*2.5. Screening of Antibacterial Agents in Water Using High Performance Liquid Chromatography*

An aliquot of collected water samples with enrofloxacin from days ten to 14 and days 26 to 30 was used for analyses. The concentration of enrofloxacin in the water was determined using high performance liquid chromatography (HPLC) via the method described by Scherz [27]. Exactly 100 μL of the sample was injected into the system with an autosampler (System Gold 508, Beckmann, Munich, Germany). A flow of 1 mL per minute was maintained by the System Gold 126 solvent module (Beckmann, Munich, Germany). A CC250/4 NUCLEODUR 100-5C 18ec (25 cm, Macherey-Nagel, Oensingen, Germany) column was used, this being connected to a precolumn (LiChroCART® 4-4, Li- Chrospher® 100 RP-18e, 5 μm, Merck, Darmstadt, Germany). A fluorescence detector (RF-551 Shimadzu, Nakagyo-ku, Japan) with 280 nm for excitation and 450 nm for emission was used for detection. The mobile phase consisted of 85% citrate buffer pH 3.0 (citric acid monohydrate: 1.80 g/L tri-sodium-citrate-dihydrate: 0.43 g/L). The concentration of enrofloxacin in the water samples was calculated with the external standard method.
