Tetrodotoxins in French Bivalve Mollusks—Analytical Methodology, Environmental Dynamics and Screening of Bacterial Strain Collections

Abstract

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5–3 month periods (2018–2019). Concentrations of TTX ranged from ‘not detected’ to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex.

An Updated Review of Tetrodotoxin and Its Peculiarities

“…Recent works [29,55] have correlated TTX presence in shellfish to the simultaneous presence of Vibrio and Pseudomonas, although no cultures of any TTX-related bacteria were possible to obtain from the samples analyzed. The assumption that bacteria or micro-algae are linked to TTX production is further supported by the seasonality of TTX in bivalves located mostly during warmer months, that is late spring and summer, in European countries [14,[28][29][30][31][32][33]55], as well as New Zealand [37,64], indicating the involvement of TTX-producing microorganisms adapted to warmer water temperatures.…”

“…As regards TTXs occurrence in Europe, the first record dates back to digestive glands of Mytilus galloprovincialis mussels and Venus verrucosa clams harvested in 2006 and 2008, respectively, in Greece and retrospectively analyzed as retained official monitoring samples [14]. A number of further reports on TTXs presence in bivalve mollusks harvested between 2013 and 2019 from six more countries, that is, England, the Netherlands, China, Italy, Spain, and France, gradually appeared in the literature [26][27][28][29][30][31][32][33][34][35]55,78], along with further TTX records from additional species in New Zealand [38,64,79,80]. So far, among the highest TTXs concentrations detected in bi-valve mollusks were those of 1600 µg/kg in Greenshell TM mussels (Perna canaliculus) [64] and 800 µg/kg in "pipi" clams (Paphies australis) from New Zealand [79], 541 µg/kg in Mediterranean mussels (M. galloprovincialis) from Italy [32], and 253 µg/kg in Pacific oysters (Magallana gigas) from England and the Netherlands [28,55].…”

“…Later, in 2015, evidence on the occurrence of TTXs in European bivalve mollusks started to appear in the literature. So far, TTXs in shellfish have been reported in a number of European countries (UK, Greece, Netherlands, Portugal, Spain, Italy, France), with this presence detected in samples dating as early as 2006 and in a variety of species, including bivalve mollusks (mussels, oysters, venus clams) and gastropods (Gibbula umbilicalis, Monodonta lineata, and C. lampas) [7,14,[26][27][28][29][30][31][32][33][34], but no further human TTX-intoxications were associated with these aquatic organisms. Similarly, there are reports on the presence of TTXs in several bivalve mollusks species from other temperate areas of the world, including New Zealand, China and Japan [8,[35][36][37][38], with no relevant intoxications in humans.…”

Tetrodotoxin and Its Analogues in Food: Recent Updates on Sample Preparation and Analytical Methods Since 2012

“…Certified reference materials for TTX are available from many sources: Funakoshi (purity = 96%, Tokyo, Japan), Nacalai tesque (purity ≥94%, Kyoto, Japan), Wako pure chemical industries (purity >99%, Osaka, Japan), Affixscientific Corporation (purity ≥98%, Fremont, CA, USA), Sigma-Aldrich (St. Louis, MO, USA), Cifga (Lugo, Spain), ,,, Tocris Bioscience (purity ≥98.8%, Bristol, UK), ,, Biorbyt Ltd. (Cambridge, UK), Abcam plc (Cambridge, UK), Latoxan (Valence, France), , J&K Scientific Ltd. (Beijing, China), Must Bio-Tech Co., Ltd. (Chengdu, China), , among others. Sometimes, TTX standards contain its analogues, for example, a TTX standard solution from CIFGA (Lugo, Spain) contains TTX, 4,9-anhydroTTX, 11-deoxyTTX, 4-epiTTX, and trace levels of 5,6,11-trideoxyTTX and 11-norTTX-6-ol . However, almost all the analogues of TTX are not commercially available, and they have to be isolated from available sources (with further chemical modification in some cases), except 4,9-anhydro TTX, which is available from NRC (Halifax, Nova Scotia, Canada), Cifga (Lugo, Spain), or Biomol GmbH .…”

“…QTRAP, also known as triple-quadrupole linear ion trap (QqLIT), is a sensitive and versatile instrument that can be a good alternative for TTX analysis. For one thing, QTRAP retains classical QqQ functions and can be used in MRM mode for sensitive detection of TTX. , For another, it has the capacity of MS n ion accumulation and fragmentation for structural elucidation. Besides, QTRAP can be operated along with information-dependent acquisition (IDA) procedures to obtain as much as possible MS and MS/MS information on unknown compounds within the same chromatographic run.…”