Effects of Per/polyfluoroalkyl Substance Exposure on Larval Amphibians

Per/polyfluoroalkyl substances (PFAS) are a group of persistent organic pollutants of emerging concern. Widespread use, along with volatility of some PFAS, has led to global distribution in soils and surface waters. The chemical stability of these compounds assures persistence in the environment, with effects unknown to much of the chronically-exposed biota. Many toxicology studies implicate PFAS in disruption of the hypothalamus-pituitary-thyroid (HPT) axis, which is known to play a major role in control of metamorphosis in amphibians. Thus, amphibians represent a class of organisms that may be susceptible to sub-lethal effects of PFAS exposure. Three main aims were accomplished to improve our understanding of the effects of these chemicals on larval amphibians. First, kinetics and bioaccumulation of PFAS in live animal tissues exposed in vivo were examined to understand uptake and depuration in the environment. Next, the effects of PFAS exposure on growth, development, and time to metamorphosis during a chronic exposure at environmentally relevant concentrations were evaluated and dose-response relationships constructed. Finally, the effects of mixtures of PFAS compounds were evaluated for synergistic or antagonistic effects in vitro. For in vivo experiments on PFAS uptake, bioconcentration, and sublethal effects, three model species were used: northern leopard frogs (Rana pipiens), eastern tiger salamanders (Ambystoma tigrinum), and American toads (Anaxyrus americanus). Four environmentally relevant PFASs were chosen for these aqueous exposures: perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and 6:2 fluorotelomer sulfonate (6:2 FTS). All four compounds are found in part-per-billion (ppb) levels in groundwater at the federally impacted site of Wurtsmith Air Force Base in Iosco County, Michigan, following decades of fire-fighting training and use of aqueous film-forming foams (AFFFs). Instances of contamination in both surface and ground waters with these compounds have been documented globally. For in vitro experiments, an amphibian fibroblast cell line (“Speedy cell”) was used. These cells were isolated from the hind limb bud of Xenopus during metamorphosis, and represent the most relevant available cultured cell for amphibian studies. In these experiments, cell viability following a 48-hour exposure to PFASs was determined using the MTT assay. Single-chemical exposures defined an IC50 for each compound; these IC50 values were then used to determine mixture ratios. Binary mixtures of various PFASs were then tested at varying levels and isobolograms were constructed, looking for synergistic or antagonistic effects of the combinations. In sum, PFAS are an important group of contaminants capable of significant bioaccumulation and effects on growth at environmentally relevant levels. However, not all PFAS are of equal concern, as bioconcentration and sub-lethal effects on growth and development are dependent on the exposure chemical. Of the chemicals examined, PFOS was the only one to show significant bioconcentration, while all of the chemical tested resulted in negative sub-lethal effects in at least one species. Not all species respond to PFAS exposure in the same manner, but it seems that amphibians with longer larval stages may be the most at-risk species for sub-lethal toxicity. This is best shown in salamanders, which were overall the most sensitive of the three species tested. All species are capable of bioconcentration, most notably of PFOS, which may have far-reaching implications in management of the many wildlife species that utilize amphibians as a food source. Finally, while the exact mechanisms of PFAS toxicity is still not fully understood, the exposure to mixtures may yield detrimental effects at concentrations below single chemical exposures, particularly if those mixtures contain (as most do) both PFOA and PFOS. Importantly, this may decrease the threshold of toxicity for compounds that are abundant, varied, and persistent in the environment