Investigating in vivo toxicity of per- and polyfluoroalkyl substances (PFAS) mixtures on the development of zebrafish ( <i>Danio rerio</i> ) embryos as a model for environmental impact

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals widely used as surfactants and repellents across industries such as textiles, personal care products, and nonstick cookware. In India, rapid industrialization and urbanization have increased PFAS usage, raising concerns about environmental contamination. Per- and polyfluoroalkyl substances are persistent, bioaccumulative, and have been detected in multi-environmental matrices including humans. This widespread contamination poses health risks to millions through water and food chains. Because PFAS usually occur as complex mixtures, comprehensive toxicity assessments addressing mixtures rather than individual compounds are urgently needed. The zebrafish (Danio rerio) is an established model for rapid toxicity screening and provides predictive insights into human health risks. This study evaluates developmental effects of two PFAS mixtures on zebrafish embryos and larvae: a 2-compound mixture (2-mix) consisting of perfluorooctanoic acid and perfluorobutanesulfonic acid, and a 24-compound mixture (24-mix) including long- and short-chain PFAS and their precursors. Developmental endpoints monitored were survival, hatching success, heart rate, and deformities. Morphometric analyses of head, eye, yolk sac, and pericardial areas were conducted with ImageJ. Oxidative stress was assessed via reactive oxygen species (ROS) quantification, and histopathology evaluated tissue alterations. Results revealed significant developmental toxicity, with the 24-mix causing delayed hatching, growth inhibition, blood accumulation, and reduced heart rate, whereas the 2-mix showed milder effects. Elevated ROS levels indicated oxidative stress in both groups, and histopathology confirmed damage to the eye, brain, and muscles. These findings demonstrate that prolonged exposure to environmentally relevant PFAS concentrations can induce significant biological effects. This study provides critical insights into PFAS mixture toxicity, informing risk assessments and guiding regulatory policy development to protect public health.