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Arsenic and antimony species in the terrestrial environment Koch, Iris 1998

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ARSENIC AND ANTIMONY SPECIES IN THE TERRESTRIAL ENVIRONMENT by IRIS K O C H B . S c , The University o f Waterloo, Waterloo, 1992  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y  in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department o f Chemistry) We accept this thesis-as conforming to the^3^ed/standiB5i  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A October 1998 © I r i s K o c h , 1998  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  University  of  British  Columbia,  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his  and study. scholarly  or  her  thesis  for  of  CH6  fAl  The University of British Vancouver, Canada  Date  DE-6 (2/88)  QrA-  S"  -, W  ^ T ^ V  Columbia  %  requirements  gain shall  that  agree  may  representatives.  financial  the  I agree  I further  purposes  permission.  Department  of  It not  be is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  The determination o f arsenic and antimony species in environmental samples can be used to assist in toxicity assessment, as well as to yield information about environmental processes. Such information about samples from the terrestrial environment was sought. Existing methods for speciation were adapted, including high-performance liquid chromatography ( H P L C ) coupled with inductively coupled plasma-mass spectrometry ( I C P - M S ) , for the determination o f both arsenic and antimony species, and hydride generation-gas chromatography ( H G - G C ) with atomic absorption spectrometric ( A A S ) detection and mass spectrometric ( M S ) detection, for the determination o f antimony species. Arsenate, when added to mycelial cultures o f Scleroderma citrinum and Macrolepiotaprocera,  was reduced to arsenite, but no further processes (i.e.,  methylation or formation o f arsenosugars or arsenobetaine) were observed. This may indicate that the presence o f more complex arsenicals in environmental mushroom specimens is dependent on symbiotic interactions between the fungus and its surroundings, rather than resulting from independent synthesis by the fungus. Pleurotus flabellatus oxidized antimony (III) to antimonate ( S b ( O H ) ) , and formed an antimony-containing metabolite o f unknown identity. Water soluble 6  arsenic species were determined in a host o f terrestrial and freshwater biota from a hot springs environment (Meager Creek, B C ) and from an area impacted by mining and smelting activities (Yellowknife, N W T ) . Arsenate and arsenite (the more toxic forms o f arsenic) were the predominant species extracted from plants, mosses, microbial mats, algae and lichens. Small amounts o f arsenosugars and methylated arsenic were detected as well. Arsenobetaine was discovered for the first time in lichens, and it was also the major form o f arsenic in freshwater fish. The majority o f detectable arsenic in freshwater mussels and snails was as arsenosugars and the tetramethylarsonium ion, respectively. Large amounts o f arsenic, o f an unknown ii  toxicological and chemical nature, remained unextracted or undetected in all samples. A dimethylantimony compound was found in moss samples from Yellowknife, confirming that methylation of antimony takes place in the environment.  T A B L E OF CONTENTS  Abstract  ii  Table o f Contents  iv  List o f Tables List of Figures  viii x  List o f Abbreviations  xiii  Acknowledgment  xv  Chapter 1. I N T R O D U C T I O N 1.1. Flistorical aspects o f arsenic and antimony 1.2. Chemistry o f arsenic and antimony 1.3. Toxicity o f arsenic and antimony compounds 1.4. Environmental chemistry o f arsenic 1.4.1. Arsenic sources, uses and exposure to humans 1.4.2. Arsenic species in the marine environment 1.4.3. Arsenic species in the terrestrial environment 1.5. Environmental chemistry o f antimony 1.5.1. Antimony sources, uses and exposure to humans 1.5.2. Antimony species in the environment 1.5.3. Biological and chemical transformations o f antimony 1.6. Objectives and scope o f work References  Chapter 2. M E T H O D S F O R T H E A N A L Y S I S O F A R S E N I C A N D A N T I M O N Y 2.1. Methods for the Analysis o f Arsenic 2.1.1. Introduction 2.1.2. Experimental 2.1.2.1. Chemicals and reagents 2.1.2.2. Instrumentation and methods o f analysis  2.1.2.2.1. HPLC-ICP-MS 2.1.2.2.2. ESI-IT-MS 2.1.3. Results and Discussion 2.1.3.1. Speciation o f arsenic compounds by H P L C  2.1.3.1.1. Anion exchange chromatography  1 1 2 6 7 7 8 14 16 16 17 18 20 22  28 28 28 34 34 34  34 36 38 38  38 iv  r 2.1.3.1.2. Cation exchange chromatography 2.1.3.1.3. Ion-pairing chromatography  40 43  2.1.3.2. Characterization o f arsenosugars and compounds in kelp powder extract 45 2.2. Methods for the Analysis o f Antimony 65 2.2.1. Introduction 65 2.2.2. Experimental 67 2.2.2.1. Chemicals and reagents 67 2.2.2.2. Method o f analysis for H G - G C - A A S 67 2.2.2.3. Sample preparation 70 2.2.3. Results and Discussion 71 2.2.3.1. Demethylation of trimethylantimony species in aqueous solution during analysis by using G C - H G - A A S 71  2.2.3.1.1. 2.2.3.1.2. 2.2.3.1.3. 2.2.3.1.4. 2.2.3.1.5.  The effect of acid The effect of concentration The effect of sample matrix Other studies to determine causes of demethylation Suggested reasons for demethylation  References  Chapter 3. A R S E N I C A N D A N T I M O N Y I N M U S H R O O M S 3.1. Introduction 3.2. Experimental 3.2.1. Chemicals and reagents 3.2.2. Apparatus and method o f analysis  3.2.2.1. HG-GC-AAS analysis for antimony speciation 3.2.2.2. HPLC-ICP-MS analysis for antimony and arsenic speciation 3.2.2.3. ICP-MS analysis for total arsenic and antimony concentrations 3.2.3. Cultivation of Pleurotusflabellatus fruiting bodies on a solid substrate 3.2.4. Preparation o f pure submerged cultures of fungi 3.2.5. Sample preparation and analysis. 3.2.6. Isolation o f an unknown compound containing antimony 3.3. Results and Discussion 3.3.1. Arsenic species in edible mushrooms 3.3.2. The interaction o f arsenic species with pure submerged cultures of fungi  3.3.2.1. Culture experiments with Scleroderma citrinum 3.3.2.2. Culture experiments with Macrolepiota procera and Sparassis crispa 3.3.2.3. Summary of the interaction of arsenic with fungi that can produce mushrooms 3.3.3. The interaction o f antimony species with fungi  3.3.3.1. Cultivation of Pleurotus flabellatus fruiting bodies 3.3.3.2. Culture experiments with Pleurotus flabellatus 3.3.3.2.1. ICP-MS and HG-GC-AAS analysis of biomass extracts and media 3.3.3.2.2. HPLC-ICP-MS analysis of biomass extracts and media 3.3.3.3. Culture experiments with Scleroderma citrinum  71 74 75 77 .78 81  86 86 88 88 90  90 91 91 93 94 97 98 100 100 104  104 108 Ill 112  112 114 114 117 126  3.3.3.4. Summary of the interaction of antimony with fungi that can produce mushrooms References  131  Chapter 4. A R S E N I C I N T H E M E A G E R C R E E K H O T S P R I N G S E N V I R O N M E N T 4.1. Introduction 4.2. Experimental 4.2.1. Chemicals and reagents 4.2.2. Sampling 4.2.3. Sample preparation and analysis 4.3. Results and Discussion 4.3.1. Total concentrations o f arsenic in water samples 4.3.2. Total concentrations o f arsenic in biota 4.3.3. Arsenic speciation in biota samples  4.3.4. Summary References  5.3.3. Freshwater shellfi sh  5.3.3.1. Total arsenic in shellfish 5.3.3.2. Speciation of arsenic in shellfish 5.3.4. Plants  5.3.4.1. Total arsenic in plants 5.3.4.2. Arsenic speciation in plants 5.3.5. Algae and microbial mats  5.3.5.1. Total arsenic in algae 5.3.5.2. Arsenic speciation in algae 5.3.6. Mosses  147 152 155 157 158 161 162  Chapter 5. A R S E N I C I N T H E Y E L L O W K N I F H E N V I R O N M E N T  5.3.2.1. Total arsenic in 5.3.2.2. Arsenic speciation in  133 133 135 135 135 137 140 140 141 145  4.3.3. 1. Algae and microbial mat samples 4.3.3.2. Vascular plants (sedge, cedar, fleabane, monkey flower) 4.3.3.3. Moss 4.3.3.4. Fungi including lichens 4.3.3.5. Extraction efficiency for arsenic species  5.1. Introduction 5.2. Experimental 5.2.1 Chemicals and reagents 5.2.2. Sampling 5.2.3. Sample preparation and analysis 5.3. Results and Discussion 5.3.1. Water and sediment samples 5.3.2. Freshwater  129  166 166 167 167 168 171 174 175 176  fish  fish fish  177 179 187  187 188 194  196 199 204  205 206 209 vi  5.3.6.1. Total arsenic in mosses 5.3.6.2. Arsenic speciation in mosses 5.3.7. Lichens and mushrooms 5.3.7.1. Total arsenic in lichens and mushrooms 5.3.7.2. Arsenic speciation in lichens andfungi 5.4. Summary References  209 211 214 215 217 229 231  Chapter 6. A N T I M O N Y I N E N V I R O N M E N T A L S A M P L E S  236  6.1. Introduction 236 6.2. Experimental 237 6.2.1. Chemicals and reagents 237 6.2.2. Sampling and sample preparation 237 6.2.3. I