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Ecotoxicological information

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:
07-13 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 and the EC Method C.3 with GLP certificate. All validity criteria were fulfilled.
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)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on July 05, 2016/ signed on October 28, 2016)
Specific details on test material used for the study:
- Stability in water: Stable (internal Firmenich data)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 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. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A nominal amount of test item (220 mg) was stirred with 2.2 liters of test water using a magnetic stirrer at a rate just fast enough to form a dimple at the water surface for 48 hours in a sealed vessel with minimal headspace. After the stirring period, the solution was settled for 24 hours prior to removing the aqueous phase by mid-depth siphon to produce a 100% v/v saturated solution of the test item. A series of dilutions was made from this saturated solution to give stock solutions of 10, 5.6, 3.2, 1.8 and 1.0% v/v saturated solution. An aliquot (2 liters) of each of the stock solutions was separately inoculated with 13.3 mL of algal suspension to give the required test concentrations of 1.0, 1.8, 3.2, 5.6 and 10% 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.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.
- 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 ± 2 °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 10^3 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 10^4 - 10^5 cells/mL.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
24 ± 1 °C throughout the test
pH:
8.1-8.5 at the start (0 hours); 9.9-10.4 at termination (72 hours) of the test
Dissolved oxygen:
No data
Salinity:
None
Conductivity:
None
Nominal and measured concentrations:
Nominal Concentrations: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks; Six flasks each completely filled with test solution were used for the control and three flasks each completely filled were used for each treatment group.
- Type: closed
- Initial cells density: At the start of the test, 5.00 x 10^3 cells per mL were inoculated.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Due to the potentially volatile nature of the test item, stirring and settling was conducted in a sealed vessel with minimal headspace. The exposure phase was conducted in completely filled and stoppered test vessels.

TEST MEDIUM / WATER PARAMETERS
- Culture Medium: The culture medium used for the definitive test was the same as that used to maintain the stock culture. The media used in the definitive test was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination provided by warm white lighting (380 – 730 nm)
- Light intensity and quality: Light intensity approximately 7000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Samples were taken at 25, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
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. The appearance of the test media was recorded daily.

TEST CONCENTRATIONS
According to a QSAR prediction obtained for this substance, the ErC50 value was expected to be close to the 48h-EC50 obtained from the Acute Toxicity to Daphnia magna study (close to 1.7 mg/L). Therefore, at the request of the Sponsor and the Study Monitor, a range-finding test was not conducted. The definitive test was performed with the same range of test concentrations used in the Acute Toxicity to Daphnia magna study: 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (Envigo Study Number FP48BQ)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.4 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Value obtained by extrapolation of the best fit line as no concentration tested resulted in greater than 20% inhibition of growth rate.
Details on results:
Growth Data
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 Based on 0-Hour Measured Concentrations
ErC10 (0 - 72 h): 1.7 mg/L
ErC20 (0 - 72 h): 2.2 mg/L*
ErC50 (0 - 72 h): 3.4 mg/L*
Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the 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, 0.13, 0.25, 0.47 and 0.86 mg/L test concentrations (P≥0.05), however the 1.7 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.86 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 1.7 mg/L.
Inhibition of Growth Rate Based on Geometric Mean Measured Concentrations
ErC10 (0 - 72 h): 1.5 mg/L
ErC20 (0 - 72 h): 2.0 mg/L*
ErC50 (0 - 72 h): 3.1 mg/L*
Where ErCx is the test concentration that reduced growth rate by x%.
There were no statistically significant differences between the control, 0.11, 0.21, 0.40 and 0.72 mg/L test concentrations (P≥0.05), however the 1.5 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.72 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 1.5 mg/L.
Inhibition of Yield Based on 0-Hour Measured Concentrations
EyC10 (0 - 72 h): 1.5 mg/L
EyC20 (0 - 72 h): 1.6 mg/L
EyC50 (0 - 72 h): 1.8 mg/L*
Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control, 0.13, 0.25, 0.47 and 0.86 mg/L test concentrations (P≥0.05), however the 1.7 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.86 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 1.7 mg/L.
Inhibition of Yield Based on Geometric Mean Measured Concentrations
EyC10 (0 - 72 h): 1.3 mg/L
EyC20 (0 - 72 h): 1.4 mg/L
EyC50 (0 - 72 h): 1.6 mg/L* *
Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control, 0.11, 0.21, 0.40 and 0.72 mg/L test concentrations (P≥0.05), however the 1.5 mg/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.86 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 1.5 mg/L.

