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Price, Roy E.
Biogeochemical cycling of arsenic in the shallow marine hydrothermal system of Tutum Bay, Ambitle Island, Papua New Guinea
h [electronic resource] /
by Roy E. Price.
[Tampa, Fla] :
b University of South Florida,
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Dissertation (Ph.D.)--University of South Florida, 2008.
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Advisor: Thomas Pichler, Ph.D.
ABSTRACT: The marine shallow-water hydrothermal vent system of Tutum Bay, Ambitle Island, PNG discharges hot, acidic, arsenic-rich, chemically reduced fluid into cool, alkaline, oxygenated seawater. Gradients in temperature, pH, total arsenic (TAs) and arsenic species, among others, are established as the two aqueous phases mix. Hydrous ferric oxides (HFO) are precipitated around focused venting, and coat the surrounding sediments visibly to 150 m away. HFO coatings, mechanical transport and weathering of volcanoclastic sediments, as well as dissolution of carbonate sediments nearer to venting, combine to alter sediment chemistry substantially. Tutum Bay surface sediments have a mean As concentration of 527 mg/kg. Arsenic at concentrations up to 50 mg/kg (mean = 19.7 mg/kg) was extracted from the easily extractable fraction of surface sediments.Arsenic is elevated in surface seawaters (8 g/L) directly over hydrothermal vents, and As(III) is substantially enriched in both surface and bottom seawater throughout Tutum Bay. Surprisingly, aqueous As(V) far exceeded aqueous As(III) at almost all distances and depths investigated for Tutum Bay pore waters. These data indicate that throughout Tutum Bay, chemical disequilibria among As species provides potential metabolic energy for arsenite oxidizing and arsenate reducing microorganisms, and that As is bioavailable from two major environments: 1) easily-exchangeable As from surface sediments, and 2) in surface seawaters, which may allow for biological uptake and trophic transfer through plankton. The soft coral Clavularia sp., the calcareous algae Halimeda sp., and the tunicate Polycarpa sp. were collected and analyzed to assess bioaccumulation and biotransformation patterns. All organisms collected from the hydrothermal area displayed higher (2 to 20 times) TAs.Concentrations of arsenic species in their tissues were also elevated compared to the control site. Increased concentrations were observed near focused venting. Distinct arsenic speciation patterns in Clavularia and Polycarpa collected from near hydrothermal venting suggest rapid methylation/detoxification of arsenic, with enhanced bioaccumulation of dimethylarsenate and arsenobetaine as products of the organisms metabolic pathways. Elevated concentrations of As(III) in Halimeda suggest that this organism is not as efficient at methylating inorganic arsenic. The presence of arsenobetaine in Halimeda suggests the biomethylation pathway for calcareous algae is different from commonly studied seaweeds.
t USF Electronic Theses and Dissertations.
Biogeochemical Cycling of Arsenic in the Ma rine Shallow-water Hydrothermal System of Tutum Bay, Ambitle Island, Papua New Guinea by Roy E. Price A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Geology College of Arts and Sciences University of South Florida Major Professor: Thomas Pichler, Ph.D. Robert H. Byrne, Ph.D. Peter Harries, Ph.D. Pamela Hallock Muller, Ph.D. Mark C. Rains, Ph.D. Date of Approval: March 31, 2008 Keywords: arsenic, biogeochemical cycli ng, shallow-water, marine, hydrothermal, bioaccumulation, speciation Copyright 2008, Roy E. Price
Dedication To Joe W. Smith, who inspired me to become a scientist.
Acknowledgements Many people have contri buted to this dissertat ion, both on a personal and professional level. In many ways, the support and guidance began years before this research was conducted. Without the suppor t and encouragement of many people during many stages in my entire life, this dissertati on would not have become a reality. First, I sincerely thank my parents, Larry and Carol Price, my brother Dean and his wife, Mandy, for their love, support, encouragement, always being there for me when I needed them, and most of all for believing in me. I owe them a debt I can never repay. Many thanks to Mr. Joe W. Smith, my hi gh school science teacher, who inspired me to become a scientist. It was he who taught me that determination is the key to success and that if I put my mind to it, I could accomplish anything. Mr. Smith is just beginning his 40th year of teaching science, a measure of his determination and dedication. For this reason, I dedicate this di ssertation to him. I mu st also thank Mrs. Jeanie Oliver, who saw potential in a shy kid and taught me that having the courage to follow your dreams is the most im portant thing in life. Many thanks to my professors at the University of Arkansas: Dr. Walter Manger, Dr. Doy Zachery, and Dr. Jon van Brahana, who inspired me to become a geologist. I thank Anya Frashuer, my best frie nd and partner, who has optimistically supported me throughout the highs and lows th at come with attempting to complete a research project of this magnitude. I sincer ely thank Anya for always believing in me.
Support for this research is from th e National Science Foundation (BE: CBC# 0221834) awarded to Dr. Thomas Pichler, my primary advisor for this research. Many thanks to Thomas for giving me the opportuni ty to do this amazing research when he could have easily chosen someone else, fo r giving me many grea t opportunities to do exciting research outside the scope of this dissertation, and for revi ewing all my papers and manuscripts, even on the weekends. Than k you also to my dissertation committee, Dr. Robert Byrne, Dr. Pamela Hallock Mulle r, Dr. Peter Harries, and Dr. Mark Rains, who reviewed this manuscript and provided many useful comments, and who have all been incredibly encouraging and confident in my abilities. I thank Pamela Hallock Muller, Brian McClosky, Jim Garey, David Karlen, Jan Amend, and Darcy Meyer-Dombard for their ad vice and considerable support with field work. I thank Dr. Kevin Francesconi for providing arsenosugar extracts described in Chapter Four. I thank David Karlen for providi ng grain-size, sorting, and percent organic and inorganic carbon data presented in Chapte r One. Thanks to Dr. Dirk Wallschlager and Jacqueline London for considerable help with the organoarsenic analyses presented in Chapter Four. Thanks to Dr. William Maher, for reviewing the first draft of the paper which became Chapter Four. Thanks to Dr. Jan Amend, who allowed me to work in his laboratory at Washington University in St. L ouis. Although that research did not make it into this dissertation, I am very grateful to have had the opportu nity to learn about microbiology. Finally, I say thank you to all the friends and family not listed here, but who have contributed to shaping me, my research, and my future.
i Table of Contents List of Tables iv List of Figures vi Chapter One. Introduction 1 1.1 Marine shallow-water hydrothermal venting 1 1.2 Description of the Tutum Bay ma rine shallow-water hydrothermal system 4 1.3 Review of arsenic speciation a nd its importance to toxicity and bioavailability 9 1.4 Rationale 14 1.5 Objectives 17 1.6 Arrangement of Dissertation 18 1.7 Lithology and geochemistry of sediments of Tutum Bay 20 1.7.1 Surface Sediments 22 1.7.2 Core Sediments 26 1.7.3 Element distributions along the Tutum Bay transect 28 1.7.4 Element distributions in core sediments 33 1.8 Controls on element distributions 33 Chapter Two. Enhanced Geochemical Gr adients in a Marine Shallow-water Hydrothermal System: Unusual Arsenic Sp eciation in Horizontal and Vertical Pore Water Profiles 39 2.1 Introduction 39 2.2 Methods 40 2.2.1 Field 40 2.2.2 Laboratory 43 2.3 Results 44 2.3.1 Horizontal Profile (10 cm Pore Water Samples) 44 2.3.2 Pore Water Profiles 45 2.4 Discussion 45 2.4.1 Are the As(III)/As(V) ratios in vertical pore water profiles unusual? 45 2.4.2 Thermodynamic and Kinetic Considerations 50 2.4.3 Possible Microbial Metabolisms 53 2.5 Summary and Conclusions 55
ii Chapter Three: Distribution, Speciation, and Bioavailability of Arsenic in a Shallow-water Submarine Hydrothermal System, Tutum Bay, Ambitle Island, PNG 59 3.1 Introduction 59 3.1.1 Arsenic Speciation and Bioavailability 60 3.2 Methods and Procedures 62 3.2.1 Sample Handling and Preparation 62 3.2.2 Lab Measurements 65 3.2.3 Sequential Extraction of As in Tutum Bay Sediments 66 3.3 Results 68 3.3.1 Temperature and pH Variation of Surface Sediment Porewater 68 3.3.2 Arsenic in Vent Fluids, Pore Waters and Ambient Seawater 70 3.3.3 Arsenic Abundance and Bioavaila bility in Precipitates and Sediments 72 3.4 Discussion 75 3.4.1 The Importance of Diffuse Ve nting on As Distribution and Speciation 75 3.4.2 Mechanism for As Enrichment in Sediments 79 3.4.3 Bioavailability of As in Tutum Bay 80 3.5 Summary 81 Chapter Four: Enhanced Bioaccumulation and Biotransformation of Arsenic in Coral reef Organisms Surrounding an Ar senic-rich Marine Shallow-water Hydrothermal Vent System 89 4.1 Introduction 89 4.2 Biota 92 4.2.1 Clavularia 92 4.2.2 Halimeda 94 4.2.3 Polycarpa 94 4.3 Methods 94 4.3.1 Field 94 4.3.2 Laboratory 95 220.127.116.11 Total Arsenic Concentration (TAs) 95 18.104.22.168 Extraction and Arsenic Speciation 96 4.3.3 Quality Assurance/Quality Control 97 4.4 Results and Discussion 99 4.4.1 General Points 99 4.4.2 Total Arsenic Concentration TAs 100 22.214.171.124 Clavularia 100 126.96.36.199 Halimeda 103 188.8.131.52 Polycarpa 104 4.4.3 Organoarsenic Speciation 105 184.108.40.206 Clavularia 106
iii 220.127.116.11 Halimeda 111 18.104.22.168 Polycarpa 117 22.214.171.124 Assessment of excluded arsenic 118 4.5 Biomethylation Pathways 119 4.5.1 Clavularia 120 4.5.2 Halimeda 122 4.5.3 Polycarpa 124 4.6 Summary and Conclusions 124 Chapter Five: Summary and Conclusions 130 References 134 About the Author End Page
iv List of Tables Table 1.1. Bulk geochemistry data for Tutum Bay surface sediments arranged by distance from focused hydrothermal venting. 35 Table 1.2. Bulk geochemistry data for Tutum Bay core sediments arranged by distance from focused hydrotherm al venting, then by depth below seafloor. 37 Table 2.1. Arsenic, H4SiO4, Mg2+ and other physicochemical parameters measured in 10-cm pore waters for the Tutum Bay hydrothermal system, sorted by distance from focused venting. 57 Table 2.2. Arsenic, H4SiO4, Mg2+ and other physicochemical parameters in all pore waters for the Tutum Bay hydrothermal system, sorted by distance from focused venting, th en by depth into sediment. 58 Table 3.1. Sequential chemical extraction procedure for As and Fe in sediments and vent precipitates from Tu tum Bay hydrothermal system. 84 Table 3.2. Temperature, pH, arsenic (As) and silica (H4SiO4) in pore-water profiles for points along the tran sect shown in Figure 1.8 and the Picnic Island control site (C S1), compared to Vent 4. 85 Table 3.3. Arsenic concen tration and speci ation in seawater for sampling points along the transect shown in Figure 1.8 in surface and bottom seawater. 86 Table 3.4. Arsenic, Fe and Ca com position in Tutum Bay sediment cores collected along the transect show n in Figure 1.8, compared to the Picnic Island control site (CS1). 87 Table 3.5. Four-step sequen tial extraction results for vent precipitates (VP), surface sediments collected along th e transect shown in Figure 1.8, compared to the Picnic Island control site (CS1). All values in mg/kg. 88 Table 4.1. Comparison of total arseni c abundance (TAs), Sum of leachable arsenic species ( ), total leachable arsenic (TLAs), and mass balance. 126
v Table 4.2. Arsenic species in methano l/water extracts. Data are arranged vertically by organism (column 1), then by distance away from focused hydrothermal venting (c olumn 2). Elution times are presented above the species ID. All values are mg/kg solid. 127 Table 4.3. Arsenic species in methanol/w ater extracts presented as percent of species. 128 Table 4.4. Cationic arsenic specia tion of methanol extracts for Clavularia and Halimeda All values are mg/kg solid. 129
vi List of Figures Figure 1.1. Location of Ambitle Island and the marine shallow-water hydrothermal vents investigat ed in this research. 5 Figure 1.2. Location of focused vent areas of the hydrothermal system in Tutum Bay. 6 Figure 1.3. Underwater photograph of focused hydrothermal venting. 7 Figure 1.4. Underwater photograph of diffuse hydrothermal venting. 8 Figure 1.5. A) Underwater photograph of HFO precipitate s. B) Scanning Electron Microscope (SEM) image of HFO. 10 Figure 1.6. Eh-pH diagram for the system As-O-H at 25C (dashed lines) and 100C (solid lines) at 1.013 bars. 12 Figure 1.7. Physical, chemical, and biologi cal trends stepping away from the area of active venti ng in Tutum Bay. 16 Figure 1.8. Location of transect along wh ich samples were collected for this study. 19 Figure 1.9. Photograph of surface sediment s collected along the transect. Note color change from orange (HFO) to white (CaCO3). 23 Figure 1.10. Surface sediment character istics for Tutum Bay hydrothermal transect and Danlam Bay reference site 24 Figure 1.11. Graphic showing sediment ch aracteristics for each core collected along the transect. 27 Figure 1.12. Photograph of a core coll ected from near hydrothermal vents showing distinct redox boundary. 29 Figure 1.13. SEM images of framboidal pyrite discovered in reduced core sediments. 30 Figure 1.14. Selected element distribu tion patterns for Tu tum Bay surface sediments. 32
vii Figure 2.1. Distribution of arsenite (As(III )) and arsenate (As(V)) in pore waters collected at 10 cm sediment dept h, stepping away fr om the area of hydrothermal venting. 48 Figure 2.2. Distribution of arsenite (As (III)) and arsenate (A s(V)) in vertical pore water profiles collected at distances of 2.5, 30, 60, 140 and 300 m from the area of focused hydrothermal venting. 49 Figure 2.3. Conceptual model of the exp ected As gradients in Tutum Bay pore water profiles exclusively invoki ng the assumption of seawater mixing and abiotic oxidation. 50 Figure 2.4. Observed vertical pore wate r profiles in Tutum Bay sediments at a distance of 30 and 60 m from the main area of hydrothermal venting. 52 Figure 3.1. Temperature and pH relationshi p in pore-waters of surface sediment (~5 cm depth) along transect vs. distance from focused venting. 69 Figure 3.2. Arsenic and Si concentrati ons in surface and bottom waters along the transect. 71 Figure 3.3. The variation of As concentra tion in the easily-extractable sediment fraction and total concentration of As in surface sediments along the transect shown in Figure 1.8. 73 Figure 4.1. Underwater photogr aphs of coral-reef or ganisms collected from Tutum Bay. 93 Figure 4.2. Total arsenic concentration for A) Clavularia, B) Halimeda, and C) Polycarpa. 101 Figure 4.3. Chromatograms showing the rela tive difference in arsenic speciation between Clavularia collected from the Picnic Island control site (CS1; top), and the Clavularia collected from the vent site (bottom). 107 Figure 4.4. Data evaluation for Clavularia sp 109 Figure 4.5. Data evaluation for Halimeda sp. 116
viii Biogeochemical Cycling of Arsenic in the Ma rine Shallow-water Hydrothermal System of Tutum Bay, Ambitle Island, Papua New Guinea Roy E. Price ABSTRACT The marine shallow-water hydrothermal vent system of Tutum Bay, Ambitle Island, PNG discharges hot, acidi c, arsenic-rich, chemically reduced fluid into cool, alkaline, oxygenated seawater. Gradients in temperature, pH, total arsenic (TAs) and arsenic species, among others, are estab lished as the two aqueous phases mix. Hydrous ferric oxides (HFO) are precipi tated around focused venting, and coat the surrounding sediments visi bly to 150 m away. HFO coati ngs, mechanical transport and weathering of volcanoclastic sediments, as well as dissolution of carbonate sediments nearer to venting, combine to alter sediment chemistry substantially. Tutum Bay surface sediments have a m ean As concentration of 527 mg/kg. Arsenic at concentrations up to 50 mg/kg (mean = 19.7 mg/kg) was extracted from the easily extractable fraction of surface sediments. Arsenic is elevated in surface seawaters (8 g/L) directly over hydrothermal vents, and As(III) is substantially enriched in both surface and bottom seawater throughout Tutu m Bay. Surprisingly, aqueous As(V) far exceeded aqueous As(III) at almost all distan ces and depths investigated for Tutum Bay pore waters. These data indicate that th roughout Tutum Bay, chem ical disequilibria among As species provides potentia l metabolic energy for arsenite oxidizing and arsenate reducing microorganisms, and that As is bi oavailable from two major environments: 1)
ix easily-exchangeable As from surface sediments, and 2) in surface seawaters, which may allow for biological uptake and tr ophic transfer through plankton. The soft coral Clavularia sp. the calcareous algae Halimeda sp. and the tunicate Polycarpa sp. were collected and analyzed to assess bioaccumulation and biotransformation patterns. All organisms co llected from the hydrothermal area displayed higher (2 to 20 times) TAs. Concentrations of arsenic species in their tissues were also elevated compared to the control site. In creased concentrations were observed near focused venting. Distinct arsenic speciation patterns in Clavularia and Polycarpa collected from near hydrothermal venting s uggest rapid methylati on/detoxification of arsenic, with enhanced bioaccumulation of dimethylarsenate and arsenobetaine as products of the organisms metabolic pathways Elevated concentrations of As(III) in Halimeda suggest that this organism is not as e fficient at methylati ng inorganic arsenic. The presence of arsenobetaine in Halimeda suggests the biomethylation pathway for calcareous algae is different fr om commonly studied seaweeds.
1 Chapter One Introduction 1.1 Marine shallow-water hydrothermal venting Marine shallow-water hydrot hermal vent systems, defi ned as occurring in less than 200 m seawater and often having a meteor ic water source, are commonly enriched in biologically toxic elements such as As, Sb, Se Cr, Co, Pb, Cd, Ag, Cu, Tl, Zn, Hg, and S, as well as possible nutrients such as Si a nd Fe (Dando et al., 1999; Varnavas and Cronan, 1988; Vidal et al., 1978). These systems have been described in many areas around the world, where they are generally associated with volcanic (e.g., Dando et al., 2000) or tectonic activity (e.g., Vidal et al., 1978), which provide the necessary heat source. Considering their location, the biotic impact of these systems may be much larger than predicted. Coastal waters are of ten breeding and nursing grounds for many organisms, in tropical areas they host coral reefs, and humans have always used these waters for fishing and recreation. The chemical composition of seawater and sediments in these coastal areas is nor mally controlled through a combination of natural and anthropogenic processes. However, in areas of volcanic activity and/or high heat flow, the discharge of fluids from marine sha llow-water hydrothermal systems may have a considerable impact on the chemical com position of the often biologically important coastal surface waters and sediments. Focu sed discharge of hydrothermal fluids at discrete vent orifices, along with diffuse venting through sediments, can occur. Diffuse
2 venting may influence benthic organisms at great distances from fo cused venting (e.g., Karlen et al., in review; Tarasov et al ., 1999; Varnavas and Cronan, 1988). Considering the global abundance of marine shallow-water hydrothermal vent systems, aqueous and gas phase emissions may well be a significant contributor to global ocean and atmosphere chemical budgets that have b een consistantly overlooked. Like their deep-sea counterparts, marine shallow-water hydrothermal systems are characterized by steep physicochemical gr adients, which can drastically affect surrounding biology, particularly microbial community composition (Amend et al., 2003a; Brinkhoff et al., 1999; Rogers and Ame nd, 2006; Rusch et al., 2005; Tarasov et al., 1986). The chemical disequilibria that ar e caused by hydrothermal venting can also have large impacts on coastal benthic communiti es, including corals (Karlen et al., in review), and steep geochemi cal gradients (e.g., for arsenic speciation) may allow for microbial communities similar to those found in terrestrial hot springs or hydrothermal lakes (Langner et al., 2001; Oremland and Stolz, 2003). The hydrothermal fluids may contain elemen ts that can generate oxide, sulfide, and precious metal ore deposits (Canet et al., 2005a; Cane t et al., 2005b; Hein et al., 1999; Prol-Ledesma et al., 2002; Stoffers et al ., 1999). In contrast to deeper hydrothermal systems, which express themselves typically with pyrite chimneys, lower pressures and consequently lower water boiling temperatures in marine shallowwater hydrothermal systems lead to the possibility of subsur face deposition of metals (e.g., Gurvich, 2006). Many minerals have been reported in mari ne shallow-water hydrothermal systems, incuding; pyrite/marcasite, cinnabar, realgar, orpiment, anhydrite, gypsum, barite, calcite,
3 aragonite, phosphates (hydroxylap atite), ferrihydrite, todoroki te, native sulfur, opaline silica and even elemental Hg (Canet et al ., 2005a; Canet et al., 2005b; Pichler and Veizer, 1999; Prol-Ledesma et al., 2005; Stoffers et al., 1999). Marine shallow-water hydrothermal ve nting can serve as compelling natural analogs for coastal anthropoge nic point source pollution, since hydrothermal systems are very often associated with pot entially toxic elements, particul arly As (Dando et al., 1999; Price and Pichler, 2005; Varnavas and Cr onan, 1988; Vidal et al., 1978). The negative effects of contaminants on tropical marine ecosystems are of increasing concern as human populations expand adjacent to these communities (Peters et al., 1997). It is therefore increasingly important to understa nd the natural, coastal biogeochemical cycle of arsenic and other transition metals and metalloids. This will allow us to better detect, predict, and evaluate changes arising fr om human activity (Maher and Butler, 1988). Despite increased scientific and public interest, As remains an element whose environmental cycling is poorly understood. Bioaccumulation and biotransformation can remove much of the arsenic from sediments and waters; nevertheless, tissue arsenic is often overlooked as a sink, even though a ppreciable concentrations are common in marine organisms (Cullen and Reimer, 1989). The marine shallow-water hydrothermal sy stem of Tutum Bay, Ambitle Island, in northeastern Papua New Guinea, may be one of the largest and most unusual hydrothermal systems studied to date. This hydrothermal system is unique in that it contains some of the highest concentrations of arsenic ever discovered for a submarine hydrothermal system, including mid-ocean ridge black smokers. A more detailed
4 description of Tutum Bay hydr othermal vent fluids and precipitates is available elsewhere (Pichler et al., 1996; 1999a; 1999b). Materials relevant to this dissertation are summarized here. 1.2 Description of the Tutum Bay marine shallow-water hydrothermal system Ambitle Island is part of the Feni Isla nds group and is located northeast of New Ireland Province, Papua New Guinea (Figure 1.1 ). The island is of volcanic origin and several terrestrial geothermal areas are present on the west side of the island associated with faulting. These faults can extend offshore, and one hydrothermal area occurs submerged off the west side of Ambitle Is land, within Tutum Bay in 5Â–10 m water depth. Two types of venting are observed: (1) Focuse d discharge of a clea r, hydrothermal fluid at discrete ports, 5-15 cm in diameter. Th ere are four main focused vents, and fluid temperatures at vent orifices are between 89 and 98 C with discharge rates are as high as 400 L/min (Figures 1.2 and 1.3). (2) Diffuse di scharge consists of hydrothermal fluids and streams of gas bubbles (94 Â– 98 % CO2) emerging directly through the sandy to pebbly unconsolidated sediment and through fr actures in volcanic rocks (Figure 1.4). The hydrothermal fluids are of mete oric origin and, compared to seawater, vent fluids are approximately one order of magnitude less saline, causing them to be quite buoyant. Of all potentially biologi cally toxic elements, As is the only one that was significantly enriched compar ed to seawater (~500 times ). Pichler et al. (1999b) estimated that the main hydrothermal vents discharge as much as 1.5 kg As per day directly into the coral-reef ecosystem of Tutum Bay.
