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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-07-02 to 2019-07-05
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
not specified
Principles of method if other than guideline:
The pH of the control group increased by more than 1.5 units during the test. It is mentioned in the Test Guideline that the pH of the control medium should not increase by more than 1.5 units during the test. Generally, the concentration of bicarbonate ion is decreased by carbon dioxide assimilation in algae. If there is very little air exchange (supply of carbon dioxide) in closed system, the pH is increased. Since algae grew exponentially in the control group, it was considered that pH increase in this test was acceptable.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Concentrations: All test solutions
- Sampling method: Collect a proper quantity of the test solution or the test cultures from the middle layer of the vessel for collecting analysis sample. The concentration of the test substance in the analysis samples were adapted to the range of calibration curve by changing the ratio of ultra pure water and the test solution or the test culture. For the test groups where the concentration of the test substance were expected within the range of calibration curve, 10 mL of the test solution or the test culture (0 mL of ultra pure water) were analyzed.
Details on test solutions:
- Method: 109 µL (equivalent to 100 mg) were collected with a microsyringe, diluted with 1000 mL OECD medium and stirred for 1 hour.
- Controls: pure OECD medium
- Test concentration separation factor: 2.19
- Evidence of undissolved material: Test solutions before inoculation were colorless, and showed no suspended solids, no floating solids, and no precipitates in all test groups.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Unicellular green algae
- Strain: ATCC22662
- Source: American Type Culture Collection
- Method of cultivation: Algae have been maintained by period subculture using Gorham medium. Algae are kept under sterile condition. Sterile test has been performed periodically, approximately every six months, to confirm that the algae are not contaminated.

- Acclimation period: 4 days
- Culturing conditions (same as test or not): Same
- Any deformed or abnormal cells observed: No
Test type:
Water media type:
OECD medium
Limit test:
Total exposure duration:
72 h
Test temperature:
22 °C, variation range over the exposure period is within ± 2 °C
Test start: 7.7 (all solutions)
Test end: 7.9-10.4
The pH of the control group increased by more than 1.5 units during the test.
Nominal and measured concentrations:
Nominal: 1.0, 2.2, 4.8, 10, 23 and 50 mg/L
Measured (mean): 0.848, 1.88, 3.96, 8.65, 18.8 and 41.8 mg/L
Individual measured values were between 68 and 97 %
Details on test conditions:
- Test vessel: 500 mL Erlenmeyer flask with stopper
- Type: closed
- Fill volume: 100 mL/vessel
- Aeration: Shaking (100 rpm)
- Initial cells density: 5000 cells/mL
- Control end cells density: 551000 cells/mL (mean of 6 replicates)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

- Standard medium used: yes, OECD medium

- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 65 µE/m2/s with white fluorescent lamp (on the surface of test culture)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (1 mL of test culture was suspended in 9.0 mL of electrolyte to count the algal cells)

- Spacing factor for test concentrations: 2.19
- Range finding study:
- Test concentrations: 1.0, 5.0, 50 and 100 mg/L

-Details on culturing apparatus used: AGP-150RL, Itoh Seisakusho, Ltd.
Reference substance (positive control):
Potassium dichromate
72 h
Dose descriptor:
Effect conc.:
1.88 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
Key result
72 h
Dose descriptor:
Effect conc.:
18.4 mg/L
95% CI:
18.1 - 18.6
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no, in the two highest test item concentrations the number of cells was not enough for observation.
- Colour differences: in the two highest test item concentrations the color of test cultures showed no tendency to get greenish

- Other:
- Any stimulation of growth found in any treatment: yes, 1.6% in the lowest test item concentration
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No. The most likely reason for the decrease of its concentration was volatilization from the test solution and test culture.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 0.737 mg/L (June 2019), 0.802 (average since June 2000)
- Other: Growth inhibition test of a reference substance (potassium dichromate) has been conducted periodically (approximately every six months) and 72-hour medium growth inhibition concentrations were estimated by comparison of growth rates (ErC50).
Reported statistics and error estimates:
EC50 is determined by the least squares linear regression analysis of points recognized as straight in the concentration-inhibition curves. 95 % confidence limits are calculated, if possible.

The NOECr value was determined by analysis of variance (ANOVA), Dunnett test, subsequent to Bartlett test for homogeneity of variances. The significance level for all tests was set at a = 0.05, except Bartlett test, which was set at a = 0.01.

Validity of the test

The biomass in the control cultures increased exponentially above 16-fold during 72-h culture. The mean CV for section-by-section specific growth rates in the control cultures and the CV of average specific growth rates during whole test period in replicate control cultures did not exceed 35 % and 7 %, respectively. This test is valid because these results met the test validity criteria. 

Temperatures, Light Intensities and Revolutions in the Incubation Chamber

Exposure Period (Hours)Temperature (°C)Light Intensity (µE/m2/s)Revolution (rpm)

pH Values of Test Solutions and Test Cultures

Test Group0 hour72 hoursVessel No.
Conc. 17.710.21
Conc. 27.710.31
Conc. 37.710.41
Conc. 47.79.81
Conc. 57.77.91
Conc. 67.78.11


Validity criteria fulfilled:
For details see 'Any other information on results incl. tables'
The ErC50 was determined to be 18.4 mg/L.

Description of key information

In the GLP study with the green algae Raphidocelis subcapitata the ErC50 was determined to be 18.4 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
18.4 mg/L

Additional information