• tPODs provide sensitive molecular effect thresholds for PFAS in zebrafish embryos. • PFOA and GenX show distinct, substance-specific transcriptomic response patterns. • tPOD-based thresholds are more sensitive than traditional apical toxicity endpoints. • Results support integrating toxicogenomic benchmarks into environmental risk assessment. Per- and polyfluoroalkyl substances (PFAS) are highly persistent and globally distributed contaminants associated with adverse effects in humans and wildlife. Legacy compounds such as perfluorooctanoic acid (PFOA) are strictly regulated, whereas emerging alternatives like GenX remain less stringently controlled despite widespread use. This study applied a transcriptomics-based zebrafish embryo test (OECD TG 236) to derive mechanistically informed, 3R-compliant effect thresholds for PFOA and GenX. Embryos were exposed to 2.5–25,000 µg/L, followed by RNA sequencing, differential expression analysis, transcriptomic point of departure (tPOD) derivation, and pathway-level sensitivity assessment. First-peak tPODs were 4,190 µg/L for PFOA and 241 µg/L for GenX, indicating transcriptomic sensitivity comparable to or more protective than chronic fish toxicity thresholds. GenX induced broad, concentration-dependent transcriptional responses, whereas PFOA caused fewer changes, reflecting distinct molecular modes of action. Pathway analyses revealed GenX effects on RNA metabolism, mitochondrial function, and developmental signaling, while PFOA impacted endocytosis, pentose and glucuronate interconversions, and neuroactive ligand–receptor interaction. These findings show that tPOD-based benchmarks provide sensitive, biologically meaningful thresholds for PFAS and support integrating next‑generation molecular endpoints into regulatory decision-making, particularly for under-regulated compounds like GenX.
Benad et al. (Fri,) studied this question.