5 Figure 1.1. Location of Ambitle Island and the marine shallow-water hydrothermal vents investigated in this research. Tutum Bay hydrothermal area and the control sites are indicated (modified from Pichler and Dix, 1996). X Non-hydrothermal control site 2 Non-hydrothermal control site 1
6 Figure 1.2. Location of focused vent areas of the hydrothermal system in Tutum Bay (modified from Pichler et al., 1999a). 100 m Ambitle Island V-1 V-4 V-3 V-2 Sediment Reef Reef 10 m
7 Figure 1.3. Underwater photograph of focused hydrothermal venting. Note proximity to corals.
8 Figure 1.4. Underwater photogra ph of diffuse hydrothermal venting. (modified from Pichler et al., 2006)
9 In addition to As, vent fluids are also si gnificantly enriched in Fe, Mn, Cs, Tl, Si, and HCO3-, and slightly elevated in Mg, Zn, S b, F, Cu, Co, Rb, Ba, Pb, Ni, and Mo relative to average seawater, whereas the ma jor ions in seawater, Cl, Na, Br, K, SO4, Ca, and Sr, are depleted (Pichler et al., 1999a). The dominant hydrothermal precipitates are 2line ferrihydrite (HFO), which readily adsorb s As (Figure 1.5; Feely et al., 1994; Pichler et al., 1999b). Tutum Bay contains a pa tchy distribution of cora lÂ–algal reefs surrounded by mediumto coarse-gra ined, mixed carbonateÂ–vol caniclastic sand and gravel (Figures 1.3 and 1.4). Despite the large amount of As released into the bay through focused hydrothermal venting, corals, clams and fish did not show an obvious response to the elevated concentrations (Pichler et al., 1999b) Fish often bathe in the hydrothermal fluid, and the health of the corals surrounding the hydrothermal vents is seemingly unaffected (Figure 1.3). 1.3 Review of arsenic speciation and its im portance to toxicity and bioavailability Recently it has been recognized that the to tal concentration of an element in an ecosystem does not necessarily represent its biol ogical availability or potential toxicity (Newman and Jagoe, 1994). This concept is known as Â‘bioavailabilityÂ’, and is a function of the abundance and chemical form of the toxi n in solution (i.e., oxidation state), and the nature of its binding to sedime nt grains. For example, the el ement may be part of a very stable mineral, such as quartz, and thus is not available for biol ogical processes (Newman and Jagoe, 1994). Alternatively, the toxin can be adsorbed to sediment grains in an
10 Figure 1.5. A) Underwater pho tograph of HFO precipitate s. B) Scanning Electron Microscope (SEM) image of HFO. (A modified from Pichler et al., 1999a) A) B)
11 easily-extractable form (Tessi er et al., 1979), which would then be more available to benthic organisms (Yoo et al., 2004). Bioavail ability is defined here as the degree to which an element or molecule is able to move into or onto an organism ( sensu Benson, 1994). Transfer of a toxin into an organism can occur by diffusion onto/across the cell membrane, or through the food web. The free-me tal ion in solution is by far the most biologically toxic, although eas ily-exchangeable forms in sediments can also be harmful (Newman and Jagoe, 1994). Several elements, su ch as As, Cr, and Sb, are more difficult to characterize because they occur in severa l oxidation states in water and sediments and therefore will have a different degree of bi oavailability, depending on the species present (Gebel et al., 1997). Oxidation-reduction, li gand exchange, precipitation, and adsorption reactions can all influence ar senic speciation, mobility, and bioavailability (Ferguson and Gavis, 1972). Thermodynamically, arsenic is stable in most natural waters primarily in two inorganic forms, arsenite (As3+ or As(III)) and arsenate (As5+ or As(V)). The oxygen content and the prevailing redox conditions ar e thus primary controls on the mobility and toxicity of As in an aqueous environmen t, because these control the oxidation state (Figure 1.6). Arsenite is more mobile a nd by far the more toxic, causing reduction in growth to marine organisms at a queous concentrations less than 3 g/L, and neurological damage in humans at concentrations as low as 100 g/L (Gebel et al., 1997; Sanders et al., 1994). Arsenate is less t oxic and less mobile, but more readily taken up by biota (Francesconi and Edmonds, 1998). Thus, determining the species present in the
12 Figure 1.6. Eh-pH diagram for the system As-O -H at 25C (dashed lines) and 100C (solid lines) at 1.013 bars Activities of As(OH)4 and HCO3 are assumed to be 10-3 and 10-4, respectively. Thermodynamic data are from Brookins (1988) and references therein. Plot from Pichler et al. (1999a).
13 environment is critical for unde rstanding the bioavailability a nd potential toxicity of the element (Newman and Jagoe, 1994). As noted by Sillen (1961) and Johnson (1972), thermodynamic calculations suggest that oxygenated surface seawater s hould contain predominantly As(V), but biological activity can reduce appreciable amou nts to As(III). These inorganic forms can be taken in and methylated by coastal or ganisms and either are excreted through a detoxification mechanism or bi oaccumulated within the orga nismsÂ’ tissue. The two major organoarsenic species, which can occasionally be detected in s eawater and sediment pore-waters as a result of pla nktonic and/or bacterial intera ctions (Andreae, 1979; Cullen and Reimer, 1989), are dimethylarsinate (D MA) or monomethylarsonate (MMA). In addition to DMA and MMA, the major methylat ed organoarsenic species found in tissues of marine organisms include trimethylarsi ne oxide (TMAO), tetramethylarsonium ion (TETRA), arsenobetaine (AB), arsenocholin e, (AC), and the four major arseniccarbohydrate compounds, referred to collectively as arsenosuga rs or arsenoribosids (AR). These are glycerol sugar (AR 1). phosphate sugar (AR 2), sulfonate sugar (AR 3), and sulfate sugar (AR 4). While these are the ma jor arsenic species encountered in coastal marine organisms, more than 30 were id entified (Francesconi and Kuehnelt, 2004). Concentrations of metals in sediments usually exceed those of the overlying water by 3 to 5 orders of magnitude (Bryan and Langston, 1992; Mountouris et al., 2002). Therefore the bioavailabili ty of even a small amount of th e total metal in sediment can be important. With respect to bioavailability, the metals in the most readily extracted fractions in the series are the most importa nt in this study, because they are the most
14 bioavailable and are potentia lly the most immediately dange rous to the biota (Bendell Young and Harvey, 1991; Bhumbla and Keefer, 1994; Sahuquillo et al., 2002; Tessier et al., 1979). However, As concentrations in all mineral phases must be determined for any long-term prediction of arsenic behavior in sediments because changes in environmental or physicochemical conditions (e.g., sediment burial, reworking by storms, etc.) may alter mineral stability. 1.4 Rationale This dissertation is an integral part of a larger NSF-funde d project entitled Â“Ecosystem response to elevated arsenic c oncentrationÂ”. This pr oject specifically investigated the impact of elevated As le vels resulting from hydrothermal discharge on the abundance and diversity of microbes, fo raminifera, and invertebrates throughout the marine shallow-water hydrothermal system of Tutum Bay. Project participants included four teams, each consisting of one PI and one Ph.D. student. The teams consisted of two geochemists, two microbiologists, two macro/meiobiologists, and two biologists specializing in foraminifera, respectively. The primary goals for the larger project were to: 1) establish a study site in an area of active hydrothermal venting; 2) establish a control site away from th e influence of hydrothermal venting; 3) determine the physico-chemical conditions in both sites; 4) determine microbial, foraminiferal, meiofaunal and macrofaunal invertebrate diversities and community structures at each site using a combination of morphological and molecular methods; 5) isolate, culture, and characteri ze microorganisms on geochemically designed growth media at high in situ As concentrations;
15 6) model in situ bioenergetics of As metabolism using thermodynamic calculations; 7) establish mathematical models th at explore ecosystem response to elevated As values and food web stru cture and dynamics using both As as a tracer and stable isotop es of carbon and nitrogen. My specific objectives as a part of the team of geochemists on this project are outlined in detail below. The geochemical ev aluation, particularly for arsenic abundances and arsenic species, contributed substantiall y to the overall project, providing data which was or will be used by each of the other team s. Great care was taken to ensure all team members worked on subsets of the same sample s. Thus, the research presented in this dissertation can link As to microbial, foraminiferal, meiofaunal, and macrofaunal invertebrate diversity and community struct ure in pore fluids, the water column, and sediments. The initial results for th e overall project are summarized in Figure 1.7, which is published in Pichler et al. (2006). Briefly, As does appear to have an impact on the biodiversity in Tutum Bay. Molecular data suggest that high temp erature and high As concentrations of the vent fluids limit ar chaeal biodiversity, whic h quickly increases a short distance from the vent (Figure 1.7). Th e absence of foraminifers and mollusks near the vents indicates that pH may be a pr edominant factor controlling abundance and distribution for organisms that rely on cal cium carbonate deposition. The influence upon foraminiferal and macrofaunal diversity to a distance as far as 150 m from the vents suggests that the hydrothermal influence is more far-reaching than was initially thought.
16 Figure 1.7. Physical, chemical, a nd biological trends stepping away from the area of active venting in Tutum Bay. From left to ri ght, the y-axes are num ber of foraminifera shells per gram of sediment, concentrations of As in milligrams per kilogram (sediment) and milligrams per liter (pore water), pH, and temperature in C. The values for As in pore waters were multiplied by a factor of 104 to fit into the plot; the value at a distance of one meter is 0.9 milligrams per liter. The macrofauna pie at a distance of 300 meters represents a sample taken at the reference site In the legend on the right side, UC and UE indicate Â‘uncultured CrenarchaeotaÂ’ and Â‘unc ultured Euryarchaeota,Â’ respectively. (from Pichler et al., 2006).
17 Thus, As may be the key factor affect ing the Tutum Bay ecosystem as pH and temperature stabilize with greater distance from focused venting. 1.5 Objectives Given the elevated As concentrations be ing discharged into Tutum Bay, there are many first-order questions which can be asked. For example, what is the source and fate of arsenic, and what conditions lead to its accumulation in surrounding sediments? How is hydrothermal venting affecting surface and subsurface sediment chemistry, and how far away from focused venting can this effect be recorded? What is the bioavailability and speciation of arsenic in sediments, surrounding seawater, core sediments and pore waters? If the arsenic is bioavailable, are nearby organisms bioaccumulating excess arsenic? How are the organisms tolerating el evated arsenic concentrations? How do their biotransformation pathways compare to the same organism from an area unaffected by hydrothermal venting? Our ability to answer these questions depends on a better basic understanding of the ecosystem surrounding hydr othermal vents. Hence, the specific objectives for this dissertation were to determine: 1) the source for the arsenic and what leads to arsenic accumulation in the sediments; 2) the extent of influence hydrothermal venting may have on the chemistry of surrounding coastal sediment, porewater seawater, and distribution and abundance of nearby biota; 3) the abundance, bioavailability, and speciation patterns of arsenic in seawater, sediments and sediment pore waters; and 4) the bioaccumulation and biotransformation patterns of arseni c in coral-reef organisms.
18 1.6 Arrangement of Dissertation This dissertation consists of five chapters. Each chapter describes some aspect of the physicochemical gradients resulting fr om hydrothermal venting, which were delineated by a roped transect leading away fr om focused venting (Figure 1.8). This first chapter introduces the hydrothermal system by first reviewing relevant past research conducted at the site, provides a detailed si te description, and concludes with a summary of the basic lithology, mineralogy, and bulk geoc hemistry of surface and core sediments collected from the hydrothermal system. Th e site description is comprehensively presented in this chapter, and is thus omitted from each subsequent chapter to reduce redundancy. Section 1.7 of this introduction will be published under th e title Â“Controls on sediment geochemistry in the marine sha llow-water hydrothermal system of Ambitle Island, Papua New GuineaÂ”. At this time, however, this section is limited to describing the general characteristics of the sediments along the transect. Chapter Two, entitled Â“Enhanced geochemical gradients in a ma rine shallow-water hydrothermal system: unusual arsenic speciation in horizontal and vertical pore water pr ofilesÂ”, was published in a special issue of Applied Geochemistry (Price et al., 2007), a nd describes the potential for microbial metabolisms based on disequilib rium between arsenic species measured in the pore waters of Tutum Bay. Chapter Thr ee details the abundan ce, distribution, and bioavailability of arsenic from sediments and water throughout Tutum Bay in an effort to determine if in fact the organisms can be affected by hydrothermal venting, and have access to abundant arsenic. The chapter is titled Â“Distribution, Speciation and Bioavailability of Arsenic in a Shallow-wa ter Submarine Hydrothermal System, Tutum
19 Figure 1.8. Location of transect along whic h samples were collected for this study. X X X Hydrothermally influenced Non-hydrothermally influenced X X X X X = sampling location 25 m V-4 Reef 10 m 30 m
20 Bay, Ambitle Island, PNGÂ”, and was published in 2005 in a special issue of Chemical Geology (Price and Pichler, 2005). Chapter Four describes how orga nisms throughout the hydrothermal system bioaccumulate and biotransform (detoxify) available As. This was achieved by measuring total arsenic abundances and speciation in tissu es from coral-reef organisms collected along the transect. The ch apter is titled: Â“Enhanced bioaccumulation and biotransformation of arsenic in coral reef organisms surrounding an arsenic-rich marine shallow-water hydrothermal vent syst emÂ”, and will be published later this year. Finally, Chapter Five summarizes and concludes this dissertation with a discussion of the major scientific contributions. 1.7 Lithology and geochemistry of sediments of Tutum Bay Detailed geochemical investigations of sediments from surface and subsurface environments are necessary to evaluate: 1) how extensive hydrothermal influence can be, 2) what controls the distribution of hydrotherm al influence, as well as 3) the extent to which surrounding biology can be affected. The role of marine shallow-water venting of hydrothermal fluids on the mass balance of coastal sediment chemistry, including the input of several hazardous elements to the sediments and seawater, has not been sufficiently evaluated, but can potentially be assessed by detailed investigations of surface and core sediment chemistry. This section, therefore, focuses on the major lithology and geochemistry of surface and core sediments throughout the mari ne shallow-water hydrothermal system of Tutum Bay. Within this hydrothermal system, high concentrations of dissolved Fe(II) (~1
21 to 1.5 mg/L) and Mn2+ (~0.3 to 0.5 mg/L) in reducing hydrothermal fluids mix with oxygen-rich seawater, thus pr ecipitating abundant hydrous ferric oxide (HFO) and Mnoxide minerals, which coat surrounding sedime nts (Pichler et al., 1999a). These minerals are known to be excellent scavengers of ma jor and trace elements (Feely et al., 1994). Previous investigations have already show n the importance of HFO in removing arsenic from this hydrothermal system (Pichler et al., 1999b). In addition, hydrothermal fluids are also elevated in H2S, possibly precipitati ng sulfides below the redox transition between oxygen-rich seawater and reduc ing hydrothermal fluids. To compare the Tutum Bay area to a Â‘Â‘nor malÂ’Â’ marine environment, two control sites were established for the current researc h. First, a coral-reef control site (Picnic Island; heretofore referred to as CS1) was established several kilometers to the north, well beyond the influence of hydrothermal ven ting (Figure 1.1). This area was chosen because the sediments at this contro l site consisted entirely of CaCO3, thus providing a suitable end-member for comparison. Another co ntrol site was delineated to the south of Tutum Bay, in Danlam Bay (CS2, Figure 1.1) This site was chosen because it is lithologically and sedimentologically more si milar to the coastal environment of Tutum Bay, although beyond the influence of Tutum Ba y hydrothermal activity. Picnic Island is isolated from the volcanoclastic sediments of Ambitle Island, and could be considered an example of a pristine, 100 % calcium carbonate, reef enviro nment, whereas the Danlam Bay control site has volcanoclastic sediment s and mineralogical composition similar to Tutum Bay, excluding the HFO precipitates and containing more calcium carbonate.
22 Terrestrial hot springs are located just in shore of CS2, and it is possible they are influencing the chemistry of sediments there (Figure 1.1). 1.7.1 Surface Sediments Surface sediment samples were collected along a roped transect leading from the area of focused hydrothermal venting to 300 m away, as well as from CS1 and CS2 (Figures 1.1 and 1.8). The sediments from the vi cinity of hydrothermal venting are coated with HFO, likely 2-line ferrihydrite descri bed by Pichler et al., (1999b). This is a characteristic unique to the hydrothermally in fluenced sediments. Thus, they are very orange colored and clearly indicate the ex tent of hydrothermal influence (Figure 1.9). Diffuse venting was evidenced by higher temp eratures as measured with our underwater temperature probe, and also as visible dens ity fronts as the lowe r salinity hydrothermal fluids seep through the sediments and mi x with the high salinity seawater. This phenomenon was observed as far away as 150 m from focused ve nting (Price et al., 2007). This was also the distance where the color of the surface sediments changed from orange to white (Figure 1.9). These orange co lored sediments predominantly consisted of fineto medium-grained sands and gravels co mposed of andesite rock fragments along with the weathered components of these rock s. This includes idiomorphic minerals Nafeldspar, quartz, amphibole ( hornblende), pyroxene (augite), with minor amounts of mica and magnetite minerals. Overall, the gr ain size immediately adjacent to focused hydrothermal venting is much larger, in some cases gravel to boulde r size (Figure 1.10 A; Karlen et al., in review). However, the gr ain size both for rock fr agments and individual
Figure 1.9. Photograph of surface sediments co llected along the transect. Note color change from orange (HFO) to white (CaCO3). control 1m 7.5m 12m 30m 60m 90m 125m 150m 175m 200m 225m 23Vent Precipitate
24 A) B) C) D) Figure 1.10. Surface sediment characteristics for Tutum Bay hydrothermal transect and Danlam Bay reference site (CS2, labeled Ref; from Karlen et al., in review). A) median Grain Size (mm); B) sorting co efficient; C) percent organic carbon; D) percent inorganic carbonates. 0m 30m 60 m 90m 120m 1 4 0m 180m 2 5 0m 300m Re f Median Grain Size (mm) 0.1 1 10 Gravel Very Coarse Sand Coarse Sand Medium Sand Fine SandGrain Size 0 m 30m 6 0m 9 0m 1 20 m 1 40 m 180m 250m 3 00 m Re f Sorting Coefficient ( 0.0 0.5 1.0 1.5 2.0 2.5 3.0 SortingVery Poorly Sorted Poorly Sorted Moderately Sorted Moderately Well Sorted Well Sorted Very Well Sorted 0m 30m 60m 90m 1 2 0m 1 40 m 1 8 0m 2 50 m 300m Re f % Organic Carbon 0 1 2 3 4 5 6 7 8 9 10 Organics 0m 30m 60m 90m 1 2 0m 1 40 m 1 8 0m 2 50 m 300m Re f % Carbonates 0 5 10 15 20 25 30 35 40 Carbonates
25 minerals from ~1 m out to ~150 m along our tr ansect is fine sand sized. These sediments are also very well sorted (Figures 1.10 A and B). The larger grain size surrounding focused hydrothermal venting is likely a result of 1) focused venting suspending smaller grains, which are then advected away, and/or 2) shallower, higher energy environment, which can concentrate coarse-grained se diments while transporting fine-grained sediments offshore. Outside of this area of hydrothermal iron-rich sediments, there is a gradual transition to a carbonate sand and r ubble with increasing di stance from Ambitle Island, with declining amounts of fine sa nd-sized volcanic sediments, and no HFO (Figure 1.9). The samples collected from 180, 250, and 300 m are medium, medium to coarse, and coarse-sand size, respectively, and reflect the increas e in biogenically produced carbonate clasts (Figures 1.10 A and B). The carbonate clasts are composed mostly of Halimeda sp fragments, a green calcareous alga, along with coral debris, mollusk shells, as well as some planktonic and benthic foraminifers. Thus, sorting of surface sediments is influenced by increasing biogenic components beyond 140 m. The transition in sorting patterns from 140 to 180 m is quite abrupt, quickly changing from well-sorted to poorly sorted by 180 m (Figure 1.10 B). The grain size and sorting patterns can th erefore be explaine d by 1) higher energy environment at the beginning of the tr ansect due to wave energy and focused hydrothermal venting, and 2) the abrupt in crease in carbonate sk eletal debris beyond ~150 m. The percent organic carbon and percen t inorganic carbonate are also indicative of this transition, changing from less than 2 % organic carbon to greater than 6 % organic carbon at 300 m, and from ~1 % inorganic ca rbonate at 0 to 140 m to greater than 30 %
26 inorganic carbonate at 300 m (Figures 1.10 C and D). The lack of a carbonate component is considered to be a result of hydrothermal venting of hot, slightly acidic fluids (pH ~ 6.0, temperatures exceeding 40C) to as fa r away as 150 m (Price and Pichler, 2005). Another possibility is mechanic al transport of hydrothermally influenced sediments out to 160 m from focused venting. Surface sediments at the Picnic Island cont rol site (CS1) consisted of essentially 100 % calcium carbonate, and were coarse to ve ry coarse sand. The Danlam Bay control site (CS2) consisted predominantly of the sa me mineral assemblage and andesite rock fragments as the 1-140 m samples in Tutu m Bay, although there was also ~6 to 8 % carbonate (aragonite) biogenic material, and no HFO coatings. Sorting for the Danlam Bay sample falls between moderately and poor ly sorted, and grain size ranged from fine to medium sand size (Figures 1.10 A and B). 1.7.2 Core Sediments The lithology and mineralogy of core sediments was highly variable, but was generally similar to surface sediments descri bed above (Figure 11). Some core sediments were composed of predominantly 100 % HFO pr ecipitates. Contrastingly, a grey, highly reduced sediment comprised of clay with is omorphic minerals of Na-feldspar, quartz, amphibole (hornblende), pyroxene (augite), with minor amounts of mica and magnetite minerals, was present in cores nearer to fo cused hydrothermal venting (1 Â– 12 m). These sediments may be chemically weathered ande site, and the green/gray clay could be a result of propylitic altera tion (a.k.a., greenstone). Howeve r, the mineralogical and chemical similarity with the overlying oxidi zed sediments suggests reductive dissolution
27 Figure 1.11. Graphic showing sedi ment characteristics for each core collected along the transect. Type 1 = 100 % HFO; Type 2 = HF O-coated volcanoclas tic sediments with lithic clasts of andesite; Type 3 = HFO-co ated, well sorted, volcanoclastic sediments; Type 4 = volcanoclastic sediments without HFO and < 20 % carbonates; Type 5 = highly reducing volcanoclastic sediments contai ning framboidal pyrite; Type 6 = carbonates with < 20 % volcanoclastic sediments. 1 2 3 4 5 6 Sediment Type 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 1 2.5 7.5 12 30 60 90 140 225 300 Distance (m) Depth (cm)
28 of HFO. One common observation is that th e green/gray clays c ontain framboidal pyrite, suggesting that the physicochemical conditions change from oxidizing (O2 from seawater) where the HFO is present, to highly reducing (ven t fluids lacking O2) where the grey clay and framboidal pyrite exists (Figur es 1.12 and 1.13). This suggests that influx of vent fluids creates a very dynamic redox environment. Finally, near the end of our transect, the core sediments were increasin gly composed of calcium carbonate (Figure 11). The core at 60 m from focused hydrotherm al venting penetrated to the greatest depth, and was unique. It illust rated the fact that hydrotherm al influence can extend to great distances away from focused venting. This core consists of HFO-coated, well sorted, volcanoclastic sediments in the upper 30 cm, and the color changes from dark red to light red in the upper 31 cm. Between 31 a nd ~42 cm depth, the color of the sediments is a dark red/black, probably due to Mn oxides (core geochemical data show this sample contains the highest concen tration of all samples, at 118050 mg/kg Mn). Below 60 cm, there is a mixture of HFO precipitates, da rk-red Mn oxide precipitates, and a yellow precipitate. The highly-a ltered nature and heterogeneity of precipitates in this core is likely caused by diffuse venting of hydrothermal fluids. 1.7.3 Element distributions along the Tutum Bay transect The bulk surface sediment chemistry and bulk core sediment chemistry are presented in Tables 1.1 and 1.2, respectively. Th e data in each table has been arranged by
29 Figure 1.12. Photograph of a core collected from near hydrothermal vents showing distinct redox boundary. 31 cm
30 Figure 1.13. SEM images of framboidal pyrite discovered in reduced core sediments.
31 distance from focused hydrothermal venti ng, and core sediment chemistry data are further arranged by depth below the seafloor. Three distinct patterns of element con centration vs. distance away from focused hydrothermal venting in Tutum Bay surface sedi ments are observed. First, some elements decrease in concentration with increasing distance from focused hydrothermal venting. Elements following this pattern include Fe, As, Sb, Cu, Cs, La and Zn. The best representation of this pattern is Fe, and thus I have presented Fe concentration vs. distance in Figure 1.14 A as a reference. The second pattern observed for some elements is first increasing, and then decreasing in concentration versus distance away from focused venting. Elements in this suite include Mn, P, Co, Ce, Cr, Eu, Mg, Ni, Yt, V, and Sm. Figure 1.14 B presents the plot for Mn vs distance as the best example of this element distribution pattern. Th e third pattern observed for element distributions along the transect was increasing in concentrati on versus increasing di stance from focused venting. Elements following this pattern in cluded Ca, Sr, Pb, and S. Calcium versus distance is presented in Figure 1.14 C as an example. Some elements, including Al, K, and Na, follow no systematic change. Figures 1. 14 A-C also include the concentrations of vent precipitates, the control site from Picnic Island (CS1, ~100 % CaCO3), the control site from Danlam Bay (CS2), and a sample of andesite taken from Ambitle Island (see legend).
