2007 Annual Report 1a.Objectives (from AD-416) Objective 1: Determine metabolic variables (rates of absorption, tissue and microbial biotransformation, excretion) that positively or negatively influence the practical use of novel pre-harvest food safety chemicals in food animals. Objective 2: Determine the fate of endogenous animal hormones, novel pre-harvest food safety compounds, and antibiotics in animal wastes, including their transport through soil and water, and develop intervention strategies that reduce their environmental impact. Objective 3: Develop sensitive and accurate analytical tools to rapidly detect and quantify agriculturally important chemicals studied under objectives 1 and 2. 1b.Approach (from AD-416) The broad objective of this proposal is to determine the fate of chemicals in food animals and in the environment (excreta, soil, water) after elimination from food animals. We will study endogenously produced steroid hormones, novel developmental oxyanions, novel developmental nitro-compounds, and antibiotics. Endogenous steroid hormones (estrogens) are highly potent endocrine-disrupting compounds that may concentrate in intensive food-animal production settings. Novel developmental compounds show promise for food-safety applications in ruminant, non-ruminant, and avian food animals. Specifically we plan to. 1)determine the metabolic variables (i.e., absorption, tissue and microbial biotransformation, rates of excretion) that positively or negatively influence the food safety (i.e., tissue residues) of developmental oxyanions such as chlorate salts and novel nitro-compounds such as 2-nitropropanol in food animals;. 2)determine the fate of steroid hormones, antibiotics, and developmental compounds in manure management systems of animals and in soils with the goal of gaining an understanding of the impact that residues of such chemicals may have in intensive food animal production settings; and. 3)we will develop analytical tools for the accurate measurement and(or) identification of these analytes or their metabolites. 3.Progress Report Optional, none. 4.Accomplishments Development of a Sodium Chlorate Feed Additive as a Food Safety Tool. Program 108, “Food Safety”, Components 1.1 “Pre-harvest Food Safety” and 1.1.3 “Ecology, Host Pathogen and Chemical Residue Relationships”. Studies were conducted in market broilers to determine the identity of chemical residues in edible tissues after treatment with an investigational chlorate-based water additive. It was demonstrated that residues of sodium chlorate in edible tissues of broilers will not limit the development of chlorate as a food safety tool. The chlorate-based product is being developed as a feed/water additive because it inhibits the growth of gram-negative pathogens such as E. coli O157:H7 and Salmonella in live animals. We determined that tissue residues fell below safe tissue concentrations estimated by the US FDA Center for Veterinary Medicine. This technology has the potential to be a safe and effective pre-harvest tool to reduce the incidence of pathogens in live animals prior to entering the human food chain. Metabolism of Sodium Chlorate in Rats. Program 108, “Food Safety”, Components 1.1 “Pre-harvest Food Safety” and 1.1.3 “Ecology, Host Pathogen and Chemical Residue Relationships”. Studies published in the early 1980’s on the fate of the chloroxyanion, chlorate, used as a “bleaching” agent during municipal water treatment plants indicated that chlorate was metabolized to, and excreted as, the toxic agent chlorite in rats. Because chlorate is not metabolized to chlorite by cattle, swine, or poultry, a study was conducted to verify or refute earlier chlorate metabolism work with rats. Using modern analytical methods, it was proven in an unambiguous manner that chlorite is not excreted by rats and that chlorite is not present in rat tissues after chlorate administration. These results clearly demonstrated that chlorate is converted to a nutrient (chloride ion) in an important lab species and that food animals such as cattle, swine, and chickens are not different from lab species in this regard. Further, it was demonstrated that earlier reports of chlorite being excreted by rats were due to faulty analytical methods used by earlier investigators. Results of this study are consistent with the notion that chlorate salts could safely be used as a pre-harvest tool to safely eliminate pathogens from animal carcasses. Fate of Sodium Chlorate in Gastrointestinal Fluids of Cattle. Program 108, “Food Safety”, Components 1.1 “Pre-harvest Food Safety” and 1.1.3 “Ecology, Host Pathogen and Chemical Residue Relationships”. Chlorate is converted to large amounts of chloride ion by cattle, exactly where this conversion took place was unknown. A study was conducted that clearly showed that substantial quantities of sodium chlorate may be converted to chloride ion in gastrointestinal fluids of cattle and that the diets of cattle did not greatly influence the conversion of chlorate to chloride ion. Conversion of chlorate to chloride ion within the digestive tract reduces the amount of chlorate available to kill pathogens, a factor that must be taken into account in the development of chlorate as a pre-harvest food safety tool. Fate of Steroidal Hormones in Waste Systems. Program 108, “Food Safety”, Components 1.1 “Pre-harvest Food Safety” and 1.1.3 “Ecology, Host Pathogen and Chemical Residue Relationships”. Steroidal hormones are constantly released into the environment by man-made and natural sources. The goal of this study was to obtain experimental evidence for the persistence and fate of 17beta-estradiol and testosterone in soil under aerobic and anaerobic, as well as, native and sterile conditions. Testosterone was metabolized to carbon dioxide to a much greater extent (10-fold) than 17beta-estradiol in native soil under aerobic conditions. Methane production was very limited for testosterone under anaerobic