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Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-10-18 to 1994-10-28
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP according to OECD guideline 209 (adopted 1984). Test duration was 30 minutes, however, for test items which do not rapidly degrade the test duration should be 3 h according to OECD guideline 209 adopted 2010.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 4 April 1984
according to guideline
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
adopted 30 May 1988
GLP compliance:
yes (incl. QA statement)

Test material

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test article was directly weighed into the test flasks and slurried with small portions of bidistilled water under stirring until a volume of about 200 mL was reached. These mixtures were treated in an ultrasonic bath for 15 minutes before adding water and 16 ml of synthetic sewage feed to reach a volume of 300 ml. Finally, 200 mL of microbial inoculum (activated sludge) was added.
- Controls: three controls
- Chemical name of vehicle: no vehicle used
- Evidence of undissolved material: The test article remained undissolved down to the lowest concentration of 10 mg/L.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Source of sludge: aerobic activated sludge from a domestic waste water treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland)
- Preparation of inoculum for exposure: The sludge was centrifuged and the supernatant liquid phase decanted. The solid material was resuspended in tap water and again centrifuged. The latter procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined. Based on this ratio, a volume of washed sludge suspension, corresponding to 4 g dry material, was made up to one litre with Soerensen buffer (pH 7). To this mixture, 50 ml OECD recommended synthetic sewage feed was added and the sludge was aerated at room temperature until use. Before use, the pH was measured to be 7.9.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
30 min
Post exposure observation period:
No data.

Test conditions

Test temperature:
Starting pH 7.9
Nominal and measured concentrations:
Nominal test concentrations: 0, 10, 20, 32, 50 and 100 mg/L
Details on test conditions:
- Test vessel: 5 flasks with the test substance, 5 flasks with the reference compound and three control flasks
- Material, size, headspace, fill volume: > 500 mL glass flasks filled up to 500 mL
- Aeration: 30 min at 22 °C; flasks aerated with an air-flow about 0.2 L/min
- No. of vessels per concentration (replicates): one
- No. of vessels per control (replicates): three
- No. of vessels per vehicle control (replicates): none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Oxygen consumption was measured over a period of about 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 0X1 91 (Wissenschaftlich-Technische Werkstaetten, Weilheim/Germany). The pH was determined with the pH 90 (WTW Weilheim/Germany).
Reference substance (positive control):

Results and discussion

Effect concentrations
30 min
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance remained undissolved down to the lowest concentration of 10 mg/L.

- The variance between the control samples measured before and after the reference compound was < 15 % (i.e. 1 %).
Results with reference substance (positive control):
- Results with reference substance valid? Yes:
- Relevant effect levels: At the two lowest concentrations of 1.0 and 3.2 mg/l, the respiration was inhibited by 4.6 and 7.8 %, respectively. At 10 and 32 mg/l, the respiration was inhibited by 25.5 and 74.2 %, respectively. At 50 mg/l, the respiration rate was inhibited by 80.0 %. The EC50 determined by linear regression was calculated to be 15.1 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:
The inhibition of the degradation activity of activated sludge is not anticipated when introduced in appropriate low concentrations.