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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 13 to March 02, 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test planned. Results available for March 2010. To be completed later on.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Batch number/Supplier: A1WX67010101

Main characteristics:
appearance: Colourless liquid
- purity: 99.79%
- water solubility: Completely soluble at 20 °C according to SDS
- relative density at 20°C: 0.716

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
Range finding test
A 0.5 g/L stock solution of the test item was prepared with ultra-pure water and diluted in order to obtain concentrations of 1, 10 and 100 mg/L.

Definitive test
A 0.5 g/L stock solution of DIISOPROPYLAMINE was prepared with ultra-pure water and diluted in order to obtain a concentration of 100mg/L.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
The inoculum comes from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64). The effluent, obtained 2 days before seeding the flasks, is centrifuged at approximately 20 °C for 20 min at 4000 g; the base is re-suspended in ultra-pure water in order to keep the concentration unchanged with respect to the sample. The amount of dry material in the inoculum, is determined by dessiccation at 105 °C until a constant weight. From this result the amount of wet sludge can be calculated in order to obtain an activated sludge with a mixed liquor suspended solids level of 2 g/L (+/-10%). The inoculum is preconditioned (aeration). Every day, 50ml/L of synthetic sewage feed are added until the day the flasks are inoculated.

Value of suspended level was 8.44g/L. Then a dilution was used for obtain 2 g/L This level corresponds to a concentration of 0.8 g/L in the test medium.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h

Test conditions

Reference substance (positive control):

Results and discussion

Effect concentrations
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
Results with reference substance (positive control):
The sensitivity of the test system and the methodology is evaluated with 3,5-dichlorophenol. An EC50-3h value of 7.3 mg/L was obtained, which is within the validity specified range from 5 to 30 mg/L.

Any other information on results incl. tables

The respiration rate is calculated from the recorder trace as mg O2/L.h between approximately 6.5 mg O2/L and 2.5 mg O2/L or over a time period of 10 min. when the respiration rate is low. The portion of the respiration curve over which the respiration rate is measured should be linear.

The inhibition effect ofthe test itemat each concentration is expressed as percentage of mean of the two control respiration rates:

% inhibition = 1- (2Rs / (Rc1 + Rc2))*100

Rs: oxygen consumption rate at the tested concentration of test substance

Rc1: oxygen consumption rate for control 1

Rc2: oxygen consumption rate for control 2

Applicant's summary and conclusion

Validity criteria fulfilled:
The following performance criteria have been met:
The difference in respiration between the two controls was 0.6 mg O2/L/h which is below 15%.
The EC50 of 3,5-dichlorophenol was 7.3 mg/L that is between 5 and 30 mg/L.

The test fulfilled the validity criteria according to OECD guideline N°209.

The test item showed only a slight effect on the respiration of activated sludge in the conditions of the test. EC50 – 3h is higher than 100 mg/L.
Executive summary:

The purpose of this study was to determine the effect of the test item on bacteria according to the OECD guideline 209 “Activated Sludge, Respiration Inhibition Test”.

The effect is assessed by measuring the micro-organisms respiration rate on a sample of activated sludge during an incubation period of 3 hours.

The method was applied with respect to the following criteria:

The difference between the 2 controls was below 15 %;

EC50of the control test with the reference item 3,5-dichlorophenol was between 5 and 30 mg/L (7.1 mg/L);

DIISOPROPYLAMINEshowed only a slight effect on the respiration of activated sludge in the conditions of the test.EC50– 3his higher than 100 mg/L.