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EC number: 952-026-5 | CAS number: -
Toxicity to aquatic algae and cyanobacteria
Administrative data
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- The study was conducted between 13 May 2010 and 28 May 2010
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Justification for type of information:
- The use of data derived for Soda-ash flux calcined kieselghur are justified for read-across to
synthetic wollastonite. Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
Considering the available data:
The source substance show no concerns for the environment.
The source substance has low acute toxicity and low toxicity in repeated dose studies, is non-irritant (skin and eye), non-sensitizing, non-mutagenic to bacteria, non-cytogenic and has low toxicity for reproductive and developmental toxicity.
Please see RAAF attached in Section 13. for further details. - Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- GLP compliance:
- yes (incl. QA statement)
- Details on sampling:
- Samples were taken from the control (replicates R1 - R6 pooled) and the 100% v/v saturated solution test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter - Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION:
PRE-STUDY MEDIA PREPARATION TRIAL:
Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Given the insoluble nature of the test item and also its high purity value the test item was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
An amount of test item (550 mg) was dispersed, in duplicate, in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for periods of 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
- Filtration through a 0.2 µm Gelman Acrocap filter (approximately 100 ml discarded in order to pre-condition the filter)
- Filtration through a 0.2 µm Gelman Acrocap filter (approximately 500 ml discarded in order to pre-condition the filter)
Whilst the results obtained from the pre-study media preparation trial proved inconclusive due to the level of silicon found in the control samples it was considered that the use of a saturated solution method of preparation stirred for a period of 24 hours was appropriate for this test item in order toensure the maximum potential for dissolution.
RANGE FINDING TEST:
An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (3 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution
DEFINITIVE TEST:
An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (1 litre) of this saturated solution was inoculated with algal suspension (23.8 ml) to give the required test concentration of 100% v/v saturated solution. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity - Test organisms (species):
- Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
- Details on test organisms:
- TEST ORGANISM
- Strain: CCAP 276/20
- Source: Obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation):
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C
ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed: - Limit test:
- yes
- Total exposure duration:
- 72 h
- Test temperature:
- Temperature was maintained at 24 ± 1ºC throughout the test.
- pH:
- The pH values of each test and control flask are given in Table 3
- Details on test conditions:
- TEST SYSTEM
- Test vessel: 250 ml glass conical flasks plugged with polyurethane foam bungs
- Fill volume: 100 ml
- Initial cells density: 4 x 10^3 cells per ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
GROWTH MEDIUM
- Refer to table 1
OTHER TEST CONDITIONS
- Photoperiod: Continuous light
- Light intensity and quality: Continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter
TEST CONCENTRATIONS
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours. Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration no effect on algal growth was observed. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
- Duration:
- 72 h
- Dose descriptor:
- other: ErC50
- Effect conc.:
- > 100 other: % v/v saturated solution
- Details on results:
- RANGE FINDING TEST:
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 2. The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
DEFINITIVE TEST:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5.
OBSERVATIONS:
- Observation of abnormalitiesAll test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions. - Reported statistics and error estimates:
- Statistical analysis of the growth rate data was carried out for the control and 100% v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution
There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution. - Validity criteria fulfilled:
- yes
- Conclusions:
- The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution.
Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite. - Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The study was conducted between 13 May 2010 and 28 May 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- GLP compliance:
- yes (incl. QA statement)
- Details on sampling:
- Samples were taken from the control (replicates R1 - R6 pooled) and the 100% v/v saturated solution test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter - Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION:
PRE-STUDY MEDIA PREPARATION TRIAL:
Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Given the insoluble nature of the test item and also its high purity value the test item was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
An amount of test item (550 mg) was dispersed, in duplicate, in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for periods of 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
- Filtration through a 0.2 µm Gelman Acrocap filter (approximately 100 ml discarded in order to pre-condition the filter)
- Filtration through a 0.2 µm Gelman Acrocap filter (approximately 500 ml discarded in order to pre-condition the filter)
Whilst the results obtained from the pre-study media preparation trial proved inconclusive due to the level of silicon found in the control samples it was considered that the use of a saturated solution method of preparation stirred for a period of 24 hours was appropriate for this test item in order toensure the maximum potential for dissolution.
RANGE FINDING TEST:
An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (3 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution
DEFINITIVE TEST:
An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (1 litre) of this saturated solution was inoculated with algal suspension (23.8 ml) to give the required test concentration of 100% v/v saturated solution. The prepared concentration was inverted several times to ensure adequate mixing and homogeneity - Test organisms (species):
- Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
- Details on test organisms:
- TEST ORGANISM
- Strain: CCAP 276/20
- Source: Obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation):
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C
ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed: - Limit test:
- yes
- Total exposure duration:
- 72 h
- Test temperature:
- Temperature was maintained at 24 ± 1ºC throughout the test.
- pH:
- The pH values of each test and control flask are given in Table 3
- Details on test conditions:
- TEST SYSTEM
- Test vessel: 250 ml glass conical flasks plugged with polyurethane foam bungs
- Fill volume: 100 ml
- Initial cells density: 4 x 10^3 cells per ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
GROWTH MEDIUM
- Refer to table 1
OTHER TEST CONDITIONS
- Photoperiod: Continuous light
- Light intensity and quality: Continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)
EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter
TEST CONCENTRATIONS
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours. Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration no effect on algal growth was observed. - Reference substance (positive control):
- yes
- Remarks:
- Potassium dichromate
- Duration:
- 72 h
- Dose descriptor:
- other: ErC50
- Effect conc.:
- > 100 other: % v/v saturated solution
- Details on results:
- RANGE FINDING TEST:
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 2. The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
DEFINITIVE TEST:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3. Daily specific growth rates for the control cultures are given in Table 4. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5.
OBSERVATIONS:
- Observation of abnormalitiesAll test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions. - Reported statistics and error estimates:
- Statistical analysis of the growth rate data was carried out for the control and 100% v/v saturated solution test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution
There were no statistically significant differences (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution. - Validity criteria fulfilled:
- yes
- Conclusions:
- The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution
Referenceopen allclose all
Table 2: Cell Densities and Percentage Inhibition of Growth from the Range-finding Test
Nominal Concentration (% v/v Saturated Solution) |
Cell Densities*(cells per ml) |
Inhibition Values (%) |
|||
0 Hours |
72 Hours |
Growth Rate |
Yield |
||
Control |
R1 |
4.62E+03 |
2.72E+05 |
|
|
|
R2 |
4.55E+03 |
3.45E+05 |
- |
- |
|
Mean |
4.59E+03 |
3.09E+05 |
|
|
0.10 |
R1 |
3.78E+03 |
5.29E+05 |
|
|
|
R2 |
4.44E+03 |
5.33E+05 |
[17] |
[73] |
|
Mean |
4.11E+03 |
5.31E+05 |
|
|
1.0 |
R1 |
4.06E+03 |
3.59E+05 |
|
|
|
R2 |
3.63E+03 |
5.21E+05 |
[14] |
[44] |
|
Mean |
3.85E+03 |
4.40E+05 |
|
|
10 |
R1 |
4.02E+03 |
4.11E+05 |
|
|
|
R2 |
4.02E+03 |
4.05E+05 |
[14] |
[33] |
|
Mean |
4.02E+03 |
4.08E+05 |
|
|
100 |
R1 |
3.91E+03 |
4.33E+05 |
|
|
|
R2 |
4.19E+03 |
4.18E+05 |
[12] |
[39] |
|
Mean |
4.05E+03 |
4.26E+05 |
|
|
*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.
