Source: UNIVERSITY OF MAINE submitted to NRP
EVALUATING THE CAPACITY OF ADSORBENTS TO BIND PERFLUOROOCTANESULFONIC ACID (PFOS) ACROSS SIMULATED RUMINAL, ABOMASAL, AND INTESTINAL CONDITIONS AND EXPANDING OUR UNDERSTANDING OF PFOS RUMINAL RELEASE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032317
Grant No.
2024-67016-42363
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-07731
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2026
Grant Year
2024
Program Code
[A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals
Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469
Performing Department
(N/A)
Non Technical Summary
Exposure to perfluorooctanesulfonic acid (PFOS) causes serious pathologies in humans. The US EPA current advisory level for PFOS in drinking water is 70 ppt. Based on this threshold, Maine was the first state to set the action level for milk PFOS at 210 ppt. Among the thousands of per- and polyfluoroalkyl substances (PFAS), PFOS contaminates milk and beef to a far greater extent than the other PFAS. This is in part because it easily bioaccumulates in cattle and is relatively abundant in contaminated sites. Because milk is a major excretion route of PFOS in animals, dairy products are especially vulnerable to contamination. Our preliminary data showed that a selected binder can effectively bind PFOS under ruminal conditions, while other binder types failed. We also know that PFOS is not immediately released from forages under ruminal conditions, and it is gradually released and then taken in part by rumen microbes from the liquid fraction.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
(N/A)
Developmental
70%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3141699200050%
3023410101050%
Knowledge Area
302 - Nutrient Utilization in Animals; 314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals;

Subject Of Investigation
1699 - Pasture and forage crops, general/other; 3410 - Dairy cattle, live animal;

Field Of Science
1010 - Nutrition and metabolism; 2000 - Chemistry;
Goals / Objectives
1) Evaluate the relative efficacy of binders to sequester PFOS across simulated ruminal, abomasal, and intestinal conditions;2) Evaluate the effects of application rate on the capacity of a selected binder to sequester PFOS across simulated ruminal, abomasal, and intestinal conditions; and3) Evaluate the PFOS release kinetics and partition between rumen fluid and microbial mass from a PFAS-contaminated grass under simulated ruminal conditions.
Project Methods
Objective 1: Evaluate the relative efficacy of a collection of binders to sequester PFOS across simulated ruminal, abomasal, and intestinal conditions.Substrate. A grass stand reporting 6 ± 1 ppb of PFOS (DM basis) will be mowed and wilted to 80-85% DM. Enough hay will be collected from 5 plots within the stand [plots= blocks], dried at 60°C for 48 h, and ground to pass a 1-mm screen. Ground hay from each of the five plots (plots= blocks) will be further divided into four piles, and treatments will be allocated randomly to each of those piles.Treatments. Three adsorbents plus a control. All adsorbents will be applied at 1.7% grass hay (w/w; DM basis) as outlined in the response to reviewers. Briefly, binders can be applied up to 2% without causing health issues (Harvey et al., 1991). We are aware that above 1% binders will dilute dietary nutrients, but the purpose of this experiment is not to recommend a dose for in vivo studies but to identify promising binder types that could be further optimized to bind PFOS in cattle.Measures. The filtrate from the ruminal, abomasal, and intestinal incubation steps will be submitted for the basic PFAS analysis package at Maine Laboratories (Norridgework, ME; see attached quote). The following 18 analytes will be included: PFOS and PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA, PFTrDA, PFTeDA, NMeFOSAA, NEtFOSAA, PFBS, PFHxS, HFPO-DA, ADONA, 9Cl-PF3ONS, 11Cl-PF3OUdS. Rumen pH, VFA profile, NH3N, and gas production data will be processed and analyzed as described by Killerby et al. (2022).Experimental design. The concentration of each unbound (soluble) PFAS will be analyzed using a randomized complete block design (RCBD, 5 blocks) with the effects of 4 treatments measured across three simulated gastrointestinal conditions, which will be considered a repeated measure (repeated in condition). Treatments will be in 5 blocks as suggested by a power analysis conducted using SAS GLMPOWER (α= 0.05 and a β= 0.85) and the data from our preliminary results. Rumen adsorption results for each of the adsorbents will also be analyzed relative to the untreated control filtrate values using the following formula:Ruminal soluble PFAS relative adsorption (%) = [(PFAScon-PFASads)/PFAScon]×100PFAScon= Given PFAS concentration in control rumen fluid; PFASads= Given PFAS concentration in adsorbent rumen fluid. The adsorption and desorption data will be analyzed using an RCBD (minus the control). Gas production and ruminal fermentation data will be analyzed using an RCBD (incl. the control). The PROC GLIMMIX of SAS 9.4 will be used to evaluate all data generated. Fisher's LSD will be used for mean separation. Significances will be declared at a P-value < 0.05.Objective 2: Evaluate the effects of application rate on the capacity of a selected binder to sequester PFOS across simulated ruminal, abomasal, and intestinal conditions.Substrate. Same as in Objective 1. Grass hay generated from 5 field blocks (blocks= plots).Treatments. Treatments will have a factorial arrangement of 4 doses (0, 0.5, 1, and 1.7%, w/w relative to grass; DM basis). The highest dose is based on our preliminary study.Experimental design. Same as in Objective 1. Each unbound (soluble) PFAS concentration will be analyzed using a randomized complete block design (RCBD, 5 blocks) with 4 doses across rumen, abomasal, intestinal simulated conditions. The dose effect will be modeled using polynomial contrasts to assess if the dose-response was linear, quadratic, or cubic. We do not see the benefit of assessing if the dose had a quartic response from 0 to 1.7% w/w. Power analysis approach and results were the same as Obj. 1.Objective 3 - Evaluate the PFOS kinetics release and partition between rumen fluid and microbial mass from a PFAS-contaminated grass under simulated ruminal conditionsSubstrate. Same as in Objective 1. Grass hay generated from 5 field blocks (blocks= plots).Treatments. A factorial arrangement of 6 ruminal incubation times (0, 2, 6, 12, 24, and 48 h) × 2 ruminal fractions (fluid and microbial mass)Measures. The filtrate from the ruminal incubation step and the microbial pellet will be analyzed for the same PFAS analysis package as Obj. 1. Same for fermentation analysis (Obj. 1).Experimental design. The ruminal fluid and microbial mass PFOS concentrations and the relative proportion to the initial grass PFOS will be analyzed using an RCBD (5 blocks). Since samples over time will not be obtained from the same unit, this will not be a repeated measures study. Treatments will be replicated in 5 blocks as suggested by a power analysis [SAS GLMPOWER (α= 0.05 and a β= 0.85)] and the data from Kowalczyk et al. (2015) and our preliminary results.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The goal of this research is to identify a safe-to-feed binder to reduce PFOS contamination in milk and beef. The target audience include farmers, ag service providers and policymakers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoctoral research associate, one doctoral student, and undergraduates were employed for this project. How have the results been disseminated to communities of interest?On July 19, 2024, the Maine Department of Agriculture, Conservation and Forestry (DACF) visited UMaine to learn about our PFAS research. Preliminary lab results on binder use were shared. DACF is at the forefront in Maine helping PFAS impacted farmers to find sustainable dairy farm solutions and ensure the production of safe animal products (i.e., meat and milk). On December 12, 2024, ag service providers--including Agrimark, Maine Dairy Industry Association, and Maine Beef Producers Association --met with UMaine researchers to discuss ongoing and future PFAS mitigation work. These providers help us align research with industry needs. Preliminary findings have also been shared directly with farmers through field visits, offering updates and guidance on future binder application steps. What do you plan to do during the next reporting period to accomplish the goals? Completion of Objective 2 activities, mainly the abomasal and duodenal phases of this experiment. Completion of Objective 3 activities, including the PFOS kinetics release and partition between rumen and microbial mass from a PFAS-contaminated grass under simulated conditions.

