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PubMed2006

Detection system of Cryptosporidium parvum oocysts by brackish water benthic shellfish (Corbicula japonica) as a biological indicator in river water.

The brackish water benthic shellfish, Corbicula japonica, was experimentally exposed to Cryptosporidium parvum oocysts at 1.51x10(4)oocysts/clam/day for 7 or 14 days. Oocysts were predominantly eliminated through the feces of Corbicula japonica in both cases by microscopic and PCR methods. The fecal excretion rates of oocysts within 4 days after the last exposure to Corbicula japonica were 87.6% for the 7-day exposure group and 86.0% for the 14-day exposure group. The tissue residue level of oocysts in the gastrointestinal tract 3 days after the last exposure was 2.7% of total exposed oocysts and that of 7 days was 1.1% for the 7-day exposure case and 1.6 and 0.5% for the 14-day exposure case, respectively, maintaining infectivity to cultured cells (HCT-8) in vitro. At the same time, field tests of Corbicula japonica for collecting oocysts showed that this clam could certainly collect Cryptosporidium parvum oocysts in the natural river and, furthermore, the gene type of C. parvum could be also identified proving its effectiveness as a biological indicator. The present study showed that the brackish water benthic shellfish Corbicula japonica may be capable of gathering and preserving Cryptosporidium parvum oocysts to a considerable extent under the natural ecological conditions, and further suggests the effectiveness of Corbicula japonica as a practical and general bioindicator for estimates of river water contamination by oocysts of Cryptosporidium parvum.

Animals↗
PubMed2004

Analysis of the importance of lipid breakdown for elimination of okadaic acid (diarrhetic shellfish toxin) in mussels, Mytilus edulis: results from a field study and a laboratory experiment.

Okadaic acid (OA) is a lipophilic phycotoxin, which accumulates in the digestive organs of mussels and may cause diarrhetic shellfish poisoning (DSP) in humans. Depuration of toxic mussels is a potential option for the shellfish industry to increase the availability of marketable mussels. To develop cost-effective depuration methods for DSP toxins, knowledge about the environmental conditions and physiological processes regulating the rate of depuration is essential. In this paper, the importance of lipid breakdown for elimination of OA in mussels was investigated by performing a field study and a manipulative laboratory experiment. First, total lipid content and concurrent concentration of OA in the digestive glands of farmed blue mussels, Mytilus edulis, was analysed on a monthly basis from January to June 2000. A significant positive correlation between levels of OA and lipid content was observed between January and March, when lipid levels were showing a decreasing trend. This supported a previously proposed model that breakdown of lipid stores may affect the release and elimination of this lipophilic toxin. To test this causal model, a laboratory experiment was performed. Mussels containing OA were exposed to experimental treatments (increased seawater temperature and/or food limitation) for 24 days in order to increase the energy requirements and need to use lipids as an energy source. It was predicted that mussels exposed to these treatments would have a faster elimination rate of OA compared to feeding mussels kept in ambient seawater temperature. The results showed that lipid content was significantly reduced in mussels exposed to an increased water temperature (24 degrees C) compared to ambient temperature (18 degrees C). The amount of lipids was not affected by food limitation. Although lipid content was reduced in 24 degrees C, the rate of depuration of OA was not faster for mussels in this treatment and no correlation was detected between lipid content and OA. Depuration rates were very similar for all treatments and followed an exponential decrease relationship (t(1/2) = 8 days). Thus, the proposed model that lipid breakdown affects the mechanism of elimination of OA was not supported. Nevertheless, the observed rates of depuration provide useful information and a potential predictive tool for large-scale depuration methods of mussels. The difficulties to influence the rate of depuration of this toxin by changing the environmental conditions suggest that processes, insensitive to short-term manipulation of the external environment, regulate depuration of OA.

Analysis of Variance↗
PubMed2005

PCBs and organochlorine pesticides (OCPs) in edible fish and shellfish from China.

A wide variety of fish and shellfish were collected from local supermarkets of Dalian, Tianjin and Shanghai in China and analyzed for the concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) such as hexachlorobenzene (HCB), hexachlorocyclohexane isomers (HCHs), chlordane compounds (CHLs, including trans-chlordane, cis-chlordane, trans-nonachlor and cis-nonachlor) and dichlorodiphenyltrichloroethane (DDT) and its metabolites (DDTs, including o,p'-DDE, p,p'-DDE, o,p'-DDD, p,p'-DDD, o,p'-DDT and p,p'-DDT). Concentrations of PCBs in samples collected from Dalian, Tianjin and Shanghai ranged from 1.11 to 8.04 ng/g, 1.26 to 5.60 ng/g and 0.83 to 11.4 ng/g on wet weight basis, respectively. The concentrations were lower compared to those in developed countries such as Japan and Italy. Average concentrations of HCB, HCHs, CHLs and DDTs were 0.38, 0.92, 0.47 and 28.9 ng/g on wet weight basis, respectively. The daily intake of PCBs and organochlorine pesticides ingested by people living in these cities in China through fish and shellfish was estimated and compared with those observed in other areas.

Animals↗
PubMed2004

Liquid chromatography--multiple tandem mass spectrometry for the determination of ten azaspiracids, including hydroxyl analogues in shellfish.

Azaspiracids (AZAs) are a group of polyether toxins that cause food poisoning in humans. These toxins, produced by marine dinoflagellates, accumulate in filter-feeding shellfish, especially mussels. Sensitive liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS(n)) methods have been developed for the determination of the major AZAs and their hydroxyl analogues. These methods, utilising both chromatographic and mass resolution, were applied for the determination of 10 AZAs in mussels (Mytilus edulis). An optimised isocratic reversed phase method (3 microm Luna-2 C18 column) separated 10 azaspiracids using acetonitrile/water (46:54, v/v) containing 0.05% trifluoroacetic acid (TFA) and 0.004% ammonium acetate in 55 min. Analyte determination using MS3 involved trapping and fragmentation of the [M + H]+ and [M + H - H2O]+ ions with detection of the [M + H - 2H2O]+ ion for each AZA. Linear calibrations were obtained for AZA1, using spiked shellfish extracts, in the range 0.05-1.00 microg/ml (r2 = 0.997) with a detection limit of 5 pg (signal : noise = 3). The major fragmentation pathways in hydroxylated azaspiracids were elucidated using hydrogen/deuterium (H/D) exchange experiments. An LC-MS3 method was developed using unique parent ions and product ions, [M + H - H2O - CgH10O2R1R3]+, that involved fragmentation of the A-ring. This facilitated the discrimination between 10 azapiracids, AZA1-10. Thus, this rapid LC-MS3 method did not require complete chromatographic resolution and the run-time of 7 min had detection limits better than 20 pg for each toxin.

Animals↗
PubMed2005

Confirmation of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish as their anthrylmethyl derivatives using UV radiation.

A rapid and simple method for confirmation of the diarrhetic shellfish poisons (DSP): okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) using fluorescence detection following derivatization with 9-chloromethylanthracene, has been established as an alternate to LC/MS. Exposure of the anthrylmethyl derivatives of OA, DTX-1 and DTX-2 to near UV light (300-400 nm) resulted in the loss of these compounds to below detection limits within 30 min, with a concurrent appearance of two additional compounds. Based on the mass spectral evidence, we propose that these newly formed compounds are the decarboxylation products of the derivatized diarrhetic shellfish poisons. UV radiation is, therefore, proposed as a rapid and simple confirmation technique for these DSP in mussel samples.

Animals↗
PubMed2006

Nutrient dynamics at the sediment-water interface in a Mediterranean lagoon (Thau, France): influence of biodeposition by shellfish farming activities.

The Thau Lagoon, a French Mediterranean shallow lagoon, is a site where extensive shellfish farming occurs. The aim of the present work is to evaluate the role of this activity on nutrient exchange at the sediment-water interface in relation to organic matter (OM) sedimentation and degradation. Two stations inside (C5) and outside (C4) of the shellfish farming areas were sampled at three seasons. Porewater chemistry surveys and calculated diffusive fluxes were used to evaluate the trophic status of the Thau lagoon. Quantitative (Particulate Organic Carbon) as well as qualitative OM (Hydrogen Index, Carbohydrates) analyses were performed on sediments to assess OM characteristics. Results emphasized that surficial sediments at C5 are always more enriched in OM. Porewater nutrient concentrations are 10-20 times higher at C5 than at C4. In June 2003, the porewater profiles exhibit a sharp gradient at the bottom waters, indicating a hypereutrophic status, leading to an anoxic crisis.

Aquaculture↗
PubMed2005

The detection of Cryptosporidium parvum and Escherichia coli O157 in UK bivalve shellfish.

Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12-34% in mussels, 48-69.5% in oysters and 30-65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 degrees C. Increasing enrichment temperatures from 37 to 42 degrees C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.

Animals↗
PubMed2004

Paralytic shellfish poisoning toxins in green mussels (Perna viridis) from the Gulf of Paria, Trinidad.

Paralytic shellfish poisoning (PSP) toxins were determined in green mussels (Perna viridis) collected from one collection site in the Gulf of Paria in Trinidad in 1999 and 2000. Aqueous extracts of PSP were purified by passage through C-18 SPE cartridges, oxidized with peroxide and periodate, respectively, then analyzed by HPLC with fluorescence detection. This procedure provided rapid and highly sensitive screening of samples for PSP toxins. Further purification of PSP-containing extracts using COOH SPE cartridges resulted in the separation and identification of individual PSP toxins. The method of analysis was validated by spike and recovery experiments, with 85-103% recoveries of mixed toxins. PSP toxins determined in our samples in both years were GTX2,3, dcGTX2,3, STX, and dcSTX, while GTX1,4 and NeoSTX were only identified in 1999 and 2000, respectively. In 1999, GTX1,4, GTX2,3 and dcGTX2,3 predominated, as compared to NeoSTX, GTX2,3 and dcGTX2,3 in 2000. However, mussel samples in 2000 contained higher total concentrations of detected PSP toxins than those of 1999. These results represent the first identification of specific PSP toxins in local shellfish and provide a basis for effective monitoring and control of these toxins in Trinidad.

Animals↗
PubMed1997

Optimization of sample cleanup procedure for determination of diarrhoeic shellfish poisoning toxins by use of experimental design.

In routine monitoring of diarrhoeic shellfish poison (dinophysistoxin-1 and okadaic acid) there appeared to be an inconsistency between the mouse bioassay and existing chemical analysis based on liquid chromatography. The sample cleanup procedure has been subject to minor modifications in an effort to overcome the problem. However, further studies have appeared necessary and in this study all experimental factors that can influence the sample cleanup using solid-phase extraction columns prior to the LC analysis have been evaluated by use of experimental statistical design in order to understand the effect of the various factors and to optimize the conditions for recovery of the toxins. Based on our experiments we suggest using a solid-phase extraction silica column of 100 mg in the sample cleanup procedure and washing solvents composed of dichloromethane instead of chloroform to minimize the effect of stabilizing alcohol. It is sufficient to apply 7.5 ml hexane-dichloromethane (1:1) to the column in the first washing step and 2.5 ml of dichloromethane in the final washing. Elution is complete by use of 2.5 ml chloroform with 3.5% methanol. Toxic shellfish tested by this procedure confirmed the mouse bioassay.

Animals↗
PubMed1999

Capillary electrophoresis with diode array detection as an alternative analytical method for paralytic and amnesic shellfish toxins.

In recent years the marine environment has been seriously damaged by the presence of several toxic phytoplanktonic species, such as dinoflagellates and other toxic algae, which contaminate shellfish and other marine products. Amnesic and paralytic shellfish toxins are examples of these contaminants. The search for sensitive methodologies for the analysis of such compounds is one of the aims of researchers working in the marine environment. High-performance liquid chromatographic methods have been used for this purpose, allowing the detection of very low levels of these toxins. Recently, capillary electrophoresis (CE) has been used as an alternative for the separation and analysis of these compounds. In this paper, we report the optimization of CE procedures for their analysis. Due to the complexity of the matrix, clean-up procedures are required for removing interferences which affect the electrophoretic resolution. The influences of electrophoretic parameters such as voltage, buffer concentrations and organic modifiers, were studied in order to optimize the electrophoretic system to achieve high resolution as well as an accurate quantitation. Extraction and other steps such as clean-up of samples prior to the electrophoretic analysis have been also studied. Different buffers and organic modifiers were used in order to improve the separation of the toxic components, and consequently to obtain accurate quantitative information about the amount of toxins present in the contaminated samples.

Amnesia↗
PubMed1999

Separation of paralytic shellfish poisoning toxins on Chromarods-SIII by thin-layer chromatography with the Iatroscan (mark 5) and flame thermionic detection.

Thin-layer chromatography (TLC) on Chromarods-SIII with the Iatroscan (Mark-5) and a flame thermionic detector (FTID) was used to develop a rapid method for the detection of paralytic shellfish poisoning (PSP) toxins. The effect of variation in hydrogen (H2) flow, air flow, scan time and detector current on the FTID peak response for both phosphatidylcholine (PC) and PSP were studied in order to define optimum detection conditions. A combination of hydrogen and air flow-rates of 50 ml/min and 1.5-2.0 l/min respectively, along with a scan time of 40 s/rod and detector current of 3.0 A (ampere) or above were found to yield the best results for the detection of PSP compounds. Increasing the detector current level to as high as 3.3 A gave about 130 times more FTID response than did flame ionization detection (FID), for PSP components. Quantities of standards as small as 1 ng neosaxitoxin (NEO), 5 ng saxitoxin (STX), 5 ng B1-toxins (B1), 2 ng gonyautoxin (GTX) 2/3, 6 ng GTX 1/4 and 6 ng C-toxins (C1/C2) could be detected with the FTID. The method detection limits for toxic shellfish tissues using the FTID were 0.4, 2.1, 0.8 and 2.5 micrograms per g tissue for GTX 2/3, STX, NEO and C toxins, respectively. The FTID response increased with increasing detector current and with increasing the scan time. Increasing hydrogen and air flow-rates resulted in decreasing sensitivity within defined limits. Numerous solvent systems were tested, and, solvent consisting of chloroform: methanol-water-acetic acid (30:50:8:2) could separate C toxins from GTX, which eluted ahead of NEO and STX. Accordingly, TLC/FTID with the Iatroscan (Mark-5) seems to be a promising, relatively inexpensive and rapid method of screening plant and animal tissues for PSP toxins.

Calibration↗
PubMed2002

Fate of paralytic shellfish poisoning toxins in purple clam Hiatula rostrata, in outdoor culture and laboratory culture.

Purple clams (Hiatula rostrata Lighttoot) accumulate paralytic shellfish poisoning (PSP) toxins produced by a toxic strain of the dinoflagellate Alexandriun minutum Halim. The results confirm the data of our previous study concerning the muscle and siphon that were not showing a gradual rise in toxicity when shellfish accumulated more A. minutum. However, muscle and siphon are intermittently toxic both in exposure and depuration period in laboratory cultured purple clams. PSP toxins were detected in outdoor cultured purple clams, whereas no A. minutum were found in the culture pond during most of the survey time. The outdoor cultured purple clams need longer time to decrease toxicity to allowable levels than laboratory cultured purple clams. It was shown that laboratory data may not predict times over which pond-cultured purple clams may prove toxic to consumers.

Adaptation, Physiological↗
PubMed1997

Occurrence of paralytic shellfish poison in Bangladeshi freshwater puffers.

Two species of freshwater puffer fish, Tetraodon cutcutia and Chelonodon patoca, collected from several locations in Bangladesh, showed lethal potency in mice ranging from 2.0 to 40.0 MU/g tissue as paralytic shellfish poison. In both species, toxicity of the skin was generally higher than the other tissues examined (muscle, liver and ovary). Water-soluble toxins from T. cutcutia were partially purified by activated charcoal treatment followed by column chromatographies using Bio-Gel P-2 and Bio-Rex 70. Analyses by cellulose acetate membrane electrophoresis and high-performance liquid chromatography with fluorometric detection demonstrated that the toxins were composed of saxitoxin, decarbamoylsaxitoxin, gonyautoxins 2 and 3, decarbamoylgonyautoxins 2 and 3, and three unidentified components which are possibly related to paralytic shellfish poison.

Animals↗
PubMed1997

Detection of genotoxicity of polluted sea water using shellfish and the alkaline single-cell gel electrophoresis (SCE) assay: a preliminary study.

We exposed two species of shellfish, Patunopecten yessoensis and Tapes japonica, for 4 h to artificial sea water in which N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl nitrosourea (EMS), 3-chloro-4-dichloromethyl-5-hydroxy-2(H)-furanone (MX), or benzo[a]pyrene (B[a]P) were dissolved. We then assessed the DNA damage in cells isolated from the gills using the alkaline single-cell gel electrophoresis (SCG) assay. A statistically significant increase in DNA damage was observed for all exposures. Therefore, the alkaline SCG assay detected DNA damage in gill cells produced by direct mutagens and promutagen dissolved in sea water. T. japonica was exposed to sea water sampled from two Pacific Ocean coasts of Japanese local cities--Hachinohe (Aomori Prefecture, Tohoku) and Nakatsu (Oita Prefecture, Kyushu)--and three bay coasts of the industrial megalopolises--Tokyo, Osaka, and Kobe. A significant increase in DNA damage was observed after the exposure to sea water from Tokyo, Osaka, and Kobe, but not from Hachinohe and Nakatsu. These results suggested the utility of the alkaline SCG assay with shellfish gill cells for monitoring sea water genotoxicity.

Animals↗
PubMed2001

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study.

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.

Animals↗
PubMed1991

Diversity of shellfish toxins of "diarrhetic" type revealed by biological and chemical assays.

Extracts of "diarrhetic" shellfish toxins from the edible mussel (Mytilus edulis) were tested with biological and chemical assays to determine toxin content. When tested with the standard mouse bioassay, a higher toxicity was detected in most samples compared to that revealed from detection of the diarrhea-causing substances okadaic acid and dinophysistoxin-1 by high-performance liquid chromatography. Routine extraction of toxins for the two assays was carried out with two different solvents, acetone versus aqueous methanol. Accordingly, we questioned whether the variation in results between the methods could be due to differences in chemical properties of these two solvents. When tested, the two solvent systems showed practically the same efficiency concerning the extraction of okadaic acid and dinophysistoxin-1. This demonstrated that toxins other than those causing diarrhea were present in the samples, and that the mouse bioassay was sensitive to these additional toxins. Subsequent testing of the samples with the mouse bioassay, employing both acetone and methanol extracts, revealed that at least two classes of toxins were present in the mussel samples in addition to okadaic acid and dinophysistoxin-1. It is unclear whether the shellfish toxins revealed in this study are partially from known, nondiarrhetic types, such as pectenotoxins or yessotoxins, or from unknown toxin groups exhibiting ichthyotoxic and hemolytic properties.

Animals↗
PubMed2005

Development of the fecal coliform total maximum daily load using Loading Simulation Program C++ and tidal prism model in estuarine shellfish growing areas: a case study in the Nassawadox coastal embayment, Virginia.

In this study, a linked model system including the Loading Simulation Program C++ (LSPC) and the tidal prism water quality model (TPWQM) was proposed as an alternative tool for total maximum daily load (TMDL) studies. The feasibility of the model system was tested by a case study in the Nassawadox Creek, a Virginia tidal water shellfish growing area. The watershed model, driven by hourly precipitation, simulates hydrology and fecal coliform accumulation and transport processes in the watershed. The simulated surface runoff and subsurface flow as well as fecal coliform loads from the watershed are discharged to the tidal creek. The tidal prism model simulates fecal coliform transport in the Creek. The model results demonstrate the effectiveness in simulating hydrology and fecal coliform concentration in the watershed and its embayment. A series of sensitivity runs was conducted to estimate the load reduction necessary for fecal coliform concentration to meet the water quality standards. The model application to the Nassawadox Creek indicates that the model system is useful in developing fecal coliform TMDLs for estuarine shellfish growing areas.

Animals↗
PubMed1980

Reduction of lipogenic enzymes by shellfish triglycerides in rat liver.

When rats were fed for 2 weeks on 3% fat diets containing 0.5 or 1%corbicula (Corbicula japonica PRIME), clam (Tapes japonica) or oyster (Callocorchina) triglycerides, serum and liver triglyceride levels were significantly lowered. The activities of hepatic glucose-6-phosphate dehydrogenase, malic enzyme and acetyl-CoA carboxylase were markedly reduced in the rats. Cholesterol synthesis by liver slices was also reduced. The results of immunochemical titrations and Ouchterlony double-diffusion analysis indicated that the decreases in the activities of acetyl-CoA carboxylase and glucose-6-phosphate dehydrogenase were due to decreases in the enzyme quantities. The shellfish triglycerides include a high percentage of long chain and polyunsaturated fatty acids, which are common to and characteristic of the three kinds of shellfish. They would be effective components in these observations.

Acetyl-CoA Carboxylase↗