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Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

long-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2000-01-19 to 2000-02-24 (experimental phase)
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study. According to ECHA guidance, a study with a read-across substance can have no reliability of higher than 2. The study itself is valid without restriction.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:

Sampling and analysis

Analytical monitoring:
Details on sampling:
For the analysis of the test item concentrations, duplicate samples from the freshly prepared test media of all test concentrations and the solvent control were taken at the first treatment period (Day 0), at a treatment period in the second week (Day 12) and at a treatment period in the last week (Day 16).

For the determination of the maintenance of the test item concentrations during the test medium renewal periods, the following additional samples were taken at the end of two treatment periods (on Days 14 and 19):
a) from sufficient volumes of the freshly prepared test media of all test concentrations and the solvent control which were incubated during the renewal periods under the same conditions as the test itself, however without food particles and test animals.
b) from the test media of all test concentrations and the solvent control out of the actual test (including the food particles) by pouring together the contents of the test beakers after treatment period.

The first of these two stability controls lasted for 48 hours, the second for 72 hours (weekend), corresponding to the two different renewal periods of the test media. All samples were deep-frozen (at about -20 °C) immediately after sampling. Based on a pretest (without GLP) for investigation of the storage stability the test item is sufficiently stable in the test media under these storage conditions.

The concentrations of the test item were analyzed in all duplicate test medium samples from the highest test concentration of nominal 0.016 µg/l, determined in the experiment as the NOEC. From the solvent control samples only one of the duplicate samples was analyzed from each of all sampling dates. The analytical methods and results are described in the attached analytical report.

Test solutions

Details on test solutions:
Due to the low water solubility of the test item a concentrated stock solution of the test item in organic solvent was prepared just before each test medium renewal date. The solvent additive was chosen based upon its solubility properties and its relative non toxicity to Daphnia.

Prior to the start of the test and prior to each test medium renewal, the stock solution and the test media and the controls were prepared as follows: First, a concentrated solution of the test item in organic solvent (dimethylsulphoxide, = DMSO) with a concentration of nominal 320 mg/l was prepared by dissolving 32 mg of the test item completely in 100 ml DMSO. This organic test item solution was used as stock solution for the preparation of the test medium with the highest test concentration.

Additionally, this organic test item solution was diluted with DMSO to obtain the stock solutions for the preparation of the test media with the lower test concentrations.

The test media with the different test concentrations were prepared by mixing an equal volume of each of the different organic stock solutions into an equal volume of test water. In this way, the concentration of DMSO was the same in all test media (50 ml DMSO per liter test water). The test media were intensively mixed for 10 minutes.

The solvent control was prepared by addition of 50 ml DMSO per liter test water. For the control test water without addition of test item or solvent was used.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Source: in house culture, originally supplied by the University of Sheffield/UK in 1992, defined from the supplier as clone 5. Since this date, the clone is bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (regarding pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age at study initiation (mean and range, SD): < 24h
- Method of breeding: parthenogenetic reproduction
- Feeding during test: yes

Study design

Test type:
Water media type:
Total exposure duration:
21 d

Test conditions

250 mg/L CaCO3
Test temperature:
Dissolved oxygen:
>=8.1 mg/L
Nominal and measured concentrations:
0, 1.0, 2.0, 4.0, 8.0, 16 µg/L
Details on test conditions:
- Test vessel: glas beaker nominal volume 100 mL, covered with glas plates
- Fill volume: approx. 80 mL
- Renewal of test solution: on Days 2, 5, 7, 9, 12, 14, 16 and 19 of the exposure period
- No. of organisms per vessel: 1 parent animal
- No. of vessels per concentration (replicates): 10

- Source/preparation of dilution water: synthetic medium M7
- After preparation the M7 medium was aerated until saturation with oxygen is reached.

- Photoperiod: Artificial Iight, day/night-rhythm = 16 / 8 hours
- Light intensity: about 300-800 lux

- Reproduction and mortality on Days 2, 5, 7, 9, 12, 14, 16 and 19 of the exposure period
Reference substance (positive control):
not specified

Results and discussion

Effect concentrations
21 d
Dose descriptor:
Effect conc.:
>= 8.8 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:

Any other information on results incl. tables

The mean measured test item concentration in the analyzed test medium samples of the highest test concentration were in the range of 11 to 12 µg/l at the start of the test medium renewal periods. At the end of the test medium renewal periods, the concentrations decreased to 5.2 and 5.4 µg/l. This might have been caused by precipitation of finely dispersed test item, since the highest test concentration was clearly above the solubility limit of the test item in test water, which was < 7.4 µg/l.


Therefore, all biological results are related to the total mean measured test item concentration of 8.8 µg/l (calculated as the average over all measurements of the test concentration during the test period of 21 days).



In the controls and all test concentrations up to and including 8.8 µg/l (nominal 16 µg/l) the survival rate of the test animals at the end of the test was at least 90% or higher. Thus, the survival rate of Daphnia magna after 21 days was not significantly (>10%) reduced up to the highest test concentration.



The mean reproduction rate of the daphnids in the control and the solvent control was 90.7 ± 13.7 (mean ± SD) respectively 93.8 ± 17.4 (mean ± SD) alive offspring per adult. Compared to the control respectively to the solvent control, no significant toxic effect of the test item on the mean reproduction rate was determined up to and including the highest test concentration of 8.8 µg/l (nominal 16 µg/l) (results of a Williams-Test, one-sided smaller, a = 0.05).


The 21-day EC50 for the reproduction rate of Daphnia magna could not be calculated due to the absence of a toxic effect, but is clearly higher than 8.8 µg/l

Applicant's summary and conclusion

Validity criteria fulfilled: