likely that the change in pH, induced by increased salinity, might

selectively drive the precipitation of DOC, and based on the 15N and 13C values of the

flocculated organic aggregates, a trend is observed.

The formation of the organic aggregates can be biological, chemical, and/or

physical. Bacteria have been shown to have a major influence on organic aggregates and

particulate organic nitrogen (PON) formed in an estuarine environment (Kemp, 1990;

Hoch et al., 1992; Alber and Valiela, 1994; Stumm and Morgan, 1996; Sondergaard et

al., 2003). The samples taken from these laboratory experiments were not filtered prior

to the addition of salt, allowing the organic aggregates to include particulate material

already in the water which may have included suspended sediment, bacteria, plant debris,

and fecal matter. Bacterial incorporation of ammonia has been shown to be a mechanism

for producing isotopically light particles (near 1.1%o), whereas microbially degraded

material tends to have signatures closer to 8%o (Voss et al., 1997). This implies that

degradation of PON (or the organic aggregates in these experiments) can result in an

increase of 6 1N. In these experiments, samples were stirred in the beakers for up to 10








days at room temperature, and not protected from light, allowing formation of organic

aggregates which could have included growth of bacterial communities and

remineralization of isotopically light nitrogen.

Salt induced flocculation is caused by electrolytic interactions between negatively

charged functionalities in humic substances and cations in seawater (Eckert and

Sholkovitz, 1976; Stumm and Morgan, 1996), especially calcium and magnesium.

Positive salt ions neutralize the negatively charged humic substances, and Van der Waals

forces form attraction forces between the neutral molecules, forming organic aggregates.

Hydrogen bonding also contributes to aggregation of humic substances, especially when

the molecules include hydroxyl or amine groups (Stumm and Morgan, 1996). Slow

mixing encourages collision of the particles. The stability of colloids in the dissolved

organic matter pool is mainly determined by the pH and ionic strength of solution as well

as the presence of specific ions (Tombicz et al., 1999). Increasing the salt content

increases the pH and the ionic strength, and humic substances exposed to salt ions will

flocculate (Stumm and Morgan, 1996). Isotopic fractionation can occur in the process of

ion exchange reactions. For example, the process of ammonium adsorption at the site of

clay minerals (or possibly humic substances) plays a primary role in determining '5N

abundance, and the heavier isotopic species is preferentially adsorbed on clay colloids

(Delwiche and Steyn, 1970).

Ammonium is found in humic substances in the Suwannee River (Malcolm et al.,

1989b). Therefore, a possible chemical explanation for the steady increase in 685N with

increasing salinity is that some fraction of the NH4+ (associated with humic substances) is

transformed to NH3 as pH increases resulting in a heavier isotopic composition of the








residual NH4+ on the organic aggregates. Ammonia volatization leads to the enrichment

of 5SN in inorganic nitrogen (Wada and Hattori, 1991).

The 63C of the organic aggregates did not show a consistent trend as did the 1s5N

data. The data, in fact, exhibited little change across the broad range of salinities (< 1%o)

and aggregates maintained a terrestrial signature (-25 to -30%o) (Figure 2-4). Therefore,

513C measures can be a good tool for determining the source of flocculated particles.

Guo et al. (1997) studied the 613C signatures of colloidal organic carbon in the

Chesapeake Bay and also determined that stable carbon isotope signatures were good

indicators of the origin of organic particles.

The C:N ratios help to characterize the laboratory-produced organic aggregates.

For example, Middleburg and Nieuwenhuize (1998) distinguished terrestrial sources of

particulate organic material (C:N =21) from riverine, estuarine, and marine material

(C:N =7.5 to 8). In our experiments, C:N ranged from 6 to 17, indicative of relatively

high quality food source based on findings by Alber and Valiela (1994a). The large

range in the C:N ratios may be explained by different environmental causes. For the

upper Suwannee River experiments, water was collected for floc study II during a

drought and for floc study VI during more normal flow conditions. During floc study II,

the water levels were extremely low in the river, and less carbon was entering the system

(groundwater inputs dominate during low flow periods). Therefore, C:N ratios were

lower than during floc study VI when water levels were higher, and higher C:N ratios of

the material were entering the river (Figure 2-7).








It is important to determine if the organic aggregates are being consumed by filter

feeders or other fauna in the estuary. In a later chapter, the possibility that bivalves in the

Suwannee River are consuming organic aggregates will be presented.

Summary

A laboratory study was performed to determine if natural abundance measures of

stable carbon and nitrogen isotope ratios might be used as markers to characterize organic

aggregates to identify their potential role as a food source for fauna in the Suwannee

River estuary. The 8i5N values of laboratory produced aggregates increased significantly

with salinity, however the 6 3C values did not change significantly with increasing

salinity. These findings indicate that the 6S5N signatures might be used as a tracer of

organic aggregate development as they are formed as a result of increasing salinity.

These findings also indicate that 6'3C signatures might be used to quantify their role as a

food source for estuarine fauna.

Conclusions

The following conclusions may be drawn from the research discussed in this

chapter:

* Formation of organic aggregates, i.e. floes, can be induced by mixing salts with
Suwannee River water rich in dissolved organic material.

* s15N signatures of aggregates increased significantly with salinity, but exhibited
pronounced variability.

* 3C signatures did not change significantly with increasing salinity indicating that
stable carbon isotope ratios might be used as a tracer of terrestrial derived dissolved
organic matter in estuarine and coastal food webs.

* The C:N ratios increased significantly with increasing salinity, but did not exceed
values of 17 indicating that salinity induced aggregates are a potentially high
quality food source for suspension feeding organisms in the Suwannee River
estuary and associated nearshore coastal waters.













