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PubMed1990

Analysis of domoic acid and related compounds by mass spectrometry and gas chromatography/mass spectrometry as N-trifluoroacetyl-O-silyl derivatives.

A method is presented for the analysis of shellfish tissue for domoic acid, a neurotoxic amino acid responsible for cases of amnesic shellfish poisoning. Tissue extracts are first taken through a two-stage solid-phase extraction clean-up, using reversed-phase and strong cation exchange cartridges. A two-stage derivatization, using N-methyl-bis-trifluoroacetamide followed by either N-methyl-tert-butyldimethylsilyltrifluoroacetamide or N, O-bis-trimethylsilyltrifluoroacetamide, is then used to produce an N-trifluoroacetyl-O-silyl derivative which can be analyzed by mass spectrometry with introduction via direct inlet probe, moving-belt liquid chromatograph/mass spectrometer interface, or capillary column gas chromatography. The N-trifluoroacetyl-O-tert-butyldimethylsilyl derivative, which has good stability towards hydrolysis, provides a spectrum well suited to gas chromatography/mass spectrometry (GC/MS) using selected ion recording. GC/MS data for two related compounds, kainic acid and dihydrokainic acid, are also reported. The latter is used as an internal standard for quantification of domoic acid, although the method reported is intended primarily for confirmation of the toxin and related compounds in shellfish tissue.

Chromatography, Gas↗
PubMedDate not supplied

Determination of brevetoxins in shellfish by the neuroblastoma assay.

A neuroblastoma assay for determination of brevetoxins in shellfish was developed together with a method for sample cleanup that allows separation of brevetoxins from most of the components that cause matrix interference in the assay. This improved assay method was applied to a range of shellfish samples with different characteristics. Extracts of naturally contaminated and nontoxic shellfish together with extracts spiked with known amounts of toxin were tested. The results demonstrated that brevetoxins could be reliably detected in shellfish extracts at concentrations below the regulatory limit. Brevetoxin activity was detected in 15 of 23 samples from 5 separate toxicity incidents in which shellfish tested positive in the neurotoxic shellfish poisoning (NSP) mouse bioassay. Twelve of these positive NSP results came from 2 toxicity incidents. Yessotoxin was the major contributor to toxicity in 2 other incidents, although some samples contained both yessotoxin and brevetoxin. The sample from the remaining incident contained an unidentified toxin bioactivity, together with gymnodimine. In contrast to earlier versions of the neuroblastoma assay, gymnodimine was not detected by this modified method.

Animals↗
PubMed2001

PSP toxins from Aphanizomenon flos-aquae (cyanobacteria) collected in the Crestuma-Lever reservoir (Douro river, northern Portugal).

The presence of paralytic shellfish poisoning (PSP) toxins in cultures of Aphanizomenon flos-aquae, isolated from the Crestuma-Lever reservoir, was found by reversed phase high performance liquid chromatography employing two isocratic elution systems for the separation of PSP toxins. With the first isocratic elution protocol, the presence of apolar toxins as saxitoxin, decarbamoyl saxitoxin and neosaxitoxin not detected. On the other hand, GTX4, GTX1 and GTX3 as well as Cs toxins were present either in the Aphanizomenon flos-aquae cells collected directly from the bloom or in the other toxic isolates priorly cultivated in laboratory conditions.

Bacterial Toxins↗
PubMed1996

Liquid chromatographic determination of okadaic acid and dinophysistoxin-1 in shellfish after derivatization with 9-chloromethylanthracene.

The reagent 9-chloromethylanthracene was evaluated for derivatization of the diarrhetic shellfish poisons, okadaic acid and dinophysistoxin-1 (DTX-1), to form fluorescent products separable by liquid chromatography. The toxins were reacted with the reagent in acetonitrile in the presence of tetramethylammonium hydroxide for 1 h at 90 degrees C. The products were purified by using two silica solid-phase extraction cartridges before being determined by reversed-phase liquid chromatography with fluorescence detection. The results are comparable to those obtained using 9-anthryldiazomethane (ADAM) for okadaic acid and DTX-1 in mussel tissue. Detection limits were estimated to be about 70-100 ng/g hepatopancreas (equivalent to 12-20 ng/g whole tissue) for each toxin.

Animals↗
PubMedDate not supplied

The use of Daphnia magna for detection of okadaic acid in mussel extracts.

Okadaic acid (OA), the main toxin responsible for diarrheic shellfish poisoning (DSP) has toxicity for Daphnia magna (EC50 = 15 +/- 1.8 micrograms/l). A Daphnia bioassay was developed and used to analyse okadaic acid in mussel extracts. A linear correlation was found between OA concentration determined by the Daphnia bioassay and by HPLC assay (r = 0.85; p < 0.001). The Daphnia bioassay can measure OA levels 10 times below the threshold of the mouse bioassay method. It is an inexpensive sensitive tool which can be used in replacement of mouse bioassay for the screening of OA and some co-extracting toxins in mussel extracts.

Animals↗
PubMed2001

Decimal reduction times of Pyrodinium bahamense var. compressum and Escherichia coli in chlorine- and ultraviolet-treated seawater.

AIMS: Decimal reduction times (D-values) of the vegetative cells of Pyrodinium bahamense var. compressum and Escherichia coli in ultraviolet- and chlorine-treated seawater were established. METHODS AND RESULTS: The cells of the test organisms were exposed to ultraviolet- and chlorine-treated seawater and maintained at 20-35 ppt salinity and 20 to 35 degrees C. The dinoflagellate cells which cause Paralytic Shellfish Poisoning (PSP) were found to be more resilient than the bacterial cells. Ultraviolet treatment was found to be more effective than chlorine to both test organisms. Irreversible morphological changes in the treated dinoflagellate cells were noted, including protoplast discoloration, cellular membrane leakage and damage to the thecal armour. CONCLUSIONS: The vegetative cells of both test organisms in seawater were more sensitive to ultraviolet treatment than to chlorine exposure. Generally, the dinoflagellate cells were less susceptible than bacterial cells to both disinfection treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study may have significant implications in depuration procedures for molluscs and cleaning protocols for ballast waters of ships.

Animals↗
PubMed1991

Light and scanning electron microscopic studies on effects of marine algal toxins toward freshly prepared hepatocytes.

Mussels exposed to dinoflagellates may represent a human health risk due to accumulation of a variety of algal toxins. In several parts of the world, algal toxins leading to diarrhea (diarrhetic shellfish poisons, DSP) are found in mussels for extended periods of the year. Routine monitoring of these toxins involves ip injections in mice. Chemical analytical methods have been developed for only some of the toxins in question, namely, those giving diarrhea. Other toxins in the DSP complex are not easily detected by analytical methods. In this report we show that freshly prepared hepatocytes from rats are a convenient means to differentiate between the toxins that give diarrhea and those that do not. Consequently, hepatocytes can be useful in both screening and as a tool in the process of developing analytical methods. Freshly prepared hepatocytes might be useful in combination either with the mouse bioassay or with chemical analytical methods.

Animals↗
PubMed1994

Effects of nitrogen concentration and cold temperature on DSP-toxin concentrations in the dinoflagellate Prorocentrum lima (Prorocentrales, Dinophyceae).

The diarrhetic shellfish poisoning toxin-producing dinoflagellate, Prorocentrum lima, isolated from Nova Scotian waters, contained both okadaic acid (OA) and dinophysistoxin-1 (DTX-1) throughout its growth cycle in culture; maximum concentrations of toxins and highest OA/DTX-1 ratios occurred during the stationary phase. Cells of P. lima survived 0 degrees C for 5 weeks and recovered when brought to a higher temperature. During the cold period, some cell damage probably occurred with concomitant losses of toxins to the medium. Nitrogen concentration in the medium was used to limit growth or stress the cells physiologically, and when growth was limited, increases in toxin associated with the cells were recorded. The relative amounts of okadaic acid were always greater than dinophysistoxin-1, but the significance of these ratios remains to be determined.

Animals↗
PubMed1995

Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography-mass spectrometry.

Direct detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and some of their related compounds in toxic mussels (Mytilus galloprovincialis) is reported using ionspray liquid chromatography-mass spectrometry (LC-ISP-MS). This was employed to analyse diarrhoetic shellfish poisoning (DSP) toxins in mussels collected from coastal areas of the northern and southern Adriatic Sea. DTX-1 was found in some samples from both the northern and southern Adriatic and this is the first report of the unambiguous identification of this toxin in Italian mussels. The low levels found indicate that this toxin did not play a significant role in toxicity in these samples. Okadaic acid was found in all the mussels examined, although its concentration was not always sufficient to account for DSP toxicity. Furthermore, two related compounds of OA were detected in all the samples and one related DTX-1 compound was observed in some samples from the northern Adriatic. All three compounds are still to be identified, but it is possible that these substances are involved in mussel DSP toxicity in the Adriatic Sea.

Animals↗
PubMed2001

Determination of domoic acid in shellfish by liquid chromatography with electrospray ionization and multiple tandem mass spectrometry.

Amnesic shellfish poisoning is a potentially lethal human toxic syndrome which is caused by domoic acid (DA) that originates in marine phytoplankton belonging to the Pseudonitzschia genus. A new sensitive liquid chromatographic-mass spectrometry (LC-MS) method has been developed for the determination of DA in various marine biological samples. The characteristic fragmentation pathways for DA were established using multiple stage MS on selected daughter ions, which were sequentially trapped and fragmented. Chromatography was performed using a gradient of acetonitrile-water (5:95 to 40:60), containing trifluoroacetic acid (0.05%), over 25 min at 0.2 ml/min with a C18 column (Luna-2, 150 x 2.0 mm, 5 microm). Using electrospray ionisation, multiple tandem MS experiments were performed with an ion-trap mass spectrometer (Finnigan MAT LCQ). The protonated DA molecule was the precursor ion, m/z 312, and the relative collision energies were optimised for multiple MS (MS(n), n = 2-4) studies. LC-MS3 using the ions, m/z 266 and 220, from the loss of two HCOOH molecules, produced the best sensitivity data. Calibration data for various MS modes were: MS (0.05-10 microg DA/ml, r2 = 0.9973); MS2 (0.025-10 microg DA/ml, r2=0.9997); MS3 (0.025-10 microg DA/ml, 0.9994). The detection limits (3:1 signal:noise) were better than 0.02 microg DA/ml for LC-MS, 0.014 microg DA/ml for LC-MS2 and 0.008 microg DA/ml for LC-MS3. This method was applied to determine DA in scallop (Pecten maximus) tissues, which subsequently led to the closure of several shellfish harvesting sites on the west coast of Ireland.

Animals↗
PubMed2000

Geographical segregation of the neurotoxin-producing cyanobacterium Anabaena circinalis.

Blooms of the cyanobacterium Anabaena circinalis are a major worldwide problem due to their production of a range of toxins, in particular the neurotoxins anatoxin-a and paralytic shellfish poisons (PSPs). Although there is a worldwide distribution of A. circinalis, there is a geographical segregation of neurotoxin production. American and European isolates of A. circinalis produce only anatoxin-a, while Australian isolates exclusively produce PSPs. The reason for this geographical segregation of neurotoxin production by A. circinalis is unknown. The phylogenetic structure of A. circinalis was determined by analyzing 16S rRNA gene sequences. A. circinalis was found to form a monophyletic group of international distribution. However, the PSP- and non-PSP-producing A. circinalis formed two distinct 16S rRNA gene clusters. A molecular probe was designed, allowing the identification of A. circinalis from cultured and uncultured environmental samples. In addition, probes targeting the predominantly PSP-producing or non-PSP-producing clusters were designed for the characterization of A. circinalis isolates as potential PSP producers.

Anabaena↗
PubMed1990

Gastrointestinal effects of contaminated mussels and putative antidotes thereof.

A recent outbreak of amnesic shellfish poisoning (ASP) in Atlantic Canada was characterized by severe gastrointestinal and central nervous system pathology. We examined the gastrointestinal effects of an acidic extract of blue mussels contaminated with domoic acid, the suspected toxin responsible for the ASP. We also tested the gastric effects pure domoic acid as well as a putative antagonist of neuroexcitant amino acid receptors, kynurenic acid. Mussel extract produced gastric (antral) ulcers, duodenal ulcers, gastric and duodenal hyperemia and bleeding, as well as peritoneal ascites. Kynurenic acid protected significantly against extract-induced gastropathy, particularly when given 60 or 75 minutes after extract. Pure domoic acid resulted in fatalities in all infant mice tested. These animals exhibited gastric bleeding and hemorrhage, especially at the higher doses employed. In otherwise untreated rats, kynurenic acid exerted significant anti-stress ulcer and anti-gastric secretory effects, but was less effective at blocking ethanol-induced gastric lesions. We suggest that there may be both peripheral as well as central effects of kynurenic acid in modulating normal and pathological gastric function.

Animals↗
PubMed2002

Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice.

Currently, yessotoxin is regulated among the toxins in the diarrhetic shellfish poisoning (DSP) complex. Yessotoxin is equally acutely toxic towards mice upon intraperitoneal injections as those algal toxins giving diarrhea, but is not diarrheagenic. Its presence in mussels may therefore lead to overestimation of risk of DSP in consumers when the standard mouse bioassay is used. Arguments are presented for the use of analytical methods instead of the mouse bioassay for the diarrheagenic DSP toxins and yessotoxin. Yessotoxin was found to be more than ten times less toxic to mice via the oral route, compared with intraperitoneal injections. Even at 10mg/kg body weight, the highest dose ever tested orally, yessotoxin did not kill the mice. By means of light microscopy of several organs, moderate changes were only observed in the heart. Ultrastructural studies revealed swelling of heart muscle cells leading to separation of the organelles. Effects were most pronounced close to the capillaries. The pathological changes were clearly dose dependent, and the lowest oral dose where any effects were seen was 2.5mg yessotoxin per kg.

Administration, Oral↗
PubMed2001

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.

Animals↗
PubMed1999

Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish.

Two acidic analogues of the polyether marine toxin, pectenotoxin-2 (PTX-2), responsible for diarrhetic shellfish poisoning (DSP), have been isolated from the toxic marine phytoplankton (Dinophysis acuta), collected in Irish waters. Liquid chromatography with fluorimetric detection (LC-FLD) analyses of the extracts of bulk phytoplankton samples, following derivatisation with 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP), showed a complex toxin profile with peaks corresponding to okadaic acid (OA) and its isomers, dinophysistoxin-2 (DTX-2) and DTX-2C, as well as other unidentified lipophilic acids. LC-UV analysis showed the presence of a diene moiety in these new compounds and two acids have been isolated. LC coupled with mass spectrometry (MS) and tandem mass spectrometry (LC-MS-MS) were used to gain structural information. Through flow injection analysis (FIA)-MS, both in positive and negative ion modes, the molecular weight of 876 for both compounds was determined. Collision Induced Dissociation (CID) from each parent ion, as performed both in positive and negative ion mode, produced mass spectra which were very similar to those obtained for authentic PTX-2 (mw 858). These new compounds have been confirmed to be pectenotoxin-2 seco acids (PTX-2SAs) and they are closely related to PTX-2 except that they contain an open chain carboxylic acid rather than a lactone ring. Toxic mussels also contained these pectenotoxin-2 analogues.

Chromatography, Liquid↗
PubMed2002

Effects of toxic dinoflagellates and toxin biotransformation in bivalves.

Attempts were made to elucidate the different responses of shellfish to paralytic shellfish poison (PSP) and the PSP donor Alexandrium minutum T1. Five species of edible bivalves (Crassostrea gigas, Meretrix lusoria, Mytilus edulis, Ruditapes philippinarum, and Soletellina diphos) were collected and examined for susceptibility to PSP and PSP donor. It was determined that all five bivalves had low susceptibility to PSP following an intramuscular injection (> 300 MU/20 g). The abnormal effects on bivalves were species-specific and varied with the concentration of A. minutum T1. Judging from the LC50 data (medium lethal concentration), the resistance of bivalves to the toxic dinoflagellate was as follows (least to most resistant): C. gigas < R. philippinarum < M. lusoria < M. edulis, S. diphos. With the exception of S. diphos, the bivalves accumulated very little toxin (< 2 MU/g edible tissue) when they were exposed to 10(7) cells/L of A. minutum for four days. The toxin levels in S. diphos increased with exposure time to the toxic dinoflagellates and accumulated primarily in the digestive gland (88-100%), followed by the gill (0-10%), and other organs (0-8%). Although the concentrations of toxin components in the digestive gland were found to be variable during the exposure period, the toxin profile in the digestive gland of S. diphos during the early exposure period was similar to that of A. minutum. Moreover, toxin components in the gills and in other organs were retained at near constant concentrations during the exposure period.

Animals↗
PubMed1999

Efficient isolation of the rare diarrhoeic shellfish toxin, dinophysistoxin-2, from marine phytoplankton.

The rare diarrhoeic shellfish poisoning (DSP) toxin, dinophysistoxin-2 (DTX-2), which is an okadaic acid (OA) isomer, has been isolated from a marine phytoplankton biomass that consisted mainly of Dinophysis acuta. Using a large double plankton net (length 5.9 m), bulk phytoplankton samples were collected off the south-west coast of Ireland and extracted with methanol and chloroform. Liquid chromatography coupled with ionspray mass spectrometry and tandem mass spectrometry (LC-MS, LC-MS-MS) showed the sample contained DTX-2 and OA, at a concentration of 80 pg/cell and 60 pg/cell, respectively. Flash chromatography using silica, sephadex LH20 and C18-silica, followed by preparative reversed-phase LC, separated DTX-2 from OA. The efficiency of the separation procedures was substantially improved by the use of a bioscreen to detect DSP toxins in eluate fractions and the application of a new derivatisation procedure for the chromatographic elucidation of toxin profiles with fluorimetric detection (LC-FLD). Thus, 1/1000th aliquots of eluate fractions were assayed using protein phosphatase-2A for the presence of inhibitory compounds. Positive fractions were further analysed for DSP toxins by LC-FLD following derivatisation using the hydrazine reagent, luminarine-3. The identity and purity of the free isolated DTX-2 was confirmed using flow injection analysis (FIA) and liquid chromatography (FIA-MS, LC-MS and LC-MS-MS).

Animals↗
PubMed2003

Monitoring of DSP toxins in small-sized plankton fraction of seawater collected in Mutsu Bay, Japan, by ELISA method: relation with toxin contamination of scallop.

Monitorings were conducted on DSP toxins in mid-gut gland of scallop (mouse assay), cell numbers of toxic dinoflagellate species of Dinophysis, and diarrhetic shellfish poisoning (DSP) toxins in small-sized (0.7-5 microm) plankton fraction of seawater collected from surface (0 m) and 20 m depth at a station in Mutsu Bay, Aomori Prefecture, Japan, in 2000. A specific enzyme-linked immunosorbent assay (ELISA) was employed for the analysis of DSP toxins in small-sized plankton fraction using a mouse monoclonal anti-okadaic acid antibody which recognizes okadaic acid, dinophysistoxin-1, and dinophysistoxin-3. DSP toxins were detected twice in the mid-gut gland of scallops at 1.1-2.3 MU (mouse units) g(-1) on 26 June and at 0.6-1.2 MU g(-1) on 3 July, respectively. Relatively high cell densities of D. fortii were observed on 26 June and 11 September, and may only contribute to the bivalve toxicity during late June to early July. D. acuminata did not appear to be responsible for the toxicity of scallops in Mutsu Bay in 2000. ELISA monitoring of small-sized plankton fraction in seawater could detect DSP toxins two weeks before the detection of the toxin in scallops, and could do so two weeks after the loss of the bivalve toxicity by mouse assay. On 17 July, toxic D. fortii was detected at only small number, <10 cells l(-1), but DSP toxins were detected by the ELISA assay, suggesting a presence of other toxic small-sized plankton in seawater. For the purpose of reducing negative impacts of DSP occurrences, monitorings have been carried out hitherto on DSP toxins of bivalve tissues by mouse assay and on cell densities of "toxic" species of Dinophysis. Here we propose a usefulness of ELISA monitoring of plankton toxicity, especially in small-sized fraction, which are possible foods of mixotrophic Dinophysis, as a practical tool for detecting and predicting DSPs in coastal areas of fisheries grounds of bivalve aquaculture.

Animals↗