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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:
01 December 2014 to 26 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study conducted to OECD and EC guidelines and in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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)
Specific details on test material used for the study:
- Expiration date of the lot/batch: 1 August 2016

Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

A further sample of each test concentration containing no algal cells was prepared at 0 hours and incubated alongside the test to provide samples for uninoculated analyses at 72 hours.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. 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. Mater cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under consistent 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.

Pseudokirchneriella subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism in freshwater ecosystems.

Test type:
static
Water media type:
other: reverse osmosis purified deionized water
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24±1°C
pH:
7.4 - 9.0
Nominal and measured concentrations:
1.0, 3.2, 10, 32, 100, 320 mg ai/L - Nominal concentrations
0.31, 1.9, 7.1, 28, 82, 265 - Measured geometric mean concentrations


72 hour No algae
1.0, 3.2, 10, 32, 100, 320 mg ai/L - Nominal concentrations
0.135, 1.52, 6.64, 28.5, 90.2, 231 - Measured concentrations
Details on test conditions:

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 Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg ai/L for a period of 72 hours. At the request of the Sponsor all test concentrations were corrected for a test item purity value of 28.6% w/w.

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 item was dissolved directly in culture medium. Prior to use the test item was heated to approximately 70 °C and mixed thoroughly in order to ensure homogeneity.

A nominal amount of test item (175 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg ai/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg ai/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg ai/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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.



Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32, 100 and 320 mg ai/L.

-Experimental Preparation
At the request of the Sponsor all test concentrations were corrected for a test item purity value of 28.6% w/w. Prior to use the test item was heated to approximately 70 °C and mixed thoroughly in order to ensure homogeneity.

Nominal amounts of test item (1119 and 350 mg) were each separately dissolved in culture medium and the volume adjusted to 1 liter to give a 320 and 100 mg ai/L stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 32, 10, 3.2 and 1.0 mg ai/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (2.2 mL) to give the required test concentrations of 1.0, 3.2, 10, 32, 100 and 320 mg ai/L.

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.

-Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation 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 1.16 x 106 cells per mL. Inoculation of 500 mL of test medium with 2.2 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.

-Evaluations
Test Organism Observations
Samples were taken at 0, 22, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

-Water Quality Criteria
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.

-Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
170 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
7.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg ai/L. However, growth was observed to be reduced at 10 and 100 mg ai/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32, 100 and 320 mg ai/L were selected for the definitive test.

Chemical analysis of the 1.0, 10 and 100 mg ai/L test preparations at 0 hours showed measured test concentrations of 75, 80 and 83% of nominal respectively were obtained. Analysis of the test preparations at 72 hours showed measured test concentrations of 11, 81 and 87% of nominal were obtained for the 1.0, 10 and 100 mg ai/L test preparations respectively. The decline in measured test concentration at 1.0 mg ai/L was considered to be due to possible adsorption of the test item to the algal cells present as at this concentration there were significantly more algal cells than at the higher concentrations of 10 and 100 mg ai/L.



-Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.70 to 305 mg ai/L (70% to 95% of the nominal test concentrations). A concentration dependant decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.069 mg ai/L) to 307 mg ai/L.

Analysis of samples prepared with the omission of algal cells showed measured concentrations in the range of 0.14 to 231 mg ai/L (19% to 120% of the 0-Hour measured concentrations) were obtained at 72 hours. These results indicated that the test item was both unstable over the test duration and was adsorbing to the algal cells present. It was therefore considered appropriate to calculate the results based on the analysis of the uninoculated test samples at 72 hours as these results more accurately reflect the concentration of test item present to which the algal cells were exposed.

Given the decline in measured test concentrations observed over the test duration 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.

-Growth Data
From the data given, it 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 Rate
ErC10 (0 - 72 h) : 3.7 mg ai/L
ErC20 (0 - 72 h) : 15 mg ai/L
ErC50 (0 - 72 h) : 170 mg ai/L; 95% confidence limits 100 - 330 mg ai/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 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 (P: greater than 0.05), between the control, 0.31 and 1.9 mg ai/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.9 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 7.1 mg ai/L.

Inhibition of Yield
EyC10 (0 - 72 h) : 3.9 mg ai/L
EyC20 (0 - 72 h) : 5.9 mg ai/L
EyC50 (0 - 72 h) : 12 mg ai/L; 95% confidence limits 10 - 14 mg ai/L

Where:

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 (P0.05), between the control, 0.31 and 1.9 mg ai/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.9 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 7.1 mg ai/L.

-Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 128 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 : 5.04 x 103 cells per mL
Mean cell density of control at 72 hours : 6.45 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 23% 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 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

-Observations on Cultures
All 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 0.31, 1.9, 7.1 and 28 mg ai/L, however cell debris was observed to be present in the test cultures at 82 mg ai/L and cell debris and no intact cells were observed in the 265 mg ai/L test cultures.

-Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was determined to be pH 7.9 at 0 and 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 Solubility
At the start of the test all control, 0.31, 1.9 and 7.1 mg ai/L test cultures were observed to be clear colorless solutions whilst the 28, 82 and 265 mg ai/L test cultures were observed to be clear colorless solutions with a slight layer of foam at the surface. After the 72-Hour test period all control, 0.31, 1.9 and 7.1 mg ai/L test cultures were observed to be green dispersions. The 28 mg ai/L test cultures were observed to be pale green dispersions whilst the 82 and 265 mg ai/L test cultures were observed to be extremely pale green dispersions with a slight layer of foam at the surface.
Results with reference substance (positive control):
A positive control (Harlan Study Number 41403074) used potassium dichromate as the reference item at concentration 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control study was conducted between 16 September 2014 and 19 September 2014.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

ErC50: 1.2 mg/L (95% confidence limits 1.1-1.4 mg/L)
EyC50: 0.63 (95% confidence limits 0.57-0.70 mg/L)

NOEC on growth rate: 0.50 mg/L
NOEC on yield: 0.25 mg/L

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

See attached fugures and tables for more details.

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 based on the geometric mean measured test concentrations gave the following results:

Growth Rate
EC50: 170 mg ai/L (95% Confidence Limits: 100-330 mg ai/L)
EC10: 3.7 mg ai/L

Executive summary:

Introduction


A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201.


 


Method


Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentration of 1.0, 3.2, 10, 32, 100 and 320 mg active ingredient (ai)/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


Analysis of the test preparations at 0hours showed measured test concentrations to range from 0.70 to 305 mg ai/L (70% to 95% of the nominal test concentrations). A concentration dependant decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.069 mg ai/L) to 307 mg ai/L.


 


Analysis of samples prepared with the omission of algal cells showed measured concentrations in the range of 0.14 to 231 mg ai/L (19% to 120% of the 0-Hour measured concentrations) were obtained at 73 hours. These results indicated that the test item was both unstable over the test duration and was adsorbing to the algae cells present. It was therefore considered appropriate to calculate te results based on the analysis of the uninoculated test samples at 72 hours as these results more accurately reflect the concentration of test item present to which the algal cells were exposed. 


 


Given the decline in measured test concentrations observed over the test duration 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.


 


Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:


 


Growth Rate      


EC50: 170 mg ai/L (95% Confidence Limits: 100-330 mg ai/L)


EC10: 3.7 mg ai/L

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:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
Source and target substances share structural similarities (predominantly linear aliphatic hydrocarbon chain) with a common functional group: (polar sulfonate group). The molecular structure is almost identical.
They are manufactured from similar resp. identical precursors under similar conditions. Therefore, common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident. Target and source substances are both a mixture of linear long chain Sulfonic acids, alkene, sodium salts and Sulfonic acids, alkane hydroxy, sodium salts and share an identical counter ion. A constant pattern in the changing of the potency of the properties across the target and source substances by chain-length and is not observed, because the distribution is too narrow.