Impacts
What was accomplished under these goals? PFOS contamination in livestock is a growing concern due to its persistence and accumulation in milk and meat, especially when animals consume contaminated forage or water. Dairy farms dealing with PFOS contamination face significant challenges as milk is a major excretion route for PFOS in lactating animals. We aim to find a safe-to-feed binder that can reduce PFOS contamination in milk and beef using lab techniques simulating ruminal, abomasal (a.k.a. stomach of ruminants) and duodenal conditions while also expanding our understanding of PFOS ruminal release kinetics from forage substrates. Objective 1 - This objective was guided by prior findings showing that cholestyramine, an anion exchange resin, can bind up to 52% of PFOS in ruminal fluid via its quaternary ammonium groups and hydrophobic moiety. As a result, we evaluated the relative efficacy of various quaternary ammonium-containing binders (QBDs) to sequester PFOS under simulated ruminal, abomasal, and duodenal conditions. The evaluated binders included cholestyramine - an anion exchange resin (AER), a polymer with quaternary ammonium groups (CPQ), a natural by-product feed (NBF), a polysaccharide with quaternary ammonium groups (PLQ), a clay binder amended with quaternary ammonium groups (CLQ), and a dioctahedral phyllosilicate clay binder (CLY). To simulate rumen conditions, PFAS-contaminated grass (3g) was mixed with 0.05g of each binder before adding 100 mL of rumen media. ANKOM bottles were shaken at 60 rpm, and incubated for 48 h at 39°C. After centrifugation, the rumen fluid was analyzed for PFOS and rumen fermentation analysis and the ruminal pellet was freeze-dried. The percentage of PFOS binding affinity of the respective binders was calculated in relation to CON. A randomized complete block design (RCBD) with a total of 4 blocks was used to analyze the data using SAS v.9.4 PROC GLIMMIX. Differences were declared at P≤ 0.05. Fisher's protected least significant difference (LSD) tesy was used for mean separation. Finding revealed that AER exhibited a high ability to sequester PFOS (89.6%), followed by CQQ (31%) and NBF (26.7%) (SEM=7.56, P<0.01). All other tested binders had binding capacities below 10%. None of the binders affected ruminal pH, except CPQ (pH=6.59 vs. x?=6.04; SEM?=?0.1, P?
Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: February 2025. Oral Poster at the Maine PFAS Research Convening at Colby College, ME. PFAS and dairy. K, Nishimwe, G.M. Pereira, J. Romero.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: " August 2024. Presentation. Forage multistate group. Fargo, ND. PFAS and dairy research at UMaine. J. Romero. https://nimss.org/seas/52604
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: " June 2025. Poster Presentation at the Annual American Dairy Science Meeting. Louisville, KY. Evaluating the efficacy of cationic feed binders to retain bound perfluorooctane sulfonate (PFOS) across simulated abomasal and duodenal conditions. A. Jimenez, M. Chusho, J. Sandro, R. Heath, M. Manning, T. Wood, K. Nishimwe, G. Pereira, Y. Jiang*, and J. Romero. University of Maine, Orono, ME; *University of Wisconsin  Madison, Madison, WI.