conditions, while it was nonexistent for 17beta-estradiol. Metabolism of these hormones was determined to be microbially-mediated because no metabolites were produced under sterile conditions. A majority of each hormone dose was tightly bound to soil particles (humic substances), even under sterile conditions. These findings suggest that previous estimates of the persistence and risk of these hormones in the environment might be overestimated due to their high aerobic metabolism by soil biota and reduced bioavailability resulting from non-extractable sorption to soils. Fate of Estrogenic Hormones in Lagoon Wetlands. Program 108, “Food Safety”, Components 1.1 “Pre-harvest Food Safety” and 1.1.3 “Ecology, Host Pathogen and Chemical Residue Relationships”. Hormonal activity in livestock waste and treated wastewater releases is of interest because of intense livestock production practices. A lagoon, constructed wetland system was evaluated for efficacy in removal of estrogenic activity from swine wastewater at a swine birthing facility. Wetlands reduced estrogenic activity 83-93%. Nutrient removals were typical for treatment wetlands; nitrogen 59-75%, and orthophosphate (a form of phosphorus) 0-18%. Expected seasonal differences were observed (limited data). Good agreement was found between the E-screen (a bioassay) values and the equivalent concentrations determined by chemical analyses. The most persistent estrogenic compound was identified as estrone, a breakdown product of estradiol. Constructed wetlands were effective in producing water with estrogenic activity below the lowest equivalent E2 concentration known to have an effect. Validation and Harmonization of Analytical Methods for Estrogens. Program 108, “Food Safety”, Components 1.1 “Pre-harvest Food Safety” and 1.1.3 “Ecology, Host Pathogen and Chemical Residue Relationships”. Project NORMAN (Network of Reference Laboratories for Monitoring of Emerging Environmental Pollutants) was created by Environment Agency of UK in part to create a network of expert reference laboratories and to encourage the validation and harmonization of analytical methods. To this end, estrogenic activity was assessed using an E-Screen assay in a variety of aqueous samples provided by the Environment Agency. Cooperating research groups independently assessed estrogenic hormones in the same samples by ELISA, and in vivo estrogenic activity by vitellogenin induction in fish exposed to samples. Results from this cooperative effort will help to establish uniform testing methods for estrogenic compounds and to establish the relative usefulness of each method. 5.Significant Activities that Support Special Target Populations A Navajo college student was hired for eight weeks under the auspices of the ARS Northern Plains Area Native American Internship Program which has the goal of introducing Native Americans to agricultural research. The student’s field of study at Arizona State University is Equine Science. To compliment the student’s academic interest, and to compliment the lab’s capabilities, a study was initiated investigating the fate of zilpaterol in horses. Zilpaterol was approved by the US FDA in 2006 as a leanness enhancing agent for use in cattle, but it has the potential for illicit use in performance horses as a doping agent. The study documented some physiological effects of zilpaterol in horses and the length of time that zilpaterol could be detected in urine of treated horses. Results of the research were presented formally to the staff at the BRL, and to the Department of Animal Sciences at the University of Arizona. In addition, the student will present the data in poster form as a portion of a regional undergraduate scientific competition. Data from this study will be published. 6.Technology Transfer Number of active CRADAs and MTAs 3 Number of new commercial licenses granted 1 Number of non-peer reviewed presentations and proceedings 10 Number of newspaper articles and other presentations for non-science audiences 6 Review Publications Oliver, C.E., Craigmill, A.L., Caton, J.S., Anderson, R.C., Smith, D.J. 2007. Pharmacokinetics of ruminally-dosed sodium chlorate in beef cattle. Journal of Veterinary Pharmacology and Therapeutics 30:358-365. Fan, Z., Casey, F.X., Hakk, H., Larsen, G.L. 2007. Persistence and Fate of 17beta-estradiol and testosterone in agricultural soils. Chemosphere 67(5):886-895. Horrocks, S.M., Jung, Y., Huwe, J.K., Harvey, R.B., Ricke, S.C., Carstens, G.E., Callaway, T.R., Anderson, R.C., Ramlachan, N., Nisbet, D.J. 2007. Effects of short-chain nitrocompounds against Campylobacter jejuni and Campylobacter coli in vitro. Journal of Food Science. 72:M50-M55. Shappell, N.W., Billey, L.O., Forbes, D., Poach, M.E., Matheny, T.A., Reddy, G.B., Hunt, P.G. 2007. Estrogenic activity and steroid hormones in swine wastewater processed through a lagoon constructed-wetland system.. Environmental Science and Technology 41(2):444-450. Smith, D.J., Anderson, R.C., Huwe, J.K. Effect of sodium [36cl]chlorate dose on total radioactive residues and residues of parent chlorate in growing swine. Journal of Agriculture and Food Chemistry 54:8648-8653. Fan, Z., Casey, F.X., Hakk, H., Larsen, G.L. 2007. Discerning and Modeling the Fate and Transport of Testosterone in Undisturbed Soil. Journal of Environmental Quality 36:864-873. Van Emon, J.M., Shelver, W.L. 2007. Journal of Agricultural and Food Chemistry 55:3749-3749. Oliver, C.E., Bauer, M.L., Caton, J.S., Anderson, R.C., Smith, D.J. 2007. The in vitro reduction of sodium [36Cl]-chlorate in bovine ruminal fluid. Journal of Animal Science 85:2059-2068. Smith, D.J., Byrd, J.A., Anderson, R.C. 2007. Total radioactive residues and residues of [36Cl]chlorate in market size broilers. Journal of Agricultural and Food Chemistry 55:5898-5903. Hakk, H., Smith, D.J., Shappell, N.W. 2007. Tissue residues, metabolism, and excretion of Na[36Cl]O3 in rats. Journal of Agricultural and Food Chemistry 55:2034-2042.