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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-14 to 2018-04-06
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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
according to guideline
EU Method C.3 (Algal Inhibition test)
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:
- Analytical purity: 99.5 %
- 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 were taken before the start of the test and after 24h and 72 hours from each test medium and from the control. Additionally, reserve samples of 1.4 mL were taken for possible analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer until 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 10 to 20 minutes of magnetic stirring (final vs combined limit/range-finding, respectively) 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.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

- Acclimation: Pre-culture 3 days before start of the test

Study design

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

Test conditions

24 mg CaCO3/L
Test temperature:
21-24 °C
t=0h: 8.1-8.3
t=72h: 8.0-8.1
Dissolved oxygen:
not applicable
not relevant
not relevant
Nominal and measured concentrations:
Combined limit-range-finding test
nominal test concentrations: 0, 0.1, 1.0, 100 mg/L
measured test concentration t = 0 h (mg/L): n.m., n.m., 0.0062°, 51.9
measured test concentration t = 72 h (mg/L): n.d., n.m. Not measured
n.d. Not detected
° estimated value, calculated by extrapolation of the calibration curve
* No test item was found in the 100-times diluted nominal 100 mg/L sample after 72 hours. The sample was, therefore, reanalysed applying a minimal dilution factor. The observed response was smaller than that of the lowest calibration standard. By the time of reanalysis, the test item was not stable in end solution anymore. The response of the lowest calibration solution was reduced for approximately 50%. Therefore, the results is reported as smaller than the limit of quantification (LOQ; corresponding to 0.1 mg/L i.e. the low concentration of the QC samples).

1st full test:
nominal test concentrations: 0, 4.6, 10, 22, 22*, 46, 100 mg/L
measured test concentration t = 0 h (mg/L): n.d., 0.0572, 0.524, 2.58, 2.07, 9.62, 36.2
measured test concentration t = 24 h (mg/L): n.d., n.d., n.d., 0.0169, 0.0194, 0.124, 0.810
measured test concentration t = 72 h (mg/L): n.d., n.d., n.d., n.d., n.d., n.d., 0.0023°
n.d. Not detected
* without algae
° estimated value, calculated by extrapolation of the calibration curve

2nd Full test:
nominal test concentrations: 0, 1, 3.2, 10, 10*, 32, 100 mg/L
measured test concentration t = 0 h (mg/L): n.d., 0.828, 2.68, 8.14, 8.28, 26.8, 77.8
measured test concentration t = 24 h (mg/L): n.d., 0.0181, 0.0887, 0.158, 0.195, 0.928, 5.20
measured test concentration t = 48 h (mg/L): n.d., 0.0014°, 0.0020°, 0.0046°, 0.0067°, 0.0311, 0.235
measured test concentration t = 72 h (mg/L): 0.0015°, 0.0014°, 0.0016°, 0.0016°, 0.0017°, 0.0027°, 0.0158
n.d. Not detected
* without algae
° estimated value, calculated by extrapolation of the calibration curve
Details on test conditions:
- Test vessel: 100 mL all-glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 251 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the test microscopic observations were performed on the undiluted SS to observe for any abnormal appearance of the algae.

- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 86 to 88 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- Effect calculated parameters: specific growth rate and yield

- Spacing factor for test concentrations: x 2.2
- Range finding study: yes
- Test concentrations: 1.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
72 h
Dose descriptor:
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L: 0.13-0.18 mg/L
Key result
72 h
Dose descriptor:
Effect conc.:
0.029 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- 72-h EC50 for yield inhibition = 0.067 mg/L (95% C.L: 0.06-0.075 mg/L)
- 72-h NOEC for yield inhibition = 0.013 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 48-h EC50 (growth rate) = 0.59 mg/L (95% C.L. 0.52 to 0.66 mg/L)
Reported statistics and error estimates:
- For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Growth rate: Williams Multiple Sequential t-test Procedure, Yield: Step-down Jonckheere-Terpstra Test Procedure; α=0.05, one-sided, smaller).
- Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.
- The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance EMME according to the OECD guideline 201 (GLP conditions). The EC50 for growth rate (72h-ErC50) based on TWA concentrations was 0.25 mg/L with a 95% confidence interval ranging from 0.23 to 0.28 mg/L and the 72-h NOEC for growth rate was 0.029 mg/L. The results of the test can be considered reliable without restrictions.