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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 July 2016 to 18 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
yes
Remarks:
The temperature in the third experiment was 20.9 – 22.2°C instead of 20.0 ± 2.0 °C. Because linearity of all regression curves was given and the correlation of the inhibition values within the replicates was good, this was stated as uncritical.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
The temperature in the third experiment was 20.9 – 22.2°C instead of 20.0 ± 2.0 °C. Because linearity of all regression curves was given and the correlation of the inhibition values within the replicates was good, this was stated as uncritical.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In the control vessels, 16 mL nutrient solution was mixed with 234 or 231 mL dilution 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. The test item was directly weighed into the test vessels. 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 in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the oxygen concentration over a maximum period of 5 minutes. The following vessels were measured in five minute intervals.
- Differential loading: Test item nominal concentrations were of 1, 10, 100, 1000 mg/L (first experiment) and of 1, 3.2, 10, 32, 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 water, without or with ATU, respectively. 3,5-Dichlorophenol (CAS 591-35-5) was used as positive control.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No
- Name and location of sewage treatment plant where inoculum was collected: The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf.
- Pretreatment: Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until used in the test and fed daily with 50 mL synthetic sewage feed /L.
- Initial biomass concentration:
Exp. 1) The dry matter was determined as 3.20 g suspended solids/L, giving a concentration of 1.60 g suspended solids/L in the test;
Exp. 2) The dry matter was determined as 2.74 g suspended solids/L, giving a concentration of 1.37 g suspended solids/L in the test;
Exp. 3) The dry matter was determined as 2.62 g suspended solids/L, giving a concentration of 1.31 g suspended solids/L in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
1.03 mmol/L
Test temperature:
First experiment: 20.8 to 21.6°C
Second experiment: 19.7 to 20.7°C
Third experiment: 20.9 to 22.2°C
pH:
First experiment: 7.5 to 7.8
Second experiment: 7.3 to 7.7
Third experiment: 7.4 to 7.6
Dissolved oxygen:
N/A
Salinity:
N/A
Conductivity:
242 µS/cm at 25°C
Nominal and measured concentrations:
Nominal concentrations of 1, 10, 100, 1000 mg/L in the first experiment (range finding test) and 1, 3.2, 10, 32 and 100 mg/L in the second and third experiment.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers used as test vesels. Narrow-neck glass bottles with flat bottoms (250 mL) were used as measuring flasks.
- 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.
- Sludge concentration (weight of dry solids per volume): 1.31 - 1.60 g suspended soilds/L
- Nutrients provided for bacteria: Yes, sludge was fed with 50 mL of nutrient solution/L sludge
- Nitrification inhibitor used: Test conducted with and without N-allylthiourea (ATU)

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

EFFECT PARAMETERS MEASURED: Inhibition of respiration rate of activated sludge measured after a contact time of 3 hours

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 3.2 was used for nominal test concentrations in both the second and third experiments.
- Range finding study: Yes, nominal test concetrations of 1, 10, 100, 1000 mg/L (first experiment)
- Test concentrations: 1, 3.2, 10, 32, 100 100 mg/L (second and third experiment)
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (CAS-No. 591-35-5)
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without ATU, 95 % CI: 21- 50 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without ATU
Duration:
3 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: without ATU
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: with ATU; 95 % CI: 12 - 37 mg/L
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 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:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Remarks on result:
other: 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 - 11 mg/L (95% C.I. 5.7- 18 mg/L), and with ATU - 13 mg/L (95% C.I. 4.1 - 33 mg/L)
Second experiment without ATU - 8.2 mg/L (95% C.I. 5.1 - 11 mg/L)
Third experiment with ATU -13 mg/L (95% C.I. 2.5 - 35 mg/L)

All values are 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 then a t-test was used to check for the significance of differences.
For the calculation of the EC10 and EC50, the percentage of inhibition was plotted versus concentration in a Gauss-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
Conclusions:
A 3h-NOEC of 3.2 mg/L, 3h-EC10 of 31 mg/L and 3h-EC50 of >100 mg/L were determined based on inhibition of total respiration (without the nitrification inhibitor ATU). A 3h-NOEC of 1 mg/L, 3h-EC10 of 20 mg/L and 3h-EC50 of >100 mg/L were determined based on inhibition of heterotrophic respiration (with the addition of ATU).
Executive summary:

Effect of platinum, 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complexes (Karstedt concentrate) on the inhibition of the respiration of activated sludge was assessed in a study according to test guidelines OECD 209 and EU-Method C.11 (Muckle 2016). The study was considered to be reliable without restrictions, as it is GLP-compliant and follows standard test guidelines. The duration of the test was 3 hours. Activated sludge 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 range finding test, the test item was tested using 4 nominal concentrations with and without the nitrification inhibitor N-allylthiourea (ATU). In the range finding test (first experiment) inhibition values in the three highest concentrations were strongly scattering; higher nominal concentrations did not result in higher inhibition but inhibition did not reach 50 % in any of the tested concentration. It is likely that this scattering was caused by the poor solubility of the test item and therefore limited bioavailability. Nevertheless, statistical significant inhibition was observed. Therefore, additionally one experiment with nitrification inhibitor and one experiment without nitrification inhibitor was performed as a main test under the same test conditions (second and third experiment).

A 3h-NOEC of 3.2 mg/L, 3h-EC10 of 31 mg/L and 3h-EC50 of >100 mg/L were determined based on inhibition of total respiration (without the nitrification inhibitor ATU). A 3h-NOEC of 1 mg/L, 3h-EC10 of 20 mg/L and 3h-EC50 of >100 mg/L were determined based on inhibition of heterotrophic respiration (with the addition of ATU).

Description of key information

A 3h-NOEC of 3.2 mg L-1, 3h-EC10 of 31 mg L-1and 3h-EC50 of >100 mg L-1were determined based on inhibition of total respiration (without the nitrification inhibitor ATU). A 3h-NOEC of 1 mg L-1, 3h-EC10 of 20 mg L-1and 3h-EC50 of >100 mg L-1were determined based on inhibition of heterotrophic respiration (with the addition of ATU).

Key value for chemical safety assessment

Additional information

The inhibition of the respiration of activated sludge when exposed to Platinum, 1,3-diethenyl-1,1,3,3-tetramethyldisiloxane complexes was assessed in a study according to OECD 209 and EU-Method C.11 test method (Muckle 2016). The study was considered to be reliable without restrictions, as it is GLP-compliant and follows standard test guidelines.

The duration of the test was 3 hours. Activated sludge 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 range finding test, the test item was tested using 4 nominal concentrations with and without the nitrification inhibitor N-allylthiourea (ATU).

In the range finding test (first experiment), inhibition values in the three highest concentrations were strongly scattering; higher nominal concentrations did not result in higher inhibition but inhibition did not reach 50 % in any of the tested concentrations. It is likely that this scattering was caused by the poor solubility of the test item and therefore limited bioavailability. Nevertheless, statistically significant inhibition was observed. Therefore, additionally one experiment with nitrification inhibitor and one experiment without nitrification inhibitor was performed as a main test under the same test conditions (second and third experiment).

A 3h-NOEC of 3.2 mg L-1, 3h-EC10 of 31 mg L-1 and 3h-EC50 of >100 mg L-1 were determined based on inhibition of total respiration (without the nitrification inhibitor ATU). A 3h-NOEC of 1 mg L-1, 3h-EC10 of 20 mg L-1 and 3h-EC50 of >100 mg L-1 were determined based on inhibition of heterotrophic respiration (with the addition of ATU).