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Toxicity to aquatic algae and cyanobacteria

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toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-26 to 2007-10-09
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 201 (Alga, Growth Inhibition Test)
according to
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
: Certificate issued by the Swiss GLP monitoring authorities.
Analytical monitoring:
Details on sampling:
- Sampling method: Duplicate samples were taken from each test medium and the control at the start and at the end of the test. At the end of the test, the contents of the respective replicates were combined prior to the sampling.
- Sample storage conditions before analysis: The samples were stored deep-frozen and protected from light until analysis was performed.
- The samples from the nominal test concentrations of 0.32 to 32 mg/L were not analyzed, since these concentrations were below the determined 72-hour NOEC. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of the test substance of 100 mg/L was prepared by dissolving 50.8 mg of the test substance completely in 500 mL of test water using intense stirring (15 minutes at room temperature). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media with the lower test substance concentrations. The test media were prepared just before the start of the test.
- Controls: yes, concurrent no treatment
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: green algae
- Strain: 61.81
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Goettingen, D-37073, Goettingen, Germany)
- Age of inoculum (at test initiation): no data
- Method of cultivation: no data

- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): Standard conditions according to guideline.
- Any deformed or abnormal cells observed: no data
Test type:
Water media type:
Limit test:
Total exposure duration:
72 h
Post exposure observation period:
0.24 mmol/L (24 mg/L as CaCO3)
Test temperature:
22 - 23 °C
no data
Dissolved oxygen:
not applicable
not applicable
Nominal and measured concentrations:
- Nominal test concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- At the start and the end of the test, the analytically determined concentration in the test medium of the highest test concentration was 156 and 111% of the nominal value, respectively.
- The mean measured concentration was 134 mg/L.
Details on test conditions:
- Test vessel: Flask
- Type (delete if not applicable): closed (covered with glass dishes)
- Material, size, headspace, fill volume: Erlenmeyer flasks, glass, 50 mL, 35 mL, 15 mL
- Aeration: not applicable
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cell density: 10000 cells/mL
- Control end cell density: 523000 +/- 57000 cells/mL (mean, n=6)
- No. of organisms per vessel: approx. 150000 cells/vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

- Source/preparation of dilution water: sterile purified dilution water
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

- Sterile test conditions: no data
- Adjustment of pH: no data
- Photoperiod: 24 hours/day light
- Light intensity and quality: 4500 Lux (mean); range 4100 to 4800 Lux (the surface of the test media). This illumination was achieved by fluorescent tubes, installed about 35 cm above the test flasks)
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth
- Determination of cell concentrations: electronic particle counter at 24, 48 and 72 hours
- Chlorophyll measurement: no
- Other: After 72 hours of exposure, a sample was taken from the control and from the highest test concentration (nominal 100 mg/L). The shape and size of the algal cells were examined microscopically in these samples.

- Spacing factor for test concentrations: Concentrations exceeding 100 mg/L were not tested in accordance with the test guidelines. An 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.
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: no data
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: The test concentrations were based on the results of a range-finding test and the results of the pre-experiment to determine the solubility of the test substance in test water (without GLP).
Reference substance (positive control):
potassium dichromate
72 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
> 100 mg/L
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):
- Unusual cell shape: no effects
- Color differences: no effects
- Any stimulation of growth found in any treatment: A stimulatory effect on growth rate was observed at 0-24 h in the 10 and 32 mg/L concentrations with 13.5 and 17.7% stimulation, respectively, and at 0-72 h in the 1.0 and 3.2 mg/L concentrations with 2.2 and 3.6% stimulation. A stimulatory effect on biomass was observed at 0-24 h in the 10, 32 and 100 mg/L concentrations with 21.3, 27.1 and 14.8% stimulation, respectively, and at 0-72 h in the 1.0 and 3.2 mg/L concentrations with 8.7 and 13.8% stimulation, respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
Results with reference substance (positive control):
- Results with reference substance valid?: The latest result of the positive control test in 2006 (RCC Study No. B13151) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 2000 to 2006).
- EC50: The latest result of the positive control test in 2006 (72-h EC50 for the growth rate: 1.70 mg/L) showed that the toxic performance was valid and within the historical range of the RCC laboratory (from 2000 to 2006: 72-h EC50: 0.71-1.74 mg/L).
Reported statistics and error estimates:
- The 72-hour EC10 and EC50 values of the test item could not be calculated because no inhibitory effect of the test item was determined on the growth of the algae at the tested concentrations.
- For the determination of the LOEC and NOEC, the calculated mean yield and the average growth rate at the test concentrations were tested for significant differences when compared to the control values by Dunnett's tests.

The mean coefficient of variation of the daily growth rates was 26% after 72 hours and, thus, lower than the maximum of 35% given by the OECD test guideline. The coefficient of variation of the average specific growth rates in the control replicates during the test period of 72 hours was 2.5% and, thus, below the maximum of 7% indicated by the OECD test guideline. Therefore, all validity criteria given by the test guidelines were fulfilled.

At the start of the test, the pH of the test media and the control was 8.4. At the end of the test, pH values of 8.2 to 8.4 were measured. The water temperature during the test was 22 to 23 deg C.

Validity criteria fulfilled:
A 72-h growth inhibition test with the freshwater green algal species Pseudokirchneriella subcapitata was performed with the test substance T002675 according to OECD guideline 201 (GLP conditions). The test substance had no statistically significant inhibitory effect on the growth (growth rate and yield) during the test period of 72 hours up to and including the highest nominal test concentration of 100 mg/L; therefore, 100 mg/L was determined to be the 72-hour NOEC. Results of the test can be considered reliable without restriction.

Description of key information

The study of Bätscher (2007), investigating the acute toxicity of T002675 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The 72-h NOEC and 72-h EC50 for growth rate were determined to be > 119 mg/L and 119 mg/L respectively (mean measured concentration). The substance can be considered as not  harmful to algae. 

Key value for chemical safety assessment

EC10 or NOEC for marine water algae:
100 mg/L

Additional information