P153 (FAR-1117-816769) Phototoxicity of oil sands derived polycyclic aromatic hydrocarbons to Japanese medaka, Oryzias latipes.
Start time: 8:00 AM
Farwell, AJ¹, Nero, V¹, Croft, M¹, Rhodes, S¹, Dixon, DG¹, ¹ University of Waterloo, Waterloo, Ontario, Canada
Alkylated polycyclic aromatic hydrocarbons are present in natural and reclaimed aquatic environments in the region of the oil sands in northern Alberta, Canada. An oil-sands derived PAH extract, rich in alkylated PAHs, has been shown to induce significant lethal and sublethal responses using the Japanese medaka embryo-larval assay. While information on exposure and effects of oil sands PAHs is available, there is little information on the impact of modifying factors. This study focuses on the effect of solar radiation on oil-sands derived PAH extract toxicity. Japanese medaka embryo larval experiments were conducted to determine the toxicity of the photomodified PAH extract following 1 and 4 day exposures to simulated solar radiation (SSR). Next, to evaluate the combined toxic effects of photomodified and photosensitized products of the oil-sands PAH extract, Japanese medaka embryos were co-exposed to the PAH extract + SSR, and compared to exposures of a photomodified and non-photomodified PAH extract. Changes in toxicity were evaluated using the following measurement endpoints: mortality, hatch length, and evidence of BSD symptoms. A one day pre-exposure of the PAH extract was sufficient to reduce the toxic effects for all measurement endpoints compared to the non-photomodified extract. A four day pre-exposure of the PAH extract was required to further reduce the effect on hatch length which was the most sensitive endpoint. PAH + SSR (16h) co-exposure enhanced lethality and the sublethal effects on embryonic development (BSD symptoms) compared to the photomodified (16h) and non-photomodified PAH exposures. Reductions in hatch length for the PAH + SSR (16h) co-exposure and the non-photomodified PAH extract exposure were similar (LOEC, 11 g/l), whereas the photomodified extract had less of an effect on hatch length (LOEC, 44 g/l). The 2-fold increase in the toxic effects to embryonic development has implications for environmental risk assessment, monitoring and reclamation strategies.

P154 (FAR-1117-816253) Modified Japanese medaka embryo larval bioassay for rapid determination of developmental abnormalities.
Start time: 8:00 AM
Farwell, AJ¹, Nero, V¹, Croft, M¹, Bal, P¹, Dixon, DG¹, ¹ University of Waterloo, Waterloo, Ontario, Canada
The Japanese medaka (Oryzias latipes) embryo larval assay has been used extensively as a laboratory model for the evaluation of toxicant impacts. However, the naturally high variability in the time of hatch is a limitation in terms of the duration of the test. To improve the economic feasibility of this bioassay, we modified an 18 day test protocol by gently agitating the test containers to synchronize hatching and induce early hatching. For non-exposed embryos, the modified protocol with agitation resulted in median hatch times of 7 - 8 days compared to 9 - 14 days for the 18 day protocol without agitation. To determine whether agitation had any effect on the sensitivity of sublethal endpoints, the 18 day and modified protocols were compared using 2 different complex mixtures containing either polycyclic aromatic hydrocarbons (PAH EXT) or naphthenic acids (NA EXT). Agitation reduced time to hatch by ≥1/3 and improved hatch success (100%). The modified protocol was generally more sensitive for a stressor (ie. PAH EXT) which caused reduced hatch length and induced symptoms of blue sac disease (BSD) including heart, yolk sac-pericardial, and cranial-skeletal deformities but had no effect on time to hatch. In contrast, the 18 day test was more sensitive for a stressor (ie. NA EXT) that had no effect on hatch length, was a weak inducer of BSD symptoms but increased time to hatch. The data suggests that differences between the protocols may be a function of the chemical properties of the test compound(s) and their ability to induce BSD symptoms. Although there was variation in the sensitivity of the measurement endpoints for the modified protocol, this protocol is recommended as a cost effective and rapid method to screen the sub-lethal impact of toxicants on the early-life stages of fish.

P155 (GRO-1117-728467) Fry production of eastern mosquitofish collected from Florida streams receiving pulp and paper mill effluents.
Start time: 8:00 AM
Noggle, J^{1, 2}, Smith, J², Bradley, W³, Gross, T^{1, 2}, ¹ USGS-FISC-CARS, Gainesville, FL, USA² University of Florida, Gainesville, FL, USA³ NCASI, Vanceboro, NC, USA
Female mosquitofish with varying degrees of male gonopodial development have been observed in pulp and paper mill effluent-receiving streams for the past two decades. Impacts of this masculinization phenomenon on reproductive success have not been directly addressed despite speculation of negative impacts. The current study evaluated fry production and masculinization in females collected from two effluent-receiving streams in Florida over two reproductive seasons (one month the first year and four months the second year). The discharging mills differed greatly in furnish, processing, and product. In addition, only one mill was subject to EPA Cluster Rule. Potential exposure was documented by GC/MS analysis of wood extractives in water samples from field sites (e.g., phytosterols, resin and fatty acids). Morphological masculinization was consistent between years for one mill (overall average of 1.45+0.03) while the response was only detected the second year of study at the other mill (1.22+0.02 year one and 1.39+0.03 year two). Fecundity appeared reduced in the first year single collection for both effluent-exposed sites (average reduction of 5 to 8 number fry per female). However, population structures implied mosquitofish at effluent-exposed sites may have started reproducing sooner than at unexposed sites. Fry production over several months the second year affirmed different reproductive patterns in females among sites and through the season. Furthermore, overall fecundities were higher in females from one exposed site relative to respective references. Variation in fecundity among all sites, regardless of exposure, ranged 2 fry per female. For both years of fry production, fecundity did not correlate with anal fin elongation. Rather than negatively impacting fecundity, pulp and paper mill effluent exposure may be stimulating modified reproductive strategies in mosquitofish influenced by changes in environmental and ecological factors as opposed to chemical exposure.

P156 (GRO-1117-749351) An evaluation of OCP effects in fathead minnows: Comparison of embryo and maternal exposure.
Start time: 8:00 AM
Huge, D^{1, 2}, Gross, T^{1, 2}, ¹ USGS-FISC-CARS, Gainesville, FL, USA² University of Florida, Gainesville, FL, USA
Previous studies from this laboratory have documented decreased reproductive efficiency and altered hormone concentrations in wild populations of American alligators (Alligator mississippiensis) and largemouth bass (Micropterus salmoides) from sites contaminated with organochlorine pesticides (OCPs). OCPs bioaccumulate significantly in fish. The purpose of this study was to determine whether adverse effects are caused by direct embryo toxicity or are due to OCP-induced effects on maternal reproductive physiology. The study investigated maternal transfer of the OCPs dichlorodiphenyldichloroethylene (DDE), dieldrin, chlordane, and toxaphene from adult fathead minnows (Pimephales promelas) to egg and larvae. Adult female fathead minnows were dosed at rates of high, middle, and low. The high dose was a complex mixture comparable to concentrations found in blue tilapia (Oreochromis aurea() from a historically contaminated lake in central Florida. The middle dose was 50% of the high dose, and the low dose was 25% of the high dose. The frequency of spawns, the number of eggs laid per spawn, the number of eggs hatched, as well as the number of larvae survived to day 14 were recorded. The second portion of the experiment investigated the effects on hatch and mortality rates of fathead minnow eggs nanoinjected with comparable concentrations of OCPs. The third portion of the experiment investigated whether fathead minnow eggs exposed aqueously to comparable doses had an adverse effect on hatch and mortality rates. Preliminary analysis indicates maternal exposure to OCPs had a decreased effect on the frequency of spawns. Preliminary analysis also indicated fathead minnow eggs nanoinjected with various concentrations of DDE had a decreased effect on hatch rate, and a decrease in survivorship. In conclusion, wild populations of fathead minnows maternally exposed to OCPs may spawn less frequently. Eggs and embryos from wild populations directly exposed to OCPs may have a decreased hatch rate and decreased survivorship.

P157 (GOO-1117-634649) Factors influencing the exchange of water between in situ exposure chambers and the surrounding environment.
Start time: 8:00 AM
Goodson, R¹, Ingersoll, C², Wang, N², ¹ Westminster College, Fulton, MO, USA² US Geological Survey, Columbia, MO, USA
The objective of this study was to evaluate the factors that may influence the exchange of water between in situ exposure chambers and the surrounding environment. In situ exposure chambers have been developed to expose aquatic invertebrates, fish, or amphibians to contaminated water or sediment in the field. Chambers were constructed out of PVC materials to contain 450 ml of water. Mesh used in the current study was 75-, 150- or 300-um stainless steel screen. A solution of 450 ml of well water containing dye (red 40 food coloring) was placed in each chamber. Individual chambers were placed in 40-L buckets containing 10 L of well water and absorbance was recorded inside or outside chambers at 0, 24, 48, 72, and 96 h. Four laboratory studies were conducted to evaluate exchange of water between chambers and the surrounding environment: (1) mesh size; (2) fouling (conditioned chambers for 48 hours in biologically-active water); (3) fouling with aeration of the surrounding water, and (4) placement of chambers in sediment. A fifth study was conducted by placing chambers in an experimental stream in sediment or in water. In the laboratory studies, water in chambers with 300-um mesh equilibrated with the surrounding environment within about 24 h; however, chambers with 75-um mesh did not reach equilibrium within 96 h. Fouling of the mesh delayed time to equilibration by 24 to 48 hours in the chambers with 75- and 150-um mesh. Gentle aeration of water in the buckets surrounding chambers with fouled mesh resulted in equilibration within 24 hours for all mesh sizes. Placement of chamber in sediment resulted in only 40 to 50% water change by 96 h across all mesh sizes. Chambers placed in water in an experimental stream reached equilibrium within 24 h (similar to the aeration treatment in the laboratory). Chambers placed in sediment in an experimental stream resulted in only limited water exchange by 96 h across all mesh sizes (similar to the sediment treatment in the laboratory). Results of these studies suggest there may be adequate exchange of water when chambers with larger mesh size are placed in water. However, chambers with smaller mesh size or chambers placed in sediment may severely limit exchange of water and may not adequately represent exposure conditions in situ.

P158 (LIA-1117-689968) Natural occurrence of Microcystin and its relative environmental factors in Dianshan Lake, China.
Start time: 8:00 AM
LIAN, M¹, YU, S², DIXON, K¹, ¹ Institute of Environmental and Human Health, Texas Tech University / TTU Health Sciences Center, Lubbock, TX, USA² Fudan University School of Public Health, Shanghai, Shanghai, P.R.China
Freshwater eutrophication is an important environmental issue worldwide. As one of its secondary outcomes, waterblooms of cyanobacteria and its hepatotoxic metabolite, microcystin, are becoming threats to the safety of drinking water. Dianshan Lake is the biggest freshwater lake in the Shanghai area of China, and the source of Huangpu River that is the source of drinking water in Shanghai City. To evaluate the safety of drinking water, we investigated the natural occurrence of microcystin in Dianshan Lake. Results of the seasonal study showed that microcystin occurred mainly in summer, and the microcystin concentration was positively related to the water temperature and total phosphorus, and negatively related to water pH value, visual degree of water body, and the ratio of nitrogen-phosphorus, but not correlated with total nitrogen because of the high concentration of nitrogen throughout the year. In the second study during summer, sampling was expanded to 11 locations, 4 depths, and once every 6 hours in a day. The result showed that microcystin concentration ranged from 0 to 4817ng/L. The distribution of microcystin was significantly different between day and night, and among locations. No difference, however, was found among different depths because depth of the lake was only 2 meters. Microcystin content was highest in the morning and afternoon. It was the lowest in the southwest of the lake because of the algal movement following the southwestward wind direction. Stepwise regression analysis showed that microcystin distribution was mainly related to total phosphorus and visual degree of water body. An investigation of seven waterworks around the lake showed that microcystin concentration of source water ranged from 132 to 1865ng/L. Microcystin was detected in one waterworks in four seasons, and in two waterworks, microcystin was detected in tap water in August.

