PHOTOCHEMISTRY of BREVETOXIN, Pbtx-2, PRODUCED by the DINOFLAGELLATE, KARENIA BREVIS

PHOTOCHEMISTRY OF BREVETOXIN, PbTx-2, PRODUCED BY THE DINOFLAGELLATE, KARENIA BREVIS Jaclyn Pitt A Thesis Submitted to the University of North Carolina at Wilmington in Partial Fulfillment Of the Requirements for the Degree of Master of Science Department of Chemistry and Biochemistry University of North Carolina, Wilmington 2007 Approved by Advisory Committee ______________________________ ______________________________ ______________________________ Accepted by _______________________________ Dean, Graduate School TABLE OF CONTENTS ABSTRACT....................................................................................................................... iii ACKNOWLEDGEMENTS.................................................................................................v LIST OF TABLES............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii INTRODUCTION ...............................................................................................................1 METHODS ..........................................................................................................................6 Reagents and Standards .......................................................................................................6 Photolysis Experiments........................................................................................................6 Sample Extraction................................................................................................................9 Liquid Chromatography Mass Spectrometry.......................................................................9 Experimental Conditions ...................................................................................................14 RESULTS ..........................................................................................................................18 Photodegradation of PbTx-2 in Filtered Coastal Seawater................................................18 Effect of Chromophoric Dissolved Organic Matter on PbTx-2 Photodegradation ...........23 Effect of Trace Metals on PbTx-2 Photodegradation ........................................................26 Effect of Oxygen on Photodegradation of PbTx-2 ............................................................28 Rate of Adsorption of PbTx-2 onto Particles.....................................................................33 DISCUSSION....................................................................................................................35 Implications........................................................................................................................37 LITERATURE CITED ....................................................................................................40* APPENDIX........................................................................................................................43 ii ABSTRACT The predominant brevetoxin produced by Karenia Brevis, PbTx-2 was added to 0.2 µm- filtered Wrightsville Beach, North Carolina seawater (WBSW) and irradiated under simulated sunlight for six hours to determine its photolability. The average first order rate constant of PbTx-2 degradation was 0.20 ± 0.05 h-1 at 25 ºC. When PbTx-2 was added to UV-irradiated WBSW, the average rate constant decreased significantly to 0.08 ± 0.03 h-1 at 25 ºC relative to that of untreated WBSW suggesting dissolved organics are involved in the photodegradation of PbTx-2 in seawater. UV-irradiated WBSW treated with Chelex-100 resin to remove trace metals caused no degradation of PbTx-2 upon exposure to sunlight suggesting that trace metals, in addition to organics, are involved in the photodegradation of PbTx-2. Irradiation of PbTx-2 in deoxygenated filtered WBSW caused the toxin to degrade almost immediately upon exposure to simulated sunlight. The effect of deoxygenation on the photodegradation of other brevetoxins including PbTx-1, 3, 6 agreed with the rate of PbTx-2. PbTx-9 and the naturally-produced compound, brevenal, degraded considerably in deoxygenated WBSW but not as rapidly. These results demonstrate that photosensitizers are involved in the degradation mechanism, suggesting that PbTx-2 degrades by secondary photochemical pathway. The mechanism suggested here is that PbTx-2 degrades in the presence of the excited chromophoric dissolved organic matter (CDOM)* and trace metal complex. Furthermore, the removal of oxygen from seawater causes the rate of degradation to increase rapidly because (CDOM)* no longer scavenges oxygen thereby scavenging solely PbTx-2. A model is also proposed which suggests that 75% of PbTx-2 in coastal seawater can be removed in 6 hours by exposure to sunlight. This represents a significant impact on the residence time of the toxin in bloom indicating photodegradation is an important and perhaps the dominant sink of the toxin in coastal seawater. iii ACKNOWLEDGEMENTS I would like to thank my advisors, Drs. Robert Kieber, Stephen Skrabal, and Jeffrey Wright for their relentless support and guidance on this research. I would like to thank Dr. Kieber for making this process less financially stressful. Also, to my mentor, Thomas Schuster, for teaching me the inner workings of mass spectrometry as well as laboratory practice, and for also being a great friend. Thanks especially to Carrie Miller, Andrea Bourdelais, and Ryan Van Wagoner for their patience and support with my random questions throughout the past year. Also, I’d like to thank the MACRL group for entertaining me and letting me entertain them during those long days at the laboratory. Without this group, this experience would not have been nearly as fulfilling and enjoyable. Finally, thanks to the Marine Science department for truly providing me with a well- rounded, challenging, and intriguing education. iv LIST OF TABLES Table Page 1. Percent sample recoveries of various tested cartridges and eluents. .....................11 2. Parameters used in analytical method of liquid chromatography mass spectrometry a) Gradient used for liquid chromatography analysis b) Source parameters for mass spectrometry ...........................................12 3. Pseudo-first order rate constants of PbTx-2 in 0.2 µm-filtered Wrightsville Beach seawater..................................................................................21 4. Pseudo-first order rate constants of PbTx-2 in UV-irradiated 0.2 µm-filtered Wrightsville Beach seawater ........................................................25 5. Pseudo-first order rate constants of PbTx-1, 2, 3, 6, and 9 in oxygenated and deoxygenated 0.2 µm-filtered Wrightsville Beach seawater ................................................................................32 v LIST OF FIGURES Page 1. Type B (PbTx-2), one of the two parent structural backbones to the family of brevetoxins ...................................................4 2. Intensity of wavelength vs. wavelength of solar simulator versus natural sunlight at high noon 35ºN ...............................................................8 3. Counts per second as a function of concentration of PbTx-2 in a typical liquid chromatography mass spectrometry calibration curve.....................................................................................................13 4. Structures of common brevetoxins used in photolysis ....................................16, 17 5. a) Loss of PbTx-2 concentration of light exposed and dark controls as a function of irradiation time (hours) of one representative experiment. b) Natural logarithm of the first order rate constant of PbTx-2 in Wrightsville Beach seawater as a function of concentration ....................................................................................20 6. Loss of PbTx-2 concentration (m/z 895) and suspected photoproduct (m/z 911) as a function of irradiation time ......................................22 7. a) Loss of PbTx-2 concentration of light exposed and dark control samples as a function of irradiation time (hours) of one representative experiment in UV-irradiated 0.2 µm-filtered Wrightsville Beach seawater b) Natural logarithm of PbTx-2 concentration as a function of irradiation time in UV-irradiated 0.2 µm-filtered Wrightsville Beach seawater ........................................................24 8. a) The concentration of light exposed and dark control samples of PbTx-2 in trace metal clean 0.2 µm-filtered Wrightsville Beach seawater as a function of irradiation time..............................27 9. Concentration of light exposed and dark control samples in deoxygenated 0.2 µm-filtered Wrightsville Beach seawater as a function of irradiation time.............................................................................29 10. a) Concentration of light exposed samples in deoxygenated 0.2 µm-filtered Wrightsville Beach seawater as a function of irradiation time (hours) in one representative experiment. b) Pseudo-first order rate constant of light exposed samples (PbTx-1, 2, 3, 6, 9) as a function of time in untreated filtered WBSW.................31 11. The loss of PbTx-2 onto particles in the dark as a function of time in unfiltered and filtered WBSW. The loss of PbTx-2 -1 to particles in unfiltered water is kunf = 0.02 ± 0.002 h ........................................34 vi INTRODUCTION The marine dinoflagellate, Karenia brevis,

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