Mechanistic insights into the comparative ecotoxicity of PFOS and its emerging alternatives in zebrafish: An...

Perfluorohexane sulfonate (PFHxS), perfluorobutane sulfonate (PFBS), and 6:2 fluorotelomer sulfonate (6:2FTSA) are increasingly detected in aquatic environments as primary alternatives to perfluorooctane sulfonate (PFOS). However, their comparative toxicological profiles and underlying mechanisms remain poorly understood, complicating ecological risk assessments. To address this, we integrated multi-endpoint bioassays with molecular simulations to systematically assess the ecotoxicity of PFOS and its alternatives, utilizing zebrafish (embryos and adults) as a sensitive in vivo model. Zebrafish embryos and adults were exposed to an environmentally relevant concentration of 50 μg L^-1, with subsequent experimental assessments conducted after 120 h and 28 days of exposure, respectively. We focused on oxidative stress, apoptosis, and immune system responses across two developmental stages. Our findings revealed that three alternatives induced toxicological response patterns similar to PFOS, triggering oxidative damage, apoptosis, and inflammatory responses in adult zebrafish. Notably, stage-specific immunotoxicity was identified in embryos: while PFOS, PFBS, and 6:2FTSA primarily impaired immune cytokine secretion, PFHxS distinctively disrupted cellular immune homeostasis. To quantitatively evaluate these multidimensional effects, the integrated biomarker response (IBR) index was applied. IBR values highlighted that PFHxS induced the most pronounced overall toxicity across both life stages (yielding maximum IBR values of 13.99 in embryos and 18.62 in adults, which were 1.29 and 1.58 times higher than those of PFOS, respectively), whereas PFBS and 6:2FTSA posed comparatively lower ecological risks than PFOS. Molecular docking analyses provided a crucial mechanistic underpinning for these physiological indicators, revealing that PFOS and its alternatives bind to critical enzyme sites, thereby altering their structural conformations and inhibiting catalytic activity. Ultimately, coupling molecular mechanisms with the macroscopic IBR index establishes a robust framework for ecotoxicological evaluation. This study provides vital mechanistic insights and quantitative data to predict the ecological impacts of emerging PFOS alternatives, thereby informing regulatory frameworks and mitigating environmental risks to aquatic ecosystems.