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Paralytic shellfish toxins (PSTs) are potent alkaloids of microalgal and cyanobacterial origin, with worldwide distribution. Over the last 20 years, the number of poisoning incidents has declined as a result of the implementation of legislation and monitoring programs based on bivalves. In the summer of 2012 and 2013, we collected a total of 98 samples from 23 different species belonging to benthic and subtidal organisms, such as echinoderms, crustaceans, bivalves, and gastropods. The sampling locations were Madeira, São Miguel Island (Azores archipelago), and the northwestern coast of Morocco. The samples were analyzed using post-column oxidation liquid chromatography with a fluorescence detection method. Our main goal was to detect new vectors for these biotoxins. After reporting a total of 59 positive results for PSTs with 14 new vectors identified, we verified that some of the amounts exceeded the limit value established in the EU. These results suggest that routine monitoring of saxitoxin and its analogs should be extended to more potential vectors other than bivalves, including other edible organisms, for a better protection of public health.

Paralytic shellfish poisoning results from consumption of seafood naturally contaminated by saxitoxin and its congeners, the paralytic shellfish toxins (PSTs). The levels of such toxins are regulated internationally, and maximum permitted concentrations in seafood have been established in many countries. A mouse bioassay is an approved method for estimating the levels of PSTs in seafood, but this is now being superseded in many countries by instrumental methods of analysis. Such analyses provide data on the levels of many PSTs in seafood, but for risk assessment, knowledge of the relative toxicities of the congeners is required. These are expressed as “Toxicity Equivalence Factors” (TEFs). At present, TEFs are largely based on relative specific activities following intraperitoneal injection in a mouse bioassay rather than on acute toxicity determinations. A more relevant parameter for comparison would be median lethal doses via oral administration, since this is the route through which humans are exposed to PSTs. In the present study, the median lethal doses of gonyautoxin 5, gonyautoxin 6, decarbamoyl neosaxitoxin and of equilibrium mixtures of decarbamoyl gonyautoxins 2&3, C1&2 and C3&4 by oral administration to mice have been determined and compared with toxicities via intraperitoneal injection. The results indicate that the TEFs of several of these substances require revision in order to more accurately reflect the risk these toxins present to human health.

The marine dinoflagellate Gymnodinium catenatum has been associated with paralytic shellfish poisoning (PSP) outbreaks in Portuguese waters for many years. PSP syndrome is caused by consumption of seafood contaminated with paralytic shellfish toxins (PSTs), a suite of potent neurotoxins. Gymnodinium catenatum was frequently reported along the Portuguese coast throughout the late 1980s and early 1990s, but was absent between 1995 and 2005. Since this time, G. catenatum blooms have been recurrent, causing contamination of fishery resources along the Atlantic coast of Portugal. The aim of this study was to evaluate the toxin profile of G. catenatum isolated from the Portuguese coast before and after the 10-year hiatus to determine changes and potential impacts for the region. Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was utilized to determine the presence of any known and emerging PSTs in sample extracts. Several PST derivatives were identified, including the N-sulfocarbamoyl analogues (C1-4), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and decarbamoyl derivatives, decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNeo) and decarbamoyl gonyautoxin 3 (dcGTX3). In addition, three known hydroxy benzoate derivatives, G. catenatum toxin 1 (GC1), GC2 and GC3, were confirmed in cultured and wild strains of G. catenatum. Moreover, two presumed N-hydroxylated analogues of GC2 and GC3, designated GC5 and GC6, are reported. This work contributes to our understanding of the toxigenicity of G. catenatum in the coastal waters of Portugal and provides valuable information on emerging PST classes that may be relevant for routine monitoring programs tasked with the prevention and control of marine toxins in fish and shellfish.

Out of control proliferation of toxic phytoplankton, called harmful algal blooms (HABs), have a significant economic impact on bivalve aquaculture and harvesting in coastal waters. Some phytotoxins, such as paralytic shellfish toxins (PSTs), are of concern due to the life-threatening symptoms they can cause. Development of rapid and low-cost screening tools would be a welcome addition to the laboratory methodologies employed in routine monitoring programs. However, most of the assays and biosensors for the screening of PSTs, are restricted to a single target, saxitoxin (STX), which is the most potent PST. The present study aimed at developing an assay for the detection of N-sulfocarbamoyl PST—GTX5, which is one of the most abundant toxins in bivalves during G. catenatum blooms as found on the Portuguese coast. Enzymatic assay employing PSTs’ transforming enzyme—carbamoylase—was proposed. Carbamoylase was extracted and purified from the surf clam S. solida. Carbamoylase displayed similar specificity to both carbamate (STX) and N-sulfocarbamate toxins (GTX5 and C1+2) converting them into decarbamoyl saxitoxin (dcSTX) and decarbamoyl gonyautoxins 2+3 (dcGTX2+3), respectively. The enzymatic assay involved hydrolysis of GTX5 by carbamoylase and quantification of the product of enzymatic reaction, dcSTX, using a potentiometric chemical sensor. A potentiometric sensor with plasticized PVC membrane that displayed sensitivity to dcSTX and selectivity in the presence of GTX5 was employed. Enzymatic assay allowed determination of GTX5 in the concentration range from 0.43 to 3.30 µmolL−1, which encompasses levels of GTX5 in contaminated bivalve extracts with toxicities above PSTs regulatory limits. The feasibility of the carbamoylase-based potentiometric assay for detection of GTX5 was demonstrated.

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12β-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12β-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12β-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.