C P - M S analysis for total arsenic and antimony concentrations 241 6.2.4. H G - G C - A A S analysis for antimony speciation 241 6.2.5. H G - G C - M S analysis for confirmation o f methylantimony species 242 6.3. Results and Discussion 244 6.3.1. Antimony species and total antimony in environmental samples 244 6.3.1.1. Inorganic antimony species 244 6.3.1.2. Methylated antimony species 247 6.3.1.3. Extraction efficiencies for biota and percent Sb species of total Sb in waters 250 6.3.1.4. Total concentrations of antimony compared with arsenic 251 6.3.2. The confirmation o f antimony in samples containing methylantimony compounds by using H G - G C - A A S 253 6.3.3. The use o f headspace H G - G C - M S for the speciation o f antimony compounds 254 6.4. Summary 263 References 264  Chapter 7. C O N C L U S I O N S A N D F U T U R E W O R K  266  vii  LIST OF TABLES Chapter 1 Table 1.1. Names, abbreviations and structures o f some arsenic compounds Table 1.2. Names, abbreviations and structures o f some antimony compounds  4 5  Chapter 2 Table 2.1. Table 2.2. Table 2.3. Table 2.4. Table 2.5.  H P L C conditions for arsenic speciation Operation parameters for I C P - M S E S I - I T - M S experiments and fragments for pure arsenosugars E S I - I T - M S experiments for kelp powder extract Comparison o f amounts of demethylation when using different concentrations o f  35 36 47 60  acids  74 77  Table 2.6. Studies to determine causes o f demethylation Chapter 3 Table 3.1. Table 3.2. Table 3.3. Table 3.4.  H P L C conditions for arsenic speciation 92 H P L C conditions for antimony speciation 92 Operation parameters for I C P - M S 93 Summary o f pure culture experiments 95 Table 3.5. Y M Broth ingredients and composition 96 Table 3.6. Arsenic species in edible mushrooms 101 Table 3.7. Comparison o f proportions o f arsenic species in mushrooms in the current study with those found in published studies 102 Table 3.8. Total concentrations o f arsenic obtained by I C P - M S analysis for experiments conducted with S. citrinum 106 Table 3.9. Proportions o f arsenic species (%) in experiments conducted with S. citrinum 107 Table 3.10. Arsenic species (% o f arsenic extracted) found in wild specimens o f M procera and S. crispa (from Slejkovec etal.) 109 Table 3.11. Concentrations o f arsenic species in experiments conducted w i t h M procera and S.  crispa.  110  Table 3.12. Antimony in mushrooms after acid digestion, analyzed by hydride generation-GCAAS  113  Table 3.13. Antimony extracted from mushrooms and soils (ppm dry weight), H G - G C - A A S analysis  114  Table 3.14. Antimony in media and biomass extracts o f Pleurotus flabellatus grown in submerged culture Table 3.15. Relative amounts o f antimony compounds (%) in some P. flabellatus samples analyzed by using Method A  116 119  Table 3.16. Concentration o f total antimony (ppm) in media and biomass extracts o f Scleroderma citrinum grown in submerged culture  126  viii  Chapter 4 Table 4.1. Table 4.2. Table 4.3. Table 4.4. Table 4.5. Table 4.6.  Operation parameters for I C P - M S H P L C conditions for arsenic speciation Some physical and chemical characteristics of Meager Creek waters Samples, sampling location, sampling times and arsenic levels in biota samples Estimated concentrations o f arsenic species in biota samples Percent amounts o f arsenic extracted  138 139 141 142 146 159  Chapter 5 Table 5.1. Operation parameters for I C P - M S Table 5.2. H P L C conditions for arsenic speciation Table 5.3. Concentrations of total arsenic and arsenic species in water samples and soil  173 173  extracts  175  Table 5.4. Total arsenic concentrations in fish from location 9 178 Table 5.5. Comparison o f arsenic concentrations by using protease and acid digestion methods for oyster tissue (NIST 1566) Table 5.6. Concentrations o f arsenic species in fish from location 9 Table 5.7. Moisture content and arsenic concentration in freshwater shellfish Table 5.8. Concentrations o f arsenic species in freshwater mussels and snails Table 5.9a. Arsenic concentrations in plants from Yellowknife Table 5.9b. Arsenic concentrations in plants from Yellowknife, previous study Table 5.10. Concentrations of arsenic species in Yellowknife plants Table 5.11. Percent arsenic species of total arsenic extracted from plants Table 5.12. Arsenic concentrations in algae from Yellowknife Table 5.13. Concentrations of arsenic species in Yellowknife algae Table 