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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 2017-12-01 to 2018-02-28
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Three parallel samples were analysed from each test concentration and control.
- Sampling method: Formulation samples were diluted with mobile phase and the control samples were directly injected. The samples were analysed at the start and at the end of the test.
- Sample storage conditions before analysis: Ambient temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 20 mg/L (nominal concentration) was prepared by dissolving an appropriate amount of the test item in OECD medium in order to obtain the concentration of 20 mg/L and this stock solution were placed into an ultrasonic bath for appr. 10 minutes. The further test solutions were prepared by appropriate dilution of this stock solution.
- Controls: Untreated control ran parallel in the test. The positive control was performed with potassium dichromate.
- Evidence of undissolved material: No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: algae
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, 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 was intended to give an amount of algae suitable for the inoculation of test cultures.

ACCLIMATION
- Acclimation period: The pre-culture was incubated for four days at this test.
- Culturing media and conditions: The culture conditions were the same as the test. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing.
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None
Post exposure observation period:
Not performed
Hardness:
No data
Test temperature:
The temperature was in the range of 23.0 – 23.5 °C measured in the flask and 22.4 – 23.9 °C measured within the climate chamber.
pH:
The pH did not vary by more than 1.5 units during the test. The range of the pH was 7.77 – 9.27 during the study.
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
No data
Nominal and measured concentrations:
- Nominal concentration: The following nominal concentrations were tested: 0.25, 0.74, 2.2, 6.7, 20.0 mg/L (loading rate)
- Measured concentration: The measured concentration of the test item in the test solutions were 0.24, 0.72, 2.18, 6.29 and 20.8 mg/L (corresponding to 94 and 104 % of the nominal value) at the start of the test and 0.22, 0.70, 2.16, 6.46 and 19.5 mg/L (corresponding to 90 and 98 % of the nominal value) at the end of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume
- Fill volume: 100 mL
- Aeration: No
- Initial cells density: The initial cell density was about 10 000 cells/ mL in each test flask.
- Control end cells density: The cell density in the controls has increased from nominal N = 1 × 10 000 cells/mL at the start of the test (0 hours) to N = 57.50 × 10 000 cells/mL (mean value) after 72 hours in the untreated control. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, temperature was continuously measured (with a min/max thermometer) within the climate chamber. The pH was checked at the beginning and at the end of the study, in the controls and at each concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks during the test was 7866 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED:
The purpose of this study was to determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The algal growth in relation to the untreated control culture was determined over a fixed test period of 72 hours and thus, over several algal generations.
- Determination of cell concentrations: 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.
- Chlorophyll measurement: Not determined