CHAPTER 3
SPATIAL GRADIENTS IN THE STABLE CARBON AND NITROGEN ISOTOPE
COMPOSITION OF SUSPENDED PARTICLES IN THE SUWANNEE RIVER
ESTUARY DURING DIFFERENT FLOW REGIMES

Introduction

River discharge provides a significant source of nutrients to estuaries. Increased

nutrient input into estuaries and marine environments, whether from point or non-point

sources, can lead to increased primary production and changes in trophic structure

(Ryther and Dunstan, 1971; Smith et al., 1999; White et al., 2004). Nitrate

concentrations in many of Florida's spring-fed rivers are rapidly increasing (Mytyk and

Delfino, 2004), especially in the Big Bend region of the northeast Gulf of Mexico (Ham

and Hatzell, 1996; Jones et al., 1997, Katz et al., 2001), where it has long been

recognized as relatively unaffected by anthropogenic forces (Bass and Cox, 1988). In

particular, the Suwannee River has such high levels of nitrate and carbon that it may alter

the biological character of the Suwannee River estuary.

Distinct seasonal patterns of the distribution of nutrients (specifically nitrate and

ammonium) occur in the Suwannee River and estuary. With the second highest mean

annual discharge in Florida (301 m3/s), the Suwannee River has its peak flows typically

in the early spring and low flows typically in the fall (Crandall et al., 1999). Nitrate

concentrations in the estuary are lower during periods of high flow than during periods of

low flow. This is a karst system where groundwater interacts with surface water, and

during the fall (low flow period), the main source of freshwater for the river is

groundwater which has high nitrate concentrations (Pittman et al., 1997; Crandall et al.,








1999). During the spring (high flow period), overland surface flow is the dominant

source of water for the river. High color in the system limits nutrient uptake and

assimilation by phytoplankton (due to light limitation). Thus, much of the otherwise

available nutrients are discharged directly to the estuary (Bledsoe, 1998). Nitrogen is the

limiting nutrient in the Suwannee River estuary (Bledsoe and Phlips, 2000), and

eutrophication is a potential problem of concern (Ryther and Dunstan, 1971; Bledsoe et

al., 2004).

Nitrogen loading can have a significant impact on biological productivity and

ecosystem structure in coastal and nearshore marine systems; however the mechanisms

and pathways involved may be difficult to resolve with traditional methods (e.g.

hydrographic and mass balance studies) because of the complexity of the nitrogen cycle

and relatively large spatial and temporal scales of interest (Owens, 1987; Peterson and

Fry, 1987; Horrigan et al., 1990a, Horrigan et al., 1990b; Montoya et al., 1990; Montoya

and McCarthy, 1995). The stable isotopic signature of inorganic and organic nitrogen

provides an integrative tool for studying the sources of nitrogen supporting production in

an ecosystem as a whole (Valiela et al., 1997). Stable carbon isotope ratios also provide

a means for determining the source of organic matter supporting production in these

ecosystems.

Spatial gradients in nutrient concentrations, particularly of nitrate and/or

ammonium, are characteristic of estuarine systems and can occur as a result of physical

mixing processes and uptake and assimilation by phytoplankton and other autotrophs

(e.g. attached macroalgae, epiphytic microalgae, and seagrasses). Isotopic fractionation

associated with the uptake and assimilation of nitrate and/or ammonium can generate








spatial gradients in the stable nitrogen isotope composition of the residual pool of

dissolved inorganic nitrogen. This will also be reflected in the isotopic composition of

nitrogen sequestered in particulate forms (Montoya and McCarthy, 1995). In essence,

stable nitrogen isotopes can serve as in situ tracers of nitrogen as it moves through an

estuarine system. Stable carbon isotopes are also used as in situ tracers of the source of

carbon in estuarine systems.

Here, data are presented from the Suwannee River and estuary in Northwest Florida

during two different high and low flow periods, hypothesizing that distinct spatial and

temporal patterns will be seen in the stable nitrogen isotopes of suspended particulate

organic matter (SPOM), and that stable carbon isotopes will allow determination of the

source of organic matter fueling production in higher-level consumers. Stable nitrogen

and carbon isotopic data for oysters and zooplankton collected in the same area are

presented to determine the relationship between oysters, zooplankton, and SPOM.

Methods, Materials, and Site Description

Site Description

The historic Suwannee River is a blackwater, tidally influenced river with its main

origin in the Okefenokee Swamp in Georgia. The river is approximately 394 km in

length and flows southwest through Florida. It discharges into the Gulf of Mexico just

north of Cedar Key, draining a watershed of approximately 25,640 km2 (Nordlie, 1990).

Main tributaries include the Alapaha, Withlacoochee, and Santa Fe Rivers. At the mouth

of the river there is a delta region which is characterized by a network of intertidal and

subtidal oyster reefs that are subject to harvest periodically throughout the year (Wolfe

and Wolfe, 1985).








Periods of peak flow in the Suwannee River occur typically in early spring and

periods of low flow occur in the fall. There is much less land development and point

source pollution in this watershed than in most other coastal regions in Florida (Bass and

Cox, 1988). Major land uses in the watershed include silviculture and agriculture (row

crops, dairy, poultry, and swine) (Katz et al., 2001). The land immediately surrounding

the river is predominantly used for pine forestry operations. Fertilizer from pine forestry

operations and waste material from dairy practices are thought to be a significant source

of non-point source nutrient loading to the river (Ashbury and Oaksford, 1997). Spring

and groundwater discharge contribute significantly to nitrate concentrations in the river,

particularly during periods of low flow. The upper part of the estuary, comprising both

oligohaline and mesohaline zones, is bordered by brackish marsh habitat and is

dominated by emergent grasses and rushes. Submerged aquatic vegetation (SAV) is less

abundant. The lower portion of the nearshore estuary is a mosaic of saltmarsh, tidal

creeks, mudflats and oyster habitats. Seagrasses are much less abundant within this

estuary when compared to other areas of Florida's Big Bend coast, as a consequence of

highly colored surface water and low light levels (Bledsoe, 1998). The main pathway of

nitrogen transfer to higher trophic levels likely occurs directly through the pelagic

compartment (i.e. phytoplankton production in the surface water) (Frazer et al., 1998;

Bledsoe et al., 2004).

