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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-04-21 to 1999-05-7
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffcat ZR-50
- Chemical name: 2-propanol, 1-(bis(3-(dimethylamino)propyl)amino)-
- Physical state: liquid
- Analytical purity: 100%
- Lot/batch No.: 184-3-0998
- Expiration date of the lot/batch: not provided
- Storage conditions: room temperature in the dark and dry
Analytical monitoring:
Details on test solutions:
- Nominal test concentrations were established in the test mixtures by the addition of appropriate volumes of a stock solution of 0.5 g/L.
- pH of stock solution was measured and adjusted to approximate neutrality before use.
- Test solutions were prepared by additions of dechlorinated tap water, synthetic sewage and microbial inoculum.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source of activated sludge: Oakley Sewage Treatment Works (treats predominantly domestic waste), sample taken the day before the start of the test
- Samples were maintained under aerobic conditions in the lab until the start of testing. Synthetic sewage (50 mL/L) was added and the mixture was aerated overnight.
- MLSS was determined on the day of collection and immediately before the start of the test.
- On the day of the test the MLSS was adjusted to 4 g/L by addition of dechlorinated tap water.
- Initial SS concentration in the test: 1.6 g/L.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
10 min for oxygen consumption rate measurements
190 mg/L (outside range specified in protocol (200-250 mg CaCO3/L), however, this was not considered to affect the results of the test)
Test temperature:
19-21 degrees C
Dissolved oxygen:
Nominal and measured concentrations:
Nominal: Control, 1.0, 10 and 100 mg/L
Details on test conditions:
- Test vessel: flask
- Material, size, headspace, fill volume: glass, 500 mL fill volume
- Aeration: yes, during 3 h, using a Pasteur pipette connected to a laboratory supply of oil-free compressed air
- No. of vessels per concentration (replicates): 1 each of 1 and 10 mg/L, 3 of 100 mg/L
- No. of vessels per control (replicates): 1
- After 3 h of exposure, samples were transferred to BOD bottles (capacity 270 mL) and the oxygen consumption rate was measured during 10 min using a Yellow Springs Instruments DO meter, with temperature probe and self-stirring bottle probe, connected to a chart recorder.

- Source/preparation of dilution water: dechlorinated tap water
- Hardness of dilution water: 190 mg CaCO3/L
- Dilution water for preparation of synthetic sewage was softened tap water treated by reverse osmosis and then purified to give resistivity of 18 Megohm/cm
- pH and temperature were measured at the start and end of the test.

- Adjustment of pH: to neutral

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): oxygen consumption rate over 10 min after 3 h of exposure

- Spacing factor for test concentrations: 10
Reference substance (positive control):
Key result
3 h
Dose descriptor:
Effect conc.:
> 100 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.:
>= 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: none
- Effect concentrations exceeding solubility of substance in test medium: none
- Specific respiration rate of control at the end of the test was 106% of the rate established at the start (32.8 mg O2/g/h vs 31.1 mg O2/g/h), showing that the activated sludge was viable.
- Variation in respiration rate in control vials was < 15% (validity criterium).
- No significant differences were observed between exposed samples and control samples.
Results with reference substance (positive control):
3-h EC50 = 10.5 mg/L (95% CI = 8.3-13.5 mg/L), which shows that the activated sludge employed was sensitive to inhibition (acceptable range 5-30 mg/L).
Reported statistics and error estimates:
The 3-h EC50 for DCP and its confidence limits were calculated by the Moving Average Method.
Validity criteria fulfilled:
The test substance had no significant inhibitory effect on the respiration rate of the activated sludge at any of the concentrations employed in the test. The 3-h EC50 of the test substance could therefore not be calculated and must be > 100 mg/L, the highest level tested. The 3-h NOEC is consequently >= 100 mg/L. The results of the study can be considered reliable.

Description of key information

An activated sludge inhibitation test (Burwood & Dickinson, 1999) was carried out according to the OECD guideline 209 and indiated that the substance had no significant inhibitory effect on the respiration rate of the activated sludge at any of the concentrations employed in the test. The resulting 3h-EC50 was >100 mg/L and the 3h-NOEC was >=100 mg/L (based on nominal concentrations). The study is given a Klimisch score of 1, was conducted under GLP and is considered as the key study for endpoint coverage.

Key value for chemical safety assessment

Additional information