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Title:
Oceanic interfaces investigations of biogeochemical changes across nutriclines and frontal boundaries
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Book
Language:
English
Creator:
Adornato, Lori R
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects / Keywords:
North Pacific
Subtropical front
Subtropical gyre
Oligotrophic
Spectrophotometric elemental analysis system
Nitrite
Nitrate
Phosphate
Nanomolar
Liquid core waveguide
Dissertations, Academic -- Marine Science -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Biogeochemical changes across oceanic interfaces, and method development to study such changes, are described in this work. The interfaces studied include the Subtropical Front in the Pacific Ocean and the boundary at the base of the euphotic zone. Both interfaces are characterized by accumulations of phytoplankton, although the forcing functions that result in increased biomass are distinctly different.The Subtropical Front, located at approximately 30°N in the Pacific Ocean, was detected during a cruise in the summer of 2002 by its diagnostic 34.8 salinity outcrop, in spite of the absence of its associated temperature signature. The front displayed elevated concentrations of large diatoms; Rhizosolenia and Hemiaulus, with concentrations penetrating deeper in the water column south of the front. Rhizosolenia species were dominant on the warmer, high salinity side of the front, while Hemiaulus prevailed on the cooler, low salinity side.^ ^While high cell counts were enumerated by net tows, the elevated biomass was not visible in satellite color imagery. Size fractionated chlorophyll data revealed > 10 um cells were found below 200 m, indicating export of large cells out of the euphotic zone. This confirms observations by other investigators that fronts represent important regions of episodic export, although such export may go undetected if the biomass is not visible in ocean color images. Another region of interest was the narrow layer at the base of the euphotic zone. During stratified conditions, the layer was characterized by a fluorescence maximum, a primary nitrite maximum, and a nutricline. While fluorescence maxima have proven easy to detect using commercial fluorometers, nutrient distributions have proven more difficult.^ ^The Spectrophotometric Elemental Analysis System (SEAS) permitted detection of low concentrations of nitrite, nitrate, and phosphate with nanomolar sensitivity and 1 Hz or better sampling frequency. Using multiple wavelength spectroscopy, the range of nitrate concentrations from 2 nM to 20 uM have been detected. Profiles of nitrite obtained across the North Pacific Subtropical Gyre revealed the close correlation between nitrite and chlorophyll fluorescence maxima, suggesting that the nitrite maximum is formed by phytoplankton when insufficient light is available to permit reduction of nitrite to ammonia.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Lori R. Adornato.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 124 pages.
General Note:
Includes vita.

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aleph - 001921899
oclc - 191221890
usfldc doi - E14-SFE0001891
usfldc handle - e14.1891
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ABSTRACT: Biogeochemical changes across oceanic interfaces, and method development to study such changes, are described in this work. The interfaces studied include the Subtropical Front in the Pacific Ocean and the boundary at the base of the euphotic zone. Both interfaces are characterized by accumulations of phytoplankton, although the forcing functions that result in increased biomass are distinctly different.The Subtropical Front, located at approximately 30¨N in the Pacific Ocean, was detected during a cruise in the summer of 2002 by its diagnostic 34.8 salinity outcrop, in spite of the absence of its associated temperature signature. The front displayed elevated concentrations of large diatoms; Rhizosolenia and Hemiaulus, with concentrations penetrating deeper in the water column south of the front. Rhizosolenia species were dominant on the warmer, high salinity side of the front, while Hemiaulus prevailed on the cooler, low salinity side.^ ^While high cell counts were enumerated by net tows, the elevated biomass was not visible in satellite color imagery. Size fractionated chlorophyll data revealed > 10 um cells were found below 200 m, indicating export of large cells out of the euphotic zone. This confirms observations by other investigators that fronts represent important regions of episodic export, although such export may go undetected if the biomass is not visible in ocean color images. Another region of interest was the narrow layer at the base of the euphotic zone. During stratified conditions, the layer was characterized by a fluorescence maximum, a primary nitrite maximum, and a nutricline. While fluorescence maxima have proven easy to detect using commercial fluorometers, nutrient distributions have proven more difficult.^ ^The Spectrophotometric Elemental Analysis System (SEAS) permitted detection of low concentrations of nitrite, nitrate, and phosphate with nanomolar sensitivity and 1 Hz or better sampling frequency. Using multiple wavelength spectroscopy, the range of nitrate concentrations from 2 nM to 20 uM have been detected. Profiles of nitrite obtained across the North Pacific Subtropical Gyre revealed the close correlation between nitrite and chlorophyll fluorescence maxima, suggesting that the nitrite maximum is formed by phytoplankton when insufficient light is available to permit reduction of nitrite to ammonia.
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Oceanic Interfaces: Investig ations of Biogeochemical Changes Across Nutriclines and Frontal Boundaries by Lori R. Adornato A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy College of Marine Science University of South Florida Major Professor: Robert H. Byrne, Ph.D. Peter R. Betzer, Ph.D. David J. Hollander Ph.D. Mary Jane Perry, Ph.D. Edward S. Van Vleet, Ph.D. Date of Approval: March 15, 2007 Keywords: North Pacific, Subtropical Front, Subtropical Gyre, oligotrophic, Spectrophotometric Elemental Analysis Syst em, nitrite, nitrate, phosphate, nanomolar, liquid core waveguide Copyright 2007 Lori R. Adornato

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Dedication I would like to dedicate this work to my husband, Brian, and my son, Sean, who make everything possible.

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Acknowledgements I am truly grateful to my advi sor, Dr. Robert Byrne, for his encouragement, support and insight. I also thank Eric Kaltenbacher for his engineering genius and friends hip. I have been fortunate to work with two such fine people. I am indebted to Tracy Villareal for his generosity as the chief scientist on the RoMP 2002 cruise and to his willingne ss to serve as committee chair at my defense. He has been a wonderful collabo rator and co-author. My committee has also been very helpful. Id like to thank Dean Peter Betzer in particular. He has always championed the work of the Co llege of Marine Science students. To the great group of people who have participated in the SEAS cruises, I owe a debt of gratitude. Id like to specifically thank Kelly Quinn, Xuewu Liu, Regina Easley, Aleck Wang, Graham Til bury, Ryan Bell, Lori Pillsbury, Eva Romero Luna, Jenny Wollschlager, and Johan Schijf for their cheerful assistance at sea, in spite of long hours and often rough sea conditions. Id like to thank my friends and colleagues Danielle Greenhow and Jim Patten for their steadfast support during the past few years. The captains and crews of the R/V Melville, R/V Suncoaster, and R/V Bellows have been very helpful. Finally, Id like to acknowledge th e friends and families of Robert M. Garrels, and Elsie and William Knight for financial support via endowed fellowships; and the Office of Naval Research (Grants N00014-96-1-5011 and N00014-02-0823) for funding the SEAS project.

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Note to Reader: The original of th is document contains color that is necessary for understanding the data. The original dissertation is on file with the USF library in Tampa, Florida.

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i Table of Contents Table of Contents i List of Figures iii Abstract viii 1 Introduction 1 1.1 Subtropical Front 1 1.2 Euphotic Zone 3 1.3 SEAS Instrument 6 2 Continuous in-situ determinati ons of nitrite at nanomolar concentrations 15 2.1 Abstract 15 2.2 Introduction 16 2.3 Methods 20 2.4 Results and Discussion 26 2.4.1 SEAS performance characteristics 26 2.4.2 Nitracline Measurements 29 2.5 Conclusions 34 3 Physical and biological features of the North Pacific Subtropical Front in summer 36 3.1 Abstract 36 3.2 Introduction 38 3.3 Materials and methods 41 3.3.1 Conductivity, temperature, depth and chlorophyll fluorescence 41 3.3.2 Particulate organic carbon (POC) 41 3.3.3 Diatom abundances 42 3.3.4 Dissolved oxygen and AOU 43 3.3.5 Photosynthetically ava ilable radiation (PAR) 43 3.3.6 Nutrient measurements 43 3.3.7 Chlorophyll a concentrations 44 3.4 Results and discussion 45 3.4.1 Temperature, salinity and density 46 3.4.2 Particulate organic carbon 53 3.4.3 Diatom distributions 55

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ii 3.4.4 Export Production 59 3.4.5 Sea Surface texture 62 3.4.6 30 N summer chlorophyll blooms 63 3.4.7 Nutrient and dissolved oxygen distributions 64 3.5 Conclusions 70 4 High-Resolution In Situ Analysis of Nitrate and Phosphate in the Oligotrophic Ocean 73 4.1 Abstract 73 4.2 Introduction 74 4.3 Experimental Section 77 4.3.1 Apparatus 77 4.3.2 Analysis of Nitrate. 82 4.3.3 Analysis of Phosphate. 86 4.3.4 Analysis of Nitrite. 88 4.4 Results and Discussion 89 4.4.1 Nitrate Measurements. 89 4.4.2 Nitrite Measurements. 92 4.4.3 Phosphate Measurements. 93 Appendices 110 Appendix A: Analytical Procedures Nitrite 111 Appendix B: Analytical Procedures Nitrate 115 Appendix C: Analytical Procedures Phosphate 119 Appendix D: SEAS Cruise Locations 123 About the Author End Page

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List of Figures Figure 1.1 Map of Eastern Centra l North Pacific showing general locations of the Subarctic, North Subtropical, and Subtropical Fronts (Lynn, 1986). 2 Figure 1.2. Diagram of euphotic zone showing depth of 1% light level and the location of the nutricline. 3 Figure 1.3. 3-D diagram of SEAS I. 7 Figure 1.4. Total internal reflection occu rs when light is introduced at an angle greater than the critical angle. Light introduced at the critical angle propagates down the waveguide/water interface, and light at less than the critical angle is lost from the system. 8 Figure 2.1. Profiles obtained on WOCE transect p03hy provided general upper and lower bounds for the depth of the PNM. Standard spectrophotometric methods precluded measurement of low nanomolar concentrations in the upper water column. 18 Figure 2.2. Geographic locations of SEAS casts during the RoMP 2002 cruise between June 20 and July 16, 2002 on the R./V. Melville 20 Figure 2.3. Schematic diagram of the SEAS instrument (11.5 cm diameter, 50 cm long). The pressure housing (rated to 500 m) contains the pump motors, lamp, sp ectrometer, and electronics. The reagent reservoir, sample intake, pump heads, and waveguide are exterior to the pressure housing. Two optical fibers are used to transmit light from the lamp into the waveguide, and from the waveguide to the spectrometer. A 12-V battery and Falmouth CTD are connected to SEAS by a wate rproof cable. Sp ecifications and additional operational characteristic s of the SEAS instrument have been previously detailed by Kaltenba cher et al. (Kaltenbacher et al., 2000) and Steimle et al. (Steimle et al., 2002). 23 Figure 2.4. A typical co ntinuous flow calibration obtained using the SEAS instrument with commercial sulfanilamide solution (LabChem). 24 iii

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iv Figure 2.5. Comparison of in situ da ta and laboratory measurements of bottle samples for station 14. The upcast for the SEAS instrument preceded the rosette cast by about tw o hours. Diamonds represent in situ data and circles represent data from bottle samples. The four discrete points below 100 m diffe r in depth from the corresponding in situ concentrations by an average of 1.3 0.9 m. 26 Figure 3.1. Underway salinity map for the Rhizosolenia Mats in the Pacific (RoMP) 2002 cruise constructed from over 52,000 latitude/longitude/salinity observation s. Station locations are shown in subsequent figures so that th e underway frontal salinity changes are not obscured. 45 Figure 3.2. Curtain plot of salinit y distributions (surface to 300 m depth) embedded in the cruise map shows the high degree of variability observed over the course of the cruise. Data from a total of 34 casts were used to generate the plot. 46 Figure 3.3. Distributions of temperat ure ( C) from the surface to 300 m. Due to summer insolation, temperature displays much less variability than that of salinity. Location of the Subtropical Front by AVHRR or MODIS satellite data dur ing summer months is rendered impossible by the disappearance of diagnostic temperature outcrops. 47 Figure 3.4. Photograph of the Subt ropical Front obtained at 29.564 N, 138.867 W (between Stations 20 and 21) demonstrates the sharp change in surface texture ofte n observed at frontal boundaries. Arrows in the photograph indicate the location of the front, which was visible over a great distance. Underway data highlight the abrupt salinity change that occurred at the fr ontal boundary depicted in the photograph. 49 Figure 3.5. Salinity distributions between Station 13 (30.154 N, 145.592W) and 14 (30.655N, 143.801W), and between 20 (29.125N, 138.312W) and 21 (29.900 N, 137.420 W) provide examples of both the magnitude of change across frontal stations and the profile of variability in frontal salinity distributions. 51 Figure 3.6. Curtain plot of sigma-t from the surface to 300 m depth shows shoaling of the pycnocline ne ar the front. The shallowest pycnocline occurs at the station (1 0) nearest the satellite-observed chlorophyll bloom. While the dept h of the pycnocline varies across the transect, the vertical density structure is highly stable. 52

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v Figure 3.7. Particulat e organic carbon ( g/L) distributions from the surface to 300 m reveal low concentr ations in the gyre and elevated levels near the front. High POC con centrations penetrate deeper into the water column on the southern, high salinity side of the front compared to those at northern, lower salinity stations. 53 Figure 3.8. Vertical distributions of Hemiaulus and Rhizosolenia cells plotted over salinity contours show the aversion of Hemiaulus to low salinity. Note the similarity of POC and cell density distributions. 57 Figure 3.9. A) Photograph of a Pacific Hemiaulus bloom taken near the sea surface by a diver during the RoMP 2003 cruise. While the photograph was not taken during this study, it is representative of the level of marine snow observed during an intense Hemiaulus bloom. 59 Figure 3.10. Elevated chlorophyll con centrations were measured near the front, although a corresponding increase in in situ fluorescence was not observed. Size-fractionated chlorophyll data indicated that high percentages of large (> 10 m) cells were found below 175 meters, particularly in the region with high Hemiaulus concentrations, indicating enhan ced export in the diatom bloom region. 61 Figure 3.11. Nutrient contours reflect low concentrations observed in the NPSG and upwelling in frontal regions and the California Current. Elevated silicate concentrations were found above the nitracline, particularly near the fr ont, thereby favoring diatom species that are capable of nitrate transport or that possess diazotrophic endosymbionts. 66 Figure 3.12. Vertical distributions of (A) fluorescence, (B) POC and (C) AOU for gyre casts 5-4 and 5-6 show the remarkable differences between data collected 5 hours and 1 nautical mile apart. Unusually high fluorescence, POC, and AOU ob served from 60 m to 125 m during cast 5-4 data suggests a high level of respira tion associated with collapse of a phytoplankton population. Distributions of fluorescence, POC and AOU at station 5-6 were representative of those found at other gyre stations. 69 Figure 4.1. A. A 3D model of the SEAS II instrument. Portions of the instruments protective housings have been removed to reveal inner components. 78

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vi Figure 4.2. Absorbance measurements for a nitrate standard solution were conducted using a single buffe r solution with a variety of sulfanilamide to NED ratios. A combined solution ratio of 5:1 was selected as it provided the highest absorbance and the least variability with changes in mixing ratios. 83 Figure 4.3. A. A schematic diagram of the nitrate procedure. In addition to the sample and reagent pumps, a main pump is used to pull the combined solution through the system. The cadmium column produces sufficient back-pressure that erratic flow rates can result if the main pump is not active. 85 Figure 4.4. Calibration curves produ ced during the same experiment show the differences between the ini tial parabolic result (a), and the linear result when the reaction is permitted to proceed for an additional 105 seconds (b). 87 Figure 4.5. A. Normalized spectrum of the azo dye produced in both the nitrate and nitrite chemistries shows relative absorbance as a function of wavelength. 90 Figure 4.6. Nitrate profile obtained in the Gulf of Mexico on November 14, 2006, 10:26 a.m. (2721.237 N, 84.510 W) constructed from multiple wavelengt h (543 nm [blue], 575 nm [red], 590 nm [green], and 600 nm [aquamarine]) absorbance spectroscopy corresponds well with discrete samples collected two hours later (black). 91 Figure 4.7. Nitrate and nitr ite profiles collected concurrently show the tight coupling observed between the nitracline and nitrite inflection (27.901 N, 84.482 W, November 15, 2006, 3:53 a.m.). 93 Figure 4.8. Intensity at 700 nm plot ted as a function of temperature shows the rapid signal attenuation that occurs at the 16.2C isotherm. Data were collected August 4, 2005, 6:40 a.m. (27.333 N, 84.806 W). 94 Figure 4.9. Phosphate profile using a heater cartridge set at 30 C (27.777 N, 84.628 W, August 10, 2006, 12:38 a.m.) compares well with bottle samples collected from the ships rosette. 96 Appendix Figure 1 Reaction of nitrit e with sulfanilamide in acidic medium to form an intermediate species which subsequently reacts with NED to form the azo dye product. 113

