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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):None
Analytical monitoring:
yes
Details on sampling:
- Concentrations:Range-Finding Test : nominal test concentrations of 1.0, 10 and 100% v/v saturated solutionDefinitive Test : Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)Preliminary Media Preparation TrialInformation provided by the Sponsor indicated the water solubility of the test item to be less than 0.2 mg/L. Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.Based on this information the test item was categorized 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.Range-Finding Test:A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters 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 and 1.0% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.3 mL) to give the required test concentrations of 1.0, 10 and 100% v/v saturated solution.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 ± 1 ºC 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.A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.Definitive test:A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters 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 stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (1 liter) of each of the stock solutions was separately inoculated with 6.7 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- Strain: CCAP 278/4.- Source (laboratory, culture collection): Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not available
Test temperature:
24 ± 1 ºC
pH:
The pH value of the control cultures was observed to range from pH 7.6 at 0 hours to pH 7.4 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 available
Salinity:
Not applicable
Nominal and measured concentrations:
Range-Finding Test : nominal test concentrations of 1.0, 10 and 100% v/v saturated solutionDefinitive Test : Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Details on test conditions:
Range-Finding Test: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 ± 1 ºC 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.A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.Definitive Test:As in the range-finding test 250 mL glass conical flasks were used. 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.The control group was maintained under identical conditions but not exposed to the test item.Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 7.45 x 105 cells per mL. Inoculation of 1 liter of test medium with 6.7 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.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.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
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:
EC50
Effect conc.:
86 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 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.:
50 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
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
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:
other: Yield
Details on results:
Range-finding TestThe results showed no effect on growth at the test concentrations of 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.Chemical analysis of the test preparations at 0 and 72 hours (see Appendix 4) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.082 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.Definitive TestVerification of Test ConcentrationsAnalysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.082 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.Growth DataIt is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.Accordingly the following results were determined from the data:Inhibition of Growth RateErC10 (0 - 72 h) : 76% v/v saturated solutionErC20 (0 - 72 h) : >100% v/v saturated solutionErC50 (0 - 72 h) : >100% v/v saturated solutionwhere ErCx is the test concentration that reduced growth rate by x%.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, 6.25, 12.5, 25 and 50% 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 50% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100% v/v saturated solutionInhibition of YieldEyC10 (0 - 72 h) : 41% v/v saturated solution EyC20 (0 - 72 h) : 54% v/v saturated solution EyC50 (0 - 72 h) : 86% v/v saturated solutionWhere:EyCx is the test concentration that reduced yield by x%.Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences between the control, 6.25, 12.5, 25 and 50% v/v saturated solution test concentrations (P≥0.05), however the 100% v/v saturated test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 50% v/v saturated solution. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100% v/v saturated solutionValidation CriteriaThe following data show that the cell concentration of the control cultures increased by a factor of 256 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 : 3.46 x 103 cells per mLMean cell density of control at 72 hours : 8.86 x 105 cells per mLThe mean coefficient of variation for section by section specific growth rate for the control cultures was 22% 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%.Observations on CulturesAll test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50% v/v saturated solution, however cell clumping and enlarged cells were observed to be present in the test cultures at 100% v/v saturated solution.Water Quality CriteriaTemperature was maintained at 24 ± 1 ºC throughout the test.The pH value of the control cultures was observed to range from pH 7.6 at 0 hours to pH 7.4 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.Observations on Test Item SolubilityAt the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 6.25, 12.5, 25 and 50% v/v saturated solution test preparations were observed to be bright green dispersions whilst the 100% v/v saturated solution test preparations were observed to be green dispersions.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:ErC50 (0 – 72 h) : 1.2 mg/L; 95% confidence limits 1.1 – 1.4 mg/LEyC50 (0 – 72 h) : 0.63 mg/L; 95% confidence limits 0.57 – 0.70 mg/LNo Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/LNo Observed Effect Concentration (NOEC) based on yield: 0.25 mg/LLowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/LLowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
Reported statistics and error estimates:
Statistical Analysis 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) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

None

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results based on nominal test concentrations:EC50 (% v/v Saturated Solution) : >100 (Growth Rate)EC50 (% v/v Saturated Solution) : 86 (Yield)No Observed Effect Concentration (NOEC) (% v/v Saturated Solution) : 50 (Growth Rate, Yield)Lowest Observed Effect Concentration (LOEC) (% v/v Saturated Solution) : 100 (Growth Rate, Yield)
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 761/2009.

Information provided by the Sponsor indicated the water solubility of the test item to be less than 0.2 mg/L. Preliminary 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.

A preliminary media preparation trial indicated that a saturated solution method of preparation was most appropriate for this test item.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 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 (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters 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.

Results:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.082 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

EC50 (% v/v Saturated Solution) : >100 (Growth Rate)

EC50 (% v/v Saturated Solution) : 86 (Yield)

No Observed Effect Concentration (NOEC) (% v/v Saturated Solution) : 50 (Growth Rate, Yield)

Lowest Observed Effect Concentration (LOEC) (% v/v Saturated Solution) : 100 (Growth Rate, Yield)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):None
Analytical monitoring:
yes
Details on sampling:
None
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: 125 mg test material was dissolved in auxiliary solvent and the volume made up to 25 ml. A 200 µl aliquot of the solvent stock solution was dispensed to 2 litres of algal suspension to give the required test concentrations.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM- Common name: Scenedesmus subspicatus (Chodat)- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Cumbria, U.K.- Method of cultivation: Sterile culture medium was innoculated from a master culture of Scenedesmus subspicatus and incubated under conditions illumination and aeration at 21 0C tp give an algal suspension in log phase growth characterised by an abasorbance of 0.894 (@665nm). The suspension was diluted to an absorbance of 0.030 prior to use. This suspension had a mean cell density of 1.86 X 104 ceels/ml.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not available
Test temperature:
24 ± 1 ºC
pH:
The pH of each test and control culture was measured at 0 and 72 hours.
Dissolved oxygen:
Not available
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
None
Details on test conditions:
TEST SYSTEM- Test vessel: 250ml conical flasks stoppered to reduce evaporation- Type (delete if not applicable): closed- Material, size, headspace, fill volume: 250ml- Initial cells density: 0.50 mg/l
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
None
Results with reference substance (positive control):
None
Reported statistics and error estimates:
None

Inhibition of growth from the exposure of Scenedesmus subspicatus to D-523 for a period of 72 hours:

Nominal Concentration (mg/l Area under curve @72h % inhibition Growth rate (24h - 48h) % inhibition
Control 17.156  - 0.059 -
Solvent control 17.168  - 0.065 -
0.5 17.378 [1] 0.063 3
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Scenedesmus subspicatus has been investigated over a 72-Hour period and gave the following results based on nominal test concentrations:EbC50 (72h) value : >0.50mg/l (Growth Rate)ErC50 (24-48h) value : >0.50mg/l (Yield)The following results are based on the 72-hour mean measured test concentrations:EbC50 (72h) value : >0.25mg/l (Growth Rate)ErC50 (24-48h) value : >0.25mg/l (Yield)No Observed Effect Concentration (NOEC) (% v/v Saturated Solution) : >=0.50mg/l (Growth Rate, Yield)
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 761/2009.

The test concentration was assigned following a preliminary range finding study whereby the test organisms were exposed to the highest attainable test concentration of 0.50 mg/l for a period of 72 hours. 0.5 mg/l was the highest test concentration that could be prepared given the limited solubility of the test material in water and auxiliary solvent.

Samples were taken at 0, 24, 48 and 72 hours and the absorbance was measured at 665nm using a Jenway 6100 Spectrophotometer.

Results:

The absorbance values were sufficiently well correlated with cell density values to be used to monitor the growth of the test cultures.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

EbC50 (72h) value : >0.50mg/l (Growth Rate)

ErC50 (24-48h) value : >0.50mg/l (Yield)

The following results are based on the 72-hour mean measured test concentrations:

EbC50 (72h) value : >0.25mg/l (Growth Rate)

ErC50 (24-48h) value : >0.25mg/l (Yield)

No Observed Effect Concentration (NOEC) (% v/v Saturated Solution) : >=0.50mg/l (Growth Rate, Yield)

Description of key information

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:
EC50 (% v/v Saturated Solution) : >100 (Growth Rate)
EC50 (% v/v Saturated Solution) : 86 (Yield)
No Observed Effect Concentration (NOEC) (% v/v Saturated Solution) : 50 (Growth Rate, Yield)
Lowest Observed Effect Concentration (LOEC) (% v/v Saturated Solution) : 100 (Growth Rate, Yield)

Key value for chemical safety assessment

Additional information

A key study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 761/2009.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

EC50 (% v/v Saturated Solution) : >100 (Growth Rate)

EC50 (% v/v Saturated Solution) : 86 (Yield)

No Observed Effect Concentration (NOEC) (% v/v Saturated Solution) : 50 (Growth Rate, Yield)

Lowest Observed Effect Concentration (LOEC) (% v/v Saturated Solution) : 100 (Growth Rate, Yield)

Another supporting study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 761/2009.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

EbC50 (72h) value : >0.50mg/l (Growth Rate)

ErC50 (24-48h) value : >0.50mg/l (Yield)

The following results are based on the 72-hour mean measured test concentrations:

EbC50 (72h) value : >0.25mg/l (Growth Rate)

ErC50 (24-48h) value : >0.25mg/l (Yield)

No Observed Effect Concentration (NOEC) (% v/v Saturated Solution) : >=0.50mg/l (Growth Rate, Yield)