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Ecotoxicological information

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:
24 February 2010 and 11 March 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and a current guideline
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
See principles of method if other than guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
See principles of method if other than guideline
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/l) of test item in culture medium for a period of 48 hours prior to removing any undissolved test item present by filtration (0.2 μm Gelman Acrocap, first approximate 500 discarded in order to pre-condition the filter) to give a saturated solution of the test item.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/2009
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

- Sampling method:
The concentration of the test item in the test solutions were verified by chemical analysis at 0 and 72 hours.

Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.

- Sample storage conditions before analysis:
Samples were stored at approximately 4°C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20ºC for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Definitive test:
An amount of test item (250 mg) was dispersed in 2.5 litres of culture medium with the aid of magnetic stirring at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 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 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (500 ml) of each of the stock solutions was separately inoculated with 12.1 ml algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (equivalent to 0.013, 0.041, 0.13, 0.41 and 1.3 mg/l as test item based on the mean measured test concentrations obtained for the 32 and 100% v/v saturated solution test preparations.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration of the test item in the test solutions was verified by chemical analysis at 0 and 72 hours (see Appendix 4 - attached background material).

- Controls:
A positive control used potassium dichromate as the reference item.











- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name:
Algae

- Strain:
Strain CCAP 276/20

- Source:
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:

Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. 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 10E4 - 10E5 cells/ml.


ACCLIMATION

- Acclimation period:
Not recorded.

- Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture Medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
Hardness:
Not reported.
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test.
pH:
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.
pH range: 7.1 - 7.5
Dissolved oxygen:
Not reported.
Salinity:
Not reported.
Nominal and measured concentrations:
Range-finding:
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.
Definitive test:
Based on the results of the pre-study media preparation trial and range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution (equivalent to 0.013, 0.041, 0.13, 0.41 and 1.3 mg/l as test item based on the mean measured test concentrations obtained for the 32 and 100% v/v saturated solution test preparations).
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type (delete if not applicable): closed: flasks were plugged with polyurethane foam bungs.
- Material, size, headspace, fill volume: 250 ml glass conical flasks containing 100 ml of solution.
- Aeration: No aeration.
- Renewal rate of test solution (frequency/flow rate): No renewal.
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.65 x 10E5 cells per ml. Inoculation of 500 ml of test medium with 12.1 ml of this algal suspension gave an initial nominal cell density of 4 x 10E3 cells per ml and had no significant dilution effect on the final test concentration.
- Six flasks each containing 100 ml of solution were used for the control and three flasks each containing 100 ml were used for each treatment group.

GROWTH MEDIUM
- Standard medium used: yes - 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
- Adjustment of pH: The culture medium pH was adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Photoperiod and light intensity: The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C 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.



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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
- Range finding study
- Test concentrations:
Due to the low aqueous solubility and high purity of the test item the test concentrations used in the range-finding test were prepared by diluting (with culture medium) a saturated solution prepared from an initial test item dispersion at a concentration of 100 mg/l.
The test concentrations to be used in the definitive test were 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.
An amount of test item (200 mg) was dispersed in 2 litres of culture medium with the aid of magnetic stirring at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 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 (4.7 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
A sample of the uninoculated control and 100% v/v saturated solution were taken for chemical analysis at 0 hours to determine the concentration of manganese, and hence the dissolved test item concentration present in the saturated solution (see Appendix 4).
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 control group was maintained under identical conditions but not exposed to the test item.
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.

- Results used to determine the conditions for the definitive study:
Based on the results of the pre-study media preparation trial and range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
32 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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 1.
The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.

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 Table3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 (see attached background material for figures 1-3).