P159 (SAM-1117-724295) The mayfly, Centroptilium triangulifer, as an indicator of aquatic toxicity.
Start time: 8:00 AM
Samel, A¹, Jackson, J², Sweeney, B², Bouchelle, L¹, Funk, D², ¹ DuPont Haskell Laboratory, Newark, Delaware, USA² Stroud Water Research Center, Avondale, Pennsylvania, USA
Aquatic toxicity testing for protecting or setting freshwater quality criteria have been based on results from predominantly fish and non-insect invertebrates (i.e., Daphnia magna, Ceriodaphnia dubia, Hyalella azteca) bioassay studies. Only a few species of sediment-dwelling aquatic insects have been routinely used in bioassay studies (i.e., Chironomus tentans, C. riparius, Hexagenia rigida, H. limbata). Mayflies are an abundant and diverse group of aquatic insects found in most natural streams and rivers worldwide, and are generally considered to be sensitive to environmental perturbation relative to other aquatic insects and non-insects. The mayfly Centroptilium triangulifer has several characteristics that make it well suited as a bioassay organism. It has a relatively short egg and larval stage and can be readily cultured under laboratory conditions. The species is widely distributed and reproduces as parthogenetic clones. Toxicity testing with chlordane was previously conducted and reported. New data presented includes results from 48-hour acute toxicity tests with copper and an insecticide. The 48-hour LC/EC50 values were compared with results from 48-hour acute toxicity studies with Daphnia magna. For both chemicals, the EC₅₀ values for C. triangulifer and D. magna were within 2x of each other. When addressing risk to aquatic fauna, data from studies with additional test organisms, like C. triangulifer can augment an existing toxicity database based on regulatory core test organisms (i.e., fish, Daphnia sp., Chironomus sp.) for new or existing chemicals The data can also be used for developing species sensitivity curves for new or existing chemicals.

P160 (LAS-1117-132326) Effects of hardness and alkalinity in culture and test waters on reproduction of Ceriodaphnia dubia.
Start time: 8:00 AM
Lasier, P¹, Winger, P¹, Hardin, I², ¹ US Geological Survey, Athens, Georgia, USA² The University of Georgia, Athens, Georgia, USA
Ceriodaphnia dubia is a freshwater organism often used for testing the chronic toxicity of municipal and industrial effluents. Standard procedures for testing whole effluent toxicity (WET) call for their culture in moderately-hard water, but many effluents and receiving waters in the southeastern United States are low in hardness concentrations. This study examined the effects of water hardness and alkalinity in culture waters and test waters on the responses of C. dubia to toxic levels of chloride, sulfate and bicarbonate. Animals were cultured for a minimum of eleven months prior to testing in one of four different laboratory-water formulations that provided waters with the following hardness and alkalinity concentrations (mg/L as CaCO3): 40/40, 40/100, 100,70 and 100/100. Two chronically toxic concentrations of each anion were evaluated with animals from each culture in each of the four test waters. Three replicate tests were conducted for each solution using the standard 7-day, 3-brood C. dubia reproduction test often mandated for permit compliance for the discharge of effluents. When C. dubia cultured in moderately-hard water were exposed to test waters with low hardness, their reproduction was significantly reduced. Animals cultured in low-hardness waters exhibited no reduction when exposed to waters with moderate hardness. The hardness of the water in which C. dubia was cultured did not significantly affect their sensitivity to the three anions. However, increased hardness in the test waters significantly ameliorated the toxicities of chloride and sulfate, but not the toxicity of bicarbonate. Differences in alkalinity among the formulated base waters had no observable effect on reproduction. The physiological stress associated with a sudden decrease in solution hardness may contribute to marginal failures of otherwise non-toxic soft-water effluents. The standard WET protocol should be broadened to allow the culture of C. dubia under site-specific criteria such as hardness that are characteristic of receiving waters.

P161 (HAN-1117-545486) Effects of caging and feeding on selected biomarkers in rainbow trout (Oncorhynchus mykiss).
Start time: 8:00 AM
Hanson, N¹, Larsson, A¹, ¹ Applied Environmental Science, Goteborg University, Goteborg, Sweden
Many human made chemicals have had adverse effects on ecosystems, something that has led to an increasing need for environmental monitoring and ecological risk assessment. Historically, monitoring and risk assessment of pollutants has been focusing on point sources and known chemicals. During the last decade, however, monitoring of environmental pollution has started to emphasize on diffuse sources and mixtures of pollutants. This has raised a need for new strategies and methodologies in pollution monitoring. Most pollutants sooner or later end up in aquatic ecosystems. The use of biomarkers in stationary fish species has previously proved useful in monitoring of water pollution. Biomarkers are useful to demonstrate exposure to various known and unknown environmental toxicants. One drawback, however, is that suitable stationary fish species not always are available. Furthermore, natural variations in fish populations could bias the results. One way to avoid these problems is to use farmed fish. In this study, the use of biomarkers in farmed Rainbow trout (Oncorhynchus mykiss), kept in cages, has been evaluated as a potential method for assessment of total environmental pollution load. The use of farmed fish has previously been used to evaluate point sources of pollution. However, when pollution levels are low, there is an increased risk that factors other than pollution level could affect the results. Here, the factors type of caging (net-cages and plastic tanks) and feeding level (8% and 2% weekly of total fish weight) has been examined for a number of biomarkers. We found significant differences between levels of feeding for plasma concentration of Na+, hepatic EROD-activity and presence of immature red blood cells. The use of caged fish may become a useful tool in pollution monitoring, especially when there are many or unknown chemicals present. However, the results of this study indicate that good experimental design is important to avoid misleading results.

P162 (CHU-1117-566958) Sensitivity of the juvenile clam, Mercenaria mercenaria , to Multiple Contaminants.
Start time: 8:00 AM
Chung, K.¹, DeLorenzo, M.¹, Hoguet, J.¹, ¹ NOAA, National Ocean Service, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC, USA
Littleneck clams (Mercenaria mercenaria) are marine filter-feeding, infaunal mollusks with great commercial importance along the East coast of the US. Previous research in our laboratory has shown that the juvenile clam, Mercenaria mercenaria, is sensitive to a variety of organic (DDT and fluoranthene) and inorganic (cadmium and copper) contaminants in acute bioassays. Aqueous bioassays were conducted to determine the sensitivity of juvenile clams (>212 m and <350 m) to six contemporary-use contaminants (resmethrin, Scourge®, permethrin, imidacloprid, simvastatin, irgarol). The goal of this research was to evaluate the relative sensitivity of this test species to these contaminants. Bioassays were conducted in Revco® Environmental Chambers at 20°C, 20-m filtered seawater (30‰), and a 12-h light:12-h dark cycle. Test chambers consisted of 16-oz glass jars. Mortality was assessed daily. Range finder assays have been conducted on all six contaminants. Juvenile clams showed low sensitivity to the three pyrethroids tested (Scourge®, resmethrin, permethrin). The highest Scourge® concentration of 100g/L elicited only 30% mortality to the clams. The highest resmethrin concentration of 100g/L elicited only 27% mortality to the clams. The highest permethrin concentration of 100g/L elicited only 27% mortality to the clams. Final 96-h LC₅₀ values will be reported for all six contaminants.

P163 (MAC-1117-577621) Effects of physical/ chemical characteristics of formulated sediments on bioassay responses by Leptocheirus plumulosus to synthetic-based drilling muds.
Start time: 8:00 AM
MacGregor, III, R.¹, Morris, Hope¹, Slimp, Karla¹, Stone , Shantel¹, ¹ Halliburton Baroid / Bioassay Lab, Houston, TX, USA
The key objective of this study was to identify controllable parameters that might reduce test variability of the 4-day sediment toxicity bioassay of synthetic-based drilling muds (SBM) required for offshore drill cuttings discharge into the Gulf of Mexico. Within this test, parameters that may affect the results include, but are not limited to, SBM-sediment blending procedures, sediment composition and test organism health, size and weight. This study was designed to assess which sediment physical/ chemical characteristics might impact the variability of the SBM-sediment toxicity test responses. The study design focused on 3 basic formulated sediments. These sediments were distinguished by particle size distribution (PSD), percent water content, wet/ dry weight ratio, wet density and total carbon. The bioassays measured the percent survival, LC50, and dose-response quality of L. plumulosus to the synthetic-based drilling muds. These bioassay responses were evaluated by multi-variant analyses for correlations or relationships with the key physical/ chemical characteristics of the sediment types. These effects or correlations between sediment characteristics and bioassay response variability could be used to identify potential improvements in the application of this test as a regulatory tool.

P164 (MOU-1117-831687) Laboratory and Field Studies to Quantify Responses of Daphnids to Combined PAH and UV Light Exposure.
Start time: 8:00 AM
Mount, D¹, Diamond, S¹, Erickson, R¹, Heinis, L¹, Highland, T¹, Hockett, J¹, Jenson, C¹, Prihoda, K¹, ¹ U.S. EPA/ORD, Duluth, MN, USA
Hazard from photo-activation of PAHs has been well documented for several aquatic organisms. Far less certain is the degree to which risk actually occurs in the field. This presentation outlines a series of laboratory and field experiments conducted to better understand the dosimetry and effects of simultaneous UV and PAH exposure in the cladoceran, Daphnia magna. In the laboratory, pyrene was used as a model PAH for establishing basic exposure-response relationships. Simultaneous exposures to pyrene and artificially-produced UV light were used to determine the relationship between aggregate UV/PAH exposure and time to mortality. Age-specific exposures indicated that 1-2d old neonates had slightly higher sensitivity than did organisms 4d and older. Chronic testing showed very little effect of UV/PAH exposure on reproduction in treatments where adult survival was not also affected. In situ exposures at a PAH-contaminated site were also conducted, with simultaneous exposure to a range of sunlight intensities. Both PAH and UV exposure varied both daily and seasonally. Exposure-dependent mortality was observed in excess of that attributable to UV exposure alone. We discuss these findings in the context of assessing risk from photo-activation at PAH-contaminated sites. This abstract does not necessarily reflect EPA policy.

