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United States Department of Agriculture

Agricultural Research Service

2010 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
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-ß-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, 17ß-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.

1. 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, produce leaner meat, and enhance athletic performance, this agent has a potential 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 more slowly after the fifth withdrawal day. Our analytical methods were able to 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.

2. Biological Activity of a-E2. Estradiol, a natural female hormone, can exist in two structural forms, 17 a-estradiol (a-E2) and 17ß-estradiol (ß-E2). Recently, a-E2 was measured in wastewater associated with dairy and swine concentrated animal operations. Because the biological effects of a-E2 on aquatic species have not been documented (those of ß-E2 are well characterized), it is impossible to assess the environmental risk associated with a-E2. Studies conducted by researchers at the ARS in Fargo, ND, and collaborators at St. Cloud State University (St. Cloud, MN) compared the effect of a-E2 to ß-E2 on sexually mature male and female fathead minnows, an organism that has a long history of use in evaluation of endocrine disruptors. Several endpoints were used to assess biological activity of the estradiol isomers. Data from this study suggest that ß-E2 is about 9-10 times more potent than a-E2 in the fathead minnow biological assay. These data will be used in risk assessments associated with agricultural practices and the presence of estrogenic compounds in groundwater.

3. 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 have 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 residues.

4. Discerning the Transport of Hormones in the Environment. Potent steroid hormones such as estrogen are frequently detected in environmental samples despite laboratory results suggesting that transport in soil/water systems 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 site 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.

5.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.

Review 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.

Last Modified: 11/30/2015
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