Paralytic Shellfish Toxins Occurrence in Non-Traditional Invertebrate Vectors from North Atlantic Waters (Azores, Madeira, and Morocco)

toxins Article Paralytic Shellfish Toxins Occurrence in Non-Traditional Invertebrate Vectors from North Atlantic Waters (Azores, Madeira, and Morocco) Marisa Silva 1,2,*,†, Verónica Rey 3,†, Aldo Barreiro 1,2, Manfred Kaufmann 2,4,5 , Ana Isabel Neto 6, Meryem Hassouani 7, Brahim Sabour 7, Ana Botana 3, Luis M. Botana 8 and Vitor Vasconcelos 1,2 1 Department of Biology, Science Faculty, University of Porto, Rua do Campo Alegre, 4619-007 Porto, Portugal; aldobarreir[email protected] (A.B.); [email protected] (V.V.) 2Interdisciplinary Center of Marine and Environmental Research-CIMAR/CIIMAR, University of Porto, Novo Edificio do Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, 4450-208 S/N Matosinhos, Portugal; [email protected] 3Department of Analytical Chemistry, Science Faculty, University of Santiago de Compostela, 27002 Lugo, Spain; veronica.r[email protected] (V.R.); [email protected] (A.B.) 4Life Sciences Faculty, Madeira University, Marine Biology Station, 9000-107 Funchal, Madeira Island, Portugal 5Center of Interdisciplinary Marine and Environmental Research of Madeira-CIIMAR-Madeira, Edificio Madeira Tecnopolo, Caminho da Penteada, 9020-105 Funchal, Madeira, Portugal 6cE3c/GBA—Centre for Ecology, Evolution and Environmental Changes/Azorean Biodiversity Group and Department of Biology, Faculty of Sciences and Technology, University of Azores, 9501-801 Ponta Delgada, São Miguel, Azores, Portugal; [email protected] 7 Phycology Research Unit-Biotechnology, Ecosystems Ecology and Valorization Laboratory, Science Faculty, University of Chouaib Doukkali, El Jadida BP20, Morocco; [email protected] (M.H.); sabour[email protected] (B.S.) 8 Department of Pharmacology, Veterinary Faculty, University of Santiago de Compostela, 27002 Lugo, Spain; [email protected] *Correspondence: [email protected]; Tel.: +351-2234-01800; Fax: +351-2233-80609 † This authors contributed equally to this work. Received: 7 August 2018; Accepted: 4 September 2018; Published: 6 September 2018   Abstract: 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. Keywords: paralytic shellfish toxins; new vectors; post-column oxidation liquid chromatography; Madeira island; São Miguel island; Morocco Toxins 2018,10, 362; doi:10.3390/toxins10090362 www.mdpi.com/journal/toxins Toxins 2018,10, 362 2 of 13 Key Contribution: Detection of PSTs in non-traditional vectors; first report of new vectors, some of them with great commercial importance, for this group of biotoxins in Portuguese and Moroccan waters. 1. Introduction Paralytic shellfish toxins (PSTs) are a type of phycotoxins which represent a serious threat to public health. Episodes of poisoning in humans are caused not only by bivalve consumption but also by other sorts of seafood. They are produced by several common genera of microalgae and cyanobacteria with worldwide distribution. They are a group of alkaloids including saxitoxin (STX) and its analogs. To date, more than 50 saxitoxin analogues have been identified and are divided into five groups [ 1 ]: (i) carbamate toxins (saxitoxin, neosaxitoxin (NEO), and gonyautoxins (GTX1-4)); (ii) decarbamoyl toxins (decarbamoyl-saxitoxin (dcSTX), decarbamoyl-neosaxitoxin (dcNEO) and decarbamoyl-gonyautoxins (dcGTX1-4)); (iii) N-sulfocarbamoyl-toxins (gonyautoxins 5-6 (GTX5-6) and C1-4) cited from higher to lower toxicity; (iv) 13-deoxydecarbamoyl toxins (13-deoxydecarbamoyl-saxitoxin (doSTX) and 13-deoxydecarbamoy-gonyautoxins (doGTX2-3)); (v) GC toxin (GC1–6), with unknown toxic properties [2,3]. Marine dinoflagellates of the genera Alexandrium,Gymnodinium, and Pyrodinium are considered the main producers, being the majority of cases of paralytic shellfish poisoning (PSP) associated with blooming events of these species [ 4 , 5 ]. Although PSTs production has also been found in freshwater and brackish cyanobacteria [ 6 ], these organisms are rarely reported as the original cause of human intoxications. PSTs are spread through the food web by the ingestion of their producers, i.e., the above-mentioned