Source: WEAVER LABS LLC submitted to
INVESTIGATION OF NOVEL FLUOR MOP ADSORBENT FOR PREVENTION AND REMEDIATION OF PFAS CONTAMINATION IN LIVESTOCK
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1030094
Grant No.
2023-51402-40224
Cumulative Award Amt.
$175,000.00
Proposal No.
2023-00804
Multistate No.
(N/A)
Project Start Date
Jul 1, 2023
Project End Date
Jun 30, 2025
Grant Year
2023
Program Code
[8.3]- Animal Production & Protection
Project Director
May, A.
Recipient Organization
WEAVER LABS LLC
1110 S INNOVATION WAY
STILLWATER,OK 74074
Performing Department
(N/A)
Non Technical Summary
1. Perfluoroalkyl substances (PFAS) are widespread environmental pollutants in the United States (US) and elsewhere in the world. Although PFAS compounds have been used for decades in industry and commercial products, such as in nonstick cookware, food packaging, waterproof jackets, carpets, furniture, as well as a component in firefighting foams used at commercial airports and military bases. PFAS are considered emerging contaminants in foods and food animals because very few regulatory thresholds exist for PFAS in foods. The US Environmental Protection Agency (EPA) has proposed to designate PFAS compounds as hazardous chemicals under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) which will potentially classify hundreds, if not thousands, of sites within the US as new clean-up sites. Current estimates suggest that up to 57,000 sites in the US are contaminated with PFAS and it is expected that the frequency of discovering on-farm contamination of food-animals will increase as testing becomes more common. At present there is no economically viable recourse for livestock producers whose animals are contaminated with PFAS. Technologies to treat PFAS contamination for live animals do not exist at this time, though it is known that humans get most of their PFAS contamination from their diet. Total remediation of PFAS will require clean up of PFAS from water, animals. and soil. This proposal is a material that targets treating and/or preventing PFAS contamination in livestock to improve the animal products for human consumption.2. Two studies will be conducted simultaneously to determine the performance of Fluor Mop material at treating PFAS contamination and determine the performance at reducing PFAS in already contaminated animals. Both of these scenarios are likely to occur outside of a controlled study and they will each provide valuable information. Broiler chickens will be used in the study for preventing PFAS contamination because they have relatively short lifespans before harvest (~7 weeks). The broiler chickens will be given contaminated water and then they will be given feed that has been mixed with the Fluor Mop material for treatment at different concentrations. At the end of 7 weeks, muscle, liver, and tissue will be collected and tested for 11 different PFAS that are known to accumulate in chickens. This study will also inform us how much Fluor Mop material is needed to keep PFAS from accumulating in the broilers. This information can be scaled to other animals and provides evidence Fluor Mop can prevent PFAS contamination.To access the ability of Fluor Mop to reduce PFAS levels in already contaminated animals, laying hens will be used since they have a longer production cycle and the eggs can be tested as well since it is known PFAS can pass from the hens to the eggs. Hens will be exposed for two weeks to contaminated water and then given clean drinking water and eggs will continually be collected and frozen until testing. After two weeks of contaminated water, clean drinking water will be provided and food with Fluor Mop added will be given to the laying hens for 28 days at which point the study will conclude. Laying hens will be euthanized and their liver, plasma, muscle tissue and previously collected eggs will be tested for the same 11 different PFAS as in the first study. This will determine the ability of Fluor Mop to reduce PFAS levels in an already contaminated animal. During both studies testing will be done to ensure Fluor Mop is not absorbed or retained by the animal.3. Completion of these studies will provide proof that Fluor Mop material can be used to prevent and/or treat PFAS in chickens/laying hens. This will allow for larger studies on other livestock like dairy and beef cows, goats, and pigs. Good performance of Fluor Mop with other animals would mean it is the first treatment/prevention method for PFAS contamination in livestock. This would mean healthier animal products for people around the world and would give farmers a simple food additive treatment option for their livestock that have PFAS contamination, a problem that is expected to become larger with more testing.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7113220102050%
3083210115050%
Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3210 - Egg-type chicken, live animal;

Field Of Science
1020 - Physiology; 1150 - Toxicology;
Goals / Objectives
Establish efficacy in animalsfor using Weaver Labs material, Fluor Mop, as a practical on-farm PFAS remediation tool in animals by showing it can prevent and/or treat PFAS contamination in animals allowing for a larger phase II study which will expand the treatment beyond broilers and laying hens to other livestock and further establish the efficacy of Fluor Mop material.Determine the efficacy of dietary Weaver Labs sorbents at preventing the accumulation of PFAS residues in broiler-chickens exposed to PFAS contaminated feed and water.Determine the efficacy of dietary Weaver Labs sorbents at remediating PFAS residues in animals already contaminated with PFAS with laying hens including testing of eggs layed by hens.
Project Methods
During objective 1, 11 PFAS (PFPeS, PFHxS, PFHpS, PFOS, 3-MePFOS, 6-MePFOS, PFNS, PFBA, PFOA, PFNA, and PFDA), which have been consistently detected in the plasma of broilers dosed with PFAS, will be tested for using the existing US FSIS analytical method for the determination of PFAS residues in animals. The broiler chickens will be dosed with contaminated drinking water that mimics the typical concentrations found in drinking water (100 ppt) and stock solutions will be prepared weekly and stored under refrigeration in polypropylene carboys. Additionally, PFAS contaminated feed will be prepared by spraying an ethanolic solution of the PFAS standards onto PFAS-free poultry grower feed within a ribbon mixer. An equivalent concentration of 100 ppt (100 ng/kg) of PFAS will be prepared. Ethanol will be allowed to evaporate from the feed prior to the beginning of the experiment. After the addition of PFAS and evaporation of ethanol, feed will be formulated with 0, 25, 50, and 100 mg of sorbent per kg of diet (w/w) and mixed in a ribbon mixer.PFAS will be added to feed prior to sorbent to allow the PFAS analytes to sorb to feed particles rather than to the sorbent. One mg of sorbent will bind approximately 3 μg of PFASs, thus sorbent levels formulated into feed treatments provide excessive binding capacity compared to daily PFAS exposures.Day old broiler chickens will be purchased and randomly allocated to 16 poultry runs (2 birds per fun) and 6 extra birds, in a single run elsewhere will provide blank tissues for analysis purposes. All runs, except for the control, will be supplied with contaminated water and four runs each will be supplied with contaminated feed at 0, 25, 50, and 100 mg of sorbent per kg of feed and allocated using a random complete block experimental design. Access to contaminated food and/or water will begin at arrival and end at ~7 weeks (market weight). At harvest, blood, liver, and skeletal muscle will be collected for the PFAS analysis. Analysis will be done by by liquid chromatography-tandem mass spectrometry using FSIS method CLG-PFAS 2.03, but with a sample concentration step that lowers limits of detection to approximately 0.1 ng/mL or ng/g. Additionally, silicates will be determined by inductively coupled atomic plasma atomic emission spectrometry as described by ECETOC, which will determine if there is any evidence of retention of the sorbent materials by the animals. Milestone 1 we will determine the dose effect of Fluor Mop material for the remediation of PFAS in animals that are being exposed to PFAS during the treatment.Milestone 2:determine if there is any adsorption or retention of the Fluor Mop materials by the test animals.For objective 2, the same PFAS will be tested for using stock solutions stored in the same way. Laying Hens will be used for this study since they have a relatively long production cycle, , because eggs can serve as a sentinel tissue for PFAS contamination (Wilson et al. 2021), and because PFOS half-lives in eggs are relatively rapid (4-7 days). Laying hens (n = 30; 20 weeks of age) will be purchased and adapted to the USDA ARS animal facilities in Fargo in individual cages until a set of 24 birds is in consistent lay (an egg every day for 3 consecutive days). The 24 birds will then be provided water containing 3 ng/L each of the PFAS mixture for two weeks, a sufficient time for egg PFAS residues to reach steady state. Eggs will be collected daily during the dosing period and 3 pools of 8 yolks each will be created to measure PFAS accumulation. Essentially 100% of PFASs are partitioned into yolk rather than white. After a two-week exposure period, individually housed hens will be provided with clean drinking water and treatments will be randomly assigned to cage so that there are 6 birds per treatment. The objective of the study is to test the effectiveness of Weaver Labs Fluor Mop sorbent at reducing or eliminating PFAS residues in contaminated animals. Feed will be prepared containing 0, 25, 50, or 100 mg of Weaver Labs sorbent per kg of feed. Sufficient sorbent material will be mixed with approximately 100 g of carrier feed and the carrier feed will be added to a ribbon mixer containing blank feed to ensure uniform distribution of sorbent. Treatments containing graded levels of Weaver Labs sorbent will be fed for 28 days (approximately 4 half-lives of PFHxS in eggs). During the treatment period time eggs will be individually collected, the yolk removed, and frozen until analysis. On study day 28 the layer hens will be euthanized and blood, liver, skeletal muscle will be collected. Analysis of liver, plasma, and muscle will be done using the same method as objective 1. Yolks will be analyzed as described by Wilson et al, 2021. The experiment is a simple randomized design. Within tissue, one-way analysis of variance will be used to determine if there means testing is warranted. Egg yolk residue depletion data will be fit by least-squares regression using linear or non-linear fits. Parameter (slope) estimates will be compared to determine if treatment related differences in depletion rates occurred. Milestone 3: we will determine the rate of depletion of PFAS at different doses for contaminated animals. Milestone 4: determine which of the 11 PFAS, if not all, are depleted from the liver, plasma, and muscles, and eggs, tested.