Thermal history modulates PFUnDA and PFTeDA toxicity in the Mediterranean mussel Mytilus galloprovincialis

Per- and polyfluoroalkyl substances (PFAS) are persistent contaminants of increasing concern in aquatic ecosystems, yet their interactions with climate-related stressors remain insufficiently understood. This study evaluated whether prior exposure to a moderately elevated temperature modifies the sub-cellular responses of the Mediterranean mussel Mytilus galloprovincialis to two long-chain perfluoroalkyl carboxylic acids, perfluoroundecanoic acid (PFUnDA) and perfluorotetradecanoic acid (PFTeDA). Mussels were pre-exposed to 23 °C for 6 days and then exposed for 14 days at 17 °C to PFUnDA or PFTeDA at a nominal concentration of 20 ng/L. A multi-biomarker approach showed that PFAS exposure at 17 °C induced moderate biochemical changes, mainly involving metabolic depression and antioxidant modulation, with limited oxidative damage. In contrast, thermally pre-exposed mussels displayed higher metabolic activity, increased energy consumption, stronger antioxidant and detoxification responses, and reduced cellular energy allocation, indicating a more energetically demanding physiological state. The strongest integrated response was observed in PFTeDA-exposed mussels after thermal pre-exposure, suggesting compound-specific effects consistent with chain-length-related physicochemical behaviour. However, because exposure concentrations were nominal and internal PFAS burdens were not measured, this interpretation should be considered a mechanistic inference rather than direct evidence of differential bioaccumulation. Overall, these findings indicate that thermal history can reshape sublethal PFAS responses in marine mussels and highlight the need to incorporate stressor sequence, internal exposure and food availability into future ecological risk assessments under climate change.