okadaic-acid and domoic-acid

Topics: Animals; Chromatography, Liquid; Guidelines as Topic; Humans; Kainic Acid; Marine Toxins; Mice; Okadaic Acid; Oxocins; Risk Management; Saxitoxin; Shellfish; Shellfish Poisoning; Spectrometry, Fluorescence; Spiro Compounds; Tandem Mass Spectrometry; Tetrodotoxin

A freeze-dried mussel tissue-certified reference material (CRM-FDMT1) was prepared containing the marine algal toxin classes azaspiracids, okadaic acid and dinophysistoxins, yessotoxins, pectenotoxins, cyclic imines, and domoic acid. Thus far, only a limited number of analogues in CRM-FDMT1 have been assigned certified values; however, the complete toxin profile is significantly more complex. Liquid chromatography-high-resolution mass spectrometry was used to profile CRM-FDMT1. Full-scan data was searched against a list of previously reported toxin analogues, and characteristic product ions extracted from all-ion-fragmentation data were used to guide the extent of toxin profiling. A series of targeted and untargeted acquisition MS/MS experiments were then used to collect spectra for analogues. A number of toxins previously reported in the literature but not readily available as standards were tentatively identified including dihydroxy and carboxyhydroxyyessotoxin, azaspiracids-33 and -39, sulfonated pectenotoxin analogues, spirolide variants, and fatty acid acyl esters of okadaic acid and pectenotoxins. Previously unreported toxins were also observed including compounds from the pectenotoxin, azaspiracid, yessotoxin, and spirolide classes. More than one hundred toxin analogues present in CRM-FDMT1 are summarized along with a demonstration of the major acyl ester conjugates of several toxins. Retention index values were assigned for all confirmed or tentatively identified analogues to help with qualitative identification of the broad range of lipophilic toxins present in the material.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Freeze Drying; Kainic Acid; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Reference Standards; Spiro Compounds; Tandem Mass Spectrometry

A novel electrochemical immunosensor for the detection of the important marine biotoxins domoic acid (DA) and okadaic acid (OA) was developed. The sensors used carbon black modified screen-printed electrodes (CB-SPE) obtained using a high-throughput method. The electrochemical performance and stability of CB modified SPEs and bare carbon SPEs (c-SPEs) were compared using cyclic voltammetry and electrochemical impedance spectroscopy. CB-SPEs showed improved long-term (at least six months) stability and electro-catalytic properties compared with c-SPEs. The CB-SPEs were bio-functionalized with DA or OA protein-conjugates and used to develop two indirect competitive immunosensors using differential pulse voltammetry (DPV). The DPV signals obtained for the OA and DA immunosensors fitted well to four-parameter dose-response curves (R

Topics: Animals; Biosensing Techniques; Electrochemical Techniques; Electrodes; Immunoassay; Kainic Acid; Marine Toxins; Okadaic Acid; Soot

Algal toxins (domoic acid, saxitoxin, okadaic acid) were monitored at seven locations off southern California using Solid Phase Adsorption Toxin Tracking. At least two types of toxins were found at all locations, with co-occurrence of two and three toxins in 12% and 10% of samples, respectively. This study expands our limited understanding of the simultaneous presence of multiple algal toxins along the coast and raises questions regarding the potential health ramifications of such co-occurrences.

Topics: Adsorption; California; Environmental Monitoring; Harmful Algal Bloom; Kainic Acid; Marine Toxins; Okadaic Acid; Saxitoxin; Seawater

An analytical procedure is proposed for determining three cyanotoxins (microcystin RR, microcystin LR, and nodularin) and two phycotoxins (domoic and okadaic acids) in seawater and algae-based food supplements. The toxins were first isolated by a salting out liquid extraction procedure. Since the concentration expected in the samples was very low, a dispersive liquid-liquid microextraction procedure was included for preconcentration. The ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate (80 mg) was used as green extractant solvent and acetonitrile as disperser solvent (0.5 mL) for a 10 mL sample volume at pH 1.5, following the principles of green analytical chemistry. Liquid chromatography with electrospray ionization and quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) was used. The selectivity of the detection system, based on accurate mass measurements, allowed the toxins to be unequivocally identified. Mass spectra for quadrupole time of flight-mass spectrometry (Q-TOF-MS) and Q-TOF-MS/MS were recorded in the positive ion mode and quantification was based on the protonated molecule. Retention times ranged between 6.2 and 17.9 min using a mobile phase composed by a mixture of methanol and formic acid (0.1%). None of the target toxins were detected in any of the seawater samples analyzed, above their corresponding detection limits. However, microcystin LR was detected in the blue green alga sample.

Topics: Acetonitriles; Borates; Chromatography, High Pressure Liquid; Dietary Supplements; Food Contamination; Imidazoles; Ionic Liquids; Kainic Acid; Liquid Phase Microextraction; Marine Toxins; Microcystins; Okadaic Acid; Peptides, Cyclic; Seawater; Solvents; Spain; Spirulina; Stramenopiles; Tandem Mass Spectrometry

The occurrence and seasonal variations of marine algal toxins in phytoplankton and oyster samples in Daya Bay (DYB), South China Sea were investigated. Two Dinophysis species, namely, D. caudata and D. acuminata complex, were identified as Okadaic acid (OA)/pectenotoxin (PTX) related species. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis demonstrated that 2.04-14.47 pg PTX2 per cell was the predominant toxin in single-cell isolates of D. caudata. D. acuminata was not subjected to toxin analysis. The occurrence of OAs in phytoplankton concentrates of net-haul sample coincided with the presence of D. accuminata complex, suggesting that this species is most likely an OA producer in this sea area. OA, dinophysistoxins-1 (DTX1), PTX2, PTX2sa, gymnodimine (GYM), homoyessotoxin (homoYTX), and domoic acid (DA) demonstrated positive results in net haul samples. To our best knowledge, this paper is the first to report the detection of GYM, DA, and homoYTX in phytoplankton samples in Chinese coastal waters. Among the algal toxins, GYM demonstrated the highest frequency of positive detections in phytoplankton concentrates (13/17). Five compounds of algal toxins, including OA, DTX1, PTX2, PTX2sa, and GYM, were detected in oyster samples. DA and homoYTX were not detected in oysters despite of positive detections for both in the phytoplankton concentrates. However, neither the presence nor absence of DA in oysters can be determined because extraction conditions with 100% methanol used to isolate toxins from oysters (recommended by the EU-Harmonised Standard Operating Procedure, 2015) would likely be unsuitable for this water-soluble toxin. In addition, transformation of DA during the digestion process of oysters may also be involved in the negative detections of this toxin. GYM exhibited the highest frequency of positive results in oysters (14/17). OAs were only detected in the hydrolyzed oyster samples. The detection rates of PTX and PTX2sa in oysters were lower than those in the net haul samples.

Topics: Animals; Bays; China; Chromatography, Liquid; Dinoflagellida; Environmental Monitoring; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Kainic Acid; Marine Toxins; Okadaic Acid; Ostreidae; Phytoplankton; Pyrans; Tandem Mass Spectrometry; Water Pollutants, Chemical

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

Topics: Animals; Chromatography, Liquid; Freeze Drying; Furans; Kainic Acid; Macrolides; Marine Toxins; Mass Spectrometry; Mollusk Venoms; Mytilus edulis; Okadaic Acid; Oxocins; Pyrans; Reference Standards; Seafood; Spiro Compounds

Different clinical types of algae-related poisoning have attracted scientific and commercial attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, mainly due to the low sensitivity and the absence of specialized variations. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins. This chapter describes state-of-the-art LC-MS/MS methods for the detection and quantitation of different classes of phycotoxins in shellfish matrices. These classes include the highly hydrophilic paralytic shellfish poisoning (PSP) toxins. Hydrophilic interaction liquid chromatography (HILIC) has been shown to be useful in the separation of PSP toxins and is described in detail within this chapter. Another important class of phycotoxins is diarrhetic shellfish poisoning (DSP) toxins. This group traditionally comprises okadaic acid and dinophysistoxins (DTXs), pectenotoxins (PTXs), and yessotoxins (YTXs). The most recently described shellfish poisoning syndrome, azaspiracid shellfish poisoning (AZP) is caused by azaspiracids, which in turn are diarrhetic, but usually are treated separately as AZP. The last group of regulated shellfish toxins is the amnesic shellfish poisoning (ASP) toxin domoic acid, produced by species of the genus Pseudo-nitzschia.

Topics: Chromatography, Liquid; Kainic Acid; Macrolides; Marine Toxins; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Shellfish; Spiro Compounds; Tandem Mass Spectrometry

Like marine diseases, harmful algal blooms (HABs) are globally increasing in frequency, severity and geographical scale. As a result, bivalves will have to face the combined threat of toxic algae and marine pathogens more frequently in the (near) future. These stressors combined may further affect the recruitment of ecologically and economically important bivalve species as HABs can affect the growth, viability and development of their larvae. To date, little is known on the specific effects of HABs on the innate immune system of bivalve larvae. This study therefore investigates whether two common harmful algae can influence the larval viability, development and immunological resilience of the blue mussel Mytilus edulis. Embryos of this model organism were exposed (48 h) to five densities of Pseudo-nitzschia multiseries or Prorocentrum lima cells. In addition, the effect of six concentrations of their respective toxins: domoic acid (DA) and okadaic acid (OA) were assessed. OA was found to significantly reduce larval protein phosphatase activity (p < 0.001) and larval viability (p < 0.01) at concentrations as low as 37.8 μg l(-1). P. multiseries (1400 cells ml(-1)), P. lima (150 cells ml(-1)) and DA (dosed five times higher than typical environmental conditions i.e. 623.2 μg l(-1)) increased the phenoloxidase (PO) innate immune activity of the mussel larvae. These results suggest that the innate immune response of even the earliest life stages of bivalves is susceptible to the presence of HABs.

Topics: Animals; Diatoms; Dinoflagellida; Embryonic Development; Harmful Algal Bloom; Immunity, Innate; Kainic Acid; Larva; Marine Toxins; Mytilus edulis; Okadaic Acid; Phosphoprotein Phosphatases

A novel multiplexed immunoassay for the analysis of phycotoxins in shellfish samples has been developed. Therefore, a regenerable chemiluminescence (CL) microarray was established which is able to analyze automatically three different phycotoxins (domoic acid (DA), okadaic acid (OA) and saxitoxin (STX)) in parallel on the analysis platform MCR3. As a test format an indirect competitive immunoassay format was applied. These phycotoxins were directly immobilized on an epoxy-activated PEG chip surface. The parallel analysis was enabled by the simultaneous addition of all analytes and specific antibodies on one microarray chip. After the competitive reaction, the CL signal was recorded by a CCD camera. Due to the ability to regenerate the toxin microarray, internal calibrations of phycotoxins in parallel were performed using the same microarray chip, which was suitable for 25 consecutive measurements. For the three target phycotoxins multi-analyte calibration curves were generated. In extracted shellfish matrix, the determined LODs for DA, OA and STX with values of 0.5±0.3 μg L(-1), 1.0±0.6 μg L(-1), and 0.4±0.2 μg L(-1) were slightly lower than in PBS buffer. For determination of toxin recoveries, the observed signal loss in the regeneration was corrected. After applying mathematical corrections spiked shellfish samples were quantified with recoveries for DA, OA, and STX of 86.2%, 102.5%, and 61.6%, respectively, in 20 min. This is the first demonstration of an antibody based phycotoxin microarray.

Topics: Animals; Cattle; Kainic Acid; Luminescence; Marine Toxins; Mice; Mice, Inbred BALB C; Microarray Analysis; Okadaic Acid; Saxitoxin; Shellfish; Shellfish Poisoning

The presence of paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP) toxins in seafood is a severe and growing threat to human health. In order to minimize the risks of human exposure, the maximum content of these toxins in seafood has been limited by legal regulations worldwide. The regulated limits are established in equivalents of the main representatives of the groups: saxitoxin (STX), okadaic acid (OA), and domoic acid (DA), for PSP, DSP, and ASP, respectively. In this study a multidetection method to screen shellfish samples for the presence of these toxins simultaneously was developed. Multiplexing was achieved using a solid-phase microsphere assay coupled to flow-fluorimetry detection, based on the Luminex xMap technology. The multidetection method consists of three simultaneous competition immunoassays. Free toxins in solution compete with STX, OA, or DA immobilized on the surface of three different classes of microspheres for binding to specific monoclonal antibodies. The IC50 obtained in the buffer was similar in single- and multidetection: 5.6 ± 1.1 ng/mL for STX, 1.1 ± 0.03 ng/mL for OA, and 1.9 ± 0.1 ng/mL for DA. The sample preparation protocol was optimized for the simultaneous extraction of STX, OA, and DA with a mixture of methanol and acetate buffer. The three immunoassays performed well with mussel and scallop matrixes displaying adequate dynamic ranges and recovery rates (around 90% for STX, 80% for OA, and 100% for DA). This microsphere-based multidetection immunoassay provides an easy and rapid screening method capable of detecting simultaneously in the same sample three regulated groups of marine toxins.

Topics: Animals; Antibodies, Monoclonal; Flow Cytometry; Immunoassay; Kainic Acid; Okadaic Acid; Saxitoxin; Shellfish; Toxins, Biological

Phycotoxins, secondary phytoplankton metabolites, are considered as an important food safety issue because their accumulation by shellfish may render them unfit for human consumption. However, the likely intakes of phycotoxins via shellfish consumption are almost unknown because both contamination and consumption data are very scarce. Thus, two 1-year surveys were conducted (through the same population: recreational shellfish harvesters and from the same geographical area) to assess: shellfish consumption and contamination by major toxins (domoic acid (DA) group, okadaic acid (OA) group and spirolides (SPXs)). Recreational shellfish harvesters had been targeted as an at-risk subpopulation because they consume more shellfish than general population and because they eat not only commercial shellfish species controlled by official authorities but also their own harvests of shellfish species may be in non-controlled areas and more over shellfish species non-considered in the official control species. Then, these two kinds of data were combined with deterministic and probabilistic approaches for both acute and chronic exposures, on considering the impact of shellfish species and cooking on phycotoxin levels. For acute risk, monitoring programs seem to be adequate for DAs, whereas OAs could be a matter of concern for high consumers (their acute intakes were up to ninefold the acute reference dose (ARfD)). About chronic risk, OAs are a matter of concern. The daily OAs intakes were close to the ARfD, which is, by definition, greater than the tolerable daily intake. Moreover, SPX contamination is low but regular, no (sub)chronic SPX toxicity data exist; but in case of (sub)chronic toxicity, SPX exposure should be considered.

Topics: Adult; Aged; Aged, 80 and over; Diet; Food Contamination; France; Humans; Kainic Acid; Marine Toxins; Middle Aged; Models, Statistical; Okadaic Acid; Phytoplankton; Shellfish; Young Adult

A multiplex surface plasmon resonance (SPR) biosensor method for the detection of paralytic shellfish poisoning (PSP) toxins, okadaic acid (and analogues) and domoic acid was developed. This method was compared to enzyme-linked immunosorbent assay (ELISA) methods. Seawater samples (n=256) from around Europe were collected by the consortia of an EU project MIcroarrays for the Detection of Toxic Algae (MIDTAL) and evaluated using each method. A simple sample preparation procedure was developed which involved lysing and releasing the toxins from the algal cells with glass beads followed by centrifugation and filtering the extract before testing for marine biotoxins by both multi-SPR and ELISA. Method detection limits based on IC20 values for PSP, okadaic acid and domoic acid toxins were 0.82, 0.36 and 1.66 ng/ml, respectively, for the prototype multiplex SPR biosensor. Evaluation by SPR for seawater samples has shown that 47, 59 and 61 % of total seawater samples tested positive (result greater than the IC20) for PSP, okadaic acid (and analogues) and domoic acid toxins, respectively. Toxic samples were received mainly from Spain and Ireland. This work has demonstrated the potential of multiplex analysis for marine biotoxins in algal and seawater samples with results available for 24 samples within a 7 h period for three groups of key marine biotoxins. Multiplex immunological methods could therefore be used as early warning monitoring tools for a variety of marine biotoxins in seawater samples.

Topics: Biosensing Techniques; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Europe; Harmful Algal Bloom; Humans; Ireland; Kainic Acid; Limit of Detection; Marine Toxins; Microalgae; Okadaic Acid; Seawater; Shellfish Poisoning; Spain; Surface Plasmon Resonance; Water Pollutants, Chemical

The occurrence and intensity of harmful algal blooms (HABs) have been increasing globally during the past few decades. The impact of these events on seawater desalination facilities has become an important topic in recent years due to enhanced societal interest and reliance on this technology for augmenting world water supplies. A variety of harmful bloom-forming species of microalgae occur in southern California, as well as many other locations throughout the world, and several of these species are known to produce potent neurotoxins. These algal toxins can cause a myriad of human health issues, including death, when ingested via contaminated seafood. This study was designed to investigate the impact that algal toxin presence may have on both the intake and reverse osmosis (RO) desalination process; most importantly, whether or not the naturally occurring algal toxins can pass through the RO membrane and into the desalination product. Bench-scale RO experiments were conducted to explore the potential of extracellular algal toxins contaminating the RO product. Concentrations exceeding maximal values previously reported during natural blooms were used in the laboratory experiments, with treatments comprised of 50 μg/L of domoic acid (DA), 2 μg/L of saxitoxin (STX) and 20 μg/L of brevetoxin (PbTx). None of the algal toxins used in the bench-scale experiments were detectable in the desalinated product water. Monitoring for intracellular and extracellular concentrations of DA, STX, PbTx and okadaic acid (OA) within the intake and desalinated water from a pilot RO desalination plant in El Segundo, CA, was conducted from 2005 to 2009. During the five-year monitoring period, DA and STX were detected sporadically in the intake waters but never in the desalinated water. PbTx and OA were not detected in either the intake or desalinated water. The results of this study demonstrate the potential for HAB toxins to be inducted into coastal RO intake facilities, and the ability of typical RO operations to effectively remove these toxins.

Topics: California; Environmental Monitoring; Harmful Algal Bloom; Kainic Acid; Marine Toxins; Okadaic Acid; Osmosis; Oxocins; Pilot Projects; Saxitoxin; Seawater; Water Purification

Okadaic acid, a diarrhetic shellfish poison, domoic acid, an amnesic shellfish poison, and saxitoxin, a paralytic shellfish poison, are three of the best-known marine biotoxins. The mouse bioassay is the method most widely used to detect many of these toxins in shellfish samples, but animal welfare concerns have prompted researchers to seek alternative methods of detection. In this study, three direct competitive enzyme-linked immunosorbent assays (ELISAs), each based on antibodies raised in rabbits against a conjugate of the analyte of interest, were developed for marine biotoxin detection in mussel, oyster, and scallop. One assay was for okadaic acid, one for saxitoxin, and one for domoic acid usually detected and quantified by high-performance liquid chromatography-ultraviolet light (HPLC-UV). All three compounds and a number of related toxins were extracted quickly and simply from the shellfish matrices with a 9 : 1 mixture of ethanol and water before analysis. The detection capabilities (CCbeta values) of the developed ELISAs were 150 microg kg(-1) for okadaic acid, 50 microg kg(-1) for domoic acid, and 5 microg kg(-1) or less for saxitoxin. The assays proved satisfactory when used over a 4-month period for the analysis of 110 real samples collected in Belgium.

Topics: Animals; Antibody Specificity; Belgium; Bivalvia; Calibration; Carcinogens; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Kainic Acid; Marine Toxins; Mice; Okadaic Acid; Ostreidae; Pectinidae; Rabbits; Saxitoxin; Sensitivity and Specificity

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Topics: Acrylamides; Animals; Cell Line, Tumor; Cnidarian Venoms; Dose-Response Relationship, Drug; Furans; Glioma; Hybrid Cells; Kainic Acid; Macrolides; Marine Toxins; Mice; Neuroblastoma; Okadaic Acid; Oxocins; Pyrans; Saxitoxin; Time Factors; Toxicity Tests

The effect of gamma-irradiation on concentrations of hydrophilic and lipophilic phycotoxins has been investigated by use of HPLC-UV and LC-MS. Pure toxins in organic solvents and toxins in mussel (Mytilus edulis) tissues were irradiated at three different doses. In solution all toxin concentrations were reduced to some extent. Most severe decreases were observed for domoic acid and yessotoxin, for which the smallest dose of irradiation led to almost complete destruction. For pectenotoxin-2 the decrease in concentration was less severe but still continuous with increasing dose. Azaspiracid-1 and okadaic acid were the least affected in solution. In shellfish tissue the decrease in toxin concentrations was much reduced compared with the effect in solution. After irradiation at the highest dose reductions in concentrations were between ca. 5 and 20% for the lipophilic toxins and there was no statistical difference between control and irradiated samples for azaspiracids in tissue. Irradiation of shellfish tissues contaminated with domoic acid led to a more continuous decrease in the amount of the toxin with increasing dose. The effect of irradiation on the viability of microbial activity in shellfish tissues was assessed by using total viable counting techniques. Microbial activity depended on the type of shellfish and on the pretreatment of the shellfish tissues (with or without heat treatment). As far as we are aware this is the first investigation of the effectiveness of irradiation as a technique for stabilising tissue reference materials for determination of phycotoxins. Our results suggest that this technique is not effective for materials containing domoic acid. It does, however, merit further investigation as a stabilisation procedure for preparation of shellfish tissue materials for some lipophilic toxins, in particular azaspiracids. Chemical structures of the toxins investigated in the study.