5.14. Arsenic concentrations in mosses from Yellowknife Table 5.15. Concentrations of arsenic species in Yellowknife mosses Table 5.16. Arsenic concentrations in lichens and fungi from Yellowknife Table 5.17a. Concentrations o f arsenic species in Yellowknife lichens Table 5.17b. Concentrations o f arsenic species in Yellowknife fungi Table 5.18. Proportions of total arsenic extracted, for lichens and mushrooms  180 181 188 189 196 197 200 201 205 207 210 212 216 218 219 222  Chapter 6 Table 6.1. Operation parameters for I C P - M S  241  Table 6.2. G C - M S parameters  243  Table 6.3a. Total antimony, extracted antimony species and estimated extraction efficiency in extracts o f environmental biota samples  245  Table 6.3b. Total antimony, antimony species and percent Sb species of total Sb in environmental samples o f water 246 Table 6.4. Comparison of total concentrations of antimony and arsenic for selected samples . 2 5 2 Table 6.5. Relative amounts for methyantimony peaks in moss and water samples 254 ix  LIST OF FIGURES Chapter 1 Figure 1.1a. The Challenger mechanism, showing alternating reducing and oxidative addition steps  9 9 11  Figure 1.1b. The structure for S-adenosylmethionine ( S A M ) Figure 1.2. Pathway showing the formation o f dimethylarsinoribosides and arsenobetaine Figure 1.3. Pathway showing the formation of a trimethylarsonioriboside followed by the formation o f arsenobetaine  13  Chapter 2 Figure 2.1. Chromatogram o f standard arsenic compounds by using Hamilton P R P - X 1 0 0 anion exchange column (15 cm) with 20 m M ammonium phosphate, p H 6  39  Figure 2.2. Chromatogram o f standard arsenic compounds by using Whatman S C X cation exchange column with 20 m M pyridinium formate, p H 2.7  41  Figure 2.3. Chromatogram o f standard arsenic compounds by using ion-pairing reversed phase chromatography, with G L Sciences C I 8 column and 10 m M T E A H / 4 . 5 m M malonic acid, p H 6.8, 0 . 1 % M e O H 44 Figure 2.4. Mass spectrum o f arsenosugar X standard, M S - M S with m/z 329 selected, positive mode 49 Figure 2.5. Mass spectrum of arsenosugar X I standard, M S - M S with m/z 483 selected, positive mode '. 51 Figure 2.6. Mass spectrum o f arsenosugar X I standard, M S - M S - M S with m/z 481 selected, then m/z 389, negative mode 52 Figure 2.7. Mass spectrum o f arsenosugar X I I standard, M S - M S with m/z 393 selected, positive mode 55 Figure 2.8. Mass spectrum o f arsenosugar X I I standard, M S - M S with m/z 391 selected, negative mode 56 Figure 2.9. Mass spectrum of arsenosugar X I I I standard, M S - M S with m/z 407 selected, negative mode 57 Figure 2.10. Mass spectrum of arsenosugar X I I I standard, M S - M S - M S with m/z 407 selected, then m/z 285, negative mode 58 Figure 2.11. Mass spectrum of precursor ion m/z 481 in kelp extract subjected to C I D , negative mode 62 Figure 2.12a. Mass spectra for precursor ion m/z 407 in kelp extract subjected to C I D , negative mode. Figure 2.12b. Precursor ion m/z 407 selected, then m/z 285 63 Figure 2.13. Schematic diagram for hydride generation o f stibines, Method 3 69 Figure 2.14. Percent amounts o f stibines generated from M e S b C l at varying p H , when using HG-GC-AAS 72 3  2  Figure 2.15a. Chromatogram o f an aqueous fungus extract analyzed at neutral p H (water only, unbuffered). Figure 2.15b. Chromatogram of the same aqueous fungus extract analyzed at p H 6.2, citrate buffer. Figure 2.15c. Chromatogram o f antimony-free aqueous fungus extract spiked with 200 ng M e S b C l , analyzed as in Figure 2.15a. Figure 2.15d. Chromatogram o f 200 ng M e S b C l in water analyzed as in Figure 2.15a 76 3  3  2  2  Chapter 3 Figure 3.1. Chromatograms o f standard antimony compounds (100 ppb each) on two H P L C I C P - M S systems 118 Figure 3.2. Chromatogram (Method A ) o f medium after 14 days o f growth for P. flabellatus amended with Sb ( V ) 120 Figure 3.3. Chromatograms of P. flabellatus media and biomass extracts (Method B ) for experiments amended with Sb ( V ) 122 Figure 3.4. Chromatograms o f unknown B 4 (Method B 124 Figure 3.5. R a w area counts for Sb(OH) ~ in media for P. flabellatus grown in Sb (Ill)-amended culture 125 Figure 3.6. Chromatograms for biomass extracts (fresh weight) for S. citrinum experiments (Method A ) 128 6  Chapter 4 Figure 4.1. M a p (not to scale) o f Meager Creek Hot Springs area showing sampling locations  136  Figure 4.2. Chromatograms for Algae 1 and laboratory