PHYCOTOXIN LOADS IN BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) FROM THE INDIAN RIVER LAGOON ESTUARY SYSTEM AND COASTAL ATLANTIC WATERS, FLORIDA: 2002 – 2011. by JEREMY ALAN BROWNING B.S., California State University Long Beach at Long Beach A thesis submitted to the Department of Biological Sciences of Florida Institute of Technology in partial fulfillment of the requirements for the degree of MASTERS OF SCIENCE in BIOLOGICAL SCIENCES Melbourne, Florida July 2016 PHYCOTOXIN LOADS IN BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) FROM THE INDIAN RIVER LAGOON ESTUARY SYSTEM AND COASTAL ATLANTIC WATERS, FLORIDA: 2002 – 2011. A THESIS By JEREMY ALAN BROWNING Approved as to style and content by: Spencer E. Fire, Ph.D., Chairperson John Trefry, Ph.D., Member Associate Professor Professor Department of Biological Sciences Management and Environmental Systems Robert van Woesik, Ph.D., Member Wendy Noke Durden, M.S., Member Professor Research Scientist Department of Biological Sciences Hubbs-SeaWorld Research Institute Rich B. Aronson, Ph.D. Professor and Head Department of Biological Sciences July 2016 ABSTRACT PHYCOTOXIN LOADS IN BOTTLENOSE DOLPHINS (TURSIOPS TRUNCATUS) FROM THE INDIAN RIVER LAGOON ESTUARY SYSTEM AND COASTAL ATLANTIC WATERS, FLORIDA: 2002 – 2011. by Jeremy Alan Browning, B.S., CSU Long Beach Chairperson of Advisory Committee: Spencer E. Fire, Ph.D. Harmful algal blooms (HABs) are dramatic increases of algae that produce phycotoxins, and these toxins lead to subsequent neurological dysfunction with symptomatic illness leading, in extreme cases, to mass mortalities of marine mammals. This presents a conservation challenge since the frequency and severity of HABs are increasing on a global scale. Two HAB species that impact marine mammals residing in and utilizing an eastern Florida estuary and surrounding coastal Atlantic waters are Karenia brevis (K. brevis) and Pyrodinium bahamense (Pyrodinium); these HAB species produce the neurotoxins brevetoxin (PbTx) and saxitoxin (STX), respectively. These toxins are present at times in high concentrations during a HAB and in almost all trophic levels in the food web of bottlenose dolphins (Tursiops truncatus), a sentinel marine mammal species in the Florida’s Indian River Lagoon estuary system (IRL) and coastal Atlantic waters. Ecological impacts of present HAB toxins in this region are not well understood. In 2007 a rare and toxic K. brevis HAB event in the coastal Atlantic waters bordering Mosquito Lagoon (a water basin bordering the northern iii Indian River Lagoon) resulted in the detection of PbTx (liver; 11 – 89 ng·g-1) in most of the dead-stranded dolphins analyzed in this region. However, lack of baseline concentrations for HAB toxins in dolphins from this region prevented a full understanding of the impact of phycotoxins in this event on a regional scale. The IRL is also an emerging area of concern for STX contamination of other marine organisms. In this study, we used an enzyme-linked immunosorbent assay (ELISA) method to analyze PbTx and STX in liver tissue from bottlenose dolphins recovered from the IRL from 2002–2011 (n = 119). This tissue aided in completing our primary objective establishing preliminary baseline concentration values and concentrations for HAB toxin exposure in these animals. For dolphins recovered during baseline conditions, toxin concentrations ranged between 0.27 – 1.2 ng·g-1 and 0.41 – 1.9 ng·g-1 for PbTx and STX, respectively. For liver samples from dolphins recovered during exposed conditions detected maximum concentrations of 12.1 ng·g-1 and 9.9 ng·g-1 for PbTx and STX. This research also involved attempting to model the probability of detecting HAB toxins in dolphins at a given cell abundance; which is a key component when determining a dolphins chance of receiving a toxin if its distribution overlaps with a ‘hot-spot’ for HABs within a period of time. The detection probability may be useful in predicting future HAB impacts on dolphins in this region. Although HABs in eastern Florida are infrequent, cell concentrations approach densities large enough to pose significant health risks for both dolphins and human inhabiting this region. Continued biomonitoring of HAB toxins is therefore warranted and can aid resource managers in assessing future impacts and risks to bottlenose dolphins, a sentinel species which inhabits both the IRL estuary and adjacent coastal Atlantic waters iv ACKNOWLEDGEMENTS My thanks go out to the staff at Hubbs-SeaWorld Research Institute: Wendy Noke Durden, Megan Stolen, and Teresa Jablonski, for retrieving and organizing for transport my samples for this research. For establishing certain aspects of this research I had help from Marilyn Mazzoil (Florida Atlantic University) and Wendy Noke Durden, with their knowledge of IRL dolphins, their experience with dolphin behavior, and a passion for ecology conservation they were a huge asset. For much of the HAB historical phytoplankton data, help with querying and unpacking the data came from my initial contact Karen Atwood (FWRI) and was then carried on through Jim Ivey and Alina Corcoran (FWC). Both St. Johns River Water Management District (Ali Simpson & Wendy Tweedale) and FWC staff helped and aided when necessary in my GIS work as well. I want to acknowledge my committee members, especially Spencer Fire my advisor; thank you all for helping me realize this research in guidance, wisdom, and experience. To my other professors, colleagues, family, friends, and ones I miss daily; without you who would I learn from and share with. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………......iii ACKNOWLEDGMENTS………………………………………………………….v TABLE OF CONTENTS…………………………………………………………..vi LIST OF TABLES………………………………………………………………..viii LIST OF FIGURES………………………………………………………………...ix INTRODUCTION………………………………………………………………......1 METHODS AND MATERIALS………………………………………………….12 STUDY AREA…………………………………………………………….12 DOLPHIN SAMPLE COLLECTION………………………………….....12 TOXIN EXTRACTION…………………………………………………...15 Brevetoxin (PbTx)………………………………………………...15 Saxitoxin (STX)…………………………………………………...16 ANALYSIS OF EXTRACTS…………….…………………………….....16 Brevetoxin………………………………………………………...16 Saxitoxin………………………………………………………….17 HAB PHYTOPLANKTON MONITORING SAMPLES…………………18 GIS DATA MANAGEMENT………………………………………….....19 DEFINING EXPOSURE……………………………………………….....22 DATA ANALYSIS………………………………………………………..26 RESULTS…………………………………………………………………………28 vi PHYTOPLANKTON DATA……………………………………………...............28 K. brevis…………………………………………………………...28 Pyrodinium………………………………………………………...29 COMBINING IRL AND COASTAL SAMPLES………………………...30 ELISA RESULTS…………………………………………………………31 Brevetoxin………………...…………………………………….....31 Saxitoxin………………..………………………………………….32 Modeling toxin detection for PbTx………………………………..34 Modeling toxin detection for STX………………………………...35 DISCUSSION……………………………………………………………………..36 LITERATURE CITED……………………………………………………………47 APPENDIX………………………………………………………………………..56 vii LIST OF TABLES PAGE Table 1. Summary of grouping dolphins into Kb-Baseline vs. Kb-Exposed for PbTx………………………………………………………………….32 Table 2. Summary of grouping dolphins into Pb-Baseline vs. Pb-Exposed for STX…………………………………………………………………..33 viii LIST OF FIGURES PAGE Figure 1. Mass mortality events (MMEs) by year and causes……………………..3 Figure 2. Spread of HABs and toxin type globally………………………………...4 Figure 3. Study Area: Indian River Lagoon Estuary System (IRL) and coastal Atlantic waters………………………………………………5 Figure 4. IRL and coastal Atlantic recovered dolphin carcasses 2002-2011…………………………………………...14 Figure 5. Historical HAB data displayed as contours of color for densities using a Kriging interpolation raster (ArcMap 10.2)…………21 Figure 6. Example map of rendered dolphin-digital buffer (ArcMap 10.2) and toxic phytoplankton data………………………..…25 Figure 7. Historical HAB data and PbTx toxin positive dolphin livers plotted; K. brevis abundance data on log scale……………………………………………….................................28 Figure 8. Historical HAB data and STX toxin positive dolphin livers plotted; Pyrodinium abundance data on log scale……………………………………………….................................29 Figure 9. Bar chart of Welch’s two sample t-test between Kb-exposed and Kb-Baseline dolphins for PbTx concentrations (ng·g-1)………….………………………………32 Figure 10. Bar chart of Welch’s two sample t-test between Pb-exposed and Pb-Baseline dolphins for STX concentrations (ng·g-1)..………………………………………...33 ix Figure 11. Logistic model showing increase in probability of detection of PbTx as cell abundance increases for K. brevis…………..34 Figure 12. Logistic model showing increase in probability of detection of STX as cell abundance increases for Pyrodinium….…….35 Figure 13. Historical K. brevis Kriging interpolation raster for densities displayed with recovered dolphins and their detected levels of PbTx………………………………………………………….37 Figure 14. Historical Pyrodinium Kriging interpolation raster for densities displayed with recovered dolphins and their detected levels of STX…………………………………………………………..42 x 1 INTRODUCTION Marine mammals are frequently described as sentinel marine organisms and serve as indicators of ocean health typically because of their long life spans, coastal residence, role as top predators, and propensity to accumulate bioavailable contaminants and toxins in their tissues (Wells et al. 2004; Stolen & Barlow 2003; Odell et al. 1990; Wells et al. 2004; Durden et al. 2007, Fire et al. 2007, Wang 2008, Bossart 2011, Twiner et al. 2011). Unfortunately, marine mammal populations that live in close proximity to growing human coastal populations are