VALIDATION CRITERIA
The following data show that the cell concentration of the control cultures increased by a factor of 42 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 : 4.28 x 103 cells per ml
Mean cell density of control at 72 hours : 1.78 x 105 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 32% 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 2% 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 2 and 4, it is clear that the growth rate (r) and yield (y) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test item over the 72 Hour exposure period.
Accordingly the following results were determined from the data expressed in terms of % v/v saturated solution:

Inhibition of growth rate:
ErC10 (0 - 72 h) : 56% v/v saturated solution
ErC20 (0 - 72 h) : 81% v/v saturated solution
ErC50 (0 - 72 h) : >100% v/v saturated solution*

*It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate
where ErCx is the test concentration that reduced growth rate by x%.


EyC10 (0 - 72 h) : 41% v/v saturated solution
EyC20 (0 - 72 h) : 51% v/v saturated solution
EyC50 (0 - 72 h) : 81% v/v saturated solution
where EyCx is the test concentration that reduced yield by x%.


Observations 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.

Observations on test item solubility:
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, 1.0, 3.2 and 10% v/v saturated solution test cultures were observed to be pale green dispersions. The 32% v/v saturated solution test cultures were observed to be very pale green dispersions whilst the 100% v/v saturated solution test cultures were observed to be clear colourless solutions.

Physico-chemical measurements:
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 7.2 at 0 hours to pH 7.5 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.

Verification of test concentrations:
The test item contained a theoretical manganese content of 77% w/w. The test samples were analysed for manganese only. Analysis of the test preparations at 0 hours (see Appendix 4) showed measured test concentrations to range from less than the limit of quantitation (LOQ) of the analytical method employed to 1.3 mg/l as test item. Analysis of the test preparations at 72 hours showed measured test concentrations to range from less than the LOQ to 1.3 mg/l as test item.
Given that no decline in measured concentration was observed over the test period, it was considered appropriate to determine the theoretical measured concentrations of the 1.0, 3.2 and 10% v/v saturated solution test concentrations based on the mean measured test concentrations obtained for the 32 and 100% v/v saturated solution test preparations. The theoretical measured test concentrations were determined to be:

Nominal Test Concentration (% v/v saturated solution) Theoretical/Mean Measured Test Item Concentration (mg/l)
1.0 0.013
3.2 0.041
10 0.13
32 0.41
100 1.3

Accordingly the following results were determined from the data based on the theoretical/mean measured test concentrations:
Growth rate:
ErC10 (0 - 72 h) : 0.73 mg/l
ErC20 (0 - 72 h) : 1.1 mg/l
ErC50 (0 - 72 h) : >1.3 mg/l*
* It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate.

No Observed Effect Concentration (NOEC) = 0.41 mg/l
Lowest Observed Effect Concentration (LOEC) = 1.3 mg/l

Yield:
EyC10 (0 - 72 h) : 0.53 mg/l
EyC20 (0 - 72 h) : 0.66 mg/l
EyC50 (0 - 72 h) : 1.1 mg/l
No Observed Effect Concentration (NOEC) = 0.41 mg/l
Lowest Observed Effect Concentration (LOEC) = 1.3 mg/l
Results with reference substance (positive control):
A positive control used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h) : 0.49 mg/l*
EyC50 (0 – 72 h) : 0.18 mg/l, 95% confidence limits 0.16 – 0.21 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

*It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Reported statistics and error estimates:
Inhibition of growth rate:
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 1.0, 3.2, 10 and 32% v/v saturated solution test concentrations (P≥0.05), however the 100% v/v saturated solution test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32% v/v saturated solution.

Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100% v/v saturated solution.

Inhibition of yield:
Statistical analysis of the yield data was carried out as in Section 5.2.2.1. There were no statistically significant differences between the control, 1.0, 3.2, 10 and 32% v/v saturated solution test concentrations (P≥0.05), however the 100% v/v saturated solution test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 32% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100% v/v saturated solution.

