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Paramagnetic microspheres can be used in planar array fluorescence immunoassays for single or multiplex screening of food contaminants. However, no confirmation of the molecular identity is obtained. Coated blade spray (CBS) is a direct ionization mass spectrometry (MS) technique, and when combined with triple quadrupole MS/MS, it allows for rapid confirmation of food contaminants. The lack of chromatography in CBS, though, compromises the specificity of the measurement for unequivocal identification of contaminants, based on the European Union (EU) regulation. Therefore, a rapid and easy-to-use immuno-magnetic blade spray (iMBS) method was developed in which immuno-enriched paramagnetic microspheres replace the coating of CBS. The iMBS-MS/MS method was fully optimized, validated in-house following the EU 2021/808 regulation, and benchmarked against a commercial lateral flow immunoassay (LFIA) for on-site screening of DA. The applicability of iMBS-MS/MS was further demonstrated by analyzing incurred mussel samples. The combination of immunorecognition and MS/MS detection in iMBS-MS/MS enhances the measurement's selectivity, which is demonstrated by the rapid differentiation between the marine toxin domoic acid (DA) and its structural analog kainic acid (KA), which cannot be achieved with the LFIA alone. Interestingly, this first-ever reported iMBS-MS/MS method is generic and can be adapted to include any other immuno-captured food contaminant, provided that monoclonal antibodies are available, thus offering a complementary confirmatory analysis approach to multiplex immunoassay screening methods. Moreover, thanks to its speed of analysis, iMBS-MS/MS can bridge the logistics gap between future large-scale on-site testings using LFIAs and classical time-consuming confirmatory MS analysis performed in official control laboratories.

The marine toxin tetrodotoxin (TTX) poses a great risk to public health safety due to its severe paralytic effects after ingestion. Seafood poisoning caused by the consumption of contaminated marine species like pufferfish due to its expansion to nonendemic areas has increased the need for fast and reliable detection of the toxin to effectively implement prevention strategies. Liquid chromatography-mass spectrometry is considered the most accurate method, although competitive immunoassays have also been reported. In this work, we sought to develop an aptamer-based assay for the rapid, sensitive, and cost-effective detection of TTX in pufferfish. Using capture-SELEX combined with next-generation sequencing, aptamers were identified, and their binding properties were evaluated. Finally, a highly sensitive and user-friendly hybrid antibody–aptamer sandwich assay was developed with superior performance compared to several assays reported in the literature and commercial immunoassay kits. The assay was successfully applied to the quantification of TTX in pufferfish extracts, and the results obtained correlated very well with a competitive magnetic bead-based immunoassay performed in parallel for comparison. This is one of the very few works reported in the literature of such hybrid assays for small-molecule analytes whose compatibility with field samples is also demonstrated. info:eu-repo/semantics/publishedVersion

Pufferfish is a major source of tetrodotoxin (TTX), a potent neurotoxin that can lead to death in humans if consumed. The content of TTX and its analogs in different pufferfish species and the distribution in various fish organs are poorly understood. In this study, we compared TTX analysis methods, including mouse bioassay, enzyme-linked immunosorbent assay, high-pressure liquid chromatography (HPLC) coupled with a fluorescence detector, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS) methods. Different HPLC columns (e.g., amide, hydrophilic interaction liquid chromatography, and C18 columns) and mobile phases were compared for TTX analysis using LC-MS. Additionally, published extraction methods were compared for LC-MS analysis. The content of TTX and its analogs in different pufferfish species and their distribution in various organs (e.g., liver, muscle, intestine, testis, ovary, and skin) were summarized. This information can help understand the available TTX analysis methods, variation in TTX contents in pufferfish species from different regions, and TTX distribution in various organs.

In this contribution, we propose a new synthetic approach to tetrodotoxin (TTX), one of the most famous marine toxins that, after first preparing a functionalized linear substrate, forms a cyclohexane core from the substrate utilizing our mercuric triflate (Hg(OTf)2)-catalyzed cycloisomerization reaction. The concept was applied to the synthesis of 11-nor-6,7,8-trideoxyTTX and 11-nor-4,9-anhydro-6,7,8-trideoxyTTX, which are unnatural TTX analogues, demonstrating the validity of our new approach.

Cyclic imine marine toxins have attracted considerable attention from the synthetic community in the past two decades due to their unique chemical structures and clinically relevant biological activities. This review presents recent efforts of our group in the development of various strategies to efficiently construct the common spirocyclic imine fragments of the cyclic imine toxins. In particular, the use of α,β-unsaturated N-acyl iminium ion dienophiles in Diels–Alder reactions are highlighted, whereby direct access to spirocyclic imine motifs was obtained and important mechanistic details were discovered. Alternative approaches to spirocyclic imine systems involving hydroamination of amino alkynes are also summarized. One such approach led to serendipitous access to N-vinyl amide products, while our most recently ­reported approach involving an intermolecular Diels–Alder/cross-­coupling sequence using novel 2-bromo-1,3-butadienes to access 5,6-spirocyclic imines is also discussed. Additionally, the development of a novel method to construct another challenging motif present in the portimines is also introduced.1 Introduction2 Strategies towards the Spirocyclic Imine Fragment of Cyclic Imine Toxins2.1 Diels–Alder Cycloadditions of α,β-Unsaturated N-Acyl Iminium Dienophiles2.2 Early Studies Using in situ-Generated Iminium Ion Dienophiles2.3 Use of More Stable Iminium Ion Dienophiles for Diels–Alder Reactions2.4 Other Notable Strategies towards Spirocyclic Imines2.5 Recent Efforts towards the 5,6-Spirocyclic Imine Marine Toxin Portimine A2.6 Construction of Another Challenging Motif of Portimine A3 Conclusion and Future Perspectives

Saxitoxin (STX) is one of the most important marine toxins which affects the safety of domestic water. Rapid, sensitive and selective recognition of STX is crucial in environment monitoring. Here, we demonstrate a facile and ultrasensitive colorimetric sensor based on gold nanoparticles (Au NPs) and aptamer (Au NPs-aptamer biosensor) for specific and quantitative detection of STX. The aptamer reacts specifically with STX, resulting in the aggregation of Au NPs and the color change of the Au NP solution. The lowest detection concentration of the colorimetric sensor is 10 fM (3 fg mL−1), and a good linear relationship (R2 = 0.9852) between the absorbance ratio and STX concentrations (10 fM to 0.1 μM) indicates that our Au NPs-aptamer biosensor can be used for quantitative sensing of STX. The detection time of STX is 30 minutes, and the sensor is successfully applied in the specific detection of STX in seawater. The Au NP-aptamer biosensor shows great potential in practical applications to monitor environmental pollution, marine aquaculture pollution, and seafood safety.

Paralytic shellfish poisoning (PSP) occurs when shellfish contaminated with saxitoxin or equivalent paralytic shellfish toxins (PSTs) are ingested. In British Columbia, Canada, documented poisonings are increasing in frequency based on 62 investigations identified from 1941–2020. Two PSP investigations were reported between 1941 and 1960 compared to 31 since 2001 (p &lt, 0.0001) coincident with rising global temperatures (r2 = 0.76, p &lt, 0.006). The majority of PSP investigations (71%) and cases (69%) were linked to self-harvested shellfish. Far more investigations involved harvests by indigenous communities (24%) than by commercial and recreational groups. Single-case-exposure investigations increased by more than 3.5 times in the decade 2011–2020 compared to previous periods. Clams (47%), mussels (26%), oysters (14%), scallops (6%), and, in more recent years, crabs (4%) were linked to illnesses. To guide understanding of self-harvesting consumption risks, we recommend collecting data to determine when PST-producing algae are present in high concentrations, improving the quality of data in online shellfish harvest maps to include dates of last testing, biotoxin testing results, and a description of bivalve species tested. Over reliance on toxin results in biomonitored species may not address actual consumption risks for unmonitored species harvested from the same area. We further recommend introducing phytoplankton monitoring in remote indigenous communities where self-harvesting is common and toxin testing is unavailable, as well as continuing participatory education about biotoxin risks in seafoods.

