Lipophilic marine toxins in sediments from Arrábida marine protected area, Portugal (NE Atlantic).

During the development and senescence of harmful algal blooms (HAB), most of the algae cells not ingested by grazers or filter-feeding organisms sink to the bottom, making sediments important reservoirs of algae toxins. In this study, lipophilic marine toxins were determined in the sediments collected from depths ranging from 5 to 145 m depth in the marine protected area of Arrábida (southwest Portuguese coast). Sediments were characterized in terms of granulometry, water and organic matter content. The toxins were determined by liquid chromatography with tandem mass spectrometry. Okadaic acid (OA), dinophysistoxin-2 (DTX2), and azaspiracid-2 (AZA2), reaching concentrations up to 3.4, 1.3, and 0.13 ng/g, respectively, were found. A trend in the occurrence of DTX2 and AZA2 with sediment water and organic matter content was observed, as well as with AZA2 and depth. This study highlights the need to further investigate sediment deposition of toxins and their availability for bottom-dwelling organisms and its contamination.

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.

Occurrence and risk assessment of okadaic acid, dinophysistoxin-1, dinophysistoxin-2, and dinophysistoxin-3 in seafood from South Korea.

The okadaic acid (OA)-group toxins, including OA, dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), and dinophysistoxin-3 (DTX3), cause diarrheic shellfish poisoning in humans. To manage OA-group toxins more strictly, Korean regulations were recently revised to consider OA, DTX1, DTX2, and DTX3 combined. Thus, our study characterized the occurrence of OA, DTX1, DTX2, and DTX3 in seafood distributed across South Korea, and a risk assessment of seafood consumption was conducted. Two hundred and seventeen samples from 16 bivalve and 7 non-bivalve species collected from three representative coastal areas in 2021 were analyzed via liquid chromatography-tandem mass spectrometry. OA, DTX1, and DTX3 were detected in 2.3%, 4.1%, and 9.2% of the examined samples, with positive mean levels of 11.3, 16.4, and 40.9 µg/kg, respectively. DTX2 was not detected in any of the samples. At least one OA-group toxin was detected in the bivalve samples, including blood clams, pan shells, hard clams, mussels, and scallops, whereas none were detected in non-bivalves. The estimated acute exposure to OA-group toxins through the intake of seafood in the Korean population and consumer groups was low, ranging from 24.7 to 74.5% of the recommended acute reference dose (ARfD) of 0.33 µg OA equivalents/kg body weight. However, for the scallop consumers aged 7-12 years, acute exposure to OA-group toxins exceeded the ARfD, indicating a possible health risk. These results suggest that including DTX3 in the new regulatory limits is appropriate to protect Korean seafood consumers from exposure to OA-group toxins.

Seasonal Single-Site Sampling Reveals Large Diversity of Marine Algal Toxins in Coastal Waters and Shellfish of New Caledonia (Southwestern Pacific).

Algal toxins pose a serious threat to human and coastal ecosystem health, even if their potential impacts are poorly documented in New Caledonia (NC). In this survey, bivalves and seawater (concentrated through passive samplers) from bays surrounding Noumea, NC, collected during the warm and cold seasons were analyzed for algal toxins using a multi-toxin screening approach. Several groups of marine microalgal toxins were detected for the first time in NC. Okadaic acid (OA), azaspiracid-2 (AZA2), pectenotoxin-2 (PTX2), pinnatoxin-G (PnTX-G), and homo-yessotoxin (homo-YTX) were detected in seawater at higher levels during the summer. A more diversified toxin profile was found in shellfish with brevetoxin-3 (BTX3), gymnodimine-A (GYM-A), and 13-desmethyl spirolide-C (SPX1), being confirmed in addition to the five toxin groups also found in seawater. Diarrhetic and neurotoxic toxins did not exceed regulatory limits, but PnTX-G was present at up to the limit of the threshold recommended by the French Food Safety Authority (ANSES, 23 µg kg-1). In the present study, internationally regulated toxins of the AZA-, BTX-, and OA-groups by the Codex Alimentarius were detected in addition to five emerging toxin groups, indicating that algal toxins pose a potential risk for the consumers in NC or shellfish export.

Okadaic Acid Detection through a Rapid and Sensitive Amplified Luminescent Proximity Homogeneous Assay.

Okadaic acid (OA), a marine biotoxin produced by microalgae, poses a significant threat to mariculture, seafood safety, and human health. The establishment of a novel, highly sensitive detection method for OA would have significant practical and scientific implications. Therefore, the purpose of this study was to develop an innovative approach for OA detection. A competitive amplified luminescent proximity homogeneous assay (AlphaLISA) was developed using the principle of specific antigen-antibody binding based on the energy transfer between chemiluminescent microspheres. The method was non-washable, sensitive, and rapid, which could detect 2 × 10-2-200 ng/mL of OA within 15 min, and the detection limit was 4.55 × 10-3 ng/mL. The average intra- and inter-assay coefficients of variation were 2.54% and 6.26%, respectively. Detection of the actual sample results exhibited a good correlation with high-performance liquid chromatography. In conclusion, a simple, rapid, sensitive, and accurate AlphaLISA method was established for detecting OA and is expected to significantly contribute to marine biotoxin research.

Rat tight junction proteins are disrupted after subchronic exposure to okadaic acid.

Okadaic acid (OA), a lipophilic phycotoxin distributed worldwide, causes diarrheic shellfish poisoning and even leads to tumor formation. Currently, the consumption of contaminated seafood is the most likely cause of chronic OA exposure, but there is a serious lack of relevant data. Here, the Sprague-Dawley rats were exposure to OA by oral administration at 100 µg/kg body weight, and the tissues were collected and analyzed to assess the effect of subchronic OA exposure. The results showed that subchronic OA administration disturbed colonic mucosal integrity and induced colitis. The colonic tight junction proteins were disrupted and the cell cycle of colonic epithelial cells was accelerated. It is inferred that disruption of the colonic tight junction proteins might be related to the development of chronic diarrhea by affecting water and ion transport. Moreover, the accelerated proliferation of colonic epithelial cells indicated that subchronic OA exposure might promote the restitution process of gut barrier or induce tumor promoter activity in rat colon.

Differences in Toxic Response Induced by Three Variants of the Diarrheic Shellfish Poisoning Phycotoxins in Human Intestinal Epithelial Caco-2 Cells.

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Diel Variations in Cell Abundance and Trophic Transfer of Diarrheic Toxins during a Massive Dinophysis Bloom in Southern Brazil.

Dinophysis spp. are a major source of diarrheic toxins to marine food webs, especially during blooms. This study documented the occurrence, in late May 2016, of a massive toxic bloom of the Dinophysis acuminata complex along the southern coast of Brazil, associated with an episode of marked salinity stratification. The study tracked the daily vertical distribution of Dinophysis spp. cells and their ciliate prey, Mesodinium cf. rubrum, and quantified the amount of lipophilic toxins present in seston and accumulated by various marine organisms in the food web. The abundance of the D. acuminata complex reached 43 × 104 cells·L−1 at 1.0 m depth at the peak of the bloom. Maximum cell densities of cryptophyceans and M. cf. rubrum (>500 × 104 and 18 × 104 cell·L−1, respectively) were recorded on the first day of sampling, one week before the peak in abundance of the D. acuminata complex. The diarrheic toxin okadaic acid (OA) was the only toxin detected during the bloom, attaining unprecedented, high concentrations of up to 829 µg·L−1 in seston, and 143 ± 93 pg·cell−1 in individually picked cells of the D. acuminata complex. Suspension-feeders such as the mussel, Perna perna, and barnacle, Megabalanus tintinnabulum, accumulated maximum OA levels (up to 578.4 and 21.9 µg total OA·Kg−1, respectively) during early bloom stages, whereas predators and detritivores such as Caprellidae amphipods (154.6 µg·Kg−1), Stramonita haemastoma gastropods (111.6 µg·Kg−1), Pilumnus spinosissimus crabs (33.4 µg·Kg−1) and a commercially important species of shrimp, Xiphopenaeus kroyeri (7.2 µg·Kg−1), only incorporated OA from mid- to late bloom stages. Conjugated forms of OA were dominant (>70%) in most organisms, except in blenny fish, Hypleurochilus fissicornis, and polychaetes, Pseudonereis palpata (up to 59.3 and 164.6 µg total OA·Kg−1, respectively), which contained mostly free-OA throughout the bloom. Although algal toxins are only regulated in bivalves during toxic blooms in most countries, including Brazil, this study indicates that human seafood consumers might be exposed to moderate toxin levels from a variety of other vectors during intense toxic outbreaks.

Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.

Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure-activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2-epi-DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 >> 2-epi-DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs' activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors.

Persistent Contamination of Octopuses and Mussels with Lipophilic Shellfish Toxins during Spring Dinophysis Blooms in a Subtropical Estuary.

This study investigates the occurrence of diarrhetic shellfish toxins (DSTs) and their producing phytoplankton species in southern Brazil, as well as the potential for toxin accumulation in co-occurring mussels (Perna perna) and octopuses (Octopus vulgaris). During the spring in 2012 and 2013, cells of Dinophysis acuminata complex were always present, sometimes at relatively high abundances (max. 1143 cells L-1), likely the main source of okadaic acid (OA) in the plankton (max. 34 ng L-1). Dinophysis caudata occurred at lower cell densities in 2013 when the lipophilic toxins pectenotoxin-2 (PTX-2) and PTX-2 seco acid were detected in plankton and mussel samples. Here, we report for the first time the accumulation of DSTs in octopuses, probably linked to the consumption of contaminated bivalves. Perna perna mussels were consistently contaminated with different DSTs (max. 42 µg kg-1), and all octopuses analyzed (n = 5) accumulated OA in different organs/tissues: digestive glands (DGs) > arms > gills > kidneys > stomach + intestine. Additionally, similar concentrations of 7-O-palmytoyl OA and 7-O-palmytoly dinophysistoxin-1 (DTX-1) were frequently detected in the hepatopancreas of P. perna and DGs of O. vulgaris. Therefore, octopuses can be considered a potential vector of DSTs to both humans and top predators such as marine mammals.

Single laboratory validation of a ready-to-use phosphatase inhibition assay for detection of okadaic acid toxins.

A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC(50) values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78-101%; king scallop, 98-114%), DTX-1 (king scallop, 79-102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS.

Detection of shellfish toxins from scallops in Guangzhou seafood market

To evaluate scallop safety in the Guangzhou seafood market, contents of shellfish toxins in adductor muscle, mantle skirts, gills and visceral mass of scallops were examined using enzyme-linked immunosorbent assay (ELISA) and mouse unit assay. The results showed that: paralytic shellfish poisoning contents were up to 37.44 µg/100 g by ELISA and 319.99 MU/100 g by mouse unit assay, which did not exceed the limits of national standards (80 µg/100g and 400 MU/100 g); the contents of diarrhetic shellfish poisoning were 142.04 µg/100g and 0.2 MU/100 g, which exceeded the national standard limits (60 µg/100g); neurotoxic shellfish poisoning was undetectable; the contents of amnesic shellfish poisoning reached 220.12 µg/100g (no limit value could be referred to) . In addition, these poisons were present mainly in visceral mass and gills rather than adductor muscle and mantle skirts, suggesting that these toxins accumulate in a tissue-specific manner.(AU)

