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Dinoflagellate species of the genera Gambierdiscus and Fukuyoa are known to produce ciguatera poisoning-associated toxic compounds, such as ciguatoxins, or other toxins, such as maitotoxins. However, many species and strains remain poorly characterized in areas where they were recently identified, such as the western Mediterranean Sea. In previous studies carried out by our research group, a G. australes strain from the Balearic Islands (Mediterranean Sea) presenting MTX-like activity was characterized by LC-MS/MS and LC-HRMS detecting 44-methyl gambierone and gambieric acids C and D. However, MTX1, which is typically found in some G. australes strains from the Pacific Ocean, was not detected. Therefore, this study focuses on the identification of the compound responsible for the MTX-like toxicity in this strain. The G. australes strain was characterized not only using LC-MS instruments but also N2a-guided HPLC fractionation. Following this approach, several toxic compounds were identified in three fractions by LC-MS/MS and HRMS. A novel MTX analogue, named MTX5, was identified in the most toxic fraction, and 44-methyl gambierone and gambieric acids C and D contributed to the toxicity observed in other fractions of this strain. Thus, G. australes from the Mediterranean Sea produces MTX5 instead of MTX1 in contrast to some strains of the same species from the Pacific Ocean. No CTX precursors were detected, reinforcing the complexity of the identification of CTXs precursors in these regions. European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03 Xunta de Galicia | Ref. ED481A-2018/207 Generalitat de Catalunya | Ref. CERCA

The Selvagens Islands, which are a marine protected area located at the southernmost point of the Portuguese maritime zone, have been associated with fish harboring ciguatoxins (CTX) and linked to ciguatera fish poisonings. This study reports the results of a field sampling campaign carried out in September 2018 in these remote and rarely surveyed islands. Fifty-six fish specimens from different trophic levels were caught for CTX-like toxicity determination by cell-based assay (CBA) and toxin content analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Notably, high toxicity levels were found in fish with an intermediate position in the food web, such as zebra seabream (Diplodus cervinus) and barred hogfish (Bodianus scrofa), reaching levels up to 0.75 µg CTX1B equivalent kg−1. The LC-MS/MS analysis confirmed that C-CTX1 was the main toxin, but discrepancies between CBA and LC-MS/MS in D. cervinus and top predator species, such as the yellowmouth barracuda (Sphyraena viridis) and amberjacks (Seriola spp.), suggest the presence of fish metabolic products, which need to be further elucidated. This study confirms that fish from coastal food webs of the Selvagens Islands represent a high risk of ciguatera, raising important issues for fisheries and environmental management of the Selvagens Islands. European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03 Fundação para a Ciência e a Tecnologia | Ref. PTDC/CTA-AMB/30557/2017 Fundação para a Ciência e a Tecnologia | Ref. UID/Multi/04326/2020 Xunta de Galicia | Ref. ED481A-2018/207 Generalitat de Catalunya | Ref. CERCA

Over the last decade, knowledge has significantly increased on the taxonomic identity and distribution of dinoflagellates of the genera Gambierdiscus and Fukuyoa. Additionally, a number of hitherto unknown bioactive metabolites have been described, while the role of these compounds in ciguatera poisoning (CP) remains to be clarified. Ciguatoxins and maitotoxins are very toxic compounds produced by these dinoflagellates and have been described since the 1980s. Ciguatoxins are generally described as the main contributors to this food intoxication. Recent reports of CP in temperate waters of the Canary Islands (Spain) and the Madeira archipelago (Portugal) triggered the need for isolation and cultivation of dinoflagellates from these areas, and their taxonomic and toxicological characterization. Maitotoxins, and specifically maitotoxin-4, has been described as one of the most toxic compounds produced by these dinoflagellates (e.g., G. excentricus) in the Canary Islands. Thus, characterization of toxin profiles of Gambierdiscus species from adjacent regions appears critical. The combination of liquid chromatography coupled to either low- or high-resolution mass spectrometry allowed for characterization of several strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and the Canary Islands. Maitotoxin-3, two analogues tentatively identified as gambieric acid C and D, a putative gambierone analogue and a putative gambieroxide were detected in all G. australes strains from Menorca and Mallorca (Balearic Islands, Spain) while only maitotoxin-3 was present in an F. paulensis strain of the same region. An unidentified Gambierdiscus species (Gambierdiscus sp.2) from Crete (Greece) showed a different toxin profile, detecting both maitotoxin-3 and gambierone, while the availability of a G. excentricus strain from the Canary Islands (Spain) confirmed the presence of maitotoxin-4 in this species. Overall, this study shows that toxin profiles not only appear to be species-specific but probably also specific to larger geographic regions. European Food Safety Authority | Ref. EuroCigua (GP/EFSA/AFSCO/2015/03) Xunta de Galicia | Ref. ED481A-2018/207 IRTA-URV-Santander | Ref. 2016 PMF-PIPF-74

