ARSENIC TRANSFORMATION IN MARINE MACRO ALGAE SOPHIA CATARINA REINEKE GRANCHINHO. B.Sc., The University of British Columbia, 1998

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1 ARSENIC TRANSFORMATION IN MARINE MACRO ALGAE by SOPHIA CATARINA REINEKE GRANCHINHO B.Sc., The University of British Columbia, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 2000 Sophia Catarina Reineke Granchinho, 2000

2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date al Septemher ^ooo

3 ABSTRACT A high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) system was utilized for the determination of arsenate (As(V)), arsenite (As(III)), monomethylarsonate (MMA), dimethylarsinate (DMA) and arsenosugars X- XIII. In marine algae and in a marine fungus, the system was used to study pathways for the biotransformation of arsenicals. Fucus gardneri, Nereocystis luetkeana and Fusarium oxysporum melonis were grown in media enriched with arsenicals. Arsenosugars in the algae were identified by comparing the retention times with the organoarsenic compounds previously identified in oyster tissue standard reference material (NIST-1566a) and Fucus sample standard (IAEA-140/TM). Two HPLC columns and two mobile phase conditions were used. Arsenate, when added to Fucus gardneri under different environmental conditions, was reduced to As(III) and methylated to DMA and several different arsenosugars. The amount of arsenic species varied depending on the environmental condition. High levels of salinity and high levels of phosphate resulted in lower amounts of the arsenic species compared to low levels of salinity and phosphate. The presence or absence of antibiotics in the medium did not result in any major changes in the amount of arsenic species produced. This may indicate that the presence of more complex arsenicals in environmental algae samples is dependent on symbiotic interactions between the algae and its surroundings, rather than resulting from independent synthesis by the algae. The bull kelp Nereocystis luetkeana reduced and methylated As(V) to DMA. Significant amounts of more complex arsenic species, such as arsenosugars, were not observed in the cells ii

4 or medium. The bioaccumulation and biomethylation of arsenic species by Nereocystis was found to be different than Fucus gardneri. The Fusarium fungus, which grows with Fucus gardneri in a parasitic relationship produces arsenite and DMA when incubated with arsenate. The amounts, however, were about 1000 times lower than produced by Fucus. Water soluble arsenic species were determined in a variety of marine algae collected from two sampling areas in British Columbia. Arsenate, MMA and arsenosugars X, XII and XIII were the predominant species extracted from the samples. The total arsenic content was determined by ICP-MS and the water content was determined by freeze-drying for all the samples collected. It was found that the kelp samples contained the highest amounts of total arsenic as well as the highest water content. The extraction efficiency varied between samples. iii

5 TABLE OF CONTENTS Abstract Table of Contents List of Tables List of Figures List of Abbreviations Acknowledgments ii iv ix xi xiii xiv Chapter 1. GENERAL INTRODUCTION ARSENIC IN THE MARINE ENVIRONMENT TOXICITY OF ARSENIC MARINE ALGAE General Information Commercial Use of Marine Algae Arsenosugars in Marine Algae Previous Studies of Arsenic Transformation in Marine Algae FUNGI OBJECTIVE OF PRESENT WORK 14 Chapter 2. EXPERIMENTAL METHODS FOR THE ANALYSIS OF ARSENIC INSTRUMENTATION High Performance Liquid Chromatography (HPLC) Apparatus Conditions Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 17 iv

6 2.2 GENERAL CULTURE MAINTENANCE Conviron Environmental Chamber (Model CMP 3023) Reagents and Chemicals Medium and Antibiotics SAMPLE PREPARATION FOR ANALYTICAL ANALYSIS Sample Storage Extraction Acid Digestion of Samples SPECIATION OF STANDARD COMPOUNDS BY HPLC-ICP-MS Standard Reference Materials for Ion-Pairing HPLC-ICP-MS Condition Analytical Standards for Ion-Pairing HPLC-ICP-MS Condition Analytical Standards for Anion-Exchange HPLC-ICP-MS Condition Analytical Standards for Total Analysis by ICP-MS 32 Chapter 3. THE BIOMETHYLATION AND BIOACCUMULATION OF ARSENICALS BY MARINE ALGAE FUCUS GARDNERI UNDER DIFFERENT ENVIRONMENTAL CONDITIONS INTRODUCTION SAMPLE COLLECTION AND TREATMENT Sample Collection Sample Treatment Treatment of Fucus gardneri before the Acclimation Period Treatment of Fucus gardneri during the Acclimation and Exposure Periods Fucus gardneri collected October 1998, acclimated in seawater or artificial seawater, and exposed to As(V) in seawater or artificial seawater Fucus gardneri collected February 1999, acclimated in seawater, and exposed to As(V) in artificial seawater Fucus gardneri collected October 1999, acclimated in artificial seawater, and exposed to As(V) in artificial seawater at different phosphate levels Fucus gardneri collected June 1999 and acclimated in seawater in the absence of antibiotics Fucus gardneri collected August 1999, acclimated in seawater, and exposed to As(V) in seawater in the absence of antibiotics Sample Preparation Culture and Medium Conditions 44

7 3.3 RESULTS OF EXPOSURE OF FUCUS GARDNERI TO ARSENIC(V) IN DIFFERENT MEDIA Introduction Results from Fucus gardneri Acclimated in Seawater or Artificial Seawater, and Exposed to Arsenic(V) in Seawater or Artificial Seawater Acclimation Period Exposure Period Results from Fucus gardneri Acclimated in Seawater and Exposed to Arsenic(V) in Artificial Seawater Acclimation Period Exposure Period RESULTS OF EXPOSURE OF FUCUS GARDNERI TO ARSENIC(V) IN DIFFERENT PHOSPHATE CONCENTRATION MEDIA Introduction Results and Discussion Acclimation Period Exposure Period RESULTS OF EXPOSURE OF FUCUS GARDNERI TO ARSENIC(V) IN THE ABSENCE OF ANTIBIOTICS Introduction Results and Discussion Acclimation Period Exposure Period SUMMARY 77 Chapter 4. BIOMETHYLATION AND BIOACCUMULATION OF ARSENICALS BY MARINE ALGAE NEREOCYSTISLUETKEANA INTRODUCTION SAMPLE COLLECTION AND TREATMENT Sample Collection Sample Treatment Treatment of Nereocystis luetkeana before the Acclimation Period Treatment of Nereocystis luetkeana during the Acclimation and Exposure Periods ; 82

