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Prorocentrum lima is a widely distributed toxigenic benthic dinoflagellate whose production of diarrhetic shellfish toxins threatens the shellfish industry and seafood safety. Current research primarily assesses the difference between free and post-hydrolysis total toxin methods, ignoring the impact of different detection methods on technical accuracy. After removing matrix interference with SPE extraction, a thorough HRMS strategy was created in this study. Alkaline hydrolysis could release the diol esters and played a crucial role in obtaining an accurate assessment of toxin levels, achieving satisfactory recoveries (74.0-147.0%) and repeatability (relative deviation <12.3%). The HRMS approach evaluated toxin profile variation during the growth of three P. lima strains from China. A total of 24 toxin contents varying in composition, content, and a high proportion were detected. The SHG, HN, and 3XS strains had total toxin contents of 23.3 ± 1.74, 19.8 ± 1.25, and 19.5 ± 1.58 pg cell -1 , respectively. The diol esters proportion varied among the strains, with SHG having 58.9-69.9, HN having 75.4-86.5, and 3XS having 91.0-91.7%. The variety of toxins produced by distinct P. lima strains highlighted the significance of this method for appropriately measuring the risks connected with DSTs manufacturing. The proposed approach provides a technical basis for gathering comprehensive and accurate data on the potential risks of P. lima DSTs production, with significant implications for ensuring food safety and preventing harmful toxins from spreading in the marine ecosystem., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Saxitoxin (STX) is a potent shellfish toxin found in freshwater and marine ecosystems which threatens human health by contaminating drinking water and shellfish. The formation of neutrophil extracellular traps (NETs) is a defense mechanism employed by polymorphonuclear leukocytes (PMNs) to destroy invading pathogens, and also plays a critical role in the pathogenesis of various diseases. In this study, we aimed to investigate the role of STX on human NET formation. Typical NETs-associated characteristics were detected from STX-stimulated PMNs using immunofluorescence microscopy. Moreover, NET quantification based on PicoGreen® fluorescent dye revealed that STX triggered NET formation in a concentration-dependent manner, and NET formation peaked at 120 min (with a total time of 180 min) after induction by STX. Intracellular reactive oxygen species (iROS) detection showed that iROS were significantly elevated in STX-challenged PMNs. These findings present insight into the effects of STX on human NET formation and serve as a basis for further investigations of STX immunotoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mediterranean waters have undergone environmental changes during the last decades leading to various modifications of the structure of phytoplankton populations, especially Harmful Algal Blooms (HABs) species. Monitoring of the potentially toxic phytoplankton species was carried out biweekly in the western Mediterranean coast of Morocco from March 2018 to March 2019. Lipophilic Shellfish Toxins (LSTs) using LC-MS/MS and Domoic Acid (DA) using HPLC-UV were measured in the exploited mollusks, the cockle Acanthocardia tuberculata and the smooth clam Callista chione. We also determined the prevailing environmental factors in four surveyed sites (M'diq bay, Martil, Kaa Asras, and Djawn) selected to cover a variety of coastal ecosystems. Results showed that Pseudo-nitzschia spp. a DA producer species, was abundant with a pick of 50 × 10 3  cells l -1 on October 2018 in Djawn. Dinophysis caudata was the dominate Dinophysis species and showed a maximum density of 2200 cells l -1 on July in Djawn. Prorocentrum lima, an epibenthic dinoflagellate, appeared rarely in the water column with densities <80 cells l -1 . Gonyaulax spinifera and Protoceratium reticulatum were found occasionally with a maximum density of 160 cells l -1 . Karenia selliformis was detected only five times (<80 cells l -1 ) throughout the survey period. LC-MS/MS analyses revealed the presence of OA/DTX3, PTX-2, PTX-2 sa, and PTX-2 sa epi in the cockle at concentrations of up to 44.81 (OA/DTX-3+PTXs) ng g -1 meat. GYM-A was detected in the clam at concentrations of up to 4.22 ng g -1 meat. For the first time, AZAs and YTXs were detected in the southwestern Mediterranean with maximum values of 2.49 and 10.93 ng g -1 meat of cockle, respectively. DA was detected in moderate concentrations not exceeding 5.65 μg g -1 in both mollusks. Results showed that the observed toxic algae in the water column were responsible from the analysed toxins in the mollusks. It is likely that the southwestern Mediterranean waters could see the development of emergent species producing potent toxins (YTXs, AZAs, GYM-A). These dinoflagellates have to be isolated, ribotyped, and their toxin profiles determined., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are well known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Monitoring of PSTs is typically performed using chemical methods which quantify the concentration of the individual PST analogues, of which there are many. However, since the toxicities of analogues are different, they do not equally contribute to the overall toxicity of the sample. To account for these differences, toxicity equivalency factors (TEFs) need to be determined for each analogue and applied. Currently there are no established TEFs for decarbamoyl gonyautoxin 1&4 (dcGTX1&4), which occurs in some clam species such as Mactra chinensis contaminated with PSTs due to metabolism within the shellfish. In this study the median lethal dose of purified, equilibrated epimeric mixture of dcGTX1&4 has been determined by intraperitoneal injection (i.p.) (4.75 μmol/kg) and by feeding (34.9 μmol/kg). The most relevant route of exposure is orally with feeding being more representative of human consumption and more reliable than gavage. Based on the median lethal dose by feeding, a TEF of 0.1 is recommended for dcGTX1&4. Receptor binding activity and i.p. toxicity results showed dcGTX1&4 to be much less toxic than STX (140-170-fold). However, by feeding a much smaller difference in toxicity was observed with dcGTX1&4 being only 11-fold less toxic than STX. Analysis of the gut contents of mice dosed with dcGTX1&4 showed the presence of decarbamoyl gonyautoxin 2&3, decarbamoyl saxitoxin and decarbamoyl neosaxitoxin, all of which are of greater toxicity. This conversion of dcGTX1&4 within the digestive track to more toxic congeners may explain the high relative toxicity of dcGTX1&4 by feeding compared to that determined by i.p. and by sodium channel activity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A total of 1338 samples were analyzed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to study the toxin profiles of lipophilic marine toxins in bivalve mollusks collected from the southeast coast of China from 2017 to 2020. The most abundant toxin was HomoYTX, followed progressively by YTX and PTX2. Low proportions of OA, DTX-1, and DTX-2 were found. No AZA1, AZA2, and AZA3 were quantified above limit of quantitation (LOQ). The highest concentrations of HomoYTX, YTX, PTX2, OA, DTX-1, and DTX-2 were 429, 98.0, 40.3, 33.0, 22.6, and 26.5 μg/kg, respectively. Mussels (Mytilus galloprovincialis, Perna viridis), scallop (Chlamys farreri) and clam (Atrina pectinate) accumulated higher toxin levels than clams (Sinonovaculla Constricta, Ruditapes philippinarum), oyster (Crassostrea gigas) and scallop (Arca granosa). Homo YTX and PTX2 levels reached the maximum in July and June, respectively, and the OA-group peaked in August. The results provide a reliable basis for monitoring marine toxins and protecting the health of aquatic consumers., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Economic bivalve ingested toxic algae causes frequent human poisoning events. To explore new compounds that can accelerate the depuration of toxins in shellfish, we investigated the detoxification of the paralytic shellfish toxins (PSTs) and the biotransformation pathway of PSTs during detoxification by the application of three treatments to a toxic bloom, Alexandrium minutum (A. minutum). The detoxification effect of Platymonas subcordiformis (PS) mixed with carboxymethyl chitosan (CMC) group is significantly better than the starving group in each oyster tissues. The toxicity of viscera which occupied 78.95% of total toxicity reduced to 155 MU/100g after 13 days' depuration experiment. And adding CMC could significantly achieve rapid detoxification and effectively reduce the STX to 0.07 μmol/100 g in viscera. Meanwhile, PSTs underwent biotransformation during the depuration process, which mainly manifested as GTX1/4→GTX2/3→STX, GTX2→dcGTX2. This study explored a new strategy for toxin depuration in shellfish., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Blooms of the dinoflagellate Alexandrium catenella, which produces Paralytic Shellfish Toxins (PST), generate serious socio-economic consequences for mariculture in Chile, especially for the production of Mytilus chilensis and other bivalves. Palliative strategies, such as the depuration of mussels in enriched water with chitosan offer encouraging prospects against the advance of contaminated areas and toxin persistence. Adult mussels were fed with A. catenella for 20 days and then were allowed to depurate using chitosan as facilitator, for the same period. Intoxicated mussels showed a reduction in feeding activity and rapid PST accumulation in 20 days (C = 451.5t + 1,673.6, R 2  = 0.55 p = 0.008). Not enough evidence was found to indicate a positive effect of chitosan in mussel depuration after 20 days (C = -311.1t + 8,462.4, R 2  = 0.8 p = 0.001). At the end of the study, toxicity was higher than 800 μg STX eq kg -1 . C2 and GTX4 analogues were the most abundant in the dinoflagellate strain, while C2 and C1 were the most accumulated in mussels. The presence of C1 was notorious during depuration, as the persistence of GTX2,3. GTX5 was only detected in A. catenella, while STX was only present in mussels. Mussel sensitivity to the presence of the toxic dinoflagellate was observed in the present study. The biotransformation, selective elimination and epimerization processes were deduced from intoxication and depuration experiments., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A receptor binding assay (RBA) for the determination of paralytic shellfish poisoning toxicity is formally validated through collaborative study and approved for regulatory monitoring use in the US for mussels and clams. However, to date, the method has not been tested on bivalve molluscs originating from European waters and no validation studies have been conducted for oysters, a shellfish species of great importance globally. This study firstly reports the work conducted to assess the performance of the assay in comparison with a regulatory chemical detection method for a range of shellfish species originating from Great Britain. Data obtained showed a complete absence of false negative RBA results, with a tendency to over-estimate PSP toxicity for some shellfish species in comparison with liquid chromatography with fluorescence detection. Secondly, the performance of the RBA was assessed for oysters, with the analysis of a dilution series of oyster matrix certified reference materials. Method trueness, sensitivity and precision were found to compare well with results reported previously for other species. In addition, the RBA analysis of untreated and demetallated oyster extracts, provided good evidence that the RBA is not suppressed in the presence of high concentrations of zinc as reported previously for the mouse bioassay. Consequently, there is strong evidence from this study, that the RBA would be suitable for determination of PSP toxicity in bivalve molluscs from GB, with acceptable method performance in oysters. Further validation studies would be required for other shellfish species of interest before the method can be considered suitable for implementation in Europe., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In October 2012, paralytic shellfish toxins (PST) were detected in the hepatopancreas of Southern Rock Lobsters (Jasus edwardsii) collected from the east coast of Tasmania, Australia. This resulted in the first commercial closure in Australia for this species. Questions were raised on how the toxins were transferred to the lobsters, how long the toxins would persist, whether PST-contaminated hepatopancreas posed a risk to human health, and what management strategies could be applied. The aim of this study was to investigate whether PST-contaminated mussels are a potential vector enabling toxin accumulation in J. edwardsii and to collect information on toxin uptake, distribution and depuration rates and toxin profiles under controlled experimental settings. Lobsters were fed mussels naturally contaminated with PST for a period of 28 days in an experimental setting; following this, lobsters were allocated to either fed or starved treatment groups. PST were not detected in the tail tissue of lobsters at any stage of the experiment. Lobster hepatopancreas contained mean levels of 2.4 mg STX.2HCl eq/kg after 28 days of uptake, although substantial variability in total toxicity was observed. The PST profile of the hepatopancreas was similar to that of the contaminated mussels used as feed. Significant differences were noted in the PST depuration rates between fed and starved treatment groups. The daily depuration rate for total PST was estimated to be 0.019 and 0.013 mg STX.2HCl eq/kg for the fed and starved treatment groups respectively using a constant-rate decay model. After 42 days of depuration, total PST (STX equivalents) levels in the hepatopancreas of all lobsters were below 0.8 mg STX.2HCl eq/kg, which represents the regulatory level applied to bivalves. This result indicates that long-term holding to depurate PST may potentially be used as a risk management tool., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The south west coast of India has been showing a steady increase in shellfish cultivation both for local consumption and fishery export, over recent years. Perna viridis and Crassostrea madrasensis are two species of bivalve molluscs which grow in some selected regions of southern Karnataka, close to the city of Mangalore. In the early 1980s, shellfish consumers in the region were affected by intoxication from Paralytic Shellfish Poison present in local bivalves (clams and oysters) resulting in hospitalisation of many, including one fatality. Since then, there have been no further reports of serious shellfish intoxication and there is little awareness of the risks from natural toxins and no routine monitoring programme in place to protect shellfish consumers. This study presents the findings from the first ever systematic assessment of the presence of marine toxins in mussels and oysters grown in four different shellfish harvesting areas in the region. Shellfish were collected and subjected to analysis for ASP, PSP and lipophilic toxins, as well as a suite of non-EU regulated toxins such as tetrodotoxin and selected cyclic imines. Results revealed the presence of low levels of PSP toxins in oysters throughout the study period. Overall, total toxicities reached a maximum of 10% of the EU regulatory limit of 800 μg STX eq/kg. Toxin profiles were similar to those reported from the 1980 outbreak. No evidence was found for significant levels of ASP and lipophilic toxins, although some cyclic imines were detected, including gymnodimine. The results indicated that the risk to shellfish consumers during this specific study period would have been low. However, with historical evidence for extremely high levels of PSP toxins in molluscs, there is a strong need for routine surveillance of shellfish production areas for marine toxins, in order to mitigate against human health impacts resulting from unexpected harmful algal blooms, with potentially devastating socio-economic consequences., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Low concentrations of azaspiracids have been found in bivalve molluscs all over the Northern and Northwestern coast of the Iberian Peninsula. The detections started in June 2016 and lasted until March 2017. The observed toxin profile was dominated by AZA2, followed by AZA1 and some other AZAs that were detected only in some samples. Some compounds producing fragments characteristics of AZAs but that do not fit with any of the known ones were also found. The causative agent has not been identified but, in sight of the toxin profile in the bivalves, it seems that it should be a new species or strain. The detections of AZAs in bivalves in the Northern Coast was linked to downwelling or upwelling relaxation and, in the Galician Rías, took place (with only a few exceptions) in the outer (more oceanic) part, suggesting that the responsible species develops at the open sea and that the populations are advected to the shore., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. YTX content in shellfish is regulated by many food safety authorities to protect human health, although currently no human intoxication episodes have been unequivocally related to YTX presence in food. The immune system has been proposed as one of the target organs of YTX due to alterations of lymphoid tissues and cellular and humoral components. The aim of the present study was to explore subacute immunotoxicity of YTX in rats by evaluating the haematological response, inflammatory cytokine biomarkers and the presence of YTX-induced structural alterations in the spleen and thymus. The results showed that repeated administrations of YTX caused a decrease of lymphocyte percentage and an increase of neutrophil counts, a reduction in interleukine-6 (IL-6) plasmatic levels and histopathological splenic alterations in rats after four intraperitoneal injections of YTX at doses of 50 or 70 μg/kg that were administered every 4 days along a period of 15 days. Therefore, for the first time, subacute YTX-immunotoxicity is reported in rats, suggesting that repeated exposures to low amounts of YTX might also suppose a threat to human health, especially in immuno-compromised populations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lipophilic shellfish toxins (LSTs) produced by diverse microalgae have become a potential threat to the marine aquaculture industry in coastal regions throughout the world. In this study, phytoplankton, scallop, and seawater samples were collected and monitored in the aquaculture zone of Liuqing Bay located in the coast of Qingdao, Yellow Sea, in 2014 and 2015. Dissolved LSTs in seawater were adsorbed by solid phase adsorption toxin tracking (SPATT) bags and solid phase extraction (SPE) cartridges, respectively. PTX2 was found to be the predominant LSTs, while OA and DTX1 were detected at trace levels in the three species of Dinophysis and Phalacroma genera picked from the investigation zone. The highest quota of PTX2 occurred in D. fortii (0.28 pg cell -1 ), followed by D. acuminata complex (0.08 pg cell -1 ) and P. rotundatum (=D. rotundata) (0.02 pg cell -1 ). PTX2 (nd∼5.7 μg kg -1 ), OA (nd∼2.8 μg kg -1 ) and DTX1 (nd∼1.6 μg kg -1 ) were also observed in cultivated scallops, but in addition to the former, PTX2sa, 7-epi-PTX2sa and an isobaric form of DTX1 suspected as DTX1b were also found in the SPATT bags. The isobaric compound of DTX1 only appeared in the SPATT bags deployed in field samples of seawater. A significant higher quantity of OA was adsorbed by SPATT bags compared to DTX1, although similar concentrations of both toxins were adsorbed by SPE cartridges from the aquaculture zone. AZA2 was also found in the concentrated phytoplankton cells, which demonstrated that AZA-producing microalgae were present in this investigated area. The variation in concentrations of PTX2 adsorbed by SPATT bags could reflect the change of Dinophysis density in seawater, but there was a time lag behind the peaks of Dinophysis density. Comparative results did not support the SPATT technology as a useful warning tool for toxic algal blooms and toxin accumulation in bivalves. The data suggest that the contamination of LSTs was prevalent in the coast of Qingdao and the environmental behavior of LSTs should be explored further., (Copyright © 2016. Published by Elsevier Ltd.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The neurotoxin β-N-methylamino-l-alanine (BMAA) is naturally present in some microalgal species in the marine environment. The accumulation of BMAA has widely been observed in filter-feeding bivalves that are known to consume primary producers constituting the base of complex aquatic food webs. This study was performed to assess the occurrence of BMAA and isomers in mollusks collected from nine representative shellfish production areas located on the three French coasts (Channel, Atlantic and Mediterranean sites). The use of a highly selective and sensitive HILIC-MS/MS method, with D5DAB as internal standard, revealed the systematic detection of BMAA and DAB, in concentrations ranging from 0.20 to 6.7 μg g(-1) dry weight of digestive gland tissues of mollusks. While we detected BMAA in four strains of diatoms in a previous study, here BMAA was only detected in one diatom species previously not investigated out of the 23 microalgal species examined (belonging to seven classes). The concentrations of BMAA and DAB in mussels and oysters were similar at different sampling locations and despite the high diversity of phytoplankton populations that mollusks feed on at these locations. Only small variations of BMAA and DAB levels were observed and these were not correlated to any of the phytoplankton species reported. Therefore, extensive research should be performed on both origin and metabolism of BMAA in shellfish. The levels observed in this study are similar to those found in other studies in France or elsewhere. A previous study had related such levels to a cluster of Amyotrophic Lateral Sclerosis in the South of France; hence the widespread occurrence of BMAA in shellfish from all coasts in France found in this study suggests the need for further epidemiological and toxicological studies to establish the levels that are relevant for a link between the consumption of BMAA-containing foodstuffs and neurodegenerative diseases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Okadaic acid (OA) and its derivatives, DTX-1 and DTX-2, are marine biotoxins associated with diarrhetic shellfish poisoning. Routine monitoring of these toxins relies on the mouse bioassay. However, due to the technical unreliability and animal usage of this bioassay, there is always a need for convenient and reliable alternative assay methods. A panel of monoclonal antibodies against OA was generated and the most suitable was selected for biosensor-based assay development using surface plasmon resonance. The cross reactivity of the selected antibody with DTX-1 was found to be 73%, confirming the antibody suitability for both OA and DTX detection. The OA and derivative assay was designed as an inhibition assay covering the concentrations 1-75 ng/ml, with a sensitivity of 22.4 ng/ml. The assay was highly reproducible and preliminary validation showed no matrix interference from mussel extracts and good recovery of added standard in mussel extracts, with %CV of <9.3%. This assay could provide a useful and convenient screening tool for OA and its derivatives with a comprehensive extraction protocol for shellfish monitoring programmes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The presence of phytoplankton responsible for the production of lipophilic marine biotoxins is well recognised throughout parts of South America. To date, the quantitation of lipophilic toxins in Argentinean shellfish has been limited to select and highly focussed geographical studies. This work reports the analysis for lipophilic marine biotoxins in shellfish harvested across five regions of Argentina between 1992 and 2012. LC-MS/MS analysis was used for the quantitation of all regulated lipophilic toxins. High concentrations of okadaic acid group toxins were quantified, with a clear dominance of the parent okadaic acid and more than 90% of the toxin present as esters. Results showed DSP toxins in shellfish from the Buenos Aires Province during 2006 and 2007, earlier than previously described. There was also strong evidence linking the presence of okadaic acid to human intoxications. Other lipophilic toxins detected were yessotoxin, pectenotoxin-2 and 13-desMeC spirolide. With evidence published recently for the presence of azaspiracid producers, this work reports the detection of low concentrations of azaspiracid-2 in shellfish. As such the data provides the first published evidence for yessotoxins and azaspiracids in Argentinean shellfish and further evidence for the continuing presence of lipophilic marine toxins in Argentinean waters., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Development of novel analytical tools to detect marine biotoxins has been warranted in view of the apparent global pervasiveness of algal-derived shellfish poisoning, and the limitations of existing methods. Here, we describe the initial phase in the development and evaluation of a tyrosine-containing analog of μ-conotoxin (μ-CTX) GIIIA as an alternative to saxitoxin (STX) in a receptor binding assay (RBA) for paralytic shellfish poisons. The peptide analog was synthesized and characterized for structure and bioactivity. The major product of oxidation elicited paralytic symptoms in mice at a minimum dose of 1.31 mg kg(-1) (i.p.). Mass spectrometry analysis of the bioactive peptide gave a molecular mass of 2637.52 Da that was close to the predicted value. Iodination via chloramine-T produced non-, mono- and di-iodinated peptides (respectively, NIP, MIP and DIP). Competition assays against (3)H-STX revealed higher Ki and EC50 (P < 0.0001, ANOVA) indicating reduced affinity for the receptor, and limited displacement of receptor-bound STX. However, subsequent use of MIP may extend the application of RBA to detect small changes in toxin levels owing to its likely enhanced displacement by STX. This may be useful in analyzing samples with toxicities near the regulatory limit, or in establishing baseline values in high risk environments., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The need for homogenous reference materials stable for paralytic shellfish toxins is vital for the monitoring and quality assurance of these potent neurotoxins in shellfish. Two stabilisation techniques were investigated, heat treatment through autoclaving and the addition of preserving additives into the tissue matrix. Short and long-term stability experiments as well as homogeneity determination were conducted on materials prepared by both techniques in comparison with an untreated control using two LC-FLD methods. Both techniques improved the stability of the matrix and the PSP toxins present compared to the controls. A material was prepared using the combined techniques of heat treatment followed by spiking with additives and data is presented from this optimised reference material as used over a two year period in the Irish national monitoring program and in a development exercise as part of a proficiency testing scheme operated by QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) since 2011. The results were indicative of the long-term stability of the material as evidenced through consistent assigned values in the case of the proficiency testing scheme and a low relative standard deviation of 10.5% for total toxicity data generated over 24 months., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Seasonal dynamics and the distribution of dinophysistoxin-3 (DTX-3) in organs of mussel Crenomytilus grayanus, collected in 2013 in Peter the Great Bay, Sea of Japan, were determined. Active toxin forms (OA, DTX-1/2) concentrated in digestive glands (60-80%), whereas non-active 7-O-acyl derivatives (DTX-3) were more abundant in edible soft tissues (more than 80%). Possible mechanism of fast removing of DTX-3 from the digestive gland and accumulation of DTX-3 in the soft tissues is supposed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cyanobacteria produce the neurotoxic amino acid β-N-methylamino-l-alanine (BMAA), which in contaminated marine waters has been found to accumulate in shellfish. Exposure to BMAA has been associated with an increased risk of neurodegenerative disease. Analysis of blinded samples found BMAA to be present in neuroproteins of individuals who died from ALS and ALS/PDC, but generally not in the brains of patients who died of causes unrelated to neurodegeneration or Huntington's disease, an autosomal dominant neurodegenerative disease. We here report support for a link between a patient with ALS and chronic exposure to the cyanobacterial neurotoxin BMAA via shellfish consumption. The patient had frequently eaten lobsters collected in Florida Bay for approximately 30 years. LC-MS/MS analysis of two lobsters which this ALS patient had placed in his freezer revealed BMAA at concentrations of 27 and 4 μg/g, respectively, as well as the presence of 2,4-diaminobutyric acid (DAB), a BMAA isomer. Two additional lobsters recently collected from Florida Bay also contained the neurotoxins BMAA and DAB. These data suggest that invertebrates collected in water where cyanobacterial blooms are present, if consumed, may result in direct human exposure to these neurotoxic amino acids. The data support the assertion that prolonged exposure to BMAA may have played a role in the etiology of ALS in this patient., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A toxic dinoflagellate, Karenia brevisulcata, devastated almost all marine life in Wellington Harbour, New Zealand during the late summer of 1998. Brevisulcatic acids (BSXs) and brevisulcenals (KBTs), both polycyclic ether toxins, have been identified as the causative agents. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the sensitive and specific determination of BSXs and KBTs in culture medium, seawater and shellfish. Acidified algal culture, or seawater, was extracted using reverse phase solid phase extraction cartridges. Shellfish tissue homogenate was blended with methanol-water (9:1) and partitioned with hexane to remove non-polar lipids. This extraction protocol is similar to that used for analysis of lipophilic shellfish toxins. LC-MS/MS (triple quadrupole) was used for quantitative analysis with gradient elution (acidic buffer), positive electrospray ionization and multiple-reaction monitoring. Purified toxins were available for 4 KBTs (KBT-F, -G, -H and -I) and 4 BSXs (-1, -2, -4, and -5), and were used to calibrate the instrument responses. Relative response factors were used for semi-quantitative analysis of BSX-3 and BSX-6, using BSX-1 and BSX-4 respectively. Calibration curves for all toxins monitored were linear over the concentration range tested (5-200 ng mL(-1)) with r(2) values >0.99. The method limit of quantitation was determined to be 2 ng mL(-1) for BSXs and KBTs, except KBT-I, which was 5 ng mL(-1). Validation data was generated for culture medium and shellfish. Toxin recoveries were typically >70% with relative standard deviations <20% across all of the matrices tested. In addition, toxins specific to K. brevisulcata were able to be detected in seawater at a cell concentration of 10,000 cells L(-1), which represents the suggested trigger level for this harmful algal species. This method shows suitable performance characteristics to be regarded a useful tool to monitor toxin levels in a variety of sample matrices during future bloom events., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The eastern oyster (Crassostrea virginica) and northern quahog (= hard clam, Mercenaria mercenaria) are two species of economic and ecological significance in east coast waters of the United States and the Gulf of Mexico. Commercial industries for these species, especially within the state of Florida, are significant. The current study was undertaken to build upon the already established body of knowledge surrounding effects of the toxic dinoflagellate Karenia brevis on shellfish, to provide an understanding of the kinetics of brevetoxins within shellfish tissues, and to provide an estimate of brevetoxin retention times in these shellfish after a bloom event. Individual clams and oysters were exposed to the toxic dinoflagellate, K. brevis at a bloom concentration of 5 × 10(5) cells·L(-1) for eight days and then transferred to filtered water for depuration. Individuals were sampled periodically to determine depuration rates. Concentrations of brevetoxins (and/or their metabolites measured as PbTx-3 equivalent) in tissues were determined using an Enzyme Linked Immunosorbent Assay (ELISA). After five days of exposure, brevetoxin levels in tissues of both species reached concentrations well above the regulatory limit of 800 ng g(-1) (Pb-TX3 equivalent). Averaged concentration of brevetoxins in clams was 1000 ng g(-1), while the oysters averaged 1986 ng g(-1). After two weeks of depuration, tissue concentrations in both species were below regulatory levels with clams averaging ~204 ng g(-1) and oysters averaging ~437 ng g(-1). Toxins (or their metabolities) remained detectable in both clams (139 days) and oysters (82 days) for the duration of the experiment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Yessotoxin (YTX) and okadaic acid (OA), algal toxins accumulated in edible shellfish, were previously shown to induce a specific and reversible T Cell Receptor (TCR) down-regulation in T lymphocyte EL-4 cells, in a time and concentration-dependent manner, via protein kinase C (PKC) and serine/threonine protein phosphatase 2A (PP2A) activities. In this study we have evaluated the development of other signs of toxicity induced by low concentrations of YTX or OA for 3 days of treatment. Concentrations of YTX as low as 1 nM decreased a 35% the concentration of viable cells after 48 h exposure to the toxin, while concentrations as little as 5 nM YTX or OA were sufficient to induce membrane blebbing. The concentration of YTX that produced after 24 h of incubation a 50% reduction in maximum cell viability (EC50₂₄) was approximately 46 nM, whereas with OA over 75% of the cells were still viable after exposure to 100 nM OA. According to our results, the cytoskeleton of EL-4 cells seems to be a cell component particularly sensitive to YTX and OA with disruption of F-actin cytoskeleton in these cells treated with concentrations of YTX or OA as low as 5 nM at 48 h incubation. Toxicity by YTX or OA involved typical hallmarks of apoptosis and an increase of reactive oxygen species (ROS) production. The cytotoxic effects of YTX and OA reported here, and the previously demonstrated potential of these toxins to regulate the activity of EL-4 cells through the regulation of TCR expression, rise reasonable concern about possible risks for human health associated to the chronic exposure to low amounts of YTX or OA itself or enhanced by the presence of other shellfish toxins specially by a population potentially at risk such as immunocompromised patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction and SPE clean-ups steps may also contribute. By contrast, 9 samples presented a much higher total toxicity by HPLC-FLD than by MBA. These higher results obtained by HPLC-FLD could not only be due to the use of the highest toxicity equivalency factor (TEF) for isomers oxidated into products that coelute when total toxicity of these samples were calculated. Further analyses of results obtained by HPLC-FLD and by MBA with both extracts were done separately., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤ 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required., (Copyright © 2012. Published by Elsevier Ltd.)

Chinese shellfish samples were harvested from different locations along the Chinese coast. These shellfish were analyzed by liquid chromatography in combination with mass spectrometry to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), petenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirlides (SPXs) and gymnodimines (GYM). The results revealed the lipophilic toxin profiles varied with shellfish sampling locations. In addition to OA, GYM and YTX derivatives, PTX-2 and its derivatives were found for the first time in the following Chinese shellfish: Crassostrea gigas, Mactra chinensis and Mytilus galloprovincialis. The presence of GYM, YTXs, OA and PTXs in Chinese shellfish collected from regions where no previous record of DSP-neutral toxic compounds was reported. Serious efforts should therefore be made to conduct a phycotoxin monitoring program to detect the presence of lipophilic toxins in biological materials of marine origin, which may ensure that Chinese seafood products do not present a health risk. With respect to suspected carcinogenicity, further research on the distribution and concentrations of toxic compounds are needed, in order to carry out long-term risk assessments, particularly sub-acute and chronic toxicity tests associated with of lower doses., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pre-column oxidation liquid chromatography with fluorescence detection is a chemical method for analyzing paralytic shellfish toxins. In order to improve the sample throughput and efficiency of AOAC Method 2005.06, solid core particle column technology was evaluated. We demonstrate that supplanting the original fully porous particle column with a solid core particle column reduces sample analysis time from 15 to 5 min per sample and improves resolution., (Published by Elsevier Ltd.)

Domoic acid is a potent neurotoxin that is naturally produced by several diatom species of the genus Pseudo-nitzschia. The toxin acts as a glutamate agonist and is excitotoxic in the vertebrate central nervous system and other glutamate receptor-rich organs. Human exposure to domoic acid occurs via the consumption of contaminated shellfish that have accumulated the toxin while filter feeding on toxigenic phytoplankton during blooms. The first reported human domoic acid poisoning event occurred in Canada in 1987 during which clinical signs of acute toxicity such as gastrointestinal distress, confusion, disorientation, memory loss, coma and death were observed. The illness was named amnesic shellfish poisoning (ASP) and due to effective seafood monitoring programs there have been no documented ASP cases since 1987. However, domoic acid poisoning has a significant effect on marine wildlife and multiple poisoning events have occurred in marine birds and mammals over the last few decades. Currently, domoic acid producing diatom blooms are thought to be increasing in frequency world wide, posing an increasing threat to wildlife and human health. Of particular concern are the potential impacts of long-term low-level exposure in "at risk" human populations. The impacts of repetitive low-level domoic acid exposure are currently unknown. This review provides a basic description of the mechanism of action of domoic acid as well as a synthesis of information pertaining to domoic acid exposure routes, toxin susceptibility, and the importance of effective monitoring programs. The importance of investigating the potential human health impacts of long-term low-level domoic acid exposure in "at risk" human populations is also discussed., (Published by Elsevier Ltd.)

Herein, we describe advancements in monitoring of brevetoxins in molluscan shellfish, with respect to exposure management and control of neurotoxic shellfish poisoning (NSP). Current knowledge of the fate of brevetoxins in molluscan shellfish, and the toxic potency of brevetoxin metabolites, is presented. We review rapid assays for measuring composite brevetoxins, and methodology for measuring constituent brevetoxins, in contaminated shellfish. The applicability of in vitro methods for estimating brevetoxin burden and composite toxicity in shellfish is assessed. Specific and measurable biomarkers of brevetoxin exposure and toxicity in shellfish, and of human intoxication, are described. Their utility in regulatory monitoring of toxic shellfish and in clinical diagnosis of NSP is evaluated., (Published by Elsevier Ltd.)

Diarrhetic Shellfish Poisoning (DSP) is a specific type of food poisoning, characterized by severe gastrointestinal illness due to the ingestion of filter feeding bivalves contaminated with a specific suite of toxins. It is known that the problem is worldwide and three chemically different groups of toxins have been historically associated with DSP syndrome: okadaic acid (OA) and dinophysistoxins (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). PTXs and YTXs have been considered as DSP toxins because they can be detected with the bioassays used for the toxins of the okadaic acid group, but diarrhegenic effects have only been proven for OA and DTXs. Whereas, some PTXs causes liver necrosis and YTXs damages cardiac muscle after intraperitoneal injection into mice. On the other hand, azaspiracids (AZAs) have never been included in the DSP group, but they cause diarrhoea in humans. This review summarizes the origin, characterization, structure, activity, mechanism of action, clinical symptoms, method for analysis, potential risk, regulation and perspectives of DSP and associated toxins produced by marine dinoflagellates., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Outbreaks of human illness caused by the consumption of contaminated seafood, continues to be a major problem particularly for the shellfish industry. Toxins from marine, brackish and freshwater environments, which are often produced as a result of harmful algal blooms, have been implicated as the causative agents of these poisonings. Commonly, poisoning events have been grouped into one of six classes, Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), Neurotoxic Shellfish Poisoning (NSP), Ciguatera Fish Poisoning (CFP), Azaspiracid Shellfish Poisoning (AZP), and Amnesiac Shellfish Poisoning (ASP). The causative agents of these specific poisonings along with their biosyntheses are discussed in this review. The highly unusual and complex structures of most common seafood toxins have made them interesting targets for biosynthetic studies. Many of the toxins presented are biosynthesized via complex pathways that have been elucidated either through isotope labelled precursor feeding studies and/or characterization of the genes encoding the producing organism's biosynthetic machinery. Feeding studies key to our understanding of a particular toxin's biosynthesis, such as the incorporation of unusual precursors, as well as unique biosynthetic pathways and rare chemical mechanisms involved in the assembly process are highlighted. More recently, however, modern genomics-based techniques have been used for the elucidation of biosynthetic pathways and these are presented in the context of polyketide, non-ribosomal peptide, and hybrid pathway derived, toxin assembly., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Several recombinant antibodies against one of the most potent marine toxins, Palytoxin (PlTX), were obtained using two naive human semi-synthetic phage display libraries (Tomlinson I and J) as an effective method for generating specific anti-toxin single-chain variable fragment (scFv) antibodies. After four rounds of panning and selection on free palytoxin adsorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Four phage-antibody clones specifically recognized the toxin. A competitive ELISA assay was optimized with one of these phage antibodies giving a very reproducible standard curve with a linear regression (R(2)=0.9945), showing a working range of 0.0005-500ngmL(-1). Several spiked shellfish samples were analysed by competitive ELISA to determine the accuracy of the assay, with a mean recovery rate of 90%. This study demonstrates that phage display libraries provide a valuable system for the easy and rapid generation of specific antibody fragments directed against difficult antigenic targets, such as free small molecules. Large-scale, low-cost production of anti-palytoxin scFv antibodies in Escherichia coli (E. coli) is an exciting prospect for the development of rapid and simple detection methods. Our results suggest that anti-palytoxin phage antibodies could be a valuable tool with competitive ELISA to detect palytoxin in natural shellfish samples.

The presence of ASP toxins in Luanda Bay, an area 2700 km apart from the closest record of this type of toxicity and with a different hydrographic regime, was studied. Two outbreaks were confirmed by LC/MS/MS with presence of domoic acid and some isomers both, in plankton and in three of the most important bivalve species from the area. Domoic acid levels in the studied bivalves were below the regulatory limits for most countries and the first estimations indicate that they depurated the toxin quickly. It is, therefore, unlikely that any intoxication would have taken place by consumption of these bivalve species. Notwithstanding, the relatively high annual frequency of the blooms together with possibility that other bivalve species could retain this compound more strongly, suggest that this kind of intoxication might pose a significant risk in Angola., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The Alagados Reservoir (Brazil) is an important source for the supply of water, recreation and fishery. Since 2002, the occurrence of cyanobacterial blooms (paralytic shellfish toxins - PST producers) have been noted. This study was aimed at the monitoring of PST occurrence in the Reservoir's water and fish. Biomarkers such as ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), catalase (CAT), and acetylcholinesterase (AchE) activities, lipoperoxidation (LPO), histopathology, and comet assay were analyzed in fish. Water and fish were sampled in spring, summer and autumn. The PST concentrations in water were 5.15, 43.84, and 50.78 ng equiv Saxitoxin/L in the spring, summer and autumn, respectively. The PST muscle concentration was below the limit for shellfish. Gonyautoxins (GTX) were found in water samples and fish muscle, and GTX 5 was the major analogous found in muscle. In the summer samples, the LPO, genetic damage, and the GST and AchE activities increased while in the autumn an increase in EROD activity and genetic damage were observed. In all samplings, histopathological alterations in the fish gills and liver were found. The results showed a seasonal variation in the fishes health, which could be related also to farming activities and to the contaminants bioavailability during the year., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In an attempt to evaluate the risk for human consumption associated to the accumulation of lipophilic toxins by two commercially important bivalves: macha (Mesodesma donacium) and clam (Mulinia edulis) in Coquimbo Bay (Chile), monitoring of these species was carried out from March to September 2008. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to detect okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins and spirolides. Low levels of Azaspiracid-1 and 13-desmethyl C spirolide were found in both species. The toxins were detected at different dates throughout the monitoring period and in some cases both toxins were detected in the same sample. In all cases, the concentration of the toxins was below the limit of quantification of the technique used and therefore these detections are only indicative of a potential risk. This is the first report of the occurrence of these groups of toxins in Chile and suggests that it is necessary to monitor routinely these substances to warrant public health and shellfish exportations., (Copyright 2009 Elsevier Ltd. All rights reserved.)

We have investigated some characteristics of antibodies in the lateral flow format for detecting paralytic shellfish poisoning (PSP) toxins and compared them with the mouse bioassay (MBA). The MBA is still the most reliable test for toxicity in shellfish because it provides an estimate of toxicity directly and can include more than one contaminant. Most other methods, including those involving antibodies, provide estimates of toxin concentration from which toxicity is implied or calculated using conversion factors. Antibody methods suffer from an additional deficiency as sensitivities to the different PSP analogues are unequal. Furthermore, these differences in cross-reactivity are unrelated to differences in specific toxicities. We have addressed the question of what is the toxicity of a sample at the limit of detection (LOD) of the Jellett Rapid Testing Ltd (JRT) lateral flow immunochromatographic (LFI) test. A way to calculate sensitivity to toxicity from toxin profiles is presented and used to examine a variety of PSP toxin mixtures. The calculated values for the sensitivity of the JRT (toxicity at the LOD) for separate PSP toxin analogues may vary over a wide range, but for complex mixtures, typical of natural samples, the range is much narrower. An analysis of PSP toxin profiles of 339 samples from Alaska, Britain, Canada (BC), and USA (Maine) shows the distribution of calculated toxicities at the LOD. The majority (76%) falls within the range 20-50 microg STX eq/100g with a mean at 32 microgSTXeq/100g which is similar to that of the MBA. Observed data from independent parallel studies with the JRT and MBA with a total of 3492 samples from regulatory laboratories in different countries in the period 2003-2007 show close agreement between the two methods. All samples that were found to be positive with the MBA were also positive with the JRT except for one which indicated a false negative rate of less than 0.03% of all samples tested. The JRT for PSP was designed to be more sensitive than the MBA in order to be used as a screen to reduce the high proportion of negative samples encountered in routine monitoring. Toxicity at the LOD varies depending on the mixture of PSP toxins and false positives are therefore inevitable. In this study false positives accounted for between 1.4% and 55% of the total number of samples tested. This would also depend on whether contamination was endemic or rare in the different locations. The data from regulatory monitoring for PSP show that in all areas the majority of samples are negative and so the use of a screen would result in a significant reduction in the use of mice., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Portuguese bivalves are recurrently contaminated with okadaic acid (OA) and dinophysistoxin-2 (DTX2), found mainly in esterified forms. Throughout the years different conditions have been reported in the literature for releasing the parent toxins through an alkaline hydrolysis step, in order to simplify their detection by HPLC-FLD or LC-MS. In order to clearly understand toxin stability and reaction end-point the binominous temperature/time course and base concentration were studied using naturally contaminated bivalve samples. The results showed a strong temperature dependence of the reaction. At 60 degrees C and 70 degrees C the hydrolysis was fast, and 40min were sufficient for maximal recovery of OA and DTX2, while at 40 degrees C and 50 degrees C it was only complete after 100min and 60min, respectively. At room temperature the reaction was slow and incomplete even after 2h. Stability of OA and DTX2 in semi-purified bivalve matrix at 70 degrees C for 2h was demonstrated. Concentrations of sodium hydroxide lower than 2.5M, corresponding to a final incubation concentration of 0.23M, resulted in incomplete release of parent toxins, demonstrating that high concentrations are needed when taking into account the dilution in the supernatant extract.

