Early Life Stage Exposure to Environmentally Relevant Concentrations of TCPP Induces Multigenerational Reproductive Toxicity in Estuarine Fish: A Study Based on Whole-Genome Methylation and Steroidogenesis Pathway

Abstract Tris (1-chloro-2-propyl) phosphate (TCPP) is ubiquitous in estuarine environments. Parental TCPP exposure impaired reproduction and offspring developmental in zebrafish; yet its multigenerational and transgenerational effects and epigenetic mechanisms in estuarine fish remain unknown. Here, Mugilogobius chulae embryos were exposed to TCPP until 25 days posthatching and reared in clean seawater until F2 hatching. Reproductive and gonadal end points were assessed across generations, while gonadal steroidogenesis and whole-body DNA methylation in larvae were analyzed in F0 and F1 to investigate potential endocrine and epigenetic mechanisms. TCPP induced multigenerational reproductive toxicity that was transmitted to F2 embryos, with F1 and F2 lowest observed effect concentrations 1 order of magnitude lower than that in F0. In F0, 100 μg/L TCPP reduced fecundity and male gonadosomatic index. In F1, 10, and 100 μg/L prolonged spawning latency and affected male gonadal development. In F2, 10 μg/L reduced embryonic hatching. Disruption of steroidogenesis gene expression and hormone levels occurred in F0 and F1 male gonads but only in F1 females. Whole-genome DNA methylation revealed persistent hypermethylation of nerve growth factor receptor (NGFR) and calcium voltage-gated channel auxiliary subunit γ 2 (CACNG2) in the mitogen-activated protein kinase (MAPK) pathway across F0 and F1. Their hypermethylation and reduced expression were associated with decreased steroidogenic acute regulatory protein (STAR) and cytochrome P450 family 11 subfamily A member (CYP11A) in male gonads, suggesting NGFR and CACNG2 may represent candidate epigenetically regulated genes associated with TCPP-induced multigenerational reproductive toxicity. This study reveals transgenerational risks of environmental concentrations of TCPP to estuarine fish and provides epigenetic evidence for multigenerational endocrine disruptors.