Local population frequently consumes moray eels and dusky groupers from the Canary Islands. These species are top predators and the interactions between them include predation but also, in some cases, collaborative hunting. These fish are well known to cause ciguatera (CFP) outbreaks in several marine areas such as Japan, Hawaii, French Polynesia and Caribe. Groupers have been involved in CFP events in the Canary Islands, however, moray eels have not yet been well studied in this regard. The present research seeks to describe the finding of a black moray in the stomach of a positive dusky grouper during its necropsy, and to clarify the implication of groupers and moray eels in the food webs, accumulating CTXs in the Canarian environment. The study also updates statistics on the presence of toxic groupers in this archipelago. For these purposes, 248 grouper samples from the CFP official control in the Canary Islands (2018–2019) were analysed and 36 moray eels (5 species) were collected under the EuroCigua project and one was obtained during a dusky grouper necropsy. All samples were analysed with the Neuro-2a cell-based assay (CBA) to evidence CTX-like toxicity. Regarding the necropsied grouper and the moray eel found in its stomach content, the LCMS/MS method allowed the identification and quantification of CCTX1 in both fish at similar levels while none of the P-CTXs for which standards were available were detected. Among groupers, 25.4 % displayed CTX-like toxicity with differences between islands. For moray eels 38.9 % showed toxicity, involving 4 species. Black moray exhibited a high proportion of positives (9/12) and a positive correlation was found between CTX-like toxicity quantification and the black moray weight. Regarding the grouper, and the moray eel found in its stomach, the LCMS/MS method allowed the identification and quantification of C-CTX1 in both fish at similar levels. This found suggests a trophic interaction between these species and their role in maintaining CTXs in the Canary waters where local population commonly demand those species for consumption. The island of El Hierro stands out above all the other Canary Islands with the concerning percentage of positive grouper samples and the high CTX toxicity levels obtained in moray eel specimens analysed in this marine area. This is the first report of CTX-like toxicity in flesh of moray eels fished in the Canary archipelago and the confirmation of the presence of C-CTX1 by LCMS/MS in a black moray from this marine area. info:eu-repo/semantics/acceptedVersion

Nuts are important components of a healthy diet since they provide nutritional value and bioactive components. Melatonin is a well-known molecule in plants, and its relevance in foodstuffs is increasing. This study investigated the presence of melatonin in nuts using chromatographic techniques and optimized extraction procedures according to the high oil content of nuts. Melatonin was detected in four walnut cultivars with levels similar to those previously reported. Moreover, the melatonin content in walnut seeds decreased sharply during the ripening process from the ripe green stage to the mature dehydrated fruit and increased after harvesting when the fruits were edible. A number of other commercial nuts were also measured, with melatonin contents varying markedly, and generally being lower than in walnuts. The presence of melatonin was lower in commercial roasted nuts than in raw nuts, with the exception of peanuts, where melatonin content increased with roasting. It seems that this industrial processing negatively alters the structure of this molecule and its availability, which should be taken into account when estimating its levels during nut consumption. Therefore, this study reveals new data on the presence of melatonin in walnut seeds in a natural format and its evolution with maturation as well as in other commercial nuts. We also highlight the importance that processing has on melatonin and other antioxidants in the nuts that reach the consumer. Financiado para publicación en acceso aberto: Universidade de Vigo/CISUG Xunta de Galicia | Ref. ED481A-2017/387

