First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum

Abstract

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.

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety

“…could be detected in TTX-contaminated mussel samples. In the case of Pseudomonas , several species are confirmed by 99% sequence homology and for the first time they can be clearly linked to TTX and contaminated mussels17.…”

“…Levels of 222.9 μg/Kg and 206.3 μg/Kg of TTXs respectively were reported when these samples were previously analysed by mass spectrometry17. Sequence reads were clustered at 97% identity resulting in 297 unique Operational Taxonomic Units (OTUs).…”

“…Before 2014 only one highly toxic TTX-accumulation in scallops from Japan had been reported14. However, from 2014 to date three reports have identified the presence of TTX in shellfish and it was also detected in several edible gastropods1516172223. Therefore, the presence of TTXs in shellfish as well as the source of these toxins should be taken into account, since TTX-contaminated samples will not be detected by official analytical methods, such as LC-MS/MS or HLPC with fluorescence detection, because TTX is not required to be included in these methods.…”

“…Aliquots of 2 g of DG (digestive glands) of sample 1786/2012 collected in Greece were extracted as previously described17. The extract obtained was used for patch clamp recordings.…”

“…This atypical toxicity coincided with the absence of known dinoflagellates and other toxins, while only P. minimum was present in seawater. After mass-spectrometry analysis, both TTX and TTX analogues were confirmed17. Therefore a potential TTX presence-dinoflagellate link was hypothesized.…”