Fate of the veterinary antibiotic 14C-difloxacin in soil including simultaneous amendment of pig manure with the focus on non-extractable residues

The fate of C-14-labeled difloxacin (C-14-DIF) was studied in time course experiments after application on soil (Ap horizon of silt loam) and amendment of authentic DIF containing pig manure (146 mL kg(-1); 4.17 MBq kg(-1); 0.85 mg kg(-1)) or water (124 mL kg(-1); 0.42 MBq kg(-1); 0.09 mg kg(-1)) for 56 and 120 days of incubation, respectively. Mineralization of C-14-DIF was below 0.2% in both experiments after 56 days or 120 days. In the course of the experiments, portions of extractable radioactivity (Accelerated Solvent Extraction (ASE); acetonitrile-water) decreased to 19-21% depending only little on manure amendment. Non-extractable residues of C-14-DIF increased to 70-74% after 56 days and 120 days, respectively, and therefore were the main route of C-14-DIF in soil. According to radioanalytical HPLC and LC-MS/MS, only the parent compound was found in all extracts over the whole time of the experiment. According to fractionation of the non-extractable residues (NER) into particle size fractions, C-14 portions were associated to the water used for fractionation, the silt and clay fractions, whereas no radioactivity was detected in the sand fraction. The majority of C-14 was found within the clay fractions. Fractionation of humic components showed that radioactivity derived from C-14-DIF was associated with humic acids, fulvic acids, humins and minerals and very little with soluble, non-humic HCl fraction. The highest portions of radioactivity were found in the fulvic acid fraction. Results obtained by size exclusion chromatography (SEC) of the purified fulvic acids were similar for every sample analyzed. One large portion of C-14 co-eluted with fulvic acids of a molecular weight below 910 g mol(-1). Both fractionation methods demonstrated that the parent compound DIF or initial metabolites were rapidly integrated into humic materials and, thus, were major components of NER.