32 Figure 1.14. Selected element distribution patte rns for Tutum Bay surface sediments. A). decreasing concentration versus distance fr om focused venting; B). increasing, then decreasing concentration, versus distance; C) increasing versus dist ance. Concentrations for vent precipitates are presen ted at the top of each plot. A) C) B) Legend 0 250 500 750 1000 1250 1500 1750 0100200300Distance (m)Mn (mg/kg)Vent precipitate = 1330 mg/kg Transect CS2 CS1 Andesite 0 5 10 15 20 0100200300Distance (m)Fe (%)Vent precipitate = 31.1 % 0 10 20 30 40 0100200300Distance (m)Ca (%)Vent precipitate = 1.3 %
33 1.7.4 Element distributions in core sediments The chemistry of core sediments in horizontal profile overall reflects similarities with surface sediment elemen tal distributions. Again, Fe, As Sb, Cu, Cs, La and Zn are much more elevated in core sediments n earer to focused hydrothermal venting, and progressively decrease systematically with distance along the trans ect to 300 m. Ca and Sr follow an inverse pattern, a nd increased along the transect. When assessing the chemistry of core sedi ments in vertical profiles, there are no systematic trends. However, element en richments can correlate with lithology. For example, HFO-rich layers are s ubstantially enriched in As and Sb. Grey layers have very similar concentrations of Fe compared to overlying oxidized zones of HFO, but less As and Sb, while being enriched in Cu, Ni, and Ti. 1.8 Controls on element distributions Several factors could be controlling horizon tal and vertical element distributions. 1) Precipitation of hydrothermal minerals (HFO and Mn-oxides) in areas of focused or diffuse venting: these minerals readily adsorb elements such as As, Sb, etc (Berner, 1980; Feely et al., 1994). 2) Mech anical transport of hydrothe rmal precipitates surrounding focused hydrothermal venting downslope towa rds the continental shelf to areas not directly influenced by venting. 3) Alteratio n/chemical weathering of the normal marine sediments, including clay formation due to alte ration of the dominant feldspar grains, and reduction of Fe and Mn oxides. This process is reported to release As and other toxins. 4) Dissolution of carbonates due to focused a nd diffuse venting or alternatively the
34 increasing abundance of biogenic compone nts beyond 150 m. 5) Precipitation of hydrothermal minerals (sulfides) in the near subsurface where slowly discharging hydrothermal fluids mix with seawater.
35 AsAlBaBeBrCaCeCoCrCsCuEuFeKLaLuMgMn mg/kg%mg/kgmg/kgmg/kg%mg/kgmg/kgmg/kgmg/kgmg/kgmg/kg%%mg/kgmg/kg%mg/kg Vent337000.253196.81.317115731.10.370.681330 0.57394.05125512.33.3719268913560.59.782.6126.96.36.199861 114003.26282.1515291041610719.31.758.90.411.08683 7.539118.95741.90.09 123211909.51911381.08.70.12 203725.1417.75.56132784490.78.431.632.661020 303834.5112.46.8714361575500.99.8188.8.131.52.311300 405415.85261114.87.191829127500.98.661.6184.108.40.2061130 604824.0513.27.932038197501.09.881.057.60.263.511390 806274.9314.57.511828121431.07.671.4220.127.116.111200 9031726014.9181518.104.22.168 1003773.917711.09.392535186371.18.371.038.20.253.091390 1203964.5120026.57.0920271214370.86.931.322.214.171.1241150 1403645.3116518.37.8422271103390.87.261.617.00.233.141200 1602775.421659.57.891722106370.85.991.77.00.182.99999 1802254.4731513.511.11620943126.96.36.199188.8.131.5223 2001583.613518.814.5162195340.55.881.2184.108.40.20610 22072.82.8916.4171619851260.45.010.94.90.111.95672 22578.022520.41712220.127.116.11 24051.02.8921013.720.41319912218.104.22.16822.214.171.124680 26050.82.8816.721.51014672240.63.610.834.40.092.46559 28058.53.0616.719.3141264250.33.420.935.2-0.052.63501 300351.8711012.222.381151150.42.710.573.50.082.33416 Danlam45.25.4271.037.67.318.716.682.82.070.40.126.96.36.199.21.5533.2 Picnic2.275.745.4188.8.131.522.00.41.4 Host Rock7.44.4834013.95.4343178302131.24.873.620.10.280.881560 Table 1.1. Bulk geochemistry data for Tutum Bay surface sediments arranged by distance from focused hydrothermal venting. Distance
36MoNaNdNiPPbSSbScSmSrTi U V W YYbZn mg/kg%mg/kgmg/kg%mg/kg%mg/kgmg/kgmg/kgmg/kg%mg/kgmg/kgmg/kgmg/kgmg/kgmg/kg Vent11.990.12200.124461.90.37260.0322349 0.542.65300.15100.2271.815.42.111000.441.93051591.072 1232.29320.11320.1915815.62.68270.322872681.575 184.108.40.206.60.6 121333.826.13.460.8 2022.69340.1350.0332.426.22.911700.491.13315100.964 3032.24420.1440.0312.631.23.19350.664508121.785 4042.8511350.1450.0416.728.03.012100.51346131.362 6041.94430.1440.04220.127.116.11000.66451131.582 8032.49340.140.0313.731.42.99900.452.1302131.454 90177.318.104.22.168 10011.86440.1550.039.240.63.67630.54366151.364 12032.587340.1340.058.329.42.78850.42278121.151 14032.8410370.1440.048.430.52.810500.43283141.253 16032.9300.1360.047.325.42.412700.34217120.741 18012.5910250.130.076.618.92.120100.32.020291.139 2002.1270.1390.134.617.02.224700.361.725170.945 2201.76250.120.121.912.81.526000.312.222960.738 22522.214.171.124.9 2401.59260.13126.96.36.199.728700.31.520771.032 2601.626220.12110.191.112.01.530000.221.915170.825 2801.83210.110.171.012.21.725900.22.313561.021 3001.18188.8.131.52.91.324600.171.411750.514 Danlam2.42.821.60.119.00.20.810.52.01646.00.32.32184.108.40.2067.4 Picnic220.127.116.11.20.45726.72.4 Host Rock23.161260.1918.104.22.168.921500.41300141.491 Table 1.1 continued: Bulk geochemistry data for Tutum Bay surface sediments arranged by distance from hydrothermal venting. Distance
. 37DistanceDepthAsBaBrCaCeCoCrCsEuFeLaLuNaNdSbScSmSr U W Zn m c m mg/kgmg/kgmg/kg%mg/kgmg/kgmg/kgmg/kgmg/kg%mg/kgmg/kg%mg/kgmg/kgmg/kgmg/kg%mg/kgmg/kgmg/kgVent03370096.81.371131.1 1.94461.90.3726 49 134551102.55215519640.922.214.171.124.571137.923.33.10.080.711210 196341506304215491.611.710.00.242.332050.3126.96.36.199.014212 115810225522177191.07.3910.80.122.701049.5188.8.131.52.011 12810103550.863128131111.69.2714.30.233.011472.4184.108.40.206.419136 13480.13101.47483415852.57.2516.80.272.303214.9220.127.116.11.310227 2.53635-5012.6618249741.28.218.104.22.1681436.922.214.171.124.2967 2.596632554.963169252121.517.014.40.202.841553.5126.96.36.199.513294 2.51511502457.763442194121.612.313.30.233.071766.3188.8.131.52.520173 2.521.542337014.37252220161.36.7512.00.162.321918.8184.108.40.206.7573 2.52875438513.773833262101.98.7116.90.293.432220.127.116.11.187.816255 2.53011503109.673532196151.89.718.104.22.168222.214.171.124.215.618148 2.54013903559.673732152161.810.814.20.073.0214126.96.36.199.195.324239 2.5466703705.36413118982.28.3218.60.302.922247.5188.8.131.52.913191 7.5124351506.052243131130.99.049.80.152.051654.423.03.30.1310100 7.5251494900.752447175121.211.011.20.171.901656.731.04.10.1211190 7.5351753204.862765230131.613.712.50.202.001746.3184.108.40.206.813273 12167472756162266100.78.9220.127.116.11818.104.22.168.1118 122042113011172175100.88.287.00.101.75946.115.02.40.251190 122610641709182993141.322.214.171.124.7812143126.96.36.1992136 123828124542752169131.614.713.00.211.881749.7188.8.131.52.617156 301070635516.17164216461.89.78184.108.40.2061324.134.04.30.132.88243 303053722012.3818248151.27.2220.127.116.11913.019.82.70.132.1 30506243105.7723257121.06.718.104.22.168117.522.214.171.124.03 30688001459.5616268361.06.9126.96.36.199188.8.131.52.123.0767 Table 1.2. Bulk geochemistry data for Tutum Bay core sediments arranged by distance from focused hydrothermal venting, then by depth below seafloor.
38DistanceDepthAsBaBrCaCeCoCrCsEuFeLaLuNaNdSbScSmSr U W Zn m c m mg/kgmg/kgmg/kg%mg/kgmg/kgmg/kgmg/kgmg/kg%mg/kgmg/kg%mg/kgmg/kgmg/kgmg/kg%mg/kgmg/kgmg/kg6025102012522.272044149184.108.40.206.202.931719.8220.127.116.11.63110 60313570195012.472042312940.915.310.10.201.921618.104.22.168.166.810293 6045200060518.762682195181.618.39.90.072.6612117.533.84.00.097.3102226 606470427.553044182171.813.511.43.351886.722.214.171.124.837234 6075122022029.162039150181.011.912.00.163.511153.035.73.50.144.627110 6080153037524.851633126160.910.99.60.133.061251.027.12.70.104.827 6085391024029.332056115171.314.910.00.133.171465.4126.96.36.199.440 6092.5521120013.123260111291.77.0188.8.131.52936.3184.108.40.206.726138 90255148012.99255921041.9220.127.116.111611.848.75.10.091.3245 905057327514.837254817741.89.8610.93.002113.318.104.22.168.5 9079121037518.58294916861.710.5012.70.274.011422.214.171.124.114.34 1402051926010.16173213041.37.5126.96.36.1991610.432.53.91.0137 1403847128813.15192810541.26.939.02.75149.428.63.70.09 1406243829713.35203111341.06.809.00.172.881310.527.53.50.081.4 14073.549725013.26182610351.16.7188.8.131.52110.8184.108.40.206 1502040727012.311223012541.66.908.50.212.961338.7220.127.116.113164 1503536126517.213242811741.36.029.40.203.131418.104.22.168.311.02132 1505036442016.912243112631.56.4710.10.213.491015.134.74.10.151.74154 2251348.513017.3261422722.214.171.124.101.6671.014.82.00.402.382 2252344.113015.3267301010.95.5126.96.36.199188.8.131.52.382.7103 300831.813.523916570.53.763.50.071.1060.710.01.70.28 53 3002820.813.826915670.53.314.30.081.090.810.21.70.301.4 3004815.712518.1311113560.32.8184.108.40.2060.88.41.40.281.9 Danlam 45.2271.037.67.318.716.682.82.00.65.220.127.116.11.810.52.01646.02.357.4 Picnic 2.275.745.418.104.22.1682.0 0.41.40.80.21.20.45726.72.4 Host Rock7.43403.95.4343178301.24.8720.10.283.1622.214.171.124150 91 Table 1.2 continued: Bulk geochemistry data for Tutum Bay core sediments arranged by distance from focused hydrothermal venting then by depth below seafloor.
39 Chapter Two Enhanced Geochemical Gradients in a Mari ne Shallow-water Hydrothermal System: Unusual Arsenic Speciation in Horizontal and Vertical Pore Water Profiles 2.1 Introduction Most research on hydrothermal activity ha s focused on deep, mid-ocean ridge and back-arc basin systems; marine shallow-wa ter hot springs (<200 m), which occur in coastal environments, have b een largely overlooked. Like th eir deep-sea counterparts, the marine shallow-water hydrothermal systems are characterized by steep physicochemical gradients, which can drastically affect surrounding biology, part icularly microbial community composition (Amend et al., 2003a; Brinkhoff et al., 1999; Rogers and Amend, 2006; Rusch et al., 2005; Tarasov et al., 1986). They can be found worldwide, commonly on the flanks of active volcanoes, near the tops of seamounts, or in areas of tectonic activity (Dando et al., 2000; Johnson and Crona n, 2001; Pichler and Dix, 1996; Tarasov et al., 1986; Vidal et al., 1978). The chemical disequilibria that are caused by hydrothermal venting may have large impacts on coastal biota, including corals (Price and Pichler, 2005), and steep geochemical gr adients (e.g., for arsenic speciation) may allow for microbial communities similar to those found in terrestrial hot springs or hydrothermal lakes (Langner et al., 2001; Or emland and Stolz, 2003). Considering their occurrence in coastal waters, which are the breeding and hatching grounds for many
40 species of fish, marine shallow-water hot spri ngs may be of greater importance than their restricted geographica l occurrence suggests. Marine shallow-water hydrothermal syst ems are often characterized by gradients in temperature, pH, bicarbonate (HCO3 -), and an array of biologically toxic elements (e.g., As, Sb, Pb, Cd, Hg; Price and Pichler, 2005). This article explores the geochemical gradients associated with the hydrothermal system in Tutum Bay, Ambitle Island, Papua New Guinea. There, arsenic (as As(III)) is the only one of these potential toxins that was demonstrably enriched in the hydrothermal fluid. Here, we present data for total As abundance (TAs), As speciation (As (III) and As(V)), aqueous silica (H4SiO4), Mg2+, and other physicochemical parameters related to the hydrothermal system. We present analyses first for pore water samples colle cted at 10 cm sedime nt depth along a 300 m horizontal transect, and sec ond for pore waters profiles down to 100 cm, which were collected at distances of 2.5, 30, 60, 140, and 300 m along the same transect. The organoarsenicals, DMA and MMA, were not det ected in any of the pore water samples. 2.2 Methods 2.2.1 Field To investigate the transiti on from hydrothermal to Â“ normalÂ” marine conditions a sampling transect was established, which bega n at Vent 4 of Pich ler et al., (1999a) and extended out to beyond 300 m (Figures 1.2 and 1 .8). Pore water temperature and pH were measured every meter at a sediment depth of ~10 cm, using an IQ Scientific Instruments #IQ 150 pH/mV/temperature probe, with auto matic temperature compensation to 100C,
41 in an underwater housing. Vent fluids were collected by placing an inverted Teflon funnel over focused venting. The temperature fr om the top of the funnel, along with the appearance of density fronts, i ndicated that the flow of hydr othermal fluid discharge had displaced all seawater from the funnel. 60 mL syringes were attached to the funnel, and the vent fluid was slowly drawn into the syringe at a rate slower th an vent discharge to decrease seawater entrainment. The collection of pore waters in Tutum Bay required specialized, easy to use and reliable equipment designed to endure very high temperatures (~ 100C), low pH (2-6), and which must be submersible and light-weight for transport to remote areas. With those requirements in mind we developed a 10-port porewater sampler ( 10 cm spacing), which consisted of a 1Â” diameter aluminum pipe with Teflon tubing extending through the pipe to all individual sample ports. Up to 10 syringes could be attached and filled simultaneously to ensure equal flow to all po rts. In-situ temperatures for the pore water profiles were measured by using a Fisher Scientific Traceable digital thermometer in an underwater housing with a probe that extended up to 1 m into the sediment. In addition to the vertical profiles we also collected pore wa ters at 10 cm sediment depth from 16 sites along the transect. Those samples were collecte d using a small plastic tube connected via Tygon tubing to a 60 mL syringe. A pipette ti p with small holes punched into the end served as a filter to prevent sediment entrainment. Vent fluids, pore waters, and surrounding seawater we re collected and brought on board for immediate measurement of sensitive field parameters such as pH and alkalinity. Total alkalinity was measured as equivalent CaCO3 using a HACH digital titrator, titrated
42 to an endpoint of pH 4.5 and is expressed as bicarbonate (HCO3 -). On board, the pH was measured using a Myron-L pH meter with temperature compensation. Onboard pH was very similar to in situ pH measurements using the underwater-housed pH meter. Water samples were preserved by following two proce dures for later laboratory analysis. The first procedure was for preservation of majo r cations, total arseni c abundance (TAs), and arsenic species (As(III) and As(V)). This cons isted of syringe filtering the water sample through 0.45 m filters into 30 mL nalgene bottles and acidifying to a pH < 2 using 1 % optima HCl. All Nalgene bottles used for As speciation were capped tightly without headspace. Decreasing the pH to less than 2 prevents Fe(II) from precipitating and thus helps to preserve As speciation (McCleskey et al., 2004). The second procedure was for preservation of major anions, and consisted of syringe filtering th e water sample through 0.45 m filters into 30 mL Nalgene bottles without acidification. All water sample bottles were sealed with electr ical tape and kept cool and aw ay from light until laboratory analysis. Sample collection for As speciation analysis specifically consisted of the following step by step procedure. This pr ocedure was strictly followed to ensure preservation of As speciation: 1) Sample collection into 60 mL syringes using the pore water sampler with multisyringe puller described above. 2) Transporting syringes to the ship, di sconnecting them from the sampler, immediately attaching to syringe filters (0.45 m) and filtering into 30 mL Nalgene bottles containing 0.3 mL optima HCl. 3) The lids were screwed on tight with no h eadspace by slightly squeezing the bottle. 4) Sample bottles were immediately placed in a refrigerator until it was time to leave the ship.
43 5) For transport from the ship to the lab, samples were placed into a cooler with ice packs, which maintained a low temperatur e until reaching the laboratory, at which time they were transferred to the refrigerator. 6) Arsenic speciation was then performed in the lab following the methods described below. 2.2.2 Laboratory Magnesium and silica were analyzed by inductively coupled plasma-optical emission spectrometry (ICP-OES) on a Perkin Elmer Optima 2000 DV. Arsenic abundance and speciation was determined by hydride generation-atomic fluorescence spectrometry (HG-AFS) on a PSAnalytical 10.05 5 Millennium Excalibur system (Cai, 2000). In preparation for the determination of TAs, 10 mL of sample were combined with 15 mL concentrated HCl and 1 mL saturate d potassium iodide (KI) solution to reduce As(V) to As(III), and diluted with deioni zed water (DI) to a fi nal volume of 50 mL. Arsenic speciation analysis was carried out by high pressure liquid chromatography (HPLC) separation of As(III), DMA, MMA a nd As(V) prior to detection by HG-AFS. A sample volume of 200 L was injected without pretreatment and separated in a Hamilton PRP-X100 cation exchange column using a KH2PO4/K2HPO4 buffer at a pH of 6.00. Analytical and instrumental quality assurance an d quality control (QA/QC) was evaluated for all laboratory analyses by including sa mple duplicates and certified reference standards from NIST and Fisher Scientific, which indicated a prec ision of better than 5 % for HG-AFS and ICP-OES. Background signal drift was consistently <1 % for all instruments. Because the TAs analysis is considered to be more accurate, we used the
44 ratio of the concentration obtained from the As speciation analysis and applied this to the TAs concentration to get a final c oncentration of each As species. 2.3 Results 2.3.1 Horizontal Profile (10 cm Pore Water Samples) With increasing distance from the vent, temperature, HCO3 -, TAs, and H4SiO4 decreased considerably, alt hough occasional spikes were observed (Table 2.1). The temperature decreased from 90.5 C close to th e vents (0.5 m) to near ambient seawater temperature (~30 C) at 300 m. Bicarbonate decreased from 683 mg/L in the vent fluids to near seawater concentration (~140 mg/L) at 300 m. H4SiO4, a very good indicator of hydrothermal fluid mixing, dropped from 100 mg/L in the vent fluid to ~2 mg/L both at the end of the transect and at the Picnic Is land reference site (CS1). On the other hand, pH and Mg2+ concentrations increased with distance from the vent (Table 2.1); pH values from 6.1 to 8.0, and Mg2+ from ~130 mg/L to ~1200 mg/L. In the vent fluid and in pore waters n ear the area of focu sed venting (<2.5 m), As(III) was the dominant valence state (Table 2.1). However, As(III) levels decreased rapidly, leveling off to near ambient values of less than 10 g/L at a distance of 12 m. One notable spike in As(III) (51 g/L) was observed at 150 m. At distances from 2.5 to 300 m, As was, with one exception, predomin antly in the oxidized, pentavalent form (As(V)) (Table 2.1). Although the As(V) levels fluctuated considerably, they were higher than those of ambient se awater and CS1 (1 2 g/L). With increasing distance from the
45 vent, TAs decreased exponentially from ~900 g/L at the source to <20 g/L beyond 150 m. TAs gradients correlat e well with those of H4SiO4, and inversely with those of Mg2+. 2.3.2 Pore Water Profiles The TAs concentration in pore water prof iles generally increased with sediment depth, although only the two profiles at 30 and 60 m showed a continuous increase (Table 2.2). However, TAs correlated well with H4SiO4 and Mg2+, indicating that mixing between hydrothermal fluid and seawater controlled its abunda nce. Unexpectedly, however, As(V) was the dominant oxidation stat e at all depths from 10 to 100 cm in all Tutum Bay profiles (Table 2.2). The As(III)/As (V) ratio ranged from 0.01 to 0.23, with one anomalous value of 0.59 (2.5 m at 10 cm depth). To the contrary, the pore water profile at CS1 had an As(III )/As(V) ratio > 1 (Table 2.2). 2.4 Discussion 2.4.1 Are the As(III)/As(V) ratios in vertical pore water profiles unusual? The measurements of H4SiO4, Mg2+, HCO3 -, temperature, and pH (Table 2.1) demonstrated (1) the increase of hydrother mal influence with sediment depth on pore water chemistry and (2) the decrease of that influence with distance from the vent area. The changes in TAs concentration along the tran sect and with depth told the same story. On the other hand, the As speciation data did not seem to reflect hydrothermal influence, because As in the vent fluid was predominantly As(III). Hence, in pore waters with elevated temperatures and at sites with obvious diffuse venting, a relatively high As(III)/As(V) ratio might be expected.
