Daphnia magna: Reproduction Test (OECD 211: 2012).           

This test aims to evaluate the effects of chemicals on the reproduction of Daphnia magna. To do this, young Daphnia females less than 24 hours old at the beginning of the test are exposed to the test product added to the water in a range of concentrations. The duration of the trial is 21 days. At the end of the test, the total number of live pups produced is evaluated. For this, the offspring produced by the adults that die during the test must be excluded from the calculations.

Animal reproduction can be expressed in various ways (for example, as the number of live pups produced by each animal per day from day one) but this should be reported in addition to the total number of new live pups produced at the end of the trial. The reproductive capacity of the animals exposed to the tested product is compared with that of the controls to determine the minimum concentration with effect (LOEC: Lowest Observed Effect Concentration) and likewise the concentration without effect (NOEC: No Observed Effect Concentration). In addition, if possible, the data is analyzed using a regression model to estimate the concentration that would cause a given percentage reduction in reproductive capacity e.g. EC (e.g. EC50, EC20 or EC10). Survival of breeding animals and the period until the appearance of the first calf should also be reported. Other effects on parameters such as growth (e.g. length) or the intrinsic rate of population increase can also be examined.

The OECD Guide indicates that the semi-static or continuous flow test can be performed. In our laboratory, only the semi-static test is carried out, which uses at least 10 animals maintained individually per test series and at least ten animals in the control series.

To select a suitable range of test concentrations, it is useful to have the results of the acute immobilization test (OECD 202: Daphnia spp. Acute Immobilization Test) with Daphnia magna. When necessary, a range-finding test can be performed with at least 5 concentrations and 2 replicates per concentration and control. The definitive test must be performed with at least 5 concentrations arranged in geometric series with a factor not exceeding 3.2. If it is desired to estimate the LOEC and NOEC, the lower test concentration should not produce a significant effect on reproduction relative to the control and the higher concentration should produce a significant effect. When the range-finding test shows no effect on reproduction at the highest concentration or when the test substance is suspected to be of no or low toxicity, a borderline test can be performed with a test concentration and a control, with 10 replicates in each group. If reproductive effects are observed in this test, the full test should be performed.

Test substances must not be tested above their solubility limit in the test medium. Test sample concentrations should preferably be prepared in the test medium without using solvents or dispersants. The client must provide the solubility of the test product. Also, it is convenient to have the structural formula, purity of the substance, light stability, stability under test conditions, pKa, POW and results of biodegradability tests.