Controlled Spread of a Ag Layer from the Core to the Tip along the Branches of AuAg Nanostars for Improved SERS Detection of Okadaic Acid in Shellfish.

Plasmonic Au-Ag nanostars are excellent surface-enhanced Raman scattering (SERS) probes due to bimetallic coupling and the tip effect. However, the existing preparation methods of AuAg nanostars cannot achieve controlled growth of the Ag layer on the branches of nanostars and so cannot display their SERS to the maximum extent, thus limiting its sensitivity in biosensing. Herein, a novel strategy "PEI (polyethylenimine)-guided Ag deposition method" is proposed for synthesizing AuAg core-shell nanostars (AuAg@Ag NS) with a tunable distribution of the Ag layer from the core to the tip, which offers an avenue for investigating the correlation between SERS efficiency and the extent of spread of the Ag layer. It is found that AuAg@Ag NS with a Ag layer coated the whole branch has the strongest SERS performance because the coupling between the tips and Ag layer is maximized. Meanwhile, as a completely closed core-shell structure, AuAg@Ag NS can confine and anchor 4-ATP inside the Ag layer to avoid an unstable SERS signal. By connecting the aptamer, a reliable internal standard nanoprobe with a SERS enhancement factor (EF) up to 1.86 × 108 is prepared. Okada acid is detected through competitive adsorption of this SERS probes, and the detection limit is 36.6 pM. The results gain fundamental insights into tailoring the nanoparticle morphologies and preparation of internal standard nanoprobes and also provide a promising avenue for marine toxin detection in food safety.

Spatial distribution of lipophilic shellfish toxins in seawater and sediment in the Bohai Sea and the Yellow Sea, China.

Lipophilic shellfish toxins (LSTs) are widely distributed in marine environments worldwide, potentially threatening marine ecosystem health and aquaculture safety. In this study, two large-scale cruises were conducted in the Bohai Sea and the Yellow Sea, China, in spring and summer 2023 to clarify the composition, concentration, and spatial distribution of LSTs in the water columns and sediments. Results showed that okadaic acid (OA), dinophysistoxin-1 (DTX1) and/or pectenotoxin-2 (PTX2) were detected in 249 seawater samples collected in spring and summer. The concentrations of ∑LSTs in seawater were ranging of ND (not detected) -13.86, 1.60-17.03, 2.73-17.39, and 1.26-30.21 pmol L-1 in the spring surface, intermediate, bottom water columns and summer surface water layers, respectively. The detection rates of LSTs in spring and summer seawater samples were 97% and 100%, respectively. The high concentrations of ∑LSTs were mainly distributed in the north Yellow Sea and the northeast Bohai Sea in spring, and in the northeast Yellow Sea, the waters around Laizhou Bay and Rongcheng Bay in summer. Similarly, only OA, DTX1 and PTX2 were detected in the surface sediments. Overall, the concentration of ∑LSTs in the surface sediments of the northern Yellow Sea was higher than that in other regions. In sediment cores, PTX2 was mainly detected in the upper sediment samples, whereas OA and DTX1 were detected in deeper sediments, and LSTs can persist in the sediments for a long time. Overall, OA, DTX1 and PTX2 were widely distributed in the water column and surface sediments in the Bohai Sea and the Yellow Sea, China. The results of this study contribute to the understanding of spatial distribution of LSTs in seawater and sediment environmental media and provide basic information for health risk assessment of phycotoxins.

Spatiotemporal distribution of lipophilic shellfish toxins in plankton and shellfish in the offshore regions of Shandong province, China.

Lipophilic shellfish toxins (LSTs) threaten the ecosystem health and seafood safety. To comprehensively investigate the spatiotemporal distribution of common LSTs in phytoplankton, zooplankton and economic shellfish, three cruises were conducted in five typical offshore aquaculture regions of Shandong province, China, including Haizhou Bay, Jiaozhou Bay, Sanggou Bay, Sishili Bay and Laizhou Bay, in spring (March-April), summer (July-August) and autumn (November-December). This study revealed significant variability in the composition and content of LSTs in phytoplankton samples collected from different regions. Pectenotoxin-2 (PTX2), dinophysistoxin-1 (DTX1) and okadaic acid (OA) were mainly detected in the ranges of not detected (nd)-5045 pmol g-1 dry weight (dw), nd-159 pmol g-1 dw, and nd-154 pmol g-1 dw, respectively. In zooplankton, DTX1 and OA were the predominant components of LSTs, with the highest levels of ∑LSTs in spring ranging from nd to 406 pmol g-1 dw. Spearman's correlation analysis between LSTs and environmental factors indicated significant correlations for the contents of homo-yessotoxin (hYTX), gymnodimine-A (GYM-A), and spirolide-1 (SPX1) with these factors. Totally relatively low levels of LSTs with dominative DTX1 were detected in economic shellfish, which showed a low risk to seafood safety for human health.

