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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-01-04 to 2008-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (OECD 201), GLP compliant including analytical monitoring

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF Test Guidelines, 2-7-7, Algae growth inhibition, 2005
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: WAFs with the following loading rates of the test item were tested: 1.0, 3.2, 10, 32, and 100 mg/L plus a control
- Sampling method: For determination of the actual concentrations of the test item, duplicate samples were taken from the test media of all loading rates at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses. A volume of 400 mL per sample was necessary for analytical purposes. The concentrations of the test item were determined in the duplicate test medium samples with the loading rate of 100mg/L taken at the start and the end of the test. The samples from the loading rates of 1.0 - 32 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
- Sample storage conditions before analysis: All samples were deep-frozen immediately after sampling and stored at about -20 °C until analysis. In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
In order to assess the toxicity of the test item containing different components, water accommodated fractions (WAFs) were prepared.

- Method: For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared using ultrasonic treatment for 15 minutes. The dispersions were stirred for 3 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. The stirring period of 3 hours was chosen based on the results of a pre-test (without GLP) in which the maximum concentration of dissolved test item was reached after the stirring period of 3 hours.
Then, the dispersions were filtered through membrane filters (0.45 μm) and the undiluted filtrates were tested as WAFs.

- Differential loading:
Loading Rate (mg/L) / Preparation:
3.2 / 9.72 mg test item in 3000 mL test water
10 / 20.25 mg test item in 2000 mL test water
32 7 64.08 mg test item in 2000 mL test water
100 / 200.65 mg test item in 2000 mL test water

- Due to technical reasons, the WAF with the lowest loading rate of 1 mg/L was prepared as dilutions of the WAF with the loading rate of 3.2 mg/L.
- The test media were prepared just before the addition of the algae (i.e. start of the test).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: The test organism used for the study was Pseudokirchneriella subcapitata, (formerly Selenastrum capricornutum),
- Strain: No. 61.81 SAG,
- Source: supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen / Germany).
- Method of cultivation: The algae were cultivated in RCC’s laboratories under standardized conditions according to the test guidelines.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3)
Test temperature:
22 +/- 0 °C
pH:
8.1 - 8.9
Nominal and measured concentrations:
Loading rate: 0, 1.0, 3.2, 10, 32, 100 mg/L;
The analytically measured test item concentration in the analyzed test medium (loading rate of 100 mg/L) was 7.8 and 2.3 μg/L at the start and at the end of the test duration, respectively.
The samples from the loading rates of 1.0 - 32 mg/L were not analyzed.
Details on test conditions:
TEST CONCENTRATIONS

- Range finding study: Results used to determine the selection of the loading rates was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item (non-GLP).
WAFs with the following loading rates of the test item were tested: 1.0, 3.2, 10, 32, and 100 mg/L. Additionally, a control was tested in parallel (test water without test item).
- Spacing factor for test concentrations: The enlarged spacing factor of 3.2 between the test concentrations was chosen, because according to the results of the range-finding test the concentration-effect relationship was rather flat and, thus, a large concentration range had to be tested.

TEST SYSTEM AND CONDITIONS

- Illumination: The test flasks were illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks.
- Light intensity: The mean measured light intensity at the level of the test solutions was approximately 7400 Lux (range: 6090 to 8160 Lux, measured at nine places in the experimental area).
- No. of vessels per concentration: The test design included three replicates per test concentration and six replicates of the control.
- Initial cells density: The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline.
- Design: A static test design was applied.
- Duration: The duration of the test was 72 hours.


Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No acute toxic effects within the range of solubility.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No acute toxic effects within the range of solubility.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No acute toxic effects within the range of solubility.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: No acute toxic effects within the range of solubility.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: No acute toxic effects within the range of solubility.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: No acute toxic effects within the range of solubility.
Details on results:
- Growth rate: The test item had no significant inhibitory effect on the growth of the algae (growth rate and yield) during the test period of 72 hours up to and including the highest loading rate of 100 mg/L (results of Dunnett’s tests, one-sided, α = 0.05.

The loading rate of 100 mg/L was, therefore, determined to be the 72-hour NOEC, since up to and including this loading rate the growth rate and yield of the algae after 72 hours were not significantly lower than in the control.

- Measured concentration: The analytically measured test item concentration in the analyzed test medium (loading rate of 100 mg/L) was 7.8 and 2.3 μg/L at the start and at the end of the test duration, respectively.

- Observation of abnormalities: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the loading rate of 100 mg/L and the algal cells in the control. The shape and size of the algal cells was obviously not affected by the test item up to at least this concentration.

- Exponential growth in the control (for algal test):
In the control the biomass increased by a factor of 138 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 13 %. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 3.2 %.

- Colour differences: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
With high probability not acutely harmful to aquatic algae. No acute toxic effects within the range of solubility.