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Dietary Exposure to Organochlorine Pesticides p,p'-DDE and Dieldrin and Their Effects on Steroidogenesis and Reproductive Success in Florida Largemouth Bass (Micropterus salmoides floridanus)

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Dietary Exposure to Organochlorine Pesticides p,p'-DDE and Dieldrin and Their Effects on Steroidogenesis and Reproductive Success in Florida Largemouth Bass (Micropterus salmoides floridanus)
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JOHNSON, KEVIN GEORGE
Copyright Date:
2008

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Dosage ( jstor )
Eggs ( jstor )
Female animals ( jstor )
Freshwater bass ( jstor )
Gonadal steroid hormones ( jstor )
Gonads ( jstor )
Oocytes ( jstor )
Pesticides ( jstor )
Plasmas ( jstor )
Sex hormones ( jstor )
City of Tallahassee ( local )

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University of Florida
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University of Florida
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Copyright Kevin George Johnson. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2006
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436098784 ( OCLC )

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DIETARY EXPOSURE TO ORGANOCHLORINE PESTICIDES p,p’-DDE AND DIELDRIN AND THEIR EFFECTS ON STEROIDOGENESIS AND REPRODUCTIVE SUCCESS IN FL ORIDA LARGEMOUTH BASS ( Micropterus salmoides floridanus ) By KEVIN GEORGE JOHNSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Kevin George Johnson

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iii ACKNOWLEDGMENTS I extend my gratitude to Dr. Timothy S. Gross for providing the resources and guidance necessary for me to succeed as a gr aduate researcher, and to Dr. Daniel E. Canfield, Jr. for providing me the opportunity to succeed as a graduate student. I give special thanks to all the staff at the US GS-FISC-CARS Ecotoxicology Lab (Gainesville, FL); without their help my research would not have been possible. Special thanks go to Dr. Maria S. Seplveda, Dr . Richard H. Rauschenberger, Carla Weiser, Janet Scarborough, Travis Smith, Jessica Grosso, Jon Wiebe, Kevin Kroll, and everyone in Dr. Nancy Denslow’s lab who assisted with my research. I thank my other committee member, Dr. Charles E. Cichra, for his invaluable assistance in revising my thesis. I acknowle dge Jennifer Muller and Dr. Christopher J. Borgert for their grant editorial assistance. My research was supported by a grant to Dr . Timothy S. Gross and Dr. Christopher J. Borgert from the American Chemistry Council. I thank my family, friends, and fellow students who supported me throughout my career as a student and my father Bill Johnson, who gave me the inspiration to pursue an education in fisheries science.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 The Situation.................................................................................................................1 Largemouth Bass Reproductive Biology......................................................................3 Endocrine Disruption and Reproducti ve Effects of OCP Exposure.............................8 Research Significance.................................................................................................11 2 DIETARY SUBACUTE EXPOSURE TO P,P ’-DDE AND DIELDRIN AND THEIR EFFECTS ON REPRODUCTIVE AND HEALTH BIOMARKERS IN FLORIDA LARGEMOUTH BASS...........................................................................13 Introduction.................................................................................................................13 Materials and Methods...............................................................................................14 Largemouth Bass.................................................................................................14 Feed Preparation..................................................................................................14 Experimental Design...........................................................................................15 Feeding Rate........................................................................................................16 Fish Collection and Bleeding..............................................................................16 Gonad Histology..................................................................................................17 Determination of Circulating Sex Steroid Hormones.........................................18 OCP Analysis......................................................................................................19 Statistical Analysis..............................................................................................19 Area 7 Largemouth Bass.....................................................................................19 Results and Discussion...............................................................................................20 3 DIETARY CHRONIC EXPOSURE TO P,P ’-DDE AND DIELDRIN AND THEIR EFFECTS ON REPRODUCTIVE SUCCESS IN LARGEMOUTH BASS..........................................................................................................................3 5

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v Introduction.................................................................................................................35 Materials and Methods...............................................................................................35 Largemouth Bass.................................................................................................35 Feed Preparation..................................................................................................36 Experimental Design...........................................................................................36 Feeding Rate........................................................................................................37 Fish Collection and Bleeding..............................................................................37 Day 120 Spawning..............................................................................................38 Determination of Circulating Sex Steroid Hormones.........................................39 OCP Analysis......................................................................................................39 Statistical Analysis..............................................................................................40 Area 7 Largemouth Bass.....................................................................................40 Results and Discussion...............................................................................................40 4 GENERAL CONCLUSIONS.....................................................................................65 LIST OF REFERENCES...................................................................................................69 BIOGRAPHICAL SKETCH.............................................................................................74

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vi LIST OF TABLES Table page 2-1. Day-30 GC-MS mean SD results of both female and male largemouth bass p,p ’-DDE and dieldrin concentrations (ng/ g) in both the carcass and gonads per treatment (n = 2 samples per treatment, for each sex).............................................24 2-2. Day-30 mean SD results of fema le and male weight, total length, condition index (K), GSI, and HSI for each treatment, for largemouth bass fed p,p ’-DDE diets. Treatments with the same lo wer case letter were not significantly different ( p > 0.05), with a sample size of 10 largemouth bass per treatment.........29 2-3. Day-30 mean SD results of fema le and male weight, total length, condition index (K), GSI, and HSI for each treatm ent, for largemouth bass fed dieldrin diets. Treatments with the same lo wer case letter were not significantly different ( p > 0.05), with a sample size of 10 largemouth bass per treatment.........30 3-1. Day-30 mean SD results of fema le and male weight, total length, condition factor (K), GSI, and HSI per treatment (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide.............................................................................................................48 3-2. Day-60 mean SD results of fema le and male weight, total length, condition factor (K), GSI, and HSI per treatment (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide.............................................................................................................49 3-3. Day-90 mean SD results of fema le and male weight, total length, condition factor (K), GSI, and HSI per treatment (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide.............................................................................................................50 3-4. Day-120 mean SD results of fema le and male weight, total length, condition factor (K), GSI, and HSI per treatment (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide.............................................................................................................51

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vii 3-5. Day-120 GC-MS mean SD results of both female and male largemouth bass p,p ’-DDE and dieldrin concentrations (ng/ g) in the carcass per treatment (n = 3 carcasses per treatment, for each sex)......................................................................60 3-6. Day-120 mean SD resu lts of percent hatch for the p,p ’-DDE and dieldrin treatments (n = 6 clutches per treatmen t). Treatments with the same upper case letter were not signi ficantly different ( p > 0.05)......................................................64

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viii LIST OF FIGURES Figure page 2-1. Female largemouth bass mean SD carcass concentrations (shaded bars) of p,p ’-DDE and dieldrin treatments (n = 2 largemouth bass per treatment), as compared to the target carcass concentrati ons (black bars). Included is the mean carcass concentration of p,p ’-DDE and dieldrin for the five female largemouth bass sampled from Area 7 (shaded bar) on February 26, 2003................................25 2-2. Male largemouth bass mean SD carcass concentrations (shaded bars) of p,p ’DDE and dieldrin treatments (n = 2 larg emouth bass per treatment), as compared to the target carcass concen trations (black bars)......................................................26 2-3. Female largemouth bass mean SD gonad concentrations of p,p ’-DDE and dieldrin treatments (n = 2 largemouth ba ss per treatment). Included is the mean gonad concentration of p,p ’-DDE and dieldrin for the five female largemouth bass sampled from Area 7 on February 26, 2003.....................................................27 2-4. Male largemouth bass mean SD gonad concentrations of p,p ’-DDE and dieldrin treatments (n = 2 largemouth bass per treatment).......................................28 2-5. Mean female estradiol concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 largemouth bass per treatment. Treatments with the same lower case letter were not significantly different ( p > 0.05)....................................31 2-6. Mean male estradiol concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 largemouth bass per trea tment. Treatments with the same lower case letter were not significantly different ( p > 0.05)....................................32 2-7. Mean female 11-ketotestoste rone concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 larg emouth bass per treatment. Treatments with the same lower case letter were not significa ntly different ( p > 0.05).............33 2-8. Mean male 11-ketotestostero ne concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 larg emouth bass per treatment. Treatments with the same lower case letter were not significa ntly different ( p > 0.05).............34 3-1. Female estradiol concentrations , on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. As terisk represents significant difference ( p 0.05) from the Control......................................................................................52

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ix 3-2. Female estradiol concentrations , on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. As terisk represents significant difference ( p 0.05) from the Control......................................................................................53 3-3. Female 11-ketotestosterone con centrations, on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control.....................................................................54 3-4. Female 11-ketotestosterone con centrations, on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treat ments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control...................................................55 3-5. Male estradiol concentrations, on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. As terisk represents significant difference ( p 0.05) from the Control......................................................................................56 3-6. Male estradiol concentrations, on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. As terisk represents significant difference ( p 0.05) from the Control......................................................................................57 3-7. Male 11-ketotestoste rone concentrations, on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Contro l. Asterisk represents significant difference ( p 0.05) from the Control.....................................................................58 3-8. Male 11-ketotestoste rone concentrations, on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control.....................................................................59 3-9. Female (white bars) and male (sha ded bars) largemouth ba ss mean SD carcass concentrations of p,p ’-DDE and dieldrin treatments (n = 3 carcasses per treatment, for each sex). Included is the mean carcass concentration of each organochlorine for the five female largemouth bass sampled from Area 7 (white bar) on February 23, 2004........................................................................................61 3-10. Change in female GSI (%) over the en tire 120-day sampling period for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day). Sample days with the same lower case le tter were not significantly different ( p > 0.05).......................................................................................................................... 62

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x 3-11. Change in female GSI (%) over the en tire 120-day sampling period for the 0.8, 0.4, and 0.04 g/g Dieldrin treatments (n = 6 largemouth bass per sample day). Sample days with the same lower case le tter were not significantly different ( p > 0.05).......................................................................................................................... 63

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xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DIETARY EXPOSURE TO ORG ANOCHLORINE PESTICIDES p,p ’-DDE AND DIELDRIN AND THEIR EFFECTS ON STEROIDOGENESIS AND REPRODUCTIVE SUCCESS IN FL ORIDA LARGEMOUTH BASS ( Micropterus salmoides floridanus ) By Kevin George Johnson August 2005 Chair: Daniel E. Canfield, Jr. Cochair: Timothy S. Gross Major Department: Fisher ies and Aquatic Sciences Previous work has indicated that high organochlorine pesticide (OCP) concentrations in tissues of Florida largemouth bass ( Micropterus salmoides floridanus ), sampled from reclaimed agriculture lands w ithin the St. Johns River Water Management District’s Emeralda Marsh Conservation Ar ea (EMCA) have been associated with reproductive abnormalities, including depresse d hormone concentrations. Two of the OCPs found in highest concentration at this site are p,p ’-DDE and dieldrin. For my first study, hatchery-reared Florida largemouth bass were fed for 30 days using chemically treated floating pelletted fee d. Twenty largemouth bass, 10 males and 10 females per tank, were placed into each of nine treatme nts in replicate: Control; 1, 7, 35, and 136 g/g p,p ’-DDE; and 0.03, 0.1, 0.6, and 5 g/g Dieldrin. After day 30, five males and five females per replicate were sacrificed, had th eir blood and plasma collected for circulating sex steroid hormone analysis, and gonads collected for contaminant analysis and

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xii calculation of GSI. Gonads a nd carcasses from one male and one per replicate were also analyzed for OCPs, revealing a consistent co rrelation between the ad ministered doses and the concentrations found in the gonads and carcasses for both p,p ’-DDE and dieldrin. Final carcass concentrations for both p,p ’-DDE and dieldrin were similar to those found in largemouth bass from the EMCA. Histologi cal analysis of gonadal tissue indicated that all examined fish were sexually ma ture. GSI did not vary with dose of p,p ’-DDE or dieldrin. Analysis of sex steroid hormones also revealed no consistent relationships between p,p ’-DDE or dieldrin dosages and ci rculating concentrations of 17 -estradiol or 11-ketotestosterone. For my second study, p,p ’-DDE and dieldrin exposure length was extended to a 120-day period, between the months of November and March, encompassing a larger portion of the steroidogenic and gametogenic portions of the reproductive cycle. One hundred largemouth bass were plac ed into each of seven tr eatments: Control; 5, 46 and 50 g/g p,p ’-DDE; and 0.04, 0.4, and 0.8 g/g Dieldrin. On day 0 of the experiment, 24 fish were sampled to collect background measurements; then approximately every 30 days, six males and six females per treatment were sampled to collect measurements on the same reproductive biomarkers. Exte nsion of exposure length demonstrated reductions in female E2 concentrations, a lack of exp ected seasonal incr easing trend in female E2 concentrations, and abnormal increases in female 11-KT concentrations, similar to sex steroid hormone abnormalities reported for largemouth bass from the EMCA. Attained OCP carcass concentrations and achieved depressions of female E2 concentrations did not transl ate into a reduction of percen t hatch of eggs produced by these largemouth bass with high p,p ’-DDE and dieldrin ti ssue concentrations.

