The use of aqueous film-forming foams containing poly- and perfluoroalkyl substances (PFAS) at Defense Environmental Restoration Program (DERP) fire, crash, and training sites has potentially contributed to PFAS contamination of soil, water, and sediment. The overall objective of this SERDP effort is to develop amphibian toxicity reference values (TRVs) to support ecological risk assessment of PFAS in impacted DERP sites. Such data are needed for supporting cleanup and/or exposure mitigation decisions. Specifically, the project team aims to: 1) develop toxicity reference values for perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in amphibians, and 2) determine the potency of two additional PFAS in amphibians: 6:2 fluorotelomer sulfonate and perfluorohexane sulfonate.

Project Summary

Technical Approach

TRVs will be developed following standard published toxicity test procedures for Northern Leopard Frogs (Lithobates pipiens), American Toads (Anaxyrus americanus), and Eastern Tiger Salamanders (Ambystoma tigrinum). Toxicity testing will be conducted under steady state conditions, which will be determined prior to testing through range finding studies. Route of exposure, length of exposure, and life stage for these toxicity tests will include: 1) Aquatic Chronic Larvae 21 d; 2) Sediment Subchronic Larvae 10 d; 3) Terrestrial Dermal Chronic Adults 21 d; and 4) Terrestrial Oral Chronic Adults 42 d. Each assay will be validated through a partnering laboratory. Experiments will be conducted using larvae and juveniles reared from egg masses collected from natural amphibian populations located in West Lafayette, Indiana. To generate juveniles for the experiments, a subset of the species will be moved into outdoor mesocosms. Exposure levels will be determined from the range-finding studies (from 10X above background concentrations to no greater than 1,000 μg/L). Based on these results, 5 concentrations will be selected and each treatment will be replicated 4 – 8 times for a total of 20 – 40 experimental units per assay. Experiments will begin at Gosner stage 30 for the anurans and Harrison stage 46 for the salamanders. Although PFAS are expected to be very stable, multiple water and/or sediment samples will be collected over time to assess the amount of breakdown that has occurred. Dose-response curves will be calculated for growth, development (time to, and effects on metamorphosis), and survival. Then TRVs will be calculated and compared across species and compounds. At the end of the chronic exposures, chemical concentrations will be quantified from whole-animal tissue on a set of the experimental samples for expression of TRVs based on body burdens. PFAS extraction will be conducted following protocols established and modified for optimal recoveries and extracts analyzed by ultra-high pressure liquid chromatography tandem mass spectrometry in negative electrospray ionization.


The core outcome of these studies will be the development of amphibian TRVs for PFAS that can be used in the derivation of surface water, sediment and soil risk-based environmental criteria for assessments at PFAS-impacted sites. The ultimate goal is not only to develop TRVs that are protective of the most sensitive amphibian species, but also that can be used for a more refined identification of surrogate species for the evaluation of specific amphibian taxa. The project team will work to answer the following questions:

  1. What amphibian species and what life stages are most sensitive to PFAS?
  2. What is the range of amphibian TRVs for PFAS varying in environmental persistence and bioavailability?
  3. What is the range of bioaccumulation factors for PFAS varying in environmental persistence and bioavailability; and
  4. Are there differences in TRVs between different exposure routes (oral, dermal, gills)?

(Anticipated Project Completion - 2023)


Abercrombie, S.A., C. de Perre, Y.J. Choi, M. Iacchetta, R.W. Flynn, M.S. Sepúlveda, L.S. Lee, and J.T. Hoverman. 2020. Sublethal Effects of Dermal Exposure to Poly- and Perfluoroalkyl Substances on Post-Metamorphic Amphibians. Environmental Toxicology and Chemistry, 40(3):717-726. doi.org/10.1002/etc.4711.

Abercrombie, S.A., C. de Perre, Y.J. Choi, B.J. Tornabene, M.S. Sepúlveda, L.S. Lee, and J.T. Hoverman. 2019. Larval Amphibians Rapidly Bioaccumulate Poly- and Perfluoroalkyl Substances. Ecotoxicology and Environmental Safety, 178:137-145. doi.org/10.1016/j.ecoenv.2019.04.022.