8 4.2.3 Sample Preparation Culture and Medium Conditions RESULTS AND DISCUSSION Acclimation Period Exposure Period SUMMARY 93 Chapter 5. ARSENIC TRANSFORMATION BY THE FUNGUS FUSARIUM OXYSPORUM MELONIS INTRODUCTION SAMPLE COLLECTION AND TREATMENT Agar Conditions and Treatment of Fusarium oxysporum melonis before the Exposure Period Treatment of Fusarium oxysporum melonis during the Exposure Period Sample Preparation Culture and Medium Conditions RESULTS AND DISCUSSION Analysis of the Extracts of the Fusarium oxysporum melonis after the Exposure Experiments Results after Exposure to As(V) Results after Exposure to DMA Analysis of the Culture Media from the Exposure Experiments Results of the Medium Collected after Exposure to As(V) Results of the Medium Collected after Exposure to DMA SUMMARY 106 vii

9 Chapter 6. ARSENIC SPECIATION OF DIFFERENT MARINE ALGAE FOUND IN THE BRITISH COLUMBIA COAST INTRODUCTION WATER CONTENT ARSENIC SPECIATION ASSESSMENT OF ACCURACY OF EXTRACTION METHOD 113 Chapter 7. SUMMARY AND FUTURE CONSIDERATIONS 116 BIBLIOGRAPHY 119 viii

10 LIST OF TABLES Table 1.1 Arsenic Species found in the Marine Environment 3 Table 1.2 Arsenosugars found in some common Marine Algae 8 Table 2.1 Summary of Experimental HPLC Conditions 16 Table 2.2 Operating Parameters of ICP-MS 18 Table 2.3 Operating Parameters for the Conviron Environmental Chamber 19 Table 2.4 Contents of Artificial Seawater, ASP6 F2 21 Table 2.5 A3 Antibiotics for Axenation of Bacteria and Fungus 22 Table 3.1 Medium Conditions for the Exposure Period for Fucus gardneri collected October Table 3.2 Medium Conditions for the Exposure Period for Fucus gardneri collected February Table 3.3 Medium Conditions for the Acclimation Period for Fucus gardneri collected October Table 3.4 Medium Conditions for the Exposure Period for Fucus gardneri collected October Table 3.5 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) following Acclimation and As(V) Exposure in Seawater or Artificial Seawater 52 Table 3.6 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) following Acclimation in Seawater and Exposure to As(V) in Artificial Seawater 59 Table 3.7 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after Acclimation in different Phosphate Concentration Media 65 Table 3.8 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after As(V) Exposure in different Phosphate Concentration Media 67 Table 3.9 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after Acclimation in Seawater with no added Antibiotics 73 Table 3.10 Arsenic Speciation of Seawater Samples (ppb, wet weight) and Fucus gardneri Extract Samples (ppm, dry weight) after As(V) Exposure in the absence of Antibiotics 75 ix

11 Table 4.1 Medium Conditions for the Exposure Period for Nereocystis luetkeana 83 Table 4.2 Arsenic Speciation of Nereocystis luetkeana Extracts (ppm, dry weight) before and after Acclimation in Seawater 88 Table 4.3 Arsenic Speciation of Nereocystis luetkeana Extracts (ppm, dry weight) after As(V) Exposure in ASP6 F2 Medium 91 Table 5.1 Medium Conditions for Fusarium oxysporum melonis 98 Table 5.2 Arsenic Speciation of Fusarium oxysporum melonis Extracts after As(V) Exposure (ppb, wet weight) 102 Table 6.1 Water Content of Marine Algae from British Columbia Coast 110 Table 6.2 Relative Amounts of Arsenicals found in some Marine Algae (ppm, dry weight) Table 6.3 Determination of Extraction Efficiency by using Total Digestion and Determination of Detection Efficiency by using the Extraction Method 114 x

12 LIST OF FIGURES Figure 1.1 Proposed Pathway for the Biogenesis of Arsenosugars in Marine Algae 10 Figure 2.1 Schematic Diagram of ICP-MS (VG Elemental, Fisons Instrument) 18 Figure 2.2 Oyster Tissue SRM Standard 26 Figure 2.3 Fucus Sample Standard 27 Figure 2.4 Kelp Powder Standard 27 Figure 2.5 Five Arsenic Standards 28 Figure 2.6 Arsenate Calibration Curve for Ion-Pairing HPLC-ICP-MS 29 Figure 2.7 Standard Solution of Four Arsenic Compounds 30 Figure 2.8 Arsenate Calibration Curve for Anion-Exchange HPLC-ICP-MS 31 Figure 2.9 Arsenate Calibration Curve for Total Digestion 32 Figure 3.1 Fucus gardneri Silva Figure 3.2 Map of Collection Sites 35 Figure 3.3 Fucus Extract before and after 14 days of Acclimation in Seawater 48 Figure 3.4 Fucus Extract before and after 14 days of Acclimation in ASP6 F2 Medium 48 Figure 3.5 Fucus gardneri Exposed to As(V) in Seawater Medium 50 Figure 3.6 Controls for the Exposure Experiment 50 Figure 3.7 Fucus gardneri Exposed to As(V) in ASP6 F2 Medium 51 Figure 3.8 Seawater Samples collected during the Exposure of Fucus gardneri to Arsenic(V).. 53 Figure 3.9 ASP6 F2 Medium Samples collected during the Exposure of Fucus gardneri to Arsenic(V) 54 Figure 3.10 Fucus Extracts before and after 14 days of Acclimation in Seawater 57 Figure 3.11 Fucus gardneri Exposed to Arsenic(V) in ASP6 F2 Medium 58 xi