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:This phase effort focuses on testing the efficacy of Fluor Mop material to treat PFAS contamination and reduce PFAS in broiler chickens and laying hens. This will provide important Fluor Mop dose-dependent and PFAS remediation information needed to establish proof of concept for a larger phase II study that will expand to more animals. Our ultimate target is to provide a safe, economical, and effective food additive treatment for PFAS contamination in agricultural animals that are used for human consumption which significantly contributes to increased levels of PFAS in humans. This project is a pioneering initiative for PFAS treatment in animals, beginning with broiler chicken studies. Upon successful proof-of-concept, it will be extended to other animals, such as dairy cows. If fully successful, this initiative will result in a feed additive that farmers worldwide can use to produce PFAS-free food products like meat, dairy, and eggs. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided an opportunity for our in-house staff at Weaver Labs to develop our material testing skills for PFAS in other mediums otherthan water, which was the only area we formerly hadexpertise in. We have also gotten the opportunity to visit the USDA facility in Fargo,our CRADA partners on this project, togive a presentation, have discussions, and learn from other scientists doing great work over there to equip ourselves with new insight and shape the future direction of our research. How have the results been disseminated to communities of interest?Currently, we are yet to disseminate any of our result to any community of interest becase that would be premature. However, we plan to do so in the nearest future. What do you plan to do during the next reporting period to accomplish the goals?Although the plasma analysis for PFAS residue after sorbent treatment is one of the most critical analyses, which we have completed ahead of schedule, we are still working on other tissue analyses. The tissue analysis has taken longer than anticipated. Generally, tissue analysis is time-consuming because tissues need to be homogenized, extracted, and subjected to numerous validation steps. Additionally, the LC-MS instrument used for most PFAS analyses is very sensitive, requiring regular maintenance and availability, which further slows down the process. The tissue analyses we are currently working on include the liver and muscle. We believe that everything is still on course as planned but we needed more time due to unforeseen challenges. The extension we have obtained will give us more time to complete the analyses. In addition, fixing the sorbent contamination problem would will yield more reliable results for Objective 2.

Impacts
What was accomplished under these goals? Introduction: In the execution of objective 1, we produced 20 g scaled-up batch of our superior performing PFAS binding sorbent, FMV8. Both product and intermediates from the 3-step synthetic process were characterized by 1H-,13C-, and 19F-NMR, TGA, and FTIR. The sorbent was then sent to our CRADA partner at the USDA Animal Metabolism-Agricultural Chemicals Research Unit in Fargo, North Dakota for the live and analytical phases of the project. Method: A broiler chicken model developed by Lupton et al. (USDA ARS, Fargo) was used over a seven-week period to demonstrate PFAS accumulation in the plasma and tissues of the broilers. All plasma analysis has been completed, while analysis of tissues, including the liver, skeletal muscle, are still ongoing. Dosing Feed and Water: For the water dose, an equimolar stock solution containing 100 ppt of seven PFAS sulfonates (PFPeS, PFHxS, PFHpS, PFOS, 3-MePFOS, 6-MePFOS, and PFNS) and four PFAS carboxylic acids (PFBA, PFOA, PFNA, and PFDA) was prepared. For the feed dose, an equivalent concentration of 100 ppt (100 ng/kg) of each of the 11 PFAS was prepared by spraying an ethanolic solution of the PFAS standards onto PFAS-free poultry grower feed in a ribbon mixer, allowing the ethanol to evaporate. Four treatment levels of sorbent-PFAS contaminated feed were formulated: T0, T1, T2, and T3, with 0, 25, 50, and 100 mg of sorbent per kg of diet (w/w), respectively, mixed in a ribbon mixer. Broilers: Day-old broiler chickens (n = 38) were purchased and randomly allocated to 16 poultry runs (2 birds per run), equally distributed in four pens (four runs per pen). Six extra birds, housed separately, were used to provide blank tissues for analytical purposes. All runs, except the control, were supplied with PFAS-contaminated water. Four runs each were supplied with feed formulated to contain 100 ppt of each PFAS and 0, 25, 50, or 100 mg of sorbent per kg of feed. Treatments (sorbent levels) were randomly allocated to pens in a randomized complete block experimental design, with each run representing an experimental unit. The birds were fed both the contaminated drinking water and feed from arrival until tissue harvest at approximately 7 weeks of age (market weight). Blood, liver, and skeletal muscle samples were then collected to determine PFAS residues. Analyses: Plasma and ongoing tissue analyses for PFAS content were conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the FSIS method CLG-PFAS 2.03 (FSIS, 2021), with an added sample concentration step to lower the limits of detection to approximately 0.1 ng/mL or ng/g.5,6 This method involves fortifying tissue samples with heavy-isotope internal standards, followed by simple extraction with methanol or acetonitrile, filtration, and analysis by LCMS/MS. Calibration is performed using matrix-matched standard curves with internal standardization. The analyte/internal standard ion ratios of samples are regressed against the calibrant/internal standard ratios to quantify PFASs in the unknown samples. Result and discussion: Of the 11 PFAS residues observed in the plasma analysis, a general trend of a mild reduction of perfluorosulfonates was observed for PFHpS, PFOS, 3Me-PFOS, and 6Me-PFOS. Over the four treatment levels, a reduction in concentration level ranging from the lowest, 0.12 ng/mL in PFHpS, to the highest 0.60 ng/mL in 6Me-PFOS was observed. No significant difference was observed for PFHxS over the four treatment levels T0- 0.696 (±0.09) ng/mL, T1- 0.708 (±0.09) ng/mL, T2- 0.646 (±0.07) ng/mL, and T3- 0.695 (±0.06) ng/mL. PFPeS also didn't show any significant difference with the average of each treatment level measurement staying below 0.03ng/g. On the other hand, PFNS showed an increase of 0.11 ng/mL in concentration over the four treatment levels. Although, this indicates some level of contamination but we believe this contamination is mild as it only affected the PFNA analysis which might have resulted from several sources due to the ubiquitous nature of PFAS. However, perfluorocarboxylic acids, PFBA, PFOA, and PFNA showed a very significant steady jump between the four treatment levels. The highest increase was accounted for in PFOA analysis which showed an increase of 33.44 ng/mL from T0 to T3 shown in Figure 3. This amount was very high and wasn't expected. PFNA also followed suit having a steady increase from T0 to T3 with an increase of 23.07 ng/mL from T0 to T3. The short-chain PFAS, PFBA had the lowest overall increase from T0 to T3 with a 1.41 ng/mL difference. However, PFDA showed the opposite trend, with a mild decrease of 0.18 ng/mL over the four treatment levels. These results indicated a contamination issue likely originating from the sorbents. As the amount of sorbents added to the feed increased, the levels of PFAS carboxylic acids also increased. We decided to investigate the PFAS levels of the carboxylic acids in both our sorbent and the feed mix. The treatment levels in the sorbents-feed mix for T0, T1, T2, and T3 were 0 mg/Kg feed, 25 mg/Kg feed, 50 mg/Kg feed, and 100 mg/Kg feed respectively. PFOA had the highest solid-phase concentration at 10,310 ng/g in the sorbent, followed by PFHxA. This confirms our initial suspicion that the source of the PFAS increase across the four treatment levels was the sorbent. PFDA showed the lowest levels, with 17.07 ng/g in the sorbent. The sorbent-feed mix retained these level trends. Although, plasma analysis for PFNA was recorded to have a higher concentration than PFBA, which had a higher concentration in the initial sorbent-feed mix, which indicates the difference in dose-response and bioaccumulation of different PFAS. The short-chain PFAS, PFBA, has less bioaccumulative potential compared to long-chain PFAS. This agrees with previous literature reports on the bioaccumulation potentials of different PFAS. To rectify the problem of contamination in our sorbents we went back to the drawing board at Weaver Labs to fix this problem. Firstly, we conducted a series of PFAS analyses focusing on PFOA using the EPA 1633 method. We tested the PFAS-binding sorbent sent to USDA (Sorbent-FMV8), an older batch of the PFAS-binding sorbent that we've made previously (Old sorbent batch), and the starting precursor we used in making our fluorous tag (perfluoroiodide) which was our prime suspect for the introduction of PFOA. The perfluoroiodide reagent showed a contamination level of 11,637 ng/g, while the old batch of sorbent showed a much lower PFOA level of 2,868 ng/g, and may have resulted from a change in our supplier. We resolved the problem of contamination by washing our sorbent material with a saturated ethanolic brine solution. We conducted a performance validation test for the sorbent using PFAS and recorded a significant performance improvement post-washing. Consequently, we have decided to include PFAS contamination check analysis and validation as crucial steps in FluorMop (FM) preparation and characterization to prevent future PFAS contamination issues. Reference: Lupton, S. J.; Smith, D. J.; Scholljegerdes, E.; Ivey, S.; Young, W. S.; Genualdi, D. L. Plasma and Skin Perfluoroalkyl Substance (PFAS) Levels in Dairy Cattle with Lifetime Exposures to PFAS Contaminated Drinking Water and Feed. submitted 2022.

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