124 (IRW-1116-377343) Relationships between reproductive endpoints measured in flatfish collected near an outfall in Orange County, California.
Start time: 1:50 PM
Irwin, M¹, Reyes, J², Steinert, S³, Hwang, W¹, Armstrong, J⁴, Sakamoto, K⁴, Kelley, K², Schlenk, D¹, ¹ University of California Riverside, Riverside, CA, USA² California State University Long Beach, Long Beach, CA, USA³ CSC Biomarker Laboratory, San Diego, CA, USA⁴ Orange County Sanitation District, Fountain Valley, CA, USA
Estrogenic activity in fish has primarily been evaluated using vitellogenin (vtg) expression in male and juvenile animals. Although the response has been widespread in field and laboratory studies, the relevance of the response to higher-level adverse effects, particularly in the field, is less than clear. Previous evaluations of vtg within flatfish species collected near the Orange County Sanitation District (OCSD) outfall and stations as far as 7.7 km down current indicated bioavailable estrogens within demersal flatfish populations. In order to evaluate the persistence of estrogenic activity and relationships to reproduction and development, fish were sampled in the winter and summer of 2003 and 2004 from the same locations and vtg, plasma estradiol (E2) concentrations, gonadosomatic indices (GSI), sperm DNA damage, development and gender ratios were measured in English Sole (Pleuronectes vetulus) and Hornyhead Turbot (Pleuronichthys verticalis). Variable levels of vtg were continually observed in plasma samples from fish collected in the locations analyzed. Vtg expression and plasma E2 levels were significantly correlated in female Hornyhead Turbot and both sexes of English Sole. A positive relationship was demonstrated between plasma E2 levels and sperm DNA damage. Rather than an expected feminization of populations, a trend toward masculinization was observed particularly at the OCSD outfall, as indicated by gender ratios and significantly higher GSI in males versus males from the reference station. These results are consistent with previous studies showing vtg expression in male flatfish, but no alteration in overall flatfish abundance at the sampled sites.

125 (LER-1117-843870) Alterations in thyroid hormone status in Atlantic croaker exposed to Aroclor 1254 and selected PCB congeners.
Start time: 2:10 PM
LeRoy, K¹, Thomas, P¹, Khan, I¹, ¹ University of Texas Marine Science Institute, Port Aransas, TX, USA
Many studies in animal and human populations have demonstrated that PCB exposure interferes with the normal function of the thyroid system. In this study, Atlantic croaker (Micropogonias undulatus) were exposed to a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) or one of four individual congeners (two planar, PCB 77 and 126; two non-planar, PCB 47 and 153) in the diet for 30 days during gonadal recrudescence to investigate the effects of PCBs on thyroidal status. Thyroid hormones were extracted from plasma samples, and thyroid hormones triiodothyronine (T₃) and thyroxine (T₄) were measured with an enzyme-linked immunosorbent assay. Both male and female croaker exposed to the mixture Aroclor 1254 or individual congeners PCB 77, 126, or 153, showed decreased levels of T₃, but not T₄. By examining the effects of PCB mixtures and individual congeners on thyroidal status in Atlantic croaker, we can further develop our understanding of the mechanisms of action of these synthetic endocrine-disrupting chemicals and better predict values for both human, fish, and wildlife risk assessment.

126 (ROY-1117-829614) Vitellogenin levels in three-spine sticklebacks (Gasterosteus aculeatus) to detect effects of pesticide exposure.
Start time: 2:30 PM
Roy, R.¹, Maltais, D.¹, Couillard, C.¹, Lebeuf, M.¹, ¹ Fisheries and Oceans Canada, Mont-Joli, Quebec, Canada
As part of a project investigating effects of pesticides on fish, we isolated vitellogenin (VTG) from threespine sticklebacks (Gasterosteus aculeatus). In June - July 2003, sticklebacks were captured from a site on the South shore of the St Lawrence Estuary (SLE). In the laboratory, mature females (n = 120) were anaesthetised, injected with a proteolysis inhibitor (aprotinin) and sampled for blood. Plasma was obtained by centrifugation. The protein was precipitated from plasma with MgCl₂and EDTA, following modifications of the method described by Norberg (1995) and further purified by gel filtration. The purity of the pooled VTG fractions was confirmed by Native-PAGE (3-8% tris-acetate gel). Molecular weight (MW) was determined by gel filtration run with MW standards. Stickleback VTG has a relatively high MW of approximately 637 kDa (vs 520 kDa for smooth flounder VTG, Roy et al. 2004). Precipitated plasma from mature female sticklebacks and the presumptive VTG stained with Coomassie blue, suggesting that the latter is a female-specific protein. Staining with methyl green (phosphoprotein), Sudan black B (lipoprotein, Prat et al. 1969) and Periodic Acid-Schiff reagent (glycoprotein) on Native-PAGE identifies the purified protein as a phospho-lipo-glycoprotein - further evidence that it is VTG. Finally, Western blot with commercially available antibodies confirmed that the protein is stickleback VTG. We developed a homologous enzyme immunoassay for stickleback VTG, using the purified protein for standards, and analysed plasma from mature sticklebacks from 3 other SLE sites. The results suggest that VTG levels in these populations are not affected by pesticides.