13 Figure 3.12 ASP6 F2 Medium Samples collected during the Exposure of Fucus gardneri to Arsenic(V) 60 Figure 3.13 Arsenic Species found in Fucus gardneri Extracts before and after 14 days of Acclimation 64 Figure 3.14 Arsenic(V) Speciation of Fucus gardneri Samples following Exposure in different Phosphate Concentration Media 66 Figure 3.15 Arsenate (As(V)) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 68 Figure 3.16 Arsenite (As(III)) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 68 Figure 3.17 Dimethylarsinate (DMA) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 69 Figure 4.1 Nereocystis luetkeana Postels and Ruprecht 80 Figure 4.2 Nereocystis luetkeana Bulb Extracts before and after 9 days of Acclimation in Seawater 87 Figure 4.3 Nereocystis luetkeana Blade Extracts before and after 9 days of Acclimation in Seawater 87 Figure 4.4 Nereocystis luetkeana Bulb Extracts after Exposure to As(V) in ASP6 F2 Medium. 89 Figure 4.5 Nereocystis luetkeana Blade Extracts after Exposure to As(V) in ASP6 F2 Medium 90 Figure 4.6 ASP6 F2 Medium Samples collected during the Exposure of Nereocystis luetkeana to Arsenic(V) 92 Figure 5.1 Micrograph of Fusarium oxysporum melonis 96 Figure 5.2 Fusarium oxysporum melonis Extracts after Exposure to Arsenic(V) in ASP6 F2 Medium 102 Figure 5.3 Short Term Effects of the Fusarium oxysporum melonis Exposed to As(V) over 45 days 104 Figure 5.4 Long Term Effects of the Fusarium oxysporum melonis Exposed to As(V) over 45 days 105 xii

14 LIST OF ABBREVIATIONS ADP AsB AsC As(III) As(V) ATP cps DMA HPLC IAEA ICP MMA MS m/z NIST ODS PDA PDB ppb ppm ppt PTFE r.f. SAM Sc sp. SRM TEAH TM TRA X-XIV adenosine diphosphate arsenobetaine arsenocholine arsenite arsenate adenosine triphosphate counts per second dimethylarsinate, also dimethylarsinic acid high performance liquid chromatography International Atomic Energy Agency inductively coupled plasma monomethylarsonate, also monomethylarsonic acid mass spectrometry mass to charge ratio National Institute of Standards and Technology octadecylsilica potato dextrose agar potato dextrose broth parts per billion, ng/g or ng/ml parts per million, pg/g or pg/ml parts per thousand, mg/g or mg/ml polytetrafluoroethylene (Teflon) radio frequency S-adenosylmethionine standard deviation from calibration curve species standard reference material tetraethylammonium hydroxide trace metals time resolved analysis arseno sugars X through XIV xiii

15 ACKNOWLEDGMENTS I would like to give my deepest appreciation to my research supervisor Dr. W.R. Cullen for his guidance, advice, encouragement and financial support throughout the course of this project. I would also like to thank Dr. Elena Polishchuk for her time, patience and expert advice on growing and maintaining the cultures for this project. I'm particularly grateful to Bert Mueller for the technical assistance in ICP-MS and to Catherine Franz for preparing the fungus samples for identification. I am indebted to my past and present colleagues for their continual help and useful discussions. They include Vivian Lai, Corinne Lehr, Bianca Kiupers, Iris Koch, Paul Andrewes, Lixia Wang, Changqing Wang, Ulrik Norum and Sarah Maillefer. I would also like to thank all the people from Biological Services for always keeping me in a cheerful mood. Finally, I would like to thank my sister and my parents for their daily support, love and encouragement. xiv

16 1 GENERAL INTRODUCTION Arsenic is an element that is most commonly associated with poison; therefore it may be a surprise to learn that it is present in elevated levels in many types of seafood and edible marine algae. In the environment arsenic is found in many types of mineral deposits, particularly those containing sulphides. It accompanies many metals including Cu, Ag, Au, Pt and Fe; hence, arsenic is a good indicator in geochemical prospecting surveys for elements of commercial importance. 1 Arsenic has mainly found uses as weapons and poisons from the Middle Ages to World War I, nonetheless some arsenical compounds have been found to have therapeutic uses in treating specific types of acute leukaemia. ' Even though arsenic is now being used for various applications other than poison, it is still found in the environment from extremely toxic to relatively non-toxic forms. Therefore it is important to determine how plants and animals in the environment take up arsenic, and how the arsenic is treated and transformed by them. 1

17 1.1 ARSENIC IN THE MARINE ENVIRONMENT Arsenic is an ubiquitous element. It is found in the atmosphere, in the earth's crust, in rocks, in soils, in sediments, in organisms, in freshwater and in seawater, and even in interstellar gas. 4 The sources of arsenic in the marine environment are both natural and anthropogenic. Natural sources of arsenic in the marine environment include weathering, volcanic activity, and soils and sediments. 5 Some of the main anthropogenic sources are mine tailings; smelting of gold, silver, copper, and lead ores; fossil fuel combustion; herbicides and pesticides. 5 ' 6 In seawater, the arsenic concentrations typically range from 1.0 ppb to 8.0 ppb. ' The predominant species that is present in the ocean is arsenate [As(V)]; however, significant amounts of arsenite [As(III)], monomethylarsonic acid [MMA] and dimethylarsinic acid [DMA] have also been observed and are believed to be associated with the biological activity of marine algae. 9 ' 10 Marine animals and plants contain a higher and wider range of concentration levels of arsenic (0.78 to over 100 ppm). 11 The wide range of concentration levels depends on factors such as the species type, habitat and feeding habits. The most common non-toxic organoarsenic form found in marine animals is arsenobetaine. 11 Arsenic-containing ribofuranosides (arsenosugars) were first identified in 1981 in the * brown macroalga Ecklonia radiata. ' Further investigations of other brown algae species, in addition to algae from other classes, revealed the existence of 15 arsenosugar compounds. 13 ' 14 ' 15 Because these compounds can occur at high concentrations (several mg As/kg wet weight) in marine organisms, including those used as human food, there is considerable interest regarding their toxicological behaviour. 2

