OECD 301 D, Regulation (EC) No 440/2008,C.4-E, DIN EN ISO 10707
The Closed Bottle Test is the classical test method for " ready” biodegradability. A medium with a mineral substratum and the test substance as the only carbon source is inoculated by a relatively low amount of microorganisms taken from a mixed culture. Subsequently, the biochemical oxygen demand is determined in regular intervals over a period of 28 days, by means of test bottles prepared in parallel and withdrawn again after determination. Biodegradation is then calculated from the ratio between the oxygen consumption that is caused by the degradation of the test item (corrected by the blank values of the inoculum), and the theoretical oxygen demand (ThOD), or alternatively the chemical oxygen demand (COD).
The test concentration is about 2 - 10 mg test substance per litre. Effluents from outflow of a municipal wastewater treatment plant are used as standard inoculum. A degradation extent of > 60% ThOD within 28 days and within a "10-days window" after the end of the lag-phase is defined as criterion for classifying a particular test substance as "readily” biodegradable.
The advantage of the „closed bottle test“ is its simple performance, the possibility to study also poorly soluble and volatile test compounds, as well as the low test concentrations required. This allows getting below possible effect threshold levels that are toxic to bacteria. Disadvantageous is on the one hand the low density of inoculation of a few ml of cleaned wastewater per litre, and on the other hand the oxygen reduction that is caused by the consumption of oxygen due to nitrification. For this reason, the test application is limited if slowly degradable and/or nitrogen-rich substances have to be studied.