Although antibody immobilization on solid surfaces is extensively used in several applications, including immunoassays, biosensors, and affinity chromatography, some issues are still challenging. Self-assembling protein layers can be used to coat easily different surfaces by direct deposition. A specific biofunctional layer can be formed using genetic engineering techniques to express fused proteins acting as self-immobilizing antibodies. In this study, fusion proteins combining the self-assembling adhesive properties of a fungal hydrophobin and the functionality of the single chain fragment variables (ScFvs) of two antibodies were produced. The chosen ScFvs are able to recognize marine toxins associated with algal blooms, saxitoxin, and domoic acid, which can bioaccumulate in shellfish and herbivorous fish causing food poisoning. ScFvs fused to hydrophobin Vmh2 from Pleurotus ostreatus were produced in Escherichia coli and recovered from the inclusion bodies. The two fusion proteins retained the functionality of both moieties, being able to adhere on magnetic beads and to recognize and bind the two neurotoxins, even with different performances. Our immobilization procedure is innovative and very easy to implement because it allows the direct functionalization of magnetic beads with ScFvs, without any surface modification. Two different detection principles, electrochemical and optical, were adopted, thus achieving a versatile platform suitable for different antigen detection methods. The sensitivity of the saxitoxin optical biosensor [limit of detection (LOD) 1.7 pg/ml] is comparable to the most sensitive saxitoxin immunosensors developed until now.

Saxitoxin (STX) belongs to the family of marine biological toxins, which are major contaminants in seafood. The reference methods for STX detection are mouse bioassay and chromatographic analysis, which are time-consuming, high costs, and requirement of sophisticated operation. Therefore, the development of alternative methods for STX analysis is urgent. Electrochemical analysis is a fast, low-cost, and sensitive method for biomolecules analysis. Thus, in this study, an electrolyte-insulator-semiconductor (EIS) sensor based on aptamer-modified two-dimensional layered Ti3C2Tx nanosheets was developed for STX detection. The high surface area and rich functional groups of MXene benefited the modification of aptamer, which had specific interactions with STX. Capacitance-voltage (C-V) and constant-capacitance (ConCap) measurement results indicated that the aptasensor was able to detect STX with high sensitivity and good specificity. The detection range was 1.0 nM to 200 nM and detection limit was as low as 0.03 nM. Moreover, the aptasensor was found to have a good selectivity and two-week stability. The mussel tissue extraction test suggested the potential application of this biosensor in detecting STX in real samples. This method provides a convenient approach for low-cost, rapid, and label-free detection of marine biological toxins.

Purpose The present study aims to develop a forensic toxicological library to identify 56 natural toxic substances by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (LC–QTOF-MS/MS). Methods For setting up the library of product ion spectra, individual substances (31 plant toxins, 7 mushroom toxins, 5 marine toxins, 5 frog venoms, 4 mycotoxins, and 4 substances derived from plants) were analyzed by LC–QTOF-MS/MS with positive and negative ionization. The product ion spectra were acquired at the collision energies (CEs) of 20, 35, and 50 eV in single enhanced product ion mode and then in collision energy spread mode in which the CE ramp range was set to 35 ± 15 eV. Results To test the performance of the library, human blood plasma samples were spiked with a mixture of lycorine and domoic acid, extracted by acetonitrile deproteinization and analyzed by LC–QTOF-MS/MS. Identification by our library search could be achieved for these toxins at the purity scores of 79.1 and 67.2, respectively. The method was also applied to postmortem blood from a death case with an aconite intake, and showed that four toxins in an aconite could be identified in the blood sample at the purity scores of 54.6–60.3. Conclusions This library will be more effective for the screening of natural toxic substances in routine forensic toxicological analysis. To our knowledge, there are no reports dealing with development of library for natural toxic substances by LC–QTOF-MS/MS.

Efficient synthesis of the partial structure of marine toxin 13-desMe spirolide C was achieved via the selective Diels-Alder reaction and C-C bond formation with the use of a silatrane substituent and the subsequent facile formation of a γ-butenolide ring. The comparison of NMR data between the synthesized diastereomers and the natural product strongly suggested that the relative configuration at the C4 position was S configuration with respect to the 7,6-spirocycle.

The sea as a habitat for organisms, is very characteristic and extreme compared to the terrestrial environment. This extreme environment allows the metabolic processes of living things within to become characteristic and produce a number of secondary metabolites that are characteristic for defense. Secondary metabolites are certain compounds that are only possessed by certain orgasms, and these class of compounds are very valuable for pharmaceuticals such as raw materials for medicines, nutraceuticals, cosmetics, to medical devices. A number of types of compounds with biological activity very important for pharmaceuticals originating from marine organisms are (a) Cytotoxic activities sourced from Sinularin compounds, Crassin Acetate, Cytarabine, Fludarabine, Aplysistatin, Geranylhydroquinone, Asperidol (b) Cardiovascular activities for (i) Activities for Cardiotonic namely compounds Laminin, Octopamine, Saxitoxin, Autonomium Chlorides (ii) Activities for Hypotensive namely Spongosine, Doridosine, Aaptameine, Hymenin (c) Marine Toxin, namely compounds of the group Polytoxin, such as Palytoxin; Red Tide Toxin compounds such as Tetrodotoxin, Brevetoxin in the form of Pb Tx-1 and Pb Ts-1; and Ciguatera Toxin groups, namely Ciguatoxin (d) Antimicrobial Activity such as Zonarol, Prepacifenol, Polyhalo-3-Butene-2-one; Tetrabromo-2-heptanone; 2-Cyano-4,5-dibromopyrrole, Aeroplysinin (+) and (-); Eunicin (e) Activities for Antibiotics namely Okadaic Acid, Acanthfolicin, Norhalichondrin A; and (f) Active Miscellaneous for Pharmaceuticals, namely Latrunculin A and B; Kainic Acid, Domoic Acid, Vidarabine, Aplysinopsin, Barettin, Nereistotoxin and 28-Deoxyzoanthenamine compounds.

Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine species of shellfish periodically collected from five representative aquaculture zones throughout a year were analyzed with a method of liquid chromatography-tandem mass spectrometry (LC–MS/MS). Lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), homo-yessotoxin (homo-YTX), azaspiracids (AZA2 and AZA3), gymnodimine (GYM), and 13-desmethyl spirolide C (13-DesMe-C), were detected in more than 95 percent of the shellfish samples. Toxins PTX2, YTX, 13-DesMe-C and GYM were predominant components detected in shellfish samples. Scallops, clams and mussels accumulated much higher level of lipophilic marine toxins compared to oysters. Toxin content in shellfish samples collected from different sampling locations showed site-specific seasonal variation patterns. High level of toxins was found during the stages from December to February and June to July in Hangu, while from March to April and August to September in Laishan. Some toxic algae, including Dinophysis acuminata, D. fortii, Prorocentrum lima, Gonyaulax spinifera and Lingulodinium polyedrum, were identified as potential origins of lipophilic marine toxins in the Bohai Sea. The results will offer a sound basis for monitoring marine toxins and protecting the health of seafood consumers.

