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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with relevant OECD TG

Data source

Reference Type:
secondary source
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 201 (Alga, Growth Inhibition Test)
yes The initial cell density was 10000 cells/mL instead of 2000 to 5000 cells/mL, but this has no influence on the test results
GLP compliance:

Test material

Constituent 1
Reference substance name:
Details on test material:
- Name of test material (as cited in study report): Menthone-L/D-Isomenthone
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: all test concentrations were sampled
- Sampling method: duplicate sampling at the beginning of the test (without algae) and at the end of the test (containing algae)
- Sample storage conditions before analysis: aliquots of 10 mL were diluted with 3 mL acetonitrile and samples were stored deep-frozen until analysis

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test medium with the highest nominal test concentration was prepared by dissolving an appropriate amount of test substance in water in a glass stoppered 1000 mL Erlenmeyer flask using intense stirring for 20 minutes at room temperature. The lower test concentrations were then obtained by diluting the stock solution.
- Controls: test vessels with algae and medium, but without test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subspicata
- Strain: No 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures, SAG Institute for Plant Physiology, University of Göttingen, Germany
- Age of inoculum (at test initiation): an inoculum culture was set up four days before the start of exposure; the inoculum culture was diluted threefold one day before the start of the test
- Method of cultivation: standard conditions according to guideline

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
not applicable

Test conditions

Test temperature:
22 °C
Initial pH was 7.5; pH value of the test medium was in the range from 7.8 to 8.4
Nominal and measured concentrations:
Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
- Test vessel: all
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks were completely filled with about 60 mL test medium to minimise the air space in the flasks
- Aeration: no
- Initial cells density: 10000 cells/mL
- Control end cells density: 1033333 cells/mL
- No. of vessels per concentration (replicates): 3 per concentration
- No. of vessels per control (replicates): 6 controls

- Standard medium used: yes

- Source/preparation of dilution water: AAP medium according to guidelines; NaHCO3 was added to provide an additional carbon source

- Sterile test conditions: yes
- Adjustment of pH: 6 mmol/L HEPES buffer (corresponding to 1430 mg/L) was added to the test medium to keep the pH value in the closed vessels as stable as possible
- Photoperiod: continuous
- Light intensity and quality: mean intensity was 6300 Lux (range from 5890 to 6670 Lux) from fluorescent tubes (Philips TLD 36W-1/840)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : development of biomass and growth rate
Reference substance (positive control):
potassium dichromate (tested twice per year)

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
58 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 56-61 mg/L
72 h
Dose descriptor:
Effect conc.:
31 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 29-33 mg/L
Results with reference substance (positive control):
The result of the latest positive control test performed in February 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.97 mg/L (Harlan Study Number D50552), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.71-1.7 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
The substance has relatively low toxicity to the freshwater alga Pseudokirchneriella subspicata with an EC50 value of 58 mg/L over 72 hours.
Executive summary:

The toxicity of the test substance Menthone-L / D-Isomenthone to the freshwater alga Pseudokirchneriella subspicata was studied under GLP over a period of 72 hours in accordance with OECD TG 201. The tests were performed with standard medium prepared in accordance with the guideline in closed bottles due to the volatility of the test substance avoiding air space in the bottles. The test temperature was 22 °C. Tests were conducted under continuous illumination with an average intensity of 6300 Lux. The inoculum was set up four days before the experiment and the cultures were diluted one day before start of exposure to ensure that the cells were in the exponential growth phase. The initial nominal cell density was 10000 cells/mL. Nominal concentrations of 0.32, 1, 3.2, 10, 32 and 100 mg/L were tested. These concentrations were confirmed by HPLC-UV analysis, showing the stability of test substance over the test period and good agreement of nominal and analytical test concentrations. The EC50 value for the inhibition of growth rate and yield was calculated by Weibull analysis. The LOEC and NOEC were determined by comparing the growth rate and yield obtained with the test concentrations to the control values using the Williams t-test and Welch t-test, where appropriate. The EC50 value for growth rate was 58 mg/L (based on nominal concentrations), the EC10 was 31 mg/L and the NOEC was 10 mg/L after 72 hours.

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