Progress 02/03/06 to 02/02/11

Outputs
Progress Report 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. 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. This is the final report of the 5 year project 5442-32000-012-00D, which was terminated in 2011 and replaced with project 5442-32000-014-00D. Substantial progress was made during the life of the project. For example, the metabolic fate and tissue residues of chlorate salts in swine, broilers, and lactating sheep were determined under anticipated commercial use conditions. A major finding was that a presumed metabolite of toxicological concern, chlorite, is not present in animal wastes or in tissues used for food and that tissue chlorate residues should fall well below thresholds for food safety concern being considered by the US FDA CVM. The major product of chlorate metabolism in live animals and in animal waste systems is chloride, a nutrient. Larger than expected levels of estradiol were detected in well water from fields receiving swine manure, but surface waters collected from fields amended with dairy manure contained estrogen levels below proposed regulatory action limits. It was hypothesized that soil reservoirs of estrogen are associated with small particles or as water soluble metabolites. Subsequent studies demonstrated that soil particles enhance estradiol persistence and that water soluble metabolites of estrogen have significant capacity for water transport and for conversion to parent estradiol. Estradiol is detected more frequently in soil layers nearer to the water table than in the upper, more highly organic layers, providing evidence of how estradiol contamination of groundwater can occur. Laboratory studies of composting, anaerobic and aerobic digesters, and chemical treatments and on-farm studies with constructed wetlands suggest that application of these technologies in animal waste process streams could reduce estrogen releases into the environment. Analytical methods were developed for chlorate salts in diverse matrices including serum, urine, feces, intestinal fluids, and milk. For chlorate analyses requiring high sensitivity, a liquid chromatography-mass spectrometric method was developed utilizing an 18-oxygen stable isotope internal standard. Immunologically based assays for sulfonamides were used to analyze effluents from wastewater treatment plants and swine rearing facilities and a multi-residue method was developed to simultaneously analyze structurally related sulfonamides in soil and water. The methods could be used to assist in monitoring the environmental fate of sulfonamides. Immunochemical methods to detect the beta-adrenergic agent zilpaterol were generated and validated using animal systems and these reagents were made commercially available through license agreements. A liquid chromatography-mass spectrometry method was also developed and validated for the measurement of exquisitely small concentrations of estradiol in surface and groundwater. This assay is being used in several studies investigating the fate of and remediation of estradiol in agricultural settings. The limitation of commercially available estrogen analysis kits for application to environmental estrogen analysis was also evaluated and compared to mass-spectral and cell-based analyses. Accomplishments 01 Safe residues after vegetable fumigation to eliminate pathogens. Chlorin dioxide gas fumigation of fruits, vegetables, and melons effectively kil pathogen contaminants and increase produce shelf-life. The technology ha not been used commercially because any residues remaining on fruits, vegetables, or melons after chlorine dioxide treatment have not been quantified or characterized. ARS scientists in Fargo, ND, in collaborati with Purdue University determined that essentially all of the residues remaining on chlorine dioxide treated vegetables are non-toxic, natural products. The data have been submitted to the US-Environmental Protectio Agency in support of a product registration for the use of chlorine dioxide gas for decreasing pathogen content and increasing shelf-life of vegetables. Decreasing pathogen loads on fruits and vegetables, without increasing risks of chemical residues, increases the safety of food for consumers.

Impacts
(N/A)

Publications

• Shappell, N.W., Hyndman, K., Bartell, S.E., Schoenfuss, H. 2010. Comparative biological effects and potency of 17a- and 17�-estradiol in fathead minnows. Aquatic Toxicology. 100:1-8. DOI:10.1016/j.aquatox.2010. 07.005.
• Schuh, M.C., Casey, F.X., Hakk, H., Desutter, T.M., Richards, K. G., Khan, E., Odour, P. 2011. An on-farm survey of spatial and temporal stratifications of 17�-estradiol concentrations. Chemosphere. 82:1683-1689.
• Derby, N.E., Hakk, H., Casey, F.X.M., Desutter, T.M. 2011. Effects of composting swine manure on nutrients and estrogens. Soil Science. 176(2) :91-98.
• Zitnick, K.K., Shappell, N.W., Hakk, H., DeSutter, T.M., Khan, E., Casey, F.X.M. 2011. Effects of liquid swine manure on dissipation of 17�- estradiol in soil. Journal of Hazardous Materials. 186:1111-1117.
• Smith, D.J., Taylor, J.B. 2011. Chlorate analysis in matrices of animal origin. Journal of Agricultural and Food Chemistry. 59:1598-1606.
• Shrestha, S.L., Bai, X., Smith, D.J., Hakk, H., Casey, F.X.M., Larsen, G.L. , Padmanabhan, G. 2011. Synthesis and characterization of radiolabeled 17�- estradiol conjugates. Journal of Labelled Compounds and Radiopharmaceuticals. Vol. 54(5):267-271.
• Shelver, W.L. 2011. Generation of antibody and development of an enzyme- linked immunosorbant assay for the feed additive roxarsone. Food and Agricultural Immunology. 22(2):171-184.
• Fan, Z., Casey, F.X., Hakk, H., Larsen, G.L., Khan, E. 2011. Sorption, fate, and mobility of sulfonamides in soils. Water, Air, and Soil Pollution. 218(1):49-61.
• Shelver, W.L., Smith, D.J. 2011. Immunochemical-based zilpaterol measurement and validation in urine and tissues. Food and Agricultural Immunology. 22(3):247-258.
• Hakk, H., Sikora, L. 2011. Dissipation of 17B-estradiol in composted poultry litter. Journal of Environmental Quality. 40:1560-1566.
• Schuh, M.C., Casey, F.X.M., Hakk, H., DeSutter, T.M., Richards, K.G., Khan, E., Odour, P.G. 2011. Effects of field-manure applications on stratified 17B-estradiol concentrations. Journal of Hazardous Materials. 192:748-752.
• Wang, J., Li, H., Shelver, W.L., Wang, Z., Li, Q.X., Li, J., Xu, T. 2011. Development of a monoclonal antibody-based, congener-specific and solvent- tolerable direct enzyme-linked immunosorbgent assay for the detection of 2, 2',4,4'-tetrabromodiphenyl ether in environmental samples. Analytical and Bioanalytical Chemistry. doi: 10.1007/s00216-011-5283-x.

