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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. 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

Pufferfish are some of the most poisonous marine organisms due to their capability to accumulate potent neurotoxins such as tetrodotoxins (TTXs) and paralytic shellfish toxins (PSTs). Several species are consumed, mainly in Asiatic countries, for the quality of their flesh, which may cause severe human poisoning every year due to errors in preparation. Lagocephalus lagocephalus the oceanic pufferfish, is a cosmopolitan species present in European Atlantic and Mediterranean coasts. Although considered toxic, only a few studies have previously addressed the occurrence of toxic compounds in this species. In the present study, 29 individuals of L. lagocephalus caught in Madeira Island (NE Atlantic) were dissected and analysed by HPLC-FLD for PSTs detection. Saxitoxin (STX) and decarbamoyl STX (dcSTX) were detected in liver, intestine and gonads but not in the muscle. Neosaxitoxin (NEO) could also be detected in a liver sample. The total STX equivalent (eq) concentrations in liver ranged from < LOQ to 973.4 μg STX eq kg−1. The presence of toxins in the intestines and gonads points to the fish capability to acquire toxins through feeding or by association with endosymbiotic, toxin-producing microbes as well as the ability to accumulate them in specific organs. Although pufferfish trade in Europe is forbidden by European Union regulations, the increasing occurrence of this species in new areas and the expected raise of blooms of PSTs-producer organisms claims for a better knowledge on oceanic pufferfish toxicity and biology. This will be needed to provide accurate information to stakeholders and population to prevent illegal trade of these species and poisoning incidents. PTDC/CTA-AMB/30557/2017; European Commission: Lisboa-01-0145-feder-030557; FCT: UIDB/Multi/04326/2020. info:eu-repo/semantics/publishedVersion

Commonly affected by changes in climate and environmental conditions, coastal areas are very dynamic environments where shellfish play an important ecological role. In this study, the oxidative stress and genotoxic responses of mussels (Mytilus galloprovincialis) exposed to paralytic shellfish toxin (PST) - producing dinoflagellates Gymnodinium catenatum were evaluated under i) current conditions (CC: 19 degrees C; pH 8.0), warming (W: 24 degrees C; pH 8.0), acidification (A:19 degrees C; pH 7.6) and iv) combined effect of warming and acidification (WA: 24 degrees C; pH 7.6). Mussels were fed with G. catenatum for 5 days, and to a non-toxic diet during the following 10 days. A battery of oxidative stress biomarkers and comet assay was performed at the peak of toxin accumulation and at the end of the post-exposure phase. Under CC, gills and hepatopancreas displayed different responses/vulnerabilities and mechanisms to cope with PST. While gills presented a tendency for lipid peroxidation (LPO) and genetic damage (expressed by the Genetic Damage Indicator - GDI), hepatopancreas seems to better cope with the toxins, as no LPO was observed. However, the mechanisms involved in hepatopancreas protection were not enough to maintain DNA integrity. The absence of LPO, and the antioxidant system low responsiveness, suggests DNA damage was not oxidative. When exposed to toxic algae under W, toxin-modulated antioxidant responses were observed in both gills and hepatopancreas. Simultaneous exposure to the stressors highlighted gills susceptibility with a synergistic interaction increasing DNA damage. Exposure to toxic algae under A led to genotoxicity potentiation in both organs. The combined effect of WA did not cause relevant interactions in gills antioxidant responses, but stressors interactions impacted LPO and GDI. Antioxidant responses and LPO pointed out to be modulated by the environmental conditions in hepatopancreas, while GDI results support the dominance of toxin-triggered process. Overall, these results reveal that simultaneous exposure to warming, acidification and PSTs impairs mussel DNA integrity, compromising the genetic information due to the synergetic effects. Finally, this study highlights the increasing ecological risk of harmful algal blooms to Mytilus ganoprovincianis populations. CESAM [UIDP/50017/2020] FCT/MCTESPortuguese Foundation for Science and Technology [UIDP/50017/2020] Portuguese Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology [UID/Multi/04326/2020] Portuguese Science and Technology Foundation (FCT)Portuguese Foundation for Science and Technology [PD/BD/113484/2015] info:eu-repo/semantics/publishedVersion