Palytoxin analogs are marine toxins with large complex polyol structures. A benthic dinoflagellate Ostreopsis siamensis produces more than ten palytoxins (ostreocins, OSTs). The limited sample availability of minor OSTs restricts the definition of their chemical structures. The present investigation characterizes structures of two minor OSTs, i.e., ostreocin-A (OSTA) and ostreocin-E1 (OSTE1), using ostreocin-D (OSTD) as a reference compound, by liquid chromatography/quadrupole-time-of-flight mass spectrometry. The molecular formulas of OSTA and OSTE1 were C127H219N3O54 and C127H217N3O52, respectively. Compared to OSTD, OSTA has an extra oxygen atom whereas OSTE1 lacks one oxygen atom and two hydrogen atoms. The MS/MS experiments (precursor ions: [M + H]+ and [M-H]−) suggested a hydroxyl substitution at C82 in OSTA and alteration(s) between C53 and C100 in OSTE1. Further analysis of structural details in OSTE1 was performed through a pseudo-MS3 experiment (precursor ion: m/z 1432.748). Accordingly, the planar structures of OSTA and OSTE1 were assigned to 42,82-dihydroxy-3,26-didemethyl-19,44-dideoxypalytoxin and 42-hydroxy-3,26-didemethyl-19,44,73-trideoxypalytoxin-72-ene, respectively. Abbreviations:CID: collision induced dissociation; HR-LC/MS/MS: high-resolution liquid chromatography/tandem mass spectrometry; LC/ESI/Q-TOF MS: liquid chromatography/quadrupole time-of-flight mass spectrometry equipped with an electrospray ionization source; NMR: nuclear magnetic resonance; OSTs: ostreocins; OSTA: ostreocin-A; OSTB: ostreocin-B; OSTD: ostreocin-D; OSTE1: ostreocin-E1; OVTX-a: ovatoxin-a; OVTXs: ovatoxins; PLTX: palytoxin Structure elucidation of ostreocins, novel palytoxin analogs, using complementary positive and negative ion LC/Q-TOF MS.

PubMedID: 30529858 Tetrodotoxin (TTX) levels in Lagocephalus sceleratus (Silverstripe blaasop), Lagocephalus spadiceus (Half-smooth golden pufferfish) and Lagocephalus suezensis (Suez puffer) caught in Mersin Bay in the Northeastern Mediterranean Sea were analysed using Q-TOF LC/MS. Pufferfish were caught using trawl fishing, longlining and fishing line from December 2015 to October 2016. The TTX changes in the gonads, livers, intestines, skins and muscle tissues were evaluated according to sex and season. TTX levels for L. sceleratus and L. suezensis for all tissues were in the range of 0.69–35.6 µg/g and 0.67–3.09 µg/g, respectively. The highest TTX levels were observed in the gonads of female L. sceleratus caught in the autumn, and in the skin of L. suezensis caught in the spring but no quantifiable levels of TTX were found for L. spadiceus. In conclusion, L. sceleratus and L. suezensis species caught in the North-Eastern Mediterranean are toxic, and their consumption is unsafe. © 2018 Elsevier Ltd TOVAG-115O679 This research was financially supported by the Scientific and Technological Research Council of Turkey (TUBITAK); TOVAG-115O679 . We would like to thank graphic designer Yeter MERCAN for the support for the preparation of the Graphical Abstract.

Okadaic acid (OA), a potent polyether marine toxin, accumulates in the digestive glands of marine mollusks and therefore can severely threaten the health of humans after ingestion of contaminated shellfish. In vivo and in vitro studies have revealed that exposure of various cells, including human embryonic amniotic cells, hepatocytes, neuroblastoma cells, to OA induces morphological and functional modifications as well as the death of cells. As the number of reports on OA poisoning has increased, this toxin has gradually attracted the public's attention, and researchers are trying to study it. This review summarizes the current literature on the toxicity effects of OA, in addition to its detection and detoxification.

The increasing demand by citizens and environmental organization for the protection, preservation, and possible restoration of the marine environment has made seawater protection one of the urgent priorities of the European Union. In this contest, extensive monitoring and surveillance are required to correctly assess the current status of marine environment, paying attention not only to traditional chemical parameters, but also to the so-called “emerging pollutants”. Among these, marine biotoxins represent a serious hazard, because they can accumulate in fish and mussels and enter the food chain. The detection of marine toxins released by algae has mostly been directed on the analysis of fish/shellfish homogenate rather than seawater samples. To fill this gap, in this work, we propose the re-modeling of ELISA kits to set-up non-automated direct competitive ELIMC (Enzyme-Linked Immuno-Magnetic Colorimetric) assays for the detection of Domoic Acid (DA), Okadaic Acid (OA) and Saxitoxin (STX) in seawater. For each toxin, linear working range, sensitivity and detection limit (0.03, 0.05, 0.01 ng/mL for DA, OA and STX, respectively) of the ELIMC assays were calculated. After evaluation of seawater matrix effect and recovery study, ELIMC assays were integrated within a novel automated networked system (ASMAT - Analytical System for Marine Algal Toxins) based on the micro Loop Flow Reactor (μLFR) technology, suitable for on-line monitoring of marine toxins. Once established the best operational conditions, ASMAT was calibrated towards DA, OA and STX verifying its capability to detect sub-ppb levels of the target toxins. Analysis of DA, OA and STX in real samples of marine water, sampled in Alonnisos (Greece) were carried out in laboratory using ELIMC assays, ASMAT and ELISA Kits, for a comparative evaluation. Finally, on-line suitability of ASMAT as an early warning alarm system was demonstrated by a field installation within a floating platform in the port of La Spezia (Italy). © 2018 Elsevier B.V.

The neurotoxic complex saxitoxin, is a group of marine toxins that historically has significantly impacted human health and the ability to utilize marine resources. A steady increase in the distribution and intensity of Alexandrium catenella blooms in Chile, and around the world, has caused major ecological and socioeconomic impacts, putting this type of dinoflagellate, and its toxicity, in the spotlight. Ostrea chilensis is a commercially and ecologically important resource harvested from wild populations and farmed in centers of southern Chile, where it is exposed to large harmful algal blooms of the type that can cause paralysis in humans. This study contributes to our understanding about the transfer of toxins from A. catenella cells to juvenile and adult Ostrea chilensis by tracking transformations of the neurotoxic complex until it reaches its most stable molecular form in the intracellular environment of O. chilensis tissues. These biotransformations are different in O. chilensis juveniles and adults, indicating a differentiated response for these two life stages of this bivalve species. These studies can be used for similar analyses in other ecologically and commercially important species of filter feeding organisms, providing greater understanding of the specific interactions of bivalves in scenarios of toxic dinoflagellate proliferations (e.g. A. catenella blooms).

A simple QuEChERS method coupled with ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed to improve the extraction efficiency of lipophilic marine toxins (yessotoxins, dinophysistoxins, okadaic acid, azazspiracids, and spirolides) in fresh and processed shellfish products. The proposed method included freezing and dispersive solid-phase extraction with graphene oxide as the sorbent to clean complex matrices containing lipids (e.g., free fatty acids) and pigments. Quantification was performed using matrix-matched calibration curves. Recoveries were 85%–117.4% and the relative standard deviation for precision was less than 10% for marine toxins in fresh and processed shellfish products. The limits of detection (signal-to-noise = 3) and quantification (signal-to-noise = 10) were 0.10–1.47 and 0.32–4.92 μg/kg, respectively. The validated QuEChERS method, coupled with UPLC–MS/MS, was applied successfully to determine lipophilic marine toxins in fresh and processed shellfish samples.

