Risk Assessment of Pectenotoxins in New Zealand Bivalve Molluscan Shellfish, 2009-2019.

Pectenotoxins (PTXs) are produced by Dinophysis spp., along with okadaic acid, dinophysistoxin 1, and dinophysistoxin 2. The okadaic acid group toxins cause diarrhetic shellfish poisoning (DSP), so are therefore regulated. New Zealand currently includes pectenotoxins within the DSP regulations. To determine the impact of this decision, shellfish biotoxin data collected between 2009 and 2019 were examined. They showed that 85 samples exceeded the DSP regulatory limit (0.45%) and that excluding pectenotoxins would have reduced this by 10% to 76 samples. The incidence (1.3%) and maximum concentrations of pectenotoxins (0.079 mg/kg) were also found to be low, well below the current European Food Safety Authority (EFSA) safe limit of 0.12 mg/kg. Inclusion within the DSP regulations is scientifically flawed, as pectenotoxins and okadaic acid have a different mechanism of action, meaning that their toxicities are not additive, which is the fundamental principle of grouping toxins. Furthermore, evaluation of the available toxicity data suggests that pectenotoxins have very low oral toxicity, with recent studies showing no oral toxicity in mice dosed with the PTX analogue PTX2 at 5000 µg/kg. No known human illnesses have been reported due to exposure to pectenotoxins in shellfish, a fact which combined with the toxicity data indicates that they pose negligible risk to humans. Regulatory policies should be commensurate with the level of risk, thus deregulation of PTXs ought to be considered, a stance already adopted by some countries.

Toxinas Algales en el Mar Argentino: nuevos hallazgos, nuevos desafíos / Algal Toxins in the Argentine Sea: new findings, new challenges

Resumen Las floraciones de algas nocivas son un problema cada vez más frecuente a nivel mundial que ocasiona severos daños sobre la salud pública, pérdidas económicas en acuicultura, perjuicios al turismo y episodios de mortalidad de poblaciones naturales de peces, aves y mamíferos marinos. Las toxinas son producidas por el fitoplancton y se acumulan en moluscos bivalvos que se alimentan por filtración del agua siendo estos los principales vectores de intoxicación humana. En el Mar Argentino, se han reportado toxinas marinas de origen microalgal asociadas con cuatro síndromes de intoxicación por moluscos. Los síndromes más graves por su extensión, frecuencia, toxicidad y organismos afectados, son los originados por el dinoflagelado Alexandrium cate-nella responsable de la Intoxicación Paralizante por Moluscos la cual ha ocasionado numerosas muertes humanas. Seguidamente, la más leve, en cuanto a gravedad y frecuencia, ha sido la Intoxicación Diarreica por Moluscos. En contraste, el ácido domoico, conocido como toxina amnésica de moluscos, no ha producido hasta ahora intoxicaciones humanas. Recientemente, se amplió el rango de toxinas para la región al registrarse las toxinas y los dinoflagelados productores de la Intoxicación Azaspirácidos por Moluscos. Además, se han detectado las potencialmente tóxicas Yessotoxinas y Espirolidos, cuyos mecanismos de acción y toxicidad están siendo aún evaluados a nivel mundial. Estas toxinas emergentes para la región, representan un riesgo potencial para la salud e inconvenientes socioeconómicos por el cierre de los sitios de explotación de moluscos. Ciertamente presentan un nuevo desafío, pues la detección y cuantificación sólo puede realizarse por medio de métodos basados en HPLC - espectrometría de masas, lo cual dificulta el monitoreo en laboratorios regionales en el país. La herramienta clave de manejo es la prevención, a través de políticas, regulaciones y sistemas de monitoreo y control de cada grupo de toxinas. A través de estas mejoras, se anticipa que no sólo disminuirá el número de afectados por estas intoxicaciones, si no que se podrán realizar vedas más eficientes, asegurando un equilibrio que proteja tanto la salud pública como el desarrollo de la industria pesquera.

