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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:
from 2014-09-25 to 2014-11-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The highest concentration samples (50 mg/L) were tenfold diluted; 20 mg/L samples were fivefold diluted; 8 mg/L samples were twofold diluted with water before the derivatization. The other samples were directly derivatized with FMOC-Cl. Then the samples were analysed with the previously validated HPLC method.

- Stock solution: A solution of the test item with a nominal concentration of 50 mg/L in OECD Medium was prepared by suspending 0.050 g test item in 1000 mL OECD Medium, thereafter stirred for 20 hours at 300 rpm. The resulted “stock solution” was the highest test item solution and it was further diluted accordingly. The test solutions were freshly prepared in the testing laboratory just before introduction of the algae (start of the experiment).

- Sampling method: At the start and at the end of the study three replicates samples were taken from the test solutions, and one sample was taken from the control solution.

- Sample storage conditions before analysis: At room temperature, tightly closed
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: dissolving the test item in OECD medium, thereafter stirred for 20 hours at 300 rpm. The resulted stock solution was the highest test concentration. The further test solutions were prepared by appropriate dilution of this stock solution.

- Algal Mineral Salts Test Medium (Dilution Water): Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.

- Separate stock solutions were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

- Control:
Untreated Control: The dilution water (without addition of the test item) was used as untreated control.
Toxic Reference Control: For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of algal cultures, D-37073 Göttingen, Germany
- Breeding conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with algal mineral salts culture medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. The pre-culture was incubated for four days at this test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
In the range of 22.1 – 22.4 °C
pH:
Control: Start: 7.74 End: 9.23;
Sample: 50 mg/L: start: 8.12 End: 8.02
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
- Nominal concentration: Based on the results of the preliminary tests, the following nominal concentrations were tested in the main study: 0.512; 1.28; 3.2; 8; 20 and 50 mg/L (spacing factor for test concentrations: 2.5).
- Measured concentrations: The measured concentration of the test item in the test solution was in the range of 0.554 – 48.0 mg/L at the start and 0.526 – 47.7 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values.
Details on test conditions:
TEST SYSTEM
- Test vessel: Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks for an exposure time of 72 hours.
- Type: The flasks were covered with air-permeable stoppers
- No. of organisms per vessel: The test was started (0 hours) by inoculation of 0.1 mL algal biomass (10 000 000 algal cells per mL) to 100 mL test item solution.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Initial cell density: ~10 000 cells/ mL in each testing flask. These cells were taken from an exponentially growing pre-culture, set up four days prior to the test start using the same conditions as in the test.
- Replicates: The test was performed with three replicates per test concentrations and six replicates in the untreated control group

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A solution of the test item with a nominal concentration of 50 mg/L in OECD Medium was prepared by suspending 0.050 g test item in 1000 mL OECD Medium, thereafter stirred for 20 hours at 300 rpm. The resulted “stock solution” was the highest test item solution and it was further diluted accordingly. The test solutions were freshly prepared in the testing laboratory just before introduction of the algae (start of the experiment).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was measured at the beginning and at the end of the study in the control and test item group. The pH was not adjusted throughout the test period. The pH of the control medium did not increase by more than 1.5 during the study.
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity: The light intensity was checked and recorded at the start of the test. The average light intensity measured at the position occupied by algal culture flasks at the start of the experiment was 8938 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED:
- Determination of cell number: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological changes of algal cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: Pre-experiments were performed to determine the solubility of the test item in test water and to select suitable methods for the preparation of a stock solution and the dosage of the test item into the test media as well to provide data for the analytical method validation. The concentration levels are chosen based on the results of the preliminary range-finding tests. In both of the preliminary tests a stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving the test item in OECD medium, thereafter stirred for 20 hours at 300 rpm. The resulted stock solution was the highest test concentration. The further test solutions were prepared by appropriate dilution of this stock solution. 100 % of the inhibition of the growth rate was found with a nominal concentration of the test substance of 50 mg/L.
- Test concentrations:
Nominal concentration: Based on the results of the preliminary tests, the following nominal concentrations were tested in the main study: 0.512; 1.28; 3.2; 8; 20 and 50 mg/L (spacing factor for test concentrations: 2.5).
Measured concentrations: The measured concentration of the test item in the test solution was in the range of 0.554 – 48.0 mg/L at the start and 0.526 – 47.7 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.786 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
11.098 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits (9.225-13.352 mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.512 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.269 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.157 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits (1.763-2.639 mg/L))
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.512 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.512 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with SIKA Hardener AI (SIKA Härter AI), the 72-h EC50 based on the growth rate was determined to be 11.098 mg/L and based on the yield the 72-h EC50 was determined to be 2.157 mg/L.
The overall NOEC was determined to be < 0.512 mg/L while the overall LOEC was calculated to be 0.512 mg/L (all calculations were based on the nominal concentrations).
Results with reference substance (positive control):
The 72h ErC50 of the reference substance was determined to be 1.48 mg/L, (95 % confidence limits: 1.30 – 1.69 mg/L) and the 72h EyC50 was determined to be 0.69 mg/L, (95 % confidence limits: 0.61 – 0.78 mg/L).
Reported statistics and error estimates:
Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using Bonferroni t-Test (α = 0.05) by TOXSTAT software.
The results of the statistical evaluation showed that the 0-72 h average specific growth rate was statistically significantly different from that of the untreated control at the test item concentrations of 1.28; 3.2; 8; 20 and 50 mg/L. The 72- h yield was statistically significantly different from that of the untreated control in the whole test item concentration range. Accordingly, the overall No Observed Effect Concentration (NOEC) was determined as < 0.5 mg/L, while the overall 72h-LOEC was 0.5 mg/L test item concentration.
Validity criteria fulfilled:
yes
Conclusions:
In a static growth inhibition test, exponentially-growing cultures of unicellular green algal species Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) were exposed to the test item for 72 hours according to OECD 201 (2011). The 72-h EC50 based on the growth rate was determined to be 11.1 mg/L, the 72-h EC10 based on the growth inhibition was determined to be 1.8 mg/L and the NOEC was determined to be < 0.5 mg/L (all calculations were based on the nominal concentrations).
Executive summary:

