Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
yes
Remarks:
see Principles and method other than guideline
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
see Principles and method other than guideline
Principles of method if other than guideline:
Due to a technical oversight, the pH of the pooled solvent control cultures was not determined at 72 hours. This was considered to have had no adverse effect on the outcome of the test given that the pH of the pooled control test cultures where a similar increase in cell density occurred did not deviate by more the 1.5 pH units after 72 hours exposure. It was therefore considered that the pH of the solvent control would not have deviated by more than 1.5 units after 72 hours either.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
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:
yes
Details on sampling:
Samples were taken from the solvent control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
yes
Remarks:
triethylene glycol
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A nominal amount of test item (167 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg ai/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 3.2, 1.0, 0.32 and 0.10 mg ai/L. An aliquot (500 mL) of each of the 0.10, 0.32, 1.0, 3.2 and 10 mg ai/L stock solutions was stabilized by the addition of 50 µL of triethylene glycol prior to inoculation with algal suspension (5.0 mL).
- Controls: blank cobtrol 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. A white precipitate, determined to be bacterial cells was observed in all preparations at 72 hours.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: liquid

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
24 ± 1 ºC
pH:
pH 8.2 at 0 hours to pH 7.7 at 72 hours.
Nominal and measured concentrations:
nominal control, solvent control, 0.10, 0.32, 1.0, 3.2 and 10 mg ai/L
geometric mean measured: 0.078, 0.25, 0.77, 2.3, 7.3 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): The flasks were plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: 5000 cells/mL
- Control end cells density: Control: 5.32E+05 cells/mL, solvent control 5.41E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24h/day
- Light intensity and quality: intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter or haemocytometer and light microscope
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in the control, solvent control or test cultures at 0.078 mg ai/L. Cell clumping was observed in the 0.25 and 0.77 mg ai/L test cultures, cell clumping and small cells were observed in the 2.3 mg ai/L test cultures whilst no viable cells were observed in the 7.3 mg ai/L test cultures.
- 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. A white precipitate, determined to be bacterial cells was observed in all preparations at 72 hours.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the solvent control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
72h-ErC50: 0.94 mg a.i./L
72h ErC10: 0.18 mg a.i./L
Executive summary:

In the Klimisch 1 GLP study from Vryenhoef (2018) the toxicity of C18 unsaturated fatty acids, reaction products with 1-aminopropan-2-ol, maleic anhydride and sodium bisulfite to Pseudokirchneriella subcapitata was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201 and EU Method C.3. The nominal concentrations of the test item of 0.10, 0.32, 1.0, 3.2 and 10 mg active ingredient (ai)/L were tested in parallel with a control and a solvent control. Dose verification analysis was performed. The measured concentrations of the test item were 0.078, 0.25, 0.77, 2.3 and 7.3 mg ai/L. Therefore, the biological results were based on geometric mean measured test concentrations. In order to determine the growth of the cultures, the cultures were measured with the Coulter counter. The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC10 and ErC50 are 0.18 and 0.94 mg a.i./L based on geometric mean measured test concentrations.

This information is considered to be relevant and reliable for the further risk assessment.