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United States Department of Agriculture

Agricultural Research Service


Location: Animal Metabolism-Agricultural Chemicals Research

2009 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
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.

1. Analytical Method for Sulfonamides for Use in Waste Systems. Long-term exposures of water systems to animal and human pharmacueticals have raised environmental health concerns. Because of their high volume usage, sulfonamides are one of the most commonly detected pharmaceuticals in environmental matrices. An UPLC -MS/MS method was developed that allows for the simultaneous measurement of 14 sulfonamides during a six minute chromatographic run. The method has been used to measure sulfonamide concentrations in soils, surface and ground water, as well as slurry manure from hog farms. Sulfamethazine was found in all the matrices at variable concentrations. Slurry manure concentrations are roughly one-thousand fold those measured in surface and ground waters. Sulfamethazine was found in top soils but not sub-soils. Sulfonamides were still present in the soil one year after the lagoon application demonstrating that sulfonamides are more persistent than previously reported. Sulfathiazole was found in the slurry manure but not in the ground water or soil. Use of this method allows the simultaneous tracking of various sulfonamide antimicrobials and can be used to generate data that will be employed to develop strategies that minimize the impacts of sulfonamides in the environment.

2. Impact of Dairy Farming on Estrogenicity of Surface Waters. The potential impact of a large confinement dairy (> 2,000 milking head) employing best-management practices on the estrogenicity and estrogen content of surface waters and drain-tile run-off was evaluated. Fifty-four thousand tons of dairy wastes were applied to approximately 2,000 acres of surrounding land. Estrogenic activities from pre- and post-application water samples were similar. In addition, all surface waters had activity below the proposed no observable effect concentration (NOEC) for estradiol. Estrogenicity of drain tile water samples after manure application increased less than 2-fold relative to pre-application water samples, but were still well below the proposed NOEC for estradiol. Chemical analyses of the applied dairy wastes allowed the determination of the chemical forms of estrogen present and which were present in the highest relative concentrations. Our data indicate that field application of animal wastes using best management practices, did not increase the biological estrogenic activity of surrounding surface waters.

3. Fate of Water Soluble Estrogen Metabolites in the Environment. Estrogens and androgens are natural hormones excreted in all vertebrate species; however, as animal agriculture becomes more intensive, excretion of large amounts of these natural compounds may cause endocrine disruption in wildlife. Laboratory studies have shown that steroid hormones should readily absorb to the organic matter of soil, however, they are frequently detected in environmental samples. We proposed that very water-soluble metabolites of these hormones, which are excreted from animals, are transported through the soil in the water fraction and are subsequently converted back to parent compound after transport. We synthesized a common estradiol metabolite, estradiol glucuronide, and used it in studies designed to determine its potential to move in various soil-water systems. The results indicate that the first 48 hours after urinary elimination is critical for the estrogen metabolite’s transport through soil into water systems. The study suggests that retention of animal wastes for at least 48 hours will likely eliminate water-soluble forms of estrogenic steroids to levels below those of environmental concern.

5.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.

6.Technology Transfer

Number of the New/Active MTAs (providing only)1
Number of Other Technology Transfer3

Review 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.

Last Modified: 4/19/2014
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