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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
Study period:
Study test phase 2014-02-10 to 2014-02-25
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets the criteria for classification as reliable without restriction according to Klimisch et al (1997).

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
lithium hydrogen 7-{(E)-[2-amino-4-(2-naphthyl)-1,3-thiazol-5-yl]diazenyl}naphthalene-1,3,5-trisulfonate (2:1:1)
EC Number:
Molecular formula:
lithium hydrogen 7-{(E)-[2-amino-4-(2-naphthyl)-1,3-thiazol-5-yl]diazenyl}naphthalene-1,3,5-trisulfonate (2:1:1)
Test material form:
solid: particulate/powder
migrated information: powder

Sampling and analysis

Analytical monitoring:
Details on sampling:
Water samples were taken from the control and the 120 mg/l test group at 0 (from the bulk test preparation) and 48 (pooled replicates) hors for analysis. Samples were frozen prior to analysis.

Test solutions

Details on test solutions:
A nominal amount of test item (240 mg) was dissolved in test water with the aid of ultrasonication for approximately 1 minute and the volume adjusted to 2 liters to give the 120 mg/L test concentration.

The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

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

Test conditions

Test temperature:
7.2 - 8.8
Dissolved oxygen:
8.6 - 8.8 mg O2/L
Nominal and measured concentrations:
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of 92% and 97% of nominal value respectively and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.
Details on test conditions:
250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20°C with a photoperiod of 16 hours light (439 to 494 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

Based on the results of a range-finding test a "Limit test" was conducted at a concentration of 120 mg/L to confirm that at the maximum concentration no immobilization or adverse reactions to exposure were observed.
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
48 h
Dose descriptor:
Effect conc.:
> 120 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
120 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
There was no immobilization in 20 daphnids exposed to a test concentration of 120 mg/L for a period of 48 hours.
Results with reference substance (positive control):

A positive control (Harlan Study Number 41303936) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

48h EC50 0.77 mg/L (0.73 - 0.82 95 % confidence limits)
48h NOEC 0.56mg/L

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Throughout the duration of the test, the test preparations were observed to be dark orange/red solutions.

The test was considered to be valid given that none the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Applicant's summary and conclusion

Validity criteria fulfilled:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of greater than 120 mg/L. The No Observed Effect Concentration was 120 mg/L.
Executive summary:


A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Results & Conclusions

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna gave a 48-Hour EC50 value of greater than 120 mg/L. The No Observed Effect Concentration was 120 mg/L.