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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:
between 29 September 2008 and 16 October 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no/or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
A study to determine the General Physico-Chemical properties of the test material (Project Number: 2551/0013) determined the water solubility of the test material to be 0.224 mg/l. Pre-study solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A test concentration of 0.20 mg/l (by visual inspection) was obtained using a preliminary solution in dimethylformamide. At higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water.
Based on this information the test material 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 material under test conditions.


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 0.0070 and 0.070 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test material was prepared using a preliminary solution in dimethylformamide.
An amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 50 ml to give a 100 mg/50 ml solvent stock solution. An aliquot (100 µl) of this solvent stock solution was dispersed in 1 litre of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to removal of any undissolved test material by centrifugation at 10000 g for 30 minutes to produce a 0.070 mg/l stock solution. A dilution was made from the 0.070 mg/l stock solution to give a further stock solution of 0.0070 mg/l. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (5 ml) to give the required nominal test concentrations of 0.0070 and 0.070 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/l of dimethylformamide.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive test
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 0.070 mg/l to confirm that at the highest attainable concentration no effect on algal growth was observed.
Chemical analysis of the test preparations at 0 hours (see Appendix 2) showed measured concentrations to be higher than 0.070 mg/l; this was considered to be due to slight differences in media and/or sampling techniques used to remove the supernatant after centrifugation between the media preparation trial and the definitive test.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For the purpose of the definitive test, the test material was prepared using a preliminary solution in dimethylformamide.
An amount of test material (50 mg) was dissolved in dimethylformamide and the volume adjusted to 25 ml to give a 50 mg/25 ml solvent stock solution. An aliquot (250 µl) of this solvent stock solution was dispersed in 2.5 litres of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to removal of any undissolved test material by centrifugation at 10000 g for 30 minutes to give the test concentration of 0.18 mg/l.
The stock solutions and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: no recorded
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Dinstaffnage Marine laboratory
- Age of inoculum (at test initiation): Not recorded
- Method of cultivation:Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1C until the algal cell density was approximately 104 - 105 cells/ml.

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not recorded.
Hardness:
Not recorded
Test temperature:
24 +/- 1 deg C
pH:
The pH values of the control cultures were observed to increase from pH 7.5 at 0 hours to pH 7.7 – 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines
Dissolved oxygen:
Not recorded
Salinity:
not recorded
Nominal and measured concentrations:
Main Test: geometric mean measured test concentration of 0.11 mg/l.
Details on test conditions:
TEST SYSTEM
The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1C.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1C until the algal cell density was approximately 104 - 105 cells/ml.
Culture Medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.
- Photoperiod and light intensity: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.


TEST CONCENTRATIONS
- Spacing factor for 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 0.0070 and 0.070 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test material was prepared using a preliminary solution in dimethylformamide.
An amount of test material (100 mg) was dissolved in dimethylformamide and the volume adjusted to 50 ml to give a 100 mg/50 ml solvent stock solution. An aliquot (100 µl) of this solvent stock solution was dispersed in 1 litre of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to removal of any undissolved test material by centrifugation at 10000 g for 30 minutes to produce a 0.070 mg/l stock solution. A dilution was made from the 0.070 mg/l stock solution to give a further stock solution of 0.0070 mg/l. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (5 ml) to give the required nominal test concentrations of 0.0070 and 0.070 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/l of dimethylformamide.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.11 mg/L
Nominal / measured:
meas. (geom. 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 abnormalilities were observed in any control or test culture at 72 hours.

Results with reference substance (positive control):
- Results with reference substance valid?
The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control are recorded. Daily specific growth rates for the control cultures and growth rate, yield and biomass integral values are recorded. Percentage inhibition values are plotted against test concentration.
Accordingly the following results were determined from the data:

ErC50 (0 - 72 h) : 0.57 mg/l; 95% confidence limits 0.48 - 0.66 mg/l
EyC50 (0 - 72 h) : 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l
EbC50 (0 - 72 h) : 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral : 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral : 0.125 mg/l

The results from the positive control with potassium dichromate were within the normal range for this reference material.



Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the solvent control and 0.11 mg/l 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 decreases in growth rate, between the solvent control and 0.11 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.11 mg/l.

Pre-study media preparation trial: A Determination of the General Physico-chemical Properties study conducted on the test material showed that the water solubility value of the test material was 0.224 mg/l.  Preliminary solubility work showed that the highest attainable test concentration (by visual inspection) that could be prepared was 0.20 mg/l using a preliminary solution in dimethylformamide.  At higher test concentrations precipitation of the test material was observed on addition of the test material solvent stock solution to water. Based on this information a pre-study media preparation trial was conducted using both saturated solution and solvent spike methods of preparation.  The results from the saturated solution method of preparation were significantly in excess of the water solubility value indicating that neither filtration nor centrifugation was removing the excess test material present.  The results obtained from the solvent spike preparations indicated that the test material (at a significantly lower loading rate compared to the saturated solution preparations) may have been passing through the filter matrices.  The results of the centrifuged test samples showed consistent results between the 10000 and 40000 g samples indicating that all the undissolved test material had been removed.  A decline in measured concentrations was observed after 24 and 48 hours which was inline with the preliminary stability analyses conducted which indicated that the test material was unstable.  As such the use of a solvent spike preparation followed by removal of any undissolved test material by centrifugation at 10000 g for 30 minutes was considered appropriate for this material.

Range-finding test: The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1 below:

Nominal Concentration

(mg/l)

Cell Densities* (cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield/Biomass Integral

Control

R1

4.62E+03

4.59E+05

R2

4.56E+03

5.07E+05

-

-

Mean

4.59E+03

4.83E+05

Solvent Control

R1

4.71E+03

6.03E+05

R2

4.72E+03

5.87E+05

-

-

Mean

4.71E+03

5.95E+05

0.0070

R1

4.66E+03

5.07E+05

R2

4.72E+03

4.12E+05

4

23

Mean

4.69E+03

4.59E+05

0.070

R1

4.13E+03

4.70E+05

R2

4.06E+03

4.97E+05

1

19

Mean

4.10E+03

4.84E+05


* Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 and R2 = Replicates 1 and 2

The results showed no effect on growth rate at the nominal test concentrations of 0.0070 and 0.070 mg/l.

Based on this information a single test concentration of six replicates, of 0.070 mg/l was selected for the definitive test.  This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration of 0.070 mg/l no effect on growth was observed.

The concentration of 0.070 mg/l was below the water solubility value of 0.224 mg/l and also the 0-Hour measured test concentrations obtained during the definitive test.  This difference was considered to be due to differences in the solubility of the test material in different test media and also the technique used to remove the supernatant following centrifugation. 

Definitive test:

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2.  Daily specific growth rates for the control cultures are given in Table 3.  Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

Table 2: Cell Densities and pH values in the definitive test

Geometric Mean Measured Test Concentration (mg/l)

pH

Cell Densities* (cells per ml)

pH

0 h

0 h

22 h

45 h

72 h

72 h

Control

R1

7.5

4.29E+03

1.21E+04

3.79E+04

3.50E+05

7.7

R2

7.5

3.98E+03

1.44E+04

3.68E+04

3.72E+05

7.7

R3

7.5

4.00E+03

1.23E+04

3.63E+04

3.47E+05

7.8

R4

7.5

4.23E+03

1.45E+04

3.96E+04

2.07E+05

7.7

R5

7.5

4.13E+03

1.22E+04

3.68E+04

3.25E+05

7.7

R6

7.5

4.19E+03

1.24E+04

3.55E+04

3.45E+05

7.7

Mean

4.14E+03

1.30E+04

3.71E+04

3.24E+05

Solvent Control

R1

7.4

4.31E+03

1.27E+04

3.87E+04

4.09E+05

7.8

R2

7.4

3.96E+03

1.39E+04

3.75E+04

3.25E+05

7.8

R3

7.4

4.12E+03

1.27E+04

3.78E+04

3.56E+05

7.7

R4

7.4

4.06E+03

1.45E+04

3.92E+04

3.17E+05

7.7

R5

7.4

4.12E+03

1.43E+04

3.58E+04

3.31E+05

7.7

R6

7.4

4.16E+03

1.30E+04

3.61E+04

3.31E+05

7.7

Mean

4.12E+03

1.35E+04

3.75E+04

3.45E+05

0.11

R1

7.4

4.39E+03

1.25E+04

3.17E+04

3.79E+05

7.8

R2

7.4

4.02E+03

1.18E+04

3.01E+04

3.75E+05

7.8

R3

7.4

3.62E+03

1.33E+04

3.15E+04

4.51E+05

7.9

R4

7.4

4.02E+03

1.27E+04

3.33E+04

3.99E+05

7.9

R5

7.4

4.15E+03

1.24E+04

3.39E+04

4.00E+05

7.9

R6

7.4

4.21E+03

1.37E+04

3.99E+04

4.18E+05

8.0

Mean

4.07E+03

1.27E+04

3.34E+04

4.04E+05


* Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 - R6 = Replicates 1 to 6

Table 3: Daily specific growth rates for the control cultures in definitive test

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.050

0.050

0.082

R2

0.058

0.041

0.086

R3

0.051

0.047

0.084

R4

0.059

0.044

0.061

R5

0.051

0.048

0.081

R6

0.051

0.046

0.084

Mean

0.053

0.046

0.080

Solvent Control

R1

0.052

0.049

0.087

R2

0.057

0.043

0.080

R3

0.052

0.047

0.083

R4

0.058

0.043

0.077

R5

0.058

0.040

0.082

R6

0.053

0.044

0.082

Mean

0.055

0.044

0.082


R1 - R6 = Replicates 1 to 6

Table 4: inhibition of growth rate, yield and biomass integral in the definitive test