Abstract Harmful algal blooms are an increasingly common problem worldwide, causing severe damage to public health, economic losses in aquaculture, damage to tourism and mortality events of natural populations of fish, birds and marine mammals. The toxins are produced by phytoplankton and accumulated in bivalve molluscs that feed on water filtration, being these main vectors of human intoxication. In the Argentine Sea marine toxins of microalgal origin have been reported associated with four shellfish poisoning syn-dromes. The most serious due to their extension, frequency, toxicity and affected organisms are those caused by the dinoflagellate Alexandrium catenella responsible for the Paralytic shellfish poisoning that has caused numerous human deaths. Then, the mildest, in severity and frequency, is the Diarrhetic shellfish poisoning. In contrast, domoic acid, known as Amnesic shellfish toxin, has not produced human intoxications yet. Recently, toxins and dinoflagellate species causing Azaspiracid shellfish poisoning have been re-corded, expanding the range of toxins for the region. In addition, the potentially toxic Yessotoxins and Spirolides have been detected, whose mechanism of action and toxicity is still being evaluated worldwide. These emerging toxins represent a potential risk to public health and socioeconomic activities due to the eventual closure of mollusc exploitation sites. They certainly present a new challenge, since detection and quantification can only be carried out using methods based on HPLC - mass spectrometry, which makes monitor-ing in regional laboratories difficult. Prevention through policies, regulations, and monitoring and control systems of each toxin group is the key management tool. These preventive measures are expected to contribute to reducing the number of poisonings and to ap-plying more efficient fisheries closures, ensuring a balance that protects both public health and the development of the fishing industry.

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish.

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)-m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)-m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)-m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)-m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.

Effects of marine harmful algal blooms on bivalve cellular immunity and infectious diseases: A review.

Bivalves were long thought to be "symptomless carriers" of marine microalgal toxins to human seafood consumers. In the past three decades, science has come to recognize that harmful algae and their toxins can be harmful to grazers, including bivalves. Indeed, studies have shown conclusively that some microalgal toxins function as active grazing deterrents. When responding to marine Harmful Algal Bloom (HAB) events, bivalves can reject toxic cells to minimize toxin and bioactive extracellular compound (BEC) exposure, or ingest and digest cells, incorporating nutritional components and toxins. Several studies have reported modulation of bivalve hemocyte variables in response to HAB exposure. Hemocytes are specialized cells involved in many functions in bivalves, particularly in immunological defense mechanisms. Hemocytes protect tissues by engulfing or encapsulating living pathogens and repair tissue damage caused by injury, poisoning, and infections through inflammatory processes. The effects of HAB exposure observed on bivalve cellular immune variables have raised the question of possible effects on susceptibility to infectious disease. As science has described a previously unrecognized diversity in microalgal bioactive substances, and also found a growing list of infectious diseases in bivalves, episodic reports of interactions between harmful algae and disease in bivalves have been published. Only recently, studies directed to understand the physiological and metabolic bases of these interactions have been undertaken. This review compiles evidence from studies of harmful algal effects upon bivalve shellfish that establishes a framework for recent efforts to understand how harmful algae can alter infectious disease, and particularly the fundamental role of cellular immunity, in modulating these interactions. Experimental studies reviewed here indicate that HABs can modulate bivalve-pathogen interactions in various ways, either by increasing bivalve susceptibility to disease or conversely by lessening infection proliferation or transmission. Alteration of immune defense and global physiological distress caused by HAB exposure have been the most frequent reasons identified for these effects on disease. Only few studies, however, have addressed these effects so far and a general pattern cannot be established. Other mechanisms are likely involved but are under-studied thus far and will need more attention in the future. In particular, the inhibition of bivalve filtration by HABs and direct interaction between HABs and infectious agents in the seawater likely interfere with pathogen transmission. The study of these interactions in the field and at the population level also are needed to establish the ecological and economical significance of the effects of HABs upon bivalve diseases. A more thorough understanding of these interactions will assist in development of more effective management of bivalve shellfisheries and aquaculture in oceans subjected to increasing HAB and disease pressures.

Conversion and Stability of New Metabolites of Paralytic Shellfish Toxins under Different Temperature and pH Conditions.

A number of new C-11 hydroxyl metabolites (so-called M-toxins) of paralytic shellfish toxins (PSTs) have been discovered in contaminated shellfish, and trace amounts have also been detected in some strains of PST-producing microalgae. To investigate the chemical conversion and stability of M-toxins, mussel extracts were purified with solid-phase extraction cartridges (Oasis HLB) and Biogel P-2 resin columns and four partially purified M-toxin fractions were stored at different temperatures (-20, 4, and 20 °C) and pH values (3, 4, and 5). The concentrations and profiles of M-toxins in these fractions were analyzed using liquid chromatography coupled with tandem mass spectrometry for 27 weeks. Results further confirmed the chemical conversion pathway M1 → M3 → M5 and determined for the first time two new transformation pathways: M2 → M4 → M6 and neosaxitoxin (NEO) → M10. The half-lives of M1, M2, M4, and M10 were calculated using a first-order degradation kinetics model, which indicated that the degradation of all M-toxins was dependent upon the temperature and pH, increasing with rising temperature and pH. In comparison to M4 and M10, M1 was more sensitive to the temperature, followed by M2. Results suggest that M-toxins should be maintained at a low temperature (-20 °C) and low pH (3) for their prolonged storage. M-toxins were less stable than all of the common analogues of PSTs, which may be beneficial for shellfish to achieve rapid detoxification through transformation of PSTs to M-toxins. These new findings are of significance because they enable further understanding of the metabolism of PSTs and their detoxification mechanisms in contaminated shellfish.

Oil pipelines and food sovereignty: threat to health equity for Indigenous communities.

Energy projects may profoundly impact Indigenous peoples. We consider effects of Canada's proposed Trans Mountain oil pipeline expansion on the health and food sovereignty of the Tsleil-Waututh Nation (TWN) through contamination and impeded access to uncontaminated traditional foods. Federal monitoring and TWN documentation show elevated shellfish biotoxin levels in TWN's traditional territory near the terminus where crude oil is piped. Although TWN restoration work has re-opened some shellfish-harvesting sites, pipeline expansion stands to increase health risk directly through rising bioaccumulating chemical toxins as well as through increased hazardous biotoxins. Climate change from increased fossil fuel use, expected via pipeline expansion, also threatens to increase algae blooms through higher temperature and nutrient loading. As the environmental impact assessment process failed to effectively consider these local health concerns in addition to larger impacts of climate change, new assessment is needed attending to linked issues of equity, sustainability and Indigenous food sovereignty.

Effects of Temperature, Growth Media, and Photoperiod on Growth and Toxin Production of Azadinium spinosum.

Azaspiracids (AZAs) are microalgal toxins that can accumulate in shellfish and lead to human intoxications. To facilitate their study and subsequent biomonitoring, purification from microalgae rather than shellfish is preferable; however, challenges remain with respect to maximizing toxin yields. The impacts of temperature, growth media, and photoperiod on cell densities and toxin production in Azadinium spinosum were investigated. Final cell densities were similar at 10 and 18 °C, while toxin cell quotas were higher (~3.5-fold) at 10 °C. A comparison of culture media showed higher cell densities and AZA cell quotas (2.5-5-fold) in f10k compared to f/2 and L1 media. Photoperiod also showed differences, with lower cell densities in the 8:16 L:D treatment, while toxin cell quotas were similar for 12:12 and 8:16 L:D treatments but slightly lower for the 16:8 L:D treatment. AZA1, -2 and -33 were detected during the exponential phase, while some known and new AZAs were only detected once the stationary phase was reached. These compounds were additionally detected in field water samples during an AZA event.

Novel Insights on the Toxicity of Phycotoxins on the Gut through the Targeting of Enteric Glial Cells.

In vitro and in vivo studies have shown that phycotoxins can impact intestinal epithelial cells and can cross the intestinal barrier to some extent. Therefore, phycotoxins can reach cells underlying the epithelium, such as enteric glial cells (EGCs), which are involved in gut homeostasis, motility, and barrier integrity. This study compared the toxicological effects of pectenotoxin-2 (PTX2), yessotoxin (YTX), okadaic acid (OA), azaspiracid-1 (AZA1), 13-desmethyl-spirolide C (SPX), and palytoxin (PlTX) on the rat EGC cell line CRL2690. Cell viability, morphology, oxidative stress, inflammation, cell cycle, and specific glial markers were evaluated using RT-qPCR and high content analysis (HCA) approaches. PTX2, YTX, OA, AZA1, and PlTX induced neurite alterations, oxidative stress, cell cycle disturbance, and increase of specific EGC markers. An inflammatory response for YTX, OA, and AZA1 was suggested by the nuclear translocation of NF-κB. Caspase-3-dependent apoptosis and induction of DNA double strand breaks (γH2AX) were also observed with PTX2, YTX, OA, and AZA1. These findings suggest that PTX2, YTX, OA, AZA1, and PlTX may affect intestinal barrier integrity through alterations of the human enteric glial system. Our results provide novel insight into the toxicological effects of phycotoxins on the gut.

Combined effects of okadaic acid and pectenotoxin-2, 13-desmethylspirolide C or yessotoxin in human intestinal Caco-2 cells.

Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filtering shellfish and can cause human intoxications. Humans can be exposed to combinations of several phycotoxins. The toxicological effects of phycotoxin mixtures on human health are largely unknown. Published data on phycotoxin co-exposure show that okadaic acid (OA) is simultaneously found with pectenetoxin-2 (PTX-2), 13-desmethylspirolide C (also known as SPX-1), or yessotoxin (YTX). Therefore, the aim of this study was to examine the effects of three binary mixtures, OA/PTX-2, OA/SPX-1 and OA/YTX on human intestinal Caco-2 cells. A multi-parametric approach for cytotoxicity determination was applied using a high-content analysis platform, including markers for cell viability, oxidative stress, inflammation, and DNA damage. Mixtures effects were analyzed using two additivity mathematical models. Our assays revealed that OA induced cytotoxicity, DNA strand breaks and interleukin 8 release. PTX-2 slightly induced DNA strand breaks, whereas SPX-1 and YTX did not affect the investigated endpoints. The combination of OA with another toxin resulted in reduced toxicity at low concentrations, suggesting antagonistic effects, but in increased effects at higher concentrations, suggesting additive or synergistic effects. Taken together, our results demonstrated that the cytotoxic effects of binary mixtures of lipophilic phycotoxins could not be predicted by additivity mathematical models. In conclusion, the present data suggest that combined effects of phycotoxins may occur which might have the potential to impact on risk assessment of these compounds.

A Strategy to Replace the Mouse Bioassay for Detecting and Identifying Lipophilic Marine Biotoxins by Combining the Neuro-2a Bioassay and LC-MS/MS Analysis.

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.

Phycotoxins in Marine Shellfish: Origin, Occurrence and Effects on Humans.

Massive phytoplankton proliferation, and the consequent release of toxic metabolites, can be responsible for seafood poisoning outbreaks: filter-feeding mollusks, such as shellfish, mussels, oysters or clams, can accumulate these toxins throughout the food chain and present a threat for consumers' health. Particular environmental and climatic conditions favor this natural phenomenon, called harmful algal blooms (HABs); the phytoplankton species mostly involved in these toxic events are dinoflagellates or diatoms belonging to the genera Alexandrium, Gymnodinium, Dinophysis, and Pseudo-nitzschia. Substantial economic losses ensue after HABs occurrence: the sectors mainly affected include commercial fisheries, tourism, recreational activities, and public health monitoring and management. A wide range of symptoms, from digestive to nervous, are associated to human intoxication by biotoxins, characterizing different and specific syndromes, called paralytic shellfish poisoning, amnesic shellfish poisoning, diarrhetic shellfish poisoning, and neurotoxic shellfish poisoning. This review provides a complete and updated survey of phycotoxins usually found in marine invertebrate organisms and their relevant properties, gathering information about the origin, the species where they were found, as well as their mechanism of action and main effects on humans.

Identification of 21,22-Dehydroazaspiracids in Mussels ( Mytilus edulis) and in Vitro Toxicity of Azaspiracid-26.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 µg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

Preliminary Results on the Evaluation of the Occurrence of Tetrodotoxin Associated to Marine Vibrio spp. in Bivalves from the Galician Rias (Northwest of Spain).

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

Proposed Biotransformation Pathways for New Metabolites of Paralytic Shellfish Toxins Based on Field and Experimental Mussel Samples.

A seafood poisoning event occurred in Qinhuangdao, China, in April 2016. Subsequently, the causative mussels (Mytilus galloprovincialis) were harvested and analyzed to reveal a high concentration [∼10 758 µg of saxitoxin (STX) equiv kg-1] of paralytic shellfish toxins (PSTs), including gonyautoxin (GTX)1/4 and GTX2/3, as well as new metabolites 11-hydroxy-STX (M2), 11,11-dihydroxy-STX (M4), open-ring 11,11-dihydroxy-STX (M6), 11-hydroxy-neosaxitoxin (NEO) (M8), and 11,11-dihydroxy-NEO (M10). To understand the origin and biotransformation pathways of these new metabolites, uncontaminated mussels (M. galloprovincialis) were fed with either of two Alexandrium tamarense strains (ATHK and TIO108) under laboratory conditions. Similar PST metabolites were also detected in mussels from both feeding experiments. Results supposed that 11-hydroxy-C2 toxin (M1) and 11,11-dihydroxy-C2 (M3) are transformed from C2, while 11-hydroxy-C4 toxin (M7) and 11,11-dihydroxy-C4 (M9) are converted from C4. In addition, the metabolites M2, M4, and M6 appear to be products of GTX2/3, and the metabolites M8 and M10 are likely derived from GTX1/4.

