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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-09-09 to 1997-09-12
1 (reliable without restriction)
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
equivalent or similar to guideline
other: EC Directive 87/302, 'Biodegradation - 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 ZF-10
- Physica state: Clear liquid
- Lot/batch No.: 186-2-05/95
Analytical monitoring:
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance, synthetic sewage, water, and inoculum
- Controls: Yes (Syntheic sewage, water, and inoculum)
Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: A sample of activated sludge was obtained a day before the start of the test from Oakley sewage treatment works, a sewage plant treating predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: Aliquots (25 mL) of the activated sludge were filtered through dried and preweighed Whatman's GFC filter papers which were then dried at 110 °C for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added and the mixture aerated overnight. MLSS content was determined and adjusted to 4 g/L by the addition of dechlorinated tap water.
- Pretreatment: The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
Test type:
other: aerobic
Water media type:
Limit test:
Total exposure duration:
3 h
Test temperature:
18.7 - 20.0 °C (preliminary test)
18.7 - 20.4 °C (final test)
7.3 - 7.9 (preliminary test)
7.3 - 8.1 (final test)
Nominal and measured concentrations:
Preliminary test : 1, 10, 100 mg/L (nominal)
Final limit test with triplicate vessel containing test substance at a nominal concentration of 100 mg/L
Details on test conditions:
Mixtures were placed into one-litre glass beakers and aerated using a Pasteur pipette connected to a laboratory supply of oil-free compressed air for three hours. Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (capacity; 270 mL) and its rate of oxygen consumption over a period of approximately ten minutes was measured using a Yellow Springs Instruments (YSI) dissolved oxygen meter, with temperature probe and self-stirring bottle probe, connected to a chart recorder. The pH and temperature of the samples were measured at the start and end of the test.
Reference substance (positive control):
3.5-dichlorophenol (3.0, 10.0 and 32.0 mg/L)
Key result
3 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
In the preliminary test, the respiration rate of the sludge was decreased by 2% at a nominal concentration of 10 mg/L. In the final limit test, the respiration rates of sludges in three replicates at a nominal concentration of 100 mg/L were increased by 3, 7 and 11%. These effects are not thought to be biologically significant.
Results with reference substance (positive control):
Results show that the tests were valid and that the samples of activated sludge employed were sensitive to inhibition.
Validity criteria fulfilled:
The three-hour EC50s for 3,5-dichlorophenol fulfilled the validity criterion relating to sensitivity to inhibition.
The test substance had no significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the tests, including at a concentration of 100 mg/L which was the highest concentration tested.

Description of key information

The Activated sludge, respiration inhibition test of test substance was conducted according to OECD guideline 209 and GLP (Huntsman 1997).Test substance had no significant inhibitory effect on the respiration rate of activated sludge at any of the concentrations employed in the tests, including at a concentration of 100 mg/L which was the highest concentration tested. IC50 (3h) was determined was greater than 100 mg a.i /L.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information