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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

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
according to
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Regulation (EC) No. 440/2008
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
Details on test material:
Identification: C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite
Batch: S016318441
Purity: 30%
Physical state/Appearance: Yellow liquid
Expiry Date: 22 January 2018
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
Samples were taken from the control, solvent control and each test group from the bulk freshly prepared test preparation at 0 and 24 hours and from the old or expired pooled replicates at 24 and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

triethylene glycol.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (40 mg) was dissolved in test water and the volume adjusted to 2 liters to give the 20 mg/L test concentration from which a series of dilutions was made to give the required test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 mg test item/L. After preparation of the test solutions, triethylene glycol was added to stabilize the test item in solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- Controls: blank control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): triethylene glycol
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain/clone: in house laboratory cultures
- Feeding during test: no

-Adult daphnids were maintained in 150 mL glass beakers containing 100 mL AAP medium (see Annex 2) 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: 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.

Study design

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

Test conditions

Test temperature:
7.4 - 7.9
Dissolved oxygen:
8.2 - 9.6 mg/L
Nominal and measured concentrations:
Nominal 1.0, 1.8, 3.2, 5.6 and 10 mg test item/L
Measured: 0.13, 0.088, 0.91, 1.8 and 3.9 mg a.i./L
Details on test conditions:
- Test vessel:150 mL glass jars
- Material, size, headspace, fill volume: approximately 100 mL
- jars were covered to reduce evaporation
- Renewal rate of test solution (frequency): renewal every 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

- Source/preparation of dilution water: AAP medium
- Culture medium different from test medium: No
- Intervals of water quality measurement: every 24 hours

- Adjustment of pH: no
- Photoperiod: photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity:(between 200 and 1200 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC50


- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilization was observed at the test concentrations of 0.10 and 1.0 mg/L, however, 100% immobilization was observed at 10 and 100 mg/L. Sub lethal effects of exposure were observed at the test concentration of 100 mg/L. This response was reduced mobility.
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
Details on results:
- Behavioural abnormalities: Sub-lethal effects of exposure were observed in test concentrations of 0.13, 0.91 and 1.8 mg a.i./L. These responses were trapping at surface and reduced mobility, however, no sub lethal effects of exposure were observed throughout the test in the test concentrations of 0.088 and 3.9 mg a.i./L.
- Mortality of control: no mortality in the control and solvent control
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Dose-response test: yes
- ECx: 48h-EC50: 0.75 mg/L
Reported statistics and error estimates:
An estimate of the EC50 value, NOEC and LOEC at 24 hours was given by inspection of the immobilization data.
The EC50 value and associated confidence limits at 48 hours and the slope of the response curve and its standard error were calculated by Logit analysis using Linear Maximum Likelihood regression. The LOEC and the NOEC at 48 hours were calculated using the Step-down Cochran Armitage Test Procedure. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Applicant's summary and conclusion

Validity criteria fulfilled:
48h-EC50: 2.6 mga.i./L (Daphnia magna)
Executive summary:

In the Klimisch 1 GLP study from Sacker (2018) the acute toxicity ofC18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfiteon Daphnia magna was determined in an 48 hour semi-static test according to OECD 202 and EU method C.2. The test was performed with1.0, 1.8, 3.2, 5.6 and 10 mg test item/L and a blank and solvent control. Four replicates with 5 daphnids each were set up. After 48 hours 0, 0, 0. 5, 5 0 and 95% immobilization was observed in the blank control, solvent control and at nominal test concentrations of control, solvent control,1.0,1.8, 3.2, 5.6 and10 mg test item/L, respectively. The nominal concentrations correspond to mean measured concentrations of < LOQ, < LOQ, 0.13, 0.088, 0.91, 1.8 and 3.9 mg a.i./L. The EC50 is 2.6 mg a.i./L based on mean measured concentrations.

The results are considered relevant and reliable for the risk assessment.