46 Unfortunately, literature on arsenic speci ation in marine pore waters, whether hydrothermally influenced or not, is surprisingly sparse. On e of the first publications describing As speciation in marine pore waters reported an As(III)/As(V) ratio of 0.25 for samples from the Santa Barbara Basin, California (Andreae, 1979). The sedimentary environments studied spanned a wide range of redox conditi ons and biologi cal activity. Nevertheless, As(V) dominated As(III) even in the reducing sediments, although not to the extent of the Tutum Bay hydrothermal system. Reimer and Thompson (1988) reported As(III)/As(V) ratios, ranging from 0.3 to 1.8 (mean = 0.9), for pore waters from two British Columbia coastal sites which were influenced by mine-tailings discharge. Peterson and Carpenter (1985), reported As( III)/As(V) ratios between 0.21 and 1.5 for pore waters in Puget Sound (estuary), 0.026 to 0.76 for the Washington coast (coastal ocean), and 2.8 to 5.0 for Saanich Inlet (interm ittently anoxic fjord), but controls on the arsenic speciation were unclear. These i nvestigations suggest that pore water As(III)/As(V) are highly variable, and that th ey are controlled by a combination of redox conditions, pH and adsorption/release from solid phases, such as iron and manganese (hydr)oxides. Andreae (1979) suggested that ar senic was released (as As(V) desorption) to the pore water during organic matter degr adation or iron and ma nganese dissolution. The conditions in Tutum Bay differ from thos e of the previous st udies in that TAs concentrations in the pore waters were much higher and that mixing between an As(III)dominated hydrothermal fluid and seawater occurred. Also, iron (hydr)oxides are much more elevated throughout the sediments of Tu tum Bay. At the Picnic Island control site (CS1) the As(III)/As(V) ratios in 10-cm pore waters and ambient seawater were 1.3 and
47 1.1, respectively. These ratios are simila r to those previously reported for nonhydrothermally influenced sediments. The As (III)/As(V) ratio for pore waters from our reference site at 20 cm depth however is 7.36, indicating predominance of As(III) and reducing conditions. Freshly deposited carbonate sediments, however have a high organic matter content, which causes a rapid change from oxidizing to reduc ing conditions (Berry Lyons et al., 1973). This could explain the observed dominance of As(III) deeper in the sediment at the reference site. Along the tran sect in Tutum Bay th e As(III)/As(V) ratios in pore water at 10 cm depth are similar ( 0.76), if the 0.5 and 1 m samples are excluded. These two samples were almost identical to th e hydrothermal fluid (Table 2.1). To better illustrate the arsenic speciation at Tutum Ba y, we have plotted the As(III)/As(V) ratio represented as percent species versus distan ce away from focused venting in 10-cm pore waters and vertical pore water prof iles (Figures 2.1 and 2.2, respectively). Unexpectedly, the arsenic speciation ratio in the vertical pore water profiles (i.e., below 10-cm depth) is an orde r of magnitude lower than at the Picnic Island control site (CS1), with values consistently below 0.1 (range = 0.01 to 0.59; Figure 2.2; Table 2.2). Comparing previously reported ratios and reference site ra tios to Tutum Bay pore waters, the arsenic speciation ratio in hydrothermally influenced pore waters below 10-cm were clearly skewed towards As(V) (Figure 2.2) If mixing with seawater, and thus O2, would have caused oxidation of the As(III) to As(V ), the ratio of As(III)/As(V) should be somewhere between that of the hydrothermal fluid and seawater. However, in the
48 Figure 2.1. Distribution of arsenite (As(III)) and arsenate (As(V )) in pore waters collected at 10 cm sediment depth, stepping away from the area of hydrothermal venting. 0% 20% 40% 60% 80% 100% 0.52.5172060140180240Distance (m)%As species AsV AsIII
49 Figure 2.2. Distribution of arseni te (As(III)) and arsenate (As(V)) in vertical pore water profiles collected at distances of 2.5, 30, 60, 140 and 300 m from the area of focused hydrothermal venting. 0% 20% 40% 60% 80% 100%%As species AsV AsIII 10 20 30 40 10 20 30 60 100 10 20 30 60 100 10 20 30 40 50 60 90 10 20 30 40 50 60 70 2.5 30 60 140 300 Distance (m)Depth (cm)
50 hydrothermal system at Tutum Bay, the As(III) /As(V) ratio is far below that found in local seawater. Mixing between hydrothermal fl uid and seawater is demonstrated by pH and temperature, and by increasing /decreasing concentrations of HCO3 -, H2SiO4, Mg2+ and TAs in the pore water profiles (Tab les 2.1 and 2.2). Thus one would expect As(III)/As(V) ratios to increase with sediment depth, i.e., greater sediment depth leads to higher hydrothermal fluid/seaw ater ratio, more reducing se diments, and thus greater As(III) (Figure 2.3). That scenario was not encountered in the Tutum Bay pore water profiles. All profiles showed the expected increase in TAs with sediment depth, but unexpectedly As(V) was always the dominant As speci es. The depth profiles of sample locations 30 m and 60 m are good examples of th is enigma (Figure 2.4). In those profiles As(III) is more or less absent and As(V) is equal to TAs, decreasing towards the sediment-seawater interface. 2.4.2 Thermodynamic and Kinetic Considerations Smedley and Kinniburgh (2002) suggest burial of organic matter and slow diffusion of O2 out of the sediment can lead to reducing conditions just below the sediment-water interface, thus encouraging the reduction of As(V). Other studies have shown that Â‘typicalÂ’ coastal waters become reducing just below the sediment-water interface, and that in organi c-rich sediments it is common to observe oxygen reduction at the sediment surface, nitrate, manganese, and iron reduction within the next few centimeters, and sulfate reducti on over another meter or so (e.g., Berner, 1980). These reactions are almost certainly microbially me diated. Cullen and Reimer (1989) provide an
51 Figure 2.3. Conceptual model of the expected As gradients in Tutum Bay pore water profiles exclusively i nvoking the assumption of seawater mixing and abiotic oxidation. As(III) Hydrothermal Fluid Seawater O2 O2 Seawater As(III) As V TAs Arsenic Abundance (A) (B) Hydrothermal Fluid Sediment
52 Figure 2.4. Observed vertical po re water profiles in Tutum Bay sediments at a distance of 30 and 60 m from the main ar ea of hydrothermal venting.
53 excellent review on arsenic speciation in ocean environments, and thermodynamic calculations suggest that under slightly re ducing conditions and/or lower pH, As(III) is stable, mainly as neutral H3As03. Based on thermodynamic data alone however it is widely accepted that As(V) should strongly dominate over As(III) in Â“normalÂ” seawater (pH ~8, O2 ~ 8 mg/L) with As(III)/As(V) ratios as low as 10-26 predicted (Cullen and Reimer, 1989). However, thermodynamic predictions of As(III)/As(V) ra tios are tricky at best, and calculated concentrations rarely match the observed. Th e oxidation kinetics for As(III) in seawater are slow. Johnson and Pilson (1975 ) report a half-life on the scale of weeks to months for various temperatures and salinit ies. Thus, in the pore waters of the hydrothermal system at Tutum Bay the oxidation of As(III) by oxyge n cannot be completely ruled out, but according to the observed As(III)/As(V) ratios, oxidation must have happened at a sediment depth of more than 1 m (Figure 2.4) Considering results from previous studies of marine pore waters (e.g., Berner, 1980; Berry Lyons et al., 1973) it is very unlikely that dissolved oxygen (DO) would be avai lable at that dept h, either by downward circulating seawater or some other mechan ism. Unpublished data (Hsia-Akerman et al., in process) suggest that the hydrothermal flui ds and pore waters from Tutum Bay contain several other redox sensitive species, such as dissolved iron (Fe(II)) and sulfide, which are much more easily oxidized by DO in seawater (e.g., Millero et al., 1987). We therefore make the assumpti on that little to no abioti c (thermodynamically driven) oxidation of As(III) by seawater derived DO has occurred. 2.4.3 Possible Microbial Metabolisms
54 One critical variable th at thermodynamic calculations fail to incorporate are biologically mediated redox reactions. Th e oxidation rate described above was empirically derived, ignoring th e potential role of microorgani sms. In fact, slow reaction kinetics are precisely wh at microorganisms use to obtain metabolic energy. In a recent paper, Oremland and Stolz (2003) reviewed available data on microbially mediated arsenic redox reactions, and several studies confirmed the presence of a microbial As cycle, wher e As(III) oxidation in the oxic z one is coupled to arsenate reduction in the anoxic zone (Macur et al., 2004; Oremland et al., 2005; Rhine et al., 2005). While dissimilatory arsenate reduci ng prokaryotes have received far more attention (e.g., Hoeft et al., 2004; Hollib augh et al., 2006; Niggemeyer et al., 2001; Saltikov and Newman, 2003), several bacteria l strains are known to gain metabolic energy from As(III) oxidation (e.g., Santini et al., 2000). The terminal electron acceptor is generally O2, but NO3 has also been implicated (O remland et al., 2002). Several studies have shown that the rates of As oxi dation increase dramatically when catalyzed by microorganisms (e.g., Salmassi et al., 2002; Weeger et al., 1999). As an example, the oxidation of As(III) was 100 times faster in an experiment with the bacterium Thermus HR13 than in the abiotic control (Gihring and Banfield, 2001). A similarly enhanced biotic rate for As(III) oxidation was repor ted by Skudlark and Johnson (1981). Wilke and Hering (1998) showed that in the pres ence of submerged macrophytes that hosted oxidizing bacteria, the half-life for As oxidation was 0.3 h. For comparison, Langner et al. (2001) reported a half-life fo r As oxidation of 1 min in the presence of a brown Fe/Asrich microbial mat in a hot spring at Yellowst one National Park. The data presented in the
55 current study support the inte rpretation that microorganisms played a major role in oxidizing the As(III) found in the vent flui d. We posit that microorganisms, perhaps including yet unidentified thermophilic archaea and bacteria, use As(III) as an electron donor in a respiratory process, as the vent fl uid and seawater mix in the water column and in the shallow subsurface. For example, mi croorganisms in Tutum Bay sediments could use oxygen or nitrate as terminal electron acceptors. Mapping of energetics for these reactions ca n be used to help target specific metabolism, and perhaps show how As(III) oxidation is being enhanced. However, calculating the energetics for possible microbial metabolisms in hydrothermal systems is very involved (e.g., Amend et al., 2003b; McCollom and Shock, 1997). These calculations are currently being conducted, and will be presented in a future publication dedicated to energetics calculations. In a ddition, the microbial life of Tutum Bay is currently being characterized, with culturing efforts targeted towards arsenite-oxidizing microbes. 2.5 Summary and Conclusions The pore waters in Tutum Bay seemed to be influenced by hydrothermal venting out to more than 300 m away from the main ar ea of venting. With respect to total arsenic concentration (TAs) two gene ral trends could be observe d: (1) a TAs increase with sediment depth and (2) a TAs decrease with distance from the main area of venting. Those trends were caused by mixing between the hydrothermal fluid and seawater, which was corroborated by the abundance of H4SiO4 and Mg2+, two excellent tracers of
56 hydrothermal influence (Bishoff and Dick son, 1975; Pichler et al., 1999a). The TAs concentrations in seawater and in pore waters at the Picnic Island control site fell into the range expected for Â“normalÂ” marine c onditions (e.g., Smedley and Kinniburgh, 2002). To the contrary, the distribution of the two i norganic As species, arsenite (As(III)) and arsenate (As(V)) showed neith er the expected influence of hydrothermal venting nor what could be expected for Â“normalÂ” mari ne conditions (e.g., Cullen and Reimer, 1989; Smedley and Kinniburgh, 2002). The oxidized As species, As(V) was always dominant, except at the reference site. Several things may be cont rolling the oxidation state of arsenic in Tutum Bay pore waters. These include, but are likely not limited to, microbial interactions, mixing with seawater, and the influence of iron-rich sediments. We propose that microorganisms likely play a role in the transformation of As(III) to As(V) in pore waters of Tutum Bay. Possible microbial metabolic reactions include the use of O2 or NO3 as electron acceptors and As(III) as the electron donor. Re search continues which will hopefully allow a full understanding of the role microbes play in affecting the redox chemistry of pore waters throughout Tutum Bay and other marine sh allow-water hydrothermal systems.
57 HCO3 -(mg/L) Vent 4986683.2950<1~945.6945.6100130 0.590.56.26101031.2<1~1031.21031.285.2230 177.66.1610900<1~90090097220 2.5816.1439.295.2160.30.59255.561.5710 1290.16.1197.42.334.30.0736.6n.a.144 1733.86.5218.28.611.40.75207.91290 1833.16.5126.96.36.199.0428.310.71220 2029.77.6188.8.131.52.796.513.61100 3031.76.1385.51.7230.0725211090 6033.36.82443.1130.2316.4151140 9031.17.51222.4200.1221.921320 140346.12441.845.40.0447.216.41075 150 n.a.a7.224451124.2262.821310 18032.87.8141.54.914.70.3319.621260 225n.a.7.81184.108.40.2064.521330 240n.a.7.9220.127.116.11.867.80.41260 300n.a.7.1167.1212.10.1714.111305 Picnic Islan d n.a.7.618.104.22.168.33.421289 SWb30.28.122.214.171.124.122.411330an.a. = not availablebSW = ambient seawater Table 2.1. Arsenic, H4SiO4, Mg2+ and other physicochemical parameters measured in 10-cm pore waters for the Tutum Bay hydrothermal system, sorted by distance from focused venting Distance (m)T (C)pH As(III) ( g/L) Mg2+ (mg/L) As(V) ( g/L) As(III)/A s(V) TAs ( g/L) H4SiO4 (mg/L)
58. Distance (m) Depth (cm)T (C)pH HCO3 (mg/L) As(III) ( g/L) As(V) ( g/L) As(III)/ As(V) TAs ( g/L) H4SiO4 (mg/L) Mg2+ (mg/L) Vent 4-986683950<1~945.6945.6100129 2.510816.143995.2160.30.59255.561.5710 2.52073.65.956121.3599.60.04620.968.2592 2.53069.55.956177.3430.50.18507.863.7678 2.54063.55.853712.8358.40.04371.167.8864 301031.76.13861.7230.0725211090 3020336.23121.1420.0342.9211010 303034.56.23221.2610.0262.619987 306037.96.14404.53550.01359.581582 3010040.8n.a.2050.6950.0195.5271170 601033.36.82443.1130.2316.4151140 602034.96.22201.5380.0439.2121150 603036.66.22492630.0365.2161130 606040.5n.a.2601.2650.0266.5161350 60100n.a.n.a.473201300.15150561314 14010346.12441.845.40.0447.216.41075 14020n.a.6.22781.921.70.0923.612.41190 14030n.a.6.11852.131.80.0733.913.21225 14040n.a.6.3254215.90.1217.811.21180 14050n.a.6.11901.729.40.0631.19.21215 14060n.a.6.31002.314.40.1616.68.41200 14090n.a.6.51662.4200.1222.46.71220 30010n.a.7.1167212.10.1714.111305 30020n.a.7.215126.96.36.1992.80.91260 30030n.a.6.71371.611.60.1313.20.71280 30040n.a.6.6831.214.10.0915.41.41300 30050n.a.6.82052.517.90.1420.41.51310 30060n.a.7.11462.515.20.1617.71.91290 30070n.a.6.915188.8.131.524.62.21280 SW b -30.28.1184.108.40.206.122.41289 Picnic Island10n.a.a7.6220.127.116.11.33.421330 Picnic Island20n.a.7.818.104.22.168.362.121300an.a. = not availablebSW = ambient seawaterTable 2.2. Arsenic, H4SiO4, Mg2+ and other physicochemical parameters in all pore waters for the Tutum Bay hydrothermal system, sorted by distance from focused venting, then by depth below seafloor
59 Chapter Three Distribution, Speciation and Bi oavailability of Arsenic in a Shallow-water Submarine Hydrothermal System, Tutum Bay, Ambitle Island, PNG 3.1 Introduction Submarine shallow-water hydrothermal sy stems, which are defined here as occurring in less than 200 m seawater and often having a meteoric water source, are frequently enriched in biologi cally toxic elements such as As, Sb, Se, Cr, Co, Pb, Cd, Ag, Cu, Tl, Zn, Hg, and S, as well as possible nutr ients such as Si and Fe (Dando et al., 1999; Varnavas and Cronan, 1988; Vidal et al., 1978). These systems have been described in many areas around the world, where they are ge nerally associated with volcanic (e.g., Dando et al., 2000) or tectonic activity (e .g., Vidal et al., 1978), which provide the necessary heat source. The impact of these systems may be much larger than expected, considering their location. Co astal waters are often br eeding and nursing grounds for many organisms, in tropical areas they host coral reefs and humans have always used these waters for fishing and recreation. The ch emical composition of seawater in coastal areas is controlled through a combination of natural and anthr opogenic processes. In areas of volcanic activity and/ or high heat flow, the discha rge of fluids from marine shallow-water hydrothermal systems may have a considerable impact on the chemical composition of the often biologically important coastal surface waters. Diffuse venting may also influence benthic organisms at very large distances away from focused venting
60 (Tarasov et al., 1999; Varnavas and Crona n, 1988). Considering their abundance, emissions from shallow-water hydrotherm al systems may well be a significant contributor to global geochemical budgets that has been consistently overlooked. The shallow-water hydrothermal vents in Tutum Bay, Ambitle Island, PNG discharge approximately 1.5 kg of As per da y into an area of approximately 50 by 100 m that has an average depth of 6 m (Pichl er et al., 1999a; 1999b). Of all potentially biologically toxic elements, As is the only one that is significantly enriched compared to seawater (~ 500 times). Despite the amount of As released into the bay, corals, clams and fish do not show an obvious response to the elevated concentrations (Pichler et al., 1999b). Fish have been observed to hover over vent orifices bathing in the hydrothermal fluid. The diversity and health of the coral reef itself is indistinguishable from reefs that are not exposed to hydrothermal discharge, although the surrounding sediments are quite different. The skeletons of sclerac tinian corals and the shells of Tridacna gigas clams do not show elevated concentrations of As or other trace metals when compared to specimens collected from outside Tutum Bay (Pichler et al., 2000). While the general geochemistry of As in the Tutum Bay fluids and precipitates is known (Pichler, 2004; Pichler and Veizer, 1999 ; Pichler et al., 1999a; 1999b), only little knowledge exists about its distribution, speci ation, cycling and bioavailability. The primary objective of this research, therefore, was to assess the availability of As for biological uptake from Tutum Bay sediments (cores and surface samples), seawater, pore water and vent fluids. 3.1.1 Arsenic Speciation and Bioavailability
61 Elements such as As, S, Mn, Zn, Cr, Co, Cu, and Se can play a very biologically complex role. These elements are essent ial for many biological processes, but concentrations above a certain level have adve rse effects, thereby c onverting an essential element to a toxin (Emsley, 1991). Recently, it has been recognized that the total concentration of an element in an ecosystem does not necessarily represent its biological availability or potential t oxicity (Newman and Jagoe, 1994). This concept is known as Â‘bioavailabilityÂ’, and is a function of the abundance and chemical form of the toxin in solution (i.e., oxidation state) and the nature of its bindi ng to sediment grains. For example, the element may be part of a very stab le mineral, such as quartz, and thus is not available for biological processes (Newma n and Jagoe, 1994). Alternatively, the toxin can be adsorbed to sediment grains in an easily-extractable form (Tessier et al., 1979), which would then be more available to benthic organisms (Yoo et al., 2004). Bioavailability is defined here as the de gree to which an element or molecule is able to move into or onto an organism ( sensu Benson, 1994). Transfer of a toxin into an organism can occur by diffusion onto/across th e cell membrane, or through the food web. The free-metal ion in solution is by far th e most biologically toxic, although easilyexchangeable forms in sediments can also be harmful (Newman and Jagoe, 1994). Several elements, such as As, Cr, and Sb, ar e more difficult to characterize because they occur in several oxidation states in wate r and sediments and therefore will have a different degree of bioavailabi lity, depending on the species present (Gebel et al., 1997). Arsenic, for example, can occur as arsenite (As3+ or As(III)) and arsenate (As5+ or As(V)), as well as several methylated forms (e.g., DMA and MMA; Francesconi and
62 Edmonds, 1994). Of the two commo n oxidation states of arsenic, As(III) is more mobile and by far the more toxic, causing reduction in growth to marine organisms at aqueous concentrations less than 3 g/L, and neurological damage in humans at concentrations as low as 100 g/L (Gebel et al., 1997; Sanders et al., 1994). Therefor e, the oxygen content and the prevailing redox condition are primary c ontrols on the mobility and toxicity of As in an aqueous environment, because these c ontrol the oxidation state. Thus, determining the species present in the environment is necessary for understanding the bioavailability of the element (Newman and Jagoe, 1994). Concentrations of metals in sediments usually exceed those of overlying water by 3 to 5 orders of magnitude (Bryan an d Langston, 1992; Mount ouris et al., 2002). Therefore the bioavailabili ty of even a small amount of th e total metal in sediment can be important. With respect to bioavailability, the metals in the most readily extracted fraction are the most important, because they are the most bioavailable and are potentially the most immediately dangerous to th e biota (Bendell Young and Harvey, 1991; Bhumbla and Keefer, 1994; Sahuquillo et al., 2002; Tessier et al., 1979). However, As concentrations in all mineral phases must be determined fo r any long-term prediction of arsenic behavior in sediments because ch anges in environmental or physicochemical conditions (e.g., sediment burial, reworking by storms, etc.) may alter mineral stability. 3.2 Methods and Procedures 3.2.1 Sample Handling and Preparation
63 Field work was conducted in November 2003 and consisted of SCUBA diving over a period of 14 days to collect samples of vent fluid, vent precipitates, sediments, pore water, and ambient seawater from Tutu m Bay (Figures 1.1 and 1.2). In addition to the Tutum Bay samples, we collected a car bonate sediment approximately 1.6 km north at a location unaffected by hydrothermal activ ity (Picnic Island c ontrol site (CS1)). Sampling was conducted along a transect stepping away from the area of focused venting to an area where the character of the sedime nt (i.e., color, mineralogy) seemed to be unaffected by hydrothermal activity, 225 m away (Figure 1.8). The transect was marked by aluminum stakes approximately every 30 m that were connected by a metered rope. Temperature and pH of waters just be low the sediment/water interface (~5cm) were measured along the transect at a spacing of 1 m using an IQ Scientific Instruments #IQ150 pH/mV/temperature pr obe, with automatic temp erature compensation to 100 C in an underwater housing, and a Forestry S uppliers waterproof digital thermometer, respectively. Vent fluids were collected following the procedure of Pichler et al. (1999a). This consisted of placing an inverted Teflon funnel over focused venting. The temperature from the top of the funnel, along with the appe arance of density fronts, indicated that the flow of hydrothermal fluid discharge had disp laced all seawater from the funnel. 60 mL syringes were attached to the funnel, and the vent fluid was slowly drawn into the syringe at a rate slower than vent discharge so as not to contaminate the hydrothermal fluid sample with seawater. Vent precipitates we re collected in the im mediate vicinity of focused venting. Although vent fluid and prec ipitates from this locality have been
64 collected in the past (Pichler and Dix, 1996; Pichler et al., 1999a), they were collected again to assess consistency and temporal va riability in the hydrothermal system. The direct comparison with data from th is research is presented below. Surface sediments were collected alo ng the transect at 1, 7.5, 12, 30, 60, 90, 125, 150, 175, 200, and 225 m away from focused venti ng to assess the extent of hydrothermal influence on sediment chemistry (Figure 1.8). Sediment cores up to 1 m deep were taken to assess hydrothermal influence wi th depth at 1, 7.5, 12, 30, 60, 90, 150, and 225 m (Figure 1.8). Cores were de scribed on-board and sample d at approximately 10 cm intervals. At the same locations where sediment cores were taken we obtained pore-water profiles down to a depth of 1 m when possible, using Teflon tubing inside a 1Â” aluminum pipe with screened openings every 10 cm. The pore fluids were collected into syringes and, to reduce vertical mixing, a multi-syringe puller was used to extract the sample slowly and simultaneously fr om up to six different depths. Ambient seawater was collected at approximately 15 cm below the ocean surface, and 1 m above the seafloor every 15 m al ong the entire transect. This was done simultaneously by two divers swimming al ong the transect using 60 mL syringes. As soon as vent fluids, pore waters, and ambient seawaters were brought on board, unstable parameters, such as pH and alkalinity were measured. Total alkalinity was measured using a HACH digital titrato r, and titrating to an endpoint of pH 4.5 (HACH, 1997). The pH was measured usi ng a Myron-L pH meter with temperature compensation. Water samples were preserve d for analysis of As abundance, As
65 speciation, major cations and anions, and major elements by filtering through 0.45 m filters, and acidified using ultrapure HCl. A ll water sample bottles were sealed with electrical tape and kept in i ced coolers until lab analysis. 3.2.2 Lab Measurements Sediment samples were described by op tical microscopy, and using a Hitachi S3500N variable pressure Scanning Electr on Microscope (SEM), equipped with a Robinson backscatter detector and a PGT En ergy Dispersive X-ray (EDX) system housed at the USF Electron Microscopy Center. Th e EDX analysis of hydrothermal vent precipitates was used in combination with sequential extraction to verify the primary arsenic phases. Arsenic abundance and specia tion in vent fluids, seawat er, and pore-waters were determined by hydride generation-atomic fluorescence spectrometry (HG-AFS) on a PSAnalytical 10.055 Millennium Excalibur syst em at the USF Center for Water and Environmental Analysis. In preparation for th e determination of bulk (total) As, 10 mL of sample were combined with 15 mL concen trated HCl and 1 mL saturated potassium iodide (KI) solution to reduce As (V) to As(III) and diluted with deionized water (DI) to a final volume of 50 mL. Arsenic speciation analyses were carried out by high-pressure liquid chromatography (HPLC) separation of As(III), DMA, MMA and As(V) prior to detection by HG-AFS. A sample volume of 100 L wa s injected without pretreatment and separated in a Hamilton PRP-X100 ca tion exchange using a K-P buffer.
66 Following homogenization and acid digesti on, Fe, Ca and As in sediments and precipitates were measured by a combinati on of AFS, NAA and ICP-MS at Actlabs, Ontario, Canada and at USF. The element Si was analyzed by inductively coupled plasma Â– optical emission spectrometry (ICP-OES) on a Perkin Elmer Optima 2000 DV, also housed at the Center for Water a nd Environmental Analysis, USF. 3.2.3 Sequential Extraction of As in Tutum Bay Sediments The As fraction in sediments that is most important for bioavailability studies is the easily extractable surface-complexed fraction (Newman and Jagoe, 1994). We extracted this fraction through a cation-ex change reaction using a solution of KH2PO4/K2HPO4 at a pH of 7.2, following Gleyzes et al. (2002). This extraction step consisted of weighing 0.5 g of sediment or pr ecipitate into a 50 mL centrifuge tube, then adding 20 mL of the reagent. The reaction was allowed to continue for ~16 hours at room temperature with periodic shaking. The sample was centrifuged, and the supernatant was transferred from the centrifuge tube using a dropper. 10 mL of DI water was added to the remaining sediment, shaken, and then the ri nse was added to the existing supernatant. Overall, this experiment was performed three times: 1) as a single extraction; 2) at the beginning of the four-step complete sequential extraction (see below); and 3) to test the completeness of this reaction, the PO4 extraction was repeated three times consecutively on the same sample. A four-step sequential extraction, tailored specifically to the mineralogy and geochemical attributes of Tutum Bay sedi ments was carried out to evaluate As concentrations in the surface-bound, carbonate Fe-oxyhydroxide (hydrous ferric oxide or
67 HFO), and residual fractions. This extraction scheme is a combination and modification of methods developed by Tessier et al., (1979), Pich ler et al. (2001), We nzel et al. (2001), and Gleyzes et al. (2002). Often MgCl2 is used on the easily extr actable fraction (Tessier et al., 1979). We did not use the MgCl2 reagent based on research by Gleyzes et al. (2002), who showed Mg2+ cannot be exchanged efficiently with the anionic species, the univalent chloride ion has low anion exchange power, and, more importantly, the pH is not buffered during MgCl2 extraction procedures. Low fina l pH values could contribute to attack the carbonate fraction, which coul d overestimate the exchangeable fraction while underestimating the carbonate fraction. Our sample collection, preparation, and extraction procedure consisted of the following: 1) Each sediment or precipitat e sub-sample was air dried. 2) 0.1 to 1.0 g of homogenized sample, depending on the quantity of HFO and CaCO3 present, was weighed into 50 mL Teflon centrifuge tubes. 3) An amount of each extracting reagent was added sequentially for the listed time and conditions (Table 3.1). 4) Once the reaction was complete, the sample was centrifuged at 8600 rpm for 15 min and the supernatant liquid was decanted into separate bottles for analysis following methods outlined above. 5) DI was added to the sample, and the rinse was added the supernatant. The reagents and conditions (pH, temp erature, reaction time, agitation, and rinsing) selected for each fraction are pr esented in Table 3.1. Additional reagent was added when necessary to the carbonate and HFO extraction steps (e.g., for vent precipitates or contro l-site samples, which consist of nearly 100 % HFO and CaCO3, respectively).
68 Analytical and instrumental quality a ssurance and quality control (QA/QC) was evaluated for all lab analyses by including sample duplicates and certified reference standards from Fisher Scientific, which indicate a precision of better than 5 % for HGAFS and ICP-OES. Background signal drift was consistently <1 % for all instruments. Acid blanks for digestions and sequentia l extractions were tested and showed no contamination for the analyzed elements. No reference material exists for the fractionation of arsenic in sedi ments, thus we used the marine sediment reference, PACS2, from the Institute for National Measuremen t Standards National Research Council of Canada, to track the reproducibility of our extractions. The supernatant fluids were analyzed by a combination of ICP-OES and HG-AFS. The sum of the individual arsenic fractions was also compared to a total aqua regia digestion. 3.3 Results 3.3.1 Temperature and pH Variation of Surface Sediment Pore-water The temperature and pH of pore-waters in surface sediments show a hydrothermal influence extending up to 150 m away from the vent site (Figure 3.1). The temperature of these pore-waters decreases from 94 C near the vents to a constant 31 C (ambient seawater) 100 m away from the vents. For th e first 20 m the temperature shows dramatic variations of up to 60 C between sampling points only a meter apart (Figure 3.1). From 20 to 100 m there is still some variation of up to 5 C. The pH of the pore-fluids increases from approximately 6 near the vents to 8.2 al ong the transect. Large variations for pH of up to 1 unit were observed between 10 and 20 m. The pH values then gradually increase
69 Figure 3.1. Temperature and pH relationship in pore-waters of surface sediment (~5 cm depth) along transect vs. di stance from focused venting. 25 35 45 55 65 75 85 95 105 050100150Distance (m)Temp (C)6 6.5 7 7.5 8 8.5pH Temp pH
70 to ambient seawater values along the transe ct, with a pH lower than seawater (pH 8) extending to >30 m (Figure 3.1). The temperatur e and pH at the control site are 30.2 C and 8.02, respectively. 3.3.2 Arsenic in Vent Fluids, Pore Waters and Ambient Seawater The transect starts at vent 4 of Pichler et al. (1999a) and its arse nic concentration of 950 g/L, exclusively present as As (III), closely matches previous data (Pichler et al., 1999a). Stepping away from vent 4, As concentrations at the seawater-sediment interface (porewaters at 0 to 10 cm) remained high (900.4 g /L) near the vent area, but then dropped consistently to a minimum of 4.5 g/L at 225 m (Table 3.2). Beginning with the porewater profile at 30 m, As(V) is the domina nt species and concentrations generally increased with depth (Table 3.2). Pore-waters closer to the vent site contained higher concentrations of As(III), and at 1 m, no As (V) was detected. A good correlation exists between As and Si in the pore-waters (R2 = 0.84; Table 3.2). Ambient surface seawater in Tutum Bay ranges from 8.4 g/L above the vent area to 2.5 g/L at the end of the tr ansect (Figure 3.2 A). This valu e is still significantly above the value of 1.35 g/L, which is reported for surface seawater (Cutter, 2002). Bottom seawater shows uniformly lower As concen trations than the corresponding surface samples (Figure 3.2 A). Concentrations decr ease from 3.1 to 1.8 g/L. The abundance of Si along the transect mirrors that of As in both the surface and bottom samples, with a
71 0 2 4 6 8 10 04080120160200240Distance (m)As (ppb) surface bottom 0 1 2 3 04080120160200240Distance (m)Si (ppm) surface bottom Figure 3.2. Arsenic and Si concen trations in surface and bottom waters along the transect.
72 correlation coefficient of 0.98 and 0.93 for surface and bottom waters, respectively (Figure 3.2 B). Both inorganic As species, As(III) and As(V) are present in ambient seawater (Table 3.3). The species distribution is surprisingly constant along the transect, without variation beyond the analytical uncer tainty. In surface samples, As(V) is the dominant species (~70 %) whereas As(III) is the dominant species in the bottom samples (~60 %). Neither of the two methylated As species, DMA and MMA, were detected in any of the samples that were selected for As speciation, i.e., vent fluid, pore waters, ambient seawater. 3.3.3 Arsenic Abundance and Bioavailability in Precipitates and Sediments The highest As concentrations in Tutu m Bay are found in hydrothermal HFO that precipitates around vent orifices. Arsenic values for HFO collect ed directly at vent 4 are as high 33,200 mg/kg (Table 3.4, Figure 3.3) although concentrations as high as 76,500 mg/kg were previously reported (Pichler a nd Veizer, 1999). The As concentration in the surface sediments decreases from a maxi mum of 1482 mg/kg at the beginning to 52 mg/kg at the end of the transect (Figure 3.3, Table 3.4). Iron exhibits a similar trend, decreasing from 18.4 % to 6.6 %. Calcium values increase along the transect, reflecting the increasing amount of calcium carbonate in the sediment (Table 3.4). The calcium carbonate sediment that was collect from outside Tutum Bay (CS1) contains 2.2 mg/kg As (n = 11; = 0.25), 33.4 % Ca and 0.4 % Fe. The sediment cores displayed substantia l vertical and horiz ontal heterogeneity, reflecting hydrothermal alteration, including precipitation of HFO a nd framboidal pyrite
73 Figure 3.3. The variation of As concentration in the easily-ex tractable sediment fraction and total concentration of As in surface sediments along the transect shown in Figure 1.8. 0 100 200 300 400 500 600 700 800 900As (ppm) 017.512306090125150175200225 Distance (m) Total As Extracted 23 16 4.3 6.1 4.0 4.8 3.8 4.1 2.6 3.3 1.9 13.3 % extractable PI 33200 *VP *VP = vent precipitate +PI = Picnic Island Control 8.1 %
74 in highly altered sediment. Arsenic concentrations range from 159 to 1483 mg/kg near the vent, and reach 44 to 72 mg/kg 225 m away from the vent site (Table 3.4). The highest As total concentrations are concen trated in HFO and Mn-oxide layers. For example, the highest concentration of As for all core-sediments was 4025 mg/kg, located in a zone of extensive HFO coating in the core collected at 60 m (Table 3.4). This is also reflected in the sequential extract ion, which is discussed below. The bioavailability of As from Tutum Ba y surface sediments and precipitates was estimated using the single PO4 extraction (Table 3.1). Arse nic concentrations for the easily extractable fraction ra nged from 444 mg/kg in HFO pr ecipitates, to 1.5 mg/kg in sediments at 225 m, and 0.7 mg/kg from the se diments at the control site (CS1). The easily extractable portion of arsenic in Tutu m Bay surface sediments can be as high as 50 mg/kg, with a mean of 19.7 mg/kg when excl uding the vent precip itates and control end members (Table 3.5, Figure 3.3). This averages 3.6 % of the total concentration, with a range of 1.6-4.1 % (Figure 3.3). Vent precipitate s and the control sample had extractable As of 1.3 and 32.1 %, respectively (Table 3.5, Figure 3.3). The PO4 extraction for the surface-bound arsenic that was performed on the same sediments three times consecutively showed near-perfect reproducib ility and values were in close agreement with the single extractions. The four-step sequential ex traction showed that of the easily extractable, carbonate, HFO, and residual fractions, the As was predominantly associated with the HFO. In fact, for the HFO precipitates, 98.6 % of the As was leached along with 99.7 % of the Fe during this step (Table 3.5). The easily-extractable, carbonate, and residual
75 fractions for vent precipit ates contained 1.4, 0.03, and 0.1 % As, respectively. The mean easily-extractable fraction fo r this experiment was 21.5 mg/kg, which is in very good agreement with the other extraction experiment explained previously. It is important to note that the sum of the fractions in most, but not all, cases were equal to the bulk As concentration determ ined by NAA. This would suggest that the residual fraction was not completely represente d (digested) in some of the samples, and that the percent values would therefore be effected. However, the HFO fraction obviously contains the majority of As in Tutum Bay se diments, and ~20 mg/kg As is available from the easily-extractable fraction of the sedime nts. Residual minerals included quartz, hornblende and feldspars. 3.4 Discussion 3.4.1 The Importance of Diffuse Venting on As Distribution and Speciation Diffuse discharge seems to play a critical role in the distri bution of As throughout Tutum Bay waters and sediments. The As c oncentration in Tutum Bay surface sediments is elevated to >200 m, with a mean value of 527 mg/kg. This is approximately one order of magnitude more As than observed at th e end of the transect and two orders of magnitude higher than the As concentration at the control site (2.2 mg/kg). Discharge of hydrothermal fluid through the sediment is vi sible to at least 30 m away from focused venting, while temperature and pH is highly variable along the tran sect to almost 100 m (Figure 3.1). However, the most compelling evidence that suggests diffuse venting is a major contributor of As in Tutum Bay sedi ments is the high As concentration in pore
76 fluids up to 150 m away fr om the vent site (62.8 g/L; Table 3.2). Altered core sediments with HFO coatings contain elevated As concentr ations to the end of the transect at 225 m. Hydrothermal discharge through the sediments is also reflected in the lack of calcium carbonate near the vent, only appearing in the se diment at 175 m. This is likely a result of the low pH of hydrothermal fluids preventi ng the formation and/or prolonged existence of calcium carbonate in the sediment. In a ddition to high As concentrations in surface sediments at the end of the transect, elevated Fe concentrations al so exist (e.g., 6.6 % at 225 m compared to 0.4 % at the control site). Vent fluids at Ambitle Island are charac terized by very high As concentrations. Interestingly, however, the ambient seawater immediately surrounding the vent site has As values which are three orders of magn itude lower and are near commonly reported values for normal seawater of ~ 2 g/L (Andreae, 1977; Plant et al., 2003). Arsenic speciation in bottom waters seems to be slig htly affected by diffu se flow, with higher As(III) concentrations when compared to aver age seawater (5-10 %; Table 3.5; Plant et al., 2003). The use of Si as a tracer of hydrothe rmal venting is valid provided there is a sufficient concentration difference between the hydrothermal fluid and seawater, which is the case at Ambitle Island (Pichler et al ., 1999a; Sedwick and Stuben, 1996). The low Si and As concentrations in bottom waters show that there is lit tle hydrothermal fluid leaving the sediments, although pore water and sediment chemistry reflects diffuse flow (Tables 3.2, 3.4, 3.5). This likel y reflects the absorption cap abilities of HFO at depth (Pichler et al., 1999b).
77 The As content in Tutum Bay surface seaw ater is elevated. This enrichment can be explained by the buoyancy driven rise of hydrothermal fluids due to differences in temperature and salinity (Pichler et al., 1999a). If the As in surface waters of Tutum Bay is hydrothermally derived, the As speciation sh ould be characterized by elevated As(III), in particular at the surfac e. About 30 % of the As occurs as As(III), which is approximately one order of magnatitude higher than the concentration of As(III) typically found in average seawater (Table 3.5; Plant et al., 2003). However, As in surface waters still have higher As(V) concentr ations than expected if the As is hydrothermally derived (~100 % As(III)). As(III) will be moderately uns table in the presence of oxygen, which is enriched more in surface waters. Photoor biological oxidation of As(III) to As(V) can also occur within a few hours in coastal surf ace waters (Cutter, 1992; McCleskey et al., 2004; Sanders et al., 1994). This might explai n the elevated As(V) concentration in the surface waters of Tutum Bay. The pore-waters of Tutum Bay are enriched in As with depth, suggesting that diffuse discharge of hydrothermal fluid o ccurs throughout Tutum Bay. As a comparison, higher concentrations of As frequently occur in pore-waters extracted from unconsolidated sediments than in overlying surface waters (see Plant et al., 2003). However, the relative concentration increas e between pore-water and the overlying water column are much more enhanced in hydrotherm al areas. High concentrations of As are found in pore waters from geothermal ar eas (e.g., 6.4 mg/l in Lake Ohakuri, New Zealand; Aggett and Kriegman, 1988). However, if the higher As concentrations in porewaters of Tutum Bay are from hydrothermal fluids, we would expect predominantly
78 As(III). To the contrary however, pore-waters are dominated by As(V). An important aspect to consider is the role that microbes and bacteria may play in oxidizing As(III) to As(V) in the sediments, in particular in t hose areas with hydrothermal diffuse discharge where mixing of reduced fluids and seawater may create redox grad ients. Microbes that obtain energy through oxidation or reduction of As have been described in several hydrothermal systems (Oremland and Stol z, 2003; Plant et al., 2003). In addition, abundant HFO coatings occur in the core, and are associated with elevated As concentrations (e.g., 4000 mg/kg As in the co re at 60 m; Table 3.4). The enrichment is likely due to diffuse hydrothermal discharg e, precipitation of HFO upon mixing with seawater, and adsorption of As. Compared to coastal regions unaffected by hydrothermal input, that show As concentrations in the range from 3 to 15 mg/kg (Sanders et al., 1994), the As concentrations of sediments at Tutum Bay ar e two orders of magnatitude higher (mean = 527 mg/kg). Similar enrichments have also been reported in the sediments from Santorini hydrothermal field, with a maximum concentr ation of As = 927 mg/kg and a mean of ~460 mg/kg (Varnavas and Cronan, 1988). Varnav as and Cronan (1988) also suggest that hydrothermal As is scavenged into the se diments by freshly precipitating HFO. Johnson and Cronan (2001) show a maximum concen tration of 997 mg/kg As in shallow submarine hydrothermally influenced sediments at Champagne Hot Springs of Dominica (Lesser Antilles), but provide no transect data to examine the extent of hydrothermal influence. McCarthy et al. (2004) has also s hown decreasing As with increasing distance away from focused hydrothermal venting at Champagne Hot Springs.
79 3.4.2 Mechanism for As Enrichment in Sediments We suggest the main control on the distri bution of As in Tutum Bay sediments is the upward diffusion of hydrothermal flui d, precipitation of HFO upon contact with oxidizing seawater, and adsorption of As ont o HFO. Mixing of the hydrothermal fluids and seawater causes rapid oxidation of Fe(II) an d therefore the precip itation of HFO, and possibly the subsequent complete removal of As from the hydrothermal fluids. The scavenging of As by HFO has been well docum ented (Feely et al., 1994). In oxidizing environments, the primary mechanism for atte nuation of As is its adsorption onto HFO (Sracek et al., 2004). For long term predictions, our sequential extractions ha ve shown that >98 % of the As is associated with coprecipitated HF O in vent precipitates, and a mean of 93.5 % for surface sediments (range = 88.2 to 96.3; Ta ble 3.4). Long term stability of this adsorbed As can be effected by abiotic r eactions (oxidation, reduc tion, precipitation, and dissolution) and biotic reactions (microbial transformations), as well as the physical disturbance of sediments (Mok and Wai, 1994; Sanders et al., 1994). If redox conditions remain oxidizing, the HFO will remain stable. Upon burial, however, oxidizing sediments could potentially be subjected to reducing c onditions. Previous work has shown that the reduction of HFO upon burial can cause the rele ase of any adsorbed or coprecipitated As (Plant et al., 2003). Recent work also confirms the associat ion of As with HFO in oxic sediments, its release to the interstitial wa ter when Fe(III) is reduced to Fe(II) upon burial of the sediments, and its upwar d diffusion to the sediment-wat er interface (Sanders et al.,
80 1994). There it is reprecipitated, under oxic c onditions, with newly formed HFO, or is released to the water column unde r prevalent reducing conditions. Dissolved As can also be co-precipitated with pyrite (Sanders et al., 1994), and we have observed framboidal pyrite in some sediments of Tutum Bay. In reduced environments, sulfides control the distribu tion of As (Plant et al., 2003). Physical disturbance of the sediments by storm, typhoon, or flooding may move the underlying sediments to an oxidizing environment wh ere the sulfides under go oxidation, releasing the As. 3.4.3 Bioavailability of As in Tutum Bay The concentration of bioavailable As from surface sediments in Tutum Bay excluding vent precipitates ranges from 5.5 to 54 mg/kg, w ith a mean of 19.7 mg/kg (Table 3.5). We can see that because As but no Fe, was leached during the easilyextractable step of the sequential extracti on, that this portion of As is surface-bound, whereas the rest is likely co-precipitated with the HFO. Solid-phase toxicity to marine organisms is low when compared to the potenti al toxicity of the fr ee-metal in the aqueous phase (Benson, 1994; Newman and Jagoe, 1994). Th erefore, the elevated concentrations of As and the abundance of the more toxic As(III) in surface waters of Tutum Bay may have the most impact on biota. Arsenic is an example of an element that effects mainly the base of the food chain (Sanders et al., 1994). Exposure of an ecosyst em to above normal levels of As may induce stress and results in a reduction in diversity and the incr eased abundance of a limited group of opportunistic taxa (E ngle, 2000; Pearson and Rosenberg, 1978).
81 However, from the viewpoint of toxicity, the most sensitive link in the chain is phytoplankton, with reduction in growth exhibited at concentrations of arsenite as low as 3 g/L (Sanders et al., 1994). This is very significant given the fact that the surface waters of Tutum Bay have approximately 8 g/L As, and one order of magnitude more As(III) when compared to average seawater. While invertebrates appear to be more resistant to dissolved As (death at >100 g /L), they may be effected by changes in phytoplankton community structure that are changed due to high As concentrations. Future work should therefore include measurem ents of the plankton community structure, abundance, and diversity as compared to a control area. However, it appears as though the majority of As is rapidly locked up into the solid phase due to mixing of hydrothermal wa ters with seawater throughout Tutum Bay, and therefore biota is essentially buffered from the potential deleterious effects. 3.5 Summary The discharge of potentially toxic el ements such as As by shallow-water hydrothermal systems has to date received li ttle attention, despite the large number of vents reported from around the world. By studying the abundance, distribution, and bioavailability of As in Tutum Bay, Ambitle Island, PNG, we assessed the potential impact of a hydrothermal system discharging large amounts of a single toxin to biota for the first time. We have found that: 1) As concentrations in surface sediments along a transect ranges from ~1500 mg/kg to 50 mg/kg, and decrease exponentially away from focused venting, with the highest enrichments in vent precipitates. 2) The bioavailable
82 portion of As in Tutum Bay surface sediments is an average of 3.6 % of the total, which equals a mean bioavailable concentration of 19.7 mg/kg. 3) The As abundance in surface waters of Tutum Bay is elevated by four tim es above average seawater as far as 170 m away from focused venting, with a maxi mum of 8.4 g/L. Low salinity and high temperature cause the hydrothermal fluids to quickly rise to the surface of Tutum Bay. The As concentration in bottom waters is sim ilar to average seawater concentrations of 2 g/l (Andreae, 1977; Cullen and Reimer, 1989; Pl ant et al., 2003). As species in surface waters are about 70 % As(V), and 30 % As (III). The methylated species DMA and MMA were not detected. Bottom waters are even more enriched in As(III), with only approximately 40 % As(V). The enrichment of As(III) in bottom waters is likely caused by diffuse venting of hydrothermal fluids, wh ile As speciation in surface waters could be controlled by photoor bio-oxi dation. 4) The As concentrati on increases with depth in sediment pore-waters, and is characterized by As(V) as the major As species, possibly due to microbial activity. Arsenic in core se diments is enriched in HFO layers which precipitate upon mixing of reducing hydrothermal fluids with oxic seawater (max = 4025 mg/kg). The water seeping through the sediment s is enriched in As, Fe, and Si, and the As is being locked up in hydrous ferric oxi des before the fluid reaches the sedimentseawater interface. These findings emphasize the importance of measuring the distribution, bioavailability, and element cycling throughout an entire hydrothermal system, and not just from vent fluid and preci pitates. The most important c ontrol on this di stribution, at least with respect to As, is the discha rge and precipitation of HFO upon mixing of
83 hydrothermal fluids with seawater. However, the most bioavailable As is in surface waters where uptake by phytoplankton, which forms the base of the food chain for the coastal ecosystem, may cause bioaccumul ation of As in reef organisms.
84 StepFractionExtractant/reagentsConditionsReference 1Easily 0.1 M KH2PO4/K2HPO4pH=7.2, 20 C, 16hGleyzes et al., 2002 extractableshaking periodically rinsed with DI 2Carbonate1.0 M NaOAc/HOAcpH=5.0, 20 C, 4hTessier et al., 1979 shaking periodically rinsed with DI 3Amorphous 0.2 M NH4 +-oxalate pH=3.25, 20 C, 4hWenzel et al., 2001 and crystalline shaking periodically metal-oxidesrinsed with DI 4ResidualAqua regia96 C, 2.5hPichler et al., 2001 3:1 HCl to HNO3rinsed with DITessier et al., 1979 Table 3.1 Sequential chemical extraction procedure for As and Fe in sediments and vent precipitates from Tutum Bay hydrothermal system
85 Distance (m) Depth (cm) T (C) pH As(T) (g/L) As(III) (g/L) As(V) (g/L) H4SiO4 (mg/L) Vent 40986.0950950n.d.a100 1038.76.2n.a. b n.a.n.a.n.a. 1077.66.1900900n.d.97 7.5042.26.281324914 1045.56.2n.a.n.a.n.a.n.a. 12045.57.4362.1344 1090.16.1n.a.n.a.n.a.n.a. 30030.17.1101.48.75 1031.76.1251.72321 20336.2431.14221 3034.56.2631.26119 6037.96.13594.535581 10040.8950.69527 60030.77.19.21.57.84 1033.36.8163.11315 2034.96.2391.53812 3036.66.2652.06316 6040.5661.26516 1001502013056 90030.58.1 1031.17.5222.4202 15007.97.31.36.01 107.26351122 307.6277.9193 22508.2 22.214.171.124.22 CS1030.28.0n.a.n.a.n.a.n.a. 126.96.36.199.42 188.8.131.52.32 Note: The methylated species, DMA and MMA, were not detectedan.d. = not detected b n.a. = not analyzed Table 3.2 Temperature, pH, arsenic (As) and silica (H4SiO4) in pore-water profiles for points along the transect shown in Figure 1.8 and the Picnic Island control site (CS1), compared to Vent 4.
86 Distance (m)LocationTAsAs(III)As(V)As(III)As(V) g/Lg/Lg/L%% 0surface184.108.40.2068.371.7 15surface220.127.116.113.366.7 30surface7.8n.a.an.a.n.a.n.a. 45surface8.1n.a.n.a.n.a.n.a. 60surface8.4n.a.n.a.n.a.n.a. 75surface18.104.22.1683.576.5 90surface7.4n.a.n.a.n.a.n.a. 105surface7.3n.a.n.a.n.a.n.a. 120surface8.0n.a.n.a.n.a.n.a. 135surface7.5n.a.n.a.n.a.n.a. 150surface6.91.95.027.572.5 165surface5.8n.a.n.a.n.a.n.a. 180surface22.214.171.1242.157.9 195surface126.96.36.1996.853.2 210surface188.8.131.52.963.1 225surface184.108.40.2066.753.3 0bottom220.127.116.117.642.4 15bottom18.104.22.1681.748.3 30bottom2.0n.a.n.a.n.a.n.a. 45bottom1.6n.a.n.a.n.a.n.a. 60bottom22.214.171.1240.539.5 75bottom1.8n.a.n.a.n.a.n.a. 90bottom126.96.36.1997.043.0 105bottom1.7n.a.n.a.n.a.n.a. 120bottom1.9n.a.n.a.n.a.n.a. 135bottom1.5n.a.n.a.n.a.n.a. 150bottom1.6n.a.n.a.n.a.n.a. 165bottom1.4n.a.n.a.n.a.n.a. 180bottom188.8.131.527.932.1 195bottom1.3n.a.n.a.n.a.n.a. 210bottom1.5n.a.n.a.n.a.n.a. 225bottom1.51.00.564.935.1 Note: an.a. = not analyzed Table 3.3 Arsenic concentration and speciation in seawater for sampling points along the transect shown in Figure 1.8 in surface and bottom seawater.
87 Distance (m)Depth (cm)As (mg/kg)Fe (%)Ca (%) 003320029.73.0 10148318.44.8 1515912.36.4 2520312.86.4 7.5078312.25.9 587211.96.9 124319.05.0 2514811.04.9 3517813.75.7 1206808.46.0 24738.76.8 167479.06.0 204168.310.9 26106413.18.6 3827314.74.5 3005398.36.9 248311.27.1 107199.86.7 305377.38.0 506246.87.2 687886.96.0 60061410.58.2 563511.58.0 25104311.27.1 31366815.37.2 45205318.36.1 6473613.55.2 75125511.95.8 80158010.94.8 85402514.93.1 925307.01.9 90044310.311.0 546411.89.0 2552411.48.9 505909.922.6 79124510.57.3 12504686.05.8 15004026.57.6 54116.58.9 204116.911.1 353696.012.7 503746.510.4 17503608.85.6 20001636.212.6 2250526.619.7 5726.519.4 13484.325.4 23445.624.8 CS1 averagesa2.20.433.4an=11 Table 3.4 Arsenic, Fe and Ca composition in Tutum Bay sediment cores collected along the transect shown in Figure 1.8, compared to the Picnic Island control site (CS1)
88Distance (m)AsFe AsFe AsFe AsFe VP4440.19.3134317832814793.5719 15467a4.7 13 461360243.7 7769 7.512 n.d. b 0.7 10 340359160.6 7406 1229 n.d. 2.7 14 446310482.3 3951 3030 n.d. 2.3 14 43335981n.d.3892 6027 n.d. 1.3 9.5 37532574n.d.5116 9018 n.d. 0.9 6.5 25728340n.d.5046 12516 n.d. 0.7 8.7 282218950.4 1864 15014 n.d. n.d. n.d. 270230900.6 1572 17510 n.d. 0.02 62 195235700.1 1841 2005.5 n.d. n.d. 49 104211440.2 1555 2251.5 n.d. n.d. 19 1721715n.d.2761 CS10.7 n.d. n.d. 87 n.d.1928n.d. 65 bn.d. = not detected Table 3.5 Four-step sequential extraction results for vent precipitates (VP), surface sediments collected along the transect sh own in Figure 1.8, compared to the Picnic Island control site (CS1). All values in mg/kg Notes: aThis value suggests the HFO fraction may have contributed some As to the easily extractable fraction. However, repeat experimen ts showed approximately the same amount of As being extracted from this sample (54 3 mg/kg). EASILY EXTRACTABLECARBONATEHFORESIDUAL
89 Chapter Four Enhanced Bioaccumulation and Biotransforma tion of Arsenic in Coral reef Organisms Surrounding an Arsenic-rich Marine Sha llow-water Hydrothermal Vent System 4.1 Introduction The negative effects of contaminants on tropical marine ecosystems are of increasing concern as human populations expand adjacent to these communities (Peters et al., 1997). It is therefore incr easingly important to unders tand the natural, coastal biogeochemical cycle of arsenic and other co ntaminants which will allow us to better detect, predict, and evaluate changes aris ing from human activity (Maher and Butler, 1988). The chemical behavior and speciation of arsenic in marine environments depends on the physicochemical conditions of surroundi ng seawater, sediments, and sediment pore water (e.g., Cullen and Reimer, 1989; Fr ancesconi and Edmonds, 1998; Maher and Butler, 1988; Neff, 1997; Sadiq, 1992; Smed ley and Kinniburgh, 2002) Briefly, the two major inorganic arsenic species present in coastal marine environments are arsenite (As(III)) and arsenate (A s(V)). As noted by Sillen (1961) and Johnson (1972), thermodynamic calculations suggest that oxyge nated surface seawat er should contain predominantly As(V), but biol ogical activity can reduce appr eciable amounts to As(III). These inorganic forms can be taken in and me thylated by coastal organisms and either are
90 excreted through a detoxification mechanism or bioaccumulated within the organismsÂ’ tissue. The two major organoarsenic species which can occasionally be detected in seawater and sediment pore-waters as a result of planktonic and/or bacterial interactions (Andreae, 1979; Cullen and Reimer, 1989), are dimethylarsinate (DMA) and monomethylarsonate (MMA). In addition to DMA and MMA, the major methylated organoarsenic species found in tissues of marine organisms include trimethylarsine oxide (TMAO), tetramethylarsonium ion (TETRA), arsenobetaine (AB), arsenocholine, (AC), and the four major arsenic-carbohydrate compounds, referred to collectively as arsenosugars or arsenoribosids (AR). These are glycerol sugar (AR 1). phosphate sugar (AR 2), sulfonate sugar (AR 3), and sulfat e sugar (AR 4). While these are the major arsenic species encountered in coastal mari ne organisms, more than 30 have been identified (Francesconi and Kuehnelt, 2004). Despite an increased scientific and public interest, As is an element whose environmental cycling is still poorly unde rstood. Bioaccumulation and biotransformation can remove much of the arsenic from sediment s and waters; nevertheless, tissue arsenic is often overlooked as a sink, even though a ppreciable concentrations are common in marine organisms (Cullen and Reimer, 1989). Shallow-water, near-shore, marine coastal hydrothermal vents are compelling natural analogs for coastal anthropogenic pol lution. These hydrothermal systems are very often associated with potentially toxic elem ents such as As, but can also often be enriched in other biologically toxic elements such as Sb, Se, Cr, Co, Pb, Cd, Ag, Cu, Tl, Zn, Hg, and S, as well as possible limiting nu trients such as Si and Fe (Dando et al.,
91 1999; Varnavas and Cronan, 1988; Vidal et al., 1978). The di scharge of fluids from marine shallow-water hydrothermal system s can have a considerable impact on the chemical composition of the often biologically important coastal surf ace waters (Pichler et al., 1999a; Price et al., 2007). Diffuse ve nting may also influence benthic species diversity and abundance at very large distances away from fo cused venting (Karlen et al., in review). Coastal hydrothermal system s were described in many areas around the world, where they are primarily associated with volcanic (Dando et al., 2000; Johnson and Cronan, 2001; Tarasov et al., 1986) or te ctonic activity (Vidal et al., 1978), which provide the necessary heat sources. In an effort to better understand the po tential toxicity, biogeochemical cycle, bioaccumulation and biotransformation pattern s in a coastal area affected by arsenic pollution, this paper focuses on the marine shallow-water hydrothermal system off Ambitle Island, Papua New Guinea. As previous research has shown, as much as 1.5 kg of arsenic was estimated to be discharged in to Tutum Bay on a daily basis (Pichler et al., 1999a). In addition, arsenic is the only potentia lly toxic element which is highly enriched in this system, allowing for the first time the investigation of the effect of a single toxin on a coastal coral reef ecosystem. Thus, hydrothermal systems provide an excellent opportunity to investigate a natural system impacted by high arsenic concentrations. Here, we report our investigation of the uptake, bioaccumulation, and biotransformation of hydrothermally derived arsenic in the Â“biologi calÂ” compartment of the coastal environment. This paper is a follow-up and is complementary to Â“ Distribution, speciation and bioav ailability of arsenic in a shallow-water submarine
92 hydrothermal system, Tutum Bay, Ambitle Island, PNG Â” by Price and Pichler (2005), which investigated the abiotic (inorganic) compartmental fate of the hydrothermal arsenic and its bioavailability (Chapter Three of th is dissertation). Due to the very elevated bioavailable concentrations of arseni c throughout the hydrothermal environment, enhanced bioaccumulation and biotransform ation were suspected in surrounding reef organisms. 4.2 Biota 4.2.1 Clavularia Clavularia is a genus of soft coral, co mmonly called star polyp, which is widespread in the Indo-Pacific and Atlantic (Figure 4.1 A; Fabric iuis and Alderslade, 2001). This organism is found throughout Tu tum Bay in abundance, not only at the control site (CS1), but also very near fo cused hydrothermal venting, particularly where focused hydrothermal venting and abundant ga s bubbles are being discharged through the rocks and sediments. Clavularia are zooxanthellate, and thus obtain most of their fixed carbon through photosynthes is byproducts of symbiotic alga e and direct uptake from the water column, although filter feeding is possible (Borneman, 2001). For example, these soft corals are common in salt-water aquaria, where feeding is not required, and calcium, strontium, and iron, along with adequate li ght, are the only key elements required for Clavularia to grow.
93 Figure 4.1.Underwater photographs of coral-r eef organisms collected from Tutum Bay. A) Clavularia sp B) Halimeda sp C) Polycarpa sp surrounded by Clavluaria sp A) B) C) ~5 cm ~ 2.5 cm ~5 cm
94 4.2.2 Halimeda Halimeda is a green calcareous macroalg ae very common in tropical marine environments, particularly in lagoonal areas between coral reefs and the shore, and the remains of Halimeda and other calcareous algae ofte n make up the primary sediment component in these areas (Figure 4.1 B). Halimeda are found throughout the coastal environment of Ambitle Island, particularly wh ere there is abundant coral growth. Due to low pH conditions and lack of a suitable hard substratum, Halimeda were not very common surrounding focused hydrothermal venti ng. Samples were collected from rubble and reefs near diffuse and/or fo cused venting when possible. 4.2.3 Polycarpa Polycarpa (a.k.a. tunicate or sea squirt) is a filter feeder with incurrent and excurrent siphons, whose primary food sour ce is plankton (Figure 4.1 C; Borneman, 2001). The incurrent siphon is used to inta ke food and water and the excurrent siphon expels waste and water. The Polycarpa in Tutum Bay were primarily located on elevated reefs, above the influence of diffuse ven ting and hydrothermal sediments. They are members of the phylum Chordata. 4.3 Methods 4.3.1 Field Tissue samples were collected by SCUBA al ong a transect beginning at Vent 4 of Pichler et al. (1999a) and extending out to 300 m, well beyond the influence of hydrothermal venting (Figures 1.2 and 1.8). A control site located approximately 1.6 km
95 north of the main vent area (Figure 1.1), unaffected by hydrothermal activity, was also sampled (Picnic Island control site (CS1)). Once samples were on board, the organisms were dissected and washed thoroughly with DI water, placed in 1.5 mL centrifuge tubes and frozen. Samples were kept frozen during transport to th e University of South Florida. 4.3.2 Laboratory Once at the University of South Florida, each sample was thawed, and then placed in 1.5 mL centrifuge tubes, freeze-dried, and powdered using an agate mortar and pestle. Samples were then taken to the Environm ental & Resource Studies Center at Trent University in Canada for arsenic abunda nce and arsenic speciation measurements. 184.108.40.206 Total Arsenic Concentration (TAs) Complete digestion of tissues was carried out by weighing fre eze-dried tissue (50 Â– 200 mg) into 50 mL digestion vials from Environmental Express, adding 1 mL HNO3 (16 mol/L) and 0.5 mL H2O2 (10 mol/L), and letting the reaction proceed for 1 h at room temperature, followed by 1 h at 80 C in a water bath. This two-step procedure was employed to reduce sample foaming during digestion. After cooling, the digest was diluted to 50 mL with Milli -Q water, filtered through 0.2 m filters. A second dilution was needed to normalize carbon content (see below), and consisted of diluting with HNO3 (0.3 mol/L) to the degree th at corresponded to 25 mg solid tissue sample (e.g. 1+1 for 50 mg sample weight before digestion) All reagents used for this study (e.g., HNO3, H2O2) were ultrapure grade and obtained from Fisher Scientific and High Purity Standards.
96 Total As (TAs) in the tissue digests was determined by inductively-coupled plasma-dynamic reaction cell-mass spectrometry (ICP-DRC-MS) using O2 to eliminate the 40Ar35Cl+ interference on 75As+ (Bandura et al., 2001). It was important to normalize the amount of tissue sample represented by th e final diluted digest aliquot because the measured As concentration (both for sample s and analytical spikes) was proportional to the digested sample weight. This suggests that while the dige stion procedure was effective at dissolving the ti ssue material, it did not break down all organic carbon into solution. Since it is known that organic carbon enhances the signal for As in ICP-MS (Larsen and Sturup, 1994), the measured concentration depende d on the amount of Â“digestedÂ” tissue in the final solution analyzed. 193Ir was used as an internal standard to compensate for other matrix interferences. 220.127.116.11 Extraction and Arsenic Speciation For our study, an 4:1 methanol/water ex traction was carried out on the DI-rinsed, homogenized, freeze-dried sample splits (Ackle y et al., 1999; Cullen and Reimer, 1989; Yeh and Jiang, 2005). Twenty to 200 mg of sa mple was weighed into 50 mL centrifuge tubes, and 10 mL of the 80 % methanol so lution was added. The samples were placed on a spinning shaker for 14 to 16 hours (Goe ssler et al., 1997; Ku ehnelt et al., 2001), removed from the shaker, diluted to 50 mL, and passed through 0.45 m filters. After the extraction procedure was compet ed, total leachable arsenic (TLAs) for each extract solution was measured by ICPMS following the procedure for TAs outlined above.
97 Arsenic speciation analysis was ca rried out by high-pressure liquid chromatography (HPLC) separation of anioni c species prior to de tection by ICP-MS (Kirby et al., 2004). A sample volume of 200 L was injected without pretreatment and separated in a Hamilton PRP-X100 anion-ex change column. Asse ssment of excluded species, which are either neutrally or positivel y charged and therefore not retarded by the anion exchange column, was assessed on sample splits for Clavularia and Halimeda to determine the majority of excluded species This was conducted us ing an ion-paired column and measurement by ICP-MS. An arsenoribose extract was provided by Dr Kevin Francesconi of Karl-Franzens University, Graz, Austria. Arsenoriboses were evaluated during each analysis by simultaneous analysis of this al gal extract from the brown alga Fucus serratus known to contain glycerol, phosphate, sulfonate, and sulfate arsenosugars (Madsen et al., 2000). Time of elution for each AR peak was matche d to peaks in the chromatograms for each sample, and quantified using As(III) as a calibration reference, assuming a relatively similar signal for As(III) and each AR species. Our chromatography conditions were similar to Madsen et al. (2000), and the orde r of elution for their study (AR 1 = glycerol, AR 2 = phosphate, AR 3 = sulfonate, and AR 4 = sulfate) were appl ied. This is logical given that the glycerol arsenosugar behaves as a cation with mobile pha ses at pH < 3, and that the other three arsenosugars show in creasing acid strength from the phosphoric acid ester 2 through the su lfuric acid ester 4 (Madsen et al., 2000). 4.3.3 Quality Assurance/Quality Control
98 Procedural, analytical and instrumental quality assurance and quality control (QA/QC) was evaluated for all lab analyses by including sample duplic ates and certified reference standards. Arsenic abundance analys is was assessed by using certified external standards NIST 1566b, NRCC DORM-2, NRCC TORT-1, and IAEA 392, a brown fucus algae, Scenedesmus obliquus Spiking of samples and certif ied reference materials was carried out using As(III), As(V), DMA, MMA, AC, and AB purchased from High Purity Standards and Fisher Scientific. Spikes were also performed with the AR extract to assess/discount co-elution. Samples were prep ared in duplicate to assess repeatability throughout each analysis. These duplicates in cluded two samples from Ambitle Island, and the standard DORM-2. Method (preparation) and instrument blanks were prepared and analyzed for arsenic abundance and arse nic speciation analyses, and all sample results were then blank corrected. An extern al standard (CRM-TMDW) from High-Purity Standards was used to evaluate instrument accuracy. Our QA/QC procedures for TAs analysis show analytical (i nstrument) percent relative standard devi ation (RSD) averaged 2.0 with a range of 1.0 to 3.8. The complete digestion method and analysis percent RSD for 10 duplicate samples, split in the field, averaged 8.4, and ranged from 0.0 to 26.8. Percen t recovery for blank and matrix spikes ranged from 102.9 to 104.2, with a percent RS D of 1.4. TAs in certified reference materials show recoveries of 95.2 % for DORM-2, 91.6 % for TORT-2, 87.1 % for NIST 1566b, and 107.4 % for IAEA 392. Thus, for As abundance, the uncertainty intervals of the measured and certified concentrations overlapped for three out of four certified reference materials; only for NIST 1566b (oyste r tissue), the measured concentration was
99 slightly below the certified range (87 % recove ry). This suggests that accuracy of the measured As concentrations in tissues was ge nerally acceptable. The instrument detection limit for As was estimated to be approximately 0.002 g/L. QA/QC for anion speciation s how instrument RSD was consistently better than 0.1, with an estimated method detection limit of 0.006 g/kg. Method RSD for duplicate samples of Clavularia for the most abundant As species averaged 1) excluded (i.e., species not retained on the column): 15.4 with a range of 4.4 to 33.3. 2) As(V): 53.1 with a range of 31.2 to 79.0. 3) AR4: 16.8 with a range of 8.7 to 30.1. 4) DMA: 16.9 with a range of 2.8 to 28.2. Analytical percent RSD for Halimeda was 1) As(III): 19.6 with a range of 6.5 to 44.7. 2) excluded: 67.6 with a range of 10.9 to 117.2. 3) DMA: 22.9 with a range of 15.7 to 28.5. Percent RSD for Polycarpa was 1) excluded: 5.6 with a range of 3.4 to 7.9. 2) DMA: 1.0 to 4.4. Percent recovery for blank and matrix spikes ranged from 81.4 to 114.7. 4.4 Results and Discussion 4.4.1 General Points Arsenic is toxic at elevated concentr ations and almost universally marine organisms have evolved mechanisms for det oxification. Marine organisms can take in arsenic by two major pathways: 1) cell diffusi on from the water column and 2) trophic transfer. In the case of cell diffusion, the organi sm can either take in As(V), substituting it for phosphate using specific transporters f ound in the cell wall, or as As(III), via aquaglyceroporins, pore proteins that facilitate the efficient and selective flux of small
100 solutes across biological membranes (Hub and de Groot, 2008; Oremland and Stolz, 2003). While organisms take in arsenic by these pathways, in normal coastal environments their detoxification (methylation) and excretion of the arsenic is more rapid than uptake, allowing theoretically for cons tant tissue concentrations. However, when environmental concentrations are elevated, the chance for bioaccumulation is increased because the organism cannot methylate and excrete the incoming arsenic quickly enough, and there is a net increase in total arsenic concentration (TAs). Given the very elevated, bioavailable concentrations of arsenic thr oughout Tutum Bay, we pred icted the potential for increased bioaccumulation and enhanced bi otransformation (Price and Pichler, 2005). 4.4.2 Total Arsenic Concentration TAs 18.104.22.168 Clavularia Total arsenic concentration in Clavularia tissue samples ranged from 2.9 to 20.9 mg/kg dry weight. Values are generally higher in samples co llected closer to focused hydrothermal venting (Figure 4.2 A), although there is significant scatter. This scatter is likely a combination of specimen age (incr eased time to accumulate As), and the presence of microenvironments where diffuse venting occurs. Clavularia grow preferentially on rubble and rocks very near to the sediment/water interface. However, in some cases it was necessary to sample from slightly above the sedi ment/water interface when rubble was not present (i.e., from a nearby reef). Due to the very steep physicochemical gradients which develop wher e hydrothermal fluids mix with overlying seawater, uptake of arsenic will primarily occur in organisms in direct contact with hydrothermal fluids and arsenicrich sediments. Therefore, th e highest concentrations are
101 Figure 4.2. Total arsenic concentration for A) Clavularia, B) Halimeda, and C) Polycarpa A) B) C) 0 1 2 3 4 5 6 7 8 050100150200250300Distance (m)As (mg/kg) Control 0 5 10 15 20 25 050100150200250300Distance (m)As (mg/kg) Control 0 5 10 15 20 25 050100150200250300Distance (m)As (mg/kg) Control
102 considered the most important when assessi ng the extent of bioaccumulation of arsenic. Even though some samples collected from the hydrothermal area contained arsenic concentrations similar to c ontrol site concentrations, sa mple collection can account for this discrepancy. Specimen age may also play a role. It is important to note that our control site sample datum for Figure 4.2 A is an average value where n = 2, and the standard deviation is 0.1 mg/ kg (i.e., 2.1 0.1 mg/kg). Given th e fact that these values are well above analytical uncertainty, it is probable that bioaccumulation to up to 10 times above background concentrations can occur in Clavularia from Tutum Bay as a result of hydrothermal venti ng of arsenic-rich fluids. Unfortunately, Clavularia has not been analyzed for TAs or speciation by other researchers, making the comparison of our data with the literature difficult. In fact, TAs in the soft tissue of either hard corals (with a skeleton) or soft cora ls (without a skeleton) has, to our knowledge, only rarely been investigated. This is because most research of As in marine biota was conducted almost exclus ively for edible species. Reichelt-Brushett and McOrist (2003) determined the distribution of trace metals between tissue, zooxanthellae, gametes, and coral skeleton s, finding that tissue of the hard coral Acropora tenuis contained between 1.12 and 2.69 mg/kg As. These values are similar to the concentration of arsenic in Clavularia from the control site of this study. Miao et al. (2001) reported an arsenic concentration of 17 mg/kg (12-19, n = 4) in homogenized skeleton/tissue samples for the coral Porites evermanni collected from French Frigate Shoals, Hawaii.