Evaluación de los Niveles de Mercurio en Productos Pesqueros en Costa Rica, Durante 2003-2013, como insumo para recomendar una ingesta semanal tolerable / Evaluation of Mercury Levels in Fishery Products in Costa Rica, During 2003-2013, as an Input to recommend a tolerable weekly intake

ResumenLos peces y mariscos pueden acumular mercurio en sus tejidos. En este trabajo se establecen recomendaciones de consumo de productos pesqueros para los residentes de Costa Rica, a partir de la evaluación de los niveles de mercurio en pescados y mariscos analizados durante los años 2003-2013.Objetivo: Evaluar los niveles de mercurio (en mg/kg) en muestras de productos pesqueros, analizadas por el Laboratorio Nacional de Servicios Veterinarios (LANASEVE) del Servicio Nacional de Salud Animal (SENASA) de Costa Rica, durante el período 2003-2013, para sugerir un valor de Ingesta Semanal Tolerable Recomendada (ISTR) de pescados y mariscos.Materiales y métodos: Los ensayos de mercurio se realizaron, bajo un esquema de análisis de riesgo determinado por el Programa Nacional de Residuos del SENASA, en pescados y mariscos. Las muestras de músculo fueron recolectadas por personal del SENASA y enviadas al LANASEVE para evaluar los niveles de Hg. Las concentraciones de mercurio fueron luego comparadas con los dos valores límite establecidos por la normativa nacional e internacional para productos pesqueros: 1 mg Hg/kg para pescados depredadores y 0,5 mg Hg/kg para otros productos pesqueros.Resultados: Se encontró que aproximadamente el 85 % de las muestras de pescados depredadores cumplió con el valor límite de 1 mg Hg/kg. Por su parte, el 93 % de los otros productos pesqueros evaluados, mostraron niveles de mercurio por debajo de 0,5 mg Hg/kg.Conclusión: Para mujeres embarazadas se establecieron valores de ISTR de 171 g de pescados depredadores o 889 g de otros productos pesqueros, en tanto para niños los ISTR recomendados fueron 100 g de pescados depredadores y 519 g de otros productos pesqueros.

AbstractFish and seafood can accumulate mercury in their tissues. This work establishes fishery products intake recommendations for the residents of Costa Rica, based upon the evaluation of mercury levels on fish and seafood analyzed during years 2003-2013.Objective: To evaluate mercury levels (in mg/kg) in fishery samples, as analyzed by the National Veterinary Services Laboratory (LANASEVE) of the National Animal Health Service (SENASA) of Costa Rica, during the 2003-2013 period, to suggest a Tolerable Weekly Intake Recommendation (TWIR) for fish and seafood.Methods: Mercury assays were performed, under a risk-analysis scheme determined by the National Residue Program of SENASA, in fish and seafood. Muscle samples were collected by SENASA staff and sent to LANASEVE for evaluation of Hg contents. Mercury concentrations were then compared to two limit values established by national and international regulations for seafood: 1 mg Hg/kg for predatory fish, and 0,5 mg Hg/kg for other fishery products.Results: It was found that, approximately, 85 % of predatory fish samples complied with the 1 mg Hg/kg limit value. Meanwhile, 93 % of the other fishery products analyzed, showed mercury levels below 0,5 mg/kg.Conclusion: For pregnant women TWIR values were established at 171 g for predatory fish and 889 g for other fishery products, while for children the TWIR values were 100 g for predatory fish and 519 g for other fishery products.

Effect of the industrial canning on the toxicity of mussels contaminated with diarrhetic shellfish poisoning (DSP) toxins.

The effect of canning in pickled sauce and autoclaving on weight, toxin content, toxin concentration and toxicity of steamed mussels was studied. Weight decreased by 25.5%. Okadaic acid (OA) and DTX2 content of mussel meat decreased by 24.1 and 42.5%, respectively. The estimated toxicity of the mussel remained nearly unchanged (increased by 2.9%). A part of the toxins lost by the mussels was leached to the sauce but the remaining part should have been thermally degraded. DTX2 underwent more degradation than OA and, in both toxins, free forms more than conjugated ones. This process, therefore, cannot be responsible for the large increments of toxicity of processed mussels -relative to the raw ones-sometimes detected by food processing companies. The final product could be monitored in several ways, but analysing the whole can content or the mussel meat once rehydrated seems to be the most equivalents to the raw mussel controls.

