Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test substance at a range of concentrations (at least five concentrations) for a period of 48 hours. Immobilisation is recorded at 24 hours and 48 hours and compared with control values. The results are analysed in order to calculate the EC50 at 48h. Determination of the EC50 at 24h is optional. At least 20 animals, preferably divided into four groups of five animals each, should be used at each test concentration and for the controls. At least 2 ml of test solution should be provided for each animal (i.e. a volume of 10 ml for five daphnids per test vessel). The limit test corresponds to one dose level of 100 mg/L. The study report should include the observation for immobilized daphnids at 24 and 48 hours after the beginning of the test and the measures of dissolved oxygen, pH, concentration of the test substance, at the beginning and end of the test.