Paralytic Shellfish Toxins (PST) are endemic components found in filter bivalves in Southern Chile. Post-mortems analysis of fluid and tissue samples has shown biotransformation of PST in humans. The Gonyautoxin 3 (GTX3) and Gonyautoxin 2 (GTX2) are the major PST components in the toxin profile found in Chilean shellfish extracts, being as much as 65% of the total content of PST in filter bivalves. Therefore, they are the major accountable components of the human intoxication by shellfish consumption. The aim of this study is to show in vitro glucuronidation and biotransformation of GTX3 and GTX2 when they are incubated with microsomal fraction isolated from healthy human livers. Microsomes fractions isolated from human livers were incubated with GTX3 and GTX2 purified from contaminated mussels. After different incubation times, incubated samples were extracted and analyzed by HPLC with fluorescent on line detection and HPLC-MS analysis. The results revealed that GTX3 and GTX2, only when they were incubated with microsomal fraction and appropriated cofactors, showed to be enzymatic transformed in vitro. The glucuronidation of GTX3 and GTX2 followed typical Michaelis-Menten kinetics, resulting in apparent kinetic parameters of Km=39.4+/-0.24 microM and Vmax=6.0x10(-3) pmol/min/mg protein. In addition, the microsomes fraction also oxidized GTX3 and GTX2 into Gonyautoxin 4 (GTX 4) and Gonyautoxin 1 (GTX 1) resulting in 0.339x10(-3) pmol/min/mg protein. In conclusion, this study reports oxidation and glucuronidation of GTX3 and GTX2 when they are incubated with human liver microsomal fraction. The metabolism occurs via a glucuronidation reaction, the basis first step of biotransformation in human liver. Also it is showed that GTX4 and GTX1 came by biotransformation from GTX3 and GTX2 in humans. This data confirm human biotransformation found in human post-mortem fluid and tissue samples described previously. This data is the first evidence of in vitro glucuronidation of PST, given a metabolic pathway of detoxification and excretion of PST in human.

A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at -20 degrees C for at least 4h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313 microg STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89 microg STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC-MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC-MS/MS analyses.

Urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) were examined for biomarkers of brevetoxin intoxication. Brevetoxins were concentrated from urine by using solid-phase extraction (SPE), and analyzed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine extracts were fractionated by LC, and fractions analyzed for brevetoxins by ELISA. In subsequent LC-MS/MS analyses, several brevetoxin metabolites of B-type backbone were identified, with elution profiles consistent with those of ELISA. The more abundant brevetoxin metabolites in urine were characterized structurally by LC-MS/MS. With the exception of BTX-3, brevetoxin metabolites in urine differed from those found in shellfish and in shellfish meal remnants. Proposed structures of these major urinary metabolites are methylsulfoxy BTX-3, 27-epoxy BTX-3, and reduced BTX-B5. BTX-3 was found in all specimens examined. BTX-3 concentrations in urine, as determined by LC-MS/MS, correlated well with composite toxin measurements by ELISA (r(2)=0.96). BTX-3 is a useful biomarker for confirmation of clinical diagnosis of NSP.

The French Phytoplankton and Phycotoxins monitoring network (REPHY) recently found positive or dubious negative shellfish samples using lipophilic toxins mouse bioassay. These samples were analyzed by liquid chromatography (LC) in combination with mass spectrometry (MS) to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirolides (SPXs) and gymnodimines (GYMs). Over the 2006-2007 period, chemical analyses revealed various lipophilic toxin profiles according to shellfish sampling locations. In addition to OA and/or PTX-2 and their derivatives, several other compounds were found for the first time in France: (1) during the summer of 2006, AZA-1 and AZA-2 in Queen scallops (Aequipecten opercularis) from Northern Brittany; (2) during the summer of 2007, YTX and its major metabolites (45-hydroxy-YTX, homo-YTX, carboxy-YTX) in shellfish from the Mediterranean coast. Regarding YTX-group, the toxin profiles evolution in mussels during summer showed that: (i) the carboxy-YTX depuration rate was much slower than the YTX and 45-hydroxy-YTX ones; (ii) the homo-YTX concentration, which was initially very weak, increased significantly during the last depuration phase, which seems to reveal a YTX-group high metabolisation level in mussels. This paper reports for the first time on AZA and YTX-groups detection in French shellfish.

Shellfish samples were collected from coastal and offshore aquaculture sites and harvesting areas in Scottish waters between March 2003 and September 2004. Samples were analysed for the presence of algal toxins using traditional mouse bioassays for the detection of paralytic shellfish poisoning (PSP) toxins and diarrhetic shellfish poisoning (DSP) toxins; immuno-lateral flow chromatography for the detection of PSP toxins in the form of the Jellett Rapid Test; high-performance liquid chromatography (HPLC) with UV diode-array for the detection of amnesic shellfish poisoning (ASP) toxins; and liquid chromatography with mass spectrometry (LC-MS) for the detection of multiple lipophilic shellfish toxins (LSTs) including pectenotoxins (PTXs), yessotoxins (YTXs), azaspiracids (AZAs) and toxins from the 'traditional' DSP toxin group, okadaic acid (OA) and dinophysistoxins (DTXs). In order to investigate the presence of OA esters, alkaline hydrolysis was performed. All toxin groups were detected with a geographically widespread distribution. ASP toxins were the most prevalent occurring in 69% of samples. Using the PSP mouse bioassay, PSP toxins were detected in 5% of shellfish samples from coastal waters around the islands and the east coast. The Jellett Rapid Test for PSP toxins revealed a wider distribution (24% of samples) including the west coast of Scotland. Toxins from the 'traditional' DSP toxin group (OA/DTXs) and/or other LST groups (PTXs, YTXs and AZAs) were detected by LC-MS in 63% of the shellfish analysed. PSP, ASP toxins and LSTs occurred concurrently in a limited sample set, highlighting the importance of using methods capable of detecting multiple algal toxin groups in Scottish shellfish monitoring programmes.