Geometric Mean Measured Test Concentration (mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

Biomass Integral

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

0 – 72 h

% Inhibition

Control

R1

0.062

3.45E+05

5.70E+06

R2

0.063

3.68E+05

6.02E+06

R3

0.062

3.43E+05

5.63E+06

R4

0.055

-

2.02E+05

-

3.86E+06

-

R5

0.061

3.21E+05

5.34E+06

R6

0.062

3.41E+05

5.59E+06

Mean

0.061

3.20E+05

5.35E+06

SD

0.003

5.96E+04

7.64E+05

Solvent Control

R1

0.064

4.04E+05

6.52E+06

R2

0.061

3.21E+05

5.40E+06

R3

0.062

3.52E+05

5.79E+06

R4

0.061

-

3.13E+05

-

5.34E+06

-

R5

0.061

3.27E+05

5.44E+06

R6

0.061

3.27E+05

5.42E+06

Mean

0.062

3.41E+05

5.65E+06

SD

0.001

3.38E+04

4.57E+05

0.11

R1

0.063

[2]

3.75E+05

5.95E+06

[5]

R2

0.063

[2]

3.71E+05

5.83E+06

[3]

R3

0.066

[6]

4.48E+05

6.94E+06

[23]

R4

0.064

[3]

3.95E+05

6.27E+06

[11]

R5

0.064

[3]

3.96E+05

6.28E+06

[11]

R6

0.065

[5]

4.14E+05

6.70E+06

[19]

Mean

0.064

[4]

4.00E+05

[17]

6.33E+06

[12]

SD

0.001

2.82E+04

4.25E+05


* In accordance with the OECD test guideline only the mean value for yield 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 78 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 84 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                             :    4.14 x 103 cells per ml
Mean cell density of control at 72 hours                           :    3.24 x105 cells per ml
Mean cell density of solvent control at 0 hours                  :    4.12 x 103 cells per ml
Mean cell density of solvent control at 72 hours                :    3.45 x 105cells per ml

The mean coefficients of variation for section by section specific growth rate for the control and solvent control cultures were 30% and 33% respectively and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficients of variation for average specific growth rate for the control and solvent control cultures over the test period (0 – 72 h) were 5% and 2% respectively and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Verification of test concentrations:

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.18 to 0.19 mg/l.  A decline in measured test concentrations was observed at 72 hours in the range of 0.057 to 0.068 mg/l.  This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium. 

The 0-Hour measured concentrations were in excess of those obtained during the pre-study media preparation trial conducted (0.070 mg/l).  This was considered to be due to differences in the test media and also the technique used to remove the supernatant following centrifugation.  Given that the values obtained were similar to the predicted water solubility value of 0.224 mg/l this was considered to have had no adverse effect on the outcome of the test.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values.  It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.  The geometric mean measured test concentrations were determined to be:

0-Hour Measured Test Concentration (mg/l)

Geometric Mean Measured Test Concentration (mg/l)

Expressed as a % of the Nominal Test Concentration

0.18 R1-R3

0.11

61

0.18 R4-R6

0.10

56

Growth data:

From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material at a geometric mean measured test concentration of 0.11 mg/l over the 72-Hour exposure period.

The test concentration of 0.11 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline. Other various recognised auxiliary solvents were used during preliminary solubility work, however, dimethylformamide was found to give the best testable dispersion of the test material in water. At higher test concentrations there was a marked precipitation of the test material on addition of the solvent stock solution to water.

Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:

Inhibition of growth rate:

ErC10 (0 - 72 h)              : > 0.11 mg/l
ErC20 (0 - 72 h)              : > 0.11 mg/l
ErC50 (0 - 72 h)              : > 0.11 mg/l

where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the solvent control and 0.11 mg/l test group using a Student’s t-test incorporating 's test for homogeneity of variance (Sokal and Rohlf 1981).  There were no statistically significant decreases in growth rate (P³0.05), between the solvent control and 0.11 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.11 mg/l.

Inhibition of yield:

EyC10 (0 - 72 h)             : > 0.11 mg/l
EyC20 (0 - 72 h)             : > 0.11 mg/l
EyC50 (0 - 72 h)             : > 0.11 mg/l

where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 5.3.3.1.  There were no statistically significant decreases in yield (P³0.05), between the solvent control and 0.11 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.11 mg/l.

Inhibition of biomass integral:

EbC10 (0 - 72 h)             : > 0.11 mg/l
EbC20 (0 - 72 h)             : > 0.11 mg/l
EbC50 (0 - 72 h)             : > 0.11 mg/l

where EbCx is the test concentration that reduced biomass integral (area under the growth curve) by x%.

Observation on cultures: All test and control cultures were inspected microscopically at 72 hours.  There were no abnormalities detected in any of the control or test cultures at 72 hours.

Observations on test material solubility: At the start of the test all control, solvent control and test cultures were observed to be clear colourless solutions.  After the 72-Hour test period all control, solvent control and test cultures were observed to be green dispersions

Physico-chemical measurement:

The pH values of each test and control flask are given in Table 2.  Temperature was maintained at 24 ± 1ºC throughout the test.

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.5 at 0 hours to pH 7.7 – 7.8 at 72 hours.  The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Validity criteria fulfilled:
yes
Conclusions:
Based on the geometric mean measured test concentrations the EC50 values were estimated to be greater than 0.11 mg/l. Correspondingly the No Observed Effect Concentration was 0.11 mg/l.
Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Methods. 

A Study to Determine the General Physico-Chemical Properties of the test material gave a water solubility value for the test material of 0.224 mg/l. Based on this information the test material 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 media preparation trials were conducted in order to determine the solubility of the test material under test conditions. 

Pre-study media preparation trials indicated that the use of preliminary solution in auxiliary solvent to spike the test medium followed by centrifugation at 10000gto remove any undissolved test material was the most suitable method of preparation. 

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a geometric mean measured test concentration of 0.11 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

Results

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.18 to 0.19 mg/l. A decline in measured test concentrations was observed at 72 hours in the range of 0.057 to 0.068 mg/l. This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium. 

The 0-Hour measured concentrations were in excess of those obtained during the pre-study media preparation trial conducted (0.070 mg/l). This was considered to be due to differences in the test media and also the technique used to remove the supernatant following centrifugation. Given that the values obtained were similar to the predicted water solubility value of 0.224 mg/l this was considered to have had no adverse effect on the outcome of the test.

Given the decline in measured test concentrations over the test period it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

The EC50values based on the geometric mean measured test concentrations were greater than 0.11 mg/l and correspondingly the No Observed Effect Concentration was 0.11 mg/l.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an ErC50(0 - 72 h) of 0.57 mg/l; 95% confidence limits 0.48 – 0.66 mg/l, an EyC50(0 - 72 h) of 0.32 mg/l; 95% confidence limits 0.29 ‑ 0.35 mg/l, and an EbC50(0 - 72 h) of 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/l respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/l respectively. 


 

Description of key information

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC.

Methods. 

A Study to Determine the General Physico-Chemical Properties of the test material gave a water solubility value for the test material of 0.224 mg/l. Based on this information the test material 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 media preparation trials were conducted in order to determine the solubility of the test material under test conditions. 

Pre-study media preparation trials indicated that the use of preliminary solution in auxiliary solvent to spike the test medium followed by centrifugation at 10000 g to remove any undissolved test material was the most suitable method of preparation. 

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a geometric mean measured test concentration of 0.11 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Results. 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.18 to 0.19 mg/l. A decline in measured test concentrations was observed at 72 hours in the range of 0.057 to 0.068 mg/l. This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium. 

The 0-Hour measured concentrations were in excess of those obtained during the pre-study media preparation trial conducted (0.070 mg/l). This was considered to be due to differences in the test media and also the technique used to remove the supernatant following centrifugation. Given that the values obtained were similar to the predicted water solubility value of 0.224 mg/l this was considered to have had no adverse effect on the outcome of the test.

Given the decline in measured test concentrations over the test period it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

The EC50 values based on the geometric mean measured test concentrations were greater than 0.11 mg/l and correspondingly the No Observed Effect Concentration was 0.11 mg/l.

Exposure of Desmodesmus subspicatusto the reference material, potassium dichromate, gave an ErC50(0 - 72 h) of 0.57 mg/l, an EyC50(0 - 72 h) of 0.32 mg/l, and an EbC50(0 - 72 h) of 0.31 mg/l. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/l respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/l respectively. 

Key value for chemical safety assessment

EC50 for freshwater algae:
0.11 mg/L
EC10 or NOEC for freshwater algae:
0.11 mg/L

Additional information

The test concentration of 0.11 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.