Role of turbulence in Dinophysis spp. growth, feeding, and toxin leakage in culture.

Dinophysis, a mixotrophic dinoflagellate that is known to prey on the ciliate Mesodinium rubrum, and retain its chloroplasts, is responsible for diarrhetic shellfish poisoning (DSP) in humans and has been identified on all U.S. coasts. Monocultures of Dinophysis have been used to investigate the growth of Dinophysis species in response to variations in environmental conditions, however, little is known about the roles of system stability (turbulence) and mixotrophy in the growth and toxicity of Dinophysis species in the U.S.. To begin to address this gap in knowledge, culturing experiments were conducted with three species (four strains) of Dinophysis, that included predator-prey co-incubation (Dinophysis spp.+ M. rubrum) and prey-only (M. rubrum) flasks. Cultures were investigated for effects of low or high turbulence on Dinophysis spp. growth, feeding, and amounts of intra- and extracellular toxins: okadaic acid and derivatives (diarrhetic shellfish toxins, DSTs) and pectenotoxins (PTXs). Turbulence did not have a measurable effect on the rates of ingestion of M. rubrum prey by Dinophysis spp. for any of the four strains, however, effects on growth and particulate and dissolved toxins were observed. High turbulence (ε = 10-2 m2s-3) significantly slowed growth of both D. acuminata and D. ovum relative to still controls, but significantly stimulated growth of the D. caudata strain. Increasing turbulence also resulted in significantly higher intracellular toxin content in D. acuminata cultures (DSTs and PTXs), but significantly reduced intracellular toxin content (PTXs) in those of D. caudata. An increase in turbulence appeared to promote toxin leakage, as D. ovum had significantly more extracellular DSTs found in the medium under high turbulence when compared to the still control. Overall, significant responses to turbulence were observed, whereby the three strains from the "Dinophysis acuminata complex" displayed a stress response to turbulence, i.e., decreasing growth, increasing intracellular toxin content and/or increasing toxin leakage, while the D. caudata strain had an opposite response, appearing stimulated by, or more tolerant of, high turbulence.

A multi-omics approach to elucidate okadaic acid-induced changes in human HepaRG hepatocarcinoma cells.

Okadaic acid (OA), a prevalent marine biotoxin found in shellfish, is known for causing acute gastrointestinal symptoms. Despite its potential to reach the bloodstream and the liver, the hepatic effects of OA are not well understood, highlighting a significant research gap. This study aims to comprehensively elucidate the impact of OA on the liver by examining the transcriptome, proteome, and phosphoproteome alterations in human HepaRG liver cells exposed to non-cytotoxic OA concentrations. We employed an integrative multi-omics approach, encompassing RNA sequencing, shotgun proteomics, phosphoproteomics, and targeted DigiWest analysis. This enabled a detailed exploration of gene and protein expression changes, alongside phosphorylation patterns under OA treatment. The study reveals concentration- and time-dependent deregulation in gene and protein expression, with a significant down-regulation of xenobiotic and lipid metabolism pathways. Up-regulated pathways include actin crosslink formation and a deregulation of apoptotic pathways. Notably, our results revealed that OA, as a potent phosphatase inhibitor, induces alterations in actin filament organization. Phosphoproteomics data highlighted the importance of phosphorylation in enzyme activity regulation, particularly affecting proteins involved in the regulation of the cytoskeleton. OA's inhibition of PP2A further leads to various downstream effects, including alterations in protein translation and energy metabolism. This research expands the understanding of OA's systemic impact, emphasizing its role in modulating the phosphorylation landscape, which influences crucial cellular processes. The results underscore OA's multifaceted effects on the liver, particularly through PP2A inhibition, impacting xenobiotic metabolism, cytoskeletal dynamics, and energy homeostasis. These insights enhance our comprehension of OA's biological significance and potential health risks.

Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).

This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L-1, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.

Assay for okadaic acid O-acyl transferase using HPLC-FLD.

Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins (DSTs), which may cause gastric illness in people consuming such as bivalves. Both compounds convert to dinophysistoxin 3 (DTX3, 3; generic name for 1 and 2 with fatty acids conjugated at 7-OH) in bivalves. The enzyme okadaic acid O-acyl transferase (OOAT) is a membrane protein found in the microsomes of the digestive glands of bivalves. In this study, we established an in vitro enzymatic conversion reaction using 4-nitro-2,1,3-benzoxadiazole (NBD)-OA (4), an OA derivative conjugated with (R)-(-)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APy) on 1-CO2H, as a substrate. We detected the enzymatically produced 3, NBD-7-O-palmitoyl-OA (NBD-Pal-OA), using high-performance liquid chromatography-fluorescence detection. We believe that an OOAT assay using 4 will facilitate the fractionation and isolation of OOAT in the future.

A graphene oxide-assisted protein immobilization paper-tip immunosensor with smartphone and naked eye readout for the detection of okadaic acid.

BACKGROUND: Okadaic acid (OA), as a diarrhetic shellfish poisoning, can increase the risk of acute carcinogenic or teratogenic effects for the ingestion of OA contaminated shellfish. At present, much effort has been made to graft immunoassay onto a paper substrate to make paper-based sensors for rapid and simple detection of shellfish toxin. However, the complicated washing steps and low protein fixation efficiency on the paper substrate need to be further addressed. RESULTS: A novel paper-tip immunosensor for detecting OA was developed combined with smartphone and naked eye readout. The trapezoid paper tip was consisted of quantitative and qualitative detection zones. To improve the OA antigen immobilization efficiency on the paper substrate, graphene oxide (GO)-assisted protein immobilization method was introduced. Meanwhile, Au nanoparticles composite probe combined with the lateral flow washing was developed to simplify the washing step. The OA antigen-immobilized zone, as the detection zone Ⅰ, was used for quantitative assay by smartphone imaging. The paper-tip front, as the detection zone Ⅱ, which could qualitatively differentiate OA pollution level within 45 min using the naked eye. The competitive immunoassay on the paper tip exhibited a wide linear range for detecting OA (0.02-50 ng∙mL-1) with low detection limit of 0.02 ng∙mL-1. The recovery of OA in spiked shellfish samples was in the range of 90.3 %-113.%. SIGNIFICANCE: These results demonstrated that the proposed paper-tip immunosensor could provide a simple, low-cost and high-sensitivity test for OA detection without the need for additional large-scale equipment or expertise. We anticipate that this paper-tip immunosensor will be a flexible and versatile tool for on-site detecting the pollution of marine products.

An electrochemiluminescent dual-signal immunosensor based on a Ru(bpy)32+-doped silica nanoparticle/copper nanocluster composite for okadaic acid detection.

In this study, a sensitive dual-signal electrochemiluminescence (ECL) immunosensor was developed for okadaic acid (OA) detection utilizing copper nanoclusters (CuNCs) and Ru(bpy)32+-doped silica nanoparticles (RuSiNPs). Interestingly, the CuNCs could simultaneously enhance both cathodic (-0.95 V) and anodic (+1.15 V) ECL signals of RuSiNPs, forming a dual-signal ECL sensing platform. Further, RuSiNPs@CuNCs were used as immunomarkers by covalently conjugating them with an anti-OA monoclonal antibody (mAb) to form probes. Finally, dual ECL signals of the immunosensor were fabricated and showed good linear relationships with OA concentrations in the range of 0.05-70 ng mL-1, having a median inhibitory concentration (IC50) of 1.972 ng mL-1 and a limit of detection of 0.039 ng mL-1. Moreover, the constant ratio of the cathodic and anodic ECL peaks achieved self-calibration of the detection signal and improved the reliability of the results. Finally, we successfully applied the ECL sensor to detect OA in spiked oyster samples.

Development of a Multiplexed Electrochemical Aptasensor for the Detection of Cyanotoxins.

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.

Polystyrene microplastics exacerbated the toxicity of okadaic acid to the small intestine in mice.

Microplastics (MPs) and okadaic acid (OA) are known to coexist in marine organisms, potentially impacting humans through food chain. However, the combined toxicity of OA and MPs remains unknown. In this study, mice were orally administered OA at 200 µg/kg bw and MPs at 2 mg/kg bw. The co-exposure group showed a significant increase in malondialdehyde (MDA) content and significant decreases in superoxide dismutase (SOD) activity and glutathione (GSH) level compared to the control, MPs and OA groups (p < 0.05). Additionally, the co-exposure group exhibited significantly higher levels of IL-1ß and IL-18 compared to other groups (p < 0.05). These results demonstrated that co-exposure to MPs and OA induces oxidative stress and exacerbates inflammation. Histological and cellular ultrastructure analyses suggested that this combined exposure may enhance gut damage and compromise barrier integrity. Consequently, the concentration of OA in the small intestine of the co-exposure group was significantly higher than that in the OA group. Furthermore, MPs were observed in the lamina propria of the gut in the co-exposure group. Transcriptomic analysis revealed that the co-exposure led to increased expression of certain genes related to the NF-κB/NLRP3 pathway compared to the OA and MPs groups. Overall, this combined exposure may disrupt the intestinal barrier, and promote inflammation through the NF-κB/NLRP3 pathway. These findings provide precious information for the understanding of health risks associated with MPs and phycotoxins.

Protective effects of 2,4-dinitrophenol in okadaic acid-induced cellular model of Alzheimer's disease.

Alzheimer's disease (AD) research started several decades ago and despite the many efforts employed to develop new treatments or approaches to slow and/or revert disease progression, AD treatment remains an unsolved issue. Knowing that mitochondria loss of function is a central hub for many AD-associated pathophysiological processes, there has been renewed interest in exploring mitochondria as targets for intervention. In this perspective, the present study was aimed to investigate the possible beneficial effects of 2,4 dinitrophenol (DNP), a mitochondrial uncoupler agent, in an in vitro model of AD. Retinoic acid-induced differentiated SH-SY5Y cells were incubated with okadaic acid (OA), a neurotoxin often used as an AD experimental model, and/or with DNP. OA caused a decrease in neuronal cells viability, induced multiple mitochondrial anomalies including increased levels of reactive oxygen species, decreased bioenergetics and mitochondria content markers, and an altered mitochondria morphology. OA-treated cells also presented increased lipid peroxidation levels, and overactivation of tau related kinases (GSK3ß, ERK1/2 and AMPK) alongside with a significant augment in tau protein phosphorylation levels. Interestingly, DNP co-treatment ameliorated and rescued OA-induced detrimental effects not only on mitochondria but also but also reinstated signaling pathways homeostasis and ameliorated tau pathology. Overall, our results show for the first time that DNP has the potential to preserve mitochondria homeostasis under a toxic insult, like OA exposure, as well as to reestablish cellular signaling homeostasis. These observations foster the idea that DNP, as a mitochondrial modulator, might represent a new avenue for treatment of AD.

Generalized ratiometric surface-enhanced Raman scattering biosensor for okadaic acid in food based on Au-triggered signal amplification.

BACKGROUND: Reliability and robustness have been recognized as key challenges for Surface-enhanced Raman scattering (SERS) analytical techniques. Quantifying the concentration of an analyte using a single characteristic peak from SERS has been a controversial topic because the Raman signal is susceptible to highly concentrated electromagnetic hotspots, inhomogeneity of SERS substrate, or non-standardization of measurement conditions. Ratiometric SERS strategies have been demonstrated as a promising solution to effectively balance and compensate for signal fluctuations caused by matrix heterogeneity. However, it is not easy to construct ratiometric SERS sensors with monitoring the ratio of two different signal intensities for target analysis. RESULTS: An attempt has been made to develop a novel ratiometric biosensor that can be applied to detect okadaic acid (OA). Aptamer-anchored magnetic particles were first combined with gold-tagged short complementary DNA (Au-cDNA) to create heterogeneous nanostructures. When the target was present, the Au-cDNA was dissociated from nanostructures, and 4-nitrothiophenol (4-NTP) was initiated to reduce to 4-aminothiophenol (4-ATP) in the presence of hydrogen sources. The SERS ratio change of 4-NTP and 4-ATP was finally detected by AuNPs-coated film. OA was successfully quantified, and the detection limit was as low as 2.4524 ng/mL. The constructed biosensor had good stability and reproducibility with a relative standard deviation of less than 4.47%. The proposed method used gold nanoparticles as an intermediate to achieve catalytic signal amplification and subsequently increased the sensitivity of the biosensor. SIGNIFICANCE AND NOVELTY: Catalytic reaction-based ratiometric SERS biosensors combine the multiple advantages of catalytic signal amplification and signal self-calibration and provide new insights into the development of stable, reproducible, and reliable SERS detection techniques. This ratiometric SERS technique offered a universal method that is anticipated to be applicable for the detection of other targets by substituting the aptamer.

