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PubMed1978

Analytical methodology for the determination of Kepone residues in fish, shellfish, and Hi-Vol air filters.

The recent discovery of the pollution of the environment with Kepone has resulted in a tremendous interest in the development of residue methodology for the compound. Current multiresidue methods for the determination of the common organochlorinated pesticides do not yield good quantitative analytical results for Kepone. The extracting solvents are usually of insufficient polarity to extract Kepone from the various media. This article describes some of the recently developed methodology for Kepone in air filters, finfish, finfish livers and entrails, shellfish, and archival oyster samples. The finfish and archival oyster samples were Soxhlet extracted using diethyl ether/petroleum ether (1:1 v/v) as the extracting solvent. The finfish livers and entrails were macerated in a Duall tissue grinder containing acetonitrile followed by partitioning of the Kepone into benzene. Shellfish samples were analyzed after extracting the sample with acetonitrile and partitioning the Kepone into benzene. The Hi-Vol air samples were extracted with methanol/benzene (1:1 v/v). The coextracting contaminants were removed by micro-Florisil column chromatography and/or acid digestion. The procedural recovery of Kepone from fortified samples averaged 82%.

Air↗
PubMed2006

Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins.

The need for reference materials for quality control of analysis of foodstuffs has been stressed frequently. This has been particularly true in the phycotoxins field, where there is a great shortage of both pure calibration standards and reference materials. Worldwide there are very few independent bodies that produce certified reference materials for phycotoxins, the main producers currently being the National Research Council Canada and the Japanese Food Research Laboratory. Limited availability of contaminated shellfish and algae, as well as the time and knowledge necessary for the production of adequate reference materials, continuously lead to limited editions of certified reference materials and even more limited production of in-house reference materials. The restricted availability of in-house quality control materials promotes the rapid use of the limited certified reference materials, which in turn hampers the production of the suite of materials required globally for complete protection of public health. This paper outlines the various options that analysts can pursue in the use of reference materials for internal and external quality control, with a view to optimising the efforts of both reference materials users and reference materials producers. For this purpose, the logical sequence is reviewed from the discovery of a new bioactive compound in shellfish, through initial method development up to regulation for food safety purposes including accepted reference methods. Subsequently, the requirements for and efforts typically spent in the production and characterisation of laboratory reference materials, certified reference materials and other test materials used in inter-laboratory studies or proficiency testing, in the area of marine biotoxins are evaluated. Particular emphasis is put on practical advice for the preparation of in-house reference materials. The intricate link between reference material characterisation and method performance is outlined to give guidance on the appropriate in-house method validation in the rapidly developing field of phycotoxins.

Animals↗
PubMed1998

Detection of microbial pathogens in shellfish with multiplex PCR.

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55 degrees C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelextrade mark 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was </=10(1)-10(2) cells following a "double" multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneCombtrade mark (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneCombtrade mark assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneCombtrade mark DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish.

Animals↗
PubMed2000

Foodborne infections vectored by molluscan shellfish.

Foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5000 deaths each year in the United States. The authors present and analyze information derived from epidemiologic investigations and surveillance systems on foodborne infections caused by consumption of molluscan shellfish. This review focuses on the bias in reporting of shellfish-vectored illness, prevention and control of such infections, the origin of recognized viral and bacterial etiologic agents, and a new potential public health threat of a food-and-waterborne protozoan contaminant, Cryptosporidium parvum.

Animals↗
PubMed1993

Analysis of paralytic shellfish poisoning toxins by automated pre-column oxidation and microcolumn liquid chromatography with fluorescence detection.

Periodate oxidation of the toxins responsible for paralytic shellfish poisoning (PSP) yields fluorescent purines suitable for trace analysis by reversed-phase LC. Mobile phases containing perfluorinated acids, such as heptafluorobutyric acid, as ion-pair agents were found to provide high capacity factors for the oxidized products. Gradient elution on a microbore column with large volume injections and fluorescence detection permitted the detection of femtomole quantities of PSP toxins. A fully automated pre-column oxidation procedure was developed for an LC autosampler system in order to improve precision and allow unattended analyses. The complete method was applied successfully to various samples, including shellfish and toxic phytoplankton.

Autoanalysis↗
PubMed1985

A combination fluorescence assay and Folin-Ciocalteau phenol reagent assay for the detection of paralytic shellfish poisons.

