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Administrative data

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

Data source

Reference Type:
study report

Materials and methods

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

Test material

Test material form:
liquid: viscous
Specific details on test material used for the study:
Batch/Lot number: 0002293298
Appearance: High-viscous, colourless to yellowish liquid
Purity: Considered as 100%
Expiry date: 22 November 2019
Storage condition: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials will be applied to assure personnel health and safety.

Test solutions

Details on test solutions:
The stock formulation (3200 mg/L) and the test vessels will be freshly prepared at the beginning of the experiment, in the testing laboratory.

Preparations of the test flasks:
One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) will be prepared per treatment in a glass flask.
A volume of 12.8 mL synthetic sewage and an adequate amount of test item will be filled up with deionised water before the start of the incubation. At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3 g per litre of suspended solids will be added to all flasks (except the abiotic controls). The activated sludge concentration in all the test, reference and blank (but not abiotic control) mixtures will nominally be 1.5 g/L of suspended solids.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
Species: Activated sludge, microorganisms from a domestic waste water treatment plant.

Source: The activated sludge will be supplied by the sewage plant for domestic sewage in Veszprém, Hungary

Conditioning: The activated sludge will be supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge will be fed daily with 50 mL synthetic sewage (see section 3.7.3.) per litre and kept aerated at 20 ± 2°C until use.
The activated sludge used for this study will be washed by centrifugation and the supernatant liquid phase will be decanted. The solid material will be re-suspended in chlorine-free tap water and again centrifuged. (This procedure will be repeated if appropriate.)
Before use, the dry weight of the activated sludge will be measured. An aliquot of the final sludge suspension will be weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amounts of wet sludge will be suspended in chlorine-free tap water to yield a concentration equivalent to about 2 - 4 g per litre (on dry weight basis).
The pH of the activated sludge inoculum will be checked and if necessary adjusted to pH 7 – 8 with diluted sulphuric acid or sodium bicarbonate solution.

Study design

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

Test conditions

Details on test conditions:
Surrounding type: Controlled environment room (during the formulation, incubation and during the oxygen measuring) was used.
Temperature: 20 +/- 2°C
Aeration: With compressed air (0.5 to 2 litre per minute)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.
pH of the test mixtures: The pH of the test mixtures were checked at the beginning and at the end of the incubation period. If necessary, solutions/suspensions of the test item in water will be neutralised prior to addition of inoculum.
Reference substance (positive control):

Results and discussion

Effect concentrations
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
Remarks on result:
other: no inhibition effect

Applicant's summary and conclusion

According to OECD 209, the test item had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L (nominal), no further testing is necessary.