Comparison of sub-lethal metabolic perturbations of select legacy and novel perfluorinated alkyl substances (PFAS) in Daphnia magna

Per- and polyfluoroalkyl substances (PFAS) are a class of pollutants of concern due to their ubiquitous presence, persistence, and toxicity in aquatic environments. Legacy PFAS pollutants such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) have been more widely studied in aquatic environments. However, replacement PFAS, such as ammonium perfluoro (2-methyl-3-oxahexanoate; GenX) are increasingly being detected with little known information surrounding their toxicity. Here, Daphnia magna, a model organism for freshwater ecotoxicology was used to compare the acute sub-lethal toxicity of PFOS, PFOA, GenX, and PFAS mixtures. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), the targeted polar metabolic profile extracted from single Daphnia was quantified to investigate perturbations in the exposure groups versus the unexposed organisms. Multivariate statistical analyses demonstrated significant non-monotonic separation in PFOA, GenX, and PFAS mixture exposures. Sub-lethal exposure to concentrations of PFOS did not lead to significant separation in multivariate analyses. Univariate statistics and pathway analyses were used to elucidate the mode of action of PFAS exposure. Exposure to all individual PFAS led to significant perturbations in many amino acids including cysteine, histidine, tryptophan, glycine, and serine. These perturbations are consistent with biochemical pathway disruptions in the pantothenate and Coenzyme A (CoA) biosynthesis, thiamine metabolism, histidine metabolism, and aminoacyl-tRNA biosynthesis pathways. Overall, the collected metabolomic data is consistent with disruptions in energy metabolism and protein synthesis as the primary mode of action of sub-lethal PFAS exposure. Secondary modes of action among individual pollutant exposures demonstrated that the structural properties (carboxylic acid vs. sulfonic acid group) may play a role in the metabolic perturbations observed. Sub-lethal exposure to PFAS mixtures highlighted a mixed response when compared to the individual pollutants (PFOS, PFOA, and GenX). Overall, this study emphasizes the niche capability of environmental metabolomics to differentiate secondary modes of action from metabolic perturbations in both single pollutant and pollutant mixtures within the same chemical class.