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vii Appendix Figure 2 Nitrite undergoes dr amatic changes over a diel cycle (November 15-16, 2005, 27 23.738 W, 84 50.238 W). Depth is from 0 to 250 meters and nitrite conc entration is from 0 to 250 nM. The color of the background indicate s time of day, with the darkest at midnight and the lightest at noon. 114 Appendix Figure 3 Calibrations conducted during and after the November 2006 cruise show main tenance of cadmium reductor efficiency. 118 Appendix Figure 4 Optimization of reagent to sample ratios is accomplished by varying the reagent pump speed while the main pump speed is held constant (25 rpm). 120 Appendix Figure 5 The Keggin structur e includes a phosphorus atom at the center surrounded by ten molybda te and two antimonate ions, each in an octahedral structure. Spheres represent oxygen atoms. Figure from Barteau et al. (Barteau et al., 2006). 121 Appendix Figure 6 Map of the West Fl orida Shelf with the locations of the August 2006 and November 2006 cruises marked with pin icons. 124

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viii Oceanic interfaces: Investigat ions of biogeochemical changes across nutriclines and frontal boundaries Lori R. Adornato Abstract Biogeochemical changes across oceanic interfaces, and method development to study such changes, are de scribed in this work. The interfaces studied include the Subtropical Front in the Pacific Ocean and the boundary at the base of the euphotic zone. Both interf aces are characterized by accumulations of phytoplankton, although the forcing functions that result in increased biomass are distinctly different. The Subtropical Front, located at ap proximately 30 N in the Pacific Ocean, was detected during a cruise in the summer of 2002 by its diagnostic 34.8 salinity outcrop, in sp ite of the absence of its associ ated temperature signature. The front displayed elevated conc entrations of large diatoms; Rhizosolenia and Hemiaulus with concentrations penetrating deeper in the water column south of the front. Rhizosolenia species were dominant on the warmer, high salinity side of the front, while Hemiaulus prevailed on the cooler, low salinity side. While high cell counts were enumer ated by net tows, the elevated biomass was not visible in satellite color imagery. Size fractionated chlorophyll data revealed > 10

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ix m cells were found below 200 m, indicati ng export of large cells out of the euphotic zone. This confirms observati ons by other investigators that fronts represent important regions of epis odic export, although such export may go undetected if the biomass is not visible in ocean color images. Another region of interest was the narr ow layer at the base of the euphotic zone. During stratified conditions, the layer was characterized by a fluorescence maximum, a primary nitrite maximum, and a nutricline. While fluorescence maxima have proven easy to detect us ing commercial fluorometers, nutrient distributions have proven more difficult. The Spectrophotometric Elemental Analysis System (SEAS) permitted detection of low concentrations of nitrite, nitrate, and phosphate with nanomolar se nsitivity and 1 Hz or better sampling frequency. Using multiple wavelength spectroscopy, the range of nitrate concentrations from 2 nM to 20 M have been detected. Profiles of nitrite obtained across the North Paci fic Subtropical Gyre reveal ed the close correlation between nitrite and chlor ophyll fluorescence maxima, suggesting that the nitrite maximum is formed by phytoplankton when insufficient light is available to permit reduction of nitrite to ammonia.

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1 1 Introduction Oceanic interfaces, defined for this work as narrow regions with abrupt changes in seawater properties, provide biological niches whic h can be exploited by a variety of organisms. Such interf aces can be caused by changes in salinity, temperature, density, chemical gradients, or light. The two explored in this work are (1) the North Pacific Subtropical Fr ont, characterized in the summer by an abrupt change in salinity; and (2) the base of the euphotic zone, characterized generally by light limitation, a nut ricline, and a pycnocline. 1.1 Subtropical Front The Subtropical Front spans the centra l north Pacific at approximately 30 N. In the winter months, the front is characterized by 34.8 salinity and 18 C (Roden, 1980) (or 17 C (Seki et al., 2002)) temperature outcrops, but in summer months the 18 C temperature outcrop migrat es ~10 latitude to the north. The Transition Zone Chlorophyll Front (TZC F), defined as the edge between low chlorophyll (<0.15 mg/m3) surface waters and the 0.2 mg/m 3 chlorophyll surface signature (Polovina et al., 2001) tends to o ccur at approximately the same latitude as the 18C outcrop and migrates seasonally as well. Other semi-permanent fronts in the region include the South Subtropical Front (35 salinity and 20 C winter

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outcrops, ~28N) (Seki et al., 2002) and the Northern Subtropical Front (34.4-34.5 salinity and 16 C winter outcrops, ~34 N (Lynn, 1986). North of the transition zone lies the Subarctic Front which can be determined by the southern extent of the 33.8 surface isohaline (Lynn, 1986). In the absence of satellite-visible surface temperature or chlorophyll signatu res, summer front locations must be determined using in situ techniques. Subtropical Front North Subtropical Front Subarctic Front Subtropical Front North Subtropical Front Subarctic Front Figure 1.1 Map of Eastern Central North Paci fic showing general locations of the Subarctic, North Subtropical, and Subtropical Fronts (Lynn, 1986). While a general location for a front ca n be determined, the actual structure can be very dynamic. Eddies, meanders and jets typify frontal boundaries (Kase and Siedler, 1982). In the northern hemi sphere, cyclonic eddies are generally found to the north of the front and anticycl onic eddies to the south (Roden, 1981). The temperature/salinity structure across the Subtropical Fron t is often almost completely density compensated (Rudnick and Ferrari, 1999). 2

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Intensification and weakening of fr ontal boundaries (frontogenesis and frontolysis) occur on time scales of ~ 30 days which provides sufficient time for biological response (Olson et al., 1994). Increased pr oductivity can occur when phytoplankton and nutrients are upwelled into ar eas of increased light intensities. 1.2 Euphotic Zone The euphotic zone is defined as th e region between the water surface and the depth of the 1% surface light level. Because this value does not take into account the amount of light actually reach ing phytoplankton in the water column (see Banse, 2004), it can only re present an estimate based on water clarity. On a cloudy day, for instance, the euphotic zone may be much shallower than the 1% light level; or much deeper if 1% inci dent light is much greater than the compensation depth. Depth Nutrient Concentration (NO3 -, PO4 3-) Euphotic Zone Depth Nutrient Concentration (NO3 -, PO4 3-) Euphotic ZoneNutricline 1% Light Level Depth Nutrient Concentration (NO3 -, PO4 3-) Euphotic Zone Depth Nutrient Concentration (NO3 -, PO4 3-) Euphotic ZoneNutricline 1% Light Level Figure 1.2. Diagram of euphotic zone showing depth of 1% light level and the location of the nutricline. 3

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4 For the purpose of this discussion, the base of the euphotic zone is assumed to occur over the range of depths encompassing the deep chlorophyll maximum and the nutricline in stratified waters. Both of these phenomena are thought to be light-related. In the spring, increased insolation sp urs the spring bloom, since winter mixing introduces nutrients to the up per water column. During the summer months, as upper euphotic zone nutrients are depleted and the water column becomes stratified, near-surface chlorophy ll concentrations drop to very low levels (< 0.15 mg/m 3 ). The summer chlorophyll maxi mum then occurs at the top of the nutricline, which is often loca ted near the 1% light level and the pycnocline. The deep chlorophyll maximum (DCM) is comprised primarily of phytoplankton adapted to low light levels (Fennel and Boss, 2003; Letelier et al., 1993). In the oligotrophic open ocea n, phytoplankton community structure is dominated by the pico size fraction (0.2 2 m), since their high surface to volume ratio provides an advantage under low nutrient conditions. The dominant phytoplankton genus in the oligotrophic North Pacific Subtropical Gyre is Prochlorococcus, with the water column partit ioned between low-light and highlight species (Moore et al., 2002). The DC M is also comprised of a variety of picoeucaryotes, although their size ma kes them extremely difficult, if not impossible, to identify to the sp ecies level (Campbell et al., 1997). Depth distributions of chlorophyll ar e typically determined by collecting discrete seawater samples via a ships rosette, filtering the samples, extracting the

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5 pigment, and determining the chlorophyll concentrations using fluorometry. The values are plotted against depth to fo rm a profile. As with any other method utilizing discrete bottle samples, the resolution is limited by the number of bottles on the rosette. In situ fluorometers provide another tool for determining the depth of the DCM, but the fluorescence maximum and the extracted chlorophyll maximum often occur slightly apart, with the chlorophyll maximum slightly shoaler in the water colum n. In addition, fluorescen ce profiles obtained during daylight hours are affected by suninduced quenching of near-surface fluorescence (Kolber and Fal kowski, 1993). Nevertheless, these effects are welldocumented and fluorometers provide prof iles with much higher resolution than can be obtained with bottle samples. While the DCM suggests a phytoplankton biomass maximum, this is not necessarily the case. Phytoplankton resi ding near the surface do not require as much chlorophyll as those living deeper in the water column so the chlorophyll content per cell changes with depth (Falkowski and Owens, 1980). This proves particularly troublesome with the pico size-fraction, and Prochlorococcus in particular (Monger and Landry, 1993). The near-surface population of Prochlorococcus is often counted as bacteria. Transmissometers can aid in dete rmining the depth distribution of microscopic organisms, however they cannot distinguish between bacteria, phytoplankton, small heterotrophs, and detr ital particulate organic carbon. Because of the high ratio of phytoplankton relative to grazers, and because the vertical distribution of bacteria tends to be somewhat constant in the euphotic

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6 zone, transmissometer data can provide clues to the distribution of phytoplankton populations. Another feature commonly found near the DCM is the primary nitrite maximum. Nitrite represents an intermed iate species in the oxidation-reduction cycle between nitrate and ammonia. Nitr ate levels are often below the detection limits of standard spectrophotometric systems in the euphotic zone, but quickly rise to micromolar levels at the nitraclin e below the DCM. Nitrite concentrations are similarly depleted in near-surface wate rs, but rise to a narrow spike just below the nitracline. Study of n itrite concentration changes versus depth and time have been hampered by discrete sampling stra tegies and the high detection limits of standard spectrophotometric systems. Use of in situ long pathlength spectrometry has provided a means to study nutrient dyna mics in the oceanic water column. 1.3 SEAS Instrument The first Spectrophotometric Elemen tal Analysis System (SEAS) was designed as a single reagent in situ nutrient analyzer (Byrne et al., 2000; Waterbury et al., 1998). It provided distinct advantages over other nutrient sensors because of its sensitivity, multiple wavelength capability, and depth rating. Spectrophotometry is based on the reaction of essentially clear, colorless reagents with a specific an alyte in solution. The colo red product of the reaction absorbs light over a known range of wavele ngths and this absorbance is directly proportional to the concentration of the analyte in solution. Some reagents do absorb in the visible region, and such abso rbance can result in elevated baseline

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measurements, termed the reagent blank. This blank, if well-characterized, can be subtracted from the apparent analyte concentr ation without seri ous consequence, but this is often difficult to achieve. Figure 1.3. 3-D diagram of SEAS I. Spectrophotometry relies on the relationship esta blished in the Beer Lambert Law, A = bc, Equation 1.1 where A is the wavelength dependent absorbance, is the molar absorptivity (M 1 cm -1 ), b is pathlength (cm), and c is analyte concentration (M). Standard 7 spectrophotometric systems use a 1 10 cm pathlength, providing a detection

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8 ell terface, 1.2 1e ind veguide ce greater than the critical a ngle. Light introduced at th e critical angle propagates t limit on the order of 0.05 1.0 M, depending on the molar absorptivity of the product. SEAS provides lower detection li mits because it uses a novel optical c (0.040 o.d. x 0.032 i.d.) made of a flex ible fluoropolymer (Teflon AF 2400) with a refractive index (1.29) less than wa ter (1.33) and seawater (1.34). The lower index of refraction permits the Teflon tube to perform as a liquid core waveguide, with water acting as the core (Fig. 1.2). Snells Law describes refraction through an in n 1 sin 1 = n 2 sin 2 Equation where n and n 2 are the refractivices of the two media (i.e. wa material and liquid), and 1 and 2 are the angles of light relative to the surfa normal (Fig. 1.2). Figure 1.4. Total internal reflection occurs when light is introduced at an angle down the waveguide/water interface, and light at less than the critical angle is los from the system.

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9 hen the equation is rearranged and solved for 2, W 11 1 2sin sin n 2nEquation 1.3 the angle of refraction through the second m value in the an c 90 and e dium is valid when the parentheses is equal to or less th an one. The critical angle, c is the angle of incidence ( 1 ) for which the value in the pa rentheses equals one, whereby 2 equals 90. Thus, light introduced at th e critical angle is refracted to travel parallel to the waveguide/wat er interface. Light incident at angles greater th will experience total internal reflection and will propagate solely in the liquid core. Light incident at angles less than c will be refracted into the tube wall. The value for the critical angle can be determined by setting 2 equal to solving for 1 : Csin 1 2 n n Equation 1.4 In the case of water-filled less than 15 d the ty), waveguide, light introduced at an angle relative to the water/waveguide interface (or greater than 75 relative to the normal) is totally internally reflected. Using the refractive index values liste above, the critical angle for the wave guide/water system is 75.9 and for waveguide/seawater is 74.3. However, due to uncertainty that exists for refractive index values (e.g. the refractive index of seawater depends on salini an approximate value of 75 is used throughout this manuscript.

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10 As an added complexity, differences occur between use of the waveguide in air and in water. This is because the refractive index of the material external to the waveguide affects the fate of light th at passes through the wa veguide material. When the waveguide is used in air, the refractive index of air (1.0, which is lower than that of the waveguide material) ca n cause total internal reflection at the waveguide/air interface. This reflected light can re-ent er the waveguide core and actually reach the detector at the end of the waveguide. Since this light has not traveled solely through the liquid core, its attenuation is distinct from that of the liquid, providing an error on th e overall absorption measurement. However, this error is only problematic when absorban ce values associated with chemical concentration becomes very low. In situ the waveguide is surrounded by water and any light that is refracted into the waveguide is subsequently refracted into the water and lost to the system, thus eliminating this source of error. Another effect that can alter the abso rbance signal is the loss of light due to scattering. This is most apparent when particulates are present in a water sample. High turbidity samples, for instance, can cause partial or total attenuation of the signal. While inlet filters may be used, they tend to clog over time, resulting in a reduction of sample flow. When this occurs, the sample to reagent ratio changes and system calibrations are altered. SEAS is therefore best suited for limpid limnological system s and the oligotrophic open ocean. The spectrometer used in the SEAS in strument provides the capability to monitor multiple wavelengths anywhere from 350 850 nm. This provides two main benefits. The first is the ability to monitor a non-absorbing wavelength.

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11 by nce Since most colored products abso rb over a known and limited range of wavelengths, there may exist a range of wavelengths that are unaffected chemical reactions. Such wavelengths can be monitored for changes in cell performance to make appropriate correc tions to concentration calculations. Absorbance changes from such sources can be removed from sample absorba calculations as follows, ng nonabsorbi dark dark sample dark darkII II II II A 2 002 1 001log log Equation 1.5 where 1 is the sample wavelength and 2 is the non-absorb ing wavelength. The ity e rometer is the ability to perform multiple wavelength spectroscopy. Depending on the absorbance dark value (I dark ) is a measurement from a detector element, masked from all sources of light, which represen ts background electrical noise. I 0 is the intens without reagent (reference in tensity), and I is the inte nsity with reagent (sample intensity) of the various wa velengths monitored. The fi rst term represents the absorbance measured at the sample wa velength and the second is the absorbanc measured at the nonabsorbing wavelength. This second term is an effective absorbance related to changes in intens ity from sources unrelated to analyte absorbance. Possible sources include air bubbles, lamp drift, inherent optical properties of the sample solution, and physical effects that can change the efficiency of light propaga tion through the waveguide. The second advantage of a broad-spectru m spect

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12 spectru are r ded. ded motors were operational. as in pressure housings. The individual pump pressure housings ere fi m of a given colore d product, there exists a range of wavelengthdependent molar absorptivities that can be used when various wavelengths monitored. In the case of the azo dye formed in nitrate analysis, the mola absorptivities vary by orders of magnitude. When multiple wavelengths are monitored concurrently, the linear dynamic range of the system can be exten Although the pressure housing was ra ted to 500 meters, SEAS I provi reliable data to approximately 200 meters depth. The primary source of difficulty in the SEAS I system was the pump configuration. The main pump tended to lose torque under the pressures experienced belo w 200 meters. This resulted in either reduced pump speed or stoppage at de pth. Fewer problems were experienced with the stepper motor used for the dye pump. The other difficulty associated with the pumps was the possibility of seawater leakage along the pump shafts when the Because the pump assemblies penetrated into the main pressure housing, the possibility of catastrophic failure was alwa ys a concern. While every effort w made to avoid such events, the occurr ence of leakage was frequent enough to require a solution. Reconfiguration of the instrument (SEAS II) resulted in separation of pumps from the ma wlled with oil to reduce the pressu re differential between the ambient and the internal chamber. This solution ha s proven less satisfactory than anticipated, and a move towards a system where the pump shaft does not penetrate into the pressure housing is underway.

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13 grated ba ttery housing, a heater cartridge, valves ed blished as a single er e cts are two-fold. The first effect is Additional improvements included conn ectivity to auxiliary instruments improved user interface, an inte and the ability to use multiple pumps. The ability to use SEAS II to collect data from a suite of peripheral instruments stre amlined post-cruise data analysis since auxiliary data could be collected on the same cast as the SEAS instrument. In prior instances, data had to be adjusted for differences in water column hydrology between casts. The ability to select peri pheral instruments from a list has allow the collection of high-resoluti on custom-designed data sets. The other main advantage of SEAS II is the capability of quickly and easily changing instrument configurations. SEAS I was esta analyte system. SEAS II was designed for ease in programmability. This was accomplished by creating a list of user comm ands so that the user could create and store a variety of methods. During s ea trials, if one instrument failed, anoth could quickly be converted to measure th e analyte of interest without opening th pressure vessel or repl acing electronic boards. Another consequence of deep water sa mpling is the range of temperatures experienced over the course of a cast. The effe that of reduced temperature on the instrument itself. A ny defect in the electronics of the instrument is exacerbated by a reduction in temperature. This has been observed in improperly soldered pins and the fit of an incompatible connector. Both operate well under room temperature conditions but lose electronic connectivity when the metal pins cont ract under low temperature conditions. Diagnosing such problems is difficult because they do not occur in the lab

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14 It Typica oth ics Particularly, the ea rliest version of SEAS is discussed in Chapte The second effect is that of reduced temperature on reaction chemistry is common for reaction kinetics to cha nge as a function of temperature. lly, reactions slow as temperatures dr op. Another effect is the solubility of reagent components under various temper ature and salinity conditions. B effects can drastically alter the apparent c oncentration of the an alyte of interest. The addition of a heater unit provided a so lution to the issues of reaction kinet and reagent solubility. Further details regarding SEAS confi gurations and uses are described in the following chapters. r 2 and the reconfigur ed version, SEAS II, is described in Chapter 4. Nutrient chemistries successfully adapted to in situ SEAS analyses are presented in these chapters as well as in the appendices.

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15 2 Continuous in-situ determinations of nitrite at nanomolar concentrations The following chapter has been peer-review ed and published essentially in this form: Adornato, L.R.; Kaltenbacher, E.A.; Villareal, T.A.; and Byrne, R.H. (2005) Deep-Sea Research I 52, 543-551 2.1 Abstract Sharp gradients of chemical distributions in the nutricline are poorly resolved via conventional sampling techniques. Resolution of the fine structure of chemical distributions in the wa ter column requires the use of in situ procedures. We describe here a high-resolution me thod for the measurement of nitrite concentrations in the upper 200 meters of the water column. A long-pathlength Teflon AF-2400 liquid core waveguide pr ovides the low nanomolar detection limits required for observations of nitrite in near-surface waters. Our spectrophotometric elemental analysis sy stem (SEAS) has a two second sampling period. Coupled with an 11 cm/s descent rate, SEAS is able to accurately identify the depth of the primary nitrite maximu m and provide a detailed inventory for nitrite in the upper ocean.

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16 2.2 Introduction Recent investigations of phytoplankt on niche partitioning and thin layer formation have changed the requirements of the oceanographic community with regard to measurement of chemical di stributions in the water column. Many phytoplankton species thrive in narrow environments defined by factors such as light level, nutrient avai lability, temperature, and physical forcing (CavenderBares et al., 2001; Deksheni eks et al., 2001; DuRand et al., 2001; Franks, 1995; Riegman and Kraay, 2001). Genomic studies of the related genera Synechococcus and Prochlorococcus reveal very different mechanisms for the transport and usage of nutri ents, reflecting adaptation to specific niches found in the euphotic zone (Rocap et al., 2003). In conjunction with st udies of microbial distributions, it is also cr itical to understand both the detailed water column chemical distributions that favor the dominance of certa in species and the effect of organisms on these distributions. Op timally, a suite of instruments collecting chemical profiles would serve this pur pose, however, only dissolved oxygen is routinely monitored in situ Standard sampling protocols require wa ter collection at discrete depths, and analysis of casts that typically co ntain from 12 to 36 samples spanning several hundred to several thousand me ters. While some shipboard nutrient analyses are prompt, a great deal of sample processing occurs either post-cruise or on a time scale that precludes further ad aptive on-site investigation. Once the samples are analyzed, concentrations are plotted against dept h, and profiles are

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17 generated by linear interpolation. While this method provides general assessments of chemical gradients at th e nutricline, it cannot resolve important small-scale variations in chemical distri butions that are required to more fully understand phytoplankton community structure. Characterization of nitrite distributions in the upper ocean is particularly challenging. Except for a narrow concentra tion spike at the base of the euphotic zone, nitrite is generally present at low nanomolar le vels. As a result, the distribution of nitrite obtai ned using standard sampli ng techniques is coarsely represented (Fig. 2.1). Furthermore, using conventional spectrophotometric procedures, wherein pathlengths are less than or equal to 10 cm, near surface concentrations are ofte n undetectable (Fig. 2.1). Methods for the determination of nitrogen species at low nanomolar concentrations have been develope d only recently (Dore and Karl, 1996b; Garside, 1982; Yao et al., 1998). While desktop chemiluminescence analysis and long-pathlength spectrometry provide the required sensitivity for observations of low nutrient concentrations, characterizati ons of nutrient distributions are still limited by sample collection methodology (Dore and Karl, 1996b). In addition, nitrite can oxidize to nitr ate over time, so analyses should either be conducted in situ or as soon as possible after sampling (Goyal and Hafez, 1995; van Standen et al., 1996).

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Figure 2.1. Profiles obtained on WOCE tran sect p03hy provided general upper and lower bounds for the depth of the PNM. Standard spectrophotometric methods precluded measurement of low na nomolar concentrations in the upper water column. Steimle et al. (2002) described an in situ long-pathlength, Spectrophotometric Elemental Analysis System (SEAS) capable of real-time nitrite analysis at nanomolar levels. Data acquisition was fast, simple, and free from the nitrox gas contamination suscep tibility that often troubles laboratory analyses of nitrogen species at low na nomolar concentrations. In its initial configuration, the instrument was depl oyed at a number of depths, where it 18

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19 flushed itself with ambient seawater, colle cted dark and reference spectra, added colorimetric reagent, allowed 120 s for color development, and collected five absorbance spectra. The data were uploaded post-cast and the apparent concentrations were determined base d on a stopped-flow calibration line. Although this process produced prompt resu lts with excellent detection limits, the profile suffered from the same dearth of data as previous methods. Johnson et al. (1986, 1989) developed an in situ spectrophotometric instrument capable of continuous analysis of various ionic species in seawater. While the method provided the data density required to determine detailed nutrient distributions, th e detection limit of 0.1 M precluded detection of the low nanomolar nitrite concentrations typi cally found in oligotrophic waters. We present here an alternative m easurement procedure that provides a much more thorough characterization of nitrite distributions in the upper 200 meters of the water column. This method is applicab le to any flow injection colorimetric analysis, as long as signif icant color development occurs within several minutes. Whether or not this requi rement is met for a given analyte is a function of both the analytes in situ concentration and the inherent reaction kinetics of colored species formation.

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Figure 2.2. Geographic locations of SEAS cas ts during the RoMP 2002 cruise between June 20 and July 16, 2002 on the R./V. Melville 2.3 Methods Nitrite profiles were obtained at vari ous stations along a transect on the R./V. Melville during the Rhizosolenia Mats in the Pacific (RoMP) 2002 cruise (June 20 to July 16, 2002) in the North Pacific Subtropical Gyre (NPSG) (Fig. 2.2). Independent conductivity, temper ature, salinity, and fluorescence measurements were obtained using a Sea-Bird SBE9 attached to the ships rosette. PAR Sensors, models QSP-200L and QSR 240L, provided photosynthetically active radiance (PAR) profile s on rosette casts. Locati on of the deep chlorophyll maximum (DCM) was determined from the fluorescence signal, and verified by chlorophyll a measurements. Except as noted, reagent and standard preparati on procedures followed those previously described by Steimle et al. (after Grasshoff, 1983). Initial 20

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21 standard dilutions were prepared with Nanopure Infinity Ultrapure system water (17.8-18.2 M ), and calibration standards were made using uncontaminated seawater from the shipboard flow-thr ough system. A 10.0 mM nitrite standard was prepared by dissolving 0.345g of NaNO 2 in 500ml of deionized water. A pellet of NaOH was added to avoid production of nitrous acid, and 1 ml of chloroform was added as a preservative. The solution was refrigerated (t = 4 C) in a brown glass bottle. A 2 nitrite standard was prepared by dilution prior to each calibration. Calibration standards, prepared by spiking nitrite-depleted surface seawater to 25, 50, 75, 100, and 150 nM nitrite concentrations, were run immediately after preparation. Deionized water and surface s eawater blanks, run prior to each calibration, were indisti nguishable. Surface seawater nitrite concentrations were consistently below th e 1 nM detection limit, defined as three times the standard deviation of the bla nk signal. Standards were analyzed at surface seawater temperatures, which averaged 23.4 1.5 C in the gyre and 18.8 0.4 C in the California Current. The minor influence of temperature on nitrite measurements is discussed in the results section. The analyses utilized the Griess me thod, in which nitrite reacts with sulfanilamide to form a diazonium ion, which subsequently reacts with N(1naphthyl)ethylenediamine, dihydrochlor ide (NED) to form a pink azo dye (maximum absorbance at 541 nm) (Fox, 1979). A 5:1 ratio of sulfanilamide to NED was selected for the color reagent solution. Transmitted light at a nonabsorbing wavelength (700 nm) was monito red to reduce potential error created

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22 by lamp intensity fluctuations or changes in optical cell performa nce. Due to the relationship between concen tration and pathlength in the Beer-Lambert Law, A = bc, Equation 2.1 where A is the wavelength dependent absorbance, is the molar absorptivity (M 1 cm -1 ), b is pathlength (cm), and c is analyte concentration (M); the 97 cm pathlength used for this analysis exte nded the detection limit by an order of magnitude compared to a standard 10 cm spectrophotometric cell. The detection limit, defined at the 95% confidence level, was 1.0 nM, and the linear dynamic range exceeded 500 nM (Skoog et al., 1998). The Type I liquid core waveguide (LCW ) employed in this work is made of a flexible AF-2400 fluoropolymer (Dupont ) with a refractive index (n = 1.29) smaller than that of pure water (1.33) and seawater (1.34) (Abbott et al., 2000; Byrne and Kaltenbacher, 2001). Light introdu ced axially at an angle equal to or smaller than 15 relative to the waveguide/water boundary is internally reflected through the water-filled wave guide and detected by a spectrometer coupled to the waveguide via an optical fiber (Callahan et al., 2002). To lessen possible changes in signal due to movement of the wavegui de as the system traverses the water column, the waveguide is immobilized in a Teflon case (i.d. 7.6 cm) during deployment. An opaque cover over the top of the instrument minimizes stray light. Signal quality was maintained between deployments by flushing the waveguide sequentially with DI water, dilute Micro 90 surfactant/cleaner (Cole-Parmer), DI water, 1.0 M HCl, and DI water. In a ddition to removing any adsorbed material

PAGE 36

from the waveguide, the surfactant aided in the removal of microbubbles trapped in the system. In order to ensure optim al performance of the waveguide, a custom designed interface program monitored abso rbance spectra throughout the cleaning procedure. Figure 2.3. Schematic diagram of the SEAS instrument (11.5 cm diameter, 50 cm long). The pressure housing (rated to 500 m) contains the pump motors, lamp, spectrometer, and electronics. The reagent reservoir, sample intake, pump heads, and waveguide are exterior to the pressure housing. Two optical fibers are used to transmit light from the lamp into the waveguide, and from the waveguide to the spectrometer. A 12-V battery and Falmouth CTD are connected to SEAS by a waterproof cable. Specifications and a dditional operational ch aracteristics of the SEAS instrument have been previously detailed by Kaltenbacher et al. (2000) and Steimle et al. (2002). The instrument (Fig. 2.3) was powered fr om a 12V battery, interfaced with a Falmouth Scientific Inc. CTD (Model MD TD-DBP-D), and secured on a custommade frame. During a programmed 5-minute delay time, the instrument was lowered to 10 m depth, where it pumped ambient seawater for 270 seconds. The 23

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pressure at 10 m collapsed any bubbles re maining in the waveguide. After dark and reference spectra were taken, the r eagent pump was activated for 80 seconds prior to initiation of data collection. After this period, the instrument was lowered at a speed of seven m/min while data were collected every two seconds. To ensure consistent mixing between sample and reagents, the main pump and reagent pump were programmed to operate at constant speeds for the duration of the deployment. Figure 2.4. A typical continuous flow calib ration obtained using the SEAS instrument with commercial sulfanilamide solution (LabChem). Since the instrument was deployed in a continuous flow mode, the in situ nitrite concentration was determined by running a calibration in the lab immediately before or after each deploy ment (Fig. 2.4). As full development of the azo dye requires several minutes (F ox, 1979), the slope of the calibration line 24

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25 reflected both the fractional completion of the reaction under the chosen experimental conditions and the molar absorbance of the azo dye (Steimle et al., 2002). The calibration slope provides a multiplicative constant that relates the extent of color development in the co ntinuous flow mode to the full color development that occurs after the reacti on proceeds to completion. During the course of the 28 day cruise, th e calibration slope averaged 0.59 0.05. Calibration slopes obtained early in the cruise us ing laboratory-composed sulfanilamide solutions averaged 0.63 0.04 with an intercept of 8.3 2.3. A mid-cruise change to commercially pr oduced sulfanilamide solution (LabChem) provided an average calibration slope of 0.57 0.03 and intercept of 2.7 0.9. Because of a reduced reagent blank and improved calibration stability, the commercially made sulfanilamide was us ed throughout the remainder of the cruise. The time required for a sample to travel from the sample inlet to the waveguide inlet was 78 4 seconds. The overall 7.7 cm 3 /min (sample plus reagent) flow rate included a 0.6 cm 3 /min (7.5%) contribution from the reagent. Since the internal volume of the 97 cm waveguide was 0.50 cm 3 the nominal filltime of the waveguide was 4 seconds. At the conclusion of each cast, the data were immediately uploaded and plotted ag ainst depth. Because the instrument was either descending or ascending thr oughout the period of sample collection, the depth of each measurement was ad justed based on the observed 78-second delay between sample acquisition and meas urement. Thus, the location of each

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concentration observation reflected the depth of each water sample as it entered the in situ instrument. 2.4 Results and Discussion 2.4.1 SEAS performance characteristics A nitrite profile acquired using the SEAS instrument is compared in Figure 5 with measurements performed on samples obtained from a rosette cast two hours later. The rosette samples we re analyzed in the lab immediately following the cast using the same instrume ntal procedure that had been employed in situ There was excellent agreemen t between the two data sets. Figure 2.5. Comparison of in situ data and laboratory measurements of bottle samples for station 14. The upcast for th e SEAS instrument preceded the rosette cast by about two hours. Diamonds represen t in situ data an d circles represent data from bottle samples. The four di screte points below 100 m differ in depth from the corresponding in situ concentrations by an average of 1.3 0.9 m. 26

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27 In continuous flow instruments, intersample mixing can become problematic. However, the comparisons shown in Figure 2.6 indicate that sample carryover was insignificant in our system. Were this not the case, upcast and downcast concentration discrepancies would be part icularly evident at both the inflection depth and at the peak. It should be no ted, as well, that significantly greater resolution can be obtained via more slowly descending and ascending instrumental systems. Ultimately this would involve the use of untethered systems that are not influenced by ships motions.