Purification, structural characterization, and neuroprotective effect of 3,6-diisobutyl-2,5-piperazinedione from Halomonas pacifica CARE-V15 against okadaic acid-induced neurotoxicity in zebrafish model.

Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.

The Potential of Chitosan-Based Composites for Adsorption of Diarrheic Shellfish Toxins.

Okadaic acid (OA) is one of the most potent marine biotoxins, causing diarrheal shellfish poisoning (DSP). The proliferation of microalgae that produce OA and its analogues is frequent, threatening human health and socioeconomic development. Several methods have been tested to remove this biotoxin from aquatic systems, yet none has proven enough efficacy to solve the problem. In this work, we synthesized and characterized low-cost composites and tested their efficacy for OA adsorption in saltwater. For the synthesis of the composites, the following starting materials were considered: chitosan of low and medium molecular weight (CH-LW and CH-MW, respectively), activated carbon (AC), and montmorillonite (MMT). Characterization by vibrational spectroscopy (FTIR), X-ray diffraction (XRD), and microscopy revealed differences in the mode of interaction of CH-LW and CH-MW with AC and MMT, suggesting that the interaction of CH-MW with MMT has mainly occurred on the surface of the clay particles and no sufficient intercalation of CH-MW into the MMT interlayers took place. Among the composites tested (CH-LW/AC, CH-MW/AC, CH-MW/AC/MMT, and CH-MW/MMT), CH-MW/MMT was the one that revealed lower OA adsorption efficiency, given the findings evidenced by the structural characterization. On the contrary, the CH-MW/AC composite revealed the highest average percentage of OA adsorption (53 ± 11%). Although preliminary, the results obtained in this work open up good perspectives for the use of this type of composite material as an adsorbent in the removal of OA from marine environments.

Okadaic acid enhances NfKB, TLR-4, caspase 3, ERK ½, c-FOS, and 8-OHdG signaling pathways activation in brain tissues of zebrafish larvae.

This study was designed to investigate the potential neuronal damage mechanism of the okadaic acid (OA) in the brain tissues of zebrafish embryos by evaluating in terms of immunofluorescence of Nf KB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG signaling pathways. We also evaluated body malformations. For this purpose, zebrafish embryos were exposed to 0.5 µg/ml, 1 µg/ml and 2.5 µg/ml of OA for 5 days. After application, FITC/GFP labeled protein-specific antibodies were used in immunofluorescence assay for NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG respectively. The results indicated that OA caused immunofluorescence positivity of NfKB, TLR-4, caspase 3, ERK ½, c-FOS and 8-OHdG in a dose-dependent manner in the brain tissues of zebrafish embryos. Pericardial edema (PE), nutrient sac edema (YSE) and body malformations, tail malformation, short tail and head malformation (BM) were detected in zebrafish embryos. These results suggest that OA induces neuronal damage by affecting the modulation of DNA damage, apoptotic, and inflammatory activities in the brain tissues of zebrafish embryos. The increase in signaling pathways shows that OA can cause damage in the structure and function of brain nerve cells. Our results provide a new basis for the comprehensive assessment of the neural damage of OA and will offer enable us to better understand molecular the mechanisms underlying the pathophysiology of OA toxicity.

Determination of lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) and domoic acid in mussels by solid-phase extraction and reversed-phase liquid chromatography with tandem mass spectrometry.

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8-49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values.

Occurrence and distribution of phycotoxins in the Antarctic Ocean.

