Surveillance of algal toxins in shellfish from Scottish waters.

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.     

Investigation of the toxin profile of Greek mussels Mytilus galloprovincialis by liquid chromatography-mass spectrometry.

Samples of Mytilus galloprovincialis were harvested from five different locations in Thermaikos gulf, Greece after harmful algae bloom. All of the mussel samples were found positive by mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins. Liquid chromatography (LC) coupled with mass spectrometry (MS) was used to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and yessotoxins (YTXs). In order to investigate the presence of okadaic acid esters, alkaline hydrolysis was performed for all the samples, and LC-MS analyses were carried out on the samples before and after hydrolysis. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) analyses were also carried out to investigate the presence of domoic acid and paralytic shellfish poisoning (PSP) toxins at trace levels. All of the samples were found to be contaminated only with okadaic acid at levels 0.10-0.20 microg/g.     

Lipophilic toxin profile in Galicia (Spain): 2005 toxic episode

By the end of 2005, a toxic episode of phytoplankton origin in bivalve shellfish led to the closing down of several shellfish production areas in Galicia (northwestern region of Spain). During this time, different kinds of shellfish were collected and analysed by LC–MS/MS to search for the following lipophilic toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and spirolides. Samples were analysed before alkaline hydrolysis in order to investigate the presence of free OA and DTXs, AZAs, PTXs and spirolides, and after alkaline hydrolysis to detect OA and DTXs esters. All of the samples were found to be contaminated with OA and/or DTX-2, as well as esterified forms of these diarrhetic shellfish poison (DSP) toxins, at levels around and above European regulatory limit (160 μg of okadaic acid equivalents/kg). The analyses of mussels and razor clam also revealed the presence of 13-desmethyl spirolide C (SPX-1) at levels below 31 μg/kg. Likewise, in many of the samples different levels of pectenotoxin-2 secoacid (PTX-2sa) were detected. DSP toxin esters represent practically the 100% of the total OA equivalents for scallops, clams, razor clams and cockles.     

Acute Cardiotoxicity Evaluation of the Marine Biotoxins OA,DTX-1 and YTX

Phycotoxins are marine toxins produced by phytoplankton that can get accumulated in filter feeding shellfish. Human intoxication episodes occur due to contaminated seafood consumption. Okadaic acid (OA) and dynophysistoxins (DTXs) are phycotoxins responsible for a severe gastrointestinal syndrome called diarrheic shellfish poisoning (DSP). Yessotoxins (YTXs) are marine toxins initially included in the DSP class but currently classified as a separated group. Food safety authorities from several countries have regulated the content of DSPs and YTXs in shellfish to protect human health. In mice, OA and YTX have been associated with ultrastructural heart damage in vivo. Therefore, this study explored the potential of OA, DTX-1 and YTX to cause acute heart toxicity. Cardiotoxicity was evaluated in vitro by measuring hERG (human èter-a-go-go gene) channel activity and in vivo using electrocardiogram (ECG) recordings and cardiac damage biomarkers. The results demonstrated that these toxins do not exert acute effects on hERG channel activity. Additionally, in vivo experiments showed that these compounds do not alter cardiac biomarkers and ECG in rats acutely. Despite the ultrastructural damage to the heart reported for these toxins, no acute alterations of heart function have been detected in vivo, suggesting a functional compensation in the short term.     

DTX5c, a new OA sulphate ester derivative from cultures of Prorocentrum belizeanum

Prorocentrum belizeanum is a dinoflagellate known for its okadaic acid (OA) and dinophysitoxins (DTXs) production, both OA and DTX are polyether toxins of the Diarrhetic Shellfish Poisoning (DSP) group. We have recently published the isolation of a new diol-ester of okadaic acid from cultures of P. belizeanum. On this occasion we present a new sulphated water-soluble derivative, DTX-5c, isolated from this microalga, whose structure was established on the basis of its spectroscopical data.     

Profiles and levels of fatty acid esters of okadaic acid group toxins and pectenotoxins during toxin depuration, Part I: Brown crab (Cancer pagurus)

In 2002, two outbreaks of diarrhetic shellfish poisoning (DSP) occurred in Norway, which was later confirmed to be caused by the consumption of brown crab (Cancer pagurus) contaminated predominantly by esters of okadaic acid (OA) after feeding on toxic blue mussels (Mytilus edulis). In addition to OA-group toxins, pectenotoxins (PTXs) are commonly detected in the toxin-producing algae (i.e. Dinophysis). In this paper, an experiment was set up to study the fatty acid ester profiles and depuration rates of OA-group toxins and PTXs from C. pagurus after feeding on M. edulis containing these toxin groups. OA, DTX1, DTX2 and PTX2 SA were all detected primarily in the form of fatty acid esters in the crab hepatopancreas (HP). Crabs preferentially assimilated toxins of the OA group after feeding on the mussels for 1 week. Detailed analysis of the fatty acid ester profile in crabs and mussels showed that the ester profiles in the crabs differed slightly from profiles of the fatty acid esters in M. edulis, but neither ester profile nor ester to free toxin ratio appeared to change in the crabs during the first 2 weeks of depuration. Calculations of depuration rates of the free forms of toxins resulted in similar reduction rates for OA and DTX2, whereas the depuration rate of DTX1, PTX2 and PTX2 SA was considerably faster. From the industrial perspective, the PTX-compounds are of minor importance compared to the OA group toxins in crabs, considering (1) the uncertainty regarding the oral toxicity of the PTXs, (2) the preferential ingestion of OA-group toxins compared to PTXs and (3) the faster depuration of PTXs.     

Studies of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland using multiple tandem mass spectrometry

Diarretic shellfish poisoning (DSP) is a toxic syndrome associated with the consumption of bivalve molluscs. The DSP toxins are polyether compounds, which include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs) and pectenotoxin seco acids (PTX2SAs). These toxins originate in marine dinoflagellates, including Dinophysis spp. Phytoplankton samples were collected from the southwest coast of Ireland and D. acuta was the predominant species. Monocultures of D. acuta cells were prepared by hand picking from microscope slides in order to confirm their toxin profiles. There was a remarkable consistency in the toxin profiles in all of the phytoplankton samples collected during the summer months, irrespective of location, depth or mesh size. Analysis using liquid chromatography—multiple tandem mass spectrometry (LC-MS/MS) revealed that DTX2 and OA were the predominant toxins at a consistent ratio. The average toxin composition was: DTX2 (53±5%), OA (26.5±2.3%) and total pectenotoxins (20.8±4.7%). Toxin profiles in D. acuta from Europe were distinctly different from those found in New Zealand, where PTX2 was the predominant toxin and DTX2 was absent.     

First report on azaspiracid and yessotoxin groups detection in French shellfish.

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.     

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.     

Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC-MS/MS analysis.

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.     

Differences in Susceptibility to Okadaic Acid,a Diarrhetic Shellfish Poisoning Toxin,between Male and Female Mice

The mouse bioassay (MBA) for diarrhetic shellfish poisoning (DSP) toxins has been widely used in many countries of the world. In the Japanese and EU methods, male mice are designated to be used for MBA. Female mice were described to be less susceptible than male mice. To the best of our knowledge, however, there have been no reports on the details of sex differences in susceptibility to DSP toxins. In this study, we investigated whether, and to what extent, female mice are less sensitive to DSP toxins. A lethal dose of okadaic acid (OA), one of the representative DSP toxins, was injected intraperitoneally into mice. The mice were observed until 24 hours after injection. Both male and female mice of ICR and ddY strains, which are designated in the Japanese official method, were compared. All the mice were four weeks old and weighed 18–20 g. The experiments were repeated twice. The lethality was 70%–100%. Survival analysis showed no sex differences in susceptibility to OA, but ICR female mice showed significant resistance compared with other groups in one out of two trials. These results indicate that sex differences were not clear but, nonetheless, male mice showed more stable results.     

Comparison between HPLC and a commercial immunoassay kit for detection of okadaic acid and esters in Portuguese bivalves.

Liquid chromatography (HPLC), used to identify diarrhoeic shellfish poisoning (DSP) toxins in Portuguese shellfish, has detected okadaic acid (OA), dinophysistoxin-2 (DTX2) and esters of both of these. In Donax clams, a surprisingly high level of esters has been recently associated with some outbreaks of diarrhoea in shellfish consumers. In view of these events, we have proposed that screening for esters must be included in monitoring programmes for DSP toxins. HPLC is laborious, time-consuming and suffers from some interferences at low detection levels in total meat extracts. An enzyme-linked immunosorbent assay (ELISA) based on the procedure of Usagawa et al. ('DSP-Check' kit) was tested against HPLC. The 'DSP-Check' kit was capable of quantitatively detecting DSP toxins in all the tested contaminated samples containing only okadaic acid, provided that the parent toxins were within the range of detection and were not in the ester form. A high correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit tested appeared to be more sensitive, specific and faster than HPLC for determination of DSP in total shellfish meat extracts. No problems were found when using hydrolysed semi-purified extracts in order to detect esters of okadaic acid. In view of the results obtained so far, Donax clams appeared to be an excellent indicator of shellfish contamination with diarrhoeic toxins. On sandy beaches of the Portuguese southern coast, were rock mussels are not so abundant, they should be screened more often than other species in order to prevent diarrhoea in humans.     

Diarrhetic shellfish poisoning by okadaic acid esters from Brown crabs (Cancer pagurus) in Norway.

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.     

Esterification of okadaic acid in the mussel Mytilus galloprovincialis

Okadaic acid and other toxins of the diarrheic shellfish poisoning (DSP) group are transformed mainly to their acyl-derivatives in bivalves. Some recent studies suggest that bacteria present in the bivalve gut could contribute substantially to the acylation of the toxins. By feeding microcapsules containing okadaic acid to mussels we have shown unequivocally that the ingested okadaic acid is nearly completely transformed to its fatty acid esters (acyl-derivatives). Treating mussels with antibiotics did not have any significant effect on the acylation of the supplied okadaic acid, suggesting that bacteria do not play any significant role in this process. The microsomal and mitochondrial subcellular fractions of the cells of the digestive gland have been shown to have contain enzymes that are able to transfer a fatty acid molecule from Coenzyme A to okadaic acid (so, that have Acyl-CoA:OA acyltransferase activity). This activity was related to that of the enzyme Cytochrome C reductase (NADPH), a marker of endoplasmic reticulum, suggesting that this organelle is the main responsible for the acylation process. Acylation of DSP toxins seems to be a key step in the depuration of these toxins from mussels, as these compounds are found in feces as acyl-derivatives. This is probably true for most bivalves. The proportion of acyl-derivatives accumulated can point to the key process of the depuration: acylation or excretion of acylated derivatives. In the mussels Mytilus galloprovincialis, Mytilus edulis and in Donax trunculus, the first process seems to be the most important, but in most bivalve species it seems to be the second one. Other aspects of the relationship between depuration and acylation are also discussed.     

Okadaic acid, useful tool for studying cellular processes

One of the most interesting groups of substances of marine origin, from structural and pharmacological points of view are polyether toxins, which generally present a great diversity in size and potent biological activities. The subject of this review is limited to okadaic acid (OA). It was the first example of a group of polyether toxins produced by marine microalgae, which is responsible for the natural phenomena known as Diarrhetic Shellfish Poisoning, DSP red tides. These toxins are accumulated in the digestive glands of the shellfish with a disastrous effect upon the shellfish industry in many parts of the world. Thus, it has been demonstrated that OA is a highly selective inhibitor of protein phosphatases type 1 (PP1) and 2A (PP2A), subsequently that it causes dramatic increases in phosphorylation of numerous proteins as well as being a potent tumour promoter. For that reason, OA is an extremely useful tool for studying the cellular processes that are regulated by reversible phosphorylation of proteins as signal transduction, cell division and memory.     

First U.S. report of shellfish harvesting closures due to confirmed okadaic acid in Texas Gulf coast oysters

Between March 7 and April 12, 2008, several bay systems on the east (Gulf of Mexico) coast of Texas, USA were closed to the harvesting of oysters (Crassostrea virginica) due to the presence of the DSP (Diarrheic Shellfish Poisoning) toxin okadaic acid in excess of the 20 μg/100 g tissue FDA regulatory guidance level. This was the first shellfish harvesting closure due to the confirmed presence of DSP toxins in US history. Light microscopic cell counts were performed on water samples collected from numerous sampling sites along the Texas Gulf coast where shellfish harvesting occurs. Ultra performance liquid chromatography, electrospray ionization, selected reaction monitoring, mass spectrometry (UPLC/ESI/SRM/MS) was used to detect DSP toxins in oysters. The closures were associated with an extensive bloom of the dinoflagellate Dinophysis cf. ovum. Only okadaic acid (OA) and OA acyl esters were found in shellfish tissues (max. OA eq. levels 47 μg/100 g tissue). OA was also confirmed in a bloom water sample. No illnesses were reported associated with this event. DSP toxins now add to a growing list of phycotoxins, which include those responsible for PSP (paralytic shellfish poisoning), NSP (neurotoxic shellfish poisoning), and ASP (amnesic shellfish poisoning) which must now be monitored for in US coastal waters where shellfish are harvested.     

Monthly variations in diarrhetic toxins and yessotoxin in shellfish from coast to the inner part of the Sognefjord, Norway.

Monthly concentrations of diarrhetic shellfish poisoning (DSP) toxins and yessotoxin (YTX) in mussels from the coast to the inner part of the Sognefjord were determined. Mussels from nine locations were sampled from March to November 1997. The DSP toxins and YTX were analysed by a colorimetric protein phosphatase 2A (PP2A) inhibition assay or fluorometric HPLC, respectively. The mouse bioassay for DSP toxins was performed including either chloroform or diethyl ether in the final step of extraction. Using ether in the final step normally facilitated extraction of the DSP toxins, okadaic acid (OA) and dinophysis toxin-1 (DTX-1), while chloroform extraction included a wider spectrum of toxins, including YTX and a fast acting toxin(s) with neurotoxic effects. The concentrations of DSP toxins and YTX in mussels increased with distance from the coast. The highest concentrations of YTX (574 microg YTX/100 g mussel meat) and diarrhetic toxins (349 microg OA equivalents/100 g mussel meat) were measured in May and August, respectively, at locations in the inner part of the fjord. Since concentrations of DSP toxins and YTX in mussels increased with distance from the coast, the locations for mussel farming in the Sognefjord close to the coast, seem to be preferable.     

Study of cytoskeletal changes induced by okadaic acid in HL‐7702 liver cells and development of a fluorimetric microplate assay for detecting diarrhetic shellfish poisoning

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal illness with symptoms such as diarrhea, nausea, vomiting, headache, chills and moderate to severe abdominal pain. DSP has been recognized as a worldwide public health problem, causing great concern to the shellfish industry. Accumulation of DSP in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread collection and thorough monitoring program for mollusk toxicity. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would be necessary. In this study we investigated cytoskeletal changes induced by okadaic acid in HL‐7702 Liver Cells and developed a new cytotoxicity assay for detection and quantitation of DSP. This assay is based on fluorometric of F‐actin depolymerization induced by okadaic acid (OA) compounds in HL‐7702 liver cell line. The measurable range of OA was 2.5 ～ 40 nmol/L. The detection limit of the F‐actin assay for OA was 2.01 μg/100 g muscles in shellfish extracts. The performance of this assay has been evaluated by comparative analysis of shellfish samples by the fluorescent assay, mouse bioassay, and ELISA assay. Comparison of the results by all three methods revealed excellent consistency, the results of fluorescent assay were in significant correlation with ELISA assay (R² = 0.830). Examination of F‐actin assay is very convenient, rapid, and sensitive, which can be used to quantify the amount of OA in shellfish samples. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

First confirmation of human diarrhoeic poisonings by okadaic acid esters after ingestion of razor clams (Solen marginatus) and green crabs (Carcinus maenas) in Aveiro lagoon, Portugal and detection of okadaic acid esters in phytoplankton.

A new outbreak of human diarrhoeic poisonings (DSP) with esters of okadaic acid (OA) was confirmed after ingestion of razor clams (Solen marginatus) harvested at Aveiro lagoon (NW Portugal) in the summer of 2001. Accumulation of marine toxins in second order consumers was investigated in the edible parts of a shellfish predator abundant at Aveiro lagoon, the green crab Carcinus maenas. Okadaic acid was found, also in a predominant esterified form. Levels in edible parts (comprising mainly viscera) surpassed 16microg/100g. We suggest that one patient may have developed profuse diarrhoea after ingestion of a large number of green crabs contaminated with okadaic acid esters. At least 32microg OA/100g were found in a remaining sample of its meal. Domoic acid was also found but under the allowable level in force in USA of 30microg/g crab viscera. In cooked crabs, significant losses of domoic acid were found and it is not suspected to have contributed to the poisoning event, although being a vector for this toxin. The low percentage of free okadaic acid found is in accordance with a predation predominantly on benthonic shellfish (razor clams, clams and common cockle) rather than on rock mussels. These last ones present usually higher percentages of free okadaic acid.Okadaic acid was confirmed with full-scan mass spectra either in plankton and mussel extracts. Okadaic acid esters were also found in plankton extracts. Percentages between 40-60% of esterified OA were found in samples freshly extracted. Ester's percentage diminished drastically if after sonication the extract was kept at room temperature. The major part of the esters was water-soluble.     

Naturally contaminated shellfish samples: quantification of diarrhetic shellfish poisoning toxins in unhydrolysed and hydrolysed extracts and cytotoxicity assessment

Contamination of shellfish from the Portuguese coast with diarrhetic shellfish poisoning (DSP) toxins is a recurrent event, with most of the commercial bivalves contaminated with high percentages of esters of okadaic acid (OA) and dinophysistoxin‐2 (DTX2). This report describes the quantification of DSP toxins in unhydrolysed and hydrolysed extracts of several cockle and mussel samples naturally contaminated and the evaluation of their cytotoxicity profiles in V79 cells. The quantification of the acyl esters in the shellfish samples involved the cleavage of the ester bond through alkaline hydrolysis and the release of the parent toxins OA and DTX2. Unhydrolysed and hydrolysed extracts were then analyzed by liquid chromatography (LC) coupled with mass spectrometry (MS) for the detection and quantification of DSP toxins. The cytotoxicity of the analysed extracts was evaluated using the MTT reduction assay and compared with the cytotoxicity presented by different concentrations of OA standard (1–100 nm ). OA exhibited marked cytotoxic effects and decreased cell viability in a dose dependent mode, with an IC₅₀ of 27 nm . The cytotoxicity pattern of unhydrolysed extracts was clearly dependent on the concentration of free toxins. Moreover, the cytotoxicity of the esterified toxins present was revealed after their conversion into free toxins by alkaline hydrolysis. For the hydrolysed extracts of cockles and mussels, the cytotoxicity presented was mainly related to the concentration of OA and DTX2. Copyright © 2010 John Wiley & Sons, Ltd.