Ankley, G.T., P. Cureton, R.A. Hoke, M. Houde, A. Kumar, J. Kurias, R. Lanno, C. McCarthy, J. Newsted, C.J. Salice, B.E. Sample, M.S. Sepulveda, J. Steevens, and S. Valsecchi. 2020. Assessing the Ecological Risks of Per- and Polyfluoroalkyl Substances: Current State-of-the Science and a Proposed Path Forward. Environmental Toxicology and Chemistry, 40(3):564-605. doi.org/10.1002/etc.4869.

Brown, S.R., R.W. Flynn, and J.T. Hoverman. 2020. Perfluoroalkyl Substances Increase Susceptibility of Northern Leopard Frog Tadpoles to Trematode Infection. Environmental Toxicology and Chemistry, 40:689-694. doi.org/10.1002/etc.4678.

Flynn, R.W., M.F. Chislock, M.E. Gannon, S.J. Bauer, B.J. Tornabene, J.T. Hoverman, and M.S. Sepúlveda. 2019. Acute and Chronic Effects of Perfluoroalkyl Substance Mixtures on Larval American Bullfrogs (Rana catesbeiana). Chemosphere, 236:124350. doi.org/10.1016/j.chemosphere.2019.124350.

Flynn, R.W., T.D. Hoskins, M. Iacchetta, C. Perre, L.S. Lee, J.T. Hoverman, and M.S. Sepúlveda. 2020. Dietary Exposure and Accumulation of Per- And Polyfluoroalkyl Substances Alters Growth and Reduces Body Condition of Post-Metamorphic Salamanders. Science of the Total Environment, 765:142730. doi.org/10.1016/j.scitotenv.2020.142730.

Flynn, R.W., M. Iacchetta, C. De Perre, L.S. Lee, M.S. Sepúlveda, and J.T. Hoverman. 2020. Chronic PFAS-Exposure Under Environmentally Relevant Conditions Delays Development in Northern Leopard Frog (Rana pipiens) Larvae. Environmental Toxicologyand Chemistry, 40(3):711-716. doi.org/10.1002/etc.4690.

Flynn, R.W., G. Hoover, M. Iacchetta, S. Guffey, C. de Perre, B. Huerta, W. Li, J.T. Hoverman, L. Lee, and M.S. Sepúlveda. 2022. Comparative Toxicity of Aquatic Per- and Polyfluoroalkyl Substance Exposure in Three Species of Amphibians. Environmental Toxicology and Chemistry, 41(6):1407-1415. doi.org/10.1002/etc.5319.

Foguth, R.M., R.W. Flynn, C. de Perre, M. Iacchetta, L.S. Lee, M.S. Sepúlveda, and J.R. Cannon. 2019. Developmental Exposure to Perfluorooctane Sulfononate (PFOS) And Perfluorooctanoic Acid (PFOA) Selectively Decreases Brain Dopamine Levels in Northern Leopard Frogs. Toxicology and Applied Pharmacology, 377:114623. doi.org/10.1016/j.taap.2019.114623.

Foguth, R., T.D. Hoskins, G.C. Clark, M. Nelson, R.W. Flynn, C. DePerre, J.T. Hoverman, L.S. Lee, M.S. Sepúlveda, and J. Cannon. 2020. Single and Mixture Per- And Polyfluoroalkyl Substances Accumulate in Developing Northern Leopard Frog Brains and Produce Complex Neurotransmission Alterations. Neurotoxicology and Teratology, 81:106907. doi.org/10.1016/j.ntt.2020.106907.

Foguth, R., M.S. Sepúlveda, and J. Cannon. 2020. Per- And Polyfluoroalkyl Substances (PFAS) Neurotoxicity in Sentinel and Nontraditional Laboratory Model Systems: Potential Utility in Predicting Adverse Outcomes in Human Health. Toxics, 8(2):42. doi.org/10.3390/toxics8020042.

Hoover, G.M., M.F. Chislock, B.J. Tornabene, S.C. Guffey, Y.J. Choi, C. De Perre, J.T. Hoverman, L.S. Lee, and M.S. Sepúlveda. 2017. Uptake and Depuration of Four Per/Polyfluoroalkyl Substances (PFASs) in Northern Leopard Frog Rana pipiens Tadpoles. Environmental Science and Technology Letters, 4:399-403. doi.org/10.1021/acs.estlett.7b00339.s001.

Hoover, G., S. Kar, S. Guffey, J. Leszczynski, and M.S. Sepúlveda. 2019. In Vitro and In Silico Modeling of Perfluoroalkyl Substances Mixture Toxicity in an Amphibian Fibroblast Cell Line. Chemosphere, 233:25-33. doi.org/10.1016/j.chemosphere.2019.05.065.

Hoskins, T.D., E.B. Allmon, R.W. Flynn, L.S. Lee, Y. Choi, J.T. Hoverman, and M.S. Sepúlveda. 2022. An Environmentally Relevant Mixture of Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Does Not Conform to Additivity in Northern Leopard Frogs Exposed Through Metamorphosis. Environmental Toxicology and Chemistry, 41(12):3007-3016. doi.org/10.1002/etc.5486.

Lech, M.E., Y.J. Choi, L.S. Lee, M.S. Sepúlveda, and J.T. Hoverman. 2022. Effects of Per- and Polyfluoroalkyl Substance Mixtures on the Susceptibility of Larval American Bullfrogs to Parasites. Environmental Science and Technology, 56(22):15953-15959. doi.org/10.1021/acs.est.2c04574.

Pandelides, Z., J. Conder, Y. Choi, E. Allmon, T. Hoskins, L, Lee, J. Hoverman, and M. Sepúlveda. 2023. A Critical Review of Amphibian PFAS Ecotoxicity Research Studies: Identification of Screening Levels in Water and Other Useful Resources for Site‐Specific Ecological Risk Assessments. Environmental Toxicology and Chemistry, 42:2078-2090. doi.org/10.1002/etc.5695.

Tornabene, B., M. Chislock, M. Gannon, M.S. Sepúlveda, and J, Hoverman. 2021. Relative Acute Toxicity of Three Per- And Polyfluoroalkyl Substances on Nine Species of Larval Amphibians. Integrated Environmental Assessment and Management, 17(4):684-690. doi.org/10.1002/ieam.4391.

Wasel, O., K.M. Thompson, Y. Gao, A.E. Godfrey, J. Gao, C. Mahaptra, L.S. Lee, M.S. Sepúlveda, and J.L. Freeman. 2020. Comparison of Zebrafish In Vitro and In Vivo Developmental Toxicity Assessments of Perfluoroalkyl Acids (PFAAs). Journal of Toxicology and Environmental Health, Part A, 84(3):125-136. doi.org/10.1080/15287394.2020.1842272.

Theses and Dissertations

Abercrombie, S.A. 2017. The Uptake and Effects of Poly- and Perfluoroalkyl Substances on Larval and Juvenile Amphibians (Master's Thesis). Purdue University.

Hoover, G.M. 2017. Effects of Per/Polyfluoroalkyl Substance Exposure on Larval Amphibians (Ph.D. Dissertation). Purdue University.

Lech M.E. 2021. Investigating the Effects of Per-/Polyfluoroalkyl Substances (PFAS) on Host-Parasite Interactions in a Larval Amphibian-Echinostome System. (Master's Thesis). Purdue University.

Perez, E. 2021. An Environmentally Relevant Binary Mixture of PFOS and PFHxS Results in Antagonism and Reduced Body Condition in Northern Leopard Frogs. (Master's Thesis). Purdue University.


Strathmann, T. J., C. Higgins, B. Wu, and S. Hoa. 2023. Hydrothermal Technology for Decontamination and Mineralization of Perfluoro- and Polyfluoroalkyl Substances (PFAS) in Wastes, Concentrate Solutions, and Chemical Stockpiles. U.S. Patent 11577111-B2.