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1 CHAPTER 1 INTRODUCTION The Situation Sawgrass marshes surrounding a number of cen tral Florida lakes were drained for farming (muck farms) between the mid-1900s and the 1980s. In the 1990s, the State of Florida began purchasing muck farms in order to restore the farms back to their original floodplain marsh ecosystems. Between 1991 and 1994, the St. Johns River Water Management District (SJRWMD) acquired a 2,630-hectare portion of muck farms along the north-east shore of Lake Griffin. Th e site was designated the Emeralda Marsh Conservation Area (EMCA) and was to be used as a tool to reduce nutrient loading into Lake Griffin (Marburger et al ., 1999). Once flooding of the EMCA started in 1992, the Florida Fish and Wildlife Conservation Commission (FFWCC) began stoc king forage and game fish into these systems in an attempt to establish pub lic-accessible sport fish populations. FFWCC reported limited success establishing reproduc ing game fish populat ions. Of great concern was the apparent limited reproduction by stocked adult Florida largemouth bass ( Micropterus salmoides floridanus ) or recruitment of largem outh bass to the fingerling stage. FFWCC fish population surveys, conduc ted on several of the flooded properties in 1995 and 1996, however, revealed excellent gr owth rates for adult and fingerling largemouth bass (Benton and Douglas, 1996; Marburger et al ., 1999). The reason or reasons for the low largem outh bass recruitment at the EMCA were unclear, but there was speculation that poor re productive success might be related to the

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2 presence of residual organochlorine pesticides (OCPs). OCPs were produced after World War II and were widely used throughout the Un ited States for crop pest control on muck farms. According to standard operating prac tices, OCPs were selec tively applied as preemergence soil insecticides for vegeta ble production. Pesticides included DDT derivatives, dieldrin, al drin, endrin, chlordane, and hept achlor. The U.S. Environmental Protection Agency (USEPA), however, began to restrict or ban the use of many of these OCPs on agricultural lands between 1978 and 1983 because of their environmental persistence and their ability to biomagnify in food webs (USEPA, 1990). A study of OCP levels, in soil and largemouth bass ti ssues from the EMCA, demonstrated soil concentrations of p,p ’-DDE, dieldrin, and toxaphen e to be over 3,000, 500, and 40,000 ng/g, respectively. The same study also reveal ed concentrations of OCPs in largemouth bass ovaries and fat reached over 4,000 and 17,000 ng/g, respectively, for total DDT derivatives, over 100 and 700 ng/g for di eldrin, and over 4,000 and 20,000 ng/g for toxaphene (Marburger et al ., 2002). This evidence advanced the hypothesis th at the low largemouth bass reproductive success in the EMCA could be related to OCPs. Research, therefore, became focused on determining the extent of pesticide contamin ation in largemouth bass found in the waters of the reclaimed muck farm s adjacent to both Lakes Apopka and Griffin. Marburger et al . (1999) suggested that largemouth bass were bioaccumulating OCPs due to their top predator status and the persistence of OCPs in the muck farm soils. In addition to high OCP concentrations in tissu es of largemouth bass sampled from the EMCA, depressed sex steroid hormone (i.e., 17 -estradiol and 11-ketotestostero ne) concentrations were also reported (Marburger et al ., 1999). In addition, monthly sex steroid hormone values of

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3 both male and female tagged largemouth bass, captured from the EMCA, demonstrated that sex steroid hormone concentrations rema ined low throughout the year and showed no seasonal trends (Marburger et al ., 1999). However, there have been no experimental studies directly demonstrating any causal relationship between low sex steroid hormone concentrations and reproductive success (Benton and Douglas, 1996). Largemouth Bass Reproductive Biology Reproductive processes of teleost fishes, in cluding the Florida largemouth bass, are well defined. Chew (1974) described the earl y life history traits of Florida largemouth bass and found that sexual maturity is genera lly achieved at a tota l length (TL) of 250 mm, a length that can be obtai ned in only 1 year. Mature largemouth bass in Florida are capable of spawning between mid-November to August, with a peak spawning period in February and March (Clugston, 1966). Larg emouth bass are synchronous spawners, an act that is mainly triggered by a rise in wa ter temperature during the spring months to a level between 20 and 24C. It is also repor ted that spawning generally ceases at water temperatures below 18C, and above 27C (C lugston, 1966). Largemouth bass fecundity is highly variable, ranging from 2,000 to 145,000 eggs per female, but is generally accepted that females average about 4,000 eggs per pound of body weight (Tidwell et al ., 2000). Fecundity also appears to be directly related to age, condition, size, and to some environmental factors such as water temperature (Chew, 1974). The reproductive biology of teleost fishes follows a well-defined cycle that is regulated by exogenous environmental cues such as photoperiod and temperature, and endogenous hormonal cues (Gross et al ., 2002). This process is dependent on the coordinated actions of hormones associated with the brain-hypothalamus-pituitary-gonad axis (Van Der Kraak et al ., 1998). The hypothalamus controls the synthesis and release

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4 of gonadotropin-releasing hormone (GnRH), re sulting from neural stimulation of the central nervous system. This messenger hormo ne controls the synthesis and release of the primary teleost gonadotropin hormones GTH-I and GTH-II from the pituitary. These two gonadotropins are the re gulators of reproduction and are analogous to mammalian follicle-stimulating hormone (FSH) and luteinizing hormone (LH), respectively (Redding and Patino, 1993). GTH-I is typically involv ed in stimulating events leading to vitellogenesis or spermatogenesis and early gonadal development, whereas GTH-II is typically involved in stimulating events l eading to final oocyte maturation and ovulation in females and spermiation in males. Despite the difference with re gards to the role of GTH-I and GTH-II in female and male fish, these gonadotropins are known to be responsible for stimulating st eriodogenesis or the synthe sis of sex steroid hormones (androgens, estrogens, and progestins), which, in turn, act on target tissues to regulate gametogenesis (Van Der Kraak et al ., 1998). In the majority of female and male teleosts, 17 -Estradiol and 11-Ketotestosterone are the primary sex steroid hormones respons ible for regulating gametogenesis, and increases in plasma concentrations of thes e hormones are associated with the onset of seasonal reproductive activity (Gross et al ., 2002). 17 -estradiol and 11-ketotestosterone are the same hormones reported to have depre ssed concentrations th roughout the year for largemouth bass from the EMCA (Marburger et al ., 1999). In female fish, the development of oogene sis is controlled by GTH-I (Redding and Patino, 1993). Plasma concentrations of GTH-I increase during early oocyte development and bind to receptors on follicle ce lls. The cells synthesize testosterone and allow for aromatization to result in the formatio n of estradiol. Subs equently, estradiol is

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5 released by the follicle cells into the blood, where it binds to estrogen receptors in the liver, initiatimg a cascade of events resu lting in the production of vitellogenin (vitellogenesis), a precursor to egg yo lk protein, produced by the liver (Wahli et al ., 1981). Vitellogenin is released from the live r into the blood and binds to receptors on the oocytes which incorporate the protein as a nutrient source. As development of the oocytes continues, concentrations of GT H-I begin to decrease and are replaced by increasing concentrations of GTH-II (Van Der Kraak et al ., 1998). Receptors for GTH-II are found predominately on the granulosa cells of the follicles and binding stimulates the synthesis and release of proge stins, which play a role in final gamete maturation and stimulates ovulation (Redding and Patino, 1993; Van Der Kraak et al ., 1998). Similarly, in male fish, GTH-I is typically elevated th roughout spermatogenesis and decreases at the time of spawning, whereas GTH-II is typically low throughout the growth process and is elevated at spawning. These gonadotropins stimulate proliferation of spermagonia as well as the synthesis of androgens required fo r gametogenesis in male fish (Nagahama, 1994; Van Der Kraak et al ., 1998). Vitellogenesis in oviparous fish is the pr inciple event contributing to the massive growth of oocytes, due to a rapid uptake of the egg yolk precurso r vitellogenin (Wallace and Selman, 1981). Once vitellogenin is take n up by the vitelloge nin receptors on the surface of an oocyte, it is cleaved into sma ller yolk proteins. These proteins are then incorporated into yolk granules, which account for about 90 percent of the protein content of mature oocytes. The yolk granules are stored during oogenesis and serve as a nutrient source for embryonic development (Wahli et al ., 1981).

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6 Vitellogenesis ceases once the oocytes reac h their fully developed size, and is followed by a period of maturation (Wallace and Selman, 1981). During this time, follicles increase in volume due to hydration and accumulation of other vital proteins. Protein uptake stops at the time of germinal vesicle breakdown. The follicle however, continues to increase in volume by hydration. It is during this time that the chorion, the cellular envelope which surrounds an egg in pr eparation for ovulation, begins to develop. The timing of ovulation is species specific and takes place when follic les reach a specific size (Nelson, 2001; Wallace and Selman, 1981). Vitellogenesis represents a critical process in the development of teleost oocytes. This proc ess is initiated by seasonal changes in estradiol concentrations, however , monthly sex steroid hormone values of female largemouth bass from the EMCA de monstrated that sex steroid hormone concentrations remained low throughout the year and showed no seasonal trends (Marburger et al ., 1999). A lack of exp ected seasonal trends in estradiol concentrations could have detrimental effects on vitellogenesis. Gross et al . (2002) characterized the annual cy cles of circulating sex steroid hormones, vitellogenin, and gonad developm ent over a one-year period for pond-reared Florida largemouth bass in the st ate of Florida. Plasma samp les for both male and female largemouth bass were analyzed for 17 -estradiol (E2), 11-tetotestosterone (11-KT), testosterone (T), and vite llogenin (VTG). For males, 11-KT was the predominate androgen, and the only sex steroid observed to show a strong seasonal pattern which had a peak concentration of about 2,800 pg/mL in February. Even though T did show a seasonal pattern, the peak concentration of th is steroid in March was less than one-half that of the February peak for 11-KT, suppor ting the idea that 11-KT is the predominate

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7 androgen synthesized for endocrine function in male teleosts. E2 was detected in male largemouth bass, but at concentrations of about one-third that of females (Gross et al ., 2002). Females showed distin ct seasonal patterns for E2, T, and VTG. E2 showed the strongest pattern with circulat ing concentrations nearly twic e those of T, with a peak concentration of almost 4,000 pg/mL in Fe bruary; however, T did follow a similar seasonal pattern and peaked at the same time that E2 peaked. As previously mentioned, follicles must first synthesize testosterone befo re estradiol is formed, and may explain the similar seasonal trends demonstrated by both of these hormones. 11-KT was detected in females, but at concentrations nearly one -half that of males. Circulating VTG concentrations closely mimicked those of E2, rising in November and peaking in January at about 6 mg/ml. 17 -estradiol is one of the sex st eroid hormones reported to have depressed concentrations throughout the ye ar for largemouth bass from the EMCA. Vitellogenin is synthesized by the liver in re sponse to estradiol pr oduction; however, it is believed that changes in seasonal concentrations of this sex steroid hormone could lead to decreased vitellogenin production, causing impair ed female oocyte development (Muller, 2003). Interruption of the vite llogenic process could then have detrimental effects on oogenesis, ultimately leading to developmenta l abnormalities, increased sac fry mortality, and even spawning inhibition (Burdick et al ., 1964; Cross and Hose, 1988; Hose et al ., 1989; Macek, 1968; Monod, 1985; Smith and Cole, 1973). Gross et al . (2002) also calculated annual change s in gonadosomatic index (GSI), a number that reflects the per cent total body weight which the gonad comprises for a fish for pond-reared Florida largemouth bass in the state of Florida. Largemouth bass GSI was found to show similar seasonal changes re gardless of sex. GSI began to rise in

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8 November, peaking between January and May at a mean of approximately 5 and 2% for female and male largemouth bass, respectively. Dramatic decreases in GSI were reported between July and October. Reproductive seasonal changes in gonad maturation, as indicated by tissue histology, were also correla ted to changes in GSI and sex steroids in both sexes, and to fluctuati ons in VTG in females (Gross et al ., 2002). In female largemouth bass, gonadal stage was characteri zed as undeveloped (stage1) between May and July, as (stage 2) prev itellogenic between August and Oc tober, and as (stages 3 and 4) vitellogenic ovaries between November a nd April. In male largemouth bass, gonadal stage was generally characteri zed as having low spermatogeni c activity between June and November and moderate to high spermat ogenic activity between December and May (Gross et al ., 2002). Endocrine Disruption and Reproduc tive Effects of OCP Exposure In both fish and wildlife, the majority of research concerning potential biological effects of OCPs has been focused on endocrine disruption (Gallagher et al ., 2001; Gross et al ., 1994; Guillette et al ., 1994; Mills et al ., 2001; Muller et al ., 2004). Altered sex steroid concentrations in fi sh, environmentally and experi mentally exposed to OCPs, could result from the disruptiv e effects of these chemicals on hypothalamic or pituitary gonadotropin hormones (Gore, 2002; Shukl a and Pandey, 1986; Spies and Thomas, 1995). Gonadotropin hormones serve as stim ulating hormones for the synthesis and secretion of sex steroid horm ones, and any alteration in normal concentrations of these hormones could ultimately lead to decrea sed sex steroid production. Largemouth bass from the EMCA have demonstrated depressed sex steroid hormone (17 -estradiol and 11-ketotestosterone) concentr ations, and could be the re sult of a disruption in the