Lipophilic phycotoxins (LPTs) and domoic acid (DA) in Antarctic seawater, as well as parts of the South Pacific and the Southern Indian Oceans were systematically investigated. DA and six LPTs, namely pectenotoxin-2 (PTX2), okadaic acid (OA), yessotoxin (YTX), homo-yessotoxin (h-YTX), 13-desmethyl spirolide C (SPX1), and gymnodimine (GYM), were detected. PTX2, as the dominant LPTs, was widely distributed in seawater surrounding Antarctica, whereas OA, YTX, and h-YTX were irregularly distributed across the region. The total concentration of LPTs in surface seawater ranged from 0.10 to 13.57 ng/L (mean = 2.20 ng/L). ∑LPT levels were relatively higher in the eastern sea areas of Antarctica than in the western sea areas. PTX2 was the main LPT in the vertical profiles, and the PTX2 concentration was significantly higher in the epipelagic zone than water depths below 200 m. The predominant sources of PTX2 and OA in Antarctic sea areas are likely to be Dinophysis.

Assessment of the health risks associated with the consumption of bivalve mollusks potentially contaminated with phycotoxins from the coastal ecosystem of the Ebrié lagoon, Côte d'Ivoire.

This work focused on assessing of the risk associated with the consumption of bivalve mollusks, potentially contaminated with phycotoxins. The studied phycotoxins are saxitoxin (STX), okadaic acid (OA), dinophysistoxins (DTXs), yessotoxins (YTXs), pectenotoxins (PTX), azaspiracids (AZAs), and domoic acid (DA). These toxins were investigated in three species of bivalve mollusks (Anadara senilis, Crassostrea gasar, and Perna perna), originating from the Ebrié lagoon. Chemical analyses were carried out by LC-MS/MS, HPLC-FLD, and HPLC-UV. The level of OA and DTXs, STX, and DA was 10.92 µg OA eq./kg, 9.6 µg STX eq./kg, and 0.17 mg DA eq./kg, respectively. The level of PTXs and AZAs was 3.3 µg PTX-2 eq./kg and 13.86 µg AZA-1 eq./kg; that of YTXs was 0.01 mg YTX eq./kg. The daily exposure dose (DED) was 0.019 µg OA eq./kg bw for OA and DTXs; 0.285 µg DA eq./kg bw for DA; 0.006 µg PTX-2 eq./kg bw for PTXs; 0.016 µg STX eq./kg bw for STX; 0.01 µg YTX eq./kg bw for YTXs; and 0.024 µg AZA-1 eq./kg bw for AZAs for the oyster Crassostrea gasar. These estimated values are lower than the acute reference dose (ARfD) of each phycotoxin recommended by the European Food Safety Agency (EFSA). The risk of harmful effects is acceptable. The absence of risk is valid only for the study period (11 months) and concerns coastal populations living near the sampling points.

A 2D carbon nitride-based electrochemical aptasensor with reverse amplification for highly sensitive detection of okadaic acid in shellfish.

Okadaic acid (OA) is one of the main virulence factors of diarrheal shellfish toxins (DSP). It is of great significance to detect OA with an accurate, specific and cost-effective technique in the fields of seafood safety and water quality control. In this work, an electrochemical aptasensor with reverse amplification was developed for the sensitive detection of OA. A two-dimensional graphite-phase nanomaterial (carbon nitride) modified with an anti-OA aptamer and thionine (Th) was immobilized onto the surface of the electrochemical electrode as the sensitive element to capture target OA molecules. ssDNA-modified carbon nitride was used as the reverse amplification element by hybridizing with non-OA linked aptamers. The preparation of the electrochemical aptasensor was well characterized by Scanning Electron Microscopy (SEM), zeta potential detection, UV-Vis absorption, Brunner-Emmet-Teller (BET) measurements, and electrochemical measurements. The quantitative assessment of OA was achieved by differential pulse voltammetry (DPV). Experimental results indicated that this aptasensor showed a concentration-dependent response to OA with a good detection performance including in terms of selectivity, repeatability, reproducibility, and stability. It exhibited 100-fold selectivity between OA and other toxins including dinophysistoxins (DTX), pectenotoxins (PTX), and yessotoxins (YTX). In addition, it showed a much wider quantification range, which is 10-13 M-10-10 M (0.080-80.50 pg mL-1). The detection limit was as low as 10-13 M (0.080 pg mL-1). The aptasensor also successfully achieved significant practicality on real shellfish samples contaminated by OA. All these results demonstrated that the reverse amplification strategy for marine toxin detection may provide a label-free and rapid detection approach for portable applications in the fields of environmental monitoring and food security.