The Genoa 2005/2006 outbreaks. Determination of putative palytoxin and its analogue in Ostreopsis ovata by a new LC-MS/MS method.

Over the last few years a new treat due to harmful algal blooms has been impending in the Mediterranean Sea: microalgae belonging to Ostreopsis genus have spread along the Italian coasts, mostly in the Tyrrhenian Sea, causing serious concerns to both environment and public health. In fact, concurrently to the algal blooms, environmental sufferings involving mostly epibenthos were observed and symptoms of rhinorrhoea, conjunctivitis, dermatitis, cough, dyspnoea, wheezes and severe fever arouse in people exposed to marine aerosols by recreational or working activities. Such a phenomenon was recorded for the first time during summer 1998 along the north-western Tuscany coasts,1 but reached alarming proportions in late July 2005, when about 200 people exposed to marine aerosols on the beach and promenade of Genoa required extended hospitalization. Respiratory distress observed in humans was concurrent with a massive bloom of Ostreopsis ovata along Genoa coasts and disappeared when the O. ovata population decreased. Human illness were recorded also in summer 2006, when O. ovata populations spread along the whole Tirrenian coasts from Liguria to Sicily and in the Southern Adriatic Sea. In the present work, we report on the investigation of plankton samples collected along Liguria coasts during the 2005/2006 outbreaks. The application of LC-MS/MS methods we have developed so far for the analyses of a number of liposoluble and hydrosoluble phycotoxins2-5 prompted us to exclude the presence of such toxins in the samples. Then, since some Ostreopsis strains are regarded as the producing organisms of palytoxin,6 one of the most potent marine toxin so far known, a new LC-MS/MS method for detection of palytoxin was set up.7 It allows the rapid and specific determination of palytoxin in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. The minimum detection levels for matrix-free toxin on-column were estimated to be 125 pg and 200 pg in MRM and SIM mode, respectively. Spiking experiments before and after extraction allowed to assess limits of detection and quantitation for palytoxin in matrix, accuracy, intra-day and inter-day reproducibility. Relying on the newly developed method, we were able to detect the presence of putative palytoxin and its analogue in the plankton samples and to infer them as the likely cause of the Genoa 2005/2006 outbreaks.