dinoflagellate genera, and bioaccumulation in seafood. Contaminated seafood is found mainly in filter-feeding mollusks like bivalves, in particular mussels and clams [ 4 ]. Although bivalves are considered the traditional vectors and bioindicators for PSTs, reports of non-traditional vectors are being increasingly recognized. These include marine gastropods (carnivorous and grazers), crustaceans, echinoderms, tunicates, and ascidians, which can accumulate toxic levels of PSTs [ 7 – 11 ]. Certain fish such as sardines [ 12 ], salmon [ 13 , 14 ], herring [ 15 ], and mackerel [ 16 , 17 ] were also reported to accumulate PSTs, though only sub-toxic levels have been detected so far [8]. Even though a large number of PSP cases have been reported all over the world [ 4 , 5 , 18 ], the successful implementation of monitoring programs for the presence of PSTs in both marine microalgae and shellfish and an action level or regulatory limit defined as 800 µ g STX equivalents (eq)diHCl/kg tissue [19] have helped to minimize public health risks in many countries. For the last 70 years, the mousse bioassay [ 20 ] has been used worldwide as an official method in monitoring programs. While this method is considered reliable for regulatory purposes, it is being replaced by chemical methods that provide better results in terms of reproducibility and toxin profile detection. There are two methods for PSTs analysis (based on the Association Official Analytical Chemists (AOAC)-validated High Pressure Liquid Chromatography with Fluorescence Detection (HPLC-FLD)) that have been successfully validated in a collaborative study, according to the harmonized protocol of ISO/IUPAC/AOAC [ 21 ] for detection and quantification of PSTs in mussels, clams, scallops, and oysters. These two methods consist in a pre-column oxidation method [ 22 ] and a post-column oxidation method (PCOX) [ 23 ]. Although the pre-column oxidation method (method of Lawrence) may also be used as an alternative method to the mouse bioassay [ 24 ], the PCOX has been proposed as a better routine option [25]. European reference methods for the determination of PSTs were developed and optimized for the analysis of bivalve mollusks. Nevertheless, the increase in the production and capture of certain species for which monitoring programs have not been properly designed is being gradually recognized Toxins 2018,10, 362 3 of 13 because of commercial demands in the fishing industry. However, for non-bivalve vectors, no specific monitoring plans have been established to date [26]. The production or capture of echinoderms and tunicates in the EU is small compared to those of fish and other seafood [ 13 ]. The commercialization of gastropods and crustaceans has been growing in the last few years [ 14 ]. According to FAO reports from recent years [ 27 ], crustacean fisheries exceeded the production of bivalve mollusks in the EU, and marine gastropod fisheries have also increased considerably, duplicating in the last two decades. Therefore, bearing in mind the growing consumers’ interest in these species, it is of special importance to evaluate the exposure to marine biotoxins through the ingestion of these new vectors as well as the adjustment of the existing reference methods for this new scenario. In this work, samples of different invertebrate and fish species from the North Atlantic waters were analyzed using the PCOX method in order to detect new vectors for these biotoxins and evaluate their potential as a threat to public health. 2. Results and Discussion A total of 98 samples were analyzed using the PCOX method [ 10 ] with slight modifications [ 28 ] to determine and quantify PSTs. The samples were collected from three different sites: Madeira Island, São Miguel Island (Azores archipelago), and the Moroccan Atlantic coast (the sampling sites are described in detail in the Material and Methods section). Several edible (with commercial interest) and non-edible species were selected to search for potential new vectors and the prevalence of the screened biotoxins in the food web: gastropods (Stramonita haemastoma, Phorcus lineatus,Cerithium vulgatum, Gibbula umbilicalis,Aplysia