103 Unfortunately, comparison of arsenic c oncentrations between the tissues of different coral species is ques tionable given that the differe nce in bioaccumulation varies greatly between organisms. Wide variation in TAs is found even for the same species (Cullen and Reimer, 1989). Higher As concen trations in marine organisms can be correlated to elevated As concentrations in the environment (Klumpp and Peterson, 1979; Langston, 1984; Maher and Butler, 1988; Pe nrose et al., 1975). Zooxanthellae can enhance the accumulation of As in mari ne organisms (Benson and Summons, 1981). Arsenate uptake can be either proportional to the arsenate concen tration in surrounding waters until a threshold value is reached, at which time arsenate uptake is inhibited, or uptake is independent of arsenate concentra tion (Maher and Butler, 1988 and references therein). Arsenic uptake can also be a function of salinity, te mperature, light availability, and/or exposure (Maher and Butler, 1988). Th is obviously has important implications for our research; predicting tissue concentrations of arsenic based on organisms collected from different environments is not good practice. Ther efore, comparing the same organism from the As-rich hydrothermal site to a control site should be the appropriate way to demonstrate bioaccumulation. 22.214.171.124 Halimeda Total arsenic concentrations in the Halimeda collected along the roped transect ranged from 8.5 to 20.2 mg/kg dry weight. These concentrations are graphically displayed in Figure 4.2 B. Halimeda were collected from sediments along the transect, but approaching hydrothermal venting the sample s were collected from slightly above the sediment/water interface because the Halimeda were not growing in direct contact with
104 hot, acidic hydrothermal fluids. This could explain data scatte r, and account for the lower TAs concentration for the 0 m sample. Regard less, the maximum concentration is most important, and the Halimeda collected from the hydrothermal area contained a maximum arsenic concentration of 20.2 mg/kg, while c ontrol site samples contained an average arsenic abundance of 0.8 mg/kg. Thus, bioaccumu lation of arsenic into calcareous algae seems to be occurring. There is also an increas e in arsenic concentra tion closer to focused hydrothermal venting. Concentrations in macroalgae as measured by other researchers have a very large range. For example, Maher (1983) reported tota l arsenic abundances for three macroalga species with a mean range of 50 to 145 mg /kg. The review by Maher and Butler (1988) reported arsenic concentrations ranging from 6.3-179 mg/kg with a means.d. of 3737 for macroalgae. Likewise, the review by Ne ff (1997) reported 0.1 Â– 382 mg/kg for algae (mean = 43.70). Lunde (1973) reported a total arsenic concentration of 14.2 mg/kg for a single brown alga Laminaria hyperborean Thus, higher concentra tions of arsenic in algae are quite common. However, these concen trations are almost always reported for edible types of brown or green Fucas (seaweed) species and not calcareous algae. Extensive literature searches revealed no st udies of arsenic total concentrations in calcareous algae. 126.96.36.199 Polycarpa Arsenic abundance in the tunicate Polycarpa collected along the roped transect ranged from 4.2 to 7.0 mg/kg dry weight. These concentrations are graphically displayed in Figure 4.2 C, which shows no clear increas e in arsenic abundance in samples collected
105 closer to focused hydrothermal venting. Howe ver, the control site samples contain an average arsenic abundance of 2.2 mg/kg. Thus arsenic may be bioaccumulated into tunicates surrounding hy drothermal venting. Polycarpa were only growing on the reef and on coral mounds above the sediment/wat er interface; thus the opportunity for enhanced bioaccumulation as a result of dire ct contact with diffuse hydrothermal fluids was lower. Sea squirts are the only organisms in th is study for which published A abundance data actually exists. For example, Shiomi et al. (1983) published an average arsenic concentration of 25 mg/kg for Halocynthia roretzi Comparing our values with those published concentrations menti oned above, it seems that the Polycarpa within the hydrothermal system at Tutum Bay are not much different from other tunicates. Concentrations of TAs in seawater 1 m a bove the sediment contains concentrations similar to normal seawater, although higher As(III) (Price and Pich ler, 2005). However, comparison of TAs concentrations to our cont rol site suggests that some bioaccumulation may be occurring in Polycarpa 4.4.3 Organoarsenic Speciation Clavularia Halimeda and Polycarpa contained elevated concentrations of nearly all organoarsenic species present, as well as several unknown peaks. To illustrate the elevated concentrations graphically, I presen t two chromatograms with the same scale for comparison; one chromatogram represen ts the organoarsenic speciation for Clavularia tissue sample collected from the hydrothermal area, and the other chromatogram shows
106 the organoarsenic spec iation pattern for a Clavularia sample collected from the Picnic Island control site (CS1; Figure 4.3). 188.8.131.52 Clavularia Evaluating the mass balance between TAs, TLAs, and the sum ( ) of all arsenic species separated by liquid chromatography is im portant to assure data integrity (Table 4.1). For Clavularia the mass balance between the sum of species and TLAs ranged from 70.0 to 90.8 %, suggesting a fairly good oncolumn recovery of arsenic compounds following separation by liquid chromatogra phy. This conclusion is supported by very good spike recoveries (102.9 to 104.2 %, with a RSD of 1.4 %). While the objective would be total extraction of all species, this is not realistic (Francesconi, 2003; Francesconi and Kuehnelt, 2004). The Clavularia extraction efficien cies were only 12.4 % for the control site, and ranged from 19.9 to 82.4 % for samples from the hydrothermal vent site (Table 4.1). Our da ta suggest, therefore, that Clavularia converted much bioaccumulated As into a non-extractable fo rm. Extraction of arse nic species varies depending on the nature of the compound of interest (i.e., wh ether the compound is water-, methanolor lipid-soluble). Very polar arsenicals ar e poorly extracted by methanol (Francesconi, 2003), and these, along with any lipid-bound As, may represent the missing As. However, higher extracti on efficiencies for samples from the hydrothermal transect suggests that some As species bioaccumulated as a result of hydrothermal activity are in an extracta ble, and perhaps bioavailable, form.
107 Figure 4.3. Chromatograms showin g the relative difference in arsenic speciation between Clavularia collected from the Picnic Island control site (CS1; top), and the Clavularia collected from the vent site (bottom). Ch romatograms are the same scale for direct comparison. 0 1000 2000 3000 4000 03006009001200time (sec)IntensityClavularia sp. collected from the Picnic Island Control Site ( CS1 ) 0 1000 2000 3000 4000 03006009001200time (sec)intensityClavularia sp. collected from area of focused hydrothermal venting
108 An interesting observation to note for Clavularia is that TLAs for hydrothermal samples are all approximately the same con centration, whether the TAs for the sample was 2 or 20 mg/kg (e.g., for n = 5 hydrothermal site samples, the mean is 2.6 with a standard deviation of 0.2; Table 4.1). Thus, it seems the organism may be bioaccumulating excess arsenic, but has only a specific concentration of that arsenic which is extractable. This also suggests that only a fraction of the organismÂ’s As would be bioavailable to other organisms feeding upon it (e.g., gastropods), regardless of the TAs concentration. Finally, there is an inve rse correlation between lower concentrations overall (TAs) and improved extrac tion efficiencies (TLAs/TAs; r2 = 0.82; Figure 4.4 A). Therefore, this suggests that the organism co llected from the hydrothermal site with the most extractable arsenic (e.g., 82.4 % for the 30 m sample), can approximately represent the complete suite of species prior to excess bioaccumulation (i.e., this organism has efficiently been able cope with elevated arsenic concentrations quickly enough without bioaccumulation occurring up to that point). Organoarsenic species measured above this concentration threshold would then re present the species which are being bioaccumulated. The As species in extracts of Clavularia tissue were, in order of elution: excluded, DMA, AR 2, AR 3, As(III) (for the control-si te only), AR 4, an unknown peak at 7.7 min, an unknown peak at 8.5 minutes, MMA, and As(V) (Table 4.2). Arsenic species extracted from Clavularia tissue had consistently much highe r concentrations overall in the transect samples compared to the control site, with the exception of As(III), which is only
109 Figure 4.4. Data evaluation for Clavularia sp A) X-Y coordinate plot showing correlation between TAs and extraction efficien cy (TLAs/TAs). B) XY coordinate plot showing decrease in AR 3 with distance away from focused hydrothermal venting. C) XY coordinate plot showing correlation be tween TAs and extracted As(V). D) X-Y coordinate plot showing correlation between TAs and extracted excluded (arsenobetaine). A) B) C) D) R2 = 0.76 0 5 10 15 20 25 020406080Excluded (%)TAs (mg/kg) R2 = 0.84 0 5 10 15 20 25 0.00.20.40.60.81.01.2 As(V) (mg/kg)TAs (mg/kg) R2 = 0.900.006 0.009 0.012 0.015 050100150Distance (m)AR 3 (mg/kg) R2 = 0.82 0 20 40 60 80 100 051015TAs (mg/kg)TLAs/TAs (%)
110 present in the control-site sample, alt hough near the detection limit (Table 4.2). MMA was only slightly more elevated. Excluded (i.e., species not retained on the column), AR 4, As(V), and DMA are the four most abundant species extracted from Clavularia tissue. Excluded arsenic concentrations in hydrothermal samples are approximately 1 mg/kg (0.753 to 1.642) compared to 0.101 mg/kg for the control site sample. DMA concentrations are ~ 0.2 (0.165 to 0.337) vs. 0.016. AR 4 concentrations ar e very elevated comp ared to the control site, being up to two orders of magnit ude higher, ranging from 0.168 to 0.645 (at 0 m), compared to 0.006 mg/kg in the control site sample. As(V) was more similar to the control site sample, with 0.071 mg/kg in the control site sample vs. a range of 0.094 to 1.133 mg/kg for the transect samples. These f our species comprise 92.8 % of the sum of species. AR 3 was only present in samples n earer to focused hydrot hermal venting (0, 30, 60, and 120). This species did not exist in samples collected beyond 120 m from focused venting, nor at the control site, suggesting its presence is a direct result of microenvironments where elevated As concen trations exist due to hydrothermal venting. The concentration of AR 3 is highest in th e 0 m sample (0.014), decreasing to 0.007 at 120 m (r2 = 0.91 vs distance; Figure 4.4 B). Also, two unknown peaks, eluting at 7.7 and 8.5 minutes, which did not coincide with th e elution times for any of the standards analyzed, only existed in the samples from the hydrothermal transect, and were not present in the control site sample. It can be very useful to assess each sp ecies data in terms of percent sum of species. For Clavularia the percent for excluded arse nic in the hydrothermal samples
111 (taking the average of 0-300 m percent values) is approximately the same for the controlsite samples (46.2 % for the control site sa mples vs. a mean of 48.4 % with a range of 23.7 to 71.3 % for the hydrothermal site sample s). Most species follow a similar pattern, although the fraction of DMA a nd AR 4 species in tissue sa mples from the hydrothermal site is higher than the control si te. Because DMA and AR 4 species in Clavularia are substantially elevated (as percent) compared to the control site, it is likely these species may be bioaccumulated. Comparing TAs vs th e concentration for each As species can help us determine if one part icular species is responsible for overall higher abundances. Of all species detected in Clavularia As(V) was the only species which is relatively well correlated with TAs (r2 = 0.85; Figure 4.4 C), suggesti ng that As(V) comprises an important part of the TAs. AR 3 in the 0 Â– 120 m samples were 0.3 to 0.6 % of the sum of species. Although As(III) was not detected in hydrothermal samples, it makes up 3.4 % for the control site. The percent MMA for th e control site sample was 5.3 % vs. 0.6 to 1.1 % for the hydrothermal transect samples. It is interesting to note th at percent excluded arsenic species vs. TAs is in fact inversely correlated (i.e., neg slope; r2 = 0.77; Figure 4.4 D), while As(V) as percent vs. TAs is correlated (r2 = 0.87). 184.108.40.206 Halimeda The mass balance between the sum of species and TLAs for Halimeda is similar to Clavularia ranging from 76.2 to 99.3 % for hydrothermal samples, and 58.6 % for the control site sample, suggesting good on-colu mn recoveries of each arsenic species extracted from this organism (Table 4.1). Lowe r recovery in samples from the control site
112 suggested there is an additiona l non-extractable arsenic species, or alternatively that the arsenic in hydrothermal samples is more extractable. The ra tio of TLAs/TAs was generally higher for Halimeda around 70 to 80 %, although th e control site had much less extractable species ( 21.1 %). For comparison, it would be ideal to have 100 % extraction for all samples, but the fact that th e arsenic species in the control site sample are not extractable, while most of the arsenic is extracted from hydrothermal samples, is important. This suggests that the organisms from the hydrothermal site contain arsenic which is in a different form, is overall more extractable, and theref ore more bioavailable. In addition, although the percent mass balance is similar to Clavularia directly comparing TLAs with TAs as mg/kg shows clea rly that much more of the arsenic was extracted for samples collected from the hydrot hermal area. Concentr ations exceeded the 2.5 mg/kg range found for Clavularia and were highly variable for Halimeda ranging from 1.93 to 15.72 mg/kg (Table 4.1). The arsenic species in Halimeda tissues were, in orde r of elution: excluded, DMA, AR 2, AR 3 (only in the 0 m sample), As(III), an unknown peak eluting at 7.7 minutes, MMA, an unknown peak eluting at 14.5 minutes (only in the 0 m and 300 m samples), and finally As(V). The most abundant species in Halimeda collected from the hydrothermal vent transect was As(III) by far, ranging as high as 11.7 mg/kg and as much as 82.1 % of the sum of species (Table 4.2). In contrast, no As(III) was detected for the control site sample. The other tw o most abundant As species in Halimeda were excluded (ranging from 6.6 to 57 %) and DMA (ranging from ~1 to 4 % in transect samples). These three species comprise up to ~90 % of the sum of species. All arsenic species were
113 elevated in hydrothermal transect samples co mpared to the control site sample; up to two orders of magnitude for excluded, DMA, AR 2, As(III), one order of magnitude for UNK 1 and MMA, and slightly elevated for As(V). The Halimeda sample from 0 m, collected very near focused hydrothermal venting and living on an elevated reef, seems to be unique in its arsenic speciation pattern. This sample contains an AR 3 peak, although near the detection limit, and a la rge concentration (6 8.3 % of the sum of species) of an unknown peak eluting at 14.5 minutes. If we excl ude this species from our calculations, As(III), excluded, and DMA are the majority sp ecies for this sample, and there is much more similarity with the other hydrothermal samples. A small concentration of this unknown peak was also detected in the 300 m sample. When assessing the Halimeda data as percent of the sum of species, the excluded peak for the control sample is similar to th e average of hydrothermal samples. However, DMA, AR 2, UNK 1, MMA, and As(V) are all mu ch more elevated in the control site sample compared to the hydrothermal samp le, while no As(III) was detected in the control site sample. In addition, the percent TLAs for excluded arsenic seems to increase along the transect while moving to greater di stances from focused hydrothermal venting. This is actually caused by two distinct groupings which show increasing trends: 1) samples collected from 30, 60, and 120 comp rise one group, and the excluded arsenic was ~ 16 % of the TLAs, and 2) samples from 140, 180, and 300 are a second group, with excluded arsenic ~50 % of the TLAs. The second most abundant species, DMA, follows an identical trend as the exclude d species, while the mo st abundant species present, As(III) follows an inverse pattern; the 30, 60, and 120 m samples of Halimeda is
114 comprised of ~80 % As(III), while the 140, 180, and 300 m sample contains ~40 % of the TLAs. Therefore, it is clear that for Halimeda samples collected nearer to focused hydrothermal venting (0 excluding the peak at 14.5 min, plus 30, 60, and 120 m samples), there is a very high relative percent As(III), while there are very low relative percent of organoarsenicals excluded, DMA, AR 2, a nd UNK 1. Then, this relative abundance switches for the samples collected from 140, 180, and 300 m, which show lower relative abundances of As(III) and higher relative abund ances of organoarsenicals compared to samples from the beginning of the transect. These percent trends, however, are controlled by the concentration of As(III), which is very elevated in the 30 to 120 m samples. The concentrations of the organoarsenic species mentioned are all approximately the same along the transect. The elevated concentrations of As(III) can be explained by the spatial distribution and extent of diffuse venti ng, which was observed to end at approximately 140 m (Price and Pichler, 2005). The significance of this result is that the Halimeda nearer to focused hydrothermal venting is taking in more arsenic than it can methylate, and thus has higher concentrations of As(III) relative to organoarsenicals, while the Halimeda outside of hydrothermal influence does not. Halimeda is also a calcareous alga, and the calcification process may also have an e ffect on arsenic speciation. When we exclude the As(III) from the per cent calculation, the percent values for organoarsenic are much more similar (Table 4.3 ). After removal of As(III), there is much more excluded arsenic for hydrothermal samp les relative to the c ontrol site samples,
115 indicating that although As(III) is the dominant species, other organoarsenicals are also being bioaccumulated. As(III) concentrations correlate with distance away from focused venting (r2 = 0.72; Figure 4.5 A). When we assess TAs vs. As(III) in percent, there is a correlation coefficient of 0.67, supporting the concept that this was the dominant bioaccumulated species. TAs versus DMA and As(V) extracted from Halimeda have correlation coefficients of 0.67 and 0.68, respec tively (Figures 4.5 B and C). Assessing increasing/decreasi ng trends vs. distance al ong our transect from a mg/kg perspective, DMA, As(III), and As(V ) possibly decrease along the transect, though not distinctly. This sugge sts that these species are th e most abundant extractable arsenic for the organism, and thus may be bioaccumulated as a result of hydrothermal venting. As(V) and As(III) are readily (bio)available in seawater for uptake by algae. Previous researchers have shown that As(V) did not accumulate, but was rapidly detoxified by methylation and alkylation, a nd organisms would then accumulate the end products of this detoxification, namely ar senoriboses (Francesc oni and Edmonds, 1998). Most edible macroalgae (seaweeds) investigated to date contain predominantly the four major arsenosugars discussed in the introduction, although up to 15 were detected, with minor amounts of AB and DMA (Francesconi and Edmonds, 1998; Mads en et al., 2000). Several publications reported relative abunda nces of inorganic vs. organic arsenic in extracts, and according to Maher (1983), ed ible macroalgae can contain a mean arsenic
116 Figure 4.5. Data evaluation for Halimeda sp. A) X-Y coordinate pl ot showing correlation between As(III) and TAs. B) X-Y coordinate plot showing correlation between TAs and DMA. C) X-Y coordinate plot showi ng correlation between TAs and As(V). A) B) C) R2 = 0.72 0 5 10 15 20 25 051015As(III) (mg/kg)TAs (mg/kg) R2 = 0.67 0 5 10 15 20 25 000000DMA (mg/kg)TAs (mg/kg) R2 = 0.66 0 5 10 15 20 25 0000AsV (mg/kg)TAs (mg/kg)
117 abundance of 79-114 mg/kg total arsenic compar ed to 1.7-3.6 mg/kg inorganic arsenic for the three brown macroalgae mentioned above. Shinagawa et al. (1983) report two brown alga with 3.2-7.5 % inorganic arsenic, but one alga, Hizikia fusiforme with 61 % inorganic arsenic. However, Yasui et al. ( 1978) reported mean per cent inorganic arsenic for brown alga between 1.6 and 9.8, sugges ting brown alga typically have lower abundances of inorganic arsenic. The very high As(III) values for Halimeda cannot be compared to brown, edible fucus alga. 220.127.116.11 Polycarpa Polycarpa species analysis indicates a pproximately 55 to 65 % on-column recovery, which suggests that some of the species extracted remained on the column during liquid chromatography (Table 4.1) Since this was not a problem for Clavularia and Halimeda there could an arsenic species unique to Polycarpa The mass balance of TLAs/TAs suggests near complete extraction of arsenic from this organism (Table 4.1). The control site sample had a TLAs/TAs ra tio of 73 %, suggesting more relative nonextractable forms in the control site. Thus, th e majority of the ex cess arsenic taken in by Polycarpa from the hydrothermal site is converted into a form that is extractable, and thus more bioavailable. The arsenic species extracted from Polycarpa tissue collected from the hydrothermal transect were, in order of elution: excluded, DMA, AR 2, AR 4, an unknown peak at 7.7 minutes, with several sa mples containing unknown peaks at 8.5 and 14.5 min, as well as MMA. No As(V) was dete cted. The most abundant species detected in Polycarpa extracts were excluded and DMA, which was >90 % of the sum of species.
