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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October 2021 - 4 November 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0291 to 100 mg sodium chlorite/L (nominal, corrected for purity) and control.
- Sampling method: Analytical samples including retain samples were taken at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions.
- Sample storage conditions before analysis: All samples were stored ambient until they were transferred to the analytical laboratory.
Vehicle:
yes
Remarks:
Test medium (APP-Medium)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The necessary amount of test item for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask (corrected for purity). Test medium was added up to the bench mark and the solution was treated with 5 minutes of ultrasonication. Afterwards the solution was clear and transparent. Lower test solutions were prepared by dilution of appropriate solutions with test medium.
- Test concentration separation factor: 3.2
- Evidence of undissolved material: No.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.

ACCLIMATION
- Acclimation period: 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: No.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.6 – 23.3 °C
pH:
7.75 – 8.44
Nominal and measured concentrations:
Nominal concentrations: 100, 31.3, 9.77, 3.05, 0.954, 0.298, 0.0931, 0.0291, 0.00909 mg/L sodium chlorite.
Measured concentrations (mean recoveries): 91.8, 93.9, 104.4, 109.0, 85.0, 82.1, 85.1 mg/L sodium chlorite (nominal doses of 0.0291 and 0.00909 mg/L not measured since below LOQ).
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): clossed (aluminium caps)
- Material, size, headspace, fill volume: Glass, 100 mL, fill volumn ~50 mL.
- Aeration: By continous agitation.
- Initial cells density: 0.5 x 10E4 cells/mL
- Control end cells density: 74.57 x 10E4 cells/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
- APP-Medium (OECD 201)

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was adjusted to 7.5  0.1 with NaOH or HCl
- Photoperiod: Continuously
- Light intensity and quality: 94.7 µEm-2s-1 (mean light intensity)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Fluorescence measurement (fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 2.0.10.0), at 0, 24, 48 and 72h (in duplicates).
- Other: morphological appearance of the algae cells was observed microscopically at the end of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 100, 31.3, 9.77, 3.05 and 0.954 mg/L
- Results used to determine the conditions for the definitive study:
Growth inhibition (72h): >100, 42.7, 3.2, 2.9 and -2.6% respectively.
Yield inhibition (72h): >100, 86.2, 12.5, 11.5 and -14.8% respectively.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.8 mg/L
95% CI:
>= 3.79 - <= 6.08
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
8.04 mg/L
95% CI:
>= 6.23 - <= 10.5
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21.5 mg/L
95% CI:
>= 13.3 - <= 34.2
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.954 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.72 mg/L
95% CI:
>= 1.19 - <= 2.49
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.61 mg/L
95% CI:
>= 1.83 - <= 3.72
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.76 mg/L
95% CI:
>= 3.72 - <= 8.84
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The cells were considered normal for the control and up to and including a nominal concentration of 9.77 mg sodium chlorite/L. No cells were observed at 31.3 and 100 mg sodium chlorite/L.
- Flocculation: No.
- Adherence to test vessels: No.
- Aggregation of algal cells: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50: 1.55 mg/L
- Dates of work: 11 Jan 2021 - 14 Jan 2021

Average cell numbers for each sampling time and concentration:






















































































Nominal
sodium chlorite
conc.
Average cell numbers [104/mL]
[mg/L]0 h24 h48 h72 h
Control0.562.0915.0874.57
0.009090.562.1214.6368.53
0.02910.562.0014.0471.19
0.09310.562.2615.9277.35
0.2980.562.1014.6169.09
0.9540.562.2015.0767.57
3.050.561.9511.6355.89
9.770.561.606.4822.14
31.30.560.470.420.20
1000.560.050.000.00

 


Percentage inhibition of growth rate:











































































Nominal
sodium chlorite
conc.
% Inhibition of growth rate
[mg/L]0 h – 24 h0 h – 48 h0 h – 72 h
Control0.00.00.0
0.00909-0.41.11.9
0.02913.82.51.1
0.0931-5.6-1.4-0.5
0.2980.11.31.8
0.954-3.50.32.2
3.055.48.16.1*
9.7720.425.925.0*
31.3114.9108.8130.5*
100266.1≥ 100≥ 100*

*statistically significant.


 


Percentage inhibition of yield:











































































Nominal
sodium chlorite
conc.
% Inhibition of yield
[mg/L]0 h – 24 h0 h – 48 h0 h – 72 h
Control0.00.00.0
0.00909-2.03.18.2
0.02915.97.24.6
0.0931-11.1-5.8-3.7
0.298-0.73.27.4
0.954-7.20.19.5
3.059.223.825.2*
9.7732.059.270.8*
31.3105.9101.0100.5*
100133.3103.9100.8*

*statistically significant.

Validity criteria fulfilled:
yes
Remarks:
Cell numbers in controls increased by a factor of 133.16 (>16). Mean coefficient of variation for the section-by-section specific growth rates in controls was 21% (<35%). Coefficient of variation of average growth rate in replicate controls was 1.7% (<7%)
Conclusions:
The EC50 of sodium chlorite in green algae was 21.5 mg/L based on growh rate inhibition. The EC10 was determined to be 4.80 mg/L and the NOEC 0.954 mg/L.
Executive summary:

The toxicity to the Single Cell Green Alga Pseudokirchneriella subcapitata Hindák was investigated with sodium chlorite. Based on preliminary tests, initial cell densities of 0.5 × 10E4 cells/mL were exposed to 100, 31.3, 9.77, 3.05, 0.954, 0.298, 0.0931, 0.0291, 0.00909 mg/L sodium chlorite.Six replicates were employed for the control and three for each test item concentration. Analytical samples were taken from control and concentrations of 0.0931 to 100 mg sodium chlorite/L at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions. The recoveries were between 80-120% and thus, the endpoints were calculated based on nominal concentrations. All validity criteria were fulfilled. Significant inhibitory effects were determined for growth rate and yield at concentrations of 3.05 mg sodium chlorite/L and above. The corresponding NOEC was set at 0.954 mg sodium chlorite/L. The EC10 value for growth rate was determined to be 4.80 mg sodium chlorite/L. The EC10 value for yield was 1.72 mg sodium chlorite/L. The EC20 value for growth rate was determined to be 8.04 mg sodium chlorite/L. The EC20 value for yield was 2.61 mg sodium chlorite/L. The EC50 value for growth rate was determined to be 21.5 mg sodium chlorite/L. The EC50 value for yield was 5.76 mg sodium chlorite/L.

Description of key information

Key study: OECD Guidance 201 (GLP study). The EC50 of sodium chlorite in green algae was 21.5 mg/L based on growh rate inhibition. The EC10 was determined to be 4.80 mg/L and the NOEC 0.954 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
21.5 mg/L
EC10 or NOEC for freshwater algae:
4.8 mg/L

Additional information