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study: Yes. A non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
- Test concentrations: Algal cells were exposed to the concentration of 100, 50, 10, 1, 0.1 and 0.01 mg/L test item, plus untreated control, for 72 hours.
- Results used to determine the conditions for the definitive study: In the pre-experiments the 72h-EC50 was determined to be between 0 and 10 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits (0.292 - 4.858 mg/L)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits (4.767 - 28.145 mg/L)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.82 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Remarks on result:
other: 95 % conf. limits (0.271 - 1.426 mg/L)
Duration:
72 d
Dose descriptor:
EC50
Effect conc.:
3.07 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Remarks on result:
other: 95 % conf. limits (1.863 - 5.244 mg/L)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
yield
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): Not observed
- Unusual cell shape: Not observed
- Colour differences: Not observed
- Flocculation: Not observed
- Adherence to test vessels: Not observed
- Aggregation of algal cells: Not observed
- Any stimulation of growth found in any treatment: Not observed
- Any observations that might cause a difference between measured and nominal values: No. The measured concentration of the test item in the test solutions was in the range of 94 – 104 % at the start and 90 – 98 % 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 biological results was based on the nominal concentration values.
- Effect concentrations exceeding solubility of substance in test medium: No
- Toxic effects: In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata at the concentrations of 6.7 and 20.0 mg/L. After 72 hours, the Er10 and the ErC50 were determined to be 2.50 mg/L and 9.31 mg/L respectively.The EyC10 and the EyC50 were determined to be 0.82 mg/L and 3.07 mg/L respectively and the 72h-NOEC was determined to be 2.2 mg/L.
All validity criteria were met. The results are based on nominal test item concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: The 72h ErC50 was determined to be 0.82 mg/L, (95 % confidence limits: 0.57 – 1.25 mg/L) and the 72h EyC50 was determined to be 0.51 mg/L, (95 % confidence limits: 0.45 – 0.57 mg/L)
Reported statistics and error estimates:
- Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, 24 h and 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software.
- Percentage inhibition of growth rate (µ) and yield (y) were calculated using EXCEL for Windows software.
- The ErCx, and EyCx values of the test item and their confidence limits were calculated using Probit analysis by SPSS+ software.
- For the determination of the LOEC and NOEC, the calculated mean growth rate (µ) and yield (y) at the test concentrations were tested on significant differences to control values by Dunnett’s Test using SPSS+ software.
Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata) was determined according to the principles of OECD 201 (2011) under GLP conditions. After 72 hours the ErC10 was determined to be 2.5 mg/L and the ErC50 was determined to be 9.31 mg/L.
Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata) was determined according to the principles of OECD 201 (2011). The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 10 000 cells/mL. 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. 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. Based on the results of the non-GLP Preliminary Range-Finding Test the following five test concentrations in a geometric series (with a spacing factor of 3.0) were tested: 0.25, 0.74, 2.2, 6.7 and 20.0 mg/L (nominal concentrations). Untreated control ran parallel in the test and a positive control with potassium dichromate was performed. An appropriate amount of test item was dissolved in the dilution water (OECD medium) in order to obtain the concentration of 20 mg/L. The further test solutions were prepared by appropriate dilution of this stock solution. Samples were taken from the test concentrations and the control at the start and at the end of the experiment and analyzed by HPLC method with MS detection. Three parallel samples were analyzed from each test concentration and control at the start and at the end of the test. The measured concentration of the test item in the test solutions were 0.24, 0.72, 2.18, 6.29 and 20.8 mg/L (corresponding to 94 and 104 % of the nominal value) at the start of the test and 0.22, 0.70, 2.16, 6.46 and 19.5 mg/L (corresponding to 90 and 98 % of the nominal value) at the end of the test. The concentration of the tested substance was maintained within ± 20 % of the nominal concentrations throughout the test. Therefore, the analysis of the results was based on the nominal concentration values. The 24h-ErC50 and the EyC50 of potassium dichromate was determined to be 0.82 and 0.51 mg/L respectively. The validity criteria were fulfilled. After 72 hours the ErC10 was determined to be 2.5 mg/L and the ErC50 was determined to be 9.31 mg/L.

Description of key information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata) was determined according to the principles of OECD 201 (2011) under GLP conditions. After 72 hours the ErC10 was determined to be 2.5 mg/L and the ErC50 was determined to be 9.31 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.31 mg/L
EC10 or NOEC for freshwater algae:
2.5 mg/L

Additional information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata) was determined according to the principles of OECD 201 (2011). The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 10 000 cells/mL. 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. 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. Based on the results of the non-GLP Preliminary Range-Finding Test the following five test concentrations in a geometric series (with a spacing factor of 3.0) were tested: 0.25, 0.74, 2.2, 6.7 and 20.0 mg/L (nominal concentrations). Untreated control ran parallel in the test and a positive control with potassium dichromate was performed. An appropriate amount of test item was dissolved in the dilution water (OECD medium) in order to obtain the concentration of 20 mg/L. The further test solutions were prepared by appropriate dilution of this stock solution. Samples were taken from the test concentrations and the control at the start and at the end of the experiment and analyzed by HPLC method with MS detection. Three parallel samples were analyzed from each test concentration and control at the start and at the end of the test. The measured concentration of the test item in the test solutions were 0.24, 0.72, 2.18, 6.29 and 20.8 mg/L (corresponding to 94 and 104 % of the nominal value) at the start of the test and 0.22, 0.70, 2.16, 6.46 and 19.5 mg/L (corresponding to 90 and 98 % of the nominal value) at the end of the test. The concentration of the tested substance was maintained within ± 20 % of the nominal concentrations throughout the test. Therefore, the analysis of the results was based on the nominal concentration values. The 24h-ErC50 and the EyC50 of potassium dichromate was determined to be 0.82 and 0.51 mg/L respectively. The validity criteria were fulfilled. After 72 hours the ErC10 was determined to be 2.5 mg/L and the ErC50 was determined to be 9.31 mg/L.