Metabolism and excretion kinetics of 14 C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites $, $$ Premasis Sukul, Marc Lamsho ¨ ft, Souvik Kusari, Sebastian Zu ¨ hlke, Michael Spiteller à Institut fu ¨r Umweltforschung (INFU), Technische Universita ¨t Dortmund, Otto-Hahn-Strabe 6, 44221 Dortmund, Germany article info Article history: Received 24 July 2008 Received in revised form 20 November 2008 Accepted 10 December 2008 Available online 31 January 2009 Keywords: Veterinary drug LC-MS/MS Accurate mass measurement Radiochemical methods abstract Fluoroquinolones are amongst the most important antibiotics used in veterinary medicine. On this account the behavior of difloxacin (DIF) and its metabolites was investigated by administering the 14 C- labeled and non-labeled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Sarafloxacin (SAR) was found to be the major metabolite, three further trace metabolites were also recovered, applying high-resolution (HR) mass spectrometric technique. The identification of DIF and SAR was confirmed by comparison with the spectroscopic and chromatographic data of the authentic references. The identification of the three trace metabolites was performed by HR- MS/MS. Only 8.1% of the administered radioactivity remained in the pig after 10 days and DIF accounted for 95.9% of the radioactivity excreted. More than 99% of the labeled compounds were detected and identified in the manure. The mean recoveries for all single electrolytes were X94%. Linearity was established over concentration range 10–10,000 mg/kg manure with a correlation coefficient X0.99. By using in vitro antimicrobial activity tests against a group of standard pathogenic control strains, the results showed that the residual antibiotic concentrations in the manure of pigs are high enough to exhibit antibacterial activity. & 2008 Elsevier Inc. All rights reserved. 1. Introduction Antibiotics are widely used to treat and prevent diseases in humans and animals. Antibiotics are also used in animal husbandry as feed additives to increase the animal mass (Hassouan et al., 2007). Antibiotics together with their metabo- lites are excreted and reach the environment via different routes depending on pharmacokinetic and pharmacodynamic para- meters in the organisms (Kemper, 2008). Application of liquid manure in agricultural land from livestock and sewage sludge from wastewater treatment plants is a general practice to enrich soil with nutrients and/or for sustainable nutrient recycling. However, this practice unintentionally encourages the contam- ination of different environmental compartments through the entry of hazardous materials contained in the liquid manure. Out of many hazardous substances, potential candidate could be antibiotics. The antibiotics, being biocidal, may pose threat to the soil- and water-dwelling organisms when they reach the environ- ment. Once administered to animals, the drugs are absorbed and partially metabolized before being excreted in urine and feces. Upon application of the resulting liquid manure and slurry the antibiotics with their metabolites are released to the land, they may interact with different soil components and may enter the food chain via uptake by plants, translocation into groundwater, or soil surface runoff into surface waters (Boxall et al., 2002; Thiele-Bruhn, 2003). Several antibiotics have previously been detected in soil and occasionally in ground water (Boxall et al., 2004; Sukul and Spiteller, 2006, 2007; Kemper, 2008). The incidence of antibiotic resistance among strains of aquatic sources has already been reported (Schwartz et al., 2003; Messi et al., 2005). The exhibition of antimicrobial activity by many metabo- lites of antibiotics, with a special reference to fluoroquinolones (FQs), cannot be ruled out (Marengo et al., 1997; Wetzstein et al., 2000). Therefore, the fate of metabolites should also be con- sidered in order to establish the environmental risk assessment of ARTICLE IN PRESS Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/envres Environmental Research 0013-9351/$ -see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2008.12.007 $ Funding source: This study has been performed within the frame of the research project ‘‘Veterinary medicines in soils: basic research for risk analysis’’, funded by the German Research Foundation [Deutsche Forschungsgemeinschaft (DFG)]. $$ Animal research approval: The experiment on animals was carried out in NOTOX Safety and Environmental Research B.V., ‘s-Hertogenbosch, The Nether- lands. The study type was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of NOTOX (DEC NOTOX no. 07-84) as required by the Dutch Act on Animal Experimentation (February 1997). The study procedures were based on ‘‘Guideline on Environmental Impact Assessment (EIAs) for Veterinary Medicinal Products (VMPs): EMEA/VICH/592/98, 1 July 2001’’. à Corresponding author. Fax: +49 2317554085. E-mail address: M.Spiteller@infu.uni-dortmund.de (M. Spiteller). Environmental Research 109 (2009) 225–231