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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:
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-)
EC Number:
EC Name:
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-)
Cas Number:
Molecular formula:
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-)
Test material form:
Details on test material:
- physical state: solid
- color: brown
Specific details on test material used for the study:
- Storage condition of test material: The test substance was stored at ambient temperature.

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is a poorly water soluble mixture; therefore a water accommodated fraction (WAF) was prepared following general guidance provided in OECD 23. Each test solution was prepared separately (differential loading) by directly adding test substance to test medium according to the loading rate described in the table below and stirring for approximately 1 day. In concentrations 10 - 220 mg/L undissolved test substance was visible in the solution and at the wall and in 100 - 220 mg/L additionally at the bottom. The 10 and 22 mg/L concentration were colored beige, 46 mg/L was colored orange and 100 and 220 mg/L were colored brown. Undissolved test substance was removed by filtration with a Whatman nylone filter (pore width 0.2 µm). The first 50-100 mL of filtered solution was discarded (used to condition the filter). The exposure was started after separation of the undissolved material. The aqueous fraction of the test solution, after separation of the undissolved material, is considered the water saturated fraction (WSF) in test media.
In preliminary experiments attempts to separate the undissolved fraction by settling and centrifugation were unsuccessful. Therefore filtration was the only suitable option.
Fresh test solutions were prepared daily.
The filtrated solutions (test concentration and control) were stirred for appox. 10 minutes on a magnet stirrer to ensure that the oxygen content was not lower than 3 mg/L.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Strain/clone: Daphnia magna STRAUS
- 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.
- Culture conditions: 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.
- Age at start of exposure: <24 hours (at least 3rd brood progeny)

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 – 20.2 °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), 10, 22, 46, 100, 220 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), covered with glass Petri plates to slow evaporation
- Test volume: 50 mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): In order to insure constant exposure conditions this study will be conducted as a static-renewal exposure. The renewal period will be 24 hours
- No. of organisms per vessel: 5 animals / test vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 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

- Adjustment of pH: No
- Photoperiod: 16 hours light : 8 hours darkness
- Light intensity: 161 – 677 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. The test vessels were maintained as described above.
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 solution.
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 concentration.
The TOC (Total organic carbon) values of the test concentrations and the control were analyzed at the start and the end of one renewal interval as an additional water quality parameter.
In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels.

In a preliminary test a loading rate of 100 mg/L caused 60% immobilisation after 48 hours.
A loading rate exceeding 100 mg/L was included in the definitive test in order to capture the full range of response.
The raw data of the range finding test 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.:
41.6 mg/L
Nominal / measured:
Conc. based on:
other: loading rate
Basis for effect:
Details on results:
Identical effects (55% immobilization) were recorded between 22 -100 mg/L. Due to this unusual response the calculated ECx values are not statistically valid.
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.

This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• ≤10% immobilisation in the control
• O2 concentration ≥3 mg/L in control and test vessels

Concentration control analysis was not performed because a sufficiently sensitive method for analyses in the required concentration range was not available. However all reasonable efforts were taken to produce a saturated solution of all soluble components of the test substance in test media. Since the test substance is a mixture, the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD 23 [Ref. 7], for tests with chemicals that can not be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures).

All of the test solutions were visibly clear and colorless after preparation and remained so after 24 and 48 hours of exposure in the test. No undissolved test substance was visible and there were no other remarkable observations.

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 4.54 g/L (experiment date: 12 Jul 2016, project number: 50E0789/12E060).
This result is within the range of 3.88 – 7.22 g/L (Ref. 12) and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.
Reported statistics and error estimates:
Tables and/or figures of measured parameters presented in the report were produced using relevant computing software. All reported data values are rounded to the appropriate significant figures based on the precision of the analytical method and/or consistent with the requirements in the pertinent test guideline(s).
For the statistical evaluation of the data and calculation of the EC50 the probit method was used and performed with the commercial software "TOXRAT Professional 2.10” (ToxRat Solutions GmbH, Alsdorf, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance is acutely harmful for aquatic invertebrates.