Progress 10/01/09 to 09/30/10

Outputs
Progress Report 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. 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. To support cooperative studies investigating the fate of chlorate salts in food animals, a colorimetric procedure was developed and validated for measuring high concentrations of chlorate residues in serum and milk. The assay was used to analyze samples (>300) from a pharmacokinetic study in sheep, a ewe lactation study, and a sheep yearling study. In addition, an ion chromatography-based assay was developed to measure chlorate salts in urine of sheep. A study was initiated which tested the hypothesis that the kinetics of flunixin (a non-steroidal anti-inflammatory agent) in cattle differ after intravenous and subcutaneous administration. Violative flunixin residues are being detected in beef cattle carcasses and it is thought that cattle are being treated by subcutaneous or intramuscular drug administration. The FDA-approved method for flunixin administration to beef cattle is by intravenous infusion. EPA has requested data to use for the assessment of risks of application of animal manures as fertilizers to frozen fields. In collaboration with the USDA-ARS Coshocton laboratory, runoff samples from control or �treated� watersheds (turkey or swine manure applied to frozen fields) were analyzed for nutrients and estrogenic activity. Data are currently being processed, with initial observations indicating that all estrogenic activity is below the proposed �Lowest Observable Effect Concentration� for estradiol. Swine manure was applied again this winter, and samples have been collected for analyses. In a collaborative effort with the Aquatic Toxicology Laboratory, St. Cloud State University (St. Cloud, MN) the biological activities of 17 a- and 17-�-estradiol (E2) isomers in fat-head minnows were compared. This work has established the relative potency of the estradiol isomers, and the relative environmental risk of 17 a-E2. We conducted a controlled lab study suggesting that a window of time exists for estradiol sulfate, an endogenous urinary metabolite of estradiol, to move through the environment before soil bacteria degrade it. Results of these studies indicate that whereas parent compound is poorly transported in soil/water systems mammalian metabolites readily move through soil. Thus, a mechanism has been discovered which may explain the frequent detection of these endocrine disrupting compounds in the environment. Despite frequent detection of the potent steroid hormone, 17�-estradiol, in the environment, laboratory results indicate it should be strongly and rapidly bound to soil. Field waters contained dissolved substances and undissolved particulate matter of various sizes. We developed a method to quantify particulate matter in water and then conducted studies to measure the amount of dissolved and particulate-bound estradiol in field waters. Upwards of 20% of the applied estradiol was associated with particulate matter. These results have provided a putative mechanism for the transport of this endocrine disruptor in the environment. Significant Activities that Support Special Target Populations Navajo and Sioux student interns from the University of Arizona (Tucson, AZ) and United Tribes Technical College (Bismarck, ND), respectively, were hired under the auspices of the ARS Northern Plains Area Native American Internship Program. The program has the goal of introducing Native Americans to the Agricultural Research Service and to a research setting. To compliment the student�s academic interests in Animal Science, and our lab�s capabilities, a study was conducted investigating the fate of the anti-inflammatory agent, flunixin in cattle. Violative flunixin residues in beef cattle tisses are increasingly being detected by the USDA Food Safety and Inspection Service. The interns participated in the live phase of the study and made significant contributions this portion of the study. Data from this study will be published. Accomplishments 01 Depletion of Illicit Drug Residues from Horses. Zilpaterol is a growth promoter approved for use in cattle in Mexico, South Africa, and the United States. Because of its ability to improve feed efficiency, produc leaner meat, and enhance athletic performance, this agent has a potentia to be used illegally. Zilpaterol use is forbidden in many countries and consequently it is necessary to have valid analytical methods capable of demonstrating the animal products are free from Zilpaterol. Since the beneficial effects of using growth promoters are also useful in species other than cattle, a study conducted by ARS researchers at Fargo, ND, examined the depletion of zilpaterol residues in urine of horses fed a supplement containing zilpaterol. The study demonstrated that zilpaterol levels in urine dropped rapidly for the first five days, but declined mo slowly after the fifth withdrawal day. Our analytical methods were able detect the presence of zilpaterol in horse urine for up to 21 days after the zilpaterol exposure. These analytical methods could be very powerful tools for detecting the off-label use of zilpaterol in horses and other species. 02 Biological Activity of a-E2. Estradiol, a natural female hormone, can exist in two structural forms, 17 a-estradiol (a-E2) and 17�-estradiol ( E2). Recently, a-E2 was measured in wastewater associated with dairy and swine concentrated animal operations. Because the biological effects of E2 on aquatic species have not been documented (those of �-E2 are well characterized), it is impossible to assess the environmental risk associated with a-E2. Studies conducted by researchers at the ARS in Far ND, and collaborators at St. Cloud State University (St. Cloud, MN) compared the effect of a-E2 to �-E2 on sexually mature male and female fathead minnows, an organism that has a long history of use in evaluatio of endocrine disruptors. Several endpoints were used to assess biologica activity of the estradiol isomers. Data from this study suggest that �-E is about 9-10 times more potent than a-E2 in the fathead minnow biologic assay. These data will be used in risk assessments associated with agricultural practices and the presence of estrogenic compounds in groundwater. 03 Multiresidue Sulfonamide Assay. Sulfonamides are commonly used in food animals to treat infection, prevent disease, and improve animal growth. However, sulfonamides believed to be associated with agricultural use ha been found in surface waters within the United States. An analytical method was developed by ARS Researchers in Fargo, ND, using ultra performance liquid chromatography-triple quadrupole-tandem mass spectrophotometry (UPLC-TQ-MS/MS) to simultaneously analyze 14 sulfonamides in six minutes. This method was applied to water, soil, and manure slurry from swine rearing facilities and the results demonstrated some sulfonamides were present in the manure slurry. When the manure slurries were used as liquid fertilizer, sulfonamides were measured in soil while water samples generally had non-detectable sulfonamide residu 04 Discerning the Transport of Hormones in the Environment. Potent steroid hormones such as estrogen are frequently detected in environmental sampl despite laboratory results suggesting that transport in soil/water syste would be improbable. Two mechanisms were discovered by ARS and North Dakota State University researchers (Fargo, ND) that help to explain hormone transport. One involves hormone transport as water-soluble conjugates, which may readily be transported off an animal production si to be subsequently degraded back to parent hormone. The other involves association of steroid hormones with suspended organic particles, which are highly mobile in the environment. These results are very significant to understand the fate and transport behavior of these compounds, and provide the basis needed to devise means to reduce their environmental release.

Impacts
(N/A)

Publications

• Smith, D.J., Oliver, C.E., Shelver, W.L., Caesar, T., Anderson, R.C. 2009. Clorate Metabolism in Pure Cultures of Escherichia Coli 0157:H7 Pretreated with Either Nitrate or Chlorate. Journal of Agricultural and Food Chemistry. 57:10216-10224. DOI:10.1021/jf901513f
• Shelver, W.L., Hakk, H., Larsen, G.L., DeSutter, T.M., Casey, F.X.M. 2010. Development of an Ultra-High-Pressure Liquid Chromatography-Tandem Mass Spectrometry Multi-Residue Sulfonamide Method and Its Application to Water, Manure Slurry, and Soils from Swine Rearing Facilities. Journal of Chromatography A. 1217:1273-1282. DOI:10.1016/j.chroma.2009.12.034.
• Shelver, W.L., Thorson, J.F., Hammer, C.J., Smith, D.J. 2010. Depletion of Urinary Zilpaterol Residues in Horses as Measured by ELISA and UPLC-MS/MS. Journal of Agricultural and Food Chemistry. 58:4077-4083. DOI:10. 1021/jf904253t.
• Shappell, N.W., Elder, K.H., West, M.S. 2010. Estrogenicity and Nutrient Concentration of Surface Waters Surrounding a Large Confinement Dairy Operation Using Best Management Practices for Land Application of Animal Wastes. Environmental Science and Technology. 44:2365-2371.
• Anderson, R.C., Huwe, J.K., Smith, D.J., Stanton, T.B., Krueger, N.A., Callaway, T.R., Edrington, T.S., Harvey, R.B., Nisbet, D.J. 2010. Effect of nitroethane, dimethyl-2-nitroglutarate and 2-nitro-methyl-propionate on ruminal methane production and hydrogen balance in vitro. Bioresource Technology. 101:5345-5349.
• Oliver, C.E., Beier, R.C., Hume, M.E., Horrocks, S.M., Casey, T., Caton, J. S., Nisbet, D.J., Smith, D.J., Krueger, N.A., Anderson, R.C. 2010. Effect of chlorate, molybdate, and shikimic acid on Salmonella enterica serovar Typhimurium in aerobic and anaerobic cultures. Anaerobe. 16:106-113.
• Willard, R.R., Shappell, N.W., Meekin, J.H., Talbot, N.C., Caperna, T.J. 2009. Cytochrome P450 expression erofile of the PICM-19H pig liver cell line: Potential application to rapid liver toxicity assays. In Vitro Cellular and Developmental Biology - Animals. 46(1):11-19.
• Duringer, J.M., Craig, A.M., Smith, D.J., Chaney, R.L. 2010. Uptake and transformation of soil [14C]-trinitrotoluene by cool-season grasses. Environmental Science and Technology. 44:6325-6330.

Progress 10/01/08 to 09/30/09

Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations An isotopically-labeled internal standard was synthesized and a mass- spectral based analytical method for sodium chlorate residues developed for matrices from swine and sheep. The method has been used in support of studies examining the absorption of chlorate salts from food animals. Studies investigating the fate of [14C]-labeled nitro-analogs of amino acids were initiated in ruminal fluid, but technical issues have temporarily stalled the progress. A gas chromatographic assay was developed to investigate fate of unlabeled nitro-amino acid analogs in ruminal fluid. Lysimeters underneath three animal waste treatment plots and an untreated plot were sampled across a single growing season. Estradiol detections were frequent, and it was concluded that prior groundwater exposures to estradiol existed, and that colloidal transport may be the mechanism of transport. Lagoon waste digesters were designed and constructed to allow the fate of steroid hormones to be determined under anaerobic conditions. Steroids were rapidly, although not completely, degraded. A study to compare three methods of steroid hormone measurement was conducted with several matrices. Immunoassays overestimated the concentration of steroid hormones in common environmental matrices, while LC/MS results were accurate in relatively clean matrices. Soil core samples collected over two years in fields treated or not treated with manure slurry as a fertilizer were analyzed. Manure slurry treatment did not lead to elevated steroid hormone detections. However, in lower soil layers, hormones were detected more frequently. Therefore, the movement into lower, anaerobic soil layers reduces the degradation potential of hormones. The frequent detection of estrogens in field samples suggests that they may be transported through water as polar (water-soluble) metabolites. This hypothesis was tested using polar estradiol glucuronide metabolites. A short window of opportunity (48h) exists for the glucuronide to be transported in the environment before its degradation back to parent estradiol. Run-off water samples collected from control fields or from fields that had manure applied when they were frozen were assayed for estrogenic activity and for nutrient content. Analyses of these samples using E- Screen are essentially complete. These data will be the first report of estrogenic activity in run-off collected from well-characterized watersheds. Spring samples have been collected and analyzed in a cooperative study designed to compare the estrogenic activity of surface waters associated with non-animal agricultural sites and sites receiving agricultural animal wastes. In collaboration with EPA, and USGS, three assays for estrogenicity (two mammalian- and one yeast-cell based) will be also compared. Summer and fall sampling points are in progress. Dairy waste often contains significant concentrations of the alpha- estradiol, relative to concentrations of beta-estradiol. Biological potency of each isomer on fat head minnows is being evaluated. Extraction and chemical analyses of test water is in progress. St. Cloud State University has completed the exposure experiments. Significant Activities that Support Special Target Populations Two Navajo and Choctaw student interns from the University of Arizona were hired for eight weeks under the auspices of the ARS Northern Plains Area Native American Internship Program. The program has the goal of introducing Native Americans to the Agricultural Research Service and to a research setting. To compliment the student�s academic interests in Animal Science, and our lab�s capabilities, a study was initiated investigating the kinetics of chlorate salts in sheep. Chlorate salts are being developed as a potential pre-harvest food safety tool to reduce the occurrence of human pathogens in and on animals at slaughter. The interns participated in all phases of the study and made significant contributions to the conduct of the live phase and analytical portions of the study. Data from this study will be published. Technology Transfer Number of New/Active MTAs(providing only): 1 Number of Other Technology Transfer: 3

Impacts
(N/A)

Publications

• Thompson, M.L., Casey, F.X., Khan, E., Hakk, H., Larsen, G.L. 2009. Occurrence and Pathways of Manure-borne 17beta-Estradiol in Vadose Zone Water. Chemosphere. 76:472-479.

Progress 10/01/07 to 09/30/08

Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations This report serves to document progress conducted for CRIS project 5442- 32000-012 entitled �Metabolic variables affecting the efficacy, safety, and fate of agricultural chemicals�. A series of experiments was performed to determine the effects of chlorate salts on pure cultures of E. coli O157:H7 and to determine the metabolic fate of chlorate within these cultures. The formation of the putative toxic intermediate, chlorite, was directly measured using ion chromatography with radiochemical detection. Studies were also initiated to develop a mass- spectral based analytical method sufficient to measure chlorate concentrations in the gastrointestinal contents and feces of swine. The general method should be applicable to a variety of matrices. A series of experiments was initiated to determine the metabolic fate of a nitro substituted amino acid within ruminal cultures. This amino acid analog has been shown to be a potent inhibitor of ruminal methane production and understanding its ruminal fate will provide an indication of whether it might be safe for use in commercial livestock systems. An experiment was conducted with laboratory scale lagoons to measure the fate of steroid hormones under anaerobic conditions similar to those found in swine waste lagoons. Parent steroids were degraded to methane gas, two water-soluble metabolites, and to intermediates that bind tightly to lagoon solids. Anaerobic digestion of lagoon waste provided an effective tool to reduce the introduction of steroid hormones into the environment from commercial livestock systems. Estrogenic activity was measured in a variety of aqueous samples provided by the Environment Agency (UK) using an E-Screen assay. Cooperating research groups independently measured estrogens in the same samples using ELISA and an in vivo bioassay using fish. Results from all three methods were reproducible at higher estrogen concentrations (>1 ng/L), but at the lowest concentrations (0.5ng/L) results were more variable (>20%). This research will help to establish uniform testing methods for estrogens and help to establish the relative usefulness of each analytical method. All of the accomplishments relate to National Program 108, "Food Safety" component 1.1, "Pre-harvest food Safety", 1.1.1, "Methodology"; 1.1.3 "Ecology, Host Pathogen and Chemical Residue Relationships"; and 1.1.4 "Intervention Strategies". Technology Transfer Number of New/Active MTAs(providing only): 1 Number of New Patent Applications filed: 1

Impacts
(N/A)

Publications

• Wagner, S.A., Mostrum, M.S., Hammer, C.J., Thorson, J.S., Smith, D.J. 2008. Adverse effects of zilpaterol administration in horses: three cases. Journal of Equine Veterinary Science 28(4):238-243.
• Smith, D.J., Craig, A.M., Duringer, J.M., Chaney, R.L. 2008. Absorption, tissue distribution, and elimination of residues after 2,4,6- trinitro[14C]toluene administration to sheep. Environmental Science and Technology 42(7):2563-2569.
• Shappell, N.W., Vrabel, M.A., Madsen, P.J., Harrington, G.E., Billey, L.O., Hakk, H., Larsen, G.L., Beach, E.S., Horwitz, C.P., Ro, K.S., Hunt, P.G., Collins, T.J. 2008. Destruction of estrogens using Fe-TAML/peroxide catalysis. Environmental Science and Technology 42:1296-1300.
• Casey, F.X., Oduor, P., Hakk, H., Larsen, G.L., Desutter, T.M. 2008. Transport of 17beta-estradiol and testosterone in a field lysimeter. Soil Science 173:456-467.
• Shelver, W.L., Shappell, N.W., Franek, M., Rubio, F.R. 2008. ELISA for sulfonamides and its application for screening in water contamination. Journal of Agricultural and Food Chemistry 56:6609-6615.
• Oliver, C.E., Magelky, B.K., Bauer, M., Cheng, F., Caton, J.S., Hakk, H., Larsen, G.L., Anderson, R.C., Smith, D.J. 2008. Fate of chlorate present in cattle wastes and its impact on Salmonella typhimurium and E. coli 0157:H7. Journal of Agricultural Chemistry 56:6573-6583.
• Fan, Z., Casey, F.X., Hakk, H., Larsen, G.L. 2008. Modeling of Coupled Degradation, Sorption, and Transport of 17beta-Estradiol in Undisturbed Soil. Water Resources Research 173:456-467.

Progress 10/01/06 to 09/30/07

Outputs
Progress Report 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. 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. 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. 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. 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,Presentations for NonScience Audiences: 6

Impacts
(N/A)

Publications

• 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.
• 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.
• 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.
• 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.
• 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.

Progress 10/01/05 to 09/30/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Consumers in the United States increasingly expect inexpensive meat products that are free of chemical and (or) bacterial contamination; they also demand that livestock production systems have minimal to negligible impacts on the environment. These expectations are difficult to achieve because of the continued development of large farms having extraordinary animal densities. These intensive food-animal production systems generally require the use of agricultural chemicals to reduce the negative impact of pests and disease, to increase animal efficiencies, and to decrease the incidence of human pathogens in slaughter animals. While the use of these chemicals is beneficial for animals and producers, their residues in meat products must be safe to consumers. In addition, extremely dense numbers of animals in localized areas excrete quantities of steroid hormones that may result in ground water contamination. Data are not available to support or refute the safe use of new chemical entities (oxy-anions, nitro-compounds) being developed for use in food animals. Furthermore, the impacts that some established chemicals (antibiotic feed additives) and endogenous hormones (steroids) have in soils and water systems are unknown. Research conducted within the auspices of this CRIS project falls within Program 108, Food Safety has the specific objectives of: 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. 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. Develop sensitive and accurate analytical tools to rapidly detect and quantify agriculturally important chemicals studied under objectives 1 and 2. These studies are relevant to animal producer groups such as the National Cattlemens Beef Association, The Pork Board, the National Turkey Federation, and the US Poultry and Egg Association because their members benefit from the applied aspects of the project. Industrial concerns (i.e., EKA Chemicals, Neogen, Abraxis LLC, Testing Components Corportation) have provided material support of the research through CRADA agreements, Trust Agreements, by providing technical expertise, and(or) by licensing technologies gained from the research. Data obtained from studies within the project will be reported to regional, national, and international academic, regulatory, industrial, and governmental scientists. 2. List by year the currently approved milestones (indicators of research progress) 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 Year 1 (2006) o Conduct chlorate metabolism study in rats o Validate an assay for the measurement of oxidized myoglobin and oxidized hemoglobin o Synthesize sodium [36Cl]chlorite o Conduct a chlorate residue and metabolism study in swine o Initiate synthesis of [14C]nitro-compounds Year 2 (2007) o Conduct in vitro studies investigating the stability of chlorate in blood o Conduct a chlorite metabolism study in rats o Investigate the ruminal metabolism of chlorate o Conduct a chlorate residue and metabolism study in broilers o Continue chemical synthesis of nitro-compounds Year 3 (2008) o Conduct and in vivo study investigating the kinetics of chlorate uptake and formation of met-hemoglobin o Initiate studies on the site of chlorate absorption o Conduct a chlorate residue and metabolism study in turkeys o Initiate in vitro nitro-compound metabolism study Year 4 (2009) o Conduct in-vivo metabolism of nitro compound in ruminants o Conduct chlorate residue and metabolism study in sheep Year 5 (2010) o Conduct follow-up studies on chlorate and nitro-compounds in test species 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. Year 1 (2006) o Conduct study investigating the fate of steroid hormones in ground water beneath a commercial hog farm o Conduct laboratory fate and transport studies for chlorate salts. Year 2 (2007) o Conduct estrogenicity analysis using an e-screen assay of ground water samples o Conduct ELISA analysis of ground water samples o Initiate lab-scale composting studies investigating the kinetics of chlorate degradation o Conduct laboratory fate and transport studies on sulfathiazole Year 3 (2008) o Initiate studies on the fate of endogenous hormones in a laboratory- scale lagoon system o Conduct laboratory fate and transport studies on neomycin o Determine rate of chlorate degradation in mixed excreta of cattle Year 4 (2009) o Investigate the fate of chlorate in a laboratory scale lagoon o Determine the rate of chlorate degradation in mixed excreta of swine o Initiate laboratory scale composting studies with sulfathiazole Year 5 (2010) o Determine lab-scale degradation study of endogenous hormones in manure/slurry storage system Objective 3. Develop sensitive and accurate analytical tools to rapidly detect and quantify agriculturally important chemicals studied under objectives 1 and 2. Year 1 (2006) o Validate the LC/MS/MS steroid assay for use in environmental matrices o Initiate chlorate LC/MS/MS method Year 2 (2007) o Develop and validate NMR method for 35Cl o Validate LC/MS/MS chlorate method in animals tissues o Compare quantitative results for endogenous hormones obtained from LC/MS/MS method to ELISA and E-screen assay Year 3 (2008) o Validate 35Cl NMR method to quantify and speciate chlorine residues in biological matrices o Develop chlromatographic methods for nitro-compounds Year 4 (2009) o Develop and validate LC/MS/MS method for target nitro-compounds 4a List the single most significant research accomplishment during FY 2006. Destruction of estrogenic compounds catalyzed by Fe-TAMLRG. Program 108, Food Safety, Component 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships. Estrogenic residues have been found in surface water as a result of both agricultural and municipal wastewater release. Fe-TAMLRG is a compound developed by Carnegie Melon scientists that, in the presence of hydrogen peroxide, effectively catalyses the destruction of environmental contaminants such as dyes, drugs, and colored paper-mill effluent. We demonstrated that Fe-TAMLRG rapidly catalyzed the destruction of four natural and one synthetic estrogens that are of potential concern in agricultural and municipal wastes. Utilization of this treatment has the potential to reduce negative environmental impacts from agricultural and municipal wastewater. 4b List other significant research accomplishment(s), if any. Development of 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 swine 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 swine 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 enterica 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. 4c List significant activities that support special target populations. A Navajo college student was hired for eight weeks under the ARS/NPA Native American Internship Program which has the goal of introducing Native Americans to the field of agricultural science. The student initiated studies on the anaerobic digestion of 17?-estradiol in laboratory-scale lagoon simulators and successfully obtained samples, purified and analyzed them, and summarized the data. The research will be presented formally to the staff at the BRL, and to Department of Animal Sciences at the University of Arizona. Additionally, the data will presented (by the student) as a portion of a regional competition for under-represented minorities in the sciences. The ND Turkey Federation expressed concern about antibiotic usage in their industry and potential environmental impacts of antibiotics excreted in turkey wastes. In response to these concerns work was initiated to evaluate the antiobiotic activity of poultry litter from birds reared by industry standards using antibiotics. As this type of litter is typically applied to fields as fertilizer it is important to assess its biological activity in light of potential microbial resistance. 4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and the US Pork Board. Each year thousands of US consumers become ill because they have eaten food products that are contaminated with pathogenic bacteria. Intense efforts have been made to eliminate pathogenic organisms from food animals before they are slaughtered and to remove contamination after slaughter. A new pre- harvest food safety strategy utilizing chlorate salts has been developed that has been shown to greatly reduce, or even eliminate gram-negative pathogens from live swine. Use of this new feed additive has not yet been approved by regulatory organizations because it is not known whether residues present in edible tissues of treated animals would represent a health risk. The purpose of this study was to quantify residues of a novel chlorate based feed additive in edible tissues of swine. For all of the doses tested, chlorate residues in liver, kidney, muscle, and fat fell well below amounts that the FDA have estimated to be safe. The major metabolite of chlorate was chloride, a nutrient already present in almost all human food sources. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A Material Transfer Agreement (MTA) has been in place with Dr. Joseph Wang, Bio Scientific Co. to evaluate ractopamine monoclonal antibodies (November, 2005). A non-funded cooperative agreement (No. 58-5442-6-082FN) has been established with Scientists from Chinese Academy of Inspection & Quarantine (Drs. Xiaohua Qi & Yiyang Dong) to serve as a co-investigator for the National Natural Science Foundation of China Major International (Regional) Joint Research Project, Microfluidic Immunochemical Research for the Analysis of Growth Promoters in Foods of Animal Origin. (June, 2006) Ractopamine and Zilpaterol immunoassay reagents have been requested by Bruce Ritter, President, ELISA technologies, Inc., FL. A material transfer agreement was established on 7/18/2006. A Trust Fund Cooperative Agreement has been established between the ARS and the National Cattlemens Beef Association to determine variables affecting the efficacy of chlorate salts proposed for use in beef cattle production as a food safety tool (No. 58-5442-6-416; June 2006). As part of a Material Transfer Agreement (MTA) with USDA-ARS and Carnegie Mellon Institute, Fe-TAMLRG was evaluated as a catalyst in the destruction of estrogenic compounds. Provided data to GreenOx Catalysts, Inc. as part of a Premanufacturing Notice package submitted to the EPA as required to obtain permission for sales and distribution of Fe-TAMLRG for unlimited field use in closed systems and discharge into water streams. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presented data on chlorate residues in tissues of swine at the Pre- and Post-harvest Pork Safety Meetings sponsored by the US Pork Board; Des Moines, IA May 24-25, 2006. Requested by the Deputy Director of Public Prosecutions, Department of Justice, Hong Kong to provide a written deposition or to serve as an expert witness regarding a case alleging the illegal marketing of doped hogs. (November, 2005). Request declined. Presented data on environmental fate and transport of steroid hormones originating from a farm setting at the Public Health and Worker Safety Meetings sponsored by the National Pork Board in Des Moines, IA May 23, 2006. Crop, Soil and Agronomy News, Vol 51:4-5. Feb. 2006, First Profile of Estrogenic Activity in Regional Surface Waters. Inquiries about use of TAML in the destruction of estrogens after USDA- ARS press release and talk at American Chemical Society 10th Annual Green Chemistry & Engineering Conference from: Nature, Associate Editor, Risk Policy Report, New Scientist, Frontiers (Ecological Society of America), Pittsburgh Tribune-Review, Water Environment Federation, and National Public Radio (Living on Earth). In addition, information from the press release has been posted on over 14 sites on the world wide web.