standards showing the presence o f arsenosugars X and X I  148  Figure 4.3. Chromatograms o f a microbial mat extract (top layer, microbial mat) and extract spiked with arsenosugar X I  149  Figure 4.4. Seasonal arsenic speciation in higher plants, sedge (Scirpus sp.) and fleabane (Erigeron sp.)  154  Figure 4.5. Seasonal and spatial arsenic speciation in moss (Fumaria hygrometrica)  157  Chapter 5 Figure 5.1a. M a p o f Royal Oak Giant mine property and surrounding area 169 Figure 5.1b. M a p o f Yellowknife, showing Royal Oak Giant Mine and Miramar C o n Mine.... 170 Figure 5.2. Chromatogram o f protease digest (PD) o f a Yellowknife fish 184 Figure 5.3. Relative amounts o f arsenic species in moss and associated organisms from Yellowknife ( Y K ) and Meager Creek ( M C ) 213 Figure 5.4. Arsenobetaine ( A B ) in lichens 220 Figure 5.5. Relative amounts o f arsenic species in the puffball mushroom Lycoperdon sp. from location 7 (Giant Mine tailings pond) and location 15 (Con Mine tailings pond) 223  xi  Figure 5.6. Chromatogram ofPaxillus involutus extract (diluted lOx), showing the presence o f unknown compounds  225  Chapter 6 Figure 6.1a. M a p (not to scale) of Meager Creek H o t Springs area showing sampling locations  Figure 6.1b. M a p of Yellowknife sampling locations Figure 6.2. Chromatogram obtained by using H G - G C - A A S (217.6 nm) showing stibines  239 240  generated at neutral p H from 100 m L o f a sample o f standing water from location 4 in Yellowknife 248 Figure 6.3. Chromatogram mass spectrum following H G - G C - M S for M e S b generated from 30 ngMe SbCl 255 Figure 6.4. Chromatogram mass spectrum following H G - G C - M S o f stibines generated from 100 ng M e S b C l (1 M HC1) 257 Figure 6.5. Chromatogram mass spectrum following H G - G C - M S of moss extract (June, Y K Location 1) ..259 Figure 6.6. Chromatogram mass spectrum following H G - G C - M S of moss extract (August, Y K Location 1) 260 Figure 6.7. Chromatogram and mass spectra, following H G - G C - M S of a moss extract (August, Y K Location 4) 261 Figure 6.8. Chromatogram and mass spectra, following H G - G C - M S of a snail extract 262 3  3  2  3  2  xii  LIST OF ABBREVIATIONS  AB AC AsS BCF BTT CE CEPA CID CR CRM Da DE DMA DMAA DMAE DSA dw EDTA EE ESI-IT-MS FAAS FAB FID fw GFAAS HG-GC-AAS HPLC i.d. ICP-MS IS LOD M+H M-H M/W m/z MC MeOH MMA MMAA MS MSA MS" NIST o.d.  arsenobetaine arsenocholine extract containing arseno sugars bioconcentration factor B i l l ' s Toxic Team capillary electrophoresis Canadian Environmental Protection Agency collision induced dissociation Campbell River certified reference material Dalton (atomic mass unit) digestion efficiency dimethylarsinate dimethylarsinic acid dimethylarsinylethanol dimethylstibinic acid dry weight ethylenediaminetetraacetic acid extraction efficiency electrospray ionization-ion trap-mass spectrometry flame atomic absorption spectrometry fast atom bombardment flame ionization detection fresh weight graphite furnace atomic absorption spectrometry hydride generation-gas chromatography-atomic absorption spectrometry high performance liquid chromatography inner diameter inductively coupled plasma-mass spectrometry ionspray limit o f detection molecular ion + H molecular ion - H methanol/water (1:1) extraction mass to charge ratio Meager Creek methanol methylarsonate methylarsonic acid mass spectrometry methylstibonic acid tandem mass spectrometry National Institute for Science and Technology outer diameter xiii  OES PC PD ppb ppm PTFE R RBF SAM SD SFC SUDS TEAH TMA TMAO TRA TSbO UK US-EPA US-FDA UV v/v w/v WHO X-XIII YK  optical emission spectrometry pixie cups protease digestion parts per billion, ng/g, pg/kg, or ug/L parts per million, ug/g, mg/kg, or mg/L poly(tetrafluoroethylene) ratio obtained from (fresh weight mass)/(dry weight mass) round bottom flask S-adenosylmethionine standard deviation supercritical fluid chromatography Sudden Infant Death Syndrome tetraethylammonium hydroxide trimethylarsenic (unspecified structure) trimethylarsenic oxide time resolved analysis trimethylantimony oxide unknown compound United States Environmental Protection Agency United States Food and Drug Administration ultra violet volume per volume weight per volume W o r l d Health Organization arsenosugars X through X I I I Yellowknife  xiv  ACKNOWLEDGMENT I will never be able to express how deeply grateful and warmly attached I feel towards all the people who have