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9 hypothalamus-pituitary-gonad axis, responsible for stimulating synthesis and secretion of sex steroid hormones (Gross et al ., 2003). Competitive binding of these exogenous co mpounds to estrogen receptors could severely affect normal estrogen function, lead ing to depressed hormone concentrations, impaired gonadal development, decreased vite llogenesis, and ultimately poor egg quality (Muller, 2003). This hypothesis is based on re cent studies that dem onstrated a significant depression of genes responsible for vitell ogenesis and egg chorion development in largemouth bass, when exposed to p,p ’-DDE (Larkin et al ., 2002). Cultured ovarian cells, treated with p,p ’-DDE, exhibited an inhibition of st eroid synthesis as a consequence of FSH receptor interference (Chedrese and Feyles, 2001). DDT deri vatives have also demonstrated the ability to displace es trogen by competitively binding with estrogen receptors (Danzo et al ., 2002; Larkin et al ., 2002; Matthews et al ., 2000; Spies and Thomas, 1995; Vonier et al ., 1996). Other prominent OC Ps, including dieldrin, methoxychlor, and endosulfan, have also dem onstrated the ability to bind to estrogen receptors (Matthews et al ., 2000; Tollefsen et al ., 2002). Alternatively, dieldrin and DDT derivatives have demonstrated the ability to inhibit androgen bi nding to the androgen receptors, thereby preventing the transcri ption of testosterone, resulting in demasculinization (Baatrup and Junge, 2001; Bayley et al ., 2002; Danzo et al ., 1997; Foster et al ., 2001; Kelce et al ., 1995; Wells and Van Der Kraak, 2000). The effect of OCPs on reproduction and early-l ife stages of development in fish has also been a focus of research. As previ ously mentioned, vitelloge nesis, represents a critical process in the developm ent of teleost oocytes. This process is responsible for the major source of nutrition during embryonic a nd early-life stage development. It is

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10 hypothesized then, that any effect on this process by an anthropogenic compound could have detrimental effects on oogenesis, em bryonic development, hatching and larval survival. Decreased egg production and survival of early-life stages could in turn affect recruitment and have significant pop ulation-level effects (Muller, 2003). Exposure of several fish species to DDT and its derivatives have resulted in decreased fertilization and increase d embryo and fry mortality (Burdick et al ., 1964; Macek, 1968; Monod, 1985). White croaker Genyonemus lineatus , environmentally exposed to DDT at ovarian DDT concentratio ns of 4 mg/L and above, demonstrated an inability to spawn (Cross and Hose, 1988; Hose et al ., 1989). Exposure to aqueous sublethal concentrations of DDT have also resulted in fry vertebral deformities at time of hatch. Deformities included erosion and he morrhaging at the vertebral junctions, and were found in fry which hatched from eggs containing DDT concentrations equal to or exceeding 2.39 mg/L (Smith and Cole, 1973). Exposure of fish and wildlife to OCPs can result in a myriad of negative endocrine effects including inhibition or suppressi on of GnRH and gonadotropin hormones, necrosis of gonadotroph cells responsible fo r the secretion of gonadotropin hormones, and estrogen and androgen receptor binding; all of which could ultimately lead to decreased sex steroid production (Baatrup and Junge, 2001; Bayley et al ., 2002; Chedrese and Feyles, 2001; Danzo et al ., 1997; Danzo et al ., 2002; Foster et al ., 2001; Gore, 2002; Kelce et al ., 1995; Larkin et al ., 2002; Matthews et al ., 2000; Shukla and Pandey, 1986; Spies and Thomas, 1995; Tollefsen et al ., 2002; Vonier et al ., 1996; Wells and Van Der Kraak, 2000). Depressed hormone concentratio ns could then lead to impaired gonadal development and decreased vitellogenesis. In terruption of the vitellogenic process could

PAGE 23

11 then have detrimental effects on oogenesi s, ultimately leading to developmental abnormalities, increased sac fry mortality, a nd even spawning inhibition. Decreased survival of early-life stages could in turn affect recr uitment and have significant population-level effects. Definitive causes and mechanisms of endocrine disruption and reproductive effects following OCP exposure are still under inve stigation (Muller et al ., 2004); however, it is for these reasons th at reproductive abnormalities reported for largemouth bass in the EMCA may be occurring. Research Significance Because largemouth bass are an important sport fish in Florida and nationally, much effort is being placed into the restor ation of a viable fishery in the EMCA and Upper Ocklawaha River Basin (Benton et al ., 1991; Benton and Douglas, 1996; Marburger et al ., 1999). For this reason, I chose the Florida largemouth bass as an animal model for laboratory st udies of OCP exposure. This research aims to determine whole carcass and gonad OCP concentrations, a nd to compare several health parameters and reproductive biomarkers [weight, length, condition index (K), hepatosomatic index (HSI), gonadosomatic index (GSI), circulati ng sex steroid hormones, and percent hatch] for Florida largemouth bass following dietary exposure to p,p ’-DDE and dieldrin, two predominant OCPs found in the EMCA. Si ngle chemical exposures were therefore performed to assess the potential contributi on of each pesticide to overall reproductive function and steroidogenic declines found to occur in largemouth bass environmentally exposed to these contaminants in the EMCA (Marburger et al ., 1999). Because these pesticides have been shown to act as endocrine system modulators in other animals (Gallagher et al ., 2001; Gross et al ., 1994; Guillette et al ., 1994; Mills et al ., 2001; Muller et al ., 2004), it is hypothesized that dietary exposure, of largemouth

PAGE 24

12 bass to either p,p ’-DDE or dieldrin at increasing concentrations, will cause a doseresponse decrease in circulating sex ster oid hormones, for both male and female largemouth bass. This, in turn, should alter gonad development and cause a doseresponse decrease in GSI. Altered female largemouth bass oocyte gonad development, in turn, should cause a dose-response decrease in the number of fry that hatch from clutches produced by these treated larg emouth bass administered pesticide doses at sub-lethal concentrations, similar to wild caught adul t largemouth bass, at contaminated sites (Marburger et al ., 1999). Therefore, no dose-response decreases on weight, length, and condition index (K) of the largemouth ba ss should be seen for either sex.

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13 CHAPTER 2 DIETARY SUBACUTE EXPOSURE TO P,P ’-DDE AND DIELDRIN AND THEIR EFFECTS ON REPRODUCTIVE AND HEAL TH BIOMARKERS IN FLORIDA LARGEMOUTH BASS Introduction Levels of organochlorine pe sticide (OCP) concentrations in tissues of Florida largemouth bass ( Micropterus salmoides floridanus ) and American alligators ( Alligator mississippiensis ) sampled from reclaimed agricultur e lands within the Ocklawaha River Basin and St. Johns River Water Management District’s Emeralda Marsh Conservation Area (EMCA) have been associated with reproductive abnormalities, including depressed and/or reversed sex steroid hormone concentrations (Gross et al ., 1994; Marburger et al . 1999). Two of the OCPs found in highest concentration at these sites are p,p ’-DDE and dieldrin (Marburger et al . 2002). The objectives of this study were to determine whole carcass and gonad OCP concentrations for Florida largemouth bass, a nd to compare several health parameters and reproductive biomarkers [weight, length, c ondition index (K), hepatosomatic index (HSI), gonadosomatic index (GSI), and circ ulating sex steroid hormones] following a 30day dietary exposure period to p,p ’-DDE and dieldrin. p,p ’-DDE and dieldrin, were chosen to evaluate single chemical dose -response relationships for the health and reproductive biomarkers listed above. Doses fo r both pesticides were chosen to create whole carcass concentrations similar to t hose reported for wild largemouth bass in reclaimed agricultural areas in central-Florida (Marburger et al . 1999 and 2002), and were based on a feeding rate and percen t accumulation for a 30-day largemouth bass

PAGE 26

14 p,p ’-DDE and dieldrin dietary e xposure study described in Muller et al . (2004). Characterization of the possible effects th at these two pesticides have on these biomarkers will assist in determining if thes e chemicals act alone as a causative agent to impair the endocrine system of largemouth bass. Materials and Methods Largemouth Bass Hatchery-reared two-year-old Florida largemouth bass, w ith a mean body weight of 150 g, were obtained from American Sportf ish Hatchery, Montgomery, AL on March 10, 2003. Feed Preparation Chemically treated floating pelletted feed was developed using methods modified from those described by Muller et al . (2004). Organochlorine pesticides p,p ’-DDE (2,2-bis(4-chlorophenyl)-1,1-di chloroethylene, Lot # 09020KU, 99% purity) and dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4,5,8dimethanonaphthalene, Lot # 77H3578, 90% purity ) were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). The pesticides were mixed into menhaden fish oil supplied by Zeigler Brothers, Inc. (Gardners, PA) to form two separate concentrated stock solutions (2.5 g p,p ’-DDE/50 mL fish oil and 1.6 g dieldrin/100 mL fish oil) The stock solutions were then shi pped to Zeigler Brothers, Inc., where they were used as a top-dressing to coat Silver Finfish floating pelletted feed. The pesticide-laden floating feed was manufactured by first diluting a m easured amount of the stock solution into menhaden fish oil. This fish oil/stock solution mixture was then added into a mixer containing the pelleted feed to achieve a c onsistent coating of a ll pellets. Continued dilutions of both the p,p ’-DDE and dieldrin stock solutio ns allowed for pesticide-laden

PAGE 27

15 feed of both chemicals to be manufactured at different concentrati ons. Control feed was also manufactured by combining the floating pe lletted feed with a top dressing of pure menhaden fish oil. All pesticide-laden feeds and one sample of the control feed were sent to New Jersey Feed Laboratory, Inc. (Trenton, NJ) by Zeigler Brothers, Inc. for a chlorinated pesticide OCP screen analysis. The cont rol feeds had no detectable levels of organochlorine pesticides. Target feed doses of 1, 7, 37, and 185 g/g p,p ’-DDE had actual concentrations of 1, 7, 35, and 136 g/g p,p ’-DDE, respectively. Target feed doses of 0.02, 0.1, 0.6, and 3 g/g dieldrin had act ual concentrations of 0.03, 0.1, 0.6, and 5 g/g dieldrin, respectively. Experimental Design The largemouth bass were housed in groups of 20 fish (10 males and 10 females) in 18 separate 700-liter round plastic tanks, equipped with a flow-though water system supplied by on-site well water and aeration. Fish sex was determined by external examination of the urogenital pore or by palpating to evaluate the release of eggs or milt. Water quality parameters: temperature, dissolved oxygen, pH, and ammonia were measured twice a week for every tank. Temperature ranged from 20.6 to 22.7 C, dissolved oxygen ranged from 6.74 to 8.60 mg/L , percent saturation ranged from 77 to 102%, pH ranged from 7.6 to 8.0, and ammoni a content remained below 1 mg/L. Largemouth bass were fed for 30 days using the chemically treated floating pelletted feed beginning March 21, 2003. Larg emouth bass were randomly placed into each of nine feed treatm ents in replicate: Control; 1, 7, 35, and 136 g/g p,p ’-DDE; and 0.03, 0.1, 0.6, and 5 g/g Dieldrin. After day 30, subsets of 10 fish (5 males and 5 females) per replicate were sampled to collect measurements of health parameters

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16 (weight, length, condition index, and HSI) and reproductive biomarkers (GSI and circulating sex steroid hormones). Feeding Rate The mean fish body weight of 150 g, at th e beginning of the study, was used to determine the feeding rate. Feed was administered to each tank for 30 days at 1% mean body weight per day. Target feed doses of 1, 7, 37, and 185 g/g p,p ’-DDE were chosen to create final theoretical carcass co ncentrations of 0.1, 0.63, 3, and 17 g/g p,p ’-DDE, respectively. Target feed doses of 0.02, 0.1, 0.6, and 3 g/g dieldrin were chosen to create final theoretical carcass con centrations of 0.003, 0.015, 0.09, and 0.45 g/g dieldrin, respectively. Estimations of fi nal carcass concentrations were based on a p,p ’DDE and dieldrin feeding study described in Muller et al . (2004), which characterized percent accumulation of both pesticides after 30 days of dietary exposure for pond-reared Florida largemouth bass. Fish Collection and Bleeding On April 20, 2003, ten largemouth bass (5 males & 5 females) per replicate were weighed to the nearest gram using a portable di gital scale and measured (total length) to the nearest millimeter for determination of condition index (K) (Anderson and Neumann, 1996). Fish were then bled from the cauda l vein with a hepari nized 20-gauge 3.81-cm needle and a 3-mL syringe to collect approxi mately 1 mL of blood. Blood samples were then dispensed into 3-mL heparinized vacuta iners and kept on ice until centrifuged at 1,000 g at 4 C for 15 minutes to separate red blood cells from the plasma. Plasma was removed with transfer pipettes, placed into cryovials, and stored in a C freezer for later analysis of circulating sex steroid horm ones. After bleeding, fish were dissected and sex was determined by gonad morphology. The gonads and livers were excised from all

PAGE 29

17 fish and weighed on a portable scale to the nearest 0.01 g for determination of gonadosomatic index (GSI) and hepatosoma tic index (HSI) (Anderson and Neumann, 1996). After removal, a cross section of one lobe of each gonad was collected, placed into a histological cassette a nd fixed in 10% buffered formalin for later histological analysis. Remaining gonad tissue and the fish carcass was then wr apped in aluminum foil, placed into a labeled whorl pack, and se t into a freezer for later GC-MS analysis of p,p ’-DDE and dieldrin. However, only one male and one female carcass and gonad composite from each replicate was used for analyses. Data for the males and females from identical replicates were pooled for graphical represen tation to form an n = 2 for every treatment. Gonad Histology Gonad tissue samples were embedded in pa raffin, sectioned at 5 m, mounted on glass slides, air dried, and stained with Mayer’s hematoxylin and eosin (H&E) by Histology Tech Services (Gainesville, FL ). Slides were observed under a light microscope at 40X and stages of sexual maturation were assigned according to Gross et al . (2002). Ovaries were classi fied into four stages of sexual maturation: (stage1), ovaries were undeveloped with mostly peri nucleolar oocytes at various stages of previtellogenic growth; (stage 2), ovaries were previtellogeni c with perinucleolar oocytes and cortical alveoli oocytes; (stage 3), ova ries were early vitellogenic with some vitellogenic oocytes of different sizes, w ith low to moderate amounts of vitelline granules, and few to no fully developed eggs; an d (stage 4), ovaries were late vitellogenic with the majority of the oocytes fully de veloped and containing numerous vitelline granules. Similarly, testes were classified into four stages of sexual maturation: (stage 1), no sperm present with an extremely thin germinal epithelium; (stage 2), presence of

PAGE 30

18 scattered spermatogenic activity with a thin ge rminal epithelium; (stage 3), presence of moderate spermatogenic activity (diffuse to mo derate presence of mature sperm) with a reasonably thick germinal epithelium; and (s tage 4), presence of high spermatogenic activity (heavy presence of mature sperm) with a thick germinal epithelium. Determination of Circulating Sex Steroid Hormones Plasma samples from largemouth bass were analyzed for sex steroid hormones 17 Estradiol (E2) and 11-Ketotestosterone (11-KT) with a validated 3H radioimmunoassay (RIA) procedure, using methods modi fied from those described by Gross et al . (2002) and Muller et al . (2004). All plasma samples were a ssayed in duplicate, and values were reported as pg/mL of plasma. Plasma sample s (50 mL) were extracted twice with diethyl ether prior to RIA analysis. Standa rd curves (1, 5, 10, 25, 50, 100, 250, 500, and 1000 pg/mL) were prepared in phosphate buffered saline plus gelatin and sodium azide (PBSGA) with known amounts of radioinert E2 (ICN Biomedicals, Costa Mesa, CA) or 11-KT (Sigma Chemicals, St. Louis, MO) and 3H-E2 or 3H-11-KT. PBSGA buffer and antibodies specific to each sex steroid hormone were also added to each sample tube and incubated overnight at 4 C. Antibodies were purchased from ICN Biomedicals (E2) or Helix Biotech, Richmond, BC, Canada (11KT). After incubation, unbound sex steriod was removed by the addition of dextran-co ated charcoal and centrifugation for 10 minutes at 1,000 g . Four hundred L of sample supernatant was removed and added to a scintillation vial with 4 mL of Scintiverse scintillation cocktail (Fisher Scientific, Pittsburg, PA). Each sample vial was then placed into a liquid scintillation counter (Packard Tricarb, Model 1600) and counted for tw o minutes. Cross-reactivities of the E2 antiserum with other steroids were: 11.2% for estrone, 1.7% for estriol, and < 1.0% for 17 -estradiol and androstene dione. Cross reactivity of the 11-KT antiserum

PAGE 31

19 with other steroids were: 9.7% for testosterone, 3.7% for -dihydrotestosterone, and < 1.0% for androstenedione. The minimum concen tration distinguishable from zero for all assays were (mean SD) 66.8 15.2 pg/mL for E2 and 45.8 19.5 pg/mL for 11-KT. OCP Analysis One male and one female carcass and gona d composite from each replicate was analyzed for p,p ’-DDE and dieldrin at the Cent er for Environmental and Human Toxicology, University of Florida, usi ng methods described by Rauschenberger et al . (2004). First, the largemouth bass carcass/ gonad tissue was homogenized to eliminate any variability within the sample. Then, a 2-g portion of each sample was extracted into ethyl acetate. The sample was then prepar ed for analysis by solid phase extraction on C18 and NH2 SPE cartridges. Total OC P content was determined using gas chromatography-mass spectrometry (GC-MS). Percent recovery for p,p ’-DDE ranged between 90-98%, with a limit of detection of 0.11-1.5 ng/g. Percent recovery for dieldrin ranged between 70-89%, with a li mit of detection of 0.46-1.5 ng/g. Statistical Analysis Parameters were analyzed using the Statis tical Analysis System (SAS), version 9. Data were analyzed using the univariate procedure to determin e if the data were normally distributed. ANOVAs were then performe d and significance was declared at a p value equal to or lower than 0.05. Duncan’s Multiple Range test followed as a multiple comparison procedure to determine which trea tments differed. Results are presented as means SD. Area 7 Largemouth Bass Five female largemouth bass were collect ed from Emeralda Marsh Conservation Area 7 using electofishing on February 26, 2003 for whole carcass and gonad GC-MS

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20 contaminant analysis of p,p ’-DDE and dieldrin. These fis h, which had a mean weight of 1470 g, were collected to compare current wild largemouth bass OCP concentrations to the pesticide concentrations achieved in this study. Results and Discussion The results of this study demonstrated that a 30-day exposure to diets containing the OCPs p,p ’-DDE and dieldrin at varying doses can result in internal carcass and gonad concentrations of these two pesticides (Table 2-1), at levels similar to those found in largemouth bass from the EMCA. The five female largemouth bass from Area 7 had mean carcass p,p ’-DDE and dieldrin concentratio ns of 270 120 ng/g and 8.5 5.8 ng/g respectively. These fish had mean gonad p,p ’-DDE and dieldrin concentrations of 4900 1000 ng/g and 11.4 2.6 ng/g, re spectively. Graphical an alysis of these data demonstrates a consistent dose accumulati on in the whole carcass and gonads for both males and females, across all treatment levels, for both p,p ’-DDE and dieldrin (Figures 21, 2-2, 2-3, and 2-4). Carcass concentrations for all p,p ’-DDE and dieldrin treatments were greater than predicted target concentr ations (Figures 2-1 and 2-2). Achieved p,p ’DDE and dieldrin female carcass and gonad con centrations for the treatments fell within mean p,p ’-DDE and dieldrin concentrations of the five female largemouth bass from Area 7 (Figures 2-1 and 2-3), thereby ach ieving eco-relevant loads of both OCPs. Achieved concentrations of both pesticid es did not induce biologically significant dose-response decreases in weight, length, K, HSI, GSI (Tables 2-2 and 2-3), or circulating sex steroid ho rmone concentrations, E2 and 11-KT (Figures 2-5, 2-6, 2-7, and 2-8), for either female or male largemouth ba ss. Histological analys is demonstrated that 72% of females and 100% of male largemouth bass at the end of the study were at their fully developed stage of gonad maturation. Th e presence of fully developed gonads prior

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21 to this study may have been the reason why the pesticides we re not able to induce doseresponse decreases in GSI or circulating sex steroids. Fully devel oped gonads indicate that the endocrine system processes of larg emouth bass that initiate biological changes leading to gonad maturati on, including surges in E2 and 11-KT sex steroid production, had already taken place and were already on a seasonal decline by the time pesticide exposure in this study began. Endocrine events of a largemouth bass follow a well-established cycle beginning with the secretion of gonadotropins in th e pituitary, which bind estrogen or androgen receptors, stimulating gonad sex steroid hormone production in preparation for testicular maturation (spermatogenesis) in males a nd oocyte maturation and vitellogenesis in females (Van Der Kraak et al ., 1998). Increasing E2 concentrations in females stimulate the liver to produce vitellogenin, a protein that serves as a yolk precursor in oviparous vertebrates (Wahli et al ., 1981). Vitellogenin produced in the liver is released into circulation to travel to th e gonad where it is sequestered as a nutrient source in developing oocytes. Gross et al . (2002), who characterized the annual cycles of circulating sex steroid hormones for pond-reared Florida larg emouth bass, found that female E2 concentrations began to increase in September and peaked in February at a concentration of 4,000 pg/mL. E2 then declined sharply to a concen tration of approximately 2,600 pg/mL in March and then to a concentration of appr oximately 2,000 pg/mL in April. For male largemouth bass, 11-KT concentrations began to increase in October, with a peak concentration of 2,800 pg/mL in February. 11-KT then declined to a concentration of approximately 2,300 pg/mL in March and then to a concentration of approximately 1,900

PAGE 34

22 pg/mL in April. Gross et al . (2002) also found that GSI, which is an index correlated with an increase in gonad ma turation, began to rise in October for both males and females, peaking in February to March, respectively. Pesticide exposure for my study did not begi n until March 21 because of delays in the manufacturing of the feed. By that date, sex steroids used to trigger the endocrine processes that lead to gona d maturation for both sexes, had probably peaked and were on the decline by the time pesticide dietary expos ure for this experiment began. Histological analyses demonstrated that a majority of the largemouth bass had gonads that were fully developed. Pesticide exposure, beginning th is late in the re productive cycle of a largemouth bass therefore, led this study to miss critical events in the endocrine processes, such as estrogen or androgen receptor binding (Garcia et al ., 1997; Larkin et al ., 2002). The ability of any exogenous co mpound to bind a sex steroid hormone receptor and agonize and/or antagonize the action of an endogenous hormone can severely affect normal endocrine function. This is because normal estrogen or testosterone concentrations and actions are critical for development of both male and female gonads. Largemouth bass, from reclai med agriculture areas reported to have impaired endocrine function, are exposed to these pesticides year round, thus spanning the entire duration of their annual reproductive cycle. Circulating concentrations of both E2 for females and 11-KT for males were on average 1,500 pg/mL less than what was repor ted for pond-reared largemouth bass in the Gross et al . (2002) study, sampled during the sa me time of the calendar year. Largemouth bass used in the Gross et al . (2002) study were on average 3-4 years old and were from a different population of largemout h bass. This may have attributed to the

PAGE 35

23 differences in hormone concentrations between these two studies because the fish used in this study were 2 years of age. These fi sh may have been going through their first reproductive season, which could have also increased the variability in hormone concentrations. In my study, achieved OCP concentrations were not able to induce dose-response decreases in GSI or circ ulating sex steroids E2 and 11-KT for both female and male largemouth bass. This is likely attributable to OCP exposure whic h took place during a portion of the annual reproducti ve cycle of the largemouth bass, after their reproductive organs were already fully deve loped, causing this experiment to miss critical events in the reproductive system that could have facilita ted endocrine disruption. Endocrine system changes that initiate gonad ma turation, including surges in E2 and 11-KT sex steroid production, had already taken place and were already on a seasonal decline by the time pesticide exposure in this study began. De spite a lack of response in measured reproductive biomarkers, exposing largemouth bass to floating pelletted feed coated with OCP contaminated fish oil served as an effective and accurate dosing method, achieving OCP concentrations in measured tissues c onsistent with those found in largemouth bass from reclaimed agriculture lands. Future research needs to focus on p,p ’-DDE and dieldrin dietary exposure over a larger portion of the reproductive cycle of the largemouth bass to see if these pesticides can induce endocrine system and reproductive function changes similar to those reported fo r largemouth bass, exposed to these OCPs in the wild.

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24 Table 2-1. Day-30 GC-MS mean SD results of both female and male largemouth bass p,p ’-DDE and dieldrin concentrations ( ng/g) in both the carcass and gonads per treatment (n = 2 samples per treatment, for each sex). Treatments (g/g) p,p ’-DDE Control 1 7 35 136 Female Carcass 8 2 239 21 1276 331 9226 3065 44502 20176 Male Carcass 10 1 251 39 1436 83 7231 1445 39093 12840 Female Gonad 10 1 397 29 1474 493 8913 33 57208 724 Male Gonad 10 2 476 270 953 395 19721 5093 83701 15659 Treatments (g/g) Dieldrin Control 0.03 0.1 0.6 5 Female Carcass 0.5 0 7.3 1.9 28.7 3.4 108.5 6.7 876.6 463.3 Male Carcass 0.5 0 6.5 1 24.6 2.6 103.3 46.3 1155 221.3 Female Gonad 2 1.3 5 0.9 28.9 9.6 84.3 12.6 793.6 289.4 Male Gonad 2.1 2 5.9 4.2 10.1 3.6 169.6 70.6 1488.1 421.3

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25 0 10000 20000 30000 40000 50000 60000 70000 Control1 g/g7 g/g35 g/g136 g/gArea 7 Treatment GroupsCarcass Concentration p,p '-DDE (ng/g) 0 200 400 600 800 1000 1200 1400 1600 Control0.03 g/g0.1 g/g0.6 g/g5 g/gArea 7 Treatment GroupsCarcass Concentration Dieldrin (ng/g) Figure 2-1. Female largemouth bass mean SD carcass concentrations (shaded bars) of p,p ’-DDE and dieldrin treatments (n = 2 largemouth bass per treatment), as compared to the target carcass concentr ations (black bars). Included is the mean carcass concentration of p,p ’-DDE and dieldrin for the five female largemouth bass sampled from Area 7 (shaded bar) on February 26, 2003. dieldrin p,p ’-DDE

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26 0 10000 20000 30000 40000 50000 60000 Control1 g/g7 g/g35 g/g136 g/g Treatment GroupsCarcass Concentration p,p '-DDE (ng/g) 0 200 400 600 800 1000 1200 1400 1600 Control0.03 g/g0.1 g/g0.6 g/g5 g/g Treatment GroupsCarcass Concentration Dieldrin (ng/g) Figure 2-2. Male largemouth bass mean SD carcass concentrations (shaded bars) of p,p ’-DDE and dieldrin treatments (n = 2 largemouth bass per treatment), as compared to the target carcass concentrations (black bars). p,p ’-DDE dieldrin

PAGE 39

27 0 10000 20000 30000 40000 50000 60000 70000 Control1 g/g7 g/g35 g/g136 g/gArea 7 Treatment GroupsGonad Concentration p,p '-DDE (ng/g) 0 200 400 600 800 1000 1200 Control0.03 g/g0.1 g/g0.6 g/g5 g/gArea 7 Treatment GroupsGonad Concentration Dieldrin (ng/g) Figure 2-3. Female largemouth bass m ean SD gonad concentrations of p,p ’-DDE and dieldrin treatments (n = 2 largemouth bass per treatment). Included is the mean gonad concentration of p,p ’-DDE and dieldrin for the five female largemouth bass sampled from Area 7 on February 26, 2003. p,p ’-DDE dieldrin

