(the parent animals), aged less than 24 hours at the start of the test, are exposed to the test substance added to water at a range of concentrations. The test duration is 21 days. At the end of the test, the total number of living offspring produced per parent animal alive at the end of the test is assessed. This means that juveniles produced by adults that die during the test are excluded from the calculations. Reproductive output of parent animals can be expressed in other ways (e.g. number of living offspring produced per animal per day from the first day offspring were observed) but these should be reported in addition to the total number of juveniles produced per parent alive at the end of the test. The reproductive output of the animals exposed to the test substance is compared to that of the control(s) in order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC). In addition, and as far as possible, the data are analysed using a regression model in order to estimate the concentration that would cause a x % reduction in reproductive output (i.e. the EC
50
, EC
20
, or EC
10
).
The survival of the parent animals and time to production of first brood must also be reported. Other substance-related effects on parameters such as growth (e.g. length) and possibly intrinsic rate of increase, may also be examined.
1.4. INFORMATION ON THE TEST SUBSTANCE
Results of an acute toxicity test (see Method C.2, Part I) performed with
Daphnia magna
should be available. The result may be useful in selecting an appropriate range of test concentrations in the reproduction tests. The water solubility and the vapour pressure of the test substance should be known and a reliable analytical method for the quantification of the substance in the test solutions with reported recovery efficiency and limit of determination should be available.
Information on the test substance which may be useful in establishing the test conditions includes the structural formula, purity of the substance, stability in light, stability under the conditions of the test, pKa, P
ow
and results of the test for ready biodegradability (see Method C.4).
1.5. VALIDITY OF THE TEST
For a test to be valid, the following performance criteria should be met in the control(s):
— the mortality of the parent animals (female
Daphnia
) does not exceed 20 % at the end of the test,
— the mean number of live offspring produced per parent animal surviving at the end of the test is ≥ 60.
1.6. DESCRIPTION OF THE TEST METHOD
1.6.1. Apparatus
Test vessels and other apparatus which will come into contact with the test solutions should be made entirely of glass or other chemically inert material. The test vessels will normally be glass beakers.
In addition, some or all of the following equipment will be required:
— oxygen meter (with microelectrode or other suitable equipment for measuring dissolved oxygen in low volume samples),
— adequate apparatus for temperature control,
— pH meter,
— equipment for the determination of the hardness of water,
— equipment for the determination of the total organic carbon concentration (TOC) of water or equipment for the determination of the chemical oxygen demand (COD),
— adequate apparatus for the control of the lighting regime and the measurement of light intensity.
1.6.2. Test organism
The species to be used in the test is
Daphnia magna
Straus. Other
Daphnia