An efficient chiral liquid chromatography - high resolution - mas spectrometry method has been developed for the determination of climbazole (CBZ) enantiomers in wastewater / sludge. The obtimized analytical method has been validated with good quality parameters including resolution > 1.1 and method quantification limits down to the ng/L range. On the basis of this newly developed analytical method, the stereochemisttry of CBZ was investigated over time in effluent / sludge biotic and steril microcosms under anaerobic dark and light conditions and in influents and effluents of five different subsurface constructed wetlands (CWs). CBZ stereoselective degradation was exclusively observed under biotic conditions, confirming the specificity of enantiomeric fraction variations to biodegradation processes. CBZ was always biotransformed into CBZalcohol due to the specific and enantioselective reduction of the ketone function into a secondary alcohol function. This transformation was almost quantitative and biodegradation gave a good first order kinetic fit. We investigated the possibility to apply the Rayleigh equation to enantioselective processes by replacing the isotope ratio by the enantiomer ratio for quantitative biodegradation assessment of CBZ in biological treatment processes and in receiving surface water or soil. The results of enantiomeric enrichment pointed the way for a quantitative assessment of in situ biodegradation processes due to a good fit (R2 > 0.98) of the anaerobic CBZ biodegradation to the Rayleigh dependency in all the biotic microcosms and in CWs. These results demonstrate that enantiomeric fractionation constitutes a very interesting quantitative indicator of CBZ biodegradation under anaerobic conditions. The enantiomeric fractionation tool does not imply to achieve a mass balance of the contaminant, which constitutes a relevant advantage over the conservative tracer approaches. A prediction of the extent of biodegradation is also allowed as fars as the enantiomeric enrichment factor is known.