101 | Physiological and cellular responses of the Mediterranean mussel to antifouling biocides 2-n-octyl-4-isothiazolin-3-one and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one, alone and in mixture

Federica Impellitteri¹, Maria Giovanna Rizzo¹, Giuseppe Piccione², Paolo Pastorino³, Cristiana Roberta Multisanti², Caterina Faggio^1|4 | ¹Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; ²Department of Veterinary Sciences, University of Messina, Messina, Italy; ³Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Torino, Italy; ⁴Department of Eco-sustainable Marine Biotechnology, Stazione Zoologica Anton Dohrn, Naples, Italy.

Abstract

Antifouling biocides are widely used to prevent biofouling on marine infrastructures, yet their release into coastal environments raises concerns about potential sub-lethal effects on non-target organisms (1). Among these compounds, 2-n-octyl-4-isothiazolin-3-one (OIT) and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) are commonly detected (2) in marinas and harbors, although their combined biological effects remain poorly understood. In this study, the Mediterranean mussel Mytilus galloprovincialis was used as a model organism to investigate physiological responses to environmentally relevant concentrations of OIT and DCOIT, tested individually and in mixture. Mussels were exposed under controlled laboratory conditions, and a multi-biomarker approach was applied to assess hemocyte and tissue-level responses. Endpoints included haemocyte viability, regulatory volume decrease (RVD), phagocytic activity, and gene expression profiles of markers involved in oxidative stress, detoxification (MnSOD, Cu/ZnSOD, CYP4Y1), and inflammatory response (TNF-α, IL-17) pathways. Overall, exposure to both biocides induced a clear transcriptional activation, with OIT generally eliciting stronger responses than DCOIT. The mixture produced the most pronounced effects, suggesting additive or synergistic interactions between the two compounds. This pattern was mirrored at the cellular level, where hemocyte viability was significantly reduced, and RVD was markedly impaired in the MIX group, indicating compromised volume regulation and early cellular dysfunction. Antioxidant and phase I detoxification genes were particularly modulated, supporting the digestive gland as a primary target compartment for biocide-induced stress. Alterations in hemocyte functionality and immune-related parameters were consistent with early stress responses, potentially preceding overt toxicity. Taken together, these results highlight the importance of integrating molecular and functional biomarkers to elucidate the mechanisms underlying antifouling biocide toxicity.