The recent emergence of ciguatera in the eastern Atlantic, particularly in the Canary Islands (Spain) and Madeira (Portugal) prompted the development and implementation of liquid chromatography tandem-mass spectrometry (LC/MS-MS) methods for the detection of ciguatoxins in fish. The complexity of fish tissue matrices, low concentrations of ciguatoxins in hazardous fish, and the scarcity of ciguatoxin standards present challenging issues for successful implementation of routine ciguatoxin analysis. A laboratory reference material of Caribbean Ciguatoxin-1 (C-CTX1), which was previously confirmed in fish responsible for ciguatera outbreaks in the Canary Islands, was used to assess the toxin's stability under strongly acidic conditions and solvent systems commonly used in LC-MS/MS. It was observed that strongly acidic conditions caused the transformation of C-CTX1 to a C56 methoxy congener, C-CTX1-Me. C-CTX1 was structurally characterised by LC-MS/MS and fragmentation pathways are presented showing the same fragmentation pattern as C-CTX1-Me. These results suggest that the use of strongly acidic conditions during sample pretreatment for C-CTX analysis, might produce significant artefacts, and risks failing to detect the presence of C-CTX1.

The emergence of marine biotoxins in geographical areas where they have never been reported before is a concern of considerable impact on seafood contamination, and consequently, on public health [...]

Ciguatoxins (CTXs), endemic from tropical and subtropical regions of the Pacific and Indian Ocean and the Caribbean Sea, have caused several human poisonings during the last decade in Europe. Ciguatera fish poisonings (CFP) in Madeira and Canary Islands appear to be particularly related with consumption of fish caught close to Selvagens Islands, a Portuguese natural reserve composed of three small islands that harbor high fish biomass. In this study, fish specimens considered as potential vectors of CTXs were caught in Madeira and Selvagens archipelagos for toxins determination via sensitive liquid chromatography with tandem mass spectrometry detection (LC&ndash, MS/MS). CTXs were found in most of the fish samples from Selvagens and none from Madeira. Caribbean ciguatoxin-1 (C-CTX1) was the only toxin congener determined, reaching the highest value of 0.25 &micro, g C-CTX1 kg&minus, 1 in a 4.6 kg island grouper (Mycteroperca fusca). This study indicates that a diversity of fish from different trophic levels contains CTXs, Selvagens appear to be one of the most favorable locations for CTXs food web transfer and finally, this study highlights the need of further research based on intensive environmental and biological sampling on these remote islands.

Vitamin B12 (cobalamin), a large and complex organometallic molecule composed by a corrin ring with a central cobalt atom, is an essential coenzyme for nearly half of phytoplankton species but its production in marine ecosystems is restricted to prokaryotic organisms. Vitamin B 12 auxotrophy is widespread in the marine microbial community not being linked to any species lineage. Knowledge about the role of this organic compound on the control of the structure, composition and growth of natural marine microbial assemblages is scarce, but recent studies carried out in shelf waters off NW Iberian Peninsula have demonstrated that growth of phytoplankton and heterotrophic bacteria can be affected by the availability of this vitamin. Liquid Chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been set up as an analytical tool for the first direct determination of vitamin B 12 present in these coastal waters enabling the separation of two different B 12 analogs: hydroxocobalamin (HyB12) and cyanocobalamin (CyB12). In this study, samples were collected from shelf (coastal station) and shelf-break waters of the NW Iberian Peninsula in February, April and August 2016 in order to encompass both the spatial and temporal variability of vitamin B 12 concentrations in seawater. Concentrations of dissolved vitamin B 12 varied from 0.15 pM, at the shelf-break station, to 2.7 pM at the coastal station, HyB12 being the most abundant congener. Overall, the low concentrations of dissolved vitamin B 12 and the significant and positive relationship between depth-weighted water column averages of Chl-a and vitamin B 12 (r 2 = 0 . 80 , p>0.001), suggest that this micronutrient might play a significant role in phytoplankton biomass production during the spring productive period of this coastal ecosystem. The temporal cycle elucidated from this data described an exhaustion of vitamin B 12 winter inventory by direct consumption during the phytoplankton productive period and a stock recovery in August.

