Domoic acid (DA) was identified as the toxin responsible for an outbreak of illness in Canada in 1987, caused by eating blue mussels that had accumulated DA as a result of the presence of Pseudo-nitzschia pungens. It is named amnesic shellfish poisoning (ASP). Since a regulatory level of 20 mg DA/kg of shellfish meat was established. The methods of determination of domoic acid are reviewed: biological assays in vivo and in vitro, biochemical and chemical assays. The AOAC mouse bioassay, which is used for PSP, cannot detect domoic acid at the regulatory level of 20 mg/kg tissue and no interlaboratory validation of this method for domoic acid has been carried out. The Quillian method, a liquid chromatography-UV detection method is approved as European Norm by CEN and recommended by Codex Alimentarius as reference method. The European Commission Regulation stipulate that the reference method shall be the HPLC method and the Community Reference Laboratory on Marine Biotoxins (CRLMB) recommends Quillian method as routine method. Others protocols such as receptor binding assay, immunoassays and thin layer chromatography can be used as alternative for routine monitoring. The liquid chromatography-mass spectrometry method allows to confirm the presence of domoic acid isomers.

Paralytic shellfish poisoning (PSP) is caused by consumption of a wide variety of shellfish which accumulated saxitoxins (STXs) from marine dinoflagellates (Alexandrium minutum, A. tamarense, Gymnodinium catenatum, Pyrodinium bahamense) and affect a wide variety of shellfish. There are about 20 saxitoxin analogs with closely related structures. A regulatory level of 0.8 mg/kg shellfish meat as STX equivalents has existed in North America and Europe for many years. The methods of determination of saxitoxins are reviewed: biological assays in vivo and in vitro, biochemical and chemical assays. The mouse bioassay protocol has been widely used and has protected public health for over 50 years using an action level of 0.8 mg/kg STX.2HCL equiv. It is a routine method, and in EU it is the reference method if the results are challenged (Commission Regulation 2005/274/CE). However ethical issues, relating to the use of live animals, affect the acceptance and use of mouse bioassay in some countries. A precolumn-derivatization liquid chromatography method (Lawrence method) is approved as European Norm by CEN and by AOAC as official method of analysis. Others techniques such as receptor binding assay (in vitro bioassay), immunoassay (commercial test kits) and postcolumn-derivatization liquid chromatography can be used as alternative method for routine monitoring. Capillary electrophoresis and liquid chromatography with mass spectroscopic detection are studied in research laboratories. SEAFOODplus – Traceability – Valid – Saxitoxin analysis in bivalve molluscs Monique Etienne, Ifremer, Nantes, France Nov. 2006

Paralytic shellfish poisoning (PSP) is caused by consumption of a wide variety of shellfish which accumulated saxitoxins (STXs) from marine dinoflagellates (Alexandrium minutum, A. tamarense, Gymnodinium catenatum, Pyrodinium bahamense) and affect a wide variety of shellfish. There are about 20 saxitoxin analogs with closely related structures. A regulatory level of 0.8 mg/kg shellfish meat as STX equivalents has existed in North America and Europe for many years. The methods of determination of saxitoxins are reviewed: biological assays in vivo and in vitro, biochemical and chemical assays. The mouse bioassay protocol has been widely used and has protected public health for over 50 years using an action level of 0.8 mg/kg STX.2HCL equiv. It is a routine method, and in EU it is the reference method if the results are challenged (Commission Regulation 2005/274/CE). However ethical issues, relating to the use of live animals, affect the acceptance and use of mouse bioassay in some countries. A precolumn-derivatization liquid chromatography method (Lawrence method) is approved as European Norm by CEN and by AOAC as official method of analysis. Others techniques such as receptor binding assay (in vitro bioassay), immunoassay (commercial test kits) and postcolumn-derivatization liquid chromatography can be used as alternative method for routine monitoring. Capillary electrophoresis and liquid chromatography with mass spectroscopic detection are studied in research laboratories.

Domoic acid (DA) was identified as the toxin responsible for an outbreak of illness in Canada in 1987, caused by eating blue mussels that had accumulated DA as a result of the presence of Pseudo-nitzschia pungens. It is named amnesic shellfish poisoning (ASP). Since a regulatory level of 20 mg DA/kg of shellfish meat was established. The methods of determination of domoic acid are reviewed: biological assays in vivo and in vitro, biochemical and chemical assays. The AOAC mouse bioassay, which is used for PSP, cannot detect domoic acid at the regulatory level of 20 mg/kg tissue and no interlaboratory validation of this method for domoic acid has been carried out. The Quillian method, a liquid chromatography-UV detection method is approved as European Norm by CEN and recommended by Codex Alimentarius as reference method. The European Commission Regulation stipulate that the reference method shall be the HPLC method and the Community Reference Laboratory on Marine Biotoxins (CRLMB) recommends Quillian method as routine method. Others protocols such as receptor binding assay, immunoassays and thin layer chromatography can be used as alternative for routine monitoring. The liquid chromatography-mass spectrometry method allows to confirm the presence of domoic acid isomers.

The aim of this study was to develop a new behavioral pain test based on the evaluation of cognitive capacity impairments in rats with colitis and to determine the impact of different acute analgesic treatments. Colitis was induced in rats by an enema containing 2,4,6-trinitrobenzen sulfonic acid. Visual non-selective, non-sustained attentional level was assessed by a new behavioral testing procedure. Animals were familiarized on three consecutive days with an open field containing four small, similar, familiar objects. On the day of testing, one of the objects was randomly replaced by a new one. Attentional level was determined by the ability of the rat to perceive this small modification to its familiar environment. The effect of morphine, acetaminophen, aspirin or ibuprofen treatment was assessed on testing day and compared with that observed during a Von Frey test to assess referred tactile hypersensitivity of the skin of the lower back. Rats with colitis had decreased attentional level but no change in their locomotor activity, interest in the environment or memory encoding. Morphine (1 mg/kg, s.c. and 10 μg/rat, i.t.) and acetaminophen (200 mg/kg, p.o.) had a beneficial effect on attentional level and on referred tactile hypersensitivity. Testing for the latter showed that aspirin and ibuprofen (400 mg/kg, p.o.) were ineffective. The decrease in visual non-selective, non-sustained attention induced by chronic inflammatory painful state can be relieved by effective analgesic treatments. This finding could lead to the development of a new behavioral test to assess spontaneous pain in chronic painful subjects.

A direct sequencing method based on a 103 bp diagnostic sequence derived from a species-specific mitochondrial DNA cytochrome b sequence of 150 bp obtained by Polymerase Chain Reaction was tested for the identification of 47 commercial canned sardine and sardine-type products from various countries. Multiple alignment of 14 analyzed reference samples belonging to Clupeomorpha species was performed versus the canned samples. Low intraspecific variability was observed for canned sardine (less than or equal to0.03), whereas mean interspecific variability was 0.23. A phylogenetic tree was constructed, and the calculated bootstrap values (BP, 88-99%) were used as indicators of the correct assignment of unknown canned samples to reference species. According to this methodology, the 26 commercial canned sardines analyzed were grouped in the same clade as the Sardina pilchardus reference and identified unequivocally. These assignments were confirmed by the high BP value of 99%.

The DNA sequence diversity of Sardina pilchardus (Walbaum, 1792) and some closely related species of Clupeomorpha was investigated using the mitochondrial DNA gene encoding cytochrome b. The nucleotide sequences of complete and partial mtDNA cytochrome b were determined in numerous specimens. Sequence divergence between species and genera was evenly distributed in the cytochrome b gene but rather high compared to reports for other fish species. Phylogenetic analyses on complete cytochrome b were used to study the relationships among the considered species. S. pilchardus was easily differentiated, showing a genetic distance of 0.25 with respect to Clupeidae species and 0.26 with respect to the other species. A species-specific short fragment (< 150 bp) was isolated by polymerase chain reaction (PCR) using primers designed for Clupeomorpha. A rapid and reliable PCR method using restriction fragment length polymorphism (RFLP) with two restriction enzymes (MnII/HinfI) was optimized for unambiguous differentiation of S. pilchardus from the other species tested (raw and canned products).

The FAO/WHO Codex Alimentarius Commission has laid down a Codex standard for salted and dried salted fish which requires an official determination method of the salt content. A draft of an analytical method has been elaborated by a Codex working group and the WEFTA working group on analytical methods and was asked to validate this method. Seventeen laboratories out of 10 countries participated in a WEFTA collaborative trial on salt determination in fish and fishery products. Four samples containing different salt contents were distributed, including two spiked samples and two typical commercial products. All laboratories applied a common method involving the precipitation of fish proteins by Carrez I and II followed by titrimetric determination of the salt content according to Mohr. They also carried out salt determination by their own "home" methods. The following results were obtained: The recovery rate of the common method was nearly quantitative and the relative standard deviation for reproducibility was between 1.96 and 2.64%, depending on the sample. Larger variations were obtained between different laboratories when applying their home methods. In one sample a significant difference of the measured salt content was found between the average results of the common and the home methods. Application of ashing of the sample before salt determination always yielded in lower salt contents compared to other determination procedures. The common method is judged to be suitable as a standard method for salt determination in fish and fishery products. Key Words: Fishsalt determinationcollaborative study

The identification, by SDS-PAGE, of four red algae used as sea vegetables or ingredients by the food industry, was performed. They were Palmaria palmata (Dulse), Chondrus crispus (Pioca), Porphyra umbilicalis (Nori), Gracilaria verrucosa (Ogo-nori). For each species, variations in protein patterns were observed, according to the season. However, for all species, some protein bands were always present during the yearly cycle of the plant. The reference pattern of P. palmata was composed of six protein bands with apparent molecular weights between 59.6 and 15.2 kDa. The G. verrucosa pattern was constituted of eight permanent bands. Two pattern bands, with apparent molecular weights of 49.1 and 45.9 kDa,differentiated the G. verrucosa profile from other seaweed patterns. C. crispus could be identified by a reference pattern composed of seven bands; three, with close molecular weights (49.3; 46.2 and 43.2 kDa), were characteristic of this species. Finally, the P. umbilicalis pattern showed seven bands with molecular weights between 73.1 and 15.9 kDa. The presence of a band with a molecular weight above 70 kDa appeared to be specific to the Porphyra pattern. So the SDS-PAGE seemed able to identify the four red species used by the food industry, but this analytical method appeared to be applicable only to raw material dried in mild conditions.

SDS PAGE was tested as an analytical tool for the identification of fourgreen algae (Ulva rigida, U. rotundata, Enteromorphaintestinalis, E. compressa) used as food ingredients. A referencepattern composed of the bands present all year long was performed foreach species. The pattern for Ulva rotundata consists of 7 bandslocated between 69.9 and 15.5 kDa with the presence of triplicate bandsat 29.5, 26.3 and 22.9 kDa. The pattern for Ulva rigida is consistsof three bands with apparent molecular weights of 68.5, 56.4 and 44.7kDa. The Enteromorpha compressa pattern is characterised by sixbands located between 65.8 and 19.8 kDa. A double band withmolecular weights of 23.1 and 23.9 distinguished this pattern from theothers. Six bands situated between 66.4 and 19.4 kDa with a specifictriplicate band of 25.9, 23.9 and 22.5 kDa constituted the specific patternof Enteromorpha intestinalis. SDS PAGE appears to be suitable forthe identification of green seaweed foods.

Within the framework of an experimental longline fishing campaign at sea in the Bay of Biscay, a first evaluation of the adequacy of the bluefin tuna catches with the requirements of the Japanese market for the preparation of sashimi has been carried out. The criteria to define a higher quality tuna are: a high level of freshness, signs of a fast bleeding of tuna after the landing, the absence of "yake" or burn of the flesh due to lactic acid formation, the absence or a very low content in histamine and a high fat content. The tunas caught during the seven trips were all classified as Extra quality fishes in the auction. Very low level of histamine was detected (15 mg/kg). The analyses done on six fishes of different sizes in several points (four per fish) confirmed the distinction of the flesh in two classes. The "toro" is rich in fat (minimum 15% from 40 kg) and the "akami" is clearly leaner (below 7% on average); a rapid increase of the fat content linked to the weight has been observed for the fishes of more than 50 kg. In the perspective of a development of the catches for the sashimi market, the handling of the product onboard could be appreciably improved.

The suitability and reliability of three electrophoretic methods of fish species identification, urea isoelectric focusing (IEF), sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and native IEF, were evaluated on formed fish fillets and high pressure fish flesh by a collaborative study among four institutes. By following optimized standard operation procedures, the protein patterns of processed fish were compared to patterns of raw reference samples. The method to use depended of the effect of processing on the protein pattern. The proteins obtained from formed products were not denatured and therefore any of the three methods proved to be adequate, with a preference for native IEF which had a better discriminatory power for the species used. The high pressure process altered the proteins, and so only urea IEF and SDS-PAGE methods could be used. For these products, the chosen method should then be the one with the better discriminating power for the species being examined.

Three electrophoretic techniques, isoelectric focusing (IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and urea-IEF were used for species identification of "baby-clams" from Vietnam (Meretrix lyrata), grooved carpet shell (Ruditapes decussatus) and the common edible cockle (Cerastoderma edule). Analysis were performed on proteins extracted from different parts of the mollusks, adductor muscle. mantle, foot, of raw and cooked samples. The electrophoretic patterns of adductor muscle were the most discriminative. Although all three methods tested could be used for bivalve species identification, the electrophoretic profiles with IEF were modified after cooking, whereas those with SDS-PAGE or urea-IEF were identical in raw and cooked samples and thus more suitable for sample authentication.

A collaborative study, to validate the use of SDS-PAGE and urea IEF, for the identification of fish species after cooking has been performed by nine laboratories. By following optimized standard operation procedures, 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Some differences in the recoveries of proteins from cooked fish flesh were noted between the urea and the SDS extraction procedures used. Generally, the urea extraction procedure appears to be less efficient than the SDS extraction for protein solubilization. Except for some species belonging to the Salmonidae family (Salmo, Oncorhynchus), both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled identification of the species of the samples to be established. With urea IEF, two laboratories could not differentiate Salmo salar from Salmo trutta. The same difficulties were noted for differentiation between Oncorhynchus gorbuscha and Oncorhynchus keta samples. With SDS-PAGE, three laboratories had some difficulties in identifying the S. trutta samples. However, in the contrast with the previous technique, SDS-PAGE allows the characterization of most of the Oncorhynchus species tested. Only Oncorhynchus mykiss was not clearly recognized by one laboratory. Therefore, SDS-PAGE (Excel gel homogeneous 15%) appears to be better for the identification, after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) is better for the gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.

A collaborative study on the use of sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), urea-isoelectric focusing (urea-IEF) and native isoelectric focusing for the identification of species of smoked salmonids, gravad salmonids and smoked eels was carried out by eight laboratories. With SDS-PAGE, minor changes took place in the profiles of the processed salmonid species making it impossible or very difficult to identify closely related species. With urea-IEF, there were fewer changes in the profiles due to processing and the system generally had greater species-discriminating power for the processed salmonids than SDS-PAGE. The profiles of the eel species as obtained on SDS-PAGE or urea-IEF were not affected by smoking. Urea-IEF had greater species-discriminating power than SDS-PAGE for the eel species. Native IEF was useful in providing supplementary identification on species difficult to identify by SDS-PAGE or by urea-IEF in the case of cold smoked products.