In this Guideline six methods are described that permit the screening of chemicals for ready biodegradability in an aerobic aqueous medium.


  1. 301 A: DOC Die-Away
  2. 301 B: CO2 Evolution (Modified Sturm Test)
  3. 301 C: MITI (I) (Ministry of International Trade and Industry, Japan)
  4. 301 D: Closed Bottle
  5. 301 E: Modified OECD Screening
  6. 301 F: Manometric Respirometry


A solution, or suspension, of the test substance in a mineral medium is inoculated and incubated under aerobic conditions in the dark or in diffuse light. The amount of DOC in the test solution due to the inoculum should be kept as low as possible compared with the amount of organic carbon due to the test substance. Allowance is made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without test substance, although the endogenous activity of cells in the presence of a chemical will not exactly match that in the endogenous control. A reference compound is run in parallel to check the operation of the procedures.


In general, degradation is followed by the determination of parameters such as DOC, CO2 production, and oxygen uptake and measurements are taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation. With automatic respirometers the measurement is continuous. DOC is sometimes measured in addition to another parameter but this is usually done only at the beginning and end of the test. Specific chemical analysis can also be used to assess primary degradation of the test substance and to determine the concentration of any intermediate substances formed. It is obligatory in the MITI method (301 C).

Normally, the test lasts for 28 days. Tests however may be ended before 28 days, i.e. as soon as the biodegradation curve has reached a plateau for at least three determinations. Tests may also be prolonged beyond 28 days when the curve shows that biodegradation has started but that the plateau has not been reached by day 28. However, in such cases the chemical would not be classed as readily biodegradable.