P165 (KIN-1116-959849) Evaluating developmental effects of three xenobiotic chemicals on Xenopus laevis.
Start time: 8:00 AM
Kumsher King, M¹, Dickson, K¹, La Point, T¹, Venables, B¹, Waller, W¹, Irizarry, E¹, ¹ University of North Texas, Denton, Texas, USA
The Frog Embryo Teratogenesis Assay – Xenopus (FETAX) is useful for screening the developmental toxicity potential of pure chemicals and complex mixtures. In this study, FETAX was used to assess the teratogenic potential of two anti–microbial agents and one herbicide. Triclosan (2,4,4′–trichloro–2′–hydroxydiphenyl ether) is a broad-spectrum anti–microbial agent found in a variety of personal care products including soaps, detergents, cosmetics, pharmaceuticals, plastics and fabrics. In a survey by the U.S. Geological Survey, triclosan was one of the most frequently detected compounds in U.S. streams, with a median concentration of 0.14 g/L. Triclocarban (3,4,4–Trichlorocarbanilide) or TCC is used in North America as an anti–microbial and preservative in bar and liquid soaps and body washes. The TCC Consortium reported that TCC concentrations ranged from <0.074 g/L to 0.228 g/L in U.S. streams. Atrazine [6–chloro–N–ethyl–N′–(1–methylethyl)–1,3,5–triazine–2.4–diamine] is an herbicide used extensively throughout the United States to control weeds in corn fields. Environmental concentrations of atrazine vary with season and location, but are typically measured in the tenths of a part per billion range. At environmentally relevant concentrations, none of the three xenobiotics tested induced teratogenic effects using FETAX. Triclosan was tested in concentrations ranging from 0.007 g/L to 100 g/L. TCC was tested at concentrations ranging from 12.5 g/L to 50.0 g/L. For both anti–microbials, mortality and malformation rates did not differ significantly from controls at any concentration. Future studies with triclosan and TCC will be performed to estimate their LC50 and EC50 in the FETAX model. Atrazine was tested at concentrations ranging from 0.0003 mg/L to 30 mg/L. We estimated an LC50 of 12.8 mg/L and an EC50 of 4.6 mg/L. Although these values are similar to recently reported results, we intend to confirm these initial LC50 and EC50 estimates.

P166 (EIC-1117-049760) Solvent Toxicity to Topsmelt Eggs.
Start time: 8:00 AM
Serben, K¹, Eickhoff, C¹, Pinturier, L², ¹ Vizon SciTec Inc., Vancouver, British Columbia, Canada² Total E&amp;P Norge AS, Stavanger, Norway
When conducting toxicity tests with poorly water soluble chemicals, it is often necessary to use organic solvents to prepare concentrated stock solutions. The available guidance limits the final concentration of co-solvents in the test solutions to 0.01% or 0.1 mL/L. However, there is no background information or rationale for this value. In some cases, it may be conservative, in others, toxic. Recent toxicity tests conducted at Vizon used dimethyl formamide (DMF) as a solvent in early life stage tests with topsmelt, Atherinops affinis. Significant toxicity (hatching success) was observed in the solvent control, even at lower solvent concentrations (0.005%). An experiment was designed to assess the toxicity of different solvents to topsmelt embryos of varying ages. Tests were conducted with embryos of <2 days old, <4 days old, <6 days old, and <8 days old at test initiation. Four different solvents were tested at 0.005% in seawater: DMF, diethyl ether (Ether), acetone, and dimethyl sulfoxide (DMSO). The test solutions were renewed every second day, and the tests completed when all of the control embryos were hatched. Mean percent viability (embryos that are hatched and normal at test completion) was assessed as the toxicity endpoint. The results indicated that DMF caused significant mortality in young embryos compared to the other solvents tested, and that the sensitivity of the embryos decreased with age at test initiation.

P167 (CAP-1117-086351) Impacts of Suspended Sediment on Aquatic Organisms.
Start time: 8:00 PM
Capper, N¹, Klaine, S¹, ¹ Clemson Institute of Environmental Toxicology, Pendleton, SC, USA
Land use change may result in significant amounts of soil migrating into aquatic streams during storm events. This soil, even in the absence of adsorbed contaminants, may be a significant stressor to aquatic organisms. In many parts of the US, total suspended solids (TSS) concentrations surpass 100,000 mg/L during storm events. The limited data on effects of suspended sediment concentrations has mainly focused on salmonids or ecosystem level consequences such as habitat alteration. Few studies have quantified the effects of suspended solids on water column organisms. These studies are needed since little physiological data is available on this subject. The purpose of this research was to determine the effects of suspended sediment concentrations on Daphnia magna and Pimephales promelas. Results of 7 d toxicity tests with D. magna in Kaolin suspensions indicated an LC₅₀ of approximately 100 mg/L. This value was considerably less than TSS concentrations common in many areas of the US and can be sufficient to elicit feeding inhibition and reproductive effects in Cladocerans. The experimental design for this research also included the use of natural sediments collected during storm events to facilitate the extrapolation of laboratory results to predict response in the field. Results of this research underscore the need to treat eroded soil as a water column contaminant.

P168 (HIR-1116-322145) 'Point of no return' on formation of testis-ova in Japanese medaka exposed to 17-Estradiol.
Start time: 8:00 AM
Hirai, N¹, Nanba, A¹, Abe, R¹, Koshio, M¹, Kondo, T¹, Tatarazako, N¹, ¹ National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan
Several fish assays have being conducted to evaluate the dose-dependent influence of estrogen activity, but there are few study related to evaluate the period-dependent effects of estrogens. Point of no return (PNR), which is an indicator to evaluate the lethal starvation period (Blaxter and Hempel 1961) in study on early life mortality of fishes, is expected as a suitable endpoint to evaluate the time influence when fish are exposed to estrogens. We examined that PNR on formation of testis-ova was examined in Japanese medaka exposed to 17-estradiol (E2: 171 ng/L) during egg and larval period. The 10 series of eggs within six hours after fertilizations were transported to 2-liter glass tanks by 20 eggs, and each series was exposed to E2 for 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 days after fertilization, respectively. Testis-ova in genetic-male gonad were observed in larvae exposed to E2 for over 18 days after fertilization, which is 6 days post-hatch. Males only exposed during egg period do not have testis-ova in their testis. Our previous study indicates testis-ova are first found at 12 days post hatch larvae when medaka is transferred to E2 during egg and larval period. Taken together with this study, the value PNR for 171 ng/L E2 was assessed as 18 days after fertilization, and suggesting that exposure in larval period may be related to the formation of testis-ova in genetic-males.

P169 (CHO-1116-414827) Identification of hemoglobin in Chironomus riparius and Daphnia magna as a potential biomarker of freshwater monitoring.
Start time: 8:00 AM
LEE, S¹, LEE, S¹, HA, M¹, CHOI, J¹, ¹ University of Seoul, Faculty of Environmental Engineering, Seoul, Korea
In an attempt to identify invertebrate hemoglobin (Hb) as a biomarker of freshwater monitoring, Hb alterations were studied in two invertebrate species, Chironomus riparius and Daphnia magna, exposed to various environmental pollutants (nonylphenol, bisphenol-A, bis(2-ethylhexyl)phthalate, endosulfan, paraquat dichloride, chloropyriphos, fenitrothion, cadmium chloride, lead nitrate, potassium dichromate, benzo[a]pyrene and carbon tetrachloride). Chemical-induced Hb alterations were evaluated, in terms of gene expression by RT-PCR, total contents by cyanometHb procedure and oxidation status by multi-wavelength rapid-scanning spectrophotometry. The results suggest that Chironomus and Daphnia Hb could give useful information to diagnose freshwater quality. Considering the potential of Chironomus riparius and Daphnia magna as biomonitoring species, and the physiological particularities of their respiratory pigments, Hb in these species seems to be a promising biomarker.

P170 (FOR-1116-627498) Inhibition of Oocyte Maturation in Xenopus by Polybrominated Diphenyl Ethers, Perfluorooctane Sulfonate, and Perfluorooctanoic Acid.
Start time: 8:00 AM
Fort, D¹, Rogers, R¹, Thomas, J¹, Guiney, P², Weeks, J², ¹ Fort Environmental Laboratories, Stillwater, OK, USA² SC Johnson & Son, Racine, WI, USA
Under normal conditions, Xenopus oocytes undergo final maturation which is induced by progesterone via a membrane bound receptor (OMPR) or androgens via a classical intracellular receptor (AR). Final maturation is marked morphologically by germinal vesicle breakdown (GVBD). The anti-progestin and anti-androgen activities of a series of persistent halogenated substituted polybrominated diphenyl ethers (BDEs) including: pentabromodiphenyl ether (PBDE) [DE-71], octabromdiphenyl ether (OBDE), and decabromodiphenyl ether (DBDE); and the ubiquitous perfluorochemicals (PFCs), perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) were screened using an in vitro Xenopus oocyte GVBD assay. Results suggested that each of the BDEs, but not PFOS or PFOA inhibited progesterone-induced GVBD in vitro in a concentration-dependent manner. BDE potency for progesterone-induced GVBD, expressed as an IC25, ranged from 0.08 ug/L for PBDE to 8.45 ug/L for DBDE. PFOS, PFOA, and to a lesser extent PBDE, inhibited androstenedione-induced GVBD with potencies (IC25) ranging from 1.98 ug/L for PFOS to 8.14 ug/L for PBDE. Radioreceptor binding studies with isolated OMPR indicated that inhibition of progesterone-induced GVBD was proportional to BDE binding affinity for the OMPR. These results suggested that the inhibitory effects of the BDEs on GVBD may be mediated primarily through the OMPR, whereas, the inhibitory nature of PFOS and PFOA is postulated to be a result of inhibition of the AR pathway. Furthermore, the inhibitory potency of the BDEs appeared to be inversely related to the degree of bromination of each mixture. Ultimately, these results suggested that these substances were capable of interfering with endocrine systems and reproductive processes in amphibians.

P171 (EVA-1116-000795) Are we underestimating the toxicity of hydrophobic organic contaminants to marine organisms?
Start time: 8:00 AM
Evans, A¹, Nipper, M¹, ¹ Texas A&M University-Corpus Christi, Corpus Christi, TX, USA
Hydrophobic organic contaminants (HOCs), including petroleum-derived organics (e.g., polycyclic aromatic hydrocarbons (PAHs) and alkanes) and organochlorines (e.g., polychlorinated biphenyls (PCBs) and chlorinated pesticides) enter the marine environment through industrial, urban and agricultural runoff and accumulate in the sediments. Methods for the measurement of sediment toxicity include porewater tests using sea urchin fertilization and embryological development assessments, and the copepod Schizopera knabeni 96-hour survival and nauplii hatching success test. In previous studies investigating the toxicity of sediments from Boston Harbor, it was determined that significant binding of contaminants to organic matter and particles led to insufficient evidence of the bioavailability of HOCs. It is hypothesized that excessive biomass in porewater toxicity testing systems prevents a critical mass of HOCs to be achieved and bioaccumulated, thus curbing toxic effects. Therefore, standard ecotoxicological methods for examination of the effects of HOCs of varying solubility are in question. In this study the toxicity of three categories of HOCs were assessed: a PAH (phenanthrene), an alkane (decane) and a chlorinated pesticide (lindane). The results of toxicity tests with these HOCs using different amounts of organisms (biomass) in the test system are under investigation using sea urchin early life stage tests. The additive effect of these HOCs, which are expected to exert a narcotic effect, in binary and trinary combinations is also under assessment with the same methods as well as with the copepod S. knabeni. The results are under investigation and will be discussed.

