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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 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:
Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
EC Number:
Molecular formula:
C32H22CrN10O8.C10-14H21-29NH2 / C34H24CrN8O6.C10-14H21-29NH2 / C33H23CrN9O7.C10-14H21-29NH2
Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1) and Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Test material form:
Details on test material:
- Physical state/Appearance: Solid / red
- Total Organic Carbon: 592 mg/g
Specific details on test material used for the study:
- Storage condition of test material: Storage at room temperature

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
The test substance is a poorly water soluble mixture; therefore a water accommodated fraction (WAF) was prepared following general guidance provided in OECD 23. The test solutions were prepared by directly adding test substance to test medium and stirring for about 2 days. After this time, undissolved test substance was removed by filtration with a membrane filter (pore width 0.2 μm). The first 50-100 mL of filtered solution was discarded (used to condition the filter). The aqueous fraction of the test solution, after separation of the undissolved material, was considered the water saturated fraction (WSF) in test media. The exposure was started after separation of the undissolved material.
Fresh test solutions were prepared and renewed daily to maximize the exposure to all soluble components of the test substance.
The control solutions were visibly clear over the exposure period.
The test concentrations were visibliy clear and light yellow up to yellow over the exposure period.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Strain/clone: Daphnia magna STRAUS 1820
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.

Daphnia brood stock are kept in mass cultures consisting of approx. 20 – 30 parthenogenetically reproducing females for a maximum of 4 weeks and fed live unicellular algae (Desmodesmus subspicatus), cultured continuously at the test facility. All individuals in each mass culture originate from a single female and are thus genetic clones. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at start
of the exposure. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing.

The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, and temperature (20 ±1°C).

Study design

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

Test conditions

2.20 – 3.20 mmol/L
Test temperature:
19.7 – 20.1 °C (continuous monitoring)
7.5 – 8.5
Dissolved oxygen:
Must remain ≥ 3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 hours prior to use.
550 - 650 μS/cm
Nominal and measured concentrations:
0 (control), 4.6, 10, 22, 46 and 100 mg/L as loading rate based on test substance mass without a correction for purity or composition
Details on test conditions:
- Test vessel: Numbered glass beakers (nominal volume 100 mL)
- Test volume: 50 mL:
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): In order to insure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal period was 24 hours.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 animals / test vessel (0.1 animals/mL)

A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 202. The general properties of this medium are as follows.
Total hardness: 2.20 – 3.20 mmol/L
Acid capacity up to pH 4.3: 0.80 – 1.00 mmol/L
Molar ratio Ca:Mg: about 4 : 1
pH value: 7.5 – 8.5
Conductivity: 550 - 650 μS/cm
Total organic carbon: <2 mg/L
Dissolved oxygen: Must remain ≥3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 hours prior to use.

- Adjustment of pH: No
- Photoperiod: 16 hours light : 8 hours darkness
- Light intensity: 122 – 693 lux at a wave length of 400-750 nm

The exposure was started by impartially distributing 20 neonate (< 24 hours old) Daphnia magna evenly among the 4 test vessels per test group. The neonate daphnids all originated from the same mass culture and were thus genetic clones. The Daphnia were transferred into the test vessels with minimal culture water by capturing each daphnid in a pipette then gently expelling it onto a Teflon mesh (70 μm) held by forceps. Each daphnid was
then immediately transferred from the mesh into the corresponding test vessel from lowest to highest concentration.
Each test vessel was visually checked for immobilized daphnids after 0, 24 and 48 hours. In addition any abnormal behavior or appearance was documented. For test renewal, daphnids in each replicate were captured with a pipette and transferred into test vessels with fresh test
Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily in old and new test solutions. The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, TOC and conductivity) were determined after aeration and prior to use in the test.
The pH and dissolved oxygen content of the test solutions was measured after 0h and 24h (new test solutions) and after 24 h and 48 h (old test solutions) in replicate 1 of each tested concentrations and the control.
In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels.

In the first experiment, 65% immobilisation in the limit test concentration 100 mg/L was observed, therefore a full concentration range was performed. According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤2.2 should be used, preferably encompassing the range from no effect to 100% immobilization.
The raw data of the range finding test and the first experiment are archived together with the raw data of this study.
Reference substance (positive control):
sodium chloride

Results and discussion

Effect concentrations
48 h
Dose descriptor:
Effect conc.:
5.04 mg/L
Nominal / measured:
Conc. based on:
other: loading rate
Basis for effect:
Details on results:
ELx values are based on loading rate and calculated using the same statistical methods as for ECx. According to OECD 23, ELx values for WAFs are comparable to ECx values for pure substances within their solubility range.
Results with reference substance (positive control):
In order to verify that the Daphnia magna culture is responding normally to toxic stress, tests with a reference substance, sodium chloride, are conducted monthly. Reference substance tests are conducted according to OECD 202 guidelines and in accordance with GLP, but without a GLP status.
The EC50(48h) of the reference substance sodium chloride was 4.46 g/L (experiment date: 12 Oct 2016, project number: 50E0789/12E060).
This result is within the range of 3.88 – 7.22 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Any other information on results incl. tables


he following observations were recorded for the dissolution behavior of the test substance in the test water:

Test Group [mg/L] 0 hour 24 hours 48 hours
Old test solution New test solution
0 (control) 0 0 0 0
4.6 1 1 1 1
10 1 1 1 1
22 1 1 1 1
46 1 1 1 1
100 1 1 1 1
Explanation of abbreviations:
0: no remarkable observations, clear test medium
1: coloration caused by the test substance (light yellow up to yellow, coloration depends on test

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance is acute toxic for aquatic invertebrates.