In a growth inhibition test, exponentially-growing cultures of unicellular green algal species Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) were exposed to the test item for 72 hours according to OECD 201 (2011) over several generations under defined static conditions. The test item hydrolyzes very quickly, therefore concentration of the hydrolysis product was determined. The hydrolysis product, 5-Amino-1,3,3-trimethylcyclohexanemethylamine was derivatized with FMOC-Cl (9-Fluorenylmethyl chloroformate) and than the samples were analyzed by a reverse phase HPLC method using UV detection. The effects are based on the hydrolyzed product. Nevertheless the results can be extrapolated to the test substance itself. Based on the results of the preliminary tests, the following nominal concentrations were tested in the main study: 0.512; 1.28; 3.2; 8; 20 and 50 mg/L (spacing factor for test concentrations: 2.5). The measured concentration of the test item in the test solution was in the range of 0.554 – 48.0 mg/L at the start and 0.526 – 47.7 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The 72 h EC50 based on the growth rate was determined to be 11.1 mg/L and based on the yield the 72 h EC50 was determined to be 2.2 mg/L. The 72 h EC10 based on the growth rate was determined to be 1.8 mg/L and based on the yield the 72 h EC10 was determined to be 0.3 mg/L. The overall NOEC was determined to be < 0.5 mg/L while the overall LOEC was calculated to be 0.5 mg/L (all calculations were based on the nominal concentrations).

Description of key information

In a static growth inhibition test, exponentially-growing cultures of unicellular green algal species Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) were exposed to the test item for 72 hours according to OECD 201 (2011). The 72-h EC50 based on the growth rate was determined to be 11.1 mg/L, the 72-h EC10 based on the growth inhibition was determined to be 1.8 mg/L and the NOEC was determined to be < 0.5 mg/L (all calculations were based on the nominal concentrations).

Key value for chemical safety assessment

EC50 for freshwater algae:
11.1 mg/L
EC10 or NOEC for freshwater algae:
1.8 mg/L

Additional information

In a growth inhibition test, exponentially-growing cultures of unicellular green algal species Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) were exposed to the test item for 72 hours according to OECD 201 (2011) over several generations under defined static conditions. The test item hydrolyzes very quickly, therefore concentration of the hydrolysis product was determined. The hydrolysis product, 5-Amino-1,3,3-trimethylcyclohexanemethylamine was derivatized with FMOC-Cl (9-Fluorenylmethyl chloroformate) and than the samples were analyzed by a reverse phase HPLC method using UV detection. The effects are based on the hydrolyzed product. Nevertheless the results can be extrapolated to the test substance itself. Based on the results of the preliminary tests, the following nominal concentrations were tested in the main study: 0.512; 1.28; 3.2; 8; 20 and 50 mg/L (spacing factor for test concentrations: 2.5). The measured concentration of the test item in the test solution was in the range of 0.554 – 48.0 mg/L at the start and 0.526 – 47.7 mg/L at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The 72 h EC50 based on the growth rate was determined to be 11.1 mg/L and based on the yield the 72 h EC50 was determined to be 2.2 mg/L. The 72 h EC10 based on the growth rate was determined to be 1.8 mg/L and based on the yield the 72 h EC10 was determined to be 0.3 mg/L. The overall NOEC was determined to be < 0.5 mg/L while the overall LOEC was calculated to be 0.5 mg/L (all calculations were based on the nominal concentrations).