Detecção de ácido ocadaico em cultivo de mexilhões Perna perna, Angra dos Reis, RJ / Okadaic acid detection in mussel cultivation Perna perna, Angra dos Reis, RJ

A ficotoxina ácido ocadaico (AO) é produzida por um grupo de microalgas conhecidas como dinoflagelados. Os mexilhões, ao se alimentarem dessas microalgas, acumulam a toxina em sua glândula digestiva, desencadeando a Síndrome ou Envenenamento Diarreico por Moluscos (EDM) no ser humano. Os sintomas se apresentam em torno de 30 minutos após o consumo do molusco contaminado, variando entre náuseas, dores abdominais, vômitos e diarreia. Quando a ingestão da toxina acontece em quantidades inferiores a 48µg g-1, os sintomas não se desenvolvem, porém seu consumo continuado favorece o surgimento de tumores no trato gastrointestinal em razão do poder carcinogênico do AO. Este estudo pretendeu detectar e quantificar a toxina diarreica AO em mexilhões Perna perna coletados entre os meses de maio e outubro de 2006. A detecção do AO nos mexilhões foi realizada por Cromatografia Líquida de Alta Eficiência com Detecção Fluorimétrica (CLAE-DF). Os resultados cromatográficos indicaram a presença da toxina AO em baixas concentrações, em todas as amostras de mexilhões obtidas de maio a outubro de 2006. Os resultados indicam a necessidade da elaboração e aplicação efetiva de um programa de controle higiênico-sanitário dos moluscos, assim como o monitoramento do ambiente aquático, objetivando, acima de tudo, a segurança da saúde pública.

Okadaic acid (OA) is a phycotoxin produced by a group of microalgae known as Dino-flagellates. When mussels feed themselves from this micro seaweed the toxin accumulates in their hepatopancreas, triggering the Syndrome or Diarrhetic Shellfish Poisoning (DSP) in the human being. The symptoms appear around 30 minutes after the consumption of contaminated mussels and include abdominal nauseas, pains, vomits and diarrhea. When the toxin ingestion happens in amounts lower than 48µg g-1, the above described symptoms do not develop. However, the continued consumption favors the emergence of tumors in the gastrointestinal tract because of the high carcinogenic power of OA. This study aimed to detect and quantify the diarrheic toxin OA in Perna perna mussels collected between May and October 2006. The detection of OA in the mussels was carried out through High Performance Liquid Chromatography with Fluorimetric Detection (HPLC-FD). The chromatographic results indicate the presence of OA toxin in low concentrations in all the mussel samples gathered from May until October 2006. The results suggest the necessity of elaboration and effective application of a hygienic-sanitary mussel control program as well as environment monitoring, with the main aim of enhancing public health safety.

Detection of diarrheic shellfish poisoning and azaspiracid toxins in Moroccan mussels: comparison of the LC-MS method with the commercial immunoassay kit.

Diarrheic shellfish poisoning (DSP) is a recurrent gastrointestinal illness in Morocco, resulting from consumption of contaminated shellfish. In order to develop a rapid and reliable technique for toxins detection, we have compared the results obtained by a commercial immunoassay-"DSP-Check" kit" with those obtained by LC-MS. Both techniques are capable of detecting the toxins in the whole flesh extract which was subjected to prior alkaline hydrolysis in order to detect simultaneously the esterified and non esterified toxin forms. The LC-MS method was found to be able to detect a high level of okadaic acid (OA), low level of dinophysistoxin-2 (DTX2), and surprisingly, traces of azaspiracids 2 (AZA2) in mussels. This is the first report of a survey carried out for azaspiracid (AZP) contamination of shellfish harvested in the coastal areas of Morocco. The "DSP-Check" kit was found to detect quantitatively DSP toxins in all contaminated samples containing only OA, provided that the parent toxins were within the range of detection and was not in an ester form. A good correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit appeared to be more sensitive, specific and faster than LC-MS for determination of DSP in total shellfish extract.