Low oxygen stress alters response to sublethal copper exposure without inducing cross-tolerance in the blue mussel Mytilus edulis.

The Baltic Sea's limited water exchange and densely populated catchment area contribute to the frequent co-occurrence of hypoxia and chemical pollutants such as copper (Cu), raising concerns about the effects of sequential stressors on marine organisms. This study tested whether pre-exposure to intermittent oxygen stress alters the physiological and immune responses of Mytilus edulis to subsequent Cu exposure. Mussels were preconditioned for 21 days to either mild intermittent hypoxia (7 h/day at ∼2 mg/L DO) or severe intermittent anoxia (2 days/week at <0.01 mg/L DO), followed by a 7-day exposure to sublethal Cu (20 µg/L) under normoxia. Hypoxia preconditioning impaired Cu homeostasis, leading to elevated Cu accumulation. Hypoxia-exposed mussels maintained immune function (phagocytosis, lysosomal integrity, acid phosphatase activity), while anoxia disrupted phagocyte-lysosome balance. Cu exposure alone reduced hemocyte abundance and increased acid phosphatase activity, indicating typical immunotoxic effects that were not alleviated by prior oxygen stress. Lipid reserves declined under both oxygen regimes, while carbohydrate and protein levels were reduced by hypoxia. Anoxia preconditioning mitigated Cu-induced energy depletion, as reflected by elevated lipid and carbohydrate levels. Mitochondrial electron transport system activity increased following low oxygen preconditioning and further during Cu exposure, suggesting elevated energy demands at post-stress recovery/detoxification. Despite these bioenergetic adjustments, oxidative stress markers (total antioxidant capacity, lipid peroxidation) and stress gene expression (hsp70, hsp90, GADD45A, nrf2, NfκB) remained stable across treatments. In conclusion, while oxygen stress modulated mussel responses to Cu, it did not confer cross-tolerance, highlighting the complex and context-dependent nature of multiple stressor interactions in coastal environments.