Photoperiod perturbations impact perfluorohexane sulfonate (PFHxS) induced developmental toxicity.

Perfluorohexane sulfonate (PFHxS) is a persistent perfluoroalkyl substance with increasing scrutiny due to its widespread occurrence and potential for developmental neurotoxicity. In this study, we investigated how photoperiod - a critical regulator of circadian physiology- modulates PFHxS toxicity in zebrafish embryos. Photoperiods are highly relevant in today's context, where artificial lighting, shift work, and time-zone travel increasingly disrupt natural light-dark cycles. To evaluate this variable, embryos were raised under either a standard 14-hour light:10-hour dark (14 L:10D) cycle, constant darkness (24D) or constant light (24 L) and simultaneously exposed to PFHxS across a concentration range (0.0025-25 µM) from 2 to 120 h post-fertilization. Since 24 L did not elicit any significant phenotypic differences, this photoperiod was excluded from further studies. LC-MS analysis revealed comparable PFHxS uptake across 24D and 14 L:10D photoperiods, ruling out bioaccumulation-induced differences. However, photomotor behavioral assays showed photoperiod-specific phenotypes, with hyperactivity during light epochs under 24D. PFHxS elevated melatonin levels under 14 L:10D but reduced them under 24D, indicating circadian endocrine disruption. Transcriptomic analysis revealed greater PFHxS-induced dysregulation under 24D, including suppressed DNA replication and cell cycle pathways and increased oxidative stress. These effects were concordant with elevated ROS, increased DNA damage, and reduced proliferation. Eye-specific studies revealed reduced retinal proliferating cells and increase in cone Arrestin3a under 24D, but not 14 L:10D, suggesting that aberrations in eye development may be a driver of photomotor phenotype. Collectively, our findings highlight the critical role of environmental lighting in shaping toxicological outcomes and emphasize the need to incorporate photoperiod context into risk assessments.