First occurrence of tetrodotoxins in bivalve mollusks from Northern Adriatic Sea (Italy)

Highlights

• Tetrodotoxins are neurotoxic and potentially fatal for humans

• Tetrodotoxins were found in shellfishes collected in Northern Adriatic sea

• The positive samples were from the same sampling point both in 2017 and 2018

• The tetrodotoxin amount for one sample of 2017 was the highest recovered in Europe

Abstract

Tetrodotoxins are a potent neurotoxic class of toxins with potentially fatal effects if ingested by humans, and thus, these toxins are a dangerous threat for food safety. Tetrodotoxins are widely distributed among various organisms of different taxa, i.e. pufferfish, blue-ringed octopuses and marine gastropods. Lately the presence of these toxins has been reported in mussels (Mytilus sp.). Countries like the United Kingdom, Greece, the Netherlands, and Italy have increasingly notified the detection of tetrodotoxins in these bivalves, which are widely diffused in European shellfish farming. In this context we report, for the first time, the detection of substantial amounts of tetrodotoxins in Mytilus galloprovincialis collected in Italy in a single area of the Northern Adriatic Sea (Marano Lagoon). Tetrodotoxin-positive mussels were identified in samples collected from that geographic area at the end of May in the context of official monitoring programs both in 2017 and 2018. After the positive results were obtained by means of mouse bioassay screening, tetrodotoxins were detected and quantified by HILIC-MS/MS, revealing the bioaccumulation in the mussels of 541 and 216 μg/kg in 2017 and 2018 respectively, the highest amount ever found in mollusks in Europe. The presence of tetrodotoxins at the concentration of 413 μg/kg was further confirmed with Liquid Chromatography coupled to High Resolution Mass Spectrometry in the mussels sampled in 2017.

Introduction

Tetrodotoxins (TTXs) are commonly known as puffer fish toxins, as Tetraodontidae is the fish family in which they were characterized for the first time (Suehiro, 1994). They are a class of potent neurotoxins, with effects in humans that are similar to those of Paralytic Shellfish Toxins (PSTs). Tetrodotoxins are able to block voltage-gated Na⁺ channels, impeding the extrusion of monovalent cations through the cell membrane. In humans this leads to a sequence of acute symptoms from perioral numbness and paraesthesia, lingual numbness, early motor paralysis, incoordination, slurred speech to generalized flaccid paralysis, aphonia and fixed/dilated pupils to hypoxia, hypotension, bradycardia, cardiac dysrhythmias and unconsciousness, and in the end death due to respiratory failure and cardiac collapse (EFSACONTAM Panel (EFSA Panel on Contaminants in the Food Chain) et al., 2017). Since their discovery, different marine taxa other than Tetraodontidae, have been reported to accumulate TTXs within their tissues. These include among others the Nemertinea (Miyazawa et al., 1988; Turner et al., 2018); Anellidae (Yasumoto, Yotsu, Endo, Murata, & Naoki, 1989), Crustacea (Yasumura, Oshima, Yasumoto, Alcala, & Alcala, 1986), Gasteropoda, and Cephalopoda (Narita et al., 1981; Noguchi et al. 1981, 1984; Sheumack, Howden, Spence, & Quinn, 1978; Yotsu-Yamashita, Mebs, & Flachsenberger, 2007). The wide diversity of TTX-bearing taxa, however, means the possibility that they possess a common gene encoding for TTX production is controversial, so the toxin's origins could be attributable to an exogenous source, perhaps involving an absorption via the food chain and consecutive accumulation within the animal tissues (Noguchi & Arakawa, 2008). In fact, the trumpet shell Charonia sauliae was reported to acquire the toxin from a TTX-bearing starfish, on which the gastropod fed (Noguchi, Maruyama, Hashimoto, & Narita, 1982). Additionally, the TTX-bearer pufferfish Takifugu rubripes, when fed in a controlled environment with TTX-free diets, exhibited no toxicity whatsoever (Noguchi, Arakawa, & Takatani, 2006). To shed some light on the possible exogenous origin of TTX in distant related taxa, Noguchi et al. (1986) were the first to isolate bacteria of genus Vibrio from the crab Atergatis floridus (a TTX-bearing species) and to detect TTX and anhydro-TTX in a cellular extract and culture medium by means of HPLC-FLD and GC-MS assays, while Simidu, Noguchi, Hwang, Shida, and Hashimoto (1987), by culturing a series of marine-bacteria strains (mostly ATCC and NCMB strains), were able to induce the production of TTX and/or anhydro-TTX in many of them. Since then, the amount of TTX-producing bacteria isolated from marine TTX-bearing species has grown consistently, accounting for at least 150 strains, divided in 31 genera (Magarlamov, Melnikova, & Chernyshev, 2017). It is noteworthy that TTX concentrations reported in bacterial cultures are usually very low, as compared to those detected in higher trophic species (Biessy et al., 2019) and, to date, the biosynthetic pathways regarding TTX production are not completely understood (Chau, Kalaitzis, & Neilan, 2011).