depilans,Charonia lampas,Onchidella celtica,Patella gomesii,Patella aspera, Umbraculum umbraculum, Patella ordinaria), crustaceans (Pollicipes pollicipes), bivalves (Mytilus spp.), starfish (Ophidiaster ophidianus,Marthasterias glacialis,Echinaster sepositus), sea-cucumber (Holothuria (Platyperona)sanctori), sea-urchins (Paracentrotus lividus,Arbacia lixula,Sphaerechinus granularis, Diadema africanum), and fish (Sphoeroides marmuratus). Since the PCOX method is not validated for these different matrices, optimizations were needed and made for echinoderms and gastropods species, thus adding an additional step prior to the HPLC-FLD analyses and enhancing the reliability of the results [29]. 2.1. Madeira Island (Madeira Archipelago) From a total of 22 samples collected during the summer of 2012, 15 were positive for PSTs, with 7 above the maximum legislated value [30,31] (Table 1). Table 1. Quantified paralytic shellfish toxins (PSTs) samples in Madeira island, Madeira Archipelago. Sampling Data Sampling Site Sample Species Code µg STX.diHCleq/Kg SM 8 August 2012 Northern coast of Madeira Limpet Patella ordinaria 336 1123.3 Limpet Patella aspera 337 122.4 16 September 2012 Reis Magos Sea urchin Paracentrotus lividus 339#1 <LOQ Starfish Ophidiaster ophidianus 341#1 2071 Starfish O. ophidianus 341#2 2224.1 Starfish O. ophidianus 341#3 4625.4 Limpet P. aspera 344 866.4 Sea urchin Arbacia lixula 345 <LOQ Sea snail Stramonita haemastoma 346 964.5 18 September 2012 Caniçal Limpet P. aspera 350 12.2 Limpet Umbraculum umbraculum 351 536.8 Starfish Echinaster sepositus 353 668 Sea snail Charonia lampas 354 1423.4 Sea urchin Diadema africanum 355#1 276.3 Sea urchin D. africanum 355#2 227.9 Values in bold are above the legal limit; STX: saxitoxin; eq: equivalents; SM: Shellfish Meat; LOQ: Limit of quantification. Regarding toxin uptake, the highest values were detected in echinoderms, more specifically, in the red velvet starfish O. ophidianus (4625.4 µ g STX.diHCleq/Kg), followed by gastropods C. lampas Toxins 2018,10, 362 4 of 13 (1423.4 µ g STX.diHCleq/Kg SM), P. ordinaria (1123.3 µ g STX.diHCleq/Kg SM), and S. haemostoma (964.5 µg STX.diHCleq/Kg SM). Concerning monitoring, bivalves are the selected key sentinel species for which all analytical methods have been validated [ 22 , 23 , 26 ]. Being the Madeira archipelago located in oligotrophic waters where mussels are quite rare, echinoderms and gastropods showed a good potential as bio-indicators for toxin monitoring, as suggested in previous works [11,32,33]. 2.2. São Miguel Island (Azores Archipelago) From a total of 38 samples from June 2013, 22 were positive for PSTs, with 7 above the maximum legislated value [30,31] (Table 2). Table 2. Information on Azores samples. Sampling Data Sampling Site Sample Species Code µg STX.diHCleq/Kg SM 7 June 2013 Lagoa Sea urchin Sphaerechinus granularis 409#3 43.4 Sea urchin S. granularis 409#4 42.5 Starfish O. ophidianus 412 1689.6 Sea snail S. haemastoma 413 939.4 Limpet Patella gomesii 415 1192.4 Mosteiros Limpet P. gomesii 420 902.3 8 June 2013 Etar Sea urchin A. lixula 421 <LOQ Ilhéu S. Roque Sea urchin A. lixula 423 111.7 Starfish O. ophidianus 424 2588.4 Sea urchin S. granularis 425#1 <LOQ Sea urchin S. granularis 425#2 <LOQ Sea urchin S. granularis 425#3 <LOQ Starfish Marthasterias glacialis 426#2 3.8 9 June 2013 Cruzeiro Starfish M. glacialis 428 7744.3 Sea snail S. haemastoma 431 678.3 Caloura Sea urchin A. lixula 432 <LOQ Starfish M. glacialis 433#1 47.5 Starfish M. glacialis 433#2 24.9 Sea snail S. haemastoma 434 544.7 Starfish O. ophidianus 435 920.3 10 June 2013 Caloura Starfish O. ophidianus 440 245 10 June 2013 Caloura Sea snail S. haemastoma 443 128.6 Values in bold are above the legal limit. Regarding toxin uptake (Table 2), similarly to Madeira, we detected seven values above the legal limit in starfish (O. ophidianus and M. glacialis), followed by mollusks S. haemostoma and P. gomesii (939.4 and 902.3 µ g STX.diHCleq/Kg SM, respectively). The maximum uptake value detected was in the yellow spiny starfish M. glacialis from Cruzeiro, with 7744.3 µg STX.diHCleq/Kg. 2.3. Moroccan Coast The northwestern Moroccan