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PubMed1992

Phycotoxins in seafood--toxicological and chromatographic aspects.

Two typical clinical types of algae-related seafood poisoning have attracted medical and scientific attention: paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP). Therefore, it became necessary to establish methods for the evaluation of possible hazards caused by contamination of seafood with these phycotoxins. Bioassays with mice or rats are the common methods for the determination of the toxin content of seafood. However, biological tests are not completely satisfactory because of a lack of sensitivity and pronounced variations. Additionally, there is growing opposition against animal testing. Therefore, many efforts have been undertaken to determine phycotoxins by chromatographic methods. PSP determination is mainly based on high-performance liquid chromatographic (HPLC) separation by ion-pair chromatography followed by postcolumn oxidation of the underivatized toxins in alkaline solution and fluorescence detection. HPLC methods for the determination of the DSP toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are characterized by precolumn derivatization with 9-anthryldiazomethane (ADAM) and/or 4-bromomethyl-7-methoxycoumarin (Br-Mmc), followed by chromatographic separation of the DSP esters formed and fluorescence detection. The chromatographic methods discussed in this review allow the rapid, sensitive and non-ambiguous determination of individual species of the two most important phycotoxins in seafood, PSP and DSP.

Animals↗

PubMed1988

Protogonyaulax cohorticula, a toxic dinoflagellate found in the Gulf of Thailand.

Two clones of Protogonyaulax cohorticula were isolated from the Gulf of Thailand. The extracts of these clones killed mice with typical signs of paralytic shellfish poisoning. The toxicities corresponded to those of strongly toxic clones of P. tamarensis. In the HPLC and electrophoretic analyses, gonyautoxins and saxitoxin were detected. About 80% of the toxins consisted of gonyautoxin I. These results show that P. cohorticula is a toxic species of Protogonyaulax and that it is at least one of the causative organisms of paralytic shellfish poisoning in Thailand.

Animals↗

PubMed1994

Comparative study of the stability of saxitoxin and neosaxitoxin in acidic solutions and lyophilized samples.

Paralytic shellfish poison (PSP) has historically been a problem for the shellfish industry. In order to prevent the marketing of contaminated seafood products, governments have implemented monitoring programs where standards of toxins are necessary. The stability of these standard toxins is very important. In this paper we analysed the stability of saxitoxin (STX) and neosaxitoxin in acidic solution and lyophilized samples. Individual toxins were determined in each sample using a high-performance liquid chromatographic procedure employing post-column oxidation of the toxins to form fluorescent derivatives. Our results demonstrate that STX is very stable in solution samples and could be adopted as a reference standard. This toxin can be kept in dilute acidic solutions for 18 months without loss of potency. However, neosaxitoxin is unstable, possibly due to transformation to other toxins.

Animals↗

PubMed1986

Transfer of environmental plutonium and americium across the human gut.

Data on gut transfer factors for environmental forms of radionuclides are essential for estimates of public radiation exposures following ingestion, and thus in decisions on controlling waste discharges. Dose estimates for transuranic nuclides are particularly sensitive to uncertainties stemming from gut transfer data being related to non-environmental forms and/or derived from animal experiments. We have measured human gut transfer factors for plutonium and americium in two experiments using marine foods obtained near Sellafield, Cumbria. Firstly, the urine of volunteer members of the critical group of shellfish consumers was analysed for transuranics and the results related to their consumption rates. Secondly, remotely-based volunteers ate single quantities of shellfish obtained near Sellafield, and their urine was analysed. An overall result for the gut transfer factor for environmental plutonium of 0.8 X 10(-4) indicates no need to increase the value of 1 X 10(-4), currently used by the International Commission on Radiological Protection (ICRP) for soluble forms. Results for americium show that the ICRP value of 5 X 10(-4) is maximising, and that a value of 1 X 10(-4) would be supportable. The results from the study of critical group members provide confidence in our habits survey techniques and reassurance that there are no significant pathways for intake of transuranics by these people that have not been recognised.

Adult↗

PubMed1991

Incidence of Listeria species in seafood and seafood salads.

A total of 128 samples of seafood on the Icelandic market were tested for the presence of Listeria monocytogenes and other Listeria species. The samples included raw, smoked and dried fish, frozen shellfish and shrimps as well as several fish salads. These products are generally consumed without heating. Listeria spp. were present in 56% of the samples of raw fish, 29% of the smoked fish, 9% of the shrimps and 32% of the salads. No Listeria spp. were present in the shellfish or dried fish. In 46% of the positive samples L. monocytogenes could be demonstrated, either alone or together with L. innocua. The other positive samples contained L. innocua and, in one sample, L. welshimeri. All products sampled had been processed and packed in Iceland, mostly for use on the domestic market. It is suggested that consuming certain fish products and fish salads may form an additional risk factor for listeriosis in humans.

