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
Study period:
From 14 September, 2017 to 17 October, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:6K0001
- Purity test date:97.5%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:The test sample was stored in a dark storage place at room
temperature.
- Stability under test conditions: IR spectrum of the test item after the completion of the experiment
was the same as that before the start of the experiment, and it indicated that the test item was stable
under the storage conditions.
- Solubility and stability of the test substance in the solvent/vehicle: Dimethylsulfoxide;>50 mg/mL,
Acetone;>50 mg/mL

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Stock solution content of 100, 31.6, 10.0, 3.16 and 1.00% (all test levels)
- Sampling method: Another solution sampled separately from the preparation container (at the start
of exposure)
The mixed solution taken out with equal volume of the test solution from the test vessels in each test
level (at the end of exposure)
- Sample storage conditions before analysis: immidiately pretreated

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:test sample and medium were mixed to prepare the nominal concentration of 100 mg/L and
stirred for 48 hours. The test solution was prepared by diluting the stock solution with medium.
- Controls:medium without the test item, which was treated in the same manner as the stock solution

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source; American Type Culture Collection
Strain number; ATCC 22662

Study design

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

Test conditions

Test temperature:
23.0-23.2 derees(Celsius)
pH:
7.8-9.4
Nominal and measured concentrations:
Stock solution content of 100, 31.6, 10.0, 3.16 and 1.00% (2.76, 0.813, 0.271, 0.0774 and 0.0247 mg/
L as measured concentrations)
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilized 500 mL Erlenmeyer flask (closed vessel)
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Volume of the test solution; 100 mL (headspace more than
400 mL)
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate):none
- Initial cells density: 5000 cells/mL
- Control end cells density: 350000 cells/mL(mean)
- No. of organisms per vessel: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates):0
GROWTH MEDIUM
- Standard medium used: yes(OECD TG medium)
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:none
- Photoperiod:continuous illumination
- Light intensity and quality:nominal 90μmol∙m-2∙s-1 (87 to 89 μmol∙m-2∙s-1)
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : every 24 hours after
the start of exposure
- Determination of cell concentrations: [ electronic particle counter; ]
- Chlorophyll measurement:none
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study
- Test concentrations: stock solution content of 100, 32.0, 10.0, 3.20, 1.00, 0.320%
- Results used to determine the conditions for the definitive study:test range
Reference substance (positive control):
not specified
Remarks:
There are periodically reference substance data.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.077 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Unusual cell shape:Some bloat cells were observed in 2.76 mg/L level.
- Colour differences: none
- Flocculation: none
- Adherence to test vessels: none
- Aggregation of algal cells: none
- Any stimulation of growth found in any treatment: 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:none
Results with reference substance (positive control):
- EC50:1.1 mg/L(Potassium dichromate)
Reported statistics and error estimates:
Regarding the growth rate, Bartlett’s test was done to determine the homogeneity of variance for the
data. Then one-way analysis of variance and Dunnett’s multiple comparison test was used to determ
ine the significant difference between the control and the exposure levels. Higher concentration level
than ErC50 on respective parameter were excluded for each analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
ErC50 was 1.43 mg/L and NOEC was 0.0774 mg/L.
Executive summary:

This study was conducted in order to estimate the effect on the test organisms under the solubility of

test item in medium.The test item has a smell. In preliminary study, it was found that the test item of test

solution in closed vessel during the exposure was more stable than the test vessel with gas-permeable

Silicosen®. Therefore, definitive study was conducted using the closed vessel.As a result, ErC50 was

1.43 mg/L and NOEC was 0.0774 mg/L. The test item concentration in test solution was reduced slightly

during the exposure, and that at the end of exposure was 63.6-87.8% of the concentration at the start. So,

the results of study were estimated by geometric mean of the measured concentrations of test item in

test solution during the exposure.

The environmental conditions were within the suitable range. Therefore, it is concluded that this study

complied with the applied test guideline.