RP056 (HAN-1117-728259) Comparison of Acute and Chronic Daphnia Ecotoxicity Data for Selected Pharmaceuticals.
Start time: 8:00 AM
Hannah, R¹, ¹ GlaxoSmithKline, King of Prussia, PA, USA
GlaxoSmithKline as part of an overall material hazard assessment strategy has been routinely developing ecotoxicity and environmental fate data on pharmaceuticals, isolated intermediates and key starting materials. As part of an initiative to improve environmental hazard assessments, GSK has been applying chronic ceriodaphnia ecotoxicity screening assays to a variety of active pharmaceuticals across a range of therapeutic classes. Initial results will be presented and assessed.

RP057 (DRA-1117-740908) Toxicity of histamine antagonists on aquatic organisms.
Start time: 8:00 AM
La Bella, N¹, Draper, A¹, ¹ Trinity College, Hartford, CT, USA
Pharmaceutical contamination of surface water has become an increasing concern due to the potential negative implications for aquatic organisms and human health. The design of once-a-day pharmaceuticals to enhance compliance has resulted in drugs that resist breakdown in wastewater treatment systems and thus, release pharmaceuticals into surface water. Because some of the most commonly prescribed pharmaceuticals are the histamine antagonists, the following drugs were used in toxicity tests: cimetidine, famotidine, nizatidine, ranitidine, astemizole, brompheniramine, diphenhydramine, fexofenadine, hydroxyzine, loratadine, promethazine, and pyrilamine. The toxicity of these drugs was examined on Selenastrum capricornutum, Lemna minor, Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas, all cultured according to Standard Methods. Exposures were performed using 10 ppm of each drug in the appropriate medium. A two week static assay was performed on S. capricornutum and growth was no different in exposed cultures than in control cultures. In a 96 hr static assay, L. minor also exhibited little difference in growth when exposed to drugs compared with the control. A 48 hr static assay was conducted on C. dubia and D. magna and there was no difference in the mortality rate in treated organisms compared to controls. P. promelas underwent a 96 hr static renewal assay in which the drugs also had little effect on the mortality rate. Based on this research, detrimental effects from histamine antagonists at these low, environmentally relevant concentrations are improbable, but environmental effects may be observed with higher concentrations or more susceptible organisms.

RP058 (KIM-1117-826873) Acute, reproduction toxicity and sublethal effects after multigeneration exposure of molinate on Moina macrocopa.
Start time: 8:00 AM
Kim, Byung Seok^{1, 2}, Park, Yeon Ki¹, Park, Kyung Hun¹, Shin, Jin Sup¹, Kim, Jin Hwa¹, Ahn, Young Joon², ¹ National Institute of Agricultural Science and Technology, Suwon, Kyunggido, Korea² Seoul National University, Seoul, Korea, Korea
Acute and reproduction toxicity tests with Molinate were conducted on Moina macrocopa. The 24 and 48h-EC50 in the acute toxicity test were 14.1 and 8.3mg/l respectively. In the 10-day reproduction test, the effect of molinate on survival, reproduction and growth of M. macrocopa were monitored at sublethal concentrations (0.25, 0.5, 1.0, 2.0, 4.0, 8.0mg/l). Total offspring and brood size, parameters used to evaluate the effects on reproduction, were significantly reduced when molinate concentration increased. The survival and growth also decreased in M. macrocopa exposed to 4mg/L and 8mg/L concentration medium. The NOEC of molinate on reproduction was 2mg/l. To evaluate sublethal effects during multigenerational exposure with molinate, F₀ M. macrocopa were exposed to the lower concentrations (0.016, 0.16, 1.6mg/l) than the NOEC of the 10-day reproduction toxicity test. The neonates (F ₁-3rd brood) from the parentals (F₀) preexposed to the herbicide were exposed to the same concentrations for 10 days. The neonates (F₂-3rd brood) produced from F₁ were exposed to the same concentrations also. The Effects on survival and reproduction of F₀ and F₁ M. macrocopa exposed to molinate were not significantly different from the control. The reproduction of F₂ M. macrocopa preexposed to 0.16, 1.6mg/l medium through 2 generations were significantly different from the control. Although M. macrocopa experienced adverse effects on reproduction when they were exposed to high concentrations of, M. macrocopa seems likely to experience no adverse effects in real environment, because the environmentally relevant concentrations of molinate in Korea are 5-28 times lower than the concentrations that showed adverse effects at present study. To investigate the development of resistance of M. macrocopa to molinate, the neonates (F₃-3rd brood) produced from F₂ preexposed to the herbicide during 3-generational periods were used in acute toxicity test. There were not any differences in their sensitivity between control groups and preexposed organisms.

RP059 (ROE-1117-804759) Standardisation of test methods with dung organisms: The work of DOTTS.
Start time: 8:00 AM
Roembke, J.¹, Barrett, K.², ¹ ECT Oekotoxikologie GmbH, Floersheim, Germany² Huntingdon Life Sciences, Huntingdon, United Kingdom
According to testing requirements for veterinary medicines as agreed-on by VICH initiative (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Products) the potential effects of these pharmaceuticals on dung organisms has to be assessed. However, there are currently no internationally recognised guidelines for laboratory testing for effects of veterinary medicines on dung flies and dung beetles. Therefore, the Dung Organism Toxicity Testing Standardisation (DOTTS) Group was founded in 2002. Currently it has more than 50 members from different institutions in 17 countries. Up to now, draft guidelines for the testing of the flies Scathophaga stercoraria and Musca autumnalis have been developed. Currently, these methods are ring-tested by members of the DOTTS group using Ivermectin as a model substance. In addition, two further draft guidelines, covering the beetle species Onthophagus taurus and Aphodius constans are under preparation. According to the current work programme, ring-testing with these species will start in 2006. The final aim of this work is the preparation of test guidelines according to OECD format. In this presentation the work of DOTTS as well as first results of the practical work are presented. In addition, an overview of the activities of this advisory group, which is affiliated to SETAC, is given, highlighting the importance of voluntary work for the further progress on the scientific as well as the applied level of ecotoxicology.