Paralytic shellfish toxins (PSTs) are naturally occurring marine compounds which in some instances result in significant consumer sickness following consumption of contaminated shellfish products. The toxins are found in shellfish grown in marine waters throughout the world, and many instances of human intoxication are reported annually. This chapter describes the evolution of instrumental analytical methods for the determination of PSTs in shellfish, in the context of the global move away from reliance on live animal testing for food safety testing. Key methods are described and assessed, describing the major aspects and challenges associated with each. Fluorimetric methods, particularly those conducted following liquid chromatography separation of toxin congeners, have been formally validated and in an increasing number of countries applied to official control monitoring of shellfish harvesting zones. While these provide a reliable alternative to biological assays, these methods are complex and are unable to detect all the currently known toxin threats. While capillary electrophoresis has shown some potential, the development of rapid chromatography, particularly in tandemwith mass spectrometry, has shown perhaps the greatest promise for the development of reliable, accurate, rugged, and reproducible monitoring tools. Nevertheless, further work and interlaboratory validation studies are still required to ensure such methods are fit for purpose and continue to improve the quality of the monitoring conducted to mitigate the serious risk from these highly toxic compounds.

Neurotoxicity induced in fish by domoic acid (DA) was assessed with respect to occurrence of neurotoxic signs, lethality, and histopathology by light microscopy. Sparus aurata were exposed to a single dose of DA by intraperitoneal (i.p.) injection of 0, 0.45, 0.9, and 9.0 mg DA kg(-1) bw. Mortality (66.67 ± 16.67%) was only observed in dose of 9.0 mg kg(-1) bw. Signs of neurological toxicity were detected for the doses of 0.9 and 9.0 mg DA kg(-1) bw. Furthermore, the mean concentrations (±SD) of DA detected by HPLC-UV in extracts of brain after exposure to 9.0 mg DA kg(-1) bw were 0.61 ± 0.01, 0.96 ± 0.00, and 0.36 ± 0.01 mg DA kg(-1) tissue at 1, 2, and 4 hours. The lack of major permanent brain damage in S. aurata, and reversibility of neurotoxic signs, suggest that lower susceptibility to DA or neuronal recovery occurs in affected individuals.

Aspartic acid, glutamic acid, gamma-amino-n-butyric acid (GABA) and 2-aminoethanesulfonic acid are neuroactive amino acids. They are found in the central rat nervous system. Here, we have studied if a relationship exists between the presence of saxitoxin (STX) a paralytic poisoning shellfish (PSP) and the neuroactive amino acids. Samples of striatum (S), hypothalamus (H), mid brain (MB), frontal cortex (FC), brain stem (BS), right hemisphere (RH) and left hemisphere (LH) of rat brain were collected and analyzed for neuroactive amino acids (AAn(t)) by Aswad method (1984). Experiments, consisting of intraperitoneal injection of SXT (5 and 10microgkg(-1) body weight) to young male rats, evoked significant changes in AAn(t) above basal values. Aspartic and glutamic acid significantly increased for RH and LH (after 30min the increased was 116% and 210%, Por =0.001 over basal values, respectively). On the other hand, aspartic, glutamic, taurine and GABA significantly decreased for S (after 30min the decreased was 77.4%; 84%; 93.8% and 95.3%, Por =0.001 over basal values, respectively). These results suggest that STX alters AAn(t). It is produced at least in part, because STX blocks voltage-gated sodium channels and this blockade could decrease AAn(t) release by exocytotic dependent mechanism of depolarization.

Toxic microalgae outbreaks have caused significant economic losses in the Mexican aquaculture industry. Blooms that involve PSP and NSP phycotoxins are two of the most dangerous, causing harmful effects to the environment, economy and public health. The exact metabolic mechanism of these toxins in shrimp still remains unknown. Because shrimp consume microalgae their edible tissues are clearly possible vectors for human toxic syndrome. This study examined and verified the toxicological effects for white leg shrimp (Litopenaeus vannamei) exposed to different cell densities of Gymnodinium catenatum and Karenia brevis. Acute assays demonstrated good survival rates of shrimp at low densities of dinoflagellates (103 cell/L), while mortality and abnormal behavior were observed with higher densities (>104 cell/L). Chronic assays showed significant differences in survival rates, percentage of feed and weight gain of organisms exposed to the dinoflagellates with respect to controls. Furthermore, PSP and NSP toxins were detected in all the edible tissues. Gastric glands and muscle retained toxins for a longer period of time compared to other tissues, even after a depuration period. Histology damages were observed in the heart, gastric gland and brain. This study strongly supports that shrimp represent a potential risk for humans as unconventional vectors of phycotoxins.

The presence of cyanobacterial toxins (microcystins) in waters and food increases the risk of toxicity to animal and human health. These toxins can degrade in the human gastrointestinal tract before they are absorbed. To evaluate this possible degradation, water samples spiked with known concentrations of microcystins MC-LR, MC-RR, and MC-YR, which are the toxins most commonly produced by such toxic cyanobacteria as Microcystis aeruginosa, Oscillatoria spp., and Nostoc spp., were submitted to a dissolution test that used gastric and intestinal fluids according to U.S. Pharmacopeia conditions. HPLC with UV detection was used to determine the toxins before and after treatments. This study revealed enzymatic alterations in gastric conditions for all the toxins assayed. MC-RR was the toxin most affected: its range of inactivation was 49-64%. The percentage of degradation for MC-YR and MC-LR was around 30%. However, none was degraded by intestinal digestion.

The presence of algal toxins in the aquatic environment represents an important socioeconomic concern in many places worldwide, due to the toxicity that these compounds can induce in seafood or freshwater organisms at very low levels. Several analytical alternatives have been proposed over the last years for the control of these contaminants, which acute or chronic toxicity requires low detection levels and demands for the search of sensitive methods for their detection and determination. HPLC has been widely used for this purpose, although several alternatives such as CE or capillary electrochromatography (CEC) are being lately developed with this aim. In this work we report on the application of improved HPLC, as well as CE and CEC, for the analysis of diarrhetic shellfish poisoning toxins, amnesic shellfish poisoning (ASP) toxins and microcystins (MCs) present in different matrices such as water, shellfish or algae. Improvements in sample preparation for increasing sensitivity and selectivity are also shown. While UV and fluorimetric detection are the detection methods generally used, mass spectrometric detection was also applied for ASP toxins and MCs, especially for confirmatory purposes. From the results obtained it can be concluded that both HPLC and CE offer a good potential for a sensitive and selective determination of these algal toxins in such complex matrices. The results obtained for CEC allow also to conclude that this technique can result in a promising technique for such application.

This paper shows the results of an intralaboratory validation of a fast method for the determination of lipophilic shellfish toxins working under acidic conditions using ultra-high performance LC (UHPLC) with MS/MS. Fourteen lipophilic marine toxins and domoic acid were acquired with fast polarity switching.Whereas azaspiracids (AZAs), pecenotoxins, 13-desmethyl spirolide C (SPX1), and gymnodimine were analyzed in the positive mode, yessotoxins (YTXs) were measured in negative mode. The okadaic acid (OA) group compounds were analyzed in both positive and negativeionization modes, and the accuracy of the results for both were compared. When using dynamic multiple reaction monitoring (MRM) in fast polarity switching, LODs were lower and reproducibility and linearity were better compared to static MRM. The UHPLC separation allowed for higher sample throughput in routineuse. Compared to the previously used HPLC/MS/MS method, LODs were improved up to a factor of 10 in mussel extract. Matrix effects were evaluated by comparing standards prepared in solvent with matrix-matchedcalibrations in blank mussel extract. For accurate quantification matrix-matched calibrations were used when analyzing reference mussel materials, providingrecoveries for OA, Dynophysis toxins (DTX)1, DTX2, YTX,AZA1, and SPX1 between 80 and 120% with RSDs below 8% over a 3-day validation procedure.