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Figure 2.6.A. The nitrite profile for stati on 8, including upcast and downcast data, shows a slight depth discre pancy caused by internal waves. Figure 2.6.B. Profiles plotted vs. density remove the in fluence of internal waves on the nitrite distribution. Nitrite depth profiles often reflected the movement of internal waves, whereby apparent peak depths were somewhat offs et for downcasts and upcasts. In order to remove this influence on the profiles, nitrite distributions were plotted against 28

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29 density. The resultant upcast and downcas t profiles aligned well along isopycnals (Fig. 2.6B). It has been demonstrated that the reacti on rate of the Griess reaction decreases at temperatures below 20 C (Fox, 1979). As such, one factor that potentially affects the profiles is water-column therma l gradients. The temperature at the primary nitrite maximum (PNM) was 17.4 1.4 C in the gyre, with the first seven stations averaging 18.6 0.2 C and the next six averaging 16.0 0.7 C. Maxima for stations 24 and 26 in th e California Current occurred at 15.1 C and 12.0 C. However, using a three-minute dwe ll time, Steimle et al. (2002) reported only a 4.5% difference in calculated n itrite concentrati ons between 16.8 and 21.2 C. Given the small range of temp eratures at the PNM across the gyre, errors attributable to reacti on kinetics should therefore be minimal. Inclusion of a thermostatted heating unit on future versi ons of the instrument should alleviate potential problems with temperature depe ndent reaction kinetics that may result from deployment in colder regimes. 2.4.2 Nitracline Measurements In terms of peak concentration and depth, the nitrite profiles obtained in this work correspond well with previously pub lished profiles for the North Pacific Subtropical Gyre (NPSG) (Dore and Karl, 1996 b). It is evident, however, that the profiles obtained in situ exhibit far greater detail. In general, the upper water column nitrite gradient (between the su rface and the depth of a sharp inflection) was 0.08 0.03 nM/m. The nitrite inflecti on depth is well defined by the

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intersection of the upper wate r column gradient and the much steeper gradient of the primary nitrite maximum (PNM) (F ig. 2.7). The average PNM nitrite concentration gradient (inflection to peak) for sixteen stations in the North Pacific was 10 3 nM/m, and the average distance be tween the inflection depth and the nitrite maximum was 25 8m. Below the peak, nitr ite concentrations decreased to low nanomolar levels, albeit at slow er rates than observed for the shallower inflection-to-peak gradient. All but two casts of seve nteen contained a single, well-defined peak. Figure 2.7. Nitrite inflection depths are well defined by the intersection between the best fit upper water column gradient (0.08 0.03 nM/m) and that of the PNM gradient (10 3 nM/m). The nitracline has variously been defi ned as (1) the depth of the first detectable nitrate concentration usin g standard spectrophotometric methods (Herbland and Voituriez, 1979) (2) the depth that is halfway between the first 30

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31 depth at which nitrate plus nitrite is great er than or equal to 100 nM and the depth of the sample immediately above it (D ore and Karl, 1996b), (3) the shallowest depth at which nitrate reaches micromolar concentrations (Villa real et al., 1999), and (4) the depth at which the nitrate plus nitrite concentration gradient surpasses 2 nmol kg -1 m -1 (Letelier et al., 2004). Letelier et al. (2004) found th at the depth of the nitracline was 117 5 m during the summer m onths from 1998 to 2001 at Station ALOHA in the NPSG. Using SE AS, the nitrite inflection depth across the NPSG in the summer of 2002 was 117 10 m, and 116 11 m including two casts in the California Current. Given th e sharp inflections th at were typically observed using SEAS in the North Pacific, the nitrite inflection depth may serve as a well-defined maximum depth for the nitracline. As the nitrite maximum is tightly coupled with, and situated just be low, the top of the nitracline, and the PNM is only observed in the presence of nitrate, the nitrite inflection must be located at or just beneath the top of the nitracline as defined by Letelier et al. (Dore and Karl, 1996b; Herbland and Voituri ez, 1979; Letelier et al., 2004). An in situ nitrate instrument, currently under deve lopment, will facilitate investigations of the relationship between the nitrite inflection and the nitracline.

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Figure 2.8. Comparison of nitrite and fluorescen ce profiles for station 7, one of two stations containing multiple nitrite peak s. Close correlations between nitrite and fluorescence profiles were observed throughout the cruise. 32

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Figure 2.9 Contour plot of nitrite distri bution vs. longitude and depth. Diamonds represent the depths of fluores cence maxima. For the sake of clarity, only fluorescence data collected within seve ral hours of nitrite casts are included. Shoaling of the nitrite maximum occu rred at a front located between 150 W 140 W, and in the California Current. Because of incomplete reduction of nitrate by phytoplankton at low irradiance, the primary nitrite maximum is often located only slightly below the deep chlorophyll maximum (Brzezinski, 1988; Collos, 1998; French et al., 1983; Kiefer et al., 1976). 33

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34 A comparison of the nitrite and fluorescence distributions relative to density for station 7 (Fig. 2.8) show s the close correlation often observed between the two profiles over the course of the cruise. Fo r all stations in the NPSG and California Current transect, the fluorescence maximu m was observed at depths intermediate to the well defined PNM and nitrite inflection depths. The average PNM depth determined using SEAS in the NPSG in the summer of 2002 was 138 10 m. The average depth of the fluorescence maximum was 125 11 m in the gyre, and 123 12 m including stations in the Californi a Current (Fig. 2.9). This is similar to the average depth of the su mmer fluorescence maximum, 125 9 m, reported by Letelier et al. (2004) for Station ALOHA. 2.5 Conclusions We have demonstrated the utility of in situ continuous flow measurements for characterization of nitrite distributions in the natural environment. SEAS-nitrite instrume nts are sufficiently compact to allow deployment on either a ships rosette sampling system or autonomous profiling systems, and can be used to specify both the nitrite infl ection and nitrite maximum depths with great precision. Th e resolution that can be achieved using this instrumental system should greatly facilitate studies of upper-ocean nitrite dynamics. Accurate integration of nitrite concentrations over a given depth range is now possible, making between-cast comp arisons much more meaningful. The method described here can be adapted to any colorimetric analysis, providing that

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35 the rate of color development is suffici ently rapid to allow a continuous flow configuration.

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36 3 Physical and biological features of the North Pacific Subtropical Front in summer The following chapter has been submitted for publication: L.R. Adornato, T.A. Villareal, E.A. Kaltenbacher, C.A. Schoenbaechler, and R.H. Byrne, Deep-Sea Research I 3.1 Abstract The North Pacific Subtropical Fr ont (NPSF) is a biologically dynamic region created by convergence of warm, hi gh salinity North Pacific Subtropical Gyre (NPSG) waters and cooler, less saline waters originating in the Subpolar Gyre. The winter and spring frontal boundary can be readily detected from surface temperature and sea surface hei ght anomalies. Because summer insolation typically obscures the diagnostic 17/18 C winter temperature outcrop of the Subtropical Front, existence of the front in summertime is generally demonstrated only through use of in situ techniques. Otherwise, in the absence of satellite-visible surface expression s, the frontal region is typically not distinguished from th e oligotrophic NPSG.

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The NPSF and its associated physical and biological features were examined during a June/July 2002 cruise Underway data provided clear evidence of the diagnostic NPSF 34.8 sa linity outcrop that defined frontal boundaries. Vertical temperat ure and salinity distributions on the southern frontal margin reflected the nutricline doming t ypical of convergent zones. Salinity profiles north of the front exhibited a high degree of variability due to subduction of cold, low salinity water into the warmer saltier waters to the south. A sharp change in sea surface texture was obs erved at a frontal boundary, where the warmer, high salinity water was glassy and the cooler, low salinity water appeared rough. In contrast to the picoplankton-domin ated NPSG, frontal regions near 30 N displayed high concen trations of large Rhizosolenia and Hemiaulus diatom species, with highest concentrations of Rhizosolenia cells south of the front and highest Hemiaulus counts north of the front Vertically migrating Rhizosolenia mats also displayed a preference for warm, south frontal waters. Particulate organic carbon (POC) concen trations shifted from low bimodally distributed levels in the gyre to high surface leve ls near the front. Elevated POC concentrations penetrated deeper into the water column from south to north, echoing the distribution of Rhizosolenia and Hemiaulus cells. A large percentage of the deep-water (> 200 m) chlorophyll near the front was in the >10 m size fraction, indicating enhanced export of larger cells. The increased biomass and elevated cell abundance at the front was not observed via SeaWiFS, although a chlorophyll bloom was detected by sate llite north of the cruise track. 37

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If these diatom blooms are a characteri stic feature of the NPSF, then export production in this region has been underestimated and is likely to account for some of the flux that benthic respiration rates suggest is missing from current estimates. 3.2 Introduction North Pacific Subtropical Gyre (N PSG) waters are bounded to the north by a series of semi-permanent fronts, co llectively called the Subtropical Frontal Zone, that include the S outh Subtropical Front (SSTF), the Subtropical Front (STF), and the Transition Zone Chlor ophyll Front (TZCF) (Bograd et al., 2004; Roden, 1980; Roden, 1981). Winter frontal boundaries typi cally include sharp gradients in thermohaline properties, sea surface height anomalies, and increases in primary productivity that can often be determined by remote sensing methods (Archer et al., 1998; Seki et al., 2002). Because th e oligotrophic gyres re present vast areas of low trophic transfer, these narrow, hi gh chlorophyll regions provide important forage and migration pathways for a la rge variety of pelagic predators and seabirds (Hyrenbach et al., 2002; Polovina et al., 2000; Pol ovina et al., 2000). The TZCF, easily identified by its surface 0.2 mg/m 3 chlorophyll signature, migrates from 30-35 N in th e winter to 40-45 N in the summer (Polovina et al., 2001). Ot her North Pacific summer frontal locations prove difficult to detect by remote sensing since increased insolation warms the surface water above the diagnostic frontal temp erature and surface chlorophyll expression does not indicate persistent, sharp in creases in primary productivity. 38

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Although the salinity outcr op that characterizes each front may be located by in situ CTD measurements, the occurrence of oceanographic expeditions to fronts in this area is comparativel y rare. As an alternative to in situ observations, it has been noted that ocean surface texture, such as a marked change from smooth to rough conditions, or the presence of a debris line, may provide another means whereby frontal locations can be determined (Archer et al., 1998; Flament and Armi, 2000; Stommel, 1965). The impact of highly localized region s of increased productivity may be grossly underestimated in trophic transfer calculations; partic ularly when surface chlorophyll concentrations ar e insufficient for detection by SeaWiFS ocean color measurements. While elevated concentrations of Richelia -containing Rhizosolenia, Hemiaulus diatom species, and vertically-migrating Rhizosolenia mats were measured near 30 N in the P acific in the summer of 2002 (Pilskaln et al., 2005; Wilson et al., in press), elevated chloro phyll was not observed by SeaWiFS. The only remotely observe d signal was a chlorophyll bloom (> 0.15 mg/m 3 ) somewhat north of the cruise tr ansect (Wilson et al., in press). Nevertheless, summer chlorophyll blooms near 30 N have been observed in the majority of the years that ocean color da ta have been available, implying that frontal mechanisms commonly generate bl ooms in this region (Wilson et al., in press). The species composition of these fr ontal blooms was noteworthy since nitrogen-fixing diatom symbionts were abundant and Montoya et al. (2004) reported high N 2 -fixation rates by unicellular di azotrophs. Since the NPSG has 39

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been considered nitrogen-limited over the relatively short time that such measurements have been made, organisms that bring new nitrogen to near-surface waters, either by nitrogen fixation or by ve rtical transport of nitrate from the nitracline, are extremely important. In contrast to upwelling, nitrogen fixation and vertical nitrogen transport introduce new nitrogen into the euphotic zone without concomitant carbon input. Several investigators have examined changes in oceanographic parameters over large-scale meridional studies (Lynn, 1986; Pak et al., 1988; Roden, 1980; Roden, 1981; Seki et al., 2002). The c ontribution of the present study includes sampling parallel to the front, both nort h and south of the boundary, including data on differences in phytoplankton community structure. Although the temperature and salinity differences acro ss oceanic fronts are often too small to affect cell growth or physiology directl y, phytoplankton specie s typically exhibit temperature and salinity preferences a ssociated with particular hydrographic provinces (Braarud, 1962; Ga o et al., 2000; Moore et al ., 1995; Venrick, 1971). Sharp changes in temperature and salinity can result from convergent flow at fronts, and the distributions of phytoplankton can change rapidly across a front as a result of physical concentration or s hoaling of the nutricline (Seki et al., 2002; Yoder et al., 1994). Thirteen casts performed in the central gyre provided a basis of comparison with casts performed in frontal regions. Most gyre stations displayed typical oligotrophic charact eristics that included low near-surface nutrient concentrations, seasonal and permanen t thermoclines, shallow oxygen maxima, 40

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and fluorescence and nitrite maxima situ ated at the top of the nutricline. Deviations from the norm were obs erved at two gyre stations. 3.3 Materials and methods 3.3.1 Conductivity, temperature, dept h and chlorophy ll fluorescence Data were collected during the Rhizosolenia Mats in the Pacific (RoMP) cruise aboard the R/V Melville from June 20 to July 16, 2002. A SeaBird SBE 9 CTD provided salinity, temperature and pressure measurements on each rosette cast. Chlorophyll fluorescence was measured with a Seapoint fluorometer. CTD measurements, processed using Seasoft, were averaged and binned into 1 meter depth intervals. Cast depths ranged from 300 to over 4,000 meters, with most averaging 400 meters. 3.3.2 Particulate organic carbon (POC) Percent transmittance (T r ) data were collected with a WetLabs C-Star Sea Tech transmissometer (25cm pathlengt h, 6km depth range, calibrated for 100% transmittance in pure water) interfaced with the Sea Bird CTD. The beam attenuation coefficient, c was determined using the equation c = (c p + c CDOM ) = [ln (T r /100)]/ r (m -1 ) Equation 3.1 Where c is the attenuation of light at 660 nm due to absorbance and scattering of particles (c p ) and colored dissolved organic matter (c CDOM ); and r is the pathlength of the transmissometer. Given that the absorbance due to colored dissolved organic matter at 660 nm is insignificant (c CDOM 0), c can be equated with attenuation by particulate organic carbon (POC) (Brica ud et al., 1981; Nelson et 41

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al., 1998). Pursuant to Wals h et al. (1995), the cruise minimum beam attenuation coefficient value, determined at approxi mately 4000 meters depth, was subtracted from each of the profiles and the partic ulate organic carbon concentration at the deep minimum was set to a nominal value of 4 g/L (Walsh, pers. comm.). The cruise minimum subtraction provided a means whereby contributions to beam attenuation from the optical windows coul d be excluded from the calculation. Furthermore, since the primary focus of the POC determination was comparison of surface water distributions between cas ts, a small error in the assumed deep water value should not affect the overall assessment. POC concentrations ( g/L) were calculated by re arranging the equation of Fennel and Boss derived at Station ALOHA POC = 630( g/L)c P + 4 Equation 3.2 (Fennel and Boss, 2003). Use of this relationship produced values near Station ALOHA that corresponded well with those previously published for this location (Campbell et al., 1994). 3.3.3 Diatom abundances Transmissometry quantifies the abun dance of particles with diameters ranging from 0.5-20 m (Behrenfeld and Boss, 2003). Abundances of large diatoms, such as Hemiaulus and Richelia -containing Rhizosolenia were enumerated using a Multiple Opening/Closing Net and Environmental Sensing System (MOCNESS) with 64 micron mesh nets. Neither of these taxa is completely retained by this mesh size; however, they ar e retained semiquantitatively due to their colony size. Filtered water volumes and cast depth 42

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ranges were monitored by an on-board computer. Individual counts were conducted by hand in triplicate and averaged for each bin. Plots of bottlecollection versus MOCNESS counts indicate d the two agreed within a factor of two. 3.3.4 Dissolved oxygen and AOU Dissolved oxygen was measured with a SeaBird SBE 43 oxygen sensor. Apparent Oxygen Utilization (AOU) wa s calculated by subtracting observed dissolved oxygen concentra tion from saturation oxygen content (SOC) as defined by Boyer et al. (1999) such that oxygen supersaturation appeared as a negative value. 3.3.5 Photosynthetically avai lable radiation (PAR) Surface photosynthetically avai lable radiation (SPAR) and in situ PAR were measured using Biospherical Licor instruments. 3.3.6 Nutrient measurements High resolution in situ nitrite measurements were collected by the Spectrophotometric Elemental Analysis System (SEAS) instrument as described by Adornato et al. (2005). With a data collection frequency of 0.5 Hz and a detection limit of 1.0 nM, SEAS provided high ly detailed descriptions of nitrite concentrations in the upper 250 m of the water column on the 2002 cruise, and accurately established the depths of th e nitrite inflection (ave. 116 11 m) and primary nitrite maximum (ave. 138 12 m). It has been suggested that the depth of the nitrite inflection may provide a proxy for the nitracline (Adornato et al., 43

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2005). Subsequent in situ measurements of nitrite and nitrate concentrations collected using SEAS instruments in th e Gulf of Mexico (Adornato et al., accepted) revealed that the nitrite inflect ion depth and the nitracline were very tightly correlated. Furthermore, Dore and Karl (1996a) found that the location of the upper primary nitrite maximum (UPNM, average depth 126 18 m) has always been found just below the nitracline (average depth 115 18 m) at Station ALOHA. Letelier et al. ( 2004) reported the depth of the nitracline occurred at 117 5 m at Station ALOHA in the summer. Because the bottle samples did not provide the spatial resolution necessary to pinpoint locations of the nitracline at all stations, applicable nitrite inflecti on depths were used for this purpose. Seawater samples for macronutrient determination were collected in 12 mL snap-cap tubes. The tubes were ri nsed three times with sample prior to filling, frozen at -20 C, and analyzed postcruise. Nitrate plus nitrite and silicate analyses were conducted on a Lachat Quikchem 8000 ion analyzer using the manufacturers recommended chemistries. Reactive phosphate was measured manually on a spectrophotometer (Parsons et al., 1984). 3.3.7 Chlorophyll a concentrations Chlorophyll a concentrations were determined by filtration onto 0.4 and 10.0 micron pore size membrane filters (200 and 250 ml filtered, respectively), extraction overnight in methanol at -20 C, and fluorometric analysis using the non-acidification technique de veloped by Welschmeyer (S ingler and Villareal, 2005; Welschmeyer, 1994). 44

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3.4 Results and discussion Figure 3.1 Underway salinity map for the Rhizosolenia Mats in the Pacific (RoMP) 2002 cruise constructed from over 52,000 latitude/longitude/salinity observations. Station locati ons are shown in subseque nt figures so that the underway frontal salinity changes are not obscured. 45

