Tetraammonium decachloro-μ-oxodiruthenate(4-)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Few deviations occurred (i.e. T°C and measurement period differed from the study plan). Both reported to be uncritical, normal respiration activity of the control was observed and correlation of the inhibition values within the test replicates was good.

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Due to the poor solubility of the test item, the test item was weighed directly into the test vessels
- Method: In the control vessels, 16 mL nutrient solution was mixed with both 234 and 231 mL dilution water (i.e. tap water), without and with the addition of the nitrification inhibitor N-allylthiourea (ATU), respectively. Both the positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution, test item nominal concentrations, and ATU into the respective test vessel (concentrations of the test item and positive control were calculated using the concentration of the respective stock solution and the dilution factor). Then, 16 mL nutrient solution and water to 250 mL were added. Lastly, 250 mL inoculum was added in 5 minute intervals and the mixtures were aerated.
After 3 hours, the content of the first vessel was poured into a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the oxygen concentration over a period of max 5 minutes. The following vessels were measured in five minute intervals.
- Differential loading: Test item nominal concentrations were of 1000, 100, 10, 1 mg/L (first experiment), and of 1000, 560, 320, 180, 100 mg/L (second and third experiment)
- Controls: Both blank and positive controls were used. Blank controls contained 16 mL nutrient solution, 250 mL inoculum, and either 234 mL or 231 mL, according to the absence or addition of ATU, respectively. 3,5-Dichlorophenol (CAS 591-35-5) was used as positive control. A stock solution in deionised water containing 500 mg/L (nominal) was freshly prepared for each experiment.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None
- Nutrient solution: Synthetic sewage that was frozen immediately after preparation.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from a predominantly domestic sewage treatment plant was collected on the day before treatment. Sludge was filtrated, washed in tap water, resuspended in tap water, aerated until usage in the test, and fed daily with 50 mL synthetic sewage feed/L. The dry matter was determined, and volume was adapted to the desired content of dry matter. The dry matter content of the inoculum was also determined on the day of each experiment, and the following results were obtained, respectively:
Exp. 1) The dry matter was determined as 3.18 g suspended solids/L, giving a concentration of 1.59 g suspended solids/L in the test;
Exp. 2) The dry matter was determined as 2.80 g suspended solids/L, giving a concentration of 1.40 g suspended solids/L in the test;
Exp. 3) The dry matter was determined as 2.66 g suspended solids/L, giving a concentration of 1.33 g suspended solids/L in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

-

Test temperature:

First experiment: range from 21.7 to 22.6 °C
Second experiment: range from 21.2 to 23.2 °C
Third experiment: range from 21.0 to 22.1 °C

pH:

First experiment: pH range from 6.0 to 8.0
Second experiment: pH range from 5.7 to 8.2
Third experiment: pH range from 6.5 to 8.2

Dissolved oxygen:

-

Salinity:

-

Nominal and measured concentrations:

Test item nominal concentrations of 1000, 100, 10, 1 mg/L were used in the first experiment, and similarly nominal concentration of 1000, 560, 320, 180, 100 mg/L were used in the second and third experiments.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Aeration: Purified air, using Pasteur pipettes
- No. of vessels per concentration (replicates): 1 replicate per treatment (first experiment), 5 replicates per treatment (second and third experiment).
- No. of vessels per control (replicates): 2 replicates before and at the end of the exposure period, both for the test item and positive control treatments.
- No. of vessels per positive control (replicates): 1 replicate per treatment.
- Biomass loading rate: 250 mL activated sewage inoculum

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

TEST CONCENTRATIONS
- Spacing factor for test concentrations: A spacing factor of 10 was used for nominal test concentration in the first experiment. A spacing factor of about 1.8 was used for nominal test concentrations in both the second and third experiments.
- Test concentrations: Nominal test concentrations of 1000, 100, 10, 1 mg/L, and 1000, 560, 320, 180, 100 mg/L used for the first experiment, and the following two experiments, respectively.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS n. 591-35-5)

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: Without ATU

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

190 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 160 -210 mg/L. Without ATU

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 450 - 550 mg/L. Without ATU

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: With ATU

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: 220 - 280 mg/L. With ATU

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

740 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: 650 - 850 mg/L. With ATU

Details on results:

The oxygen consumption rate was calculated from the slope of the linear part of the oxygen consumption curve using linear regression.

Results with reference substance (positive control):

The calculated 3h-EC50 values for the positive control were:
First experiment without ATU - 10 mg/L (95% C.I. 8.2 - 12 mg/L), and with ATU - 13 mg/L (95% C.I. 8.2 - 19 mg/L)
Second experiment without ATU - 14 mg/L (95% C.I. 10 - 18 mg/L)
Third experiment with ATU -14 mg/L (95% C.I. 7.9 - 26 mg/L)
All values lay within the recommended range of 2 - 25 mg/L (total respiration without ATU), and 5 - 40 mg/L (heterotrophic respiration with ATU)

Reported statistics and error estimates:

A statistical determination of the NOEC was carried out, in order to test whether the differences between the related effect level and the control were significant. For this determination, the values of the oxygen consumption were used. Firstly, it was checked whether equality of variance was given, and afterwards a t-test was applied. No significant differences were found between the respective NOEC values and controls.
For the calculation of the EC10 and EC50, the percentage of inhibition was plotted versus concentration in a Gaub-logarithmic diagram. EC10 and EC50 were determined from the x values of the regression line at y=10% and y=50%. The data calculated were evaluated using a linear fit on a probability-logarithmic scale.

Validity criteria fulfilled:

yes

Remarks:

EC50 values for the positive control were within the required range. Coefficient of variation of O2 uptake rate in controls was <30%. Oxygen uptake rate for blank control was >20 mg O2/g activated sludge in 1 hour

Conclusions:

The 3h-NOEC for total respiration (without ATU) was 100 mg/L, 3h-EC10 was 190 mg/L and 3h-EC50 was 500 mg/L. The 3h-NOEC for heterotrophic respiration (with ATU) was 180 mg/L, 3h-EC10 was 250 mg/L and 3h-EC50 was 640 mg/L.

Executive summary:

Muckle (2015) assessed the inhibition of the respiration of activated sludge when exposed to tetraammonium decachloro-µ-oxodiruthenate. The study is GLP compliant and followed the standard test guidelines OECD 209 and EU-Method C.11.

The duration of the test was 3 hours. Activated sludge, which was collected from a predominantly domestic sewage treatment plant, was used as inoculum. The substance 3,5-dichlorophenol was used as a positive control. Three experiments were carried out. In the first experiment, which was performed as the range finding test, the test item was tested at 4 nominal concentrations with and without the nitrification inhibitor N-allylthiourea (ATU). Since significant inhibition was observed, two additional experiments were performed under the same test conditions, but with and without the addition of ATU, respectively. In the test series without ATU, inhibition was slightly higher than in the test series with ATU. This explains that the test item acted as a slight nitrification inhibitor. All validity criteria were met.

The 3h-NOEC for total respiration (without ATU) was 100 mg/L, 3h-EC10 was 190 mg/L and 3h-EC50 was 500 mg/L. The 3h-NOEC for heterotrophic respiration (with ATU) was 180 mg/L, 3h-EC10 was 250 mg/L and 3h-EC50 was 640 mg/L.

Description of key information

The 3h-NOEC for total respiration (without ATU) was 100 mg L-1, 3h-EC10 was 190 mg L-1 and 3h-EC50 was 500 mg L-1. The 3h-NOEC for heterotrophic respiration (with ATU) was 180 mg L-1, 3h-EC10 was 250 mg L-1 and 3h-EC50 was 640 mg L-1.

Additional information

Muckle (2015) assessed the inhibition of the respiration of activated sludge when exposed to tetraammonium decachloro-µ-oxodiruthenate. The study is GLP compliant and followed the standard test guidelines OECD 209 and EU-Method C.11.

The duration of the test was 3 hours. Activated sludge, which was collected from a predominantly domestic sewage treatment plant, was used as inoculum. The substance 3,5-dichlorophenol was used as a positive control. Three experiments were carried out. In the first experiment, which was performed as the range finding test, the test item was tested at 4 nominal concentrations with and without the nitrification inhibitor N-allylthiourea (ATU). Since significant inhibition was observed, two additional experiments were performed under the same test conditions, but with and without the addition of ATU, respectively. In the test series without ATU, inhibition was slightly higher than in the test series with ATU. This explains that the test item acted as a slight nitrification inhibitor. All validity criteria were met.

The 3h-NOEC for total respiration (without ATU) was 100 mg L-1, 3h-EC10 was 190 mg L-1 and 3h-EC50 was 500 mg L-1. The 3h-NOEC for heterotrophic respiration (with ATU) was 180 mg L-1, 3h-EC10 was 250 mg L-1 and 3h-EC50 was 640 mg L-1.