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. This work was funded by Portuguese Science Foundation (Fundação para a Ciência e a Tecnologia, FCT) and under the Projects MOREBIVALVES (PTDC/ASP-PES/31762/2017) and UID/Multi/04423/2013 co-financed by NORTE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT. AM Almeida acknowledges funding from unit program LEAF–Linking Landscape, Environment, Agriculture and Food Research Centre (ISA–University of Lisbon) funded by the FCT Strategic Project (UID/AGR/04129/2019). This work had support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125).

Marine biotoxins, produced as a secondary metabolite by certain phytoplankton species, can alter cellular processes of other organisms, from plankton to top predators. Phytoplankton cells, including those from harmful algal blooms (HABs) are the main nutritional source of zooplankton grazers and filter-feeding shellfish. The ability to metabolize and detoxify HAB toxins is critical to their survival, which most likely evolved to acquire resources that enable them to tolerate these toxins. Filter-feeding shellfish and zooplankton grazers, although susceptible to biotoxins, often act as potent toxin vectors in the marine ecosystem and not as victims. On the other hand, top predators at higher trophic levels that are less frequently exposed to HAB-toxins, may experience more severe effects. Accumulation, biotransformation, sequestration and transfer throughout the marine food web can affect different organisms causing a wide range of effects, from innocuous transient to sublethal or lethal effects, that in extreme cases generate events of mass mortality in higher vertebrates. Due to the global increase in frequency and intensity of HABs during the last decades, it is extremely important to understand the extent of the effects elicited by HAB toxins on marine organisms, therefore, an update on the effects of HAB toxins is needed. The present work reviews the currently available information regarding the effects of paralytic, amnesic, diarrhetic and neurotoxic shellfish toxins on marine organisms.

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 &deg, C). PST were detected in fish at the peak of the exposure period (day five, 0.22 &micro, g g&minus, 1 N-sulfocarbamoylGonyautoxin-1-2 (C1 and C2), 0.08 &micro, 1 Decarbamoylsaxitoxin (dcSTX) and 0.18 &micro, 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 &micro, g STX eq. kg&minus, 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.

This study aimed to assess the bioaccessibility of different marine biotoxins in naturally contaminated shellfish and fish gonads using an in vitro digestion methodology. In general, hydrophilic toxins (domoic acid, paralytic shellfish poisoning toxins and tetrodotoxins) showed higher bioaccessibility than lipophilic ones (okadaic acid and azaspiracids). The bioaccessibility of toxins from the okadaic acid group ranged from 69% (raw European razor clams) to 74% (raw donax clams). Regarding azaspiracids, 47% of the initial content was bioaccessible in steamed blue mussel. As for hydrophilic toxins, 100% of the initial content was bioaccessible after digestion in raw shellfish and puffer fish gonads. The total tetrodotoxin bioaccessibility in puffer fish gonads decreased significantly after steaming. The profile of tetrodotoxins changed during the digestion process: TTX and 11-norTTX-6S-ol analogues decreased significantly after digestion, but the 5,6,11-trideoxy TTX analogue increased in both raw and steamed puffer fish gonads. These preliminary findings confirm the need to consider bioaccessibility data in future seafood risk assessment, as such information enables a more accurate and realistic estimation of potential seafood hazards, particularly in what concerns lipophilic toxins, therefore, constituting a crucial tool in the refinement of regulatory limits for the presence of biotoxins in seafood. © 2019, The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under the ECsafeSEAFOOD project (grant agreement n° 311820) and through the European Regional Development Fund. IRTA members acknowledge support from CERCA Programme/Generalitat de Catalunya. L.R. acknowledges a scholarship from the ECsafeSEAFOOD project. Ana C. Braga has a Doctoral Grant (PD/BD/113484/2015) from the Portuguese Science and Technology Foundation (FCT), whereas Antonio Marques and Pedro R. Costa are supported through the FCT Investigator program (IF). Lagocephalus sceleratus was kindly provided by Andrés Izquierdo-Muñoz from Centro de Investigación Marina de Santa Pola (CIMA) (Spain).

