Preliminary Results on the Evaluation of the Occurrence of Tetrodotoxin Associated to Marine Vibrio spp. in Bivalves from the Galician Rias (Northwest of Spain)

Abstract

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

Evaluation of the Occurrence of Tetrodotoxin in Bivalve Mollusks from the Portuguese Coast

“…Among these samples, TTX trace levels were observed in three mussel samples collected in Tavira (Formosa lagoon) in July and August ( Figure 2). These results are in agreement with the recent findings in Spain, the nearby country where no TTX have been found in bivalve mollusks in two TTX survey studies [16,18].…”

“…A gradient started at 98% B at 0.4 mL/min for the first 5 min, 98-50% B for the next 2.5 min, and this composition was kept for 1.5 min but the flow rate was linearly increased to 0.5 mL/min until 9.0 min, and then B reverted to 98% by 9.5 min, flow rate ramped to 0.8 mL/min at 10.0 min, held until 10.6 min, and dropped back to 0.4 mL/min until 11.0 min. Two multiple-reaction-monitoring (MRM) transitions from the protonated ions were monitored for TTX and TTX derivatives as described in [14, 16,17]: For TTX and 4-epiTTX the transitions monitored were the m/z 320.1 > 302. 1…”

First Report on the Occurrence of Tetrodotoxins in Bivalve Mollusks in The Netherlands

“…The organism responsible for producing TTX in these areas is not known yet and still under investigation. In other areas such as the UK and Spain, vibrio was positively identified as one of the producers of TTX [ 15 , 21 ]. In literature a relationship is suggested between the occurrence of TTX and phytoplankton, which should thus be kept in mind as a possible factor for TTX transport to shellfish too.…”

First Detection of Tetrodotoxin in Bivalves and Gastropods from the French Mainland Coasts

“…Several European studies have been conducted since the EFSA risk assessment [12][13][14][15][16]. All these new data were obtained using hydrophilic interaction liquid chromatography associated with tandem mass spectrometry (HILIC-MS/MS).…”