Location: Food Animal Metabolism Research
2021 Annual Report
Objectives
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.
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.
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.
Objective 4: Develop and/or validate rapid screening assays for the detection of environmental chemicals relevant to U.S. food production.
Objective 5: Determine levels and sources of dioxins and related compounds in the domestic food supply. Provide food safety agencies with data to confirm or refute the wholesomeness and competitiveness of beef, pork, chickens, turkeys and/or catfish.
Objective 6: Determine the uptake, metabolism (in vitro or in vivo), distribution, excretion, and fate of environmental contaminants with the goal of developing pharmacokinetic rate and volume constants pertinent to residue depletion, selection of marker compounds, and calculation of withdrawal intervals.
Approach
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.
Progress Report
This is the final report for project 3060-32420-001-00-D which began in February 2016 and terminated in January 2021. See the report for the replacement project 3060-32420-003-00-D, “Detection and Fate of Environmental Chemical and Biological Residues and their Impact on the Food Supply” for additional information.
Objectives 1 and 4, which are very similar due to the consolidation of two project plans, focus on the development and/or validation of analytical tools to rapidly detect and quantify chemicals in food-animal matrices. To this end, substantial effort was expended developing atmospheric pressure-based mass-spectrometric assays for the detection and quantitation of environmental and veterinary chemicals. Specifically, atmospheric solids analysis probe (ASAP), modified desorption electrospray ionization (mDESI), direct analysis in real time (DART), and electrospray ionization inlet ionization (ESII) mass spectrometric technologies were pursued to determine their suitability for the rapid and semi-quantitative chemical residues in urine and tissues of food animals. These techniques are very rapid (typically less than 10 minutes per sample, including sample preparation time) and are selective for a wide range of environmental contaminants and for chemicals used in veterinary practice, either legally or illegally. In testing each technology, results were validated using animal matrices which contained incurred residues that had been previously quantified using ‘gold standard’, quantitative liquid chromatography-mass spectrometric methods. Depending upon the atmospheric pressure method tested, sheep exposed to even trace-level ractopamine and/or zilpaterol exposures (commensurate with accidental residual contamination) were correctly identified by testing either tissues or urine. Sheep exposed to as little as one-thousandth of the normal zilpaterol dose could be positively identified. Similarly, sheep fed at one-five hundredth of the regular dietary ractopamine were reliably detected using atmospheric methods and urine analysis. Conversely, unexposed animals were correctly identified as having no residue. A variety of chemical analytes (perfluoroalkyl compounds, brominated flame retardants, beta-agonists, antibiotics, and anti-inflammatory drugs) have been tested using atmospheric ionization technologies as have a variety of animal-derived matrices including plasma, urine, oral fluid, lung, liver, kidney, and skeletal muscle.
Significant effort was also expended on testing and characterizing field-based detection methods. For example, a sensitive, selective, inexpensive, and rapid lateral flow assay (similar to an over-the-counter pregnancy test) was developed for the beta-agonist zilpaterol. The test was designed to be used on-site with minimal training, with results available in about 10 minutes. The accuracy and sensitivity of the assay was verified in tissues and urine from animals exposed to zilpaterol. This simple and inexpensive assay could be used to determine accidental, illegal, or purposeful zilpaterol exposure. In a similar vein, we demonstrated that an inexpensive lateral flow test strip could rapidly detect penicillin residues in urine of commercial sows, and that penicillin’s presence in urine accurately predicted violative penicillin residues in edible tissues. The use of an inexpensive and easy to use test strip could allow the differential marketing of penicillin-free sows and sows retaining drug residues.
Objectives 2 and 6 of the completed project plan focused primarily on the uptake, metabolism, distribution, and/or elimination of chemicals in food animals or food systems. To this end, the absorption of two important classes of dust-borne brominated flame retardants was demonstrated to occur in rats (a model species); but absorption was not equal within, or across, flame retardant class. A finding of importance to risk assessors was that after absorption, flame retardants generally migrated to fat (a component of red meat), but not liver (which has much lower consumption rates than red meat). An additional study demonstrated that some, but not all, polybrominated flame retardants (PBDEs), an important class of environmental pollutants, are readily absorbed by laying hens and transferred to eggs. Highly brominated PBDEs (having 10 bromine atoms) were poorly absorbed and were rapidly excreted by hens with relatively little opportunity for transfer into eggs. In contrast, PBDEs containing four or five bromine atoms were well absorbed and up to 27% –an extraordinary amount– of the doses were eliminated in eggs during a short 7-day study period. These data will inform risk assessors on relevant human background exposures to BDEs and possible exposure scenarios in the event of a contamination event.
A series of studies clearly demonstrated that when low dose chlorine dioxide is properly used for sanitation, chemical residues in edible cantaloupe or tomato cannot be distinguished from untreated produce, even when very sensitive analytical techniques are used. These studies were conducted with both [36Cl]-labeled (radioactive) chlorine dioxide and non-labeled chlorine dioxide. The Environmental Protection Agency reviewed residue data generated from these studies and approved the use of low dose chlorine dioxide gas to preserve and sanitize cantaloupes and tomatoes.
Accomplishments
1. Persistent organic pollutant transfer to chicken eggs. Polybrominated diphenyl ethers (PBDEs) were used as flame retardants in consumer products for decades but are now considered long-lasting environmental pollutants. Although PBDE levels in environmental samples have generally declined in recent years, specific PBDEs remain at elevated levels. Food animal exposures to these PBDEs are a concern because PBDEs in meat, milk, or eggs could serve as sources to consumers. Unfortunately, very little is known about PBDE absorption and elimination in food animals. Experiments conducted by ARS researchers in Fargo, North Dakota, were designed to determine the absorption, distribution, and excretion of three major PBDEs in laying hens and to determine if PBDEs are transferred to eggs. Researchers learned that PBDE absorption was not uniform across compound; PBDEs having high number of bromines (10) were relatively poorly absorbed compared to PBDEs with moderate numbers (5 or 6) of bromines which were highly absorbed. Some PBDEs were readily transferred to eggs, especially in the yolk. Fatty tissues including meat and skin of chickens also contained relatively high PBDE levels. The study demonstrated that individual PBDEs are not equivalent with respect to absorption after dietary exposure and that chicken eggs and meat could serve as sources of human exposure if an on-farm PBDE contamination were to occur. Regulators and risk assessors will use these data to model human exposures in instances of problematic environmental contamination.
2. PFOA incorporation into alfalfa and bioavailability. Perfluorooctanoic acid (PFOA) was a chemical surfactant used for decades in many consumer products such as textiles and cooking pan coatings. As a result of its long-term use, PFOA is ubiquitous in the environment and humans, but the understanding of PFOA transfer from soil to plants to animals is poor. Experiments conducted by researchers in Fargo, North Dakota, were designed to determine the uptake of PFOA by alfalfa from soil and the bioavailability of PFOA in rats after consumption of PFOA residues incurred into alfalfa. PFOA uptake by alfalfa was fast and it accumulated mainly in the leaves. PFOA residues in alfalfa were readily absorbed by rats and were quickly excreted in urine. After a 2-day depuration period less than 0.5% of the dosed PFOA was measured in tissues such as the liver, blood, kidney, and skin. The study demonstrated that PFOA residue in alfalfa are quickly absorbed after consumption, but are also rapidly eliminated so that minimal tissue accumulation occurs. These data will be used by risk assessment agencies and policy makers in the possibility of PFOA contamination events.
Review Publications
Hakk, H., Pfaff, C.M., Lupton, S.J., Singh, A. 2020. Absorption, distribution, metabolism, and excretion of three [14C]PBDE congeners in laying hens and transfer to eggs. Xenobiotica. 51:335-344. https://doi.org/10.1080/00498254.2020.1860269.
Chakraborty, P., Shappell, N.W., Mukhopadhyay, M., Onanong, S., Rex, K., Snow, D. 2020. Surveillance of plasticizers, bisphenol A, steroids and caffeine in surface water of River Ganga and Sundarban wetland along the Bay of Bengal: occurrence, sources, estrogenicity screening and ecotoxicological risk assessment. Water Research. https://doi.org/10.1016/j.watres.2020.116668.
Shappell, N., Shipitalo, M., Billey, L.O. 2020. Estrogenicity of agricultural runoff: A rainfall simulation study of worst-case scenarios using fresh layer and roaster litter, and farrowing swine manure. Science of the Total Environment. 750:141188. https://doi.org/10.1016/j.scitotenv.2020.141188.
Lupton, S.J., Hakk, H. 2020. Perfluorooctanoic acid (PFOA) uptake by alfalfa (Medicago sativa) and bioavailability in Sprague-Dawley rats. Journal of Food Protection. 84:688–694. https://doi.org/10.4315/JFP-20-389.
