Location: Food Animal Metabolism Research
2021 Annual Report
Objectives
Objective 1: Determine the absorption, distribution, metabolism, and excretion of emerging and legacy chemicals in food animals.
Sub-objective 1.A: Determine the metabolism and disposition of [14C]-nitrofurazone in broiler chickens.
Sub-objective 1.B: Determine the ADME of [14C]-PBDEs 47, 99, and 153 in laying turkeys.
Sub-objective 1.C: Determine the ADME of 1,3,7,8-tetrabromo [14C]-dibenzo-p-dioxin in laying hens.
Sub-objective 1.D: Determine the ADME of a defined mix of PFAS, including perfluorohexane sulfonic acid (PFHxS) in lactating cattle.
Sub-objective 1.E: Determine the fate of PFAS originating in a contaminated water source during the life cycle of laying hens.
Sub-objective 1.F: Determine the ADME of [14C]-(-)-trans-'9-tetrahydrocannabinol (THC) and/or [14C]-cannabidiol (CBD) in lactating dairy goats.
Sub-objective 1.G: Determine the accumulation and depuration kinetics of THC and CBD in feedlot cattle supplemented with dietary hemp.
Sub-objective 1.H: Evaluation of cellular uptake, translocation, and toxicity of microplastics using cell models.
Sub-objective 1.I: Determination of the fate of microplastics in laying hens.
Sub-objective 1.J: Determination of the uptake and depuration of microplastics in lactating dairy goats.
Objective 2: Develop and validate sensitive and accurate rapid analytical tools to detect emerging and legacy residues in food animals and food animal systems.
Sub-objective 2.A: Develop ambient ionization mass spectrometric detection and quantitation techniques of chemicals in matrices easily collected from live animals (blood, hair, urine, saliva).
Sub-objective 2.B: Develop ambient ionization mass spectrometric detection and quantitation techniques of chemicals in postmortem matrices (blood, tissues).
Objective 3: Determine levels and sources of emerging and legacy chemical or biological residues in the domestic food supply.
Sub-objective 3.A: In cooperation with regulatory agencies, determine the levels of dioxins, furans, and PBDEs in the U.S. meat supply.
Sub-objective 3.B: Determine the source(s) contributing to high background levels of PBDEs in commercial turkey.
Approach
Consumers loathe the idea of chemical residues in milk, meat, and eggs even though quantifiable risk of harm from chemicals in U.S. livestock products is exceedingly low. Regardless, consumers equate trace-levels of chemical residues in food with poor product quality and safety. Consequently, producers, regulatory officials, industry representatives, and consumers agree that chemical residues in food should be minimized to the greatest extent possible.
We propose to conduct absorption, distribution, metabolism, and excretion (ADME) studies on legacy and emerging chemicals for which significant data gaps exist. These chemicals include hemp-derived cannabinoids, a legacy antibiotic (nitrofurazone), halogenated persistent pollutants, and environmentally relevant microplastic contaminants (Objective 1). Basic ADME studies will allow the science-based selection of target matrices (saliva, urine, milk, liver, kidney, fat, etc.) and ‘marker compounds’ (parent compound or metabolites) of critical importance to the development of practical rapid screening technologies (Objective 2). ADME studies also provide data from which pre-harvest residue accumulation rates and post-exposure depuration rates can be calculated. Such data will facilitate the marketing of essentially residue-free animals in instances of known animal exposures. In some cases, especially for highly potent halogenated hydrocarbons and emerging contaminants, the U.S. government has a vested interest in ensuring that residues in remain well below regulatory thresholds. Under Objective 3, we propose a continuation of a 25-year cooperative effort with the USDA Food Safety and Inspection Service (FSIS) to survey the U.S. meat supply for dioxins and dioxin-like chemical residues. This survey has been critical to the discovery of environmental sources of dioxins and has been critical to reducing food animal exposures. We also propose to continue discovery efforts to elucidate contamination sources of livestock-based foods.
Collectively, the goal of this proposal is to develop science-based solutions that minimize consumer exposures to chemical residues in food animal products.
Progress Report
Research efforts relating to Objective 1, “Determine the absorption, distribution, metabolism, and excretion of emerging and legacy chemicals in food animals”, included studies on the fate of 1- and 2-monobutyrins in simulated intestinal fluids. 1- and 2 Monobutyrin are feed additives used in Europe to improve gastrointestinal health in poultry. These studies demonstrated that hydrolysis of monobutyrin occurs in intestinal, but not gastric fluids, but that hydrolysis was not so extensive as to preclude absorption of intact monobuytrins. Work was also conducted to describe the in vivo fate of carbon-14 labelled 1- and 2-monobutryin in broiler chickens. The analytical phase of a study initiated in Fiscal Year (FY) 2020 to determine the fate of sodium chlorite in live broiler chickens was completed. In cooperation with ARS researchers in Nebraska and California, analytical methods were developed, and samples analyzed, to quantify chlorate residues in beef and almond products after sanitation with chlorine dioxide. Studies specifically related to Sub-objective 1G, “Determine the accumulation and depuration kinetics of tetrahydrocannabidiol (THC) and cannabidiol (CBD) in feedlot cattle supplemented with dietary hemp”, were designed, organized, and executed in collaboration with North Dakota State University (NDSU). Plasma, urine, and tissue samples were collected for the duration of the 112-day feeding study and samples are now being analyzed.
A study has been designed and a protocol is in draft form to investigate the absorption, distribution, and metabolism of perfluoroalkyl substances (PFAS; 9 perfluorinated carboxylic acids and 7 perfluorinated sulfonates) in laying hens (Sub-Objective 1E). The study is designed to investigate laying hen exposure to PFAS during the entire life cycle via drinking water exposure. That is, laying hens will be exposed from 1 or two days after hatch through growth and maturation and into the egg laying production cycle. A depuration phase is also included in the study design to determine whether PFAS elimination occurs in eggs after removal from PFAS sources. Institutional Animal Care and Use Committee review of the protocol is expected in the 4th quarter of FY2021. Additional work was conducted on method development and validation for the extraction and quantification of 2 branched isomers for perfluorooctane sulfonate (PFOS) in plasma, milk, and muscle. This analytical method is being applied to approximately 200 samples obtained from a PFAS contaminated dairy herd to determine the contribution of the branched isomers to the overall concentration of PFOS previously measured in the same samples. Data from these analyses were shared with risk assessors at the USDA-Food Safety and Inspection Service for the construction of kinetic models useful for risk and exposure assessments.
Micro/nanoplastics of different sizes, surface properties, and doses on were tested on gastric and liver cells for different exposure periods. The toxicities, percentages of micro/nanoplastics uptake, and mechanisms that caused cellular toxicity were studied. Studies on gastric cells have been completed, and studies on liver cells are in progress. Efforts on producing different shapes of microplastics in a controlled environment, to simulate properties of microplastic particulates found in the environment, are ongoing. Once sufficient quantities of characterized microplastic particles have been generated, cell studies will be performed. Animal protocols to determine the fate and transfer of micro/nanoplastic in chickens and goats have been written and approved by the Institutional Animal Care and Use Committee. The commercial synthesis of [14C]-polystyrene particles has been contracted; once the material is received animal studies will commence.
Objective 2 is focused on the development of analytical methods capable of rapidly and sensitively measuring chemical analytes in food-animal matrices. A major effort has been focused on the utility of electrospray ionization rapid screening (RS-ESI-MS) and direct analysis in real-time (DART) techniques in conjunction with very simple sample extraction methods. Using a straightforward ethyl acetate extraction scheme, internal standardization, and RS-ESI-MS a series of 4 avermectins, 4 beta-agonists, and 10 cannabinoids have been quantified in plasma, serum, oral fluid, plant, and/or animal tissue matrices in support of a number of internal and cooperative studies. For example, the analytical technique was used to measure cannabinoids in plasma of cattle dosed with hemp flower meal (in cooperation with Kansas State University) or hempseed cake (in cooperation with North Dakota State University); avermectins in cattle dosed with moxidectin, eprinomectin, or doramectin (in cooperation with ARS researchers in Kerrville, Texas); and animal health drugs including clenbuterol, ractopamine, salbutamol, and zilpaterol.
Commercially available lateral flow tests were utilized to test hog oral fluids collected for the beta-agonist feed additive ractopamine to determine the cause of false positive assay results. Particulates in saliva and timing of exposure may cause ractopamine false positive tests using the commercial rapid screening test. Different extraction methods were tested for a multi-residues analysis of urine using a single quadrupole mass spectrometer; the experiments results proved unsuccessful.
Research efforts relating to Objective 3 included dioxin (20 dioxin, furan, and polychlorinated biphenyl compounds) and brominated diphenyl ether (BDE) (9 different congeners) analyses obtained from the 2018-19 Dioxin survey. That is, the last samples of suliriform catfish (n=20), dairy cow livers (n=16), and beef livers (n=60) were extracted and analyzed. However, for these tissues additional method validation and laboratory quality assurance criteria were established prior to the analyses of survey samples. All extractions have been completed, final dioxin analyses are being conducted, and data compiled for sharing with the USDA-Food Safety Inspection Service.
Accomplishments
1. Allergens in ready-to-eat foods. It is estimated that 30,000 emergency room visits, and 150 deaths occur annually within the U.S. due to food allergens. Further, 30-40% all food recalls in the U.S are due to undeclared allergens. The incidence of undeclared allergens, or undetected allergens in pre-cooked frozen meals or meals ready-to-eat (MREs) was unknown. ARS scientists in Fargo, North Dakota, utilized individual tests and an immunoassay capable of detecting 7 common food allergens simultaneously (7-plex assay) to screen meat or poultry containing MREs and frozen meals for allergen content. A low number of undeclared allergens were detected in the surveyed food items, but both assay methods performed poorly for the detection of allergens in MREs. These data indicate that product labeling of frozen foods is mostly accurate, however, better methods would be beneficial for the detection of allergens in both frozen meals and MREs.
2. Fate of sodium chlorite in broilers chickens. Sodium chlorite has been proposed as a pre-harvest feed ingredient because of its activity in against several important pathogens. Practical use of sodium chlorite in poultry feed, however, is precluded by the dearth of data describing the presence or absence of chlorite-related chemical residues in meat products of poultry. ARS scientists in Fargo, North Dakota, conducted in vitro and in vivo residue studies which demonstrated that chlorite is transformed in gastric fluids and blood mostly to chloride ion, a major component of table salt. Chlorate, a minor but stable transformation product of chlorite, was either absent from edible meat, or present in very low quantities when broilers were fed sodium chlorite at very high feed levels. These data suggest that residues of sodium chlorite would not be a major obstacle for its development as a tool for pathogen mitigation in poultry production.
Review Publications
Mahdi, O.S., Greenlee, K.J., Rose, E., Rinehart, J.P., Smith, D.J. 2021. The sporicidal activity of chlorine dioxide gas on Paenibacillus larvae spores. Journal of Apicultural Research. https://doi.org/10.1080/00218839.2021.1920761.
Shelver, W.L., McGarvey, A.M., Yeater, K.M. 2021. Performance of allergen testing in a survey of frozen meals and meals ready-to-eat (MREs). Food Additives & Contaminants. 38:1249-1259. https://doi.org/10.1080/19440049.2021.1914870.
Banerjee, A., Shelver, W.L. 2020. Micro- and nanoplastic induced cellular toxicity in mammals: A review. Science of the Total Environment. 755:142518. https://doi.org/10.1016/j.scitotenv.2020.142518.
