Zync pyrithione induces immunotoxicity in the colonial ascidian Botryllus schlosseri

After TBT ban by many countries in the world, due to its severe impact to coastal ecosystems, mainly related to immunosuppressive effects on both invertebrates and vertebrates, alternative biocides have been massively introduced in formulations of antifouling paints. Zinc pyrithione (ZnP) is one of these new generation biocides, used in dermatology for its antidandruff and antimycotic action. However, up to now, no data are available, concerning its potential target organisms, long-term toxic effects on biocoenoses, mechanisms of action, bioaccumulation and environmental fate. Our interest in the study of ascidian defence reactions led us to investigate the effects of ZnP on cultured phagocytes of the colonial ascidian Botryllus schlosseri, a model organism for immunobiology investigations, widespread in shallow waters, already used in the study of tributyltin (TBT) immunotoxicity. We set up short-term haemocyte cultures (60 min at 20°C) exposed to various concentrations (0.1 to 10 µM) of ZnP (LC50 = 82.5 µM). The fraction of cells with amoeboid morphology, expressed as the amoebocytic index, and the percentage of haemocytes containing phagocytised yeast cells were significantly (p < 0.05) reduced after exposure to 0.1 and 0.5 µM ZnP, respectively. These effects were dose- and time-dependent, and irreversible, similarly to those reported for TBT. Detection of microfilaments and microtubules at fluorescence microscope by FITC-phalloidin and anti-α-tubulin antibody, respectively, revealed thorn-shaped, cytoplasmic projections due to cytoskeletal alterations only in the actin component. Isodynamic mixtures of ZnP and TBT showed and antagonistic interaction on their effects on the amoebocytic index, although no cytoskeletal disassembly was observed, and significant (p < 0.05) detachment of cells from the substrate occurred after incubation at 0.5 µM. Moreover, like TBT, ZnP induced apoptosis which was detected both as chromatin condensation with acridine orange at 0.1 µM and chromatin fragmentation with TUNEL reaction at 0.5 µM. The latter concentration also significantly (p < 0.001) affected both oxidative phosphorylation and lysosomal activities through the inhibition of cytochrome-c-oxidase and acid phosphatase activity, respectively. Differently to TBT, no effect was observed on Ca2+ homeostasis, since no decrement in Ca2+-ATP ase activity occurred, although a small decrease in cytosolic Ca2+ was detected after incubation at the highest concentration. In conclusion, our results demonstrate that ZnP shows a strong toxicity on cultured haemocytes at very low concentrations and interferes with fundamental cell activities. Therefore, this substituent biocide results as much toxic as TBT and, since many mechanisms of actions are unknown, it represents a potential risk for the environmental health.