Characterization of cylindrospermopsin chlorination Sylvain Merel a , Michel Clément a , Annick Mourot b , Valérie Fessard b , Olivier Thomas a, ⁎ a Environment and Health Research Laboratory (LERES), French School of Public Health (EHESP), Avenue du Professeur Léon-Bernard—CS 74312, 35043 Rennes Cedex, France b Unit of Genetic Toxicology of Food Contaminants, French Food Safety Agency (AFSSA), La Haute Marche, BP 90203, 35302 Fougères Cedex, France abstract article info Article history: Received 21 September 2009 Received in revised form 9 April 2010 Accepted 17 April 2010 Available online 21 May 2010 Keywords: Cyanotoxin Cylindrospermopsin Chlorination Drinking water treatment Disinfection by-products Cytotoxicity In temperate countries, the occurrence of cyanobacterial blooms threatens drinking water resources. Consequently, cyanotoxins are increasingly considered in water treatment, and their reactions with chlorine used to disinfect drinking water are particularly investigated. This study presents new elements for further understanding of cylindrospermopsin chlorination, through reactants and by-products monitoring, UV spectrum examination, and cytotoxicity assessment on human intestinal Caco-2 cells. On the one hand, the evolution of mixture UV spectrum indicated that cylindrospermopsin was quickly transformed at least into one intermediate by-product. While mass spectrometry experiments confirmed that cylindrospermopsin was almost totally transformed within 5 min, chlorine was consumed up to 20 min after the beginning of the reaction with a rate of 5 mol per mol of toxin. Then, LC-MS analysis gave rise to the formation of a third cylindrospermopsin by-product in addition to 5-chloro-cylindrospermopsin and cylindrospermopsic acid previously identified. Thanks to the accurate mass measurement provided by the LTQ-Orbitrap mass spectrometer, this new and stable chlorination by-product was assigned the chemical formula C 13 H 18 N 4 O 7 S. On the other hand, both of the mitochondrial and lysosomal activities measured on Caco-2 cells revealed that cylindrospermopsin chlorination significantly decreases mixture cytotoxicity. © 2010 Elsevier B.V. All rights reserved. 1. Introduction In temperate countries, the occurrence of algae crisis in surface water is a growing environmental and public health concern. In addition, climate change and anthropic pressure may enhance their frequency and magnitude (Dale et al., 2006; Paul, 2008). Among microorganisms forming blooms, cyanobacteria are particularly considered because some species can produce harmful metabolites called cyanotoxins that have been associated with various intoxications worldwide (Edwards et al., 1992; Mez et al., 1997; Kuiper-Goodman et al., 1999; Griffiths and Saker, 2003). Cyanotoxins include a wide range of compounds, such as cylindrospermopsin (CYL), mainly produced by Cylindrospermopsis raciborskii and mostly reported in Australia (Saker et al., 1999; Griffiths and Saker, 2003), New Zealand (Stirling and Quilliam, 2001), Florida (Yilmaz et al., 2008), Israel (Banker et al., 1997) and Thailand (Carmichael et al., 2001; Li et al., 2001). Although CYL has often been considered as a tropical toxin, recent studies have identified its occurrence in temperate countries, like Germany (Fastner et al., 2003; Rücker et al., 2007; Wiedner et al., 2008), Italy (Messineo et al., 2009) and France (Brient et al., 2009). This 415 Da toxin encloses a tricyclic guanidine unit and a uracil moiety which could be the origin of its toxicity (Banker et al., 2001). CYL primarily affects the liver (Hawkins et al., 1985) inhibiting protein synthesis and leading to cell death (Froscio et al., 2003; Metcalf et al., 2004; Froscio et al., 2008), but other effects like tumour initiation are known too (Falconer and Humpage, 2001). Humans are potentially exposed to cyanotoxins through drinking water produced from contaminated resources. For example, the most famous intoxication by CYL, known as the Palm Island mystery disease, occurred in 1979 in Palm Island, northern Queensland, Australia (Byth, 1980; Bourke et al., 1983; Griffiths and Saker, 2003). The algicidal treatment of a dense cyanobacterial bloom in the water supply of the area led to CYL release and, some days later, over 100 children suffering from gastroenteritis attributed to the consumption of contaminated drinking water were admitted to the local hospital. Consequently, CYL has to be carefully considered in water treatment, and a guideline of 1 μgL -1 as a maximum concentration in drinking water has been proposed (Humpage and Falconer, 2003). Chlorination is a common process to disinfect drinking water whose effect on cyanotoxins has been particularly investigated (Acero et al., 2005; Ho et al., 2006; Xagoraraki et al., 2006; Rodríguez et al., 2007a; Rodríguez et al., 2008; Merel et al., 2009). Although CYL has not been extensively studied, chlorine twice in excess was shown to provide efficient toxin transformation with 2 by-products identified: 5-chloro- cylindrospermopsin and cylindrospermopsic acid (Banker et al., 2001; Science of the Total Environment 408 (2010) 3433–3442 Abbreviations: CYL, cylindrospermopsin; MTT, methylthiazolyldiphenyl-tetrazolium bromide; NRU, neutral red uptake. ⁎ Corresponding author. Tel.: +33 2 99 02 29 20; fax: +33 2 99 02 29 29. E-mail address: Olivier.Thomas@ehesp.fr (O. Thomas). 0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2010.04.033 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv