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The knowledge of the routes of excretion of the toxins accumulated by molluscs is a key step in designing methods that accelerate depuration. In this work, the excretion route, in mussels and cockles, of the main diarrhetic shellfish poisoning (DSP) toxins in Europe (okadaic acid and dinophysistoxin-2) after natural intoxication were studied. During depuration, the amounts of free toxins and their derivatives were quantified in bivalves, faeces, and water. Most toxins (>98%) were excreted through faeces as acyl derivatives (most likely 7-O-acyl esters), independent of the ratio between these derivatives and free toxins in soft tissues. The small proportion of toxins excreted into water mostly constituted the free forms of the toxins. Both species shared the same route even though they contained very different proportions of free toxins in their soft tissues. No substantial changes in this general pattern were observed during the experiment. The esters of fatty acids with 16 carbon atoms were the most abundant in both soft tissues and faeces, but they were not the same in mussels and cockles. Most of the variability in ester proportions can be attributed to the species more than to their differential excretion (water or faeces) suggesting that there are not large differences in the depuration of the different esters.

Okadaic acid (OA) is a low-molecular-weight marine toxin from shellfish that causes abdominal pain, vomiting and diarrhea, i.e., diarrheic shellfish poisoning. In this study, a ssDNA aptamer that specifically binds to OA with high affinity was obtained via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assisted by graphene oxide (GO). This aptamer was then applied to fabricate a novel direct competitive enzyme-linked aptamer assay (ELAA). At the optimized conditions, this ELAA method showed a low detection limit (LOD of 0.01 ng/mL), wide linear range (from 0.025 to 10 ng/mL), good recovery rate (92.86–103.34% in OA-spiked clam samples) and repeatability (RSD of 2.28–4.53%). The proposed method can be used to detect OA in seafood products with high sensitivity and can potentially be adapted for the determination of other small molecular analytes.

Background/Aims: Okadaic acid (OA) and the structurally related compounds dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine phycotoxins that cause diarrheic shellfish poisoning (DSP) in humans due to ingestion of contaminated shellfish. In order to guarantee consumer protection, the regulatory authorities have defined the maximum level of DSP toxins as 160 µg OA equivalent kg -1 shellfish meat. For risk assessment and overall toxicity determination, knowledge of the relative toxicities of each analogue is required. In absence of enough information from human intoxications, oral toxicity in mice is the most reliable data for establishing Toxicity Equivalence Factors (TEFs). Methods: Toxins were administered to mice by gavage, after that the symptomatology and mice mortality was registered over a period of 24 h. Organ damage data were collected at necropsy and transmission electron microscopy (TEM) was used for ultrastructural studies. Toxins in urine, feces and blood were analyzed by HPLC-MS/MS. The evaluation of in vitro potencies of OA, DTX1 and DTX2 was performed by the protein phosphatase 2A (PP2A) inhibition assay. Results: Mice that received DSP toxins by gavage showed diarrhea as the main symptom. Those toxins caused similar gastrointestinal alterations as well as intestine ultrastructural changes. However, DSP toxins did not modify tight junctions to trigger diarrhea. They had different toxicokinetics and toxic potency. The lethal dose 50 (LD 50 ) was 487 µg kg -1 bw for DTX1, 760 µg kg -1 bw for OA and 2262 µg kg -1 bw for DTX2. Therefore, the oral TEF values are: OA = 1, DTX1 = 1.5 and DTX2 = 0.3. Conclusion: This is the first comparative study of DSP toxins performed with accurate well-characterized standards and based on acute toxicity data. Results confirmed that DTX1 is more toxic than OA by oral route while DTX2 is less toxic. Hence, the current TEFs based on intraperitoneal toxicity should be modified. Also, the generally accepted toxic mode of action of this group of toxins needs to be reevaluated.

A competitive electrochemiluminescence (ECL) immunosensor is proposed to accurately and rapidly assess okadaic acid (OA) levels in shellfish using a novel self-reinforced solid-state ECL marker, which is essential for ensuring seafood safety. Graphene quantum dots doped with nitrogen and sulfur ( N,S -GQDs) were synthesized, for the first time, through the electrolysis of graphite in 3-( N -morpholine) propane sulfonic acid solution. Intriguingly, these N,S -GQDs exhibited exceptional co-reactant properties, significantly enhancing the anodic ECL performance of Ru(bpy) 3 2+ in a phosphate-buffered saline solution. Following the functionalization of Ru(bpy) 3 2+ -doped silica nanoparticles (RuSiNPs) with poly(diallyldimethylammonium) chloride, we achieved a well-dispersed assembly of N,S -GQDs on the exterior of the RuSiNPs through electrostatic interactions. Importantly, the core–shell structure of RuSiNPs@ N,S -GQDs efficiently encapsulated both the luminophore and co-reactant, thus improving the transfer rates of electrons, shorting interaction distances, and reducing energy loss during light emission. Leveraging this “bright” ECL beacon, the ECL immunosensor demonstrated remarkable analytical performance, yielding a low half maximal inhibitory concentration (IC 50 ) of 0.14 ng mL −1 , an extensive linear range spanning 0.003–40 ng mL −1 , and impressively low limit of detection of 0.001 ng mL −1 for OA determination. Graphical Abstract

