Cyanobacterial toxins profiling in the Subalpine lakes

Cyanobacteria have a key role in all aquatic ecosystems. However, the presence of toxic strains of cyanobacteria can have negative impact on all water uses and can pose serious health risks for humans and animals. Toxic species belong mainly to the genera Microcystis, Planktothrix and Anabaena, which have the capability of producing toxic metabolites potentially lethal for aquatic organisms. Toxins can also accumulate in tissues and transfer to other animals and humans through the food chain (Lehman et al. 2010). Microcystins are the most abundant and dangerous toxins produced by cyanobacteria. In addition to microcystins, many other toxins are known, such as nodularins, anatoxins, saxitoxins, cylindrospermopsins. Microcystins are potent hepatotoxins, and are also endowed with tumor promotion. Anatoxins and saxitoxins are potent neurotoxins. Cylindrospermopsins are endowed with generic cytotoxic effects although their main target organs are liver and kidneys. From a chemical point of view, cyanotoxins can be divided in two major groups: cyclic peptides (microcystins and nodularins) and alkaloids (anatoxins, saxitoxins, cylindrospermopsins). (Aráoz et al., 2009; Chorus and Bartram 1999; Zurawell et al. 2005; Welker and von Dohren 2006) The World Health Organization (WHO 1998) has set a tolerable daily intake (TDI) for Microcystin-LR of 0.04 mgkg-1 body weight day-1 for humans. A provisional limit of 1 gL-1 in drinking water has been suggested, until further studies regarding the carcinogenicity of MCs are completed. The Italian law has set 0.84 gL-1 total MCs as the tolerable limit in freshwaters (Italian Ministry of Health 1998) to prevent possible intoxication due to swimming and recreational activities. Exposure guidelines have not been estimated for other cyanotoxins, due to insufficient data. Although cyanobacteria are widespread in Italian lakes, very limited informations are available in the scientific literature about cyanotoxins distribution (Messineo et al. 2009). The aim of present investigation was to determine the degree of variability and diversity of cyanobacterial toxins in a group of lakes located in the Italian subalpine region (Garda, Maggiore, Como, Iseo, Lugano, Idro, Pusiano, Ledro and Levico) which are characterized by the presence of diverse populations of cyanobacteria (Salmaso et al. 2006). Many of these lakes are particularly important for the economy of Northern Italy because of their size and volume, and tourist attractivity. We conducted a sampling campaign for collecting cyanobacteria biomass; then we extracted toxins from the biomass and performed LC-MS analysis. A major difficulty from an analytical point of view was the lack of analytical standards: in literature more than one hundred variants of cyanobacterial toxins are reported (at least 70 microcystins and 40 alkaloids) but only a dozen of commercial analytical standard is available on the market, thus making a comprehensive analysis of cyanotoxins virtually impossible. We restricted the search to a selected pool of variants which could be confidently identified and quantified with LC-MS techniques by using commercial standards. Hence we set up a method for the screening of twenty-one cyclic peptides and thirteen alkaloids. The quali- and quantitative analysis of toxins was accomplished by means of an LC-MS equipment composed by a Waters Acquity UPLC coupled with AB Sciex 4000 QTRAP mass spectrometer. Multiple reaction monitoring (MRM) scan mode was employed in the analysis. The results of the survey showed that microcystins were constantly present in all water bodies although with very different concentrations. The highest values were recorded in summer periods in connection with the highest development of cyanobacterial biomass. Microcystins therefore represent a constant threats in aquatic environments. Moreover, the different lakes were characterized by different microcystins variants; this aspect is very important from a sanitary point of view since among the 70 and more microcystins variants, toxicities values span over a 2 orders of magnitude range. Four variants of microcystins were by far the most represented in all lakes.