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.

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&rsquo, 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.

Ciguatoxins (CTXs) are fish metabolism products and a result of biotransformation of precursor gambiertoxins produced, in the first instance, by benthic dinoflagellates Gambierdiscus and Fukuyoa. Ciguatoxins are potent neurotoxins that selectively open voltage gated sodium channels in excitable cells causing the human food poisoning known as Ciguatera (CFP). Endemic from tropical areas in central Pacific and West Indian Ocean, and the Caribbean Sea, CTX may affect up to 500,000 people annually due to fish consumption. Their recent occurrence in European waters highlights the need for a multidisciplinary approach of CTX research in order to better understand the diversity and transformation of microalgae products through food webs. This article intends to review available information on chemistry, toxicity, distribution and fate of known CTX compounds from a critical perspective to provide an overview of future trends and needs on ciguatera research.

Fish are frequently exposed to paralytic shellfish toxins (PSTs) derived from harmful algal blooms (HAB). PSTs are potent neurotoxins with strong effects on fish at multiple trophic levels. Here, we examine the historical and contemporary mass mortality events combining with the available data on response of fish to PSTs’ exposure. The data show that fish are negatively affected by these natural toxins on a recurring basis. Transient effects of PSTs have also been observed in fish and although difficult to quantify, these sublethal effects can affect foraging and predation avoidance. Additionally, the spatiotemporal overlapping of fish spawning and HAB can also be critical for fish survival and fisheries recruitment. Exposure of fish in their early life stages to dissolved toxins in the water column has also been identified as a risk factor for fish survival. In the context of global climate change, where PSTs are likely to become increasingly important, new insights and a synthesis of up-to-date information on fish response to PSTs and the risk associated with toxic dinoflagellate blooms are presented.

This is the first report of a bloom of the toxic marine dinoflagellate Alexandrium minutum in the Azores Archipelago (Northeastern Atlantic Ocean). In September 2013, high cell concentrations (1.3 × 10(7) cells L(-1)) of this species were recorded in Santo Cristo coastal lagoon causing an orange-brown water discoloration, death of small pelagic fish and toxification of shellfish resources (Ruditapes decussatus). Levels of Paralytic Shellfish Poisoning toxins in clams exceeded 30 times the regulatory limit, and were associated with the intoxication of four people.

The neurotoxin β-N-methylamino- l -alanine (BMAA) and its putative role in multiple neurodegenerative diseases have been intensely studied since 2005 when the toxin was discovered to be produced by worldwide-distributed cyanobacterial species inhabiting terrestrial, marine, brackish, and freshwater ecosystems. Recently, BMAA production was also associated with one eukaryotic group, namely, diatoms, raising questions about its production by other phytoplanktonic groups. To test for BMAA bioavailability in ecosystems where abundant phytoplanktonic blooms regularly occur, samples of filter-feeding shellfish were collected in two Portuguese transitional water bodies. BMAA content in cockles ( Cerastoderma edule ) collected weekly between September and November 2009 from Ria de Aveiro and at least once a month from May to November from Ria Formosa, fluctuated from 0.079 ± 0.055 to 0.354 ± 0.066 μg/g DW and from below the limit of detection to 0.434 ± 0.110 μg/g DW, respectively. Simultaneously to BMAA occurrence in cockles, paralytic shellfish toxins were detected in shellfish as a result of Gymnodinium catenatum blooms indicating a possible link between this marine dinoflagellate and BMAA production. Moreover, considerable high BMAA levels, 0.457 ± 0.186 μg/g DW, were then determined in a laboratory grown culture of G. catenatum . This work reveals for the first time the presence of BMAA in shellfish from Atlantic transitional water bodies and consubstantiate evidences of G. catenatum as one of the main sources of BMAA in these ecosystems.

