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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1989.10.19 to 1989.11.08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP test with all relevant information reported and validity criteria fulfield.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Minor deviations: instead of 3,5-dichlorophenol, 2,4,5-trichlorophenol was used as a reference compound and preliminary experiments to determine the concentration range are performed but not reported according to the guidelines.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
Minor deviations. See above.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
reported 1990.05.17
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
The test substance was added to the vessels using a stock solution of 177 mg/l. This stock solution was prepared by means of ultrasonic dispersion.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Secondary activated sludge was collected from the RZWI Nieuwgraaf in Duiven (1989.11.28). Prior to use, the activated sludge was diluted with tap water (1.5 *) and homogenized for two minutes in a lab blender 400. The activated sludge concentration in the incubation vessels was 0.6 g dry weight/litre.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Post exposure observation period:
None
Hardness:
Not specified.
Test temperature:
20° C
pH:
The pH of the reaction mixture before the respiration measurement varies from 7.5 to 7.9.
See details in Table 1 in the "Remarks on results including tables and figures" field.
Dissolved oxygen:
Measured parameter.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: two controls, 6.3, 12.5, 25, 50 and 100 mg/l.
Details on test conditions:
The homogenized activated sludge was incubated in a shaking water bath (100 hz, 20°C) for 30 mn with various concentrations of the test compound and synthetic sewage.
Reference substance (positive control):
yes
Remarks:
2, 4, 5-trichlorophenol was choosen for its structural resemblance and is expected to give the same result as with 3, 5-dichlorophenol which was not commercially available.
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 33.2-61.5 mg/l
Key result
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
None
Results with reference substance (positive control):
See Table 2 in the "Remarks on results including tables and figures"
Reported statistics and error estimates:
None

Table 1. Respiration rates of the activated sludge (Rs) and the inhibition percentage calculated at various concentrations of the test compound

Concentration (mg/l)

pH start

Respiration rate (mg O2 l/ min)

Inhibition (%)

Control (Rc1)

7.9

0.45

-

Control (Rc 2)

7.7

0.47

-

6.3

7.8

0.42

9

12.5

7.8

0.34

26

25

7.7

0.27

41

50

7.6

0.22

52

100

7.5

0.15

68

Table 2. Respiration rates of the activated sludge and inhibition percentages calculated at various concentrations of 2, 4, 5 trichlorophenol

Concentration (mg/l)

pH start

Respiration rate (mg O2 l/ min)

Inhibition (%)

Control

7.9

0.43

-

Control

7.9

0.46

-

5

7.8

0.39

12

10

7.7

0.24

46

20

7.7

0.17

62

40

7.8

0.08

82

Validity criteria fulfilled:
yes
Remarks:
The validity of the test is shown by two important criteria. Firstly, the control respiration rates are within 9% of each other and secondly, the EC50 of the reference compound is within the described range (5-30 mg/l).
Conclusions:
The EC50 of tert-butyl-peroxybenzoate for activated sludge is 43 mg/l with 95% confidence limits of 33.2 and 61.5 mg/l. The EC10, EC20 and EC80 are 6, 11 and 167 mg/l, respectively.
Executive summary:

In order to predict effects of tert-butyl-peroxybenzoate in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed according to the OECD 209 and the EEC Method C.11 Guidelines . The test is reliable including an GLP certificate with relevant informations reported and all validity criterias fulfilled. The homogenized activated sludge (at 0.6 g dry weight /L; collected from the RZWI Nieuwgraaf in Duiven) was incubated for 30 min with various concentrations of the test compound (two controls, 6.3, 12.5, 25, 50 and 100 mg tert-butyl-peroxybenzoate/L) and synthetic sewage. Prior to use, the activated sludge was diluted with tap water and homogenized for two minutes in a lab blender 400. The temperature was 20°C and the pH ranged from 7.5 to 7.9. A reference substance was used during the test : 2,4,5 -trichlorophenol at 5, 10, 20 and 40 mg/L.

The EC50 of tert-butyl-peroxybenzoate for activated sludge is 43 mg/l with 95% confidence limits of 33.2 and 61.5 mg/l. The EC10, EC20 and EC80 are 6, 11 and 167 mg/l, respectively.

Description of key information

3h-EC50 based on the Activated Sludge Respiration Inhibition test (OECD 209) is 43 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
43 mg/L
EC10 or NOEC for microorganisms:
6 mg/L

Additional information

In order to predict effects of tert-butyl-peroxybenzoate in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed according to the OECD 209 and the EEC Method C.11 Guidelines. The test is reliable including an GLP certificate with relevant informations reported and all validity criteria fulfilled.

The EC50 of tert-butyl-peroxybenzoate for activated sludge is 43 mg/L with 95% confidence limits of 33.2 and 61.5 mg/L. The EC10, EC20 and EC80 are 6, 11 and 167 mg/L, respectively.