PAGE 40

28 0 20000 40000 60000 80000 100000 120000 Control1 g/g7 g/g35 g/g136 g/g Treatment GroupsGonad Concentration p,p '-DDE (ng/g) 0 500 1000 1500 2000 2500 Control0.03 g/g0.1 g/g0.6 g/g5 g/g Treatment GroupsGonad Concentration Dieldrin (ng/g) Figure 2-4. Male largemouth bass m ean SD gonad concentrations of p,p ’-DDE and dieldrin treatments (n = 2 largemouth bass per treatment). p,p ’-DDE dieldrin

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29 Table 2-2. Day-30 mean SD results of fe male and male weight, total length, condition index (K), GSI, and HSI for each treatment, for largemouth bass fed p,p ’-DDE diets. Treatments with the same lo wer case letter were not significantly different ( p > 0.05), with a sample size of 10 largemouth bass per treatment. p,p ’-DDE Treatments (g/g) Female Control 1 7 35 136 Weight (g) 183 32a 176 21a 193 23a 188 27a 180 12a Length (mm) 234 12a 231 9a 234 9a 234 10a 228 5a K 1.41 0.11c 1.42 0.08b,c 1.50 0.08a,b 1.47 0.08a,b,c 1.51 0.08a GSI (%) 2.05 0.96b 4.26 1.87a 2.80 1.39a,b 3.06 1.96a,b 3.87 1.58a HSI (%) 3.68 0.74a 4.19 0.43a 3.83 0.84a 3.79 0.80a 3.74 0.52a p,p ’-DDE Treatments (g/g) Male Control 1 7 35 136 Weight (g) 184 28a 178 23a 194 22a 183 26a 190 25a Length (mm) 237 9a 234 8a 238 8a 234 9a 237 9a K 1.38 0.07a 1.39 0.09a 1.43 0.06a 1.42 0.07a 1.43 0.06a GSI (%) 0.68 0.40a 0.65 0.16a 0.64 0.20a 0.66 0.12a 0.58 0.16a HSI (%) 3.46 0.61a 3.44 0.58a 3.46 0.78a 3.17 1.08a 3.16 0.66a

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30 Table 2-3. Day-30 mean SD results of fe male and male weight, total length, condition index (K), GSI, and HSI for each treatm ent, for largemouth bass fed dieldrin diets. Treatments with the same lo wer case letter were not significantly different ( p > 0.05), with a sample size of 10 largemouth bass per treatment. Dieldrin Treatments (g/g) Female Control 0.03 0.1 0.6 5 Weight (g) 186 21a,b 199 23a 188 24a,b 196 16a,b 179 28b Length (mm) 228 17a,b 237 10a 233 8a,b 232 8a,b 227 11b K 1.66 0.73a 1.49 0.11a 1.48 0.13a 1.58 0.12a 1.52 0.13a GSI (%) 3.00 2.06a,b 4.53 1.73a 2.82 1.26b 4.19 2.34a,b 3.90 1.88a,b HSI (%) 3.55 1.02b 4.17 0.64a,b 3.76 0.46a,b 2.89 0.90c 4.20 0.80a Dieldrin Treatments (g/g) Male Control 0.03 0.1 0.6 5 Weight (g) 193 42a 190 30a 189 23a 201 39a 203 27a Length (mm) 239 10a 236 10a 236 8a 233 10a 237 11a K 1.39 0.19c 1.43 0.09b,c 1.43 0.08b,c 1.58 0.12a 1.52 0.11a,b GSI (%) 0.71 0.30a 0.67 0.16a 0.68 0.24a 0.65 0.18a 0.60 0.12a HSI (%) 4.07 1.29a 3.37 0.56a,b 3.10 0.74b 3.81 1.14a,b 3.91 0.57a

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31 0 100 200 300 400 500 600 700 800 900 Control1 g/g7 g/g35 g/g136 g/gEstradiol (pg/mL) 0 100 200 300 400 500 600 700 800 900 Control0.03 g/g0.1 g/g0.6 g/g5 g/gTreatment GroupsEstradiol (pg/mL) Figure 2-5. Mean female estradio l concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 largemouth bass per treatment. Treatments with the same lower case letter were not significantly different ( p > 0.05). a,b a b a b b a,b c dieldrin p,p ’-DDE a,b a,b

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32 0 50 100 150 200 250 300 350 400 450 500 Control1 g/g7 g/g35 g/g136 g/gEstradiol (pg/mL) 0 50 100 150 200 250 300 350 400 450 500 Control0.03 g/g0.1 g/g0.6 g/g5 g/gTreatment GroupsEstradiol (pg/mL) Figure 2-6. Mean male estradio l concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 largemouth bass per treatment. Treatments with the same lower case letter were not significantly different ( p > 0.05). b b b a b a,b a a,b a,b b dieldrin p,p ’-DDE

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33 0 100 200 300 400 500 600 Control1 g/g7 g/g35 g/g136 g/g11-Ketotestosterone (pg/mL) 0 100 200 300 400 500 600 Control0.03 g/g0.1 g/g0.6 g/g5 g/gTreatment Groups11-Ketotestosterone (pg/mL) Figure 2-7. Mean female 11-ketotestos terone concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 larg emouth bass per treatment. Treatments with the same lower case letter were not significa ntly different ( p > 0.05). b b b a b a b b a a dieldrin p,p ’-DDE

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34 0 100 200 300 400 500 600 700 Control1 g/g7 g/g35 g/g136 g/g11-Ketotestosterone (pg/mL) 0 100 200 300 400 500 600 700 Control0.03 g/g0.1 g/g0.6 g/g5 g/gTreatment Groups11-Ketotestosterone (pg/mL) Figure 2-8. Mean male 11-ketotestoste rone concentrations at day 30 for p,p ’-DDE and dieldrin, with a sample size of 10 larg emouth bass per treatment. Treatments with the same lower case letter were not significa ntly different ( p > 0.05). a a a a a a b,c c a,b b,c dieldrin p,p ’-DDE

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35 CHAPTER 3 DIETARY CHRONIC EXPOSURE TO P,P ’-DDE AND DIELDRIN AND THEIR EFFECTS ON REPRODUCTIVE SUCCESS IN LARGEMOUTH BASS Introduction A lack of dose-response effect s from dietary exposure to p,p ’-DDE and dieldrin on measured reproductive biomarkers (GSI and circulating sex steroi d hormones) in the 30day study (see Chapter 2) led to the need for extending the exposure period. Exposure length for this study was extended to a 120-da y period, between the months of November and March, encompassing a larger part of the steroidogenic and gametogenic portion of the reproductive cycle of the largemouth bass. Extending exposure length for this study aimed to reevaluate single chemical dose -response effects of dietary exposure to p,p ’DDE and dieldrin on health parameters and reproductive biomarkers [weight, length, condition index (K), hepatosomatic index (HSI), gonadosomatic index (GSI), and circulating sex steroid hormones] for Florida largemouth bass ( Micropterus salmoides floridanus ) and to determine if p,p ’-DDE or dieldrin doses affe ct clutch hatchability of eggs produced by the spawning of the treated fish. Materials and Methods Largemouth Bass Hatchery-reared two-year-old Florida largemouth bass, w ith a mean body weight of 159 g, were obtained from American Sportf ish Hatchery, Montgomery, AL on October 20, 2003.

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36 Feed Preparation Chemically treated floating pelletted feed was developed using methods described in Chapter 2. Stock solutions contained 20 g p,p ’-DDE/400 mL fish oil and 6.4 g dieldrin/400 mL fish oil. Control feed had no detectable levels of organochlorine pesticides. Target feed doses of 7, 37, and 185 g/g p,p ’-DDE had actual concentrations of 5, 46, and 50 g/g p,p ’-DDE, respectively. Target doses of 0.1, 0.6, and 3 g/g dieldrin had actual concentrations of 0.04, 0.4, and 0.8 g/g dieldrin, respectively. Experimental Design Largemouth bass were housed in groups of 100 fish in seven separate 6,000-liter outdoor concrete raceways (366 cm x 183 cm x 91 cm) with flow-through pond water and aeration. Water temperature and dissolv ed oxygen were measured twice a week for every tank. Temperature ranged from 12.2 to 22.4 C, dissolved oxygen from 7.06 to 11.75 mg/L, and percent saturation from 67 to 140%. One hundred largemouth bass were randomly placed into each of seven feed treatments: Control; 5, 46 and 50 g/g p,p ’DDE; and 0.04, 0.4, and 0.8 g/g Dieldrin. Larg emouth bass were fed the seven diets, five days a week beginning on November 4, 2003, represented as day 0. On day 0 of the experiment, 24 largemouth bass were sampled fr om the Control to collect measurements of health parameters (weight, length, condition index, and HSI) and reproductive biomarkers (GSI and circulat ing sex steroid hormones). Approximately every 30 days, six males and six females per treatment were sampled to collect measurements on the same health parameters and reproductive biomarkers. Fish sex was determined by external examination of the urogenital pore or by palpating to evaluate the release of eggs or milt. Sample days took place on Decem ber 1, 2003 and January 6 and February 3, 2004 for all treatments. The last sample was on March 10, 2004 for the Control and p,p ’-

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37 DDE treatments, and on March 26, 2004 for the dieldrin treatments and a second set of Control fish. Largemouth bass in the p,p ’-DDE treatments were housed in their corresponding raceways for a total of 128 days, and were fed their corresponding diets for a total of 86 days. Largemouth bass in the Control and dieldr in treatments were housed in their corresponding raceways for a total of 144 days, and were fed their corresponding diets for a total of 98 days. There was only one mortality throughout the entire duration of this study, which occurred in the 0.8 g/g Dieldrin treatment raceway. Feeding Rate All feed was administered to each ta nk at 1% mean body weight for (100) largemouth bass. Feeding rate was adjusted every thirty days according to changes in mean body weight and the number of largemouth bass remaining in each raceway. Fish Collection and Bleeding Twenty four largemouth bass (12 males & 12 females) on day 0, and a subset of 12 largemouth bass (6 males and 6 females) per treatment were sampled on days 30, 60, 90, and 120 to collect measurements of the same health parameters and reproductive biomarkers as described in Chapter 2. On the last sample day (day 120), carcasses of sampled largemouth bass from every treatment were wrapped in aluminum foil, placed into a labeled whorl pack, and set into a freezer for later GC-MS analysis of p,p ’-DDE and dieldrin. However, carcasses of only three males and three females from every treatment were used for contaminant analys is. The final concentrations from the contaminant analysis were then pooled by sex fo r graphical representation to form an n = 3 for every treatment.

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38 Day 120 Spawning After the last sample days in March for both p,p ’-DDE and dieldrin, one group of eight males and eight females from each treat ment and a set of control diet largemouth bass were placed into four separate 0.10-acre experimental ponds, which contained spawning mats. Daily snorkeling was used to locate largemouth bass nests along the bottom of each pond. If a fertilized nest was found on the spawning mat, that portion of the mat was cut out, folded over, gently raised to the surface, and pl aced into a cooler of pond water for transport to the lab. The first si x clutches collected per pond were used to characterize differences in percent hatch of eggs produced by the spawning of these treated largemouth bass. Spawning mat sections contai ning egg clutches were removed from the cooler and placed into a 1.5% sodium sulfite solution for 5-7 minutes to loosen the eggs from the spawning mat. The mat was then carefully re moved from the solution and placed into a nalgene container of pond water. The sodium sulfite solution was th en poured through a series of sieves to collect any eggs that may have fallen off of the mat. If any eggs were collected, they were placed into a pyrex dish containing pond water. To continue the egg removal process, the mat sections placed into the nalgene container, were sprayed with water to remove the remaining eggs. Water from the nalgene container was then poured through the same series of siev es to collect the remaining e ggs. These eggs were added to the pyrex dish to keep an entire clutch together. Once an entire clutch was separated from the spawning mat, three separate live 100-embryo sets from each clutch were count ed and placed randomly into three separate McDonald jars to allow for hatching of the eggs. Progress of the embryos was recorded once daily. Temperature and dissolved oxygen of the head tank, that supplied on-site

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39 well water to the jars, was recorded once a day. Temperature ranged from 20.1 to 21.3 C, with a mean of 20.7 C and dissolved oxygen from 7.13 to 8.49 mg/L, with a mean of 7.76 mg/L. The eggs were treated daily w ith a hydrogen peroxide (500 mg/L of 35% active ingredient) static bath for 30 min to prevent fungal growth. Once hatching of the embryos in each jar was complete, the fry were removed and set in 10% buffer formalin. The number of fry produced by the three separa te 100-embryo sets for each clutch were counted, added together, and di vided by 300. The resulting value represented the percent hatch for each clutch. The mean percent hatc h values of the six clutches, collected for each treatment, were used to characteri ze differences in percent hatch among the treatments. Determination of Circulating Sex Steroid Hormones Plasma samples from the largemouth bass were again analyzed for sex steroid hormones 17 -Estradiol (E2) and 11-Ketotestosterone (11-KT) with a validated 3H radioimmunoassay (RIA) procedure, using methods described in Chapter 2. The minimum concentration distinguishable from zero for all assays were (mean SD) 89 32.9 pg/mL for E2 and 72.3 17.6 pg/mL for 11-KT. OCP Analysis Carcasses of three males and three females from every treatment were analyzed for p,p ’-DDE and dieldrin content at the Center for Environmental and Human Toxicology, University of Florida, using methods desc ribed in Chapter 2. Percent recovery for p,p ’DDE was 87%, with a limit of detection of 0.1-1.5 ng/g. Percent recovery for dieldrin was 95%, with a limit of detection of 0.6-1.5 ng/g.

