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Diss Factsheets

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:
07-apr-2009 to 10-apr-2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
according to guideline
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
-- Name of test material (as cited in study report): Custom Red #2
- Substance type: Violet powder
- Physical state: Solid
- Lot/batch No.: C50APR0805D
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
Details on sampling:
Not applicable.

Test solutions

Details on test solutions:
Preparation of test solutions started with a loading rate of 100 mg/l applying 1 day of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a membrane filter (Whatman, 0.45 µm) where after the filtrate was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:

Test conditions

0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
between 22.3 and 23.1°C
At t=0 h: 8.1
At t=72 h: 7.8
Dissolved oxygen:
No data.
Not applicable.
Nominal and measured concentrations:
Nominal concentrations: 0.10, 1.0, 10 and 100 mg/ml

Measured concentrations: The study did not include sampling for determination of actual exposure concentrations because no analytical method could be validated to measure test concentrations due to the low solubility of Custom Red #2.
Details on test conditions:
- Test vessel: 100 ml, all glass
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1176000 cells/ml
- No. of vessels per concentration (replicates): 6 replicates of the highest concentration, 3 replicates for in-between concentrations
- No. of vessels per control (replicates): 1 replicate of each test concentration without algae.
- No. of vessels per vehicle control (replicates): 6

- Standard medium used: yes

- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 94 to 96 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with cuvettes (pathlength =10 mm). Algal medium was used as blank and the extra replicates without algae as background for the treated solutions.]
- Other: 72 h NOErC, 72 h NOEbC, 72 h ErC50, 72 h EbC50

- Spacing factor for test concentrations: 10
- Test concentrations: 0.10, 1.0, 10 and 100% of a filtered solution prepared at a loading rate of 100 mg/l
Reference substance (positive control):
potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
> 100 other: % of the filtrate
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
100 other: % of the filtrate
Nominal / measured:
Conc. based on:
test mat.
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 stimulation of growth found in any treatment: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50:
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.6 mg/l with a 95 % confidence interval ranging from 1.2 to 2.1 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the present batch corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.73 mg/l with a 95 % confidence interval ranging from 0.51 to 1.1 mg/l. Historical ranges are not yet available.

Applicant's summary and conclusion

Validity criteria fulfilled:
Due to the very low solubility of Custom Red #2 in test medium, concentration levels that might be toxic for algae could not be reached.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) was above a loading rate of 100 mg/l. Actual test concentrations could not be determined but were expected to be much lower.

The NOEC for both growth rate reduction and yield inhibition was above a loading rate of 100 mg/l.