1a. Objectives (from AD-416):
Objective 1: Develop and (or) validate sensitive and accurate analytical tools to rapidly detect and quantify chemicals in food animals, food animal products, or other foods. Sub-objective 1.A: Validate the usefulness of rapid screening tests in ante-mortem matrices (urine, plasma, serum, saliva, milk, etc.) for predicting post-mortem tissue residues and(or) animal exposures to target compounds. Sub-objective 1.B: Develop and validate on-site detection methods for new and emerging xenobiotic residues. Objective 2: Investigate the kinetics of uptake, metabolism, distribution, and (or) the elimination of chemicals in and from food animals and (or) produce with the goal of reducing public exposure to chemical residues in foods. Sub-objective 2.A: Determine the effect of mediators of inflammation on drug metabolizing enzymes, drug clearance, and violative residues in food animals using flunixin meglumine as a model compound. Sub-objective 2.B: Determine the fate and distribution of chlorine dioxide gas in foods treated for pathogen or rot-organism remediation. Objective 3: Determine the fate of endogenous reproductive hormones, antibiotics, and or other chemicals, including biologically-active metabolites or degradation products in wastes of food animal or in food processing systems. Sub-objective 3A: Determine the partitioning of chemical residues into cream, curd, whey, and water fractions during milk processing. Sub-objective 3B: Determine the fate of estrogens in animal wastewater systems.
1b. Approach (from AD-416):
The broad objective of this project is to determine the fate of natural and manmade chemicals in food animals and in food animal systems (wastes, soil, water). Three broad classes of chemicals will be targeted for study: (1) veterinary drugs or feed additives administered to food animals under extra-label use conditions, (2) endogenous steroid hormones, and (3) novel developmental chemicals of potential utility to the livestock industry. Use of veterinary chemicals in an extra-label manner without knowledge of residue depletion kinetics has led to unsafe residues in meat products. Endogenous steroid hormones excreted by livestock are highly potent endocrine-disrupting compounds that are thought to disrupt the development of aquatic species after their entry into surface waters. Finally, chemical technologies developed by the ARS, e.g., chloroxyanions and nitro compounds, are active against Salmonella and E. coli pathogens in livestock immediately prior to slaughter, but the impacts of chemical residues in meat products have not been fully investigated for these compounds. Regardless of the chemical class being investigated, the development of sensitive and accurate analytical tools is critical completion of the objectives. Therefore, a significant portion of the project is devoted to developing the analytical tools required to ensure success of the project. The overall project goal is to understand the broad impact that chemical residues play in influencing food and environmental safety.
3. Progress Report:
Objective 1. An Atmospheric Solids Analysis Probe (ASAP) mass spectrometry method was developed and validated for the analysis of zilpaterol and ractopamine in biological matrices. The method is currently being developed for incurred residues of penicillin in swine tissues. An antibody towards the veterinary antibiotic paromomycin was generated in rabbits. The sensitivity and selectivity of the antibody towards paromomycin has been tested and an enzyme linked immunosorbent assay (ELISA) is being developed for use in measuring the paromomycin in animal matrices. The paramomycin antibody has been evaluated for accuracy, repeatability, selectivity, and sensitivity in an ELISA format. The paramomycin ELISA works well in pork muscle matrix, but not kidney matrices. Objective 2. A live phase zilpaterol feeding study was conducted in which sheep were exposed to trace levels of dietary zilpaterol. The presence of zilpaterol in urine and tissue samples was tested using three types of rapid screening tests (lateral flow test strips, ELISA, and Atmospheric Solids Analysis Probe mass spectrometry) and by UPLC-MS/MS. The sensitivity of each analytical method for accurately detecting and(or) quantifying zilpaterol was determined by comparing analytical results derived from each method to those obtained by UPLC-MS/MS. The fate of trace levels of zilpaterol in sheep was determined. Subcellular fractions were prepared from bovine liver to investigate the potential for sex and/or age to alter flunixin metabolism via alterations in liver enzyme activity and cytochrome P450 levels. Altered enzymatic activity may explain violative tissue residues. Assays were validated and fractions analyzed. Older cows were found to have statistically lower microsomal enzyme activity and P450 concentrations compared to younger heifers and steers. These data may explain the presence of violative flunixin residues, as they are almost exclusively found in older dairy cull cows. A study was completed on the fate of chlorine dioxide used for sanitizing alfalfa seeds and the presence of chloroxyanion residues on seeds and sprouts during the sprouting process. Studies were initiated and completed on testing the efficacy of chlorine dioxide gas to remediate fumonisin contaminated corn. Objective 3. A study investigating the partitioning of numerous pharmaceuticals and environmental contaminants into milk fractions (skim, cream, whey, and protein) was completed. Sample analysis was completed from the second year of a rainfall simulator study investigating the fate of estrogen compounds and estrogenic activity present in poultry manure. The field portion of the third year of study was completed comparing runoff post-application of farrowing swine manure vs. layer litter. Sample analyses are in process.
1. Residue-free alfalfa seed sanitation. Pathogenic bacteria, if present on seeds used to produce sprouts for human food, proliferate greatly during the sprout-making process. Chlorine dioxide gas is very effective at eliminating pathogens from a variety of fruits, vegetables, melons, seeds, and food preparation surfaces. Researchers at the Agricultural Research Service in Fargo, North Dakota demonstrated that even when high concentrations of chlorine dioxide gas are used to sanitize alfalfa seed used for edible sprout preparation, chemical residues, including chlorate and perchlorate, are absent in sprouts from those seeds. Further, chlorine dioxide treatment eliminated coliform bacteria and did not affect germination of seeds. The study suggests that chemical residues are not a major obstacle for the development of chlorine dioxide gas as a safe sanitizing agent for use in edible sprout production.
2. Immediate detection of drug residues. Zilpaterol is a ß-adrenergic feed additive that has been approved for cattle production in North and Central America and in South Africa, but its use is strictly banned by the European Union, China, and many other countries. Because zilpaterol use is legal in only a few countries, the rapid and sensitive detection of zilpaterol in live animals or in animal tissues is of interest to regulatory and trade officials worldwide. Researchers from the Agricultural Research Service in Fargo, North Dakota optimized two mass-spectrometry based techniques to allow the rapid, sensitive, and semi-quantitative screening of zilpaterol residues in animal urine and tissues in about 30 seconds per sample with little to no sample preparation needed. The new instrument-based techniques have the potential to be adapted for field use, and to be used for the rapid detection of numerous other chemical residues of importance to animal agriculture.
Feifarek, D.J., Shappell, N.W., Schoenfuss, H.L. 2018. Do environmental factors affect male fathead minnow (Pimephales promelas) response to estrone? Part 1. Dissolved oxygen and sodium chloride. Science of the Total Environment. 610-611:1262-1270. http://dx.doi.org/10.1016/j.scitotenv.2017.07.251.
Smith, D.J., Herges, G.R. 2018. Chloroxyanion residue on seeds and sprouts after chlorine dioxide sanitation of alfalfa seed. Journal of Agricultural and Food Chemistry. 66(8):1974-1980. https://doi.org/10.1021/acs.jafc.7b05953.
Shelver, W.L., Smith, D.J. 2018. Development of an immunochromatographic assay for the ß-adrenergic agonist feed additive zilpaterol. Food Additives & Contaminants: Part A. 35(8):1519-1529. https://doi.org/10.1080/19440049.2018.1463568.