Multigenerational and persistent toxicity of environmentally relevant concentrations of Benzo[a]pyrene in Marine Medaka (Oryzias melastigma)

Benzo[ a ]pyrene (BaP) is a well-known environmental pollutant with extensively documented toxicological hazard. While transgenerational BaP effects have been documented, the reversibility of these effects following exposure cessation at different generational stages remains unexplored. This study employed a novel exposure methodology on marine medaka ( Oryzias melastigma ) with two key regimens: including continuous multi-generational BaP exposure (G1F1-G3F3) and post-exposure recovery (non-BaP, G1F2-G1F3 and G2F3). Integrated analyses were conducted across phenotypic, developmental, reproductive, and molecular biological endpoints. The results showed that: (1) BaP significantly inhibited growth, development, and reproduction of marine medaka, increasing offspring deformity rate and mortality rate. Most notably, 100 % offspring mortality occurred in both G2F4 and G3F4 generations, as well as the post-exposure recovery in G2F4; (2) 200 μg·L −1 BaP damaged male gonads, decreasing sperm counts and increasing testicular non-cellular areas, and the non-cellular areas found in G2F3 and G3F3 were exacerbated; (3) molecular analyses demonstrated systematic disruption of the hypothalamic-pituitary-gonadal (HPG) axis, with significant downregulation of key genes of steroidogenic enzymes ( cyp17a , 17βhsd , cyp19a ) and reproductive hormone receptors ( erα , arα ). These findings demonstrate that environmentally relevant concentrations of BaP induce irreversible transgenerational reproductive impairment that persists beyond exposure termination. This underscores the critical need to reassess environmental quality criteria for BaP, while also emphasizing the importance of comprehensive ecological risk assessment and the necessity of incorporating trans/multigenerational testing into standard ecotoxicological frameworks.