P172 (HEM-1116-274640) Water and Sediment Quality Survey of Threatened and Endangered Freshwater Mussel Habitat in the Ochlockonee River Basin.
Start time: 8:00 AM
Hemming, Jon¹, Winger, Parley², Gierhart, Wendy¹, Jarvis, Robert¹, ¹ U. S. Fish and Wildlife Service, Division of Ecological Services, Panama City, FL, United States² U. S. Geological Survey, Patuxent Wildlife Research Center Athens, Athens, GA, United States
The U.S. Fish and Wildlife Service Freshwater Mussels Recovery Plan (USFWS 2003) called for the evaluation of freshwater mussel habitat in the Ochlockonee River of Florida and Georgia to identify areas with factors potentially limiting to mussel life history. Identification of these areas will allow for the implementation of measures to maximize recovery efforts and ensure adequate water quality for these imperiled species. To meet the recovery plan criteria a coordinated effort among the Service's Environmental Contaminants Program, the U.S. Geological Survey's Patuxent Wildlife Research Center, and the Service's Fisheries Resources Office was conducted. The combined effort of these programs allowed for performance of water chemical analyses, porewater and whole sediment chemical analyses, and porewater and whole sediment toxicity testing. These analyses allowed for the identification of impaired sites and determination of differences between sites that currently support mussels and those that have ceased to support populations. The results further provided an evaluation of potential risk to aquatic species which was used as a preliminary risk estimation for freshwater mussels. The preliminary risk estimation based on degraded water and sediment quality was used for identification and ranking of mussel habitat in need of restoration. Cumulatively, sites posing potential risk to freshwater mussel species achieved an estimated potential risk score of five or greater (below the dam, Little River, and ∼3 miles SSE of Cairo, GA). These sites corresponded well with areas that no longer support their historic freshwater mussel populations. Factors contributing to the high risk estimation for these sites were sediment toxicity (porewater and whole sediment), sediment lead, sediment manganese, sediment ammonia and low dissolved oxygen.

P173 (MUK-1117-750504) Acute and sub-acute toxicity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in zebrafish (Danio rerio).
Start time: 8:00 AM
Mukhi, sandeep¹, Patino, Reynaldo ², ¹ Department of Environmental Toxicology and Texas Cooperative Fish & Wildlife Research Unit,Texas Tech University, Lubbock, Texas, USA² U.S. Geological Survey Texas Cooperative Fish & Wildlife Research Unit, Lubbock, Texas, USA
Hexahydro-1,3,5-trinitro-1,3,5-triazine is a cyclonitroamine commonly known as RDX that is used in the production of military munitions. Geographically widespread contamination of soil, sediment, and ground and surface waters with RDX has been reported. Acute and sub-acute toxicities of RDX have been relatively well documented in terrestrial vertebrates, but the information available for aquatic vertebrates is limited. The objectives of this study are to characterize the acute and sub-acute toxicity of RDX in zebrafish. Mortality (LC50) and incidence of vertebral column deformities (EC50) were two of the end points measured in the acute study. The 96-h LC50 was estimated at 22.98 and 25.64 mg L-1 in two separate tests. The estimated no-observed-effective-concentration (NOEC) levels for lethality were 13.27 ± 0.05 and 15.32 ± 0.30 mg L-1; and the lowest-observed-effective-concentration (LOEC) levels were 16.52 ± 0.05 and 19.09 ± 0.23 mg L-1, in the two tests respectively. The 96-h EC50 for vertebral deformities in survivors from one of the LC50 tests was estimated at 20.84 mg L-1, with NOEC and LOEC of 9.75 ± 0.34 and 12.84 ± 0.34 mg L-1, respectively. Behavioral aberrations were also noted in the acute toxicity study, including the occurrence of whirling movement and lethargic behavior. The acute effects of RDX on survival, incidence of deformities, and behavior of larval zebrafish occurred at the high end of the RDX concentration range reported in aquatic environments. For the sub-acute toxicity test, sub-adult zebrafish were exposed to 0, 1 and 10 ppm (nominal) of RDX for a period of 12 weeks. Bioaccumulation of RDX and its metabolites were observed at 4, 8 and 12 weeks of exposure. The average bioaccumulation factor was estimated at 1.4. RDX at 10 ppm affected condition factor (growth index) of the fish after 4 and 8 weeks of exposure but not after 12 weeks, possibly indicating an adaptive response. RDX at 1 ppm did not affect condition factor at any time during the exposure. Sub-acute effects of RDX on organ histopathology are currently under investigation. This research was supported by funding from U.S. Department of Defense through the Strategic Environmental Research and Development Program.

P174 (AIR-1117-795730) Effects of temperature and oxygen concentration in sediment toxicity testing.
Start time: 8:00 AM
Airas, S¹, Leppanen, M¹, Kukkonen, J¹, ¹ University of Joensuu, Joensuu, Finland
Sediment toxicity tests are usually performed in the standardized laboratory conditions to minimize external variation. This system produces useful and comparable results about sediment toxicity for the risk assessment work world wide. Still, site specific evaluation of contaminated sediments creates new challenges because several abiotic factors, like temperature, organic content of sediments and oxygen concentration in water can have a large impact on the actual sediment toxicity at a site. In this study we wanted to focus on the effects of temperature and oxygen concentration in water for the sediment toxicity tests. The tests were performed with two typical test species: an oligochaete Lumbriculus variegatus and a midge Chironomus riparius. Growth, lipid content, feeding rate and reproduction of L. variegatus and growth, survival and emergence of C. riparius were tested in the laboratory in four treatment combinations (I: +20°C 100% O₂ saturation, II: +20°C <40% O₂ saturation, III: +10°C 100% O₂ saturation, IV: +10°C <40% O₂ saturation). Two different sediments were used, LH sediment from a relatively unpolluted lake Hoytiainen and SP sediment from a sawmill pool contaminated with several PAHs, metals and dioxins. Our results indicate that abiotic factors have significant effect on sediment toxicity test results. For example feeding rate and reproduction of L. variegatus were significantly lower in the low temperature treatments (II and IV) than in the treatments I and III. O₂ concentration did not have any observed effect on the endpoints measured with L. variegatus. C. riparius was more sensitive to low O₂ concentration and the growth and survival were significantly affected by the lowered O₂ concentration. We suggest that temperature and other abiotic factors should be taken into account in sediment toxicity testing especially at the sites where the actual conditions largely differ from the laboratory conditions to add environmental realism to sediment assessment work.

P175 (ARI-1117-790219) Effects of nonylphenol on the molting cycles of estuarine mysid shrimp Americamysis bahia.
Start time: 8:00 AM
Hirano, M¹, Ishibashi, H², Watanabe, N¹, Iwasaki, Y¹, Miyazaki, H¹, Arizono, K¹, ¹ Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto² Center for Marine Environmental Studies, Ehime University
The present study aimed to examine the effects of exposure to a xenoestrogen, nonylphenol (NP) on a marine crustacean, the mysid shrimp Americamysis bahia. Seven-days-old mysids were exposed to the nominal NP concentrations of 0.3, 1, 3, 10 and 30 microg/L for 14 days. The body length of the A. bahia was significantly decreased relative to the controls when exposed to 1, 3, 10 or 30 microg/L NP for 14 days. Moreover, the carapace length was significantly decreased in the 10 or 30 microg/L NP treatment groups, and the body weight exposed to NP of 30 microg/L for 14 days was also significantly decreased relative to the controls. Although the induction of immature gonad found in the 30 microg/L NP treatment group, no significant differences were observed in sex ratio with the appearance of secondary sex characteristics. The total number of molting exposed to NP for 14 days was significantly decreased in the treatment groups at 10 or 30 microg/L relative to the controls. However, no significant differences were observed in the 20-hydroxyecdysone levels in A. bahia at the end of NP exposure for 14 days in the control, solvent control and all NP treatment groups. Moreover, no abnormal sexual differentiation in gonads of mysid was observed in any of groups treated with NP. These results suggest that exposure to environmentally relevant concentrations of NP affected the development of A. bahia that were linked to delays in molting cycles.

P176 (VER-1117-803938) Endocrine disruption in natural fish populations in Belgium.
Start time: 8:00 AM
Versonnen, B¹, De Coen, W², Janssen, C³, ¹ Euras, Ghent, Belgium² University of Antwerp, Antwerp, Belgium³ Ghent University, Ghent, Belgium
Since the observation of higher incidences of intersex in fish living in waters receiving effluents from sewage treatment works (STWs), effects of estrogenic compounds in the environment have been widely studied. However, to date no information was available on the occurrence of endocrine disruption in natural fish populations in Belgium (Western Europe). Moreover, in the very densely populated (440 inhabitants/km²) northern part of Belgium only 57 % of the household sewage water is treated prior to release in surface waters. Compounds with estrogenic activity are thus likely to occur in the surface waters. Therefore, we sampled, over a 3-year period, 797 roach (Rutilus rutilus), rudd (Scardinius erythrophtalmus) and tench (Tinca tinca) in 12 rivers, canals and ponds in Flanders (northern Belgium). Different conditional and endocrine parameters of these fish were measured: condition factor (CF), gonadosomatic index (GSI), blood or plasma vitellogenin (VTG) content and gonad histology. Elevated VTG levels in blood plasma were detected in fish collected at 2 sampling stations, 25 % of the male roach sampled for histology showed signs of intersex: 2 - 80 primary oocytes per testis slice in most fish and completely affected gonads in one fish. Further, degraded, vacuoled oocytes were observed in severely affected fish and overall sex-ratios of roach were skewed towards females. These results are compared with calculated and measured concentrations of natural hormones (excretions by cattle, human population) and other endocrine disrupters. The findings suggest that endocrine disrupting compounds are present in surface waters in Flanders in concentrations that adversely affect roach populations. However, compared with some of our neighbouring countries the effects observed appear less severe.

P177 (HOR-1117-817005) Temporal Changes of Species Composition and Biomass of Benthic Community in Tokyo Bay, Japan.
Start time: 8:00 AM
Horiguchi, T.¹, Ochiai, S.², Yamaguchi, A.², Kume, G.¹, Kodama, K.¹, Shibata, Y.¹, Shiraishi, H.¹, Morita, M.¹, Shimizu, M.³, ¹ National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan² Nagasaki University, Nagasaki, Nagasaki, Japan³ The University of Tokyo, Bunkyo-ku, Tokyo, Japan
In the chain of the study to analyze the relationship between environmental pollution and qualitative/quantitative changes in biological community, temporal changes of species composition and biomass of benthic community were investigated in Tokyo Bay, Japan, from December 2002 to October 2004. Specimens (fishes, crustaceans and mollusks) were quarterly collected by 10-minute trawling at 20 stations in Tokyo Bay. Catch per unit effort (CPUE: the number of individuals and the weight per a trawl) was calculated for each species, and compared with the similar data of benthic community in Tokyo Bay from 1977 to 1995. Cluster analyses were applied using the weight CPUE data. Totally, 156 species (78 fish, 44 crustacean, 9 cephalopod and 25 bivalve/gastropod species) were collected between December 2002 and October 2004. The total number of individuals collected in the period was 28,159: 8,794 fish (31.2 %), 12,346 crustaceans (43.8 %), 854 cephalopods (3.0 %) and 6,165 bivalves/gastropods (22.0 %). There were 5 elasmobranches among 10 predominant species in the trawling surveys between December 2002 and October 2004. Although the total number of elasmobranch individuals collected in the previous surveys from 1977 to 1995 was 337, corresponding to only 0.3 % of the total number of fish (103,548 individuals), 671 elasmobranch individuals (7.6 % of total fish individuals) were collected from December 2002 to October 2004. The marked increase of elasmobranch in benthic community seemed to have recently occurred in Tokyo Bay. Results on cluster analyses showed 4 groups of duration: 1977-83, 1984-88, 1989-95 and 2003-2004 as phases I to IV, respectively. Both individual and weight CPUE increased from phases I to II, and reached at maximum in 1987 of phase II. Both individual and weight CPUE, however, decreased from phases II to III. Although the individual CPUE has remained low level in phase IV, the weight CPUE has markedly increased due to the increase of elasmobranch species as well as the Japanese sea bass. Previous predominant species, such as the mantis shrimp and the marbled sole, have markedly decreased in Tokyo Bay.

P178 (CAS-1117-816121) Toxicity of stormwater pond sediments for embryo and larval amphibians.
Start time: 8:00 AM
Snodgrass, J¹, Joseph, D¹, Casey, R¹, ¹ Towson University, Towson, MD, USA
Stormwater management ponds are common features of newer development projects. Because stormwater ponds may attract pond-breeding amphibians and the sediments of ponds often contain a complex mixture of pollutants, these ponds may represent ecological traps for amphibians. To assess the toxicity of stormwater pond sediments to early developmental stages of amphibians potentially utilizing stormwater ponds as breeding sites we exposed eggs and larvae to pond sediments and clean sand controls in laboratory microcosms. Developing wood frog eggs (Rana sylvatica) exposed to pond sediments experienced reduced hatching success in comparison to controls and no larvae exposed to pond sediments survived to metamorphosis. In contrast, hatching success of American toad eggs (Bufo americanus) exposed to pond sediments was high and similar to eggs exposed to clean sand. Furthermore, while metamorphs showed sublethal effects of exposure to pond sediments, metamorphic success was similar between larvae exposed to pond sediments and those exposed to clean sand. Analyses of trace metal levels and water chemistry in the microcosms suggested contamination of sediments from road salting was responsible for the lethal effects observed among developing wood frogs embryos and larvae. Overall, our results suggest contamination of stormwater ponds with road salt is a factor in reducing the wildlife habitat quality of ponds and the role of ponds as ecological traps for pond-breeding amphibians warrants further investigation.

P179 (ZIE-1117-827034) A simple and efficient water-renewal system for large volume sediment toxicity testing.
Start time: 8:00 AM
Ziegler, G¹, Fisher, D¹, ¹ University of Maryland Wye Research and Education Center, Queenstown, Maryland, USA
Sediment toxicity tests are a valuable screening tool for potentially toxic sediments and an important component of the sediment quality triad approach used to assess benthic habitats. When used as a screening tool sediment tests are often simultaneously conducted on a large number of sites at a specific time of the year. Guidelines for conducting whole-sediment toxicity tests require the exchange of overlying water in test vessels to maintain acceptable water quality. The siphoning and renewal of overlying water is a tedious task taking several minutes per beaker compounded by 5 replicate beakers per site. Previously described renewal systems were evaluated and considered too expensive and complicated or were unable to deliver the required volumes. Here we describe a water renewal system based on plexiglass modules that accurately deliver water (500 ml ± 5%) at a controlled flow rate (1.8 ml per second ± 10%). Each module contains 10 chambers, enough for 2 tests, with the option to add more modules for additional tests. Modules are simple and inexpensive (approx. $70.00 / module) while also being flexible with an adjustable flow rate delivered through vinyl tubing. These units have been used in 28-day chronic sediment tests with Leptocheirus plumulosus .

P180 (LOT-1117-824376) Evaluation of Gonadal Histopathology in Spawning vs. Non-Spawning Medaka.
Start time: 8:00 AM
Lothenbach, D.¹, Ankley, G.¹, Johnson, R.¹, ¹ USEPA Mid-Continent Ecology Division, Duluth, MN, USA
Recently, the U.S. Environmental Protection Agency has been involved in an international cooperative effort with the Organization of Economic Cooperation and Development (OECD) to develop fish screening assays capable of detecting endocrine-disrupting chemicals. One proposed OECD assay involves a non-spawning protocol with adults for the detection of androgenic or estrogenic chemicals. Responses considered to be indicative of estrogenic and androgenic effects include changes in vitellogenin, secondary sex characteristics, gonadosomatic index (GSI) and/or gonadal histopathology. To initiate the test, actively-spawning fish are placed in each aquarium but are prevented from further spawning by isolating males from females. After two weeks of acclimation without chemical, fish are exposed for 21-days, euthanized and sampled. In several non-spawning tests, gonad histopathology was ambiguous because of substantial gonadal pathology in the controls. It was hypothesized that much of the pathology observed was caused by retention of mature oocytes following the cessation of spawning. This study assessed the effect of spawning termination by gender segregation on gonad histopathology. Six aquaria, each containing 6 male and 6 female medaka (Oryzias latipes) were setup as specified by the OECD non-spawning assay. Fish in three aquaria were maintained in the non-spawning state and fish in the remaining three aquaria were not segregated and allowed to continue spawning. After 21 days, the fish were sacrificed and their gonads examined for histopathology. The ovaries of non-spawning females contained a large number of ovulated oocytes and only a few stage four oocytes. Many of the ovulated oocytes were at the initial stages of atresia. Ovaries from spawning females were primarily at stage 3 or 4, and contained oocytes at all stages of development. These results suggest that the experimental design used for the non-spawning protocol can cause artifactual alterations suggestive of endocrine disruption in ovarian tissue.

P181 (HEA-1117-821395) Stress Responses of Juvenile Zebrafish Exposed to -Naphthoflavone.
Start time: 8:00 AM
Heather, B¹, Lux, S¹, Weber, L¹, Janz, D^{1, 2}, ¹ Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada² Veterinary Biomedical Science, University of Saskatchewan, Saskatoon, SK, Canada
Many ubiquitous environmental contaminants such as halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons are aryl hydrocarbon receptor (AhR) agonists. The hypothesis of this study is that low-level exposure to AhR agonists during development will cause stress at the cellular and whole animal levels, resulting in alterations in growth, bioenergetics and gonadal development. In zebrafish (Danio rerio), exposure to toxicants such as AhR agonists during the period of sexual differentiation (20-40 days post-hatch (dph)) may interfere with normal gonadal development and gametogenesis. To test this hypothesis, juvenile zebrafish were exposed aqueously to -naphthoflavone (BNF) (0.001 mg/L, 0.01 mg/L, or 0.1 mg/L), an AhR agonist, or vehicle control (DMSO; 0.1% v/v) from 10 to 40 dph. The specific objectives of this research were to determine the potential effects of BNF exposure on bioenergetics, HSP70 expression and histopathology in juvenile zebrafish collected at 20, 30 and 40 dph. At 40 dph, mean fish length was significantly decreased in the high dose group (0.1 mg BNF/L) compared to the control group. Weight-at-length was significantly increased in the high dose group at 40 dph compared to the control group. Further work will focus on relating the observed changes in growth with bioenergetics (whole body RNA/DNA ratio, protein and triglycerides), whole body HSP70 expression, and gonadal histopathology and cell death.

P182 (KAV-1117-833387) Reproductive impairment observed in fish exposed to oil sands tailings.
Start time: 8:00 AM
Kavanagh, R¹, Van Der Kraak, G¹, ¹ Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada
Approximately 20 % of the oil that Canada produces comes from Alberta's oil sands. As a result of the Clarke hot water extraction method, 4 m³ of fluid tailings are produced for each m³ of oil sand processed. Over 3 x 10⁸ m³ of fluid tailings are currently being stored in settling ponds. Developers plan to eventually transfer these tailings to mined-out pits and cap them with a layer of clean water and an aquatic ecosystem is then expected to be established in these end pit lakes. Previous work suggests that the oil sand constituents that are present in these lakes may alter the reproductive physiology of fish. In order to determine if fluid tailings would impair reproduction, a 21 day fathead minnow reproduction assay was conducted at Syncrude Canada Ltd. Fathead minnows were held in Gregoire Lake water (reference site) for 14 days to gather baseline data on fecundity, fertility, and hatching success. Fathead minnows were then held in water from different tailings ponds and in Gregoire Lake water for 21 days and fecundity, fertility, and hatching success were monitored. A total cessation of spawning was observed in fathead minnows that had been held in tailings pond water. Female fathead minnows also had significantly reduced GSIs and male fish had tubercles that were reduced both in size and number. Naphthenic acids, which are natural constituents of oil sands, are one of the contaminants suspected of being responsible for the reproductive impairment observed in fathead minnows. In vitro studies provide some support for this theory as fish testes stimulated with hCG and incubated with naphthenic acid extract (10 mg/l)showed a significant reduction in testosterone levels compared to controls. Collectively these studies show that oil sands tailings have the potential to affect the reproductive physiology of fish.

P183 (PET-1117-829478) In vitro steroidogenesis in Golden Shiner (Notemigonus crysoleucas) collected from a Lake Ontario tributary surrounded by pesticide usage.
Start time: 8:00 AM
Peters, L^{1, 2}, Park, B², Struger, J³, Palace, V², ¹ University of New Brunswick, Saint John, NB, Canada² Fisheries and Oceans Canada, Winnipeg, MB, Canada³ Environment Canada, Burlington, ON, Canada
Reductions in circulating levels of sex steroids can indicate exposure to compounds impacting the reproductive system of fish. Effects correlated with such reductions include delayed maturation, reduced gonad size and fecundity, and the inhibited expression of secondary sex characteristics. Assessing plasma sex steroid levels in small-bodied species is problematic due to insufficient plasma volumes and potential confounding effects of acute capture stress. Therefore, the use of an in vitro incubation technique to assess the biosynthetic capacity of fish gonadal tissue to produce sex steroids has become a reliable alternative. This in vitro technique was used to examine potential reproductive effects of pesticide exposure in golden shiner (Notemigonus crysoleucas), a small bodied forage fish species, collected from four locations on Twenty Mile Creek, ON. This creek is a tributary of Lake Ontario, and is surrounded by moderate to heavy pesticide usage. Previous laboratory studies have shown significantly lower gonado-somatic indices in female fathead minnows exposed to Twenty Mile Creek water, and an overall delay in maturation. Clopyralid, dicamba, MCPA, 2,4-D, simazine, atrazine, metribuzin and metalochlor residues have routinely been detected in surface waters in Twenty Mile Creek. Results of this field study indicate that the in vitro technique is suitable for evaluating steroidogenic capacity in golden shiners. Correlations with other reproductive parameters that are used to investigate potential impacts of pesticides at different sites will be made.

P184 (KAJ-1117-828487) High ambient ammonia promotes growth in a ureogenic goby, Mugilogobius abei.
Start time: 8:00 AM
Kajimura, Makiko^{1, 3}, Iwata, Katsuya ¹, Sakamoto, Tatsuya ², Iwata, Ichiko ¹, Nishiguchi, Eri¹, Smith, Richard ³, Wood, Chris ³, ¹ Biological Laboratory, Faculty of Education, Wakayama University, Wakayama, Wakayama, Japan³ Department of Biology McMaster University, Hamilton, Ontario, Canada² Ushimado Marine Laboratory, Okayama University, Setouchi, Okayama, Japan
While ammonia is generally viewed as a toxicant which inhibits growth during sublethal exposure, recent reports in salmonids suggest that chronic, very low level exposure may actually promote growth. Mugilogobius abei lives naturally in elevated ammonia environments and has the ability to produce large amounts of urea under these conditions. Despite this metabolically costly approach, M. abei exposed to high levels of ammonia (2mM NH₄Cl, pH=7.5-8.0) in a pilot experiment exhibited no adverse effects on growth. To further investigate this observation the growth of M. abei was measured at room temperature for 8 weeks at a constant ration level under solitary and grouped conditions, in 20% SW with or without (control) 2mM NH₄Cl (pH = 7.5-8.0). Furthermore, pituitary mRNA levels of growth hormone, oxygen consumption, incorporation of external ¹⁵N-ammonia into amino acid and protein fractions as well as behavioral activities were also examined. The specific growth rates of ammonia-exposed fish under grouped condition over the 8 weeks were significantly higher than those of control, while those rates under solitary condition were not significantly different between the treatments. The pituitary of ammonia-exposed fish had higher growth hormone mRNA than in control fish. The use of ¹⁵N isotope revealed that M. abei can actively use external ammonia as a supplementary nitrogen source. Oxygen consumption of ammonia-exposed fish was significantly lower than that of control fish. Locomotor activity and aggressive behavior under grouped condition were significantly reduced in ammonia-exposed fish as compared those of control. These combined alterations in the ammonia-exposed fish may result in the higher growth rates. We are currently measuring the protein synthesis rates of control and ammonia-exposed fish.

P185 (OLI-1117-865167) Polycyclic Aromatic Hydrocarbons in Sediments and Histopathological Findings in Fish from Cartagena Bay.
Start time: 8:00 AM
Olivero-Verbel, J¹, Johnson-Restrepo, B^{1, 2}, Guette-Fernandez, J, Baldiris-Avila, R¹, O'Byrne-Hoyos, I¹, Kannan, K², ¹ Environmental and Computational Chemistry Group, University of Cartagena, Cartagena, Colombia² Wadsworth Center, New York State Department of Health and Department of Environ. Health Sci, State University of New York, Albany, NY, USA
Tropical ecosystems are of great importance due to their key role on high productivity and as habitat for many species. In the Atlantic coast of Colombia, several ecosystems are being polluted by anthropogenic sources and their overall health quality is rapidly decreasing. In this study, levels of polycyclic aromatic hydrocarbons (PAHs) were measured in sediments, by gas chromatography-mass spectrometry, as well as pathological alterations in the liver of Lisa (Mugil incilis) collected in Cartagena Bay, an industrialized estuary in northern Colombia, were compared to those found in Caimanera marsh and Totumo marsh, which are relatively low-polluted sites located in the south and north of Cartagena Bay, respectively. Sediments of Cartagena Bay had total PAH concentrations of 3206, 1738 and 1334 ng /g dry wt, in 40, 30 and 20 mesh size fractions, respectively, whereas the same compounds in Caimanera marsh and Totumo marsh were lower than 283 and 146 ng/g, dry wt, respectively. Compound-specific ratio analysis showed that PAHs from Cartagena Bay are different in sources from those collected in the other sites, being the principal sources originated from petrochemical activities and pyrolitic processes. The most important histopathological finding in liver from Lisa collected at Cartagena Bay was the high prevalence of parasite cysts. In addition, hepatomegaly, granuloma, steatosis and focal necrosis were also found although almost always in great proportion than those observed in fish from the low-polluted sites. Based on these data, Cartagena Bay can be considered to be highly polluted in terms of PAHs, which together with other environmental factors might be decreasing the health status of fish living in this ecosystem, increasing the risk for health-related problems in those people who feed on these resources. Sponsored by Colciencias, Bogota, Colombia, Grant: 1107-1312576, and the University of Cartagena, Cartagena, Colombia.

P186 (ING-1117-740089) Development of an ASTM standard guide for conducting laboratory toxicity tests with freshwater mussels.
Start time: 8:00 AM
Ingersoll, C¹, Augspurger, T², Barnhart, C³, Cope, G⁴, Dwyer, F⁵, Bishop, C⁶, Neves, R⁷, Newton, T⁸, Roberts, A⁵, Wang, N¹, ¹ US Geological Survey, Columbia, MO, USA² US Fish and Wildlife Service, Raleigh, NC, USA³ Missouri State University, Springfield, MO, USA⁴ North Carolina State University, Raleigh, NC, USA⁵ US Fish and Wildlife Service, Columbia, MO, USA⁶ EA Engineering Science and Technology, Sparks, MD, USA⁷ US Geological Survey, Blacksburg, VA, USA⁸ US Geological Survey, LaCrosse, WI
A draft guide for conducting laboratory toxicity tests with glochidia and juvenile mussels in water-only exposures is being balloted through ASTM Committee E47 on Biological Effects and Environmental Fate. The draft guide describes procedures for collecting adult mussels to obtain glochidia and to propagate juvenile mussels for testing. Toxicity tests with glochidia are typically started within 2 h after glochidia are isolated from the gill of female mussels. The test endpoint for glochidia is survival (viability) as determined by shell closure after the addition of a solution of NaCl. For most species, the duration of a toxicity test with glochidia is up to 24 h with survival measured at 6 and 24 h; however, longer exposures may be appropriate for glochidia of some species. Acceptable control survival is >90% at the end of toxicity tests conducted with glochidia. Toxicity tests with juvenile mussels are typically started with organisms <5 d after release from the host fish; however, some toxicity tests are started with 2- to 4-month-old juvenile mussels. Acute toxicity tests with juvenile mussels are typically conducted for 96 h with survival measured at 48 and 96 h. Chronic toxicity tests started with 2- to 4-month-old juvenile mussels have been conducted for 21 to 28 d with measures of survival (based on foot movement) and growth (shell length). For tests conducted with juvenile mussels, acceptable control survival is >90% at the end of tests conducted for 96 h and is >80% at the end of tests conducted for 10 to 28 d. Early life stages of mussels of most species tested are more sensitive to some metals and ammonia in water exposures compared to many commonly tested species of other invertebrates, fish, or amphibians that are commonly used to establish USEPA Water Quality Criteria (WQC). Importantly, results of these previous studies indicate that WQC for several individual chemicals established for the protection of aquatic organisms may not be adequately protective of sensitive stages of freshwater mussels.

P187 (IVE-1117-742440) Influence of water hardness and chloride on the acute toxicity of sulfate to fatmucket (Lampsilis siliquoidea) and fingernail clams (Sphaerium simile).
Start time: 8:00 AM
Ivey, C¹, Ingersoll, C¹, Soucek, D², Wang, N¹, ¹ US Geological Survey, Columbia, MO, USA² Illinois Natural History Survey, Champaign, IL, USA
Sulfate can reach concentrations in wastewater discharges that can severely impair sensitive macroinvertebrates. Previous laboratory toxicity tests with the amphipod Hyalella azteca and with the cladoceran Ceriodaphnia dubia indicate sulfate toxicity can be reduced with increased level of water hardness or increased concentration of chloride. The objective of this study was to assess the effects of water hardness and chloride on the acute toxicity of sulfate in 96-h water-only exposures to laboratory-cultured juvenile freshwater mussel, fatmucket, and field-collected fingernail clams. Sulfate toxicity tests with fatmucket juveniles were conducted at three hardness levels (100, 300, and 500 mg/L as CaCO3 with Ca:Mg ratio of 2.33) and at two chloride concentrations (5 and 33 mg/L) at one hardness (100 mg/L with Ca:Mg ratio of 1.46). Sulfate toxicity tests with fingernail clams were conducted at two hardness levels (100 or 300 mg/L as CaCO3; varying ratios of calcium to magnesium within a hardness level) and at two chloride concentrations (5 and 33 mg/L). Fatmucket juveniles tended to be less acutely sensitive to sulfate exposure compared to amphipods, cladocerans or fingernail clams. Sulfate LC50s for fatmucket ranged from 2100 to 4500 mg/L and neither water hardness nor chloride had a substantial influence on sulfate toxicity. However, sulfate was more toxic to juvenile fatmucket in water with a lower Ca:Mg ratio at the same water hardness. Fingernail clams tended to exhibit a similar sensitivity to sulfate exposure compared to amphipods and cladocerans. Sulfate LC50s for fingernail clams ranged from 2200 to 3900 mg/L and the toxicity of sulfate tended to be lower at increased water hardness levels or at decreased chloride concentrations. However, in contrast to the results of the exposures with fatmucket juveniles, sulfate toxicity to fingernail clams was higher at the highest Ca:Mg ratio tested at the same water hardness. Additional studies are needed to determine how other species of freshwater mussels respond to sulfate in acute and chronic exposures.

P188 (BRE-1118-085846) The Development of Scoring Methodologies for the Evaluation of Ecotoxicity Studies Submitted Under the New Substances Notification Regulations in Canada.
Start time: 8:00 AM
Breton, R.¹, Thompson, R.¹, Atkinson, A.², Lortie, M.², Lewis, M.², Teed, S.¹, Gilron, G.³, Taylor, L⁴, Scroggins, R⁴, ¹ Cantox Environmental Inc., Ottawa, Ontario, Canada² New Substances Branch, Environment Canada, Gatineau, Quebec, Canada³ Golder Associates Ltd., Mississauga, Ontario, Canada⁴ Environmental Technology Centre
New substances nominated for import or manufacture in Canada must be notified to Environment Canada and Health Canada under the New Substances Notification Regulations (NSNRs), implemented July 1, 1994. The New Substances Program then conducts ecological and human health risk assessments based on information supplied by the notifier, and other complementary information available to the two Departments. Over the past 10 years, more than 650 ecotoxicity studies have been submitted to the New Substances Branch of Environment Canada under the NSNRs. Most of these experimental studies are not publicly available, but are useful in the ecological risk assessment of new and existing substances and for quantitative structure-activity relationships (QSARs) development. This poster presentation will describe a computer-based scoring system and our approach in the development of scoring methods used to assess the quality and reliability of ecotoxicity studies with fish, Daphnia and algae generated following either OECD test guidelines or Environment Canada test methods. Results of ranking exercises conducted using these methodologies will be described and discussed, together with potential use of these results in a regulatory context.

P189 (GRE-1117-748577) Effect of the estrogen mimic genistein on sex differentiation and EROD activity in channel catfish.
Start time: 8:00 AM
Green, C¹, Kelly, A¹, ¹ Fisheries and Illinois Aquaculture Center, Southern Illinois University, Carbondale, IL, USA
Several chemicals present in aquatic environments are known to serve as endocrine disruptors by mimicking or antagonizing the actions of steroid hormones. In fish, one consequence of endocrine disruption maybe an alteration in phenotypic sex differentiation. Soybean meal, proposed as an alternative protein source in aquaculture feeds, contain significant levels of the phytoestrogen genistein, which mimics estrogen activity. Channel catfish (Ictalurus punctatus) were fed four dietary concentrations of the phytoestrogen genistein (0, 2, 4, and 8 mg/g) to determine its effect on gonadal sex differentiation. Additionally, the relation between ethoxyresorufin-O-deethylase (EROD) activity and chronic exposure to dietary phytoestrogens during sexual differentiation was examined. The four treatment diets were fed to sexually undifferentiated channel catfish between 30 and 150 days of age. Phenotypic sex was determined by histological examination of the gonads. Phenotypic sex was significantly dependant on dietary phytoestrogen concentration (p = 0.014). Additionally, logistic regression showed a significant relation between genistein concentration in the diet and gonadal sex (p = 0.038). Intersex individuals and greater number of male phenotypes were present in the 4 and 8 mg/g treatment groups. No significant differences in EROD activity among treatments and control were observed during or after dietary exposure. These findings establish that relevant dietary concentrations of genistein could alter sex ratios in cultured channel catfish populations, and demonstrate the uncertainty of EROD activity as a biomarker of chronic exposure to phytoestrogens.

P190 (AND-1117-853412) The use of terminal restriction fragment length polymorphism (T-RFLP) in fecal bacterial source tracking.
Start time: 8:00 AM
Anderson, J¹, Farmer, J², Johnson, T², Bailey, F¹, ¹ Middle Tennessee State University, Murfreesboro, TN, USA² Tennessee State University, Nashville, TN, USA
Fecal pollution of environmental waters is a serious health problem that cannot be remedied until sources are identified. This study examines the hypothesis that terminal restriction fragment length polymorphism (T-RFLP) can be used in fecal source tracking. T-RFLP is a PCR-based method used in determining the genetic diversity within communities of organisms. Generally, each group within the community contributes at least one unique terminal DNA fragment. The T-RFLP procedure has been successful in profiling certain bacterial populations within fecal communities. However, in terms of fecal source tracking, most molecular methods employ the use of fecal bacterial libraries. We are exploring the use of T-RFLP as a non-library dependent method for aquatic fecal source tracking. A non-library dependent method will provide a quicker and less expensive way of determining the source of fecal contamination and may be less region-specific. Fecal bacterial DNA has been extracted from human, bovine, and sewage samples. Labeled and unlabeled 8F primers, labeled 516F, and unlabeled 1541R primers were used for PCR amplification. The samples were cut with the restriction enzymes Hae III and Hha I. After restriction cutting, differences were found among the human, bovine and sewage groups. T-RFLP analysis of the samples is now in progress. We expect that the terminal fragments of the three samples will be unique enough to allow differentiatiation between the source groups, thus making T-RFLP a tool for use in fecal source tracking.

P191 (RUD-1117-867855) Stormwater Toxicity Evaluation of Major Rivers Entering the Southern California Bight.
Start time: 8:00 AM
Rudolph, J¹, Stransky, B¹, Bailey, H¹, Bay, S², Greenstein, D², ¹ Nautilus Environmental, San Diego, CA, USA² Southern California Coastal Water Research Project, Westminster, CA, USA
In an effort to assess the overall ecological condition of the Southern California Bight, this project was designed to address the magnitude and persistence of toxicity in nine river discharges into Southern California coastal waters using gametes of the purple urchin Strongylocentrotus purpuratus. Magnitude of runoff toxicity was determined by collecting and testing samples from each river near its ocean discharge point. Samples were collected during three storm events (≥0.25 in.), which occurred on February 23, 2004 (eight rivers), February 12, 2005 (two rivers), and March 24, 2005 (seven rivers). Persistence of toxicity following a storm event was evaluated by testing samples collected one, three, and five days following each storm. Sample holding time effects were evaluated by re-testing selected samples on multiple occasions after collection. Test results and data analyses (e.g., among rivers and between years) are presented. Toxicity was observed in several rivers, but the degree of toxicity, when present, often varied substantially between samples collected immediately following each storm, between storm events, and during sample holding.

P192 (WAT-1117-840572) Computational Model of the Hypothalamic-Pituitary-Gonadal Axis in Female Fathead Minnows: Baseline state of unexposed fish.
Start time: 8:00 AM
Watanabe, K¹, Jensen, K², Villenueve, D², Ankley, G², Orlando, E³, Sepulveda, M⁴, Bencic, D⁵, Whitehead, T⁶, Miller, D⁷, Denslow, N⁸, ¹ Oregon Health & Science University, Beaverton, OR, USA² U.S. EPA, ORD, NHEERL, MED, Duluth, MN, USA³ Florida Atlantic University, Boca Raton, FL, USA⁴ Purdue University, West Lafayette, IN, USA⁵ U.S. EPA, ORD, NERL, EERD, Cincinnati, OH, USA⁶ U.S. EPA, ORD, NERL, ERD, Athens, GA, USA⁷ U.S. EPA, ORD, NHEERL, MED, Grosse Ile, MI, USA⁸ University of Florida, Gainesville, FL, USA
The fathead minnow (FHM, Pimephales promelas) has a widespread distribution throughout the United States, and has been identified by the U.S. Environmental Protection Agency as an excellent model for evaluating the effects of several endocrine disrupting chemicals. The normal reproductive biology of FHM has been investigated by Jensen et al. (Comp Biochem Physiol C, 128(1): 127-141.); however, there is very little known about the neuroendocrine regulation of reproduction in this species. To better understand the reproductive system of this species, we developed a physiologically based model of the hypothalamic-pituitary-gonadal axis in female FHM. The model structure included compartments representing the gill, brain, gonad, liver and other tissues. Within these compartments, more detailed biochemical processes were included such as steroidogenesis in the gonad, the formation of vitellogenin in the liver, and regulatory signaling in the control of reproduction in the brain. Measurements of the number of eggs spawned, and plasma concentrations of 17- estradiol, testosterone, and vitellogenin from 113 unexposed, control fish were used to calibrate the model. These fish were used in experiments conducted in the laboratories of the Mid-Continent Ecology Division (MED), U.S. Environmental Protection Agency. A hierarchical statistical model and Markov Chain Monte Carlo simulations were used to estimate the unknown model parameters.

P193 (CHO-1117-840033) Thyroid disruption in Xenopus by microsomal enzyme inducers: a multi-endpoint study with phenobarbital and PCN.
Start time: 8:00 AM
Chowdhury, M.¹, Holcombe, G.¹, Korte, J.¹, Kosian, P.¹, Linnum, A.¹, Bugge, M.¹, Tietge, J.¹, Degitz, S.¹, ¹ US Environmental Protection Agency, Duluth, MN, U.S.A.
Environmental chemicals can disrupt thyroid hormone (TH) homeostasis in animals by interacting with hypothalamus-pituitary-thyroid axis at various biochemical and molecular levels. The US Environmental Protection Agency (EPA) is developing an amphibian-based protocol for screening thyroid active chemicals. In mammals, induction of the microsomal enzyme uridine diphosphate glucuronosyltransferase (UDPGT) by xenobiotics results in increased thyroid hormone glucuronidation and elimination. However, it is unclear if the amphibian model shows a similar response. The objective of the present study was to test the model T₄ UDPGT-inducers, phenobarbital and pregnenolone-16- carbonitrile (PCN), to determine if the African clawed frog Xenopus laevis is sensitive to this mode of thyroid axis disruption. Stage-51 Xenopus larvae were exposed to phenobarbital (0, 125, 250, 500, 1000, and 1500 mg/L) or PCN (0, 37.5, 75, 150, 300, and 600 g/L) via the water. At day 21, we investigated developmental stages, thyroid gland histology, and mRNA steady state levels of proteins involved in thyroid hormone synthesis. Phenobarbital treatment resulted in concentration dependent delay in development and caused enlarged irregular follicles and follicular cell hypertrophy and hyperplasia in the thyroid gland. Treatment also resulted in increased expression of NIS (sodium iodide symporter), TPO (thyroperoxidase), TG (thyroglobulin), D2 (type II deiodinase), D3 (type III deiodinase) in the thyroid gland, although no changes in the pituitary TSH (thyroid stimulating hormone) were observed. Preliminary analysis of PCN data shows mild effects on larval development and thyroid morphology. Thus, the thyroid axis of X. laevis is influenced by phenobarbital as observed in mammals, but it is relatively less sensitive to PCN. We are currently developing biochemical and molecular tools to determine thyroid hormone glucuronidation in adult and larval Xenopus. (M. J. Chowdhury is a US NRC Research Associate. This abstract does not necessarily reflect US EPA policy).

P194 (ELP-1117-843801) Evaluation of inter- and intra-laboratory variability of toxicity test methods using Pacific herring, Clupea pallasi.
Start time: 8:00 AM
Elphick, J¹, Dinnel, P², Bergmann, K¹, Bailey, H¹, Paisano, L², Marshall, R³, ¹ Nautilus Environmental, Tacoma, WA, USA² Shannon Point Marine Center, Anacortes, WA, USA³ Washington State Department of Ecology, Olympia, WA, USA
Efforts to evaluate and protect Pacific herring stocks in Washington State have resulted in a demand for aquatic toxicity tests using this species for application in NPDES permits to ensure that discharges from industries and other point sources are not having adverse effects on this fish species. Consequently, test methods using early life-stages of Pacific herring have been developed, including a 96-hr survival test, an 18-day embryonic development test, and a 10-day larval growth and survival test. This presentation summarizes the results of tests conducted in a commercial laboratory for each of the three test methods using three reference toxicants, and compares them with results from tests conducted previously in the laboratory where the methods were developed. Thus, these data provide an indication in inter- and intra-laboratory variability associated with the test methods. In addition, the sensitivity of the herring tests to the three reference toxicants, as well as two other toxicant mixtures, are compared with results from a suite of other standardized toxicity tests that have been applied in NPDES permits.

P195 (OGL-1117-844371) An in situ evaluation of the effects of dredge-related activity on Pacific herring (Clupea pallasi) embryo development.
Start time: 8:00 AM
Jahn, A¹, Ogle, R², Salinas, E², Pincetich, C², Bradley, J², ¹ Port of Oakland, Oakland, CA, USA² Pacific EcoRisk, Martinez, CA, USA
Pacific herring (Clupea pallasi) support an important fishery in San Francisco Bay (SFBay). Because of their economic and recreational value, there are significant management efforts in place to maintain the health and abundance of the SFBay herring population. Due to concerns about the potential effects of dredging activities on herring reproduction, California Department of Fish and Game has placed seasonal restrictions on these activities. However, there have been no previous scientific investigations aimed at evaluating the effect of dredging-related activity on any aspect of Pacific herring reproduction; thus it is uncertain whether or not these restrictions are indeed necessary to maintain the health and well-being of the herring population. The present study was intended to begin to provide the necessary data with which to assess whether or not dredging activities affect Pacific herring success in SFBay. The performance of a routine maintenance dredging project during March, 2005, presented a unique opportunity to assess the effects of dredging-related stressors on Pacific herring embryo development under real-world conditions. Herring embryos (<24-hr old) were outplanted on horizontally- and vertically-oriented glass slides in in situ exposure chambers along a distance gradient from an active dredging project. After approximately 4 days exposure, the embryos were recovered from the field and allowed to continue development to hatching in the laboratory. Normal development and hatching success were evaluated. Qualitative examination of the slides confirmed some degree of siltation on the outplanted embryos, being markedly more pronounced for the horizontally-oriented slides. There were no significant differences in normal development between the Control treatments and outplanted embryos for either vertical or horizontal orientation. Hatching success was not affected on vertically-oriented slides; however, decreased hatching success was observed for some of the outplanted embryos on the horizontally-oriented slides.

