LINCOMYCIN AND SPECTINOMYCIN: PERSISTENCE IN LIQUID SWINE MANURE AND THEIR TRANSPORT FROM MANURE-AMENDED SOIL A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Master of Science In the Toxicology Graduate Program University of Saskatchewan Saskatoon, Saskatchewan Canada SANDRA LOUISE KUCHTA © Copyright Sandra Louise Kuchta, February, 2008. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Chair of the Toxicology Graduate Program Toxicology Centre University of Saskatchewan 44 Campus Drive Saskatoon, SK, Canada S7N 5B3 i ABSTRACT Antimicrobials administered to livestock can be excreted up to 80% in the feces and urine. Liquid swine manure from confined animal feeding operations is generally retained in lagoon storage until it is applied as a nutrient source to cropland. Thus, the applied manure becomes a possible source of antimicrobials to aquatic ecosystems. Veterinary antimicrobials have been detected in surface and ground waters in Canada, the United States and Europe, however, their environmental fate is not well known. Lincomycin and spectinomycin are two antimicrobials administered as a mixture to swine in the prairie region of Canada for the prevention of post- weaning diarrhea. In order to assess the potential for contamination of prairie wetlands, concentrations of both antimicrobials were monitored in the liquid manure from the nursery area of a commercial-scale barn during a 5-week study, and their persistence during simulated manure storage investigated. The potential for transport of lincomycin and spectinomycin to surface waters via surface runoff and to leach to groundwater was also assessed. This was achieved by monitoring manure-amended soil, simulated rainfall runoff, snow melt runoff and groundwater over a two-year period at two study sites in Saskatchewan, Canada following fall application of liquid swine manure from two commercial barns to crop and pasture land. Liquid chromatography coupled with tandem mass spectrometry was used to quantitate these antimicrobials in all matrix extracts. In the nursery area of a commercial-scale barn, concentrations of lincomycin and spectinomycin in the cumulating liquid manure at the end of the study were equivalent to 32 and 3.0%, respectively, of doses administered in the feed. In a laboratory study, using fortified liquid manure, concentrations of both antimicrobials showed a rapid initial decrease during simulated lagoon storage, followed by a slower dissipation over a period of 5 months. The average time required for 50% dissipation of lincomycin was greater than one year (365 d) and was approximately 90 d for spectinomycin. Lincomycin concentrations in soil (46.3 to 117 µg kg-1) collected immediately after fall manure application, decreased to non-detectable levels by mid-summer the following year. Lincomycin was present in simulated rainfall runoff (0.1 to 2.7 µg L-1) immediately after manure application ii with similar concentrations present in snow meltrunoff the following spring. Concentrations in groundwater were generally <0.005 µg L-1. Spectinomycin was not detected in the manure applied at the study sites nor in soil, runoff water or groundwater samples. This study confirms that some antimicrobials, including lincomycin, may be present in lagoon manure. Thus, the management practice of utilizing livestock manure from confined animal feeding operations as a plant nutrient source on cropland may result in antimicrobial transport to surface and ground waters. iii ACKNOWLEDGMENTS I would like to thank my supervisor Dr. Allan Cessna for all his time, help and guidance during my Master’s project. I would also like to thank the members of my advisory committee Dr. Barry Blakley and Dr. Mark Wickstrom as well as my external examiner Dr. Les Dickson for their time and advice. I would like to acknowledge that funding for this research came from the Agricultural Development Fund of Saskatchewan Department of Agriculture and Food, Environment Canada, Agriculture and Agri-Food Canada and the Toxicology Graduate Program. I would like to acknowledge the assistance received from Environment Canada, especially from Kerry Peru for sample analysis, and Dr. Jane Elliott and Dr. John Headley for their time and guidance. I would like to thank David Gallén, Jonathan Bailey and Sumith and Chandi Priyashantha for technical support. I would also like to thank the Prairie Swine Centre Incorporated and River Lakes Stock Farm barn personnel for information on antimicrobial use and times of manure application and sample collection, the collaborating producers for permission to sample on their farms, and the operators of the manure applicators for manure sample collection. I would also like to especially thank my friends and family for their help and support. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................... II ACKNOWLEDGMENTS ................................................................................................IV TABLE OF CONTENTS..................................................................................................V LIST OF TABLES ........................................................................................................VIII TABLE OF FIGURES ....................................................................................................IX LIST OF ABBREVIATIONS............................................................................................X 1.0 INTRODUCTION AND BACKGROUND................................................................... 1 1.1 Antimicrobial use in the livestock industry .........................................................................................................1 1.2 Potential sources of antimicrobials to the environment .....................................................................................5 1.3 Chemistry and analysis of antimicrobials in the environment ..........................................................................8 1.4 Toxicological consequences of antimicrobial residues in the environment.......................................................8 1.5 Lincomycin and spectinomycin ..........................................................................................................................11 1.5.1 Lincomycin ....................................................................................................................................................11 1.5.2 Spectinomycin ...............................................................................................................................................13 1.5.3 L-S 20 Premix................................................................................................................................................15 2.0 OBJECTIVES ......................................................................................................... 16 3.0 DEVELOPMENT OF ANALYTICAL METHODS FOR ANALYSIS OF LINCOMYCIN AND SPECTINOMYCIN IN SURFACE AND GROUND WATERS, MANURE-TREATED SOIL AND LIQUID SWINE MANURE........................................................................... 17 3.1 Preface ..................................................................................................................................................................17 3.2 Introduction ........................................................................................................................................................17 3.3 Experimental........................................................................................................................................................20 3.3.1 Chemicals and reagents..................................................................................................................................20 3.3.2 Preparation of standards and solutions...........................................................................................................20 3.3.3 Sample preparation and extraction.................................................................................................................20 3.3.4 Fortification studies .......................................................................................................................................22 3.3.5 Liquid chromatography coupled with tandem mass spectrometry analysis...................................................23 3.4 Results and discussion .........................................................................................................................................24