322 (LIU-1117-743763) Sodium arsenate enhances thyrotoxicity of sodium perchlorate in zebrafish Danio rerio.
Start time: 1:50 PM
Liu, F-J¹, Wang, J-S¹, Hooper, M¹, Cobb, G¹, Collie, N², Theodorakis, C¹, ¹ TIEHH, Texas Tech University, Lubbock, TX, US² Biology Department, Texas Tech University, Lubbock, TX, US
Both perchlorate and arsenic are contaminants in water ecosystem and tightly regulated. These two anions were found to co-exist in aquatic systems. Perchlorate is a thyroid disruptor acting as an inhibitor of iodide uptake into thyroid gland. Thyroid histopathology is widely used as endpoint in evaluating the damage by perchlorate and monitoring the perchlorate contamination. It was hypothesized that arsenic disrupts the homeostasis of deiodinase, which is responsible for the transformation from T4 to T3 thyroid hormone, as can be manifested in thyroid histopathological responses. In order to evaluate their effects on thyroid function and possible interaction between them in terms of histopathology, zebrafish Danio rerio were exposed to sodium perchlorate (10 and 100 ppm), sodium arsenate (1 and 10 ppm), and the mixture-sodium perchlorate + sodium arsenate (10+1 and 100 +10 ppm) plus control for 90 days with triplicates. At day 10, 30, 60, and 90, fish were sampled and processed for histology analysis. Epithelial cell height and colloid area were used as endpoints. Sodium perchlorate exposure resulted in apparent hypertrophy with 100 ppm more effective. However, 1 ppm sodium arsenate showed higher epithelial cell height than 10 ppm, and both concentrations caused hypertrophy. Interaction between these two anions resulted in more pronounced hypertrophy. Colloid depletion (decreased colloid area) was observed at day 10 in all treatments, but over time a compensatory response occurred in fish resulting in partial reversal of colloid depletion. A delay of a compensatory response was observed in the arsenate exposure compared with the perchlorate exposure. Due to the interaction between these two anions, the compensatory response was not observed in the mixture. Further work on other endpoints will give more clues about their effects and interaction.

323 (MUS-1117-833109) Selenium toxicosis in northern pike collected along a gradient of uranium milling effluent.
Start time: 2:10 PM
Muscatello, J^{1, 2}, Janz, D^{1, 2}, ¹ Toxicology Centre, Saskatoon, Saskatchewan, CANADA² Western College of Veterinary Medicine, Biomedical Sciences, Saskatoon, Saskatchewan, CANADA
Uranium mining in northern Saskatchewan, Canada are among the most productive uranium mines, generating 30% of the world′s uranium output. Elevated concentrations of selenium have been reported in water, sediments and fish tissues downstream of certain uranium mining and milling operations, as a consequence of uranium ore extractions, dewatering and milling. Most of the cases of selenium toxicosis in native fish populations have been reported for warm-water fish. Consequently, the applicability of guidelines and indices developed from warm-water systems to cold-water fishes, such as those in northern Saskatchewan, is controversial. The objective of the present study was to determine potential selenium toxicosis in early life stages of a native fish species, northern pike (Esox lucius) exposed to uranium milling effluent at Key Lake uranium mine. In Spring 2004, spawning northern pike were collected from a reference site and an exposure site 10 km downstream of uranium milling effluent discharge. Eggs and milt were stripped from adult female and male northern pike, fertilized in the field, and immediately transported to the University of Saskatchewan. A two-way ANOVA design was employed with embryos originating from reference and exposure sites incubated in both reference and exposure water. Deformity analysis (edema, craniofacial, skeletal, and fin deformities) showed that northern pike fry originating from the exposure site (mean egg selenium concentration 31.28 g/g dry weight) exhibited a significantly higher incidence of deformities compared to those originating from the reference site (mean egg selenium concentration 2.97 g/g dry weight). Furthermore, there were significant positive linear relationships between egg selenium concentrations and the frequencies of deformities observed in northern pike fry, as well as between egg and muscle selenium concentrations in adult female pike. Further fish collections will be conducted in Spring 2005 to increase sample sizes, sample additional sites, and confirm the results found in 2004. To date, our results suggest that maternal transfer of selenium to eggs is the causative factor for the increased frequency of deformities found in northern pike fry at this uranium milling operation.

