Final Report: Rapid Test Kit for Quantifying Hormonal Activity in Animal Feeding Operation WastewaterEPA Contract Number: EPD07051
Title: Rapid Test Kit for Quantifying Hormonal Activity in Animal Feeding Operation Wastewater
Investigators: Fort, Douglas J.
Small Business: Fort Environmental Laboratories Inc.
EPA Contact: Manager, SBIR Program
Project Period: March 1, 2007 through August 31, 2007
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2007) RFA Text | Recipients Lists
Research Category: SBIR - Animal Waste and Waste to Energy , Small Business Innovation Research (SBIR)
A number of man-made environmental pollutants have the potential to interfere with endocrine function, and there is evidence of reproductive or endocrine dysfunction in wildlife species that have been exposed environmentally to such endocrine-disrupting contaminants. Equally alarming is the widespread discovery of pharmaceuticals in wastewater effluents and surface waters across the United States. For example, clofibric acid, ketoprofen, carbamazepine, and fluoxetine were found in Lakes Ontario and Erie in 2000 and 2002. Estrogenic activities have been found in several effluents, which altered vitellogenin induction, hormone titers, gonadal histology, and secondary sexual characteristics in caged fathead minnows. Because of the pharmaceuticals and hormones used in the confined animal feeding operations (CAFOs) for cattle, sheep, swine, and poultry, the potential release into the environment is high due to the highly refractory nature of these materials. Combined with the high potencies of the pharmaceuticals and potential positive interactions with other waste materials, these substances have the potential to seriously impact resident aquatic life.
Results from the present research program enabled development and validation of a short-term, in vitro Xenopus oocyte maturation test kit for evaluating the chronic impact of hormonal supplements from CAFO waste on aquatic wildlife. Three test compounds and three complex mixtures were evaluated during the proposed project. A two-tiered approach was used in which the six test materials or environmental test samples from CAFOs were tested in tier 1. Tier 1 culminated in the preparation of a standard guidance document for the performance of the proposed test method. In tier 2, joint binary mixtures studies were used to evaluate the joint responses of the steroids used in tier 1 and the complex wastewaters, as well as potential for interaction. A radio-fluororeceptor (oocyte membrane progesterone receptor [OMPR] and androgen receptor [AR]) binding assay was used to determine if the effects induced were the result of an alteration in steroid hormone receptor binding.
Results from the validation testing suggested that both ethinyl estradiol and dexamethasone inhibited germinal vesicle breakdown, whereas the androgen, β-trenbolone, potently stimulated oocyte maturation. β-trenbolone was appreciably more potent than ethinyl estradiol, and both were markedly more potent than the synthetic glucocorticoid, dexamethasone. Receptor binding studies using the kit indicated that ethinyl estradiol was bound primarily to the OMPR. Both dexamethasone and β-trenbolone bound to both the OMPR and AR, although β-trenbolone had a much greater binding affinity for both receptors than dexamethasone, which corresponded to their differing potencies. β-trenbolone is a strong androgen, but was significantly bound to the OMPR and AR, suggesting that the OMPR is somewhat precocious. Ethinyl estradiol bound only to the OMPR, indicating that materials with estrogenic or anti-estrogenic activities will primarily bind to the OMPR and inhibit germinal vesicle breakdown (GVBD). Materials with androgenic or anti-androgenic activity could bind to both receptors and either stimulate GVBD (androgenic activity) or inhibit GVBD (anti-androgenic properties), respectively. Dexamethasone, which bound to both receptors, inhibited GVBD by acting as an antiprogestin or anti-androgen. Tests with CAFO runoff mixtures suggested that trace levels of feed additives or subdermal livestock steroids released to the environment via waste runoff were capable of interfering with normal GVBD processes in the cultured oocytes. Further, samples collected from cattle, sheep, and hog CAFOs each contained varying endocrine disrupting chemicals (EDCs) activity and potency. Based on benchmarks, the following preliminary potency assessment was established:
- Progesterone-Induced: Cattle(+)≥hog(+)>>sheep(-)
- Androgen-Induced: Cattle(+)>>hog(+)>>sheep(-)
(+) = induced GVBD; (-) = inhibited GVBD
Joint mixture studies with CAFO mixture 1 (cattle) and ethinyl estradiol, dexamethasone, and β-trenbolone produced mean total toxic unit (TU) values of 2.32, 1.34, and 1.07, which were characteristic of strong antagonism, response addition, and concentration addition, respectively. Joint mixture studies conducted with CAFO mixture 2 (hog) and ethinyl estradiol, dexamethasone, and β-trenbolone generated mean total TU values of 2.14, 1.18, and 1.05, which were characteristic of antagonism, response addition, and concentration addition, respectively. Joint mixture studies conducted with CAFO mixture 3 (sheep) and ethinyl estradiol, dexamethasone, and β-trenbolone resulted in mean total TU values of 1.08, 1.44, and 2.24, which were characteristic of concentration addition, response addition, and strong antagonism, respectively. Again, mixtures with TU values near 1.0 (fiducial intervals overlap 1.0) have concentration additive frequencies of GVBD inhibition. Thus, the toxicity of the mixture was equal to the sum of toxicities of each component when expressed in equivalent terms and adjusted for relative potencies. The concentration additive response is indicative of toxicants that have identical modes of action. Mixture TU values slightly and substantially greater than 1.0 represent response addition (toxicants that acted similarly but not identical and were noninteractive) and antagonism (negative interaction), respectively. Overall, the joint mixture interaction studies suggested that mechanisms by which the cattle, and to a lesser extent swine, CAFO wastewater samples stimulated GVBD were consistent with both AR (trenbolone-like) and OMPR (progesterone-like) response. These results suggested the presence of strong androgens and possibly progestins in the wastewater. However, joint mixture studies with sheep CAFO wastewater were similar to ethinyl estradiol activity (inhibitory) and dissimilar to progestin or androgen activity, suggesting an estrogenic impact.
Results from the present studies suggested that each of the steroids tested in tier 1 was capable of either stimulating or inhibiting GVBD in a concentration-dependent manner. The potency of the stimulatory effects of CAFO sample 1 (cattle) was remarkably potent. Further, depending on the livestock in the CAFO, a different signature response might be observed based on the hormone supplements used. Radio-fluororeceptor binding studies suggested that the steroids alone and unknown steroids in CAFO wastewater bound to either the OMPR or the AR, or in some cases both receptors. Regardless of which supplements were used, each of the CAFO samples was capable of interfering with oocyte maturation. The EDC test kit appears to fit the criteria established for emerging tier 1 screening assays, which are currently being sought as potential additions to or replacements for the existing screening assay proposed for the endocrine disruptor screening program (EDSP), and for screening complex environmental samples thought to have EDC activity, such as CAFOs. The kit is relatively straightforward and can be commercialized following validation and final proofing. Standard addition joint mixture studies have demonstrated an additional attribute of the test kit, which is that the kit is capable of modes of action. Combined with the radio-fluororeceptor assay, the kit provides the ability to determine the type of activity (estrogen, progestin, or androgen), the extent of binding to the target receptors, and the potency of response. Because the test is relatively inexpensive compared to many of the other screening tests, samples can be tested simultaneously, and the data are acquired quickly; the proposed frog oocyte EDC test kit model is ideal for high-throughput testing of environmental samples from CAFOs and other nonpoint sources.