Location: Food Animal Metabolism Research
2023 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”. The analytical phase of a cooperative study on the fate of 1- and 2-monobutyrins in broiler chickens was completed and a draft manuscript describing this work is being shared with cooperators. Under the same cooperative agreement, the synthesis of monocaprylic- and monocapric-glyceride esters, including heavy-isotope labelled internal standards, was completed. Synthetic (unlabeled) monocaprylic- and monocarpic glyceride esters were used in a live phase study with broilers chickens to determine whether they are absorbed intact. Manuscripts describing the residues of cannabinoids in beef tissues after consumption of hemp seed cake, the effects of hemp seed cake on digestive enzymes, and the effects of hemp seed cake on the respiratory and reproductive microbiota of cattle were prepared and submitted for publication.
A series of radiolabeled nitrofurazone test articles, heavy isotope labeled nitrofurazone internal standards, and nitrofurazone metabolites and internal standards were chemically synthesized. Internal standards and metabolite standards were shared with cooperators. The live phase of a [14C]-nitrofurazone metabolism study in broiler chickens was completed. Day-old broiler chicks were grown to maturity and then dosed for 7 consecutive days with feed containing [14C]-nitrofurazone labeled either of two positions (furan ring or semicarbazide). Tissues were then harvested with 0, 4, 7, and 14-day withdrawal periods. Harvested tissues are being analyzed to quantify the amounts of total radioactive residue, extractable residue, and bound residue. Extractable and bound residues are undergoing characterization to determine if a unique metabolite is present that can be used as a marker compound.
The analytical phase of a study investigating tissue residues of perfluoroalkyl substances (PFAS; 13 perfluorinated carboxylic acids and 12 perfluorinated sulfonates) in broiler chickens (n = 72) is being completed. Residues in plasma and liver have been quantified; skeletal muscle, gizzard, and skin have been processed and are being extracted for PFAS analyses. The live phase of a 9-month laying hen study, started in FY2022, was completed in FY23. Laying hens (n = 72), exposed to PFAS containing water, were harvested during growth, maturation, and during the egg-laying period. A depuration phase was included in the laying hen study so that PFAS depletion in tissues and eggs could be measured. Analysis of tissues from both studies, and eggs from the laying hen study, is in progress.
An animal protocol was developed and approved to investigate the effect of including a proprietary PFAS-specific sorbent on residues of PFAS in a model farm-animal species (broilers). The live phase of the experiment, being conducted in cooperation with a Cooperative Research and Development Agreement partner, is expected to occur in the 4th quarter of 2023 with the analytical phase extending into FY2024.
A study was initiated to evaluate the biological effects of poly(methyl methacrylate) (PMMA) micro/nanoplastics in liver cells. Initial results indicated that PMMA is less toxic than the polystyrene micro/nanoplastics that were previously studied in this laboratory. Studies confirming that the alteration of cellular endpoints is dependent upon the polymer type, size, surface charge in cell culture system are in progress.
The analytical phase of a [14C]-labeled polystyrene microparticle (microplastics) absorption and fate study in laying hens is in progress with radioactive residues characterized in tissues and eggs. Analyses of excreta is in progress. The live phase portion of a [14C]-labeled polystyrene microparticle fate and disposition study in lactating sheep was completed in early FY2023 with the analytical phase of tissues, milk, and excreta expected to last into FY2024. Strategies for [14C]-incorporation into other types of microplastics are being pursued. For example, synthetic methodologies to procure micro/nanosized polyethylene terephthalate (PET) particles have been initiated. Optimization of reaction parameters and their impacts on particle size and morphology is in progress with the goal of producing [14C]-labeled PET for use in a range of scientific studies.
Objective 2 is focused on the development of analytical methods capable of rapidly and sensitively measuring chemical analytes in food-animal matrices. Liquid chromatography-mass spectrometry (LC-MS) methods were developed to simultaneously quantify the feed additive ractopamine and its metabolite ractopamine-glucuronide in hog oral fluid. The LC-MS method was used to validate a commercial rapid screening assay developed for use on swine oral fluids. A manuscript has been submitted that describes the analytical method and which indicates that results of the commercial lateral flow assay are generally reliable.
The goal of Objective 3 is to determine the levels of dioxins, furans, and polybrominated diphenyl ethers (PBDEs) in the U.S. meat supply. An Interagency Agreement has been developed with the USDA Food Safety and Inspection Service that will allow the collection of samples in FY24. A new gas chromatograph mass spectrometer was procured in FY22 and is currently being validated with respect to the dioxin analytical assay. Analytical work on the analysis of PBDEs from the 2018-19 survey was completed and one manuscript describing those results is being drafted along with a second manuscript describing estimates of consumer exposures to dioxin.
Accomplishments
1. Microplastic absorption. Micro or nanoplastics have increasingly been detected in the environment and in food and water consumed by animals and humans. Numerous adverse health effects of micro or nanoplastics after ingestion have been hypothesized, but the extent of microplastic absorption in live animals is largely unknown. ARS scientists in Fargo, North Dakota, studied the transfer of specially labeled microparticulate styrene into tissues and eggs of laying hens. Although most of the microparticulate matter was excreted without absorption, small quantities of the labeled microparticulate plastic were measured in eggs. This study provides some of the first direct evidence that microplastics may be absorbed in farm animals and that they may be transferred, albeit in small quantities, to edible products such as eggs.
2. Cannabinoid residues in beef. Hempseed cake is a highly nutritious byproduct of oil extraction from industrial hempseed. Although hempseed oil is commonly used in food, health care, and cosmetic products, hempseed cake cannot be used in food animal rations because the magnitude of cannabidiol (CBD) and tetrahydrocannabinol (THC) residues remaining in edible tissues were not previously characterized. ARS scientists in Fargo, North Dakota, incorporated hempseed cake into a ration fed to cattle for approximately 16 weeks. Cannabinoid residues were sporadically detected in urine and plasma during the feeding period. Low levels (10 parts per billion) of CBD/THC were measured in adipose tissue (fat) of animals harvested with no withdrawal period, but CBD/THC were below detectable levels in liver, kidney, and muscle. Exposure estimates indicated that it would be very difficult for a human to consume enough fat from cattle fed hempseed cake to exceed conservative regulatory thresholds for dietary THC. These data will be valuable to state and federal officials who regulate the use of industrial hemp byproducts.
3. PFAS levels in dairy cows. Levels of poly- and perfluorinated substances (PFAS) in food has become a growing issue with consumers nationwide. PFAS contamination of water and/or forage has resulted in several incidences of livestock contamination, including dairy cows. ARS scientists in Fargo, North Dakota, analyzed perfluoro carboxylic acids (PFCAs) and perfluoro sulfonates (PFSAs) in plasma and ear notch samples collected from dry and milking cattle (> 160) of various ages from a PFAS contaminated farm. PFCA residues were essentially not detectable in plasma or ear-notch samples; in contrast, PFSA levels clearly accumulated and plateaued in animals after about 1.5 years of continued PFAS exposure. Data generated in the study clearly show that cattle do not bioaccumulate PFCAs as do other species. Data can be used by risk assessors and modelers to predict human exposures to PFAS after inadvertent food animal exposures to contaminated food and(or) water.
Review Publications
Smith, D.J., Serum, E.M., Winders, T.M., Neville, B.W., Herges, G.R., Dahlen, C.R., Swanson, K.C. 2023. Excretion and residue depletion of cannabinoids in beef cattle fed hempseed cake for 111 days. Food Additives & Contaminants. 40(4):552-565. https://doi.org/10.1080/19440049.2023.2187645.
Wang, W., Smith, D.J., Lew, H.N., Jin, Z.T., Mitchell, A., Fan, X. 2023. Lipid oxidation and volatile compounds of almonds as affected by gaseous chlorine dioxide treatment to reduce salmonella populations. Journal of Agricultural and Food Chemistry. 71(130):5345-5357.
Winders, T.M., Holman, D.B., Schmidt, K.N., Luecke, S.M., Smith, D.J., Neville, B.W., Dahlen, C.R., Swanson, K.C., Amat, S. 2023. Feeding hempseed cake alters the bovine gut, respiratory and reproductive microbiota. Scientific Reports. https://doi.org/10.1038/s41598-023-35241-1.
Lupton, S.J., Pfaff, C.M., Singh, A., Chakrabarty, S., Hakk, H. 2022. Bioavailability of hexabromocyclododecane (HBCD) in rats from dust and oil vehicles. Environmental Research. 218. Article 114853. https://doi.org/10.1016/j.envres.2022.114853.
Lupton, S.J., Smith, D.J., Scholljegerdes, E., Ivey, S., Young, W., Genualdi, S., Dejager, L., Snyder, A.W., Esteban, E., Johnston, J. 2022. Plasma and skin per- and polyfluoroalkyl substance (PFAS) levels in dairy cattle with lifetime exposures to PFAS contaminated drinking water and feed. Journal of Agricultural and Food Chemistry. 70:15945-15954. https://doi.org/10.1021/acs.jafc.2c06620?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as.
Mia, Md GK., Winders, T.M., Serum, E.M., Amat, S., Neville, B.W., Dahlen, C.R., Smith, D.J., Swanson, K.C. 2023. The effects of feeding hempseed cake on pancreatic and jejunal digestive enzymatic activity in finishing heifers. Canadian Journal of Animal Science. https://doi.org/10.1139/cjas-2023-0033.
