PARALYTIC SHELLFISH TOXINS CHARACTERIZED FROM LYNGBYA WOLLEI DOMINATED MATS COLLECTED FROM TWO FLORIDA SPRINGS By AMANDA FOSS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011 1 © 2011 AMANDA FOSS 2 To my wonderful parents 3 ACKNOWLEDGMENTS I appreciate the support provided me by GreenWater Laboratories, Mark Aubel for his wonderful direction and aid with toxin analysis and Andrew Chapman for his expertise in phycology and sampling. I thank the chair, Ed Phlips, and members of my committee, Nancy Szabo and Karl Havens, for their priceless advice and keen research assistance. I thank Mete Yilmaz for his work on the molecular portion of this study, as well as expertise. I thank Andrew Reich with the Florida Department of Health for collaboration with GreenWater Labs and the research that kick started this project, and, of course a big thank you to Alicia Plakotaris and Johnny May for their help in the field. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 12 Objectives ............................................................................................................... 12 Background ............................................................................................................. 13 Lyngbya Growth in Florida Springs .................................................................. 13 Lyngbya Toxin Production ................................................................................ 14 Paralytic Shellfish Toxins.................................................................................. 15 Properties and toxicity ................................................................................ 15 Potential risks and trophic transfer ............................................................. 16 Toxin Extraction ................................................................................................ 17 Toxin Analysis Techniques ............................................................................... 18 Molecular Biology of Lyngbya and Saxitoxin gene ........................................... 19 2 MATERIALS AND METHODS ................................................................................ 21 Sampling ................................................................................................................. 21 Site Description ................................................................................................ 21 Collection .......................................................................................................... 22 Standards and Reagents ........................................................................................ 24 Standards for Instrument Calibration and Quantification .................................. 24 Reagents .......................................................................................................... 24 Sample Preparation ................................................................................................ 25 Qualitative Analysis of Filamentous Macroalgae Samples ............................... 25 DNA Isolation, Polymerase Chain Reaction, and Sequencing ......................... 25 Toxin Samples .................................................................................................. 27 Paralytic Shellfish Toxin Extraction ......................................................................... 28 Extraction Assessment ..................................................................................... 28 SPE and Lyophilization Assessment ................................................................ 28 Final Extraction Protocol................................................................................... 29 Paralytic Shellfish Toxin Analysis ........................................................................... 29 HPLC/ Fluorescence ........................................................................................ 29 Peroxide Oxidation ..................................................................................... 29 Periodate Oxidation ................................................................................... 30 Chromatographic and Fluorescence Conditions ........................................ 31 5 Detection Limits ......................................................................................... 32 Sample Analysis ........................................................................................ 32 HPLC/MS/(MS) ................................................................................................. 32 Chromatographic and Mass Spectrometry Conditions ............................... 32 Detection Limits ......................................................................................... 33 Sample Analysis ........................................................................................ 34 3 RESULTS ............................................................................................................... 48 Field Data ............................................................................................................... 48 Water Quality .................................................................................................... 48 Field Observations ........................................................................................... 50 Algal Analysis ......................................................................................................... 50 Molecular Biology ................................................................................................... 51 HPLC/Fluorescence ................................................................................................ 52 Peroxide Oxidation Products ............................................................................ 52 Extraction Assessment ..................................................................................... 53 SPE and Lyophilization Assessment ................................................................ 55 Sample Analysis ............................................................................................... 56 HPLC/MS ................................................................................................................ 56 Extraction Assessment ..................................................................................... 56 SPE and Lyophilization Assessment ................................................................ 57 Sample Analysis ............................................................................................... 57 4 DISCUSSION ......................................................................................................... 87 Toxin Extraction ...................................................................................................... 87 Analytical Methods .................................................................................................. 88 Toxin Risk Assessment ........................................................................................... 89 LIST OF REFERENCES ............................................................................................... 94 BIOGRAPHICAL SKETCH ............................................................................................ 97 6 LIST OF TABLES Table page 2-1 List of sample collections .................................................................................... 42 2-2 Saxitoxin structural variants ................................................................................ 43 2-3 LC/FL gradient .................................................................................................... 45 2-4 MDLs for PSTs via fluorescence in elution order ................................................ 45 2-5 LC/MS gradient ................................................................................................... 45 2-6 MDLs and retention times of PSTs analyzed via LC/MS/(MS) ........................... 46 2-7 Molecular and fragmentation ions monitored LC/MS .......................................... 47 3-1 Water quality and sample collection data for sample sites ................................. 61 3-2 Water quality data for Blue Hole Spring and Silver Glen Springs from FDEP Initiative Report 2011 .......................................................................................... 62 3-3 Mat thickness measured at Silver Glen Springs ................................................. 63 3-4 Qualitative algae list from all samples ................................................................ 64 3-5 pH of each extraction solution before and after boiling ....................................... 69 3-6 Decarbamoylgonyautoxin 2&3 (dcGTX2&3) concentrations (µg g-1) for all sites and sampling months ................................................................................