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40 Statistical Analysis Parameters were again analyzed using th e Statistical Analysis System (SAS), version 9. Data were analyzed using the uni variate procedure to determine if the data were normally distributed. ANOVAs were then performed and significance was declared at a p value equal to or lower than 0.05. Dun can’s Multiple Range test followed as a multiple comparison procedure to determin e which treatments differed. Results are presented as means SD. Area 7 Largemouth Bass Five female largemouth bass were collect ed from Emeralda Marsh Conservation Area 7 using electofishing on February 23, 2004 for whole carcass and gonad GC-MS contaminant analysis of p,p ’-DDE and dieldrin. These fis h, with a mean weight of 1020 g, were collected to compare current wild largemouth bass OCP concentrations to the pesticide concentrations achieved in this study. Results and Discussion The outcome of this study demonstrated that attained car cass concentrations following 120 days of dietary exposure to p,p ’-DDE and dieldrin di d not result in any meaningful dose-response decr eases across all treatment leve ls, for both female and male weight, length, condition index (K), and HSI, at the four different sampling days (Tables 3-1, 3-2, 3-3, and 3-4). Exposure to p,p ’-DDE and dieldrin led to depressed concentrations of plasma E2 for female largemouth bass (Figures 3-1 and 3-2), increases in 11-KT concentrations for female largemout h bass (Figures 3-3 and 3-4), and a lack of consistent increases and/or depr essions of male largemouth bass E2 and 11-KT concentrations (Figures 3-5, 3-6, 3-7, and 3-8) over the 120-day period.

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41 Changes in female plasma E2 concentrations over the sampling period followed a similar pattern regardless of pesticide or dose, and demonstr ated a lack of an expected seasonal increasing trend in E2 concentrations demonstrated by the Control. Not only were their significant redu ctions in treated group E2 concentrations from the Control concentrations on at least two of the three sa mple days (Figures 3-1 and 3-2), including a day-30 reduction shown by all treatm ents, but a day-60 recovery in E2 concentrations back to a concentration not statistically diffe rent from the Control was also demonstrated by all treatments (Figures 3-1 and 3-2). The 5 g/g p,p ’-DDE treatment demonstrated a recovery in E2 concentrations back to a concentrati on not statistically different from the Control on day 90 (Figure 3-1). The rec overy shown by all treatments was also immediately followed by a si gnificant reduction in E2 concentrations on day 120 (Figures 3-1 and 3-2). Reductions in plasma E2 concentrations demonstrated by the treatments also averaged 2 to 3 times less than the C ontrol, indicating that both OCPs induced not only statistically significant changes, but that biologicall y significant reduction in E2 concentrations were shown by the treatments. Even though 11-KT is believed to be a male specific androgen, one that is responsible for spermatogenesis, it was de tected on every sample day in female largemouth bass plasma. Gross et al . (2002) also found 11-KT pr esent in the plasma of hatchery reared female largemouth bass, simila r in concentrations a nd depicting a lack of seasonal pattern, comparable to the Control in this study. Dietary exposure to either p,p ’DDE or dieldrin manifested as an increase in female 11-KT concentrations on at least two of the four sample days (Figures 3-3 and 34). Most notably was the day-90 peak in 11-

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42 KT concentrations demonstrated by all three dieldrin treatments, to a concentration 4-fold greater than the Cont rol (Figures 3-4). Male largemouth bass 11-KT concentrations did not follow a similar pattern of reduction over the 120-day sampling period (Fig ures 3-7 and 3-8), as was demonstrated by female largemouth bass E2 concentrations (Figures 31 and 3-2). An expected, seasonal increasing pattern in 11-KT concentrations wa s shown by all treatments, comparable to that of the Control (Figures 3-7 and 3-8). Day 120, on which a significant reduction by all three dieldrin treatments o ccurred, was the only sample day when 11-KT concentrations departed from the seasonal increasing pattern show n by all six treatments (Figures 3-7 and 3-8). This may have been attributed to a threshold whole-body concentration of dieldrin, reached by these th ree treatments prior to the last sample day, enough to initiate a mechanism res ponsible for endocrine disruption. The purpose of this study was not to test for reported mechanisms of endocrine disruption in fish and other w ildlife exposed to OCPs, but was intended to re plicate in a laboratory setting reproductive abnormalities reported for largemouth bass sampled from Emeralda Marsh Conservation Area. Similar to this study, female largemouth bass sampled monthly from these floode d muck farms had depressed 17 -estradiol concentrations, elevated 11-KT concentrations , and demonstrated a lack of seasonal trend in E2 concentrations (Marburger et al ., 1999). Despite non-detectable levels of dieldrin in the five female largemouth bass sampled from Area 7 in 2004 for my study, achieved p,p ’-DDE and dieldrin carcass concentrati ons (Table 3-5) were within carcass concentrations reported for largemouth bass sampled from the EMCA by Marburger et al . (1999). The five female largemouth bass, sampled from Area 7, had a mean carcass

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43 p,p ’-DDE concentration of 2300 510 ng/g. Graphical analysis of p,p ’-DDE carcass concentrations demonstrated that both fema le and male largemouth bass accumulated a consistent dose from dietary exposure for the 46 and 50 g/g p,p ’-DDE treatments, while both female and male carcasses in the 5 g/g p,p ’-DDE treatment averaged about 8,000 ng/g less p,p ’-DDE (Figure 3-9). Achieved p,p ’-DDE female and male carcass concentrations, for 46 and 50 g/g p,p ’-DDE treatments, were above the mean p,p ’-DDE concentration of the five female largemouth bass sampled from Area 7, while fish in the 5 g/g p,p ’-DDE treatment fell below this concentra tion (Figures 3-9). Graphical analysis of dieldrin carcass concentr ations demonstrated that bot h female and male largemouth bass accumulated a consistent dose from dietary exposure for the 0.4 and 0.8 g/g Dieldrin treatments, while both female a nd male carcasses in the 0.04 g/g Dieldrin treatment were just above detectable limits of dieldrin (Figure 3-9b). My study also sought to address the issue th at poor recruitment that has limited the development of the EMCA into a quality la rgemouth bass fishery might be related to reported reproductive abnormalities (Benton a nd Douglas, 1996). The ability of these OCPs to induce reductions in female largemouth bass seasonal E2 concentrations could lead to decreased vitellogenesis, or egg yol k synthesis. Vitellogenins are proteins synthesized liver in response to changes in es tradiol concentrations, and serve as a major source of nutrition during embryonic and early -life stage development in all oviparous vertebrates (Wahli et al ., 1981). It is hypothesized that reduced estrogen function could lead to decreased vitellogenesis and impaired gonad development; ending in the production of poor quality eggs and decrea sed reproductive success (Muller, 2003). Decreased egg production and survival of early-life stages coul d in turn affect

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44 recruitment and have significant populati on-level effects. Re ductions in female largemouth bass E2 concentrations for all treatments did not transl ate into dose-response reductions in female GSI (Tables 3-1, 3-2, 33, and 3-4), indicating that alterations in female E2 concentrations did not have an effect on ovarian development. Female GSI values demonstrated an expected seasona l increasing trend between the months of November and March regardless of pesticid e or dose (Figures 3-10 and 3-11). The p,p ’DDE or dieldrin treatments did not translat e into a dose-response reduction in percent hatch values, as compared to the control treatment (Table 3-6). The 50 p,p ’-DDE and 0.04 g/g Dieldrin treatments demonstrated the highest percent hatch values (Table 3-6), compared to all other treatments. Hatch values of all treatments were within reported ranges for controlled spawning and hatching of largemouth bass eggs under laboratory conditions (Carlson, 1973; Jack son, 1979). Numerous field and laborat ory studies have linked DDT egg concentrations to decreases in fecundity and fertility, early oocyte loss, sac fry mortality, and developmental alterations (Burdick et al .,1964; Macek, 1968; Smith and Cole, 1973; Hose et al ., 1989). White croaker Genyonemus lineatus , environmentally exposed to DDT residues with ovarian concentrati ons of 4,000 ng/g or greater demonstrated the inability to sp awn (Cross and Hose, 1988). The ovarian DDT concentrations reported for that study ar e comparable to measured ovarian DDT concentrations from largemouth bass sample d from the EMCA, where poor recruitment has been reported (Marburger et al ., 1999; 2002). The lack of data concerning concentrations in the ovaries of fe male largemouth bass, treated with p,p ’-DDE in this study, does not allow for a comparison to be made.

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45 Despite a lack of research into the possi ble mechanisms of endocrine disruption in largemouth bass and other teleosts following exposure to either p,p ’-DDE or dieldrin, the results of endocrine disruption in other animal models may help to provide insight into what occurred in this study. The dos e-response decreases in female E2 plasma concentrations, coupled with a lack of dose-response decreases in male 11-KT concentrations, points toward a mechanism of reduced aromatase activity found in alligators and human cells exposed to OCPs. Crain et al . (1997) found significantly decreased aromatase activity in female juvenile alligators sampled from OCP contaminated aquatic systems in central Fl orida. Toxaphene, another prominent OCP found in the soil and tissues of largemouth bass sample from Emeralda marsh, was found to decrease aromatase activity in human female breast tissue (Chen et al ., 2001). Aromatase is an enzyme essential for the conversion of testosterone to E2, and if the activity of aromatase is decr eased or inhibited in any ma nner, the end product would manifest as a reduction in the amount of E2 produced. Since male largemouth bass did not show a similar pattern of reduction in plasma 11-KT concentrations, the femalespecific reductions, follo wing dietary exposure to p,p ’-DDE or dieldrin, indicates that endocrine disruption in this study was specific to th e production of E2 in female largemouth bass. The recovery of plasma E2 concentrations on day 60 and the build up of 11-KT in female largemouth bass may have been the result of positive and negative feedback actions, two mechanisms that help to regulat e the release of hormones in the endocrine system of teleosts. Estradiol and testosterones levels exert positive and negative feedback on GTH release mediated by indir ect effects on GnRH release (Van Der Kraak

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46 et al ., 1998). Low or high sex steroid concentr ations either stimulate or cease the production of steroids by agonizing (pos itive feedback) or antagonizing (negative feedback) the release of GnRH. The seasonably low E2 concentrations demonstrated by the treatments on day 30 may have exerted a “ positive feedback” mechanism, stimulating the synthesis of more E2, so much so that E2 concentrations were able to attain a concentration comparable to that of the Control. As dietary exposure to p,p ’-DDE or dieldrin continued between days 60 and 90, increasing whole-body OCP concentrations may have mediated an increase in ar omatase inhibition, to a point that E2 production was unable to recover, cau sing reductions in E2 concentrations on days 90 and 120. The increase in 11-KT concentrations, in p,p ’-DDE and dieldrin treated female largemouth bass, could have been the result of arom atase inhibition not allo wing for the conversion of testosterone to E2. A lack of E2 synthesis resulted in a c ontinued “positive feedback” for the production of more E2, but since the OCPs were actin g to inhibit the aromataztion of testosterone to E2, this mechanism of e ndocrine disruption manifested as a build up of testosterone and then 11-ketotestosterone. My study was successful in replicati ng reproductive abnormalities found in largemouth bass sampled from the EMCA. Dietary exposure, to p,p ’-DDE and dieldrin during the reproductive season, manifested in reductions of female largemouth bass E2 concentrations, abnormal increases in 11-KT c oncentrations, and demonstrated a lack of seasonal increasing trend in E2 concentrations. Attained p,p ’-DDE and dieldrin carcass concentrations and achieved depressions of female E2 concentrations did not translate into a reduction of percent hatch. My study only sought to characterize single chemical dose-response effects for tw o of the predominate OCPs, p,p ’-DDE and dieldrin, found in

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47 the soils and various tissues of largemouth bass from the EMCA. Fish in this system are environmentally exposed to multiple pesticides that may not only contribute to reductions in hormone concentrations, but also to decr eased reproductive success. Future research may be the study of pesticide mixtures and the effects that multiple pesticide exposure may attribute to hormone depression and reproductive success.

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48 Table 3-1. Day-30 mean SD results of fe male and male weight, total length, condition factor (K), GSI, and HSI per treatmen t (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide. p,p ’-DDE Treatments (g/g) Female Control 5 46 50 Weight (g) 176 18a 183 41a 176 41a 193 20a Length (mm) 227 6a 229 14a 230 18a 239 18a K 1.50 0.06a 1.50 0.26a 1.43 0.06a 1.45 0.36a GSI (%) 1.26 0.25a 1.15 0.14a 1.29 0.42a 1.14 0.22a HSI (%) 4.04 0.60a 3.27 0.68a 3.51 0.69a 3.77 0.67a p,p ’-DDE Treatments (g/g) Male Control 5 46 50 Weight (g) 200 33a 224 18a 212 36a 193 54a Length (mm) 235 11a 245 8a 238 9a 233 19a K 1.53 0.06a 1.53 0.03a 1.56 0.15a 1.48 0.11a GSI (%) 0.53 0.18a 0.59 0.16a 0.56 0.18a 0.55 0.08a HSI (%) 4.29 0.63a 3.46 0.83a 3.86 0.96a 4.09 0.81a Dieldrin Treatments (g/g) Female Control 0.04 0.4 0.8 Weight (g) 176 18a 205 12a 188 25a 193 46a Length (mm) 227 6a 237 5a 225 20a 232 11a K 1.50 0.06a 1.53 0.11a 1.70 0.45a 1.53 0.17a GSI (%) 1.26 0.25a,b 1.35 0.17a 1.00 0.28b 0.95 0.30b HSI (%) 4.04 0.60a 3.01 0.50b 3.37 0.80a,b 3.27 0.85a,b Dieldrin Treatments (g/g) Male Control 0.04 0.4 0.8 Weight (g) 200 33a 191 37a 199 31a 167 23a Length (mm) 235 11a 229 12a,b 234 12a 221 9b K 1.53 0.06a 1.57 0.09a 1.56 0.19a 1.54 0.04a GSI (%) 0.53 0.18a 0.66 0.19a 0.67 0.06a 0.56 0.14a HSI (%) 4.29 0.63a 3.64 0.72a,b 3.45 0.48b 3.67 0.65a,b

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49 Table 3-2. Day-60 mean SD results of fe male and male weight, total length, condition factor (K), GSI, and HSI per treatmen t (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide. p,p ’-DDE Treatments (g/g) Female Control 5 46 50 Weight (g) 207 40a 222 39a 188 56a 208 38a Length (mm) 238 11a 244 10a 232 20a 237 14a K 1.52 0.14a 1.51 0.10a 1.47 0.09a 1.55 0.10a GSI (%) 1.64 0.57a 1.65 0.62a 1.68 0.29a 2.50 1.63a HSI (%) 4.79 0.95a 4.16 0.88a 4.24 0.94a 4.71 0.89a p,p ’-DDE Treatments (g/g) Male Control 5 46 50 Weight (g) 209 53a 202 24a 204 19a 215 38a Length (mm) 239 15a 243 13a 243 10a 245 10a K 1.50 0.13a 1.42 0.21a 1.42 0.09a 1.46 0.06a GSI (%) 0.53 0.12a 0.52 0.07a 0.50 0.08a 0.64 0.17a HSI (%) 4.83 1.34a 5.16 1.10a 3.98 0.91a 4.72 0.63a Dieldrin Treatments (g/g) Female Control 0.04 0.4 0.8 Weight (g) 207 40a 224 40a 231 30a 189 29a Length (mm) 238 11a 240 10a 245 10a 233 11a K 1.52 0.14a 1.61 0.14a 1.57 0.06a 1.49 0.10a GSI (%) 1.64 0.57a 1.89 0.37a 1.78 0.18a 1.49 0.80a HSI (%) 4.79 0.95a 4.08 0.92a 3.88 1.27a 4.42 0.85a Dieldrin Treatments (g/g) Male Control 0.04 0.4 0.8 Weight (g) 209 53a 227 48a 209 47a 205 49a Length (mm) 239 15a 246 12a 240 14a 238 16a K 1.50 0.13a 1.51 0.13a 1.50 0.11a 1.50 0.09a GSI (%) 0.53 0.12a 0.44 0.05a 0.48 0.11a 0.50 0.04a HSI (%) 4.83 1.34a 4.54 1.13a 4.94 1.48a 4.94 0.67a

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50 Table 3-3. Day-90 mean SD results of fe male and male weight, total length, condition factor (K), GSI, and HSI per treatmen t (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide. p,p ’-DDE Treatments (g/g) Female Control 5 46 50 Weight (g) 227 33a 243 65a 267 39a 242 36a Length (mm) 245 9a 250 20a 257 11a 246 11a K 1.53 0.07a 1.53 0.09a 1.56 0.06a 1.61 0.06a GSI (%) 2.05 0.20a 2.11 0.50a 1.93 0.32a 1.91 0.55a HSI (%) 5.13 0.68a 4.57 1.14a 4.47 1.90a 5.55 0.83a p,p ’-DDE Treatments (g/g) Male Control 5 46 50 Weight (g) 221 22a 217 25a 221 23a 220 30a Length (mm) 241 7a 240 6a 242 7a 242 8a K 1.57 0.03a 1.57 0.11a 1.56 0.09a 1.54 0.05a GSI (%) 0.61 0.09a 0.46 0.15a 0.52 0.06a 0.54 0.14a HSI (%) 4.95 1.00a 5.06 1.28a 4.84 0.80a 4.98 1.09a Dieldrin Treatments (g/g) Female Control 0.04 0.4 0.8 Weight (g) 227 33a 224 35a 210 26a 234 49a Length (mm) 245 9a 245 11a 239 9a 244 15a K 1.53 0.07a 1.51 0.11a 1.54 0.04a 1.59 0.10a GSI (%) 2.05 0.20a 1.99 0.32a 2.02 0.31a 2.04 0.30a HSI (%) 5.13 0.68a 4.64 0.63a 5.12 1.01a 5.09 1.05a Dieldrin Treatments (g/g) Male Control 0.04 0.4 0.8 Weight (g) 221 22a 217 23a 213 62a 214 49a Length (mm) 241 7a 245 9a 237 16a 241 15a K 1.57 0.03a 1.47 0.07a 1.56 0.16a 1.50 0.11a GSI (%) 0.61 0.09a 0.49 0.08b 0.51 0.11a,b 0.58 0.05a,b HSI (%) 4.95 1.00a 4.70 0.72a 4.45 0.81a 5.36 0.52a

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51 Table 3-4. Day-120 mean SD results of fe male and male weight, total length, condition factor (K), GSI, and HSI per treatmen t (n = 6 largemouth bass per treatment for each sex), for the p,p ’-DDE and dieldrin diets. Means for treatments with the same lower case letter were not significantly different ( p > 0.05) within each sex and pesticide. p,p ’-DDE Treatments (g/g) Female Control 5 46 50 Weight (g) 204 24a 222 52a 252 40a 254 49a Length (mm) 243 12a 244 16a 250 8a 254 13a K 1.42 0.13b 1.51 0.10a,b 1.61 0.15a 1.53 0.12a,b GSI (%) 2.27 1.09a 2.49 0.68a 3.04 0.61a 2.99 0.62a HSI (%) 2.71 1.21b,c 3.73 0.90a,b 2.51 0.29c 3.96 0.95a p,p ’-DDE Treatments (g/g) Male Control 5 46 50 Weight (g) 220 34a 246 37a 243 53a 230 52a Length (mm) 248 7a 253 9a 251 15a 246 18a K 1.43 0.13a 1.51 0.08a 1.51 0.10a 1.53 0.07a GSI (%) 0.67 0.06a 0.70 0.20a 0.63 0.19a 0.61 0.12a HSI (%) 2.95 0.84a 3.39 1.13a 2.62 0.74a 3.80 0.92a Dieldrin Treatments (g/g) Female Control 0.04 0.4 0.8 Weight (g) 204 24a 251 56a 233 48a 238 43a Length (mm) 243 12a 256 13a 251 18a 257 14a K 1.42 0.13a 1.47 0.11a 1.46 0.05a 1.39 0.06a GSI (%) 2.27 1.09a 3.07 1.22a 3.19 1.78a 3.14 0.62a HSI (%) 2.71 1.21a 3.32 1.10a 2.76 0.52a 2.90 0.73a Dieldrin Treatments (g/g) Male Control 0.04 0.4 0.8 Weight (g) 220 34a 258 38a 253 25a 236 37a Length (mm) 248 7a 262 12a 256 16a 253 10a K 1.43 0.13a 1.43 0.07a 1.53 0.33a 1.45 0.07a GSI (%) 0.67 0.06a 0.71 0.23a 0.67 0.22a 0.66 0.11a HSI (%) 2.95 0.84a 3.06 0.78a 2.31 0.50a 2.98 0.68a

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52 0 100 200 300 400 500 600 700 800 900 0306090120Estradiol (pg/mL) Controls DDE 50 g/g 0 100 200 300 400 500 600 700 800 900 0306090120Estradiol (pg/mL) Controls DDE 46 g/g 0 100 200 300 400 500 600 700 800 900 0306090120Sample DaysEstradiol (pg/mL) Controls DDE 5 g/g Figure 3-1. Female estradiol concentrat ions, on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Contro l. Asterisk represents significant difference ( p 0.05) from the Control. * * * * * * * *

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53 0 200 400 600 800 1000 1200 0306090120Estradiol (pg/mL) Controls Dieldrin 0.8 g/g 0 200 400 600 800 1000 1200 0306090120Estradiol (pg/mL) Controls Dieldrin 0.4 g/g 0 200 400 600 800 1000 1200 0306090120Sample DaysEstradiol (pg/mL) Controls Dieldrin 0.04 g/g Figure 3-2. Female estradiol concentrat ions, on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Contro l. Asterisk represents significant difference ( p 0.05) from the Control. * * * * * * * * *

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54 0 100 200 300 400 500 600 700 800 900 1000 030609012011-Ketotestosterone (pg/mL) Controls DDE 50 g/g 0 100 200 300 400 500 600 700 800 900 1000 030609012011-Ketotestosterone (pg/mL) Controls DDE 46 g/g 0 100 200 300 400 500 600 700 800 900 1000 0306090120Sample Days11-Ketotestosterone (pg/mL) Controls DDE 5 g/g Figure 3-3. Female 11-ketotestosterone c oncentrations, on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control. * * * * * * *

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55 0 100 200 300 400 500 600 700 800 900 1000 030609012011-Ketotestosterone (pg/mL) Controls Dieldrin 0.8 g/g 0 100 200 300 400 500 600 700 800 900 1000 030609012011-Ketotestosterone (pg/mL) Controls Dieldrin 0.4 g/g 0 100 200 300 400 500 600 700 800 900 1000 0306090120Sample Days11-Ketotestosterone (pg/mL) Controls Dieldrin 0.04 g/g Figure 3-4. Female 11-ketotestosterone c oncentrations, on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treat ments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control. * * * * * * *

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56 0 100 200 300 400 500 600 700 0306090120Estradiol (pg/mL) Controls DDE 50 g/g -100 0 100 200 300 400 500 600 700 0306090120Estradiol (pg/mL) Controls DDE 46 g/g 0 100 200 300 400 500 600 700 0306090120Sample DaysEstradiol (pg/mL) Controls DDE 5 g/g Figure 3-5. Male estradio l concentrations, on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control. * * * * *

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57 0 50 100 150 200 250 300 350 400 450 500 0306090120Estradiol (pg/mL) Controls Dieldrin 0.8 g/g 0 50 100 150 200 250 300 350 400 450 500 0306090120Estradiol (pg/mL) Controls Dieldrin 0.4 g/g 0 50 100 150 200 250 300 350 400 450 500 0306090120Sample DaysEstradiol (pg/mL) Controls Dieldrin 0.04 g/g Figure 3-6. Male estradio l concentrations, on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control. * * * * * * * * * *

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58 0 200 400 600 800 1000 1200 1400 1600 030609012011-Ketotestosterone (pg/mL) Controls DDE 50 g/g 0 200 400 600 800 1000 1200 1400 1600 030609012011-Ketotestosterone (pg/mL) Controls DDE 46 g/g 0 200 400 600 800 1000 1200 1400 1600 0306090120Sample Days11-Ketotestosterone (pg/mL) Controls DDE 5 g/g Figure 3-7. Male 11-ketotestosterone c oncentrations, on days 0, 30, 60, 90, and 120 for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control. * * * *

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59 0 200 400 600 800 1000 1200 030609012011-Ketotestosterone (pg/mL) Controls Dieldrin 0.8 g/g 0 200 400 600 800 1000 1200 030609012011-Ketotestosterone (pg/mL) Controls Dieldrin 0.4 g/g 0 200 400 600 800 1000 1200 0306090120Sample Days11-Ketotestosterone (pg/mL) Controls Dieldrin 0.04 g/g Figure 3-8. Male 11-ketotestosterone c oncentrations, on days 0, 30, 60, 90, and 120 for the 0.8, 0.4, and 0.04 g/g Dieldrin treat ments (n = 6 largemouth bass per sample day, per treatment), compared to the Control. Asterisk represents significant difference ( p 0.05) from the Control. * * *

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60 Table 3-5. Day-120 GC-MS mean SD results of both female and male largemouth bass p,p ’-DDE and dieldrin concentrations (ng/ g) in the carcass per treatment (n = 3 carcasses per treatment, for each sex). Treatments (g/g) p,p ’-DDE Control 5 46 50 Female Carcass 19 2 589 614 8545 1108 9015 1042 Male Carcass 170 222 1417 372 7279 1980 6763 1430 Treatments (g/g) Dieldrin Control 0.04 0.4 0.8 Female Carcass 2.3 0.6 0.6 0 216.7 34.3 276.3 57.1 Male Carcass 1.4 1.4 1.4 1.4 190.3 44.8 254.3 50.3

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61 -2000 0 2000 4000 6000 8000 10000 12000 ControlDDE 5 g/gDDE 46 g/gDDE 50 g/gArea 7 Treatment GroupsCarcass Concentration p,p '-DDE (ng/g) 0 50 100 150 200 250 300 350 400 ControlDieldrin 0.04 g/g Dieldrin 0.4 g/g Dieldrin 0.8 g/g Area 7 Treatment GroupsCarcass Concentration Dieldrin (ng/g) Figure 3-9. Female (white bars) and male (shaded bars) largemouth bass mean SD carcass concentrations of p,p ’-DDE and dieldrin treatments (n = 3 carcasses per treatment, for each sex). Included is the mean carcass concentration of each organochlorine for the five female largemouth bass sampled from Area 7 (white bar) on February 23, 2004. dieldrin p,p ’-DDE

