Evaluation and Modification of the OECD 301F Respirometry Biodegradation Test Method with Regard to Test Substance Concentration and Inoculum

Abstract

The majority of OECD (Organisation for Economic Co-operation and Development) ready biodegradation test methods require a relatively high concentration of test substance. Such high dosage concentrations are unsuitable for screening of inhibitory or biocidal compounds. Unfortunately, the method with the lowest dosage concentration (Closed Bottle Test, 301D) also has the lowest biodegradation potential. Therefore, we have evaluated the ability of the Manometric Respirometry (301F) test method to study biodegradation of test substances at concentrations of a few mgl−1, using sodium acetate as a model substance. Results have been compared to those obtained from closed bottle tests (301D). Problems with using activated sewage as an inoculum have been demonstrated, as respirometry curves were biphasic, and the recorded biological oxygen demand (BOD) was higher than the calculated theoretical oxygen demand (ThOD). A consortium of bacteria isolated from activated sewage was used as an alternative inoculum. Using the alternative inoculum for respirometry tests, monophasic growth curves were observed with recorded BOD values at expected levels. Sensitivity thresholds for the respirometer tests were comparable to those in closed bottles, with biodegradation being detected at concentrations as low as 2.5 mgl-1 ThOD. Comparison of whole community carbon source utilisation profiles for both inocula (obtained using BIOLOG microplates) revealed their catabolic potential to be similar. Therefore, use of an activated sewage-derived inoculum is recommended as an alternative inoculum for respirometer-based biodegradation testing.