been a part o f my life for the last five years. M y life would not have been enjoyable or productive without them. First, I would like to thank my supervisor, B i l l Cullen, who kept me inspired when I most needed it. I am also very grateful to my supervisory committee, including M i k e Blades, Guenther Eigendorf, T o m Pederson, and especially David Chen. M y second supervisor, K e n Reimer, gave me many opportunities to travel and expand my skills, and I thank him for all his help. Other people at R M C have been invaluable to me, including Chris Ollson, Ian Mace, Chris Knowlton, Wayne Ingham, Dave Pier and many o f my other friends there. The people in Yellowknife without whom the Yellowknife sampling trips would not have happened are Steve Harbicht at Environment Canada and Steve Schultz from Giant Mine, among others. I have learned much about fields other than chemistry and I am very grateful to Elena Polishchuk and Paul Haden for their expertise and excellent teaching skills. I am also indebted to the experts who helped me with identification o f biological samples, including D r . W . B . Schofield, Olivia Lee, Julie Oliveira, James Black and M i k e Fournier. I would also like to thank Martin Tanner and Paul Morgan for the use of the French press. I am particularly grateful to Bert Mueller and his expertise with the I C P - M S . I am indebted to the present and former members o f my research group, for many fruitful discussions and collaborations, as well as friendship. They include Bianca Kuipers, Vivian Lai, Corinne Lehr, Paul Andrewes, L i x i a Wang, Changqing Wang, Kirsten Falk, Dietmar Glindemann, Sepp Lintschinger, Jorg Feldmann, Andrew M o s i , Chris Harrington, Spiros Serves, and especially Chris Simpson. Summer students who have been a great help are Meghan Winters and L i n Tran. Some o f the above people were kind enough to read through parts o f my thesis and I thank them, as well as Graham Cairns, for it. Finally I would like to thank my friends, my sister and my parents for their love and support, and without whom I would not have reached my goal.  XV  Chapter 1  INTRODUCTION  " A n y wild place is filled with incredible things happening." David Cavagnaro, This Living Earth  The chemistry o f the environment is a complex area o f study, reaching into the fields o f biology, geology, physics, toxicology and medicine, among others. T w o crucial aspects o f environmental chemistry are the identification o f chemical contaminants and the determination o f their fate in natural and anthropogenic environments. This knowledge is often used to discover and solve problems caused by the presence o f chemical contaminants. Such problems become of utmost concern when they include adverse health effects in humans, animals or plants. Metals or metalloids are often found in the environment as chemical contaminants. T w o examples are arsenic and antimony, which are closely related in chemical behaviour.  1.1. Historical aspects of arsenic and antimony  B o t h arsenic and antimony have had paradoxical uses throughout history, as poisons and as panaceas. Arsenic trioxide, known as "white arsenic" is historically one o f the most common poisons. Strangely, this same compound was used until recently in a region in Austria by the "arsenic eaters", who attempted to increase their strength, virility and longevity by ingesting arsenic trioxide daily . Hippocrates (460-377 B . C . ) recommended the use o f a paste o f realgar 1  ( A s S ) to treat ulcers . In the early part o f this century (1900-1950), organoarsenic compounds, 2  4  4  such as salvarsan, neosalvarsan and atoxyl, were used for the treatment o f syphilis and sleeping sickness . Lewisite ( C l C H = C H A s C l ) , a severe blistering agent, was developed for chemical 2  2  warfare purposes. Arsenic trioxide is thought to be the active ingredient in current traditional Chinese treatments o f arthritis, skin disorders and even cancer . 3  Antimony also has a fascinating history. It is mentioned in the Bible as a cosmetic and it was commonly used up until the mid-18 century as a medicine to produce sweating, as an th  emetic, as a purgative, or all three . Many preparations o f antimony are described in the well4  known "The Triumphal Chariot o f Antimony" by Basil Valentine, first published in 1604 . 