This International Standard describes a procedure for the determination of the acute toxicity of chemicals, waters and waste waters to the water flea Daphnia magna Straus. The evaluation of harmful effects on water quality has for several years involved the performance of biological tests. Crustaceans are of interest from the ecotoxicological point of view because they are primary consumers and a major component of the zooplankton in aquatic ecosystems. The method in this International Standard involves the determination of the immobilization of the water flea Daphnia magna Straus after 24 h or 48 h exposure to the test sample under the conditions specified in this International Standard. This International Standard describes a method for the determination of the acute toxicity to Daphnia magna Straus (Cladocera, Crustacea). The method is applicable to: - chemical substances which are soluble under the conditions of the test, or can be maintained as a stable suspension or dispersion under the conditions of the test; - industrial or sewage effluents; - treated or untreated waste water; - aqueous extracts and leachates; - fresh water (surface and ground water); - eluates of fresh water sediment; - pore water of fresh water sediment. If the method described in this International Standard is used for biotesting of chemical substances, a limit test can be performed at 100 mg/l or at a lower concentration, at which the substance is soluble or is in stable dispersion under the conditions of the test. If this information is required, a limit test may also be performed at concentrations above 100 mg/l as long as the substance is soluble or in stable dispersion. This standard differs from DIN EN ISO 6341:1996-06 as follows: a) the scope of the method has been extended to include: treated or untreated waste water, aqueous extracts and leachates, eluates of fresh water sediment, pore water of fresh water sediment; b) clause "Terms and definitions" has been included; c) the test can be carried out based on the duration of the test period of 24 h or 48 h; d) a limit test may now be performed at concentrations above 100 mg/l as long as the substance is soluble or in stable dispersion; e) in clause "Reagents, test organisms and media": requirements to meet the culture health criteria of the Daphnia stock have been included, neonates hatched from the ephippia may also be used as test organisms, dechlorinated tap water is acceptable as culturing and dilution water, approval and description of the M4 medium as a hatching medium have been included, the pH of the synthetic dilution water has been changed from 7,8 ± 0,2 to 7,8 ± 0,5, for the preparation of the stock solution for testing the reference substance, it is recommended to use a ready-made potassium dichromate solution; f) in clause 7 "Apparatus and materials": before use, it is no longer required to wash test containers with diluted water, pipettes for sampling the test organisms have been included, g) in clause "Treatment and preparation of samples": sampling, transportation and storage of the samples should be performed as specified in ISO 5667-16, modifications and supplements related to the sample treatment: the cooled sample may be stored for 24 h instead of 48 h, until the beginning of the test, the samples are to be stored at a temperature between 0 °C and 5 °C, the times and temperatures of the sample storage until the beginning of the test have been modified, information relating to the thawing of the samples has been included, the treatment of environmental samples by way of sedimentation, centrifugation or filtration to separate residual suspended matter has been included, guidance on the aeration of the sample has been included, the test is carried out without pH adjustment of the sample, further information relating to the preparation of test solutions with poorly soluble substances has been included, information concerning test design and performance for tests with poorly soluble substances has been included; information on the selection of dilution series in the case of chemical substances and on environmental samples for test solutions has been included, information on the test batch for the determination of the LID has been included; for the definitive test on chemical substances, the separation factor for the serial dilutions has been limited to 3,2; h) modifications with regard to the procedure: the requirement for the oxygen content of the test batches has been expanded: the oxygen content shall be measured both in the control batches and in the test batches having the highest test concentration at the end of the test, only test batches having a dissolved oxygen concentration of more than 2 mg/l are to be considered for the final calculations, in "Validity criteria": the frequency of reference tests has been determined, the EC50 of the potassium dichromate has been changed to be within the range 1,7 mg/l to 2,1 mg/l, limit test: it is recommended to conduct a full study if the percentage of immobilization exceeds 10 % in the limit test with 100 mg/l test substance solution; i) in "Interpretation and validity of the results", estimation of the EC50: if the nominal and the measured concentration of the test substance in the test medium differ significantly, it is recommended to repeat the test with a new test medium or to carry out a flow-through test; j) the expression of results now includes the LID value, if determined; k) the test report has been supplemented to include: information on the treatment of environmental samples, results of the reference substance testing; l) Annex A now includes guidance for the preparation of the M4 medium; m) Annex B now includes the option to estimate the confidence levels from the graph; n) a new Annex C relating to general recommendations for stock culturing has been added; o) a new Annex D relating to the culturing of Daphnia magna for production of dormant eggs has been added; p) a new Annex F relating to the preparation of the dilution series for the determination of the LID has been added for testing waste water by means of a graduated dilution, D; q) the standard has been editorially revised. This document has been prepared by Technical Committee ISO/TC 147 "Water quality", in collaboration with Technical Committee CEN/TC 230 "Water analysis" the secretariat of which is held by DIN. The responsible German committee is Working Group NA 119-01-03-05-01 AK "Biotests" ("Biotests") of Working Committee NA 119-01-03 AA "Wasseruntersuchung" ("Water Analysis") at the Water Practice Standards Committee (NAW).