127 (PAR-1118-086490) Lifecycle exposure to municipal wastewater effluents decreases egg production and male sex characteristics of fathead minnows.
Start time: 2:50 PM
Parrott, J¹, Blunt, B¹, Sullivan, C, Bennie, D¹, ¹ National Water Research Institute, Environment Canada, Burlington, ON, Canada
Assessing the effects of municipal wastewater effluents (MWWEs) on fish poses a challenge, as the effluents are complex mixtures of nutrients, hormones and pharmaceutical drugs. Some pharmaceuticals discharged in MWWEs can have profound endocrine disrupting effects on fish. Assessment of the effects of very low concentrations (1-10 ng/L) of pure endocrine disrupting substances (estrogens and androgens) allows the development of sensitive and predictive fish tests that can assess the potential reproductive effects of complex mixed municipal effluents. Male fathead minnows exposed in situ to 100 % model scale MWWE for a lifecycle had increased liver-somatic indices and decreased male secondary sex characteristics (smaller dorsal fatpads and fewer nuptial tubercles). Laboratory lifecycle exposure to an Ontario MWWE (100 %) caused a decrease in egg production and decreases in male secondary sex characteristics of fathead minnows, although fish grew as well as or better than control fish. For assessing the endocrine disrupting potential of pharmaceuticals, long term fish tests of real MWWEs provide clues that will link to the health and reproductive performance of wild fish.

(58354) Break.
Start time: 3:10 PM

128 (BLA-1117-818764) Intersex in Fish: Species and Site Comparisons.
Start time: 3:50 PM
Blazer, V¹, Hinck, J², Schmitt, C², ¹ USGS, National Fish Health Research Laboratory, Leetown Science Center, Kearneysville, WV, 25430² USGS, Columbia Environmental Research Center, Columbia, MO
Intersex or ovotestis is a gonadal change that has been used as an indicator of exposure to reproductive endocrine disruptors in gonochorist fishes. This change is most often detected microscopically in male gonads and is characterized by the presence of oocytes. Although a wide variety of natural and manufactured chemicals are know to possess estrogenic activity in fishes, a high prevalence of intersex has most commonly been associated with domesticate sewage treatment discharges or runoff from agricultural/confined animal feeding operations. As part of the U.S. Geological Survey′s Biomonitoring of Environmental Status and Trends (BEST) program, gonads from 1400 common carp and 773 bass (primarily largemouth bass, but including smallmouth and spotted) have been collected from sites in the Mississippi, Rio Grande, Columbia and Colorado river drainages and examined microscopically. Using the complete data set we examined species differences and identify sites at which intersex was observed. The target sample size was 10 fish of each sex for each species per site. However, this is not always obtained. Hence, we compared prevalence of intersex only at those sites at which six or more male fish of a particular species were collected. In addition, using gonads collected from smallmouth and largemouth bass in the Potomac drainage we assessed the current BEST protocols for tissue collection: cut 1-cm pieces from the posterior end (bottom tips) of the gonads and place a maximum of five in fixative, and statistically evaluated the probablility of finding intersex when present depending on number and size of pieces examined.

129 (VIJ-1117-701228) Ah receptor activation impairs the rate limiting steps in steroidogenesis in rainbow trout.
Start time: 4:10 PM
Aluru, N¹, Vijayan, M¹, ¹ University of Waterloo, Waterloo, Ontario, Canada
Anthropogenic stressors activating aryl hydrocarbon (Ah) receptor signaling, including polychlorinated biphenyls, impair the adaptive corticosteroid response to stress, but the mechanisms involved are far from clear. Using Ah receptor agonist (beta-naphthoflavone; BNF) and antagonist (resveratrol; RVT), we tested the hypothesis that steroidogenic pathway is a target for endocrine disruption by xenobiotics activating Ah receptor signaling. Trout (Oncorhynchus mykiss) were fed BNF (10 mg/kg/day), RVT (20 mg/kg/day) or a combination of both (RBNF) for 5 days, and subjected to a handling stress. BNF induced cytochrome P4501A1 (CYP1A1) expression in the interrenal tissue and liver, while this response was abolished by RVT, confirming Ah receptor activation. In control fish, handling stress transiently elevated plasma cortisol and glucose levels and transcript levels of interrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage (P450scc) and 11 beta-hydroxylase over a 24 h period. BNF treatment attenuated this stress-induced plasma and interrenal responses; these BNF-mediated responses were reverted back to the control levels in the presence of RVT. We further examined whether these in vivo impact of BNF on steroidogenesis can be mimicked in vitro using interrenal tissue preparations. BNF depressed ACTH-mediated cortisol production and this decrease corresponded with lower StAR and P450scc, but not 11 beta-hydroxylase mRNA abundance. RVT eliminated this BNF-mediated depression of interrenal corticosteroidogenesis in vitro. Altogether, xenobiotics activating Ah receptor signaling are corticosteroid disruptors, and the mode of action includes inhibition of StAR and P450scc, the rate limiting steps in steroidogenesis.