Gymnodinium catenatum, a dinoflagellate species with a global distribution, is known to produce paralytic shellfish poisoning (PSP) toxins. The profile of toxins of G. catenatum is commonly dominated by sulfocarbamoyl analogs including the C3+4 and GTX6, which to date has no commercial certified reference materials necessary for their quantification via chemical methods, such as liquid chromatography. The aim of this study was to assess the presence of C3+4 and GTX6 and their contribution to shellfish toxicity. C3+4 and GTX6 were indirectly quantified via pre-column oxidation liquid chromatography with fluorescence detection after hydrolysis conversion into their carbamate analogs. Analyses were carried out in mussel samples collected over a bloom of G. catenatum (>63×103cellsl-1) in Aveiro lagoon, NW Portuguese coast. Concentration levels of sulfocarbamoyl toxin analogs were two orders of magnitude higher than decarbamoyl toxins, which were in turn one order of magnitude higher than carbamoyl toxins. Among the sulfocarbamoyl toxins, C1+2 were clearly the dominant compounds, followed by C3+4 and GTX6. The least abundant sulfocarbamoyl toxin was GTX5. The most important compounds in terms of contribution for sample toxicity were C1+2, which justified 26% of the PSP toxicity. The lesser abundant dcSTX constitutes the second most important compound with similar % of toxicity to C1+2, C3+4 and GTX6 were responsible for approximately 11% and 13%, respectively. The median of the sum of C3+4 and GTX6 was 27%. These levels reached a maximum of 60% as was determined for the sample collected closest to the G. catenatum bloom. This study highlights the importance of these low potency PSP toxin analogs to shellfish toxicity. Hydrolysis conversion of C3+4 and GTX6 is recommended for determination of PSP toxicity when LC detection methods are used for PSP testing in samples exposed to G. catenatum.

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.

Here we summarize the current knowledge on the transfer and accumulation of harmful algal bloom (HAB)-related toxins in cephalopods (octopods, cuttlefishes and squids). These mollusks have been reported to accumulate several HAB-toxins, namely domoic acid (DA, and its isomers), saxitoxin (and its derivatives) and palytoxin (and palytoxin-like compounds) and, therefore, act as HAB-toxin vectors in marine food webs. Coastal octopods and cuttlefishes store considerably high levels of DA (amnesic shellfish toxin) in several tissues, but mainly in the digestive gland (DG)—the primary site of digestive absorption and intracellular digestion. Studies on the sub-cellular partitioning of DA in the soluble and insoluble fractions showed that nearly all DA (92.6%) is found in the cytosol. This favors the trophic transfer of the toxins since cytosolic substances can be absorbed by predators with greater efficiency. The available information on the accumulation and tissue distribution of DA in squids (e.g., in stranded Humboldt squids, Dosidicus gigas) is scarcer than in other cephalopod groups. Regarding paralytic shellfish toxins (PSTs), these organisms accumulate them at the greatest extent in DG >> kidneys > stomach > branchial hearts > posterior salivary glands > gills. Palytoxins are among the most toxic molecules identified and stranded octopods revealed high contamination levels, with ovatoxin (a palytoxin analogue) reaching 971 μg kg−1 and palytoxin reaching 115 μg kg−1 (the regulatory limit for PlTXs is 30 μg kg−1 in shellfish). Although the impacts of HAB-toxins in cephalopod physiology are not as well understood as in fish species, similar effects are expected since they possess a complex nervous system and highly developed brain comparable to that of the vertebrates. Compared to bivalves, cephalopods represent a lower risk of shellfish poisoning in humans, since they are usually consumed eviscerated, with exception of traditional dishes from the Mediterranean area.

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 10 3 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.

