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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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Administrative data

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

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:
Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate
EC Number:
EC Name:
Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate
Cas Number:
Molecular formula:
trisodium 2-({6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)(methyl)amino]-1-hydroxy-3-sulfonato-2-naphthyl}diazenyl)naphthalene-1,5-disulfonate
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
Details on sampling:
Samples were taken from the control and 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Details on test solutions:
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to1 liter to give the 100 mg/L test concentration.The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM - Common name: Daphnia Magna - Source: In-house laboratory cultures - Feeding during testDaphnids were not fed during the main test 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 maintaining the water temperature at 18 to 22°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

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
Test temperature:
Nominal: 18 to 22°C with a maximum deviation of ±1°C.Measured: 22°C (all vessels at each measurement timepoint; 0, 24, and 48 hours).
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.Measured values: 7.6 - 7.7
Dissolved oxygen:
Measured: 8.0 - 8.8 mg O2/L
Nominal and measured concentrations:
Nominal: 0 (control), 100 mg/L.Measured (treated group only; control samples were
Details on test conditions:
TEST SYSTEM - Test vessel: Glass beakers - Type (delete if not applicable): Covered to reduce evaporation - Material, size, headspace, fill volume: 150 mL beaker, 100 mL fill volume - Aeration: No - Type of flow-through: Not applicable - Renewal rate of test solution: Not applicable - No. of organisms per vessel: 5 - No. of vessels per concentration: 4 - No. of vessels per control (replicates): 4 TEST MEDIUM / WATER PARAMETERSReconstituted Water – ISO MediumIngredientFinal Concentration (mg/L)CaCl2.2H2O294MgSO4.7H2O123NaHCO365KCl5.8The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. OTHER TEST CONDITIONS - Adjustment of pH: As noted above, the pH of the reconstituted water was adjusted to pH 7.8±0.2; the pH of the test solutions was not adjusted. - Photoperiod: 16 hours light (200 to 1200 Lux), 8 hours dark, with 20 minute dawn and dusk transition periods. EFFECT PARAMETERS MEASURED: 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 within 15 seconds after gentle agitation. VEHICLE CONTROL PERFORMED: Not applicable RANGE-FINDING STUDY - Test concentrations: 0.1, 1.0, 10 and 100 mg/L. - Results used to determine the conditions for the definitive study: Yes (no immobilisation was observed in the 100 mg/L level, so the definitive test was run as a Limit Test at 100 mg/L).
Reference substance (positive control):
A positive control was run as a separate study under similar conditions to those used during the definitive test. Daphnia were exposed to a potassium dichromate control. The date of the positive control study was 29 February to 02 March 2016.

Results and discussion

Effect concentrationsopen allclose all
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Details on results:
No sub-lethal effects of exposure were observed throughout the test.
Reported statistics and error estimates:

Applicant's summary and conclusion

Validity criteria fulfilled:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna was investigated and gave a 48-Hour EC50 value of greater than 100 mg/L. The No Observed Effect Concentration was 100 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.


Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.


Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 99% to 101% of nominal and so the results are based on nominal test concentrations.

Exposure ofDaphnia magnato the test item gave EC50values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.