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 μg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

Lipophilic marine biotoxins (LMBs) are one of the main risks associated with the consumption of mussels and oysters. Sanitary and analytical control programs are developed to detect the occurrence of these toxins in seafood before they reach toxic levels. To ensure quick results, methods must be easy and fast to perform. In this work, we demonstrated that incurred samples were a viable alternative to validation and internal quality control studies for the analysis of LMBs in bivalve mollusks. These samples were used to optimize, validate, and monitor a simple and fast ultrasound-assisted extraction (UAE) procedure. An internal quality control material containing okadaic acid (227 ± 46 µg kg −1 ) was produced and characterized. This material had its homogeneity and stability verified and was included as a quality control in all batches of analytical routine. Besides, a sample pooling protocol for extracts analysis was developed, based on tests for COVID-19. Up to 10 samples could be analyzed simultaneously, reducing the instrumental time of analysis by up to 80%. The UAE and sample pooling approaches were then applied to more than 450 samples, of which at least 100 were positive for the okadaic acid group of toxins. Graphical abstract

Okadaic acid (OKA), a marine toxin produced by dinoflagellates, is responsible for most human diarrhetic shellfish poisoning-associated health disorders. A competitive displacement assay for OKA is described here. An OKA-binding aptamer was truncated with two sequences, one labeled with 6-carboxyfluorescein (FAM), and one with a quencher. On addition of OKA, it will bind to the aptamer and green fluorescence pops up because label and quencher become spatially separated. One of the truncated aptamers exhibis an excellent binding capability (Kd 2.77 nM) for OKA compared to its full-length aptamer (526 nM). The selectivity of the assay was proven by the successful fluorometric determination of OKA in the presence of common diarrhoetic toxins and in shellfish extracts. The detection limit is as low as 39 pg·mL −1 . Graphical abstract Schematic representation of the competitive displacement assay for okadaic acid (OKA). The OKA-binding aptamer is truncated with two parts, one labeled with 6-carboxyfluorescein (FAM), and one with a quencher. On addition of OKA, green fluorescence pops up because label and quencher become spatially separated.

In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical printed (DEP) microarray composed of eight individually addressable carbon electrodes. The electrodeposition of gold nanoparticles on the carbon surface offers high conductivity and enlarges the electroactive area. The immobilization of thiolated aptamers on the AuNP-decorated carbon electrodes provides a stable, well-orientated and organized binary self-assembled monolayer for sensitive and accurate detection. A simple electrochemical multiplexed aptasensor based on AuNPs was designed to synchronously detect multiple cyanotoxins, namely, microcystin-LR (MC-LR), Cylindrospermopsin (CYL), anatoxin-α, saxitoxin and okadaic acid (OA). The choice of the five toxins was based on their widespread presence and toxicity to aquatic ecosystems and humans. Taking advantage of the conformational change of the aptamers upon target binding, cyanotoxin detection was achieved by monitoring the resulting electron transfer increase by square-wave voltammetry. Under the optimal conditions, the linear range of the proposed aptasensor was estimated to be from 0.018 nM to 200 nM for all the toxins, except for MC-LR where detection was possible within the range of 0.073 to 150 nM. Excellent sensitivity was achieved with the limits of detection of 0.0033, 0.0045, 0.0034, 0.0053 and 0.0048 nM for MC-LR, CYL, anatoxin-α, saxitoxin and OA, respectively. Selectivity studies were performed to show the absence of cross-reactivity between the five analytes. Finally, the application of the multiplexed aptasensor to tap water samples revealed very good agreement with the calibration curves obtained in buffer. This simple and accurate multiplexed platform could open the window for the simultaneous detection of multiple pollutants in different matrices.

For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (Mytilus galloprovincialis) as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5–75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves Magallana gigas (21 µg kg−1) and Tellina donacina (63 µg kg−1), respectively. However, mean concentrations for the bivalve group were shallow (2–6 µg kg−1). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6–44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg−−1, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-β-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1–5, the 24-O-β-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4–6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.