5  Fatalities resulting from its use did not prevent its mention in Martindale's Extra Pharmacopoeia, as recently as 1941  4  Antimony was o f great interest to alchemists, and one o f its properties was  that it "united with or devoured all the metals then known, with the exception o f gold" . Some 6  cases o f alleged deliberate antimony poisoning also have been documented . 4  1.2. Chemistry of arsenic and antimony  Arsenic and antimony are found in Group 15 o f the periodic table. A s metalloids, they possess characteristics o f both metals and non-metals. Antimony is more metallic than arsenic because it can exist as aqueous cationic species, [SbO ] and [ S b 0 ] (but not free [Sb ] or +  +  3+  2  [Sb ]), and its trioxide is amphoteric, dissolving in both strong acid and strong base. Arsenic, on 5+  the other hand, forms oxy-anionic species only. Its trioxide is acidic and hence dissolves in base, which is characteristic o f non-metals . Both arsenic and antimony form hydrides, implying that, 7  like non-metals, they can also exist in a negative oxidation state.  2  Arsenic and antimony possess five valence electrons like other group 15 elements. Their four oxidation states are -3, 0, +3 and +5. A n antimony compound with composition SD2O4 has been observed, implying that a +4 oxidation state is possible, but this oxide has been shown to contain only S b and Sb v  ffl  atoms . 8  The arsenic and antimony compounds that are pertinent to this thesis are either hydrides, organometallic compounds (possessing a A s - C or Sb-C bond), oxyanions, or complexes o f the inorganic cations. Table 1.1 and Table 1.2 show names, abbreviations and structures for some arsenic (Table 1.1) and antimony (Table 1.2) compounds.  3  Table 1.1. Names, abbreviations and structures o f some arsenic compounds.  Name  Abbreviation  Structure/Formula  As(V)  As(0)(OH)  A s (III)  As(OH)  Monomethylarsonic acid"  MMA  CH AsO(OH)  Dimethylarsinic acid  DMA  (CH ) AsO(OH)  TMAO  (CH ) AsO  Arsenic acid, arsenate  a  Arsenous acid, arsenite  3  a  Trimethylarsine oxide  b  3  3  3  3  3  MeAsH  Dimethylarsine  Me AsH  (CH ) AsH  Trimethylarsine  Me As  (CH ) As  Arsenobetaine  CH AsH  2  3  2  3  3  Me As  b  3  2  2  3  (CH ) As  +  4  3  2  2  Methylarsine  Tetramethylarsonium ion  3  +  4  AB  (CH ) As CH COO"  AC  (CH ) As CH CH OH  Dimethylarsinylethanol  DMAE  (CH ) As(0)CH CH OH  Arsenosugars  X-XIII  See figure below  b  Arsenocholine  b  c  +  3  2  +  3  3  3  2  2  2  2  2  OH  o  I  II (CH ) AsCH 3  3  2  OCH CHCH — R 2  2  Sugar  R  X XI XII XIII  -OH -OP0 HCH CH(OH)CH OH -S0 H -OS0 H 3  2  2  2  3  3  These compounds are commercially available. These compounds are available in our lab, having been synthesized according to common methods (see Chapter 2). These compounds are available in our lab; they are found in a laboratory reference material (kelp powder) and have also been donated (see Chapter 2). Numbering system (X-XIII) according to Shibata et al  a  b  c  d  9  Table 1.2. Names, abbreviations and structures of some antimony compounds. Name  Abbreviation  Structure/Formula  Sb(V)  Sb(OH)<f  Pentavalent inorganic antimony  Sb ( V )  inorganic S b (complexed)  Antimony trioxide"  Sb (III)  S b 0 , Sb(OH)  Antimonate  3  v  2  Antimony trisulfide Antimony potassium tartrate  3  Sb S 2  3 ( a q )  3  Sb (III)  3  \ / \ / CH-O O-CH  K  2  0  ^  C  CH-O O-CH / w_ \ - 0 - 0 ^ ^ S  b  C  0  Trivalent inorganic antimony  Sb (III)  inorganic S b (complexed)  Methylstibonic acid  MSA  CH SbO(OH)  Dimethylstibinic acid  DSA  (CH ) SbO(OH)  Trimethylantimony oxide  TSbO  (CH ) SbO  Trimethylantimony dihydroxide Trimethylantimony dichloride  b  b  m  3  3  3  Me Sb(OH) 3  Me SbCl 3  2  2  2  3  (CH ) Sb(OH) 3  3  (CH ) SbCl 3  3  Methylstibine  MeSbH  Dimethylstibine  Me SbH  (CH ) SbH  Trimethylstibine  Me Sb  (CH ) Sb  2  3  2  2  CH SbH 3  3  3  2  2  2  2  3  These compounds are commercially available. These compounds are available in our lab, having been synthesized according to common methods (see Chapter 2).  a  b  5  1.3. Toxicity of arsenic and antimony compounds  Although the word "arsenic" is usually immediately associated with poison, the actual toxicity o f arsenic in a sample is dependent on the chemical form that it takes. The same principle applies for antimony. Arsenic and antimony compounds in the -3 oxidation state are more toxic than those in the +3 oxidation state, which are more toxic than those in the +5 oxidation state. Organometallic compounds in the +5 oxidation state are less toxic than inorganic ones and some compounds, like arsenosugars and arsenobetaine, have not exhibited any toxic behaviour at all in the systems tested ' ' . 