In order to investigate the toxicity of Ostreopsis species present in Greek coastal waters, cultures of Ostreopsis sp. and Ostreopsis ovata, mixed Ostreopsis field populations and shellfish collected from coastal waters of North Aegean Sea during late summer and autumn periods of 2004, 2005 and 2006 were examined by both mouse bioassay (MBA) and hemolysis neutralization assay (HNA). MBA testing was based on two different extraction protocols, while HNA also included the use of ouabain, a known palytoxin (PLT) antagonist. Results indicated the presence of a compound in both Ostreopsis cells and shellfish tissues, which was strongly toxic to mice. This compound exhibited characteristic symptomatology in mice (death, numbness, waddling gait and blindness) to that of PLT, as well as delayed hemolytic activity, which was neutralized by ouabain. HNA indicated that Ostreopsis cells contained a PLT-like compound (putative PLT, p-PLT) at concentrations ranging between 0.4 and 0.9 pg/cell, whereas concentration in shellfish tissues was estimated to range from about 33.3 to 97.0 microg p-PLT/kg tissue. To our knowledge, this is the first report of p-PLT contamination of shellfish by natural Ostreopsis species populations in European coastal waters and possibly globally, and also the first evidence on Ostreopsis cells' toxicity in the Eastern Mediterranean Sea.

Soft shell crab is a seafood delicacy in many parts of the world. In Denmark, it has been investigated whether a commercial production of soft shell European green crabs (Carcinus maenas) would be feasible. In relation to this, a feeding study was performed to examine if occurrence of DSP toxins in the product could be a food safety problem. The crabs were fed with mussels containing DSP toxins (2500 microg total okadaic acid equivalents/kg) for 17 days and then fasted for 19 days. The content of total okadaic acid equivalents in the digestive organs was on average 27 times higher than the corresponding content in the body meat. The highest level of total okadaic acid equivalents measured was 12 microg/kg in body meat and 503 microg/kg in digestive organs. The results show that the content of DSP toxins in a commercial product of soft shell European green crab (without digestive organs) could be regarded as negligible.

Some lipophilic shellfish toxins (LSTs) can cause human illness due to eating shellfish that have become naturally contaminated following filter feeding on toxin producing algae. A mouse bioassay (MBA) is widely used to detect LSTs in regulatory monitoring of shellfish. However, the MBA is imprecise giving only a positive or negative result and is prone to interference from other compounds. In this study, liquid chromatography-mass spectrometry (LC-MS) was compared to the MBA, with the aim of substituting the in vivo assay for monitoring shellfish from Scottish waters. Overall, it was not feasible to demonstrate equivalence of LC-MS with the MBA, but due to the detection of a range of LSTs, it is considered that LC-MS methods capable of detecting multiple analogues are accepted by international markets of shellfish to assure consumer protection.

In 2002 several hundred people were taken ill after eating self-harvested brown crabs (Cancer pagurus) in the southern part of Norway. The symptoms were similar to diarrhetic shellfish poisoning (DSP) although with a somewhat delayed onset. This happened at the same time as an unusual early bloom of Dinophysis acuta had lead to high amounts of DSP toxins in blue mussels (Mytilus edulis) in the same area. The proposed cause of the intoxication was that crabs had accumulated toxins by eating blue mussels. Analyses of crab material from the area revealed very little free toxin in the form of okadaic acid (OA). However, after alkaline hydrolysis of the material, the amounts of OA found in the crabs were above the toxic level. MS/MS analysis of a sample from one intoxication episode indicated presence of the 14:0, 16:1, 16:0 and 18:1 fatty acid esters of okadaic acid. Esterified OA constituted more than 90% of total identified DSP toxins in crabs, indicating that not only esterified toxin from mussels was accumulated, but also that appreciable transfer of OA to OA-esters occurred in the crabs.

Azaspiracid poisoning (AZP), the most recently discovered human gastrointestinal illness resulting from consumption of contaminated shellfish, so far has been found in coastal areas of northern Europe. This is the first report of a survey carried out for contamination of shellfish harvested in costal areas of Portugal for the presence of azaspiracids. The study design covered the commercial species usually more contaminated by toxins from dinoflagellates (blue mussel, common cockle, donax clam) in coastal areas representative of the NW, SW and south coasts. A method based on liquid chromatography-mass spectrometry was setup for the first time for this purpose. No azaspiracids were found on 300 samples tested between 2002 and 2003. On at least three samples a peak with a retention time matching that of AZA2 was found, never surpassing one tenth of the current EU limit. Unambiguous identification of any known AZA did not occur yet. The risk for human outbreaks of AZP seems to be very low, comparatively with amnesic shellfish poisoning (ASP), where levels close to the allowance level are found sparsely, or to diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP), where high levels and registered human outbreaks have been found in recent years.

We report for the first time, the presence of saxitoxin (STX) in a common cephalopod, Octopus (Abdopus) sp. 5, collected from Cooke Point on the northern coastline of Western Australia. Sodium channel and saxiphilin based radio-receptor assays detected saxitoxin-like binding in octopi extracts. Further analysis by liquid chromatography-fluorescence detection (LC-FLD) identified STX as the major contributing toxin in these samples. The presence of STX was confirmed by LC-mass spectrometry and comparison of fragmentation patterns with an authentic STX standard. LC-FLD quantitation and conversion of the Octopus sp. 5 extracts revealed toxin concentrations as high as 246 microg STX/100g tissue, more than three times the US, European and Australian regulatory limit for human consumption of shellfish of 80 microg STX/100g tissue. There was no evidence of tetrodotoxin or other paralytic shellfish toxin derivatives. This level and distribution of STX in octopi poses a potential public health risk, particularly when routine toxin screening of wild catch is not regulated.

In July 5, 2002 fishermen working in harvesting sea urchin (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated by consumption of filter-feeder bivalve Aulacomya ater. After the ingestion of 7-9 ribbed mussel, two fishermen died 3-4 h after shellfish consumption. The forensic examination in both victims did not show pathological abnormalities with the exception of the lungs conditions, crackling to the touch, pulmonary congestion and edema. The toxic mussel sample showed a toxicity measured by mouse bioassay of 8575 microg of STX (saxitoxin) equivalent by 100 g of shellfish meat. Using post-column derivatization HPLC method with fluorescent on line detection was possible to measure mass amount of each paralytic shellfish poisoning (PSP) toxin yielding individual toxin concentrations. These PSP toxins were identified in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The toxin profiles of each body fluid and tissue samples and the amount of each PSP toxin detected are reported. The PSP toxins found in the gastric content, were STX and the gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2) which showed to be the major amount of PSP toxins found in the victims biological samples. The PSP toxin composition in urine and bile showed as major PSP toxins neoSaxitoxin (neoSTX) and GTX4/GTX1 epimers, both STX analogues with an hydroxyl group (-OH) in the N(1) of the tetrahydropurine nucleus. The neoSTX was not present in the gastric content sample, indicating that the oxidation of N(1) in the STX tetrahydropurine nucleus resulted neoSTX, in a similar way that GTX3/GTX2 epimers were transformed in GTX4/GTX1 epimers. Beside this metabolic transformation, also the hydrolysis of carbamoyl group from STX to form its decarbomoyl analogue decarbamoylsaxitoxin was detected in liver, kidney and lung. These two findings show that PSP toxins went under metabolic transformation during the 3-4 h of human intoxication period, in which PSP toxins showed enzymatic oxidation of N(1) in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. This study conclude, that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound.