RP029 (AGA-1117-854926) Contamination of Private Drinking Water Wells by Sulfonamide Antimicrobials from a Confined Animal Feeding Operation.
Start time: 8:00 AM
Aga, D.¹, Batt, A.¹, ¹ Chemistry Department, University at Buffalo, Buffalo, NY, USA
Samples from six private wells formerly used as source for drinking water by the residents of Washington County (Weiser, Idaho) were collected to assess the impact of a nearby confined animal feeding operation (CAFO) on the quality of the local groundwater. All six samples were found contaminated by two veterinary antibiotics, sulfamethazine (at concentrations between 0.076 to 0.22 to micrograms per liter) and sulfadimethoxine (at concentrations between 0.046 to 0.068 micrograms per liter). Analysis was performed using a validated solid phase extraction (SPE) and liquid chromatography with ion-trap mass spectrometry (LC/IT-MS/MS) method. Further, these groundwater samples contained elevated concentrations of nitrate and ammonia. Three of the sampled wells have nitrate levels that exceeded the maximum contaminant level set by the US Environmental Protection Agency for drinking water, with nitrate concentration as high as 39.1 mg/L. All but one well showed nitrate, which instead contained ammonia at 1.22 mg/L. Analysis of the nitrate and ammonia in these samples by isotopic ratio mass spectrometry indicated ¹⁵N characteristic of an animal or human waste source. Results from this study underscores the role of CAFO as an important source of antibiotic contamination of the groundwater. Prolonged exposure to low levels of antibiotics in the environment could result in a selective pressure which in turn favors the proliferation of antibiotic resistant bacteria. The detection of sulfonamides in groundwater previously used as source for domestic water supplies have important implications in the increasing emergence ofantibiotic resistance among pathogenic bacteria that affect human health.

RP030 (SHA-1117-823541) Estrogenic activity of swine wastewater treated by a lagoon constructed wetland system.
Start time: 8:00 AM
Shappell, N¹, Billey, L¹, Poach, M², Matheny, T³, Reddy, G⁴, Hunt, P³, ¹ Biosciences Research Laboratory, USDA-ARS, Fargo, ND, USA² Formerly Coastal Plain Soil, Water, and Plant Research Center USDA-ARS, Florence, SC, USA³ Coastal Plain Soil, Water, and Plant Research Center, USDA-ARS, Florence, SC, USA⁴ NC Agricultural &Technical State University, Greensboro, NC, USA
The estrogenic activity in livestock waste and treated wastewater effluents is of increased interest because of modern intense livestock production practices. We determined estrogenic activity(in vitro E-screen assay) along with Kjeldahl nitrogen (TKN) for swine wastewater as it was sequentially treated by a two stage anaerobic lagoon and a constructed wetland system. The wetlands were marsh-pond-marsh in design. Samples were collected from the manure pit through the wetland outlet in April, July, and November, 2004. Nitrogen treatment was similar to that reported for other treatment wetland systems. The wetlands were loaded with lagoon effluent at rates of 10 to 20 kg N ha^-1d^-1, and the N removal rates were 57 to 89%. However, the N treatment did not track estrogenic activity. Estrogenic activity of water from the manure pit was 900 pM estradiol equivalents(E₂ Eq)in both April and July. The E₂ Eqs dropped dramatically in the primary lagoons to 1% of pit values (12 to 14 pM). Furthermore, the estrogenic activity of lagoons wastewater was so low as it entered the wetlands in the April and July samples (3 pM E₂ Eq) that little to no change in activity was found across the wetlands. In the cooler temperatures of November, the E₂ Eq entering the wetlands was 100 pM, yet 94% of the activity was removed by the wetlands, while the TKN was reduced by 75% for wetlands. These data indicate that wetlands are useful in reducing both N and estrogenic activity from animal waste.

RP031 (HEN-1117-831067) Bioavailability of tylosin in environmental matrices.
Start time: 8:00 AM
Henderson, K.¹, Moorman, T.², Coats, J.¹, ¹ Iowa State University, Ames, IA, USA² USDA-ARS National Soil Tilth Laboratory, Ames, IA, USA
Tylosin is a veterinary antibiotic commonly used as a feed additive in livestock production for growth promotion and disease prevention. Tylosin is excreted by the livestock in urine and feces, and enters the environment via manure application. Little is known about the fate of tylosin in terrestrial and aquatic ecosystems. Understanding the bioavailability of pharmaceuticals in environmental matrices is particularly important considering they are often in an active form. In this study, the bioavailability of tylosin in soil and water was evaluated using Empore SDB-RPS ™ extraction disks. Incubation systems consisted of 100 ml of autoclaved pond water or 40 g agronomic soil, each spiked with tylosin to achieve a range of concentrations of tylosin (0.01 to 100 ppm). Empore disks were conditioned and added to treatment vessels for 24 hours. Disks were then extracted with 98:2 acetonitrile:glacial acetic acid, and water was extracted using Waters Oasis HLB ™ solid phase extraction cartridges. Soil was extracted with 85:15 acetonitrile:phosphate buffer pH 2.3, and concentrated under nitrogen flow. Determination of tylosin factors and metabolites was achieved using LC/MS/MS. Data suggest that tylosins remain tightly bound to soil particles, thus decreasing their bioavailability. In an aquatic system, sediments would likely become the sink for the tylosin residues, which would have implications for benthic organisms.

