Registration Dossier

Administrative data

short-term toxicity to fish
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 guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
according to guideline
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4-[1-methyl-2-[(1-oxooctadec-9-enyl)amino]ethyl] 2-sulphonatosuccinate
EC Number:
EC Name:
Disodium 4-[1-methyl-2-[(1-oxooctadec-9-enyl)amino]ethyl] 2-sulphonatosuccinate
Cas Number:
Molecular formula:
disodium 4-[1-methyl-2-(oleoylamino)ethoxy]-4-oxo-2-sulfonatobutanoate
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:
Water samples were taken from the control, solvent control and the test group, from fresh and old media throughout the 96 Hour test period, for quantitative analysis. Duplicate samples at 0 and 72 hours (fresh media) and 24 and 96 hours (old media) were taken and stored frozen for further analysis if necessary.

Test solutions

triethylene glycol, at a concentration of 100 µL/L
Details on test solutions:
- Method: A nominal amount of test item (78.3 mg) was dissolved in test water and the volume adjusted to 500 mL to give a 47 mg a.i./L stock solution. The stock solution was inverted several times to ensure adequate mixing and homogeneity. An amount of the test item stock solution (80 mL) was added to a final volume of 4 liters of test water to give the test concentration of 0.94 mg a.i./L. Immediately after preparation, the test solution was stabilized by the addition of 400 µL triethylene glycol to give a 100 µL test concentration.
- Controls: blank control: dilution water only. A solvent control was prepared by dispersing 400 µL of triethylene glycol to 4 liters of AAP medium to give the test concentration of 100 µl/L.
- 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):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: Zebra fish (Danio rerio)
- Source: Fish were obtained from Envigo and maintained in house since 15 January 2018. Fish were maintained in a glass tank with an activated carbon recirculation filter system. Fish were acclimatized to test conditions from 06 March 2018 to 13 March 2018.
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): the fish had a mean standard length of 2.1 cm (standard deviation = 0.2) at the end of the definitive test.
- Weight at study initiation (mean and range, SD): mean body weight of 62.0 mg (standard deviation = 22.7) at the end of the definitive test.
- Maintenance of the brood fish: comparable to test conditions

- Acclimation period: THe fish were adapted to the test concditions prior to testing.
- Acclimation conditions (same as test or not): comparable to test conditions
- Type and amount of food during acclimation: The stock fish were fed commercial GM 300 which was discontinued approximately 26 hours prior to the start of the definitive test.
- Feeding frequency during acclimation: commercial GM 300
- Health during acclimation (any mortality observed): There was no mortality in the 7 days prior to the start of the test.

Study design

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

Test conditions

Test temperature:
7.2 to 7.9
Dissolved oxygen:
7.4 - 8.8
Nominal and measured concentrations:
Nominal: control, solvent control, 0.94 mg a.i./L
geometric mean measured: 0.85 mg a.i./L
Details on test conditions:
- Test vessel: 5 liter glass exposure vessels
- Type (delete if not applicable): The test vessels were then covered to reduce evaporation
- Material, size, headspace, fill volume: 4 liters of test media
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution (frequency): renewal every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates):1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate:

- Source/preparation of dilution water: The test was conducted in AAP Medium. The AAP was prepared according to OECD Test Guideline No. 201.
- Culture medium different from test medium: no

- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : LC50

- Spacing factor for test concentrations: NA
- Justification for using less concentrations than requested by guideline: limit test
- Test concentrations: 0.94 mg a.i./L.
- Results used to determine the conditions for the definitive study: In accordance with the recommendations of REACH, the test was conducted according to the threshold approach recommended by ECHA. Using this approach the lowest EC50 value from either the Algal Growth Inhibition study or Acute Toxicity to Daphnia magna study is set as the threshold concentration and a “Limit test” is conducted at this threshold concentration. If no mortalities are observed this indicates that fish are not the most sensitive species and that the LC50 is greater than the threshold concentration. The ErC50 value obtained from the Algal Growth Inhibition study (Envigo study number FD55TW) was the lowest of these two EC50 values and hence the test was conducted at a single concentration of 0.94 mg a.i./L.

Results and discussion

Effect concentrationsopen allclose all
96 h
Dose descriptor:
Effect conc.:
>= 0.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
96 h
Dose descriptor:
Effect conc.:
> 0.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: none
- Mortality of control: none
- 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

Applicant's summary and conclusion

Validity criteria fulfilled:
The 96-hour NOEC and LC0 was >= 0.85 mg a.i./L (0.94 mg a.i./L nominal) and the LC50 was > 0.85 mg a.i./L (0.94 mg a.i./L nominal)
Executive summary:

In the Klimisch 1 GLP study from Sacker (2018) the acute toxicity of C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite to zebra fish (Danio rerio) was determined in a 96hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992). The following nominal concentration was tested: 0.94 mg a.i./L. Additionally, a control (test water without test item) and a solvent control was tested in parallel.

The measured concentrations of the test item in the test media of the test concentrations of 0.94 mg a.i./L was between 111 and 115% of the nominal values at the start of the test and 45to 102% at the end of each renewal period. Thus, the correct dosage of the test item was confirmed. Due to the decline of the test material concentration. Therefore, the biological results were based on teh mean measured concentration of 0.85 mg a.i./L.

In the blank control, solvent control and at the test concentration of nominal 0.94 mg a.i./L, all fish survived until the end of the test and no visible abnormalities were observed in the test fish. Thus, the 96hour NOEC and LC0 was >= 0.85 mg a.i./L (0.94 mg a.i./L nominal) and the LC50 was > 0.85 mg a.i./L (0.94 mg a.i./L nominal).

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