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ARS Home Plains Area Fargo, North Dakota Edward T. Schafer Agricultural Research Center Food Animal Metabolism Research Research





- Smith, David
- Singh, Anuradha
- Lupton, Sara
- Shelver, Weilin
- 2023
- 2022
- 2021
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Detection and Fate of Environmental Chemical and Biological Residues and their Impact on the Food Supply **Location:
                            Food Animal Metabolism Research** 2022 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 live phase of a cooperative study on the fate of 1- and 2-monobutyrins in broiler chickens was completed late in Fiscal year (FY) 2021 and the analytical phase of the study is being completed. A draft manuscript describing this work is nearing completion and will be shared with cooperators soon. Synthesis of monocaprylic- and monocarpic-glyceride esters is in progress; these compounds will be used in broiler chickens to determine whether they are absorbed intact and contribute to the beneficial gut- and immune-health effects hypothesized for monoglycerol esters. Cooperative studies with ARS researchers at Clay Center, Nebraska, to measure residues of chlorate in beef carcasses exposed to chlorine dioxide, have been completed and a draft manuscript from the cooperators is anticipated. Two manuscripts were submitted describing 1) the production effects of including hempseed cake in the diets of cattle, and 2) an analytical method for measuring hempseed cake cannabinoids in cattle tissues and excreta. Synthetic techniques to produce radiocarbon-labeled cannabidiol (CBD) are being developed so that a CBD metabolism and fate study can be conducted in a ruminant animal.
The live phase of a nitrofurazone residue study in broiler chickens was completed. Day-old broiler chicks were grown to maturity using feed containing the antibiotic nitrofurazone and tissues were harvested with 0, 1, 2, 4, 7, 10, and 14-day withdrawal periods. Harvested tissues were frozen and sent to an ARS cooperator in Wyndmoor, Pennsylvania, for analysis of current and potential marker residues.  In addition, internal standards of nitrofurazone and its cyano- metabolite were synthesized in isotopically pure form and shared with collaborators in the ARS and at the Food and Drug Administration, Center for Veterinary Medicine. Organic synthesis of radiolabeled nitrofurazone is being pursued for use in a nitrofurazone metabolism study to be conducted in the first quarter of fiscal 2023.
Two studies investigating the absorption, distribution, and metabolism of perfluoroalkyl substances (PFAS; 13 perfluorinated carboxylic acids and 12 perfluorinated sulfonates) in broiler chickens and in laying hens (Sub-objectives 1D and 1E) were started in FY2022. The live phase of the broiler study is complete; the live phase of the laying hen study was started in March 2022 but will not be completed until the first quarter of FY2023. Both studies were designed to investigate accumulation of PFAS in tissues during the entire life cycle via exposure to PFAS-contaminated drinking water. Broiler chickens were harvested throughout the growth period, but laying hens have been, and will be, harvested during growth, maturation, and during the egg-laying period. A residue depletion phase which occurs when hens are no longer exposed to PFAS contaminated water is also included in the study. Analysis of tissues from both studies, and eggs from the laying hen study, is in progress.
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, and surface charge in cell culture system are in progress.
The fate of 14C-labeled polystyrene microparticles (microplastics) in laying hens was conducted. Birds were provided a single, oral dose of 14C-lableled microplastic and excreta, eggs, and tissues were collected from all animals during the 7-day study period. Tissues were processed and analyzed for total radioactive residues. The analytical phase of the study will continue into early fiscal year 2023. An animal protocol was developed and approved for a similar 14C-labeled polystyrene microparticle fate and disposition study in lactating sheep, to be initiated in late FY2022 or early FY2023. Strategies for 14C incorporation into other types of microplastics are being pursued.
Objective 2 is focused on the development of analytical methods capable of rapidly and sensitively measuring chemical analytes in food-animal matrices. A rapid electrospray ionization method developed in FY2021 was used to simultaneously quantify 10 cannabinoids in cattle urine, plasma, muscle, kidney, and liver from 16 hempseed cake fed cattle and 15 control cattle (over 900 individual samples) in a time-efficient manner. The method accurately detected a suite of cannabinoids in treated animals, but did not detect cannabinoids in control animals (a very low false-positive rate).
A liquid chromatography-mass spectrometry (LC-MS) method was 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 results of hog oral fluids that were tested with a commercially available on-site rapid screening assay. Preliminary results from the newly developed LC-MS method verified that the commercially available screening test did not produce false-positive results. Although a limited number of hog oral fluid samples have been assayed to date, work continues to validate oral fluid samples that tested positive by the rapid screen.
The goal of Objective 3 is to determine the levels of dioxins, furans, and polybrominated diphenyl ethers (PBDEs) in the U.S. meat supply. This objective is accomplished in coordination with the USDA Food Safety and Inspection Service. Analytical work on the analysis of PBDEs from the 2018-19 survey is in its final stages but was delayed significantly because of instrument failure (which is in its final stages of resolution). The next survey is due to start in Quarter 3 of FY 23; a draft budget for the 2023-24 Dioxin Survey has been drawn up and shared with Food Safety and Inspection Services. Additionally new instrumentation has been procured for the survey to replace an older instrument that is nearing an ‘obsolete’ designation by its manufacturer. **Accomplishments**
                                

1. Microplastic uptake and toxicity by intestinal and liver cells. Micro or nanoplastics have increasingly been detected in the environment and in food and water consumed by animals and humans, but the adverse health effects of micro or nanoplastics after ingestion are largely unknown. ARS scientists in Fargo, North Dakota, studied the interactions of polystyrene micro/nanoplastics with gastric and hepatic cells grown in the laboratory to identify any toxicities that might occur and to elucidate potential causes of the identified toxicities. Small particle sizes, particularly those with positive charges on the surface, were more toxic than larger sized particles and increased concentrations and longer exposure periods exacerbated cellular toxicity. The studies confirmed that microplastic toxicity is not uniform across the multitude of plastics available for ingestion, and that cell models may be highly useful for determining the most critical microplastic properties contributing to toxicities 2. Survey of dioxins in the U.S. meat supply. The USDA Food Safety and Inspection Service (FSIS) and the ARS conduct a survey of the U.S. meat supply every 5 years to determine current dioxin levels, temporal trends in dioxin levels, and potential dioxin exposure sources. Dioxins present in exported meat, even at trace levels, may cause trade issues because regulations established by importing countries limit concentrations that are allowable in meat supplies. The USDA-FSIS collected tissue samples from U.S. beef, swine, chicken, turkey, and catfish slaughter facilities for analyses by ARS scientists in Fargo, North Dakota. During the last three decades dioxin levels in beef, swine, and poultry samples have generally declined to very low concentrations. During the most recent survey, concentrations in beef and swine tended to level and concentrations in poultry continued to decline. Collectively, the data suggest that continued monitoring of beef and swine will be needed to ensure continued low tissue burdens of dioxins. 3. Rapid quantification of cannabinoids. Hempseed cake is a byproduct of hempseed oil production that is potentially useful as a feed source for cattle, but hemp ingredients are not allowed in diets of food animals because it is not known whether biologically active residues (cannabinoids) are retained in meat. A major limitation to investigating such residues is the lack of a rapid and sensitive analytical method capable of quantifying cannabinoids in food-animal products. ARS scientists in Fargo, North Dakota, developed and validated a rapid mass-spectrometric method capable of simultaneously quantifying 10 cannabinoid residues in cattle tissues, plasma, and urine. The method was sensitive, accurate (no false positives), and rapid, requiring mass spectrometric instrument time of only 1 minute per sample. The method is currently being used in a study to evaluate residues of cannabinoids in tissues of cattle fed finishing diets containing 20% hempseed cake. Such data are important for regulatory organizations to assess risks associated with cannabinoid residues. **Review Publications**
                            *Shelver, W.L., Chakrabarty, S., Young, J.M., Byrd, C.J., Smith, D.J. 2021. Evaluation of rapid and standard tandem mass spectrometric methods to analyze veterinary drugs and their metabolites in hog oral fluid and cattle urine. Food Additives & Contaminants. 39:462-474. https://doi.org/10.1080/19440049.2021.2006801.*
                                *Banerjee, A., Billey, L.O., Shelver, W.L. 2021. Uptake and toxicity of polystyrene micro/nanoplastics in gastric cells: Effects of particle size and surface functionalization. PLoS ONE. https://doi.org/10.1371/journal.pone.0260803.*
                                *Smith, D.J., Scapanski, A.R., Herges, G.R. 2021. The fate of sodium chlorite in simulated gastric and intestinal fluids and residues of chlorate in broiler chickens after oral administration of sodium chlorite. Food Additives & Contaminants. 39(2):242-255. https://doi.org/10.1080/19440049.2021.1992513.*
                                *Lupton, S.J., Ochoa, C., Domesle, A., Duverna, R. 2022. Survey of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and non-ortho-polychlorinated biphenyls in U.S. meat and poultry from 2018-2019: Toxic equivalency levels, temporal trends, and implications. Food Additives & Contaminants. 39:560-571. https://doi.org/10.1080/19440049.2021.2022769.*
                                *Shelver, W.L., Chakrabarty, S., Smith, D.J. 2021. A rapid screening method for ß-adrenergic agonist residues incurred in animal urine using direct analysis in real time mass spectrometry (DART-MS). ACS Food Science and Technology. 2:195-205. https://doi.org/10.1021/acsfoodscitech.1c00427.*
                                *Lupton, S.J., Casey, F., Smith, D.J., Hakk, H. 2021. Perfluorooctanoic acid (PFOA) transport in soil and absorption and distribution in alfalfa (Medicago sativa). Journal of Food Protection. 85:164-172. https://doi.org/10.4315/JFP-21-276.*
                                *Ilozumba, M., Shelver, W.L., Hong, C., Ambrosone, C.B., Cheng, T.D. 2022. Urinary concentrations of triclosan, bisphenol A, and brominated flame retardant in the association of triclosan with demographic characteristics and body fatness among women with newly diagnosed breast cancer. International Journal of Environmental Research and Public Health. 19. Article 4681. https://doi.org/10.3390/ijerph19084681.*
                                *Winders, T.M., Serum, E.M., Smith, D.J., Neville, B.W., Mia, G.K., Amat, S., Dahlen, C.R., Swanson, K.C. 2022. Influence of hempseed cake inclusion on growth performance, carcass characteristics, feeding behavior, and blood parameters in finishing heifers. Journal of Animal Science. 100:1-8. https://doi.org/10.1093/jas/skac159.*
                                *Banerjee, A., Billey, L.O., Mcgarvey, A.M., Shelver, W.L. 2022. Effects of polystyrene micro/nanoplastics on liver cells based on particle size, surface functionalization, concentration and exposure period. Science of the Total Environment. 836:155621. https://doi.org/10.1016/j.scitotenv.2022.155621.*


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