ABSTRACT FLOOD, STACIE LYNN. Ecotoxicology of Estuarine Phytoplankton Growth and Toxicity in Response to Atrazine Exposures. (Under the direction of committee chair JoAnn M. Burkholder). Estuarine phytoplankton play integral roles in ecosystem functioning by providing a basis for energy fluxes (through their carbon fixation capacity) and nutrient cycling, and by influencing community structure (through oxygen and food biosynthesis, and through both competitive interactions and interactions with other trophic levels). Some phytoplankton populations (strains) proliferate to such an extent that they can form nearly monospecific blooms, with devastating impacts on ecosystems. Anthropogenic inputs of chemical environmental contaminants such as toxic substances and nutrient pollution are frequently associated with developing harmful algal blooms (HABs). A critical knowledge gap in estuarine ecology concerns how phytoplankton assemblages respond to stressors in chronically disturbed habitats, and why some populations respond by producing phycotoxins. The available evidence suggests that herbicides such as atrazine can significantly affect phytoplankton assemblage composition, but little is known about how estuarine phytoplankton respond to multiple stressors in chronically disturbed habitats, and why some strains respond by producing toxins that can kill fish and other aquatic life. The goals of this research were to establish a robust protocol for testing the effects of a ubiquitous herbicide, atrazine, on estuarine phytoplankton, and then to use that protocol to compare the effects of atrazine with versus without nutrient enrichment on selected benign and harmful species. The benign species used was the cosmopolitan estuarine/marine alga, Dunaliella tertiolecta (Chlorophyta), whereas the HAB species included the ichthyotoxic haptophyte Prymnesium parvum (Haptophyta) and the toxigenic raphidophyte Chattonella subsalsa (Heterokontophyta, Raphidophyceae). The relative toxicity of atrazine to the three species in salinity 20 nutrient- replete media was D. tertiolecta > P. parvum > C. subsalsa (mean 96-hr IC50 = 139.3, 126.5, and 111.3 µg atrazine L-1, respectively). Response estimates were influenced by the strain, the geographic origin, the salinity, and the timing of the analysis endpoint. Nutrient limitation alleviated the growth-inhibiting effects of this herbicide for all three algal taxa to varying extent. Production of hemolytic substances in the HAB species increased under co-occurring nutrient stress and herbicide exposure, and the greatest toxicity was observed under combined imbalanced nitrogen/phosphorus levels and high atrazine exposures. These findings advance knowledge about how nutrient regimes and herbicides interact to control harmful estuarine phytoplankton population dynamics. The test platform that was developed here can be extended for use with other herbicides and other estuarine phytoplankton species. © Copyright 2017 by Stacie Lynn Flood All Rights Reserved Ecotoxicology of Estuarine Phytoplankton Growth and Toxicity in Response to Atrazine Exposures by Stacie Lynn Flood A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Plant Biology Raleigh, North Carolina 2017 APPROVED BY: ______________________________ ______________________________ Dr. JoAnn M. Burkholder Dr. W. Gregory Cope Committee Chair ______________________________ ______________________________ Dr. Stacy A. C. Nelson Dr. Parke A. Rublee ______________________________ Dr. Thomas R. Wentworth ii DEDICATION This work is dedicated to my mother, Cindy Flood, who always supported my efforts to grow and encouraged my ambitions. This work is also dedicated to the memory of my grandmothers Pat Walton, who always believed in me and taught me to live with dignity and grace, and Mary Wilkinson, who kept me grounded, kept me close, and taught me to love fish, and to my great-grandmother Thelma Seals, who became a Ph.D. chemist under much more challenging circumstances than I will ever know. You are my heroes, and together you brought me to this point. This work is also dedicated to the memory of Charlie, who was by my side throughout it all, making me laugh and keeping me focused on the beauty of the world around us. The wind has taken you from my sight, but my heart will always see you. iii BIOGRAPHY Stacie Flood was born and raised in Tulsa, Oklahoma where she attended Edison High School and developed a long-abiding interest in keeping and breeding aquarium fishes. She owned and operated a local aquarium supply shop in Tulsa and specialized in the building and care of planted aquaria and in the husbandry of Symphysodon species (Discus fish) before moving to the Greater St. Louis area to work at a tropical fish hatchery. While living in Missouri, Stacie’s primary interests shifted from the captive care of tropical fish to the ecology of North American native fishes, and she began to devote her attention to environmental sciences and the factors influencing aquatic ecosystems in United States. After earning an Associate’s Degree in General Sciences at John A. Logan College in Carbondale, Illinois, she enrolled in an undergraduate zoology program at Southern Illinois University where she was fortunate to work, in turns, on projects with or in the labs of Dr.s Matt Whiles, Frank Wilhelm and Brooks Burr studying aquatic invertebrate ecology, limnology and ichthyology, respectively. Following graduation, she applied to North Carolina State University and was accepted into the Center of Applied Aquatic Ecology under the direction of Dr. JoAnn Burkholder, who has guided her through an advanced level examination of the ecology of surface and ground waters of North Carolina, the U.S., and throughout globe. These discoveries and discussions were often challenging and always fascinating, and have provided Stacie with a greater appreciation for the scale of economic, political, cultural and civic influences on natural aquatic ecosystems - and never dulled the brilliance of the small moments of wonder found glancing through snorkel or microscope at the beauty that lies hidden under the surface of a stream. iv TABLE OF CONTENTS LIST OF TABLES ................................................................................................................. vi LIST OF FIGURES ............................................................................................................. viii LIST OF ABBREVIATIONS .................................................................................................x CHAPTER 1: Assessment of Atrazine Toxicity to the Benign Estuarine Phytoplankter, Dunaliella tertiolecta (Chlorophyta) Under Varying Nutrient Conditions .........................1 1.1 INTRODUCTION .................................................................................................................... 1 1.2 MATERIALS AND METHODS ........................................................................................... 5 1.2.i Basic Platform Design ................................................................................................. 5 1.2.ii Culture Conditions ...................................................................................................... 7 1.2.iii Optical Density Versus Direct Cell Counts ............................................................ 8 1.2.iv Population Growth Rate Measurements .................................................................. 9 1.2.v Algal Bioassays ............................................................................................................ 9 1.2.vi Statistical Analysis .................................................................................................... 12 1.3 RESULTS ................................................................................................................................ 13 1.4 DISCUSSION ......................................................................................................................... 15 1.5 FIGURES AND TABLES .................................................................................................... 20 1.6 REFERENCES ........................................................................................................................ 35 CHAPTER 2: Persistence Under Sub-Optimal Conditions; Imbalanced Nutrient Regimes Increase Prymnesium parvum Resilience to Herbicide Exposure.......................46 2.1 INTRODUCTION .................................................................................................................. 46 2.2 MATERIALS AND METHODS ......................................................................................... 49 2.2.i Experimental Organism, Strains, and Culture Conditions .................................... 49 2.2.i.a. Toxic Prymnesium parvum .......................................................................................... 49 2.2.i.b. Strains and Culturing .................................................................................................. 51 2.2.ii Optical Density Versus Direct Cell Counts ........................................................... 52 2.2.iii Population Growth Rate Measurements ............................................................... 53 2.2.iv Algal Bioassays ......................................................................................................... 53 2.2.v Hemolytic Activity Assay Protocols