Paralytic shellfish poison (PSP) profiles of crude shellfish extracts were determined by linear gradient elution from a Bio-Rad AG-50-X4 strong cation-exchange resin mini-column. STX, GTX2 and GTX3 were detected by fluorescence assay. NeoSTX and GTX1/GTX4 were detected with a Folin-Ciocalteau phenol reagent assay. The major toxicity associated with extracts of Mytilus edulis and Mya arenaria collected during a 1972 red tide off Hampton, New Hampshire, was due to the presence of GTX1/GTX4, with some activity associated with neoSTX, GTX2 and GTX3. STX was also present. Correlations to mouse toxicity are provided.

Amino Acids↗
PubMed1992

Cross-reacting antigens in the butter clam (Saxidoma giganteus) and their relationship to total paralytic shellfish poison toxicity.

A specific protein with an apparent mol. wt of 23,000 was identified in foot homogenate derived from paralytic shellfish poisoning (PSP) contaminated butter clams and was found to cross-react with crab-saxitoxin-induced protein (SIP) antiserum. Antiserum, once cross-absorbed against non-toxic shellfish material, was incubated with tissue homogenate derived from 52 butter clams with varying total PSP toxicities in a prototype ELISA. A significant (r = 0.83; P less than 0.001) correlation existed between soluble clam antigen content in foot homogenate and total PSP toxicity; the latter measured by the mouse lethality bioassay. From the ELISA results, a soluble antigen threshold of 0.1% total protein was successfully used to distinguish between PSP toxic and non-toxic butter clams. It is proposed that this type of screening assay could be used in conjunction with the standard mouse bioassay to increase PSP monitoring and potentially reduce unnecessary animal testing.

Animals↗
PubMed1993

A unified bioscreen for the detection of diarrhetic shellfish toxins and microcystins in marine and freshwater environments.

Capillary electrophoresis (CE) coupled with liquid chromatography (LC)-linked protein phosphatase (PPase) bioassay was used to detect sensitivity both diarrhetic shellfish toxins and hepatotoxic microcystins in marine and freshwater samples. This procedure provided a quantitative bioscreen for the rapid optical resolution of either of these toxin families in complex mixtures such as cultured marine phytoplankton, contaminated shellfish and cyanobacteria (natural assemblages). Following detection, identified toxins were purified by an enzyme bioassay-guided two-step LC protocol. Using the latter approach, at least four microcystins were rapidly isolated from a cyanobacteria bloom (largely Microcystis aeruginosa) collected from a Canadian drinking-water lake, including a novel microcystin termed microcystin-XR, where X is a previously unidentified hydrophobic amino acid of peptide residue molecular mass 193 Da. The unified CE/LC-linked PPase bioscreen described provides a powerful capability to dissect multiple toxin profiles in marine or freshwater samples contaminated with either okadaic acid or microcystin classes of toxin.

Animals↗
PubMed1994

Effect of cooking on the concentration of toxins associated with paralytic shellfish poison in lobster hepatopancreas.

The hepatopancreases from lobsters (Homarus americanus) obtained from two locations in eastern Canada (Gaspé and Bay of Fundy) were analysed for paralytic shellfish poisons (PSP) before and after the shellfish were cooked by boiling or steaming. Forty-five lobsters from each location were divided into three groups of 15. Two of the groups were boiled or steamed while the third was uncooked for comparison purposes. The hepatopancreases of all lobsters were individually analysed for total PSP toxicity using the standard mouse bioassay procedure. Individual toxins were determined in each sample using a high-performance liquid chromatographic procedure employing pre-chromatographic oxidation of the toxins to form fluorescent derivatives. The results demonstrated that boiling or steaming reduced total toxicity (measured as saxitoxin equivalents per hepatopancreas) by approximately 65% compared to values obtained from raw lobsters. Of the individual toxins studied, saxitoxin decreased by about 60% with both the cooking treatments while gonyautoxins 2 and 3 (combined) decreased by almost 100% in the Gaspé samples and by about 90% in the Fundy samples with the same cooking treatments. Trace amounts of saxitoxin or gonyautoxins 2 and 3 were detected in some samples of tail or claw meat before or after cooking. In vitro boiling of raw hepatopancreas for up to 30 min led to no change in total or individual PSP concentration, indicating that the toxins in cooked lobster are not removed through chemical decomposition but are leached out during the loss of water.

Animals↗
PubMed2004

Disposable immunosensor for the determination of domoic acid in shellfish.

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.

Alkaline Phosphatase↗
PubMed2004

Liquid chromatography/quadrupole time-of-flight mass spectrometry for determination of saxitoxin and decarbamoylsaxitoxin in shellfish.

Saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) were determined by liquid chromatography with quadrupole time-of-flight mass spectrometry (Q-TOF MS). A shellfish tissue was extracted with 0.1 mol/l HCl under ultrasonication, and cleanup of extract was accomplished by solid-phase extraction with a C18 cartridge. Chromatographic separation was carried out on a C18 column (150 mm x 2.1 mm, 3.5 microm) with gradient elution of MeOH-H2O (20:80) containing 0.05% heptafluorobutyric acid and MeOH-H2O (15:85) containing 0.05% acetic acid. The protonated molecule [M + H]+ ions at m/z 257 for dcSTX and 300 for STX were selected in precursor ion scanning for Q-TOF MS in the positive electrospray ionizaion mode. Average recoveries and relative standard deviations, by analyzing samples spiked at a level of 0.1, 0.8 or 1.6 microg/g, were 84-92 and 8-14%, respectively. Identification of the presence of the toxins in shellfish tissues was based on the structural information offered by Q-TOF MS.

Animals↗
PubMed2006

Suitability of tandem-in-time mass spectrometry for polybrominated diphenylether measurement in fish and shellfish samples: comparison with high resolution mass spectrometry.

The first part of the present study focused on the development of an alternative automated sample preparation method for the measurement of selected polybrominated diphenyl ethers (PBDEs) in fish and shellfish. A previously developed automated method has been further optimized and simplified to decrease blank levels and cost. Sorbent and solvent quantities have been significantly reduced without altering extract quality. The second part of the study consisted of comparing tandem-in-time quadrupole ion storage mass spectrometry (QISTMS/MS) to high resolution mass spectrometry (HRMS) for the isotope dilution (ID) measurement step after gas chromatography (GC) separation. Both mass spectrometric methods performed similarly in terms of accuracy but better precision was observed for HRMS. Although better sensitivity can be attained with the high resolution sector instrument, method limits of quantification (mLOQs) were very similar for both approaches as they were dependent on the procedural blanks levels. The mLOQ values ranged between 0.04 and 3.56 ng/g fat, depending on the congener. They allowed the unambiguous identification and quantification of all target analytes, except for BDE-183, in most considered fish extracts. An analytical procedure based on rapid automated sample preparation and QISTMS/MS appeared to be suitable for the measurement of PBDEs in fish and shellfish speciment under quality assurance/quality control (QA/QC) criteria.

Animals↗
PubMed2006

Detailed profiles of 7-O-acyl esters in plankton and shellfish from the Portuguese coast.

In bivalve mollusks from the Portuguese coast contaminated by diarrhetic shellfish poisoning (DSP), most of the parent toxins, okadaic acid (OA) or dinophysistoxin-2 (DTX2), are found esterified, and toxicity assessment is only performed after an alkaline hydrolysis step to recover the parent molecules in their free form. The presence of 7-O-acyl esters with fatty acids (FAs) has already been confirmed previously in Mytilus galloprovincialis and Donax trunculus samples. This paper reports the presence of acyl esters in a wider range of estuarine and offshore bivalve species found by direct analysis in LC-MS. The total of acyl esters found in each species represented the percentages commonly found by hydrolysis in those species in previous years, justifying the majority of the esters commonly found in shellfish. This implies that any diol esters remaining after digestion of toxic microalgae would represent only a minor contribution to the ester's contents. Esters with C14:0, C16:0, C16:1, C20:5 and C22:6 FAs were the most abundant, followed by esters with C18:0, C18:1, C18:2, C18:3 and C18:4. This is the first report of OA and DTX2 esters with odd FAs: C15:0, C17:0, C17:1, and probably a branched FA: iso-C16:0. Esters with iso-C16:0 where found in high percentages particularly in two species of estuarine clams, where they represented 13-34% of total esters found. Esters were also found in plankton, predominantly with C16:0. Total esters in plankton were not higher than 10%, not enough to justify per se the high levels found in bivalves.

Animals↗
PubMed2004

Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR.

A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.

Animals↗
PubMed2005

Real-time RT-PCR for norovirus screening in shellfish.

Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively, were detected in artificially contaminated oysters. Validation of the new method on 150 archived naturally contaminated shellfish confirmed the utility of the genogroup II primer set to detect a large range of different strains circulating in France since 1995, but genogroup I strains were detected infrequently.

Animals↗
PubMed2005

Adenovirus detection in shellfish and urban sewage in Morocco (Casablanca region) by the polymerase chain reaction.

Human enteric viruses are largely excreted in faeces. These resistance of these viruses in the environment makes their faecal-oral transmission easier. Filter feeder organisms such as the mussels are bio-accumulators of viruses contaminating their aquatic environment. Thus, undercooked shellfish consumption involves sanitary risks. Thirty samples of mussels (Mytilus sp.), were tested, half were from an aquaculture origin, the others were from an area more exposed to faecal pollution. Fifteen sewage samples from this last area were also examined. Viruses were extracted from the digestive tissue by direct elution method in a glycine/NaCl pH 9.5 buffer followed by PEG 8000 precipitation. The PEG pellets were used for DNA extraction by proteinase K and phenol/chloroform. The molecular characterization, by PCR using specific adenovirus primers revealed that shellfish growing on Mohammedia (a town in the Casablanca outskirts) littoral are contaminated whereas those chosen from aquaculture and bought in the central market were not contaminated.

Adenoviridae↗
PubMed2006

An RNA extraction protocol for shellfish-borne viruses.

The GPTT virus RNA extraction method, originally developed for extraction of human norovirus and hepatitis A virus RNAs from contaminated shellfish, was evaluated for extraction of RNA from Aichi virus strain A846/88 (AiV), coxsackievirus strains A9 (CAV9) and B5 (CBV5), murine norovirus (strain MNV-1), and the norovirus surrogate, feline calicivirus (FCV) strain KCD, for the purpose of RT-PCR detection within seeded oyster (Crassostrea virginica) extracts. The RT-PCR equivalent sensitivities observed within seeded oysters as compared to virus stocks were 0.68, 6.8, 26, 5.6, and 14.5 RT-PCR(50) units when assaying 10% of total RNA extracted from seeded oyster extracts for CAV9, CBV5, AiV, FCV, and MNV-1, respectively. For oysters exposed to virus-contaminated seawater, the detection equivalent sensitivities observed were 680, 68, 2600, 560, and 14.5 RT-PCR(50) for CAV9, CBV5, AiV and FCV, and MNV-1, respectively. These results indicate that the GPTT method can be used as a general viral RNA extraction method for multiple picornaviruses and caliciviruses that could potentially contaminate shellfish.

Animals↗
PubMed2005

Low-level cadmium exposure in Toyama City and its surroundings in Toyama prefecture, Japan, with references to possible contribution of shellfish intake to increase urinary cadmium levels.

OBJECTIVES: This study was initiated to examine if exposure to cadmium (Cd) was high also outside of the previously identified Itai-itai disease endemic region in the Jinzu River basin in Toyama prefecture in Japan. METHODS: Morning spot urine samples were collected in June-August 2004 from 651 adult women (including 535 never-smokers) in various regions in Toyama prefecture, and subjected to urinalyses for cadmium (Cd), alpha1-microglobulin (alpha1-MG), beta2-microglobulin (beta2-MG), N-acetyl-beta-D-glucosaminidase (NAG), specific gravity (SG or sg) and creatinine (CR or cr). Three months later, the second urine samples were collected from those with elevated Cd in urine (e.g., > or =4 microg/g cr), together with answers to questionnaires on shellfish consumption. RESULTS: The geometric mean (GM) Cd, alpha1-MG, beta2-MG and NAG (after correction for CR) for the total participants were 2.0 microg/g cr, 2.4 mg/g cr, 104 microg/g cr and 2.8 units/g cr, respectively; further analysis with never-smoking cases only did not induce significant changes in these parameters. Analyses of the second urine samples from the high Cd subjects showed that there was substantial decrease (to about a half) in Cd in the 3-month period, and that the decrease was accompanied by reduction in alpha1-MG and NAG (beta2-MG did not show elevation even in the first samples). The urinalysis results in combination with the results of the questionnaire survey suggest that the high urinary Cd was temporary and might be induced by intake of shellfish that is edible whole. CONCLUSIONS: The overall findings appear to suggest that Cd exposure in Toyama populations (outside of the Itai-itai disease endemic region) was at the levels commonly observed on the coast of the Sea of Japan, and that the Cd level in urine might be modified by the intake of some types of seafood. Further studies are necessary to elucidate the relation of urinary Cd with seafood intake.

Acetylglucosaminidase↗