049 | CMIT as an emerging contaminant: physiological and biochemical effects in Mytilus galloprovincialis

Giuseppe Stracuzzi¹, Federica Impellitteri¹, Maria Giovanna Rizzo¹, Monia Perugini², Marika Cordaro³, Kateryna Yunko⁴, Oksana Stoliar⁴, Caterina Faggio^1|5 | ¹Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; ²Department of Feed and Food Scienxes, Teramo Univesity, Teramo, Italy; ³Department of Biomedical, Dental and Morphological and Functional Imaging University of Messina, Messina, Italy; ⁴Ternopil Volodymyr Hnatiuk National Pedagogical University, Ternopil, Ukraine; ⁵Department of Eco-sustainable Marine Biotechnology, Stazione Zoologica Anton Dohrn, Naples, Italy.

CMIT (5-chloro-2-methyl-4-isothiazolin-3-one) is a biocide included in the formulation of several products, used on a daily basis. Its widespread use has inevitably led to significant concentrations being detected in the aquatic environment, raising concerns about its potential implication on aquatic ecosystems and organisms(1). Therefore, this study investigated the impact of CMIT on aquatic organisms, using Mytilus galloprovincialis as a model. Mussels were exposed to two concentrations of CMIT (E1: 0.01mg L-1 and E2: 0.1 mg L-1). After 14 days of exposure, cytotoxic effects on hemocytes and digestive gland (DG) isolated cells were analyzed using the Neutral Red (NR) and Trypan blue (TB) colorimetric assays, which allow for the evaluation of cell viability. In addition, the phagocytic activity of the hemocytes was evaluated, using the yeast Saccharomyces cerevisiae (1×10⁷ cells/mL). Also, the osmoregulatory performance of DG cells subjected to an hypotonic shock, was evaluated through the decreasing volume regulation (RVD) assay2. In order to obtain information about CMIT-induced biochemical responses, biomarkers of oxidative stress, i.e., catalase (CAT) and glutathione (GSH/GSSG), cytochrome P450-related enzyme activity (EROD), and neurotoxicity effects (AChE), were measured in DG cells. Results showed a reduction of vitality in both experimental groups of mussels exposed to CMIT (E1 and E2) compared to the control, with a greater reduction in the E1 group. Results were similar for both cell populations, hemocytes and DG cells. The RVD assay further demonstrated dameges to transport membrane mechanisms, as DG cells from exposed mussels lost their ability to restore their original volume. Also, the phagocytosis ability analysis demonstrated a significant reduction in both groups of mussels exposed to CMIT, assuming a reduction in the immune response. Furthermore, biomarker analysis revealed significant alterations in antioxidant mechanisms and detoxification activity and neurotoxic effects. Ultimately, these results demonstrate the toxic potential of CMIT towards physiological and biochemical processes in M. galloprovincialis, laying the foundation for further research aimed at clarifying the consequences of its presence in aquatic environments and, therefore, the possible implications for human health.
This work has been funded by the European Union – NextGenerationEU, as part of the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.1 (PRIN) under Grant Assigment Decree n. 1375 adopted on 01-09-2023 by the Italian Ministry of University and Research (MUR) – Project PRIN 2022 PNRR Title: Investigations of emerging pollutant: ecological and human health impacts of isothiazolinone biocides – Project ministerial code: P2022BH28Y 03 – CUP: C53D23009240001 – Beneficiary: University of Teramo (UniTE).