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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:
February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. certificate)
Remarks:
Freie und Hansestadt Hamburg, Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations were analysed- Sampling method: samples of approx. 10 mL were taken at start and end of the test- Sample storage conditions before analysis: samples were retained at -20 °C or colder until shipment for analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:The test substance was dissolved in exposure medium by vigorously shaking the flask. The test started with a limit test at 100, 10, and 1.0 mg product/L (equal to 38.8, 3.9 and 0.39 mg a.i./L). For the main test, six concentrations arranged in a geometric series were selected (0.39, 1.2, 3.9, 12.2, 38.8 and 122.6 mg a.i./L).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- Common name: green algae- Strain: Pseudokirchnerella subcapitata/61.81 SAG - Source (laboratory, culture collection): Pflanzenphysiologisches Institut der Universität Göttingen, Sammlung von Algenkulturen, Göttingen, GermanyMaintanance of the culture: 23.0 – 23.4°C maintained in the air-conditioned chamberACCLIMATION- Culturing media and conditions (same as test or not): yes- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Test temperature:
23.0 – 23.4°C
pH:
Control: 7.74 - 7.86 (main study)Test bottles: 8.47 - 9.67
Nominal and measured concentrations:
Range finding test: 0, 0.39, 3.9, 38.8 mg a.i./L (nominal)Main test: 0, 0.39, 1.2, 3.9, 12.2, 38.8 and 122.6 mg a.i./L (nominal)The determination of the test item concentrations revealed actual concentrations of the test substance 82.6% to 132.1% of the nominal concentration. However, the concentration range of 0.39 to 1.2 mg/L was below the detection limit.
Details on test conditions:
TEST SYSTEM- Test vessel: 250 mL conical flasks containing 100 mL of medium, continuously shaken on an orbital bottle shaker- Aeration: no- Initial cells density: 1 x 10^4 cells/mL- Control end cells density: 1.3 x 10^8- No. of vessels per concentration (replicates): 3- No. of vessels per control (replicates): 6GROWTH MEDIUM- Standard medium used: yesTEST MEDIUM / WATER PARAMETERS- prepared according to OECD guideline 201 in 5 L distilled and demineralised waterOTHER TEST CONDITIONS- Sterile test conditions: all procedures were carried out taking the necessary sterility precautions- Adjustment of pH: no- Photoperiod: 24-hour white-type fluorescent lamp- Light intensity and quality: 7200 lux (0.63 x 1020 photons/m2*s)EFFECT PARAMETERS MEASURED:- Determination of cell concentrations: counting chamberTEST CONCENTRATIONS- Range finding study: range finding test: 0, 0.39, 3.9, 38.8 mg a.i./L (nominal)- Test concentrations: 0, 0.39, 1.2, 3.9, 12.2, 38.8 and 122.6 mg a.i./L (nominal)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.64 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 1.76 - 42.5 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.95 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes- Observation of abnormalities (for algal test): no- Unusual cell shape: no- Colour differences: no- Any stimulation of growth found in any treatment: no- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
The positive control substance potassium dichromate caused the expected effect on algal growth inhibition and demonstrated the sensitivity of the test system. An EC50 of 0.23 mg/L was calculated.

Inhibition of cell growth (main test):

Concentration

[mg a.i./L]

Inhibition of cell growth caused by C10AS

[%]

0

 

1

5.4

3.16

25.1

10

26.7

31.6

32.9

100

42.1

316

99.7

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
no guideline followed
Principles of method if other than guideline:
96 h growth inhibition study on green algae
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- Source (laboratory, culture collection): Norwegian Institute of Water Research (NIVA)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
4.1 mg CaCO3/L
Test temperature:
20 °C
pH:
7.0 - 7.5
Salinity:
not relevant
Details on test conditions:
TEST SYSTEM- Test vessel: 1L flasks- Type (delete if not applicable): open - Aeration: 0.8% CO2 in air at a flow rate of 0.5L/min- Initial cells density: 10000 - 11000 cells/mLTEST MEDIUM- Deionized water containing 0.37 mg/L Na2EDTA.2H2O, 2.1 mg/L Na2CO3OTHER TEST CONDITIONS- Light intensity and quality: 15000 luxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: optical density at 600 nm
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
117 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC90
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes, growth rate 1.23 ± 0.006/day (growth inhibition)
Reported statistics and error estimates:
Toxic responses were quantified by a simple graphical method. Relative response (absolute response devided by average response of the controls) was plotted against log concentration and EC10, EC50 and EC90 read from graph.Growth inhibition test: Algae were grown in approximately one-liter flasks submerged in temperature-controlled aquaria. Flasks were aerated continuously with 0.8% CO2 in air at a flow rate of 0.5 l/minute. pH was adjusted daily and did not exceed 8.0. Biomass was measured once daily. Six concentrations were tested.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations were analysed- Sampling method: samples of approx. 10 mL were taken at start and end of the test- Sample storage conditions before analysis: samples were retained at -20 °C or colder until shipment for analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:The test substance was dissolved in exposure medium by vigorously shaking the flask. The test started with a limit test at 100, 10, and 1.0 mg product/L (equal to 38.8, 3.9 and 0.39 mg a.i./L). For the main test, six concentrations arranged in a geometric series were selected (0.39, 1.2, 3.9, 12.2, 38.8 and 122.6 mg a.i./L).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- Common name: green algae- Strain: Pseudokirchnerella subcapitata/61.81 SAG - Source (laboratory, culture collection): Pflanzenphysiologisches Institut der Universität Göttingen, Sammlung von Algenkulturen, Göttingen, GermanyMaintanance of the culture: 23.0 – 23.4°C maintained in the air-conditioned chamberACCLIMATION- Culturing media and conditions (same as test or not): yes- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none
Test temperature:
23.0 – 23.4°C
pH:
Control: 7.74 - 7.86 (main study)Test bottles: 8.47 - 9.67
Nominal and measured concentrations:
Range finding test: 0, 0.39, 3.9, 38.8 mg a.i./L (nominal)Main test: 0, 0.39, 1.2, 3.9, 12.2, 38.8 and 122.6 mg a.i./L (nominal)The determination of the test item concentrations revealed actual concentrations of the test substance 82.6% to 132.1% of the nominal concentration. However, the concentration range of 0.39 to 1.2 mg/L was below the detection limit.
Details on test conditions:
TEST SYSTEM- Test vessel: 250 mL conical flasks containing 100 mL of medium, continuously shaken on an orbital bottle shaker- Aeration: no- Initial cells density: 1 x 10^4 cells/mL- Control end cells density: 1.3 x 10^8- No. of vessels per concentration (replicates): 3- No. of vessels per control (replicates): 6GROWTH MEDIUM- Standard medium used: yesTEST MEDIUM / WATER PARAMETERS- prepared according to OECD guideline 201 in 5 L distilled and demineralised waterOTHER TEST CONDITIONS- Sterile test conditions: all procedures were carried out taking the necessary sterility precautions- Adjustment of pH: no- Photoperiod: 24-hour white-type fluorescent lamp- Light intensity and quality: 7200 lux (0.63 x 1020 photons/m2*s)EFFECT PARAMETERS MEASURED:- Determination of cell concentrations: counting chamberTEST CONCENTRATIONS- Range finding study: range finding test: 0, 0.39, 3.9, 38.8 mg a.i./L (nominal)- Test concentrations: 0, 0.39, 1.2, 3.9, 12.2, 38.8 and 122.6 mg a.i./L (nominal)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.64 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits: 1.76 - 42.5 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.95 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes- Observation of abnormalities (for algal test): no- Unusual cell shape: no- Colour differences: no- Any stimulation of growth found in any treatment: no- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
The positive control substance potassium dichromate caused the expected effect on algal growth inhibition and demonstrated the sensitivity of the test system. An EC50 of 0.23 mg/L was calculated.

Inhibition of cell growth (main test):

Concentration

[mg a.i./L]

Inhibition of cell growth caused by C10AS

[%]

0

 

1

5.4

3.16

25.1

10

26.7

31.6

32.9

100

42.1

316

99.7

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM- Source (laboratory, culture collection): Norwegian Institute of Water Research (NIVA)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
4.1 mg CaCO3/L
Test temperature:
20°C
pH:
7.0 - 7.5
Salinity:
not relevant
Details on test conditions:
TEST SYSTEM- Test vessel: 1L flasks- Type (delete if not applicable): open - Aeration: 0.8% CO2 in air at a flow rate of 0.5L/min- Initial cells density: 10000 - 11000 cells/mLTEST MEDIUM- Deionized water containing 0.37 mg/L Na2EDTA.2H2O, 2.1 mg/L Na2CO3OTHER TEST CONDITIONS- Light intensity and quality: 15000 luxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: optical density at 600 nm
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
117 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC90
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes, growth rate 1.23 ± 0.006/day (growth inhibition)
Reported statistics and error estimates:
Toxic responses were quantified by a simple graphical method. Relative response (absolute response devided by average response of the controls) was plotted against log concentration and EC10, EC50 and EC90 read from graph.Growth inhibition test: Algae were grown in approximately one-liter flasks submerged in temperature-controlled aquaria. Flasks were aerated continuously with 0.8% CO2 in air at a flow rate of 0.5 l/minute. pH was adjusted daily and did not exceed 8.0. Biomass was measured once daily. Six concentrations were tested.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 September - 15 September 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Qualifier:
according to
Guideline:
other: DIN 38412, part 9
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: A stock solution was prepared by dissolving 0.5001 g/500 mL (medium not defined). From stock solution the required volumina for final test concentrations were taken.- Differential loading: yes
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM- Common name: Scenedesmus subspicatus, Chlorococcales- Strain: SAG 86.81- Source (laboratory, culture collection): Pflanzenphysiologisches Institut Universität Göttingen
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
22.5-24.5 °C
pH:
6.5 - 9.7
Nominal and measured concentrations:
Nominal: 0, 15, 30, 60 and 120 mg/LMeasured based on AS at test start: -, 14.7, 31.1, 62.8, 123.6 mg/LMeasured based on AS at test end: -, 15.7, 32, 59, 117.8 mg/LMeasured based on MBAS at test start: -, 17.8, 37.6, 75.9, 149.3 mg/LMeasured based on MBAS at test end: -, 19, 38.7, 71.3, 142.4 mg/L
Details on test conditions:
TEST SYSTEM- Test vessel: 300 mL Erlenmeyer flask, sterilised at 180°C- Filling volume: 100 mL test volume- Renewal rate of test solution (frequency/flow rate): static test- Initial cells density: 10,000 cells/mL- Control end cells density: 2,000,000 cells/mL- No. of vessels per concentration (replicates): 3 - No. of vessels per control (replicates): 3GROWTH MEDIUM- Standard medium used: yes (DIN 38412, part 9)TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: sterilised, distilled waterOTHER TEST CONDITIONS- Photoperiod: continuous ilumination- Light intensity and quality: 2000 Lux (Li-Cor-Li-185 B)EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : - Determination of cell concentrations: Coulter counterTEST CONCENTRATIONS- Spacing factor for test concentrations: 2 fold
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes- Observation of abnormalities (for algal test): no
Reported statistics and error estimates:
tabular calculation method using Lotus 1-2-3 software package
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose:
read-across source
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: A stock solution was prepared by dissolving 0.5001 g/500 mL (medium not defined). From stock solution the required volumina for final test concentrations were taken.- Differential loading: yes
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM- Common name: Scenedesmus subspicatus, Chlorococcales- Strain: SAG 86.81- Source (laboratory, culture collection): Pflanzenphysiologisches Institut Universität Göttingen
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
22.5 - 24.5 °C
pH:
6.5 - 9.7
Nominal and measured concentrations:
Nominal: 0, 15, 30, 60 and 120 mg/LMeasured based on AS at test start: 0, 14.7, 31.1, 62.8, 123.6 mg/LMeasured based on AS at test end: 0, 15.7, 32, 59, 117.8 mg/LMeasured based on MBAS at test start: -, 17.8, 37.6, 75.9, 149.3 mg/LMeasured based on MBAS at test end: -, 19, 38.7, 71.3, 142.4 mg/L
Details on test conditions:
TEST SYSTEM- Test vessel: 300 mL Erlenmeyer flask, sterilised at 180 °C- Filling volume: 100 mL test volume- Renewal rate of test solution (frequency/flow rate): static test- Initial cells density: 10,000 cells/mL- Control end cells density: 2,000,000 cells/mL- No. of vessels per concentration (replicates): 3 - No. of vessels per control (replicates): 3GROWTH MEDIUM- Standard medium used: yes (DIN 38412, part 9)TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: sterilised, distilled waterOTHER TEST CONDITIONS- Photoperiod: continuous ilumination- Light intensity and quality: 2000 Lux (Li-Cor-Li-185 B)EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : - Determination of cell concentrations: Coulter counterTEST CONCENTRATIONS- Spacing factor for test concentrations: 2 fold
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes- Observation of abnormalities (for algal test): no
Reported statistics and error estimates:
tabular calculation method using Lotus 1-2-3 software package

