OECD 301/310: Ready Biodegradability Tests

Study Design

Selection of adequate test

Depending on the test item specific properties such as water solubility, different tests are applicable:

General test design

Activated sludge is taken from the aeration tank of a domestic waste water treatment plant. After a conditioning procedure defined suspensions of the activated sludge microorganisms are introduced in the test vessels.
Beside the test item treatments a series of controls can be performed: control, reference item and toxicity control. The control contains only activated sludge. As a highly degradable reference item, e.g. Sodim Benzoate is used to check the activity of the sludge microorganisms. The toxicity control contains the test item and the reference item to detect inhibitory effects of the test item.

For test items containing nitrogen, the nitrification can be measured by Continuous Flow Analysis according to Annex V. For poorly souluble test items an application onto silica gel or an addition of silicon oil is possible. All tests are carried out in controlled environment rooms with constant temperature in the dark with a standard test duration of 28 days.

OECD 301D: Closed Bottle Test

A sufficient number of Karlsruher flasks are bubble-free filled with test solutions and tightly closed by glass stoppers. The biodegradation is followed by the oxygen uptake of the microorganisms during exposure. The oxygen concentrations are measured electrochemically at eight sampling dates in the airtight system with an O2-electrode under constant stirring. The percentage of degradation is determined by correction for the blank and under consideration of the theoretical oxygen demand of the test item. An application of this test is only possible for water soluble test items due to the fact that a stock solution of the test item is prepared and the test flasks are not continously stirred during the incubation time.

OECD 301F: Manometric Respirometry Test

Biodegradation is followed by the oxygen uptake of the micro-organisms. A manometric system is used to measure the pressure decrease in the test vessels. The bottles are remaining closed during the whole experimental time and are stirred continuously.
The percentage of degradation is determined by correction for the blank and under consideration of the theoretical oxygen demand of the test item. An application of this test is only possible for non toxic substances because relatively high amounts of the test substance are used.

OECD 310: CO2 in Sealed Vessels (Headspace) Test

The test vessels are filled with test solutions and are sealed with gas-tight septum caps. At eight sampling dates, the biodegradation is determined by following the CO2 evolution of the test item in the incubation flasks during exposure. The produced CO2 is converted to carbonate and subjected to TIC (total inorganic carbon) analysis. The percentage of degradation is determined by correction for the blank and under consideration of thecarbon content of the test item.

Endpoints

Test items that reach 60 % biodegradation within the 10-day window (the 10 days after passing 10%biodegradation of the test item), can be clasified as ready biodegradable. Test items that reach 60% after the 10-day window are clasified as ready biodegradable, but failing 10-day window.  

Guidelines and Literature

• OECD Guideline for Testing of Chemicals No. 301 D: "Ready Biodegradability: Closed Bottle Test", adopted July 17, 1992
• OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted July 17, 1992