coast was surveyed during July 2013, supplying a total of 38 samples, with 28 of them (74%) positive for saxitoxin and its analogs (Table 3). Table 3. Moroccan samples information. Sampling Data Sampling Site Sample Species Code µg STX.diHCleq/Kg SM 22 July 2013 Casablanca Corniche Bivalve Mytilus sp. 447 1376.9 Sea snail Phorcus lineatus 448 929.4 23 July 2013 Sidi Bouzid Sea snail P. lineatus 449 1404.5 Limpet Patella sp. 450 1090.5 Sea slug Aplysia depilans 451 <LOQ Bivalve Mytilus sp. 453 2266.4 Sea snail Cerithium vulgatum 454 158.8 El Jadida Sâada Sea snail C. vulgatum 455 2556 Sea cucumber Holothuria (Platyperona) sanctori 458#1 2.3 Limpet Patella sp. 459 8.6 Starfish M. glacialis 463 1852.4 Toxins 2018,10, 362 5 of 13 Table 3. Cont. Sampling Data Sampling Site Sample Species Code µg STX.diHCleq/Kg SM 24 July 2013 El Jadida Haras Barnacle Pollicipes pollicipes 464 17.7 Mrizika Bivalve Mytilus sp. 465 1140.4 Barnacle P. pollicipes 466 17.6 Limpet Patella sp. 467 3622.5 Bivalve Mytilus sp. 468 1080.9 Sea snail Gibbula umbilicalis 469 1.6 Sea snail P. lineatus 470 1043.9 Starfish M. glacialis 473 1325.4 Sea slug Onchidella celtica 474 38.8 Oualidia Sea snail C. lampas 475 0.02 Sea slug A. depilans 476 0.6 Sea snail S. haemastoma 477 384 Sea snail P. lineatus 482 85.7 Sea snail G. umbilicalis 483 12.1 Barnacle P. pollicipes 484 17.4 Bivalve Mytilus sp. 485 2708.9 Values in bold are above the legal limit. All positive values were found in mollusks and echinoderms. Thirteen were above the European [ 30 , 31 ] maximum legal limit. It is important to notice that the highest concentration value detected was in a limpet, with a total amount of 3622.5 µ g STX.diHCleq/Kg SM, despite the presence of mussels in this region. 2.4. Statistical Analysis The results of the generalized linear model for PST content as a function of genus and region (Patella and Paracentrotus) and region (Madeira and Morocco) did not find significant differences for region, despite the southwards increase detected in the whole data set. This could be explained by the low number of samples and organisms included and by the fact that these two sampling regions are closer to each other, relative to Azores. On the other hand, a significant difference was detected in toxin concentrations between genera, with higher concentrations in Patella than in the sea urchin (F1,16 = 10.4, p< 0.01), which seems to be a general pattern for sea urchins and limpets in all the three regions (see Tables 1–3). 2.5. General Discussion One of the primary aims of this work was to screen new vectors for PSTs in order to evaluate public health threats related to seafood consumption. From a total of 66% of positive results, we report 14 new vectors for these hydrophilic phycotoxins, belonging to three different phyla: mollusks (P. aspera, S. haemostoma,U. umbraculum,P. gomesii,P. ordinaria,C. vulgatum,O. celtica), echinoderms (O. ophidianus, A. lixula,E. sepositus,D. africanum,S. granularis,H. (Platyperona) sanctori), and crustaceans (P. pollicipes). In Figure 1are displayed some examples of the toxin elution in different matrices, showing that the toxin retention time was dependent on the analyzed matrix used in the PCOX method. We highlight the latitudinal pattern of PSTs uptake, since the percentage of positive results follows a north-south gradient: Azores (58%) < Madeira (68%) < Morocco (74%). Though many of the causes of dinoflagellate bloom formation are still to unravel, the water temperature and eutrophication play a pivotal role, which is consistent with our results. Water temperature in the Atlantic Ocean rises, in general, following a north-south gradient towards the Equator, and human anthropic pressures are higher in Morocco than in the Portuguese islands screened in this survey [ 34 – 39 ]. We also defined a qualitative pattern of toxin profiles, as shown in Figure 2. Toxins 2018,10, 362 6 of 13 Toxins 2018, 10, x FOR PEER REVIEW 6 of 13 Figure 1. Comparison of chromatographic separations of gonyautoxins and saxitoxins: (A) Sample 485, Mytillus spp. (B) Sample 443, Stramonita haemostoma. (C) Sample 484, Pollicipes pollicipes. Obtained by the