Cytotoxic and genotoxic effects of minor temperature changes and zinc oxide nanoparticles synthesized using Plantago major on Oncorhynchus mykiss gonadal cells

Temperature changes and nanoparticle exposure have emerged as critical environmental stressors with potential cytotoxic and genotoxic effects on aquatic species. This study investigates the impact of zinc oxide nanoparticles (ZnO NPs), synthesized using Plantago major leaves, on Oncorhynchus mykiss gonadal (RTG-2) cells under different temperature conditions (23 °C, 24 °C, and 25 °C). RTG-2 cells were exposed to hexagonal ZnO NPs (≤100 nm) at concentrations of 6.25, 12.5, 25, 50, 75, and 100 μg/mL for 24 and 48 h. Cytotoxicity was assessed using the sulforhodamine B assay, and genotoxicity was evaluated via the comet assay. Results demonstrated that both temperature and ZnO NP concentration significantly influenced cell viability and DNA integrity. The highest mortality rates (78.21% and 69.88%) were observed at 23 °C with 6.25 and 75 μg/mL of ZnO NPs after 24 and 48 h, respectively. At 25 °C, exposure to 12.5 μg/mL ZnO NPs resulted in 40.56% and 57.61% mortality after 24 and 48 h, respectively. Notably, at 24 °C, all ZnO NPs concentrations (except 25 and 50 μg/mL) significantly enhanced cell viability, with a 68.40% increase at 100 μg/mL after 48 h. Genotoxicity analysis showed the highest DNA damage (49.05% increase in tail DNA) at 23 °C with 75 μg/mL ZnO NPs after 24 h, and a 45.95% increase at 48 h with 50 μg/mL ZnO NPs at 24 °C. These findings highlight the temperature and dose dependent cytotoxicity and genotoxicity effects of ZnO NPs, suggesting that 24 °C may be a more favorable temperature for maintaining cell viability in the presence of such nanoparticles.