.•.•.. · • • • • • • • • • • • • • • • • • • • • • • • · · • . · ..•......... viii CHAPTER I GENERAL INTRODUCTION. •••••••••••••••••••••••••• 2 CHAPTER II XENOBIOTIC COMPOUNDS IN BLUE CRABS FROM THE ELIZABETH RIVER. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 o Introduction .• · · · · · · · · · · · · · · · · · · · · · · · · · ..........•... 10 Materials and Methods. .. • • • • • • • • • • • • • • • • .. · .......... 10 Results •.... · · · · · • · • · · · · · · · · · · · · • · · · · · · · · · · · · ........ 1 7 Discussion .. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · .......... 2 6 CHAPTER III INFLUENCE OF PAH IN FIELD ECDYSIS EXPOSED ON THE ACCUMULATION OF CRABS • ••••••••••••••••• 3 3 Introduction .•.. • · · · · • · • · · • · · • • · · · · • · · · · · • • · ......... 3 3 Materials and Methods •......•......•................. 33 Field Experiment .•.•.....•...•.••...•........... 3 3 Laboratory Analysis ...............•............. 35 statistical Analysis .....•........•.••..•....... 36 Results •..•. ·····•···•·•·•·••··•·····•·•·········· ... 37 Discussion ... ······•···········•················· .... 41 CHAPTER IV CONCLUSIONS ..•.•.•...••...................... 4 6 LITERATURE CITED. · • • • • · • · · · · · · · · • · · • · · · · · · · · · · · · · · · · • • · · · . 4 8 VITA •.••..•..•.... · • • • • · · · • · • · · · · • · • · · · · · · · · • · · · · · · • • • · ... 55

Vitellogenin (VTG) is widely used as a biomarker for environmental estrogens and reproductive disruption in fish. Vitellogenesis is the process by which yolk is formed. The endpoint is most sensitive in male fish where vitellogenesis is an abnormal process. Research examining e ffects of environmental mixtures of chemicals (e.g. creosote) on vitellogenesis is limited. This study examines plasma VTG expression in both male and female mummichog, Fundulus heteroclitus, collected from a creosote-contaminated site and two reference sites in lower Chesapeake Bay, USA, and in wild-caught male reference fish exposed in the laboratory to creosote-contaminated sediment. Further, this study uses tissue somatic indices (gonadosomatic index, hepatosomatic index, and condition factor) as organ-level indicators of reproductive status and creosote exposure. Western blotting with an anti-vitellogenin monoclonal antibody (FY 109) was used to identify an abundant (approx. 218 kDa) protein in F. heteroclitus plasma samples. Vitellogenin was not observed in any male fish collected from any of the field sites. No site­ specific differences were observed in plasma VTG of females from these sites. Overall, fish from the creosote-contaminated site appear resilient and reproductively fit. After seven days of exposure, reference fish exposed to sediment from the creosote-contaminated site suffered extensive mortality (approx. 25% ), but displayed no expression of VTG and had no alterations in other measured indices except for increased hepatosomatic index and induction of the biotransformation enzyme, cytochrome P4501A (CYPlA). Lack of response in Atlantic Wood fish could be due to: (1) physiological adaptation of the population from the creosote­ contaminated site to creosote exposure (2) species-specific sensitivity (3) creosote not exerting estrogenic effects and regulating vitellogenesis. Thus, while VTG has been used successfully as a practical and reliable screening method for wildlife toxicity, results herein do not support use of this biomarker for F. heteroclitus from creosote-contaminated sites. This study does reaffirm constituents of creosote binding to the aryl hydrocarbon receptor resulting in induction of CYP IA. More attention needs to be given to developing alternative biomarkers not sensitive to aryl hydrocarbon-mediated antiestrogens if detection of weak estrogens and reproductive disruption in creosote and other complex mixtures is to be achieved.