Therefore, read-across from the existing toxicity studies on the source substance, is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.1, 8.2, 8.3, 8.4, 8.5 as well as 9.1 and 9.2 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see cross reference to assessment report: Justification for read-across document attached to section 13

3. ANALOGUE APPROACH JUSTIFICATION
see cross reference to assessment report: Justification for read-across document attached to section 13

4. DATA MATRIX
see cross reference to assessment report: Justification for read-across document attached to section 13

Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
170 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
7.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg ai/L. However, growth was observed to be reduced at 10 and 100 mg ai/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32, 100 and 320 mg ai/L were selected for the definitive test.

Chemical analysis of the 1.0, 10 and 100 mg ai/L test preparations at 0 hours showed measured test concentrations of 75, 80 and 83% of nominal respectively were obtained. Analysis of the test preparations at 72 hours showed measured test concentrations of 11, 81 and 87% of nominal were obtained for the 1.0, 10 and 100 mg ai/L test preparations respectively. The decline in measured test concentration at 1.0 mg ai/L was considered to be due to possible adsorption of the test item to the algal cells present as at this concentration there were significantly more algal cells than at the higher concentrations of 10 and 100 mg ai/L.



-Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.70 to 305 mg ai/L (70% to 95% of the nominal test concentrations). A concentration dependant decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.069 mg ai/L) to 307 mg ai/L.

Analysis of samples prepared with the omission of algal cells showed measured concentrations in the range of 0.14 to 231 mg ai/L (19% to 120% of the 0-Hour measured concentrations) were obtained at 72 hours. These results indicated that the test item was both unstable over the test duration and was adsorbing to the algal cells present. It was therefore considered appropriate to calculate the results based on the analysis of the uninoculated test samples at 72 hours as these results more accurately reflect the concentration of test item present to which the algal cells were exposed.

Given the decline in measured test concentrations observed over the test duration 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.

-Growth Data
From the data given, it 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 Rate
ErC10 (0 - 72 h) : 3.7 mg ai/L
ErC20 (0 - 72 h) : 15 mg ai/L
ErC50 (0 - 72 h) : 170 mg ai/L; 95% confidence limits 100 - 330 mg ai/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 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 (P: greater than 0.05), between the control, 0.31 and 1.9 mg ai/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.9 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 7.1 mg ai/L.

Inhibition of Yield
EyC10 (0 - 72 h) : 3.9 mg ai/L
EyC20 (0 - 72 h) : 5.9 mg ai/L
EyC50 (0 - 72 h) : 12 mg ai/L; 95% confidence limits 10 - 14 mg ai/L

Where:

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 (P0.05), between the control, 0.31 and 1.9 mg ai/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.9 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 7.1 mg ai/L.

-Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 128 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 : 5.04 x 103 cells per mL
Mean cell density of control at 72 hours : 6.45 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 23% 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 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

-Observations on Cultures
All 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 0.31, 1.9, 7.1 and 28 mg ai/L, however cell debris was observed to be present in the test cultures at 82 mg ai/L and cell debris and no intact cells were observed in the 265 mg ai/L test cultures.

-Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was determined to be pH 7.9 at 0 and 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 Solubility
At the start of the test all control, 0.31, 1.9 and 7.1 mg ai/L test cultures were observed to be clear colorless solutions whilst the 28, 82 and 265 mg ai/L test cultures were observed to be clear colorless solutions with a slight layer of foam at the surface. After the 72-Hour test period all control, 0.31, 1.9 and 7.1 mg ai/L test cultures were observed to be green dispersions. The 28 mg ai/L test cultures were observed to be pale green dispersions whilst the 82 and 265 mg ai/L test cultures were observed to be extremely pale green dispersions with a slight layer of foam at the surface.
Results with reference substance (positive control):
A positive control (Harlan Study Number 41403074) used potassium dichromate as the reference item at concentration 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control study was conducted between 16 September 2014 and 19 September 2014.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

ErC50: 1.2 mg/L (95% confidence limits 1.1-1.4 mg/L)
EyC50: 0.63 (95% confidence limits 0.57-0.70 mg/L)

NOEC on growth rate: 0.50 mg/L
NOEC on yield: 0.25 mg/L

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

See attached fugures and tables for more details.

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 based on the geometric mean measured test concentrations gave the following results:

Growth Rate
EC50: 170 mg ai/L (95% Confidence Limits: 100-330 mg ai/L)
EC10: 3.7 mg ai/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
EPI Suite v4.11

2. MODEL (incl. version number)
ECOSAR Program (v1.11)

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
please see attachment

5. APPLICABILITY DOMAIN
please see attachment

6. ADEQUACY OF THE RESULT
The result supports the use of read-across for this endpoint.
The read-across source substance has higher or even toxicity to daphnia compared to the target substance.
For details please see attachment
Specific details on test material used for the study:
Structures used:
HO-C18H36-SO3Na and HO-C16H32-SO3Na
for details see QMRF attached
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0 mg/L
Remarks on result:
other: HO-C16H32-SO3Na for target C16 IOS-Na (ECOSAR Class: Alkyl, Sulfonates)
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0 mg/L
Remarks on result:
other: HO-C18H36-SO3Na for source C16-18 IOS-Na C18-rich (ECOSAR Class: Alkyl, Sulfonates)
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
111.731 mg/L
Remarks on result:
other: HO-C16H32-SO3Na for target C16 IOS-Na (ECOSAR Neutral Organics-acids)
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
25.382 mg/L
Remarks on result:
other: HO-C18H36-SO3Na for source C16-18 IOS-Na C18-rich (ECOSAR Neutral Organics-acids)
Conclusions:
As proved according to ECOSAR QSAR for the main structure of IOS, the toxicity to Algae of the source substance 3 (C16-18 IOS-Na C18-rich) is higher or equal to the toxicity of the target substance (C16 IOS-Na).
Executive summary:

ECOSAR for the two linear (main) structures (difference only in C-chain length) of target (C16) and source 3 (C18) shows



  • even ecotoxicity to algae for the C18 compared to the C16 structure in the ECOSAR “special classes” – surfactants – alkyl sulfonates.

  • higher ecotoxicity to algae for the C18 compared to the C16 structure in the ECOSAR neutral organics acid.


 


As a conclusion, as proved according to ECOSAR QSAR for the main structure of IOS, the toxicity to algae of the source substance 3 (C16-18 IOS-Na C18-rich) is higher or equal to the toxicity of the target substance (C16 IOS-Na).

Description of key information

A read-across was made from source substance 3 (C16-18 IOS-Na C18-rich), Sulfonic Acids, C16-18-alkane hydroxyl and C16-18-sec-alkane hydroxyl and C16-18-alkene and C16-18-sec-alkene, sodium salts (abbreviation C16-18 IOS-Na C18-rich).


The read-across is supported with ECOSAR QSAR calculations for the two main structures of source and target substance (HO-C18H36-SO3Na and HO-C16H32-SO3Na). The calculations showing for the C18-component lower resp. even effect concentrations compared to the C-18-component. The conclusion for this is, that making read-across from the source substance (with a higher amount of the C18-component compared to the target substance) is a worst case approach.


The read-across is also supported with a literature describing inter alia testing of C14-18 AOS (average C16.4). Though testing duration was only 48 hours. The resulting 48h-ErC50 45 mg/L was in a similar order of magnitude, showing that the key result is plausible for this kind of structure.


Read-across is based on a Klimisch 1 study according to guideline OECD 201 with analytical monitoring resulting in a 72h-ErC50 of 170 mg a.i./L and 72h-ErC10 of 3.7 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
170 mg/L
EC10 or NOEC for freshwater algae:
3.7 mg/L

Additional information