Description of key information

72h EC50 (growth rate) =  16.1 mg/L
72h EC10 (growth rate) = 1.76 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
16.1 mg/L
EC10 or NOEC for freshwater algae:
1.76 mg/L

Additional information

Since no reliable aquatic algae test is available for C9-12AS Na, in accordance to Regulation (EC) 1907/2006 Annex XI, 1.5, a read-across to other structurally related category member was conducted. Toxicity of alkyl sulfates is dependent on the carbon chain length and therefore read-across data from the following substances were used: C10AS Na (CAS 142-87-0) and C12AS Na (CAS 151-21-3).

One reliable study investigating the effects of C10AS Na (CAS 142-87-0) to the freshwater algae Pseudokirchneriella subcapitata is available (Huntsman, 2013). The test was performed according to OECD guideline 201 and is GLP compliant. Growth inhibition caused by the test substance was determined after 24, 48 and 72 h of exposure to seven test concentrations up to 122 mg a.i./L (nominal concentrations were shown to be in accordance with the measured ones with recoveries of 82.6% to 132.1%). The results showed that the 72-hour EC50 value for growth rate was 8.64 mg/L and the EC10 value for growth rate was 0.95 mg/L. There values were further used for the derivation of EC50 and EC10 values for C9-12AS Na.

Moreover, a GLP compliant study was performed with C12AS Na (CAS 151-2-3) according to guideline DIN 38412, part 9 (BASF, 1994). Aquatic algae Desmodesmus subspicatus were exposed for 72 hours to four test concentrations up to 120 mg/L (nominal concentrations). Analytical check of test concentrations showed that the nominal concentrations were in agreement with the measured ones and therefore the nominal concentrations were used. The 72-hour EC50 value for growth rate was above 120 mg/L (nominal concentration). The NOEC value for biomass was 30 mg/L (nominal concentration).

These results are supported by a publication of Nyholm and Damgaard (1990). The non-GLP test was performed with Pseudokirchnerella subcapitata exposed to six test concentrations. No special guideline was followed and analytical monitoring of test concentrations was not performed. Growth rate measurement after 96-h resulted in the ErC50 value of 117 mg/L (nominal concentration). EC10 for growth rate was 12 mg/L (nominal concentration). The study of Nyholm and Damgaard (1990) resulted in more conservative toxicity values, and therefore the EC10 of 12 mg/L and EC50 of 117 mg/L (both for growth rate) are considered as the reliable values for aquatic algae for C12AS Na (CAS 151-21-3). These values were further taken for the calculation of the EC50 and EC10 values of C9-12AS Na.

Due to structural and property similarities with the tested category member these results also apply to C9-12AS Na. The 72-hour EC50 and EC10 values for the C9-12AS Na were calculated based on the results for both homologues using the additivity formula as stipulated in the EC Regulation 1272/2008.

The following values were used in the calculation of the EC50: 8.64 mg/L for C9AS Na and C10AS Na. The EC50 of 117 mg/Lwas used for C11AS Na and C12AS Na. The EC10 value of 0.95 mg/L was used for C9AS Na and C10AS Na; the EC10 value of 12 mg/L was used for C11AS Na and C12AS Na. The composition of C9-12AS Na used for the calculations was based on the normalized mean values of provided ranges: C9: 15%; C10: 35%; C11: 25% and C12: 25%. The EC50 value of C9-12AS Na is 16.1 mg/L and the EC10 value is 1.76 mg/L.