P196 (HIL-1117-802329) Behavioural and biochemical effects of medetomidine on the three-spined stickleback (Gasterosteus aculeatus).
Start time: 8:00 AM
Hilvarsson, A¹, Lennquist, A², ¹ Dep. of Marine Ecology, Göteborg University, Fiskebäckskil, Sweden² Dep. of Zoology, Göteborg University, Göteborg, Sweden
Medetomidine is suggested as a new antifouling agent, and its effects on marine organisms are therefore being investigated. Effects on several species of fish have previously been found, including decreased oxygen consumption, alterations in activity of the enzyme CYP1A and decolouration due to melanophore aggregation. The aim of this study was to examine a broad range of effects and a wide concentration span to find the earliest and most relevant effects of medetomidine on the three-spined stickleback (Gasterosteus aculeatus). This is a well-studied species concerning both behavioural patterns and biochemical biomarkers, which makes it suitable as a test species. It is also wide spread in temperate areas and inhabits both fresh and marine shallow waters and harbours and may therefore be exposed to antifouling compounds. Marine living three-spined stickleback was collected on the Swedish west cost during reproductive season. Male fish were allowed to build a nest and set a territory in experimental aquaria, equipped with eelgrass and gravel, containing different concentrations of dexmedetomidine (0.1-500 nM). Their activity, appetite, aggression towards other males and courtship towards gravid females were studied. The female was removed after spawning and the male′s egg-guarding behaviour was studied for a period of time. Males were later sacrificed for determination of CYP1A (EROD) activity and DNA/RNA-ratio in a whole-body homogenate. These biomarkers were compared with the behavioural patterns in an attempt to find connections between them and to characterise the sub-lethal effects of medetomidine in this species. This investigation is one part of a broader hazard assessment within the evaluation programme of medetomidine.

P197 (KWO-1117-228313) Relationship between pre-existing body residue of polychlorinated biphenyls and toxicity to subsequent exposure to PCBs in Hyalella azteca.
Start time: 8:00 AM
Kwon, T-D¹, Fisher, S¹, ¹ Department of Entomology, The Ohio State University, Columbus, Ohio, USA
The relationship between pre-existing body residues and toxicity was determined. Hylella azteca was observed to examine the mortality delay in the presence of relatively lower pre-existing PCB body residues. Two parallel exposures were set up for comparison of an effect in the mortality. These included (1) a 10-d exposure to PCB extracts (from smallmouth bass) prior to performance a 10-d toxicity test with PCB 153 or PCBs mixture (PCB 47+126+153). (2) The second was exposed to uncontaminated algae following exposure to PCB 153. In comparison to significant mortality in the PCB 153 exposure, both in the presence or absence pre-exposure to PCBs extract, no mortality was observed in the PCB mixture in spite of statistically similar body residues. The mortality in the PCB 153 treatment after twice pre-exposure, PCBs extract and PCB mixture, was ranged from 46.7 to 60.0 %, despite the fact that exposure concentration was already showed near 100 % mortality in the PCB 153 exposure in the presence or absence pre-existing body residues. The relationship between CBR and time-to-death showed a positive correlation, suggesting an increase in adaptability against chemical stress.

P198 (ESA-1117-839778) Toxicological effects of military Fog Oil obscurant to Daphnia magna in field exposures.
Start time: 8:00 AM
Esarey, J¹, Soucek, D¹, Cropek, D², Smith, T², ¹ Illinois Natural History Survey, Champaign, IL, USA² United States Army Corps of Engineers Research Laboratory, Champaign, IL, USA
The U.S. Army must continually maintain military preparedness and concurrently maintain the various natural resources of military training lands as mandated by the Congress and expected by the public. Preparation for adverse battlefield conditions requires training activities using smokes and obscurants (S&O), and many threatened and endangered species (TES) cohabit training areas where S&O are released. Fog Oil (FO) is a middle distillate petroleum that is vaporized to form a screen during battle and in military field training maneuvers. This research focuses on the impacts of FO obscurant to aquatic TES. Field experiments quantifying and characterizing FO obscurant deposited on aquatic surfaces as well as 48-hour toxicity testing with Daphnia magna following FO exposures were conducted in spring and late summer at Aberdeen Proving Ground in Maryland. Exposure vessels were placed at stations ranging from five to 800 meters downwind of the release point, and for three-, 18-, 30-, and 60-minute exposures. Compared to controls (50 meters upwind of release), significant mortality was observed only at the 5-meter station. This was true for all exposure durations. The number of floaters (neonates caught in a surface film) observed at the 50-meter station (50%) was significantly higher than that in controls (no floaters) after an 18-minute deposition of FO. Fog oil deposition at this station was 0.35g/L total oil, which is nearly two orders of magnitude lower than previously documented effects levels in laboratory studies. Substantial but not significant numbers of floaters were observed as far as 100 meters downwind from the release point after 18 minutes of deposition. FO obscurant was not analytically detectable in water samples at this station. These data will serve to calibrate further laboratory studies investigating the chemical/physical effects of vaporized and photolyzed FO obscurant, and elucidating chronic effects and potential for trophic transfer of FO constituents.

P199 (BUH-1117-841712) Toxicity of ash from burned areas along the Rio Grande to endangered Rio Grande silvery minnow.
Start time: 8:00 AM
Buhl, K¹, ¹ U.S. Geological Survey, CERC-Yankton Field Research Station, Yankton, South Dakota, USA
Extensive fires in the Bosque along the middle Rio Grande in and around Albuquerque, NM during the summer of 2003 have created the potential for large quantities of ash to enter river by surface runoff and wind erosion. This has raised concerns about the potential impacts of the ash and associated contaminants on the endangered Rio Grande silvery minnow (Hybognathus amarus). The objective of this study was to determine the acute toxicity to young silvery minnow and chemical characteristics of ash collected from burned areas along the Rio Grande. Silvery minnow (mean weight, 59-97 mg) were exposed to a geometric series of ash solutions, control water, and one soil solution (equal to the highest ash concentration) for 96 h under static conditions. Samples of ash, soil, and the test solutions were analyzed for a number chemical parameters to assess the likely toxic components. Of the 35 semivolatile compounds measured in the ash, only phenol was present a quantifiable concentration (1,000 g/kg). Concentrations of priority metal pollutants in ash, soil, and test solutions were below published probable effect concentrations for freshwater sediments and waterborne toxic concentrations for fish. In both tests, all mortality occurred during the first 24 h of exposure and the concentration-response was identical, producing a 24-h and 96-h LC50 of 20.74 (95% CI, 17.00-26.84) g ash/L. Comparisons of water quality data to published toxicity values indicated that the most likely toxic components in the ash solutions were un-ionized ammonia, potassium, and possibly sulfate. Other parameters that may have contributed to the toxicity were elevated pH, bicarbonate-carbonate, and the ash particles. The ash solutions had high biological and chemical oxygen demands and dissolved oxygen concentrations in these solutions decreased precipitously after 24 h. Entry of large amounts of ash into the Rio Grande has the potential to degrade the water quality and adversely affect the silvery minnow.

P200 (TOL-1117-839399) The effects of polychlorinated biphenyls on gonadal differentiation in the gray tree frog (Hyla chrysoscelis).
Start time: 8:00 AM
Tolley, A¹, Bailey, F¹, ¹ Middle Tennessee State University, Murfreesboro, TN, USA
A growing number of studies are being conducted to determine the cause of amphibian declines. Polychlorinated biphenyls (PCBs) are one group of chemical contaminants that pose a health risk to amphibians. In laboratory studies, PCBs have been shown to cause increased mortality, reductions in body size, increased time to metamorphosis, and histological abnormalities in tadpoles. PCBs are known endocrine disruptors and can act as estrogen mimics by binding to the estrogen receptors. This action has been shown to affect gonadal differentiation in a number of vertebrate species. The purpose of this study was to determine if PCB contamination affects gonadal differentiation in the gray treefrog, Hyla chrysoscelis. Gray treefrog eggs were exposed to sediments dosed with either 10ppm or 100ppm Arochlor 1254, an acetone solvent control or 100ppm estradiol as a positive control for estrogenic effects. Forty eggs were placed into each of the four treatments with four replicates per treatment. Eggs were allowed to hatch and tadpoles remained in the treatments until metamorphosis. Upon metamorphosis, froglets were weighed, examined for morphological abnormalities and placed in 10% formalin until histological examination. Froglets in the 100 ppm group weighed significantly less than those in control and 10 ppm groups but not the estradiol group. Hatchability was higher in the estradiol group than in the 10 and 100ppm groups but not the control group. Morphological abnormalities were observed in the estradiol group with 20% of individuals having extra toes on limbs and in the 100 ppm group with 29% of individuals having fewer toes on limbs. No behavioral abnormalities were observed in any group. Histological examination of gonads is being conducted and five froglets from each treatment are being analyzed for PCB total body burdens.

P201 (HAR-1117-810450) Sensitivity differences among various strains of Hyalella azteca.
Start time: 8:00 AM
Haring, H¹, Markert, J², Lazorchak, J³, Smith, M¹, ¹ SoBran Inc c/o US EPA, Cincinnati, OH, USA² ORISE/EERD/MERB c/o US EPA, Cincinnati, OH, USA³ US EPA/NERL/MIRB, Cincinnati, OH, USA
The North American amphipod Hyalella azteca is widely used in toxicity testing. H. azteca has a broad geographic distribution, and genetic evidence suggests that populations diverged from each other long ago. The importance of this genetic divergence to toxicity testing is unclear. Here we report the LC50 results for two different toxicants, Cu²⁺ and NH₃, from tests with geographically isolated strains of H. azteca from 7 academic and government laboratories and one wild strain. The strains were reared and tested under identical conditions. These tests were 96 h renewed acute toxicity tests conducted in triplicate using serial dilutions up to 500 ug/l CuSO₄ and 500 mg/l (NH₄)₂HPO₄. The Cu²⁺ LC50s ranged from 105.76 ug/l to126.14 ug/l. The NH₃ LC50s ranged from 3.88 mg/l to 5.18 mg/l. Of the eight strains tested with each toxicant, none were found to differ statistically based on the LC50 data generated. Therefore, it appears no sensitivity differences exist among different strains of H. azteca tested thus far using Cu²⁺ and NH₃ as toxicants. Although this work was reviewed by EPA and approved for publication it may not necessarily reflect official agency policy.