Methods

Surface water samples were collected from the Suwannee River and its estuary in

November, 1996 (low flow period), March, 1997 (high flow period), May, 2003 (high

flow period), and December, 2003 (low flow period). During the 1996/1997 sampling

event, water was collected at 10 stations along a transect (stations marked with A, see








Figure 3-1). The initial sampling station was located in the main river approximately

5km from the mouth and subsequent sampling stations progressed seaward at

approximately 3.7km intervals (Figure 3-1). During the 2003 sampling event, water was

collected at 10 stations along a salinity transect (stations marked with B and C), ranging

from 0 ppt to approximately 30 ppt (Figure 3-1, Table 3-1). Conductivity, dissolved

oxygen, pH, salinity, and temperature were measured with a YSI field meter. Water

samples were held on ice and returned to the lab for processing.

Water samples collected at each station during each sampling event were filtered

through precombusted glass fiber filters (Whatman GF/F, 4500C, 4h) and the filtrate was

frozen for subsequent analyses (within 24h) of nitrate and ammonium in the laboratory.

Filtered particles were placed on ice and transported to the laboratory where they were

dried at 600C (>48h) in preparation for isotope analysis. The filtrate from the 2003

sampling trips was preserved with concentrated sulfuric acid (resulting in a pH<2)

(Holmes et al., 1998) and stored prior to analysis of the stable nitrogen isotope

composition of dissolved inorganic nitrogen (DIN). Additional water was collected at

each station and filtered on site for subsequent analysis of chlorophyll a concentration.

Water was also collected during the 96/96 and 2003 sampling trips to be analyzed for

DOC (2003 only), color, TN, TP, and phosphate. Zooplankton were collected with a

202um ring-net (diameter = 0.5m). Samples were placed on ice and returned to the

laboratory where they were immediately sorted by taxon (i.e., copepod, chaetognath, and

ctenophore) and prepared for stable isotope analysis. Oysters were collected along the

transect during low tide in December, 2003. They were stored on ice and transported to



































Figure 3-1. Map of the Sampling Sites in the Suwannee River and its Estuary.









Table 3-1. Location and Site Names for Sampling Sites in the Suwannee River and its
Estuary.

Sample Date Sample GPS Coordinates
ID Latitude Longitude
Nov. 1A 29.328012 -83.103267
1996 and 2A 29.306664 -83.147504
March 1997 3A 29.281708 -83.164847
4A 29.279032 -83.166859
5A 29.279264 -83.166504
6A 29.274404 -83.167420
7A 29.258845 -83.176053
8A 29.245006 -83.194144
9A 29.246172 -83.234362
10A 29.251276 -83.284666
May 2003 1B 29.327136 -83.103301
2B 29.307044 -83.148037
3B 29.279506 -83.165490
4B 29.275151 -83.169196
5B 29.266446 -83.170751
6B 29.266037 -83.171514
7B 29.260041 -83.176243
8B 29.251779 -83.189391
9B 29.243944 -83.198380
10B 29.199541 -83.251848
Dec. 2003 1C 29.328012 -83.103267
2C 29.306664 -83.147504
3C 29.281708 -83.164847
4C 29.279032 -83.166859
5C 29.279264 -83.166504
6C 29.274404 -83.167420
7C 29.258845 -83.176053
8C 29.245006 -83.194144
9C 29.246172 -83.234362
10C 29.251276 -83.284666








the laboratory where they were shucked, dried at 600C (>48h), ground to a fine powder

with a mortar and pestle, and stored in a desiccant cabinet prior to isotope analyses.

Concentrations of nitrate (NO3"+N02), ammonium (NH4+), and total nitrogen (TN)

in surface water samples were measured with a Bran+Lubbe AutoAnalyzer II within two

days of sampling. The limit of detection for each nutrient was <0.001 mg-N/L. Analyses

of phosphate (PO4) and total phosphorus (TP) were analyzed with a Perkin Elmer

Lambda 2 spectrophotometer (Greenberg et al., 1992). Color was analyzed using

platinum cobalt standards also using a Perkin Elmer Lambda 2 spectrophotometer and a

Spectronic 401 spectrophotometer. Chlorophyll a was measured on a Hitachi U-2000

spectrophotometer after extraction with hot ethanol. Total nitrogen and total phosphorus

were determined using the persulfate digestion method (Greenberg et al., 1992). Samples

for DOC analysis were filtered through 0.45 Am nylon filters (Millipore HNWP) and

measured with a Tekmar Dohrman Apollo 9000 HS Combustion Analyzer with a STS

8000 TOC Autosampler. The reproducibility of the DOC measurements was within

0.05%.

All samples for isotope analysis were analyzed with a Finnigan Delta-C

continuous-flow mass spectrometer. Results are reported in standard 6 notation where:

5"SN (%o)= [(("N/l'N)sample/(lN/'4N)standard)-l] x 1000 (3-la)

and

I3C (%o) = [(('3C/'C)sample/('1C/l1C)standard)-l] x 1000 (3-lb)

Atmospheric nitrogen and Pee Dee belemnite limestone served as reference standards.

The reproducibility of the stable carbon and nitrogen isotope measures was within 0.3%o.








Results

Physical Parameters

Salinities in the Suwannee River (station 1: 1996 and 1997 and stations 1 and 2:

2003; Table 3-2) were at or less than 0.2 ppt during all sampling periods. The two

sampling stations furthest offshore (stations 9 and 10: 1996 and 1997) showed salinities

near 35 ppt, and the station furthest offshore (station 10) during the 2003 sampling

periods showed a salinity near 30 ppt. Salinities at the other stations were less than these

higher values and varied with sampling date during the 1996 and 1997 sampling events,

whereas the salinities were at more regularly spaced intervals during the 2003 sampling

periods.

Color in the Suwannee River estuary showed considerable variation. Values for

color were greatest within the river and decreased along the transect from the river to the

Gulf of Mexico during the 1996 and 1997 sampling events (Table 3-2). During the 2003

sampling events, color was highest in the river and decreased steadily with distance from

the river's mouth. Color values were highest during periods of high river discharge

(April 1997 and May 2003).

