Submitted to: Water Environment Federation Technical Exhibition and Conference (WEFTEC)
Publication Type: Abstract Only
Publication Acceptance Date: October 6, 2004
Publication Date: October 6, 2004
Citation: Kolz, A., Moorman, T.B., Ong, S. 2004. Degradation of tylosin and metabolite production in anaerobic and aerobic swine manure slurries. [CD-ROM] Washington, DC. Water Environment Federation Technical Exhibition and Conference (WEFTEC).
Watershed contamination from agricultural activity is becoming a critical issue with the increased occurrence of both confined animal feeding operations (CAFOs) and use of antibiotics as therapeutics and growth promoters. Recent studies have established the presence of both antibiotic residues and antibiotic resistant genes in microbial fauna near CAFOs and agricultural fields amended with biosolids. The most common veterinary growth promoter is tylosin, a macrolide antibiotic. Biodegradation rates of tylosin have been studied but mainly in soil and soil-manure slurries. Little research has focused on the fate of antibiotics in manure lagoons, where they may reside for six months or more before land application. Tylosin data in the literature typically tracks only the disappearance of the major component (tylosin A) and provides little information about the rate of appearance of conjugates or metabolites. Additionally, analysis of tylosin and its metabolites is hampered by many coextractants that are difficult to remove with published solid-phase extraction (SPE) procedures. The current research has developed a successful method for tylosin cleanup from manure lagoon matrices using Oasis hydrophilic-lipophilic balance (HLB) solid phase extraction (SPE) cartridges. This procedure is then used to prepare tylosin biodegradation assays in anaerobic and aerobic swine waste lagoon samples for tandem mass spectrometry (LC-MS-MS)analysis. LC-MS-MS is used to track decreases in the primary form of tylosin and appearance of the prominent metabolites in the biodegradation assays. The reverse-phase SPE procedure uses high pH samples and solvent rinses to retain tylosin while rinsing manure matrix components into waste. This is a highly effective modification of a typical SPE procedure that has yet to be published in the literature. Manure slurry or solvent extract samples are diluted at least 10:1 ratio with purified water and potassium hydroxide to pH 9.4. Samples are then added to the SPE at a flow rate less than 5 mL / minute. Rinsing the SPEs with 59:39:2 methanol-water-ammonium hydroxide (v:v:v) removes much of the coextractants and visible coloration is removed from SPE packing during this step. Tylosin is then eluted with a mixture of 98:2 acetonitrile-glacial acetic acid (v:v). The high pH rinse in this method allows better cleanup and removal of tylosin from complex matrices such as manure slurry and produces better HPLC baseline resolution than similar published methods using acidic or non-pH-modified rinses. This may be attributed to more selective retention of manure matrix constituents at higher pH and higher solvent concentrations possible at pH 9.4. Tylosin is better retained in the SPE above pH 9.4 because at two pH units above its pKa approximately 100% should be in its non-ionized form. Laboratory evaluation has shown that tylosin will be retained on SPEs with basic methanol concentrations up to 60% but will not be retained on SPEs with acidic methanol rinses stronger than 40%. Recoveries of clean tylosin standards using the 59:2 methanol-ammonium hydroxide rinse mentioned above produced recoveries ranging between 93-113%. Further tests from manure slurries also showed highest recoveries from basic samples and basic methanol rinses. Recoveries from slurries ranged between 91-99%. Solvent extraction from samples is done with a mixture of 95:5 acetonitrile-isopropyl alcohol (v:v). Comparison of this mixture with ethyl acetate showed that acetonitrile-isopropyl alcohol produced cleaner extracts with higher recoveries. Anaerobic and aerobic assays have been set up in samples from anaerobic and aerobic waste lagoon samples. Assays have been sacrificed in triplicate with killed control samples over several months. LC-MS-MS is used to track decreases in the primary form of tylosin (tylosin A) and appearance of the prominent metabolites (tylosin B, C, D and desmycotylosin) expected over time in the biodegradation assays. Vials were incubated at 25 oC, which corresponds to the average summer temperature in Iowa. Data from these assays will indicate the extent and nature of tylosin degradation during detention in CAFO waste lagoons prior to application on agricultural fields.