Saxitoxin (STX) is a major marine toxin from shellfish, and it is responsible for paralytic shellfish poisoning (PSP). In this study, a highly sensitive and rapid aptamer assay was developed for STX detection by combining fluorescence resonance energy transfer (FRET) and nuclease-assisted target recycling signal amplification. The aptamer STX-41 conjugated with graphene quantum dots (GQDs) was adsorbed on magnetic reduced graphene oxide (MRGO) to establish a fluorescence quenching system. Then, the binding between STX and aptamer induced the desorption of GQD-aptamer from MRGO and the restoring of fluorescence for the fluorescent determination of STX. The digestion of the target bound aptamer by DNase I could release the target for recycling thus achieving signal amplification. Under the optimized conditions, the aptamer assay showed a wide detection range (0.1–100 ng·mL−1), low detection limit (LOD of 0.035 ng·mL−1), high specificity, good recovery (86.75–94.08% in STX-spiked clam samples) and repeatability (RSD of 4.27–7.34%). Combined with fluorescent detection technology, signal amplification technology, and magnetic separation technology, the proposed method can be used to detect STX in seafood products successfully.

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. Project Cigua PTDC/CTA-AMB/30557/2017; FCT: UID/Multi/04326/2020 info:eu-repo/semantics/publishedVersion

Palytoxin (PLTX) is a marine toxin that nowadays is recognized amongst the most toxic compounds isolated from natural products. Originally, the toxin was only identified in a single tidal pool of the island of Maui (Hawaii). Currently, this compound is considered as an emergent toxin in Europe and its prevalence in continental European waters has increased during the last years. The high toxicity of palytoxin is related with the binding to the Na+-K+ ATPase, converting this ubiquitously distributed enzyme in a permeant cation channel [1-3]. Several reports have shown that this toxin is responsible for human fatal intoxications, either after inhalation of toxin-containing marine aerosols or after ingestion of marine products contaminated with PLTX, such as crabs, groupers, mackerel, and parrotfish. So far, different groups have explored the acute oral toxicity of PLTX in mice however, discrepancies in the PLTX source as well as in the monitoring time for the toxic effects yielded controversial results. Although the presence of palytoxin in marine products is not yet currently regulated in Europe, the European Food Safety Authority (EFSA) expressed its opinion on PLTX toxicity and prompted the need to obtain more data regarding the in vivo toxicity of this compound [4]. Therefore, in this study, the acute and chronic toxicity of palytoxin was evaluated after oral administration of the toxin to mice either in a single dose and in a follow-up period of 96 hours or after chronic administration during a 28-day period. After chronic exposure of mice to the toxin, a lethal dose 50 (LD50) of 0.44 µg/kg of PLTX, much lower than that observed in the acute experiments, and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 µg/kg for repeated daily oral administration of PLTX were determined. Therefore, these data indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods remarking the need to further evaluate the potential teratogenic effects of this emerging marine toxin in mammals. References Artigas, P.; Gadsby, D.C. Ion occlusion/deocclusion partial reactions in individual palytoxin-modified Na+/K+ pumps. Ann N Y Acad Sci 2003, 986, 116-126. Artigas, P.; Gadsby, D.C. Na+/K+-pump ligands modulate gating of palytoxin-induced ion channels. Proc Natl Acad Sci U S A 2003, 100, 501-505. Artigas, P.; Gadsby, D.C. Large diameter of palytoxin-induced Na+/K+ pump channels and modulation of palytoxin interaction by Na+/K+ pump ligands. J Gen Physiol 2004, 123, 357-376. EFSA. Panel on contaminants in the food chain (CONTAM). Scientific Opinion on marine biotoxins in shellfish–Palytoxin group. EFSA Journal, 2009; Vol. 7, p 1393.

Tetrodotoxin (TTX) is a very toxic compound responsible for human intoxications after ingestion of contaminated fishery products. Although TTX was initially associated mainly with human fatalities occurring in Asiatic countries[1], nowadays has expanded to other regions including European countries. In Europe, the first non-fatal human intoxications by TTX was reported more than 10 years ago after the ingestion of a Charonia lampas trumpet shell captured in the Portuguese coast and commercialized in Spain[2]. Since then, during the last decade, the presence of the TTX-containing pufferfish Lagocephalus sceleratus has been reported in European coasts, mainly in the Mediterranean Sea[3,4] with some fish tissues containing TTXs amounts as high as 2 mg/kg[5]. Moreover, an increasing concern regarding food safety has been raised after the detection of TTX in mussels, oysters and clams harvested in UK, Greece, the Netherlands[6-8] and Spain. The current European legislation in marine toxins did not yet regulated the levels of TTX in fishery products, and nowadays the presence of the toxin is only regularly monitored in the Netherlands although the European Food Safety Authority (EFSA) has recommended the level of 44 µg/kg TTXs for routine monitoring since, at this dose, no adverse effects were observed in humans. Considering initial data on the acute oral toxicity of TTX and in view of the EFSA opinion remarking the need for additional chronic toxicity studies to further reduce the uncertainty of the likely future toxin regulation. In this work, the oral chronic toxicity of TTX using doses of 25, 44, 75 and 125 µg/kg and an observation period of 28 days was explored in female mice using protocols internationally validated to test the toxicity of chemicals. The data presented here indicated that 25 and 44 µg/kg of TTX did not cause neither blood biochemical nor behavioral alterations in mice, while at the dose of 125 µg/kg kidney and heart alterations were observed under electron microscopy analysis. Therefore, the data presented here indicate that the safety TTX dose proposed by EFSA is low enough to prevent human adverse effects, while caution should be taken in the presence of higher TTX doses. References: Bane, V.; Lehane, M.; Dikshit, M.; O'Riordan, A.; Furey, A. Tetrodotoxin: chemistry, toxicity, source, distribution and detection. Toxins 2014, 6, 693-755. Rodriguez, P.; Alfonso, A.; Vale, C.; Alfonso, C.; Vale, P.; Tellez, A.; Botana, L.M. First Toxicity Report of Tetrodotoxin and 5,6,11-TrideoxyTTX in the Trumpet Shell Charonia lampas lampas in Europe. Analytical Chemistry 2008, 80. Rambla-Alegre, M.; Reverte, L.; Del Rio, V.; de la Iglesia, P.; Palacios, O.; Flores, C.; Caixach, J.; Campbell, K.; Elliott, C.T.; Izquierdo-Munoz, A., et al. Evaluation of tetrodotoxins in puffer fish caught along the Mediterranean coast of Spain. Toxin profile of Lagocephalus sceleratus. Environ Res 2017, 158, 1-6. Katikou, P.; Georgantelis, D.; Sinouris, N.; Petsi, A.; Fotaras, T. First report on toxicity assessment of the Lessepsian migrant pufferfish Lagocephalus sceleratus (Gmelin, 1789) from European waters (Aegean Sea, Greece). Toxicon 2009, 54, 50-55. Leonardo, S.; Kiparissis, S.; Rambla-Alegre, M.; Almarza, S.; Roque, A.; Andree, K.B.; Christidis, A.; Flores, C.; Caixach, J.; Campbell, K., et al. Detection of tetrodotoxins in juvenile pufferfish Lagocephalus sceleratus (Gmelin, 1789) from the North Aegean Sea (Greece) by an electrochemical magnetic bead-based immunosensing tool. Food Chem 2019, 290, 255-262. Katikou, P. Public Health Risks Associated with Tetrodotoxin and Its Analogues in European Waters: Recent Advances after The EFSA Scientific Opinion. Toxins 2019, 11. Turner, A.D.; Powell, A.; Schofield, A.; Lees, D.N.; Baker-Austin, C. Detection of the pufferfish toxin tetrodotoxin in European bivalves, England, 2013 to 2014. Eurosurveillance 2015, 20, 21009. Vlamis, A.; Katikou, P.; Rodriguez, I.; Rey, V.; Alfonso, A.; Papazachariou, A.; Zacharaki, T.; Botana, A.M.; Botana, L.M. First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum. Toxins 2015, 7, 1779-1807.

