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The first ciguatera fish poisoning (CFP) in Portugal dates from 2008 when 11 people reported CFP symptoms after consuming a 30 kg amberjack caught around the Selvagens Islands (Madeira Archipelago). Since then, 49 human poisonings have been reported. The emergence of a new threat challenged scientists and regulators, as methods for toxic microalgae analyses and ciguatoxin (CTX) detection were not implemented. To minimise the risk of ciguatera, the Madeira Archipelago authorities interdicted fisheries in Selvagens Islands and banned the capture of amberjacks weighing more than 10 kg in the entire region of Madeira Archipelago. The accurate identification and quantification of the benthic toxin-producing algae species spreading to new areas require efforts in terms of both microscopy and molecular techniques. Two ciguatera-causing dinoflagellates, Gambierdiscus excentricus and Gambierdiscus australes, were identified in the Madeira Island and Selvagens sub-archipelago, respectively. Regarding the CTX analysis (N2a cell-based assay and LC-MS) in fish, the results indicate that the Selvagens Islands are a ciguatera risk area and that fish vectoring CTX are not limited to top predator species. Nevertheless, advances and improvements in screening methods for the fast detection of toxicity in seafood along with certified reference material and sensitive and selective targeted analytical methods for the determination of CTX content are still pending. This study aims to revise the occurrence of ciguatera cases in the Madeira Archipelago since its first detection in 2008, to discuss the risk management strategy that was implemented, and to provide a summary of the available data on the bioaccumulation of CTX in marine fish throughout the marine food web, taking into consideration their ecological significance, ecosystem dynamics, and fisheries relevance.

Marine heatwaves (MHWs) have doubled in frequency since the 1980s and are projected to be exacerbated during this century. MHWs have been shown to trigger harmful algal blooms (HABs), with severe consequences to marine life and human populations. Within this context, this study aims to understand, for the first time, how MHWs impact key biological and toxicological parameters of the paralytic shellfish toxin (PST) producer Gymnodinium catenatum, a dinoflagellate inhabiting temperate and tropical coastal waters. Two MHW were simulated—category I (i.e., peak: 19.9 °C) and category IV (i.e., peak: 24.1 °C)—relative to the estimated baseline in the western coast of Portugal (18.5 °C). No significant changes in abundance, size, and photosynthetic efficiency were observed among treatments. On the other hand, chain-formation was significantly reduced under category IV MHW, as was PSP toxicity and production of some PST compounds. Overall, this suggests that G. catenatum may have a high tolerance to MHWs. Nevertheless, some sublethal effects may have occurred since chain-formation was affected, suggesting that these growth conditions may be sub-optimal for this population. Our study suggests that the increase in frequency, intensity, and duration of MHWs may lead to reduced severity of G. catenatum blooms.

Bivalves are a high-value product whose production has markedly increased, reaching 9863 tonnes in Portugal in 2021. Bivalves’ habitats—lagoons, estuaries and coastal waters—are exposed to biological and anthropogenic contaminants, which can bioaccumulate in these organisms and pose a significant public health risk. The need to obtain a safe product for human consumption led to the implementation of standardised hygiene regulations for harvesting and marketing bivalve molluscs, resulting in routine monitoring of bivalve production areas for microbial quality, metal contaminants, and marine biotoxins. While excessive levels of biotoxins and metal contamination lead to temporary harvesting bans, high faecal contamination leads to area reclassification and impose post-harvest treatments. In this study, the seasonal and temporal variability of these parameters were analysed using historical data generated by the monitoring programme during the last decade. Moreover, the impact of the monitoring program on bivalve harvesting from 2011 to 2020 was assessed. This program presented a considerable improvement over time, with an increase in the sampling effort and the overall program representativeness. Finally, contamination risk, revising control measures, and defining recommendations for risk mitigation measures are given in the light of ten years’ monitoring.

Harmful algal blooms (HABs) are considered one of the main risks for marine ecosystems and human health worldwide. Climate change is projected to induce significant changes in species geographic distribution, and, in this sense, it is paramount to accurately predict how it will affect toxin-producing microalgae. In this context, the present study was intended to project the potential biogeographical changes in habitat suitability and occurrence distribution of three key amnesic shellfish toxin (AST)—producing diatom species (i.e., Pseudo-nitzschia australis, P. seriata, and P. fraudulenta) under four different climate change scenarios (i.e., RCP-2.6, 4.5, 6.0, and 8.5) up to 2050 and 2100. For this purpose, we applied species distribution models (SDMs) using four abiotic predictors (i.e., sea surface temperature, salinity, current velocity, and bathymetry) in a MaxEnt framework. Overall, considerable contraction and potential extirpation were projected for all species at lower latitudes together with projected poleward expansions into higher latitudes, mainly in the northern hemisphere. The present study aims to contribute to the knowledge on the impacts of climate change on the biogeography of toxin-producing microalgae species while at the same time advising the correct environmental management of coastal habitats and ecosystems.

Saxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels’ whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs’ profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicity.

Toxin-producing microalgae present a significant environmental risk for ecosystems and human societies when they reach concentrations that affect other aquatic organisms or human health. Harmful algal blooms (HAB) have been linked to mass wildlife die-offs and human food poisoning episodes, and climate change has the potential to alter the frequency, magnitude, and geographical extent of such events. Thus, a framework of species distribution models (SDMs), employing MaxEnt modeling, was used to project changes in habitat suitability and distribution of three key paralytic shellfish toxin (PST)-producing dinoflagellate species (i.e., Alexandrium catenella, A. minutum, and Gymnodinium catenatum), up to 2050 and 2100, across four representative concentration pathway scenarios (RCP-2.6, 4.5, 6.0, and 8.5; CMIP5). Despite slightly different responses at the regional level, the global habitat suitability has decreased for all the species, leading to an overall contraction in their tropical and sub-tropical ranges, while considerable expansions are projected in higher latitudes, particularly in the Northern Hemisphere, suggesting poleward distributional shifts. Such trends were exacerbated with increasing RCP severity. Yet, further research is required, with a greater assemblage of environmental predictors and improved occurrence datasets, to gain a more holistic understanding of the potential impacts of climate change on PST-producing species.

Tetrodotoxin (TTX) is a potent neurotoxin naturally occurring in terrestrial and marine organisms such as pufferfish. Due to the risk of TTX poisoning, fish of Tetraodontidae family and other puffer-related species must not be placed in the EU markets. This restriction applies to fish of the family Molidae even though no data on toxins’ occurrence is available. In this study, the presence of TTX and its analogues was investigated in the main edible tissue (the white muscle) and the main xenobiotics storage organ (the liver) of ocean sunfish Mola spp. (n = 13) from the South Portuguese coast. HILIC-MS/MS analyses did not reveal TTX in the analyzed samples, suggesting an inexistent or very limited risk of TTX poisoning.

Diarrhetic Shellfish Poisoning (DSP) is an acute intoxication caused by the consumption of contaminated shellfish, which is common in many regions of the world. To safeguard human health, most countries implement programs focused on the surveillance of toxic phytoplankton abundance and shellfish toxicity levels, an effort that can be complemented by a deeper understanding of the underlying phenomena. In this work, we identify patterns of seasonality in shellfish toxicity across the Portuguese coast and analyse time-lagged correlations between this toxicity and various potential risk factors. We extend the understanding of these relations through the introduction of temporal lags, allowing the analysis of time series at different points in time and the study of the predictive power of the tested variables. This study confirms previous findings about toxicity seasonality patterns on the Portuguese coast and provides further quantitative data about the relations between shellfish toxicity and geographical location, shellfish species, toxic phytoplankton abundances, and environmental conditions. Furthermore, multiple pairs of areas and shellfish species are identified as having correlations high enough to allow for a predictive analysis. These results represent the first step towards understanding the dynamics of DSP toxicity in Portuguese shellfish producing areas, such as temporal and spatial variability, and towards the development of a shellfish safety forecasting system.

The objective of this work was to screen the EU-regulated lipophilic and cyclic imine toxins in four bivalve species (Atrina vexillum, Pinctada imbricata, Anadara antiquata, and Saccostrea Cucculata) from the Mozambican coast in the Indian Ocean. Toxins were extracted and analyzed according to the EU reference method for the determination of lipophilic toxins in shellfish via LC–MS/MS, but no regulated toxins were found in the analyzed species. However, pinnatoxins (PnTX G, E, and F) were detected in A. vexillum, P. imbricata, and A. antiquata. Higher levels of the PnTX G were determined for A. vexillum (7.7 and 14.3 µg·kg−1) than for P. imbricata (1.6 and 2.4 µg·kg−1), and for A. antiquata (4.5 and 5.9 µg·kg−1) with both hydrolyzed and non-hydrolyzed extracts, respectively. The higher levels of PnTX G determined in the hydrolyzed extracts indicate the high potential of this species to esterify pinnatoxins, in particular PnTX G.

The dinoflagellate Amyloodinium ocellatum is the etiological agent of a parasitic disease named amyloodiniosis. Mortalities of diseased fish are usually attributed to anoxia, osmoregulatory impairment, or opportunistic bacterial infections. Nevertheless, the phylogenetic proximity of A. ocellatum to a group of toxin-producing dinoflagellates from Pfiesteria, Parvodinium and Paulsenella genera suggests that it may produce toxin-like compounds, adding a new dimension to the possible cause of mortalities in A. ocellatum outbreaks. To address this question, extracts prepared from different life stages of the parasite were tested in vitro for cytotoxic effects using two cell lines derived from branchial arches (ABSa15) and the caudal fin (CFSa1) of the gilthead seabream (Sparus aurata), and for hemolytic effects using erythrocytes purified from the blood of gilthead seabream juveniles. Cytotoxicity and a strong hemolytic effect, similar to those observed for Karlodinium toxins, were observed for the less polar extracts of the parasitic stage (trophont). A similar trend was observed for the less polar extracts of the infective stage (dinospores), although cell viability was only affected in the ABSa15 line. These results suggest that A. ocellatum produces tissue-specific toxic compounds that may have a role in the attachment of the dinospores’ and trophonts’ feeding process.

Ciguatoxins (CTXs) are naturally occurring neurotoxins that can accumulate in fish and cause Ciguatera Poisoning (CP) in seafood consumers. Ciguatoxic fish have been detected in tropical and subtropical regions of the world including the Pacific and Indian Oceans, the Caribbean Sea, and more recently in the northeast Atlantic Ocean. The biogeographic distribution of ciguatoxic fish appears to be expanding; however, the paucity of CTX standards and reference materials limits the ability of public health authorities to monitor for these toxins in seafood supply chains. Recent reports establish that Caribbean Ciguatoxin-1 (C-CTX1) is the principal toxin responsible for CP cases and outbreaks in the northeast Atlantic Ocean and that C-CTX congener profiles in contaminated fish samples match those from the Caribbean Sea. Therefore, in this work, C-CTX reference materials were prepared from fish obtained from the northeast Atlantic Ocean. The collection of fish specimens (e.g., amberjack, grouper, or snapper) was screened for CTX-like toxicity using the in vitro sodium channel mouse neuroblastoma cytotoxicity assay (N2a cell assay). Muscle and liver tissues from toxic specimens were pooled for extraction and purified products were ultimately profiled and quantified by comparison with authentic C-CTX1 using LC-MS/MS. This work presents a detailed protocol for the preparation of purified C-CTX reference materials to enable continued research and monitoring of the ciguatera public health hazard. To carry out this work, C-CTX1 was isolated and purified from fish muscle and liver tissues obtained from the Canary Islands (Spain) and Madeira archipelago (Portugal).

The molecular pathways behind the toxicity of diarrheic shellfish toxins (DSTs) in bivalves have been scarcely studied. Thus, a shotgun proteomics approach was applied in this work to understand bivalves’ molecular responses to the dinoflagellate Prorocentrum lima (1.0 × 106 cells/L). Protein expression along with toxins levels were analyzed in the gills and digestive gland of the mussel Mytilus galloprovincialis during and after exposure to this toxic strain. Results revealed an accumulation of OA and DTX1 only in the digestive gland with maximum amounts attained at the end of uptake phase (day 5; 2819.2 ± 522.2 μg OA/kg and 1107.1 ± 267.9 μg DTX1/kg). At the end of the depuration phase (day 20), 16% and 47% of total OA and DTX1 concentrations remained in the digestive gland tissues, respectively. The shotgun proteomic analyses yielded 3051 proteins in both organs. A total of 56 and 54 differentially expressed proteins (DEPs) were revealed in the digestive gland and gills, respectively. Both organs presented the same response dynamics along the experiment, although with tissue-specific features. The early response (3 days uptake) was characterized by a high number of DEPs, being more marked in gills, in relation to the latter time points (5 days uptake and depuration). Functional enrichment analysis revealed the up-regulation of carboxylic (GO:0046943) and organic acid transmembrane transporter activity (GO:0005342) pathways after 3 days uptake for digestive gland. Matching to these pathways are a group of proteins related to transmembrane transport and response to toxic substances and xenobiotics, namely P-glycoprotein (ABCB11), Sodium-dependent proline transporter (SLC6A7), and Sideroflexin-1 (SFXN1). According to Clusters of Orthologous Groups (GOs) categories, most of the DEPs found for digestive gland in all time-points were related with “cellular processes and signaling” and involving signal transduction mechanisms, cytoskeleton and post-translational modification, protein turnover, chaperone functions. In gills, the early uptake phase was marked by a balance between DEPs related with “cellular processes and signaling” and “metabolism.” Depuration is clearly marked by processes related with “metabolism,” mainly involving secondary metabolites biosynthesis, transport, and catabolism. Proteomic data are available via ProteomeXchange with identifier PXD022293.

The neurotoxin β-N-methylamino-L-alanine (BMAA), a non-proteinogenic amino acid produced by several species of both prokaryotic (cyanobacteria) and eukaryotic (diatoms) microorganisms, has been proposed to be associated with the development of neurodegenerative diseases. At first, BMAA appeared to be ubiquitously present worldwide in various organisms, from aquatic and terrestrial food webs. However, recent studies, using detection methods based on mass spectrometry, instead of fluorescence detection, suggest that the trophic transfer of BMAA is debatable. This study evaluated BMAA in 22 cetaceans of three different species (Phocoena phocoena, n = 8, Delphinus delphis, n = 8, and Tursiops truncatus, n = 6), found stranded in North-West Spain. BMAA analysis of the liver, kidney, or muscle tissues via sensitive liquid chromatography with tandem mass spectrometry did not reveal the presence of this compound or its isomers. The absence recorded in this study highlights the need to better understand the trophic transfer of BMAA and its anatomical distribution in marine mammals.

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.

Bivalves constitute an important source of proteins for human consumption, but some accumulate biotoxins such as diarrhetic shellfish toxins (DSTs), constituting a risk to human health. The cockle Cerastoderma edule is one of the most important species harvested in the Portuguese coast but also one of the most affected species due to recurrent DSTs exposure. However, little is known regarding the effects of the toxins produced by blooming dinoflagellates on C. edule. Herein, we explore the Differentially Expressed Genes (DEGs) of two tissues (gills and digestive gland) from wild cockles sampled in Portugal, through their whole transcriptomic response in two different seasons (exposed and not exposed to DSTs). The de novo transcriptome assembly returned 684,723 contigs, N50 of 1049, and 98.53% completeness. Altogether, 1098 DEGs were identified, of which 353 DEGs were exclusive for the digestive gland, 536 unique for the gills and 209 DEGs were common. Among DEGs were identified known DSTs-biomarkers including glutathione peroxidase, glutathione S-transferase, superoxide dismutase, cytochrome P450, ABC transporters, actin and tubulin-related proteins, Heat shock proteins and complement C1Q-like proteins. This study provides the first transcriptomic profile of C. edule, giving new insights about its molecular responses under different environmental conditions of DSTs exposure.

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.

Harmful algal blooms (HABs) are among the most severe ecological marine problems worldwide. Under favorable climate and oceanographic conditions, toxin-producing microalgae species may proliferate, reach increasingly high cell concentrations in seawater, accumulate in shellfish, and threaten the health of seafood consumers. There is an urgent need for the development of effective tools to help shellfish farmers to cope and anticipate HAB events and shellfish contamination, which frequently leads to significant negative economic impacts. Statistical and machine learning forecasting tools have been developed in an attempt to better inform the shellfish industry to limit damages, improve mitigation measures and reduce production losses. This study presents a synoptic review covering the trends in machine learning methods for predicting HABs and shellfish biotoxin contamination, with a particular focus on autoregressive models, support vector machines, random forest, probabilistic graphical models, and artificial neural networks (ANN). Most efforts have been attempted to forecast HABs based on models of increased complexity over the years, coupled with increased multi-source data availability, with ANN architectures in the forefront to model these events. The purpose of this review is to help defining machine learning-based strategies to support shellfish industry to manage their harvesting/production, and decision making by governmental agencies with environmental responsibilities.

Natural high proliferations of toxin-producing microorganisms in marine and freshwater environments result in dreadful consequences at the socioeconomically and environmental level due to water and seafood contamination. Monitoring programs and scientific evidence point to harmful algal blooms (HABs) increasing in frequency and intensity as a result of global climate alterations. Among marine toxins, the okadaic acid (OA) and the related dinophysistoxins (DTX) are the most frequently reported in EU waters, mainly in shellfish species. These toxins are responsible for human syndrome diarrhetic shellfish poisoning (DSP). Fish, like other marine species, are also exposed to HABs and their toxins. However, reduced attention has been given to exposure, accumulation, and effects on fish of DSP toxins, such as OA. The present review intends to summarize the current knowledge of the impact of DSP toxins and to identify the main issues needing further research. From data reviewed in this work, it is clear that exposure of fish to DSP toxins causes a range of negative effects, from behavioral and morphological alterations to death. However, there is still much to be investigated about the ecological and food safety risks related to contamination of fish with DSP toxins.

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.

Diarrhetic shellfish toxins (DSTs) are among the most prevalent marine toxins in Europe’s and in other temperate coastal regions. These toxins are produced by several dinoflagellate species; however, the contamination of the marine trophic chain is often attributed to species of the genus Dinophysis. This group of toxins, constituted by okadaic acid (OA) and analogous molecules (dinophysistoxins, DTXs), are highly harmful to humans, causing severe poisoning symptoms caused by the ingestion of contaminated seafood. Knowledge on the mode of action and toxicology of OA and the chemical characterization and accumulation of DSTs in seafood species (bivalves, gastropods and crustaceans) has significantly contributed to understand the impacts of these toxins in humans. Considerable information is however missing, particularly at the molecular and metabolic levels involving toxin uptake, distribution, compartmentalization and biotransformation and the interaction of DSTs with aquatic organisms. Recent contributions to the knowledge of DSTs arise from transcriptomics and proteomics research. Indeed, OMICs constitute a research field dedicated to the systematic analysis on the organisms’ metabolisms. The methodologies used in OMICs are also highly effective to identify critical metabolic pathways affecting the physiology of the organisms. In this review, we analyze the main contributions provided so far by OMICs to DSTs research and discuss the prospects of OMICs with regard to the DSTs toxicology and the significance of these toxins to public health, food safety and aquaculture.

Ciguatera poisoning (CP) is a common seafood intoxication mainly caused by the consumption of fish contaminated by ciguatoxins. Recent studies showed that Caribbean ciguatoxin-1 (C-CTX1) is the main toxin causing CP through fish caught in the Northeast Atlantic, e.g., Canary Islands (Spain) and Madeira (Portugal). The use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) combined with neuroblastoma cell assay (N2a) allowed the initial confirmation of the presence of C-CTX1 in contaminated fish samples from the abovementioned areas, nevertheless the lack of commercially available reference materials for these particular ciguatoxin (CTX) analogues has been a major limitation to progress research. The EuroCigua project allowed the preparation of C-CTX1 laboratory reference material (LRM) from fish species (Seriola fasciata) from the Madeira archipelago (Portugal). This reference material was used to implement a liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for the detection of C-CTX1, acquisition of full-scan as well as collision-induced mass spectra of this particular analogue. Fragmentation pathways were proposed based on fragments obtained. The optimized LC-HRMS method was then applied to confirm C-CTX1 in fish (Bodianus scrofa) caught in the Selvagens Islands (Portugal).

Shellfish are filter-feeding organisms that may accumulate high levels of naturally-occurring toxins, produced by harmful algal blooms, and act as marine toxin vectors to whomever consumes them [...]

Prevalence of marine biotoxins in seafood has been associated with increasing frequency, intensity, and duration of harmful algal blooms, and an increase of the geographical and temporal distribution of harmful algae [...]

Occurrence of tetrodotoxin (TTX), a potent natural neurotoxin, in marine organisms and seafood from the European Union has been of increasing concern due to its relatively recent detection in bivalve mollusks and gastropods. Following a request of the European Food Safety Authority (EFSA) to EU Member States to obtain more data on TTX occurrence, this study collected 117 samples of bivalve mollusks, including mussels (Mytilus galloprovincialis), oysters (Crassostrea gigas) and clams (Ruditapes philippinarum and Donax spp.), from the South and Southwest Portuguese coast between May and October 2018, for TTX determination and microbiological analysis. The analyses carried out by hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) did not detect TTX in any sample, and microbiological analysis did not reveal high concentrations of Vibrio spp., which has been linked to the presence of TTX. Although preliminary, results from this study, the first investigating the presence of TTX in bivalve mollusks from the Portuguese coast, suggest that TTX may not represent a risk for human consumption of bivalve mollusks.