18 Some common arsenic species that are found in the marine environment and in marine biological systems including marine algae are listed in Table 1.1. Table 1.1 Arsenic Species found in the Marine Environment Name Abbreviation Chemical Formula Arsenate Arsenite Monomethylarsonic acid Dimethylarsinic acid Trimethylarsine Arsenobetaine Arsenocholine Arsenosugars 3 As(V) As(III) MMA DMA Me3As AsB AsC X-XIV AsO(OH) 3 As(OH) 3 CH 3 AsO(OH) 2 (CH 3 ) 2 AsO(OH) As(CH 3 ) 3 (CH 3 ) 3 As + CH 2 COO" (CH 3 ) 3 As + CH 2 CH 2 OH See figure below O X (CH 3 ) As CBx^O yp~ CH 2 CHCH 2 Y X Y X -OH -OH XI -OH -OP0 3 HCH 2 CH(OH)CH 2 OH XII -OH -S0 3 H XIII -OH -OS0 3 H XIV -NH 2 -S0 3 H Numbering system (X-XIV) according to Shibata et al. 15 3

19 1.2 TOXICITY OF ARSENIC Arsenic is a Group 15 element and it is the 20 most abundant element in the earth's crust. 16 Arsenic is commonly found in the +5, +3 and 0 oxidation states. Arsenic is well known to be acutely toxic. It is also a carcinogen, long-term exposure to arsenic can cause skin, lung, liver and bladder cancer. 5 ' 17 The toxicity and carcinogenicity of arsenic depends on the chemical form of the arsenic species. Trivalent inorganic arsenic compounds are more acutely toxic than pentavalent inorganic arsenic compounds. Organometallic compounds in the +5 state are less toxic than inorganic ones and some compounds, such as arsenobetaine, are essentially non-toxic in all 5 18 systems tested. ' The dependence of toxicity on the chemical form of arsenic makes their identification necessary. The relative toxicity of arsenic compounds follows the general trend shown below. 6 ' 19 ' 20 H3As>As2O3[As(III)]>(RAsO)n>As 2 O 5 [As(V)]>R n AsO(OH)3.n>R4As + >As(0) where R=alkyl or aryl n=l,2 Trivalent arsenic compounds, such as arsenite, have a high affinity for thiol groups (S-H) and can interact with active sites of many enzymes. ' ' Arsenate inhibits ATP (adenosine triphosphate) synthesis by the formation of an unstable arsenate ester of ADP (adenosine diphosphate) instead of ATP. The formation of ATP may be inhibited by the formation of these 4

20 arsenate esters, which are not able to store energy like ATP. The energy of such an ester bond cannot be recovered metabolically. 5 ' 10 ' 21 ' 23 ' 24 ' 25 Organometallic compounds of arsenic (such as arsenosugars, arsenobetaine and arsenocholine) can occur in high concentration in the marine environment, including biota used for human food, but studies suggest that arsenosugars exhibit no cytotoxic or mutagenic effects. 26 The one common feature of the organoarsenicals that are found in nature is that they contain methyl groups attached to the arsenic. It has been suggested that the formation of methylated 77 arsenic compounds in marine organisms is a mechanism for the detoxification of arsenic. 5

21 1.3 MARINE ALGAE General Information Marine algae, or seaweeds, are the oldest members of the plant kingdom, extending back many hundreds of millions of years. They have little tissue differentiation, no true vascular tissue, no roots, stems, leaves, or flowers and thus take their mineral salt directly from their surrounding medium, i.e. from the seawater. Algae range in size from microscopic individual cells to huge plants more than 100 feet (30 meters) long. Algae are commonly classified into a number of groups according to their colour: Chlorophyta are the green algae, Phaeophyta are the brown algae, Rhodophyta are the red algae and Cyanophyta (or Myxophyta) are the blue-green algae. Under the widely used five-kingdom classification system, the brown, red and green algae belong to the Protista kingdom. The bluegreen algae belong to the kingdom Monera along with bacteria and viruses. Each species has its characteristic photosynthetic pigments in the body. ' Zonation patterns within algal assemblages are dictated by tidal exposure and wave impact, as well as by species interactions such as grazing by invertebrates, and by competition for space and light. 29 The brown algae (Phaeophyta) probable represent a very old group among the plant kingdom. Almost all Phaeophyta thrive in the sea, for example, Nereocystis luetkeana (Bull kelp), Fucus gardneri (Brown algae), Laminaria groenlandica (Kombu) and Eisenia bicyclis (Arame). ' ' ' All brown algae (Phaeophyta) are multicellular, and most are macroscopic. 28 The red algae (Rhodophyta) are the seaweeds that can be seen growing along shores and cast up on beaches. Almost all red algae are multicellular and are a large group of small to 6

22 medium-sized plants, for example: Porphyra spp., Mastocarpus pepillatus and Gigartina 28 exasperata. The large and clearly defined group of the marine algae is the Chlorophyta or green algae. They comprise forms of highly diverse lines of development and phases of greatly varying habit. The green algae inhabit, above all, fresh water regions and subsist under most varying conditions. The species found mostly on the British Columbia coast is the Ulva fenestrata. This project involves experiments with Nereocystis luetkeana and Fucus gardneri Commercial Use of Marine Algae Algae have been put to a number of uses in the past, and their applications are increasing. The kelp trade developed in the seventeenth century and, until quite recently, was an important branch of the iodine industry. Kelp (Nereocystis luetkeana) has been used to weave baskets and also make great musical instruments. 34 Brown seaweeds were collected and burned, and their ashes were used as a source of soda to make glass during the 16th and 17th century. Later it was discovered that the brown seaweed was rich in iodine and was used for iodine-extraction until after the First World War. 28,30 ' 31 In the modern marine algae industry, seaweeds are used in many products. ' Some examples of the use of seaweed as food are as follows: Nori is an ingredient for 'sushi' in Japanese food. Arame is used with soups or taken with soya-sauce in Japanese food. 30 ' 31 ' 36 Alginates from brown algae are used as stabilizers in ice creams, bakery and confectionery products, as thickening agents in soups and sauces, as alginate strings in meat sausages and as clarifying agents in beverages. 28 Seaweeds are also used in food products for other animals They are used as fertilizers and used for the production of agar. ' 7