Topics: Animals; Calibration; Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Chromatography, Liquid; Ethers, Cyclic; Gamma Rays; Kainic Acid; Macrolides; Marine Toxins; Mass Spectrometry; Mollusk Venoms; Okadaic Acid; Oxocins; Pyrans; Reference Values; Shellfish; Spectrophotometry, Ultraviolet; Spiro Compounds

Two samples of mussels (Mytilus edulis) were collected from the southwest of Ireland. One sample contained domoic acid, the other sample contained okadaic acid, dinophysistoxin-2 and azaspiracid-1, -2 and -3. Wet and freeze-dried reference materials were prepared from each of the two samples to test for differences in homogeneity, stability and extractability of the analytes in either condition. Wet materials were homogenised, aliquoted and hermetically sealed under argon and subsequently frozen at -80 degrees C. Dry materials were similarly homogenised but frozen in flat cakes prior to freeze-drying. After grinding, sieving and further homogenisation, the resulting powder was aliquoted and hermetically sealed. Domoic acid materials were characterised using HPLC-UV, while LC-MS was used for the determination of lipophilic toxins. The extractabilities of all phycotoxins studied were comparable for wet and freeze-dried materials once a sonication step had been carried out for reconstitution of the freeze-dried materials prior to extraction. Homogeneity was assessed through replicate analysis of the phycotoxins (n = 10), and was found to be similar for wet and freeze-dried materials, for both hydrophilic and lipophilic toxins. Water contents were determined for both wet and freeze-dried materials, and particle size was determined for the freeze-dried materials. Stability was evaluated isochronously over eight months at four temperatures (-20, +4, +20 and +40 degrees C). The freeze-dried material containing domoic acid was stable over the whole duration at all temperatures, while in the wet material domoic acid degraded to some extent at all temperatures except -20 degrees C. In freeze-dried and wet materials containing lipophilic toxins, okadaic acid, dinophysistoxin-2, azaspiracid-1 and azaspiracid-2 were stable over the whole duration at all conditions, while concentrations of azaspiracid-3 changed significantly in both materials at some storage temperatures. Figure Aliquots of freeze-dried and wet mussel tissue reference materials containing the various shellfish toxins examined in the study.

Topics: Animals; Chromatography, High Pressure Liquid; Freezing; Kainic Acid; Marine Toxins; Models, Chemical; Mytilus edulis; Okadaic Acid; Reference Standards; Reproducibility of Results; Specimen Handling; Spiro Compounds; Temperature; Time Factors; Water

A number of marine microalgae are known to produce toxins that can accumulate in shellfish and when eaten, lead to toxic and potentially fatal reactions in humans. This paper reports on the occurrence and seasonal variations of algal toxins in the waters, phytoplankton and shellfish of Southeast Queensland, Australia. These algal toxins include okadaic acid (OA), domoic acid (DA), gymnodimine (GD), pectenotoxin-2 (PTX-2) and pectenotoxin-2-seco acid (PTX-2-SA), which were detected in the sampled shellfish and phytoplankton, via HPLC-MS/MS. Dissolved OA, PTX-2 and GD were also detected in the samples collected from the water column. This was the first occasion that DA and GD have been reported in shellfish, phytoplankton and the water column in Queensland waters. Phytoplankton tows contained both the toxic Dinophysis and Pseudo-nitzschia algae species, and are suspected of being the most likely producers of the OA, PTX-2s and DA found in shellfish of this area. The number of cells, however, did not correlate with the amount of toxins present in either shellfish or phytoplankton. This indicates that toxin production by algae varies with time and the species present and that number of cells alone cannot be used as an indicator for the presence of toxins. The presence of OA and PTX-2s were more frequently seen in the summer, while DA and GD were detected throughout the year and without any obvious seasonal patterns.

Topics: Animals; Environmental Exposure; Geography; Kainic Acid; Marine Toxins; Mollusca; Okadaic Acid; Phytoplankton; Queensland; Regression Analysis; Risk Assessment; Seasons; Seawater; Shellfish

In this article the results of analysis of ASP, PSP and DSP phycotoxins content in aquatic organisms are presented. Methods of determination of toxins were ELISA and HPLS.

Topics: Food Analysis; Kainic Acid; Mollusk Venoms; Okadaic Acid; Saxitoxin; Seafood

The monoclonal antibody to ciguatoxin (CTX) produced from a hybridoma cell line was assayed for the detection of four congeners of CTX: Pacific ciguatoxin-1 (P-CTX-1), Pacific ciguatoxin-2 (P-CTX-2), Pacific ciguatoxin-3 (P-CTX-3), and Caribbean ciguatoxin-1 (C-CTX-1) and related marine toxins, including domoic acid, palytoxin, and okadaic acid, using a modified enzyme-linked immunosorbent assay (ELISA). Lower detection limits were assessed and linearity was statistically established (P<0.05) for P-CTX-1, P-CTX-2, and P-CTX-3 and C-CTX-1 at concentrations ranging from 0 to 5.00 ng, while the other marine toxins showed statistically insignificant cross-reactivities at similar concentrations. Thus, the monoclonal antibody to CTX is able to specifically detect various CTX congeners at levels comparable to those naturally occurring in ciguatoxic fish.