R1and R2= Replicates 1 and 2
Table 3: Cell Densities and pH Values in the Definitive Test
Nominal Concentration (% v/v Saturated Solution) |
pH |
Cell Densities*(cells per ml) |
pH |
||||
0 h |
0 h |
24 h |
48 h |
72 h |
72 h |
||
Control |
R1 |
7.2 |
4.59E+03 |
1.09E+04 |
5.73E+04 |
3.91E+05 |
7.9 |
|
R2 |
7.3 |
5.12E+03 |
1.12E+04 |
5.26E+04 |
3.14E+05 |
8.0 |
|
R3 |
7.3 |
4.67E+03 |
1.41E+04 |
4.32E+04 |
3.95E+05 |
8.0 |
|
R4 |
7.3 |
4.57E+03 |
1.54E+04 |
6.59E+04 |
4.38E+05 |
7.9 |
|
R5 |
7.3 |
4.94E+03 |
1.17E+04 |
6.01E+04 |
3.51E+05 |
7.9 |
|
R6 |
7.3 |
4.34E+03 |
1.16E+04 |
6.60E+04 |
3.31E+05 |
8.0 |
|
Mean |
|
4.70E+03 |
1.25E+04 |
5.75E+04 |
3.70E+05 |
|
100 |
R1 |
7.2 |
5.78E+03 |
1.29E+04 |
6.12E+04 |
3.83E+05 |
7.7 |
|
R2 |
7.2 |
4.58E+03 |
1.78E+04 |
6.35E+04 |
3.87E+05 |
7.8 |
|
R3 |
7.2 |
4.11E+03 |
1.47E+04 |
5.41E+04 |
3.83E+05 |
7.8 |
|
R4 |
7.2 |
4.27E+03 |
1.19E+04 |
6.40E+04 |
3.77E+05 |
7.7 |
|
R5 |
7.2 |
4.69E+03 |
1.13E+04 |
6.27E+04 |
3.72E+05 |
7.8 |
|
R6 |
7.2 |
4.56E+03 |
1.31E+04 |
6.63E+04 |
3.60E+05 |
7.7 |
|
Mean |
|
4.67E+03 |
1.36E+04 |
6.20E+04 |
3.77E+05 |
|
*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.
R1- R6= Replicates 1 to 6
Table 4: Daily Specific Growth Rates for the Control Cultures in the Definitive Test
|
Daily Specific Growth Rate (cells/ml/hour) |
|||
Day 0 - 1 |
Day 1 - 2 |
Day 2 - 3 |
||
Control |
R1 |
0.042 |
0.069 |
0.080 |
|
R2 |
0.043 |
0.065 |
0.074 |
|
R3 |
0.052 |
0.047 |
0.092 |
|
R4 |
0.056 |
0.061 |
0.079 |
|
R5 |
0.045 |
0.068 |
0.074 |
|
R6 |
0.044 |
0.073 |
0.067 |
|
Mean |
0.047 |
0.064 |
0.078 |
R1- R6= Replicates 1 to 6
Table 5: Inhibition of Growth Rate and Yield in the Definitive Test
Nominal Concentration |
Growth Rate (cells/ml/hour) |
Yield (cells/ml) |
|||
0 – 72 h |
% Inhibition |
0 – 72 h |
% Inhibition* |
||
Control |
R1 |
0.064 |
|
3.87E+05 |
|
|
R2 |
0.061 |
|
3.09E+05 |
|
|
R3 |
0.064 |
|
3.90E+05 |
|
|
R4 |
0.065 |
- |
4.33E+05 |
- |
|
R5 |
0.062 |
|
3.46E+05 |
|
|
R6 |
0.061 |
|
3.27E+05 |
|
|
Mean |
0.063 |
|
3.65E+05 |
|
|
SD |
0.002 |
|
4.61E+04 |
|
100 |
R1 |
0.063 |
0 |
3.77E+05 |
|
|
R2 |
0.063 |
0 |
3.82E+05 |
|
|
R3 |
0.063 |
0 |
3.79E+05 |
|
|
R4 |
0.063 |
0 |
3.72E+05 |
|
|
R5 |
0.063 |
0 |
3.67E+05 |
|
|
R6 |
0.062 |
2 |
3.55E+05 |
|
|
Mean |
0.063 |
0 |
3.72E+05 |
[2] |
|
SD |
0.000 |
|
9.80E+03 |
|
*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated
R1– R6= Replicates 1 to 6
SD= Standard Deviation
Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 79 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0
hours : 4.70 x 103cells
per ml
Mean cell density of control at 72 hours : 3.70 x 105cells
per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 27% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Growth data
From the data given in Tables 3 and 5, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus(CCAP 276/20) were not affected by the presence of the test item at a nominal test concentration of 100% v/v saturated solution over the 72‑Hour exposure period.
Accordingly the following results were determined from the data:
Inhibition of growth rate
ErC10(0 - 72
h) : >100% v/v saturated solution
ErC20(0 - 72 h) : >100% v/v saturated
solution
ErC50(0 - 72 h) : >100% v/v saturated
solution
where ErCxis the test concentration that reduced growth rate by x%.
Inhibition of yield
EyC10(0 - 72
h) : >100% v/v saturated solution
EyC20(0 - 72 h) : >100% v/v saturated
solution
EyC50(0 - 72 h) : >100% v/v saturated
solution
where EyCxis the test concentration that reduced yield by x%.
Table 2: Cell Densities and Percentage Inhibition of Growth from the Range-finding Test
Nominal Concentration (% v/v Saturated Solution) |
Cell Densities*(cells per ml) |
Inhibition Values (%) |
|||
0 Hours |
72 Hours |
Growth Rate |
Yield |
||
Control |
R1 |
4.62E+03 |
2.72E+05 |
|
|
|
R2 |
4.55E+03 |
3.45E+05 |
- |
- |
|
Mean |
4.59E+03 |
3.09E+05 |
|
|
0.10 |
R1 |
3.78E+03 |
5.29E+05 |
|
|
|
R2 |
4.44E+03 |
5.33E+05 |
[17] |
[73] |
|
Mean |
4.11E+03 |
5.31E+05 |
|
|
1.0 |
R1 |
4.06E+03 |
3.59E+05 |
|
|
|
R2 |
3.63E+03 |
5.21E+05 |
[14] |
[44] |
|
Mean |
3.85E+03 |
4.40E+05 |
|
|
10 |
R1 |
4.02E+03 |
4.11E+05 |
|
|
|
R2 |
4.02E+03 |
4.05E+05 |
[14] |
[33] |
|
Mean |
4.02E+03 |
4.08E+05 |
|
|
100 |
R1 |
3.91E+03 |
4.33E+05 |
|
|
|
R2 |
4.19E+03 |
4.18E+05 |
[12] |
[39] |
|
Mean |
4.05E+03 |
4.26E+05 |
|
|
*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.
R1and R2= Replicates 1 and 2
Table 3: Cell Densities and pH Values in the Definitive Test
Nominal Concentration (% v/v Saturated Solution) |
pH |
Cell Densities*(cells per ml) |
pH |
||||
0 h |
0 h |
24 h |
48 h |
72 h |
72 h |
||
Control |
R1 |
7.2 |
4.59E+03 |
1.09E+04 |
5.73E+04 |
3.91E+05 |
7.9 |
|
R2 |
7.3 |
5.12E+03 |
1.12E+04 |
5.26E+04 |
3.14E+05 |
8.0 |
|
R3 |
7.3 |
4.67E+03 |
1.41E+04 |
4.32E+04 |
3.95E+05 |
8.0 |
|
R4 |
7.3 |
4.57E+03 |
1.54E+04 |
6.59E+04 |
4.38E+05 |
7.9 |
|
R5 |
7.3 |
4.94E+03 |
1.17E+04 |
6.01E+04 |
3.51E+05 |
7.9 |
|
R6 |
7.3 |
4.34E+03 |
1.16E+04 |
6.60E+04 |
3.31E+05 |
8.0 |
|
Mean |
|
4.70E+03 |
1.25E+04 |
5.75E+04 |
3.70E+05 |
|
100 |
R1 |
7.2 |
5.78E+03 |
1.29E+04 |
6.12E+04 |
3.83E+05 |
7.7 |
|
R2 |
7.2 |
4.58E+03 |
1.78E+04 |
6.35E+04 |
3.87E+05 |
7.8 |
|
R3 |
7.2 |
4.11E+03 |
1.47E+04 |
5.41E+04 |
3.83E+05 |
7.8 |
|
R4 |
7.2 |
4.27E+03 |
1.19E+04 |
6.40E+04 |
3.77E+05 |
7.7 |
|
R5 |
7.2 |
4.69E+03 |
1.13E+04 |
6.27E+04 |
3.72E+05 |
7.8 |
|
R6 |
7.2 |
4.56E+03 |
1.31E+04 |
6.63E+04 |
3.60E+05 |
7.7 |
|
Mean |
|
4.67E+03 |
1.36E+04 |
6.20E+04 |
3.77E+05 |
|
*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.
R1- R6= Replicates 1 to 6
Table 4: Daily Specific Growth Rates for the Control Cultures in the Definitive Test
|
Daily Specific Growth Rate (cells/ml/hour) |
|||
Day 0 - 1 |
Day 1 - 2 |
Day 2 - 3 |
||
Control |
R1 |
0.042 |
0.069 |
0.080 |
|
R2 |
0.043 |
0.065 |
0.074 |
|
R3 |
0.052 |
0.047 |
0.092 |
|
R4 |
0.056 |
0.061 |
0.079 |
|
R5 |
0.045 |
0.068 |
0.074 |
|
R6 |
0.044 |
0.073 |
0.067 |
|
Mean |
0.047 |
0.064 |
0.078 |
R1- R6= Replicates 1 to 6
Table 5: Inhibition of Growth Rate and Yield in the Definitive Test
Nominal Concentration |
Growth Rate (cells/ml/hour) |
Yield (cells/ml) |
|||
0 – 72 h |
% Inhibition |
0 – 72 h |
% Inhibition* |
||
Control |
R1 |
0.064 |
|
3.87E+05 |
|
|
R2 |
0.061 |
|
3.09E+05 |
|
|
R3 |
0.064 |
|
3.90E+05 |
|
|
R4 |
0.065 |
- |
4.33E+05 |
- |
|
R5 |
0.062 |
|
3.46E+05 |
|
|
R6 |
0.061 |
|
3.27E+05 |
|
|
Mean |
0.063 |
|
3.65E+05 |
|
|
SD |
0.002 |
|
4.61E+04 |
|
100 |
R1 |
0.063 |
0 |
3.77E+05 |
|
|
R2 |
0.063 |
0 |
3.82E+05 |
|
|
R3 |
0.063 |
0 |
3.79E+05 |
|
|
R4 |
0.063 |
0 |
3.72E+05 |
|
|
R5 |
0.063 |
0 |
3.67E+05 |
|
|
R6 |
0.062 |
2 |
3.55E+05 |
|
|
Mean |
0.063 |
0 |
3.72E+05 |
[2] |
|
SD |
0.000 |
|
9.80E+03 |
|
*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated
R1– R6= Replicates 1 to 6
SD= Standard Deviation
Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 79 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0
hours : 4.70 x 103cells
per ml
Mean cell density of control at 72 hours : 3.70 x 105cells
per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 27% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
Growth data
From the data given in Tables 3 and 5, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus(CCAP 276/20) were not affected by the presence of the test item at a nominal test concentration of 100% v/v saturated solution over the 72‑Hour exposure period.
Accordingly the following results were determined from the data:
Inhibition of growth rate
ErC10(0 - 72
h) : >100% v/v saturated solution
ErC20(0 - 72 h) : >100% v/v saturated
solution
ErC50(0 - 72 h) : >100% v/v saturated
solution
where ErCxis the test concentration that reduced growth rate by x%.
Inhibition of yield
EyC10(0 - 72
h) : >100% v/v saturated solution
EyC20(0 - 72 h) : >100% v/v saturated
solution
EyC50(0 - 72 h) : >100% v/v saturated
solution
where EyCxis the test concentration that reduced yield by x%.
Description of key information
An algal growth inhibition study was not available for Synthetic Wollastonite. A study was performed on the analogue substance Kieselghur soda-ash flux calcined in accordance with OECD guideline 201. Exposure of Desmodesmus subspicatus to the test item gave EC50 values of greater than 100% v/v saturated solution
Key value for chemical safety assessment
Additional information
The key study (Vryenhoef 2010) was performed according to GLP and OECD guideline 201. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. Given the insoluble nature of the test item and also its high purity value it was considered that the most suitable method of preparation was as a saturated solution. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test item at a concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter. Exposure of Desmodesmus subspicatus to the test item gave EC50values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution
Justification for read-across is warranted given the similarities in toxicity profile and physico-chemical properties for Soda-ash flux calcined kieselghur and synthetic wollastonite.
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