Table 1              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

4.10E+03

3.78E+05

 

 

 

R2

3.99E+03

2.86E+05

-

-

 

Mean

4.05E+03

3.32E+05

 

 

0.10

R1

4.84E+03

4.40E+05

 

 

 

R2

4.61E+03

4.43E+05

[3]

[33]

 

Mean

4.72E+03

4.41E+05

 

 

1.0

R1

4.83E+03

3.25E+05

 

 

 

R2

4.39E+03

4.21E+05

0

[12]

 

Mean

4.61E+03

3.73E+05

 

 

10

R1

4.45E+03

1.88E+05

 

 

 

R2

6.17E+03

2.04E+05

18

42

 

Mean

5.31E+03

1.96E+05

 

 

100

R1

5.39E+03

2.99E+04

 

 

 

R2

5.14E+03

2.86E+04

61

93

 

Mean

5.27E+03

2.93E+04

 

 


*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.

R1and R2= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

Theoretical/Mean Measured Test Item Concentration

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.2

4.35E+03

1.05E+04

3.66E+04

1.93E+05

7.5

 

R2

7.2

3.90E+03

1.18E+04

3.30E+04

1.63E+05

7.5

 

R3

7.2

4.54E+03

8.85E+03

2.48E+04

1.54E+05

7.5

 

R4

7.2

4.35E+03

1.06E+04

3.90E+04

1.84E+05

7.5

 

R5

7.2

4.43E+03

9.77E+03

2.54E+04

2.07E+05

7.5

 

R6

7.2

4.08E+03

1.05E+04

3.89E+04

1.66E+05

7.5

 

Mean

 

4.28E+03

1.03E+04

3.30E+04

1.78E+05

 

0.013

R1

7.2

4.31E+03

9.01E+03

3.86E+04

2.33E+05

7.5

 

R2

7.2

4.05E+03

9.28E+03

3.50E+04

2.48E+05

7.5

 

R3

7.2

4.14E+03

1.01E+04

3.67E+04

2.05E+05

7.5

 

Mean

 

4.17E+03

9.47E+03

3.68E+04

2.29E+05

 

0.041

R1

7.1

4.12E+03

5.35E+03

2.98E+04

2.03E+05

7.5

 

R2

7.1

4.31E+03

6.04E+03

3.00E+04

2.39E+05

7.5

 

R3

7.1

4.37E+03

8.77E+03

3.24E+04

2.12E+05

7.5

 

Mean

 

4.27E+03

6.72E+03

3.07E+04

2.18E+05

 

0.13

R1

7.1

4.21E+03

8.72E+03

4.22E+04

2.65E+05

7.5

 

R2

7.1

4.46E+03

7.42E+03

3.12E+04

2.31E+05

7.5

 

R3

7.1

4.10E+03

6.82E+03

3.28E+04

2.21E+05

7.5

 

Mean

 

4.26E+03

7.65E+03

3.54E+04

2.39E+05

 

0.41

R1

7.1

4.13E+03

7.25E+03

3.31E+04

1.23E+05

7.5

 

R2

7.1

4.26E+03

4.94E+03

3.73E+04

1.70E+05

7.5

 

R3

7.1

4.30E+03

5.21E+03

3.81E+04

1.63E+05

7.5

 

Mean

 

4.23E+03

5.80E+03

3.62E+04

1.52E+05

 

1.3

R1

7.5

3.97E+03

7.82E+03

1.88E+04

8.32E+04

7.3

 

R2

7.5

4.52E+03

9.03E+03

1.64E+04

5.09E+04

7.3

 

R3

7.5

4.23E+03

8.89E+03

1.51E+04

5.75E+04

7.3

 

Mean

 

4.24E+03

8.58E+03

1.68E+04

6.39E+04

 


*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 the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.040

0.052

0.069

 

R2

0.045

0.043

0.067

 

R3

0.033

0.043

0.076

 

R4

0.041

0.054

0.065

 

R5

0.037

0.040

0.087

 

R6

0.040

0.054

0.061

 

Mean

0.039

0.048

0.071


R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Theoretical/Mean Measured Test Item Concentration (mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.054

 

