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Ciguatoxins (CTXs) are marine neurotoxins that cause ciguatera poisoning (CP), mainly through the consumption of fish. The distribution of CTXs in fish is known to be unequal. Studies have shown that viscera accumulate more toxins than muscle, but little has been conducted on toxicity distribution in the flesh, which is the main edible part of fish, and the caudal muscle is also most commonly targeted for the monitoring of CTXs in the Canary Islands. At present, whether this sample is representative of the toxicity of an individual is undisclosed. This study aims to assess the distribution of CTXs in fish, considering different muscle samples, the liver, and gonads. To this end, tissues from four amberjacks (Seriola spp.) and four dusky groupers (Epinephelus marginatus), over 16.5 kg and captured in the Canary Islands, were analyzed by neuroblastoma-2a cell-based assay. Flesh samples were collected from the extraocular region (EM), head (HM), and different areas from the fillet (A-D). In the amberjack, the EM was the most toxic muscle (1.510 CTX1B Eq·g−1), followed by far for the caudal section of the fillet (D) (0.906 CTX1B Eq·g−1). In the dusky grouper flesh samples, D and EM showed the highest toxicity (0.279 and 0.273 CTX1B Eq·g−1). In both species, HM was one of the least toxic samples (0.421 and 0.166 CTX1B Eq·g−1). The liver stood out for its high CTX concentration (3.643 and 2.718 CTX1B Eq·g−1), as were the gonads (1.620 and 0.992 CTX1B Eq·g−1). According to these results, the caudal muscle next to the tail is a reliable part for use in determining the toxicity of fish flesh to guarantee its safe consumption. Additionally, the analysis of the liver and gonads could provide further information on doubtful specimens, and be used for CTX monitoring in areas with an unknown prevalence of ciguatera. info:eu-repo/semantics/publishedVersion

Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scale Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The research leading to these results has received funding from the following FEDER-co-funded grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01, ED431C 2021/29, and the Centro singular de investigación de Galicia accreditation 2019–2022 ED431G 2019/03). From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX, and the EUROCIGUA project: “Risk Characterization of Ciguatera Fish Poisoning in Europe” GP/EFSA/AFSCO/2015/03, co-funded by the European Food Safety Authority (EFSA). From Ministerio de Ciencia e Innovación PID2020-115010RB-I00/AEI/10.13039/501100011033. David Castro (D.C.) financial support for the PhD studies was obtained through EUROCIGUA project: Risk characterization of Ciguatera Fish Poisoning in Europe, framework partnership agreement GP/EFSA/AFSCO/2015/03, co-funded by the EFSA. Pablo Estevez (P.E.) acknowledges financial support from the Xunta de Galicia (Regional Government, Spain) under grant ED481A-2018/207 SI

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

The primary objective of this specific grant was to characterize the risk associated to Ciguatera Poisoning (CP) in the EU by developing a sensitive methodology of LC-MS/MS. The preparation of reference materials including the main ciguatoxins (CTXs) responsible for the contamination was considered the secondary objective to facilitate the implementation of the LC-MS/MS methods in the EU laboratories to characterize this emerging risk. C-CTX1 has been identified by LC-MS/MS, and further confirmed by LC-HRMS, as the main responsible for the CTX toxicity in the samples from the areas selected for this study. The low concentrations levels of CTXs found on the samples evaluated has been a key limitation for the completion of the objectives of this specific grant (SG), being necessary to establish contingency plans, not only to overcome the problems of sensitivity that might compromise the confirmation of the toxic profiles, but also to accomplish the task of preparing reference materials. The contingency plans involved the development of two complementary LC-MS/MS approaches, as well as a methodological approach combiningLC-MS/MS, Neuroblastoma cell assay and chromatographic fractionations (HPLC and GPC) to characterize the toxins present in the contaminated extracts. This approach has been also used for the preparation of reference materials in order to confirm the presence of the C-CTX1 in both pure solutions of C-CTX1 and Fish Tissue Reference materials (FTRM) containing C-CTX1. The LC-MS/MS analysis of Dinoflagellates samples (Gambierdiscus and Fukuyoa sp) allowed to confirm the lack of correlation between the CTXs contamination and the fish samples from the areas were these dinoflagellates were collected. In these samples, the toxicity was attributed to several Maitotoxins analogues as well as gambieric acids C and D, Gambierone, 44-methyl gambierone, and gambieroxide that were identified by LC-HRMS with varying degrees of confirmation in strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and North East Atlantic Ocean.

Tetrodotoxin (TTX) is a potent neurotoxin, considered an emerging toxin in Europe where recently a safety limit of 44 µg TTX kg−1 was recommended by authorities. In this study, three specimens of the large gastropod trumpet shell Charonia lampas bought in a market in south Portugal were analyzed using a neuroblastoma cell (N2a) based assay and by LC-MS/MS. N2a toxicity was observed in the viscera of two individuals analyzed and LC-MS/MS showed very high concentrations of TTX (42.1 mg kg−1) and 4,9-anhydroTTX (56.3 mg kg−1). A third compound with m/z 318 and structurally related with TTX was observed. In the edible portion, i.e., the muscle, toxin levels were below the EFSA recommended limit. This study shows that trumpet shell marine snails are seafood species that may reach the markets containing low TTX levels in the edible portion but containing very high levels of TTX in non-edible portion raising concerns regarding food safety if a proper evisceration is not carried out by consumers. These results highlight the need for better understanding TTX variability in this gastropod species, which is critical to developing a proper legal framework for resources management ensuring seafood safety, and the introduction of these gastropods in the markets. Fundação para a Ciência e a Tecnologia | Ref. (UID/Multi/04326/2020)

25 pages, 1 table, 5 figures, Cobalamin (B12) is an essential cofactor that is exclusively synthesized by some prokaryotes while many prokaryotes and eukaryotes require an external supply of B12. The spatial and temporal availability of B12 is poorly understood in marine ecosystems. Field measurements of B12 along with a large set of ancillary biotic and abiotic factors were obtained during three oceanographic cruises in the NW Iberian Peninsula, covering different spatial and temporal scales. B12 concentrations were remarkably low (, The scintillation counter was supported by the 2006‐06 ICTS project of the Ministry of Science and Innovation at the Spanish Institute of Oceanography. This research was supported by the Spanish Ministry of Economy and Competitiveness through the ENVISION (CTM2014‐59031‐P) and INTERES (CTM2017‐83362‐R) projects. Further support was provided by the Swedish Research Council VR. Vanessa Joglar was supported by an FPI fellowship from the Spanish Ministry of Economy and Competitiveness

15 pages, 7 tables, 3 figures, Emerging risk identification is a priority for the European Food Safety Authority (EFSA). The goal of the Galician Emerging Food Safety Risks Network (RISEGAL) is the identification of emerging risks in foods produced and commercialized in Galicia (northwest Spain) in order to propose prevention plans and mitigation strategies. In this work, RISEGAL applied a systematic approach for the identification of emerging food safety risks potentially affecting bivalve shellfish. First, a comprehensive review of scientific databases was carried out to identify hazards most quoted as emerging in bivalves in the period 2016–2018. Then, identified hazards were semiquantitatively assessed by a panel of food safety experts, who scored them accordingly with the five evaluation criteria proposed by EFSA: novelty, soundness, imminence, scale, and severity. Scores determined that perfluorinated compounds, antimicrobial resistance, Vibrio parahaemolyticus, hepatitis E virus (HEV), and antimicrobial residues are the emerging hazards that are considered most imminent and severe and that could cause safety problems of the highest scale in the bivalve value chain by the majority of the experts consulted (75%). Finally, in a preliminary way, an exploratory study carried out in the Galician Rías highlighted the presence of HEV in mussels cultivated in class B production areas., This research was funded by Xunta de Galicia, grant number IN 607C 2017/04.

Simple Summary Some marine microalgae usually present in warm waters can produce ciguatoxins (CTXs); these toxins can accumulate in fish through the trophic chain, causing the food poisoning known as ciguatera in humans. It is important to understand how these compounds could be incorporated into fish muscle. For this purpose, this study was conducted using goldfish, an omnivorous freshwater species, daily fed raw fish flesh contaminated with a known toxicity concentration of CTX, seeking the accumulation profile in muscle and any signs of intoxication. Toxicity was detectable from day eight of the toxic diet and reached its maximum after two weeks. Signs of poisoning were observed after two weeks in all treated fish. However, two individuals developed strong symptoms, and one of them was separated and fed non-toxic food for 60 days; it showed recovery signs after the first week, and no toxicity was observed at the end of that non-toxic period. These results demonstrate that this toxin can accumulate in the muscle tissue of goldfish and produce associated symptomatology. Moreover, goldfish can recover and eliminate the CTX from its muscle if the toxin source is not available. Abstract Ciguatoxins (CTXs) are produced by dinoflagellates usually present in tropical and subtropical waters. These toxins are bioaccumulated and transformed in fish causing ciguatera fish poisoning (CFP) in humans. Few trials have been performed to understand how CTXs are incorporated into fish. This study developed an experimental model of goldfish (Carassius auratus) fed flesh contaminated with Caribbean ciguatoxin (C-CTX1). Fourteen goldfish were fed 0.014 ng CTX1B (Eq. g−1 of body weight) daily, and control goldfish received non-toxic flesh. CTX presence was determined by a cell-based assay on days 1, 8, 15, 29, 36, 43, and 84. Toxicity was detected in muscle from the second sampling and then seemed to stabilize at ~0.03 ng CTX1B Eq. g−1. After two weeks, all experimental goldfish developed lethargy and loss of brightness, but only two of them displayed erratic swimming and jerking movements near the sixth sampling. One of these fish had its toxic diet replaced by commercial food for 60 more days; the fish showed recovery signs within the first weeks and no CTX activity was detected. These results indicate that C-CTX1 could accumulate in goldfish muscle tissue and produce toxic symptoms, but also remarked on the detoxification and recovery capacity of this species.

This study aims to assess and compare the kinetics (accumulation/elimination) of the marine biotoxins okadaic acid (OA) and dinophysistoxin-1 (DTX1 ), between native (Ruditapes decussates) and invasive (Ruditapes philippinarum) clam species, and their genotoxic effects and DNA recover capacity after, exposure to toxic dinoflagellates Promcentrum lima. Clams were fed with P. lima for 5 days and then to non-toxic algae (post-exposure) during other 5 days. Toxin concentrations determined in clams by LC-MS/MS were related with DNA damage and repair assessment through the comet and base excision repair (BER) assays, respectively. Differential accumulation patterns were observed between the invasive and native species. The invasive species consistently and progressively accumulated the toxins during the first 24 h of exposure, while the native clams showed drastic variations in the toxin accumulation. Nevertheless, at the end of a 5 days of exposure period, the native dams presented higher toxin concentrations, nearly reaching the legal regulatory limit for human consumption. In addition, native clams were vastly affected by OA and DTX1, presenting an increment in the DNA damage since the first day, with a correspondent increase in the repair activity. On the other hand, invasive clams were not affected by the dinoflagellate toxins, exhibiting only some signs of the challenge, namely an increase in the DNA repair mechanisms in the post-exposure period. Invasive clams R. philippinanim are better adapted to cope with harmful algal blooms and OA-group toxins than native species. These results may increase farming interest and may lead to new introductions of the invasive clams. In sympatry sites, exposure to OA-group toxins may unbalance dams species biomass and distribution as exposure to toxic dinoflagellates affects the native clams from cellular to a population level, representing a significant threat to development and maintenance of R. decussates populations. (C) 2021 Elsevier B.V. All rights reserved. CESAM [UID/AMB/50017 - POCI-01-0145-FEDER-007638]; FCT/MCTES through national funds (PIDDAC); FEDER within the PT2020 Partnership Agreement and Compete 2020; Portuguese Foundation for Science and Technology (FCT).

