Per- and polyfluoroalkyl substances (PFAS) are an important class of emerging contaminants in the environment. Most studies on the impact of PFAS considered individual compounds; meanwhile PFAS mainly exist in soil as mixtures. To fill this knowledge gap, we investigated the impact of 200 ng/g of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS)as individual compounds and a binary mixture (PFOS+PFOA)on the survival, biomass, reproduction, and metabolites of earthworm (Eisenia fetida). PFAS significantly (p < 0.05) decreased the survival, biomass, and reproduction of E. fetida after 28 d of exposure, but the impact of the mixture was not additive. The bioaccumulation of PFOS after 28 d increased, while that of PFOA decreased when E. fetida was exposed to the mixture compared to the individual compounds. These bioaccumulation trends were partly attributed to changes in the soil distribution coefficient (Kd) of PFOS and PFOA when present in the mixture. Eighty percent of the statistically (p and FDR < 0.05) altered metabolites after 28 d were similarly perturbed by both PFOA and PFOS+PFOA. The pathways dysregulated are related to the metabolism of amino acids, energy, and sulfur. We showed that PFOA dominates the molecular-level impact of the binary PFAS mixture.

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