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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-04-18 to 2007-08-20
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
according to
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)
: Certificate issued by the Swiss GLP monitoring authorities.
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Using an analytical balance, 500.7 mg of the test substance was weighed and transferred to the designated test vessel with 284 mL tap water. The test substance was then dissolved by intense stirring for ten minutes at room temperature. After stirring, the appearance of the test medium was evaluated, and was found to be a clear solution. Then, 16 mL synthetic wastewater and 200 mL activated sludge inoculum were added. The inoculum had a sludge concentration of 2.4 g/L dry weight (corresponding to about 1.0 g dry material/L test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference substance, thirdly to the test solution of the test substance, and finally to the second control.
- Controls: yes, concurrent no treatment
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz 11, Fuellinsdorf, Switzerland) treating predominantly domestic wastewater.
- Laboratory culture: not applicable
- Method of cultivation: no data
- Preparation of inoculum for exposure: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
- Pretreatment: no
- Initial biomass concentration: An aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material/L. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater/L (prepared according to guidelines) and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was 6.9.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
no data
Test temperature:
Dissolved oxygen:
no data
no data
Nominal and measured concentrations:
1000 mg/L (nominal)
Details on test conditions:
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass beakers, 2000 mL, 1500 mL, 500 mL
- Aeration: During the incubation period of 3 hours, the single test medium and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test substance.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data

- Source/preparation of dilution water: tap water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH and dissolved oxygen concentrations were determined after the addition of synthetic wastewater and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic wastewater, the appearance of the test media was recorded.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- For measurement of the respiration rate, a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then, the dissolved oxygen concentration was measured with an oxygen electrode and an oxygen meter and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/min) was determined from the linear part of the respiration curve in the range 6.5-2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above, but always within the linear part of the respiration curve. In case of low oxygen consumption, the rate was determined over a period of at least ten minutes.

- Spacing factor for test concentrations: not applicable
- Justification for using less concentrations than requested by guideline: limit test
- Range finding study
- Test concentrations: not applicable
- Results used to determine the conditions for the definitive study: not applicable
Reference substance (positive control):
3,5-dichlorophenol (98.8% by gas chromatography)
3 h
Dose descriptor:
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
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
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: No. In a pre-experiment the test substance was soluble at a concentration of at least 5 g/L in tap water.
Results with reference substance (positive control):
- Results with reference substance valid?: The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming suitability of the activated sludge used.
- Relevant effect levels: The 3-hour EC50 of the reference substance 3,5-dichlorophenol was calculated to be 12 mg/L (the 95% confidence limits could not be calculated).
Reported statistics and error estimates:
- The 3-hour EC50, EC20 and EC80 values of the test substance and their 95%-confidence limits could not be calculated because of the absence of a toxic effect.
- The 3-hour EC50 of the reference item 3,5-dichiorophenol was calculated by Probit Analysis.
- The NOEC is defined as the calculated concentration at which no significant toxic effect, i.e. < 15% inhibition, is determined (i.e. EC15).

The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period. Just before measurement of the respiration rates, the dissolved oxygen concentrations were at least 8.3 mg/L. The temperature in the test media, measured in one control, was 20 deg C at the start and at the end of the incubation period.

The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 9% (guideline-recommended maximum variation: 15%).

Validity criteria fulfilled:
An activated sludge respiration inhibition test (according to OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h EC50 > 1000 mg/L. The results of the test can be consodered reliable without restriction.

Description of key information

The study of Bätscher (2007), investigating the toxicity of T002675 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h ELR50 was > 1000 mg/L. The 3-h NOELR was >= 1000 mg/L.

Key value for chemical safety assessment

Additional information