Laboratory work no.

Laboratory of Ecotoxicology and LCA Department of Environmental Chemistry, ICT Prague

Daphnia magna Reproduction Test

1. Purpose
The objective of the test is to assess the effect of chemicals on the reproductive output of Daphnia magna. Young female Daphnia, aged up to 24 hours are exposed to the test sample at a range of concentrations during 21 days. At the end of the test, the total number of living offspring produced per parent individual alive at the end of the test is assessed. Results of acute toxicity test performed with Daphnia magna should be available to select an appropriate concentration rate for the reproduction test.

2. Testing organism
The test species is Daphnia magna Straus. At the beginning of the test, animals should be up to 24 hours old and must not be from the 1st brood progeny. Individuals should be chosen from healthy stock. The stock organisms should be maintained in culture conditions similar to the test ones, especially in food, temperature, light conditions and medium volume per individual. Testing culture should be pre-cultured of 3 weeks before the test as an acclimation to the test conditions.

3. Materials
3.1 Tested chemical 3.2 Cultivation medium Elendt media M4 is used as cultivation water. Dissolve following chemicals in distilled water according Tab. 1. Tab. 1: Composition of Elendt M4 medium
Stock solution 1 2 3 4 5 6 7 8 9 Chemicals Na2EDTA. 2H2O* FeSO4.7H2O* H3BO3 MnCl2.4H2O LiCl RbCl SrCl2.6H2O NaBr Na2MoO4.2H2O CuCl2.2H2O Concentration in medium Concentration in Concentration in (ml/l; stock final medium stock solution solution/final (mg/l) (mg/l) medium) 2,5 5000 0,5 0,995 1991 2,85 5719 0,5 0,36 721 0,5 0,31 612 0,5 0,07 142 0,5 0,15 304 0,5 0,016 32 0,5 0,063 126 0,5 0,017 33 0,5

Laboratory work no. 9

Laboratory of Ecotoxicology and LCA Department of Environmental Chemistry, ICT Prague

0,013 0,01 0,0033 0,0022 0,0006 293,8 123,3 0,58 64,8 10 0,274 0,143 0,184 0,0075 0,001 0,00075 26 20 6,5 4,38 1,15 29 380 12 330 580 6480 1000 27,4 14,3 18,4 75 1 0,75 0,5 0,5 0,5 0,5 0,5 10 10 10 10 10 10 10 10 10 10 10

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

ZnCl2 CoCl2.6H2O KI Na2SeO3. NH4VO3 CaCl2.2H2O MgSO4.7H2O KCl NaHCO3 Na2SiO3.9H2O NaNO3 KH2PO4 K2HPO4 Thiamine HCl** B12** Biotin**

* mix the solutions, which results in Fe-EDTA formation, and autoclave immediately ** dissolve the chemicals and store the solution frozen

Note: pH of cultivation and tested sample should be in range of 6 9 during whole the test procedure.

4. Methods
4.1. Test start Prepare the concentration rate of tested chemical and fill the test and control beakers with the sample (medium). Put 1 daphnia per 50 ml of solution into test vessels. Follow the instructions of the assistant and put either 1 daphnia into individual 100-ml beaker with 50 ml of medium in for replicates or 4 pieces of daphnia into 200 ml in 250-ml beaker without replicates. Measure the pH, oxygen concentration, feed the organisms with algae and start the exposition put the test vessels into cultivator. The test conditions are described in Tab. 2. Tab. 2: Test conditions for Daphnia magna Reproduction Test
Testing organism: Parameter observed: Test conditions: Replicates: Volume of tested sample: Temperature: Exposition length: Illumination: Chemicals: Instruments and equipment: Daphnia (Daphnia magna) Offspring number per one adult Stable temperature and light cycle 16 h/8 h (light/dark), feeding 4 parents per concentration 50 ml per 1 parent individually grown / 200 ml per 4 parents together 22 1C 21 days light cycle 16 h/8 h (light: 1500 2000 lux / dark) sample, cultivation medium, 100- or 250-ml-beakers, cultivator, analytical weighs, pH-meter, oxygen meter, pipettes, Pasteur pipette

4.2. Feeding of the test organisms and water changing Test animals should be fed at least three-times a week. The diet consists from living onecell green algae, e.g. Desmodesmus subspicatus. A concentrated algal suspension should be

Laboratory work no. 9

Laboratory of Ecotoxicology and LCA Department of Environmental Chemistry, ICT Prague

fed to the Daphnia to minimise the volume of algal culture medium transferred to the test vessels. Concentration of the algae can be achieved by centrifugation followed by resuspension in distilled water, deionised water or Daphnia culture medium. Medium should be renewed three times per week, as well. When the medium is renewed, series of new test vessels are prepared and the parent animals transferred to them by, for example, a glass pipette of suitable diameter. The volume of medium transferred with the daphnia should be minimised. 4.3. Observations 4.3.1. Offspring number The offspring produced by each parent animal should be removed and counted daily from the appearance of the first brood, to prevent them consuming food intended for the adult. For the purpose of this method it is only the number of living offspring that needs to be counted, but the presence of aborted eggs or dead offspring should be recorded. 4.3.2. Mortality The mortality of adults should be examined by each offspring counting and reported. 4.3.3. Analytical measurements

Oxygen concentration, temperature, and pH values should be measured at least once a week, in fresh and old media, in the control(s) and in the highest test substance concentration.

5. Evaluation of the test results

The purpose of this test is to determine the effect of the test substance on the total number of living offspring produced per parent animal alive at the end of the test. The total number of offspring per parent animal should be calculated for each test vessel (i.e. replicate). If, in any replicate the parent animal dies during the test, then the replicate is excluded from the analysis. The analysis will then be based on a reduced number of replicates. Based on the test results, estimate the LOEC, NOEC, EC50 and LC values, if available.

6. References
ELENDT, B.P., BIAS, W.R. (1990): Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing. Effects of the optimization of culture conditions on life history parameters of D. magna. Wat. Res., vol. 24, p. 1157-1169. ISO 10706 (2000): Water quality - Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea) OECD Guidelines for Testing Chemicals 211 (1998): Daphnia magna Reproduction Test.