RP032 (CHE-1117-642244) Transformation of Tetracycline Antibiotics with Manganese Oxide and Mn(II) Ions.
Start time: 8:00 AM
Chen, W¹, Huang, C¹, ¹ Georgia Institute of Technology, Atlanta, GA, USA
Tetracyclines (TCs) are the group of antibiotics most widely used in veterinary medicines, nutrition and feed additives throughout the agricultural sector. Recently TCs have been detected in various aquatic environments, raising concerns of potential ecological and health risks. One the basis of TCs′ strong affinity to soils and sediments, transformation with soil minerals such as manganese oxides likely plays an important role in the fate and transport of TCs. Three members of TCs, tetracycline, oxytetracycline and chlortetracycline, were found to be highly susceptible to oxidation by MnO ₂ yielding Mn(II) ions. Kinetics of the above reactions were investigated carefully at various conditions and modeled by a surface reaction kinetic model to determine the parameters of reaction rate constant (k) and total activated reaction sites (S _T) (with r ² > 0.97 typically). The reaction rate decreases as MnO ₂ loading and the pH increase while decreases as TC loading increases. The reaction rate also slows sown in the presence of competing co-solutes such as Mg ²⁺ and Ca ²⁺ ions and dissolved natural organic matter. The experimental observations and preliminary kinetic modeling point to a complex surface reaction mechanism in which adsorption of TC to MnO ₂ surface is likely the rate-limiting step. Furthermore, TCs were found to be susceptible to degradation in the presence of Mn(II) ions and oxygen. This transformation reaction proceeds more rapidly at alkaline conditions than acidic conditions and can be of significant relevance in surface waters where appreciable concentrations of dissolved Mn(II) ions are often present. Overall this study demonstrates that degradation pathways involving manganese species will likely be important for the fate of TCs in the environment. Further studies to identify the degradation products of TCs and to better understand the above reactions are currently underway.

RP033 (MIL-1117-722556) Extrapolation of a molecular response to population impacts: vitellogenin in fish.
Start time: 8:00 AM
Miller, D.¹, Jensen, K.², Villeneuve, D.², Ankley, G.², ¹ US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, MidContinent Ecology Division, Grosse Ile, Michigan, USA² US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, MidContinent Ecology Division, Duluth, Minnesota, USA
A challenge in the field of ecotoxicology is the linkage of alterations at molecular and biochemical levels of organization to adverse outcomes in individuals and populations. In the present study, a predictive relationship between decreased vitellogenin concentration and decreased fecundity in female fathead minnows (Pimephales promelas) was derived from short-term, 21-d laboratory toxicity tests with fadrozole, 17-trenbolone, 17-trenbolone, prochloraz, fenarimol, and ketoconazole exposures. The relationship between decreased vitellogenin and decreased fecundity at the organismal level was utilized within a population model to translate changes in vitellogenin concentrations of the female fathead minnow to alterations in population growth rate. Application of the model requires only a life table, a measure of carrying capacity for a given population of interest, and estimation of vitellogenin concentrations within the fish of the study population. Example applications are provided using different projection scenarios for a population of fathead minnows exposed to chemical stressors that reduce vitellogenin concentrations in the female, decrease fecundity, and thus reduce recruitment to a population. This research approach could be applied to other species as a population-level analysis tool for use in interpreting the effects of multiple stressors that have a common measurable organismal level effect. This abstract does not necessarily reflect EPA policy.