Impacts
(N/A)

Publications

• Shelver, W.L., Keum, Y., Li, Q.X., Fodey, T.L., Elliott, C.T. 2005. Development of an immunobiosensor assay for the beta-adrenergic agonist zilpaterol. Meeting Abstract. VIIIth International Conference on AgriFood Antibodies, Sept. 6-9, 2005, Chester, UK.
• Shelver, W.L., Kim, H., Li, Q.X. 2005. Development of a monoclonal antibody based elisa for the beta adrenergic agonist zilpaterol. Meeting Abstract. VIIIth International Conference on AgriFood Antibodies, Sept. 6- 9, 2005, Chester, UK.
• Shappell, N.W., Smith, D.J. 2005. Ergovaline movement across CACO-2 cells. In Vitro Cellular and Developmental Biology - Animals 41:245-251.
• Shappell, N.W. 2006. Estrogenic activity in the environment: municipal wastewater effluent, river, ponds and wetlands. Journal of Environmental Quality 35:122-132.
• Shelver, W.L., Keum, Y., Li, Q.X., Fodey, T.L., Elliott, C.T. 2005. Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol. Food and Agricultural Immunology 16(3):199-211.
• Shappell, N.W., Billey, L.O., Poach, M.E., Matheny, T.A., Reddy, G.B., Hunt, P.G. 2005. Estrogenic activity of swine wastewater treated by a lagoon constructed wetland system. SETAC North American 26th Annual Conference, Baltimore, MD, Nov. 13-17, 2005.
• Thompson, M.L., Casey, F.X., Fan, Z., Hakk, H., Larsen, G.L. 2005. In situ subsurface soil studies on the occurrence, persistence, and pathways of 17beta-estradiol. Meeting Abstract. 2005 ASA-CSSA-SSSA International Annual Meetings, Salt Lake City, UT, Nov. 6-10, 2005.
• Smith, D.J., Oliver, C.E., Caton, J.S., Anderson, R.C. 2005. Effect of sodium [36Cl]chlorate dose on total radioactive residues and residues of parent chlorate in beef cattle. Journal of Agricultural and Food Chemistry 53:7352-7360.
• Kim, H., Shelver, W.L., Xu, T., Li, Q.X. 2005. Automated flow fluorescent immunoassay for the insecticide thiamethoxam. Meeting Abstract. Pacifichem 2005, Honolulu, Hawaii, Dec. 15-20, 2005.
• Smith, D.J., Anderson, R.C. 2005. Effect of sodium [36Cl]chlorate dose on total radioactive residues and residues of parent chlorate in swine. Proceedings of the 6th International Symposium on the Epidemiology & Control of Foodborne Pathogens in Pork (SafePork 2005), Sept. 6-9, 2005, Rohnert Park, CA, pp. 142-144.
• Shelver, W.L., Smith, D.J. 2006. Tissue residue and urinary excretion of zilpaterol in sheep treated for 10 days with dietary zilpaterol. Meeting Abstract. Fifth International Symposium on Hormone and Veterinary Drug Residue Analysis Meeting, Antwerp, Belgium, May 16-19, 2006.
• Shappell, N.W., Ro, K.S., Madsen, P., Horowitz, C., Hunt, P.G., Collins, T. J., Vrabel, M. 2006. Degradation of estradiol and ethinylestradiol with taml oxidant activator and hydrogen peroxide. Meeting Abstract. 10th Annual Green Chemistry & Engineering Conference, Washington, DC, June 26- 30, 2006.
• Wang, G., Rodrigues, W., Agrawal, A., Philips, H., Abolencia, E., Nuguyen, M., Smith, D.J., Shelver, W.L. 2006. Stabilization of a ractopamine enzyme conjugate in aqueous solution, a rapid and convenient immunoassay method for the detection of ractopamine. American Chemical Society Abstracts. San Francisco, CA, Sept. 10-14, 2006.
• Smith, D.J., Byrd Ii, J.A., Anderson, R.C. 2006. Effect of dose on residues and disposition of an experimental 36cl-chlorate product in broilers. American Chemistry Society Abstracts. San Francisco, CA, Sept. 10-14, 2006.
• Shelver, W.L., Smith, D.J. 2006. Tissue residues and urinary excretion of zilpaterol in sheep treated for 10-days with dietary zilpaterol. Journal of Agricultural and Food Chemistry 54:4155-4161.
• Kim, H., Shelver, W.L., Hwang, E., Xu, T., Li, Q.X. 2006. Automated flow fluorescent immunoassay for part per trillion detection of the neonicotinoid insecticide thiamethoxam. Analytica Chimica ACTA 571:66-73.
• Roberge, M.T., Hakk, H., Larsen, G.L. 2006. Cytosolic and localized inhibition of phosphodiesterase by atrazine in swine tissue homogenates. Food and Chemical Toxicology 44:885-890.
• Qi, X., Shelver, W.L., Dong, Y., Zou, M. 2006. Microfludic immunochemical ractopamine analysis. American Chemical Society Abstracts, September 10-14, 2006, San Francisco, CA.
• Edrington, T.S., Callaway, T.R., Smith, D.J., Genovese, K.J., Anderson, R. C., Nisbet, D.J. 2006. Effects of ractopamine HCl on Escherichia coli O157:H7 and Salmonella in vitro and on intestinal populations and fecal shedding in experimentally infected sheep and pigs. Current Microbiology. 53:82-88.