Unveiling the subchronic effects of PFAS mixtures in blue mussels (Mytilus spp.): An integrative assessment from molecular to physiological levels

Per- and polyfluoroalkyl substances (PFAS) are persistent synthetic chemicals that contaminate marine environments worldwide, yet their effects on marine bivalves under realistic scenarios remain poorly understood. This study investigated the subchronic effects of PFAS mixtures on blue mussels ( Mytilus spp.) over 28 days, using dietary and combined dietary-aqueous exposure pathways. The mixtures were representative of the chemical diversity found in contaminated environments, containing 36 and 7 PFAS for dietary and aqueous routes, respectively. Biological responses were assessed using analyses of gene expression, biochemical biomarkers, lipidomics and clearance rates. Unexpectedly, dietary exposure alone elicited more diverse and intense molecular responses than combined exposure. Notably, the mantle exhibited the strongest transcriptional response with several pro-apoptotic genes being upregulated, while growth/survival pathways were downregulated in the digestive gland. Dietary exposure also specifically disrupted biotransformation pathways in the gills through simultaneous cytochrome P450 downregulation and glutathione S-transferase activity induction. Conversely, combined exposure increased clearance rate, suggesting physiological compensation in mussels. Energy metabolism was also affected under both exposure conditions, as ATP production genes were specifically regulated across tissues, and limited but detectable lipidome changes occurred at the whole-organism level. Collectively, these findings indicate that PFAS mixtures can affect biotransformation, apoptosis-related and energy-related pathways in marine bivalves, although their environmental significance remains to be assessed under chronic, environmentally realistic conditions. This study underscores the importance of considering multiple exposure routes in PFAS research and supports the integration of multi-tissue approaches in future ecotoxicological assessments for bivalve biomonitoring.