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Covering period: up to 2019Water-soluble natural products constitute a relevant group of secondary metabolites notably known for presenting potent biological activities. Examples are aminoglycosides, β-lactam antibiotics, saponins of both terrestrial and marine origin, and marine toxins. Although extensively investigated in the past, particularly during the golden age of antibiotics, hydrophilic fractions have been less scrutinized during the last few decades. This review addresses the possible reasons on why water-soluble metabolites are now under investigated and describes approaches and strategies for the isolation of these natural compounds. It presents examples of several classes of hydrosoluble natural products and how they have been isolated. Novel stationary phases and chromatography techniques are also reviewed, providing a perspective towards a renaissance in the investigation of water-soluble natural products.

A total of 1338 samples were analyzed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to study the toxin profiles of lipophilic marine toxins in bivalve mollusks collected from the southeast coast of China from 2017 to 2020. The most abundant toxin was HomoYTX, followed progressively by YTX and PTX2. Low proportions of OA, DTX-1, and DTX-2 were found. No AZA1, AZA2, and AZA3 were quantified above limit of quantitation (LOQ). The highest concentrations of HomoYTX, YTX, PTX2, OA, DTX-1, and DTX-2 were 429, 98.0, 40.3, 33.0, 22.6, and 26.5 μg/kg, respectively. Mussels (Mytilus galloprovincialis, Perna viridis), scallop (Chlamys farreri) and clam (Atrina pectinate) accumulated higher toxin levels than clams (Sinonovaculla Constricta, Ruditapes philippinarum), oyster (Crassostrea gigas) and scallop (Arca granosa). Homo YTX and PTX2 levels reached the maximum in July and June, respectively, and the OA-group peaked in August. The results provide a reliable basis for monitoring marine toxins and protecting the health of aquatic consumers.

Azaspiracids (AZAs) are marine toxins produced by dinoflagellates belonging to the genera Azadinium and Amphidoma that caused human intoxications after consumption of contaminated fishery products, such as mussels. However, the exact mechanism for the AZA induced cytotoxic and neurotoxic effects is still unknown. In this study several pharmacological approaches were employed to evaluate the role of anion channels on the AZA effects that demonstrated that cellular anion dysregulation was involved in the toxic effects of these compounds. The results presented here demonstrated that volume regulated anion channels (VRACs) are affected by this group of toxins, and, because there is not any specific activator of VRACs besides the intracellular application of GTPγ-S molecule, this group of natural compounds could represent a powerful tool to analyze the role of these channels in cellular homeostasis. In addition to this, in this work, a detailed pharmacological approach was performed in order to elucidate the anion channels present in human HEK293 cells as well as their regulation by the marine toxins azaspiracids. Altogether, the data presented here demonstrated that the effect of azaspiracids in human cells was completely dependent on ATP-regulated anion channels, whose upregulation by these toxins could lead to regulatory volume decrease and underlie the reported toxicity of these compounds.

Okadaic acid (OA) is one of the marine toxins that are widely distributed and harmful to humans. However, the current detection methods for OA involve complex procedures, need long detection time, and rely on large-scale laboratory equipment. In this work, a multi-channel handheld automatic spectrometer (MHAS) based on a spectral sensor was developed with the advantages of small size, simple operation and low cost. It could achieve rapid detection within 30 s and a wide spectral detection range of 470–780 nm with a broadband LED as the light source and a microplate containing 8 wells as a sample cell. Moreover, through the combination of gold nanoparticles (AuNPs) and aptamer-OA34, a highly sensitive and rapid system for OA detection was established with a LOD of 1.80 μg L−1 and a wide detection range of 20–10 000 μg L−1, which is comparable to a microplate reader. Compared with other studies, the proposed MHAS realized rapid on-site detection of OA with a wider detection range, shorter detection time and higher portability. Therefore, the MHAS promises to be a stable and efficient optical detection instrument for on-site detection in the fields of food safety, disease diagnosis and environmental monitoring.

With harmful algal blooms, marine food poisoning caused by marine biotoxins frequently occurs and is life-threatening if severe. However, the conventional detection methods of marine toxins have a few limitations: low sensitivity and high-cost. Therefore, it is necessary to establish a fast and sensitive on-site detection method for real seafood sample. Biosensors based on aptamers, antibodies, and cells have been applied in marine toxins monitoring. This review presents the classification and toxic effects of marine toxins, and recent biosensor for marine toxin detection. In addition, we have compared the superiority and limitation of these biosensors. Finally, challenges and opportunities of biosensors in food safety detection were discussed. Considering the excellent results achieved by the aptasensor in the field of detection, it seems ready to be put into practical applications.