post-column oxidation (PCOX) method. We highlight the latitudinal pattern of PSTs uptake, since the percentage of positive results follows a north–south gradient: Azores (58%) < Madeira (68%) < Morocco (74%). Though many of the causes of dinoflagellate bloom formation are still to unravel, the water temperature and eutrophication play a pivotal role, which is consistent with our results. Water temperature in the Atlantic Ocean rises, in general, following a north–south gradient towards the Equator, and human anthropic pressures are higher in Morocco than in the Portuguese islands screened in this survey [34– 39]. We also defined a qualitative pattern of toxin profiles, as shown in Figure 2. Figure 2. Average toxin frequency in Madeira island (A), Azores (B), and Morocco (C). Among the different saxitoxin derivatives, dcGTX3 and GTX1 were more frequent in Madeira, dcGTX2, dcGTX3, GTX1, and STX in São Miguel island from Azores, and C1, C2, GTX1, and GTX4 in the Moroccan Coast. As explained in the introduction, the Toxicity Equivalency Factors (TEFs) of PSTs are: carbamate group > decarbamoyl group > N-sulfocarbamoyl group. According to this classification, the average PSTs toxin profiles from the Portuguese islands were more toxic than those from the Moroccan coast [40]. One of the reasons that could contribute to this trend is the vector species analyzed from each sampling site, since it is known that PSTs are subject to conversions in the digestive tracts of their vectors [41,42]. The Madeira and Azores profiles could be more similar to each other than to the Moroccan coast profile because of the higher percentage of echinoderms and gastropods sampled. Also, bivalves are quite rare in the Portuguese islands. Figure 1. Comparison of chromatographic separations of gonyautoxins and saxitoxins: (A) Sample 485, Mytillus spp. (B) Sample 443, Stramonita haemostoma. (C) Sample 484, Pollicipes pollicipes. Obtained by the post-column oxidation (PCOX) method. Toxins 2018, 10, x FOR PEER REVIEW 6 of 13 Figure 1. Comparison of chromatographic separations of gonyautoxins and saxitoxins: (A) Sample 485, Mytillus spp. (B) Sample 443, Stramonita haemostoma. (C) Sample 484, Pollicipes pollicipes. Obtained by the post-column oxidation (PCOX) method. We highlight the latitudinal pattern of PSTs uptake, since the percentage of positive results follows a north–south gradient: Azores (58%) < Madeira (68%) < Morocco (74%). Though many of the causes of dinoflagellate bloom formation are still to unravel, the water temperature and eutrophication play a pivotal role, which is consistent with our results. Water temperature in the Atlantic Ocean rises, in general, following a north–south gradient towards the Equator, and human anthropic pressures are higher in Morocco than in the Portuguese islands screened in this survey [34– 39]. We also defined a qualitative pattern of toxin profiles, as shown in Figure 2. Figure 2. Average toxin frequency in Madeira island (A), Azores (B), and Morocco (C). Among the different saxitoxin derivatives, dcGTX3 and GTX1 were more frequent in Madeira, dcGTX2, dcGTX3, GTX1, and STX in São Miguel island from Azores, and C1, C2, GTX1, and GTX4 in the Moroccan Coast. As explained in the introduction, the Toxicity Equivalency Factors (TEFs) of PSTs are: carbamate group > decarbamoyl group > N-sulfocarbamoyl group. According to this classification, the average PSTs toxin profiles from the Portuguese islands were more toxic than those from the Moroccan coast [40]. One of the reasons that could contribute to this trend is the vector species analyzed from each sampling site, since it is known that PSTs are subject to conversions in the digestive tracts of their vectors [41,42]. The Madeira and Azores profiles could be more similar to each other than to the Moroccan coast profile because of the higher percentage of echinoderms and gastropods sampled. Also, bivalves are quite rare in the Portuguese islands. Figure 2. Average toxin frequency in Madeira island (A), Azores (B), and Morocco (C). Among the different saxitoxin derivatives, dcGTX3 and GTX1 were more frequent in Madeira, dcGTX2, dcGTX3, GTX1, and STX in São Miguel