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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:

Sampling and analysis

Analytical monitoring:
Details on sampling:
For the determination of the test item concentrations, duplicate samples were taken from the test media at all test concentrations and the control at the start and at the end of the exposure (48 hours). For sampling at the end of the exposure, the contents of the replicate test vessels were combined prior to sampling.

Test solutions

Details on test solutions:
Reconstituted test water (ISO test water) according to the OECD test guideline was used in the study, consisting of analytical grade salts dissolved in purified water to achieve the following concentrations: 294 mg/L CaCl2 x 2 H2O, 123 mg/L MgSO4 x 7 H2O, 65 mg/L NaHCO3, 5.8 mg/L KCl.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test is performed with young daphnids of the species Daphnia magna Straus. A clone of the species from a population of the Daphnia Collection of the University of Basel, Switzerland, is successfully bred at the testing facility under temperature and light conditions that are also used in the test.
The cultivation of the parental daphnids was performed in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, temperature of 21 °C, main ions, and total hardness). Each stock animal was maintained separately in a 100-mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. No signs of stress were observed and the brood stock was healthy. The culture contains no ephippia, the first offspring were produced before day 12, the cultured daphnids did not shown any signs of stress during the 48 hours prior to test start (e.g., abnormal behavior or >5% mortality). Additionally, the cultured daphnids produced an average of at least 3 offspring per female per day over a 7-day
period prior to test initiation. During breeding, daphnids were generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/ Germany) and cultivated at at the test facility under standardised conditions or a mixture of the algal suspension and a commercial fish diet (supplied by TETRA-GmbH, 49324 Melle/Germany). At the start of the test, the test organisms were ≤24 hours old and were not first brood progeny.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

250 mg/L as CaCO3
Test temperature:
21 °C
7.7 to 7.8
Dissolved oxygen:
8.3 to 8.6 mg/L
Alkalinity was 1 mmol/L
Nominal and measured concentrations:
Nominal: 0, 4.6, 10, 22, 46, 100 mg/L
Measured (at experimental start): Measured (at experimental end):
Details on test conditions:
For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The volume of test solution provided for each daphnid was 10 mL. The daphnids were randomly distributed to the test vessels at the initiation of the test and the vessels were randomly placed at the test area.
The test was performed in 100-mL glass beakers filled with 50 mL of test medium. All vessels were cleaned before use in the test, following standard laboratory procedures. The test vessels were loosely covered with glass sheets to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was between 16 and 17 μmol/m2s.
Reference substance (positive control):
Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory conditions of the test.

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
15 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% confidence limits: 10 and 22 mg/L
Details on results:
At the start of the test, the measured concentrations of the test substance in the test media ranged between 93 and 105% of the nominal values and between 79 and 91% of nominal at the end of the test. Thus, the analytical results confirm the correct dosage of the test item. The test item was considered as stable during the exposure phase. Although in the lowest concentration the test item was observed to be 79% and thus below 80% of the nominal concentration at the end of the exposure, this is a minor deviation and this concentration is below all effect values. Therefore, the endpoint values were calculated based on the nominal concentrations of the test substance.
Results with reference substance (positive control):
The 48-hour EC50 obtained in the latest study with the positive control substance was 1.2 mg/L with a 95% confidence interval between 1.1 and 1.3 mg/L (performed in April 2020).

Any other information on results incl. tables

No immobilised animals were observed at the test concentration of 10 mg/L (and lower test concentrations), whereas all of the test organisms were found to be immobile at the three highest test concentrations after 24 hours of exposure.

Applicant's summary and conclusion

Validity criteria fulfilled:
The 48-hour EC50 value was 15 mg/L, based on immobilisation of the daphnids.
Executive summary:

The acute toxicity of the test substance to freshwater invertebrates (Daphnia magna) was determined under GLP in a 48-hour static test according to the OECD TG 202 (2004). Twenty daphnids, aged <24 hours, were randomly divided into groups of five and exposed to each test concentration in four replicates. The nominal concentrations were 4.6, 10, 22, 46 and 100 mg/L. A control (test water without test item) was tested in parallel. The analytically determined concentrations of the substance in the test media at the start and the end of the exposure ranged between 93 and 105% of the nominal values and between 79 and 91% of nominal at the end of the test. Although in the lowest concentration the test item was observed to be under 80% of the nominal concentration at the end of the exposure (79%), this is a minor deviation and this concentration is below all effect values. The effect levels were therefore calculated on the basis of nominal test concentrations. No immobilisation of test organisms was observed at test concentrations of 4.6 and 10 mg/L after 24 or 48 hours, whereas all test organisms were immobilised at the three higher test concentrations at these time points. The 48-hour EC50 value based on immobilisation was determined to be 15 mg/L, with 95% confidence limits of 10 and 22 mg/L.