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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-07-16 and 2011-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: loading rates of 2.2, 4.6, 10, 22, 46 and 100 mg test item/L
- Sampling method: Duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same times, duplicate samples were also taken from the negative control and solvent control.For sampling at the end of the study, the test medium of the treatment replicates was pooled.
- Sample storage conditions before analysis: Immediately after sampling, methanol (10 mL methanol per 10 mL sample volume) was added to each sample to stabilize it during storage. Thereafter, all samples were stored frozen at about -20 °C.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Water accommodated fractions (WAFs) with the following loading rates of the test item were tested: 2.2, 4.6, 10, 22, 46 and 100 mg/L. The dispersions were stirred for 30 minutes hours at room temperature in the dark. Then, the dispersions were filtered through membrane filters (Schleicher & Schuell, Type NC45, pore size 0.45 μm) and the undiluted filtrates were tested as WAFs.
- Differential loading: 2.2, 4.6, 10, 22, 46 and 100 mg/L of the test item
- Controls: solvent control (50 mg isododecane/L), negative control (only test water)

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Strain No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Method of cultivation: An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22°C
pH:
At the start of the test, the pH measured in the treatments was between 8.3 and 8.4. At the end of the test, pH values of 8.3 to 9.8 were measured. The pH value of the negative control (test water) was 7.5.
Nominal and measured concentrations:
nominal concentrations: 2.2, 4.6, 10, 22, 46 and 100 mg/L
mean measured concentrations (geometric mean): 0.775, 0.993, 1.45, 6.11, 13.8, 36.0 mg/L (based on active ingredient)
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks, completely filled with about 60 mL of the test medium
- Type: closed
- Initial cells density: 10000 cells/mL (nominal)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: To keep the pH of the test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to the test water.
- Light intensity and quality: approx. 7000 Lux

TEST CONCENTRATIONS
- Test concentrations: 2.2, 4.6, 10, 22, 46 and 100 mg/L (loading rates); 0.775, 0.993, 1.45, 6.11, 13.8 and 36.0 mg/L (mean measured concentration based on the active ingredient; geometric mean)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 3.1 - 3.3 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Remarks on result:
other: 1.5 - 1.6 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 1.7 - 1.9 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Remarks on result:
other: 0.98 - 1.0 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 1.26 - 1.4 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.81 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Remarks on result:
other: 0.77 - 0.85 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.993 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.:
1.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.775 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.775 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
growth rate
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
other: Yield
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
growth rate
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
other: Yield
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
growth rate
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
other: Yield
Remarks on result:
other: calculated from conc. based on act. ingr.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.986 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
growth rate
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
other: Yield
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
growth rate
Remarks on result:
other: calculated from conc. based on act. ingr.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
50% mixture
Basis for effect:
other: Yield
Remarks on result:
other: calculated from conc. based on act. ingr.
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the loading rate of 10 mg/L and the algal cells in the negative control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
Results with reference substance (positive control):
The result of the latest positive control test performed in April 2011 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.3 mg/L (Harlan Laboratories Study D25712), range of the 72-hour EC50 for the growth rate from 2000 to 2011: 0.71-1.7 mg/L).
Reported statistics and error estimates:
Dunnett's t-test

The solvent control (50 mg isododecane/L) showed statistically significant differences in growth rate and yield compared to those observed in the negative control. Consequently, the endpoints given in the table were calculated with the negative control as reference.

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EC50-value is 3.2 mg/L, the EC20-value is 1.8 mg/L and the NOEC is 0.993 mg/L for the growth rate based on the measured geometric mean concentrations of the active ingredient. Based on the registered substance (52% active ingredient in isodocenane), the EC50, EC20 and NOEC are calculated to be 6.4 mg/L, 3.6 mg/L and 1.986 mg/L, respectively.
Executive summary:

The influence of the test item Tert-Butyl Peroxyacetate on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No 761/2009, C.3. The test item Tert-Butyl Peroxyacetate was composed of the solvent isododecane and the active ingredient tert-butyl peroxyacetate (TBPA). In order to assess the toxicity of the test item containing these different components to algae, water accommodated fractions (WAFs) with the loading rates of 2.2, 4.6, 10, 22, 46 and 100 mg test item/L were tested. Additionally, a negative control (test water without test item) group and a solvent control group (test water with 50 mg isododecane/L) were tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 30 minutes. Then, the dispersions were filtered through membrane filters (0.45 μm) and the undiluted filtrates were tested as WAFs. As components of the test item may be volatile, closed (glass-stoppered) 50-mL Erlenmeyer flasks were used. Each test flask was completely filled with test medium to keep the headspace in the test flasks as small as possible. At the start of the test, the measured test item concentrations (based on the active ingredient TBPA in the test item Tert-Butyl Peroxyacetate) in the test media with the loading rates of 2.2, 4.6, 10, 22, 46 and 100 mg/L were 1.28, 2.10, 4.47, 9.29, 18.7 and 45.1 mg/L, respectively. At the end of the test, the measured concentrations in the test media with the loading rates of 2.2, 4.6 and 10 mg/L were below LOQ (limit of quantification, LOQ=0.937 mg active ingredient/L) and 4.01, 10.1 and 28.7 mg/L for the three highest loading rates, respectively. All reported biological results were based on the loading rates of the test item and on the mean measured concentrations of the active ingredient (calculated as the geometric means of the concentrations measured at the start and end of the test) related to the negative control.

Based on the results, the 72-h EC50 (growth rate) is 3.2 mg/L , the 72 -h EC20 (growth rate) is 1.8 mg/L and the 72-h NOEC (growth rate) is 0.993 mg/L based on the measured geometric mean concentrations of the active ingredient. Based on the registered substance (52% active ingredient in isodocenane), the EC50, EC20 and NOEC are calculated to be 6.4 mg/L, 3.6 mg/L and 1.986 mg/L, respectively.

Description of key information

The 72-hour EC50-value is 3.2 mg/L, the EC20-value is 1.8 mg/L and the NOEC is 0.993 mg/L for the growth rate based on the measured geometric mean concentrations of the active ingredient. Based on the registered substance (52% active ingredient in isodocenane), the EC50, EC20 and NOEC are calculated to be 6.4 mg/L, 3.6 mg/L and 1.986 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
6.4 mg/L
EC10 or NOEC for freshwater algae:
3.6 mg/L

Additional information

The influence of the test item Tert-Butyl Peroxyacetate on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 -hour static test according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No 761/2009, C.3. The test item Tert-Butyl Peroxyacetate was composed of the solvent isododecane and the active ingredient tert-butyl peroxyacetate (TBPA). In order to assess the toxicity of the test item containing these different components to algae, water accommodated fractions (WAFs) with the loading rates of 2.2, 4.6, 10, 22, 46 and 100 mg test item/L were tested. Additionally, a negative control (test water without test item) group and a solvent control group (test water with 50 mg isododecane/L) were tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 30 minutes. Then, the dispersions were filtered through membrane filters (0.45 μm) and the undiluted filtrates were tested as WAFs. As components of the test item may be volatile, closed (glass-stoppered) 50-mL Erlenmeyer flasks were used. Each test flask was completely filled with test medium to keep the headspace in the test flasks as small as possible. At the start of the test, the measured test item concentrations (based on the active ingredient TBPA in the test item Tert-Butyl Peroxyacetate) in the test media with the loading rates of 2.2, 4.6, 10, 22, 46 and 100 mg/L were 1.28, 2.10, 4.47, 9.29, 18.7 and 45.1 mg/L, respectively. At the end of the test, the measured concentrations in the test media with the loading rates of 2.2, 4.6 and 10 mg/L were below LOQ (limit of quantification, LOQ=0.937 mg active ingredient/L) and 4.01, 10.1 and 28.7 mg/L for the three highest loading rates, respectively. All reported biological results were based on the loading rates of the test item and on the mean measured concentrations of the active ingredient (calculated as the geometric means of the concentrations measured at the start and end of the test) related to the negative control. Based on the results, the 72-hour EC50-value is 3.2 mg/L, the EC20-value is 1.8 mg/L and the NOEC is 0.993 mg/L for the growth rate based on the measured geometric mean concentrations of the active ingredient. Based on the mixture (52% active ingredient in isodocenane), the EC50, EC20 and NOEC are calculated to be 6.4 mg/L, 3.6 mg/L and 1.986 mg/L, respectively.