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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 4th to 5th December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information): Not applicable
Analytical monitoring:
yes
Details on sampling:
Concentrations 1000 mg/L -32 mg/L, nominal
10 mL of the respective solution in medium was added to 90 mL demineralised water with 5 g NaCl; then, the solution was extracted two times with the solvent methyl t-butyl ether (9 mL, 4 mL), the organic phase was collected after drying with Na2SO4 into a 10 mL flask and the flask was filled to 10 mL with methyl t-butyl ether.
Concentration: 10 mg/L, nominal
30 mL of the solution in medium was added to 70 mL demineralised water with 5 g NaCI; then, the solution was extracted two times with the solvent methyl t-butyl ether (9 mL, 4 mL), the organic phase was collected after drying with Na2SO4 into a 10 mL flask and the flask was filled to 10 mL with methyl t-butyl ether. Threefold enrichment was achieved.
Vehicle:
no
Details on test solutions:
See the field "Details on sampling".
At the beginning and at the end samples for analytical determination were taken. For this, a 50 mL glass flask was completely filled with test solution and immediate closed.
Test organisms (species):
sewage, domestic
Details on inoculum:
On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL/L sludge.
On the day of the experiment, the dry matter was determined once more. The stock solution of the positive control was prepared.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
none
Hardness:
1.08 mmol/L
Test temperature:
20.5 — 21.2 °C
pH:
7.7 - 7.8
Nominal and measured concentrations:
During validation of the analytical method, the solutions for determination of recovery and stability in medium were prepared using methanol as solvent. In the main study, no solvent was used because usage of solvents is not described in the guideline. Therefore, due to the slow dissolution, the actual test item concentration during the test is not known. Therefore, the nominal concentrations were used for determination of the results.
Nominal Concentration in mg/L Measured Concentration in mg/L
0h 3h
10 1.5 3.9
32 5 30
100 11 60
320 15 104
1000 n.d. 304
Details on test conditions:
The study was performed in a closed system without aeration but with analytical determination. In the control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control and the test vessels were prepared by putting the appropriate amount of positive control solution respectively test item into the test vessel, adding 16 mL nutrient solution and water to give 250 mL. Then, 250 mL inoculum was added in five minute intervals. After addition of the inoculum, the flasks were closed immediately and the mixtures were stirred without aeration.
After three 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 02-concentration over a period of max. five minutes. The following vessels were measured likewise in five minute intervals.
At the beginning and at the end samples for analytical determination were taken. For this, a 50 mL glass flask was completely filled with test solution and immediate closed.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenole
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: n.d.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: n.d.
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: n.d.
Details on results:
The difference between treatment 1000 mg/L and the control can be considered as not significant because the 02 consumption in the treatment was higher than in the control. Therefore, the concentration 1000 mg/L is stated as NOEC.
Results with reference substance (positive control):
The calculated value for EC5O of the positive control 3,5-dichlorophenole was 7.8 mg/L. A 95% confidence interval of 5.2 — 50 mg/L was calculated.
EC50 of 3,5-dichlorophenole lies within the recommended range of 2 — 25 mg/L.
Validity criteria fulfilled:
yes
Remarks:
Coefficient of variation of O2-consumption of the control was 5.3 % which is far below the recommended upper limit of 30%. The blank controls oxygen uptake rate was well above the recommended lower limit of 20 mg O2.
Conclusions:
Because volatility of the test item could not be excluded, the experiment was performed in closed Schott flasks without aeration and with analytical determination of the test item in the liquid phase at the beginning and at the end of the test, as described in the OECD guideline. Because of slow dissolution of the test item, only marginal test item concentrations could be measured at the beginning. Surprisingly, no test item could be detected in the highest concentrated treatment at the beginning of the test. This might be caused by insufficient homogenisation of the sample. After three hours stirring, significantly higher test item concentrations could be measured in all treatments.
During validation of the analytical method, the solutions for the determination of recovery and stability in medium were prepared using methanol as solvent. In the main study, no solvent was used because usage of solvents is not described in the guideline. Therefore, due to the slow dissolution of the test item, the actual test item concentrations during the test are unknown. Therefore, the nominal concentrations were used for determination of the results, At no test item concentration, any inhibitory effect could be observed.
All validity criteria were met. For the estimation of the EC50 of the positive control, the fit showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 7.8 mg/L which is within the recommended range of 2 — 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30 % at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg 02 per gram activated sludge.
The result of the test can be considered valid.

Description of key information

EC10 (activated sludge, 3h, OECD209): >1000 mg/L
EC50 (activated sludge, 3h, OECD209): >1000 mg/L
NOEC (activated sludge, 3h, OECD209): >=1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A GLP study conducted according to OECD 209 and EU C11 is available.

Because volatility of the test item could not be excluded, the experiment was performed in closed Schott flasks without aeration and with analytical determination of the content of test item in the liquid phase at the beginning and at the end of the test, as described in the OECD guideline. The study was performed using five concentrations, ranging from 1000 to 10 mg/L nominal concentration. The dry matter of the activated sludge was determined as 2.04 g suspended solids/L, giving a concentration of 1.02 g suspended solids/L in the test. EC50 of the positive control was determined as 7.8 mg/L (95% confidence interval: 5.2 — 50 mg/L), which lies within the demanded range of 2 —25 mg/L. Because of slow dissolution of test item, the measured concentrations at the end of the test was much higher than at the beginning. At no test item concentration, any inhibition effect could be observed.