Combined environmental relevant exposure to perfluorooctanoic acid and zinc sulfate enhances apoptosis through binding with endogenous antioxidants in Daphnia magna.

Perfluorooctanoic acid (PFOA) is a long-chain legacy congener of the per- and polyfluoroalkyl substances (PFAS) family, notorious as a "forever chemical" owing to its environmental persistence and toxic nature. Essential elements such as zinc (Zn) can cause toxic effects when they change their metal speciation and become bioavailable, such as zinc sulfate (ZnSO). Combined toxicity assessment is a realistic approach and a challenging task to evaluate chemical interactions and associated risks. Therefore, the present study aims to elucidate the acute mixture toxicity (12-48 h) of PFOA and ZnSO in Daphnia magna at environment-relevant concentrations (ERCs, low dose: PFOA 10 μg/L ZnSO 20 μg/L; high dose: PFOA 20 μg/L ZnSO 50 μg/L) in terms of developmental impact, apoptosis induction, and interaction with major endogenous antioxidants. Our results showed that deformity rates significantly increased (p < 0.05) with increasing exposure duration and exposure concentrations, compared to the control group. Further, lack of antenna, tale degeneration, and carapace alterations were the most commonly observed deformities following combined exposure to PFOA and ZnSO, and these malformations were particularly pronounced after 48 h of exposure. Acridine orange (AO) staining was employed to examine apoptosis in D. magna, and apoptotic cells in terms of bright green fluorescence were detected in the abdominal claw carapace, heart, and post-abdominal area following exposure to a high dose of PFOA and ZnSO. The molecular docking results revealed that both PFOA and ZnSO showed strong binding affinities with endogenous antioxidants CAT and GST, where PFOA was more strongly bound with CAT and GST with higher docking scores of -9.59 kcal/mol and -7.49 kcal/mol than those with ZnSO (-6.70 kcal/mol and -6.55 kcal/mol, respectively). In conclusion, the mixture exposure to PFOA and ZnSO at the environmental level induce developmental impacts and apoptosis through binding with major endogenous antioxidants in D. magna.