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80–400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 105Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays.

Domoic acid (DA), the causative agent for the human syndrome Amnesic Shellfish Poisoning (ASP), is a potent, naturally occurring neurotoxin produced by common marine algae. DA accumulates in seafood, and humans and wildlife alike can subsequently be exposed when consuming DA-contaminated shellfish or finfish. While strong regulatory limits protect people from the acute effects associated with ASP, DA is an increasingly significant public health concern, particularly for coastal dwelling populations, and there is a growing body of evidence suggesting that there are significant health consequences following repeated exposures to levels of the toxin below current safety guidelines. However, gaps in scientific knowledge make it difficult to precisely determine the risks of contemporary low-level exposure scenarios. The present review characterizes the toxicokinetics and neurotoxicology of DA, discussing results from clinical and preclinical studies after both adult and developmental DA exposure. The review also highlights crucial areas for future DA research and makes the case that DA safety limits need to be reassessed to best protect public health from deleterious effects of this widespread marine toxin.

A novel electrochemical immunosensor for the detection of the important marine biotoxins domoic acid (DA) and okadaic acid (OA) was developed. The sensors used carbon black modified screen-printed electrodes (CB-SPE) obtained using a high-throughput method. The electrochemical performance and stability of CB modified SPEs and bare carbon SPEs (c-SPEs) were compared using cyclic voltammetry and electrochemical impedance spectroscopy. CB-SPEs showed improved long-term (at least six months) stability and electro-catalytic properties compared with c-SPEs. The CB-SPEs were bio-functionalized with DA or OA protein-conjugates and used to develop two indirect competitive immunosensors using differential pulse voltammetry (DPV). The DPV signals obtained for the OA and DA immunosensors fitted well to four-parameter dose-response curves (R2 > 0.98) and showed excellent LODs (LOD = 1.7 ng mL−1 for DA in buffer; LOD = 1.9 ng mL−1 for DA in mussel extract; LOD = 0.15 ng mL−1 for OA in buffer; LOD = 0.18 ng mL−1 for OA in mussel extract). No significant interference of the naturally co-occurring marine toxins saxitoxin, tetrodotoxin and OA was detected for the DA immunosensor. Similarly, for the OA immunosensor saxitoxin, tetrodotoxin and DA did not cross-react and very limited interference was observed for the dinophysis toxins DTX-1, DTX-2 and DTX-3 (OA congeners). Moreover, both immunosensors remained stable after at least 25 days of storage at 4 °C. This work demonstrates the potential of affordable, mass-produced nanomaterial-modified SPEs for marine toxin detection in shellfish.

An immunoassay was developed that utilized plasmonic coupling between immobilised gold nanorods and colloid gold nanospheres to detect the marine toxin domoic acid (DA). The aspect ratio of the nanorods was optimised and the effects of variation in acidity, silver to gold ratio, cetyltrimethylammonium bromide (CTAB) concentration and seed addition in the growth solution on the yield, size variance and LSPR peak position was investigated. Excellent nanorods (size variation, This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No 720325.

Diarrhetic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated by diarrhetic shellfish toxins (DSTs) such as okadaic acid (OA) and dinophysistoxins (DTXs) produced by some species of dinoflagellates. To prevent the occurrence of human intoxication cases, inspection of DSTs (OA and DTXs) in shellfish is important. An instrumental method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been recently employed in Japan for the monitoring of OA and DTXs in shellfish. For such analysis, reference materials (RMs) of OA and DTXs are essential. Demand for the reference materials, especially dinophysistoxin-1 (DTX1), is recently increasing in Japan. Production of the materials has been performed by mass cultivation of a dinoflagellate (Prorocentrum lima) strain that produces DTXs and OA, which indicates that the efficiency of production depends on the toxin production of the strain used. In this study, P. lima complex subclade 1e strain MIO12P was determined to be a high DTX1 producer among the three Japanese strains of the P. lima complex (subclades 1e, 1f, and 1i). It was clarified that the culture medium suitable for toxin production by strain MIO12P was metals mix SWII medium, and the optimal temperature and salinity for toxin production were 25 °C and salinity 30, respectively. The DTX1 yield (1265.3 ng ml−1) of strain MIO12P cultured under the conditions described above was the highest reported worldwide. Prorocentrum lima complex subclade 1e strain MIO12P is expected to be useful for the sustainable production of DTX1 as a source of RMs for chemical and biochemical methods in the future.

Harmful algal blooms produce toxins that bioaccumulate in the food web and adversely affect humans, animals, and entire marine ecosystems. Blooms of the diatom Pseudo-nitzschia can produce domoic acid (DA), a toxin that most commonly causes neurological disease in endothermic animals, with cardiovascular effects that were first recognized in southern sea otters. Over the last 20 years, DA toxicosis has caused significant morbidity and mortality in marine mammals and seabirds along the west coast of the USA. Identifying DA exposure has been limited to toxin detection in biological fluids using biochemical assays, yet measurement of systemic toxin levels is an unreliable indicator of exposure dose or timing. Furthermore, there is little information regarding repeated DA exposure in marine wildlife. Here, the association between long-term environmental DA exposure and fatal cardiac disease was investigated in a longitudinal study of 186 free-ranging sea otters in California from 2001 – 2017, highlighting the chronic health effects of a marine toxin. A novel Bayesian spatiotemporal approach was used to characterize environmental DA exposure by combining several DA surveillance datasets and integrating this with life history data from radio-tagged otters in a time-dependent survival model. In this study, a sea otter with high DA exposure had a 1.7-fold increased hazard of fatal cardiomyopathy compared to an otter with low exposure. Otters that consumed a high proportion of crab and clam had a 2.5- and 1.2-times greater hazard of death due to cardiomyopathy than otters that consumed low proportions. Increasing age is a well-established predictor of cardiac disease, but this study is the first to identify that DA exposure affects the risk of cardiomyopathy more substantially in prime-age adults than aged adults. A 4-year-old otter with high DA exposure had 2.3 times greater risk of fatal cardiomyopathy than an otter with low exposure, while a 10-year old otter with high DA exposure had just 1.2 times greater risk. High Toxoplasma gondii titers also increased the hazard of death due to heart disease 2.4-fold. Domoic acid exposure was most detrimental for prime-age adults, whose survival and reproduction are vital for population growth, suggesting that persistent DA exposure will likely impact long-term viability of this threatened species. These results offer insight into the pervasiveness of DA in the food web and raise awareness of under-recognized chronic health effects of DA for wildlife at a time when toxic blooms are on the rise.

Kainic acid (KA) is an excitotoxic glutamate analogue produced by a marine seaweed. It elicits neuronal excitotoxicity leading to epilepsy in rodents. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1), a nonselective cation channel protein, by capsaicin, prevents KA-induced seizures in a mouse model of temporal lobe epilepsy. However, the precise mechanism behind this protective effect of capsaicin remains unclear. In order to analyze the direct effect of KA on TRPV1, we evaluated the ability of KA to activate TRPV1 and analyzed its binding to TRPV1 using a molecular modeling approach. In vitro, KA activates a Ca(2+) influx into TRPV1 expressing HEK293 cells but not in contsrol HEK293 cells. Pretreatment with either capsaicin (1 M) or capsazepine (10 M; TRPV1 antagonist) prevents the effect of KA. Pharmacological inhibition of phospholipase C (PLC) by U73122 or overexpression of phosphatidylinositol 5 phosphatase (Synaptojanin 1; Synj-1) counters the effect of KA. Further, KA treatment causes actin reorganization in HEK(TRPV1) cells and PLC inhibition by U73122 prevents this. Molecular modeling data revealed that KA binds to TRPV1 and prebinding with capsaicin prevents the binding of KA to TRPV1. Consistently, the lack of effect of KA in activating chicken TRPV1, which is insensitive to capsaicin, suggests that there is a significant overlap between the sites of KA and capsaicin activation of TRPV1. However, PLC inhibition did not suppress TRPV1 activation by capsaicin. Collectively, our data suggest that KA binds to and activates TRPV1 and causes actin reorganization via PLC-dependent mechanism in vitro. We propose that KA mediates Ca(2+) induced toxicity possibly by activating TRPV1. Therefore, inhibiting TRPV1 will be a beneficial strategy in abating Ca(2+)-induced neurotoxicity.