1.89E+05

 

 

R2

0.052

 

1.59E+05

 

 

R3

0.051

 

1.49E+05

 

 

R4

0.053

-

1.80E+05

-

 

R5

0.055

 

2.02E+05

 

 

R6

0.052

 

1.62E+05

 

 

Mean

0.053

 

1.74E+05

 

 

SD

0.001

 

2.01E+04

 

0.013

R1

0.056

[6]

2.29E+05

 

 

R2

0.057

[8]

2.44E+05

 

 

R3

0.055

[4]

2.01E+05

 

 

Mean

0.056

[6]

2.24E+05

[29]

 

SD

0.001

 

2.19E+04

 

0.041

R1

0.055

[4]

1.99E+05

 

 

R2

0.057

[8]

2.35E+05

 

 

R3

0.055

[4]

2.07E+05

 

 

Mean

0.056

[5]

2.14E+05

[23]

 

SD

0.001

 

1.86E+04

 

0.13

R1

0.058

[9]

2.61E+05

 

 

R2

0.056

[6]

2.26E+05

 

 

R3

0.056

[6]

2.17E+05

 

 

Mean

0.057

[7]

2.35E+05

[35]

 

SD

0.001

 

2.29E+04

 

0.41

R1

0.048

9

1.19E+05

 

 

R2

0.052

2

1.66E+05

 

 

R3

0.052

2

1.59E+05

 

 

Mean

0.051

4

1.48E+05

15

 

SD

0.002

 

2.52E+04

 

1.3

R1

0.042

21

7.92E+04

 

 

R2

0.035

34

4.64E+04

 

 

R3

0.037

30

5.32E+04

 

 

Mean

0.038

28

5.96E+04

66

 

SD

0.004

 

1.73E+04

 


*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results expressed in terms of % v/v saturated solution:
Growth rate EC50: >100% v/v saturated solution
Growth rate NOEC: 32% v/v saturated solution
Growth rate LOEC: 100% v/v saturated solution
Yield EC50: 81% v/v saturated solution
Yield NOEC: 32% v/v saturated solution
Yield LOEC: 100% v/v saturated solution

Based on the theoretical/mean measured test items concentrations, exposure of Desmodesmus subspicatus to the test item gave the following results:
Growth rate EC50: >1.3 mg/l
Growth rate NOEC: 0.41 mg/l
Growth rate LOEC: 1.3 mg/l
Yield EC50: 1.1 mg/l
Yield NOEC: 0.41 mg/l
Yield LOEC: 1.3 mg/l
Executive summary:

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality for classification and labelling and for risk assessment.

Introduction.

A study was perford to assess the effect of the test item on the growth of the green algaDesmodesmus 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 Regulation (EC) No 440/2008.

Methods. 

Information provided by the Sponsor indicated that the test item was insoluble in water. 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. Based on information supplied by the Sponsor it was considered that the most appropriate method of preparation for the test item was as a saturated solution.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test item at concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solutions were prepared by stirring an excess (100 mg/l) of test item in culture medium using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 ml discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter. ResultsExposure of Desmodesmus subspicatus to the test item gave the following results expressed in terms of % v/v saturated solution:

Response Variable

EC50

(% v/v Saturated Solution)

No Observed Effect Concentration (NOEC)

(% v/v Saturated Solution)

Lowest Observed Effect Concentration (LOEC)

(% v/v Saturated Solution)

Growth Rate

>100*

32

100

Yield

81

32

100


*It was not possible to calculate an ErC50value as no concentration tested resulted in greater than 50% inhibition of growth rate.

The test item contained a theoretical manganese content of 77% w/w. The test samples were analysed for manganese only. Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantitation (LOQ) of the analytical method employed to 1.3 mg/l as test item. Analysis of the test preparations at 72 hours showed measured test concentrations to range from less than the LOQ to 1.3 mg/l as test item.

Given that no decline in measured concentration was observed over the test period, it was considered appropriate to determine the theoretical measured concentrations of the 1.0, 3.2 and 10% v/v saturated solution test concentrations based on the mean measured test concentrations obtained for the 32 and 100% v/v saturated solution test preparations.

Exposure of Desmodesmus subspicatus to the test item gave the following results expressed in terms of the test item:

Response Variable

EC50

(mg/l)

No Observed Effect Concentration (NOEC)

(mg/l)

Lowest Observed Effect Concentration (LOEC)

(mg/l)

Growth Rate

>1.3*

0.41

1.3

Yield

1.1

0.41

1.3

*It was not possible to calculate an ErC50value as no concentration tested resulted in greater than 50% inhibition of growth rate.

Description of key information

The effect of the test item on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results expressed in terms of % v/v saturated solution:

Growth rate EC50: >100% v/v saturated solution

Growth rate NOEC: 32% v/v saturated solution

Growth rate LOEC: 100% v/v saturated solution

Yield EC50: 81% v/v saturated solution

Yield NOEC: 32%  v/v saturated solution

Yield LOEC: 100%  v/v saturated solution

Based on the theoretical/mean measured test items concentrations, exposure of Desmodesmus subspicatus to the test item gave the following results:

Growth rate EC50: >1.3 mg/l

Growth rate NOEC: 0.41 mg/l

Growth rate LOEC: 1.3 mg/l

Yield EC50: 1.1 mg/l

Yield NOEC: 0.41 mg/l

Yield LOEC: 1.3 mg/l

Key value for chemical safety assessment

Additional information

This was chosen as Key study as it is the only available study which is of relevance and of sufficient quality for classification and labelling and for risk assessment.

Introduction.

A study was perford to assess the effect of the test item on the growth of the green algaDesmodesmus 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 Regulation (EC) No 440/2008.

Methods. 

Information provided by the Sponsor indicated that the test item was insoluble in water. 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. Based on information supplied by the Sponsor it was considered that the most appropriate method of preparation for the test item was as a saturated solution.

Following a preliminary range-finding test,Desmodesmus subspicatuswas exposed to solutions of the test item at concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solutions were prepared by stirring an excess (100 mg/l) of test item in culture medium using a magnetic stirrer at approximately 100 rpm at a temperature of approximately 21°C for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 ml discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer ParticleCounter.ResultsExposure ofDesmodesmus subspicatusto the test item gave the following results expressed in terms of % v/v saturated solution:

Response Variable

EC50

(% v/v Saturated Solution)

No Observed Effect Concentration (NOEC)

(% v/v Saturated Solution)

Lowest Observed Effect Concentration (LOEC)

(% v/v Saturated Solution)

Growth Rate

>100*

32

100

Yield

81

32

100


*It was not possible to calculate an ErC50value as no concentration tested resulted in greater than 50% inhibition of growth rate.

The test item contained a theoretical manganese content of 77% w/w. The test samples were analysed for manganese only. Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantitation (LOQ) of the analytical method employed to 1.3 mg/l as test item. Analysis of the test preparations at 72 hours showed measured test concentrations to range from less than the LOQ to 1.3 mg/l as test item.

Given that no decline in measured concentration was observed over the test period, it was considered appropriate to determine the theoretical measured concentrations of the 1.0, 3.2 and 10% v/v saturated solution test concentrations based on the mean measured test concentrations obtained for the 32 and 100% v/v saturated solution test preparations.

Exposure ofDesmodesmus subspicatusto the test item gave the following results expressed in terms of the test item:

Response Variable

EC50

(mg/l)

No Observed Effect Concentration (NOEC)

(mg/l)

Lowest Observed Effect Concentration (LOEC)

(mg/l)

Growth Rate

>1.3*

0.41

1.3

Yield

1.1

0.41

1.3

*It was not possible to calculate an ErC50value as no concentration tested resulted in greater than 50% inhibition of growth rate.