Comparative toxicity of seven per- and polyfluoroalkyl substances (PFAS) on viability, oxidative stress and lipid profiles of rainbow trout gill cells

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants increasingly associated with adverse effects in aquatic organisms. Fish gills are a primary site of PFAS uptake; however, the cellular mechanisms underlying PFAS toxicity in gill tissue remain poorly characterised. Here, we investigated the cytotoxic, oxidative, and lipidomic effects of seven PFAS (PFDA, PFOA, PFHxA, PFBA, PFOS, PFHxS, and PFBS) in the rainbow trout gill cell line RTgill-W1. Cell viability and reactive oxygen species (ROS) assays revealed clear concentration- and time-dependent responses, with long-chain PFAS (PFDA and PFOS) inducing greater cytotoxicity and more sustained oxidative stress than short-chain analogues. Median effective concentrations (EC50, mg/L) ranged from 51.9 ± 1.7 for PFDA to 7107 ± 3.9 for PFBS, demonstrating pronounced differences in PFAS potency linked to chain length and functional group. Untargeted lipidomic profiling following exposure to 20 mg/L PFAS identified 202 significantly altered lipids across six major lipid classes. Glycerophospholipids and sphingolipids were most strongly affected, with perfluoroalkyl carboxylic acids (PFCAs) inducing more pronounced lipidomic disturbances than perfluoroalkyl sulfonic acids (PFSAs) and controls. Alterations in glycerolipid and fatty acyl metabolism, including disrupted acylcarnitine profiles, suggested changes in cellular energy metabolism and oxidative stress responses that may be associated with altered fatty acid utilisation and storage pathways. Collectively, these results demonstrate that PFAS toxicity in RTgill-W1 cells is strongly structure- and chain length-dependent, highlighting associations between oxidative stress and lipidomic perturbations in fish gill cells. However, these findings primarily provide insight within a short-term in vitro exposure framework and should be interpreted cautiously in relation to environmentally realistic exposure scenarios.