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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Jan - 01 Feb, 2001
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
according to guideline
other: Commission Directive 88/302/EEC; Official Journal of the EC L 133 Part C
Version / remarks:
Commission Directive 88/302/EEC; Official Journal of the EC L 133 Part C (1988)
sludge concentration, number of replicates, pH.
GLP compliance:
Analytical monitoring:
Details on sampling:
not applicable
Details on test solutions:
The test substance has been added to about 130 mL deionized water and stirred overnight before testing (equilibration phase).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Type: mixed population of aquatic microorganisms.
Origin: aeration tank of a waste water treatment plant treating predominantly domestic sewage.
Because of strong respiration of the activated sludge only 0.36 g/L suspended solid (ss) were used.
Pre-treatment: aeration of the activated sludge; daily feed with synthetic medium
pH of the suspension before application 6.9
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Not reported
Test temperature:
19.5 - 19.9°C
7.4 - 8.3
Dissolved oxygen:
5.7 - 6.5 mg O2/L
Not applicable
Not reported
Nominal and measured concentrations:
Nominal concentrations: 1000, 1800, 3200 5600 and 10000 mg/L
Details on test conditions:
The respiration rate of an activated sludge is measured after a contact time of 3 hours with permanent aeration.
Reference substance (positive control):
3.5-DichIorophenoI; purity of 97%
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
Details on results:
The test material showed 27.9 % respiration inhibition of activated sludge at a test substance concentration of 10000 mg/L.
Results with reference substance (positive control):
Toxicity of the reference substance 3.5-Dichlorophenol to bacteria: EC50 = 14 mg/L
The EC values are determined by statistical methods (probit analysis)

Please refer to "overall remark/ attached background material" field for result tables.

Table 1: Validity criteria for OECD 209

Criterion from the guideline


Validity criterion fulfilled

The blank controls (without the test substance or reference substance) oxygen uptake rate should not be less than 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

Lowest consumption in control is 30 mg O2/L/hour

considering 360 mg ss activated sludge/L,

the lowest oxygen uptake per one gram of sludge (dry weight of suspended solids) /hour was about 83%.


The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30% at the end of definitive test.



EC50 of the reference substance has to lie in a specific range

3,5-DCP: 14 mg/L (total respiration)



Validity criteria fulfilled:
For further details please refer to “Any other information on results incl. tables”.
The present guideline study was conducted in compliance with GLP. Under the test conditions used, the 3-hour EC50 for activated sludge was > 10000 mg/L.

Description of key information

From the activated sludge key study, the 3-hour EC50 for microorganisms exposed to the present test material was > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
10 000 mg/L

Additional information

A GLP-study dated 2001 is available for toxicity towards microorganisms, which was retained as key study. In this study, the effects of the substance towards microorganisms were examined in a 3-h toxicity test with activated sludge under static conditions. The study was carried out according to GLP and OECD guideline 209. Activated sludge was exposed to nominal concentrations of 1000, 1800, 3200 5600 and 10000 mg/L, alongside with a control. The test material resulted in 27.9 % respiration inhibition of the activated sludge at a test substance concentration of 10000 mg/L. Based on nominal concentrations an EC50 of > 1000 mg/L was determined.