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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25th June to 2nd July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and test item concentration at 0 and 48 hours.
- Sampling method: For the determination of the actual test item concentration, duplicates samples were taken from the test medium and the control at the start and at the end of the test after 72 hours (stability samples).
For the 72-hour stability samples, additional flasks with adequate volumes of the freshly prepared test medium and the control were incubated over the test period under the same conditions as in the actual test but without algae.
The concentration of the test item was analytically determined in the duplicate test medium samples taken at the start and the end of the test. From the control samples, only one of the duplicate samples was analysed from each sampling time.
- Sample storage conditions before analysis:
The samples were analysed immediately after sampling without prior storage.
Vehicle:
no
Details on test solutions:
- Method: In order to assess the toxicity of the test substance containing different siloxanes and silicones to algae, a water accommodated fraction (WAF) with a loading rate of 100 mg/L was tested. Additionally, a control (test water without test item) was tested in parallel. Thus, a limit test was performed in accordance with the EU Commission Directive 92/69/EEC to demonstrate that WAFs of the test item have no toxic effects on the algae up to the loading rate of 100 mg/L. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.
The WAF was prepared as follows: A dispersion with a loading rate of 100 mg/L was prepared by dispersing 230.2 mg of the test item in 2300 mL of test water using intense stirring on a magnetic stirrer. The dispersion was stirred for 3 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. Ultrasonic treatment was not applied to avoid accelerating hydrolysis.
The short stirring period of 3 hours was chosen because the components of the test item are not stable in water (results of a pre-experiment to determine the solubility and stability of the test item in test water, without GLP). Then, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 pm) after the 3 hour stirring period. The test medium was prepared just before the start of the test (= addition of algae).
The actual concentration of the test item in the test medium was analytically determined.
The limit test was based on the results of a range-finding test and the results of a pre-experiment to determine the solubility of the test item (without GLP). However, loading rates above 100 mg/L were not tested according to the EU Commission Directive 92/69/EEC.
- Controls: a control (test water without test item) was tested in parallel.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures, (SAG, Institute for Plant Physiology, University of Gottingen, D-37073 Gottingen, Germany). The algae had been grown in the RCC laboratories under standardised conditions according to the test guidelines.
- Age of inoculum (at test initiation): The algal cells were taken from an exponentially growing pre-culture which was set up three days prior to the test under the same conditions as in the test.
- Method of cultivation: Not reported

ACCLIMATION
- Acclimation period: A pre-culture was set up three days prior to the test under the same conditions as in the test.
- Culturing media and conditions (same as test or not): Same
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol/l (24 mg/l as CaCO3)
Test temperature:
23 - 24 °C
pH:
Start of test: 8.0 - 8.2
End of test: 8.9
Dissolved oxygen:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 100 mg/l loading rate WAF
Measured: 2.5 µg/l at the start of the test. At the end of the test after 72 hours, the concentration of the test item was below the limit of quantification (LOQ) of 0.7 µg/l. Thus, the test item (i.e. the quantified component) was not stable over the test period of 72 hours under the conditions of the test. The mean measured test item concentration was calculated to be 1.4 µg/l, taking half of the LOQ into calculation.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open / closed: covered with glass dishes.
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks filled with 15 mL of algal suspension.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): n/a
- Renewal rate of test solution (frequency/flow rate): n/a
- Initial cells density: Inoculation of 10000 algal cells per ml of test medium
- Control end cells density: 81.01 x 10000/ml
- No. of organisms per vessel: n/a
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water: analytical grade salts were dissolved in purified water.
- Total organic carbon: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Culture medium different from test medium: not reported
- Intervals of water quality measurement: The pH was measured at the start and at the end of the test in the single test concentration and the control. The water temperature was measured daily. The appearance of the test medium was also recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: Fluorescent tubes at 6660 - 8370 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were measured using an electronic particle counter: Coulter counter
- Chlorophyll measurement: Not measured
- Other: The shape of the algal cells was examined microscopically

VEHICLE CONTROL PERFORMED: yes/no: n/a

RANGE-FINDING STUDY
- Test concentrations: The limit test was based on the results of a range-finding test and the results of a pre-experiment to determine the solubility of the test item (without GLP). However, loading rates above 100 mg/L were not tested according to the EU Commission Directive 92/69/EEC. Results of the range-finding test are not reported in the study report.
- Results used to determine the conditions for the definitive study: Results of the range-finding test are not reported in the study report.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.001 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.001 mg/L
Nominal / measured:
meas. (arithm. mean)
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 abnormalities reported
- Unusual cell shape: None
- Colour differences: None
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: None
- Other:
- Any stimulation of growth found in any treatment: No
- 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: N/a
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 72-h ErC50 0.69 mg/l

Table: Algal cell densities during the test period of 72 hours

 Loading rate (mg/l)  Mean measured concentration (µg/l) Mean density of algal cells (cell number x 10000/ml) from 6 replicates  Mean density of algal cells (cell number x 10000/ml) from 6 replicates  Mean density of algal cells (cell number x 10000/ml) from 6 replicates
     24 hours  48 hours  72 hours
 Control  -  4.65  15.44  81.01
 100  1.4  4.57  16.10  86.27
Validity criteria fulfilled:
yes
Conclusions:
A 72-hour ErL50 value of >100 mg/l and a NOELR value of >=100 mg/l (nominal, WAF loading rate) (highest concentration tested) have been determined for the effects of the submission substance (CAS 352230-22-9) on growth rate of Scenedesmus subspicatus, in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test).

Description of key information

72-hour ErL50: >100 mg/l; NOELR: >=100 mg/L (nominal, WAF loading rate) (highest concentration tested); effects of the submission substance (CAS 352230-22-9) on growth rate of Scenedesmus subspicatus.

Key value for chemical safety assessment

Additional information

A 72-hour ErL50 value of >100 mg/L and a NOELR value of ≥100 mg/L (nominal, WAF loading rate) (highest concentration tested) have been determined for the effects of the submission substance (CAS 352230-22-9) on growth rate of Scenedesmus subspicatus, in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test).

The study reports an analytically determined test substance concentration of 1.4 µg/L (mean measured). This value reflects the low water solubility of the test substance, but losses may also have occurred as a result of filtering of the test solution prior to addition of test organisms, and the potential for adsorption of the test substance onto the filter matrix.

Each constituent of the test substance has a slow hydrolysis half-life at pH 7 (predicted to be >200 to >630 hours). However, the hydrolysis rate greatly increases with higher pH; 2 to 5.3 hours at pH 9. This test was conducted at pH 8.0 - 8.9. Hydrolysis is likely to have occurred in the timescale of the test. In view of the test media preparation method and the static exposure regime it is likely that the test organisms were exposed to a mixture of the parent substance and the hydrolysis products of each constituent of the tested substance.