Mussel aquaculture is an important industry for the Galician Rias, located in northwestern Atlantic coast of Spain. Since 1976 this region has been seriously affected by incidents of paralytic and diarrhetic shellfish poisoning (PSP and DSP). A particularly bad episode occurred in 1993, when the toxic event lasted for an unusually long period. Many people were stricken ill with unusual symptoms. In this paper we report on the chemical analysis of toxic 1993 mussel samples, using the techniques of liquid chromatography and capillary electrophoresis coupled with mass spectrometry. These analyses revealed a very complex toxin profile, with both PSP and DSP toxins present. Two DSP toxins, okadaic acid and DTX2, were observed, while the primary PSP toxins were B1 and the decarbamoylated derivatives of saxitoxin, GTX2 and GTX3. Small amounts of saxitoxin and other as yet unidentified PSP toxins were observed.

Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse.

11 pages, [EN] The purpose of the study was to determine if paralytic shellfish poisoning (PSP) toxins are present in extracellular bacteria isolated from a toxic strain of the dinoflagellate Alexandrium minutum. A quantitative analysis was carried out of viable culturable bacteria attached to the surface of dinoflagellates and of bacteria present in dinoflagellate culture medium. A numerical taxonomy study was undertaken for presumptive identification of bacteria attached to the surface of dinoflagellates. Members of the following genera were detected on the cell surface of A. minutum: Cellulophaga, Marinomonas, Pseudoalteromonas, and Vibrio. The presence of intracellular PSP toxins in bacteria isolated from the cell surface of dinoflagellates was analysed by high-performance liquid chromatography with fluorescence detection (HPLC–FLD). Compounds that eluted at the same time as the standards of the PSP toxins GTX-2, GTX-3, GTX-4, dcGTX-2, and dcGTX-3 were present in some of the bacterial cell extracts. Natural fluorescent bacterial compounds, coeluting with some PSP toxins, were also detected. The results obtained showed that the fluorescent compounds, identified as putative PSP toxins by HPLC–FLD, did not correspond to any PSP analogue. This allowed us to reject the hypothesis that extracellular bacteria attached to the surface of dinoflagellates produce PSP toxins, [FR] Le but de cette étude était de déterminer si les toxines PSP (« paralytic shellfish poisoning ») étaient présentes chez les bactéries extracellulaires isolées d’une souche toxique du dinoflagellé Alexandrium minutum. Une analyse quantitative a été réalisée chez les bactéries viables cultivables attachées à la surface des dinoflagellés et les bactéries présentes dans le milieu de culture des dinoflagellés. Une numération taxonomique a été réalisée pour identifier par présomption les bactéries attachées à la surface des dinoflagellés. Les membres des genres suivants ont été détectés à la surface cellulaire de A. minutum : Cellulophaga, Marinomonas, Pseudoalteromonas et Vibrio. La présence de toxines PSP chez les bactéries isolées de la surface des dinoflagellés a été analysée par chromatographie liquide haute performance couplée à une détection en fluorescence. Des composés qui étaient élués en même temps que les toxines PSP GTX-2, GTX-3, GTX-4, dcGTX-2 et dcGTX-3 étaient présents dans quelques extraits cellulaires bactériens. Des composés bactériens naturellement fluorescents co-élués avec certaines toxines PSP ont aussi été détectés. Les résultats obtenus ont montré que les composés fluorescents, identifiés comme toxines PSP présumées, ne correspondaient à aucun analogue de PSP. Ceci nous a permis de rejeter l’hypothèse que les bactéries extracellulaires attachées à la surface des dinoflagellés produisent des toxines PSP.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na + channel and for this reason, it has been widely used in the study of Na + channel. The aim of this study was to analyze STX distribution in different rat brain regions after its acute intraperitoneal (i.p.) administration. Male rats (150–200 g) were injected i.p. with STX (5 and 10 μg/kg of body weight). After three time intervals of 30, 60, and 120 min (for 5 μg/kg STX dose) and 30 min (for 10 μg/kg STX dose) animals were sacrificed by cervical dislocation. Brains were removed and dissected in seven regions. STX concentration was measured using a precolumn oxidation high-performance liquid chromatographic method with fluorescence detection (HPLC/FLD). STX was found in all the regions evaluated at ppm levels meaning that STX peripherical administered across the blood–brain barrier and is distributed along the whole brain.