Assessment of sodium channel mutations in Makah tribal members of the U.S. Pacific Northwest as a potential mechanism of resistance to paralytic shellfish poisoning.

The Makah Tribe of Neah Bay, Washington, has historically relied on the subsistence harvest of coastal seafood, including shellfish, which remains an important cultural and ceremonial resource. Tribal legend describes visitors from other tribes that died from eating shellfish collected on Makah lands. These deaths were believed to be caused by paralytic shellfish poisoning, a human illness caused by ingestion of shellfish contaminated with saxitoxins, which are produced by toxin-producing marine dinoflagellates on which the shellfish feed. These paralytic shellfish toxins include saxitoxin, a potent Na+ channel antagonist that binds to the pore region of voltage gated Na+ channels. Amino acid mutations in the Na+ channel pore have been demonstrated to confer resistance to saxitoxin in softshell clam populations exposed to paralytic shellfish toxins present in their environment. Because of the notion of resistance to paralytic shellfish toxins, the study aimed to determine if a resistance strategy was possible in humans with historical exposure to toxins in shellfish. We collected, extracted and purified DNA from buccal swabs of 83 volunteer Makah tribal members and sequenced the skeletal muscle Na+ channel (Nav1.4) at nine loci to characterize potential mutations in the relevant saxitoxin binding regions. No mutations of these specific regions were identified after comparison to a reference sequence. This study suggests that any resistance of Makah tribal members to saxitoxin, if present, is not a function of Nav1.4 modification, but may be due to mutations in neuronal or cardiac sodium channels, or some other mechanism unrelated to sodium channel function.

Distribution of Marine Lipophilic Toxins in Shellfish Products Collected from the Chinese Market.

To investigate the prevalence of lipophilic marine biotoxins in shellfish from the Chinese market, we used hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure levels of okadaic acid (OA), azaspiracid (AZA1), pectenotoxin (PTX2), gymnodimine (GYM), and spirolide (SPX1). We collected and analyzed 291 shellfish samples from main production sites along a wide latitudinal transect along the Chinese coastline from December 2008 to December 2009. Results revealed a patchy distribution of the five toxins and highlighted the specific geographical distribution and seasonal and species variation of the putative toxigenic organisms. All five lipophilic marine biotoxins were found in shellfish samples. The highest concentrations of OA, AZA1, PTX2, GYM, and SPX1 were 37.3, 5.90, 16.4, 14.4, and 8.97 µg/kg, respectively. These values were much lower than the legislation limits for lipophilic shellfish toxins. However, the value might be significantly underestimated for the limited detection toxins. Also, these toxins were found in most coastal areas of China and were present in almost all seasons of the year. Thus, these five toxins represent a potential threat to human health. Consequently, studies should be conducted and measures should be taken to ensure the safety of the harvested product.

Determining the Advantages, Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST).

The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST). Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI) or wild-type isoforms (PWI), while most individuals express relatively equal amounts of each (EI). There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR), ingestion rate (I), and gross growth efficiency (GGE) for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed.

New insights into the causes of human illness due to consumption of azaspiracid contaminated shellfish.

Azaspiracid (AZA) poisoning was unknown until 1995 when shellfish harvested in Ireland caused illness manifesting by vomiting and diarrhoea. Further in vivo/vitro studies showed neurotoxicity linked with AZA exposure. However, the biological target of the toxin which will help explain such potent neurological activity is still unknown. A region of Irish coastline was selected and shellfish were sampled and tested for AZA using mass spectrometry. An outbreak was identified in 2010 and samples collected before and after the contamination episode were compared for their metabolite profile using high resolution mass spectrometry. Twenty eight ions were identified at higher concentration in the contaminated samples. Stringent bioinformatic analysis revealed putative identifications for seven compounds including, glutarylcarnitine, a glutaric acid metabolite. Glutaric acid, the parent compound linked with human neurological manifestations was subjected to toxicological investigations but was found to have no specific effect on the sodium channel (as was the case with AZA). However in combination, glutaric acid (1 mM) and azaspiracid (50 nM) inhibited the activity of the sodium channel by over 50%. Glutaric acid was subsequently detected in all shellfish employed in the study. For the first time a viable mechanism for how AZA manifests itself as a toxin is presented.