In March 1997 and May 2003, the observed high flows were approximately 400

m3/s and 320 m3/s respectively. In November 1996 and December 2003, recorded flows

were approximately 130 m'/s and 157 m3/s, respectively. Average monthly streamflows

for 1996, 1997, and 2003 are presented in Figure 3-2.

Chemical Parameters

Nitrate concentrations (Table 2-3) were highest in the river during periods of low

discharge (November, 1996 and December, 2003). Beyond the river, nitrate






60


Table 3-2. Chemical and Biological Parameters in the Suwannee River and its Estuary
for Each Sampling Event.
Station
1 2 3 4 5 6 7 8 9 10
SALINITY (ppt)
Nov-96 0.0 21 23 25 30 31 33 33 35 35
Mar-97 0.0 22 22 23 28 30 31 33 34 35
May-03 0.1 0.1 3.5 6.5 9.0 16 17 21 25 30
Dec-03 0.2 0.2 3.8 6.1 10 13 17 22 25 30
COLOR (mg pt/L)
Nov-96 110 27 29 32 22 19 32 27 10 8
Mar-97 150 44 46 34 19 12 14 21 12 12
May-03 188 187 163 148 122 97 84 65 46 22
Dec-03 63 66 57 54 45 41 36 25 17 9
DISSOLVED ORGANIC CARBON (ppm)
May-03 12.1 12.4 14.8 13.8 13.6 10.4 9.6 7.8 7.2 4.5
Dec-03 5.5 5.8 6.8 6.6 6.1 5.8 5.8 4.7 5.0 2.6
CHLOROPHYLL A (mg/m3)
Nov-96 0.4 2.3 2.1 2.9 9.8 9.9 7.8 2.9 1.2 0.9
Mar-97 0.2 4.2 5.6 3.7 3.0 2.0 2.2 1.3 1.5 0.7
May-03 0.3 0.3 2.2 6.1 9.9 13.9 18.9 13.7 9.8 2.8
Dec-03 0.1 BDL 1.3 2.7 5.1 3.7 11.8 4.3 5.3 2.5











600



tI ----1996
500 / --*-1997

19
S\ 2003


S400 i





4200 ;
.o- \






100




0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Time


Figure 3-2. Monthly Streamflow Data for the Suwannee River near Wilcox, Florida (USGS, 2004).








concentrations decreased rapidly along the transect during the 1996 and 1997 sampling

events. Further offshore, during the 1996 and 1997 sampling events, nitrate

concentrations were generally below the limit of analytical detection (Table 3-3). During

the May and December 2003 sampling events, nitrate concentrations also declined with

distance offshore.

Ammonium levels mirrored nitrate patterns with the exception of the December,

2003 sampling event (Table 3-3). During this event, ammonium remained in the narrow

range of about 46 to 56 jg/L with the exception of 72 gg/L at station 5C and exhibited a

peak concentration at station 6C, i.e. 111 ig/L. In November, 1996, the concentration of

ammonium in the river and nearshore of the estuary varied considerably (Table 3-3), but

was undetectable seaward of station 3A.

Total nitrogen (TN) was highest in or near the river mouth for all sampling dates

(Table 3-3). During the 1996 and 1997 sampling events, TN concentrations decreased

substantially outside the river mouth. During the 2003 sampling event, TN

concentrations declined steadily with distance offshore (Table 3-3).

Total phosphorus (TP) and soluble reactive phosphorus (SRP) were highest in or

near the mouth of the river for all sampling dates (Table 3-4). Total phosphorus

concentrations decreased markedly beyond the river mouth (station 2A) during the 1996

and 1997 sampling events. During the 2003 sampling period, TP concentrations

decreased steadily with distance offshore (Table 3-4). Higher concentrations of TP and

SRP were observed during periods of high river flow (March 1997 and May 2003). SRP

was not detectable beyond station 6 during the 1996 and 1997 sampling events.









Table 3-3. Concentrations of Nitrogen Components (pg/L) in the Suwannee River and its
Estuary for Each Sampling Event.
Station
Nov-96 1A 2A 3A 4A 5A 6A 7A 8A 9A 10A
N-N03 810 60 180 180 BDL* BDL BDL BDL BDL BDL
N-NH4* 40 320 20 BDL BDL BDL BDL BDL BDL BDL
Total N 1190 380 480 430 340 300 320 200 160 120

Mar-97 1A 2A 3A 4A 5A 6A 7A 8A 9A 10A
N-N03 360 80 80 60 BDL BDL BDL BDL BDL BDL
N-NH4' 40 320 20 BDL BDL BDL BDL BDL BDL BDL
Total N 850 380 290 350 280 170 170 100 130 150

May-03 1B 2B 3B 4B 5B 6B 7B 8B 9B 10B
N-NO3- 514 513 598 587 490 369 314 185 129 60
N-NH4' 47 52 79 73 63 60 37 29 36 BDL
Total N 1170 1170 1270 1050 1150 945 825 660 510 245

Dec-03 1C 2C 3C 4C 5C 6C 7C 8C 9C 10C
N-N03 1300 1275 935 860 640 520 450 250 115 BDL
N-NH4' 47 53 56 56 72 111 54 54 55 46
Total N 1320 1325 1100 1015 875 780 735 515 500 265


*BDL below detection limit






64


Table 3-4. Concentrations of Phosphorus Components (qg/L) in the Suwannee River and
its Estuary for each Sampling Event.
Station
Nov-96 1A 2A 3A 4A 5A 6A 7A 8A 9A 10A
SRP 50 10 BDL BDL BDL BDL BDL BDL BDL BDL
Total P 130 10 30 30 20 10 BDL BDL 10 BDL

Mar-97 1A 2A 3A 4A 5A 6A 7A 8A 9A 10A
SRP 80 20 10 10 BDL BDL BDL BDL BDL BDL
Total P 90 20 10 10 BDL BDL BDL BDL BDL BDL

May-03 1B 2B 38 4B 5B 6B 7B 8B 9B 10B
SRP 91 89 81 63 45 28 18 8 3 BDL
Total P 122 116 134 105 99 68 49 33 24 11

Dec-03 1C 2C 3C 4C 5C 6C 7C 8C 9C 10C
SRP 79 78 61 57 40 30 24 12 3 BDL
Total P 84 80 70 68 59 47 52 33 32 18








Dissolved organic carbon (DOC) was only determined for the two 2003 sampling

events. DOC concentrations decreased steadily with increasing salinity after reaching

their highest values at station 3 on both 2003 sampling dates (Table 3-2). Dissolved

organic carbon concentrations exhibited a slight increase from station 1 to station 3.