324 (SHI-1117-586407) Effects of salinity on selenomethionine toxicity in Japanese medaka eggs.
Start time: 2:30 PM
Shi, D¹, Schlenk, D¹, ¹ Department of Environmental Sciences, University of California Riverside
Although mechanisms of organoselenium toxicity in fish are still unclear, previous studies have suggested that hypersaline conditions, occurring not only in estuarine ecosystems but also as a result of agricultural runoff carrying selenium and solutes (e.g., sulfate and calcium), protect against its acute toxicity in fish. Hypersaline conditions can induce expression and activity of the enzyme flavin-containing monooxygenase (FMO), which presumably plays a role in organoselenium toxicity in fish by oxidizing accumulated organoselenides to selenoxides which subsequently consume the antioxidant, gluthathione (GSH), in sensitive target tissues such as the developing embryo. Although salinity protected fish from acute toxicity, the effects on reproductive and developmental toxicity are not known. In this study, GSH and selenium concentrations in Japanese medaka eggs were determined following a 24-hour exposure of the eggs to 0.05mM selenomethionine (SeMet) under different hypersaline conditions (i.e., 4.2, 6.7, and 16.8 dS/m). The hatch-out ratios of the eggs following the treatment were also monitored. The results show that concentrations of GSH and the hatch-out ratio of the SeMet-pretreated medaka eggs decreased in a salinity dependent manner. Selenium concentrations in the eggs were however unaltered by salinity treatment, which implies that the metal tissue burden may not be a direct function of the observed toxicity as reflected by the decreasing GSH and hatch-out ratio. Instead, a significant correlation was observed between GSH and hatch-out ratios indicating oxidative stress as a potential mechanism of SeMet toxicity in fish embryos.

325 (BRI-1118-264598) The Effects of Elevated Total Dissolved Solids on Fertilization and Water Hardening of Salmonid Embryos.
Start time: 2:50 PM
Brix, K^{1, 2}, Curry, H², Kasper, A², Gerdes, R², Grosell, M², ¹ EcoTox, Key Biscayne, FL, USA² RSMAS, Miami, FL, USA
We investigated the effects of elevated Total Dissolved Solids (TDS, primarily as Ca²⁺ and SO₄^2-) on the fertilization success and water hardening of Arctic grayling (Thymallus arcticus) and Dolly Varden (Salvelinus malma) embryos. The high TDS water is characteristic of many hard rock mine wastewater effluents that may discharge to environments where salmonids spawn. Our studies indicate fertilization success when exposed to elevated TDS is highly variable. For Arctic grayling, EC20s for fertilization success ranged from 202 to >1381 mg l^-1 TDS, while for Dolly Varden EC20s were all >1700 mg l^-1, but some tests exhibited inverse dose response relationships. Possible reasons for the high variability include natural biological variability and artifacts of the test methods employed. No significant influx of Na⁺, K⁺, Mg²⁺ and Cl^- was observed during water hardening. Transport of Ca²⁺ into eggs was observed at all concentrations tested including the control while SO₄^2- transport only occurred at elevated ambient SO₄^2- concentrations. Elevated TDS also had a direct impact on water transport into the eggs, with an approximate 50% reduction in water uptake at 1800 mg l^-1 TDS. The ecological significance of this reduced water hardening on egg survival and hatchability is unknown.

(58160) Break.
Start time: 3:10 PM

326 (SOU-1117-839612) Chloride and hardness influence the acute and chronic toxicity of sodium sulfate to freshwater crustaceans.
Start time: 3:50 PM
Soucek, D¹, ¹ Illinois Natural History Survey, Champaign, Illinois, USA
Based on the results of previous research indicating that dilution water plays an important role in the toxic response of Hyalella azteca to sodium sulfate, the purpose of this study was to quantify the influence of both chloride and water hardness on acute and chronic toxicity endpoints for Hyalella and Ceriodaphnia dubia. When chloride concentration was held constant, increasing hardness, tested at six levels ranging from 100 to 600 mg/L (as CaCO₃) at constant Ca:Mg ratios, was related to decreased toxicity of sodium sulfate (increased LC50s) in a linear fashion for both Hyalella (R²= 0.706) and Ceriodaphnia (R²= 0.720). The response of these two organisms was consistent to the degree that similar increases in mean LC50s (in terms of SO₄^2-) were observed when testing at hardness level of 300 mg/L compared to 100 mg/L. For Ceriodaphnia, the mean LC50 increased from 2,050 (hardness = 100 mg/L) to 2,946 mg/L (at hardness = 300), an increase of 896 mg/L, while for Hyalella the mean LC50 increased from 1,854 to 2,841 mg/L (an increase of 987 mg/L) at the same hardness levels. This suggests that hardness has a similar mechanism of toxicity reduction for both organisms, perhaps tightening of epithelial cells by calcium. The response to increasing levels of chloride was not the same for the two crustaceans. In the range of 5 to 33 mg Cl/L, sulfate LC50s for Hyalella were positively correlated (R²= 0.685, p = 0.042) with chloride concentrations. Over the same range of chloride concentrations, sulfate LC50s for Ceriodaphnia were not related to chloride (R²= 0.171, p = 0.486). This suggests, that Hyalella may be more dependent on chloride than Ceriodaphnia for osmoregulation. Sulfate LC50s progressively decreased for both organisms when chloride was increased to 100, 300, and 500 mg/L.