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Figure 3.2 Curtain plot of salinity distri butions (surface to 300 m depth) embedded in the cruise map shows the high degree of variability observed over the course of the cruise. Data from a to tal of 34 casts were used to generate the plot. 3.4.1 Temperature, salinity and density The cruise track (Fig. 3.1) comprised several regions defined by temperature and salinity distributions (Fig. 3.2, 3.3). Centra l gyre stations (4 through 8) displayed typical summer pr ofiles that included both seasonal and permanent thermoclines, with little vari ability between casts except as noted. South frontal stations 10 through 13 disp layed slightly lower average surface salinities and temperatures than the central gyre stations, and permanent thermoclines that shoaled to the base of the seasonal thermoclines so that no relict winter mixed layer could be discerned. North frontal stations (14 through 18) 46

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displayed highly variable salinity distri butions produced by subduction of fresher, colder waters from the northern gyre unde r the warmer higher salinity waters of the central gyre (Roden, 1980). The salin ity distribution of the northernmost station (16) most cl osely resembled those of the Ca lifornia Current stations (24, 25), but with a higher value at the surface. Salinity distributions for stations 19 though 20 demonstrated gyre characteristics, with surface values in excess of 35.2 and smooth decreases with depth to approximately 34 at 200 meters, but with surface waters 2C colder than observed at stations in the central gyre. The remaining stations, 21 through 26, displa yed transitional properties between warmer, high salinity gyre waters and the colder, low salinity waters of the California Current, with marked drops in both temperature and salinity values between stations 23 and 24. Figure 3.3. Distributions of temperature ( C) from the surface to 300 m. Due to summer insolation, temperature displays much less variabil ity than that of salinity. Location of the Subtropical Front by AVHRR or MODI S satellite data during summer months is rendered impossi ble by the disappearance of diagnostic temperature outcrops. 47

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Although the location of the Subtropical Front shifts over time, it can be detected by several means. Niiler and Reynolds (1984) reported that a welldefined local salinity minimum at approximately 180 meters depth characterized stations north of a front at 31 N, 152 30W, while a weak minimum at about 260 meters typified stations south of the front. Shallow salinity minima typical of those found north of the front were ob served at stations 12 and 14 though 18, although the salinity minimum at stati on 12 was 0.2 0.4 more saline than observed at the north frontal stations. Salinity minima corresponding to those found south of the front were observed at stations 13 and 19 though 21. Roden (1980, 1981) identified the winte r-time location of the subtropical front by its surface salinity and temperature signatures of 34.8 and 18 C. Seki et al. (2002) determined that 34.8/17C outcrops characterized the STF in late winter and early spring. Because summer insola tion causes the sea surface temperature to rise above the frontal diagnostic value, summertime location of the STF must be determined by salinity al one (Niiler and Hall, 1988). 48

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Figure 3.4. Photograph of the Subtropical Front obtained at 29.564 N, 138.867 W (between Stations 20 and 21) demonstrates the sharp change in surface texture often observed at fronta l boundaries. Arrows in the photograph indicate the location of th e front, which was visible over a great distance. Underway data highlight the abrupt salin ity change that occurred at the frontal boundary depicted in the photograph. 49

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The ships underway salinity data prov ided clear evidence of frontal boundary crossings that corresponded well with sa linity data collected during CTD casts (Fig. 3.1). Salinity and temperature fe ll between stations 13 (35.26, 23.42C) and 14 (34.63, 22.72C), rose between stations 18 (34.34, 21.78C) and 19 (35.24, 22.25C), and fell between stations 20 (35.32, 22.43C) and 21 (34.85, 21.9 C), indicating that frontal boundaries were crossed between each of these pairs of stations. In addition, a sharp change in sea surface appearance was observed at 29.564 N, 138.867 W, between stations 20 and 21. The warm side of the front at this location appear ed glassy and the cooler side appeared choppy (Fig. 3.4), similar to observations of frontal bounda ries near 2 N in the eastern Pacific (Archer et al., 1998). Salinity profiles nor th of the front e xhibited interleaving lamina of fresher and more saline waters with a high degree of variability between profiles when compared to central gy re profiles (Fig. 3.5) (Lynn, 1986). 50

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0 50 100 150 200 250 300 33.6 34.1 34.6 35.1 35.6 SalinityDepth (m) Station 13 Station 14 0 50 100 150 200 250 300 33.6 34.1 34.6 35.1 35.6 SalinityDepth (m) Station 20 Station 21 Figure 3.5. Salinity distributions between Station 13 (30.154 N, 145.592W) and 14 (30.655N, 143.801W), and between 20 (29.125N, 138.312W) and 21 (29.900 N, 137.420 W) provide examples of both the magnitude of change across frontal stati ons and the profile of variability in frontal salinity distributions. 51

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In spite of the intricate layering observed in frontal salinity profiles, a plot of sigma-t showed a highly stable vertical density st ructure (Fig. 3.6). Salinity variations compensated by temperature ha ve been observed both vertically and across frontal regions (Roden, 1980). Shoaling of isopycnals was noticeable at stations 10 through 13, and again at the California Current (Fig. 3.6). Central gyre stations exhibited the deep pycno clines typical of highly-stratified oligotrophic watersWhile stations 19 and 20 also displayed deep pycnoclines, the surfacedensities ( t = 24.34) were the highest of any station. Figure 3.6. Curtain plot of sigma-t from the surface to 300 m depth shows shoaling of the pycnocline near the front. The shallowest pycnocline occurs at the station (10) nearest the satellite-observe d chlorophyll bloom. While the depth of the pycnocline varies across the transect, the vertical density structure is highly stable. 52

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3.4.2 Particulate organic carbon Derived particulate organic carbon (POC) concentrations (Fig. 3.7) displayed a bimodal distribution in the central gyre lo cations (stations 4-8) that included a peak centered at 50 meters and another at the deep chlorophyll maximum (DCM). At several of the stations, POC concentr ations in the DCM were equal to or higher than those near the surface. This contrasts with distributions observed by Fennel and Boss (2003) at Station ALOHA where the POC concentrations at 50 meters far exceeded those in the remainder of the water column. Figure 3.7. Particulate organic carbon (g/L) distributions from the surface to 300 m reveal low concentrations in the gyre and elevated levels near the front. High POC concentrations penetrate deeper into the water column on the southern, high salinity side of the front compared to those at northern, lower salinity stations. 53

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POC distributions at stations 8 through 13 displayed higher deepwater (below 200 m) concentrations than those of stations 4 through 7, as well as a gradual shift from the bimodal particle dist ributions of the centr al gyre to particle maxima at or near the surface. This is similar to the POC distributions across the Subtropical Front in the Atlantic report ed by Vezzulli et al. (2002). Integrated chlorophyll a concentrations increased by 27% between stations 7 and 8, indicating that the rise in POC correlated with an increase in phytoplankton biomass, although other factors may have c ontributed as well. For example, low wind velocities and convergent surface curr ents at the latitude of the subtropical front during the summer months favor accu mulation of materials including plastic debris, marine snow, and Rhizosolenia mats (Kubota, 1994; Montoya et al., 2004; Wilson et al., in press; Zhurbas and Oh, 2004). The highest POC concentration detected in this study occurred at station 12 (61 g/L at 32 m depth) in conjunction with a subsurface chlorophyll bloom, with measured near-surface chloro phyll concentrations of 0.11-0.16 mg/m 3 and a deep chlorophyll maximum of 0.83 mg/m 3 Species associated with the bloom were not determined, although relatively depleted silicate concentrations (0.45 M) at the depth of the POC maximum implied that diatoms may have been responsible for the increase in chlorophyll biomass. Stations north of the front had slightly lower deepwater POC concentrations than those observed at st ations 8 through 13, but a higher level of particulate material at the surface than th e rest of the water column. Stations 13, 15 and 16 displayed surface POC concentrations on the order of 50 g/L. East of 54

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station 18, near-surface POC concentrations dropped but rose again at stations 22 and 23. California Current station POC concentration distributions most closely resembled those of the central gyre, with peaks occurring just beneath the surface mixed layer and at the DCM. The average deep POC maximum occurred at 126 13 m in the gyre, 115 9 m at the front, and 119 12 m overall. The light level at the deep POC maximum was 0.5 0.2% of surface radiance averaged over all casts where light measurements were obtained. 3.4.3 Diatom distributions The MOCNESS samples were dominated by the diatom Rhizosolenia with its diazotrophic symbiont Richelia and the diatom Hemiaulus spp. Hemiaulus contains a diazotrophic symbi ont as well, although at fre quencies that vary widely in the central Pacific (Wilson et al., in press). We could not enumerate the Hemiaulus symbiont in our preserved sample s, and cannot confirm that it was present. Concentrations of Richelia-containing Rhizosolenia cells, averaged over the upper 60 meters of th e water column, rose from approximately 200 cells/m 3 in the gyre to over 130,000 cells/m 3 at stations 10 and 12. The highest near-surface concentration of Rhizosolenia-Richelia cells observed during the cruise was 318,000 cells/m 3 within the 15-40 meter stratu m at station 10. The highest surface concentration was 230,000 cells/m 3 found in the 05 meters stratum at station 13. Rhizosolenia mat abundance determined by divers and ROV deployments also peaked at station 13 (P ilskaln et al., 2005). Yoder et al. (1994) found very high free-living (non-mat forming) Rhizosolenia cell concentrations 55

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along the warm side of a front located at 2 N in the Pacific. Although the stunning cross-front color change observe d by Yoder was not seen in this study (the frontal boundary between stations 13 and 14 was crossed at night), the obvious Rhizosolenia-Richelia preference for the warm edge of the front was confirmed by cell count. While Rhizosolenia mats and Rhizosolenia-Richelia cell counts peaked south of the front, Hemiaulus cell concentrations reached highest values north of the front. Hemiaulus cells increased from 2,000 cells/m 3 in the gyre to over 400,000 cells/m 3 at station 14. As evidenced by a concentration of 840,000 cells/m 3 in the upper 15 meters of the water column, the surface Hemiaulus biomass maximum also occurred at station 14. Elevated Hemiaulus cell counts were also observed at stations 10 thro ugh 13, and 15, albeit at lower values than observed at station 14. High concentrations of both Rhizosolenia-Richelia and Hemiaulus cells penetrated deeper into the water column south of the front (Fig. 3.8). Observed differences in near-surf ace community structure across the frontal boundary may also be responsible for the abrupt change in POC distribution demonstrated in Figure 3.7. 56

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Figure 3.8. Vertical distributions of Hemiaulus and Rhizosolenia cells plotted over salinity contours show the aversion of Hemiaulus to low salinity. Note the similarity of POC and cell density distributions. Hemiaulus surface (0 15 m) concentrations dropped from 80,000 cells/m 3 at station 15 (S = 34.5) to 32,500 cells/m 3 at 16 (S = 33.7); and from 83,000 cells/m 3 at station 23 (S = 34.2) to 500 cells/m 3 at California Current station 24 (S = 33.3). Both Hemiaulus and Rhizosolenia-Richelia surface counts fell to very low levels by station 25. The cross-frontal salinity gradient, while significant for the open sea, is unlikely to be directly responsible for the observed 57

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phytoplankton distribution. Venr ick (1971) did not observe changes in recurrent phytoplankton groups in this zone, and the small gradients noted across the front are unlikely to have a significant eff ect on growth or physiology (see (Smayda, 1980). Rather, it is a proxy for differing physical or chemical regimes separated by the front. Diatom-diazotroph blooms ar e also reported at the Hawaii Ocean Time Series (HOT) station (Scharek et al., 1999) and from the tropical Atlantic Ocean (Carpenter et al., 1999). The common features of these disparate areas that lead to such blooms remain unknown. 58

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3.4.4 Export Production B A Figure 3.9. A) Photograph of a Pacific Hemiaulus bloom taken near the sea surface by a diver during the RoMP 2003 cr uise. While the photograph was not taken during this study, it is representativ e of the level of marine snow observed during an intense Hemiaulus bloom. B) Photograph of Hemiaulus aggregate collected near 30 N during the RoMP 1996 cruise. In addition to providing new nitrogen to the euphotic zone, Rhizosolenia and Hemiaulus spp. either excrete substances or form assemblages that contribute 59

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to production of marine snow. Rhizosolenia mats produce a sticky transparent exopolymer (TEP) that can provide a nucleus for accumulation of mesozooplankton, bacteria, and other partic ulate material. The levels of TEP production measured by Pilskaln et al. (2005) closely resembled those of high productivity areas rather th an those typically observed in oligotrophic regions. Rhizosolenia-Richelia associations often produce millimeter-sized rafts (Mague et al., 1974), see Fig. 3a). High Hemiaulus spp. abundance is characterized by large quantities of particulate matter (Fig. 3.9) that can also act as a locus of flocculation; high fluxes associated with Hemiaulus have been observed at HOT (Scharek et al., 1999). Because large part icles can sink rapidly, or be consumed by zooplankton and repackaged as fecal pellets, all of thes e aggregates may contribute episodic carbon export to the d eep ocean (Michaels and Silver, 1988). Size fractionated chlorophyll measurements indicated elevated levels of large (> 10 m) phytoplankton at stations 7, 10 th rough 13, and 15 (14 was not sampled) relative to central gyre stati ons (Fig. 3.10). Particularly high percentages occurred at approximately 200 meters depth south of the front, indicating export of cells in the larger size fraction. 60

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Figure 3.10. Elevated chlorophyll concentrations were measured near the front, although a corresponding increase in in situ fluorescence was not observed. Sizefractionated chlorophyll data indicated that high per centages of large (> 10 m) cells were found below 175 meters, part icularly in the region with high Hemiaulus concentrations, indicating enhan ced export in the diatom bloom region. 61

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Recent evidence from sediment core data indicates that large diatom accumulation and export at frontal areas are common occurrences in the world oceans and that some of the diatom species found in the sedimentary record tend to form blooms that may not be visibl e at the sea surface (K emp et al., 2006). Laminated Rhizosolenia spp. deposits have been found in Neogene sediments beneath the South Equatorial Front (Pacific) and in Ho locene sediments near the Subarctic Front (Atlantic) (Andersen et al., 2004; Kemp et al., 1995). Fresh Rhizosolenia detritus was found on the seafloor two months after a bloom formed at a front located at 2 N in the Pacifi c (Archer et al., 1998; Smith et al., 1996; Yoder et al., 1994). Measurements of associated bacterial activity suggest that the detritus was labile and subject to ra pid degradation (Smith et al., 1996). 3.4.5 Sea Surface texture Changes in sea surface texture sim ilar to the change observed in the current study have been obser ved at frontal boundaries at 2 N in the Pacific and at the western boundary of the Gulf Stream in the Atlantic (Archer et al., 1998; Govoni et al., 2000). Processe s that contribute to the ch ange in texture are not clear, but it is probable that sea surf ace smoothing, or the damping of capillary waves ( < 0.5 cm), is accomplished by biological processes. Surface slick damping of capillary waves is a welldocumented phenomenon (Davies and Vose, 1965). Slicks, usually made up of inso luble organic materials, are common features at convergent fronts (Le Fevre, 1986) and often can be detected using satellite based synthetic aper ture radar (SAR) (Espedal et al., 1997; Espedal et al., 1996). Recent studies reveal that plankton blooms correlate positively with 62

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surface slicks that result in underestimation of QuikSCAT scatterometer-derived wind speed estimates (Hashizume and Li u, 2004; Lin et al., 2003). In addition, TOPEX radar altimeter data often show areas of high re turn cross sections near 30 N in the Pacific during the summer a ssociated with smooth surface conditions that may not be correlated with low wi nd speeds (Mitchum et al., 2004). Since Rhizosolenia-Richelia cultures have been observed to excrete materials that increase the viscosity of the surrounding medium (Villareal, in prep.), it is possible that high cell numbers cause surface slicks that result in damping of capillary waves, but this remains an open question. Knowledge of the temporal and spatial extent of the textural boundary would greatly aid in future studies of the front since it may be possible to identify the frontal boundary by remote sensing methods. 3.4.6 30 N summer chlorophyll blooms For 11 out of 16 years that satellite ocean color data were available, summer chlorophyll blooms occurred near 30 N between 135 to 155 W, including a bloom just north of statio n 10 during the present study (Wilson, 2003; Wilson et al., in press). While we di d not sample this 30 N bloom, it seems likely that frontal processes at this latitu de were linked to the formation of the bloom (Wilson et al., in press). Front s typically provide areas of higher productivity, species abundance, and di versity (Worm et al., 2005). The area along 30 N is exploited by l ongline fisheries for swordfish during late winter and spring months, possibly due to increased pr ey species associated with the front (Seki et al., 2002). Dandonneau et al. (2003) proposed that chlorophyll anomalies 63