Biological Indicator

Chlorophyll a concentrations in the river were consistently low (<1 mg/m3) during

the 1996 and 1997 sampling events (Table 3-2). During the 2003 sampling period,

chlorophyll a concentrations were low near the river mouth and increased to a

chlorophyll maximum near stations 6 and 7 (Table 3-2). Chlorophyll maxima were also

observed during the 1996/1997 sampling periods. A maximum value (9.9 mg/m3) was

observed at station 6A in November 1996 and a maximum value (5.6 mg/m3) was

observed at station 3A in March 1997.

Stable Isotope Analyses

Spatial Gradients in the stable nitrogen isotope composition (6'5N) of suspended

particulate organic matter (SPOM) collected from the surface waters of the Suwannee

River and its estuary were apparent during both low and high flow regimes (Figures 3-3

and 3-4, Table 3-5). During the November 1996 sampling event, there was an initial

decrease in the 61SN signature from station 1 to station 3 with a sharp drop at station 4.

The 5isN signatures of SPOM during the March 1997 sampling event exhibited a sharp

decline at station 2A. The 1s5N signatures of SPOM were higher during the March 1997

sampling event than during the November 1996 sampling event. A similar pattern was

observed in May 2003; there was a sharp drop at stations 3B and 4B. The December

2003 sampling event showed a more uniform, increasing pattern for the SPOM (Figure 3-

4).












*--*-Mar-97
-| Nov-96


B.

i.


High Flow

",


0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-3. 815N Signatures of Surface Suspended Particulate Organic Matter (SPOM)
for the 1996 and 1997 Sampling Events.


. .. -.. ...-- .






67



7


High Flow
6 *



o



4



3 '.



2 Low Flow












-1
0
-1.




-- +-- May-03
-- Dec-03

0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-4. 5' N Signatures of Surface Suspended Particulate Organic Matter (SPOM)
for the Two 2003 Sampling Events.









Table 3-5. Stable Isotope Data of Suspended Particulate Organic Matter (SPOM) and
Dissolved Inorganic Nitrogen (DIN) for the 1996, 1997, and Two 2003
Sampling Events.
Station
1 2 3 4 5 6 7 8 9 10
615N of SPOM (per mil)
Nov-96 3.80 3.51 3.43 2.37 4.77 4.90 5.33 5.55 5.24 4.92
Mar-97 9.75 6.68 7.31 7.63 7.51 7.64 7.33 6.65 ND ND
May-03 2.87 2.40 -0.69 0.24 2.96 3.79 5.42 5.77 5.29 5.99
Dec-03 1.94 1.96 1.72 2.92 2.23 2.46 3.99 3.35 2.13 5.57
613C of SPOM (per mil)
Nov-96 -26.70 23.87 24.46 23.71 22.85 22.74 22.65 22.70 22.44 22.05
Mar-97 -25.84 24.78 25.95 25.31 22.97 22.41 22.26 21.84 ND ND
May-03 -27.90 27.61 24.42 26.91 26.07 24.13 24.18 24.08 22.11 20.39
Dec-03 -27.53 25.79 25.78 25.90 26.61 25.49 28.03 23.02 23.53 22.26
6sN DISSOLVED INORGANIC NITROGEN (per mil)
Nov-96
(Nitrate) 2.2 4.1 3.4 2.8 4.7 5.4 3.8 3.5 1.8 4.9
Mar-97
(Ammon) 8.2 9.9 11.4 3.0 11.5 11.9 14.8 12.0 11.7 14.5
May-03
(Nitrate) 7.0 8.0 7.5 6.8 6.9 5.4 5.3 5.1 2.1 2.1
Dec-03
(Nitrate) 3.7 5.3 4.8 6.4 6.3 7.0 7.0 7.3 5.8 -0.8

ND Not Determined








Spatial gradients in the stable nitrogen isotope composition of dissolved inorganic

nitrogen were apparent during the March 1997 sampling event. A sharp dip was present

at station 4 for the 6tSN of the DIN-ammonium, and the 615N of the DIN-nitrate was

lower than the DIN-ammonium (Figure 3-5, Table 3-5). The N of the DIN-nitrate for

the 2003 sampling event showed a variation between the different flow regimes (Figure

3-6). During the low flow period, 1'5N values increased steadily, whereas during the

high flow period, "5N values decreased steadily.

Zooplankton were collected only at stations 3 through 10 during the 2003 sampling

events (Figures 3-7 and 3-8). Only calanoid copepods, chaetognaths, and ctenophores

were retained for stable isotope analysis. The 61 N signatures of the zooplankton were

very similar and ranged from ca. 8 to ca. 10%o chaetognathss and ctenophores) and from

ca. 5 to ca. 10%o copepodss) during the high and low flow periods (Figure 3-7 and 3-8).

Oysters were sampled opportunistically near the transect sampling sites where they could

be found, and exhibited a marrow range in their '5N signatures, ca. 7.5 to 8%o (Figure 3-

9).

Spatial gradients in the stable carbon isotope composition (13C) of SPOM

collected from the surface waters of the Suwannee River estuary were apparent during

the 96/97 and 2003 sampling periods irrespective of the flow regimes (Figures 3-10 and

3-11). The 13C signatures of the SPOM from the 96/97 sampling events were similar to

the 613C signatures of the SPOM during the 2003 sampling event, ranging from -28%o in

the river to ca. -21%o at the farthest offshore stations (Figures 3-10 and 3-11).

The range of 613C signatures of the zooplankton were similar during the May and

December 2003 sampling events, with the copepods exhibiting slightly lower signatures










16




14




12




10




z
8




6




4




2


-.- Deli5N Nitrate
- Del15N Amm


/ x
I -,


At
\ i
S\ i

4* /

\ I
\ I

'4


0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-5. 815N Signatures of Surface Dissolved Inorganic Nitrogen (DIN) for the
March 1997 Sampling Event.






71



9
--*-. May-03
--- Dec-03
8 .




Low Flow

6


5


High Flow
4


3



2






0


-1



-2
0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-6. 68'N Signatures of Surface Dissolved Inorganic Nitrogen (DIN-Nitrate) for
the 2003 Sampling Events.










10



9



8



7



6



S 5



4



3



2



1



0


--+-.COPE
- CHAET
-C- CTEN


0 1 2 3 4 5
Station


6 7 8 9 10


Figure 3-7. S15N Signatures of Zooplankton Collected during the May 2003 (High Flow)
Sampling Event (Cope = Calanoid Copepods, Chaet = Chaetognaths, Cten =
Ctenophores).


rA











11



10






8



7
-----...

6



5



4



3



2



1 *-*- COPE
-- CHAET
-*- CTEN
0
0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-8. 685N Signatures ofZooplankton Collected during the December 2003 (Low
Flow) Sampling Event (Cope = Calanoid Copepods, Chaet = Chaetognaths,
Cten = Ctenophores).













10

8

6

4

2

0

-2

-4

-6

- -8

z -10

-12

-14

-16

-18

-20

-22

-24

-26

-28


** .. +-. -**-+- *+










S 1 2 3 4 5 6 7 8 9 10 1

DeI15N
L-U- DeMl3C
























-- U-----,


Station


Figure 3-9. Stable Carbon and Nitrogen Isotopes of Oysters Collected During the
December 2003 Sampling Event.






75


-20



-21


High Flow
-22



-23
Low Flow


-24



S-25 ,



-26



-27



-28



-29
Nov-96
"5-. Mar-97
-30
0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-10. 63C of the Suspended Particulate Organic Matter (SPOM) for the 1996 and
1997 Sampling Events.






76



-20



-21


High Flow
-22



-23


-24 Low Flow
-24 .



S-25



-26



-27



-28



-29

*- *- May-03
---Dec-03
-30
0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-11. 13C of the Suspended Particulate Organic Matter (SPOM) for the Two
2003 Sampling Events.








than the chaetognaths and ctenophores at any given sampling station (Figures 3-12 and 3-

13). Oysters maintained a 813C signature of approximately -24 to -25%o (Figure 3-9).

Statistics

Analysis of variance (ANOVA) was used to test for effects of salinity on stable

isotopic signatures (15 N and 6"C) of SPOM, DIN and other water quality parameters

(Tables 3-6 through 3-9). Values were determined to be significantly different if p-values

were less than 0.05.

Discussion

The results of this study suggest differences in the physical, chemical, and

biological parameters during high and low flow regimes. The Suwannee River

discharges high concentrations of nutrients into the coastal region of west Florida,

compared to other rivers in the same area (Suwannee River Water Management District,

2003). Outside the mouth of the river, chemical analyses show a decline in nutrients with

distance offshore (Tables 3-3 and 3-4). Samples collected in the river during periods of

low flow (November 1996 and December 2003) exhibited higher concentrations of

inorganic nitrogen relative to those collected during high flow periods (March 1997 and

May 2003). In other systems such as New York Harbor, nitrogen concentrations increase

during rainy seasons due to surface water exportation into the system from the watershed

(Ryther and Dunstan, 1971), however, the opposite occurred in the Suwannee River

system. Nitrate increased in the river during low flow periods and decreased during high

flow periods (Suwannee River Water Management District, 1995, 1996, 2003). The

Suwannee River is a karst, spring-fed river system, and groundwater contributes

significantly to the river discharge during low flow periods (Pittman et al., 1997; Crandall

et al., 1999). Springs discharge is prevalent in the middle reaches of the Suwannee River






78



-15




-17




-19




-21




I -23 -

/ /
A--




-25




-27




-29

--+-*COPE
--CHAET
CTEN
-31
0 1 2 3 4 5 6 7 8 9 10 11
Station


Figure 3-12. 8'3C of Zooplankton Collected During the May 2003 Sampling Event
(Cope = Calanoid Copepods, Chaet = Chaetognaths, Cten = Ctenophores).











-20




-21




-22




-23




-24




S-25




-26




-27




-28




-29




-30


r
I
I



I
I
I


I
I

I
I
I
I .,
I
1










r


--*--COPE
--CHAET
--- CTEN


0 1 2 3 4 5 6 7 8 9 10 11

Station



Figure 3-13. 86C of Zooplankton Collected During the December 2003 Sampling Event
(Cope = Calanoid Copepods, Chaet = Chaetognaths, Cten = Ctenophores).






80


Table 3-6. ANOVA Tables for the Effects of Salinity on the 86N and 6'C of Suspended
Particulate Organic Matter during Novermber 1996.
NOVEMBER 1996 Sampling Event

Oneway Analysis of SPOM 815N By Salinity
Mean
Source of Variation DF* Sum of Squares Square F Ratio Prob > F "
Salinity 7 19.3663 2.7666 16.8960 0.00002
Error 12 1.9649 0.1637
Total 19 21.3312

Oneway Analysis of SPOM 8"C By Salinity
Mean
Source of Variation DF Sum of Squares Square F Ratio Prob > F
Salinity 7 33.6221 4.8032 87.3301 2.44E-09
Error 12 0.6600 0.0550
Total 19 34.2821

Degrees of freedom
If the probability is less than 0.05 (95% confidence) then there is significant variance






81


Table 3-7. ANOVA Tables for the Effects of Salinity on the 15N and 6"tC of Suspended
Particulate Organic Matter during March 1997.
MARCH 1997 Sampling Event

Oneway Analysis of SPOM 815N By Salinity
Mean
Source of Variation DF Sum of Squares Square F Ratio Prob> F
Salinity 7 7.3362 1.0480 2.9228 0.1062
Error 6 2.1514 0.3586
Total 13 9.4876

Oneway Analysis of SPOM 613C By Salinity
Mean
Source of Variation DF Sum of Squares Square F Ratio Prob> F
Salinity 7 36.9084 5.2726 5.2498 0.0219
Error 7 7.0305 1.0044
Total 14 43.9388









Table 3-8. ANOVA Tables for the Effects of Salinity on the Physical, Chemical, and
Biological Indicators of Suspended Particulate Organic Matter during
December 2003.
DECEMBER 2003 Sampling Event

Oneway Analysis of SPOM 815N By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob> F
Salinity 9 25.7998 2.8666 2.4135 0.0931
Error 10 11.8777 1.1878
Total 19 37.6775
Oneway Analysis of SPOM 813C By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 65.5696 7.2855 24.1083 0.00001245
Error 10 3.0220 0.3022
Total 19 68.5916
Oneway Analysis of DIN 81N-NO3 By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 172.1822 19.1314 3.5323 0.0310
Error 10 54.1615 5.4161
Total 19 226.3437
Oneway Analysis of N03 By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob> F
Salinity 9 3723545 413727 3065 5.34E-16
Error 10 1350 135.0
Total 19 3724895
Oneway Analysis of NH4 By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 6534.00 726.00 11.56 3.40E-04
Error 10 628.00 62.80
Total 19 7162.00
Oneway Analysis of Chi a By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob> F
Salinity 9 211.3423 23.4825 39.7049 1.18E-06
Error 10 5.9143 0.5914
Total 19 217.2566
Oneway Analysis of Color By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob> F
Salinity 9 6844.2000 760.4667 7604.6667 5.68E-18
Error 10 1.0000 0.1000
Total 19 6845.2000
Oneway Analysis of DOC by Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob> F
Salinity 9 25.49718 2.83302 15.31693 9.88E-05
Error 10 1.8496 0.18496
Total 19 27.34678









Table 3-8 Continued. ANOVA Tables for the Effects of Salinity on the Physical,
Chemical, and Biological Indicators of Suspended Particulate Organic Matter
during December 2003.
Oneway Analysis of TN by Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 2262920.0 251435.6 867.0 2.92E-13
Error 10 2900.0 290.0
Total 19 2265820.0
Oneway Analysis of TP by Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 8707.4500 967.4944 152.3613 1.66E-09
Error 10 63.5000 6.3500
Total 19 8770.9500
Oneway Analysis of SRP By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 15495.2 1721.6889 1565.1717 1.53E-14
Error 10 11.0 1.1000
Total 19 15506.2
Oneway Analysis of C-SPOM By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 135.2867 15.0319 19.6729 3.18E-05
Error 10 7.6409 0.7641
Total 19 142.9276
Oneway Analysis of N-SPOM By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 2.3578 0.2620 28.6788 5.52E-06
Error 10 0.0914 0.0091
Total 19 2.4492
Oneway Analysis of C:N-SPOM By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 42.4307 4.7145 96.5498 1.58E-08
Error 10 0.4883 0.0488
Total 19 42.9190
Oneway Analysis of N-DIN By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 9 102.2519 11.3613 484.8015 5.31E-12
Error 10 0.2344 0.0234
Total 19 102.4863









Table 3-9. ANOVA Tables for the Effects of Salinity on the Physical, Chemical, and
Biological Indicators of Suspended Particulate Organic Matter during May
2003.
MAY 2003 Sampling Event

Oneway Analysis of SPOM 81SN By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 118.7969 14.8496 3.7383 0.0234
Error 11 43.6950 3.9723
Total 19 162.4919
Oneway Analysis of SPOM 8"'C By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 103.4872 12.9359 40.4563 4.41E-07
Error 11 3.5173 0.3198
Total 19 107.0045
Oneway Analysis of DIN 851N-NO3 By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 433.3482 54.1685 2.6594 6.76E-02
Error 11 224.0538 20.3685
Total 19 657.4020
Oneway Analysis of N03 By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob> F
Salinity 8 691979.0500 86497.3813 917.0807 2.00E-14
Error 11 1037.5000 94.3182
Total 19 693016.5500
Oneway Analysis of NH4 By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 9718.5500 1214.8188 28.1771 2.88E-06
Error 11 474.2500 43.1136
Total 19 10192.8000
Oneway Analysis of Chi a By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 752.2718 94.0340 178.0946 1.56E-10
Error 11 5.8080 0.5280
Total 19 758.0798
Oneway Analysis of Color By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 62196.2000 7774.5250 2327.0687 1.20E-16
Error 11 36.7500 3.3409
Total 19 62232.9500
Oneway Analysis of DOC by Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 199.7360 24.9670 48.9091 1.63E-07
Error 11 5.6153 0.5105
Total 19 205.3513









Table 3-9 Continued. ANOVA Tables for the Effects of Salinity on the Physical,
Chemical, and Biological Indicators of Suspended Particulate Organic Matter
during May 2003.
Oneway Analysis of TN by Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 2028145.0000 253518.1250 369.3642 2.92E-12
Error 11 7550.0000 686.3636
Total 19 2035695.0000
Oneway Analysis of TP by Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 35881.8000 4485.2250 573.6916 2.62E-13
Error 11 86.0000 7.8182
Total 19 35967.8000
Oneway Analysis of SRP By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 23486.8000 2935.8500 949.8338 1.65E-14
Error 11 34.0000 3.0909
Total 19 23520.8000
Oneway Analysis of C-SPOM By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 159.4177 19.9272 4.2376 1.51E-02
Error 11 51.7277 4.7025
Total 19 211.1454
Oneway Analysis of N-SPOM By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 5.7842 0.7230 11.9665 1.96E-04
Error 11 0.6646 0.0604
Total 19 6.4488
Oneway Analysis of C:N-SPOM By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 77.4207 9.6776 37.4608 6.60E-07
Error 11 2.8417 0.2583
Total 19 80.2624
Oneway Analysis of N-DIN By Salinity
Source of Variation DF Sum of Squares Mean Square F Ratio Prob > F
Salinity 8 136.5347 17.0668 227.3580 4.14E-11
Error 11 0.8257 0.0751
Total 19 137.3604








and many of those springs have elevated concentrations of nitrate (up to 8.0 mgfL)

(Suwannee River Water Management District, 1995, 1996, 2003). During high flow

periods, precipitation and surface flow dilute the concentration of nitrates coming into the

river through the springs.

