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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
None.
Analytical monitoring:
no
Details on sampling:
n.a.
Vehicle:
no
Details on test solutions:
The synthetic sewage feed was prepared to contain the following constituents per liter:

- 16 g peptone
- 11 g meat extract
- 3 g urea
- 0.7 g sodium chloride
- 0.4 g calcium chloride dihydrate CaCl2, 2H2O
- 0.2 g magnesium sulfate heptahydrate MgSO4, 7H2O
- 2.8 g di-potassium hydrogen phosphate K2HPO4

The final volume was filled up with ultra-pure water.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The inoculum was obtained on 31 August 2015 from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64) which handles predominantly domestic sewage. On arrival at the laboratory, the activated sludge (around 10L) was centrifuged (1000g for 10 minutes) and the solid sewage inoculum pellet was re-suspended in 10L French source water (Volvic®). 500 mL synthetic sewage feed was then added. Sewage sludge was maintained under aerated conditions for a maximum of 4 days and fed daily with synthetic sewage feed at a rate of 50 mL/L. In order to obtain the required sludge suspended solids concentration of 3 g/L in the stock solution to be used in the experiments, the solid content of the 10-L sludge solution was determined by removing a sub-sample of homogenised sludge and drying it an oven at approximately 105°C. The suspended solids concentration was subsequently adjusted to the recommended guideline value of 3 g/L ± 10%.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
None.
Hardness:
Not reported.
Test temperature:
19.6°C - 21.2°C (min - max)
pH:
7.4 - 7.6 (min - max)
Dissolved oxygen:
Around 8 mg/L during the 3-h incubation and around 8 mg/L at the beginning of the 10-min incubation period with oxygen depletion monitoring.
Salinity:
Freshwater.
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal test item concentrations: 0 ; 1 ; 3.2 ; 10 ; 32 ; 100 mg/L
Nominal reference item concentrations: 0 ; 3.2 ; 10 ; 32 mg/L
Details on test conditions:
At the start of the test, 16 mL of synthetic sewage feed were made up to 250 mL with ultra-pure water and 250 mL activated sludge inoculum with a sludge concentration of nominally 1.5 g/L (dry weight) were added to the first control vessels couple. Thereafter, at time intervals of about fifteen minutes, the procedure was repeated with test vessels couples in which the 16 mL of synthetic sewage were made up to 250 mL with the appropriate amounts of reference or test item diluted in ultra-pure water to obtain a volume of 250 mL. As for the controls, 250 mL of activated sludge inoculum (1.5 g/L) were then added. Fifteen minutes after the preparation of the last treatment, the second control vessels couple was prepared at last. The fifteen minutes interval was done so that the 10 min incubation period with O2 depletion monitoring could take place exactly 3 hours after start of incubation of each test vessel. The addition of the inoculum was considered as the start of the 3-hours incubation time period.

All solutions were incubated at 20 +/- 2°C in darkness for 3 hours in a temperature controlled room. Solutions were mixed by stirring and aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during 3 hours. At the end of the test incubation period, a well-mixed sample of each solution (following their order of preparation) were poured into a BOD bottle in which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min or until the oxygen concentration felt below 1 mg/L.

The temperature was monitored throughout the test period.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol ; CAS 951-35-5
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Details on results:
1,3-dibutylthiourea did not cause any significant respiratory inhibition up to the highest tested concentration. The NOEC is therefore considered superior or equal to 100 mg/L. This test met the validity criteria of the guideline:

- The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: the maximum was determined to be 8.1%
- The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: min. 29.80 mg O2/g of activated sludge (dry weight of suspended solids)/h
Results with reference substance (positive control):
The 3h-EC50 for 3,5-dichlorophenol to activated sludge was estimated under the conditions of the test as follows: 17.0 mg/L for total respiration. The test with 1,3-dichlorophenol met the validity criteria of the guideline:

- The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: the maximum was determined to be 1.3%
- The 3h-EC50 for 3,5-dichlorophenol (17.0 mg/L) was between 2 and 25 mg/L for total respiration
- The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: 34.72 and 35.36 mg O2/g of activated sludge (dry weight of suspended solids)/h
Reported statistics and error estimates:
LOEC and NOEC for respiration inhibition were calculated using appropriate statistical methods with the software ToxRatPro Version 3.1.0®. ECx-values and their associated lower and upper 95% confidence limits could not be determined because the test item actually had no effects.