RP060 (MIN-1117-569852) Consequences in mallards of sub-lethal exposure to the second generation anticoagulant rodenticide Brodifacoum.
Start time: 8:00 AM
Mineau, P¹, Trudeau, S¹, Knopper, L², Smits, J³, Gallagher, S⁴, Beavers, J⁴, Jaber, M⁴, ¹ National Wildlife Research Center, Canadian Wildlife Service, Ottawa, ON, Canada² Jacques Whitford, Ottawa, ON, Canada³ University of Saskatchewan, Saskatoon, SK, Canada⁴ Wildlife International, Easton, MD, USA
Second generation anticoagulant rodenticides (e.g. brodifacoum) have become important worldwide for rodent control. Secondary poisoning in birds of prey from these compounds appears to be increasing, and a large proportion of individuals carry multiple residues at sub-lethal levels. Published studies suggest the possibility of hepatotoxicity and disruptions of osteocalcin-dependant processes after exposure, and due to slow clearance from the liver and their additive mode of action, the sensitivity of exposed birds following re-exposure is of concern. We are conducting an on-going toxicology study using mallard ducks (Anas platyrinchos) dosed with brodifacoum (BRF) to elucidate the significance of sub-lethal exposure. Preliminary results (sub-lethal effects with single dose, anticoagulation times upon re-exposure, and reproductive effects measured during a modified OECD 206 avian reproduction study) are presented. Sub-lethal effects: Treatment related mass loss in adults (0.05 mg/kg BRF) was observed, no treatment related bone density or strength relationship in ducklings (0.1 mg/kg BRF) was observed. Clotting: Clotting time in adults measured 8 weeks after dosing (0.05 mg/kg BRF). Groups of birds then given 0.05-0.8 mg/kg BRF and coagulation tested 3, 7 and 14 days after dosing. Eight weeks later, birds dosed (0.8 mg/kg BRF) and coagulation tested again. Clotting in dosed birds was significantly faster 8 weeks post-initial dosing. Following the first challenge dose, a dose-related increase in clotting time was seen 3 and 7 days post-dosing. Following the second challenge dose, a clear increase in coagulation time was seen 3 and 7 days post-dose. Reproduction: 72 pairs given either sham or 0.05 mg/kg BRF. In the 8 week pre-induction level, treatment related mass loss in males was observed. Dosed pairs exhibited a slight lag in early egg production but this was reversed in favour of dosed birds after 3 weeks. Other reproductive variables, including bone density, did not show any difference in favour of control birds.

RP061 (GRU-1117-927301) Brain cholinesterase inhibition in juvenile Chinook exposed to carbaryl in seawater-Implications for Willapa Bay, Washington.
Start time: 8:00 AM
Cabarrus, Jennifer¹, grue, christian¹, Grassley, James¹, Major, Walter¹, ¹ University of Washington, seattle, WA, USA
Efforts to restrict the use of carbaryl to control burrowing shrimp in Willapa Bay, Washington have been in part driven by concerns over potential effects on salmonids. In 2003, we studied the use of treated areas in the Bay by salmonids in an effort to quantify their actual exposure to carbaryl. The only salmonid captured before and after treatment were juvenile Chinook (Oncorhynchus tshawytsha). Brain AChE inhibition (ca. 10%) occurred during the first tide post treatment in one of the two spray events with recovery to pre-spray levels of AChE by the second tide post-treatment. Both spray events showed significant increases (ca. 5-10%) in AChE levels by the third tide post treatment. This increase in enzyme activity, particularly in the more soluble fraction, was also documented in juvenile rainbow trout exposed to 6.3 ppb carbaryl as the formulated product (Sevin®80 WSP) used in the Bay. To determine if we could also document this apparent hormetic effect in Chinook smolts in seawater under laboratory conditions, we exposed smolts to concentrations of carbaryl (ai, in the formulated product) from 0 to 3,800 ppb for 6 h or 0 to 1,000 ppb for 96 h under static conditions. We then compared levels of brain AChE inhibition in the soluble fraction of the enzyme (no Triton X-100) with those when the detergent was added yielding total AChE activity (soluble + membrane bound fractions). A marked increase in the soluble fraction (> controls) was observed in smolts exposed to 6.3 ppb carbaryl for 96-h. Otherwise, inhibition was similar between the two assays to 39 ppb, but then diverged with inhibition in the total greater than that in the soluble fraction. Results suggest that, based on concentrations of carbaryl reported in the Bay, Chinook smolts would not be expected to suffer enzyme inhibition of greater than ca. 10%.

RP062 (GRU-1117-929053) Exposure of fishes to carbaryl following applications to control burrowing shrimp in Willapa Bay, Washington.
Start time: 8:00 AM
Monson, Christopher¹, Bush, Kristin¹, Cabarrus, Jennifer¹, grue, christian¹, Grassley, James¹, Curran, Catherine¹, ¹ University of Washington, seattle, WA, USA
Efforts to restrict use of carbaryl to control burrowing shrimp in Willapa Bay, Washington have been driven by concerns over potential effects on non-target species. In 2003, we studied the use of treated areas in the Bay by fishes in an effort to quantify their exposure to carbaryl. Use of the water column above oyster beds by fishes was determined on the first daylight high tide at each of three oyster beds (10-25 ac) preceding two carbaryl spray events (2 and 14 July 2003), and during each of the three subsequent daylight high tides (ca. 6, 30, and 54 h after treatment) using a two-boat trawl net. Exposure was assessed by determining the activity of brain actylcholinesterase (AChE) expressed as total activity and, if possible, in the soluble fraction. The only salmonid captured before and after treatment were juvenile Chinook (Oncorhynchus tshawytsha); additional species of fish included Pacific herring (Clupea pallasi) and shiner perch (Cymatogaster aggregata). Pacific herring (>10) were collected at one site before and after Spray Event 1 and three sites in Spray Event 2. Perch (>10) were only collected at two sites before and after the first spray event. Because of differences in life history (site fidelity and food habits), we predicted that exposure to carbaryl would be greater in the perch than in herring or juvenile Chinook. Maximum inhibition in Chinook was ca. 10%, 15% in herring, and 28% in perch. AChE inhibition in perch increased with time and was greatest in the membrane-bound fraction, whereas inhibition in the herring occurred only during the first tide post spray and differential inhibition was not detected. Results suggest maximum levels of inhibition are less than those associated with overt effects and that the biological significance of forms of the enzyme needs to be determined.

RP063 (BAR-1117-841296) A labortaory testing method for the dung beetle Onthophagus taurus.
Start time: 8:00 AM
Barrett, K¹, Gray, J¹, Taylor, K¹, ¹ Huntingdon Life Scienecs, Huntingdon, Cambridgeshire, UK
Under the proposed Environmental Impact Assessment (EIA) for Veterinary medicinal products (VMPs) phase II, evaluation of the toxicity to dung fauna is proposed for products which may be used in animals kept at pasture where there is the potential for residues of VMPs to be present in the excreted manure. These studies are also required for and for endo and ectoparisiticides. At present there are no agreed guideline methods for this testing. To address this issue an expert group Dung Organism Toxicity Testing Standardisation (DOTTS) has been set up under the auspices of the Society of Environmental Toxicology and Chemistry (SETAC), to develop and ring test proposed methodologies. In this poster a test method developed for the dung beetle Onthophagus taurus will be described, and results from preliminary validation studies conducted using Ivermec (1% w/v ivermectin) presented.

RP064 (STR-1117-854093) Effects of the herbicide atrazine on the gray treefrog (Hyla versicolor) in a microcosm system.
Start time: 8:00 AM
Street, R¹, Bailey, F¹, ¹ Middle Tennessee State University, Murfreesboro, TN, USA
The concern over declining amphibian populations has led to the introduction of many hypotheses that explain a decrease in fitness. One hypothesis suggests that the introduction of chemical pesticides damages the individuals and impairs their ability to reproduce. The selective herbicide atrazine has been named as a possible contributor to amphibian declines by acting as an endocrine disruptor and adversely affecting reproduction. Atrazine is one of the most popular and widely used herbicides in the world and is a ubiquitous contaminant in both surface and ground waters. Some studies suggest that atrazine poses little or no threat to amphibians at typical environmental concentrations, while other researchers claim that atrazine acts as an endocrine disrupting chemical at concentrations previously thought to be safe and consistently present in flowing water. The gray treefrog (Hyla versicolor) is one species that may come in contact with atrazine applied in the US Southeast and Midwest. The objective of this study is to examine the effects of atrazine exposure on the hatchability, survival, and development of gray treefrogs. Gray treefrog eggs were exposed to nominal concentrations of 0 ppb, 0.01 ppb, 1 ppb, and 10 ppb atrazine in glass microcosms. Thirty eggs were placed into each of 10 replicates for each treatment. After hatching, tadpoles were left in the treatments until metamorphosis. Upon metamorphosis, froglets were weighed, examined for gross morphological abnormalities and placed in 10% formalin until histological examination. No treatment had significantly higher mortality or a decrease in hatchability and no external morphological abnormalities were seen. Although atrazine had no significant effects on hatchability or survivability, determination of sex ratios is in progress and histological sectioning of gonads is now underway and may show sub-lethal effects of atrazine on the gonadal development and morphology of the frogs.

RP065 (WAR-1117-819273) Effects of estrogen exposure on vertebral development in the fathead minnow (Pimephales promelas).
Start time: 8:00 PM
Warner, K¹, Jenkins, J¹, ¹ Dept. of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, USA
Natural and synthetic estrogens have been detected in surface waters, and have been linked to reproductive signaling disruption in several species. The sublethal effects of endocrine disrupting compounds may also include perturbation of homeostatic signaling affecting growth, fitness, and survival in wildlife and humans. This study focuses on the impacts of exogenous estrogen exposure on growth factor signaling in a teleost model. Estrogen is a proliferation-inducing compound in many cell types, including osteoblasts; estrogen and growth factor signaling regulate cartilage and bone deposition during development in vertebrates. We hypothesize that xenobiotics with estrogenic activity may have an adverse impact on bone formation. This study seeks to characterize vertebral anomalies as morphological markers of endocrine disruption in fish exposed to an estrogenic agent. Fathead minnows (Pimephales promelas) were exposed to 100 ppt to 100 ppb 17- ethinyl estradiol (EE2) for 30 days (egg stage to 25+ days post hatch). Fish were analyzed microscopically for vertebral malformations using a fluorescein stain to illuminate calcified tissue. Vertebral deformities were observed in up to 44% of exposed fish; deformities ranged from vertebral compression and bone fusion to severe scoliotic curvatures. These data suggest vertebral bone development is a potential endpoint of endocrine disruption from estrogenic compounds in surface waters.

RP066 (DUI-1117-819511) ERAPharm: a European project on environmental risk assessment of pharmaceuticals.
Start time: 8:00 AM
Duis, K.¹, Ternes, T.², Fenner, K.³, Escher, B.³, Schmitt, H.⁴, Roembke, J.¹, Garric, J.⁵, Hutchinson, T.⁶, Boxall, A.⁷, Knacker, T.¹, ¹ ECT Oekotoxikologie GmbH, Floersheim/M., Germany² Federal Institute of Hydrology, Koblenz, Germany³ EAWAG, Duebendorf, Switzerland⁴ RIVM-LER, Bilthoven, The Netherlands⁵ Cemagref, Lyon, France⁶ AstraZeneca Global SHE, Brixham, U.K.⁷ Central Science Laboratory, York, U.K.
Within a process of international harmonization, guidance on the environmental risk assessment (ERA) of veterinary pharmaceuticals has been established. A European guideline on ERA of human pharmaceuticals is currently being discussed. Yet, there are still a number of gaps in our understanding of specific exposure pathways and environmental effects of pharmaceuticals. The objective of the EU-funded research project 'Environmental risk assessment of pharmaceuticals' (ERAPharm) is to advance the existing knowledge and to contribute to the improvement of procedures for environmental risk assessment of human and veterinary pharmaceuticals. Fate studies are focusing on exposure routes that have so far not been adequately studied, such as input from pasture animals and through the application of manure and sewage sludge to land. Factors and processes affecting the behavior of pharmaceuticals in water, sediment, soil and manure are studied in laboratory, semi-field and field experiments. A scenario-based exposure assessment system for predicting concentrations of pharmaceuticals in soils, surface waters and sediments is developed. In effect studies, it is evaluated how in vitro and low complexity bioassays can be used to provide a first hazard characterization, and how data on pharmacokinetics and -dynamics in mammals and molecular-biological studies in fish can be used to guide the evaluation of potential sub-lethal effects of human pharmaceuticals in fish. Higher-tier ecotoxicity tests are employed for detecting possible effects of long-term exposure to pharmaceuticals in aquatic and terrestrial invertebrates and fish. The effects of antibiotics on structure and function of microbial communities are studied with special emphasis on the spread of genetically encoded resistance. Based on the results of the fate and effect studies, recommendations on the environmental risk assessment procedures for human and veterinary pharmaceuticals will be provided.

