ISO DIS 21286

Description / Abstract: This International Standard specifies a protocol to identify ecotoxicological test specimens (mainly invertebrates and plants) to the species level, based on the DNA barcoding technique. This protocol can be used by laboratories performing DNA barcoding in order to standardise both the wet-lab and data analysis workflows as much as possible, and make them compliant with community standards and guidelines.

It is not intended by this International Standard to specify one particular strain for each test method, but to accurately document the species/strain which was used.

NOTE 1 This does not imply that DNA barcoding is performed in parallel to each test run, but rather regularly (e. g., once a year, such as reference substance testing) and each time a new culture is started or new individuals are added to an ongoing culture.

This International Standard does not aim at duplicating or replacing morphological-based species identifications. On the contrary, DNA barcoding is proposed as a complementary identification tool where morphology is inconclusive, or to diagnose cryptic species, in order to ensure that the results obtained from different ecotoxicological laboratories are referring to the same species or strain.

This International Standard is applicable to identifications of immature forms which lack morphological diagnostic characters (eggs, larvae, juveniles), as well as the streamline identification of specimens collected in field monitoring studies, where large numbers of organisms from diverse taxa are classified.

NOTE 2 In principle, all species regularly used in ecotoxicological testing can be analysed by DNA barcoding. Besides the earthwoms Eisenia fetida and E. andrei, further examples for terrestrial species are Lumbricus terrestris, L. rubellus, Allolobophora chlorotica, Aporrectodea rosea and A. caliginosa, Enchytraeus albidus and E. crypticus (Haplotaxida); Folsomia candida, F. fimetaria and Sinella curviseta (Collembola); Hypoaspis aculeifer and Oppia nitens (Acari); Aleochara bilineata and Poecilus cupreus (Coleoptera); Scathophaga stercoraria, Musca autumnalis (Diptera) or Pardosa sp. (Arachnida). Also nematodes or snails and even plants can be added to this list.

NOTE 3 The efficiency of DNA barcoding depends on the availability of reliable and taxonomicallycomprehensive databases reference databases for comparison. International databases, such as GenBank or the Barcode of Life Data Systems (BOLD), allow access to all public DNA barcode sequence data.

NOTE 4 However, some of the species regularly used in ecotoxicological testing (e.g., Enchytraeus crypticus or Sinella curviseta) are poorly represented or even absent from those reference databases. In these cases, DNA barcoding can be heavily biased by Type II errors (misidentifications of queries without conspecific in the database) and therefore, species-level identifications should be confirmed by qualified taxonomists.