AbstractThe antibiotic sulfamethoxazole (SMX) is frequently detected in aquatic systems, raising environmental concerns. Therefore, it is necessary to investigate its transformation pathways under diverse environmental conditions. Here, we explored information from stable isotope fractionation during reductive transformation of SMX with Fe(II)-amended goethite suspensions vs oxidative transformation with MnO2. Transformation of SMX by surface-bound Fe(II) showed significant carbon isotope fractionation (εC = −3.6‰ ± 0.1‰ and εC = −2.9‰ ± 0.2‰, with and without buffer, respectively), pronounced nitrogen isotope fractionation (εN = −12.8‰ ± 0.6‰ and εN = −13.0‰ ± 0.2‰, with and without buffer, respectively), and evidence of transformation products that are consistent with initial reductive N–O bond cleavage. In contrast, reaction with MnO2 caused normal carbon but inverse nitrogen isotope fractionation (εC = −1.7‰ ± 0.1‰ and εC = −2.7‰ ± 0.2‰, with and without buffer, respectively, εN = +1.9‰ ± 0.2‰ without buffer) indicative of an initial oxidative electron transfer. Oxidation products were not easily identified by high-resolution mass spectrometry, emphasizing the importance of compound-specific isotope analysis (CSIA) as an alternative for detecting ongoing transformation. Our study thus highlights the potential of CSIA to detect transformation in settings where concentration and product analysis would be inconclusive and to distinguish between reductive and oxidative transformation pathways of SMX in the environment.