Elucidation of the upper pathway of alicyclic musk RomandolideA® degradation in OECD screening tests with activated sludge

The degradation of RomandolideA (R) ([1-(3',3'-dimethyl-1'-cyclohexyl)ethoxycarbonyl] methyl propanoate), a synthetic alicyclic musk, by activated sludge inocula was investigated using both the manometric respirometry test OECD 301F and the CO2 evolution test. In addition to measuring its biodegradability, key steps of the upper part of the metabolic pathway responsible for RomandolideA (R) degradation were identified using extracts at different time points of incubation. Early metabolism of RomandolideA (R) yielded ester hydrolysis products, including CyclademolA (R) (1-(3,3-dimethylcyclohexyl)ethanol). The principal metabolites after 31 days were identified as 3,3-dimethyl cyclohexanone and 3,3-dimethyl cyclohexyl acetate. Formation of 3,3-dimethyl cyclohexanone from CyclademolA (R) by sludge was confirmed in subsequent experiments using CyclademolA (R) as a substrate, indicating the involvement of an oxygen insertion reminiscent of a Baeyer-Villiger oxidation. Further mineralization of 3,3-dimethyl cyclohexanone was also confirmed in subsequent studies. Three steps were thus required for complete biodegradation of the alicyclic musk: (1) successive ester hydrolyses leading to the formation of CyclademolA (R) with concomitant degradation of the resulting acids, (2) conversion of CyclademolA (R) into 3,3-dimethyl cyclohexanone, and (3) further mineralization via ring cleavage.