RP034 (YON-1117-826411) Reconnaissance of 17 -estradiol in Delmarva watersheds receiving runoff from poultry litter-amended agricultural fields.
Start time: 8:00 AM
Yonkos, Lance¹, Fisher, Daniel¹, Van Veld, Peter², ¹ University of Maryland Wye Research and Education Center, Queenstown, Maryland, USA² College of William and Mary Virginia Institute of Marine Sciences, Gloucester Point, Virginia, USA
Nearly 1.6 billion lbs of poultry manure or litter are generated annually as a by-product of the Delmarva poultry industry. Disposal of this material is accomplished by application to regional agricultural fields as organic fertilizer, usually in excess of crop requirements, and often results in runoff of litter-associated contaminants into receiving waters following rain events. Of particular concern are steroid hormones, 17 -estradiol (E2), estrone (E1), and testosterone (T), excreted naturally by birds during development and persistent in dry manure until the time of field application. E2 concentrations in runoff can exceed 300 ng/L while effects concentrations (i.e. inhibition/disruption of sexual development and reproductive physiology) in fish and amphibians can occur at ≤20 ng/L. In a 2004 reconnaissance of agriculturally-influenced surface waters (streams, rivers) on Maryland's Eastern Shore, 52 of 93 sites investigated (55%) had detectable E2 levels (radioimmunoassay; method detection limit 18 ng/L). Concentrations ranged from 19 to 75 ng/L (average 29 ng/L). Samples were collected during or immediately after spring rain events from watersheds known to receive substantial inputs of poultry litter-associated agricultural runoff. Given the prevalence of litter application to fields in the selected watersheds it is highly likely that E2 in these samples had its origins in poultry litter. In follow-up sampling during fall low-flow conditions only 11 of 62 agricultural sites (17%) had detectable E2. An ephemeral pond associated with a large corn field, prime amphibian breeding habitat, had an E2 level of 245 ng/L immediately following the first rain event after poultry litter application. In addition, several sites within discharge plumes of municipal wastewater treatment plants had E2 at levels of 32 to 112 ng/L.

RP035 (JEN-1117-829785) Assessing the Ecological Risk of Androgenic Growth Promoters.
Start time: 8:00 AM
Jensen, K¹, Kahl, M¹, Durhan, E¹, Makynen, E¹, Wilson, V², Lazorchak, J³, Miller, D⁴, Gray, L², Ankley, G¹, ¹ U.S. Environmental Protection Agency, Duluth, MN, USA² U.S. Environmental Protection Agency, Research Triangle Park, NC, USA³ U.S. Environmental Protection Agency, Cincinnati, OH, USA⁴ U.S. Environmental Protection Agency, Grosse Ile, MI, USA
There is significant interest in environmental contaminants that adversely impact fish through disruption of the hypothalamic-pituitary-gonadal (HPG) axis. Although most work has focused on changes in the axis caused by chemicals that bind to and activate estrogen receptors, there are a variety of contaminants that can affect HPG function through interactions with the androgen receptor. For example, recent research with water associated with cattle feeding operations in the US have associated morphological alterations in fish collected from the field with in vitro androgenic activity in water samples from affected sites. Some of these responses could be related to naturally-excreted steroids; however, much of the beef production in the US utilizes anabolic androgenic materials to promote muscle mass growth in the animals. One of the most commonly used chemicals for this purpose is the synthetic androgen precursor trenbolone acetate. Two metabolites of the acetate, 17- and 17-trenbolone, are comparatively stable in animal waste and the environment (half-lives of about 260 d). Recent studies from our labs have shown that both isomers bind with high affinity to the fathead minnow (Pimephales promelas) androgen receptor, and are highly potent in 21-d reproduction studies with this species, masculinizing females and decreasing fecundity at water concentrations in the low ng/L range. In other studies by our group, androgenic activity and the occurrence of 17- and 17-trenbolone were evaluated in water samples adjacent to a beef feedlot in the Midwest. Whole water samples from the site induced androgenic activity in vitro, and contained 17- and 17-trenbolone at ng/L concentrations comparable to those that adversely affected fathead minnow reproduction in the lab. This presentation will provide an integrated overview of these studies, and identify research and monitoring needs for a broader-scale assessment of the ecological risk of androgenic growth promoters. Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy.

RP037 (KOL-1117-576974) The Occurrence and Fate of Steroidal Hormones in Surface Waters Impacted by Cattle Grazing and Animal Agriculture.
Start time: 8:00 AM
Kolodziej, E¹, Sedlak, D¹, ¹ Department of Civil and Environmental Engineering, University of California, Berkeley, Berkeley, CA, USA
To assess the occurrence and fate of steroid hormones arising from common animal agriculture practices, a suite of estrogens, androgens, and progestins was quantified in samples from surface waters impacted by cattle grazing at several locations in California. Steroid hormones were only detected in surface waters near dairy farms during wet weather periods, when overland flow occurred in fields where lagoon pond waste had been applied. Steroid hormones were detected in surface waters in rangeland where cattle had access to streams during the dry season. Steroid hormone concentrations in the small rangeland streams usually ranged from 0.1 to 5 ng/L. However, the estrogens estrone and 17a-estradiol were occasionally detected at concentrations above 10 ng/L. Samples collected from tributaries (i.e., total discharge <2 m3/s) downstream of the rangeland areas contained significantly lower concentrations of steroid hormones, indicating that dilution and attenuation are important factors controlling steroid hormone concentrations. To develop approaches for predicting the concentrations of steroid hormones from more readily accessible data, coliform bacteria were measured in samples in which steroid hormones were measured. Our data on steroid hormone occurrence suggest that measures such as fencing of streams to limit direct access by cattle and the use of riparian buffer strips may be the most effective approaches for reducing the concentrations of steroid concentrations in surface waters. Additional research is needed to assess the temporal and spatial variations in steroid hormone concentrations in surface waters and to assess the effect on aquatic organisms of short pulses of relatively high concentrations of steroid hormones.

