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PubMed1984

The detection of mutagenic chemicals in the tissues of shellfish exposed to oil pollution.

Two species of shellfish (mussels and limpets ) were collected from two sites in West Wales (U.K.) extracted in nitric acid and monitored for the presence of mutagenic chemicals using bacterial fluctuation tests over a period of an oil pollution incident. Prior to the observation of oil pollution at the two sites no mutagenic activity could be detected in the tissue extracts. However, within three days of the observation of oil pollution, chemicals capable of inducing both base-substitution and frameshift mutation could be detected in both shellfish species. At one of the sites (St. Brides ) mutagenic activity could still be detected in samples collected 12 months after the oil pollution incident, whereas at the other sites (Dale) no activity was detectable after 12 months. The results obtained demonstrate shellfish exposed to crude oil (and dispersants ) accumulate mutagenic chemicals that persist for periods longer than pollution could be observed by visual inspection.

Animals↗
PubMed1989

Neurotoxicity and lethality of toxic extracts from Atlantic coast shellfish.

1. HCl-extract of poisonous shellfish was injected i.p. into Swiss-Webster mice. 2. Behavioral effects (sluggishness, scratching, huddling, clonic convulsions, respiratory distress and mortality) were noted. 3. Infant mice were more sensitive than were the adults to XTRT toxicity. Estimated LD50 values were; adult: 34.8 ml/kg i.p., infant: 9.6 ml/kg i.p. 4. The mouse model of shellfish-toxicity offers the advantages of a degree of similarity with the human clinical situation and a rapid time course for screening antagonists against shellfish poisoning.

Aging↗
PubMed2004

Biacore biosensor immunoassay for 4-nonylphenols: assay optimization and applicability for shellfish analysis.

A rapid Biacore biosensor immunoassay of 4-nonylphenols was developed. Two types of antibodies were used in the study: polyclonal antibodies with high cross-reactivity towards technical 4-nonylphenol and a monoclonal antibody very specific to 4-n-nonylphenol. 9-(p-Hydroxyphenyl)nonanoic acid was immobilized onto surface of a sensor chip. The best assay sensitivity was achieved using a flow rate of 50 microl min(-1) and injection time of 2 min. For the assay incorporating monoclonal antibodies a limit of detection 2 ng ml(-1) for 4-n-nonylphenol was achieved. With polyclonal antibodies one order lower sensitivity was observed for 4-nonylphenols. High background level of calibration curve for technical 4-nonylphenol was decreased by using IgG fraction of polyclonal antibodies in combination with lower amount of immobilised 9-(p-hydroxyphenyl)nonanoic acid. Sensitivity of the assay was improved by using a chip with a new derivative on a surface-N-aminobutyl [2-(4-hydroxyphenyl)ethylamine] (limit of detection--5 ng ml(-1)). Applicability of the developed assays to ecological monitoring was checked in experiments using shellfish samples. 4-n-Nonylphenol from spiked samples was extracted into hexane followed by clean-up on NH2 SPE columns. Calibration curves generated for cockles, mussels and oyster samples were identical (limit of detection about 10 ng g(-1)) whereas for scallop samples a slight decrease (about 5-10%) of absolute response was observed. In the assay using the monoclonal antibody specific to 4-n-nonylphenol 31 shellfish samples were found to be negative. Results obtained with polyclonal antibodies indicated that two scallop samples contained a quantity of 4-nonylphenols. The developed biosensor assay could be applied for shellfish analysis as a preliminary screening method.

Animals↗
PubMed2004

Amnesic shellfish poison.

Amnesic shellfish poisoning (ASP) is caused by consumption of shellfish that have accumulated domoic acid, a neurotoxin produced by some strains of phytoplankton. The neurotoxic properties of domoic acid result in neuronal degeneration and necrosis in specific regions of the hippocampus. A serious outbreak of ASP occurred in Canada in 1987 and involved 150 reported cases, 19 hospitalisations and 4 deaths after consumption of contaminated mussels. Symptoms ranged from gastrointestinal disturbances, to neurotoxic effects such as hallucinations, memory loss and coma. Monitoring programmes are in place in numerous countries worldwide and closures of shellfish harvesting areas occur when domoic acid concentrations exceed regulatory limits. This paper reviews the chemistry, sources, metabolism and toxicology of domoic acid as well as human case reports of ASP and discusses a possible mechanism of toxicity.

Amnesia↗
PubMed2006

Assessment of human enteric viruses in shellfish from the northern Adriatic sea.

Incidence and circulation of different strains of hepatitis A and Norovirus in shellfish were studied on 235 samples (Tapes philippinarum, Mytilus galloprovincialis, Ostrea spp. and Chlamys spp.) obtained from different sites, representing the shellfish production areas of the northern Adriatic sea. Shellfish were harvested in the period of one year and, after depuration, were examined for bacterial (Escherichia coli and Salmonella) and viral (HAV and NoV) contamination. Viral contamination was present on average in 22% of samples: specifically, 6% of samples tested positive for HAV, 14% for NoV and 2% for both viruses. None of the samples revealed the presence of Salmonella, and in most of them (93%) the number of E. coli was below the European legislation limit of 230 MPN/100 g. T. philippinarum was the species most often contaminated, as well as being the only species in which the legal limit for E. coli was, in some cases, exceeded. Both HAV and NoV contamination were detected throughout the year; NoV detection was slightly more frequent during winter months, but positive samples were also present in summer. The sequencing of the PCR products showed the circulation of only one HAV genotype (IA) and four different NoV genotypes (Hawaii, Melksham, Lordsdale and GGIIb) with a prevalence of the GGIIb genotype in the second period of the monitoring.

Animals↗
PubMed2006

[Comparison of cell culture and RT-PCR for the detection of enterovirus in sewage and shellfish].

Enteric viruses contaminating the environment represent a danger for public health notably enteroviruses that are excreted in stools and can contaminate wastewater and shellfish. The ability of enteroviruses to grow in cell culture and the development of techniques of molecular biology applied to their detection make these viruses a good marker of viral pollution of aquatic environment. The aim of our study was to develop a rapid and sensitive RT-PCR technique, able to detect enterovirus RNA in wastewater and shellfish. From 26 samples of wastewater and 56 samples of shellfish, 50.0 and 42.8% were found positive by RT-PCR, respectively, whereas 38.4 and 28.5% were positive by culture, respectively (P=0.077 by chi square test). The two techniques were found concordant in 57.3% of the 82 combined samples, whereas 23 samples (28.0%) were positive only by RT-PCR and that 12 samples (14.6%) were positive only by culture. These discrepancies illustrate the fact that the two techniques are not equivalent: the molecular technique allows the detection of not cultivable samples but is sensitive to PCR inhibitors that are present in large amounts in environmental samples.

Animals↗
PubMed2004

Confirmation of brevetoxin metabolism in cockle, Austrovenus stutchburyi, and greenshell mussel, Perna canaliculus, associated with New Zealand neurotoxic shellfish poisoning, by controlled exposure to Karenia brevis culture.

We examined metabolism of PbTxs in New Zealand cockle, Austrovenus (A.) stutchburyi, and greenshell mussel, Perna (P.) canaliculus, by means of liquid chromatography coupled with tandem mass spectrometry. PbTx-2, PbTx-3 and BTX-B5 were detected in Karenia (K.) brevis culture medium in the ratio of ca. 50:2:5. The amounts of PbTx-3 and BTX-B5 were greatly increased in both seawater and shellfish exposed to K. brevis cultures or supernatant prepared by disruption of K. brevis under appropriate condition, while those of PbTx-2 were decreased. Some PbTx-2 was present in P. canaliculus, but not in A. stutchburyi. Low levels of BTX-B1 were detected in A. stutchburyi, but not P. canaliculus. Levels of PbTx-3 and BTX-B5 were highest immediately after exposure and then declined rapidly in both shellfish. BTX-B1 increased in concentration after exposure, and was then gradually eliminated from A. stutchburyi. Three successive exposures of A. stutchburyi to K. brevis cultures resulted in similar initial levels of PbTx-3 and BTX-B5, while BTX-B1 accumulated after each dose. In P. canaliculus, initial levels of PbTx-3 were similar, while PbTx-2 and BTX-B5 accumulated after each dose. PbTx-3 and BTX-B5 are proposed to be suitable markers for monitoring shellfish toxicity after a red tide event.

Animals↗
PubMed2005

A rapid detection method for paralytic shellfish poisoning toxins by cell bioassay.

We report here a rapid detection method for paralytic shellfish poisoning (PSP) toxins using a cultured neuroblastoma cell line, modified from the bioassay system previously established by Manger et al. [Manger, R.L., Leja, L.S., Lee, S.Y., Hungerford, J.M., Kirkpatrick, M.A., Yasumoto, T., Wekell, M.M., 2003. Detection of paralytic shellfish poison by rapid cell bioassay: antagonism of voltage-gated sodium channel active toxins in vitro. J. AOAC Int. 86 (3), 540-543]. In the present study, we made two major modifications to the previous method. The first is the use of maitotoxin, a marine toxin of ciguatera fish poisoning, which enables the incubation period to be reduced to 6 h when applied to the microplate 15 min prior to the end of the incubation. The second is the use of WST-8, a dehydrogenase detecting water-soluble tetrazolium salt for determining the target cell viability, which permits the omission of a washing step and simplifies the counting process. In addition, we attempted to reduce the required materials as much as possible. Thus, our modified method should be useful for screening the PSP-toxins from shellfish.

Animals↗
PubMed2006

First evidence of spirolides in Spanish shellfish.

During the months of November and December 2005, a harmful algal bloom in the northwestern region of Spain caused the accumulation of different algal toxins in several types of shellfish. Many of the shellfish were contaminated at levels above European regulatory limit, presenting serious risks for human health. The analysis of mussels sampled in the affected areas to search for lipophilic toxins, using liquid chromatography tandem mass spectrometry (LC-MS/MS), showed the presence of free okadaic acid (OA) and dinophysistoxin-2 (DTX-2) as well as esters of these toxins. The results also revealed the presence of minor amounts of 13-desmethyl spirolide C (SPX-1) in the analysed samples, although this toxin has never been reported before in Spain. The combination of different MS modes of operation, just as enhanced MS (EMS) and MS(3) experiments, allowed to confirm the first occurrence of spirolides in Spanish shellfish.

Animals↗
PubMed2000

Development and validation of a high-performance liquid chromatographic method using fluorimetric detection for the determination of the diarrhetic shellfish poisoning toxin okadaic acid without chlorinated solvents.

A modification of the high-performance liquid chromatographic method with fluorimetric detection method for the determination of diarrhetic shellfish poisoning toxins was developed to completely avoid the use of dangerous chlorinated solvents. The method was validated for the toxin okadaic acid (OA) over a period of 6 months where 12 calibrations were performed and 72 samples were analyzed. Analysis of toxic and non-toxic mussels, clams and scallops demonstrated its selectivity. Linearity was observed in the tested range of interest for monitoring purposes of edible shellfish, from the limit of detection (0.3 microg OA/g hepatopancreas) to 13 microg OA/g hepatopancreas. Intra-assay precision of the method was 7% RSD at the quantification limit (0.97 microg OA/g hepatopancreas at S/N=10). Accuracy was tested in triplicate recovery experiments from OA-spiked shellfish where recovery ranged from 92 to 106% in the concentration range of 0.8 to 3.6 microg OA/g hepatopancreas. Useful information on critical factors affecting calibration and reproducibility is also reported. Good correlation (R=0.87) was observed between the results of the method and those of the method of Lee, after the analysis of 45 samples of mussels from the galician rias.

Calibration↗
PubMed2002

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of yessotoxins in shellfish.

Yessotoxins are a group of large polyether toxins, produced by marine dinoflagellates, which cause widespread contamination of filter-feeding shellfish. A new, sensitive liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of yessotoxin (YTX) and 45-hydroxy-yessotoxin (45-OHYTX), a major metabolite in shellfish. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap MS in negative mode. The molecular related ion species at m/z 1141 [M-2Na+H]- was used as the parent ion for multiple MS experiments. MS-MS and MS3 gave major fragment ions at m/z 1061 [1141-SO3H]- and m/z 945 [1061-C9H12O]-. Predominant ions, that are due to the fragmentation of the backbone structure of YTXs, were observed at the MS4 stage. Reversed-phase LC using a C16 amide column was preferable to C18 phases for the separation of YTX and 45-OHYTX. Optimum calibration and reproducibility data were obtained for YTX using LC-MS-MS; r 2=0.9960, RSD < or = 6.3% at 0.25 microg YTX/g (n=5). The detection limit (S/N=3) was 30 pg YTX on-column which corresponded to 3 ng/g shellfish tissue.

Animals↗
PubMed1996

Development of a manganese dioxide solid-phase reactor for oxidation of toxins associated with paralytic shellfish poisoning.

Using manganese dioxide as a solid-phase oxidant, a post-column HPLC reactor for paralytic shellfish poison toxins was constructed and evaluated. Operating parameters such as reaction temperature and pH, flow-rate, reactor column size, and MnO2 particle size were studied. Based on a 3:1 signal-to-noise ratio the limits of detection for the non-hydroxylated toxins were in the sub-nanogram range, while the hydroxylated toxins were ten times less detectable. The limit of detection for saxitoxin was about 0.1 ng per injection. The repeatability of replicate injections was < or = 10% (relative standard deviation). The system was used to analyze extracts of shellfish and plankton yielding results that were in agreement with those obtained by established analytical methods. Extracts of shellfish contaminated at the regulatory limit of 0.8 microgram/g were successfully analyzed.

Animals↗
PubMed2002

Residue levels of HCHs, DDTs and PCBs in shellfish from coastal areas of east Xiamen Island and Minjiang Estuary, China.

Shellfish samples were collected from the coastal cultivating areas of east Xiamen Island and Minjing Estuary, China and analyzed for hexachlorocyclohexanes (HCHs), DDTs and polychlorinated biphenyls (PCBs) by GC. Concentration ranges of HCHs, DDTs and PCBs in the coastal area of east Xiamen Island were 0.18-345, 75.2-2143 and ND-23 ng g(-1) dry wt., respectively; those for Minjiang Estuary were ND-5.07, 21.5-2396 and ND-6.78 ng g(-1) dry wt., respectively. The results showed that the organochlorines were evidently accumulated in marine bivalve molluscs, particularly in oyster. The concentrations of organocholorine pollutants in shellfish samples varied with the sampling locations, the season of collection and organism species, but were generally higher than those in the sediments. The concentrations of DDTs in the shellfish were high as compared with HCHs and PCBs, so that DDTs can be considered as the typical organochlorines in the survey areas. The residue levels and compositions of HCHs, DDTs and PCBs are discussed.

Animals↗
PubMed2000

Paralytic shellfish toxins in the freshwater cyanobacterium Aphanizomenon flos-aquae, isolated from Montargil reservoir, Portugal.

Montargil reservoir, located in a dry flat area in the centre of Portugal, was filled in 1958 to fulfil agricultural, electric and industrial requirements. In May 1996, an intensive bloom of phytoplankton was detected. The algal community was strongly dominated by cyanobacteria with predominance of Aphanizomenon flos-aquae from May to June and Microcystis aeruginosa from July to August. Extracts of samples collected during the bloom period showed high toxicity by mouse bioassay. During the M. aeruginosa predominance period, the toxicity was ascribed to the presence of hepatotoxins, but clear symptoms of paralytic shellfish poison were observed when A. flos-aquae was the dominant species. In order to confirm the production of neurotoxins a strain of A. flos-aquae was isolated and established in culture. In this manuscript, we show the morphological characteristics and confirm paralytic shellfish toxins production by the strain isolated and maintained in culture. Identification of the saxitoxin analogs was achieved using high performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD) and liquid chromatographic mass spectrometry technique (LC-MS). The toxins found in the culture extract were GTX5 (64.5 mol%), neoSTX (23.0 mol%), dcSTX (6.1 mol%), STX (5.4 mol%) and GTX6 (1.1 mol%). This is, to our knowledge, the first report of unambiguous evidence of paralytic shellfish toxins produced by freshwater cyanobacteria in Portugal. The toxin profile is rather different from the previously reported PSP producing A. flos-aquae and demonstrates its diversity in terms of toxin production.

Animals↗
PubMed2001

Domoic acid in Portuguese shellfish and fish.

The presence of domoic acid (DA) in Portuguese shellfish is a recurrent event that affects shellfish resources several times a year, mainly in spring and autumn. Levels of domoic acid as high as twice the regulatory level of 20 microg DA/g tissue are not unusual. When several shellfish species are exploited in the same restricted area, common cockle (Cerastoderma edule) and carpet shell (Venerupis pullastra) are usually amongst the most toxic, followed by peppery furrow shell, clam, mussel, oyster and razor clam. In whole sardine, domoic acid was detected in levels exceeding sometimes the regulatory limit. Fortunately, toxicity is restricted to the gut content, and does not accumulate in muscle tissue. In brain tissue, domoic acid was detected in levels generally below 1 microg/g. Domoic acid was confirmed by spectra acquired with a diode-array detector. The most toxic samples were confirmed by mass spectrometry.

Chromatography, High Pressure Liquid↗
PubMed2001

A cytotoxicity assay for the detection and differentiation of two families of shellfish toxins.

There is an urgent need for an alternative to the mouse bioassay for the detection of algal toxins in shellfish on both analytical and animal welfare grounds. Several alternative methodologies have been described, but have not gained widespread acceptance to date, because each assay measures only one or a small number of related phycotoxins out of the increasing range that needs to be detected. A simple cytotoxicity assay using either the HepG2 or ECV-304 cell lines is described with two end-point measurements, which can detect and distinguish between two unrelated classes of phycotoxins. Morphological examination following 3h exposure to the sample enables the detection of the diarrhetic shellfish poisons, including okadaic acid and related toxins. Viability testing using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), following 24h exposure of the same cells to the sample, reveals a second class of toxin, which is most probably the newly-described toxin, azaspiracid. This assay should play an important role in shellfish monitoring in the future.

Animals↗
PubMed2003

Implication of brevetoxin B1 and PbTx-3 in neurotoxic shellfish poisoning in New Zealand by isolation and quantitative determination with liquid chromatography-tandem mass spectrometry.

Brevetoxin B1 (BTX-B1) was isolated from Austrovenus stutchburyi following the 1992-1993 outbreak of neurotoxic shellfish poisoning (NSP) in New Zealand. We report here the first isolation of PbTx-3 from the same shellfish and the development of a procedure for quantitative determination of PbTx-3 and BTX-B1. PbTx-3 was isolated by chromatography on columns of SiO2, ODS, and LH-20, followed by reverse-phase HPLCs. In mass spectrometry (MS) with an electrospray ionization (ESI) interface operating in the positive or negative ion mode, the abundant protonated ion [M+H]+ of PbTx-3 (m/z 897) and the de-sodiated ion [M-Na]- of BTX-B1 (m/z 1016) were generated, respectively. These served as precursor ions for collision-induced dissociation, and the product ions of m/z 725 from PbTx-3 and m/z 80 from BTX-B1 were identified, allowing unambiguous confirmation of these toxins by selected reaction monitoring liquid chromatography-tandem mass spectrometry (SRM LC-MS/MS) analysis. The determination limits were 0.4 and 2 ng/g for BTX-B1 and PbTx-3 at a signal-to-noise ratio of five, respectively. This LC-MS/MS method was successfully applied to determine BTX-B1 and PbTx-3 in the NSP-associated toxic shellfish. BTX-B1 was found in both A. stutchburyi and Perna canaliculus, but not in Crassostrea gigas, while PbTx-3 was found in all three.

Animals↗
PubMed1999

Bioaccumulation of mycotoxins by shellfish: contamination of mussels by metabolites of a Trichoderma koningii strain isolated in the marine environment.

To determine whether toxic metabolites produced by fungi could cause shellfish toxicities, mussels were contaminated in laboratory conditions by sterile filtrates of a liquid culture of a strain of the fungus Trichoderma koningii previously isolated from a shellfish, the cockle (Cerastoderma edule). Mussels were kept in aerated natural seawater and fed with a culture of the microalga Isochrysis galbana, to which a filtrate of liquid fungal culture was added. Mussels were exposed to contamination for 7 days at 16 or 20 degrees C and extractions were then performed and their activity tested on blowfly larvae. The same toxicity was found in the fungal filtrate and the shellfish, indicating bioaccumulation. The digestive gland was the most toxic part of the mussel, confirming contamination by filtration. Treated mussels produced a mucus which appeared to be a means of eliminating toxic metabolites.

Animals↗