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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:
2013-07-30 to 2013-08-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP and guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
24 August 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
23rd March 2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
April 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentration, samples were taken from each testing concentration and from the control at the start and at the end of the experiment.
Vehicle:
no
Details on test solutions:
Algal Mineral Salts Test Medium (dilution water):
Reconstituted algal growth medium (OECD medium, according to OECD 201 guideline) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water (prepared in the laboratory 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.

Test solution:
A stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving the test item in OECD medium. The test solutions were prepared by appropriate dilution of this stock solution. The test solutions were prepared immediately before introduction of algae (start of the experiment).
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Species: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Origin: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, 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:
yes
Total exposure duration:
72 h
Test temperature:
The temperature was in the range of 21.6 – 22.1 °C measured in the flask and between 21.3 and 23.0°C measured within the climate chamber.
pH:
The pH was not adjusted throughout the test period. The pH of the control medium did not increase by more than 1.5 units during the test.
Nominal and measured concentrations:
Pre-test: 1, 10, 100 mg/L (nominal)
Main-test: 100 mg/L (nominal)
The average measured concentration of the test item in the test solution was 90.78 % of the nominal at the start and 88.60 % of the nominal at the end of the test. There is evidence that the concentration of the tested substance was satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration value.
Details on test conditions:
TEST SYSTEM
- Pre-experiments:
In order to select appropriate test concentrations for use in the definitive test, preliminary range-finding test was conducted to determine the approximate toxicity of the test item.
A stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving the test item in OECD medium. The test solutions were prepared by appropriate dilution of this stock solution.
Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and control group.
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium.
- Introduction of Algae: The test was started (0 hours) by inoculation of 0.1 mL algal biomass (107 algal cells per mL) to 100 mL test item solution. The initial cell density was ~104 cells/mL in each test 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 six replicates per test concentration and six replicates in the untreated control group.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light Intensity: The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks during the test was 7985 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
- Range finding study: test concentrations: 1; 10; 100 mg/L
- Main-study: 100 mg/L (limit test)
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
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:
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:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Pre-test: Significant toxic effects were not observed during the preliminary test, therefore only one test concentration at 100 mg/L and one control group were tested in a limit test.

Analytical Results
- Test Concentration: The average measured concentration of the test item in the test solution was 90.78 % of the nominal at the start and 88.60 % of the nominal at the end of the test. There is evidence that the concentration of the tested substance was satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration value. All reported biological results are related to the nominal concentration of the test item.

- Biological Results
Growth Inhibition: Statistical comparisons of average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT 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) was not statistically significantly different from that of the untreated control. Accordingly, the No Observed Effect Concentration (NOEC) was determined to be 100 mg/L (nominal concentration).
The 72 h-EC50 and 72 h-EC10 values based on growth rate and yield were determined to be higher than the test item concentration of 100 mg/L.

- Microscopic Examinations
- 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 = 1E4 cells/mL at the start of the test (0 hours) to N = 96.33E4 cells/mL (mean value) after 72 hours of exposure. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
Results with reference substance (positive control):
The date of the last study (Study Code: 392.440.4211) with the reference item Potassium dichromate was: 11 – 14 March 2013.
The 72h ErC50: 1.40 mg/L, (95 % confidence limits: 1.27 – 1.54 mg/L)
The 72h EyC50: 0.78 mg/L, (95 % confidence limits: 0.70 – 0.87 mg/L)

Validity criteria of the study:

The experiment is valid because:

- Cell density in the control cultures increased by a factor of more than 16 (by a factor of 96.33) within 72 hours.

- The mean coefficient of variation 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: 6.40 %

- CV for section-by-section growth rate day 1 - 2: 3.47 %

- CV for section-by-section growth rate day 2 - 3: 8.90 %

The mean coefficient of variation for section-by-section specific growth rates: 6.26 %.

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

- CV for average specific growth rate day 0 - 3: 2.29 %

Validity criteria fulfilled:
yes
Conclusions:
The 72-h EC50 value based on growth rate and yield were both determined to be > 100 mg/L. The 72-h EC10 value based on growth rate and yield were determined to be > 100 mg/L.
Executive summary:

The 72-h algal growth inhibition test with Pseudokirchneriella subcapitata was conducted according to EU method C.3, OECD no.201 guideline and EPA OPPTS guideline 850.5400. The 72-h EC50 value based on growth rate and yield were both determined to be > 100 mg/L. The 72-h EC10 value based on growth rate and yield were determined to be > 100 mg/L, too. The average measured concentration of the test item in the test solution was 90.78 % of the nominal at the start and 88.60 % of the nominal at the end of the test, therefore nominal concentrations were used.

Description of key information

The 72-h EC50 value based on growth rate and yield were both determined to be > 100 mg/L. The 72-h EC10 value based on growth rate and yield weredetermined to be > 100 mg/L.

Key value for chemical safety assessment

Additional information

The 72-h algal growth inhibition test with Pseudokirchneriella subcapitata was conducted according to EU method C.3, OECD no. 201 guideline and EPA OPPTS guideline 850.5400. The 72-h EC50 value based on growth rate and yield were both determined to be > 100 mg/L. The 72-h EC10 value based on growth rate and yield were determined to be > 100 mg/L, too. The average measured concentration of the test item in the test solution was 90.78 % of the nominal at the start and 88.60 % of the nominal at the end of the test, therefore nominal concentrations were used.