During official shellfish control for the presence of marine biotoxins in Greece in year 2012, a series of unexplained positive mouse bioassays (MBA) for lipophilic toxins with nervous symptomatology prior to mice death was observed in mussels from Vistonikos Bay–Lagos, Rodopi. This atypical toxicity coincided with (a) absence or low levels of regulated and some non-regulated toxins in mussels and (b) the simultaneous presence of the potentially toxic microalgal species Prorocentrum minimum at levels up to 1.89 × 10³ cells/L in the area’s seawater. Further analyses by different MBA protocols indicated that the unknown toxin was hydrophilic, whereas UPLC-MS/MS analyses revealed the presence of tetrodotoxins (TTXs) at levels up to 222.9 μg/kg. Reviewing of official control data from previous years (2006–2012) identified a number of sample cases with atypical positive to asymptomatic negative MBAs for lipophilic toxins in different Greek production areas, coinciding with periods of P. minimum blooms. UPLC-MS/MS analysis of retained sub-samples from these cases revealed that TTXs were already present in Greek shellfish since 2006, in concentrations ranging between 61.0 and 194.7 μg/kg. To our knowledge, this is the earliest reported detection of TTXs in European bivalve shellfish, while it is also the first work to indicate a possible link between presence of the toxic dinoflagellate P. minimum in seawater and that of TTXs in bivalves. Confirmed presence of TTX, a very heat-stable toxin, in filter-feeding mollusks of the Mediterranean Sea, even at lower levels to those inducing symptomatology to humans, indicates that this emerging risk should be seriously taken into account by the EU to protect the health of shellfish consumers.

Paralytic shellfish toxins (PST) traditionally have been analyzed by liquid chromatography with either pre- or post-column derivatization and always with a silica-based stationary phase. This technique resulted in different methods that need more than one run to analyze the toxins. Furthermore, tetrodotoxin (TTX) was recently found in bivalves of northward locations in Europe due to climate change, so it is important to analyze it along with PST because their signs of toxicity are similar in the bioassay. The methods described here detail a new approach to eliminate different runs, by using a new porous graphitic carbon stationary phase. Firstly we describe the separation of 13 PST that belong to different groups, taking into account the side-chains of substituents, in one single run of less than 30 min with good reproducibility. The method was assayed in four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Pecten maximus), scallop (Ruditapes decussatus) and oyster (Ostrea edulis). The results for all of the parameters studied are provided, and the detection limits for the majority of toxins were improved with regard to previous liquid chromatography methods: the lowest values were those for decarbamoyl-gonyautoxin 2 (dcGTX2) and gonyautoxin 2 (GTX2) in mussel (0.0001 mg saxitoxin (STX)·diHCl kg⁻¹ for each toxin), decarbamoyl-saxitoxin (dcSTX) in clam (0.0003 mg STX·diHCl kg⁻¹), N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2) in scallop (0.0001 mg STX·diHCl kg⁻¹ for each toxin) and dcSTX (0.0003 mg STX·diHCl kg⁻¹ ) in oyster; gonyautoxin 2 (GTX2) showed the highest limit of detection in oyster (0.0366 mg STX·diHCl kg⁻¹). Secondly, we propose a modification of the method for the simultaneous analysis of PST and TTX, with some minor changes in the solvent gradient, although the detection limit for TTX does not allow its use nowadays for regulatory purposes.

Paralytic shellfish toxins (PSTs) are potent alkaloids of microalgal and cyanobacterial origin, with worldwide distribution. Over the last 20 years, the number of poisoning incidents has declined as a result of the implementation of legislation and monitoring programs based on bivalves. In the summer of 2012 and 2013, we collected a total of 98 samples from 23 different species belonging to benthic and subtidal organisms, such as echinoderms, crustaceans, bivalves, and gastropods. The sampling locations were Madeira, São Miguel Island (Azores archipelago), and the northwestern coast of Morocco. The samples were analyzed using post-column oxidation liquid chromatography with a fluorescence detection method. Our main goal was to detect new vectors for these biotoxins. After reporting a total of 59 positive results for PSTs with 14 new vectors identified, we verified that some of the amounts exceeded the limit value established in the EU. These results suggest that routine monitoring of saxitoxin and its analogs should be extended to more potential vectors other than bivalves, including other edible organisms, for a better protection of public health.

The separation of PSP toxins using liquid chromatography with a post-column oxidation fluorescence detection method was performed with different matrices. The separation of PSP toxins depends on several factors, and it is crucial to take into account the presence of interfering matrix peaks to produce a good separation. The matrix peaks are not always the same, which is a significant issue when it comes to producing good, reliable results regarding resolution and toxicity information. Different real shellfish matrices (mussel, scallop, clam and oyster) were studied, and it was seen that the interference is not the same for each individual matrix. It also depends on the species, sampling location and the date of collection. It was proposed that separation should be accomplished taking into account the type of matrix, as well as the concentration of heptane sulfonate in both solvents, since the mobile phase varies regarding the matrix. Scallop and oyster matrices needed a decrease in the concentration of heptane sulfonate to separate GTX4 from matrix peaks, as well as dcGTX3 for oysters, with a concentration of 6.5 mM for solvent A and 6.25 mM for solvent B. For mussel and clam matrices, interfering peaks are not as large as they are in the other group, and the heptane sulfonate concentration was 8.25 mM for both solvents. Also, for scallops and oysters, matrix interferences depend not only on the sampling site but also on the date of collection as well as the species; for mussels and clams, differences are noted only when the sampling site varies.

Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioassay, receptor binding assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction step.