Ciguatoxins (CTXs) are a group of neurotoxins responsible for the syndrome ciguatera fish poisoning (CFP) as a result of the consumption of contaminated fish. The presence of these toxins has been detected around the Pacific, Caribbean and Indian coasts. Recent reports indicate the emergence of CFP in other geographic areas, in particular in European coasts, of the Canary Islands (Spain) and Madeira (Portugal). A neuroblastoma cell line of murine origin (N2a) has been applied to assay different groups of neurotoxins, acting on voltage-gated sodium channel (VGSC) of excitable cells, N2a-MTT. The great potential of N2a-MTT as a sensitive tool for the CTXs screening is clearly recognized, notably because it allows the detection of these toxins at levels below recommended as security levels. However, the complexity of the matrix is a critical point on the application of N2a-MTT, which needs to be evaluated. The aim of this work is to provide recommendations for an implemented N2a-MTT method for CTXs determination in fish that avoids matrix effects, particularly those related to high lipid content. European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03

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

The biodiversity of lactic acid bacteria in musts and wines of Albari&ntilde, o variety has been studied. The identification of species was addressed through a combination of biochemical and genetic methods (API&reg, 50 CHL test, 16S rDNA and recA gene sequences, Amplified Ribosomal DNA Restriction Analysis -ARDRA- and 16S-26S intergenic region analysis). The results grouped the isolates into six species predominating those of the genus Lactobacillus and showing a typical biogeographical distribution. Among sixteen strains evaluated, eight of them showed malolactic activity. The study of the presence of genes hdc, odc, and tdc, along with the LC/MS-MS analysis of biogenic amines in wine, showed five strains lacking aminogenic ability. The absence of the pad gene in the above-mentioned strains discards its ability to produce volatile phenols that may adversely affect the aroma. Finally, all malolactic strains showed &beta, glucosidase activity so that they could contribute to enhance and differentiate the aromatic profile of Albari&ntilde, o wines.

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

Liquid chromatography (LC) coupled to mass spectrometry (MS) is being widely applied as an analytical tool in the field of marine biotoxins both for regulated and for new and emerging compounds. LC-MS/MS recently became the reference method for the control of lipophilic toxins in the European Union, and new methods are being developed and optimized to extend the applicability of this technique to other toxin groups. In this work, conditions for the analysis of ciguatoxins (CTXs) by LC-MS/MS were investigated using standard solutions of CTX1B and CTX3C, which are structurally representative compounds for the rest of the main congeners of Pacific group toxins (P-CTXs). Preliminary studies were carried out for the selection of precursor and product ions used for multiple reaction monitoring. Two transitions based on the chemical structures of CTXs were set up, and mass spectrometer parameters were adjusted for selected reactions monitored. The electrospray ionization source has been carefully optimized through a design of experiments that consisted of a two-level fractional factorial design of resolution IV for the screening of adequate source conditions and of response surface designs for optimization of the main interactions between factors. The statistical approach allowed maximizing the sensitivity on the MS analyzer that provides a good specificity in P-CTX detection, which can be also used for confirmation purposes.

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 presence of emerging contaminants in food and the sources of the contamination are relevant issues in food safety. The impact of climate change on these contaminations is a topic widely debated; however, the consequences of climate change for the food system is not as deeply studied as other human and animal health and welfare issues. Projections of climate change in Europe have been evaluated through the EU Commission, and the impact on the marine environment is considered a priority issue. Marine biotoxins are produced by toxic microalgae and are natural contaminants of the marine environment. They are considered to be an important contaminant that needs to be evaluated. Their source is affected by oceanographic and environmental conditions; water temperature, sunlight, salinity, competing microorganisms, nutrients, and wind and current directions affect the growth and proliferation of microalgae. Although climate change should not be the only reason for this increase and other factors such as eutrophication, tourism, fishery activities, etc. could be considered, the influence of climate change has been observed through increased growth of dinoflagellates in areas where they have not been previously detected. An example of this is the recent emergence of ciguatera fish poisoning toxins, typically found in tropical or subtropical areas from the Pacific and Caribbean and in certain areas of the Atlantic Sea such as the Canary Islands (Spain) and Madeira (Portugal). In addition, the recent findings of the presence of tetrodotoxins, typically found in certain areas of the Pacific, are emerging in the EU and contaminating not only the fish species where these toxins had been found before but also bivalve mollusks. The emergence of these marine biotoxins in the EU is a reason for concern in the EU, and for this reason, the risk evaluation and characterization of these toxins are considered a priority for the European Food Safety Authorities (EFSA), which also emphasize the search for occurrence data using reliable and efficient analytical methods.