island from Azores, and C1, C2, GTX1, and GTX4 in the Moroccan Coast. As explained in the introduction, the Toxicity Equivalency Factors (TEFs) of PSTs are: carbamate group > decarbamoyl group > N-sulfocarbamoyl group. According to this classification, the average PSTs toxin profiles from the Portuguese islands were more toxic than those from the Moroccan coast [ 40 ]. One of the reasons that could contribute to this trend is the vector species analyzed from each sampling site, since it is known that PSTs are subject to conversions in the digestive tracts of their vectors [ 41 , 42 ]. The Madeira and Azores profiles could be more similar to each other than to the Moroccan coast profile because of the higher percentage of echinoderms and gastropods sampled. Also, bivalves are quite rare in the Portuguese islands. All the crustacean samples were positive for PSTs, although the detected values were below the legal limit. These group of organisms are not considered in the European Legislation for marine toxins monitoring [ 43 ], although one of the species analyzed, P. pollicipes (see chromatogram in Figure 1), is of high commercial value. Regarding starfish, we reported 14 positive samples for PSTs, and nine of them exceeded the legal limit. In prior studies, PSTs were also detected in these matrices, though in smaller amounts and always below the legal European limit [ 11 , 44 – 46 ]. It is important to note that, to the best of our knowledge, we reported here the presence of PSTs in the holothuridae group for the first time [ 26 ]. Regarding PSTs detection in species with commercial value other than mussels, we found Toxins 2018,10, 362 7 of 13 quantifiable amounts of these alkaloids in P. gomesii,P. aspera,P. pollicipes,P. lividus, and C. lampas (values displayed in Tables 1–3). It is important to point out sample 336 purchased in a local market in Madeira island, which exhibited an amount of PSTs above the legal limit (1123.3 µ g STX.diHCl eq/Kg SM). Limpets are a much appreciated delicacy, mainly in the Madeira archipelago. Previous studies showed the need to revise and update the legislation regarding these organisms, i.e., monitoring practices should be mandatory, and analytic practices should be optimized for these vectors to avoid human poisoning incidents [ 26 ]. Our work shows real risks to human consumers, as our analyses were performed in purchased samples. In addition, since bivalves are absent from the Portuguese islands, echinoderms and gastropods would be a suitable alternative as toxin bioindicators in these geographical areas [11,32,33]. The European reference methods for PSTs determination, based on liquid chromatography with fluorescence detection [ 22 , 23 ], were developed and optimized for bivalves, though echinoderms and gastropods are also contemplated in the monitoring programs. In this work, we demonstrated that, with small amendments, it is possible to optimize with reliability the available methods for these matrices (Figure 1). Also, the application to crustaceans was straightforward. To summarize, the PCOX method [23] has proved to be a good technique for analyzing PSTs in non-traditional vectors. 3. Conclusions In this study, we used the PCOX technique to screen non-traditional vectors for paralytic shellfish toxins in three sampling areas: Madeira island, São Miguel island (Azores archipelago), and the northwestern Moroccan coast. In a total of 98 samples, 60% were positives, with the PST content of 27 samples above the maximum legislated EU value. We report for the first time 14 vectors for PSTs (P. aspera , S. haemostoma , U. umbraculum , P. ordinaria , P. gomesii , C. vulgatum , O. celtica , O. ophidianus , A. lixula, E. sepositus, D. africanum, S. granularis, H. (Platyperona) sanctori, and P. pollicipes). PST positivity was found both in edible and in non-edible species. Non-edible species are also important because they could transfer toxins to other compartments of the food web and, eventually, to edible species. Monitoring non-edible species, like mussels in the Portuguese archipelagos, could also be important, since the legislated bioindicator is rare or absent. In this sense, echinoderms and gastropods could be a good alternative for toxin monitoring in these locations. Positive hits in high-value commercial species (e.g., Patella aspera,Pollicipes pollicipes,Charonia lampas) highlight the need to reinforce monitoring programs with the inclusion of these species. 