Tetrodotoxin (TTX) is a low weight potent neurotoxin, named after the Tetradontidae fish family from where it was first isolated in 1909 by Tahara and Hirata [1]. TTX is an interesting toxin, since it was reported in several taxa genetically not close related; from bacteria; marine invertebrates; terrestrial and marine vertebrates [2]. Neither its biochemical path nor its true origin is fully clarified, since three hypotheses point to its origin: endogenous [3,4], through food-chain [5,6,7,8] or through symbionts [9,10,11,12].

TTX is an extremely potent toxin, it binds specifically to site 1 of the voltage-gated sodium channels (Nav), occluding the external pore blocking the cellular communication and causing death by cardio-respiratory paralysis [13,14,15,16]. Several poisoning incidents have occurred, especially in Asia, with Japan being the most affected country and where Fugu is considered a delicacy. Japan is the only country to have guideline values for TTX [17]. Although TTX-bearers are typical of warm waters, recent studies report the possible migration of these toxic species from the Red Sea to the Mediterranean Sea through the Suez Canal [18,19,20]. This may happen due the opening of new corridors allied to the increase of water temperature as a result of climate change. These factors all together probably influenced the bidirectional migration of species between the Red Sea and the Mediterranean Sea, resulting in the increase of poisoning incidents, especially due to the ingestion of toxic alien species, among them TTX-bearers [18,19,20,21,22]. A good example of intoxication incidents caused by TTX-bearers was the ingestion of the elongated puffer, Lagocephalus sceleratus. Its presence in the Mediterranean was first reported in 2003, causing several poisoning incidents in Egypt in the end of 2004 and in Israel between 2005 and 2008. Fortunately all patients recovered [18,20,23].

In October 2007 a case of TTX poisoning occurred in Malaga, Spain, due to the ingestion of a specimen of Charonia lampas, an autochthonous predatory gastropod from Atlantic and Mediterranean waters, caught in the southern Portuguese waters [21]. This episode was the first report TTX occurrence in autochthonous species in Atlantic and Mediterranean waters and triggered our investigation to monitor different invertebrate species in several sites of our coast [21].

In this work our goal was to detect the presence of TTX and some analogues in several marine invertebrate species collected along the continental Portuguese coast, by using UPLC-MS/MS and LC-MS/MS.

In this study, 134 samples were collected in a monthly sampling program, from July 2009 until November 2010, in 13 sites distributed along the Portuguese coast (Figure 1). The collected species belonged to different taxa and included gastropods (Monodonta lineata, Monodonta turbinata, Gibbula umbilicalis, Gibbula magus, Littorina littorea, Littorina saxatilis, Nucella lapillus, Ocenebra erinacea, Calliostoma zizyphinum, Patella intermedia, Charonia lampas), bivalves (Mytilus galloprovincialis), sea-urchins (Paracentrotus lividus) and sea-stars (Marthasterias glacialis). The naturally contaminated Charonia lampas and Lagocephalus sceleratus, obtained in former works [21,24], were used as standards in the LC-MS/MS analysis and provided us with the retention times of TTX and of the other analogues as follows; TTX (21.14 min), 4-epiTTX (20.4 min), 5,6,11-trideoxyTTX (13.7–14.2 min), monodeoxyTTX (18.08–18.80 min), 11-norTTX-6-ol (19.0–19.7 min) and 4-anhydroTTX (18.9 min) (Figure 2).

Five protonated molecules [M + H]⁺ at m/z 320, 302, 304, 290 and 272 corresponding to TTX plus its derivatives were detected. The MRM transitions selected were: TTX and 4-epiTTX: 320 > 302/162; 4,9-anhydroTTX: 302 > 256/162; monodeoxyTTX: 304 > 286/176; 11-norTTX-6-ol: 290 > 272/162 and 5,6,11-trideoxyTTX: 272 > 254/162. Quantification was done with the most abundant ion in the fragment spectra: 302 (TTX and 4-epiTTX), 162 (4,9-anhydroTTX), 286 (monodeoxyTTX), 272 (11-norTTX-6-ol) and 254 (5,6,11-trideoxyTTX) (Figure 2).

The limits of detection and quantification (LOD/LOQ) of the LC-ESI-CID-MS/MS for TTX were 16 ng/mL(S/N > 3) and 63 ng/mL (S/N > 10), respectively.

For the UPLC-MS/MS the LOD (S/N > 3) was 1.7 ng/mL, and the LOQ (S/N > 10) was 5 ng/mL. TTX and analogue contents were identified and calculated against TTX standard (Figure 3), presuming that the toxin and its derivatives had the same molar response factor in each apparatus. A sample was considered positive when the toxin levels detected were above the LOQ.

The analysis of the gastropod collected in our waters allowed us to detect some positive samples, including G. umbilicalis, collected on Memória beach in July 2009. The peaks corresponded to monodeoxyTTX, with an amount of 63.81 ng/g (Figure 4).

Two other gastropod species had positive results: M. lineata collected in April 2010 in Vila Nova de Milfontes and C. lampas collected in September 2010 in Angeiras (Figure 5). While the first revealed the co-occurrence of TTX (90.50 ng/g) and 4-epiTTX (21.48 ng/g), in C. lampas we detected low levels of 5,6,11-trideoxyTTX (6.22 ng/g). This last variant was detected previously in the same species, together with TTX [21]. Nevertheless, this is the first report on the occurrence of TTX and variants in the two small gastropods M. lineata and G. umbilicalis. These two species, regardless of their small size, are harvested and consumed by locals, being rarely found in markets. Thus, the exposure of TTX and analogues via these small gastropods is neither regulated nor controlled.

The levels of these toxins found in our samples are relatively low when compared to other species that had caused human intoxications, or even with the C. lampas that caused the intoxication episode in Malaga [21]. In Table 1 the toxin levels detected in the present work and examples of other works for comparison are displayed. Nevertheless, one should be careful when comparing data, since the levels we report are due to the toxin content in the whole animal (edible part), while the data on the C. lampas reported by Rodriguez et al. 2008 refers to levels in the digestive gland only [21].

The low levels we have found so far may also be due to the fact that TTX synthesis in warmer waters is higher than in the cold North Atlantic ones.

The “Lessepsian migration” phenomenon may play an important role in the migration of TTX-bearers to more temperate waters, such as the elongated puffer Lagocephalus sceleratus, since 1902 more than 62 Red Sea fish species have migrated via the Suez Canal to the Mediterranean [22]. Global warming may also have influenced the migration and settling of TTX-bearing species to more temperate waters, once the rise in water temperature facilitates the migration of Red Sea exotic species to the Mediterranean Sea. Since 5.7% of the Mediterranean fauna is composed of Red Sea fish species, the risk of future invasion by these alien species is potentially very high [22]. There is not yet enough epidemiological and toxicological data concerning human intoxications by TTX in the temperate waters of the Atlantic Ocean. Nevertheless, a study done in the South Pacific area concerning the possible association of ciguatera and climate change revealed interesting assumptions that may give us some hints for future studies [27]. The study projected the idea that a rise in temperature is expected to increase the incidence of ciguatera poisoning of 35–70 per thousand people in 1990 to 160–430 per thousand people in 2050 in Papua New Guinea [27]. Taking into account that we need to be cautious when using simplistic models such as these, the rise in temperature may alter the growth rate of the toxic organisms [28] and also the rates of toxin production [29]. Nevertheless, temperature may also alter accumulation, metabolism and detoxication kinetics in fish vectors as it can for TTX [30]. In fact, another work showed that the in vitro uptake of TTX into liver tissue slices of Takifugo rubripes is temperature dependent, being significantly higher at 20 °C compared to 5 °C [30].

Cellular and molecular studies on the TTX kinetics are also needed in order to better predict the potential effects of global warming on TTX levels in vector species. Taking into account that the microflora of many puffer fish may be the origin of TTX, studies on the TTX production by the main bacterial species are important. A study on the diversity of bacteria isolated from the skin, gill and intestine of Fugo niphobles showed that temperature may have an effect on the diversity and density of some bacteria [31]. The identification of Vibrio sp. species, and in particular of V. alginolyticus, was achieved when fish were exposed at 20 and 29 °C but not when reared at 10 °C. In laboratory culture, all strains of this species grew at 20 to 37 °C but very few grew at 10 °C, suggesting a preference for higher temperatures [31]. So, water temperature may have an impact on the growth rate of TTX producing bacteria, being responsible for higher bacteria counts in fish exposed to higher temperatures. More laboratory experiments are needed to support this hypothesis. All these circumstances, together with the fact that TTX travels along the food-chain [5,6,7,8], may favor the establishment of TTX in Atlantic temperate waters.

In this work, TTX plus its derivatives were detected in the Portuguese coast, not only in species already reported as TTX-bearers (Charonia lampas) but also in indigenous species not yet assigned, this being the first report of TTX presence in Gibbula umbilicalis and Monodonta lineata. Apart from the quantities detected in these animals, there is an imminent danger to the human population, since the toxin travels in the food-chain and it is unknown whether or not there is biomagnification of TTX [5,6,7,8]. This increases the potential danger, because all the species reported are edible and the toxin is water soluble and thermostable [32,33].

The species Gibbula umbilicalis and Monodonta lineata belong to the same family, Trochidae [34], and in this study we confirm their ability to accumulate TTX. Possibly there is a potential adaptation that is common for both. In addition, TTX was not detected in all specimens belonging to these two species, which could be due to different strains having different capabilities of adaptation. We can make no inferences about the seasonal intake of the toxin, due to the fact that we only had three positives (2.24% of the total sampling), although all of them were in the warmer months.

TTX is present in Portuguese waters, Angeiras being the most northern point of a TTX report in Atlantic temperate waters. Nevertheless, the low concentrations detected are not sufficient to cause a fatal outcome, since the Minimum Lethal Dose for humans is 2 mg [35]. Surveillance is advisable to avoid poisoning incidents and to understand the progress of this emergent phenomenon.

In this work we used LC-MS/MS and UPLC-MS/MS to detect TTX and several analogues in three autochthonous gastropod species of the Atlantic Portuguese continental coast. TTX, 4-epiTTX, monodeoxyTTX and 5,6,11-trideoxyTTX were detected in Monodonta lineata, Gibbula umbilicalis and Charonia lampas, being the most northern point of the Atlantic Ocean were these toxins were reported. All these species are edible, raising the probability of human health hazards. Despite the low concentrations detected, ranging from 6.22 to 90.50 ng/g, it was clearly shown that TTX and analogues should be monitored in the species reported positive, and in others that can potentially accumulate the toxins and can be used as human food.