Ciguatera Fish Poisoning is a worldwide concern caused by the consumption of fish contaminated with ciguatoxins not only in endemic regions in the Pacific Ocean or the Caribbean Sea but also in emerging areas of Macaronesia on the eastern Atlantic. The recent emergence of these toxins in other coastal areas worldwide, prompted the need for the characterization of the risk in these areas. This Ciguatera Fish Poisoning risk has been recently identified as a potential threat in subtropical areas of the Atlantic coast and scientific efforts are being focused in the identification and confirmation of the toxins involved in this potential risk. Neuroblastoma cell assay has been widely used for the evaluation of the toxicity in several marine biotoxin groups, and found to be a very useful tool for toxicity screening. LC-MS/MS has been also used for confirmatory purposes although the main limitation of the advances on LC-MS/MS development is due to commercial unavailability of reference materials and hampers method implementation and validation or even confirmation of the ciguatoxins (CTXs) responsible for the toxic profiles. While neuroblastoma cell assay (N2a) is typically used for toxicity screening as mentioned above, being necessary to confirm this N2a toxicity by LC-MS/MS, this study is designed using N2a as a tool to confirm the toxicity of the fractions obtained corresponding to potential CTXs analogues according to the analysis by LC-MS/MS. With this aim, an amberjack sample (Seriola fasciata) from Selvagen Islads (Portugal) and implicated in Ciguatera Fish Poisoning was analyzed by LC-MS/MS and Caribbean Ciguatoxins were found to be mainly responsible for the toxicity. N2a was used in this work as a tool to help in the confirmation of the toxicity of fractions obtained by HPLC. Caribbean Ciguatoxin-1 was found as the main analogue responsible for the N2a toxicity while three Caribbean Ciguatoxin-1 (C-CTX1) metabolites which contribute to the total toxicity were also identified. info:eu-repo/semantics/publishedVersion

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 [...]

Cyclic imines (CIs) are being considered as emerging toxins in the European Union, and a scientific opinion has been published by the European Food Safety Authority (EFSA) in which an assessment of the risks to human health related to their consumption has been carried out. Recommendations on the EFSA opinion include the search for data occurrence of CIs in shellfish and using confirmatory methods by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which need to be developed and optimized. The aim of this work is the application of LC-MS/MS to the analysis of gymnodimines (GYMs), spirolides (SPXs), pinnatoxins (PnTXs), and pteriatoxins (PtTXs) in mussels from Galician Rias, northwest Spain, the main production area in Europe, and therefore a representative emplacement for their evaluation. Conditions were adjusted using commercially available certified reference standards of GYM-A, SPX-1, and PnTX-G and evaluated through quality control studies. The EU-Harmonised Standard Operating Procedure for determination of lipophilic marine biotoxins in molluscs by LC-MS/MS was followed, and the results obtained from the analysis of eighteen samples from three different locations that showed the presence of PnTXs and SPXs are presented and discussed. Concentrations of PnTX-G and SPX-1 ranged from 1.8 to 3.1 µg/kg and 1.2 to 6.9 µg/kg, respectively, and PnTX-A was detected in the group of samples with higher levels of PnTX-G after a solid phase extraction (SPE) step used for the concentration of extracts. Ministerio de Economía y Competitividades | Ref. AGL2014-52403-R

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.

The increased emergence of Ciguatera Fish Poisoning in the Canary Islands and Madeira demanded the development of confirmatory methods by liquid chromatography coupled to tandem mass spectrometry to characterize the risk. Efforts were particularly focused on the optimization of sample pretreatment, especially in the sample cleanup step, to efficiently remove matrix interferences as a critical factor to consider in mass spectrometry detection. Two different LC-MS/MS approaches have been used for confirmation purposes, the first one using the sodium adduct as precursor and product ion to allow an increased sensitivity in the detection, whereas additional fragments were also monitored for further confirmation. The optimized conditions above mentioned allowed the confirmation of Caribbean Ciguatoxin-1 as the main responsible for the samples analyzed from these geographical areas, while the presence of a new hydroxyl metabolite of C-CTX1 was also confirmed in one sample analyzed in this study.

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

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.

