Phenotypic attenuation masks persistent molecular perturbations during PFOS-cadmium co-exposure in zebrafish larvae.

Perfluorooctane sulfonate (PFOS) and cadmium (Cd) are persistent contaminants that frequently coexist in aquatic environments, yet their combined effects on early neurodevelopment remain unclear. Here, zebrafish embryos were exposed to environmentally relevant concentrations of Cd, PFOS, or their mixtures from 2 to 120 h post-fertilization. Developmental endpoints, light-dark locomotor behavior, transcriptomics, and qPCR validation were assessed. Cd caused marked developmental toxicity and abnormal light-dependent behavior, whereas PFOS induced weaker and non-monotonic effects. Co-exposure partially attenuated several Cd-induced developmental and behavioral abnormalities, indicating an endpoint-dependent interaction rather than uniform enhancement of Cd toxicity. Transcriptomics analysis showed that Cd mainly affected glutathione metabolism, ferroptosis-related, tight junction, and phototransduction pathways, while PFOS affected DNA replication/repair, spliceosome function, redox regulation, and metabolic remodeling. Combined exposure retained enrichment of ferroptosis and phototransduction pathways and involved iron/transition-metal ion transport, metal-ion homeostasis, and heme/iron-binding processes. qPCR confirmed altered expression of tnfa, ptgs2b, opn1lw1, pde6ha, and cldn5a, supporting inflammatory, ferroptosis-related pathways, visual, and barrier-associated molecular responses. These findings show that PFOS modifies Cd toxicity in a pathway-specific manner, with attenuation of several phenotypic responses accompanied by persistent molecular perturbations. The coordinated perturbation of phototransduction, metal-ion homeostasis, and ferroptosis-related/inflammatory pathways provides a molecular framework for improving early-life risk assessment of mixed PFAS-metal contamination.