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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Mar - 30 May 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
Method: physico-chemical oxygen consumption
Controls: 3.5-Dichlorophenol, Purity 97%.
Test substance concentration: 1000, 1800, 3200, 5600, 10000 mg/L
Reference compound concentration: 2.5, 5, 10, 20 and 40 mg/L
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Source: aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim)
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
20 ± 2° C
7.0 to 7.5
Dissolved oxygen:
2.7 to 4.4 mg/L
Nominal and measured concentrations:
1000; 1800; 3200; 5600; 10000 mg/L (nominal concentrations)
Reference substance (positive control):
3,5-Dichloronitrophenol, 2.5; 5; 10; 20; 40 mg/L
Key result
3 h
Dose descriptor:
Effect conc.:
> 10 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
The physico-chemical oxygen consumption control is carried out, since some substances may consume oxygen by chemical reactivity. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption(respiration), at least the maximum concentration of the test item is additionally tested without activated sludge.
Results with reference substance (positive control):
Inhibition of up to 96.7 % at 40 mg/L.

Validity criteria for the measurement of the bacterial toxicity:

Target condition according to guideline:Actual condition according to the study:Validity criteria met:
In a 2004 international ring test organized by ISO using activated sludge derived from domestic sewage, the EC50 of 3,5-dichlorphenol was found to lie in the range 2 mg/L to 25 mg/L for total respiration. If the EC50 of 3,5-DCP does not lie in the expected range, the test should be repeated with activated sludge from another source. The EC50 of the reference substance 3.5-dichlorophenol is in the range 5 - 30 mg/L.Yes
The test should be performed at a temperature within the range 20 ± 2°C.The test was performed between 20 ± 2°C.Yes
The specific oxygen consumption of the controls shall be reported.The oxygen consumption of the controls is stated.Yes
Two control respiration rates are within 15% of each other.The respiratory rates of the 2 controls differ less than 15 %.Yes


Validity criteria fulfilled:
See 'Any other information on results incl. tables'.
The test substance showed 45.8 % respiration inhibition of activated sludge at a test item concentration of 10000 mg/l.
Executive summary:

A study was performed to assess the toxicity of 4-Methoxycyclohexanone to bacteria.

The study was in accordance with the Commission Directive 88/302/EEC; Official Journal of the EGL133PartC.ll (1988).

The activated sludge was exposed to 4-Methoxycyclohexanone at different concentrations.

The respiration rate of each mixture was determined after aeration periods of 3 hours.

4-Methoxycyclohexanone showed 45.8 % respiration inhibition of activated sludge at a test item concentration of 10000 mg/L.


The EC50 is higher than 10000 mg/L.

Description of key information

The test substance showed 45.8 % respiration inhibition of activated sludge at a test item concentration of 10000 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
10 000 mg/L

Additional information