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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
Study period:
2017-08-14 to 2018-03-02
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:
Diethyl ethoxymethylenemalonate
EC Number:
EC Name:
Diethyl ethoxymethylenemalonate
Cas Number:
Molecular formula:
diethyl ethoxymethylenemalonate
Test material form:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: JC170405
- Expiration date of the lot/batch: 2018-04-12 (retest date)
- Purity test date: 2017-04-18 (certificate of analysis release date)

- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not available

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t=0, t=24h and t=48h. 1.4 mL of volume was taken from the approximate centre of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: The samples were stored in a freezer. Additionally, reserve samples of 1.4 mL were taken for possible analysis.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest test concentration of 100 mg/L applying approximately 10 minutes of magnetic stirring to accelerate the dissolution of the test item in test medium. The lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water-flea
- Source: in-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): < 24 h (from parental daphnids of more than 2 weeks old, maximum age 4 weeks)
- Method of breeding: 250 newborn daphnids (i.e. less than 3 days old) are placed into 5 liters of medium in an all-glass culture vessel. Medium is M7. After 7 days of cultivation half of the medium is renewed twice a week.
- Feeding during cultivation: fresh water algae, daily
- Feeding during test: no

- Acclimation period: not relevant

Study design

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

Test conditions

180 mg/L as CaCO3
Test temperature:
At t=0h: 7.8
At t=24h (old): 7.4 - 8.0
At t=24h (fresh): 7.8 - 8.0
At t=48h (old): 7.4 - 8.0
Dissolved oxygen:
At t=0h: 9.2 - 9.5
At t=24h (old): 9.0 - 9.2
At t=24h (fresh): 9.2 - 9.3
At t=48h (old): 9.3 - 9.8
not relevant
not reported
Nominal and measured concentrations:
Range finder:
Nominal concentrations: Control, 0.1, 1.0, 10 and 100 mg/L
Measured concentrations (mg/L) at t=0h: n.a., n.a., n.a., 1.50, 79.5
Measured concentrations (mg/L) at t=48h: n.a., n.a., n.a., 0.020*, 1.2*
n.a. = not analysed
* estimated value, calculated by extrapolation of the calibration curve

Final test (1st full test):
Nominal concentrations: Control, 4.6, 10, 22, 46 and 100 mg/L
Measured concentrations (mg/L) at t=0h: n.d., 0.546, 1.35, 5.98, 23.0, 60.3
Measured concentrations (mg/L) at t=48h: n.d., 0.0076*, 0.0164, 0.0526, 0.196, 0.720
n.d. = not determined
* estimated value, calculated by extrapolation of the calibration curve

Final test (2nd full test):
Nominal concentrations: Control, 10, 18, 32, 56 and 100 mg/L
Measured concentrations (mg/L) at t=0h (fresh): n.d., 9.54, 17.3, 30.8, 51.0, 90.3
Measured concentrations (mg/L) at t=24h (old): 0.014*, 0.710, 1.33, 2.56, 4.70, 9.1*
Measured concentrations (mg/L) at t=24h (fresh): 0.0164, 7.87, 14.7, 26.3, 49.2, 85.8
Measured concentrations (mg/L) at t=48h (fresh): n.d., 0.852, 1.64, 3.09, 5.76, 10.9
n.d. = not detected
* estimated value, calculated by extrapolation of the calibration curve
Details on test conditions:
- Test vessel: beaker
- Material, size, headspace, fill volume: 60 mL, glass beaker filled with 50 mL
- Aeration: not during the test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Test medium: adjusted ISO medium

- Photoperiod: 16h light daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility was defined as those organisms which are not able to swim within 15 seconds after gentle agitation of the test container and was monitored after 24 and 48 hours of continuous treatment with the test substance.

- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L (setup: combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
7.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% C.L.: 6.9 - 8.3 mg/L
Details on results:
- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: none
- 1st full test:
No biologically relevant immobility was observed in the control and at the three lowest test concentrations throughout the test duration, while effects were complete after already 24 hours of exposure at the highest concentration tested. Chemical analysis of both the combined limit-range finding test and the 1st full test showed a high decrease in the measured exposure concentrations during the test. Additionally, concentrations
measured at the start of the test were not at the level of nominal. This was unexpected since the test item was completely soluble in test medium, and suggested a possible instability of the test item during storage period.
Based on these considerations, it was decided to perform a 2nd full test with renewal of test solutions after 24 hours of exposure and analysis of the samples on the day of sampling for determination of exposure concentrations.
- 2nd Full test
No immobility was observed in the control and at the lowest two concentrations throughout the test duration. At the end of the test, 85% of the daphnids were immobile at an average concentration of 8.9 mg/L, while complete immobility was observed at the two highest test concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Mortality: no
- EC50/LC50: 48-h EC50 = 0.54 mg/L. The sensitivity of the daphnids was within the range determined with the historical data collected at Charles River Laboratories Den Bosch.
Reported statistics and error estimates:
Calculation of EC50:
The 24 and 48h-EC50 values were calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (average exposure) using the maximum likelihood estimation method.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
The acute toxicity of EMME to Daphnia magna was determined in a 48 hour semi-static test according to OECD guideline 202. The 48-h EC50 was 7.5 mg/L based on average exposure concentrations (95% confidence interval between 6.9 and 8.3 mg/L). The results of the test can be considered reliable without restriction.