327 (MEN-1117-788379) Predicting toxicity of Alcohol Ethoxylates homologues by an artificial neural network.
Start time: 4:10 PM
Meng, YB¹, Lin, B-L¹, Tokai, A¹, Tominaga, M¹, Nakanishi, J¹, ¹ Research Center for Chemical Risk Management, National Institute of Advanced Industrial Science and Technology, Tsukuba City, Ibaraki Prefecture, Japan
The toxicity of alcohol ethoxylate (AE), a widely used non-ionic surfactant depends on its alkyl chain, polyoxyethylene (EO) chain and other conditions. The distribution of AE homologues in environment, termed as fingerprint, is usually different from that of AE products under use or upon disposal, which entails prediction of the untested homologues' toxicity from toxicity data of the tested homologues. We constructed a feed-forward artificial neural network (ANN) consisting of one hidden layer to predict the toxicity. The input of the ANN consisted of alkyl chain length, branching extent in alkyl chain, EO number, test condition, test endpoint, exposure time and species taxon. Existing toxicity data from experiments of lab and mesocosm were collected and broken down into 545 toxicity data for training of the ANN. Using early-stopping technique, six-neuron in the hidden layer was selected for good generalization. A leave-one-out cross validation process indicated that the 95% confidential interval (CI) of predicted toxicity data could include the 'true' (untrained) toxicity data with about 90% probability and the 99% CI with more than 95% probability. Simulating the ANN for AE homologues revealed that those with shorter alkyl chain, more EO, or branched alkyl chain were less toxic, but toxicity of long alkyl chain (C>14) homologues tended not to increase upon alkyl length. The predicted no-observed-effect-concentrations, NOECs, for various species, were fit to a log-normal species sensitivity distribution to infer a hazardous concentration, HC5.

328 (WAT-1117-839036) Physiologically Based Pharmacokinetic Model of Ethinylestradiol Exposed Fathead Minnows (Pimephales promelas).
Start time: 4:10 PM
Watanabe, K¹, Kroll, K², Orlando, E³, Sepulveda, M⁴, Nichols, J⁴, Knoebl, I⁶, Lazorchak, J⁶, Collette, T^{2, 7}, Szabo, N², Denslow, N², ¹ Oregon Health & Science University, Beaverton, OR, USA² University of Florida, Gainesville, FL³ Florida Atlantic University, Boca Raton, FL, USA⁴ Purdue University, West Lafayette, IN, USA⁶ U.S. EPA, ORD, NERL, EERD, Cincinnati, OH, USA⁷ U.S. EPA, ORD, NERL, ERD, Athens, GA, USA
Endocrine disrupting chemicals are known to cause adverse reproductive effects in animals, including humans. 17-ethinylestradiol (EE₂) is a potent estrogenic endocrine disruptor that is used in pharmaceutical formulations, and is discharged to the aquatic environment. To further our understanding of the impact of EE₂ on reproduction in fathead minnows (FHM, Pimephales promelas) we are developing an integrated, systems model of the hypothalamic- pituitary-gonadal axis in this fish species. One hundred five adult male FHM were exposed for 48 hours under static aqueous conditions to 0, 10 and 50 ng/L EE₂. Following exposure, fish were anesthetized, sacrificed, and tissue (blood, brain, liver, testes, and carcass) were snap-frozen and stored at -800°C until analyzed. Concentrations of EE₂, 17-estradiol (E₂), testosterone (T), and 11-ketotestosterone (11-KT) were measured using GC/MS. As a first step, we have developed a physiologically based pharmacokinetic (PBPK) model to describe the disposition of EE₂ within FHM. The model also simulates steroid hormone concentrations (i.e., E₂, T, 11-KT) because of their importance in regulating the reproductive system. Markov Chain Monte Carlo simulations were used to estimate unknown model parameters including kinetic rate constants for steroid hormone reactions and the elimination of EE₂. In this presentation, results from the PBPK model will be presented along with the uncertainty and variability in the model outputs.

329 (TUR-1117-827583) Identification of metabolites from alkyl-anthracene compounds in rainbow trout (Oncorhynchus mykiss).
Start time: 4:30 PM
Turcotte, D¹, Abudulai, N¹, Witherly, K, Hodson, P¹, Brown, R¹, ¹ School of Environmental Studies, Queen's University, Kingston, Ontario, Canada
Fish are often exposed to polycyclic aromatic hydrocarbons and the toxicity and metabolism of those toxicants is still not completely understood. The excretion mechanism of xenobiotics is composed of a two-step metabolic process. The first step (phase I), which renders compounds more polar, involves cytochrome P450 (CYP) enzymes. The second step (phase II) involves the conjugation of phase I intermediates with glucuronic acid, glutathione, sulfate or amino acid moieties. CYP enzymes, although part of a mechanism of detoxification, often enhance the general toxicity of anthracene (ANT) derivatives by forming reactive oxygen species. The purpose of this study was to investigate the metabolism of ANT and alkyl-ANTs by rainbow trout (Oncorhynchus mykiss). Phase I metabolites were investigated in vitro by incubating single ANT and alkyl-ANTs with postmitochondrial supernatant fraction (S9 fraction). Phase II metabolites were investigated in vivo by exposing rainbow trout to the same compounds through intraperitoneal injection for a period of 24 hours, after which the gall bladder was sampled. Phase I and biliary phase II metabolites were identified using HPLC coupled with a UV-diode array absorbance detector. Alkyl-chain and ring hydroxylated compounds were found as phase I intermediates. Alkyl-chain and ring glucuronide conjugates were also found for all alkyl-ANTs investigated. Ring conjugates only were found for ANT, as expected. Further metabolized glucuronide conjugates were also detected which are suspected to be diglucuronide conjugates.

330 (BAI-1117-851009) Evaluation of impacts and benefits associated with discharge of treated effluent to the Santa Clara River Estuary.
Start time: 4:50 PM
Bailey, H¹, Stransky, B¹, Magier, S¹, Kamman, G², Pfeifer, D³, ¹ Nautilus Environmental, San Diego, California, United States² Kamman Hydrology and Engineering, Inc., San Rafael, California, United States³ City of San Buenaventura, Ventura, California, United States
The objective of this study was to evaluate impacts and benefits associated with discharge of treated wastewater to the Santa Clara River Estuary. The estuary is located in a semi-arid region of California that has been subjected to intensive habitat and water development over time, and currently supports a number of species of regulatory interest, including the federally listed tidewater goby (Eucyclogobius newberryi). The analysis included evaluation of effluent toxicity and sediment quality, as well as investigations of ecological and hydrological function. The frequency and magnitude of toxicity in samples from the estuary was minimal, and not related to the discharge. In addition, the sediment quality investigation indicated that contaminants of concern were not accumulating in estuary sediments, and that the intermittent adverse effects observed in sediment toxicity tests were largely due to grain size effects. The ecological investigation relied on extensive historical information, including maps, aerial photographs, and anecdotal accounts from a variety of sources. Most changes in flora and fauna could be attributed to changes in land use that resulted in a decrease in estuary size of approximately 90% between the late 1800s and 2003. The hydrological analysis used historical data, anecdotal evidence, and modeling to develop estimates of flows that would be present in the absence of upstream diversions. This analysis showed that the discharge accounts for a portion of the river flows that would have historically reached the estuary, but which have been diverted for other uses upstream. Overall, the analysis demonstrated that the discharge has no demonstrable negative impacts on the estuary, and supports beneficial uses by maintaining water quality and habitat. In a broader perspective, this study is an example of a robust approach that integrates several environmental disciplines to evaluate the extent of impacts (both positive and negative) to a given receiving environment.