Animals↗

PubMed1995

Comparison of seven selective media for the isolation of mesophilic Aeromonas species in fish and meat.

Seven selective agar media and two enrichment broths were evaluated for their suitability for the isolation of mesophilic Aeromonas species from meat, fish, and shellfish samples. In a first trial, aeromonads were inoculated in fish and meat samples and reisolated using all selected media. For qualitative isolation, enrichment in alkaline peptone water (pH 8.7 +/- 0.1) at 28 degrees C and subsequent plating onto sheep blood agar supplemented with 30 mg/L ampicillin (ASBA 30) and bile salts-irgasan-brilliant green agar (BIBG) at 35 degrees C led to the best results. For quantitative assays, direct plating on the same agar media is recommended. In a second trial, 829 meat, fish, and shellfish samples were investigated with the same methods. The results show that BIBG is the most selective medium and that presumptive identification of aeromonads on ASBA 30 is very easy. Finally, we could confirm the opinion of other workers that optimal recovery of mesophilic Aeromonas spp. requires the use of more than one agar medium.

Aeromonas↗

PubMed1994

A competitive enzyme-linked immunoassay for domoic acid determination in human body fluids.

A polyclonal antiserum was raised in mice against domoic acid. Two of three immunogens consisted of domoic acid coupled to ovalbumin (OVA) and keyhole limpet haemocyanin at molar ratios of 47:1 and 44:1, respectively using a carbodiimide reaction. Titres of both antisera exceeded 1/35,000 against domoic acid coupled to the non-relevant carrier. Domoic acid was also conjugated to bovine serum albumin at a molar ratio of 30:1 using N-hydroxysuccinimidyl-4-azidobenzoate, a photoreactive compound. This immunogen, however, produced no measurable serum titres against domoic acid. The antiserum produced against the OVA conjugate displayed the highest affinity for free domoic acid in competitive enzyme-linked immunosorbent assay (ELISA). Furthermore, this antiserum preparation did not significantly cross-react with glutamic acid, aspartic acid, the structural analogue kainic acid, or the paralytic shellfish toxin, saxitoxin. The competitive ELISA was used to quantify domoic acid concentrations in human body fluids spiked with pure domoate. The lower limits of accurate domoic acid determinations in competitive ELISA were 0.2 micrograms/ml in urine, 0.25 micrograms/ml in plasma and 10 micrograms/ml in milk. It was concluded that the competitive ELISA described herein could be used to quantitate directly the concentration of domoic acid in the body fluids of individuals with amnesic shellfish poisoning.

Animals↗

PubMed2004

Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions.

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Determination of domoic acid by on-line coupled capillary isotachophoresis with capillary zone electrophoresis.

An on-line coupled capillary isotachophoresis--capillary zone electrophoresis (cITP-CZE) method for the determination of domoic acid in shellfish and algae is described. The optimised cITP-CZE electrolyte system was 10 mM HCl + 20 mM beta-alanine (BALA) + 0.05% hydroxyethylcellulose (leading electrolyte), 5 mM caproic acid (terminating electrolyte) and 20 mM caproic acid + 20 mM BALA + 0.1% HPMC (background electrolyte). A clear separation of the domoic acid from the other components of methanolic sample extract was achieved within 25 min. Method characteristics, i.e., linearity (0-200 microg/l), accuracy (recovery 101+/-3%), intra-assay repeatability (2.4%) and detection limit (1.5 microg/l) were determined. Speed of analysis, low laboriousness, high sensitivity and low running cost are the typical attributes of the cITP-CZE method. Developed method was successfully applied to analysis of shellfish samples and food supplements containing algae extract.

Calibration↗

PubMed2005

Heavy metal and Pb isotopic compositions of aquatic organisms in the Pearl River Estuary, South China.

The accumulation of trace metals in aquatic organisms may lead to serious health problems through the food chain. The present research project aims to study the accumulation and potential sources of trace metals in aquatic organisms of the Pearl River Estuary (PRE). Four groups of aquatic organisms, including fish, crab, shrimp, and shellfish, were collected in the PRE for trace metal and Pb isotopic analyses. The trace metal concentrations in the aquatic organism samples ranged from 0.01 to 2.10 mg/kg Cd, 0.02 to 4.33 mg/kg Co, 0.08 to 4.27 mg/kg Cr, 0.15 to 77.8 mg/kg Cu, 0.17 to 31.0 mg/kg Ni, 0.04 to 30.7 mg/kg Pb, and 8.78 to 86.3 mg/kg Zn (wet weight). High concentrations of Cd were found in crab, shrimp and shellfish samples, while high concentration of Pb was found in fish. In comparison with the baseline reference values in other parts of the world, fish in the PRE had the highest elevated trace metals. The results of Pb isotopic compositions indicated that the bioaccumulation of Pb in fish come from a wide variety of food sources and/or exposure pathways, particularly the anthropogenic inputs.

