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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to protocol
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Samples of all test concentrations were taken on preparation, just before and after changing of the test solutions and in some freshly prepared stock solutions as. Sampling frequency was increased due to poorer than expected recoveries.
At least 30 mL was sampled in each case. Samples from the actual test replicates were filtered using a 0.45 μm GHP Acrodisc filter to remove algae. Filters were primed with the relevant solution before use.
Samples from any parallels were sampled directly. Filtration was found to influence the test substance recovery. Subsequent sampling took place in parallel replicates only without algae so as to avoid the need for filtration.
Initially the samples were stored in the refrigerator until analysis. This was shown to be unsuitable and subsequent samples were analyzed immediately without storage.
Details on test solutions:
Preparation of the stock solutions:
The test substance is readily soluble in water. To prepare the stock solutions for every water renewal, on between 0.0104 and 0.0181 g of test substance was weighed on an analytical balance and dissolved directly in approx. 100 mL test medium. The stock solutions were agitated mechanically for at
least 15 minutes until the test material was completely dissolved.
The pH of the stock solution was checked and found to be between 7.6 and 7.8 and was not adjusted.
After this the stock solution was filled up to exactly 100 mL with test medium.
A fresh stock solution was prepared for each solution change.

Preparation of the test solutions:
Test solutions were prepared by further dilution of the relevant stock solutions with test medium.
The ratio between two consecutive concentrations was 2.0.
Test vessels were filled directly from volumetric flasks immediately after preparation. The solutions were renewed three times a week during the test.
The pH of the test solutions should be between 6 and 9 at refreshment and close to the value of the test medium. One control containing only test medium was included in the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: WIL Research Europe, The Netherlands) cultured in conformity with the relevant SOP. The parent animals were cultured in test medium from the day they were born.
The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use). The culture is checked half-yearly for sensitivity by a reference test with
potassium dichromate. The most recent results of which were within the range given in the guideline.

Feeding: Culture and test animals were fed a diet of 0.1 to 0.2 mg of carbon per daphnid per day, in the form of the algal strain Chlorella vulgaris. The strain is cultured in the Environmental Chemistry laboratory and total organic carbon content has previously been measured.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
12.5 °dH (75.8 mg/L Ca) which is equal to 223 mg/L as CaCO3.
Test temperature:
21.0 - 21.2 °C
7.3 - 9.0
Dissolved oxygen:
7.8 - 10.7 mg O2/L.
Nominal and measured concentrations:
nominal test concentrations: 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L
Time Weighted Average concentrations: 0.12, 0.26, 0.66, 1.21 and 3.23 mg/L
Details on test conditions:
- Test vessel: 50 mL glass beakers
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

- Source/preparation of dilution water: M4 Elendt medium
- Culture medium different from test medium: no

- Adjustment of pH: no
- Photoperiod: 16 h of light
- Light intensity: 15 .0 μmol/s/m2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Reproduction, parental weight, length and mortality


10 day range finding test in M4 medium with no effect up to 2.0 mg/L (nominal).
Reference substance (positive control):
Key result
21 d
Dose descriptor:
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
Key result
21 d
Dose descriptor:
Effect conc.:
0.98 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
Key result
21 d
Dose descriptor:
Effect conc.:
0.66 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
Details on results:
Parental mortality:
Two parent animals died during the test in the control and at 0.31 mg/L. Four parent animals died at 5 mg/L. Five parent animals died at 0.63 & 2.5 mg/L. Six parent animals died at 1.3 mg/L. Although significant mortality of parents occurred response was not dose related and hence this data will be
excluded from results calculation.

Parental Length and Weight:
For length data no significant difference from the control was detected. Only the NOEC for length could be determined as > 3.23 mg/L. Dry weight data was limited due to the levels of adult mortality in the test. Normality and Equality of variance could not be confirmed and little can be reliably calculated from this data. A NOEC / LOEC calculation was not possible due to lack of sufficient replicates. A maximum likelihood probit plot did however show a dose response trend without confidence limits. The EC10 for dry weight was 0.38 mg/L. The EC50 was 3.33 mg/L.

The first brood was in all cases released on day 7 except one that was released on day 9 at 0.63 mg/L.
Dead neonates were observed during the test. The number of dead neonates was dose response related and increased with test substance concentrations. Some dead juveniles were observed in the control however this was low in comparison to the numbers observed in the test concentrations
themselves. It was not possible in all cases to determine if affected juveniles were still born, aborted or that death had occurred shortly after birth. Endpoints for juvenile mortality were therefore not derived.
Furthermore suitable chronic endpoints were already determined for reproduction, length and weight.
Slow moving juveniles were observed at 2.5 mg/L and 5.0 mg/L (nominal concentrations).
Reported statistics and error estimates:
The data were found to be normally distributed with the exception of the dry weight data. The length and reproduction data also passed Bartlett’s tests for homogeneity of variance. Analysis of variance was performed (Dunnetts test) on the number of living neonates per parent alive at the end of the test and on the length endpoint to determine the NOEC (No observed effect concentration) and LOEC (Lowest observed effect concentration) for each endpoint. Reliability of the statistics for the dry weight endpoint should be considered low due to the sample size.
Dose response plots were generated using the maximum likelihood probit method for the reproduction and weight endpoints only. The length endpoint did not display a dose response curve due to no significant effects being detected.
Validity criteria fulfilled:
The EC10 for reproduction is 0.37 mg/L. The study was considered valid without restrictions, it was performed under GLP according to protocol and this result can therefore be used for Risk Assessment purposes.
Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211 (OECD, 2012).

The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study.

Endpoints based on parental length and dry-weight were also calculated as secondary endpoints.

The nominal concentrations used in the study were as follows: 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L.

All concentrations given refer to the technical product as supplied by the sponsor.

Analytical determinations of the test solutions were made on fresh and old solutions as well as some stock solutions throughout the test. The test substance was found not to be stable according to the guideline criteria. Time weighted mean calculations of measured concentrations meeting the required validity criteria were therefore used for expression of the tests endpoints.

The following quality criteria were respected:

· Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals, valid reference test).

· Two parent animals died in the control group over the test period, which is not more than 20%.

· The average number of juveniles per parent animal alive at the end of the test in the control was 116 after 21 days (minimum acceptable = 60)

· Analytical quality criteria were met

The EC10 and EC50 for reproduction, were calculated as 0.37 and 0.98 mg/L respectively based on time weighted mean concentrations. The NOEC and LOEC for reproduction were determined as 0.66 and 1.21 mg/L respectively. Secondary endpoints are reported in the results section.

Description of key information

The 21d-EC10 for Daphnia reproduction is 0.37 mg/L (TWA).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.37 mg/L

Additional information

One OECD 211 GLP study, semi-static, with analytical monitoring is available which meets all the quality criteria. As the test substance did not remain stable during the test, the EC10 was calculated based on time weighted average concentrations.