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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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test concentrations
- Sampling method: At test start samples were taken from the test solution preparation just before distributing it to the test replicates per treatment. At test end, samples of two representative replicates per treatment level were taken directly from the test vessels.
- Sample storage conditions before analysis: The samples were filtered through a 0.2 μm polyether sulphone membrane syringe filter at room temperature (20 – 25 °C) after collection and acidified with HNO3 (final concentration 10 %). Samples were stored at about 4°C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest test concentration was prepared by adding the required amount of the test item to 1 L sterile growth medium under sterile conditions to obtain a nominal concentration of 12 mg/L. The solution was stirred vigorously using a magnetic stirring bar for about 1.5 h at room temperature (approximately 20 °C). An aliquot of the clear test solution was then diluted with sterile growth medium to obtain the other five test concentrations.
- Controls: Test medium only
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen.
- Age of inoculum (at test initiation): 3 days old
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.3 - 22.0 °C
pH:
Test start: 7.35 - 8.23
Test end: 7.99 - 8.19
Nominal and measured concentrations:
Nominal test item concentrations: 0.038, 0,120, 0.380, 1.20, 3.80 and 12.0 mg test item/L
Mean measured (geometric mean) test item concentrations: 0.023, 0.080, 0.292, 0.962, 2.95 and 10.3 mg test item/L

At test start the measured ruthenium concentrations were 9.53, 30.2, 99.4, 325, 1037 and 3351 μg./L (82.9 – 92.3 % of nominal). After 72 h the concentrations were 5.09, 19.3, 78.6, 261, 768 and 2907 μg/L (44.3 – 80.1 % of nominal, 53.4 - 86.8 % of the initial values).
The test evaluation was based on the geometric mean concentrations of the test item, since the deviation from the measured initial concentration was greater than ± 20 %
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks covered with air-permeable silicone-sponge caps
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: 10 000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified water processed using "PURELAB Option S7 with Classic UHF”
- Culture medium different from test medium: No.
The sterilised synthetic OECD medium according to OECD 201 was used as growth medium.
In this medium, the molar ratio of iron to EDTA slightly exceeds unity with iron in excess (molar ratio Fe(III+) / EDTA = 1.14). Considering the molar ratio of Fe and EDTA and the high stability constant of EDTA with Fe(III+) ions (log Ks 25), the chelation of ruthenium remains negligible in comparison with nominal ruthenium test concentrations and therefore the EDTA containing OECD medium can be used without limitations.

- Intervals of water quality measurement: Temperature was measured daily and pH was measured at the start and end of the test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 91.31 μmol m-2 s-1 to 101.8 μmol m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were determined after 24, 48 and 72 h
- Chlorophyll measurement: Yes, using a microplate reader on the second and third day of the test

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: yes
- Test concentrations: 0.1, 1.0 and 10.0 mg test item/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.38 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.365 and 0.396 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.052 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.049 and 0.056 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.017 and 0.020 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.057 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.053 and 0.061 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.022 and 0.028 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.016 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 0.013 and 0.019 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Observation of abnormalities (for algal test): Yes
- Colour differences: Slightly blueish colour at the two highest test concentrations (2.95 and 10.3 mg/L) at the start of the test.
- Other: The amount of intact cells decreased and the cell debris increased with increasing concentrations at the end of the test for all test concentrations at 0.080 mg/L and higher.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 2.62 mg/L (95 % confidence limits: 2.52 - 2.72 mg/L)
- Other: Results of an international ring test gave an ErC50 of 3.38 ± 1.30 mg/L
Reported statistics and error estimates:
The mean value of the cell counts for each concentration plot was used for plotting growth curves. Growth responses were used for the regression analysis. Probit-analysis was used to determine the EC50, EC20 and EC10 values and their respective 95 % confidence intervals. NOEC values were determined using ANOVA followed by Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm. All statistical evaluations were performed with ToxRat software.
Validity criteria fulfilled:
yes
Remarks:
The validity criteria for the control culture increase in cell numbers within the 72 hour test period, mean % CV in the section-by-section growth rate during the test period and the % CV of average specific growth rate at test end were met.
Conclusions:
The ErC50 and ErC10 values for inhibition of growth rate were 0.380 and 0.019 mg tetraammonium-decachloro-μ-oxodiruthenate/L, respectively. The EyC50 and EyC10 values for inhibition of yield were 0.057 and 0.016 mg tetraammonium-decachloro-μ-oxodiruthenate/L, respectively. The NOEC for both, growth rate and yield was 0.023 mg tetraammonium-decachloro-μ-oxodiruthenate/L.
Executive summary:

The 72-hour toxicity of tetraammonium-decachloro-μ-oxodiruthenate to the unicellular green Pseudokirchneriella subcapitata was determined in a static system following OECD guideline 201 (Wenzel 2016). Test solutions were analysed using ICP-OES and results reported based on mean measured concentrations, as measured concentrations deviated from nominal by more than 20%. Four replicate cultures were exposed to geometric mean concentrations of 0.023, 0.080, 0.292, 0.962, 2.95 and 10.3 mg test item/L. Eight control replicates were included.

There was a concentration-related growth inhibition and the ErC50 and ErC10 values for inhibition of growth rate were 0.380 and 0.019 mg tetraammonium-decachloro-μ-oxodiruthenate/L, respectively. The EyC50 and EyC10 values for inhibition of yield were 0.057 and 0.016 mg tetraammonium-decachloro-μ-oxodiruthenate/L, respectively. The NOEC for both, growth rate and yield was 0.023 mg tetraammonium-decachloro-μ-oxodiruthenate/L.

Description of key information

No experimental study is required for ruthenium dioxide based on the outcome of a full TDp assay (cfr. IUCLID Section 4 'Water solubility') and the identified acute and chronic threshold values with other water soluble ruthenium substances.

The ErC50 and ErC10 values for inhibition of growth rate were 0.380 and 0.019 mg tetraammonium-decachloro-μ-oxodiruthenate L-1, respectively. The EyC50 and EyC10 values for inhibition of yield were 0.057 and 0.016 mg tetraammonium-decachloro-μ-oxodiruthenate L-1, respectively. The NOEC for both growth rate and yield was 0.023 mg tetraammonium-decachloro-μ-oxodiruthenate L-1.

These are the lowest acute and chronic thresholds identified with water soluble ruthenium substances, and these values serve as basis for acute and chronic ERV derivation for ruthenium.

Key value for chemical safety assessment

EC50 for freshwater algae:
115 µg/L
EC10 or NOEC for freshwater algae:
6 µg/L

Additional information

No experimental study is required for ruthenium dioxide based on the outcome of a full TDp assay (cfr. IUCLID Section 4 'Water solubility') and the identified acute and chronic threshold values with other water soluble ruthenium substances.

The 72-hour toxicity of tetraammonium-decachloro-μ-oxodiruthenate to the unicellular green algae Pseudokirchneriella subcapitata was determined in a static system following OECD guideline 201 (Wenzel 2016). Test solutions were analysed using ICP-OES and results reported based on mean measured concentrations, as measured concentrations deviated from nominal by more than 20%. Four replicate cultures were exposed to geometric mean concentrations of 0.023, 0.080, 0.292, 0.962, 2.95 and 10.3 mg test item L-1. Eight control replicates were included.

There was a concentration-related growth inhibition and the ErC50 and ErC10 values for inhibition of growth rate were 0.380 and 0.019 mg tetraammonium-decachloro-μ-oxodiruthenate L-1, respectively. The EyC50 and EyC10 values for inhibition of yield were 0.057 and 0.016 mg tetraammonium-decachloro-μ-oxodiruthenate L-1, respectively. The NOEC for both growth rate and yield was 0.023 mg tetraammonium-decachloro-μ-oxodiruthenate L-1.

These values translate to EC50 = 115 µg Ru/L and EC10 = 6 µg Ru/L.

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