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observed in oligotrophic regi ons by satellite color methods were actually made up of non-living detrital material collected at the surface by Rossby waves acting as hay rakes. However, in contrast to planetary waves which propagate east to west (Pak et al., 1988), the 30 N summer blooms remain stationary or propagate west to east. Since very high numbers of diatom cells were observed at station 10 located at the southern ti p of the bloom (Wilson et al ., in press), the evidence indicates that the 30 N summer bl ooms are due to local increases in phytoplankton abundance rather than an artifact due to absorbance by surface detrital matter. Some factors that may contribute to summer bloom appearances include shoaling of the n itracline along the STF, increased nitrogen fixation by small unicellular autotro phs and endosymbiont Richelia species, and nitrate release into the euphotic zone by buoyancy regulating Rhizosolenia mats (Montoya et al., 2004; Singler and Villareal, 2005; Villareal et al., 1999). Rhizosolenia-Richelia may release fixed N direc tly into the surrounding water (Villareal, 1987) and could contribute to a general phytoplankton increase as well as a local increase in Rhizosolenia-Richelia symbiosis. In light of the size of the bloom area and the associated potential contribution to e xport production, the phenomenon deserves further investigation. 3.4.7 Nutrient and dissolved oxygen distributions Inorganic nutrient distributions over th e course of this study revealed that the gyre was apparently nitrogen limite d, providing an advantage to organisms capable of nitrogen transport or fixation (Fig. 3.11). Concentrations integrated over the upper 200 meters showed that th e nitrogen to phosphorus ratio was 8.8:1, 64

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R 2 = 0.89. The depth of the nitracline aver aged 121 8 m in the gyre and 110 7 m near the front, resulting in an overall cruise average of 115 9 m. The lowest overall nutrient concentrations were measured at station 20 which was located at the eastern edge of a narr ow tongue of high salinity gyre water. The nitrate gradient below the nitracline was greater at near-frontal stations compared to that in the gyre. Shoaling of the nitracline would particularly benefit migrating Rhizosolenia mats, since travel time would be reduced between the nutricline and the depths where light is sufficient for photosynthesis (Villareal et al., 1996). 65

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Figure 3.11. Nutrient contours reflect low concentrations observed in the NPSG and upwelling in frontal regions and the California Current. Elevated silicate concentrations were found above the nitraclin e, particularly near the front, thereby favoring diatom species that are capable of nitrate transport or that possess diazotrophic endosymbionts. 66

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The station with the highest N:P ratio (Sta. 7, N:P = 30) was located in the central gyre. Several features distinguish this station from others in the gyre. First, the nitrite maximum displayed a diminished primary peak and several deeper peaks, rather than the typical single strong peak. Second, although deep nitrite peaks have often been attributed to bacterial de nitrification (D ore and Karl, 1996a), the deep peaks at station 7 were associated with atyp ical increases in chlorophyll fluorescence and dissolved oxygen. The reason for this is unclear but it may be that a small population of phytopl ankton was capable of persisting at the depth of the phosphocline, which was approximately 40 meters deeper than the nitracline. Hemiaulus and Rhizosolenia-Richelia cell concentrations at station 7 were twice those counted at other gyre locations, primarily due to increased abundance at the surface. Nitrogen fixa tion rates by small unicellular organisms at this station were similar to those meas ured at frontal stations rather than the much lower rates observed at gyre sta tions (Montoya et al., 2004). The ROV deployment did not reach the depth of the anomalous deep peaks (~200 m) to determine if Rhizosolenia mats (seen at the surface) were present at depth, although size fractionated extracted chlor ophyll measurements indicated that cells >10 m contributed 40% of the total at ~200 m. AOU profiles in the gyre followed a tre nd of saturation in the mixed layer, supersaturation from about 25 to 100 m, followed by a decrease at depth. The depth of maximum supersaturation (46 6 m) occurred at approximately the same depth as the upper POC maximum (46 7 m). Two instances of anomalous dissolved oxygen measurements were noted in the gyre. The first, at station 7, 67

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has been previously discussed. The s econd occurred during cas t 5-4, the first of four rosette casts completed at sta tion 5. The cast cap tured reduced oxygen saturation that correspon ded with a large spike in fluorescence and POC compared to the following cast (station 56, Fig. 3.12). Differences in integrated dissolved oxygen (50 150 m) between the two casts translated to 540 mmol/m 2 of carbon respiration assuming a Redfield ratio. Extracted chlorophyll did not account for the large observed fluorescen ce values, suggestin g that the excess fluorescence may have been due to pheopi gment degradation products. Since the two casts were completed five hours and approximately one nautical mile apart, it is possible that different water masse s were sampled. Nevertheless, the distributions observed in cast 5-4 were qui te unusual compared to others obtained in the gyre. 68

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0 50 100 150 200 250 0.00E+002.00E-024.00E-026.00E-028.00E-021.00E-01Fluorescence (AU)Depth (m) A 0 50 100 150 200 250 -20-100102030405060Apparent Oxygen Utilization ( mol/kg)Depth (m) 0 50 100 150 200 250 01 02 03 04 05POC ( g/L)Depth (m) B C 0 Figure 3.12. Vertical distributions of (A) fluorescence, (B) POC and (C) AOU for gyre casts 5-4 and 5-6 show the remark able differences between data collected 5 hours and 1 nautical mile apart. U nusually high fluorescence, POC, and AOU observed from 60 m to 1 25 m during cast 5-4 data suggests a high level of respiration associated with collapse of a phytoplankton population. Distributions of fluorescence, POC and AOU at station 5-6 were representative of those found at other gyre stations. 69

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Dissolved oxygen in both south and north frontal stations displayed greater saturation near the surface compar ed with central gyre. Deep water (>4,000 m) AOU values were measured at three stations, in cluding one in the gyre, one north of the front, and one near the California Current. AOU values mirrored POC values at these stations so that the highest deep-water AOU occurred at the same station as the highest POC values. The overall trend indicated that the highest deep-water POC values occurred near the front and that the lowest were found in the gyre. The values for the California Current station were intermediate between the two. 3.5 Conclusions The North Pacific Subtropical Front is difficult to identify during the summer without the use of in situ methods. Thermal boundaries are indistinct and salinity cannot be determined remotely. Physical properties change rapidly across the Subtropical Front, often producing a dist inct change in sea surface texture that may be due to the presence of biologica lly-produced surface sl icks. This is provocative evidence that the frontal featur e drives a biological response that can be observed by remote sensing methods su ch as sun glint observations, TOPEX radar altimeter anomalies, synthetic aperture radar (SAR), or QuikSCAT scatterometer data, however this has not as yet been demonstrated. Due to subduction of cool, low salinity northern water into warm er, saltier southern gyre water, salinity distributions in the upper 300 m at north ern front stations exhibit highly variable profiles. Gyre stat ions exhibit seasonal and permanent 70

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pycnoclines, while southern front stations display a single pyc nocline nearer the surface. Physical factors, including converg ent currents and low wind velocity, favor the accumulation of debris, marine snow, and diatom assemblages over a large area near 30 N. Anomalous ch lorophyll blooms determined by satellite occur in this area during the late summe r, although bloom compositions have not yet been determined. In view of the persis tence of blooms near 30 N, it is likely that the Subtropical Fr ont plays a major role in bloom development. Elevated concentrations of Rhizosolenia and Hemiaulus species are found near the Subtropical Front during the summer. Rhizosolenia and Hemiaulus species produce materials associated with marine snow that are likely to enhance export production by generating loci of flocculation. These diatom species exhibit preferences for different regions of the front that have characteristic T-S regimes. Rhizosolenia species increased on the warm, southern side of the front, while Hemiaulus species exhibited a biomass ma ximum on the cooler, less saline side of the front. However, both species were present on both sides of the front. Hemiaulus abundance dropped drastically at co ld, low salinity stations (i.e., northernmost and California Current stations). Both Rhizosolenia and Hemiaulus species penetrated deeper in the water co lumn south of the front, consistent with observed distributions of particulate or ganic carbon. The measured nutrient ratios suggest N limitation, and it is pr obable that input of nitrogen-fixation by diatom-diazotroph associations contri butes to bloom formation. Rich 71

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accumulations of detritus provide substr ates for microbial degradation that accelerate recycling and enhan ce vertical transport. The Subtropical Front during the summer represents a region of increased biomass relative to the North Pacific S ubtropical Gyre, although much of the biomass was not visible in ocean color measurements during our study. Due to the patchy nature of near-front diatom distributions, the contribution of bloom events to overall trophic tr ansfer is difficult to constrain. However, primary productivity for this region is almost certainly significantly underestimated, particularly if it is based on ocean color da ta. It is apparent that the frontal area maintains higher particulate organic carbon concentrations than the gyre and that these increases are maintained into d eep water. Episodic export of diatom biomass in this region may help explain so me of the reported differences between benthic respiration and measured POC fluxes (Smith, 1987; Smith and Kaufmann, 1999). Overall, gyre and frontal regions displayed surprising variability in biogeochemical characteristics. Caution should be exercised when extrapolating time series data from one station to the en tire region. Substan tial differences exist between the time-series station at HOT and the NPSF. However, the similarity in floristic composition, particularly the pul se of net-plankton biomass, suggests similar forcing functions. The nature of these processes lead ing to net-plankton blooms of diatom-diazotroph symbioses remains unclear. 72

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4 High-Resolution In Situ Analysis of Nitrate and Phosphate in the Oligotrophic Ocean Reproduced with permission from E nvironmental Science and Technology, submitted for publication. Unpublished work copyright [2007] American Chemical Society. L. R. Adornato, E. A. Kaltenbacher, D. R. Greenhow, and R. H. Byrne, authors. 4.1 Abstract Accurate, high-resolution profiles of nitrate and phosphate distributions in the open ocean are difficult to obtain using conventional techniques. Concentrations typically range from lo w nanomolar levels in the stratified euphotic zone to micromolar levels below the nutricline. With multiple pumps, a heating cartridge, a long-pathlength cell, and multiwavelength spectrometer, the reconfigured Spectrophotometric Elemen tal Analysis System (SEAS) provides the capability to fully ascer tain the distributions of nitrate and phosphate in the upper 200 m of the oligotrophic ocean. By utilizing a 15 cm pathlength and multiple wavelength spectrophotometry, SEAS can detect nitrate concentrations from 2 nM to 20 M and, with a 50 cm pathlength, can accurately measure phosphate concentrations from 1 nM to 1 M. SEAS is capable of collecting auxiliary data from up to four separate in struments, including a CTD, fluorometer, 73

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PAR sensor and a second SEAS instrument. Sampling frequency depends on peripheral instrument selecti on and ranges from 0.4 to 0.75 Hz. 4.2 Introduction Net primary productivity in oligotroph ic waters is limite d (sensu Liebig) by the first nutrient or property that falls below the level necessary to maintain growth. Limiting factors may include light, nitrate, phospha te, silicate and iron (Abbott et al., 2000; Dugdale and Wilkers on, 1998; Falkowski, 1997; Letelier et al., 2004; Martin et al., 1991; Sanudo-Wilhelmy et al., 2001). Historically, nitrogen has been deem ed to limit biomass in oligotrophic ocean waters, while phosphorus limitation has been reported in limnological systems (Hudson et al., 2000). In recent years, however, it has been determined that phosphate limits phytoplankton growth in the Western Atlantic (Wu et al., 2000) and that a shift to phosphate limitation, primarily due to an increase in diazotrophy, may be occurring in the North Pacific Subtropical Gyre (Karl et al., 2001). Monitoring such subtle changes pr oves important to understanding short and long-term ecosystem shifts since the hi storical record of nutrient levels and phytoplankton community structure is limited. Nitrate and phosphate concentrations t ypically span up to five orders of magnitude in the stratified open ocean, from low nanomolar levels at the surface to micromolar levels at depth. Standard techniques adequately determine elevated concentrations but, due to high detection li mits, fail to detect low values in the euphotic zone. A variety of methods ha ve been developed to provide greater sensitivity. These methods generally fa ll into two groups. The first involves 74

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spectrophotometry, with techniques that include preconcentration, fluorescence detection, and long-pathlength spectrophot ometry. The other group utilizes techniques that require expe nsive, delicate equipment, which are not conducive to in situ measurements (Garside, 1982; Habere r and Brandes, 2003; Yang et al., 2001). Preconcentration can be accomplished through use of adsorbents (Medvetskii et al., 2003), co-precipitation (K arl and Tien, 1992; Rimmelin and Moutin, 2005), or organic solvents (Stephens, 1963). Due to time and sample handling constraints, however, precon centration techniques are generally restricted to bench top analyses. Ot her methods suitable for highly sensitive freshwater analyses are not amenable to seawater samples. Enzyme-coupling fluorescence detection of phosphate, for inst ance, suffers from sulfate interference at concentrations above 25 mM ([SO 4 2] = 28 mM at S = 35) and, in addition, utilizes expensive reagents (Vazquez et al., 2003). Long pathlength absorbance spectrophot ometry (LPAS) uses standard spectrophotometric technique s but reduces detection limits by increasing the pathlength term in the Beer-Lambert equation, l c, Equation 4.1 where A is absorbance, is the wavelength-dependent molar absorptivity (M -1 cm 1 ), l is pathlength (cm), and c is conc entration (M) (Byrne et al., 2000). Long pathlengths are achieved through use of a flexible fluoropolymer material (AF2400, Dupont) with a refractive index (1.29) less than water and seawater (1.33, 1.34). In this case, light propagating thr ough the waveguide at angles less than 15 relative to the waveguide/water inte rface is totally internally reflected 75

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(Waterbury et al., 1997). Long pathlength spectrophotometry has been successfully used to measure low nanomolar concentrations of nitrate (Type 1, AF-2400 tube LCW) (Yao et al., 1998) and phosphate (Type 2, AF-1600 coated quartz tube LCW) (Zhang and Chi, 2002). However, due to inherent limits in linear dynamic range, LPAS measurements of high nutrient concentrations found below the base of the euphotic zone are problematic without re sorting to dilution, or variation of pathlength. All of the aforementioned methods invol ve collection of discrete samples, usually via Niskin bottles secured on a ro sette, and subsequent analysis on-board a research vessel or, more typically, in a lab onshore. Profiles are limited by the number of bottles on the rosette, so that the quantity of data making up a profile is often insufficient to accurately determine analyte distributions. Johnson et al. (2002) developed an in situ reagentless system capable of detecting nitrate with a 1 Hz sampling frequency, but the detection limit of 1.5 M for nitrate (0.2 M for a 30 s average) is insufficient to addre ss the low nanomolar concentrations often found in the stratified euphotic zones of the world oceans. A recently described instrument, the Spectrophotometric Elemen tal Analysis System (SEAS), provides the sampling frequency necessary to obtai n the detailed distribution of various analytes in the water column, and detect ion limits that allow measurements of nanomolar analyte concentrations in th e upper water column (Adornato et al., 2005; Liu et al., 2006). However, because SEAS had a maximum of two fluid lines, analytical methods were restrict ed to those using a single reagent. 76

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We describe here the newest version of SEAS (SEAS II) which includes multiple fluid lines, a heater, a valve, im proved graphical user interface (GUI) and software, and connectivity to four periphe ral instruments. SEAS II provides the means to perform in situ analyses of a wide variet y of analytes and to permit comparison of results with concurrently collected environmental data. Using multiple wavelength spectroscopy and a 15 cm waveguide, nitrate concentrations of 2 nM to 20 M can be accurately determined. A 50 cm waveguide and a heater cartridge permit detection of in situ phosphate at concentrations of 1 nM to 1 M. We present here the first highly sens itive, spatially complete profiles of nitrate and phosphate concentrations obtained in the upper 200 m of the oligotrophic ocean. 4.3 Experimental Section 4.3.1 Apparatus Developed at the Center for Ocean Technology in the College of Marine Science University of South Florida, SE AS II (Fig. 4.1A) represents a state-ofthe-art capability for in-situ colorimetr ic analyses. Utilizing a long-pathlength (0.15 meters to 10 meters) optical cell, measurements with nanomolar resolution and accuracy are readily obtained. The overall instrument measures approximately 7 in diameter by 50 l ong, is fabricated primarily in anodized aluminum, and is rated for in-situ measur ements to depths of 1000 meters. The standard battery pack made from nickel metal hydride (NiMH) batteries provides normal endurance of at least 8 hours of continuous operation. When two battery packs are connected in parallel, the endurance is doubled. In the following 77

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paragraphs, we describe electronic, fluidic and op erational aspects of the instrument in more detail. C B A Figure 4.1. A. A 3D m odel of the SEAS II instrument. Portions of the instrum e nts protective housings have b een rem oved to reveal inner com p onents. B. The heater assem b ly used in SEAS II. The com p onents on the left end of the heater are the heating cartridge, temp erature sensor and valve for evacuation. Fluid is pum ped into the heater throu gh tubing feedthroughs on the right end of the heater. C. A plastic cartridge holds the wavegui de in place during deploym e nts and prevents introduction of stray light into the spectrom e ter. Light introduced through an o p tical sapph ire in terface into th e ap erture (shown in the cen ter of the green com ponent) propagates through a wa ter-filled coiled w a veguide and out through th e second aperture. Light p a sses th roug h a second sapphire interface and into the spectrom e ter via an op tical fiber. A n optional electronic connector (shown in the center of the pink compone nt) perm its the use of transverse illum i nation f o r f l uoresc e nce m easurem ents. 78