RP067 (LIZ-1117-834370) Toxicity assessment of Mississippi delta oxbow lake sediments using Hyalella azteca.
Start time: 8:00 AM
Lizotte, R¹, Knight, S¹, Bryant, C¹, ¹ USDA-ARS National Sedimentation Laboratory, Oxford, MS, USA
Hyalella azteca was used to assess biological impairment in sediments from nine water bodies in the Mississippi delta (i.e., lower Mississippi alluvial plain). Water bodies were categorized according to land use and implementation of agricultural best management practices (BMPs). Three reference oxbow lakes were located in the White River National Wildlife Refuge (WRNWR), Arkansas; three Mississippi lakes were treated with varying BMPs to improve water quality/ecology; and two lakes and one bayou, also located in Mississippi, were listed as impaired according to USEPA section 303d Clean Water Act (303d). Two sediment samples were collected at each of three sites within each water body from June to July 2004 and analyzed for 17 current and historic-use pesticides and metabolites. Twenty-eight day H. azteca survival and growth were measured to assess the degree of biological impairment. All lake sediments examined had detectable levels of at least 14 of 17 analytes measured. Significant (P <0.05) mortality occurred in animals exposed to sediment from a single 303d lake. Significant growth impairment was observed in sediments from all three 303d water bodies and two of three BMP oxbow lakes, whereas no impairment was observed in any WRNWR lake. Attempts to associate sediment pesticide contamination with observed H. azteca responses had limited success. Historic-use pesticides and metabolites were implicated in two of five biologically impaired water bodies (303d bayou and BMP lake)while current-use pesticides were implicated in one 303d lake. Complex contaminant mixtures often limit attempts to provide clear, definitive sources of biological impairment. In this study, even accounting for sediment characteristics such as sand-silt-clay fractions and organic carbon content did not further elucidate sources of toxicity in some water bodies. Finally, results show that implementation of BMPs can mitigate biological impairment within lake sediments.

RP068 (HAN-1117-809367) Field assessment of oxytetracycline exposure to the freshwater macrophytes Egeria densa Planch. and Ceratophyllum demersum L.
Start time: 8:00 AM
Hanson, M¹, Knapp, C², Graham, D², ¹ Department of Environment and Geography, University of Manitoba, Winnipeg, Manitoba, Canada² Department of Civil, Environmental, and Architectural Engineering, University of Kansas, Lawrence, Kansas, USA
Pharmaceuticals, especially antibiotics, are widely distributed environmental contaminants, but there is a paucity of toxicity data to effectively characterize the risk posed to aquatic ecosystems. A microcosm study was conducted to describe the impacts of oxytetracycline on two species of aquatic macrophyte, Egeria densa and Ceratophyllum demersum. Plants were exposed for a period of 6 weeks at nominal concentrations of 0, 5, 20, 50 and 250 g/L oxytetracycline, plus 20 g/L oxytetracycline amended with additional nitrogen and phosphorus (all n=3), with toxicity assessment at two week intervals. The influence of species density and competition was assessed by planting the macrophytes as individuals, in pairs of the same species and as pairs of two separate species. Both plant species exhibited an overall decline in growth at the highest tested oxytetracycline concentration, and in the nutrient amended microcosms, and some intraspecies interactions were observed (e.g., root development in E. densa). Colouration in the microcosm water column resulting from elevated oxytetracycline levels appears to play the major role in the inhibition of the development of these plants. Based on the toxicity data generated in this study, we estimate that recently monitored concentrations of oxytetracycline in freshwater environments do not pose a direct risk to these two macrophytes.

RP070 (KAH-1117-746048) Effects of three fungicides on reproductive success and endocrinology of the fathead minnow.
Start time: 8:00 AM
Kahl, M.¹, Villeneuve, D.¹, Jensen, K.¹, Durhan, E.¹, Makynen, E.¹, Linnum, A.¹, Ankley, G.¹, ¹ U.S. EPA Mid-Continent Ecology Division, Duluth, MN
The toxic mechanism of action of several current-use fungicides involves inhibition of ergosterol formation by 14-demethylase, a cytochrome P450-based enzyme. However, many of these fungicides can inhibit a variety of P450s, including some involved in vertebrate steroidogenesis. A companion paper by Villeneuve et al. describes the effects of three fungicides, prochloraz, fenarimol and ketoconazole, on in vitro steroid synthesis by fathead minnow ovarian tissue. The studies in this presentation describe in vivo effects of the three pesticides on fathead minnow reproductive success (fecundity) and a suite of diagnostic measures of endocrine system status, including gonadal histopathology and plasma concentrations of vitellogenin and sex steroids (testosterone, 11-ketotestosterone, 17-estadiol). Exposure to the fungicides for 21 d significantly inhibited fecundity of the fish at water concentrations greater than 0.1 (prochloraz), 1.0 (fenarimol) and 0.4 (ketoconazole) mg/L. The suite of diagnostic responses in the fish varied among the three fungicides. Some, but not all, observed effects were suggestive of inhibition of steroid synthesis. For example, prochloraz depressed both androgen (testosterone, 11-ketotestosterone) and 17-estradiol concentrations. This was associated with decreased concentrations of vitellogenin (an estrogen-responsive protein) observed in exposed females. In contrast, fenarimol also decreased vitellogenin concentrations in female fathead minnows but the effect was not associated with a significant decrease in 17-estradiol concentrations. Results of the in vivo studies confirm that the three fungicides broadly act as endocrine-disrupting chemicals (EDCs), but suggest that they operate via modes/mechanisms of action in addition to disruption of steroid synthesis. This highlights the importance of complementary in vivo and in vitro studies to assess the mechanistic basis of adverse effects of EDCs.

