Closed aerobic biodegradation kinetics test with activated sludge and low concentration chemical mixtures

The activated sludge process at wastewater treatment plants is important to prevent discharge of organic pollutants to the environment. Determination of biodegradation kinetics in activated sludge is challenging for mixtures that cover a diverse range of structures. The aims of this study were to (1) design a closed aerobic biodegradation batch test with activated sludge and (2) develop a sample preparation procedure that is compatible with LC-MS and Solid Phase Microextraction (SPME) coupled to GC-MS. A headspace:sludge ratio of 4:1 was sufficient to ensure aerobic conditions in activated sludge for 7 days at co-solvent concentrations <0.01%. Ethanol was added to sub-samples (50%) to stop biodegradation, extract sorbed chemicals and allow storage at −18 °C without ice formation. The ethanol extracted the chemicals from the sludge before filtration (0.2 μm). The filtrate was diluted in ultrapure water to <12% ethanol before analysis by SPME GC-MS/MS and was suitable for direct injection on LC-MS/MS. Biodegradation was distinguished from sorption through abiotic controls using autoclaved poisoned sludge. Linalool, naphthalene, α-isomethylionone, phenanthrene, citronellol, drometrizole, 2-ethylhexyl 4-methoxycinnamate, dicyclohexyl phthalate, BP-1, BP-3, methyl-, ethyl-, propylparaben, alkyl sulfates and isethionates degraded within 48 h in activated sludge, while musk ketone, tonalide and 1,3,5-trichlorobenzene did not. A 10 times reduction of sludge density did not markedly affect the microbial diversity but slowed biodegradation kinetics (partly explained by theory). This study demonstrated a ‘cold’ alternative to an OECD 314b test and how biodegradation kinetics can be determined for mixtures of diverse chemicals in closed batch tests with activated sludge.