Integrated cellular, physiological, and transcriptional responses of Mytilus galloprovincialis to environmentally relevant OIT and DCOIT co-exposure.

Isothiazolinone biocides are increasingly recognized as emerging contaminants in aquatic environments. Among them, 2-n-octyl-4-isothiazolin-3-one (OIT) and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) may co-occur in receiving waters, yet their combined effects on marine organisms remain poorly understood. The Mediterranean mussel Mytilus galloprovincialis was exposed for 14 days to OIT (2.81 µg L⁻¹), DCOIT (1 µg L⁻¹), and their mixture (MIX), selected to represent environmentally relevant exposure levels. Cellular viability, regulatory volume decrease, haemocyte phagocytic activity, and the expression of oxidative stress-, xenobiotic metabolism-, and inflammation-related genes were evaluated. Overall cell viability remained high, although OIT significantly reduced haemocyte membrane integrity and lysosomal stability. All exposure conditions altered cell volume regulation and markedly reduced haemocyte phagocytic activity, with the strongest reduction observed under co-exposure. Transcriptional analyses revealed tissue-wide modulation of MnSOD, Cu/ZnSOD, CYP4Y1, TNF-α, and IL-17, with the MIX group generally showing the greatest responses. In the digestive gland, CYP4Y1 expression reached a 4.13-fold increase, while MnSOD and TNF-α increased 3.33- and 3.50-fold, respectively, compared with controls. Co-exposure to OIT and DCOIT at low concentrations was associated with integrated cellular, osmoregulatory, immune, and transcriptional responses in M. galloprovincialis. These findings indicate that limited cytotoxicity does not exclude substantial sublethal effects and highlight the ecological relevance of assessing isothiazolinones as co-occurring contaminants rather than solely as individual compounds.