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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:
8-12 November 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted following the GLP principles, and according to the OECD and Japanese guidelines.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to guideline
other: "Alga growth inhibition test, Daphnia sp. acute immobilisation test, and fish acute test" Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2, Kanpokihatsu No. 031121002, November 21, 2003; the latest revision November 20, 2006
GLP compliance:

Test material

Constituent 1
Reference substance name:
Details on test material:
- Name of test material (as cited in study report): E-C104
- Substance type: blue powder
- Physical state: solid
- Lot/batch No.: MB-1
- Storage condition of test material: room temperature, shaded, air-tight

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: 0; 10; 18; 32; 56 and 100
- Sampling method: 1.0 ml from the middle layer of all vessels and mix
- Sample storage conditions before analysis: not reported

Test solutions

Details on test solutions:
The stock solution was prepapred by addition of 100 mg test substance to 1000 ml dilution water, resulting in a concentration of 100 mg/L. From this stock solution, 100 ml were taken and added in a 300 ml Erlenmeyer flask with air-permeable silicon cap. this was considered as the highest etst concentration of 100 mg/L. Next, by diluting the stock solution, series dilutions in 100 ml medium separated by a factor of 1.78 were prepared.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection

- Acclimation period: 5-9 November 2010
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Study design

Test type:
other: static and shaking (100 rpm)
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

50 mg NAHCO3/L
Test temperature:
21.7 - 22.4
7.8 - 9.1
Nominal and measured concentrations:
Nominal: control, 10, 18, 32, 56 and 100 mg/L
Measured: 8.55; 16.3, 30.9, 56.0 and 103 mg/L
Details on test conditions:
- Test vessel: Erlenmeyer flask
- Type (delete if not applicable): open; air permeable cap
- Material, size, headspace, fill volume: glass, 300 ml
- Initial cells density: 5 x 10^3 cells/ml algae of exponential growth phase
- Control end cells density: 237 X 10^4 cells/ml (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: yes, Gorham

- Sterile test conditions: yes, every 6 months
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 110 to 120 uE/m2/s, fluorescent white

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 50% growth inhibition concentration and no observed effect concentration (0-72 h)
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no

- Spacing factor for test concentrations: for the final test only 0; 10; 18; 32; 56 and 100 mg/L. The test solutions were colorless in control and blue
- Range finding study: yes
- Test concentrations:control; 0.10; 1.0; 10 mg/L
- Results used to determine the conditions for the definitive study: 36.0 % inhibition was observed at 100 mg/L in the range finding test. As the test substance was of blue color, additional studies were conducted on effects of light absorption or interception caused by the substance on algal growth. the methods used were as follows:
1) Growth of algae under the light filtered through petri dishes filled with the 100 mg/L test solution (concentration group) was measured to investigate the effects the effect of light absorption or interception by test solutions and test cultures. Light filtered through petri dish filled with test medium was used as control.
2) The effect of light absorption or interception by test solution and test cultures was investigated by reducing the test solution volume, thus reducing the light path. One replicate 300 ml Erlenmeyer falsks were each filled with 50; 75 or 100 ml of test solutions at EC10 (10 mg/L) or EC40 (100 mg/L).

both experiments were conducted under the same conditions as the final test.

The results of the experiment 1) indicate 36.4 % growth inhibition in the 100 mg/L group compared with the control group. the light intensity at the bottom of the concentration group was 11 uE/m^2/s as compared with 43 uE/m^2/s of the control group.

Regarding experiment 2), In the Erlenmeyer flask filled with 50 ml test medium and 100 mg/L, a 11.4 % growth inhibition was measured and the light intensity at the bottom of the flask was 27 uE/m^2/s. The results of test conducted 100 ml test medium and 100 mg/L test substance indicated 15.3 % growth inhibition and a measured the light intensity at bottom of the flask of 21 uE/m^2/s. In case of the experiments conducted with 10 mg/L test substance in 50 ml and 100 ml media, the results indicated a percentage inhibition of -2.0 and -1.5 with the measured light intensities at the bottom of 70 uE/m2/s and 58 uE/m2/s, respectively.

The above results indicate a limitation of photosynthetic activity of the algae caused by the absorption or interception of lightin the test solutions colored by the test substance.
The final test was conducted in 100 ml and light intensity of 110-120 uE/m2/s in order to reduce the growth inhibition caused by the coloration of test solutions.
Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
the measured concentration was within 20% of the nominal
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
16.3 mg/L
Nominal / measured:
Conc. based on:
test mat.
NOECr was determined by ANOVA, Williams test, subsequent to Barlett test for homogenicity of variances
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- EC50: 0.698 mg/L (95% C.I. 0.660-0.739 mg/L), exposure period 8-11 Jun 2010
- Other: the average values of ErC50 since June 2000 were 0.815 +/- 0.085 mg/L, n=21
Min-max = 0.687 - 0.965 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance was a water-soluble and colored therefore causing the coloration of the test solutions. Growth inhibition resulting from the attenuation of light essential for the algal growth in the test solution was observed. the EC50 was > 100 mg/L.