CEN/TC 275/WG 14 - Marine Biotoxins

This European standard specifies a method [1] for the quantitative determination of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), neosaxitoxin (NEO), decarbamoyl neosaxitoxin (dcNEO), gonyautoxin 1 and 4 (GTX1,4; sum of isomers), gonyautoxin 2 and 3 (GTX2,3; sum of isomers), gonyautoxin 5 (GTX5 also called B1), gonyautoxin 6 (GTX6 also called B2), decarbamoyl gonyautoxin 2 and 3 (dcGTX2,3; sum of isomers), N-sulfocarbamoyl-gonyautoxin 1 and 2 (C1,2; sum of isomers) and (depending on the availability of certified reference materials (CRMs)) N-sulfocarbamoyl-gonyautoxin 3 and 4 (C3,4; sum of isomers) in (raw) mussels, oysters, scallops and clams. Laboratory experience has shown that it is also be applicable in other shellfish [2], [3] and cooked shellfish products. The method described was validated in an interlaboratory study [4], [5] and was also verified in a EURL-performance test aiming the total toxicity of the samples [6]. Toxins which were not available in the first interlaboratory study [4], [5] as dcGTX2,3 and dcNEO were validated in two additional interlaboratory studies [7], [8]. The lowest validated levels [4], [5], [8], are given in µg toxin (free base) per kg shellfish tissue and also as µmol/kg shellfish tissue and are listed in Table 1.
A quantitative determination of GTX6 (B2) was not included in the first interlaboratory study but several laboratories detected this toxin directly after solid phase extraction with ion-exchange (SPE-COOH) clean-up and reported a mass concentration of 30 µg/kg or higher in certain samples. For that reason, the present method is applicable to quantify GTX6 (B2) directly, depending on the availability of the standard substance. Currently it is possible to determine GTX6 after a hydrolysis of Fraction 2 of the SPE-COOH clean-up, described in 6.4 as NEO. The indirect quantification of GTX6 was validated in two additional interlaboratory studies [7], [8].
A quantitative determination of C3,4 was included in the first interlaboratory study. The present method is applicable to quantify C3,4 directly, depending on the availability of the standard substance. If no standard substances are available, C3,4 can only be quantified as GTX1,4 if the same hydrolysis protocol used for GTX6 (6.4) is applied to Fraction 1 of the SPE-COOH clean-up, see [10].

This European Standard specifies methods for the quantitative determination of domoic acid in raw bivalve molluscs and finfish as well as in cooked mussels. The limit of detection is about 10 ng/ml to 80 ng/ml (0,05 mg/kg to 0,4 mg/kg), depending on the UV detector sensitivity. The limit of quantification for DA by these methods is at least 2,7 mg/kg. Method A has been validated for the determination of DA in different raw matrices such as mussels, clams, scallops and anchovies, spiked and/or naturally contaminated at levels ranging from 2,7 mg/kg to 85,1 mg/kg. Method B has been validated for the determination of DA at levels ranging from 5 mg/kg to 12,9 mg/kg in cooked blue mussels.
For further information on validation data, see Clause 8 and Annex A.
Laboratory experience has shown that this standard can also be applied to other shellfish species, however, no complete validation study according to ISO 5725 has been carried out so far.

This European Standard specifies a multi-reference method for the determination of lipophilic algal toxins (fat-soluble algal toxins produced by some dinoflagellates) in raw shellfish and shellfish products including cooked shellfish, by liquid chromatography coupled to tandem mass spectrometry LC-MS/MS [1], [2], [3]. This method has been validated in an inter-laboratory study consisting of three parts via the analysis of both naturally contaminated homogenates of blue mussel and spiked extracts of blue mussel, oyster and clam. For further information on the validation, see Annex A. Additional studies have investigated further matrices (see [4], [5]).
The detection limit for toxins of the okadaic acid group, azaspiracids and pectenotoxins was determined to be 6 µg/kg shellfish meat and for yessotoxins 10 µg/kg shellfish meat.
Quantitative determination of okadaic acid (OA), pectenotoxin 2 (PTX-2), azaspiracid-1 (AZA-1) and yessotoxin (YTX) can be carried out directly by means of standard substances available commercially. Assuming an equal response factor, okadaic acid is used for the indirect quantitative determination of the two dinophysistoxins dinophysistoxin-1 (DTX-1) and dinophysistoxin 2 (DTX-2); likewise azaspiracid 1 (AZA-1) is used for the indirect quantitative determination of azaspiracid-2 (AZA-2) and azaspiracid-3 (AZA-3), while YTX is used for homo-yessotoxin, 45-OH-yessotoxin and 45-OH-homo-yessotoxin, and PTX-2 for pectenotoxin-1 (PTX-1).
The limit of quantification (LOQ) for toxins of the okadaic acid group, azaspiracids and pectenotoxins was determined to be 20 µg/kg shellfish meat and for yessotoxins 35 µg/kg shellfish meat.
By means of hydrolysis [6], the esters of okadaic acid, DTX-1 and DTX-2 can also be determined quantitatively as the corresponding free acids.

This document specifies a method [1] for the quantitative determination of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), neosaxitoxin (NEO), decarbamoyl neosaxitoxin (dcNEO), gonyautoxin 1 and 4 (GTX1,4; sum of isomers), gonyautoxin 2 and 3 (GTX2,3; sum of isomers), gonyautoxin 5 (GTX5 also called B1), gonyautoxin 6 (GTX6 also called B2), decarbamoyl gonyautoxin 2 and 3 (dcGTX2,3; sum of isomers), N sulfocarbamoyl gonyautoxin 2 and 3 (C1,2; sum of isomers) and N-sulfocarbamoyl gonyautoxin 1 and 4 (C3,4; sum of isomers) in (raw) mussels, oysters, scallops and clams. Laboratory experience has shown that this document can also be applied to other marine invertebrates [2], [3] and processed products of those species, however, no complete interlaboratory validation study according to ISO 5725 2:1994 has been carried out so far. The method described was validated in an interlaboratory study [4], [5] and was also verified in a European Union Reference Laboratory for Marine Biotoxins (EURLMB)-performance test aiming the total toxicity of the samples [6]. Toxins which were not available in the first interlaboratory study [4], [5] as dcGTX2,3 and dcNEO were validated in two additional interlaboratory studies [7], [8]. The lowest validated levels [4], [5], [8], are given in µg toxin (free base)/kg shellfish tissue and also as µmol/kg shellfish tissue and are listed in Table 1.
[Table 1]
A quantitative determination of GTX6 was not included in the first interlaboratory study but several laboratories detected this toxin directly after solid phase extraction with ion-exchange (SPE-COOH) clean-up and reported a mass concentration of 30 µg/kg or higher in certain samples. For that reason, the present method is applicable to quantify GTX6 directly, depending on the availability of the standard substance. Whenever GTX6 standard is not commercially available, it is possible to determine GTX6 after hydrolysis of Fraction 2 of the SPE-COOH clean-up, described in 6.4, as NEO. The indirect quantification of GTX6 was validated in two additional interlaboratory studies [7], [8]. A study to compare direct and indirect GTX6 quantification was conducted at the EURLMB [16].
A quantitative determination of C3,4 was included in the first interlaboratory study. The present method is applicable to quantify C3,4 directly, depending on the availability of the standard substance. If no standard substances are available, C3,4 can only be quantified as GTX1,4 if the same hydrolysis protocol used for GTX6 (6.4) is applied to Fraction 1 of the SPE-COOH clean-up [10]. A study to compare direct and indirect C3,4 quantification was conducted at the EURLMB [16].

This European Prestandard specifies a method for the quantitative determination of saxitoxin, dc-saxitoxin and the qualitative determination of neo-saxitoxin, and the gonyau toxins GTX-2 and GTX-3 in mussels. The method can also be used to identify the toxins C-1, C-2, GTX-5 and GTX-6 after hydrolysis and, if these toxins are present, to exclude false positive results for GTX-2, GTX-3, neo-saxitoxin and saxitoxin. For mussel the lowest limit of determination is for saxitoxin 0,04 mg/kg mussel meat and for dc-saxitoxin 0,03 mg/kg mussel meat (signal/noise = 10).
The upper limits of determination have not been determined. The limits of detection for C-1, C-2, GTX-2, GTX-3, GTX-5, GTX-6 and neo-saxitoxin have not been determined.

This European standard specifies a method for the quantitative determination of saxitoxin (STX) and decarbamoyl saxitoxin (dc-STX) in mussels. It may also be applicable in other shellfish, for example scallops. The limit of determination of this method (signal/noise = 10) is 0,006 mg/kg for saxitoxin and 0,02 mg/kg for dc-saxitoxin in mussel meat. The method has been tested for saxitoxin at levels at 0,4 mg/kg and 0,5 mg/kg and for dc-saxitoxin at levels at 0,4 mg/kg and 1,6 mg/kg.

This European Standard specifies a method for the determination of domoic acid in mussels using high performance liquid chromatography (HPLC).
The method has been successfully validated in an interlaboratory study according to AOAC guidelines on mussels containing 14,1 mg/g (spiked sample) to 186 mg/g (naturally contaminated sample) domoic acid.
Laboratory experiences show that the method is also applicable on the common cockle (Cerastoderma edule), the peppery furrow shell (Scrobicularia plana), clams (Venerupis pullastra, Ruditapes decussata), oyster (Crassostrea japonica) and razor clams (Ensis spp., Solen spp) [1].