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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
The study was conducted according to the test guidelines in effect at the time of study conduct.
GLP compliance:
Analytical monitoring:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance was dispersed in test water (nominal concentration 20 mg/L) by stirring overnight with the aid of a magnetic stirrer at 30ºC. The preparation was cooled to test temperature (22ºC) before use to give the stock solution.
- Controls: Aged, dechlorinated tap water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: A synthetic sewage was prepared by dissolving the following ingredients per litre of dilution water:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
- Method of cultivation:
- Preparation of inoculum for exposure: At time "0", 16 ml of synthetic sewage was made up to 300 mL with water in a 1 litre glass beaker. 200 mL inoculum were added and the mixture aerated at approximately 0.5-1 liter per minute with compressed air via an airstone.
- Pretreatment: none
- Initial biomass concentration: not reported
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Dilution water hardness was approximately 120-180 mg/L as CaCO3
Test temperature:
Activated sewage sludge pH was 8.3 and the dilution water pH was 7.5; no adjustment of pH was done.
Dissolved oxygen:
2.5-6.5 mg O2/L
not reported
Nominal and measured concentrations:
Nominal concentration: 11 mg/L, the highest concentration attainable under the conditions of the study
Details on test conditions:
- Test vessel: 1 litre glass beaker
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass, 1 litre, headspace 500 mL, fill volume 500 mL
- Aeration: 0.5-1 litre per minute with compressed air via an airstone
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

- Source/preparation of dilution water: Laboratory tap water, dechlorinated by passage through activated carbon and sofetned by reverse osmosis. Water parameters for the lab are not given in this report, but are available in other reports from approximately the same time frame.

- Adjustment of pH: not adjusted
- Photoperiod: 3 h exposure under normal laboratory lighting
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption rates for 3 hours

- Spacing factor for test concentrations: not applicable; only one concentration was used
- Justification for using less concentrations than requested by guideline: The highest concentration that could be achieved was used
Reference substance (positive control):
3,5-Dichlorophenol (purity 97%); nominal concentrations 3.2, 10, and 32 mg/L
3 h
Dose descriptor:
Effect conc.:
> 11 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
3 h
Dose descriptor:
Effect conc.:
> 11 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: not reported
- Effect concentrations exceeding solubility of substance in test medium: not reported
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: At 3.2 mg/L, the O2 consumption rate was 0.843 mgO2/L/min and inhibition was 2%. At 10 mg/L, the O2 consumption rate was 0.738 mgO2/L/min and inhibition was 14%. At 32 mg/L, the O2 consumption rate was 0.355 mgO2/L/min and inhibition was 59%.
Reported statistics and error estimates:
not reported
Validity criteria fulfilled:
The EC50 ( respiration inhibition) value for the test substance with activated sewage sludge has been determined as >11 mg/L for the 3 hour contact time.
Executive summary:

A study was performed to assess the inhibitory effect of the test substance on the respiration of activated sewage sludge. The study was conducted in accordance with the OECD Guideline for Testing of Chemicals No. 209, "Activated Sludge, Respiration Inhibition Test". Cultures of activated sewage sludge were incubated with synthetic sewage under vigorous aeration and in the presence of the test substance. Aeration was interrupted after 3 hours and the rates of respiration measured electrochemically for each culture. Percent inhibition of respiration was calculated for each culture after a 3 hour contact period by comparing oxygen depletion rates for the test substance with those for the negative control culture. A positive control, 3,5-dichlorophenol, was tested concurrently with the test substance in order to demonstrate the satisfactory performance of the procedure. The EC50 (respiration inhibition) value for the test substance has been determined as >11 mg/L for the 3 hour contact time.

Description of key information

3-hour EC10 >11 mg/L
3-hour EC50 > 11 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
11 mg/L
EC10 or NOEC for microorganisms:
11 mg/L

Additional information