Palytoxin (PLTX) is one of the most poisonous substances known to date and considered as an emergent toxin in Europe. Palytoxin binds to the Na+-K+ ATPase, converting the enzyme in a permeant cation channel. This toxin is known for causing human fatal intoxications associated with the consumption of contaminated fish and crustaceans such as crabs, groupers, mackerel, and parrotfish. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Different reports have previously explored the acute oral toxicity of PLTX in mice. Although the presence of palytoxin in marine products is currently not regulated in Europe, the European Food Safety Authority expressed its opinion on PLTX and demanded assessment for chronic toxicity studies of this potent marine toxin. In this study, the chronic toxicity of palytoxin was evaluated after oral administration to mice by gavage during a 28-day period. After chronic exposure of mice to the toxin, a lethal dose 50 (LD50) of 0.44 &micro, g/kg of PLTX and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 &micro, g/kg for repeated daily oral administration of PLTX were determined. These results indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods, pointing out the need to further reevaluate the levels of this compound in marine products.

Domoic acid (DA) is an excitatory marine neurotoxin produced by diatoms Pseudo-nitzschia spp. as a defence compound that accumulates in the food web and is associated with amnesic shellfish poisoning in humans. Although its toxicity has been well established in marine species, there is limited data on DA cytogenotoxicity in human non-target cells. Therefore, we aimed to investigate the cytogenotoxic potential of DA (0.01–10 μg/mL) in human peripheral blood cells (HPBCs) using a battery of bioassays in vitro. In addition, the influence of DA on oxidative stress parameters as a possible mechanism of action was assessed. Results revealed that DA induced dose- and time-dependent cytotoxic effects. DA significantly affected genomic instability by increasing the frequency of micronuclei and nuclear buds. Furthermore, a slight induction of primary DNA strand breaks was detected after 24 h of exposure accompanied by a significant increase in the number of abnormal size tailed nuclei. No induction of hOGG1 (human 8-oxoguanine DNA glycosylase) sensitive sites was determined upon exposure to DA. Additionally, DA induced oxidative stress by increased production of reactive oxygen species accompanied by changes in glutathione, superoxide dismutase, malondialdehyde and protein carbonyl levels. Overall, the obtained results showed adverse genotoxic effects of DA in non-target HPBCs.

Marine toxins in bivalves pose an important risk to human health, and regulatory authorities throughout the world impose maximum toxicity values. In general, bivalve toxicities due to paralytic shellfish toxins (PSTs) above the regulatory limit occur during short periods, but in some cases, it may be extended from weeks to months. The present study examines whether cockles (Cerastoderme edule), mussels (Mytilus galloprovincialis) and razor shells (Solen marginatus) naturally exposed to a bloom of Gymnodinium catenatum activated or suppressed biochemical responses as result of the presence of PSTs in their soft tissues. Toxins (C1+2, C3+4, GTX5, GTX6, dcSTX, dcGTX2+3 and dcNEO) and a set of biomarkers (ETS, electron transport system activity; GLY, glycogen; PROT, protein; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferases; LPO, lipid peroxidation; reduced (GSH) and oxidized (GSSG) glutathione contents and AChE, acetylcholinesterase activity) were determined in the three bivalve species. Specimens were harvested weekly in Aveiro lagoon, Portugal, along thirteen weeks. This period included three weeks in which bivalve toxicity exceeded largely the regulatory limit and the subsequence recovery period of ten weeks. Biochemical performance of the surveyed species clearly indicated that PSTs induce oxidative stress and neurotoxicity, with higher impact on mussels and razor shells than in cockles. The antioxidant enzymes CAT and GPx seemed to be the biomarkers better associated with toxin effects.

The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1.7 &plusmn, 0.35 pg/mL, 5.8 &plusmn, 0.9 ng/mL, 3 &plusmn, 0.5 ng/mL and 15.8 &plusmn, 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 &plusmn, 0.008 and 0.064 &plusmn, 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.

The present study tested under laboratorial conditions the sorption of okadaic acid (OA), a lipophilic toxin produced by marine phytoplankton, onto 6-mm plastic circular fragments. Fragments (4 g L−1) of polyethylene terephthalate (PET), polypropylene (PP), expanded polystyrene (EPS) and non-expanded polystyrene (PS) were exposed for 96 h to seawater spiked with 10 ng mL−1 of OA. Results of the experiments showed a broad percentage of OA removed from the water by plastics after 48 h of exposure: 30 ± 5.1% (PET), 37 ± 9.5% (PP), 62 ± 7.1% (EPS) and 83 ± 1.9% (PS). Sorption appears to be highly influenced by polymer's characteristics, such as polarity and degree of crystallinity. Further studies are needed to clarify the effect of the contact area on sorption by expanded plastics. These results point to the plausible interaction of OA and plastics in coastal waters.

Predation is one of the main sources of mortality for fish larvae. During evolution, they have developed different anti-predator behaviours, as the vibrational-evoked startle response and its habituation, for promoting survival to predator's strikes. Whereas these two behaviours can be altered by the exposure to some neurotoxicants, it is currently unknown if the exposure to environmentally relevant concentration (ERC) of neurotoxic pollutants could impair them. In this study thirty neurotoxic environmental pollutants from nine chemical groups, including: herbicides; carbamate, organophosphate (OP), organochlorine (OC), neonicotinoid and pyrethroid insecticides; toxins; metal and non-metal elements, have been screened at two concentrations, including one environmental relevant concentration (ERC), for adverse effects on anti-predator behaviours by using the Vibrational Startle Response Assay on zebrafish larvae. Significant effects over anti-predator responses were equally observed in both exposure concentrations. Focusing on the ERC scenario, it was found that the startle response was the less affected behaviour, where ten pollutants from all chemical groups except for organochlorine, neonicotinoid and pyrethroids, altered this response. Interestingly, organic and inorganic pollutants showed opposite effects on this response: whereas all organic pollutants decreased the startle response, the three remaining inorganic pollutants increased it. On the other hand, more pollutants affected habituation of the startle response of the larvae, where thirteen of the pollutants from all groups, except for herbicides, altered this behaviour at ERC, generally resulting in a faster habituation except for one OP and one marine toxin, which were able to delay this response. Ultimately, only one chemical from the OP, toxin, metal and non-metal element groups altered both the startle response and its habituation at both ERC and WSC. These results emphasize the environmental risk of the current levels of some neurotoxicants present in our aquatic ecosystems, as they are high enough to impair essential anti-predator behaviours in fish larvae. © 2020 Elsevier B.V., This work was supported by the Spanish Government (CTM2017-83242-R; D.R.). M.F acknowledges financial support from the Beatriu de Pinós programme (grant N°: 2016 BP 00233) provided by the Secretariat of Universities and Research Department of the Ministry for Business and Knowledge, Catalonia Government.