4. Materials and Methods 4.1. Sampling Sites and Selected Species The Portuguese islands of Madeira (Madeira archipelago), São Miguel (Azores archipelago), and the northwestern coast of Morocco where surveyed for non-traditional vector species for PSTs (Figure 3). Benthic organisms were harvested from the intertidal areas during low tide and by scuba diving expeditions. Madeira island was surveyed in September 2012. São Miguel island (Azores) and the Moroccan coast were sampled in June and July of 2013, respectively. The sampling sites are shown in Table 4. The samples of P. ordinaria and P. aspera were purchased in local markets in Madeira, caught in the northern coast of the island (32 ◦ 51 0 17.02 00 N; 17 ◦ 01 0 54.02 00 W). The organisms were transported to the laboratory in refrigerated containers. The samples that were not immediately processed were carefully stored at −20 ◦C. Toxins 2018,10, 362 8 of 13 Toxins 2018, 10, x FOR PEER REVIEW 8 of 13 Figure 3. Location of the sampling sites: (A) São Miguel island coast, Azores archipelago: 1, Cruzeiro; 2, Mosteiros; 3, Étar; 4, São Roque; 5, Lagoa; 6, Caloura. (B) Madeira island coast: 1, Reis Magos and 2, Caniçal. (C) Northwestern Moroccan coast: 1, Casablanca Corniche; 2, El Jadida Haras; 3, El Jadida Sâada; 4, Sidi Bouzid; 5, Mrizika; 6, Oualidia. Table 4. Sampling sites and respective geographical coordinates, surveyed during September 2012 and June and July 2013. Date Location Sampling Site Geographic Coordinates September 2012 Madeira Island Reis Magos 32°39′16.21″ N; 16°49′05.29″ W Caniçal 32°44′20.08″ N; 16°44′17.55″ W June 2013 São Miguel Island Cruzeiro 37° 50′31.19″ N; 25° 41′33.61″ W Étar 37°44′19.31″ N; 25°39′38.84″ W São Roque 37°45′15.35″ N; 25°38′31.60″ W Mosteiros 37°53′25.57″ N; 25°49′14.72″ W Lagoa 37°44′42.38″ N; 25°19′.47″ W Caloura 37°42′49.34″ N; 25°29′54.54″ W July 2013 Morocco Coast Casablanca corniche 33°36′01.2″ N; 7°39′57.5″ W El Jadida Haras 33°14′42.0″ N; 8°28′37.5″ W El Jadida Sâada 33°14′42.4″ N; 8°32′26.9″ W Sidi Bouzid 33°13′57.1″ N; 8°33′20.9″ W Mrizika 32°57′21.8″ N; 8°46′53.2″ W Oualidia 32°43′55.8″ N; 9°02′57.6″ W 4.2. PSTs Extraction and Analysis Method 4.2.1. Chemicals and Solutions Analytical reagent-grade hydrochloric acid 37%, nitric acid 65%, ortho-phosphoric acid 85%, glacial acetic acid, periodic acid, sodium hydroxide, sodium dihydrogen phosphate; HPLC-grade methanol and acetonitrile were from Panreac Quimica S.A. (Barcelona, Spain). Trichloroacetic acid, Figure 3. Location of the sampling sites: ( A ) São Miguel island coast, Azores archipelago: 1, Cruzeiro; 2, Mosteiros; 3, Étar; 4, São Roque; 5, Lagoa; 6, Caloura. ( B ) Madeira island coast: 1, Reis Magos and 2, Caniçal. (C) Northwestern Moroccan coast: 1, Casablanca Corniche; 2, El Jadida Haras; 3, El Jadida Sâada; 4, Sidi Bouzid; 5, Mrizika; 6, Oualidia. Table 4. Sampling sites and respective geographical coordinates, surveyed during September 2012 and June and July 2013. Date Location Sampling Site Geographic Coordinates September 2012 Madeira Island Reis Magos 32◦39016.2100 N; 16◦49005.2900 W Caniçal 32◦44020.0800 N; 16◦44017.5500 W June 2013 São Miguel Island Cruzeiro 37◦50031.1900 N; 25◦41033.6100 W Étar 37◦44019.3100 N; 25◦39038.8400 W São Roque 37◦45015.3500 N; 25◦38031.6000 W Mosteiros 37◦53025.5700 N; 25◦49014.7200 W Lagoa 37◦44042.3800 N; 25◦190.4700 W Caloura 37◦42049.3400 N; 25◦29054.5400 W July 2013 Morocco Coast Casablanca corniche 33◦36001.200 N; 7◦39057.500 W El Jadida Haras 33◦14042.000 N; 8◦28037.500 W El Jadida Sâada 33◦14042.400 N; 8◦32026.900 W Sidi Bouzid 33◦13057.100 N; 8◦33020.900 W Mrizika 32◦57021.800 N; 8◦46053.200 W Oualidia 32◦43055.800 N; 9◦02057.600 W 4.2. PSTs Extraction and Analysis Method 4.2.1. Chemicals and Solutions Analytical reagent-grade hydrochloric acid 37%, nitric acid 65%, ortho-phosphoric acid 85%, glacial