Passage in 1996 of the Food Quality Protection Act and amendments to the Safe Drinking Water Act reflected concerns regarding food and drinking water safety and required the U.S. Environmental Protection Agency (EPA) to develop a screening program, using appropriate validated test systems and other scientifically relevant information to determine whether certain substances may have an endocrine effect in wildlife and humans. The proposed work will result in the development of an assay that tests for hormonal substances capable of altering developmental and reproductive processes in aquatic animals exposed to waste runoff from animal feeding operations (AFOs). This project will incorporate recommendations of previous reviewers to: (1) expound on the predictability of the model and determine the propensity of false positive and false negative responses; (2) evaluate the potential for interactive effects in complex wastewaters from the AFOs; (3) demonstrate the quantitative nature of the model in relation to potential regulation of wastewater from AFOs; and (4) demonstrate the link between proposed Phase I and II activities. The primary goal of the proposed research is to develop and commercialize a rapid and cost-effective test kit to measure contaminants released into surface waters and groundwater from AFOs. Specifically, we will develop a 24-hour test model for evaluating the toxicological activity of the waste runoff from AFOs by monitoring the hormonally induced process of oocyte maturation in Xenopus laevis oocytes in vitro. Evaluation will entail testing neat chemicals with known activities to evaluate the potential for false positive and negative responses, complex wastewater samples collected from AFOs, and complex mixtures from AFOs spiked with endocrine disrupting chemicals (EDCs). The complex wastewater mixtures from the AFOs will be evaluated with and without the standard additions of known EDCs to evaluate the potential for additive, non-interactive responses and interactive responses including synergism and antagonism using a binary mixture model. Inhibition of germinal vesicle breakdown (GVBD) will be evaluated relative to the classical inhibitor ethinyl estradiol to establish a quantitative measure of estrogen equivalents. When GVBD stimulation or inhibition is detected, complementary progesterone and androgen receptor binding assays will be performed to determine if the effect is the result of receptor binding. The successful completion of the proposed technology will provide the scientific and regulatory community with an ecologically relevant, rapid means of evaluating the toxicological effects of pharmaceuticals and hormones released to the environment from AFOs. Supplemental
Keywords: small business, SBIR, EPA, animal feeding operations, AFOs, endocrine disrupting chemicals, EDCs, agricultural wastewater, waste runoff, drinking water safety, food safety, hormonal activity assay, amphibians