TTX poisoning is very common in Japan, Taiwan, Bangladesh and Southeast Asia due to the consumption of pufferfish and, occasionally, of gastropods or crabs, which are fished locally, especially in Japan where fugu (pufferfish) is a long established delicacy (Bane, Lehane, Dikshit, O'Riordan, & Furey, 2014). As for Europe, the presence of TTXs in aquatic organisms has never been considered as a potential threat (Katikou, 2019). However, the recent expansion of invasive TTX-bearing species in the Mediterranean Sea, as in the case of the pufferfish, Lagocephalus sceleratus, which has rapidly widened its area of distribution from Turkey (Akyol, Ünal, Ceyhan, & Bilecenoglu, 2005) to Spain (Katsanevakis et al., 2014), and the first record of human intoxication due to TTXs, following the ingestion of Charonia lampas lampas fished in Portuguese waters (Rodriguez et al., 2008), are of great concern, as TTXs are not monitored on a regular basis (Katikou, 2019). Additional evidence regarding the recent spread of TTXs across European waters comes from bivalve mollusks, with the first report of the toxins' presence in Mytilus edulis and Crassostrea gigas harvested in England between 2013 and 2014 (Turner, McNabb, Harwood, Selwood, & Boundy, 2015; Turner, Powell, Schofield, Lees, & Baker-Austin, 2015. Following this case, more episodes have been pointed out in recent years: UK (Turner et al., 2017), Spain (Leão, Lozano-Leon, Giráldez, Vilariño, & Gago-Martínez, 2018), The Netherlands (Gerssen et al., 2018), Greece (Vlamis et al., 2015) and Italy (Dell’Aversano et al., 2019). Among these are maritime countries (i.e. Greece, Italy and Spain) with waters encompassing the Mediterranean Sea, a crucial geographic area because of its commercial importance, especially in terms of bivalve aquaculture. As a matter of fact, of 22 countries bounding the Mediterranean basin, 14 have developed systems for marine mollusk aquaculture (Regulation EC, 2003 Spain are the three major European marine mollusks producers (FAO, 2019).

Within this context, we aim to report, for the first time, the detection of TTXs from specimens of Mediterranean mussels (Mytilus galloprovincialis) harvested in Marano Lagoon in the North Adriatic Sea (Italy).

Toxin occurrence was recorded in two distinct occasions (2017 and 2018), during routine official monitoring led by the Local Veterinary Authority for the presence of PSTs in bivalve mollusks, both times at the end of May. TTX detection was carried by mean of the Mouse bioassay screening and confirmation was performed by two distinct methods: HILIC-MS/MS and HILIC–HRMSⁿ. The amount of toxin detected (541 μg/kg in 2017) is, so far, the highest reported in bivalve mollusks in the Mediterranean basin.