acetic acid, periodic acid, sodium hydroxide, sodium dihydrogen phosphate; HPLC-grade methanol and acetonitrile were from Panreac Quimica S.A. (Barcelona, Spain). Trichloroacetic Toxins 2018,10, 362 9 of 13 acid, tetrabutyl ammonium phosphate, ammonium hydroxide 28–30%, 2-mercaptoethanol (2-ME), and heptane sulfonate were purchased from Sigma-Aldrich (Madrid, Spain). The certified standards provided by Cifga S.A. (Lugo, Spain) were: saxitoxin (STX), neosaxitoxin (NEO), decarbamoyl-saxitoxin (dcSTX), gonyautoxins 1 and 4 (GTX1,4), gonyautoxin 5 (GTX5), gonyautoxins 2 and 3 (GTX2,3), decarbamoyl-gonyautoxins 2 and 3 (dcGTX2,3), and N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2). 4.2.2. Apparatus The LC system used, composed of a binary LC-10A pump system, a degasser DGU-14A, a fluorescence detector RF-10AXL, an autoinjector SIL-20AC with a refrigerated rack, a column oven CTO-20AC, and a system controller CBM-20A, was from Shimadzu (Izasa, Barcelona, Spain). The Shimadzu LC solution software was used to control the system. The post-column reaction system used was formed by a knitted reaction coil with 1 mL of total volume (5 m × 0.50 mm i.d., Supelco, Madrid, Spain) immersed in a water bath at 80 ◦ C, and two post-column pumps, LC-20AD and LC-6A and was from Shimadzu (Izasa, Barcelona, Spain). 4.2.3. Extraction Protocol The samples were extracted following the PCOX method for the extraction of PSTs with proper amendments, adjusted to the type of sample tested [ 1 ]. Briefly, the animals were dissected and homogenized with a blender (A320R1, 700 W, Moulinex, Lisbon, Portugal) in pooled groups in order to obtain 1 g of extractable tissue, with the exception of C. lampas,M. glacialis,A. depilans,H. spp., O. ophidianus,S. granularis,U. umbraculum,D. antilarium,S. marmuratus, which were treated individually, since each animal had enough extractable biomass. A 1 g sample was homogenized with 1 mL of 0.1 M of HCl using a vortex mixer. The pH was adjusted, between 2 and 4, with 5 M of HCl or 5 M of NaOH, while stirring the mixture. The sample was heated in a boiling water bath for 5 min and cooled to room temperature. The pH was re-adjusted, and the samples was remixed and centrifuged again at 3000 × gfor 10 min (Centric150, Tehtnica, Spain). The supernatant was decanted into a 15 mL falcon, then 500 µ L were collected and deproteinated with 25 µ L of 30% (v/v) TCA. The contents were mixed in a vortex mixer and centrifuged at 16,000 × gfor 5 min (Biocen 22, Ortoalresa, Madrid, Spain). An amount of 40 µ L of 1 M NaOH was added, and the sample was mixed and centrifuged at 16,000 × g for 5 min. The supernatant was filtered through a 0.2 µ m nylon syringe filter (Whatman, Cambridge, UK) into a vial for analysis. Aliquots (5 µL for C toxins and 10 µL for the GTX and STX toxins group) were injected into the system [47]. 4.2.4. HPLC Analysis The post-column oxidation and fluorescence detection method [ 48 ] was used with the modifications reported by Rey et al. (2015) [28]. C toxins were separated using a 25 cm × 4.6 mm i.d., 5 µ m BetaBasis C8 column (Thermo, Fisher Scientific, Madrid, Spain), with a column oven at 20 ◦ C. Solvent A was composed of 2 mM of tetrabutyl ammonium phosphate aqueous solution. Solvent B was composed by 2 mM of tetrabutyl ammonium phosphate in 4% of acetonitrile. Both solvents had their pH adjusted to 5.8 with 1% of NH 4 OH. The gradient started with 0% of B during the first 8 min, which increased to 100% up to minute 15, was maintained at this concentration for one minute, then decreased to 0% in 3 min and remained at 0% for 5 min before the next injection. STX and GTX toxins were separated using a 15 cm × 4.6 mm i.d., 3.5 µ m Zorbax Bonus-RP column (Agilent Technologies, Madrid, Spain), with a column oven at 30 ◦ C. Solvent A was composed of 8.25 mM of heptane sulfonate and 5.5 mM of H 3 PO 4 aqueous solution. Solvent B was 8.25 mM heptane sulfonate and 16.5 mM H 3 PO 4 in 11.5% of acetonitrile. Both solvents had their pH adjusted to 7.1 with 28–30% of NH 4 OH. The gradient used was 0% B over 8.4 min, 100% B at 8.5 min for 10 min, and 0% B at 18.5 min for 9 min before the next injection.