Copper toxicity as stressor in Chaetoceros calcitrans under different salinity levels: Growth inhibition, ROS accumulation, and alterations in fatty acid and PUA profiles

The sensitivity of marine microalgae to metal contamination, particularly copper (Cu), is well recognized in ecotoxicology, providing key insights into predicting marine pollution impacts. This study examined the combined effects of Cu toxicity and salinity on the diatom Chaetoceros calcitrans under controlled laboratory conditions. Toxicity assays were conducted using two environmentally relevant Cu concentrations (2 and 5 μg L^-1) and three salinity levels (33, 36, and 39) over 96 h, with cell responses analyzed via flow cytometry. Specific growth rate (SGR), chlorophyll fluorescence, fatty acid methyl ester (FAME) composition, polyunsaturated aldehydes (PUAs), and reactive oxygen species (ROS) production were evaluated. Results showed that SGR increased from 1.7 d^-1 at salinity 33 to 1.9 d^-1 at 39, indicating enhanced growth at higher salinity. However, Cu exposure significantly reduced SGR, particularly under 5 μg L^-1 Cu at salinity 33, where SGR dropped to 0.86 d^-1. ROS levels were highest under the combined low salinity (33) and 5 μg L^-1 Cu treatment, suggesting strong oxidative stress. PUA profiles shifted with Cu and salinity, with stress-related compounds such as 2E,4E/Z-decadienal and 2E,4E/Z,7Z-decatrienal increasing at low salinity. Fatty acid analysis revealed marked rises in saturated fatty acids, especially palmitic acid (C16:0) and myristic acid (C14:0), under Cu exposure. In the Cu2/39 treatment, C16:0 reached 1.34 × 10⁴ fmol cell^-1 compared to 1.36 × 10³ fmol cell^-1 in the control. Monounsaturated fatty acids, notably palmitoleic acid (C16:1), also increased, reaching 7. 46 × 10³ fmol cell^-1 versus 1.71 × 10³ fmol cell^-1 in the control. Conversely, polyunsaturated fatty acids, including arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5 n-3), decreased significantly following Cu exposure. Overall, elevated salinity mitigated Cu-induced growth inhibition but intensified oxidative and biochemical stress, underscoring the complex, endpoint-specific responses of C. calcitrans to multiple environmental stressors.