118 The excluded arsenic is typically 70 to 80 % of the sum of species, with a concentration range for hydrothermal site samples of 0.293 to 3.829 mg/kg. DMA ranged from 8.4 to 14.9 % of the of species, with an average conc entration of 0.607. For both of these species, as well as AR 4, the control site samp le was an order of magnitude lower. Some of the hydrothermal samples contained AR 2, an unknown peak eluting at 7.7 minutes, and the control site sample alone contained As(III). There are no obvious increasing/decreasing tr ends in the data when assessed as mg/kg or percent, although ther e is relatively a larger fr action of excluded and DMA in the hydrothermal samples. There was inversel y higher percent AR 4 in the control site samples, as well as in the 240 and 300 m samp les. So, whereas there are overall higher concentrations, the Polycarpa appears to be efficientl y converting the arsenic to organoarsenic species, which is evidenced in relatively similar percent values. Organoarsenic speciation of sea squirts has only been performed by Shiomi et al., (1983), who suggested the Halocynthia roretzi contained one acidic and two basic arsenic compounds. No suggestions were made as to the identity of the unknown species, other than to suggest they were not AB. 18.104.22.168 Assessment of excluded arsenic Arsenic speciation by cation analysis i ndicates that the excluded peak was composed predominantly of AB. Table 4.3 presents cation data analysis for Clavularia and Halimeda This analysis was done during the prel iminary steps of analysis utilizing a different set of samples. Therefore, these data cannot be compared directly with the anion data (from a mass balance pe rspective). However, the ex traction procedure for cation
119 speciation was identical, time of elution wa s equivalent, and therefore shows which excluded arsenic species is likely represente d by the excluded void peak during the anion speciation analysis. For both Clavularia and Halimeda arsenobetaine is the dominate cationic arsenical over a wide range of distances from focused hydrothermal venting, followed by DMA. DMA has been quantified du ring the anion analysis, and therefore can be ignored. Minor amounts of an unknown peak are present, and may represent AR 1. TMAO, AC, and TETRA were not detected for any sample, either from the hydrothermal vent site or control. No cation data exist for Polycarpa. 4.5 Biomethylation Pathways It is likely that the arsenic species presen t from diffuse venting fluids can contain an abundance of not only As(III), but also As(V) (Price et al., 2007). There is also abundant As(V) adsorbed onto iron rich sedi ments which is considered bioavailable (Price and Pichler, 2005). Langston (1980; 1984) suggested enhanced bioaccumulation when arsenic is enriched in sediments. Thus, an organism living in the Tutum Bay hydrothermal environment would be expected to accumulate As(III) or As(V) from the surrounding waters, and As(V) from the sedime nts. Elevated concentrations of both species are present in Tutum Bay pore wate rs, particularly between 0 and 140 m along our transect (Price and Pi chler, 2005). Although several pathways for uptake, detoxification and excretion were proposed for marine organisms (Bentley and Chasteen, 2002; Cullen and Reimer, 1989; Maher and Butler, 1988; Mukhopadhyay et al., 2002), it is commonly suggested that, after taking in As(V ), the first step is reduction of As(V) to
120 As(III), followed by methylati on of As(III) to form less toxic methylated species. The biomethylation pathway would thus generally be As(V) As(III) MMA DMA Arsenosugars and finally AB as an end product, with intermediates (such as MeAs(III)), being produced between DMA, AR, and AB. Each of the methylation products can contain As(V) or As(III) attached to methyl groups. DMA can be converted to trimethylarsine oxide trimethylarsine, which is excret ed rather than being converted to arsenosugars (Francesconi and Edmonds, 1998). In normal coastal marine organisms, the reduction-methylation process would continue, along with excretion of As(III) or methylated arsenical s, until a constant rate of uptake/excretion (equilibrium) is reached with AB (or AR in algae) as an end product. However, if the amount of arsenic entering th e organism exceeds its capacity to detoxify and excrete the toxin, bioaccumulation may exceed normal concentrations. Thus, for organisms from the Tutum Bay hydrothermal tr ansect, hypothetically, there would be an overall increase in each As species normally present as part of each different organisms particular detoxification pathway. 4.5.1 Clavularia Clavularia may be taking in As(V) or As(III) via cell diffusion. The concentration of As(V) was elevated in our hydrothermal tr ansect samples, but it was suprising that As(III) was present only in the co ntrol. This suggests that: 1) Clavularia does not contain aquaglyceroporins, and/or 2) As(V) is met hylated directly without being reduced to As(III), and/or 3) the kinetics of methylation of As(III) is very fast. It is possible that arsenic is being taken in by Clavularia via trophic transfer from plankton (plankton
121 collected from directly over focused hydrothe rmal venting contain as much as 240 mg/kg As; Price et al., unpub. data). It may be that th e very small peak for As(III) in the control site sample is also present in the hydrotherm al samples, but is Â‘coveredÂ’ by the AR 4 peak, which is quite large for hydrothermal samples but small for the control site samples. While hydrothermal samples contain higher concentrations of As(V) compared to the control site, MMA is approximatel y the same concentration, suggesting that elevated As(V) may result due to slow met hylation kinetics as hydrothermal organisms attempt to methylate, and/or rapid reduc tion/methylation from MMA to DMA. DMA is much elevated in hydrothermal samples. The primary arsenoriboses present in the Clavularia samples are AR 2 and AR 4, with some AR 3 present only in hydrothermal samples closer to focused hydrothermal ven ting (0-120 m). Arsenosugars found in marine organisms may be formed as intermediates during the methylation process, possibly as precursors to arsenobetaine (Maher and Bu tler, 1988). The presence of AR 3 in hydrothermal samples suggests that this is an intermediate, and elevated concentrations of TAs nearer to focused hydrothermal venting ma y result in an overall increase of this species. We can only speculate as to the stru cture of the unknown compounds 1 and 3 present in Clavularia which both are only present in hydr othermal samples. They could be intermediates which are bioaccumulate d to above detection limits. Based on our chromatography conditions, these species are likely more polar (charged) than those which came earlier, but less polar compared to MMA and As(V). TMAs(III) is also an intermediate in the formation of other ar senicals, and TMAO can combine with other
122 organic molecules in the cell, and eventually degrade to produce arsenobetaine (Qin et al., 2006). It is possible that our unknown p eaks are some of these intermediates. AB is considered to be the final metabo lic product for most marine organisms, which would suggest much higher concentratio ns of this species in organisms from environments with elevated arsenic con centrations. This seems to be true for Clavularia with AB the majority species in our extracts. In summary, my data suggested that pathway mentioned above can also be applied to Clavularia and the overall concentration of arsenic which has bioaccumulated, taken in either as As(III) or As(V), is converted to methylated species MMA DMA AR and intermediates, to a primary end produc t of AB (Table 4.2). Trends in the data (i.e., increasing/decreasing along the tran sect while moving away/towards focused hydrothermal venting) are difficult to identif y, likely due to poor ex traction efficiencies. However, the presence of arsenic species in tissues from the hydrothermal environment, and their absence which are not present in the control site samples, as well as the presence of arsenic species only in the control site samples, suggests the possi bility of net bioaccumulation of methylated species because the organism is not able to excrete the newly methylated species quickly enough. 4.5.2 Halimeda Although no biomethylation pathway has been suggested for calcareous algae, it seems to be generally accepted that all algae will follow one of two paths. For example, Tukai et al. (2002) suggests that the biotra nsformation pathway for seaweed-type algae (green, red, or brown leafy algae) can eith er: 1) conclude with the formation and
123 bioaccumulation of AR, or 2) the organism can accumulate inorganic and simple methylated compounds. The latter seems to be the case for Halimeda As(V) in Halimeda is slightly elevated compared to the control site, but very elevated concentrations of As(III) are present. Halimeda is a photosynthesizing organism that can bioaccumulate in As(V) and/or As(III) via cell diffusion. Elevated concentrations of As(III) suggests either rapid reduction of As(V) and/or uptake of As(III) directly. MMA is present at approximately the same concentrations for hydrothermal vs. the control site, suggesting rapid conversion from MMA to DMA. DMA is also elevated in the hydrothermal samples compared to the control-site samples. There is no correlation between As(III) and DMA or any other organoarsenicals, suggest ing that As(III) is only slowly converted to MMA. Alternatively, it is possible that th e kinetics of methylati on of As(III) are quite slow, and given the elevated concentrations of As(V) and As(III), there is more uptake than can be methylated by the organism. The only arsenosugar present is AR 2, a phosphate arsenosugar, which suggests a direct stepwise pattern of DMA AR 2 AB. Other than As(III), AB is the most abundant of all species, sugge sting that it is an end product in the metabolic pathway (Table 4.3 ). Other researchers, however, rarely find AB in marine macroalgae, suggesting that Halimeda a calcareous alga has a novel pathway not yet described, which has an end me tabolic product of AB. It is possible that some of the excluded arsenic from our anion analysis is AR 1, but our cation analysis shows clearly that AB is present. UNK 1, wh ich elutes at 7.7 minutes, is present in all hydrothermal samples, and is two orders of magnitude elevated over the control (e.g., 0.004 mg/kg in the control compared to a range of 0.21 to 0.84 mg/kg for hydrothermal
124 samples). Again, it seems likely that this unknown peak may be an intermediate, but we can only speculate as to which organoarsenic species it is. 4.5.3 Polycarpa Bioaccumulation and biotransformation pathways in Polycarpa are less clear. There was no As(V) or As(III) present. Polycarpa however, is the only organism in our study that is distinctly a filt er feeder. Maher and Butler (1988 ) suggested that the primary As species encountered in higher trophic le vel organisms are those that are contained within the organisms being eaten. MMA only occurs in two samples from the hydrothermal site, and not at all in the cont rol site samples. DMA is the second most abundant species in Polycarpa followed by AB, suggesting th at perhaps one or both of these species are present in the plankton upon which the Polycarpa feed. TAs concentrations in plankton collected over the ve nt site are as high as 240 mg/kg (Price et al., unpub. data). Since AB is by far the most a bundant species, this is the most likely end product for Polycarpa AR 2 and UNK 1 are present, lik ely as intermediates. Thus, the metabolic pathway for arsenic biotransformation in Polycarpa proposed here is 1) uptake of DMA via trophic transfer from plankton, AR 2/UNK 1 AB. 4.6 Summary and Conclusions There is evidence of arsenic bioaccumu lation in coral-reef organisms along a gradient of increasing arsenic concentra tions in sediments and waters surrounding a hydrothermal system. For the first time total arsenic concentrations and arsenic species are presented for Clavularia Halimeda and Polycarpa TAs in all three organisms
125 investigated in this study was very elevated for samples from the hydrothermal area compared to control-site samples. Although ex traction efficiencies were not ideal, the concentration of each species present in Clavularia Polycarpa and Halimeda is at least an order of magnitude higher for hydrothermal transect samples compared to control-site samples. Biomethylation pathways for Clavularia are similar to most marine organisms. Halimeda biomethylation pathways are similar to some marine macroalgae, although AB is present as a metabolic product. Finally, Polycarpa seems to incorporate As by trophic transfer, with DMA and AB as the final metabolic products.
126 mass balancemass balance /TLAs (%)TLAs/TAs (%) 020.43.2n.a.a302.92.22.4190.882.4 6014.12.22.8077.519.9 12020.92.6n.a. 1407.02.02.8270.040.0 2404.81.92.3679.648.9 3004.22.32.5989.062.3 Picnic Island22.214.171.12485.712.4 08.54.55.8576.269.3 3019.012.313.6789.971.9 6020.214.915.7295.077.8 12013.310.411.0793.983.2 14014.74.65.7479.439.1 18011.66.78.7277.075.2 300n.a.1.91.9399.3 Picnic Island0.80.10.1758.621.1 05.22.84.561.286.7 306.83.45.858.686.0 605.53.65.664.2102.2 1126.96.36.1993.298.3 1406.93.35.857.684.2 2407.02.74.954.769.3 3005.32.54.358.480.7 Picnic Island188.8.131.523.473.1an.a. = not availableClavularia Halimeda PolycarpaTable 4.1. Comparison of total arsenic abundance (TAs), Sum of leachable arsenic species ( ), total leachable arsenic (TLAs), and mass balance. All concentrations in mg/kg dry weight. IDDistanceTAs TLAs
184.108.40.206.220.127.116.11.18.104.22.1686.1 excludedaDMAAR 2AR 3AsIIIAR 4UNK 1UNK 2UNK 3MMAUNK 4AsV 00.7530.3370.0740.0140.6450.1680.0300.0221.133 301.2920.1650.1000.0130.3000.0900.0100.0150.206 600.8010.2360.1500.0090.5210.0910.0300.0150.315 1201.0330.2150.1050.0070.1680.0630.0200.0201.002 1401.1700.2990.0780.2050.0970.0110.0220.094 2400.9700.2610.0570.4730.0820.0190.018 3001.6420.1860.0650.2180.0570.0130.0130.109 Picnic Island0.1010.0160.0060.0080.0060.0120.071 00.2930.0770.0190.0080.9790.0213.0460.021 301.7320.21310.0850.0740.0600.120 602.7290.1930.03011.7630.0840.0380.092 1201.8890.1610.0178.1220.0810.0340.089 1402.1800.2110.1021.8390.0390.1420.045 1803.8290.1630.1352.4530.0730.061 3000.8280.0820.0170.9040.0230.0130.048 Picnic Island0.0300.0100.0030.0040.0090.024 02.2170.4110.0680.064 302.9830.3070.0740.048 602.7690.5090.1540.0770.065 1202.1590.2690.0950.0730.027 1402.6810.2810.2390.0480.0270.0240.037 2402.2150.2390.0870.0360.0380.038 3001.9410.2410.1630.1100.036 Picnic Island0.6070.0720.0510.0570.0130.017aexcluded = These As species had no retention on the column, and are therefore neutral or cationic species. Our cation analysis suggests this is predominantly arsenobetaine (AB), with minor amounts of AR 1.Halimeda PolycarpaTable 4.2. Arsenic species in methanol/water extracts. Data are arranged vertically by organism (column 1), then by distance aw ay from focused hydrothermal venting (column 2). Elution times are presented above the As species ID. All values are mg/kg solid. DistanceClavularia 127
22.214.171.124.126.96.36.199.188.8.131.526.1 excluded*DMAAR 2AR 3AsIIIAR 4UNK 1UNK 2UNK 3MMAUNK 4AsV 023.710.62.30.420.35.30.90.735.7 3058.97.54.60.6184.108.40.206.79.4 6036.910.96.90.424.04.21.40.714.5 12039.28.24.00.220.127.116.11.838.1 14059.215.13.910.44.90.61.14.8 24051.613.93.025.24.31.01.0 30071.38.12.89.18.104.22.168.7 Picnic Island22.214.171.124.42.85.332.2 06.61.70.40.221.90.00.50.00.00.068.30.5 3014.11.7126.96.36.199.0 6018.31.30.27188.8.131.52.6 12018.21.50.27184.108.40.206.9 14047.84.62.240.220.127.116.11 18057.02.42.036.51.10.9 30043.34.30.9418.104.22.168.5 Picnic Island38.322.214.171.1241.130.0 080.3126.96.36.199 3087.49.02.21.4 6077.5188.8.131.52.8 12082.310.23.62.81.0 14080.38.47.21.184.108.40.206 24083.59.03.31.41.41.4 30077.99.76.54.41.5 Picnic Island220.127.116.11.71.52.0Halimeda Polycarpaaexcluded = These As species had no retention on the column, and are therefore neutral or cationic species. Our cation analysis suggests this is predominantly arsenobetaine (AB), with minor amounts of AR 1. Table 4.3. Arsenic species in methanol/water extracts presented as percent of the of species. Data are arranged vertically by organism/distance away from focused hydrothermal venting. Elution times are presented above the species ID. DistanceClavularia 128
18.104.22.1689.814.215.5 excludedaDMAbUNK1 UNK2AB TriMAs AsCTetraMAs vent2.42.350.230.18 22.214.171.1240.03 0.070.1 601.32.47 15011.40.01 Picnic Island0.30.230.01 0.050.05 vent1.11.090.29 0.13 600.50.350.02 0.030.06 1500.10.060.01 0.01 Picnic Island0.20.07 b DMA can behave as both a cation or anion, depending on eluent conditions. DMA was quantified by anion exchange chrmatography (see Tables 4.2 and 4.3) and therefore can be ignored here. Table 4.4 Arsenic speciation of methanol/water extracts for Clavularia and H alimeda separated by cation exchange. All values are mg/kg solid. TLAsaexcluded = These As species had no retention on the column, and are therefore neutral or anionic species. IDDistanceClavularia Halimeda 129
130 Chapter Five Summary and Conclusions The marine shallow-water hydrothermal sy stem of Tutum Bay, Ambitle Island, in northeastern Papua New Guinea, may be one of the largest and most unusual hydrothermal systems studied to date. This hydrothermal system is unique in that it contains some of the highest concentrations of arsenic ever discovered for a submarine hydrothermal system, including mi d-ocean ridge black smokers. Thus, the marine shallow-water hydroth ermal system of Tutum Bay, Ambitle Island, Papua New Guinea provides an excellent opportunity to study the interactions of fluid chemistry, sediment chemistry, metalloge nesis and biogeochemistry, particularly with respect to arsenic. These vents disc harge hot, slightly acidic, arsenic-rich, chemically reduced fluid into cool, slightly alkaline, oxygenated s eawater. Gradients in temperature, pH, and total arsenic (TAs), among others, are established as the two aqueous phases mix, with important implicati ons not only for sediment and seawater/pore water chemistry, but also for the surrounding reef organisms. Due to abundant Fe(II) in hydrothermal fluids, hydrous ferric oxides (HFO) precipitate around focused venting, and coat the surrounding sediments visibly to 150 m away. HFO coatings, mechanical transport a nd weathering of volcanoc lastic sediments, and dissolution of carbonate sediments nearer to venting, combine to alter sediment chemistry substantially. Of particular interest in this study was As speciation and abundance in pore waters as a function of sediment depth and as a function of distance from the area of
131 focused venting. With increasing distance, TAs concentration in the pore water decreased rapidly, but remained elevated up to 300 m from the area of focused venting when compared to a non-hydrothermal control site. Total arsenic concentrations in the pore water profiles (to ~100 cm) were elevate d, and generally incr eased with depth. Surprisingly, aqueous As(V) far exceeded a queous As(III) at almost all distances and depths investigated, while at the control si te the As(III) concentration exceeded that of As(V). Thus, in the Tutum Bay hydrothermal sy stem, chemical disequilibria among As species provide potential metabolic energy fo r arsenite oxidizing microorganisms where hydrothermal fluid mixes with seawater near the vent orifice, and for arsenate reducing microorganisms with increasing distance and depth from the hydrothermal point source. Marine shallow-water hydrothermal vent systems can introd uce large amounts of potentially toxic elements, such as arsenic (As), into coas tal marine environments. The first step to understanding and describing the potential impact of these elements throughout hydrothermally influenced coastal ec osystems is to determine the distribution, speciation, and most importantl y, the availability of the toxin for biological uptake. Submarine hot springs near Ambitle Island, Papua New Guinea, are discharging as much as 1.5 kg per day of arsenic directly into a coral-reef ecosystem. The hydrothermal fluid contained ~900 g/L TAs, almost exclusively present as the reduced, trivalent arsenite (As(III)), while local seawat er measured between 1.2 and 2.4 g/L As, with approximately equal levels of As(III) and arse nate (As(V)). Thus, we investigated the bioavailability of the As throughout Tutum Bay by studying vent fl uid, seawater, pore water, precipitates, and sediments. In addi tion to measuring As abundance, As speciation (As(III), As(V), and the methylated species DMA and MMA) was determined in various waters. The As concentration for discrete mineral phases in vent precipitates and
132 sediments was determined by sequentially extr acting arsenic from the easily extractable, carbonate, Fe-oxyhydroxide or hydrous ferric oxid e (HFO), and residual fractions, each of which have a diffe rent bioavailability. Diffuse venting has a large influence on the distribution of As in Tutum Bay surface sediments, which have a mean As concentration of 527 mg/kg in surface sediments excluding the vent pr ecipitates. Up to 50 mg/kg As were extracted from the easily extractable fraction of surface se diments (mean = 19.7 mg/kg), using a K2HPO4/KH2PO4 buffer at pH = 7.2. Arsenic from this fraction is considered to be the most available for biological processes a nd therefore the most dangerous for biota. However, sequential extraction shows that 98.6 % of the As in vent precipitates, and a mean of 93.3 % in surface sediments (range = 88.2 to 96.3 %), is coprecipitated with the hydrous ferric oxide (HFO) fraction. Thus, the bulk of the As is scavenged by the HFO, and should remain stable unless the physicochemical conditions surrounding the oxides change. We found as much as four times the average seawater concentration of As (~2 g/L) exists in surface waters of Tutum Ba y. Bottom water As abundance is near normal, although As(III) in both su rface and bottom seawater throughout Tutum Bay is substantially enriched compared to average seawater. Thus, hydrothermal venting provides bioava ilable As by two major pathways: 1) easily-exchangeable As as high as 50 mg /kg and a mean of 19.7 mg/kg throughout the sediments of Tutum Bay, and 2) in surface s eawaters, which may allow for biological uptake and transfer up th e food web through plankton. The hydrothermal fluids discharge directly into a fringing coral reef ecosystem. We have evaluated the influe nce that hydrothermal venting of arsenic-rich fluids and resulting arsenic-enriched sediments may have on surrounding biota. Total arsenic concentration (TAs) and arsenic speciation were measured in the tissue of several coral
133 reef organisms collected along a metered, ro ped transect leading away from focused hydrothermal venting. We compare these samples to a control site, a pristine coral reef unaffected by hydrothermal venting. Reef biota collected for this study included the soft flower coral Clavularia sp. the calcareous alga Halimeda sp. and the tunicate Polycarpa sp. All organisms collected from the hydrotherm al area displayed di stinctly higher (2 to 20 times) TAs compared to the control site, with increasing trends approaching focused hydrothermal venting. This is direct evidence for enhanced bioaccumulation of As in organisms living within the area of hydrothermal influence. Many of the arsenic species extracted for each organism were also typically at least one order of magnitude higher in arsenic concentration for the hydrothermal site versus the control site samples. Distinct arsenic speciation patterns in Clavularia and Polycarpa suggest rapid, adequate methylation/detoxification of hydrotherm ally-derived inorganic arsenic to organoarsenicals, with elevat ed concentrations of DMA and AB as an end product of their metabolic pathway. Very elev ated concentrations of As(III) in Halimeda (max 11.7 mg/kg, ~80 % of TAs) suggest this organism is not as efficient at methylating inorganic arsenic. AB was also detected in substantial amounts for Halimeda which suggests that the biomethylation pathway for calcareous al gae is different compared to commonly studied seaweeds.
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About the Author Roy Price grew up in beautiful northwest Arkansas, and began his geology career at the University of Arkansas in Fayettev ille, completing his B.S. in geology in the summer of 1999. Roy came to Florida in the fall of 2000, a nd began his M.S. in geochemistry at USF in spring 2001 under the tutelage of Dr Thomas Pichler. During his M.S., Roy taught several laboratory cl asses including Essentials of Geology, the Earth's Surface (Geomorphology), and Fluid Earth. Roy ta ught Introduction to Geology lecture during the last semester of his Ph.D. Roy has obtained a postdoctoral fellow ship, beginning May 1, 2008 and lasting from 2-3 years, at the University of Brem en Research Center for Ocean Margins, in Bremen, Germany