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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 2018-01-19 to 2018-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth 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)
Test material information:
Composition 1
Specific details on test material used for the study:
Batch No.: VPS9016001
Purity: 97.3 %
Analytical monitoring:
yes
Details on sampling:
For the determination of test item concentration, samples of each test concentration and control were taken from the test solutions at the start and at the end of the study. Three parallel samples were analysed from each test concentration and six samples from the control at the start and at the end of the test.
Vehicle:
no
Details on test solutions:
A stock solution of 100 mg/L (nominal concentration) was prepared by dissolving 0.08 mg of test item in 800 mL OECD Medium. The pH value (3.71) of the stock solution was adjusted to the pH of the OECD Medium (according to OECD 23; pH og OECD Medium - 7.87). The adjusted pH level of the stock solution was 7.39. Further test solutions were prepared by appropriate dilution of this stock solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 4 days
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.3 - 22.7 °C
pH:
7.82 - 9.80
Nominal and measured concentrations:
Nominal concentration: 100, 50, 25, 12.5, 6.25 mg/L
Measured concentration: 101 - 108 % of the nominal concentration at the end of the test
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: glass, ~250 mL volume, 100 mL test medium
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 93.0 x 10^4 cells/mL
- No. of organisms per vessel: 0.1 mL algal biomass (10^7 algal cells per mL)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD Medium prepared according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuously illuminated
- Light intensity and quality: 8038 lux

TEST CONCENTRATIONS
- Range finding study; yes, 2 tests, with and without pH adjustment
- Test concentrations: 0.1, 1, 10, 100 mg/L

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: microscope with counting chambe at 24, 48 and 72 hours
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
54.74 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
Remarks on result:
other: calculated EC50 = 109.52 mg/L (95 % CI: 99.367 - 123.527 mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
24.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
64.99 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
yield
Details on results:
- No morphological abnormalities were observed in the control and at the test item concentrations of 6.25, 12.5 and 25.0 mg/L during the experiment. Some thin cells among cells without sign of any effect were detected in the test group of 50.0 mg/L. In the highest test item group (100.0 mg/L) swollen and thin cells (together with cells without sign of any effect) were observed.
- The test item had a 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 concentrations of 50.0 and 100.0 mg/L (Dunnett’s Test, 2-sided, α=0.05) when compared to the control. Accordingly, the 72-hour NOEC based on growth rate and yield was determined to be 25.0 mg/L and the LOEC as 50.0 mg/L.
Results with reference substance (positive control):
The date of the last study with the reference item Potassium dichromate was: 12 – 15 September 2017. The following results were obtained:
The 72h ErC50: 0.82 mg/L, (95 % confidence limits: 0.57 – 1.25 mg/L)
The 72h EyC50: 0.51 mg/L, (95 % confidence limits: 0.45 – 0.57 mg/L)

Validity Criteria of the Study

The experiment was considered to be valid because:

- cell density in the control cultures increased by a factor of more than 16 (by a factor of 93.00) within 72 hours.

- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %.

- CV for section-by-section growth rate day 0-1: 12.68 %

- CV for section-by-section growth rate day 1-2: 15.61 %

- CV for section-by-section growth rate day 2-3: 5.51 %

The mean coefficient of variation for section-by-section specific growth rates: 11.27 %.

- the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in the test.

- CV for average specific growth rate day 0-3: 1.98 %

Validity criteria fulfilled:
yes
Conclusions:
The 72-h EC50 of the test item based on growth rate was detemined to be >100 mg/L. The EC10 and NOEC were determined to be 54.74 and 25 mg/L, respectively. The test item showed a significant toxic effects on the growth of Raphidocelis subcapitata at concentration levels of 50 and 100 mg/L.
Executive summary:

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 and under GLP. The study was conducted under static conditions with an initial cell density of 1x10^4 cells/mL for a period of 72 hours. A test with five test concentrations - 6.25, 12.5, 25, 50 and 100 mg/L, and control was performed. The measured concentrations of the test item at the start and at the end of the test were in the range of 101 - 108 % of the nominal concentration. All validity criteria were met and therefore the study can be considered as valid. The results are based on nominal test item concentrations. The test item showed a significant toxic effects on the growth of Raphidocelis subcapitata at concentration levels of 50 and 100 mg/L. The EC50-value for inhibition of growth rate (ErC50) was determined to be >100 mg/L. The EC50 value based on yield (EyC50) was determined to be 64.99 mg/L. The ErC10 and EyC10 values were determined to be 54.74 and 24.46 mg/L, respectively. The NOEC was determined to be 25 mg/L and the LOEC to be 50 mg/L.

Description of key information

The 72-h EC50 of the test item on green algae was detemined to be >100 mg/L. The EC10 and NOEC were determined to be 54.74 and 25 mg/L, respectively.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater algae:
54.74 mg/L

Additional information

The effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata was determined according to OECD 201, Regulation (EC) No. 226/2016 Method C.3 and EPA OPPTS 850.5400 and under GLP. The study was conducted under static conditions with an initial cell density of 1x10^4 cells/mL for a period of 72 hours. A test with five test concentrations - 6.25, 12.5, 25, 50 and 100 mg/L, and control was performed. The measured concentrations of the test item at the start and at the end of the test were in the range of 101 - 108 % of the nominal concentration. All validity criteria were met and therefore the study can be considered as valid. The results are based on nominal test item concentrations. The test item showed a significant toxic effects on the growth of Raphidocelis subcapitata at concentration levels of 50 and 100 mg/L. The EC50-value for inhibition of growth rate (ErC50) was determined to be >100 mg/L. The EC50 value based on yield (EyC50) was determined to be 64.99 mg/L. The ErC10 and EyC10 values were determined to be 54.74 and 24.46 mg/L, respectively. The NOEC was determined to be 25 mg/L and the LOEC to be 50 mg/L.