Fish are routinely exposed to harmful algal blooms that produce noxious compounds and impact the marine food web. This study investigates the role of phase I and II detoxification enzymes on metabolism of the novel paralytic shellfish toxins (PSTs), the hydroxybenzoate analogues recently discovered in Gymnodinium catenatum strains, in the liver of white seabream, assessing ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities. Additionally, the genotoxic potential of hydroxybenzoate PSTs was examined through the erythrocytic nuclear abnormality (ENA) assay. Fish were injected with hydroxybenzoate PSTs into the coelomic cavity and sacrificed 2 and 6 days later for biochemical and cytogenetic analyses. While the activity of EROD was unresponsive to toxins, a significant GST activity decrease was observed at 2 days after injection indicating an impairment of this line of the detoxification system. The genotoxic potential of PSTs was demonstrated by the induction of clastogenic/aneugenic effects at 2 days, as measured by the ENA assay. Overall, this study contributes to better understand the impact of toxins produced by G. catenatum blooms in fish, revealing effects that, even transitory, point out a risk associated to hydroxybenzoate analogues. published

Octopus ( Octopus vulgaris , Cuvier) plays a central role in the marine food web, being an important consumer with high metabolic rates and at the same time an important food item for higher predators. After harmful algal blooms, octopus can accumulate high levels of marine toxins trough trophic interrelationships. The aim of this study is to characterize the distribution of paralytic shellfish toxins (PSTs) in selected tissues of the O. vulgaris , in order to assess the translocation of toxins among organs with different physiological functions. Different retention times and selective elimination of particular toxin analogues were also investigated. Twenty three specimens of O. vulgaris were captured in Peniche (NW coast of Portugal) after PSTs have been detected in molluscan bivalves. Tissue matrices were dissected from organs with digestive function (digestive gland, stomach and salivary glands) and excretory function (kidneys and branchial hearts) and analyzed for toxin determination. Toxin analysis was carried out by high performance liquid chromatography with fluorescence detection (LC-FLD). PSTs were found in all tissues analyzed. The highest toxin concentrations were found in the digestive gland, reaching a maximum of 2980 μg STX equiv. kg −1 . The toxin profile was constituted by dcSTX, B1, C1 + 2, dcGTX2 + 3, dcNEO, STX and GTX2 + 3. A lower number of toxins were identified in the remaining organs, with B1 and dcSTX compromising more than 90% in molar fraction. Decarbamoyl saxitoxin was the most abundant toxin detected in digestive gland, stomach and salivary glands, while B1 was dominant in organs with excretory function. A positive correlation of concentrations of B1 and dcSTX were found in the organs analyzed. Results indicate that B1 and dcSTX are assimilated into the digestive gland in a similar proportion. Selective elimination of toxins with higher elimination of B1 and retention of dcSTX is suggested. This study contributes to better understanding of the dynamics of PSTs in O. vulgaris and the fate of PSTs in the food web.

Marine toxins generated by harmful algal blooms can be transferred through the marine food web and ultimately cause massive deaths of piscivorous predators. However, very few studies have explored the processes of accumulation and biotransformation of paralytic shellfish toxins (PSTs) within fishes. White seabream (Diplodus sargus) were orally challenged with contaminated cockles (Cerastoderma edule) containing N-sulfocarbamoyl and decarbamoyl toxins and non-contaminated cockles afterwards. Specific PSTs that occurred in low abundance in cockles (B1 7.6% and dcSTX 1.6% molar fraction) were the only toxins detected in fish viscera possibly resulting from selective elimination and transformation of the various PSTs. Concentration of toxins progressively increased in fish viscera throughout the uptake period. Toxins were then rapidly depurated (B1 0.905 day−1, dcSTX 0.467 day−1) when diet was changed to non-toxic cockles. Results indicate conversion of a precursor toxin into B1 which in turn might be converted into dcSTX at a lower extent. Low accumulation efficiency of 1.7 and 5.0% was calculated to B1 and dcSTX, respectively. This study contributes to a better understanding of dynamics of PSTs in fish and the fate of these compounds in the marine food web.

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2 + 3, GTX2 + 3 and GTX1 + 4, while an enhancement was found for C1 + 2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1 + 2 (median = 421 ng g⁻¹) exceeded largely the values for the other quantified compounds (0.09-58 ng g⁻¹). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1 + 2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218 µg STX equivalents kg⁻¹. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200-800 µg STX equivalents kg⁻¹ an increase was found between 44 and 28%.

The re-emergence of Gymnodinum catenatum blooms after a 10 year hiatus of absence initiated the present investigation. This study aims to evaluate the exposure of small pelagic fishes to paralytic shellfish toxins (PST) during blooms of G. catenatum. Sardines (Sardina pilchardus) were selected as a representative fish species. In order to assess toxin availability to fish, both intracellular PSTs (toxin retained within the algal cells) and extracellular PSTs (toxin found in seawater outside algal cells) were quantified, as well as toxin levels within three fish tissue matrices (viscera, muscle and brain). During the study period, the highest cell densities of G. catenatum reached 2.5 × 104 cells l−1 and intracellular PST levels ranged from 3.4 to 398 ng STXeq l−1 as detected via an enzyme linked immunosorbent assay (ELISA). Measurable extracellular PSTs were also detected in seawater (0.2–1.1 μg STXeq l−1) for the first time in Atlantic waters. The PST profile in G. catenatum was determined via high performance liquid chromatography with fluorescence detection (HPLC-FLD) and consisted mostly of sulfocarbamoyl (C1+2, B1) and decarbamoyl (dcSTX, dcGTX2+3, dcNEO) toxins. The observed profile was similar to that reported previously in G. catenatum blooms in this region before the 10-year hiatus. Sardines, planktivorous fish that ingest a large number of phytoplankton cells, were found to contain PSTs in the viscera, reaching a maximum of 531 μg STXeq kg−1. PSTs were not detected in corresponding muscle or brain tissues. The PST profile characterized in sardine samples consisted of the same sulfocarbamoyl and decarbamoyl toxins found in the algal prey with minor differences in relative abundance of each toxin. Overall, the data suggest that significant biotransformation of PSTs does not occur in sardines. Therefore, planktivorous fish may be a good tracer for the occurrence of offshore G. catenatum blooms and the associated PSTs produced by these algae.

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

The bloom forming marine dinoflagellate Gymnodinium catenatum Graham has been linked to paralytic shellfish poisoning (PSP) outbreaks in humans. Along the Portuguese coast (NE Atlantic), G. catenatum shows a complex bloom pattern, raising questions about the origin and affinities of each bloom population. In this work, the variability within six cultured strains of G. catenatum isolated from Portuguese coastal waters (S coast, W coast and NW coast), between 1999 and 2011, was investigated. The strains were analyzed for toxin profiling and intra-specific genetic diversity. Regarding the toxin profile, differences recorded between strains could not be assigned to the time of isolation or geographical origin. The parameter that most influenced the toxin profile was the life-cycle stage that originated the culture: vegetative cell versus hypnozygote (resting cyst). At the genetic level, all strains showed similar sequences for the D1–D2 region of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA) and shared complete identity with strains from Spain, Algeria, China and Australia. Conversely, we did not find a total identity match for the ITS-5.8S nuclear rDNA fragment. After sequence analysis, two guanine/adenine (R) single nucleotide polymorphisms (SNP 1 and 2) were detected for all strains, in the ITS1 region. This species has been reported to present very conservative LSU and ITS-5.8S rDNA regions, though with few SNP, including SNP1 of this study, already attributed to strains from certain locations. The SNP here described characterize G. catenatum populations from Portuguese waters and may represent valuable genetic markers for studies on the phylogeography of this species.

Domoic acid (DA), the amnesic shellfish toxin, is a food-web-transferred algal toxin that has been detected in many marine organisms from copepods to whales. However, cephalopods, which are important members of the food chain, have never been implicated in DA transfer or accumulation. Here, we present data showing relevant values of DA detected in the common octopus (Octopus vulgaris) from the Portuguese continental coast. Even though DA is hydrophilic and is not expected to be accumulated in the tissues, DA was always detected in our octopus tissue samples. Tissue distribution of DA revealed that the digestive gland and the branchial hearts are the main organs of DA accumulation. Highly variable DA concentrations, ranging from 1.1 to 166.2 μg DA g−1, were observed in the digestive glands. Low levels of DA were detected in the digestive tract (stomach and intestine) and could be a consequence of high digestion rates or a result of non-exposure to toxic vectors during the sampling period. In fact, octopus prey, such as bivalves, crustaceans and fishes, are known to occasionally work as DA vectors. Consequently, DA uptake into octopus tissues is likely sporadic. Similar low levels were detected in the kidney, gills, systemic heart, posterior salivary glands and mantle, and no DA was found in either the gonads or the ink sac. These data are the necessary first step towards achieving an understanding of the accumulation of phycotoxins in O. vulgaris.

Marine phycotoxins derived from harmful algal blooms are known to be associated with mass mortalities in the higher trophic levels of marine food webs. Bivalve mollusks and planktivorous fish are the most studied vectors of marine phycotoxins. However, field surveys recently showed that cephalopod mollusks also constitute potential vectors of toxins. Thus, here we determine, for the first time, the time course of accumulation and depuration of paralytic shellfish toxins (PSTs) in the common octopus (Octopus vulgaris). Concomitantly, the underlying kinetics of toxin transfer between tissue compartments was also calculated. Naturally contaminated clams were used to orally expose the octopus to PSTs during 6 days. Afterwards, octopus specimens were fed with non-contaminated shellfish during 10 days of depuration period. Toxins reached the highest concentrations in the digestive gland surpassing the levels in the kidney by three orders of magnitude. PSTs were not detected in any other tissue analyzed. Net accumulation efficiencies of 42% for GTX5, 36% for dcSTX and 23% for C1+2 were calculated for the digestive gland. These compounds were the most abundant toxins in both digestive gland and the contaminated shellfish diet. The small differences in relative abundance of each toxin observed between the prey and the cephalopod predator indicates low conversion rates of these toxins. The depuration period was better described using an exponential decay model comprising a single compartment – the entire viscera. It is worth noting that since octopuses’ excretion and depuration rates are low, the digestive gland is able to accumulate very high toxin concentrations for long periods of time. Therefore, the present study clearly shows that O. vulgaris is a high-potential vector of PSTs during and even after the occurrence of these toxic algal blooms.

Fish are recurrently exposed to paralytic shellfish toxins (PSTs) produced by Gymnodinium catenatum. Nevertheless, the knowledge regarding metabolism of PSTs and their toxic effects in fish is scarce. Consequently, the current study aims to investigate the role of phase I and II detoxification enzymes on PST metabolism in the liver of white seabream (Diplodus sargus), assessing ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities. Moreover, the genotoxic potential of PSTs was examined through the erythrocytic nuclear abnormality (ENA) assay. Fish were intracoelomically (IC) injected with a nominal dose (expressed as saxitoxin equivalents) of 1.60 μg STXeq kg⁻¹ semipurified from a G. catenatum cell culture with previously determined toxin profile. Fish were sacrificed 2 and 6 days after IC injection. PST levels determined in fish liver were 15.2 and 12.2 μg STXeq kg⁻¹, respectively, at 2 and 6 days after the injection. Though several PSTs were administered, only dcSTX was detected in the liver after 2 and 6 days. This was regarded as an evidence that most of the N-sulfocarbamoyl and decarbamoyl toxins were rapidly biotransformed in D. sargus liver and/or eliminated. This was corroborated by a hepatic GST activity induction at 2 days after injection. Hepatic EROD activity was unresponsive to PSTs, suggesting that these toxins enter phase II of biotransformation directly. The genotoxic potential of PSTs was also demonstrated; these toxins were able to induce cytogenetic damage, such as chromosome (or chromatid) breaks or loss and segregational anomalies, measured by the ENA assay. Overall, this study pointed out the ecological risk associated with the contamination of fish with PSTs generated by G. catenatum blooms, providing the necessary first data for a proper interpretation of biomonitoring programs aiming to assess the impact of phytoplankton blooms in fish. Authors greatly appreciated the helpful assistance of Pedro Lino in injection of fish. published

Paralytic shellfish poisoning (PSP), a human illness caused by the ingestion of shellfish contaminated with paralytic shellfish toxins (PSTs), has been reported in Alaska for decades. These poisoning incidents have resulted in losses to local economies due to shellfish harvest closures. Thus the development of an effective biotoxin monitoring program designed specifically for the remote regions of Alaska would provide protection for public health and allow for a viable shellfish industry. The present study provides data useful for the development of an effective toxin screening protocol by comparing PST levels quantified in shellfish by many of the currently available PST detection techniques. Seven bivalve species were collected along beaches of the Aleutian Islands from June 2006 to September 2007. The concentration of PSTs was quantified and compared using five different analytical methods: the mouse bioassay, high performance liquid chromatography (HPLC), receptor-binding assay, the commercially available Jellett Rapid PSP Test strips, and an enzyme linked immunosorbent assay technique. The Association of Official Analytical Chemists (AOAC)-approved HPLC method proved to be valuable for characterizing the suite of individual PSTs in each species for research purposes, but was not considered practical for rapid toxin screening in remote Alaskan regions due to its time-consuming nature and requirement of expensive equipment and considerable expertise. In the present study, Jellett test strips were shown to be an effective tool for rapid screening, however due to the high percentage of false positives, subsequent validation via AOAC-approved methods would be required to prevent unnecessary closures.

Traditionally, harmful algal bloom studies have primarily focused on quantifying toxin levels contained within the phytoplankton cells of interest. In the case of paralytic shellfish poisoning toxins (PSTs), intracellular toxin levels and the effects of dietary consumption of toxic cells by planktivores have been well documented. However, little information is available regarding the levels of extracellular PSTs that may leak or be released into seawater from toxic cells during blooms. In order to fully evaluate the risks of harmful algal bloom toxins in the marine food web, it is necessary to understand all potential routes of exposure. In the present study, extracellular and intracellular PST levels were measured in field seawater samples (collected weekly from June to October 2004- 2007) and in Alexandrium spp. culture samples isolated from Sequim Bay, Washington. Measurable levels of intra- and extra-cellular toxins were detected in both field and culture samples via receptor binding assay (RBA) and an enzyme-linked immunosorbent assay (ELISA). Characterization of the PST toxin profile in the Sequim Bay isolates by preMar. column oxidation and HPLC-fluorescence detection revealed that gonyautoxin 1 and 4 made up 65 ± 9.7 % of the total PSTs present. Collectively, these data confirm that extracellular PSTs are present during blooms of Alexandrium spp. in the Sequim Bay region.

The swimming crab Polybius henslowii may play an important role in the movement of the amnesic shellfish toxin, domoic acid (DA), through the marine food chain. High DA concentrations have been determined in crab samples harvested along the Portuguese coast during the summer of 2002, reaching a level of 323.1 mg DA/g crab tissue. Toxin distribution in the different crab organs showed levels as high as 571.6 mg DA/g in the visceral tissues. Levels of toxin 4 – 12 times lower were detected in the remaining tissues. This crab might be a prominent vector of the toxin to higher trophic levels, including fishes, sea birds and even humans. In Portugal P. henslowii is commercialised during the summer in some local markets. DA concentrations were found close to the legal limit of 20 mg/g in samples purchased at Figueira da Foz market. The crabs are boiled prior to reaching the consumers. The cooking process was evaluated. Determination of toxin losses during the cooking process showed a toxin reduction higher than 50%. DA was determined by HPLC-UV and confirmed by spectra acquired with diode-array detector. q 2003 Elsevier Ltd. All rights reserved.

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.