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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Sep - 05 Oct 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
July, 2010
GLP compliance:
yes (incl. QA statement)
The Department of Health of the Government of the United Kingdom (27 Jun 2016)
Analytical monitoring:
Details on test solutions:
- Method: Individually weighed quantities of test substance were added directly to the appropriate test vessels.
- Controls: Dechlorinated tap water, synthetic sewage and inoculum.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Sludge return line at Burley Menston Sewage Treatment Works, West Yorkshire, United Kingdom
- Preparation of inoculum for exposure: The sludge was transported to the test facility in a closed container with an adequate headspace to prevent the sample becoming anoxic. On arrival at the test facility, the sludge was aerated with a compressed air supply delivered through a suitable distribution device.
- Pretreatment: The sludge was maintained at 20 ± 2 °C and fed with synthetic sewage concentrate at a rate of 50 mL/L.
- Initial biomass concentration: The suspended solids concentration was 3 g/L after adjustment with dechlorinated tap water and was within the nominal range of 3 ± 0.3 g/L.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
7.01 - 7.88
Nominal and measured concentrations:
Control, 1, 10, 100, and 1000 mg/L (nominal)
Details on test conditions:
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: Material: glass; Size: 250 mL; Fill volume: 250 mL
- Aeration: At appropriate intervals, test samples were inoculated and aerated immediately. Each culture was aerated for ca. 3 h by bubbling air through the test system.
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 3
- Sludge concentration: After addition of the test substance, inoculum and additional water, the nominal suspended solids concentration was 1.5 g/L.
- Nutrients provided for bacteria: Synthetic sewage concentrate was used to feed the activated sludge during storage and to provide a uniform respiration substrate in the test cultures. The packets of pre-made synthetic sewage from Strathkelvin are added to water at 1 packet per 250 mL of water.
- Nitrification inhibitor used: N-allylthiourea (ATU)

- Source/preparation of dilution water: Dechlorinated tap water

- Adjustment of pH: The pH of the sludge was adjusted to 7.20 using 2 M NaOH.
- Details on termination of incubation: At the end of the 3-h incubation period, a portion (20 mL) of each test preparation was transferred to an appropriate sample tube containing a PTFE stirrer. For those samples requiring amendment with ATU, this was undertaken at least 10 min prior to oxygen consumption measurement. The dissolved oxygen (DO) electrode was sealed in the neck of the flask, ensuring air was completely excluded. The flask contents were stirred at a constant rate during DO measurements. Oxygen consumption was measured over a period of up to 10 min.

- Respiration Rate: Oxygen consumption was measured at the end of the 3-h incubation period over a period of up to 10 minutes.

- Spacing factor for test concentrations: 10
Reference substance (positive control):
3,5-dichlorophenol (3,5-DCP)
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:
The EC50 (3 h) is ≥ 1000 mg/L (the highest tested concentration).
Reported statistics and error estimates:
The NOEC for the test substance was based on both visual assessment of the data as well as on calculated data. The estimation of the EC50 for the test substance was not possible due to the low toxicity observed and was therefore determined as greater than the highest concentration tested (> 1000 mg/L). Statistical analysis was conducted using CETIS v

The validity criteria were met and the results of the range-finder limit test are valid.


Total, heterotrophic and nitrification respiration rates were similar across the controls and all treatment levels. There were no significant effects on total or heterotrophic respiration at any concentration. Limited inhibition (12 and 26%, respectively) of nitrification respiration was observed at 100 and 1000 mg/L respectively but determined not to be statistically significant.

Description of key information

NOEC (3 h) ≥ 1000 mg/L (nominal, OECD 209)

Key value for chemical safety assessment

Additional information

There is one study available assessing the toxicity ofN-Acetyl-DL-tryptophan (CAS 87-32-1) to microorganisms according to the OECD guideline 209 and GLP.

Activated sludge was exposed to nominal test item concentrations of 1, 10, 100 and 1000 mg/L. The nominal suspended solids concentration was 1.5 g/L. The validity of the test system was verified with a standard reference respiration inhibitor (3,5-dichlorophenol). After 3 h of exposure oxygen consumption was measured over a period of up to 10 minutes using a dissolved oxygen electrode. Nitrification inhibitor (N-allylthiourea) was added to the appropriate test and reference vessels 10 min prior oxygen measurement.

After 3 h of exposure inhibition was < 50%. Therefore, the reported EC50 (3 h) is > 1000 mg/L and the NOEC (3 h) is ≥ 1000 mg/L (nominal).