Poultry litter leachate (PLL) is known to contain a variety of contaminants including endocrine disrupting compounds (EDCs). This study analyzed the presence of steroids and contaminants in samples of poultry litter from a broiler poultry operation in Maryland, USA. Litter samples were homogenized, hydrated, incubated for two time periods (4 and 24 h) at two temperatures (20°C and 37°C), filtered, and analyzed for steroids and anthropogenic contaminants. In addition, duplicate samples were spiked with 17‐β estradiol (E2) and testosterone (T), and β-glucuronidase and aryl sulfatase, to measure steroid recovery and the presence of conjugates, respectively. A steroid recovery rate of 71 and 73% was obtained from E2 and T spiked samples, respectively. Increased incubation duration demonstrated an increased trend in E2 and a decreased trend in androgen (T and/or dihydrotestosterone [DHT]) concentrations, regardless of temperature. In contrast, increased incubation temperature displayed different trends in E2 and androgen concentrations. High temperature with a 4-h incubation resulted in an increased trend in androgen with no effect on E2. However, after 24 h of incubation at high temperature, an increased trend in E2 was observed with no effect on androgen. The presence of de-conjugating enzymes resulted in a greatly increased trend in T concentrations with a slight increased trend of E2 concentrations. Trace amounts of several metals and anthropogenic compounds were detected. Arsenic, barium, endosulfan, and bis (2-ethylhexyl) phthalate were detected at quantifiable levels. This study demonstrates that PLL contains potential EDCs and contaminants that can be toxic to, and bioaccumulate in, aquatic fauna. Determination of EDC concentrations in environmental samples is important to elucidate potential detrimental effects of agricultural runoff on aquatic wildlife.

Animal feeding operations in the United States produce more than 500 million tons of manure annually. Disposal of poultry waste via application as fertilizer results in substantial runoff of poultry litter associated contaminants (PLAC). Of particular concern are sex steroids, 17 beta-estradiol, estrone and testosterone, responsible for sex differentiation and development of reproductive structures. In a series of laboratory assays, mature male and mixed-sex larval/juvenile fish were continuously exposed to environmentally relevant PLAC solutions. Effects on gonads were assessed histologically, and vitellogenin (VTG) induction was measured as a gauge of estrogenicity. Twenty-one-day exposures to laboratory-generated PLAC solutions routinely induced VTG in mature male Pinzephales promelas. Vitellogenesis in Fundulus heteroclitus only occurred at the highest tested PLAC concentration, and Cyprinodon variegutus were unresponsive at any tested concentration. All species produced considerable VTG in response to a 17 beta-estradiol positive control. A pronounced feminization was seen in P. promelas when exposed to PLAC as larvae but not when exposed as juveniles. Runoff from a poultry litter amended field cropped under standard agronomic practices induced significant VTG in male P. promelas. Results indicate that environmentally relevant PLAC concentrations exhibit endocrine activity sufficient to induce VTG production in male fish and possibly affect sex ratios in resident fish populations. Environ. Toxicol. Chem. 2010;29:2328-2340. (C) 2010 SETAC

The brown bullhead Ameiurus nebulosus is a species of the family Ictaluridae commonly used as a sentinel of environmental contamination. While these fish have been utilized for this purpose in areas contaminated with polychlorinated biphenyls (PCBs), few controlled, laboratory-based studies have been designed to document the effects of PCB mixtures in this species. Here, brown bullhead were exposed to the PCB mixture, Aroclor 1248, via intraperitoneal injection and the effects on immune function, plasma hormones and disease resistance were evaluated. Exposure to this mixture led to a decrease in bactericidal activity and circulating antibodies to Edwardsiella ictaluri present from a previous exposure to this pathogen. A subsequent E. ictaluri disease challenge led to significantly higher mortality in A1248 treated fish compared to vehicle-control fish. The mitogenic response to the T-cell mitogen, phytohemaglutinin-P, was increased compared to vehicle-control fish. The steroid hormone, cortisol, and the thyroid hormone, T3, were also significantly lower in A1248 exposed fish. In summary, we have validated a number of functional immune assays for application in brown bullhead immunotoxicity studies. Additionally, we have demonstrated that the PCB mixture (A1248) modulates both immune function and endocrine physiology in brown bullhead. Such data may compliment the interpretation of data yielded from applied field studies conducted in PCB contaminated aquatic ecosystems.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Biosolids (treated sewage sludge) are increasingly disposed of on land. Thus particle-sorbed and dissolved constituents have the potential to enter nearby watersheds. Although organic contaminants are known to be present in biosolids these are not currently regulated and little data exist on their potential toxicity to aquatic organisms. We exposed Pimephales promelas to two concentrations of biosolids (0.5 and 2.5 g l(-1)) for 28-days (static-renewal) and characterized contaminants present and the extent of CYP1A and DNA damage induction at various time points. Many organic contaminants were detected in the biosolids, with polycyclic aromatic hydrocarbons (PAHs) being the dominant class. Substantial levels of polybrominated diphenyl ethers (PBDEs) and nonylphenols (NPs) were also present. Significant induction of hepatic CYP1A protein compared with controls (P<0.05) was observed in both low (0.5 g l(-1)) and high (2.5 g l(-1)) exposed fish from Day 7. CYP1A levels peaked at Day 21 with 21-fold and 8-fold inductions over controls in high and low dose fish respectively. Induction of DNA damage in hepatocytes (single strand breaks as measured using the COMET assay) was observed in both exposures compared with controls on Days 14 and 28 (P<0.05). A significant correlation was found between CYP1A induction and DNA damage (Pearson correlation index, P<0.05). It is plausible that activation of PAHs may be responsible for the induction of CYP1A and resulting increase in DNA damage. Our data show the potential for detrimental effects in the event of exposure of aquatic organisms to biosolids and the need for further investigations of possible impacts due to constituents not covered by current guidelines. (C) 2007 Elsevier B.V. All rights reserved.

Several populations of the estuarine killifish, Fundulus heteroclitus, also known as the mummichog, exhibit characteristics of adaptation to priority pollutants. One such population of mummichog inhabits the Elizabeth River in Virginia at the Atlantic Wood site (AW), a U.S. Environmental Protection Agency (EPA) Superfund site heavily contaminated with creosote containing a mixture of polyaromatic hydrocarbons (PAHs). Although PAHs are known to be immunotoxic in experimental animals, resident AW mummichogs seem to thrive. Mummichogs from the AW site and a reference site were subsequently examined over a 2-yr period for total immunoglobin (IgM), as well as circulating antibody levels against 5 ubiquitous marine bacteria. Expression profiles of circulating and lymphoid lysozyme and lymphoid cyclooxygenase-2 (COX-2) were also examined. Compared to relatively high total IgM and specific antibody responses in reference fish, AW mummichogs had lower circulating IgM and lower specific antibody levels against all bacteria examined, however they had higher levels of circulating lysozyme. Lymphoid cells in the AW mummichogs also expressed higher levels of lysozyme, as well as COX-2, which may indicate a state of macrophage activation. Elevated COX-2 levels may be associated with enhanced metabolism of PAHs through cooxidation-peroxidase pathways. Additional studies attempted to immunize AW mummichogs reared in uncontaminated water to compare their antibody responses to that of reference fish. AW mummichogs did not survive 40 d post culture, while reference fish thrived. Our findings suggest that the chemical environment at the AW site may be vicariously enhancing components of innate immunity, probably through oxidative stress pathways, in resident mummichogs, while actively suppressing humoral immune responses.

Studies were undertaken to examine the importance of organic proteoglycan matrices in the calcification of elasmobranch vertebral cartilage. Proteoglycans were extracted from the vertebral cartilage of the clearnose skate, Raja eglanteria Bosc, with a 3M guanidine hydrochloride/10% EDTA solution. Proteoglycan solutions (12 μg ml−1) were effective inhibitors of hydroxyapatite formation in vitro from high concentration calcium phosphate solutions. Inhibition of crystal formation appears to occur through the restriction of phase transformation from a calcium phosphate precursor to hydroxyapatite crystals. The concentration and/or degradation of proteoglycans in elasmobranch vertebral cartilage may have a physiological role in the production of alternating mineral-rich and mineral-poor growth zones, currently used in ageing studies.

Cytosolic glutathione S-transferase (GSH transferase) activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was elevated approximately three to four-fold in intestine and liver of mummichog (Fundulus heteroclitus) collected from a creosote-contaminated site in the Elizabeth River, Virginia. Intestinal GSH transferase activity at the most heavily contaminated site, at a moderately contaminated site and at a relatively clean site averaged 3.64, 2.83 and 1.11µmoles/min/mg respectively, while values for liver at these sites averaged 2.84, 1.75 and 0.93µmoles/min/mg. In addition, densitometric tracings of sodium dodecylsulfate-polyacrylamide gels of intestine and liver cytosol revealed a similar trend in the staining intensity of a 25.8 kD protein band, which lies within the molecular weight range of GSH transferase subunits. Activity in putative preneoplastic and neoplastic hepatic lesions of fish collected from the creosote-contaminated site was not significantly different from that of adjacent normal tissue. In the laboratory, dietary betanaphthoflavone (ßNF) treatment resulted in a three-fold increase in intestinal GSH transferase. Hepatic GSH transferase activity in the same fish was not affected by dietary ßNF although hepatic monooxygenase activity, measured as ethoxyresorufin O-deethylase (EROD), was. The results of this study indicate a response of the intestinal detoxification system to environmental contaminants and supports previous studies on the importance of intestinal metabolism of foreign compounds. Further, our results indicate the trend towards elevated GSH transferase in liver of feral fish could not be attributed to a cancerous disease state in these fish but indicates chemical induction in this organ as well.