Topics: Acrylamides; Antibodies, Monoclonal; Caribbean Region; Ciguatera Poisoning; Ciguatoxins; Cnidarian Venoms; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Kainic Acid; Okadaic Acid; Pacific Ocean; Seafood

Okadaic Acid (OA) the major diarrheic shellfish poisoning (DSP) toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA), the major Amnesic Shellfish Poisoning (ASP) toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN) arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA) and domoic acid (DA) to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH) using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.

Topics: Caco-2 Cells; Chromosome Aberrations; Dose-Response Relationship, Drug; Humans; In Situ Hybridization, Fluorescence; Kainic Acid; Micronucleus Tests; Mutagens; Okadaic Acid

We examine the use of external standards for quantitative measurement by 1H NMR of solution concentrations of natural products and other low molecular weight, hydrogen-containing compounds and show that precision and accuracy ca. 1% is obtainable with a commercial 11.7 T spectrometer when standards and analytes are contained in separate but identical sealed precision glass NMR tubes. Numerous factors contributing to the intensity of the NMR signals are evaluated. Precise measurements of 360 degrees pulse lengths for each sample provide direct corrections for variations in probe Q-factor that enable samples in different solvents to be compared, provided single-coil excitation and detection is used throughout. Samples need not be prepared in deuterated solvents if the 1H spectra of the solvents are simple enough for peak suppression by presaturation. The approach is particularly suitable for hazardous materials kept in sealed tubes and for the preparation of certified calibration solution reference materials for use with LC-MS and other techniques where deuterated solvents should be avoided.

Topics: Biological Products; Calibration; Chromatography, Liquid; Deuterium; Eukaryota; Heterocyclic Compounds, 3-Ring; Hydrocarbons, Cyclic; Imines; Kainic Acid; Magnetic Resonance Spectroscopy; Marine Toxins; Okadaic Acid; Reproducibility of Results; Saxitoxin; Sensitivity and Specificity; Solutions; Solvents

The frequency and scale of Harmful Algal Bloom (HAB) and marine algal toxin incidents have been increasing and spreading in the past two decades, causing damages to the marine environment and threatening human life through contaminated seafood. To better understand the effect of HAB and marine algal toxins on marine environment and human health in China, this paper overviews HAB occurrence and marine algal toxin incidents, as well as their environmental and health effects in this country. HAB has been increasing rapidly along the Chinese coast since the 1970s, and at least 512 documented HAB events have occurred from 1952 to 2002 in the Chinese mainland. It has been found that PSP and DSP toxins are distributed widely along both the northern and southern Chinese coasts. The HAB and marine algal toxin events during the 1990s in China were summarized, showing that the HAB and algal toxins resulted in great damages to local fisheries, marine culture, quality of marine environment, and human health. Therefore, to protect the coastal environment and human health, attention to HAB and marine algal toxins is urgently needed from the environmental and epidemiological view.

Topics: Amnesia; Animals; China; Ciguatoxins; Diarrhea; Dinoflagellida; Environment; Eukaryota; Eutrophication; Fisheries; Food Contamination; Foodborne Diseases; Humans; Kainic Acid; Lethal Dose 50; Marine Toxins; Neurotoxicity Syndromes; Okadaic Acid; Oxocins; Paralysis; Seawater; Shellfish Poisoning

Although pathogenesis of neuronal ischemia is incompletely understood, evidence indicates apoptotic neuronal death after ischemia. Bcl-2, an anti-apoptotic and neuroprotective protein, interacts with calcineurin in non-neuronal tissues. Activation of calcineurin, which is abundant in the brain, may play a role in apoptosis. Using co-immunoprecipitation experiments in biopsy-derived, fresh human cortical and hippocampal slices, we examined possible interactions between calcineurin and Bcl-2. Calcineuin-Bcl-2 interactions increased after exposure in vitro to excitotoxic agents and conditions of hypoxia/aglycia. This interaction may shuttle calcineurin to substrates such as the inositol-1,4,5-tris-phosphate receptor because under these experimental conditions interactions between calcineurin and inositol-1,4,5-tris-phosphate receptor also increased. A specific calcineurin inhibitor, FK-520, attenuated insult-induced increases in calcineurin-Bcl-2 interactions and augmented caspase-3 like activity. These data suggest that Bcl-2 modulates neuroprotective effects of calcineurin and that calcineurin inhibitors increase ischemic neuronal damage.

Topics: Adult; Blotting, Western; Calcineurin; Calcium Channels; Caspase 3; Caspases; Cerebral Cortex; Enzyme Inhibitors; Enzyme Precursors; Female; Humans; Hypoxia, Brain; Immunosuppressive Agents; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Kainic Acid; Male; Middle Aged; N-Methylaspartate; Neurotoxins; Okadaic Acid; Precipitin Tests; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Spectrin; Tacrolimus

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Topics: Actins; Animals; Biological Assay; Bivalvia; Cell Line, Tumor; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethers, Cyclic; Fluorometry; Humans; Kainic Acid; Marine Toxins; Methanol; Mice; Mollusk Venoms; Okadaic Acid; Oxocins; Phosphoprotein Phosphatases; Reproducibility of Results; Saxitoxin; Sensitivity and Specificity; Shellfish

The current work describes the optimisation of a screen-printed electrode (SPE) system for measurement of a variety of seafood toxins, such as okadaic acid, brevetoxin, domoic acid and tetrodotoxin. A disposable screen-printed carbon electrode coupled with amperometric detection of p-aminophenol at +300 mV vs. Ag/AgCl, produced by the label, alkaline phosphatase, was used for signal measurement. ELISA was primarily used to develop all toxin systems, prior to transferring to SPE. The sensors incorporate a relevant range for toxin detection, by which humans become ill, with detection limits achieved at SPE to the order of ng ml (-1) (ppb) or lower in some cases. The SPE system is simple and cost-effective due to their disposable nature, and analysis time is complete in 30 min. In addition, analyses can be achieved outside of a laboratory environment allowing for in-field measurements. Recovery experiments on selected toxins using the relevant working ranges highlighted the functionality of these systems yielding a +/-10% deviation for the true value.

Topics: Animals; Biosensing Techniques; Electrochemistry; Enzyme-Linked Immunosorbent Assay; Immunoassay; Kainic Acid; Marine Toxins; Okadaic Acid; Oxocins; Tetrodotoxin

Acanthocardia tuberculatum is a bivalve mollusc that presents recurrent problems of paralytic shellfish poisoning (PSP) contamination in the Mediterranean coasts of Spain and Morocco. Although not commercially exploited from the Portuguese south coast, it represents an alternative for reducing the harvest pressure on species presently exploited. Evaluation of accumulation of marine biotoxins was carried out by HPLC in this species, harvested during a campaign carried out in April 2001 aimed at evaluating bivalve's resources that covered the entire Portuguese south coast.PSP toxins were studied by automated pre-column oxidation. Toxins were found in specimens from all stations, but always under the regulatory limit of 80 microg STX eq./100g. Saxitoxin and decarbamoyl-saxitoxin were the only PSP toxins unambiguously identified. In commercially exploited species, saxitoxin and analogues were not detected. The amnesic toxin domoic acid was found, but in levels similar to those found in species commercially exploited, and always under the regulatory limit of 20 microg/g. A fast elimination, and not a prolonged retention, of domoic acid seems to occur in A. tuberculatum in view of the levels close to or higher than 20 microg/g found in commercial bivalves harvested the two preceding months. The diarrhoeic toxin okadaic acid was found at trace levels much under the allowable level, similar to what was happening with other species harvested during the same period.

Topics: Animals; Biological Assay; Chromatography, High Pressure Liquid; Eutrophication; Kainic Acid; Marine Toxins; Mollusca; Okadaic Acid; Portugal; Saxitoxin; Species Specificity

Topics: Animals; Cell Death; Cells, Cultured; Cerebellum; Colforsin; Ethers, Cyclic; Glutamates; Glutamic Acid; Kainic Acid; Marine Toxins; Models, Neurological; Neurons; Neurotoxins; Okadaic Acid; Phosphoprotein Phosphatases; Rats; Shellfish; Tetradecanoylphorbol Acetate

Liquid chromatographic analyses of extracts from shellfish and phytoplankton from the Gulf of Mexico indicated the presence of the marine toxins okadaic acid (0.162 microgram/g shellfish) and domoic acid (2.1 pg/cell phytoplankter). These toxins are causative agents of diarrhetic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. The presence of DSP and ASP toxins in a region with no previous record of outbreaks may indicate a potential for human poisoning under conditions appropriate for accumulation of these toxins in shellfish.

Topics: Animals; Ethers, Cyclic; Kainic Acid; Marine Toxins; Okadaic Acid; Phytoplankton; Shellfish