Okadaic acid (OA) is a major marine toxin that can cause diarrheic shellfish poisoning (DSP). Because of food safety concerns, simple and reliable field assays are in high demand. Herein, a highly sensitive fluorescent assay for OA in seafood was reported. For this purpose, a duplexed aptamer (DA) based on a conformational change upon binding OA was designed and immobilized on the surface of streptavidin-conjugated magnetic beads (SMBs). With the addition of OA, the DAs melted, and ss-aptamers were released into the solution. After magnetic separation, a terminal deoxynucleotidyl transferase (TdT)-mediated isothermal amplification system was formed via a free ss-aptamer acting as a template. This product with a poly(thymine) tail was directly added to a fluorescent copper nanoparticle (CuNP) formation system, and the ultralong consecutive T-rich sequence provided deposition sites for copper. Finally, the OA concentration was determined by recording fluorescence spectra and calculating the intensity change. This duplexed aptamer-isothermal amplification-based nucleic acid CuNP (DAIA-CuNP) sensor can achieve ultrahigh-sensitivity detection of OA compared to LC-MS and electrochemical methods. This DAIA-CuNP sensor can detect OA with a limit of detection (LOD) of 1.1 ng/L and a good linear range from 1.0 ng/L to 50.0 μg/L in shellfish extract. Additionally, this method is expected to be compatible with a handheld fluorescence reader and can meet field detection needs.

Domoic acid (DA) is a major marine neurotoxin, occurs frequently in most of the world's coastlines and seriously threatens ecosystem and public health. However, information on its biotransformation process in coastal anaerobic environments remains unclear. In this study, the underlying mechanism of anaerobic biotransformation of DA by marine consortium GLY was investigated using the combination of liquid chromatography-high-resolution Orbitrap mass spectrometry and comparative metatranscriptomics analysis. The results demonstrated that DA could be cometabolically biotransformed under anaerobic conditions with pseudo-first-order reaction. Anaerobic biotransformation pathway of DA was clarified, including decarboxylation, dehydrogenation, carboxylation activation with CoA and multiple β-oxidation steps occurring at aliphatic side chain, which facilitated DA detoxification. Furthermore, anaerobic cometabolic biotransformation mechanism of glycine-DA by consortium GLY was established for the first time, a number of genes related to the metabolic pathways of glycine fermentation, fatty acid synthesis and β-oxidation were responded in the consortium GLY transcriptome and involved in the anaerobic biotransformation of DA. This study could deepen understanding of interaction mechanism between toxin DA and marine microorganisms, which provides a new insight into the DA fate and its effects on benthic microbial community in marine environments.

Harmful algal bloom and marine toxins are actual and overgrowing problems. Okadaic acid (OA) is the main toxin responsible for diarrheic shellfish poisoning in humans, while Tetrodotoxin (TTX) is a tremendously strong neurotoxin. Given their high toxicity, strategies to obtain a mitigation of these toxins are every day more fundamental, in the optic of preserve consumer health, and in giving supports to shellfisheries in selling their products. In this context, the ability of lactic acid bacteria (LAB) and Paracoccus spp. to reduce OA and TTX in vitro was evaluated using HPLC/MS-MS technique. Different mechanisms of interaction were observed. Some species of LAB were able to reduce significantly the amount of toxin while in viable state (Lacticaseibacillus rhamnosus 1473 = 30% OA reduction), but also a binding effect of the toxins was observed. Contrarily, Paracoccus spp. was able to reduce significantly the amount of toxin while in a cell-free extract form, in particular Paracoccus aerius 5038 could reduce the presence of TTX of about 24%. This study provided a sound line of evidence for the possible future use of either specific bacteria or selected bacterial components to reduce the level of harmful marine toxins in aquaculture and food production.

Pseudo-nitzschia multiseries is a diatom species associated with the production of domoic acid (DA), a water soluble neurotoxin that is easily transferred up in the food web, causing devastating effects on top marine organisms and humans. Despite studies on Pseudo-nitzschia are relevant to human health safety, partitioning of marine toxins between intracellular and extracellular fractions are poorly documented. This study aimed to determine the growth rates and DA content, both intracellular and extracellular, of Pseudo-nitzschia multiseries cultures at three different light settings (15, 120 and 560 μmol m−2 s−1). The optimal conditions for cell growth were observed at 120 and 560 μmol m−2 s−1, whereas DA production was observed in P. multiseries at 15 and 120 μmol m−2 s−1, ranging between 0.18-2.56 and 0.16–3.5 pg DA cell−1, respectively. Higher intracellular DA concentrations were found during the senescence phase at low light intensity and during the exponential phase at medium light intensity, while higher concentrations of dissolved DA were found at low and medium light intensities in the senescence phase reaching 3 and 10 ng DA mL−1 respectively. The amount of toxin released into the culture medium represents the most important fraction ranging between 63 and 98% during the exponential phase and nearly 99% during the senescence phase. In contrast, under low light intensity, dissolved DA was detected in the culture medium only during the senescence phase. This study confirms the importance of light intensity on DA production and clearly shows that dissolved domoic acid is an important fraction in Pseudo-nitzschia cultures, suggesting with the careful assumptions of results from static cultures extrapolated to bloom situations that waterborne exposure of marine organism should be considered during blooms of Pseudo-nitzschia multiseries.

Domoic acid (DA) is a neurotoxin naturally produced by Pseudo-nitzschia diatoms that may be transferred through the marine food web and cause mass mortality events at higher trophic levels. Yet, the effects of the dissolved marine toxin on foraging responses and swimming performances of fish early stages are poorly known. Here we evaluated the effects of short-term exposure (24 h) to a single dose of domoic acid (136 μg DA L-1) on larvae (15-20 days post-hatch) of three commercially important fish species (the sea breams Diplodus sargus and Sparus aurata and the meagre Argyrosomus regius). Although DA exposure did not elicit significant effects on larval survival (p > 0.05) and swimming performance (p > 0.05), the toxin significantly affected the fish capture success (p

Ciguatera fish poisoning is a serious human health issue that is highly localized to tropical and sub-tropical coastal areas, affecting many of the indigenous island communities intrinsically linked to reef systems for sustenance and trade. It is caused by the consumption of reef fish contaminated with ciguatoxins and is reported as the most common cause of non-bacterial food poisoning. The causative toxins bioaccumulate up the food web, from small herbivorous fish that graze on microalgae of the genus Gambierdiscus into the higher trophic level omnivorous and carnivorous fish predating on them. The number of Gambierdiscus species being described is increasing rapidly and the role of other toxins produced by this microalgal genus in ciguatera intoxications, such as maitotoxin, remains unclear. Ciguatoxins and maitotoxin are among the most potent marine toxins known and there are currently no methods of analysis that can simultaneously monitor these toxins with a high degree of specificity. To meet this need a rapid and selective ultra-performance liquid chromatography tandem mass spectrometry method has been developed to rapidly screen Gambierdiscus cultures and environmental sample device extracts for ciguatoxins and maitotoxins. A fast sample preparation method has also been developed to allow sensitive quantification of the potent ciguatoxin fish metabolite P-CTX-1B from fish extracts, and this method has been subjected to a small validation study. Novel aspects of this approach include the use of alkaline mobile phase for chromatographic separation and specific monitoring of the various toxins. This method has good potential to help evaluate ciguatera risk associated with Gambierdiscus and related microalgal species, and to help promote method development activities for this important and analytically challenging toxin class.