130 (MAY-1117-837209) Analysis of zebrafish embryos esposed to natural and synthetic estrogen mixtures.
Start time: 4:30 PM
Mayer, G¹, Notch, E¹, ¹ University of Maine, Orono, ME, USA
Recently, much attention has been given to endocrine active compounds in the environment. One class of xenoestrogens includes the synthetic estrogens commonly found in pharmaceuticals such as birth control agents and hormone replacement therapies. Synthetic estrogens such as these mimic natural estrogens well at the receptor level, but are more resistant to degradation by natural processes (1.2 d for E2 vs. 17 d for EE2). Because of its greater stability and higher potency in vivo, EE2 may be of disproportional toxicological importance despite being found at much lower concentrations than natural steroids such as E2 and E1. In addition, combinations of estrogenic species have been shown to synergistically upregulate vitellogenin protein levels as assayed by RIA. We investigate the use of a bioassay for estrogenic potential of synthetic estrogens that utilizes the activation of the Xenopus vitellogenin promoter and the subsequent expression of luciferase as a surrogate for whole animal estrogen response. These data will be correlated to actual vitellogenin transcriptional responses in finfish embryos after exposure to E1, E2, EE2, and combinations thereof.

131 (HEC-1118-268166) Parasitism in fish - an endocrine modulator of ecological relevance?
Start time: 4:50 PM
Hecker, M¹, Karbe, L², ¹ Michigan State University, East Lansing, MI, USA² University of Hamburg, Hamburg, Germany
Wild bream (Abramis brama) were collected from the river Elbe, and the influences of parasitic infection by the tapeworm Ligula intestinalis on endocrine and related functions (vitellogenin [VTG]; plasma sex steroids: 17-estradiol [E2], 11 ketotestosterone [11KT] and testosterone [T]; relative gonad [GSI] and liver [HSI] growth; maturation stages of germ cells [MS]; prominence of spawning tubercles [STI]) were investigated. Distinct regional differences in infection rates of bream with L. intestinalis were observed along the Elbe with the highest prevalences at the Czech border (up to 45 %) and Magdeburg (up to 65 %), areas that are heavily contaminated with complex mixtures of organic chemicals and metals. Parasitized fish of both sexes had significantly lower GSIs and poorly developed gonads (low MS). In males a significant reduction in the prominence of spawning tubercles occurred. Infected females had significantly lower plasma VTG concentrations. A selective suppression of the sex steroids 11KT and E2 was observed in male and female bream, respectively. Testosterone was not affected in the same manner in fish of both sexes. At sites with an elevated prevalence of L. intestinalis, the extent of the infection of an individual was significantly correlated with the suppression of the measured biomarkers. However, when applying a linear model to compare regional differences in infection prevalence with biological parameters, not all of the observed differences could be explained by parasitization by L. intestinalis. This indicates that other factors such as pollution may have contributed to the effects on reproductive and endocrine processes that occurred along the river. Given that sites with high prevalences of L. intestinalis were also characterized by elevated pollution, it is possible that there exists a combinatory effect of both pollution and parasitization that can have a serious impact on the reproductive capacity of a population, such as was observed at the sampling site at Magdeburg.

132 (THE-1117-572188) Thyroid Endocrine Disruption in Stonerollers from Perchlorate-Contaminated Streams in East-Central Texas.
Start time: 5:10 PM
Theodorakis, C¹, Rinchard, J², Kendall, R¹, ¹ Texas Tech University, The Institute of Environmental and Human Health, Lubbock, TX, USA² The Ohio State University, School of Natural Resources, Columbus, OH, USA
In October 2001 and March 2002, a field survey of central stonerollers (Campostoma anomalum) from perchlorate-contaminated streams in central Texas was conducted to assess thyroid endocrine disruption. Perchlorate is an oxidizer primarily used in solid-fuel rockets, and many sites that processed or used perchlorate are now contaminated. Histological analysis revealed that the fish from contaminated sites had increased thyroid follicular hyperplasia, hypertrophy, and colloid depletion. Multivariate analysis was generally found to be more powerful than univariate analysis. Seasonal differences existed in the degree of thyroidal perturbation were discovered, and fish were generally less sensitive to thyroidal perturbations in March compared to October. Thyroidal histological indicators were also correlated to levels of perchlorate in the fish, water, and periphyton. Periphyton was frequently most strongly correlated to thyroidal indices, suggesting that exposure through the food chain may be of import. In addition, one of the presumed reference sites turned out to be contaminated with perchlorate, and this was reflected by thyroidal biomarkers before perchlorate was detected in the stream water or biota. This is the first known published account of perchlorate-induced thyroid disruption in fish under field situations, and also points out the value of biomarkers for contaminant biomonitoring.