Extraction and cleaning methods to detect yessotoxins in contaminated mussels.

Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from mussels that does not interfere with this detection method. YTX concentrations were measured in spiked mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction-cleaning-detection methods were applied to a naturally contaminated mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85-90% recovery.

Algal biotoxins of marine origin: new indications from the European Union.

Marine biotoxins, more or less complex molecules with various origins that can accumulate in the tissues of fish products through the food chain, are reviewed. The EU, aware of the danger incurred in eating certain fish products, has issued a set of hygiene and health directives for the purpose of preventing disease and safeguarding consumer health. In particular, directive 91/492/EEC, of 15 July 1991, lays down the sanitary norms applicable to the production and commercialization of live bivalve molluscs, echinoderms, tunicates and marine gastropods and regulates the whole system involving these products from their origin to consumption. More recently, through Commission Decision dated 15 March 2002 (EC OJ 175/62 of 16.3.2002) the EU has set new standards for the implementation of directive 91/492/EEC with respect to the maximum levels and analysis methods for some marine biotoxins.

Immunolocalization of yessotoxins in the mussel Mytilus galloprovincialis.

This study reports, for the first time, immunocytochemical evidence of the distribution of algal yessotoxins (YTXs) in the mussel Mytilus galloprovincialis. Immunopositivity to YTXs was found in immunocytes and in the digestive gland. With regards the gland, the positivity was mainly present in the lumen of both tubules and ducts. No YTXs were detected in the gonads, while the presence of toxins in the gills cannot be excluded. The data are supported by both HPLC analysis and functional assays.

High sensitivity bioassay of paralytic (PSP) and amnesic (ASP) algal toxins based on the fluorimetric detection of [Ca(2+)](i) in rat cortical primary cultures.

A high sensitivity bioassay able to recognise small amounts of paralytic and amnesic toxins in algal acetic extracts is described. The method is based on the measure of intracellular [Ca(2+)](i) in primary cultures of rat cortical neurones preloaded with Fura-2 and submitted to electrical field stimulation. Under normal conditions the basal [Ca(2+)](i) level was about 50-100 nM and was nearly doubled during the peaks induced by trains of electrical pulses at 10 Hz for 10 s. Saxitoxin (STX) 3.5 nM and tetrodotoxin (TTX) 24 nM halved the peaks height without affecting basal [Ca(2+)](i). Conversely, domoic acid increased the basal [Ca(2+)](i) (EC(50)=3. 7 microM) and decreased the calcium peaks (EC(50)=7.3 microM). CNQX (a competitive antagonist of AMPA/KA receptors) at 10 microM shifted to the right by a factor of 3 the concentration-response curves of domoic acid. The extracts of non-toxic algae were well tolerated by up to 10 microg protein/ml, whereas extracts of Alexandrium lusitanicum at 1-4 microg protein/ml reduced [Ca(2+)](i) peaks and increased basal calcium levels. This toxic effect of A. lusitanicum was unexpected since parallel HPLC analysis showed only the presence of gonyautoxins, known to act like saxitoxin. Therefore, the bioassay on rat cortical neurones revealed a complex composition of the toxins present in A. lusitanicum. The relevance of fluorimetric detection of [Ca(2+)](i) in primary neuronal cultures in the evaluation of algal risk is stressed.

High levels of yessotoxin in mussels and presence of yessotoxin and homoyessotoxin in dinoflagellates of the Adriatic Sea.

Identification of YTX and homoYTX in natural phytoplankton populations containing significant amounts of Gonyaulax polyedra and determination of detailed toxin profiles of mussels (Mytilus galloprovincialis) periodically collected from two sites of the Northern Adriatic coast from February to October 1997 was performed by LC-FLD following derivatization with ADAM or DMEQ-TAD and LC-MS and LC-MS-MS. OA and YTX concentrations were recorded in the range 0.11-2.31 and 0.18-9.02 microg per g of hepatopancreas, respectively. HomoYTX was also detected both in phytoplankton and mussel samples.

ASP, DSP, NSP and PSP monitoring in 'mucilaginous aggregates' and in mussels in a coastal area of the Northern Adriatic Sea facing Emilia-Romagna in 1988, 1989 and 1991.

In this study, monitoring of marine biotoxins in "mucilaginous aggregates" and in mussels from coastal area of Emilia Romagna (Northern Adriatic Sea) in June-August 1988, 1989 and 1991, are reported. Both "mucilaginous aggregates" and mussels were analysed for NSP and PSP in 1988, and ASP, DSP, NSP, PSP in 1989, 1991. Concerning "mucilaginous aggregate" any presence of biotoxins was never detected. In the mussels it was possible to exclude the presence of PSP, ASP and NSP, but very high levels of DSP were shown in all the considered periods, in relation to the presence in the sea water of cells of the Dinophysis genus.