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Ecotoxicological information

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 2017-10-16 to 2017-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
TOXI-COOP ZRT., 8230 Balatonfüred, Arácsi út 97. (1045 Budapest, Berlini u. 47-49.), Hungary
Analytical monitoring:
yes
Details on sampling:
- Sampling: For the determination of test item concentration, samples of test concentration and control were taken from the test solutions at the start and at the end of the study. Six parallel samples were analysed from test concentration and control at the start and at the end of the test. The samples were prepared immediately after sampling. Formulation samples were diluted with acetonitrile : water = 1 : 9 (v/v) with 0.1 % ortho-phosphoric acid and analysed by an HPLC method with UV detection.
Vehicle:
no
Details on test solutions:
- Test solution: A test solution of 100 mg test item/L (nominal concentration) was prepared by dissolving 0.1193 g of the test item in 1193 mL OECD Medium. Untreated control ran parallel in the test. The test solution was prepared just before introduction of algae (start of the experiment).
- Controls: Untreated Control: OECD Medium (without test item) was inoculated and examined in parallel in the study.
- 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: green algae
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding conditions: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD 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:
yes
Total exposure duration:
72 h
Test temperature:
in the flasks: 21.5 - 21.9 °C
in the climate chamber: 21.0 - 22.5 °C
pH:
8.22 - 8.91
Salinity:
NA
Nominal and measured concentrations:
Nominal concentration 100 mg/L
Measured concentration: 95.7 mg/L at the start and 101.5 mg/L at the end of the test
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume, Volumes of 100 mL test medium
- Type: closed, flasks with air-permeable stoppers
- Initial cells density: The test was started (0 hours) by inoculation of 0.1 mL algal biomass (1E07 algal cells per mL) to 100 mL test item solution. The initial cell density was about 1E04 cells/ mL in each test flask.
- Control end cells density: 194.17x1E04 cells/mL after 72 hours

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD Medium, according to OECD 201) was used as dilution water.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: average - 7998 lux, which was ensured with fluorescent lamps (with a spectral range of 400 - 700 nm).

EFFECT PARAMETERS MEASURED:
- 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.
- Other: 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
- Range finding study
- Test concentrations: 0, 0.1, 1, 10, 50, 100 mg/L
- Results used to determine the conditions for the definitive study: No significant toxic effect was observed during the non-GLP Preliminary Range-Finding Test, therefore, only one test concentration (equivalent to 100 mg/L nominal concentration) and one control group was tested in a limit test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Details on results:
- Analytical results: The measured concentration of the test item in the test solution was 95.7 mg/L (96 % of the nominal) at the start and 101.5 mg/L (102 % of the nominal) 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.
- Growth inhibition: The 72-h EC10, EC20 and EC50 values as well the LOEC values based on growth rate and yield were determined to be higher than 100 mg/L nominal concentration. Accordingly, the No Observed Effect Concentration (NOEC) was determined as 100 mg/L.
- Shape of the Algal Cells: Abnormal appearance of the algal cells was not observed during the experiment.
- Cell Density in Control: The cell density in the control has increased from nominal N = 1E04 cells/mL at the start of the test (0 hours) to N = 194.17E04 cells/mL (mean value) after 72 hours of exposure.
Results with reference substance (positive control):
The 72-h ErC50: 0.82 mg/L, (95 % confidence limits: 0.57 – 1.25 mg/L)
The 72-h EyC50: 0.51 mg/L, (95 % confidence limits: 0.45 – 0.57 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations were calculated for the control and test concentration at each observation period of the test using Microsoft Excel for Windows. The EC values of the test item related to growth rates and yield were determined from the raw data.
Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using Independent Samples T-Test (α = 0.05) by SPSS+ software.
The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield at the treatment group (100 mg/L nominal concentration) were not statistically significantly different from the untreated control. Accordingly, the No Observed Effect Concentration (NOEC) was determined as 100 mg/L.
The test item had no significant effect on the growth of the algae, therefore, statistical analysis was not performed to determine the ECx values.
Validity criteria fulfilled:
yes
Conclusions:
In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results show that the test item has no toxic effect at 100 mg/L nominal concentration on the algae. Accordingly, the EC10, EC20, EC50 and the LOEC values based on growth rate and yield were determined to be higher than the nominal value of 100 mg/L. The overall NOEC value of the test item was determined as 100 mg/L (nominal).
Executive summary:

In a study according to OECD TG 201, the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was determined. Exponentially growing cultures were exposed to various concentrations of the test item under static conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. Based on the results of a Range-Finding Test a limit test with test concentration of 100 mg/L was performed as main test. Untreated control ran parallel in the test. The measured concentration of the test item in the test solution was 96 % at the start and 102 % 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. The test item had no statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the tested concentration. The 72-h EC50 based on the growth rate and yield was determined to >100 mg/L. The 72-h EC10 based on the growth rate and yield was determined to be >100 mg/L. Accordingly, the 72-h NOEC based on growth rate and yield was determined to be 100 mg/L and the LOEC as >100 mg/L.

Description of key information

In a 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results show that the test item has no toxic effect at 100 mg/L nominal concentration on the algae. Accordingly, the EC10, EC20, EC50 and the LOEC values based on growth rate and yield were determined to be higher than the nominal value of 100 mg/L. The overall NOEC value of the test item was determined as 100 mg/L (nominal).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

In a study according to OECD TG 201, the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was determined. Exponentially growing cultures were exposed to various concentrations of the test item under static conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. Based on the results of a Range-Finding Test a limit test with test concentration of 100 mg/L was performed as main test. Untreated control ran parallel in the test. The measured concentration of the test item in the test solution was 96 % at the start and 102 % 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. The test item had no statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the tested concentration. The 72-h EC50 based on the growth rate and yield was determined to >100 mg/L. The 72-h EC10 based on the growth rate and yield was determined to be >100 mg/L. Accordingly, the 72-h NOEC based on growth rate and yield was determined to be 100 mg/L and the LOEC as >100 mg/L.