The presence of polychlorinated biphenyl congeners, organochlorine pesticides and marine biotoxins in the marine environment is important for the evaluation of a potential risk to human health. The purpose of the present study was to determine concentrations of polychlorinated biphenyl congeners (PCBs) and organochlorine pesticides (DDT and its metabolites) in three fish species and mussels (Mytilus galloprovincialis) and marine biotoxins in mussels from the Black Sea, Bulgaria. Concentration of six Indicator PCB congeners, DDT and its metabolites were determined by gas chromatography coupled to mass spectrometry. The mean levels of I-PCBs ranged between 6.78 ng/g ww and 16.33 ng/g ww (garfish and bluefish respectively). The sum of I-PCBs in all seafood studied did not exceed the EU maximum level. Hydrophilic marine biotoxins determination was performed by HPLC with postchromatographic oxidation. Lipophilic marine toxins were determined on liquid chromatograph coupled to mass spectrometry. The analyzed marine biotoxins were under the limit of detection.

Okadaic Acid (OA) is a marine toxin responsible for DSP (Diarrheic Shellfish Poisoning) in humans produced by dinoflagellate. The genotoxic and cytotoxic effects of OA have been well reported in mammalian experimental animals and in vitro cultured cells. However, there are no available investigations regarding the involvement of the oxidative stress pathways in OA toxicity, especially on aquatic animals such as fish. In this context, we aimed in the present work to demonstrate whether OA (7.5 μg/ml) induces oxidative stress and histopathological damages in the fish species Sparus aurata under short term exposure (2 h, 4 h and 24 h). To this end, we have assessed lipid peroxidation and anti-oxidative stress response in liver tissue, and finally ultrastructural changes were investigated in hepatic and gills tissues. Our results clearly showed that OA induced significant enhancement in all tested parameters in a time dependent manner and seems to be a strong inducer of oxidative stress in aquatic animals. The data of the present study indicate also that histology is a successful tool to reveal OA impact on liver and gill tissues of Sparus aurata since the animal showed vascular dilation and hepatocellular membrane disintegration in liver and hypertrophy in secondary lamellae and necrotic aspect in the primary lamellae in gill tissue.

A novel bioanalysis method for sensitive, rapid and field-based detection of okadaic acid (OA) is in great demand for environmental monitoring and food security. In this study, we reported a biosensing system consisting of a smartphone-based system and cell viability biosensor (CVB) for OA spot fast detection. CVB was constructed by combing living cells and cell counting kit (CCK-8). Thereinto, living cells could be used as reagent without adherent culture on the device surface, and adherent cells and suspension cells were both applicable for cell type selection. The smartphone-based system – bionic electronic eye (Bionic e-Eye) was composed of portable illumination provider and smartphone. The homemade application program (App) – iPlate Monitor integrated the software functions of real-time image acquisition and analysis, data display and storage and instant data sharing. In the measurement, the system was demonstrated to detect OA at concentration as low as 3.4083 μg/L and 13.4456 μg/L by using HepG2 and THP-1 cells, respectively. In addition, this system distinguished OA versus different marine toxins such as gonyautoxin2&3 (GTX2&3) and brevetoxin-2 (PbTx-2) in high specificity. Consequently, the smartphone-based biosensing platform provided a convenient, low-cost, easy-to-use and efficient approach for spot rapid detection of marine toxins such as OA.

Harmful algal blooms (HAB) release microtoxins that contaminate drinking water supplies and risk the health of millions annually. Crystalline ferrate(VI) is a powerful oxidant capable of removing algal microtoxins. We investigate in-situ electrochemically produced ferrate from common carbon steel as an on-demand alternative to crystalline ferrate for the removal of microcystin-LR (MC-LR) and compare the removal efficacy for both electrochemical (EC) and chemical dosing methodologies. We report that a very low dose of EC-ferrate in deionized water (0.5 mg FeO42− L−1) oxidizes MC-LR (MC-LR0 = 10 μg L−1) to below the guideline limit (1.0 μg L−1) within 10 minutes' contact time. With bicarbonate or natural organic matter (NOM), doses of 2.0–5.0 mg FeO42− L−1 are required, with lower efficacy of EC-ferrate than crystalline ferrate due to loss of EC-ferrate by water oxidation. To evaluate the EC-ferrate process to concurrently oxidize micropollutants, coagulate NOM, and disinfect drinking water, we spiked NOM-containing real water with MC-LR and Escherichia coli, finding that EC-ferrate is effective at 10.0 mg FeO42− L−1 under normal operation or 2.0 mg FeO42− L−1 if the test water has initial pH optimized. We suggest in-situ EC-ferrate may be appropriate for sporadic HAB events in small water systems as a primary or back-up technology.

Ricin, Shiga toxin, exotoxin A, and diphtheria toxin are AB-type protein toxins that act within the host cytosol and kill the host cell through pathways involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, we show a single molecule of cytosolic toxin is not sufficient for complete inhibition of protein synthesis or cell death. Furthermore, cells can recover from intoxication: cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations.

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R, 33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned 20R epimer did not.

The influence of nutritional regime and water temperature on depuration rates of OA-group toxins in the wedge shell Donax trunculus was examined by exposing naturally contaminated specimens to three nutritional regimes (microalgae, commercial paste of microalgae, and starvation) for 14 days at 16 °C and 20 °C. Total OA was quantified in the whole soft tissues of the individuals collected in days 2, 4, 6, 8, 10, 12 and 14. Mortality, dry weight, condition index, gross biochemical composition and gametogenic stages were surveyed. Low variation of glycogen and carbohydrates during the experiments suggest that wedge shells were under non-dramatic stress conditions. Wedge shells fed with non-toxic diets showed similar depuration rates being 15 and 38% higher than in starvation, at 16 and 20 °C, respectively. Depuration rates under non-toxic diets at 20 °C were 71% higher than at 16 °C. These results highlight the influence of water temperature on the depuration rate of total OA accumulated by D. trunculus, even when the increase is of only 4 °C, as commonly observed in week time scales in the southern Portuguese coastal waters. These results open the possibility of a faster release of OA in harvested wedge shells translocated to depuration systems when under a slight increase of water temperature.

Actin is one of the most abundant and highly conserved proteins in nature. The cellular pool of actin is roughly evenly divided between monomeric (G-actin) and filamentous (F-actin) forms, with the dynamic transition between these two states being tightly regulated in time and space (1). Most actin functions are linked to its filamentous form. The atomic structure of the actin monomer in complex with DNase I was determined ∼30 y ago (2) and was used to produce the first model of the filament by fitting the 8.4-A-resolution X-ray diffraction pattern of oriented gels of actin filaments (3). Today, more than 100 structures of the monomer have been determined. Crystallization has been made possible using one of several strategies that prevent polymerization, including mutagenesis, covalent modification of actin, and the formation of soluble complexes with actin-binding protein (ABPs) or marine toxins. Remarkably, the conformation of the monomer in all these structures is essentially the same. Actin has four subdomains, with subdomains 1 and 3 being structurally related, and subdomains 2 and 4 consisting of large insertions into subdomains 1 and 3. Because of their relative position in the filament, actin is also described as consisting of two major domains, with subdomains 1 and 2 forming the more exposed outer domain and subdomains 3 and 4 forming the inner domain (Fig. 1). Actin has two opposite clefts, one between subdomains 2 and 4 that harbors the nucleotide-binding site, and one between subdomains 1 and 3 that mediates most of the interactions with ABPs. The polypeptide chain crosses between the outer and inner domains only twice, at helix Q137-S145 and at loop R335-Y337, such that the nucleotide itself becomes a major bridge between the two domains. Fig. 1. Structure of the actin filament in the AMPPNP-bound state (23). ( A ) Orthogonal views of the cryo-EM structure … [↵][1]1Email: droberto{at}pennmedicine.upenn.edu. [1]: #xref-corresp-1-1