Conditions for the determination of lipophilic marine toxins, such as yessotoxins and pectenotoxins (PTX)-6, were investigated with capillary electrophoresis coupled to mass spectrometry (MS) with an electrospray ionization source. After optimization, a simple and MS compatible alkaline volatile buffer solution of ammonium acetate was selected as background electrolyte, with isopropanol/water (80/20, v/v) sheath liquid modified with ammonium acetate used at the electrospray ionization (ESI) source. Previously to capillary electrophoresis (CE) separations, the application of an on-line sample pre-concentration approach based on field-amplified sample stacking was accomplished to increase sensitivity. As a result, the limits of detection provided by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) were 0.02 microg ml(-1) (0.01 microg g(-1)), which corresponded to 1.25 pg for yessotoxin and 0.25 microg ml(-1) (0.13 microg g(-1) and 13.25 pg on capillary) for PTX-6. Accuracy tests showed 97.7% recovery from spiked blank mussel samples that showed no significant matrix influence running under optimal conditions. Intermediate precision was close to 4% relative standard deviation (RSD) for the migration time, and an RSD of 7.5% for peak areas. The method was successfully applied to naturally contaminated seafood samples in which yessotoxins and pectenotoxins-6 were clearly determined. This work demonstrated the potential of CE-ESI-MS to be applied for a sensitive determination of lipophilic toxins from the marine environment as alternative to liquid chromatography-electrospray ionization-single quadrupole mass spectrometry (LC-ESI-MS) for this purpose.

Solid phase microextraction coupled with high performance liquid chromatography with fluorescence detection has been optimized and evaluated for a simple, rapid, and selective analysis of anatoxin-a. Four kinds of fiber (100 microm polydimethylsiloxane, 60 microm polydimethylsiloxane/divinylbenzene, 50 microm Carbowax/templated resin-100, and 85 microm polyacrylate) were evaluated for an efficient extraction of the toxin. Parameters relating to the desorption step, such as desorption mode, solvent composition, time for both static and dynamic desorption, as well as carryover, have been studied and optimized. The derivatization process was investigated using NBD-F as derivatizing reagent. Anatoxin-a derivative was formed when the anatoxin-a-loaded fiber was inserted in a vial containing 5 microL of NBD-F. Variables affecting extraction such us ionic strength, temperature, and time have been also optimized. The results obtained showed linearity in the range of 10-2000 ng and a limit of detection of 0.29 ng/mL in river water. The presented method has been applied to different environmental samples.

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.

In this work, solid-phase microextraction (SPME) has been applied as an alternative for the selective extraction of 3 polybrominated diphenyl ethers (PBDEs), 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47); 2,2′,4,4′,5-pentabromodiphenyl ether (PBDE-99); and 2,2′,4,4′,6-pentabromodiphenyl ether (PBDE-100), and 2 alkylphenols, 4-tert-OP and 4-NP, prior to their analysis by gas chromatography/mass spectrometry (GC/MS). The advantages of this technique are mainly its simplicity, cost-effectiveness, and time-saving sample preparation, as well as being a solvent-free technique. With the aim of optimizing the conditions for an efficient extraction of the studied compounds, different fiber coatings and the main parameters affecting the extraction procedure have been evaluated. The results obtained showed a good linearity in the range of concentrations investigated, and adequate relative standard deviation values were found according to the range accepted for SPME. Recovery values were in the range of 78–108%, and good detection and quantitation limits at ppt levels were obtained for both methods, allowing the determination of the selected compounds in samples at trace levels. The results obtained clearly show the potential of SPME for efficient concentration of the target compounds and also demonstrate the reliability of this extraction technique for their GC/MS analysis.

In this work capillary electrophoresis (CE) with UV detection has been applied to the analysis of different natural toxins produced in the aquatic environment. This technique is presented as an alternative to other chemical techniques such as HPLC, and the optimisation of analytical methodologies was carried out for diverse marine toxins including Paralytic and Amnesic and some polyether toxins, such as Yessotoxins, as well as for certain microcystin toxins produced by cyanobacteria present in freshwaters. Sample preparation steps were optimised and adequate electrophoretic conditions developed for achieving a complete separation of compounds with similar structures involved in such contamination. The influence of the biological matrices where they are involved has also been studied and the potential use of CE-UV as a tool for monitoring these aquatic toxins is also discussed.