RP038 (KIM-1117-685486) Vitellogenin Induction by Several Pharmaceuticals to Oryzias latipes.
Start time: 8:00 AM
Kang, Hee Joo¹, Kim, Kyung Tae¹, Kim, Hyun Soo¹, Kim, Pan Gyi¹, ¹ Yongin University, Yongin, Kyoggi-Do, Repulic of Korea
Endocrine disrupting effects of four pharmaceutical products were evaluated with fish. The test pharmaceuticals, sulfamethoxazole, sulfamethazine, oxytetracycline and tetracycline have been often detected in aquatic environment of Korea (from on going study of this research team). But the ecological hazard on receptors of various trophic levels has seldom been evaluated. In the present study, we conducted acute vitellogenin induction assay with a fish, Oryzias latipes. We analyzed vitellogenin induction by qualitative (Western blot) and quantitative (ELISA) assay. We used the fish groups where more than 90% individuals has plasma vitellogenin levels below the detection limit by ELISA. 17-estradiol (Sigma E-8875) was used as a positive control. E2 was dissolved in ethanol and diluted to 10ng/ml in rearing water just before use. Male and female fish were exposed to 10ng/ml E2 for 3-5 days. Aqueous solution of chemicals were exchanged every 2 days. Sulfamethoxazole, sulfamethazine, oxytetracycline and tetracycline were dissolved in ethanol and diluted 1-20ppm in rearing water just before use. Sulfamethoxazole 10, 5, 1 ppm exposure induced vitellogenin even at male fish. Sulfamethazine 10, 5, 1ppm could induced vitellogenin at male fish. Oxytetracycline 10, 5, 1ppm could induced vitellogenin with the fish. Tetracycline 20, 10, 5 ppm exposure could induced vitellogenin at male fish. Some pharmaceuticals such as sulfamethoxazole, sulfamethazine, oxytetracycline and tetracycline could give effects to male Oryzias latipes. They could induced vitellogenin under exposure from 1-20 ppm of chemicals at male Oryzias latipes.

RP039 (YON-1117-824977) Transport dynamics of 17 -estradiol via runoff from poultry litter-amended agricultural fields to surrounding surface waters.
Start time: 8:00 AM
Yonkos, Lance¹, Fisher, Daniel¹, Staver, Kenneth¹, ¹ University of Maryland Wye Research and Education Center, Queenstown, Maryland, USA
Nearly 1.6 billion lbs of poultry manure or litter are generated annually as a by-product of the Delmarva poultry industry. Disposal of this material is accomplished by application to regional agricultural fields as organic fertilizer, usually in excess of crop requirements, and often results in runoff of litter-associated contaminants into receiving waters following rain events. Of particular concern are sex steroids, 17 -estradiol (E2), estrone (E1), and testosterone (T), excreted naturally by birds during development and persistent in dry manure until the time of field application. Poultry litter samples collected from Delmarva broiler operations (n=8) prior to field application had mean (±SD) E2 and T levels (measured via radioimmunoassay) of 126 ± 23.7 g/kg and 42 ± 16.5 g/kg, respectively. Application of this material to fields at 3 tons/ac (typical for cropping corn) introduces approximately 350 mg E2/ac. Transport dynamics of poultry litter-associated E2 under two cropping strategies (conventional-till vs no-till) were monitored by employing two 35 acre instrumented (weather station, metered discharge flumes) research fields during 2000 and 2002 growing seasons. Intense rain following litter-application produced abundant runoff (29% lateral transport of precipitation) from the no-till field (soil surface left intact) while greater infiltration of rain on the conventional-till field (surface disked to 20 cm) produced significantly less runoff (8.4%). E2 in no-till runoff, where litter was applied directly to the soil surface, averaged 125 ng/L and resulted in transport of ∼ 2.7% of initially applied E2 from the field to surrounding waters. Integration of litter into the top 20 cm of soil on the conventional-till field limited contact of precipitation such that runoff contained only 42 ng E2/L and resulted in lateral transport of only 0.26% of applied E2. Concentration in a retention pond peaked at 82 ng E2/L and persisted above the 18 ng/L detection limit for < 21 days.