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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 February 2016 - 04 February 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
according to
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Storage condition: Controlled room temperature (15-25°C, below 70 RH%)
Analytical monitoring:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because of the low solubility, the test item was added by directly weighing the test item into the bottles.

- Controls: Abiotic Control, untreated control, and reference control
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Veszprém, Hungary

- Preparation of inoculum for exposure: The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.43. The activated sludge was used directly after conditioning.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Remarks on exposure duration:
Post exposure observation period:
not specified
Test temperature:
20.8 - 21.4°C
7.31 - 8.29
Dissolved oxygen:
A well-mixed sample of each treatment was poured into a BOD flask after about 3 hours incubation time, and was not further aerated. The oxygen concentration was measured for about fifteen minutes, with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.
not specified
not specified
Nominal and measured concentrations:
Test groups (nominal concentration): 10, 100, and 1000 mg/L
Abiotic Control: 1000 mg/L nominal concentration
Reference item: 1.0, 3.2 and 10.0 mg/L
Details on test conditions:
- Test vessel: glass flask
- Material, size, headspace, fill volume: glass beakers for 500 mL volume and BOD bottles with 300 mL volume
- Aeration: With compressed air (about 2 L/min)
- No. of vessels per control (replicates): 5
- No. of vessels per vehicle control (replicates): 5
- No. of vessels per abiotic control (replicates): 5
- Sludge concentration (weight of dry solids per volume): 3 g per litre
- Nutrients provided for bacteria: daily with 50 mL synthetic sewage

Reference substance (positive control):
3,5-Dichlorophenol (3,5-DCP)
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
Details on results:
In comparison to the inoculum controls the inhibition of the respiration rate in the case of the activated sludge was between 7.10 % and 40.13 % in the examined range of 10 – 1000 mg/L of B331 test item.
The average specific respiration rate (RS) of the untreated (Blank) controls (B1-B5) was 35.05 mg O2/L/h/g solid.
The variation coefficient of oxygen uptake rate in above untreated replicates was 6.41 %.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 12.51 mg/L
Reported statistics and error estimates:
not specified
Validity criteria fulfilled:
not specified
Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of B331 was the following:
The 3-hour EC50: > 1000 mg/L
Based on the results of this study, the test item B331 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge, therefore further testing for nitrification inhibition is not necessary.

Description of key information

EC50 (3 hours) > 1000 mg/L

Key value for chemical safety assessment

Additional information

An Activated Sludge Respiration Inhibition Test (ASRIT) was conducted (CiToxLAB Hungary, 2016) to assess the short-term effects of B331 on Sewage Treatment Plant microorganisms. The study was conducted according to OECD test guideline 209 and EC method C11, and in compliance with GLP. The inhibitory effect of the test material was assessed at concentrations 10, 100 and 1000 mg/L over 3 hours.

Less than 50% inhibition of respiration was observed at the highest test concentration (3 hour EC50 > 1000 mg/L); on this basis the test material was considered to be non-inhibitory to STP microorganisms.