Warmer seawater temperatures are expected to increase harmful algal blooms (HABs) occurrence, intensity, and distribution. Yet, the potential interactions between abiotic stressors and HABs are still poorly understood from ecological and seafood safety perspectives. The present study aimed to investigate, for the first time, the bioaccumulation/depuration mechanisms and ecotoxicological responses of juvenile gilthead seabream (Sparus aurata) exposed to paralytic shellfish toxins (PST) under different temperatures (18, 21, 24 °C). PST were detected in fish at the peak of the exposure period (day five, 0.22 µg g−1 N-sulfocarbamoylGonyautoxin-1-2 (C1 and C2), 0.08 µg g−1 Decarbamoylsaxitoxin (dcSTX) and 0.18 µg g−1 Gonyautoxin-5 (B1)), being rapidly eliminated (within the first 24 h of depuration), regardless of exposure temperature. Increased temperatures led to significantly higher PST contamination (275 µg STX eq. kg−1). During the trial, fish antioxidant enzyme activities (superoxide dismutase, SOD; catalase, CAT; glutathione S-transferase, GST) in both muscle and viscera were affected by temperature, whereas a significant induction of heat shock proteins (HSP70), Ubiquitin (Ub) activity (viscera), and lipid peroxidation (LPO; muscle) was observed under the combination of warming and PST exposure. The differential bioaccumulation and biomarker responses observed highlight the need to further understand the interactive effects between PST and abiotic stressors, to better estimate climate change impacts on HABs events, and to develop mitigation strategies to overcome the potential risks associated with seafood consumption.

Harmful algal blooms are responsible worldwide for the contamination of fishery resources, with potential impacts on seafood safety and public health. Most coastal countries rely on an intense monitoring program for the surveillance of toxic algae occurrence and shellfish contamination. The present study investigates the use of near infrared (NIR) spectroscopy for the rapid in situ determination of cell concentrations of toxic algae in seawater. The paralytic shellfish poisoning (PSP) toxin-producing dinoflagellate Gymnodinium catenatum was selected for this study. The spectral modeling by partial least squares (PLS) regression based on the recorded NIR spectra enabled the building of highly accurate (R2 = 0.92) models for cell abundance. The models also provided a good correlation between toxins measured by the conventional methods (high-performance liquid chromatography with fluorescence detection (HPLC-FLD)) and the levels predicted by the PLS/NIR models. This study represents the first necessary step in investigating the potential of application of NIR spectroscopy for algae bloom detection and alerting.

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⁻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 µg C-CTX1 kg−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.

This study reports the accumulation of paralytic shellfish toxins (PSTs) in Atlantic horse mackerel (Trachurus trachurus) over a bloom of the toxigenic dinoflagellate Gymnodinium catenatum. High levels of toxins, up to 4800 μg STXeq kg–1, were registered at the peak of the bloom (5.0 103 cells l–1). The suite of individual PSTs was examined. Decarbamoylsaxitoxin (dcSTX) and B1 constituted nearly 90% of toxins (on a molar basis) determined in mackerel. This profile of toxins markedly differs from the known profile of toxins produced by G. catenatum strains isolated from the Portuguese coast, which is dominated by N-sulfocarbamoyl toxins, in particular the C1+2 toxins. The prevalence of the potent dcSTX in the pelagic environment and its transfer through the marine food web is highlighted in this study. Atlantic horse mackerel is identified as a high potential vector of PSTs along the Portuguese coast. This fish species has a central position in the marine food web, being an important predator of zooplankton and at the same time an important diet item of top predators. This study reveals bioaccumulation values that are important for evaluating potential impacts of blooms of PST-producing dinoflagellates on marine ecosystems or their components, such as fish.

Paralytic shellfish poisoning (PSP) is a severe food-borne illness, caused by the ingestion of seafood containing paralytic shellfish toxins (PST), which are naturally produced by marine dinoflagellates and accumulate in shellfish during algae blooms. Novel PST, designated as hydroxybenzoate analogues (also known as GC toxins), was relatively recently discovered in Gymnodinium catenatum strains worldwide. However, to date, there have been no studies examining their accumulation in shellfish. In this study, mussels (Mytilus galloprovincialis) were exposed to G. catenatum for five days and then exposed to a non-toxic diet for 24 h, to investigate the toxin’s accumulation/elimination dynamics. As determined by UHPLC-HILIC-MS/MS, the hydroxybenzoate analogues, GC1 to GC6, comprised 41% of the algae toxin profile and only 9% in mussels. Elimination of GC toxins after 24 h was not evident. This study highlights that a relevant fraction of PST in mussels are not routinely analysed in monitoring programs and that there is a need to better understand the toxicological potential of the hydroxybenzoate analogues, in order to properly address the risk of G. catenatum blooms.