Daphnia sp., Acute Immobilisation Test for ecotoxicity assessment of chemical substances (based on OECD Guideline for testing of chemicals 202)

Daphnia populations can be found in freshwater environments ranging from huge lakes to small temporary pools and seasonally flooded depressions.

Daphnia are filter feeders that direct small suspended particles into their mouth by a water current produced by their leaf-like legs. Daphnia's common name of 'water flea' comes from its jump-like movement, which results from the beat of the large antennae used for swimming. In a normal growth season Daphnia generates diploid eggs by asexual reproduction (parthenogenesis). These eggs develop directly into larvae in the female brood chamber and are released into the water after about 3 days. In most species the larvae go through four to six larval stages before developing into sexually mature adults. However, the Daphnia life cycle is adapted to extreme environmental conditions such as cold winters or summer droughts. If triggered by external stimuli such as high population density and a scarcity of food, Daphnia can produce haploid resting eggs by meiosis; these require fertilization and a period of extended dormancy in order to develop. Resting eggs are distributed by wind or animals and development is resumed in response to external stimuli (for example, rising temperature). Cyclic parthenogenesis, in which parthenogenesis and sexual reproduction alternate, is common in most Daphnia species, but lineages have been described that exclusively reproduce asexually (obligate parthenogenesis).

The methodology is an acute toxicity test to assess the effect of chemical substances on Daphnia species, primarily Daphnia magna or Daphnia pulex. The effect of chemical substances is tested with young - maximum 24 old- Daphnia for 48 hours.

Principle:

In the acute immobilisation test a range of concentrations of the substance investigated

exerts different degrees of toxic effects on the swimming capability of Daphnia under otherwise identical test conditions. Certain concentrations result in certain percentages of Daphnia being no longer capable of swimming at 24 hours. The test can be extended to 48 hours if desired.

Selection of species:

Daphnia magna, or any other suitable Daphnia species, not more than 24 hours old at

the beginning of the test, laboratory bred, apparently healthy and with a known history

(breeding method, pretreatment) are used in this test.

Performance of the test:

At least 20 animals, preferably divided into four groups of five animals each, should be used at each test concentration and for the controls.

The Daphnia should not be fed during the test.

Loading: at least 2 ml of test solution should be provided for each animal.

During incubation at every 24 hours the number of mobile animals within each glass is counted either macroscopically or with a stereomicroscope.

Evaluation

Reproduction inhibition (H%) is calculated according to the regular procedure in ecotoxicity using the following equation:

H (%) = 100*((Nk-Nsample)/Nk)

H –reproduction inhibition (%)
N_k –number of animals in the control sample (pcs/ml)
N_sample – number of animals in the test sample (pcs/ml)

The test results are acceptable only in case 80 % of the animals in the control sample remain mobile.

The percentage immobility at 24 hours and, if determined, at 48 hours is plotted against concentration. Normal statistical procedures are then employed to calculate the EC 50 for the appropriate exposure period.

Confidence limits (p = 0.95) for the calculated EC 50 values can be determined using the standard procedures.

Where the data obtained are inadequate for the use of standard methods of calculating the EC 50, the highest concentration causing no immobility and the lowest concentration producing 100 per cent immobility should be used as an approximation for the EC 50 (this being considered the geometric mean of these two concentrations). In this case, the ratio of the higher to the lower concentration should not exceed 2.