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Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLP study

Data source

Reference Type:
study report

Materials and methods

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

Test material

Details on test material:
Batch number: 98 043 300
Appearence: white powder
Packaging: plastic bag
Quantity received and date: approximately 750 g, received on April 2nd, 1998
Storage: in darkness and at room temperature
Identification for the study: 98021

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
a stock solution (500 mg of the test substance per litre) was prepared 52 minutes before the start of the exposure, dissolving the test substance into dilution water; pH of this stock solution was adjusted to make the test substance soluble in our experimental conditions at the request of the study monitor.

The test substance concentrations in the test media were obtained adding the appropriate volumes of the stock solution in the respective test flasks.
-for each flask, a mixture of dilution water, synthetic sewage (16mL) and inoculum (200mL), was added at 12-minutes intervals, to the substance (expected for the control flasks, without any substance), to form a final volume of 500 mL per flask.
-pH was adjusted in the test media around pH 7.5-8.0 to make the test substance soluble in our experimental condtions at the request of the study monitor.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
mixture of different strains of micro-organisms (inoculum) found in activated sludge from a sewage treatment plant treating predominantly domestic sewage (La Fouillous - Loire - FRANCE
The sludge was used on the day of collection
the sludge was mixed during 1 minute in order to destroy the coarse particles
the concentration of suspended solids was evaluated from a small amount of sludge
the sludge was centrifugated and the suspended solids obtained were washed before to be resuspended for the preparation of the inoculum (centrifugated 3 times with deminineralized water containing synthetic sewage for 5 minutes at 2000 rpm, with discarding of the supernatants)
the suspended solids were then re-suspended into synthetic sewage so as to obtain a nominal concentration of suspended solids of approximately 2 grammes per ltre in the inoculum
this inoculum was kept aerated and at thermoregulated temperature (20 +/- 2°C) until its using for the preparation of the media

Study design

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

Test conditions

Test temperature:
from 7.49 to 7.97
Nominal and measured concentrations:
1 / 10 / 100 mg/L
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:
growth inhibition
Results with reference substance (positive control):
ihnibition percentage after 3 hours:
26% at 5 mg/L
74% at 15 mg/L
77% at 30 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
EC50-3 hours > 100mg/L
Executive summary:

Inhibition of respiration in activated sludge, in compliance with OECD guideline n°209 and EEc directive 87/302


-micro-organisms from activated sludge (800 mg of suspended solids per litre of medium) were exposed to a range of concentrations of test substance for 3 hours; 1, 10 , 100mg test substance per litre of test medium

-two contro media (without test substance) were tested, one at the beginning of the test, the other one at the end of the test

-the dissolved oxygen concentration was measured in 300 mL samples from each after approximately 3 hours exposure

-a reference substance, 3,5 -dichlorophenol, was tested under the same conditions in order to control the sensitivity of the activated sludge (3 concentrations)

-the criteria of quality for the study, as notified in the OECD and EEC standards and relative to the level of oxygen consumption in the two control media and to the toxicity of 3,5 -dichlorophenol, were respected


Taking into account the results obtained under the experimental conditions employed, these ones may be expressed as follows:

EC50 -3 hours higher than 100 mg/L