* Values obtained by extrapolation of the best fit line as no concentration tested resulted in greater than 20% inhibition of growth rate or 50% inhibition of yield.
** Value obtained by extrapolation of the best fit line as no concentration tested resulted in greater than 50% inhibition of yield.

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.
Results with reference substance (positive control):
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
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).

Table 6.1.5/1: Inhibition of Growth Rate and Yield in the Definitive Test

 

Nominal Concentration (% v/v saturated solution)

Growth Rate

(cells/mL/hour)

Yield

(cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition *

Control

R1

0.062

-

4.32E+05

-

R2

0.063

4.48E+05

R3

0.063

4.57E+05

R4

0.063

4.48E+05

R5

0.063

4.48E+05

R6

0.062

4.40E+05

Mean

0.063

4.45E+05

SD

0.001

8.52E+03

1.0

R1

0.062

2

4.45E+05

[4]

R2

0.063

0

4.55E+05

R3

0.064

[2]

4.90E+05

Mean

0.063

0

4.63E+05

SD

0.001

 

2.37E+04

1.8

R1

0.062

2

4.30E+05

[3]

R2

0.063

0

4.54E+05

R3

0.064

[2]

4.93E+05

Mean

0.063

0

4.59E+05

SD

0.001

 

3.16E+04

3.2

R1

0.064

[2]

4.81E+05

[5]

R2

0.064

[2]

4.91E+05

R3

0.062

2

4.25E+05

Mean

0.063

[1]

4.66E+05

SD

0.001

 

3.53E+04

5.6

R1

0.062

2

4.23E+05

0

R2

0.062

2

4.42E+05

R3

0.063

0

4.69E+05

Mean

0.062

1

4.45E+05

SD

0.001

 

2.30E+04

10

R1

0.057

10

2.89E+05

33

R2

0.058

8

3.12E+05

R3

0.057

10

3.00E+05

Mean

0.057

9

3.00E+05

SD

0.001

 

1.18E+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

[Increase in growth as compared to controls]

 

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.13 to 1.7 mg/L. At 72 hours, the measured test concentrations were in the range of 0.094 to 1.3 mg/L (71% to 74% of the 0-Hour measured test concentrations).

The geometric mean measured test concentrations were determined to be:

 

Nominal Test Concentration

(% v/v Saturated Solution)

Measured Concentration at 0 Hours (mg/L)

Measured Concentration at 72 Hours (mg/L)

Percentage of the initial measured concentration

 

Geometric Mean Measured Test Concentration (mg/L)

Percentage of the initial measured concentration

 

1.0

0.132

0.0942

71

0.112

85

1.8

0.246

0.181

74

0.211

86

3.2

0.474

0.338

71

0.400

84

5.6

0.855

0.607

71

0.720

84

10

1.71

1.25

73

1.46

86

 

At the request of the Sponsor, all effect values were based on the 0 hour measured test concentrations as the overall geometric mean percentage of concentration during the whole test based on initial measured values are greater than 80% for all tested concentrations (84 to 86% of the 0 hour measured concentrations). Additionally, effect values based on geometric mean measured concentrations after 72 hours are given, for information purposes.

 

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 10.4 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Observations on Test Item Solubility

At 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 and test cultures were observed to be green dispersions.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 90 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.

Nominal cell density of control at 0 hours : 5.00 x 103 cells per mL

Mean cell density of control at 72 hours : 4.50 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 29% 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%.

Validity criteria fulfilled:
yes
Conclusions:
Based on the initial measured test concentrations, the 72-h EC10 and EC50 were 1.7 and 3.4 mg/L, respectively (growth rate).
Executive summary:

In a algal growth inhibition study performed according to OECD Guideline 201/ EU method C.3 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item at the nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution for 72 hours.

Due to the potentially volatile nature of the test item, stirring and settling was conducted in a sealed vessel with minimal headspace. The exposure phase was conducted in completely filled and stoppered test vessels. Following the recommendations of published data (Hermanet al1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.13 to 1.7 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of 0.094 to 1.3 mg/L. However, all effect values were based on the 0-hour measured test concentrations as the overall geometric mean percentage of concentration during the whole test based on initial measured values are greater than 80% for all tested concentrations (84 to 86% of the 0-Hour measured concentrations).

Based on the initial measured test concentrations, the 72-h EC10 and EC50 were 1.7 and 3.4 mg/L, respectively (growth rate).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
Run on 2017-06
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 limited documentation / justification
Justification for type of information:
1. SOFTWARE
ECOSAR

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

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C1(CCC=C(C1)C(=O)CCC=C)C
log Kow = 4.1 (experimental result)
Water solubility = 56.2 mg/L (experimental result)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Toxicity to aquatic algae.
- Unambiguous algorithm: Linear regression QSAR. Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253. To convert the EC50 from mmol/L to mg/L, multiply by the molecular weight.
- Defined domain of applicability: Applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of Neutral organics.
- Appropriate measures of goodness-of-fit and robustness and predictivity: N = 41+9 and the coefficient of determination R² = 0.6782.
- Mechanistic interpretation: related to the partitioning of the substance from water into the organisms lipid phase.

5. APPLICABILITY DOMAIN
Applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of Neutral organics.

6. ADEQUACY OF THE RESULT
The substance falls within the applicability domain described above and therefore the predicted value can be considered reliable.
For more information, see Neutral organics SAR document in "Attached background material".
Qualifier:
according to guideline
Guideline:
other: REACH guidance on QSARs R.6, May 2008
Deviations:
no
Principles of method if other than guideline:
See attached QPRF.
GLP compliance:
no
Remarks:
(not relevant)
Specific details on test material used for the study:
No additional information
Details on sampling:
Not applicable
Details on test solutions:
Not applicable
Test organisms (species):
other: Green algae
Details on test organisms:
None
Test type:
not specified
Water media type:
not specified
Total exposure duration:
96 h
Remarks on exposure duration:
none
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Not applicable
pH:
Not applicable
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Not applicable
Details on test conditions:
Not applicable
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.18 mg/L
Remarks on result:
other: ECOSAR Class: Neutral Organics
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1.405 mg/L
Remarks on result:
other: ECOSAR Class: Vinyl/Allyl Ketones
Details on results:
Two ECOSAR class are available for the prediction. The worst case model for the most sensitive species, Daphnid, was selected as key result.

Validity of model:
1. Defined Endpoint: Toxicity to aquatic algae.
2. Unambigous algorithm: Linear regression QSAR. Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253. To convert the EC50 from mmol/L to mg/L, multiply by the molecular weight.
3. Applicability domain: applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.
4. Statistical characteristics: N = 41+9 and the coefficient of determination R² = 0.6782.
5. Mechanistic interpretation: related to the partitioning of the substance from water into the organisms lipid phase.

Adequacy of prediction: the substance falls within the applicability domain described above and therefore the predicted value can be considered reliable. For more information, see Neutral Organics SAR document in "Attached background material".
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
N = 41+9 and the coefficient of determination R² = 0.6782.

None

Validity criteria fulfilled:
yes
Conclusions:
The substance was predicted to have an EC50 value at 2.180 mg/L to green algae after 96h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.
Executive summary:

Toxicity to green algae was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The estimated 96h-EC50 value was 2.180 mg/L (Neutral Organics class), calculated from a measured log

Kow value at 4.1, a measured water solubility value at 56.2 mg/L and a linear regression:

Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253.

The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
Run on 2017-06
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 limited documentation / justification
Justification for type of information:
1. SOFTWARE
ECOSAR

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

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC1(C)CCCC(=O)C1
log Kow = 2.0 (experimental result)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Toxicity to aquatic algae.
- Unambiguous algorithm: Linear regression QSAR. Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253. To convert the EC50 from mmol/L to mg/L, multiply by the molecular weight.
- Defined domain of applicability: Applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of Neutral organics.
- Appropriate measures of goodness-of-fit and robustness and predictivity: N = 41+9 and the coefficient of determination R² = 0.6782.
- Mechanistic interpretation: related to the partitioning of the substance from water into the organisms lipid phase.

5. APPLICABILITY DOMAIN
Applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of Neutral organics.

6. ADEQUACY OF THE RESULT
The substance falls within the applicability domain described above and therefore the predicted value can be considered reliable.
For more information, see Neutral organics SAR document in "Attached background material".
Qualifier:
according to guideline
Guideline:
other: REACH guidance on QSARs R.6, May 2008
Deviations:
no
Principles of method if other than guideline:
See attached QPRF.
GLP compliance:
no
Remarks:
(not relevant)
Specific details on test material used for the study:
No additional information
Details on sampling:
Not applicable
Details on test solutions:
Not applicable
Test organisms (species):
other: Green algae
Details on test organisms:
None
Test type:
not specified
Water media type:
not specified
Total exposure duration:
96 h
Remarks on exposure duration:
none
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Not applicable
pH:
Not applicable
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Not applicable
Details on test conditions:
Not applicable
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
43.849 mg/L
Remarks on result:
other: ECOSAR Class: Neutral Organics
Details on results:
Validity of model:
1. Defined Endpoint: Toxicity to aquatic algae.
2. Unambigous algorithm: Linear regression QSAR. Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253. To convert the EC50 from mmol/L to mg/L, multiply by the molecular weight.
3. Applicability domain: applicable to chemicals with log Kow less than 6.4 and molecular weight less than 1000 g/mol. The model can be suitable for chemicals class of neutral organic.
4. Statistical characteristics: N = 41+9 and the coefficient of determination R² = 0.6782.
5. Mechanistic interpretation: related to the partitioning of the substance from water into the organisms lipid phase.

Adequacy of prediction: the substance falls within the applicability domain described above and therefore the predicted value can be considered reliable. For more information, see Neutral Organics SAR document in "Attached background material".
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
N = 41+9 and the coefficient of determination R² = 0.6782.

None

Validity criteria fulfilled:
yes
Conclusions:
The substance was predicted to have an EC50 value at 43.849 mg/L to green algae after 96h of exposure. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.
Executive summary:

Toxicity to green algae of 3,3-dimethyl cyclohexanone (DMCH), the relevant degradation product of the registered substance, was estimated using the ECOSAR v1.11 QSAR model available from the U.S. EPA. The estimated 96h-EC50 value was 43.849 mg/L (Neutral Organics class), calculated from a measured log Kow value at 2.0 and a linear regression:

Log 96h-EC50 (mmol/L) = -0.6922*log Kow + 0.9253.

The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

Description of key information

OECD Guideline 201, EU Method C.3, GLP, key study, validity 1:

72h-ErC10 (Pseudokirchneriella subcapitata) = 1.7 mg/L based on the initial measured concentrations;

72h-ErC50 (Pseudokirchneriella subcapitata) = 3.4 mg/L based on the initial measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
3.4 mg/L
EC10 or NOEC for freshwater algae:
1.7 mg/L

Additional information

To assess the toxicity of the registered substance to aquatic algae, one valid experimental study and a QSAR prediction are available.

In the experimental study (Envigo, 2017), assessed as the key study, the freshwater green algae species Pseudokirchneriella subcapitata was exposed to the registered substance at the nominal concentrations of 1.0, 1.8, 3.2, 5.6 and 10% v/v saturated solution for 72 hours to assess the algae growth inhibition, according to the OECD Guideline 201 and EU Method C.3 with GLP statement. Due to the potentially volatile nature of the test item, stirring and settling was conducted in a sealed vessel with minimal headspace. The exposure phase was conducted in completely filled and stoppered test vessels. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.13 to 1.7 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of 0.094 to 1.3 mg/L. However, all effect values were based on the 0-hour measured test concentrations as the overall geometric mean percentage of concentration during the whole test based on initial measured values are greater than 80% for all tested concentrations (84 to 86% of the 0-Hour measured concentrations). According to the results of this study, the 72h-ErC10 and 72h-ErC50 values (growth rate) were determined at 1.7 and 3.4 mg/L, respectively, based on the initial measured concentrations.

In addition, to support the QSAR approach used for the short-term toxicity to fish endpoint, a QSAR prediction (ECOSAR, 2017), assessed as a supporting data, was provided. This QSAR prediction was performed on the registered substance to assess the toxicity of the substance to green algae, using the ECOSAR v1.11 QSAR model available from the U.S. EPA.

The estimated 96h-EC50 value was 2.18 mg/L (Neutral Organics class), calculated from a measured log Kow value at 4.1, a measured water solubility value at 56.2 mg/L and a linear regression. The substance falls within the applicability domain and therefore the predicted value can be considered reliable.

This QSAR result supports the key study performed on the same substance with an endpoint value slightly more conservative, validating the model for use with this substance.

In conclusion, according to the key experimental study (Envigo, 2017), the 72h-ErC10 and 72h-ErC50 values on Pseudokirchneriella subcapitata are 1.7 and 3.4 mg/L, respectively.

Moreover, as the registered substance is totally primarily biodegraded in the environment to form two degradation products: succinic acid (degraded also and mineralized completely) and 3,3 -dimethyl cyclohexanone (DMCH), the toxicity of the relevant degradation product of the registered substance, DMCH, to green algae was estimated using the same QSAR model presented above, ECOSAR v1.11 available from the U.S. EPA. The estimated 96h-EC50 value was 43.85 mg/L (Neutral Organics class), calculated from a measured log Kow value at 2.0 and a linear regression. The substance falls within the applicability domain and therefore the predicted value can be considered reliable. According to this result, the relevant degradation product of the registered substance, DMCH, is presumed not Toxic (T) in the PBT assessment context.