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62 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0306090120GSI (%) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0306090120GSI (%) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0306090120Sample DaysGSI (%) Figure 3-10. Change in female GSI (%) over the entire 120-day sampling period for the 50, 46, and 5 g/g p,p ’-DDE treatments (n = 6 largemouth bass per sample day). Sample days with the same lower case letter were not significantly different ( p > 0.05). 50 g/g 46 g/g 5 g/g c c b a a d c b,c b a d c,d a,b b,c a

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63 0 1 2 3 4 5 6 0306090120GSI (%) 0 1 2 3 4 5 6 0306090120GSI (%) 0 1 2 3 4 5 6 0306090120Sample DaysGSI (%) Figure 3-11. Change in female GSI (%) over the entire 120-day sampling period for the 0.8, 0.4, and 0.04 g/g Dieldrin treatments (n = 6 largemouth bass per sample day). Sample days with the same lower case letter were not significantly different ( p > 0.05). 0.8 g/g 0.4 g/g 0.04 g/g d d c b a d c,d b,c b a d c,d b,c b a

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64 Table 3-6. Day-120 mean SD re sults of percent hatch for the p,p ’-DDE and dieldrin treatments (n = 6 clutches per treatm ent). Treatments with the same upper case letter were not significantly different ( p > 0.05). p,p ’-DDE Treatments (g/g) Control 5 46 50 51 15b (36 – 74) 55 21a,b (23 – 77) 52 9b (39 – 61) 71 6a (63 – 80) Dieldrin Treatments (g/g) Control 0.04 0.4 0.8 51 15b (36 – 74) 85 6a (81 – 89) 58 10a,b (47 – 75) 61 29a,b (10 – 84)

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65 CHAPTER 4 GENERAL CONCLUSIONS Research from this study provides evid ence that dietary exposure to the OCPs p,p ’DDE and dieldrin can create internal carcass and gonad conc entrations of these two pesticides at levels similar to largemouth bass taken from reclaimed Florida agriculture areas that are believed to ha ve impaired endocrine and re productive function (Benton and Douglas, 1996; Marburger et al ., 1999). In addition, my study demonstrated that length and timing of pesticide dietary exposure is im portant in replicati ng sex steroid hormone concentrations reported for largemouth ba ss sampled from the EMCA. In my 30-day dietary exposure study, there was no evidence of a dose-response decrease in GSI or circulating sex steroid hormones. The re sults may have been influenced by OCP exposure taking place during a portion of the annual reproductiv e cycle of the largemouth bass after their reproductive orga ns were fully developed; th us, causing my first study to miss critical events in the re productive cycle that could have facilitated gametogenic and steroidogenic changes. Perhaps, endocr ine system changes that initiate gonad maturation, including surges in E2 and 11-KT sex steroid pr oduction, had already taken place and were already on a seasonal decline by the time pesticide exposure in the 30-day study began. OCP exposure length for my second study (120 days) was, therefore, extended to encompass a larger porti on of the annual reproductive cycle. Extension of exposure length demonstrated reductions in female largemouth bass E2 concentrations, a lack of expect ed seasonal increasing trend in E2 concentrations, and abnormal increases in 11-KT. These ch anges were similar to the reproductive

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66 abnormalities reported for largemouth bass sampled from the EMCA (Marburger et al ., 1999). Reductions in female plasma E2 concentrations demonstrated by my treatments ( p,p ’-DDE and dieldrin) averaged 2 to 3 times le ss than the Control, indicating that both OCPs induced significant bi ological reductions in E2 concentrations. Attained OCP carcass concentrations and achieved depressions of female E2 concentrations, however, did not cause a reduction of percent hatch between the p,p ’-DDE and dieldrin treated fish and those feed a control diet. Conseque ntly, my study did not provide any strong evidence that two of the predominate OCPs ( p,p ’-DDE and dieldrin) found in soils and largemouth bass tissues, sampled from the EM CA, cause dose-response decreases in the percent hatch of eggs produced by spawning treate d fish. This may indicate that a lack of reproductive success by adult Florida largemouth bass, stocked into the EMCA, is not the primary reason for the failure of the developm ent of the EMCA into a quality largemouth bass fishery. Future research on the reproductive effects of p,p ’-DDE and dieldrin on largemouth bass could focus on the application of a dosi ng experiment over an entire calendar year. Even when exposure length in my second study was extended to a 120-day period, effects were only demonstrated at the hormonal or biochemical level. Largemouth bass in EMCA, reported to have impaired e ndocrine and reproduc tive function, are environmentally exposed these pesticides on a yearly basis, spanning all portions of the steroidogenic and gametogenic phases of th e reproductive cycle. Extending OCP exposure length to an entire year will a llow researchers to cove r all portions of the reproductive cycle of the largemouth bass.

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67 In addition, my studies only sought to ch aracterize single ch emical dose-response effects for two of the predominate OCPs, p,p ’-DDE and dieldrin, found in the soils and various tissues of largemouth bass from th e EMCA. Fish in this system are environmentally exposed to multiple pesticid es (e.g., toxaphene and chlordane) that may not only contribute to reducti ons in hormone concentratio ns, but also to decreased reproductive success. Future research on th e reproductive effects of OCPs on largemouth bass could also focus on pesticide mixture exposure studies and the effects that multiple pesticide exposure may attribute to hormone depression and reproductive success, not just on the single chemical exposure to p,p ’-DDE and dieldrin. The application of studies using multiple single chemical doses, coupl ed with mixture exposures, will enable researchers to pinpoint what OCPs are re sponsible for causing endocrine disruption. The apparent limited reproductive success by stocked adult Florida largemouth bass or recruitment of fish to the fingerling st age in the EMCA might not be attributed to OCP endocrine disruption or t oxicity at all. An ecosystem assessment was conducted at the EMCA in 2001 to address other factors that might influence poor recruitment of largemouth bass in this system incl uding, spawning habitat and phytoplankton, zooplankton, and macroinvertebrate communities. Preliminary results of the assessment report that a low abundance of invertebrates might be contributing to poor sport fish production in the EMCA because planktonic and benthic invertebrates are important components of the diets of larval and juve nile sport fish, including largemouth bass (William E. Johnson, Florida Fish and W ildlife Conservation Commission, personal communication). There is also evidence fr om this ecosystem assessment that poor

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68 habitat (i.e., muck sediments and a scarcity of aquatic plants) might also be contributing to largemouth bass reproduction and recruitment problems in the EMCA. Lastly, it is important that biologists continue to mon itor OCP concentrations and population dynamics of largemouth bass in the EMCA. This will enable researchers to gain insight into whether or not improveme nts in largemouth bass reproduction and/or recruitment are related to changes in OCP leve ls or other environmental factors. Based on the results of my study, if m oney is a limiting factor, I would focus all research efforts on OCP mixture exposure experiments.

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69 LIST OF REFERENCES Anderson, R. O., and Neumann, R. M. 1996. Le ngth, weight, and associated structural indices. Pages 447-482 in B. R. Murphy and D. W. Willis, editors. Fisheries Techniques, 2nd edition. American Fisheries Society, Bethesda, Maryland. Battrup, E., and Junge, M. 2001. Antiandrogenic pe sticides disrupt se xual characteristics in the adult male guppy ( Poecilia reticulata ). Environmental Health Perspectives 109: 1063-1068. Bayley, M., Junge, M., and Baatrup, E. 2002. Exposure of juvenile guppies to three antiandrogens causes demasculinization and a reduced sperm count in adult males. Aquatic Toxicology 56: 227-239. Benton, J., and Douglas, D. 1996. Ocklawaha Fisheries Investigations: 1 July 1994 through 30 June 1995: Study XIII Assessment of fisheries restoration potential for reclaimed agricultural lands in the Upper Ocklawaha Basin. State of Florida Game and Fresh Water Fish Commission, Tallahassee, Florida. Benton, J., Douglas, D., and Prevatt, L. 1991. Completion report as required by federal aid in fish restoration: Wallop-Breaux project F-30-18, Ocklawaha Basin Fisheries Investigations Study XII. Lake Apopka fish eries studies. State of Florida Game and Fresh Water Fish Commission, Tallahassee, Florida. Burdick, G. E., Harris, E. J., Dean, H. J., Walker, T. M., Skea, J., and Colby, D. 1964. The accumulation of DDT in lake trout and the effect on reproduction. Transactions of the American Fisheries Society 93: 127-136. Carlson, A. R. 1973. Induced spawning of largemouth bass [ Micropterus salmoides (Lacepede)]. Transactions of the Am erican Fisheries Society 102: 442-444. Chedrese, P. J. and Feyles, F. 2001. The diverse mechan ism of action of dichlorodiphenyldichloro ethylene (DDE) and methoxyc hlor in ovarian cells in vitro . Reproductive Toxicology 15: 693-698. Chen, S. U., Zhou, D. J., Yang, C., Okubo, T., Kinoshita, Y., Yu, B., Kao, Y. C., and Itoh, T. 2001. Modulation of aromatase expr ession in human breast tissue. Journal of Steroid Biochemistry a nd Molecular Biology 79: 35-40. Chew, R. L. 1974. Early life history of the Florida largemouth bass. Florida Game and Fresh Water Fish Commission. Fish Bulle tin No. 7, Tallahassee, Florida.

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70 Clugston, J. P. 1966. Centrarchid spawning in the Florida Everglades . Quarterly Journal of the Florida Academy of Sciences 29: 137-143. Crain, D. A., Guillette, L. J., Rooney, A. A ., and Pickford, D. B. 1997. Alterations in steroidogenesis in alligators ( Alligator mississippiensis ) exposed naturally and experimentally to environmental contamin ants. Environmental Health Perspectives 105: 528-533. Cross, J. N., and Hose, J. E. 1988. Evidence for impaired reproduction in white croacker ( Genyonemus lineatus ) from contaminated areas off southern California. Marine Environmental Research 24: 185-188. Danzo, B. J. 1997. Environmental xenobiotics ma y disrupt normal endocrine function by interfering with the binding of physiol ogical ligands to st eroid receptors and binding proteins. Environmental He alth Perspectives 105: 294-301. Danzo, B. J., Shappell, H. W., Banerjee, A., and Hachey, D. L. 2002. Effects of nonylphenol, 1,1-dichloro-2,2-bis( p-chlorophenyl)ethylene ( p,p '-DDE), and pentachlorophenol on the adult female guine a pig reproductive tract. Reproductive Toxicology 16: 29-43. Foster, E. P., Fitzpatrick, M. S., Feist, G. W., Schreck, C. B., Yates, J., Spitsbergen, J. M., and Heidel, J. R. 2001. Plasma andr ogen correlation, EROD induction, reduced condition factor, and the occurrence of or ganochlorine pollutants in reproductively immature white sturgeon ( Acipenser transmontanus ) from the Colombia River, USA. Archives of Environmental Cont amination and Toxicology 41: 182-191. Gallagher, E. P., Gross, T. S., and Sheehy, K. M. 2001. Decreased glutathione Stransferase expression and ac tivity and altered sex steroi ds in Lake Apopka brown bullheads ( Ameriurus nebulosus ). Aquatic Toxicology 55: 223-237. Garcia, E. F., McPherson, R. J., Martin, T. H., Poth, R. A., and Greeley, M. S. 1997. Liver cell estrogen receptor binding in prespawning female largemouth bass, Micropterus salmoides , environmentally exposed to polychorinated biphenyls. Archives of Environmental Contam ination and Toxicology 32: 309-315. Gore, A. C. 2002. Organochlorine pesticides directly regulate gonadotropin-releasing hormone gene expression and biosynthes is in the GT1-7 hypot halamic cell line. Molecular and Cellular Endocrinology 192: 157-170. Gross, T. S., Arnold, B. S., Seplveda, M. S., and McDonald, K. 2003. Endocrine disrupting chemicals and endocri ne active agents. Pages 1033-1098 in D. J. Hoffman, B. A. Rattner, G. A. Burt on, and J. Cairns, editors. Handbook of Ecotoxicology, 2nd edition. Lewis Publishers, Boca Raton, Florida. Gross, T. S., Guillette, L. J., Percival, H. F., Masson, G. R., Matter, J. M., and Woodward, A. R. 1994. Contaminant-induced reproductive anomalies in Florida. Comparative Pathology Bulletin 26: 2-8.

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74 BIOGRAPHICAL SKETCH Kevin G. Johnson was born September 26, 1979, and grew up in Mt. Dora, FL. He attended Mt. Dora High School and gradua ted in May 1998. Kevin then attended the University of Central Florida and received his Bachelor of Science degree in August 2002, with a major in biology and a minor in environmental studies. During his undergraduate study, Kevin was a biological tec hnician for Dr. Linda Walters at UCF, working on oyster reef ecology in the Indian River Lagoon on the east coast of Florida. In the fall of 2002, Kevin began working in the ecotoxicology labor atory of Dr. Timothy S. Gross at the United States Geological Survey in Gainesville, FL. Then, in the spring of 2003, he enrolled as a graduate student at the University of Florida with Drs. Timothy S. Gross and Daniel E. Canfield, Jr. as his advisors, focusing his work on the reproductive effects of organoc hlorine pesticides on largem outh bass. Kevin’s passion for the study of fisheries and aquatic sciences has translated into his passion as a fresh and saltwater fisherman, a pastime he plan s to pursue for many years to come.