Concentrations of nutrients (nitrogen and phosphorus), chlorophyll a (estimation of

phytoplankton biomass in the water column), dissolved organic carbon, and color

contribute to light attenuation in the Suwannee River (Bledsoe, 1998). High

concentrations of nutrients (especially nitrate) enter the middle reaches of the Suwannee

River (Suwannee River Water Management District, 2003). Phytoplankton in the river

are light limited Bledsoe, 1998) and otherwise available nitrate is not utilized extensively

by phytoplankton (low chlorophyll a levels within the river). Near the mouth of the river

and in the estuary, color and DOC decrease as seawater mixes with river water, and it is

at this point that phytoplankton are no longer light-limited. Phytoplankton numbers

increase in the estuary (based on chlorophyll a concentrations, Table 3-2), and

eutrophication of this system as a consequence of increased nutrient loading is a

legitimate concern (see Frazer et al., 1998).

Natural abundance measures of stable nitrogen and carbon isotope ratios can

contribute to the study of nutrient pollution by determining the source material fueling

production in a system. Temporal and spatial patterns in 6'5N and 13C can provide

additional information on transformation processes. Temporal variation in the stable

nitrogen signature of suspended particles in the Suwannee River is reflective of relative

differences in the abundance of nitrogen sources in the river during low flow and high

flow periods (Figures 3-3 and 3-4, Table 3-5). The 65N signatures of SPOM during








March 1997 (high flow) were higher than those collected during November 1996. During

low flow periods, much of the river is being fed by springs with high nitrate

concentrations. The source of nitrate is often from synthetic fertilizers (as opposed to

remineralized organic matter) within the Suwannee River watershed (Asbury and

Oaksford, 1997; Katz et al., 2001). Typical 5i5N values of fertilizers are low, or close to

zero (Freyer and Aly, 1974; Flipse and Bonner, 1985; Macko and Ostrom, 1994; Fry et

al., 2001). This is because most fertilizers are produced by the Haber process which

converts atmospheric nitrogen to ammonium. In this process, the fertilizers retain the

isotopic signature of atmospheric nitrogen (f'5N = 0%o) (Macko and Ostrom, 1994).

During high flow periods, the river is also being fed by overland flow, and this results in

the addition of organic nitrogen from animal waste material. Nitrate produced from

animal waste typically exhibit '6SN values greater than fertilizer 6'5N (Peterson and Fry,

1987; Karr et al., 2001; Karr et al., 2003). During the two 2003 sampling events, 61sN

signatures of SPOM were nearly the same (starting at 2-3%o in the river stations and

increasing to 5-6%0 at station 10) during the high and low flow regimes. The low initial

i65N signatures of the May 2003 sampling event indicate that the source of nitrogen

coming into the system was likely from a source similar to that of synthetic fertilizers.

Differences in the 815N of SPOM in the river between 96/97 and 2003 could indicate that

input of fertilizer to the river is increasing, or the relative contribution of DIN from

animal waste is decreasing.

Spatial gradients in the '15N of SPOM collected from surface waters were present

during low and high flow regimes (Figures 3-3 and 3-4, Table 3-5). The 65N of SPOM

decreased near the mouth of the river at stations 2 (March 1997), 4 (November 1996),








and 3 (May 2003), then leveled out to a signature closer to that typical of marine

phytoplankton (6%o) (Owens, 1987). The decrease near stations 2 through 4 did not

occur during the December 2003 sampling event. It did, however, slowly increase to a

signature near 6%o by station 10.

Most reactions in which bonds with a nitrogen atom are formed or broken tend to

discriminate against the heavier isotope, resulting in a product depleted in '5N relative to

the substrate (Figure 3-14). There is a progressive increase in the 815N of the residual

pool of reactant as the reaction proceeds. If a reaction goes to completion and the entire

pool of reactants is converted to product (e.g., in a closed system), then conservation of

mass requires that the final product have the same isotopic composition as the initial pool

of reactants (Peterson and Fry, 1987; Montoya and McCarthy, 1995). In an ecological

context, the isotopic fractionation associated with the uptake and assimilation of

dissolved inorganic nitrogen (DIN) by phytoplankton and other autotrophs (i.e. attached

macroalgae, seagrasses, and epiphytic algae) should result in the production of autotroph

biomass depleted in 15N relative to the available substrate, primarily NO3- in the case of

the Suwannee River and its estuary. This, in turn, will create a spatial gradient in the

stable nitrogen isotope composition of suspended particles in the Suwannee River estuary

(Figure 3-15). Our findings are consistent with this scenario (excluding the December

2003 sampling event) and provide critical information for the interpretation of naturally

occurring variations in 15N in these and other river and estuarine systems.

The stable nitrogen isotope ratio of DIN had not been measured previously in the

Suwannee River and its estuary. These measurements are needed for a more complete

test of the spatial gradient theory. Distinct spatial gradients in the 6'SN were expected to








































100%o


Figure 3-14. Model of Spatial Gradients in the "1N Signatures of a Hypothetical Closed
System (redrawn from Montoya and McCarthy, 1995).




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ECUBYD9SZ_9VRUIM INGEST_TIME 2014-04-14T19:24:03Z PACKAGE AA00014271_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES