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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Aug 1998
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
according to OECD guideline 1984. Slightly difference from the OECD 209 (2010) regarding replicate numbers and no indication of O2 saturation%.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
The test substance was directly weighed into the test flasks. Then, the test flasks were filled each with 284 mL tap water.
One test solution with a final volume of 500 mL was tested per test concentration in 1 litre glass flasks.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
50 mL synthetic sewage feed per litre was added daily, starting two days prior to use, and the sludge was kept at room temperature under continuous aeration until use.
Just before the start of the test the dry weight was again measured and adjusted to 2 g dry weight per litre. The pH of the activated sludge was determined to be pH 8.0.
Test type:
Water media type:
other: Tap water
Limit test:
Total exposure duration:
3 h
Test temperature:
20 °C
7.6 - 8.3
Dissolved oxygen:
at least 2.5 mg/L during incubation period and at least 7.1 mg/L before measurements
Nominal and measured concentrations:
Nominal concentrations: 10, 32, 100, 320 and 1000 mg /L
Details on test conditions:
Test flasks of 1 litre volume and Karlsruher flasks of 250 mL volume were used.
Samples were prepared in about 15-minute intervals. Thereafter, aeration started using compressed air at an air-flow rate of approximately 0.6 litre per minute.
Reference substance (positive control):
Key result
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
In comparison to the inoculum controls the respiration rate of the activated sludge was only slightly inhibited by 1.2 % at the lowest test concentration of nominal 10 mg/L. At the next higher test concentration inhibition rate of 27.3 % was determined. However, at the three highest test concentrations inhibition rates between -5.7 % and 9.7 % compared to the controls were determined. Thus the inhibition rate of 27.3 % at the nominal concentration of 32 mg/L can be assumed not to be related to the test substance concentration since up to the nominal concentration of 1000 mg test substance/L no or only slight inhibition of respiration rates were determined. Thus, the respiration rate was not significantly inhibited up to the highest tested concentration of 1000 mg test substance/L. Concentrations exceeding 1000 mg/L nominal were not tested.
Results with reference substance (positive control):
In comparison to the controls the respiration rate of the activated sludge was inhibited by 30 % at the lowest concentration of nominal 10 mg/L. At the nominal concentrations of 32 and 100 mg/L, the respiration rate was inhibited by 88.1 % and 87.3 %, respectively. From these inhibition rates, the 3-hour EC50 was calculated to be 15 mg/L (95 % confidence limits could not be calculated).

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

Table 1: Validity criteria for OECD 209 (2010)

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 0.27 mg O2/L/min

considering 2 g dw activated sludge/L inoculum and 200 mL/L in final solution, the lowest O2 uptake=

40.5 mg O2/g dw sludge/hour


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 substances has to lie in a specific range

3,5-DCP: 15 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 EC50 3h for activated sludge was > 1000 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:
1 000 mg/L

Additional information

One key study (1998) is available for toxicity towards microorganisms. In this study, the effects of the substance towards microorganisms were investigated 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 10, 32, 100, 320 and 1000 mg/L, alongside with a control. Based on nominal concentrations an EC50of > 1000 mg/L was determined.