RP071 (WOJ-1117-737527) Environmental fate and effects of veterinary-derived antibiotics in the terrestrial environment.
Start time: 8:00 AM
Wojtyniak, A¹, Sibley, P¹, Solomon, K¹, ¹ University of Guelph, Guelph, Ontario, Canada
Commercial agricultural operations administer antibiotics to animals to treat and prevent disease and to promote growth. Substantial quantities of these compounds and their bioactive metabolites are excreted as waste, which may be applied to agricultural fields as fertilizer. These compounds are designed to control microorganisms in animals and are potentially harmful to native soil bacteria. While the concentrations required to elicit acute toxicity are generally higher than current environmental concentrations, chronic toxicity remains a concern. Low-level exposures to antibiotics have been linked to the development of antibiotic resistance and have been shown to interfere with fundamental microbial processes, such as nutrient cycling. Antibiotics that are neither degraded nor bound in the soil may enter surface water or groundwater, and could enter the drinking water supply. A better understanding of the fate and effects of antibiotics in soil systems is required for adequate risk assessment and risk management for both humans and the environment. We recently initiated soil column studies to assess the fate and toxicity of the veterinary antibiotics oxytetracycline, tylosin and monensin. Columns are packed with local agricultural soils, treated with antibiotics in concentrations from 0 to 1000 g/g, and watered to mimic natural rainfall. They are maintained in a controlled environment and will be monitored over eight weeks. Structural and functional endpoints being evaluated include counts of total heterotrophic, cellulose degrading and antibiotic resistant bacteria, community-level physiological profiling and denaturing gradient gel electrophoresis patterns, and effects on cellulose degradation and potential nitrification rates. Preliminary results based on structural endpoints indicate that responses are observed only at concentrations well above those typically observed in the environment. While additional endpoints remain to be fully evaluated, these results are in agreement with previous work conducted in our laboratory and suggest relatively low risk of these compounds to soil bacterial communities.

RP072 (HOW-1117-745086) Chronic effects of atrazine on the development of Northern Leopard Frog (Rana pipiens) tadpoles.
Start time: 8:00 AM
Fridgen, C¹, Pauli, B², Berrill, M¹, Doe, K³, Jackman, P³, ¹ Department of Biology, Trent University, Peterborough, Ontario, Canada² Canadian Wildlife Service, Ottawa, Ontario, Canada³ Environment Canada, Moncton, New Brunswick, Canada
Amphibians are an excellent model organism in which to observe chronic effects of contaminants, in particular effects on the thyroid and gonadal axes, because of their endocrine-mediated differentiation processes including metamorphosis and gonadal development. Atrazine exposure has been associated with abnormal development of exposed frogs, particularly demasculinization and feminization, through a possible mechanism of induced aromatase activity levels increasing conversion of androgens to estrogens. We conducted a blinded study examining the effects of chronic atrazine exposure (0.1 and 1.8 g a.i./L as Aatrex Liquid 480) on the development of Northern Leopard Frog (Rana pipiens) tadpoles. Animals were exposed from the egg stage to metamorphosis. Our main objectives were to compare our results with existing data, provide information concerning sensitive stages and endpoints in Ranid amphibians, and investigate new histological methods to quantify and stage early gonad differentiation in R. pipiens. Atrazine exposure caused significant chronic effects on both the growth and development of tadpoles and their gonads at concentrations as low as 0.1 g/L. Preliminary statistical analyses indicate that Gosner Stage 25 to 40 tadpoles exposed to the lowest concentration tested have significantly increased body length and mass, as well as an increased average stage of development over time, yet significantly fewer tadpoles being reared in both test concentrations reached metamorphic climax at test termination. Both atrazine treatments induced significantly larger female gonads, increasing both the ovary length and area. In addition, preliminary histology has revealed signs of intersex, testes with large primary oocytes, and ovaries with areas of abnormal somatic tissue. Further in-depth histological analysis of gonads to examine the influence of atrazine exposure on oocyte size, gonadal differentiation rate and stages, as well as intersex incidence is being conducted.

RP073 (ZAH-1117-731815) Recovery of acetylcholinesterase in Daphnia magna after mulitple exposures to chlorpyrifos.
Start time: 8:00 AM
Zahner, H¹, Gaworecki, K¹, Klaine, S¹, ¹ Clemson University, Clemson Institute of Environmental Toxicology, Pendleton, SC, USA
Aquatic organisms are exposed to fluctuating contaminant concentrations for varying durations of time in streams receiving run-off and effluents. Little quantitative information is available on the response of aquatic organisms to these episodic exposures. Still less information is available on the recovery of organisms between exposures. Chlorpyrifos is an organophosphorous pesticide that targets acetylcholinesterases, possibly resulting in mortality. The objective of this study was to quantify the recovery of Daphnia magna from multiple pulse exposures of chlorpyrifos using the physiological endpoint of acetylcholinesterase (AChE). Response of D. magna to double CPF exposures was characterized over 21-days. Organisms were exposed to three concentrations (0.1, 0.2, 0.4 g CPF/L), three exposure durations (3, 6, 24-hours), and four recovery periods (0, 24, 96, 144-hours) between exposures. The percent inhibition of AChE was a function of the concentration and duration of the exposure. D. magna were able to regain AChE to within control levels (85-100%) within the given recovery times. The time period between pulses had a significant effect (p<0.05) on the recovery, and ultimately survival and reproduction of D. magna. These results illustrate the existence of a concentration threshold; below which organisms are able to recover and above which results in mortality. The responses of AChE in D. magna during and after the pulse exposures were related to individual reproductive effects observed.