Health risk of human exposure to microcystin-leucine arginine (MC-LR) has drawn more and more attention in recent years. In the present study, MC-LR inhibited miR-3473g expression of mouse granulosa cells both in vitro and in vivo. By dual-luciferase reporter assay, we confirmed miR-3473g is able to bind the 3′-UTR of StAR protein (StAR) mRNA and suppress StAR expression. Thus, downregulation of miR-3473g after MC-LR exposure led to StAR overexpression. Excessive StAR probably transported much more cholesterol into the inner membrane of the mitochondria and finally resulted in overproduction of progesterone. Our results revealed that MC-LR exposure was associated with premature luteinization of granulosa cells and may adversely affect women’s fertility.

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.

Prevalence and incidence of the marine toxins (paralytic, amnesic, and lipophilic toxins) including the so-called emerging toxins (these are, gymnodimines, pinnatoxins, or spirolides among others) have increased in recent years all over the world. Climate change, which is affecting the distribution of their producing phytoplankton species, is probably one of the main causes. Early detection of the toxins present in a particular area, and linking the toxins to their causative phytoplankton species are key tools to minimize the risk they pose for human consumers. The development of both types of studies requires fast and highly sensitive analytical methods. In the present work, we have developed a highly sensitive liquid chromatography-mass spectrometry methodology (LC-MS/MS), using a column with fused-core particle technology, for the determination of fourteen lipophilic toxins in a single run of 3.6 min. The performance of the method was evaluated for specificity, linearity, precision (repeatability and reproducibility) and accuracy by analysing spiked and naturally contaminated samples. The in-house validation was successful, and the limit of detection (LOD) and quantification (LOQ) for all the toxins were far below their regulatory action limits. The method is suitable to be considered in monitoring systems of bivalves for food control.

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.

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.

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.

Discovery of novel bioactive compounds is important not only for therapeutic purposes but also for understanding the mechanisms of biological processes. To screen bioactive compounds that affect nuclear morphology in marine organism extracts, we employed a microscopy-based assay using DNA staining of human cancer cells. A crude extract from a marine sponge Mycale aff. nullarosette, collected from the east coast of Japan, induced cellular binucleation. Fractionation of the extract led to the isolation of mycalolides A and B, and 38-hydroxymycalolide B as the active components. Mycalolides have been identified as marine toxins that induce depolymerization of the actin filament. Live cell imaging revealed that low concentrations of mycalolide A produce binucleated cells by inhibiting the completion of cytokinesis. At higher concentrations, however, mycalolide A causes immediate disruption of actin filaments and changes in cell morphology, yielding rounded cells. These results suggest that the completion of cytokinesis is a process requiring high actin polymerization activity. Furthermore, luciferase reporter assays with mycalolide A treatments support the view that the level of globular actin can affect transcription of a serum response gene.

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 removal of microcystin-LR from aqueous solution using native Moringa oleifera Lam. seeds powder (MSP) and chemically pretreated M. oleifera Lam. seed powder (PMSP) was investigated in terms of equilibrium and kinetics. Optimum sorption conditions were determined as a function of pH (2–7), adsorbent dosage (0.25–1.0 g/L), initial concentration of microcystin-LR (15–120 mg/L) and contact time (15–360 minutes). The high values of regression constant, 0.98 (MSP) and 0.99 (PMSP), revealed that sorption of microcystin-LR was best fitted by the pseudo second order kinetic model. The equilibrium study was best fitted by the Freundlich model with both the adsorbents. The maximum sorption capacity by MSP and PMSP for microcystin-LR was 85.5 ± 1.1 mg/g and 92.49 ± 2.4 mg/g respectively. Fourier transform infrared spectroscopy showed the major involvement of carboxyl and hydroxyl groups for microcystin-LR sequestration either by complexation or ion exchange mechanism. The contribution of the adsorption phenomenon was confirmed by scanning electron microscopic analysis of microcystin-LR loaded and unloaded PMSP. Thus, the HCl-pretreated M. oleifera Lam. seed powder proved to be the pre-eminent biosorbent for removal of microcystin-LR from the wastewater stream.

In this work, a sensitive and selective electrochemical aptasensor for determination of microcystin-LR (MC-LR) was developed based on a dual signal amplification system consisting of a novel ternary composite and horseradish peroxidase (HRP). The ternary composite was prepared by depositing gold nanoparticles (AuNPs) on molybdenum disulfide (MoS2) covered TiO2 nanobeads (TiONBs). MoS2 nanosheet modified TiONBs provided a large surface area for immobilization of AuNPs and biomolecules. The ternary composite also possesses an improved electron transfer and catalytic capability. To construct the aptasensor, thiolated MC-LR aptamers were immobilized on the AuNP@MoS2-TiONB modified electrode through a gold-sulfur bond. Then, biotin-cDNA with a sequence complementary to the MC-LR aptamer competed with MC-LR for binding to the immobilized aptamer. The current signal catalyzed by avidin-HRP decreased with the increase of MC-LR, based on which a linear range of 0.005-30 nM and a detection limit of 0.002 nM were obtained.

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.

This study revealed the dynamics of the genus Ostreopsis in the south-western Mediterranean Sea fish farm during the 2016 and 2017 summers. This phytoplankton is known to produce palytoxin-like compounds, listed among the most potent marine toxins known, and can pose a serious concern for humans in the Mediterranean area. Principal component analysis (PCA) explained the significance of temperature, salinity, and dissolved inorganic nitrogen in the proliferation of this toxic dinoflagellate. The peak of the Ostreopsis sp. (6.34 × 103 cells L−1) was recorded at 28.4 °C, at a salinity of 38.3 PSU, and the dissolved inorganic nitrogen had a value of 0.60 μmol L−1. Our results highlight the importance of monitoring the proliferation of this harmful dinoflagellate in southern Mediterranean waters.

In the past twenty years marine biotoxin analysis in routine regulatory monitoring has advanced significantly in Europe (EU) and other regions from the use of the mouse bioassay (MBA) towards the high-end analytical techniques such as high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS). Previously, acceptance of these advanced methods, in progressing away from the MBA, was hindered by a lack of commercial certified analytical standards for method development and validation. This has now been addressed whereby the availability of a wide range of analytical standards from several companies in the EU, North America and Asia has enhanced the development and validation of methods to the required regulatory standards. However, the cost of the high-end analytical equipment, lengthy procedures and the need for qualified personnel to perform analysis can still be a challenge for routine monitoring laboratories. In developing regions, aquaculture production is increasing and alternative inexpensive Sensitive, Measurable, Accurate and Real-Time (SMART) rapid point-of-site testing (POST) methods suitable for novice end users that can be validated and internationally accepted remain an objective for both regulators and the industry. The range of commercial testing kits on the market for marine toxin analysis remains limited and even more so those meeting the requirements for use in regulatory control. Individual assays include enzyme-linked immunosorbent assays (ELISA) and lateral flow membrane-based immunoassays (LFIA) for EU-regulated toxins, such as okadaic acid (OA) and dinophysistoxins (DTXs), saxitoxin (STX) and its analogues and domoic acid (DA) in the form of three separate tests offering varying costs and benefits for the industry. It can be observed from the literature that not only are developments and improvements ongoing for these assays, but there are also novel assays being developed using upcoming state-of-the-art biosensor technology. This review focuses on both currently available methods and recent advances in innovative methods for marine biotoxin testing and the end-user practicalities that need to be observed. Furthermore, it highlights trends that are influencing assay developments such as multiplexing capabilities and rapid POST, indicating potential detection methods that will shape the future market.

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.

Molluscs provided one of the pioneering approved pharmaceuticals from the seas: the painkiller ziconotide, developed from an ω-conotoxin isolated from cone snails. As marine biotechnologists are turning towards the immense range of novel bioproducts from marine invertebrates, little attention has been given to cephalotoxins, a group of obscure proteinaceous toxins produced by the salivary glands of coleoids, i.e., octopuses, squids and cuttlefishes. These toxins, for which there is empirical evidence for acting as immobilisers at least against crustaceans, are proteinaceous substances among the many that comprise the venomous mixtures secreted by these animals. Despite the ecological and economical importance of cephalopods, little is known about cephalotoxins, beginning with the actual span of taxa that secrete them. Indeed, cephalopods are long suspected for producing specific toxins as part of their predation and defence mechanisms, making them a promising group of marine animals for the bioprospecting of novel compounds. Despite scant or absent toxicological or otherwise experimental evidence for their bioreactivity, advances in “omics” methods have shed some light in the molecular structure of cephalotoxins. There are reports of cephalotoxins being complex glycoproteins that take part in a myriad of novel compounds being produced by the salivary glands. Still, there is no consensus of cephalotoxins being a conserved form of proteins. As Blue Biotechnology and marine bioprospecting for novel bioreactives are gaining momentum, the present review will provide the state-of-the-art on cephalotoxins, highlighting old and new research and existing gaps in the current knowledge.

Shellfish toxin monitoring programs often use mussels as the sentinel species to represent risk in other bivalve shellfish species. Studies have examined accumulation and depuration rates in various species, but little information is available to compare multiple species from the same harvest area. A 2-year research project was performed to validate the use of mussels as the sentinel species to represent other relevant eastern Canadian shellfish species (clams, scallops, and oysters). Samples were collected simultaneously from Deadmans Harbour, NB, and were tested for paralytic shellfish toxins (PSTs) and amnesic shellfish toxin (AST). Phytoplankton was also monitored at this site. Scallops accumulated PSTs and AST sooner, at higher concentrations, and retained toxins longer than mussels. Data from monitoring program samples in Mahone Bay, NS, are presented as a real-world validation of findings. Simultaneous sampling of mussels and scallops showed significant differences between shellfish toxin results in these species. These data suggest more consideration should be given to situations where multiple species are present, especially scallops.

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.

10 pages, 4 figures, supporting information https://doi.org/10.1021/acs.est.0c05795, Critical research is needed regarding harmful algal blooms threatening ecosystem and human health, especially through respiratory routes. Additional complexity comes from the poorly understood factors involved in the physical production of marine aerosols coupled with complex biogeochemical processes at ocean surfaces. Here—by using a marine aerosol generation tank—five bubble-bursting experiments (with contrasting incubation times and, likely, physiological microalgal states) were run to investigate simultaneously the concentrations of the toxins, synthesized by a natural Ostreopsis cf. ovata bloom, in suspension in the water and in the atmosphere. The first two experiments (EXP1–2) were run with moderate levels of O. cf. ovata cell numbers (ca. 105 cells·L–1) and total toxin in suspension (4 × 106 pg·Lwater–1) obtained at an early phase of the bloom. After 0.75–4 h incubation, toxin concentration in the aerosols accounted for 49–69 pg·Lair–1. By striking contrast, three experiments (EXP3–5)—conducted with samples collected two weeks later with higher cell abundances and higher toxin concentration in the seston (respectively, about 1 × 106 cells·L–1 and 2 × 108 pg·Lwater–1) and incubated for 21 h—showed about 15-fold lower atmospheric concentrations (3–4 pg·Lair–1), while important foam accumulation was observed in the water surface in the tank. Offline spectroscopic analysis performed by proton-nuclear magnetic resonance spectroscopy showed that the particulate organic carbon in the water was drastically different from that of bubble-bursting aerosols from the tank experiments—suggesting a selective transfer of organic compounds from seawater into the atmosphere. Overall, the results suggest that aerosol production and diffusion of marine toxins in the atmosphere are regulated by complex interactions between biological processes and air-sea aerosol production dynamics, The authors gratefully acknowledge the financial support received by N.I.M-P. from the Spanish Ministry of Science, Innovation and Universities through the project PGC2018-095013-B-I00 and from the Agency for Administration of University and Research Grants (Government of Catalonia, Spain) through the project 2017SGR−310. N.I.M-P also thanks the PhD research financial assistance of Water Research Institute (IdRA) of the University of Barcelona. The study was supported by the CoCliME project, an ERA4CS Network (ERA-NET) initiated by JPI Climate and funded by EPA (IE), ANR (FR), BMBF (DE), UEFISCDI (RO), RCN (NO), and FORMAS (SE), with co-funding by the European Union (Grant no. 690462). CoCliME is endorsed to the internationa lprogramme on harmful algal blooms GlobalHAB (www.globalhab.info). Funds were also provided by Agencia Estatal de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER) to the PI-ICE (CTM2017−89117-R) project, and M.D.A. received a Ramón y Cajal fellowship (RYC-2012-1192 With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Globally, the livelihoods of over a billion people are affected by changes to marine ecosystems, both structurally and systematically. Resources and ecosystem services, provided by the marine environment, contribute nutrition, income, and health benefits for communities. One threat to these securities is ciguatera poisoning; worldwide, the most commonly reported non-bacterial seafood-related illness. Ciguatera is caused by the consumption of (primarily) finfish contaminated with ciguatoxins, potent neurotoxins produced by benthic single-cell microalgae. When consumed, ciguatoxins are biotransformed and can bioaccumulate throughout the food-web via complex pathways. Ciguatera-derived food insecurity is particularly extreme for small island-nations, where fear of intoxication can lead to fishing restrictions by region, species, or size. Exacerbating these complexities are anthropogenic or natural changes occurring in global marine habitats, e.g., climate change, greenhouse-gas induced physical oceanic changes, overfishing, invasive species, and even the international seafood trade. Here we provide an overview of the challenges and opportunities of the 21st century regarding the many facets of ciguatera, including the complex nature of this illness, the biological/environmental factors affecting the causative organisms, their toxins, vectors, detection methods, human-health oriented responses, and ultimately an outlook towards the future. Ciguatera research efforts face many social and environmental challenges this century. However, several future-oriented goals are within reach, including digital solutions for seafood supply chains, identifying novel compounds and methods with the potential for advanced diagnostics, treatments, and prediction capabilities. The advances described herein provide confidence that the tools are now available to answer many of the remaining questions surrounding ciguatera and therefore protection measures can become more accurate and routine.