Further studies on mussel samples from Galicia, Spain, have revealed the presence of okadaic acid (OA), dinophysistoxin 2 (DTX2), and the fatty acid acyl esters of both of these toxins as the “DTX3” complex. Measurements were performed with an improved in situ method for the formation of 9-anthryldiazomethane (ADAM) derivatives followed by liquid chromatography with fluorescence detection. Base hydrolysis of DTX3 toxins gave free OA and DTX2, which were determined following ADAM derivatization. Results were confirmed by liquid chromatography/mass spectrometry analyses, and in most of the samples, free DTX2 was the most abundant toxin. However, the OA/DTX2 ratio in the DTX3 conjugated form was different, with OA being the most abundant in all cases. This difference could be due to different rates of metabolism of OA and DTX2 to the acyl esters or due to contamination of the shellfish by the two toxins at different points in time, resulting in less acyl ester formation for one toxin versus the other. The second possibility would be reasonable if two different source organisms were producing the toxins.

Paralysing and diarrhetic shellfish poisonings (PSP and DSP) are important intoxications caused by the consumption of shellfish, mainly bivalve molluscs, contaminated by certain species of toxic dinoflagellates present in the marine phytoplankton. Their appearance and massive reproduction take place in some periods of the year, causing the phenomenon commonly known as ‘red tide’. This causes significant problems to health and to the economy of the Galician region. The AOAC mouse bioassay is the most commonly used method of analysis for these toxic compounds, being the official method in most countries. Owing to the lack of sensitivity and selectivity of the biological assay, HPLC methods were developed as an alternative methodology. In this paper work carried out on the improvement of the chromatographic conditions in order to achieve accurate information about the PSP and DSP compounds present in the studied samples is reported.

Harmful algal blooms (HAB) have been increasing in frequency and intensity most likely due to changes on global conditions, which constitute a significant threat to wild shellfish and its commercial farming. This study evaluated the impact of increasing seawater temperature and acidification on the accumulation/elimination dynamics of HAB-toxins in shellfish. Mytilus galloprovincialis were acclimated to fourenvironmental conditions simulating different climate change scenarios: i) current conditions, ii) warming, iii) acidification and iv) interaction of warming with acidification. Once acclimated, mussels were exposed to the paralytic shellfish toxins(PSTs) producing dinoflagellate Gymnodinium catenatum for 5 days and to non-toxic diet during the subsequent 10 days. High toxicity levels (1493 μg STX eq kg-1) exceeding the safety limits were determined under current conditions at the end of the uptake period. Significantly lower PSP toxicity levels were registered for warming- and acidification-acclimated mussels (661 and 761 μg STX eq kg-1). The combined effect ofboth warming and acidification resulted in PSP toxicity values slightly higher (856 μg 38 STX eq kg-1). A rapid decrease of toxicity was observed in mussels at the current conditions after shifting to a non-toxic diet, which was not noticed under the predicted climate change scenarios. Variability of each PST analogue, measured throughout the experiment, highlighted different mechanisms are associated with changes of eachenvironmental factor, although both resulting in lower toxicity. Warming-acclimated mussels showed lower accumulation/elimination rates, while acidification-acclimated mussels showed higher capability to accumulate toxins, but also a higher elimination rate preventing high toxicity levels. As different mechanisms are triggered by warmingand acidification, their combined effect not leads to a synergism of their individualeffects. The present work is the first assessing the combined effect of climate change drivers on accumulation/elimination of PSTs, in mussels, indicating that warming and acidification may lead to lower toxicity values but longer toxic episodes. PSTs areresponsible for the food poisoning syndrome, paralytic shellfish poisoning (PSP) inhumans. This study can be considered as the first step to build models for predictingshellfish toxicity under climate change scenarios.