Assessing the ecotoxicological impact of novel PFAS (GenX and PFPeA) on Mytilus galloprovincialis under different salinity conditions: a multi-biomarker approach

The introduction of next-generation per- and polyfluoroalkyl substances (PFAS), including perfluoropentanoic acid (PFPeA) and hexafluoropropylene oxide dimer acid (GenX), has raised ecotoxicological concerns due to their persistence and occurrence in coastal ecosystems. These transitional environments are also increasingly affected by climate- driven salinity fluctuations associated with extreme precipitation. This study assessed the effects of GenX and PFPeA, individually and as a mixture (MIX), on the bioindicator bivalve Mytilus galloprovincialis exposed for 28 days to salinity 30 (optimal) and 20 (hyposaline stress). Tissue analyses further revealed PFAS accumulation, confirming organismal exposure despite the generally lower bioaccumulation potential attributed to short- chain PFAS. A multi-biomarker approach evaluated metabolic, osmoregulatory, antioxidant, biotransformation, and cellular damage responses, complemented by the Integrated Biomarker Response index (IBRv2). Hyposalinity served as a major environmental stressor, increasing aerobic metabolism (ETS) and lipid peroxidation (LPO) even in the absence of PFAS exposure. At salinity 30, PFAS effects were compound-specific: PFPeA induced comparatively limited adaptive responses, whereas GenX caused stronger disruption of homeostasis, particularly through marked activation of carboxylesterases (CbEs), which also influenced mixture responses. Under hyposalinity, individual PFAS effects were partly masked, suggesting reduced physiological capacity to mount additional stress responses under high energetic demand. In contrast, MIX exposure at salinity 20 intensified disruption of the antioxidant system and redox imbalance. Overall, the results demonstrate that PFAS toxicity is strongly modulated by environmental conditions and support the need for multi- stressor approaches in ecological risk assessment to better capture interactions between chemical contamination and climate-driven salinity changes.