Animals↗

PubMed2005

Resistance of hepatitis A virus in mussels subjected to different domestic cookings.

Hepatitis A is a worldwide infectious disease. Shellfish consumption has always been one of the major risk factors for hepatitis A infection, especially when these products are eaten raw or slightly cooked. Moreover, the cooking does not always guarantee the harmlessness of shellfish. The aim of the present study was to evaluate the hepatitis A virus (HAV) resistance in experimentally contaminated mussels, subjected to domestic cooking. Three different domestic preparations (mussel hors-d'oevre, mussel au gratin, mussels with tomato sauce) were performed according to the traditional Italian cookery using different time and temperature conditions. To detect HAV-RNA, RT-nested-PCR was used; the presence of the infectious virus in the positive samples was confirmed by an integrated cell culture-RT-PCR method. The infectious virus was completely inactivated only in "mussels in tomato sauce", while it was still present, even if not quantitatively determinable, in the other preparations. The study confirmed that some factors can influence the HAV sensitivity to thermal inactivation preventing a complete decontamination of the product.

Animals↗

PubMed2004

First report of saxitoxin in octopi.

We report for the first time, the presence of saxitoxin (STX) in a common cephalopod, Octopus (Abdopus) sp. 5, collected from Cooke Point on the northern coastline of Western Australia. Sodium channel and saxiphilin based radio-receptor assays detected saxitoxin-like binding in octopi extracts. Further analysis by liquid chromatography-fluorescence detection (LC-FLD) identified STX as the major contributing toxin in these samples. The presence of STX was confirmed by LC-mass spectrometry and comparison of fragmentation patterns with an authentic STX standard. LC-FLD quantitation and conversion of the Octopus sp. 5 extracts revealed toxin concentrations as high as 246 microg STX/100g tissue, more than three times the US, European and Australian regulatory limit for human consumption of shellfish of 80 microg STX/100g tissue. There was no evidence of tetrodotoxin or other paralytic shellfish toxin derivatives. This level and distribution of STX in octopi poses a potential public health risk, particularly when routine toxin screening of wild catch is not regulated.

Animals↗

PubMed2004

Cadmium in oysters and scallops: the BC experience.

Health effects of non-occupational lifetime exposure to cadmium (Cd) are of growing concern worldwide. This overview provides some context for the current situation in coastal British Columbia, Canada, which arose in 1999 from the discovery of problematic residues of Cd in farmed Pacific oysters (Crassostrea gigas). Efforts are underway to define Cd sources and the geographical and seasonal variation of these Cd residues. The recent application by the European Community of a 1 microg Cd/g (wet weight) import limit to bivalve molluscs and the current deliberation by CODEX to adopt the same value, pose significant threats to the shellfish export trade in the Pacific Northwest (British Columbia, Washington and Alaska), where natural oceanographic conditions and coastal geology contribute to levels of Cd that usually exceed the 1 ppm limit. Human health aspects of chronic Cd exposure comprise an active field of study (this Symposium) and the validity of existing Provisional Tolerable Weekly Intake is being questioned. Bioavailability of Cd from the oyster and scallop matrix is unknown and requires study. Ramifications of this uncertainty may include damage to public perception of the safety of the cultured shellfish product, loss of export market and general undermining of an industry being encouraged by both the Province of British Columbia and Federal aquaculture initiatives. There is therefore a pressing need to redefine what the "safe" limit of lifetime Cd intake is from all sources, and determine bioavailability, specifically from bivalve molluscs. Such information would facilitate the definition of scientifically defensible Cd limits by CODEX.

Animals↗

PubMed2006

Levels of detection of Cryptosporidium oocysts in mussels (Mytilus galloprovincialis) by IFA and PCR methods.

Cryptosporidium spp. are monoxenous protozoan parasites that cause gastrointestinal diseases in humans and animals. Shellfish harvesting areas can become contaminated by the infectious stage of the parasite and humans are therefore at risk of infection either by consumption of shellfish, or by taking part in recreational activities in these areas. In the present study we determined the levels of detection, by IFA and PCR techniques, of Cryptosporidium oocysts in mussels experimentally contaminated with a theoretical number of oocysts. There was a significant correlation between the results obtained by both techniques (P<0.05). IFA and PCR were also applied to a total of 222 samples of mussels (Mytilus galloprovincialis) destined for human consumption. In the naturally contaminated samples, we detected a 31.1% of contamination and only Cryptosporidium parvum (previously denominated C. parvum genotype II) was identified.

Animals↗

PubMed2004

Influence of inter-annual variations in climatic factors on fecal coliform levels in Mississippi Sound.

Information on the effects of inter-annual variations in climatic factors on fecal coliform levels in coastal waters is scarce. We used 11 years (1990-2001) of rainfall, water temperature, Pearl River stage and salinity data to assess the effects of the 1991-1992 and 1997-1998 El Nino events on fecal coliform levels in Mississippi Sound. El Nino-Southern Oscillation had major effects on Pearl River stage and water quality in the Sound. The geometric mean fecal coliform number differed among years (P = 0.0001), being highest during 1991-1992 El Nino years (14.22 MPN per 100 ml) and lowest during 1999-2000 La Nina years (<1.8 MPN per 100 ml). Mean salinity varied among years (P = 0.0001) from 9 ppt (1991-1992) to 21 ppt (1999-2000). Mean water temperature was lowest in 1997-1998 (14.5 degrees C) and highest in 1998-2000 (19.4 degrees C). Pearl River stage differed among years (P = 0.0001), ranging from 1.96 m (1999-2000) to 3.57 m (1997-1998). Inverse relationships were observed between fecal coliform levels and salinity (r(2) = 0.74; P = 0.001) and water temperature (r(2) = 0.69; P=0.001), whereas positive relationships were obtained with total rainfall (r(2) = 0.52; P = 0.013) and Pearl River stage (r(2) = 0.90; P = 0.0001). These relationships are useful for evaluating the potential effects of climate change on water quality and classification of shellfish growing waters in order to protect humans from consuming contaminated shellfish in shallow river-influenced estuaries.

Animals↗

PubMed2000

Dinophysistoxin-2: a rare diarrhoeic toxin associated with Dinophysis acuta.

Okadaic acid and dinophysistoxin-2 have been found yearly in Portuguese shellfish. Their presence was correlated with the occurrence of Dinophysis spp: Dinophysis acuminata has until now been found to be responsible only for OA contamination, while Dinophysis acuta contributes with OA and the rare diarrhetic toxin DTX2. Differences in toxicity levels may reflect different cell toxicities and different non-toxic phytoplankton availability as food source to shellfish.

Animals↗

PubMed2001

The repeatability of two HPLC methods and the PP2A assay in the quantification of diarrhetic toxins in blue mussels (Mytilus edulis).

Repeatability in the quantification of diarrhetic shellfish toxins was investigated for two fluorometric HPLC methods and a colorimetric PP2A assay, using agreement analysis. Blue mussels (Mytilus edulis) from the Sognefjord on the southwest coast of Norway were sampled during the periods of July to October, 1996, March to November, 1997, and October to December, 1998. The results from the first and the duplicate measurement were found to be very similar for both HPLC methods using two fluorogenic reagents, 4-bromomethyl,7-methoxycoumarin and 9-anthryl diazomethane, and the colorimetric PP2A inhibition assay in detection of diarrhetic shellfish toxins. The levels of agreement between the measurements were satisfactory for all three methods. Significant correlations were found between the level of observation and the absolute difference between the two measurements and were mainly due to concentrations of OA/DTX-1 higher than 100 microg/100 g mussel meat. However, the precision of quantifying DSP toxins in the upper level was not found to be less than in the lower level. Both the HPLC methods and the PP2A assay were found to give repeatable results and thereby found to be reliable.

Animals↗

PubMed2002

First confirmation of human diarrhoeic poisonings by okadaic acid esters after ingestion of razor clams (Solen marginatus) and green crabs (Carcinus maenas) in Aveiro lagoon, Portugal and detection of okadaic acid esters in phytoplankton.

A new outbreak of human diarrhoeic poisonings (DSP) with esters of okadaic acid (OA) was confirmed after ingestion of razor clams (Solen marginatus) harvested at Aveiro lagoon (NW Portugal) in the summer of 2001. Accumulation of marine toxins in second order consumers was investigated in the edible parts of a shellfish predator abundant at Aveiro lagoon, the green crab Carcinus maenas. Okadaic acid was found, also in a predominant esterified form. Levels in edible parts (comprising mainly viscera) surpassed 16microg/100g. We suggest that one patient may have developed profuse diarrhoea after ingestion of a large number of green crabs contaminated with okadaic acid esters. At least 32microg OA/100g were found in a remaining sample of its meal. Domoic acid was also found but under the allowable level in force in USA of 30microg/g crab viscera. In cooked crabs, significant losses of domoic acid were found and it is not suspected to have contributed to the poisoning event, although being a vector for this toxin. The low percentage of free okadaic acid found is in accordance with a predation predominantly on benthonic shellfish (razor clams, clams and common cockle) rather than on rock mussels. These last ones present usually higher percentages of free okadaic acid. Okadaic acid was confirmed with full-scan mass spectra either in plankton and mussel extracts. Okadaic acid esters were also found in plankton extracts. Percentages between 40-60% of esterified OA were found in samples freshly extracted. Ester's percentage diminished drastically if after sonication the extract was kept at room temperature. The major part of the esters was water-soluble.

Adult↗