ISO 10872:2020 pdf download Water and soil quality — Determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda)
Additionally, the EC 50 of the reference substance shall be determined at least every 12 months. The EC 50 (growth) in water shall be in the range of 8 mg to 22 mg BAC/l using an EC x design as specified in ISO 5667-16. Stock solutions of BAC in the concentrations of 7,1 mg/l, 10,6 mg/l, 16 mg/l, 24 mg/l, 36 mg/l, 54 mg/l and 81 mg/l are prepared in water and tested according to the instructions for testing aqueous substrates (see 10.1, 10.2.2, 10.3).
8 Organisms 2)
8.1 Test organism Caenorhabditis elegans (Maupas, 1899) is a widespread, free-living soil nematode that feeds primarily on bacteria. Adult worms are about 1,0 mm to 1,5 mm in length and can be distinguished into hermaphrodites and rarely occurring males (see Annex B). Hermaphrodites usually reproduce by self-fertilization although they can also be fertilized by males. After hatching, C. elegans develops to the adult stage through four juvenile stages separated by moults. Under starvation conditions, a developmentally arrested stage, the dauer larva, can be formed as an alternative third larval stage. The life cycle for worms grown on E. coli is about 3 d at 20 °C. The biology of C. elegans has been extensively studied and, in many respects, it is the most thoroughly characterized animal. The “wild type” strain N2 is used as test organism.
8.2 Food organism As food organism for C. elegans, the bacterium E. coli (OP50; uracil-deficient strain) is used.
9 Stock- and pre-cultures
9.1 Stock cultures
9.1.1 Caenorhabditis elegans C. elegans is maintained on agar plates (3.1) with a bacterial lawn (E. coli OP 50; 8.2) at (20 ± 2) °C. When bacteria are used up, C. elegans forms dauer larvae (3.8) due to lack of food. These starved plates (3.14) serve as stock cultures for C. elegans that should be replenished every two months. If too many males occur (≥10 % in tests), new stock cultures should be ordered.
9.1.2 Escherichia coli Inoculate under sterile conditions 50 ml of LB-medium (5.2) in a 250 ml Erlenmeyer flask (6.4) with E. coli from a bacterial lawn on agar [as sent by the supplier] using an inoculating loop (6.25) and incubate for 17 h at (37 ± 2) °C on a shaker (6.11) (overnight culture). Transfer 200 µl glycerol (5.15) into each 1,5 ml plastic vial (6.5) and sterilize in an autoclave (6.1) at 121 °C for 20 min. Add under sterile conditions 800 µl of the E. coli overnight culture (3.13), vortex and freeze immediately at −20 °C. Bacterial stock cultures are thawed only once and discarded after use. Bacterial stock cultures should be replenished after six months.
9.2 Pre-culture Inoculate under sterile conditions an agar plate (3.1) with approximately 200 µl of an overnight culture (3.13) of E. coli, distribute equally using a Drigalski spatula (6.3) and incubate for at least 8 h at (37 ± 2) °C. Cut two or three small pieces (approximately 1 cm 2 ) out of a starved plate (3.14) and transfer them under sterile conditions to an agar plate with a fresh lawn of E. coli. After about 3 d at 20 °C, a lot of gravid hermaphrodites as well as juveniles of stages 1 and 2 are found on the plate. The test starts with worms in the first juvenile stage (approx. 250 µm to 350 µm length). In order to obtain worms synchronized to this life stage, rinse the plate with M9-medium (5.9). Then filter the suspension containing the nematodes through a cascade of filter gauze (6.6) of 5 µm and 10 µm mesh size to retain larger juveniles and adults. The filtered suspension contains only first-stage juveniles (J 1 ). Measure the length of 30 J 1 to obtain the initial length of the introduced exposed test organisms (mean value). For measuring the length of the J 1 , the organisms should be killed, e.g. by putting a sufficient number of J 1 in a drop of water placed on a microscopic slide and holding the slide for a few seconds over a flame.
10 Procedure
10.1 Preparation of food medium After thawing, vortex (6.16) a vial with the E. coli stock vigorously. ISO 10872:2020 pdf download