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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:
16 January to 19th January 1995
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study report is conducted to OECD and EEC guidelines. Contains a statement of GLP compliance and GLP certificate

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method C.3 (Algal Inhibition test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:

- Physical state: White powder
- Analytical purity: 97.8%
- Impurities (identity and concentrations): Water 2.2%
- Composition of test material, percentage of components: No information
- Lot/batch No.: A105333
- Expiration date of the lot/batch: No information
- Stability under test conditions: Considered stable
- Storage condition of test material: Clear plastic bag at room temperature
- Other:

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: approximately 15mg/l
- Sampling method: No information
- Sample storage conditions before analysis: Ambient temperature

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preliminary solution in dimethyl sulphoxide. 160 mg of test sample were of test material was dispersed in auxillary solvent and the volume adjusted to 10 ml to give a 160 mg/ 10 ml solvent stock solution. Serial dilutions were made from this initial solvent stock solution to give 80, 40, 20 and 10 mg/ 10 ml solvent stock solutions. 100 μl of each solvent stock solution was dispersed in 1 L of algal suspension to give the required test concentrations.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
- Common name: Chodat
- Strain: CCAP 276/ 20
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (CCAP), Institute of Ecology, Cumbria UK.
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: Sterile culture medium was innoculated with a master culture of scenedesmus subspicatus and incubated under continuous illumination (approx. 7000 lux) and aeration at 21 ®C to give an algal suspension in log phase growth characterised by an absorbance of 0.849 (@ 665nm). The suspension was diluted to an absorbance of 0.027 prior to use. The suspension had a mean cell density of 2.78 x 104 cells/ml

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
Not reported

Test conditions

Not reported
Test temperature:
8 - 10.4
pH values of the control and test cultures were observed to increase from pH 8.0 at 0 hours to pH 8.4-10.4 at 72 hours. This effect is considered to be due to the large number of algal cells in log phase growth respiring oxygen and producing carbonates and bicarbonates as part of photosynthesis/respiration which in solution give alkaline conditions.
Dissolved oxygen:
Not reported
Not reported
Nominal and measured concentrations:
Nominal test concentrations: 0.10, 0.20, 0.40, 0.80 and 1.6 mg/l
Measured test concentrations: 0.0997, 0.169, 0.356, 0.719 and 1.81 mg/l
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml conical flask containing 100 ml test solution
- Aeration: None
- Initial cells density: 27800 cells/ ml
- Control end cells density:
Control at 0h - 27800 cells/ ml
Control at 72h - 1070000 cells/ml
Solvent control at 0h - 27800 cells/ml
Solvent control at 72h - 1030000 cells/ ml
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): three
- No. of vessels per vehicle control (replicates): three

- Standard medium used: yes

- Range finding study
- Test concentrations: 0.10, 1.0 and 10 mg/l
- Results used to determine the conditions for the definitive study: Significant impairment of growth at 0.10 and 10 mg/l and no significant impairment of growth at 1.0 mg/l.

- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: Continuously
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Jenway 6100 spectrrophotomer
- Other:

Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
other: EbC50
Effect conc.:
0.64 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
24 h
Dose descriptor:
other: ErC50
Effect conc.:
1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
0.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Statistics
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- 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

Statistical analysis of the area under the growth curve data showed that there were no statistically significant differences (P≥ 0.05) between the solvent control, 0.10 and 0.20 mg/l test concentrations; however all other test concentrations were significantly different (P<0.001) from the other solvent control and therefore the “No Observed Effect Concentration” (NOEC) is given as 0.20 mg/l.
Results with reference substance (positive control):

Any other information on results incl. tables

The change in pH was considered to be greater numbers of viable cells in the lower test concentrations and hence a greater numbers of carbonates and bicarbonates from photosynthesis/ respiration. This is considered to not affect the integrity of the study given that the cell densities of control cultures increased by a factor of 33 -37 during the test.

Applicant's summary and conclusion

Validity criteria fulfilled:
EbC50 (72 h) = 0.64 mg/l
ErC50 (0-24 h) = 1.0 mg/l
NOEC (72 h) = 0.20 mg/l
Executive summary:

The effect of the test sample on the growth of the algal species scenedesmus subspicatus was assessed over a period of 72 hours. The test was conducted under statis conditions and with analysis of exposure concentrations measured by HPLC with a UV detector. Under the conditions of the test and in comparison with a solvent control the test sample achieved an EbC50 of 0.64 mg/l.