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.

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.

Foodborne diseases caused by pathogens and toxins are a serious threat to food safety and human health; thus, they are major concern to society. Existing conventional foodborne pathogen or toxin detection methods, including microbiological assay, nucleic acid-based assays, immunological assays, and instrumental analytical method, are time-consuming, labor-intensive and expensive. Because of the fast response and high sensitivity, cell-based biosensors are promising novel tools for food safety risk assessment and monitoring. This review focuses on the properties of mammalian cell-based biosensors and applications in the detection of foodborne pathogens (bacteria and viruses) and toxins (bacterial toxins, mycotoxins and marine toxins). We discuss mammalian cell adhesion and how it is involved in the establishment of 3D cell culture models for mammalian cell-based biosensors, as well as evaluate their limitations for commercialization and further development prospects.

Azaspiracid toxins were first identified at the end of the last century in Irish mussels, and during the last two decades considerable cytotoxic and neurotoxic effects caused by these toxins have been described. Azaspiracids are synthesized by dinoflagellates and accumulate in several species of filter-feeding bivalve mollusks, thereby incorporating into the food chain and causing human intoxications. Among the cellular effects of azaspiracids, inhibition of spikes in neurons and hyperpolarization of the neuronal membrane potential have been reported; however, the underlying processes leading to these effects were never elucidated. In this regard, initial studies reported no activity of the toxin in neuronal voltage-gated sodium channels, and a recent work described no effect of azaspiracid-1 on the inactivation kinetics of voltage-gated sodium channels; however, the relationship between the known alterations of the cytoskeleton caused by these toxins and their effects on ion channels has never been evaluated. In this work, the cytotoxic effect of azaspiracids was evaluated in human cells as well as their activity on voltage-gated sodium channels and in cell morphology in order to unravel the cellular targets involved in the mechanism of action of this group of marine toxins. The data reported here demonstrate, for the first time, that both azaspiracid-1 and azaspiracid-2 caused a rapid concentration-dependent inhibition of the amplitude of voltage-gated sodium currents without affecting their inactivation kinetics, an effect that was increased after long-term treatment of the cells with the toxin. Simultaneously, long-term exposure of the cells to azaspiracids caused a profound alteration of the cell cytoskeleton and decreased the metabolic activity of human cells. Altogether, the data presented here indicate that the partial blockade of voltage-gated sodium channels by these toxins is not related with their effect on the actin cytoskeleton. However, since azaspiracids are common toxins in European waters, their effect on voltage-gated sodium channels, first reported here, should be considered to avoid synergistic toxicity with other marine toxins that are known potent blockers of sodium channels such as the saxitoxins and tetrodotoxins, but further studies are needed in order to elucidate how these compounds alter ion homeostasis.

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

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.

Shellfish toxins, as one kind of marine toxin, have attracted worldwide attention due to their severe threat to food safety and human health. Therefore, it is highly essential and urgent to develop a low-cost and convenient method to detect these toxins. With the rapid advance in microfabrication processes, micro/nano biosensors provide novel approaches to address this issue. In addition to their features of low cost, portability, easy operation, high efficiency and high bioactivity, micro/nano biosensors have great potential to realize on-the-spot, rapid detection of shellfish toxins. This review focuses on the most recent advances in the development of micro/nano biosensors for shellfish toxin detection. These biosensors are mainly classified into five categories according to their transducer detection principles, which include optical devices, electrochemical sensors, electrochemiluminescence, field-effect transistors, and acoustic devices. Sensor strategies, toxin analytes, biosensitive elements, coupling methods and field detection performance are highlighted to discuss the applications of shellfish toxin detection. With advances in sensor technology, biomaterials, microfabrication and miniaturized electronics, micro/nano biosensors applied to in-field fast detection of shellfish toxins are expected to play a critical role in food safety, environmental monitoring, and foreign trade in the foreseeable future. Finally, the current challenges and future development trends of micro/nano biosensors for shellfish toxin detection are discussed.

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.