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As shown in Figure 4.1A, most of SE AS electronics are housed within one end of the instrument (a portion of the housing has been removed from the figure for clarity). These electronics control all operational aspects of the instrument and include the light s ource and spectrometer. A Motorola microcontroller executes the main control so ftware and acts as the bridge between the various peripherals and subsections of the instrument. Four serial (RS-232) connections are externally available for connection to commercial sensors such as CTDs, fluorometers, and PAR sensors. These data are merged with spectral data from the Ocean Optics USB2000 spectrome ter and are stored internally in FLASH memory with sufficient capacity to store 8000 measurements. Each measurement includes: intensities at selected wavelengths; data from external peripherals; calculated results such as c oncentration or pH; time stamp, positional data; diagnostic values; and a brief description of the data set. A user interface is provided to the instrument via either RS232 or Ethernet connections. The lamp (Avantes HR6000B) is mounted to the interi or endcap of the electronic housing. This section of the instrume nt also contains the optical interfaces between the waveguide, lamp and spectrometer. Common to each interface is a flat window made from sapphire providing separation between the fluid stream (and confined light) in the waveguide and components internal to the pressure housing. Sapphire was chosen because it is very hard (scratch resistant) and offers excellent transmission over a broad range of wavelengths. For the input side to the waveguide, the lamp with an integral lens is positioned to focus light through 79

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the window into the waveguide. A custom lens assembly was designed for the output side to collect light transmitted from the waveguide and direct it into the spectrometer. The middle section of the instrument is exposed to the environment and contains the fluidic systems. This expos ure is essential to equalize pressures in the fluid stream as the instrument descends. Three pumps are mounted in the center section (Fig. 4.1A). Each pump is individually packaged in a pressure housing to minimize loss in the event of a seal failure. Two channels for tubing are provided with each pump to yield a total of 6 fluid streams for sample solution and reagents. An on/off fluid valve can be installed for additional control in one fluid stream. An optional heater cartridge (Fig. 4.1B) controls the temperature of the fluid stream to a maximum of 100 C. Fluid to be heated is pumped through tubing wound around a copper core. A resistive heating cartridge in the center of the copper core provides heat to the syst em, and a resistive temperature sensor monitors the core temperature and provi des feedback to the control circuitry driving the heater. The wound tubing a nd copper core are sealed within an evacuated aluminum housing. This featur e minimizes convective and conductive heat losses. Two versions of the heater were fabricated; one with stainless steel tubing and the other with Teflon tubing. The Teflon version offers greater resistance to corrosive chemistries but limits operational depths to 200 meters. The final component in the fluid system is the optical cell. SEAS II employs a liquid core waveguide (LCW AF-2400, 0.8 mm ID) for this purpose. The submerged waveguide has a numerical aperture of approximately 0.34. Since 80

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the waveguide material is relatively flex ible, it can be coil ed to provide long pathlengths in a small volume. A speci alized cartridge and interface couplers were designed to facilitate rapid excha nge of the LCW (the holder is shown in Figure 4.1C). The LCW is securely held within positioners that engage sockets attached to the electronic housing, and properly position the ends of the LCW to interface the lamp and spectrometer optics through the sapphire windows. A simple screw clamp is utilized to secure the LCW to the instrument framework. Spare cartridges are loaded with appropr iate lengths of LCW and are easily exchanged between deployments. The instrument is operated through a custom user interface and allows either autonomous or manual direction. The graphical user interface provides the means to control the instrument manually as well as to program it for unattended deployments. All primary instrument functions: pump speed, lamp intensity, sampling rate and sampling mode are r eadily accessed. This interface also provides displays for spectral data, calcul ated results (i.e. concentration values) and external peripherals (i.e. CTD, PA R). There are a total of 28 primary directives for control of instrument functions. These commands can be grouped into any user-specified sequence and are st ored in a method. A control-system editor is provided as part of the user interface to facilitate the generation of methods. Prior to deployment, the user downloads the desired method to the instrument. Upon power up, the instru ment will sequentially execute all commands in the current method until term ination by lack of further commands or power interruption. Data presented in th e following sections were gathered using 81

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methods optimized for measurements dur ing vertical up and downcasts from a research vessel. 4.3.2 Analysis of Nitrate. Certified standards of nitrate (1000 ppm in water, Certiprep) were purchased from Fisher and stored at 4C. Water (18.2 M) was freshly obtained from a Milli-Q Gradient purification syst em prior to preparation of reagent and standard solutions. Sulfanilamide (SAN), N-(1-naphthyl)ethylenediamine dihydrochloride (NED), and ammonium chloride/EDTA buffer solutions were prepared as described by Greenbe rg et al. (1985). A 5:1 combined sulfanilamide/NED solution was selected (Fig. 4.2) since it provided the highest absorbance for a single standard nitrate solution. In addition, because the curve (Fig. 4.2) between the 4:1 and 6:1 ratios is essentially flat, small variations in the 5:1 SAN/NED ratio produced during reagen t preparation would not result in large variations in absorbance. As discussed in Yao et al. (1998), the reagent blank measured at 543 nm (~ 0.03 absorbance units) was due primarily to background nitrate in buffer reagents. The blank was determined by taking a reference spectrum of freshly obtai ned Milli-Q water (18.2 M ) and buffer solution following cadmium reduction, adding S AN/NED reagent, and measuring the apparent sample intensity. To preclude nitrox gas contamination, buffer solutions were flushed with nitrogen, pumped into me tal-coated reagent bags and stored at 4C immediately after preparation. 82

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0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 012345678Ratio of v/v Sulfanilamide to NEDAbsorbance 543 nm 575 nm 0.0513 0.623 0.660 0.672 0.675 0.671 0.668 0.297 0.358 0.376 0.381 0.382 0.379 0.378 Figure 4.2. Absorbance measurements for a ni trate standard solution were conducted using a single buffer solution w ith a variety of sulfanilamide to NED ratios. A combined solution ratio of 5: 1 was selected as it provided the highest absorbance and the least variability with changes in mixing ratios. Reduction columns were prepared w ith approximately 6 g of cadmium granules (30-80 mesh) that were rinsed with 1 M HCl, DI water, 2% copper sulfate solution (several times until the blue color remained), then DI water. The copperized granules were packed into a piece of Tygon tubing and stopped at each end with copper wool and reducing f ittings. The column was closed with loops of buffer-filled Tygon tubing and subm erged in water when not in use. Profiles were obtained during a cruise on the R/V Suncoaster from November 13 -15, 2006 in the Gulf of Mexico. A blue filter was placed between the lamp and sapphire interface to remove large differences in light intensity between the monitored wavelengths (Fi g. 4.3A). The SEAS instrument was 83

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connected to a CTD and a PAR sensor, powered on deck, and lowered to 30 meters depth. A two minute delay at th e start of the deployment and a depth trigger to terminate the program at 1 meter depth on the upcast were included to avoid introduction of air into the redu ction column. After the waveguide was flushed with a mixture of ambient seawater and buffer, a reference spectrum was taken at nine wavelengths including a nonabsorbing wavelength (700 nm), which was monitored to remove any error induced by variations in lamp intensity. The SAN/NED reagent pump was then activated to add reagent to the sample/buffer stream. Concentration measurements commenced after six minutes to allow the reagent sufficient time to mix with the sample stream and reach the waveguide. The time elapsed between sample entry into the system and analysis in the LCW was 401 9 s. The instrument package was raised to 10 meters depth, lowered to 200 meters at 5 meters per minute, held at 200 m for two minutes, raised to 10 m at 5 m/min and held at 10 m for 12 minut es. When the instrument was retrieved, the data were uploaded and plotted agains t depth. Calibrations were performed during and after sea trials using spiked su rface seawater (S = 36) with a total of eight concentrations ra nging from 50 nM to 20 M plus an unspiked sample. Sample delay calculations and calibration slope adjustments were performed as described in Adorna to et al. (2005). 84

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Spectrometer Lamp LCW Optical Fiber Waste Sample NH4Cl Buffer SAN/NED Pump ml/min Cd Column 0.25 0.65 0.95 1.85 Main A Sample Molybdate Ascorbic Acid/SDS Heater 30 C Pump ml/min Spectrometer Lamp LCW Optical Fiber Waste 1.08 0.13 0.13 B Figure 4.3. A. A schematic diagram of the n itrate procedure. In addition to the sample and reagent pumps, a main pump is used to pull the combined solution through the system. The cadmium column produces sufficient back-pressure that erratic flow rates can result if the main pump is not active. B. A schematic of the phosphate procedure. The heating unit was placed in-line prior to reagent introduction to avoid corrosion of th e stainless stee l heater tubing by the highly acidic molybdate reagent solution. 85

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4.3.3 Analysis of Phosphate. Certified standards of phosphate (10 00 ppm in water, Certiprep) were purchased from Fisher and stored at 4C. Water (18.2 M) was freshly obtained from a Milli-Q Gradient purification syst em prior to preparation of reagent and standard solutions. Reagents were pr epared as described in Zhang and Chi (2002). For field deployments, ascorb ic acid/sodium dodecyl sulfate (SDS) solution was prepared fresh daily by dissolv ing pre-measured ascorbic acid into 100 mL of sodium dodecyl sulfate solution th at had been prepared previously in the laboratory. Ammonium molybdate a nd ascorbic acid/SDS solutions were added sequentially to the sample stream (Fig. 4.3B). The total reagent contribution to the overall sample stream was 19.7 0.8% (n = 6). Optimization of reagent to sample ratios was achieved by varying th e reagent pump speed and measuring the apparent concentration of a phosphate st andard solution. Absorbance at 700 nm was monitored and sample concentra tion was calculated using a molar absorptivity of 17,000 M -1 cm -1 Initial tests revealed that when insufficient time was allowed for the reaction to proceed, calibrations were nonlinear. Subsequent tests showed that complete or near co mplete reactions were required to produce linearity (Fig 4.4). The calibration slope for the method, determined prior to and following sea trials, was 0.75 0.01 (n = 5). Reagent blanks were on the order of 0.015 absorbance units for freshly prepared molybdate solution and 0.036 for the same solution one month later. Blanks were determined by taking a reference 86

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spectrum of freshly obtained Milli-Q wate r, adding the molybdate and ascorbic acid solutions, and taking a sample spectrum. 0 100 200 300 400 500 600 700 800 0 200 400 600 800 1000Added Phosphate (nM)Apparent Concentration (nM)a b Figure 4.4. Calibration curves produced during the same experiment show the differences between the initial parabolic re sult (a), and the linear result when the reaction is permitted to proceed for an additional 105 seconds (b). During measurements at sea, the SEAS instrument was secured on a frame and connected to a CTD and a PAR sensor (August 2005 and August 2006). After a programmed two mi nute delay, the instrument was lowered to 30 meters for 12 minutes, allowed to flush with ambien t seawater, and raised to 10 meters to begin the cast. A reference spectrum was taken at 9 wavelengths, the reagent pump was activated, and sampling was initiated 690 s later. The delay between sample introduction in the system and en try in the waveguide was 473 3 s. The 87

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package was lowered at 5 m/minute to 200 meters, held for two minutes, and then raised to 10 meters at 5 m/minute where it was held for an additional 12 minutes. The phosphomolybdate complex abso rbs across the visible spectrum (Murphy and Riley, 1962; Zhang et al ., 2001), precluding the use of a nonabsorbing wavelength to remove cha nges in lamp intensity or microbubbleinduced light reduction in the waveguide. Because the instrument is lowered to 30 meters and allowed to flush with seawater prior to the start of the cast, microbubble interference in situ is rarely observed. Ho wever, care must be taken during laboratory measurements at atmos pheric pressure since introduction of microbubbles in the waveguide between th e reference and sample spectra can result in elevated baseline measurements. 4.3.4 Analysis of Nitrite. Nitrite measurements were conducted as described in Adornato et al. (2005) with slight modifications due to the ch ange in instrument configuration from SEAS I to SEAS II. A pathlength of 106 cm was selected to provide the sensitivity necessary to measure low nanom olar concentrations typical of nitrite distributions found in the Gulf of Mexico. The detection limit defined at the 95% confidence level was 0.35 nM (n = 30) and the calibration slope, determined during and following the cruise, was 0.74 0.01 (n = 5). The lower detection limit relative to that described in Adorna to et al. (2005) is due primarily to improvements in pump performance. The nitrite SEAS was connected to a CTD and fluorometer and deploye d in the same manner as the phosphate and nitrate 88

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instruments. Nitrite measurements we re performed in August and November, 2006. 4.4 Results and Discussion 4.4.1 Nitrate Measurements. Due to the wide range of concentra tions found in the upper 250 meters of the oligotrophic open ocean, develo pment of protocols for accurate in situ spectrophotometric measurements was ch allenging. Beer Lamb ert linearity at a single wavelength is typically maintained for absorbance values up to 1 to 2; depending on the quality of the spectromete r. Sample dilution permits reduction of absorbance caused by the high anal yte concentrations found below the nutricline, but is impractical for continuous flow in situ analysis. As an alternative method, multiple wavelengths may be monitored, each with a known molar absorptivity relative to the absorbance maximum (Philo, 1990; Steimle et al., 2002). The azo dye formed in the Griess reaction exhibits maximum absorbance at wavelengths ca. 541 543 nm (Fig. 4.5A). Calculation of off-peak molar absorptivities can be accomplished by linear regression of a series of concentration/absorbance measur ements (Fig. 4.5B). Because the molar absorptivity at 600 nm is approximately one order of magnitude lower than that at 541 nm, it is possible to increase the li near dynamic range of the system by monitoring both wavelengths concurrentl y. Intermediate wavelengths can be monitored for increased accuracy. 89

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0 0.2 0.4 0.6 0.8 1 1.2 400450500550600650700750800Wavelength (nm)Normalized Absorbance Molar Absorptivity for Multiple Wavelengths y = 47,900x at 541 nm y = 27,200x at 575 nm y = 11,575x at 590 nm y = 7,800x at 595 nm y = 5,100x at 600 nm0 0.2 0.4 0.6 0.8 1 1.2 0.00E+005.00E-061.00E-051.50E-052.00E-052.50E-053.00E-053.50E-05Concentration (M)Absorbance Figure 4.5. A. Normalized spectrum of the azo dye produced in both the nitrate and nitrite chemistries shows relative absorbance as a function of wavelength. B. Molar absorptivities calculated for 541 nm (blue), 575 nm (red), 590 nm (green), 595 nm (orange), and 600 nm (purple) using an HP 8453 diode array spectrometer. 90

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Selection of pathlength depends larg ely on the range of concentrations anticipated. Low nanomolar concentr ations found in the euphotic zone necessitate a pathlength of 1 meter or mo re, but measurements that extend below the nitracline require a pathlength that provides a compromise between the highest sensitivity and the widest linear dynamic range. A pathlength of 15 cm proved best suited for measurements that ranged from 2 nM 20 M. 0 50 100 150 200 0 5000 10000 15000 20000Nitrate Concentration (nM)Depth (m) 543 nm 575 nm 590 nm 600 nmGulf of Mexico, 11-14-06 10:26 a.m. Figure 4.6. Nitrate profile obtained in the Gulf of Mexico on November 14, 2006, 10:26 a.m. (27.237 N, 84.510 W) constructed from multiple wavelength (543 nm [blue], 575 nm [red], 590 nm [green], and 600 nm [purple]) absorbance spectroscopy corresponds well with discrete samples collected two hours later (black). Profiles were constructed using f our wavelengths (543 nm, 575 nm, 590 nm, and 600 nm) (Fig. 4.6). The detecti on limits, defined at the 95% confidence 91

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level as three times the standard devia tion of the blank, were 2 nM (543 nm), 3 nM (575 m), 5 nM (590 nm) and 15 nM (600 nm) (n = 30) (Skoog et al., 1998). Each concentration segment of the profile fell well within the range of linearity at the corresponding wavelength. Bottle samples collected using the ships rosette within two hours of the SEAS cast were allowed to reach room temperature and then analyzed on deck at the same flow rates that were used for in situ and calibration measurements. Nydahl (1976) re ported that nitrate to nitrite reduction was complete over a range of 10 30C as long as the flow rate through the cadmium column was sufficiently slow. Co mparison of in situ and bottle results showed good agreement without systematic bias (Fig. 4.6). Of course, some differences were expected owing to ch anges in water column hydrology between the two casts. Deployments in colder wate rs (<10 C) will require the use of a heating cartridge to maintain reaction kinetics. 4.4.2 Nitrite Measurements. Concurrent nitrate and nitrite profiles showed the close correlation observed between the two parameters (F ig. 4.7). The nitrite inflection was observed to occur at the same depth or just below the nitrate inflection in oligotrophic waters, similar to prior observat ions in the Gulf of Mexico (French et al., 1983). Tight coupling of the two para meters has also been reported for the North Pacific Subtropical Gyre, with an average vertical distance between the nitracline and the nitrite maximum of 10 meters (Dore and Karl, 1996a) which was also observed in this study. 92

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0 20 40 60 80 100 120 140 160 180 010002000300040005000600070008000Nitrate Concentration (nM)Depth (m)0 20 40 60 80 100 120 140 160 180 0 50 100 150 200Nitrite Concentration (nM)Depth (m) 543 nm 575 nm 590 nm NO2 Figure 4.7. Nitrate and nitrite profiles collect ed concurrently show the tight coupling observed between the nitrac line and nitrite inflection (27.901 N, 84.482 W, November 15, 2006, 3:53 a.m.). 4.4.3 Phosphate Measurements. Preliminary in situ phosphate measurements conducted aboard the R/V Suncoaster in August 2005 indicated low nanom olar concentrations in the euphotic zone, an increase at the nutricline, and a sec ond, much larger increase at depth. The large increase at depth did not correspond to the observed profiles of any other nutrient. It was determined th at light transmission at depth fell to essentially zero for all monitored wave lengths and that li ght transmission was restored when the instrument returned to the same depth on the upcast. The 93

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attenuation occurred at approximately the same depth on every cast during the research cruise. Initial investigations focused on a po ssible silicate interference since the blue silicomolybdate complex is similar to that formed in the phosphate reaction. Using the same operational configuration and flow rates that were employed for in situ measurements, tests in the laboratory showed that 100 M silicate did not interfere with measurements of a 1 M phosphate solution. Because the kinetics of the silicate reaction is much slower than that for phosphate, the elapsed time between reagent introduction and phos phate analysis was insufficient for formation of the silicomolybdate complex. 0 500 1000 1500 2000 2500 141618202224262830Temperature (C) Intensity (counts)16.2 C Figure 4.8. Intensity at 700 nm plotted as a function of temperature shows the rapid signal attenuation that occurs at the 16.2C isotherm. Data were collected August 4, 2005, 6:40 a.m. (27.333 N, 84.806 W). 94

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Examination of the cruise data i ndicated that strong light attenuation occurred whenever the in situ temperature was 16.2 C (Fig. 4.8). While investigating SDS as a po ssible shark repellent, Si sneros and Nelson (2001) observed precipitation in seawater at temperatures below 17 C. SEAS phosphate profiles obtained using reduced surfactant concentrations produced erratic results, probably due to small quantities of precipitate passing through the waveguide. Introduction of a heating unit between the sample intake and the reagent introduction port was tested as a possibl e remedy for surfactant precipitation. Since silicate interference can occur at elevated temperatures, it was deemed advisable to use the lowest possible temp erature that would result in a stable signal, maintain reaction kinetics, and prevent reagen t precipitation. A temperature of 30C was found to satisfy these requirements. Phosphate profiles were obtained aboard the R/V Suncoaster on August 7 11, 2006. Bottle samples obtained from the ships rosette were analyzed using the in situ method except that the samples were permitted to reach room temperature prior to analysis and run with the heater turned off. The concentrations determined using discre te samples produced good correlation with in situ data (Fig. 4.9), indicating that the reaction kinetics were maintained in situ through use of the heating cartridge. If more sensitive measurements are required in the upper euphotic zone, a so mewhat longer pathlength can be utilized. In this case, since arsenate produces a similar blue molybdate complex and can interfere when phosphate is present at low concentra tions, a third reagent line can be added 95

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to introduce either sulfite or thiosulfat e solution and reduce arsenate to the nonreactive arsenite (Johnson, 1971; Linge and Oldham, 2001). 0 20 40 60 80 100 120 140 160 180 200 020040060080010001200Phosphate (nM)Depth (m) Figure 4.9. Phosphate profile using a heater cartridge set at 30 C (27.777 N, 84.628 W, August 10, 2006, 12:38 a.m.) compares well with bottle samples collected from the ships rosette. Another difficulty often faced when analyzing phosphate is coating of optical surfaces over time by the phosphomol ybdate product (Zhang et al., 2001). This is due to the adsorption of the highly charged Keggin type anion [PSb 2 Mo 10 O 40 3, (oxidized) (Going a nd Eisenreich, 1974), or H x PSb 2 Mo 10 O 40 3, (reduced) where x = 2, 4 or 6 (Sadakane and Steckhan, 1998)] on the quartz or fused silica walls of standa rd cuvettes and flow cells. Both quartz and silica surfaces are prone to surface silanol formation when exposed to aqueous solutions. In contrast, the Type 1 LCW used in this work has a hydrophobic 96

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surface (Arcella et al., 2003), and the sapphire optical windows do not tend to form silanol surface groups (Maw et al., 2002). No signal degradation was observed during the course of phosphate an alysis using the Type 1 LCW in this work. Because turbid samples cause light attenuation in spectrophotometric systems, analyses of coastal and estuarine wa ters require the use of in-line filters. While testing SEAS in an estuarine environment, initial phosphate measurements using 0.4 m filters produced good results without observable drift. Future investigations will be conducted to determ ine the optimal conditions necessary to use the SEAS instrument in waters with reduced or variable optical clarity. During a month-long research cruise in the oligotrophic Pacific, SEAS measurements did not reveal signal attenuation due to particulate organic matter, which was highest in the upper 50 meters (Adornato et al., 2005). All continuous flow methods su ffer from some degree of sample dispersion that can blur sh arp concentration changes such as those found at the nutricline. The extent of sample disper sion increases with th e length of time that the sample spends in contact with tubing walls; an issue that is more likely to affect phosphate and nitrate analyses than methods with faster kinetics. Sample carry-over can be substantially mitigated by slowing the rate of instrument descent or ascent in large concentration gr adients. Future work will include realtime communication with SEAS II, allowi ng users to reduce ascent/descent rates in response to distributions and gr adients of particular interest. 97

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Appendices 110

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Appendix A: Analytical Procedures Nitrite Instrument Configuration : The SEAS II instrument is capable of pumping six separate sample/reagent lines, using three pump heads. The speed of each pump can be programmed, so that the flow in each of the pumps two lines can be differentially controlled by varying the diameter of the tubing. For nitrite, the second (reagent) pump is configured for addition of color reagent, using gray/gray three-stop tubing. The reagent flows to a Y connector fitted with a flow check valve, which prevents sample from backing up the reagent line when the reagent pump is off. The reagent combines with sample in a mixing column and the resultant solution is then pumped through the first (main) pump, fitted with purple/black three-stop tubing, into the liquid co re waveguide. The pump ratios, using the aforementioned tubing, are optimized to produce the greatest color development with the least sample carryover. The third pump can add a nitrite standard if the user wishes to calibrate the instrument in situ. Reagent Preparation Sulfanilamide Solution : 2.5 g of sulfanilamide (>99%, Aldrich) is dissolved in a mixture of 25 mL concentrated ( 12 M) hydrochloric ac id (Certified ACS Plus, Fisher Chemical) and 150 mL Milli-Q water (18.2 Mresistance). The resultant solution is diluted to 250 nM with Milli-Q water. N(1-naphthyl)ethylenediamine, dihydrochloride (NED) Solution : 0.25 g of NED (>98%, Aldrich) is dissolved in 250 mL of Milli-Q water and stored in a brown 111

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Appendix A (Continued) Refrigerated glass bottle (~4 C). This solution is stable for up to two months. Formation of product: Nitrite reacts first with sulfanilamide to form a diazotized intermediate, which subsequently reacts with NED to form the pink-colored azo product (Appendix Figure 1). NH2 SO2NH2 N N SO2NH2 H NO2 + Nitrite Sulfanilamide Intermediate 112

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Appendix A (Continued) HN NH2 2HCl N N SO2NH2 + HN NH2 N N SO2NH2 Intermediate N(1-naphthyl)ethylenediamine, Azo dye product (pink) dihydrochloride (NED) Appendix Figure 1 Reaction of nitrite with sulfan ilamide in acidic medium to form an intermediate species which subse quently reacts with NED to form the azo dye product. Nitrite Variability The vertical distribution of nitrite changes remarkab ly over a day/night cycle (Appendix Figure 2). 113

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Appendix A (Continued) Appendix Figure 2 Nitrite undergoes dramatic changes over a diel cycle (November 15-16, 2005, 27 23.738 W, 84 50.238 W). Depth is from 0 to 250 meters and nitrite concentration is from 0 to 250 nM. The color of the background indicates time of day, with the darkest at mi dnight and the lightest at noon. 114

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Appendix B: Analytical Procedures Nitrate Instrument Configuration : The instrument configuration for nitrate measurements is identical to that of nitrite, but with the following modifications. Orange-orange three-stop Pharmed tubing is placed in the upper slots of the main pump head to introduce buffer into the sample stream. The tubing size was selected in order to maintain the appropriate mixing ratio with the sample. The buffer line is connected to the sample lin e with a Y connector and a flow check valve is used to prevent sample from flowing into the buffer line. The sample/buffer solution is th en introduced into a coppe r coated cadmium column and thereafter connected to th e color reagen t line with a Y conn ector as detailed in the nitrite procedure. The solution is pumped through the liquid core waveguide by the main pump and monito red spectrophotometrically as described above. Reagent Preparation Ammonium Chloride/EDTA Buffer: Dissolve 6.5 g ammonium chloride (99.99%, Aldrich) and 0.85 g of ethylen ediaminetetraacetic acid (EDTA, 99.99%, Aldrich) in 400 mL Milli-Q water. Adjust the pH to 8.5 with concentrated ammonium hydroxide (99.99%, Aldrich), and dilute the resulta nt solution to 500 mL with Milli-Q water. This solution is st able for months, as long as it is not left open to the atmosphere. Copper Sulfate Solution: Dissolve 5.0 g copper sulf ate pentahydrate (98+%, ACS Reagent, Aldrich) in 250 mL Milli-Q wa ter. This solution is stable for more than six months. 115

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Appendix B (Continued) Formation of product: Nitrate is reduced to nitr ite using a copper-coated cadmi um column (Gal et al., 2004), according to the following reaction: Cd + NO 3 + 2H + NO 2 + Cd 2+ + H 2 O Equation B.1 Nitrite formed then reacts with the Gr iess reagents as discussed for nitrite detection. Reduction of oxygen by cadmium is much faster than reduction of nitrate and proceeds (Nydahl, 1976) as follows: Cd + O 2 + 2H + Cd 2+ + H 2 O Equation B.2 The effects of this are two-fold. Fi rst, the amount of cadmium removed from the reduction column can, over time, adversely affect the cadmium surface area available for reaction. Flushing the bu ffer solution with nitrogen gas reduces the dissolved oxygen content of the buffere d sample solution. Second, the pH of the solution adjacent to the cadmium metal increases. An ammonium chloride/EDTA solution is used to buffe r the pH during the reduction step. The EDTA component is also used to prev ent precipitation of cadmium and copper hydroxides that can occur at pH > 8.5 (Gal et al., 2004). Cadmium ions primarily complex with chloride ions in seawater: CdCl + 36% CdCl 2 45% CdCl 3 16% 116

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Appendix B (Continued) The remaining 3% is free (Byrne, 2002; Turner). In addition to the chloride complexes, the following equilibria ma y also occur in the presence of the ammonium chloride/EDTA buffer: Cd 2+ + H-EDTA 3Cd-EDTA 2+ H + Equation B.3 Cd 2+ + NH 3 [Cd(NH 3 )] 2+ Equation B.4 [Cd(NH 3 )] 2+ + NH 3 [Cd(NH 3 ) 2 ] 2+ Equation B.5 Copper is primarily complexed with carbona te in seawater (Byrne, 2002), but can also complex with a number of other species including ammonia and EDTA. The rates of copper and cadmium comple xation with EDTA are slowed by the presence of calcium ions in seawater (Bruland, 1992; Heri ng and Morel, 1989), but it is uncertain whether the diminish ed reaction kinetics renders EDTA less effective as a precipitation prevention agent during nitrate re duction. Calibrations conducted during and following the Novemb er 2006 cruise did not indicate any loss of reduction efficiency in the cadmium column through time (Appendix Figure 3). 117

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Appendix B (Continued) Nitrate Calibration 11-17-2006 y = 0.3879x + 110.01 R2 = 0.9998 y = 0.4216x + 91.608 R2 = 0.9994 y = 0.4219x + 121.21 R2 = 0.9999 y = 0.4451x + 75.786 R2 = 0.9997 0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 10000.0 0 500010000150002000025000 Added Nitrate (nM)Apparent Concentration (nM) 543 nm 575 nm 590 nm 600 nm Nitrate Calibration 11-22-2006 y = 0.446x + 77.772 R2 = 0.999 y = 0.4387x + 83.013 R2 = 0.9997 y = 0.4098x + 99.084 R2 = 0.9999 y = 0.4323x + 155.73 R2 = 0.9998 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 020004000600080001000012000 Added Nitrate (nM)Apparent Concentration (nM) 543 nm 575 nm 590 nm 600 nm Appendix Figure 3 Calibrations conducted during and after the November 2006 cruise show maintenance of cadmium reductor efficiency. 118

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Appendix C: Analytical Procedures Phosphate Instrument Configuration : For phosphate analysis, the second pump is configured for sequential addition of each color reagent using green/green threestop tubing. Each reagent solution flow s to a Y connector and a flow check valve, which prevent sample from back ing up the reagent line when the reagent pump is off. Molybdate solution is introduced into the sample line and, immediately thereafter, as corbic acid/SDS solution is introduced. The color reagents combine with sample in a mixing column and the resultant solution is then pumped through the first (main) pum p via green/green three-stop tubing, and thereafter into the liquid core waveguid e. The pump flow ratios, using the aforementioned tubing, are optimized to produce the greatest color development with the least sample carryove r (Appendix Figure 4). 119

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Appendix C (Continued) 0 100 200 300 400 500 600 700 0100200300400500600700Sample NumberConcentration (nM)6 rpm 5 rpm 4 rpm 3 rpm 2 rpm 1 rpm Appendix Figure 4 Optimization of reagent to sample ratios is accomplished by varying the reagent pump speed while the ma in pump speed is held constant (25 rpm). Reagent Preparation Potassium Antimony Tartrate Solution: Dissolve 1.5 g of potassium antimony tartrate (99+%, Fi sher) in 500 mL of Mill i-Q water. Store in a refrigerated brown HDPE plastic bottle (~4 C). Sulfuric Acid Solution (5 N): Slowly add 70 mL of concentrated sulfuric acid to 400 mL Milli-Q water. After the solution cools to room temperature, dilute the solution to 500 mL with Milli-Q water. Ammonium Molybdate Solution: Dissolve 2.3 g of ammonium molybdate in 192 mL of 5 N sulfuric acid solution. Add 50 mL of potassium antimony tartrate 120

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Appendix C (Continued) solution and dilute the resultant solution to 1 L with Milli-Q water. Store in a refrigerated MEK or HDPE plastic bottle. Ascorbic Acid Solution: Dissolve 0.5 g of ascorbic acid and 7 g of sodium dodecyl sulfate in 100 mL Milli-Q water. This solution should be made daily. Formation of product: Phosphate reacts with the ammonium molybdate/potassium antimony tartrate solution to form a heteropoly acid, which is then reduced by ascorbic acid to form a blue colloidal product. Although the balanced reaction proves difficult to determine, the stoichiometry of th e product has been identified as PSb 2 Mo 10 O 40 3(Going and Eisenreich, 1974) The product is an -Keggin anion with the following structure (Barteau et al., 2006): Appendix Figure 5 The Keggin structure includ es a phosphorus atom at the center surrounded by ten molybdate and two antimonate ions, each in an octahedral structure. Spheres represent oxygen atoms. Figure from Barteau et al. (Barteau et al., 2006). 121

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Appendix C (Continued) Both the oxidized and the reduced (blue) fo rms maintain the Keggin structure. At pH < 4, a multi-step, six electron redu ction of the phosphomolybdate species occurs along with addition of six hydr ogen ions, so the overall charge (-3) remains unchanged (Zhang et al., 2002). While not necessary to product formation, the addition of antimony provides additional stability and results in a product with two absorbance maxima, one at 880 nm ( = 20,000 M -1 cm -1 ) and one at 710 nm ( = 17,000 M -1 cm -1 ) (Zhang et al., 2001). This is desirable since the maximum at 710 nm falls within the range monitored by the Ocean Optics spectrometers used in the SEAS instrument s. Inclusion of th e surfactant sodium dodecylsulfate (SDS) is necessary to prevent product adsorption on waveguide surfaces (Zhang and Chi, 2002). 122

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Appendix D: SEAS Cruise Locations SEAS field tests were conducted at sites on the West Florida Shelf and at the shelfbreak (Appendix Figure 6). Th e August 2006 test station was located further west than the November 2006 cruise It became clear that the ship drift was substantially higher during the August cruise, with a current of over 1 knot (i.e. the ship drifted betw een 2.3 and 2.7 nautical miles during the course of a two-hour SEAS cast, 1 knot = 1 nautical mile/hour). The ship re turned to station at the conclusion of each cast. As a result, the parcels of water sampled were different for each cast. This effect becomes important when attempting to monitor diel changes in nutrient distributions. 123

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Appendix D (Continued) Appendix Figure 6 Map of the West Fl orida Shelf with the locations of the August 2006 and November 2006 cruises marked with pin icons. 124

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About the Author Lori Adornato earned her Bachelor of Arts degree in Chemistry from University of South Florida in 2000, where she r eceived the Outstanding Undergraduate Teaching Award. She subsequently entere d the Ph.D. program in the Department of Chemistry at the University of South Florida in 2000 and accepted the Chemistry Graduate Student Entrance Award. In 2002, she transferred to the Ph.D. program in the University of South Florida, College of Marine Science, to study high sensitivity nutrient analysis w ith Dr. Robert H. Byrne. During her Marine Science Ph.D. studies she received the Robert M. Garrels Fellowship and the William and Elsie Knight Fellowship. She participated in eighteen research cruises for a total of 91 days at sea.