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

Aristidis Vlamis,Panagiota Katikou,Angelos Papazachariou,Luis Botana,Ana Botana,Verónica Rey,Amparo Alfonso,Thetis Zacharaki,Ines Rodriguez

doi:10.3390/toxins7051779

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.

Highlights

Tetrodotoxin (TTX) is an extremely potent neurotoxin that can block sodium channels and inhibit propagation of action potentials in muscle and nerve cells [1]
During the same time period, phytoplankton counts for all known species associated with the production of regulated marine biotoxin groups were all either undetectable or present at low levels not adequate to account for any shellfish toxicity
Analysis by UPLC-MS/MS for the presence of lipophilic toxins, on the other hand, showed only trace amounts of OA, GYM, SPX and PnTXs (OA:

Summary

Introduction

Tetrodotoxin (TTX) is an extremely potent neurotoxin that can block sodium channels and inhibit propagation of action potentials in muscle and nerve cells [1]. Named after the Tetraodontidae puffer fish family, TTX is perhaps most notorious as the toxin that causes puffer fish poisoning. TTX can cause death by muscular paralysis, respiratory depression and circulatory failure [2]. Symptoms usually appear within 10–45 min of exposure, depending upon the amount of the toxin ingested; some of the reported cases were asymptomatic until as much as 3 to 6 h after exposure. The initial symptom is usually oral paresthesia, which gradually spreads to the extremities and trunk. Other early symptoms comprise taste disturbance, dizziness, headache, diaphoresis, and pupillary constriction, potentially accompanied by gastrointestinal symptoms of salivation, hypersalivation, nausea, vomiting, hyperemesis, hematemesis, hypermotility, diarrhea and abdominal pain [3,4]

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