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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 22nd to August 23rd, 1989
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted April 4, 1984
GLP compliance:
not specified
Details on test solutions:
The test article was dissolved in bidistilled water at a concentration of 500 mg/l.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: ARA, Sissach/Switzerland.
- Pretreatment: the sludge was coarsely sieved, centrifuged, the supernatant liquid phase decanted, the solid material resuspended in tap water and again centrifuged. This procedure was repeated a further two times.
- Sludge suspension: an aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (ml) to its dry weight (g) determined. Based on this ratio, a volume of washed sludge suspension corresponding to 4.0 g dry material were made up with Soerensen buffer (pH= 7) to 1 litre.
- Preparation of the inoculum: 50 ml OECD recommended synthetic sewage feed were added.
- Incubation: the sludge was incubated overnight at 20 - 25 °C under aeration.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
22 °C
control: 7.4
Test vessels: 7.4
Nominal and measured concentrations:
10 and 100 mg/l
Details on test conditions:
- Test vessel: glass-flasks (> 500 ml).
- Aeration: aerated with 0.2 l/minute instead of 0.5 l/minute to prevent possible foam development.
- No. of vessels per concentration: 1 replicate.
- No. of vessels per control: 2 replicates.
- Sludge amount: 200 ml

After 180 minutes of aeration at 22 °C, the first sample was taken for oxygen measurement over a period of up to 10 minutes. Thereafter, in 15 minutes intervals, the rest of the samples were submitted to measurement of oxygen concentration.
Sampling was started with the control 1. Thereafter, the measurements were continued with the positive controls and the test article samples. Finally, the oxygen consumption of control 2 was determined.

Oxygen consumption was measured over a period of 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The apparatus used for oxygen measurements was the WTW OXI 91 (Wissenschaftlich-Technische Werkstaetten, Weilheim/Federal Republic of Germany).

The tests are validated by testing a known standard, i.e. 3,5-dichlorophenol. The IC50-value should be in the range of 5 - 30 mg/l. Furthermore, the variation between the two control samples (start, end) should not be greater than 15 %.
Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
Tthe respiration rate of the sludge was not inhibited (0 %) at the highest concentration of test item, i.e. 100 mg/l.
Results with reference substance (positive control):
At a concentration of the standard of 3.2 mg/l the respiration rate was inhibited by 16.5 %. The IC50 was found to be about 8.3 mg/l. Almost complete inhibition (97.1 %) was observed at 50 mg/l.

Influence of test item, reference substance and control on oxygen consumption of activated sludge

Oxygen consumption (mg O2/l min.) Inhibition (%) Starting pH
Control 1.04 - 7.4
Control 1.02 - 7.4


1.03± 0.01 (2.0 %) -
Test item 10 mg/l 1.03 0 7.4
Test item 100 mg/l 1.1 -6.8 7.4
Reference substance 50 mg/l 0.03 97.1 7.5
Reference substance 10 mg/l 0.35 66 7.5
Reference substance 3.2 mg/l 0.86 16.5 7.6
Validity criteria fulfilled:
the variance between the control samples was 1.9 %. Furthermore, the inhibitory effect of 3,5-dichlorophenol was within the range of the IC50-value of 5 to 30 mg/l.
IC50 (3h) > 100 mg/l
NOEC (3h) ≥ 100 mg/l
Executive summary:

The inhibitory effect of test substance on aerobic wastewater bacteria was investigated in a respiration test, according to the OECD guideline 209.

The test article showed no inhibition (0 %) of the respiration rate at a concentration of 100 mg/l; therefore, the IC50 was determined to be higher than 100 mg/l.

In conclusion, was found to be non-toxic to waste-water bacteria.

The IC50 for dichlorophenol tested under the same conditions was about 8.3 mg/l.


IC50 (3h) > 100 mg/l

NOEC (3h) ≥ 100 mg/l

Description of key information

IC50 (3h) > 100 mg/l

NOEC (3h) ≥ 100 mg/l

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

The inhibitory effect of test substance on aerobic wastewater bacteria was investigated in a respiration test, according to the OECD guideline 209. The test article showed no inhibition (0 %) of the respiration rate at a concentration of 100 mg/l; therefore, the substance was found to be non-toxic to waste-water bacteria.