OECD 309: Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test

The purpose of this test is to measure the time course of biodegradation of a test substance at low concentration in aerobic natural water and to quantify the observations in the form of kinetic rate expressions. Degradation of the test substance will be followed for the parent substance and its potential (known) transformation products. Degradation rate, mass balancing and transformation product pattern will be determined for the test substance. Degradation kinetics will also be followed for any major transformation product, if possible.

Study Design

Test setup

Typically, the test will be carried out with 14C-labelled test substance. Incubation in surface water (“pelagic test”) will be accomplished in the dark at a temperature of 20°C under aerobic conditions and agitation (stirring). The test will be conducted using multiple flask design where each flask is connected to a flow-through system. A moderate stream of air will be used as carrier gas to collect CO2 and other volatiles in distinct traps consisting of different solutions.

Course of the test

Two concentrations of the test substance differing by a factor of 5 to 10 will be used for the test. One concentration will be ≤ 10 μg/L (concentration I) and one ≤ 100 μg/L (concentration II).

Microbial activity of the surface water will be verified by using 14C-labelled benzoic acid (control samples). For examining possible abiotic degradation or other non-biological removal of the test substance (e.g. hydrolysis or adsorption to the test vessel), sterile samples will be prepared.

The test will run for a maximum of 60 days. Apart from samples taken directly after application, at least 7 sampling time points will be included. Time intervals will be chosen in such a way that the pattern of decline of the test substance and possible transformation products can be established.

Analysis of the test substance and transformation products

In case of the 14C-labelled test substance the analytical methods will base on LSC and HPLC coupled with UV and radio detection. LC-MS/MS can be carried out as additional analytical method. The analytical method and sample preparation will be established to allow a limit of quantification of ≤ 5% of the applied radioactivity.

Special test setup

  • Non-labelled test substance can also be used for the test to investigate the degradation kinetics (e.g. DT50) of the test substance.
  • In order to provide a minimum of mineral and nutrient source for the microbial population, a small concentration of suspended solids, in the form of sediment, can be added to the surface water (≤ 0.01 g/L).
  • In general, the test samples will be agitated by stirring. Depending on the costumer needs, a setup for shaking the test samples is also available.


Kinetic analysis and calculations of DT50 and DT90 values
Transformation product pattern including characterisation/ identification of major products.

Guidelines and Literature

  • OECD Guideline for the Testing of Chemicals, No. 309, ”Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test”; adopted April 13, 2004.