Immunotoxicity in ascidians: Antifouling compounds alternative to organotins—IV. The case of zinc pyrithione Francesca Cima 1, ⁎, Loriano Ballarin 1 Department of Biology, University of Padova, Padova, Italy abstract article info Article history: Received 11 November 2014 Received in revised form 22 December 2014 Accepted 23 December 2014 Available online 6 January 2015 Keywords: Botryllus Ascidians Haemocytes Zinc pyrithione Antifouling Immunotoxicity New biocides such as the organometallic compound zinc pyrithione (ZnP) have been massively introduced by many countries in formulations of antifouling paints following the ban on tributyltin (TBT). The effects of suble- thal concentrations (LC 50 = 82.5 μM, i.e., 26.2 mg/l) on cultured haemocytes of the ascidian Botryllus schlosseri have been investigated and compared with TBT. The percentage of haemocytes with amoeboid morphology and containing phagocytised yeast cells were significantly (p b 0.05) reduced after exposure to 0.1 (31.7 μg/l) and 0.5 μM (158 μg/l), respectively. An antagonistic interaction in inducing cytoskeletal alterations was observed when ZnP and TBT were co-present in the exposure medium. ZnP affected only the actin component. As caused by TBT, ZnP induced apoptosis and inhibited both oxidative phosphorylation and lysosomal activities. In contrast to the case of TBT, a decrement in Ca 2+ -ATPase activity and a decrease in cytosolic Ca 2+ were detected after incubation at the highest concentration (1 μM, i.e., 317.7 μg/l) used. In comparison with other antifouling compounds, ZnP shows as much toxicity as TBT to cultured haemocytes at extremely low concentrations interfering with fundamental cell activities. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Beginning in the second half of the 1960s, biocides based on organotin compounds (TBT, TPT and their derivatives) were massively introduced in the formulation of the antifouling paints used for the pres- ervation of submerged structures, such as boat hulls and propellers, buoys and moles, from the settlement of various aquatic sessile organ- isms. These substances have proved to be extremely harmful to benthic marine biocoenoses, in particular to filter-feeding organisms. Moreover, they frequently persist in the environment (Bryan et al., 1986; Henderson and Salazar, 1996; Hoch, 2001; Cima et al., 2003). After the ban on the use of organotin on boats less than 25 m in length (Julian, 1989) and the proposal (2001, October) for a total ban on TBT beginning in 2003 by the International Maritime Organization (IMO) in compli- ance with the International Convention on the Control of Harmful Antifouling Systems on Ships (IMO document AFS/CONF/26), industries turned their attention to “TBT-free” biocidal combinations of new antifoulants or of compounds coming from either the pharmaceutical industry (bactericides) or agriculture (herbicides, fungicides, insecti- cides). Currently, various organic biocides are approved for use in amateur and professional antifouling products, e.g., chlorothalonil, dichlofluanid, diuron, irgarol 1051, maneb, sea-nine 211, silvex, TCMS pyridine, TCMTB, thiram, zinc pyrithione, zineb, and ziram. Their main use is to prevent both the settlement of spores and larvae of fouling macrorganisms and the formation of the bacterial and microalgal biofilm (Meadows, 1964; Railkin, 2004) required for the beginning of the ecological succession of the hard-substratum benthic biocoenosis. As a result, substantial coastal concentrations of these biocides have been found in areas of high yachting activity, particularly in marinas and recreational harbours. In terms of the toxicity and biodegradability of the new biocidal products in the aquatic environment, little or nothing has been verified prior to their introduction in antifouling paints after the banning of organotin compounds. Zinc pyrithione (ZnP) (CAS 13463-41-7, EINECS 236-671-3) is a zinc salt complex with the empirical formula C 10 H 8 N 2 O 2 S 2 Zn, molec- ular weight 317.7 g/mol, resulting from the combination of 2- mercaptopyridine-1-oxide with zinc (II). This product is used primarily in agriculture (EPA Pesticide Chemical Code 088002) for its antifungal activity and in the formulation of antidandruff shampoos. It is also known as zinc pyridinethione, bis(2-pyridylthio)zinc, 1,1′-dioxide bis(1-hydroxy-2(1H)-pyridinethionato-O,S)zinc, 2-mercaptopyridine N-oxide zinc salt and under the commercial names of Danex, De- Squaman, Zinc Omadine, and Vancide ZP. It was first synthesised in 1936 and registered in 1937 for medical use as a bactericidal, antimycotic and antiseborrheic compound (Pansy et al., 1953; Hyde and Nelson, 1984; Khattar et al., 1988). It was introduced under the Cos- metics Directive 82/368/EEC as a preservative. Under this Directive, it was authorised at a maximum concentration of 0.5%. Its allowed Comparative Biochemistry and Physiology, Part C 169 (2015) 16–24 ⁎ Corresponding author at: Department of Biology, University of Padova, Via Ugo Bassi 58/B,35131 Padova, Italy. Tel.: +39 498276198; fax: +39 498276199. E-mail address: francesca.cima@unipd.it (F. Cima). URL: https://sites.google.com/site/ascidianbiologylab/ (F. Cima). 1 These authors contributed equally to the study. http://dx.doi.org/10.1016/j.cbpc.2014.12.007 1532-0456/© 2015 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc