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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
other: experimental data on similar substance
Adequacy of study:
key study
Study period:
2 May 2016 to 6 May 2016
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
according to guideline
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
No analytical method is available for the multi-constituent test substance. See 'Details on analytical methods' for further information.
Details on test solutions:
- Method: The test solution (100 mg/L) was prepared by directly adding 100.02 mg (first interval) and 100.04 mg (second interval) of test substance to 1L of test medium and stirring for about two days. Undissolved test substance was removed by filtration with a membrane filter (Whatman OE66, pore width 0.2 μm). The first 50 - 100 mL of filtered solution was discarded (used to condition the filter). The solution was not filtered to dryness. Several mL of test solution remained over the filter. After filtration the solution was stirred for 10 min to ensure adequate dissolved oxygen levels. The control was treated in the same way. Fresh test solutions were prepared daily.
- Evidence of undissolved material: After 48 h stirring the test solution with test substance appeared dark and undissolved test substance was visible. After filtration no undissolved test substance was visible. The test solution with the test substance appeared clear and violet.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- 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.
- Health: Only young from healthy cultures without signs of stress are used for testing.
- Feeding during test: No

- Acclimation period: None. The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, and temperature (20 ± 1°C).
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Total hardness: 2.44 mmol/L
Test temperature:
19.7 - 20.1 °C
- First 24-h interval: 7.9 (start); 7.9 (end)
- Second 24-h interval: 8.0 (start); 7.8 (end)
Dissolved oxygen:
- First 24-h interval: 8.8 - 9.1 mg/L (start); 9.0 - 9.1 mg/L (end)
- Second 24-h interval: 8.6 - 8.9 mg/L (start); 8.5 - 8.9 mg/L (end)
586 μS/cm
Nominal and measured concentrations:
- Nominal loading rate: 0 (control) and 100 mg test substance/L
Details on test conditions:
- Test vessel: Numbered glass beakers (nominal volume 100 mL)
- Fill volume: 50 mL
- Renewal rate of test solution: static renewal, the renewal period was 24 hours
- Biological loading: 5 animals / test vessel (0.1 animals/mL)
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Age at the start of exposure: <24 hours (at least 3rd brood progeny)
- Application: 50 mL test solution per test vessel. 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 daphnids were inserted with a small amount of dilution water by a pipette. For test renewal, daphnids in each replicate were captured with a pipette and transferred into test vessels with fresh test solution.

- Test medium: Elendt M4
- Culture medium different from test medium: No.
- Intervals of water quality measurement: 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. Dissolved oxygen and pH were measured at the start of the exposure and at the end of the exposure in replicate 1 of all test concentrations and the control. The TOC (Total organic carbon) values of the test concentration and the control were analyzed at the start and the end of one renewal interval as an additional water quality parameter and stored in a refrigerator.

- Light intensity: About 234 - 694 lux at a wave length of 400 - 750 nm
- Photoperiod: 16 hours light : 8 hours darkness
- Diet: No feeding during the exposure period.

- Each test vessel was visually checked for immobilized daphnids after 0, 24 and 48 hours. In addition any abnormal behavior or appearance was documented.

- Results used to determine the conditions for the definitive study: In a preliminary test a concentration of 263 mg/L caused no immobilisation or symptoms after 48 hours. According to the OECD test guideline, the highest suggested test concentration is 100 mg/L for a limit test.
Reference substance (positive control):
Sodium chloride
Key result
48 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
loading rate
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
100 mg/L
Nominal / measured:
Conc. based on:
test mat.
loading rate
Basis for effect:
Details on results:
No immobilization of daphnids or behavioural abnormalities were observed.

No remarkable observations (clear test medium) were recorded for the control group. At the test substance loading rate of 100 mg/L coloration caused by the test substance (clear, violet) was observed at all time intervals (0, 24 and 48 hours).

- Water quality: TOC concentrations in the control at the start and end of the first 24-h interval were 1.5 mg/L and 1.5 mg/L, respectively. TOC concentrations in the 100 mg/L test substance loading rate at the start and end of the first 24-h interval were 10.5 mg/L and 11.3 mg/L, respectively. The higher TOC in the 100 mg/L treatment demonstrates that the test organisms were exposed to soluble organic components from the test substance and that concentrations were consistent over the renewal interval.

Results with reference substance (positive control):
The EC50(48h) of the reference substance sodium chloride was 5.08 g/L (experiment date: 12 Apr 2016). 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.
* Santos M.A.P.F., et al. (2007), Sensitivity of Four Test Organisms (Chironomus xanthus, Daphnia magna, Hydra attenuata and Pseudokirchneriella subcapitata) to NaCl: an Alternative Reference Toxicant, J. Braz. Soc. Ecotoxicol., v. 2, n. 3, 229-236
Reported statistics and error estimates:
Since the study is designed as a limit test, the data were not appropriate for the use of computerized methods for the calculation of ECx values. Any LOEC, NOEC and ECx values are estimated based on expert judgment in accordance with test guideline recommendations.
The test endpoints were calculated and reported based on the nominal test substance loading rate.
Validity criteria fulfilled:
Not toxic to aquatic invertebrates
EC50 > 100 mg/l

Description of key information

The 48-h EL50 value is >100 mg/L (based on nominal test substance loading rate) in aquatic invertebrates (D. magna) on the analogue substance. Based on the read across considerations the same resutls apply to the target substance

Key value for chemical safety assessment

Additional information

The acute toxicity to aquatic invertebrates was determined in a study according to OECD TG 202 and in compliance with GLP criteria (Stahl, 2017)on analogue substance. In this study daphnids (D. magna, 20 per concentration) were exposed to nominal loading rates of 0 (control) and 100 mg test substance/L for 48 hours under semi-static conditions (test medium renewal every 24 hours). Test concentrations were not analytically verified because a reliable method for analysis in the required concentration range could not be developed. Since all reasonable efforts were taken to produce a WAF of the test substance in the test media following the guidance of OECD 23, results are based on the loading rate. Immobilization and appearance of daphnids and test solutions was recorded after 0, 24 and 48 hours exposure. No immobilization or sub-lethal effects were observed in the control and 100 mg/L groups. The test substance loading rate of 100 mg/L was observed to be a clear, violet solution throughout the complete test. Based on these findings the 48-h EL50 value was determined at >100 mg/L.