RP074 (VIL-1117-725879) Effects of three fungicides on fathead minnow ovarian steroid synthesis in vitro.
Start time: 8:00 PM
Villeneuve, D.¹, Makynen, E.¹, Ankley, G.¹, ¹ U.S. EPA Mid-Continent Ecology Division, Duluth, MN, USA
A number of conazole and heterocyclic amine fungicides that depress fungal growth by inhibiting 14-demethylase, a cytochrome P450 that plays a key role in ergosterol biosynthesis, also have potential to disrupt the vertebrate endocrine system through effects on enzymes involved in steroid synthesis and/or metabolism. Three of these compounds, prochloraz (PRO), fenarimol (FEN), and ketoconazole (KTZ), were recently evaluated in a short-term fathead minnow reproduction test and were shown to affect spawning success to various degrees. The purpose of this study was to help define the mechanisms through which these three fungicides elicited their effect on fathead minnow reproduction using a series of in vitro bioassays relevant to steroid biosynthesis. All three chemicals inhibited in vitro testosterone (T) production in fathead minnow ovary tissue, with KTZ causing significant inhibition at concentrations greater than 3.3 g/L (6.2 nM), and PRO and FEN causing significant inhibition at 0.3-1.0 mg/L (0.9-2.6 M). In addition to reduced in vitro estradiol production concomitant with reduced T synthesis, all three chemicals were shown to inhibit aromatase activity in both brain and ovary tissue homogenates exposed in vitro, with PRO and KTZ causing significant inhibition at concentrations greater than 1.0 M, and FEN causing inhibition at concentrations greater than 75 M. Additional in vitro exposures with selective addition of steroid precursors were used to help identify the specific steroidogenic enzymes affected by each chemical. Results of this study help to establish important linkages between mechanisms of action at the biochemical level and ecologically relevant outcomes. Such linkages should help strengthen the use of mechanism-specific biomarkers and in vitro assays in ecological risk assessments related to endocrine disrupting chemicals. The contents of this abstract do not necessarily reflect U.S. EPA policy.

RP075 (TIL-1117-671639) Dithiocarbamate developmental toxicity in zebrafish.
Start time: 8:00 AM
Tilton, F^{1, 2, 3, 4}, La Du, J^{1, 2, 3, 4}, Vue, M⁴, Alzaarban, N⁴, Tanguay, R^{1, 2, 3, 4}, ¹ Department of Environmental and Molecular Toxicology² Environmental Health Sciences Center³ Marine and Freshwater Biomedical Sciences Center⁴ Oregon State University, Corvallis, OR, USA
We have demonstrated that dithiocarbamates (DTCs) from every subclass cause profound developmental toxicity involving the notochord and the surrounding tissue at environmentally relevant concentrations. Our current efforts are directed toward elucidating the mechanisms by which DTCs perturb vertebrate development. The hypothesis that metal chelation is the sole mode of developmental toxicity was tested. The membrane permeable copper chelator, neocuproine, mimicked the effects of DTCs albeit at much higher concentrations. No other metal chelator or metal caused this toxicity. Neocuproines effects were eliminated by co-treatment with copper. Moreover, copper and DTC co-treatments altered neither the developmental toxicity of DTCs nor the lethality of copper. This suggests a more complex mechanism of toxicity. The potential for DTCs to alter the redox balance within the developing embryo is currently under investigation. The thiol containing antioxidant, glutathione, will protect and recover monomethyl dithiocarbamate (MMDTC) effects but not DTCs with larger substituents. Several other thiol and non-thiol containing antioxidants were investigated with limited to no protection from the effect. There were no differences in populations of apoptotic cells between treated and control embryos during the period when the malformation becomes apparent (18 to 24 hours post fertilization). Utilizing whole mount in situ hybridization probes targeting developmentally important transcripts, similar alterations exist between DTCs, degradation products, and neocuproine exposures. Whole-mount immunohistochemistry of several neuronal cell types was also investigated to identify potential neuronal targets. Utilizing the Affymetrix zebrafish microarray, 154 genes at 18 hpf and 26 genes at 24 hpf were differentially regulated greater than 2-fold. Only 12 (18hpf) and 10 (24 hpf) genes passed the t-test and 3 genes were shared between the two time points. Genes associated with muscle development, immune function, and glutathione maintenance were well represented in the gene set. Supported by Northwest Health Foundation # 10257, NIEHS #ES00210, #ES03850, and #ES07060.

RP076 (CON-1117-723179) Environmental Fate and Ecotoxicology Testing of the Human Pharmaceutical Pregabalin.
Start time: 8:00 AM
Constantine, Lisa¹, Huggett, Duane¹, Ericson, Jon¹, Smith, Christopher¹, Ferraina, Richard ¹, ¹ Pfizer Global Research and Development, Groton, CT, USA
Scientific researchers and regulators are focusing much attention on trace quantities of pharmaceuticals being detected in natural waters as a result of release from municipal sewage treatment facilities. Environmental fate and effects studies were conducted with pregabalin, an analogue of the mammalian neurotransmitter -aminobutyric acid (GABA) shown to be effective for the management of epilepsy and chronic pain (neuropathic pain). A full summary of these environmental fate and effects data associated with pregabalin will be presented. These data assist in defining the environmental compartment pregabalin is likely to reside in if released into the environment, its mobility and degradation potential within the environment, and the potential acute and chronic effects pregabalin may have on various environmental species. The physical-chemical properties and environmental fate studies indicate that pregabalin will likely reside in the aqueous compartment. Minimal sorption (Kd sludge value of 13.3) or biotransformation was observed in experiments conducted with sludge from a wastewater treatment plant. Therefore, acute ecotoxicity tests with green algae, rainbow trout and Daphnia magna were conducted and no responses were elicited at the concentrations tested with the EC/LC50 being > 300, > 1000 and >1000 mg/L, respectively. A 7-day chronic Ceriodaphnia dubia reproduction test was conducted with NOEC and LOEC being 4.8 mg/L and 9.4 mg/L, respectively.

RP077 (MCD-1117-565133) Impacts of row crop agriculture on sexual development of anurans.
Start time: 8:00 AM
McDaniel, T.¹, Martin, P.¹, Marvin, C,², McMaster, M.², Sherry, J.², ¹ Canadian Wildlife Service, Environment Canada, Burlington, Ontario, Canada² National Water Research Institute, Environment Canada, Burlington, Ontario, Canada
Intensive row crop agriculture featuring corn and soybean production, is predominant in southwestern Ontario where the two crops account for over 50% of the total acreage of crops grown. This form of agriculture relies heavily on pesticide and nutrient inputs for continued success under conventional systems. The corn herbicides atrazine and metolachlor, are two of the most heavily applied pesticides in Ontario and are routinely detected in tributaries draining agricultural watersheds. Field and laboratory studies have indicated exposure to atrazine may disrupt normal sexual development in male anurans, causing reductions in testosterone levels, laryngeal muscle diameter, and abnormal testes development. In addition, male cane toads exposed to field levels of atrazine expressed levels of vitellogenin equivalent to those found in female toads. Pesticide levels and water quality data were collected from 33 farm ponds and agricultural drains in southwestern Ontario, two agricultural reference sites as well as four non-agricultural reference sites in August and September, 2003 and monthly from April through August in 2004. Atrazine and metolachlor were detectable in most samples, exceeding 1 ug/L at some sites. Blood samples were obtained from leopard and green frogs for analysis of sex hormones, and vitellogenin. Gonads were excised for histomorphological assessment. Analysis of frogs from late summer 2003, revealed that circulating testosterone levels were negatively correlated with atrazine concentrations in pond water in juvenile male leopard frogs (r = 0.74; p = 0.014) and adult female leopard frogs (r = 0.59; p < 0.008). No relationship was found between sex steroid levels in frog plasma and concentrations of atrazine and metolachlor in the water in 2004, contrary to results from 2003. This may be due to lower concentrations of atrazine and metolachlor in 2004. Agricultural sites, particularly those in Chatham exhibited a high percentage of males with ova-testes (45%), as compared to control sites (10%).

RP078 (MOR-1117-608178) Effects of exposure to pentachlorophenol during gestation and lactation on neuro-endocrine system in rats.
Start time: 8:00 AM
Morohoshi, K^{1, 2}, Kawaguchi, M³, Takano, H^{1, 2}, Morita, M¹, Yamamoto, H⁴, Kondo, T¹, Himi, T³, Imai, H^{1, 2}, ¹ National Institute for Environmental Studies, Tsukuba, Japan² The Master’s Program in Environmental Sciences, University of Tsukuba, Tsukuba, Japan³ Department of Toxicology and Pharmacology, Faculty of Pharmacy, Musashino University, Musashino, Japan⁴ Faculty of Integrated Arts and Studies, University of Tokusima, Tokushima, Japan
Thyroid hormone (TH) is essential for the developing central nervous system. Recently, epidemiological and experimental studies have shown that endocrine disrupting chemicals (EDCs) disturb TH systems. On the other hand, hypothyroidism in pregnant mother causes delay of child's mental development and the following low IQ. Pentachlorophenol (PCP) is one of EDCs which affect TH systems. In this study, we examined whether gestational and lactational exposure of PCP to pups affect the neuroendocrine system and the stress responses. PCP was administered to mother rats (Wistar) via drinking water (6.6 mg/L) ad libitum from gestational day 0 to postnatal day 21. Control group was given distilled water. Furthermore, a part of twelve-week-old pups was exposed to immobilization stress for five days (three hours /day). Liver, blood and brain samples were collected from three-day-old, three-week-old and twelve-week-old pups, and mothers after weaning. Blood samples were not collected from three-day-old pups. PCP concentrations in liver, plasma and brain were determined using GC-MS. Plasma levels of TH and corticosterone were determined by radioimmunoassay. Stress-induced neuronal cell death in hippocampus was estimated by counting apoptotic cells using TUNEL (Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) method. Thyroxine concentrations in plasma of PCP-treated mothers and PCP-treated pups (three-week-old) were significantly lower than those of control mothers or control pups, respectively. Significant difference of thyroxine concentration in plasma was disappeared between PCP-treated pups (twelve-week-old) and control pups (twelve-week-old). Stress-induced corticosterone levels in plasma and the number of TUNEL-positive cells in hippocampus of PCP-treated group were not significantly different from those of control for twelve-week-old pups. PCP exposure transiently affected for TH systems, however, stress responses was not affected by PCP exposure.

RP079 (HIL-1117-635948) Toxicity of selected pharmaceuticals on root organ cultures and arbuscular mycorrhizal fungi.
Start time: 8:00 AM
Hillis, D¹, Sibley, P¹, Solomon, K¹, Klironomos, J¹, ¹ University of Guelph, Guelph, Ontario, Canada
The amendment of soil with pharmaceutical-containing manure or biosolids is common practice in intensive agricultural systems. However, the impact of pharmaceuticals on the various trophic levels of soil systems is poorly understood. In particular, understanding of pharmaceutical impacts on beneficial plant-microbe interactions, which are primary determinants of plant health and soil fertility, is lacking. One of the most important plant-microbe interactions in agricultural systems is the symbiotic relationship formed between plant roots and arbuscular mycorrhizal fungi. This interaction improves plant nutrient uptake, tolerance to water, pathogenic and toxic stressors, as well as the provision of growth hormones and regulators. Consequently, arbuscular mycorrhiza should be included in the existing array of indicator organisms used in ecological risk assessment. However, arbuscular mycorrhizal fungi are obligate plant symbionts and cannot be cultured axenically. This challenge was overcome with the development of transformed root cultures, whereby root inducing transformed-DNA (Ri T-DNA) roots are cultured with symbiotic arbuscular mycorrhizal fungi. In this study, a prioritized selection of veterinary and human-use pharmaceuticals including quinolones, sulfonamides, tetracyclines and macrolides will be evaluated for toxicological impacts on the ubiquitous arbuscular mycorrhizal fungi, Glomus intraradices and Daucus carota root organ cultures. A preliminary experiment to assess the application of this test system for evaluating pharmaceuticals, was conducted using the sulfonamide, sulfamethoxazole. Results indicate significant decreases in arbuscular mycorrhizal propagules (spore and hyphal density) and root colonization at concentrations as low as 100 g/L. In addition, significant decreases in root-organ culture density were observed at this concentration indicating the potential for phytotoxic effects. These preliminary results indicate that mycorrhizal fungi may be sensitive indicators of pharmaceutical toxicity; additional experiments, using the above pharmaceutical classes, are currently being conducted to better assess this possibility.