23 Analyses of certain edible seaweeds also show that many contain significant amounts of protein, vitamins and minerals essential for human nutrition. ' Both Fucus gardneri and Nereocystis luetkeana are used in the production of vitamin supplements Arsenosugars in Marine Algae Marine algae are able to accumulate arsenic more efficiently than the higher members of the food web: in some algae, arsenic concentrations have been found to be 3000 times higher than those present in the surrounding seawater. Most of the arsenic in marine macroalgae exists in complex forms and a variety of arsenosugar derivatives have been isolated and characterized. 38 ' 39 ' 40 ' 41 The arsenosugars found in some of the common brown macroalgae are listed in Table 1.2. Table 1.2 Arsenosugars found in some common Marine Algae Algae Arsenosugars present Reference Brown Algae Fucus gardneri X, XII, XIII 42,43 Ecklonia radiata X, XI, XII 39,40 Undaria pinnatifida X, XI, XII 44,45 Red Algae Porphyra tenera (Nori) X, XI 45 Green Algae Codium fragile (Miru) X, XI 45 8

24 Edmonds and Francesconi were the first to isolate arsenosugars from the marine alga Ecklonia radiata. A total of 15 arsenosugars have been found from different species of green, brown and red algae. 25 The question of how and why arsenosugars are formed by the marine algae has been studied extensively. It is believed that the algae accumulate arsenate from the seawater, because arsenate is the predominant species found in seawater at a typical range of approximately 1.0 to 8.0 ppb. 7 ' 8 ' 10 It is believed that arsenate, being chemically similar to phosphate, is readily taken up by the algae from the water via the phosphate transport systems located in the algal cell membranes. 5 ' 10 ' 25 Although the precise mechanism involved in the formation of arsenosugars is not known, a pathway as proposed by Edmonds and Francesconi 25 ' 41 is shown in Figure 1.1. The pathway initially follows the mechanism outlined by Challenger by using SAM (S-adenosylmethionine) as the methylating agent (CFi3 + donation indicated by A in Figure 1.1). However, in the final steps the adenosyl group (indicated by B in Figure 1.1) of the methylating agent is transferred to the arsenic atom of dimethylarsinate, ultimately to form arsenosugars and arsenolipids. 8 ' 25 ' 46 9

25 Figure 1.1 Proposed Pathway for the Biogenesis of Arsenosugars in Marine Algae' The A represents methyl group donated by SAM, and B is the adenosyl group from SAM O II OH As OH I OH 1* OH As-OH CH, 4--- HOOC CH (CHJJJ S C NH, N H, <o. ; N -Vy OH S-adenosylmethionine o II OH As OH I CH 3 OH As-OH I CH, CH, HOOC CH (CH,), S CH, I ^ 2 v, y y OH NH, N N J O II H3C As OH CH, -> H 3 C As OH CH, 0 II (CH^As-CH^ ^ 70H 0 (CH 3 ) F -As-CH 2v ^O v ya NH, N N OH OH OH OH 0 OH (CHj) As CH^ yo-chjchchj- R OH OH

26 Arsenate can produce toxic effects in the algae, including inhibition of growth and phosphorus metabolism. Changes in the algal cell morphology are also seen. It has been suggested, that while the conversion of arsenate to arsenite creates a more toxic arsenic compound, arsenite is not released into the cell environment but it is pumped out immediately to avoid an excessive build-up of toxic arsenic concentrations inside the algal cell. 27 ' Previous Studies of Arsenic Transformation in Marine Algae Although arsenosugars have been isolated from seaweeds, there are only a limited number of reports that describe the transformation of arsenicals by marine macroalgae grown in culture media. Klumpp and Peterson 9 claimed that the brown macroalga, Fucus spiralis, took up the arsenic as arsenate and transformed it into water-soluble organoarsenic compounds which were then further transformed into a lipid-soluble organoarsenic compound and 12 water-soluble * 48 organoarsenic compounds. However these compounds were not positively identified as arsenosugars. Sanders and Windom 10 used arsenate, arsenite and dimethylarsinate as substrates for cultures of marine macroalgae Valonia macrophysa. Digestion of the cells in dilute nitric acid led to an increase in methylated arsenicals, suggesting that more complex arsenic compounds were produced. Cullen et al. ' found that Polyphysa peniculus transformed arsenate into arsenite and dimethylarsinate. When the alga was treated with arsenite, dimethylarsinate was the major metabolite in the cells and in the growth medium. Trace amounts of monomethylarsonate were also detected in the cells. Significant amounts of more complex arsenic species, such as 11

27 arsenosugars, were not observed in the cells or medium. Cullen et al. concluded that the arsenate taken up by Polyphysa peniculus is quickly reduced to arsenite, followed by methylation to monomethylarsonate. MMA due to its low passive diffusion coefficient is not excreted to the growth medium. The compound remains in the cells, where it is more likely to be reduced and further methylated to DMA (which is the end product for Polyphysa peniculus). DMA is then diffused into the growth medium. The fast excretion of the reduced and/or methylated arsenic Q compounds to the medium decreases the need for further detoxification. Arsenic compounds found in marine animals are generally believed to come from arsenicals produced by marine algae. It has been suggested that arsenobetaine and arsenocholine are formed from arsenosugars, which are found in marine algae

28 1.4 FUNGI The fungi (singular, fungus) are plant-like, spore-bearing organisms, which lack chlorophyll and are unable to synthesize their food. Consequently, they depend on other organisms for their nutrition. Fungi live as saprobes, which bring about the decay of organic materials, or as parasites, which attack living protoplasm, and in so doing cause diseases of plants, animals, and humans. 49 Fungi have a very broad definition, which encompasses the bacteria, the slime molds, and the so-called true fungi. The bacteria differ from the other two groups in that they possess primitive rather than the highly organized nuclei that are found among the slime molds and the fungi. 49 Fungi are often found to grow with algae in a parasitic relationship. The fungus can affect the algal cells by competing for nutrients, by changing the physical state of the medium, and by releasing substances, which inhibit the growth or kill the cells. Few studies have been done on these processes

29 1.5 OBJECTIVE OF PRESENT WORK The study of the interaction of marine algae with arsenicals is important; because arsenic compounds produced by the algae are generally believed to be one of the sources of the arsenic compounds found in marine animals. In addition to the human toxicological aspects, arsenosugars are also of interest because of their pivotal role in the cycling and biotransformation of arsenic in marine systems. Thus the first objective of this work was to determine if and how arsenosugars are produced by Fucus gardneri. The Fucus was exposed to As(V) under different environmental conditions to determine if these conditions would affect the uptake of As(V) by the algae. The different environmental conditions that were examined were variations of salinity, phosphate, and antibiotic concentrations in the medium. The second objective was to duplicate one of the above experiment using Nereocystis luetkeana in order to determine if the Nereocystis is able to accumulate As(V) from the surrounding media in a similar fashion as Fucus gardneri. The experiment that was performed was to acclimate Nereocystis luetkeana in seawater, followed by exposure to As(V) in artificial seawater The third objective was to identify a particular resilient fungus growing with Fucus gardneri and to determine if this fungus was involved in the uptake of arsenic compounds by the brown algae. The last objective was to determine the arsenosugars present in a variety of British Columbia algae samples found to grow along with Fucus gardneri and Nereocystis luetkeana. The water content of each samples was found as well as the extraction efficiency of the method used. 14

30 2 EXPERIMENTAL METHODS FOR THE ANALYSIS OF ARSENIC 2.1 INSTRUMENTATION High Performance Liquid Chromatography (HPLC) Apparatus The HPLC system consisted of a Waters Model 510 delivery pump, a Reodyne six-port injection valve with a 20 ul sample loop, an appropriate column and its corresponding guard column. The HPLC system was connected to the inductively coupled plasma mass spectrometer (ICP-MS) nebulizer by using PTFE tubing (2.5 cm) with the appropriate fittings. The analytical column used to analyze the medium samples, was an anion-exchange column (Hamilton, PRPxlOO, 250 mm x 4.6 mm) with a guard column of the same material. The mobile phase was 20 mm phosphoric acid, adjusted to ph 6.0 by using ammonium hydroxide. The column used to analyze the extract samples was a reversed-phase Ci8 column (GL Sciences Inertsil ODS, 250 mm x 4.6 mm). The guard column was packed with the same material (Supelco) as in the Inertsil ODS column for the reversed-phase conditions. The mobile phase was made up of 10 mm tetraethylammonium hydroxide, 4.5 mm malonic acid, and 0.1 % MeOH, adjusted to ph 6.8 by using nitric acid. A guard column preceded the analytical column to filter out any precipitates that might form when the injection sample combined with the mobile phase, and any other large molecules that might have been present. 15

31 Conditions The HPLC system, connected to the ICP-MS, was used to analyze the extracted samples and medium samples collected from the exposure experiments. The conditions for the columns, mobile phases and flow rates are summarized in Table 2.1. The chromatographic columns were equilibrated with the mobile phase for about 50 minutes prior to analysis. All the standard samples were filtered through a 0.45 um syringe filter (Millipore) just prior to injection onto the HPLC column, while all other samples were filtered through a 0.22 pm syringe filter before injection. Filtration of the samples removed small particles that might affect the efficiency of the column or might even contaminate the column. Most importantly, removal of the small particles will prevent the blocking of the injection loop, lines and column. The effluent from the HPLC column was fed to the nebulizer of the ICP-MS via the PTFE tubing and connections. Arsenic compounds in the samples were identified by matching the retention times of the peaks in the chromatograms with those of standard arsenic compounds. The mobile phases for the HPLC were filtered through a millipore 0.45 pm filter after they were made up. Table 2.1 Summary of Experimental HPLC Conditions Conditions Column Mobile Phase Flow Rate (ml/min) Anion exchange (medium samples) Hamilton PRPX mm phosphoric acid, ph Ion Pairing (extract samples) Inertsil ODS (GL Sciences, Japan) 10 mm tetraethylammonium hydroxide(teah), 4.5 mm malonic acid, 0.1 % MeOH ph

32 2.1.2 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) A VG Plasma Quad 2 Turbo Plus inductively coupled plasma-mass spectrometer (VG Elemental, Fisons Instrument) was used as a detector for the HPLC eluent. It was also used to measure the total arsenic, via direct injection into the system. The ICP-MS was equipped with a SX 300 quadrupole mass analyzer, a standard ICP torch, an argon plasma and a de Galan V-groove nebulizer. The mass analyzer was operated in the time resolved analysis (TRA) mode using TR Analysis software. The 75, 77, 82 and 103 mass to charge (m/z) signals were monitored. All signals were collected and the data were transferred to the computer (VG data system). The data were then exported to a Microsoft Excel 7.0 program for further processing. The TRA mode operation allowed for the monitoring of mass to charge (m/z) 75, 77 and 82; and also 103 for the total digestion analysis. The m/z ratio of 75 corresponds to that of 7 5 As (100 % isotopic abundance), but also to the possible interfering species Ar CI signal. The m/z ratio of 77 corresponds to that of Ar 3 7 Cl + signal. The interference due to Ar 3 5 Cl + can thus be accounted for by subtracting the peak area at m/z 77 from the peak area at m/z 75, having corrected for the isotopic ratio of CI to CI (75.77:24.23). There may also be a peak at m/z 77 due to 77 Se. Se has isotopes of 77 and 82, so the contribution of the 77 Se peak can be accounted for by subtracting the peak area at m/z 82 from the peak are at m/z 77, having corrected for the isotopic ratio of Se to Se (isotopic ratio: 7.63:8.74). The m/z ratio of 103 corresponds to that ofrh. A schematic diagram of the ICP-MS system is displayed in Figure 2.1 and the commonly used operating parameters for the ICP-MS are shown in Table

33 Figure 2.1 Schematic Diagram of ICP-MS (VG Elemental, Fisons Instrument) Quadrupole RF Generator Detector Quadrupole Lenses Interface Bias Voltage Supplies ICP ICP RF Generator DO 1 DOJ- Amplifier Vacuum Pumps Quadrupole RF DC Control PlasmaQuad Control MCS ^ IEEE Interlace Table 2.2 Operating Parameters of ICP-MS Feature Specific Conditions Forward r.f. power Reflected power Outer (cooling) gas flow rate Intermediate (auxiliary) gas flow rate Nebulizer gas flow rate Nebulizer type Analysis mode Quadrupole pressure Expansion pressure 1350 W <10 W 13.8 L/min 0.65 L/min L/min de Galan TRA, 1 sec time slice 9x 10" 7 mbar 2.4 mbar 18

34 2.2 GENERAL CULTURE MAINTENANCE Conviron Environmental Chamber (Model CMP 3023) The Conviron, a commercial incubator, was used to hold the samples during the growth, acclimation and exposure periods. The Conviron can be set at specific temperatures and light conditions to obtain optimal settings for the growth of the marine algae and fungus. Table 2.3 shows the operating parameters. Table 2.3 Operating Parameters for the Conviron Environmental Chamber Sample Temperature Light Intensity Photoperiod Fucus gardneri 15 C/ 7 C 100 lux 12 hour day and night cycle Nereocystis luetkeana 18 C/7 C 200 lux 12 hour day and night cycle Fusarium oxysporum 15 C none none Reagents and Chemicals All chemicals used were of at least analytical grade and obtained from commercial sources, unless otherwise stated. The chemicals and reagents used were: methanol (HPLC grade, Fisher), tetraethylammonium hydroxide (20 wt. %, Aldrich), malonic acid (BDH), nitric acid (68-71 %, sub-boiling distilled, Seastar Chemicals), phosphoric acid ( %, 85 wt. %, Aldrich) and ammonium hydroxide (30 %, Fisher). Standard solutions of arsenite (from arsenic trioxide, AS2O3, Fisher), arsenate (from sodium arsenate heptahydrate, Na2HAs04»7H20, Sigma), monomethylarsonic acid (Pfalz & 19

35 Bauer), dimethylarsinic acid (Aldrich), and arsenobetaine (synthesized as described by Edmonds, TO et ar) were made up in deionized water. Standard samples of oyster tissue SRM (NIST-1566a), Fucus sample (IAEA-140/TM) and kelp powder (laboratory standard-galloway's naturally kelp powder, Richmond, B.C., Canada) were also available to confirm the retention times of arsenosugars obtained from the HPLC-ICP-MS. Rh standard solution for total digestion was made up from RJ1CI3. Deionized water, with a resistivity better than 1 MQ, was used to make up all the standard solutions, the mobile phases for the HPLC system and the sample solutions for extraction and total digestion. Because it was necessary to avoid even trace contamination, the following precautions were taken with all the glassware and plasticware: The glassware and plasticware were cleaned by soaking in 2 % Extran solution for at least one night. They were rinsed with deionized water, and then soaked in 0.1 M HNO3 solution for at least one night. They were then rinsed with deionized water and air-dried. Any glassware used to store the standard solutions or used for the algae and fungus experiments were autoclaved Medium and Antibiotics The artificial seawater that was used in the arsenic exposure experiments was prepared according to the recipe for ASP6 F2 (see Table 2.4), which was previously used by Fries 51 to culture members of the family Fucaceae. The only deviation from the recipe was the substitution of CaCl2*2H20 for CaCb'^O (the formation with one water molecule is probably an error). In addition, the ph of the artificial seawater was adjusted from 9.72 to ~ 7.76 (which was the ph of the seawater that was collected with the algae) by addition of HC1. The ph reported for the 20

36 ASP6 F2 artificial seawater by Fries is 8.3. The seawater was prepared without the vitamins, autoclaved and stored at room temperature. The vitamin solution and the vitamin B12 solution were sterilized by using 0.22 um sterile filters and then added to the autoclaved seawater. The artificial seawater with the vitamins was then stored in the refrigerator at 4 C. Table 2.4 Contents of Artificial Seawater, ASP6 F2 51 Medium ASP6 F2 Micronutrient Solution (per ml H?0) NaCl 24 g nitrilotriacetic acid 50 mg MgS0 4 '7H 2 0 8g Fe (as chloride) 1 mg KC1 0.7 g Zn (as chloride) 0.25 mg CaCl 2 «2H 2 0** 0.55 g Mn (as chloride) 0.5 mg NaN g Co 5 Hg Na 2 -glycerophosphate 50 mg Cu 10 ug K 2 HP0 4 2mg B (as H3BO3) 1 mg KI mg Mo (Na-salt) 0.25 mg KBr 96.9 mg TRIS (hydroxy methyl) aminomethane lg Vitamin Solution (per ml H?0) Micronutrient solution 1 ml Thiamine HC1 0.2 mg Vitamin solution 1 ml Nicotinic acid 0.1 mg B Hg Ca panthothenate 0.1 mg Distilled Water 1000 ml Pyridoxin HC mg "original recipe has CaCl 2 *H 2 0 p-aminobenzoic acid Biotin Thymine Inositol Orotic Acid 0.01 mg 0.5 ug 0.8 mg 1 mg 0.26 mg The antibiotics were prepared according to the recipe for A3-antibiotics as described by Xuewu (see Table 2.5). 53 The antibiotics were sterilized by using 0.22 um sterile filters before being used. The antibiotics were used for the axenation of the bacteria and fungi in marine algae. 21

37 Table 2.5 A3 Antibiotics for Axenation of Bacteria and Fungus Antibiotics A3 Streptomycin sulphate Nystatin Erythromycin Rifampicine Distilled Water 2 g 0.5 g 1.5 g 0.02 g 100 ml Antibiotics were purchased at Sigma. Stock solutions were diluted with 100 volume of medium for treating plant materials.

38 2.3 SAMPLE PREPARATION FOR ANALYTICAL ANALYSIS Sample Storage All biomass samples collected were weighed, frozen (-20 C) immediately and then freeze-dried. Freeze-drying is a common method to preserve samples in environmental studies. It provides a convenient mode of preservation because it results in the elimination of water and the denaturation of protein. The freeze-drying procedure does not significantly alter arsenic speciation making it easier to handle the samples and also gives consistent results for quantitative analysis. All freeze-dried samples were kept at -20 C until they were extracted. All the liquid samples taken during the experiments were frozen immediately to preserve sample integrity until the time of the analysis. The liquid samples were analyzed by anionexchange HPLC-ICP-MS Extraction All the freeze-dried samples collected were extracted using a procedure similar to that described by Shibata and Morita. 44 Kelp powder (a laboratory standard), oyster tissue SRM (NIST-1566a) and Fucus sample (IAEA-140/TM) were similarly extracted as reference materials. Extractions were carried out by weighing accurately (-0.5 g) of the dried powders into 15 ml centrifuge tubes, adding 5 ml methanol/water (1:1), sonicating for 10 minutes, centrifuging for 10 minutes and transferring the liquid layer of the extracts into 250 ml round bottom flasks by using a Pasteur pipet. Each sample was extracted a total of 5 times. The combined extract solutions for each sample were evaporated to near dryness (~ 1-2 ml), and made up to 10.0 ml 23

39 using a volumetric flask. The extract solutions were frozen until just before analysis. The samples were analyzed by using the ion-pairing HPLC-ICP-MS method Acid Digestion of Samples Freeze-dried samples ( mg), the residue after extraction ( mg original for dry samples) and the extracted solution (-5 ml) were analyzed for total arsenic by using a total digestion procedure similar to that described by N0rum et al. 54 i The total digestion was carried out by placing the accurately weighed freeze-dried samples into 20 ml glass test tubes (outer diameter of 16 mm). Concentrated nitric acid (2 ml, doubly distilled in quartz, Seastar, Sidney, BC) was added to each sample together with 2-3 Teflon boiling chips. The tubes were transferred to a block heater (Standard Heatblock by VWR Scientific Products) and the temperature increased in 10 C steps every hour, starting at 70 C and reaching 150 C. At 150 C the samples were evaporated to dryness over the next 1-2 days. Once the samples were evaporated, the test tubes were removed and allowed to cool. The samples were redissolved in 4 ml "Acid-Rhodium" solution (1% nitric acid and 5 ppb Rh). The rhodium served as an internal standard during ICP-MS analysis. The redissolved digest were vortex-mixed and filtered (0.45 um) into storage vials (2 x 1.5 ml eppendorf) using disposable syringes. The samples were stored at -20 C until the time of analysis. The acid digest was analyzed by using ICP-MS and by monitoring m/z 75 and 103 for arsenic and rhodium, respectively. 24

40 2.4 SPECIATION OF STANDARD COMPOUNDS BY HPLC-ICP-MS Standard Reference Materials for Ion-Pairing HPLC-ICP-MS Condition A major problem of arsenic speciation in environmental samples is the absence of pure samples with many organoarsenic compounds. Many of the compounds important in environmental and biological systems are difficult to synthesize. This problem was solved by using oyster tissue SRM (NIST-1566a), Fucus sample (IAEA-140/TM), and kelp powder (laboratory standard) as standard reference materials for the peak identification of arsenosugars. 36 ' 44 The ion-pairing chromatography system used here is one that has been developed for the analysis of anions, particularly arsenosugars. 55 ' 56 Ion-pairing chromatography with ICP-MS detection is a technique that combines a mobile phase containing an ion-pairing reagent with a reversed-phase column. Tetraethylammonium hydroxide (TEAH) is the ionpairing reagent, and the mobile phase is adjusted to a ph of 6.8 with malonic acid and nitric acid. A small amount of methanol is added to control the chromatographic behaviour in reversedphase systems. Figure 2.2 shows a chromatogram of the oyster tissue obtained in the present study using the ion-pairing HPLC-ICP-MS condition (TEAH). The major arsenic compounds AsB, DMA, arsenosugars X and XI present in this standard reference material, were previously identified by 9 ft Shibata et al. As(V) and DMA are not baseline resolved but the individual peaks are slightly resolved making it possible to identify both peaks. The elution order shown in Figure 2.3 for the Fucus standard is DMA, As(V) and arsenosugars X, XII and XIII. This is another standard to confirm the presence and retention times of the arsenate and arsenosugars. 25

41 Figure 2.4 shows a chromatogram of the commercially available food product, kelp powder, under TEAH condition. The kelp powder contains four major peaks; arsenosugars X, XI, XII and XIII were identified by comparing the retention times with known samples by Shibata et al. 44 ' 45 Using the present chromatographic system, the arsenosugars are separated from the other ions. Although arsenosugars XI and XII are not baseline resolved from each other, their individual peaks are resolved, making it possible to identify both arsenosugars in unknown samples. Figure 2.4 shows that arsenosugar XII alone elutes slightly earlier, which allows this compound to be identified in the absence of arsenosugar XI. Likewise, arsenosugar XI can be identified in the absence of arsenosugar XII. The elution order for the sugars most likely indicates the increasing anionic character when going from arsenosugar X to XIII. 56 Figure 2.2 Oyster Tissue SRM Standard AsB DMA Retention time(s)

42 Figure 2.3 Fucus Sample Standard ^ Q XII XIII I \ DMA Retention time(s) Figure 2.4 Kelp Powder Standard Retention time(s)

43 2.4.2 Analytical Standards for Ion-Pairing HPLC-ICP-MS Condition A standard solution containing 5 compounds was used to establish the retention times for AsB, As(III), As(V), MMA and DMA under TEAH condition (Figure 2.5). The retention times for the extracts from the samples were matched with the retention times of the peaks in the standard solutions to determine the arsenic species in the extracts. In this case As(III) and AsB are not baseline resolved and MMA, DMA and As(V) are not baseline resolved, but their individual peaks are resolved, making it possible to identify As(III), AsB, MMA, DMA and As(V) in the unknown samples. Figure 2.5 Five Arsenic Standards , As(lll) AsB j I in OL O \ j Retention time(s) 28