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["Paralytic shellfish poisoning" (author's transl)].

In October 1976 an epidemic of 120 cases of paralytic shellfish poisoning was recorded in western Europe. Analysis of the 23 cases seen in Swizterland shows the following data; paresthesia of mouth and lips (100%); cerebellar syndrome (86%) with giddiness, ataxia, dysmetry and floating sensation; paresia or paralysis (62%); digestive symptoms (14%); no death. The mean incubation time is 3 hs 30 min; mean duration of the symptoms is 2 days. Half of the patients complain of asthenia and moderate memory disturbance lasting up to 3 weeks. The severity of the illness is in relation to the amount of ingested neurotoxin. The epidemiologic study shows that all European cases were due to consumption of mussels from the Atlantic coast of Spain.

Adult↗

Apparent paralytic shellfish poisoning in captive herring gulls fed commercial scallops.

This report describes an acute poisoning event observed in captive herring gull (Larus argentatus) chicks fed a batch of store-bought scallops. They developed a characteristic acute syndrome, that has not hitherto been reported in birds and the cause of which remains to be identified. We suggest that it is a variant of paralytic shellfish poisoning (PSP) insofar as it was paralytic and caused by shellfish. However, analyses by the U.S. Food and Drug Administration to identify known toxins (saxitoxins, brevetoxins, domoic acid) in the scallops were negative.

Animals↗

Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison.

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

Amphibian Proteins↗

Amnesic shellfish poisoning toxins in bivalve molluscs in Ireland.

In December 1999, domoic acid (DA) a potent neurotoxin, responsible for the syndrome Amnesic Shellfish Poisoning (ASP) was detected for the first time in shellfish harvested in Ireland. Two liquid chromatography (LC) methods were applied to quantify DA in shellfish after sample clean-up using solid-phase extraction (SPE) with strong anion exchange (SAX) cartridges. Toxin detection was achieved using photodiode array ultraviolet (LC-UV) and multiple tandem mass spectrometry (LC-MS(n)). DA was identified in four species of bivalve shellfish collected along the west and south coastal regions of the Republic of Ireland. The amount of DA that was present in three species was within EU guideline limits for sale of shellfish (20 microg DA/g); mussels (Mytilus edulis), <1.0 microg DA/g; oysters (Crassostrea edulis), <5.0 microg DA/g and razor clams (Ensis siliqua), <0.3 microg DA/g. However, king scallops (Pecten maximus) posed a significant human health hazard with levels up to 240 microg DA/g total tissues. Most scallop samples (55%) contained DA at levels greater than the regulatory limit. The DA levels in the digestive glands of some samples of scallops were among the highest that have ever been recorded (2,820 microg DA/g).

Animals↗

Liquid chromatography-electrospray ionization mass spectrometry of the diarrhetic shellfish-poisoning toxins okadaic acid, dinophysistoxin-1 and pectenotoxin-6 in bivalves.

Determination of diarrhetic shellfish-poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-6 (PTX6) was carried out by liquid chromatography (LC) followed by on-line atmospheric pressure electrospray ionization-mass spectrometric (ESI-MS) detection with a heated capillary interface. Mass spectra of authentic OA, DTXI and PTX6 standards exhibited abundant [M-H] at m/z 803, 817 and 887, respectively. Linearity of peak area obtained by selected-ion monitoring (SIM) for [M-H]- of each toxin was confirmed over a wide range of concentrations from 10 pg to 30 ng. LC-ESI-MS analysis of OA, DTX1 and PTX6 in scallops and mussels, collected at the same site (Mutsu Bay, Japan), was carried out. Scallops and mussels collected at the same site showed different toxin profiles. Although PTX6 was detected from scallops, it was not detected from mussels.

Animals↗

Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning.

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Anthracenes↗

Accumulation and depuration of paralytic shellfish poisoning toxins by purple clam Hiatula rostrata Lighttoot.

Purple clams (Hiatula rostrata Lighttoot) accumulated paralytic shellfish poisoning (PSP) toxins produced by a toxic strain of the dinoflagellate Alexandrium minutum Halim for subsequent study of toxin distribution during depuration (detoxification by a nontoxic microalgal diet or starvation). The results confirm the data in the literature concerning the high toxicity of the digestive gland, and the depuration efficiency between feed with nontoxic microalgae and starvation is similar. The toxin profile of the purple clams was similar with that of Alexandrium minutum at the end of the exposure period; GTX4 and GTX1 were dominant. However, at the end of the depuration period, GTX3 and GTX2 were dominant. The non-visceral tissues were toxic after feeding with toxic algae. The toxicity was low and the profile were also similar with that of the toxic algae. No PSP toxins other than GTX-1, 2, 3 and 4 were detected in the experimental period.

Animals↗

Simultaneous occurrence of diarrhetic and paralytic shellfish poisoning toxins in Spanish mussels in 1993.

Mussel aquaculture is an important industry for the Galician Rias, located in northwestern Atlantic coast of Spain. Since 1976 this region has been seriously affected by incidents of paralytic and diarrhetic shellfish poisoning (PSP and DSP). A particularly bad episode occurred in 1993, when the toxic event lasted for an unusually long period. Many people were stricken ill with unusual symptoms. In this paper we report on the chemical analysis of toxic 1993 mussel samples, using the techniques of liquid chromatography and capillary electrophoresis coupled with mass spectrometry. These analyses revealed a very complex toxin profile, with both PSP and DSP toxins present. Two DSP toxins, okadaic acid and DTX2, were observed, while the primary PSP toxins were B1 and the decarbamoylated derivatives of saxitoxin, GTX2 and GTX3. Small amounts of saxitoxin and other as yet unidentified PSP toxins were observed.

Animals↗

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.

Animals↗

Development of a capillary electrophoresis method for the characterization of enzymatic products arising from the carbamoylase digestion of paralytic shellfish poisoning toxins.

A sample stacking procedure is presented for the capillary electrophoretic (CE) separation of paralytic shellfish poisoning (PSP) toxins dissolved in high ionic strength buffers. The application of such a stacking procedure prior to the zone electrophoretic separation is demonstrated for the analysis of decarbamoyl toxins arising from the digestion of PSP toxins by an hydrolytic enzyme from little neck clams (Protothaca staminea). Improvements in separation efficiency facilitated identification and quantitation of substrates and enzymatic products present in the digest using CE. The separation conditions developed were found to be entirely compatible with electrospray mass spectrometry, which permitted the analysis of PSP toxins and their decarbamoyl derivatives present in the low micromolar range in crude enzyme digests. The products released during the enzymatic digestion were identified using CE combined with tandem mass spectrometry.

Animals↗

Transfer and metabolism of paralytic shellfish poisoning from scallop (Chlamys nobilis) to spiny lobster (Panulirus stimpsoni).

The transfer and transformation of paralytic shellfish poisoning (PSP) from scallop Chlamys nobilis to spiny lobster Panulirus stimpsoni were investigated in the present study. The results demonstrate that transfer and transformation of PSP toxins occurred when Panulirus stimpsoni were fed with toxic viscera of Chlamys nobilis, but depurated with non-toxic squids. Additionally, only the lobster hepatopancreas were found to contain PSP, and the toxin profiles were the same with those in the viscera of the scallop, including carbamate toxins (GTX(1-3)), N-sulfocarbamoyl toxins (C(1+2) and B(1)) and decarbamoyl toxins (dcGTX(2+3)). Unlike the lobster, the scallop contained more alpha than beta toxins. After being fed with toxic Chlamys nobili for 6 d, Panulirus stimpsoni selectively accumulated N-sulfocarbamoyl toxins with low toxicity. However, when they were depurated with non-toxic squid, N-sulfocarbamoyl toxins tended to transform into carbamate toxins with higher toxicity. The concentration of dcGTX(2+3) in Panulirus stimpsoni decreased significantly and wasn't detectable after depuration for 6 d, which was likely due to their initial low accumulation of toxins. These results reveal that PSP could be transferred and transformed in Crustaceans along the given food chain under the conditions of laboratory, but there are many questions remained to be solved, and the further studies should be carried out.

Animals↗

Liquid chromatographic determination of domoic acid in shellfish products using the paralytic shellfish poison extraction procedure of the association of official analytical chemists.

Domoic acid, the recently discovered toxic substance found in contaminated mussels from an area in eastern Prince Edward Island (Canada) was extracted from mussel tissue using the procedure of the Association of Official Analytical Chemists for paralytic shellfish poisons. This involved a 5-min boiling of the sample with 0.1 M hydrochloric acid then cooling and centrifuging. An aliquot of the supernatant was diluted ten to one-hundred times with water, filtered and analysed by reversed-phase liquid chromatography with a mobile phase consisting of acetonitrile-water (12:88) at pH 2.5 and an absorption wavelength of 242 nm. The detection limit was about 0.5 mg/kg domoic acid in seafood samples. The technique was successfully applied to a variety of commercially purchased shellfish and shellfish products.

Animals↗

First paralytic shellfish poison (PSP) infestation of bivalves due to toxic dinoflagellate Alexandrium tamiyavanichii, in the southeast coasts of the Seto Inland Sea, Japan.

The mussel Mytilus edulis and the cultured ark shell Anadara broughtonii in the southeast coasts of the Seto Inland Sea were contaminated with paralytic shellfish poison (PSP) following the appearance of the dinoflagellate Alexandrium tamiyavanichii in early December 1999. A. tamiyavanichii plankton collected around the Straits of Naruto on December 3, 1999 showed PSP toxicity, of which 83 mol% was accounted for by GTX2, GTX3 and GTX4. Its specific toxicity was 112.5 fmol/cell, and one MU was equivalent to 7,200 cells. Toxicity values at the beginning of toxification were 4.7 MU/g for the ark shell and 7.3 MU/g for the mussel. In the former, the value remained at almost 4 MU/g, resulting in prohibition of marketing for about two months. In the latter, it sharply decreased to less than 4 MU/g. These bivalves collected during the toxification period were dissected into five tissues, mantle, adductor muscle, hepatopancreas, gills and "others", and submitted to high-performance liquid chromatography (HPLC). The cultured ark shell accumulated GTX2, GTX3 and STX as major components and GTX1, GTX4, GTX5, neoSTX, dcSTX and PX1-3 (C1-C3) as minor ones. The amount of GTX3 decreased with time, while STX tended to increase. At the early stage of PSP toxification, toxins were accumulated in the gills and "others", most of which were quickly detoxified. On the other hand, PSP of the toxified mussel consisted of GTX4 as a main component, and GTX1, GTX2, GTX3, GTX5, STX and PX1-2 (C1-C2) as minor ones. Its toxin composition pattern was similar to that of the ingested causative plankton. Its total toxin decreased soon after disappearance of the dinoflagellate. During the decrease of toxicity, PSP tended to be retained in the hepatopancreas, resulting in accumulation of 50 mol% of total toxin.

Animals↗

Detection of new 7-O-acyl derivatives of diarrhetic shellfish poisoning toxins by liquid chromatography-mass spectrometry.

A novel method for the detection of acylated diarrhetic shellfish poisoning toxins is reported. Direct determination of these compounds is possible using high performance liquid chromatography coupled with ion-spray mass spectrometry. An extract, purified from the digestive glands of toxic mussels (Mytilus edulis) contaminated with okadaic acid, dinophysistoxin-1, and a recently reported analog, dinophysistoxin-2, was also shown to contain small amounts of dinophysistoxin-3, a mixture of 7-O-acyl ester derivatives of dinophysistoxin-1. In addition, acyl ester derivatives of okadaic acid and dinophysistoxin-2 were also detected by direct LC-MS analysis and confirmed by analysis of their hydrolysis products. This is the first report of the detection of other naturally occurring 7-O-acyl esters similar to dinophysistoxin-3.

Acylation↗

Chlorination for degrading saxitoxins (paralytic shellfish poisons) in water.

Chlorination was investigated as a treatment option for degrading and thus removing saxitoxins (paralytic shellfish poisons, PSPs) produced by cyanobacteria (blue-green algae) from water. It was found to be effective with the order of ease of degradation of the saxitoxins being GTX5 (B1) approximately dcSTX > STX > GTX3 approximately C2 > C1 > GTX2. However the effectiveness of chlorine was pH dependent. Degradation as a function of pH was not linear with the degree of degradation increasing rapidly at around pH 7.5. At pH 9 > 90% removal was possible provided a residual of 0.5 mg l(-1) free chlorine was present after 30 min contact time. The more effective degradation at higher pH was unexpected as chlorine is known to be a weaker oxidant under these conditions. The more effective degradation, then, must be due to the toxins, which are ionisable molecules, being present in a form at higher pH which is more susceptible to oxidation. The feasibility of using chlorine to remove saxitoxins during water treatment will therefore depend strongly on the pH of the water being chlorinated. Degradation may be improved by pH adjustment but may not be a practical solution. Although saxitoxins were degraded in that the parent compounds were not detected by chemical analysis, there is no indication as to the nature of the degradation products. However, acute toxicity as determined by the mouse bioassay was eliminated.

Animals↗

Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralytic shellfish poison (PSP), in cats.

Saxitoxin (STX) was the first known and most studied toxic component of paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel. Although the toxin's mechanism of action is well known at the molecular level, there are still many unresolved questions about its pharmacokinetics and the PSP intoxication syndrome in mammals. Some of these questions are addressed in the present paper, which describes an experimental design which allowed us to follow the dynamics of STX poisoning in vivo. Adult cats were anaesthetized and permanently coupled to artificial ventilation, they were then intravenously injected with Low (2.7 microg of STX/kg) and high doses (10 microg of STX/kg) of toxin. Cardiovascular parameters such as blood pressure and electrocardiograms were recorded, urine and blood samples were collected during the four hours of experimental time. In order to quantify mass amount of STX, we used the post-column derivatization HPLC method. Urine and blood samples were cleansed using a C-18 Sep-Pack cartridge and ultrafree microcentrifuge filters. At the end of each experiment, the animals were killed and tissue samples from brain, liver, spleen and medulla oblongata were extracted to measure the amount of STX. As compared to control period, Low doses of STX made no difference in hemodynamics parameters. In contrast, high doses drastically reduced blood pressure, produced myocardial failure and finally cardiac arrest. Administration of 2.5 microg/kg x min of dobutamine restored hemodynamics parameters and allowed the animal to overcome the shock. With high doses, the calculated STX renal clearance in cats is 0.81 ml/min x kg(-1). This valued corresponds to 20.25% of the reported inulin renal clearance. Nevertheless with Low doses the STX renal clearance is 3.99 ml/min x kg(-1). This data suggest that in cats with normal cardiovascular parameters and diuresis, the STX excretion mainly involves glomerular filtration. During experimental time, no PSP toxins other than STX was detected in the body fluids and tissue samples analyzed, indicating that the mammals can not metabolize this molecule. STX was found in intensely irrigated organs such as the liver and spleen but also in the central nervous system (brain and medulla oblongata), showing that STX was capable of crossing the blood brain barrier.

Anesthesia↗

A rapid and sensitive assay for paralytic shellfish poison (PSP) toxins using mouse brain synaptoneurosomes.

A membrane potential assay using mouse brain synaptoneurosomes was evaluated for the determination of paralytic shellfish poison (PSP) toxin content of mussels and other bivalve species important to the shellfish industry. The assay relies on the ability of PSP toxins to block veratridine-induced depolarization of synaptoneurosomes. Changes in the membrane potential of synaptoneurosomes were monitored using the voltage-sensitive fluorescent probe rhodamine 6G. Standard saxitoxin was found to be a potent inhibitor of the membrane depolarizing effects of the sodium channel activator veratridine (I(50) ca. 4 nM). Likewise, shellfish extracts containing PSP toxins inhibited veratridine-induced depolarization. Neither saxitoxin or shellfish extracts had any discernible effect on the resting membrane potential of synaptoneurosomes. When synaptoneurosomal results for extracts of mussels (n=120) and other shellfish (n=29) were correlated with official mouse toxicity assay data there was very good agreement (r(2)=0.84 and 0.86, respectively), indicating that the in vitro assay has utility for a variety of commercially relevant shellfish species. Our investigation suggests that the mouse synaptoneurosome assay is of similar sensitivity to the official CD1 mouse toxicity assay. The synaptoneurosome fraction can be prepared quickly (approx. 40 min) and an individual assay takes less than 7 min. Since 20 such assays can be performed using material from a single CD1 mouse brain, there is considerable opportunity for reducing the number of animals required in conventional PSP monitoring while retaining the same animal system.

Animals↗

Canning process that diminishes paralytic shellfish poison in naturally contaminated mussels (Mytilus galloprovincialis).

Changes in toxin profile and total toxicity levels of paralytic shellfish poison (PSP)-containing mussels were monitored during the standard canning process of pickled mussels and mussels in brine using mouse bioassays and high-performance liquid chromatography. Detoxification percentages for canned mussel meat exceeded 50% of initial toxicity. Total toxicity reduction did not fully correspond to toxin destruction, which was due to the loss of PSP to cooking water and packing media of the canned product. Significant differences in detoxification percentages were due to changes in toxin profile during heat treatment in packing media. Toxin conversion phenomena should be determined to validate detoxification procedures in the canning industry.

Animals↗