Total respiration was inhibited at each tested concentrations as described below:

 

Test item nominal concentration (mg/L)

Inhibition of total respiration (%)

1.0

6.8

3.2

1.9

10.0

4.5

32.0

3.3

100.0

8.6

 

Validity criteria fulfilled:
yes
Conclusions:
1,3-dibutylthiourea did not cause any significant respiratory inhibition up to the highest tested concentration. The NOEC is therefore considered superior or equal to 100 mg/L.
Executive summary:

This GLP OECD 209 study was carried out in order to determine the 3h-EC50 and 3h-NOEC of 1,3 -dibutythiourea on total respiration inhibition of activated sludge. The inoculum was obtained on 31 August 2015 from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64) which handles predominantly domestic sewage. The sludge was prepared so as to obtain a 10 -L stock solution at a concentration of 3g/L of suspended solids (dry weight) diluted in Volvic water medium supplemented with a synthetic sewage feed.

At the start of the test, 16 mL of synthetic sewage feed were made up to 250 mL with ultra-pure water and 250 mL activated sludge inoculum with a sludge concentration of nominally 1.5 g/L (dry weight) were added to the first control vessels couple. Thereafter, at time intervals of about fifteen minutes, the procedure was repeated with test vessels couples in which the 16 mL of synthetic sewage were made up to 250 mL with the appropriate amounts of reference or test item diluted in ultra-pure water to obtain a volume of 250 mL. As for the controls, 250 mL of activated sludge inoculum (1.5 g/L) were then added. Fifteen minutes after the preparation of the last treatment, the second control vessels couple was prepared at last. The fifteen minutes interval was done so that the 10 min incubation period with O2 depletion monitoring could take place exactly 3 hours after start of incubation of each test vessel. The addition of the inoculum was considered as the start of the 3-hours incubation time period. All solutions were incubated at 20 +/- 2°C in darkness for 3 hours in a temperature controlled room. Solutions were mixed by stirring and aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during 3 hours. At the end of the test incubation period, a well-mixed sample of each solution (following their order of preparation) were poured into a BOD bottle in which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min or until the oxygen concentration felt below 1 mg/L. The temperature was monitored throughout the test period. 1,3-dibutylthiourea did not cause any significant respiratory inhibition up to the highest tested concentration. The NOEC is therefore considered superior or equal to 100 mg/L. The 3h-EC50 for 3,5-dichlorophenol to activated sludge was 17.0 mg/L for total respiration. The test met all the validity criteria of the guideline.

Description of key information

This GLP OECD 209 study was carried out in order to determine the 3h-EC50 and 3h-NOEC of 1,3 -dibutythiourea on total respiration inhibition of activated sludge. The inoculum was obtained on 31 August 2015 from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64) which handles predominantly domestic sewage. The sludge was prepared so as to obtain a 10 -L stock solution at a concentration of 3g/L of suspended solids (dry weight) diluted in Volvic water medium supplemented with a synthetic sewage feed.

At the start of the test, 16 mL of synthetic sewage feed were made up to 250 mL with ultra-pure water and 250 mL activated sludge inoculum with a sludge concentration of nominally 1.5 g/L (dry weight) were added to the first control vessels couple. Thereafter, at time intervals of about fifteen minutes, the procedure was repeated with test vessels couples in which the 16 mL of synthetic sewage were made up to 250 mL with the appropriate amounts of reference or test item diluted in ultra-pure water to obtain a volume of 250 mL. As for the controls, 250 mL of activated sludge inoculum (1.5 g/L) were then added. Fifteen minutes after the preparation of the last treatment, the second control vessels couple was prepared at last. The fifteen minutes interval was done so that the 10 min incubation period with O2 depletion monitoring could take place exactly 3 hours after start of incubation of each test vessel. The addition of the inoculum was considered as the start of the 3-hours incubation time period. All solutions were incubated at 20 +/- 2°C in darkness for 3 hours in a temperature controlled room. Solutions were mixed by stirring and aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during 3 hours. At the end of the test incubation period, a well-mixed sample of each solution (following their order of preparation) were poured into a BOD bottle in which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min or until the oxygen concentration felt below 1 mg/L. The temperature was monitored throughout the test period. 1,3-dibutylthiourea did not cause any significant respiratory inhibition up to the highest tested concentration. The NOEC is therefore considered superior or equal to 100 mg/L. The 3h-EC50 for 3,5-dichlorophenol to activated sludge was 17.0 mg/L for total respiration. The test met all the validity criteria of the guideline.

Key value for chemical safety assessment

EC10, LC10 or NOEC for microorganisms:
100 mg/L

Additional information