OECD 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)

Microorganisms are the main contributors to biodegradation. Therefore the toxicity of chemicals to microorganisms has to be examined for example by measurement of the respiration inhibition in activated sludge according to test guideline OECD 209 (revised in 2010). The purpose of this test is to provide a rapid screening method to assess the effects of substances on the microorganisms of the activated sludge of the biological (aerobic) stage of waste water treatment plants. IBACON performs activated sludge respiration inhibition tests under GLP conditions since many years and has acquired vast experience with this test system.

Study Design

Test organisms

Activated sludge collected from the exit of the aeration tank of a well-operated wastewater treatment plant receiving predominantly domestic sewage.

Course of the test

The activated sludge with test substance and without (blank controls) is incubated with synthetic sewage under intensive aeration. Each batch of activated sludge is also tested with the toxic reference DCP (3,5-dichlorophenol) to proof the sensitivity of the test system. After a contact time of usually 3 hours the respiation rates of activated sludge fed with synthetic sewage are measured in an enclosed cell containing an oxygen electrode.

Study designs

Limit test: A limit test design is used, when the test item is assumed to be not inhibitory to the sludge microorganisms at a specific test item concentration, e.g 100 or 1000 mg test item / L. In that case, three replicates of one test item conentration ,six controls and the refernece item are tested to determine the NOEC.

Dose response test: In case of a respiration inhibition the test is carried out with five treatment concentrations in a geometric series with five replicates and six controls in order to obtain both a NOEC and an ECx (e.g. EC50). The nitrification rate of an activated sludge is determined by performing an additional test set-up, where the nitrification is blocked by the specific nitrification inhibitor N-allylthiourea (ATU). With the results of both set-ups it is possible to calculate the inhibition of three different oxyen uptakes: the total, the the heterotrophic only (without nitrification) and that due to nitrification.


The measured respiration rates of the activated sludge with test substance or reference substance are compared to that of the blank controls and expressed as % inhibition. The NOEC or ECx values are calculated.

Guidelines and literature

  • OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", adopted July 22, 2010
  • DIN EN ISO 8192: Wasserbeschaffenheit – Bestimmung des Sauerstoffverbrauchs von Belebtschlamm nach Kohlenstoff- und Ammonium-Oxidation (ISO 8192:2007)
  • Commission Regulation (EC) No 440/2008, Method C.11: "Activated Sludge Respiration Inhibition Test", Official Journal of the European Union No. L 142/559-563, dated May 30, 2008