Development of a high performance liquid chromatography method to monitor the biodegradation of naphthenic acids

Naphthenic acids are found in wastewaters from petroleum refineries and from the extraction process used to recover bitumen from large oil sands deposits found in northeastern Alberta, Canada. The naphthenic acids, comprised mainly of a complex mixture of carboxylic acids, are toxic to many forms of aquatic life. Previous studies have shown that biodegradation in natural settings or in laboratory cultures reduces the toxicity of naphthenic acids. A high performance liquid chromatography (HPLC) method was developed to follow the removal of naphthenic acids from aerobic, laboratory cultures. Small samples of the culture supernatants (100 μμL) were derivatized with 2-nitrophenylhydrazine, and the derivatized carboxylic acids were separated from the excess derivatizing agent by HPLC. The derivatized acids eluted as an unresolved hump in the chromatogram. Comparing the areas under the humps in standard preparations (containing 5–200 mg naphthenic acids/L), and in culture supernatants allowed measurements of the depletion of naphthenic acids from the microbial cultures. Three naphthenic acids preparations were studied, and up to 40% of the naphthenic acids were removed by cultures during 30- to 40-d incubation periods. Carbon dioxide formation accompanied the decrease in the naphthenic acids concentration determined by HPLC. Key words: biodegradation, high performance liquid chromatography, naphthenic acids, 2-nitrophenylhydrazine, oil sands.