RP080 (MAR-1117-228361) Reproduction and health of American kestrels breeding in agroecosystems in southwestern Ontario.
Start time: 8:00 AM
Martin, P.¹, Barrett, G.¹, Marvin, C.², Sverko, E.², ¹ Canadian Wildlife Service, Environment Canada, Burlington, Ontario, Canada² National Water Research Institute, Environment Canada, Burlington, Ontario, Canada
American kestrels are common though declining throughout the agricultural landscape of southern Ontario. They readily nest in nestboxes in agricultural land, forage on rodents, passerines and insects in farm fields and edges, typically within 1 km from their nest site, and hence provide a good upper trophic level avian model to monitor the health of terrestrial wildlife in agroecosystems. In southern Ontario, DDT was used throughout the mid-1900s and persists in soil to varying levels depending on the degree of agricultural usage; in addition, the types and intensity of use of current use pesticides vary among agroecosystems. We measured legacy organochlorine pesticides (OCs), as well as the currently used OC, endosulfan, in kestrel eggs, and atrazine in the plasma of kestrel chicks and monitored the reproductive success, growth and various biomarkers of the health of kestrel chicks produced from nestboxes on 5 trails in orchard, tobacco, corn-soy and low intensity mixed agricultural areas in 2002 to 2004. DDE levels in eggs of kestrels were highest in the Niagara orchard area relative to all others, and intermediate in a second orchard area. Clutch size, hatching and fledging success did not differ among crop type or area, although all variables were lower in 2003 than 2002. In all years however, significantly more male kestrels were raised in tobacco than orchard areas (2.15 versus 1.26 males per box). Nest success did not vary between areas; however the incidence of brood reduction was greater in orchard regions. However, pre-fledge nestlings were significantly larger in orchard than other areas. Circulating levels of thyroid hormones and retinol in nestlings did not differ among regions and did not correlate with DDE levels in sibling eggs. However, we found an decreased cutaneous T-lymphocyte-mediated response to the mitogen phytohemagglutinin in nestlings from orchard areas.

RP081 (BOU-1117-561614) Atrazine and lambda-cyhalothrin uptake in aquatic macrophytes during hydroponic laboratory exposures.
Start time: 8:00 AM
Bouldin, J¹, Farris, J¹, Moore, M², Cooper, C², ¹ Arkansas State University, State University, AR, USA² USDA-ARS-National Sedimentation Laboratory, Oxford, MS, USA
Phytoremediation encompasses an array of plant-associated processes known to mitigate contaminants from soil, sediment, and water. Modification of pesticides associated with agricultural runoff includes processes directly associated with aquatic macrophytes in addition to soil geochemical modifications and associated rhizospheric degradation. Remediation attributes of two vegetative species common to agricultural drainages in the Mississippi Delta, USA, were assessed using atrazine and lambda-cyhalothrin. Concentrations used in 8-d hydroponic exposures were calculated using recommended field applications and a 5% runoff model from a 0.65-cm rainfall event on a 2.02-ha field. While greater atrazine uptake was measured in Juncus effusus, greater lambda-cyhalothrin uptake occurred in Ludwigia peploides. Maximum pesticide uptake was reached within 48 h for each exposure and subsequent translocation of pesticides to upper plant biomass occurred in macrophytes exposed to atrazine. Sequestration of 98.2% of lambda-cyhalothrin in roots of L. peploides was measured after 8 d. Translocation of lambda-cyhalothrin in J. effusus resulted in 25.4% of pesticide uptake partitioned to upper plant biomass. These individual macrophyte remediation studies measured species- and pesticide-specific uptake rates, indicating that the seasonality of pesticide applications and macrophyte emergence might interact strongly to enhance mitigation capabilities in edge-of-field conveyance structures.

RP082 (PAR-1115-237743) Evaluation of Pesticide Use on U.S. Fish and Wildlife Service Lands.
Start time: 8:00 AM
Parson, Tiffany¹, Golden, Nancy¹, Masson, Greg¹, ¹ U.S. Fish & Wildlife Service, Arlington, VA, USA
The U.S. Fish and Wildlife Service applies pesticides on Service lands as one tool in an integrated pest management approach to control pest species that interfere with desired resource management objectives. Most of this pesticide use occurs on National Wildlife Refuges for the management of non-native invasive species, such as Canada thistle (Cirsium arvense), johnson grass (Sorghum halapense), and phragmites (Phragmites australis). However, we also use pesticides to manage for disease vectors, like mosquitoes, or to create habitat for wildlife. Due to the somewhat delicate nature of the habitat where these chemicals are being applied, all proposed uses are subjected to a site-specific evaluation of pesticide fate and effects designed to capture any potential risk to fish and wildlife trust resources. Our pesticide use proposal process is a conservative, tiered approach based on analysis of factors such as pesticide and adjuvant toxicity, application distance from water, application method, and probability of leaching. Our evaluation may corroborate risks to wildlife identified in the pesticide registration process or incorporate new data which may be specific to the species present in the application area, the proposed pattern of use, or other characteristics of the natural habitat. Through our pesticide use proposal program and pesticide analysis we can better assess risk and potential impacts to nontarget habitats and organisms to ensure protection of trust resources.

RP083 (DIL-1117-840076) Atrazine increases burrowing in the freshwater clam, Corbicula fluminea.
Start time: 8:00 AM
Dillon-White, M¹, Flynn, K¹, ¹ Adelphi University, Biology Department, Garden City, New York, USA
Atrazine, the most commonly used herbicide in the U.S., is suspected of imitating estrogen and possibly changing burrowing behavior of freshwater clams. To assess this, three tanks with atrazine-contaminated water, at concentrations analogous to 0.1, 1, and 10 times the maximum EPA level for aquatic ecosystems (1.5, 15, and 150 ug/L, respectively) were set up. Estradiol at 100mg/L and the solvent alone were positive and negative controls. The solvent was no more than 1 mL 70% ethanol in 30L tanks. In each, six laboratory-adapted clams were placed on a 7 cm gravel surface and monitored for 72 hours. Clams were classed every 6 hours as completely, partially, or not burrowed and tanks were ranked by the percent of animals completely burrowed at each time. One-way analysis of variance (ANOVA) revealed a significant difference in percent burrowed among the treatment groups (p<0.001). Post hoc t-tests compared each treatment group to control ands estradiol, mid-, and high- atrazine groups were significantly different (p<0.005). The estradiol group burrowed 72% more than control, and mid- and high- atrazine groups burrowed 44 and 103% more respectively. This data suggests that temporary exposure to ecologically-relevant concentrations of atrazine considerably effects behavior in clams.