10  11  12  The dependence o f toxicity on the chemical  form (or species) o f arsenic and antimony makes their identification (i.e., speciation analysis) necessary. Trivalent arsenic is thought to exert its toxicological effects by binding with sulfhydryl groups on enzymes ' . Inhibition o f pyruvate dehydrogenase takes place for both trivalent 2 13  arsenic and trivalent antimony ' . Pentavalent arsenic is thought to compete with phosphate 13 14  during phosphorylation to form unstable arsenyl esters, interfering with bioenergetic processes ' . Trivalent antimony binds easily in vivo to sulfhydryl groups, such as those on 2 13  enzymes, most likely constituting the toxic action o f these compounds ' . Pentavalent antimony 15  14  is excreted rapidly from the body but it has been shown that the liver can reduce Sb ( V ) to Sb (III) . 16  The International Agency for Research on Cancer ( I A R C ) has determined that there is sufficient evidence to consider arsenic a human carcinogen, and to consider antimony trioxide an animal (but not human) carcinogen . 17  6  1.4. Environmental chemistry of arsenic  1.4.1. Arsenic sources, uses and exposure to humans  Arsenic is the twentieth most abundant element in the Earth's crust, and is often associated with sulfidic ores such as arsenopyrite (FeAsS), enargite  (CU3ASS4),  orpiment ( A s S ) , 2  3  realgar (AS4S4) and proustite ( A g A s S ) ' . Arsenic is usually recovered as arsenic trioxide 13  3  18  3  from the processing o f ores, and its major present uses are in the production o f arseniccontaining agricultural pesticides (including disodium methylarsonate, sodium methylarsonate, methylarsonic acid ( M M A ) and dimethylarsinic acid ( D M A ) ) ; wood preservatives (including chromated copper arsenate ( C C A ) , ammoniacal copper arsenate ( A C A ) and fluorchrome arsenate phenol ( F C A P ) ) ; and animal feed additives (including arsanilic acid and 4nitrophenylarsonic acid) ' . Arsenic is also used as a doping agent in solid-state products . 13  19  19  Arsenic can enter the environment anthropogenically as a consequence o f its industrial use, from mining and smelting operations and through the application o f arsenic-containing pesticides. Natural inputs o f arsenic to the environment can result from the weathering o f rocks and geothermal activities, leading to open ocean arsenic concentrations typically ranging from 18 70  18  0.0056-11 ppb  and terrestrial soil and rock concentrations ranging from 0.4-100ppm ' .  Humans can be exposed to arsenic through inhalation, especially for workers in industries utilizing arsenic, through food (such as fish, shellfish, or marine algae) and through drinking water (e.g., in India, groundwater supplies contain elevated levels o f arsenic ). 21  Arsenic is considered to be a priority pollutant by the United States and the Canadian Environmental Protection Agencies ( U S - E P A and C E P A ) . U S - E P A and C E P A permit drinking water levels to be a maximum o f 5 0  22  and 25 ppb  23  (u,g/L), respectively. The W o r l d Health 7  Organization ( W H O ) recommends a daily limit through food o f 0.05 mg total arsenic/kg body weight , but for inorganic arsenic a weekly limit o f only 15 ug A s / k g body weight is suggested' 22  1.4.2. Arsenic species in the marine environment  The major species o f arsenic in seawater is A s ( V ) ' , although sometimes as much as 25  26  10-30% o f the total arsenic takes the form o f A s (III), M M A and D M A in waters from the euphotic zone, in deeper ocean waters and in interstitial waters ' ' . Arsenate is 20  25  26  thermodynamically predicted to be the major compound in oxygenated seawater ( p H 8 )  20  and  therefore the presence o f A s (III) and methylated species imply that biotransformation is taking place. This is indeed observed to be the case because marine algae , bacteria and fungi 27  20  methylate arsenic. The Challenger mechanism (Figure 1.1a), involving alternating steps o f 28  reduction and oxidative addition o f a methyl group, is considered to be a possible methylation pathway. Studies have shown that S-adenosylmethionine ( S A M ) (Figure l i b ) acts as a methyl donor . 20  8  1.1a AsO(OH)  2  CH3As(OH) (CH ) AsO 3  6  3  » As(OH) ^  2  2 e  3  " »  C  h  3  3  » CH3AsO(OH) CH3As(OH) 2  (CH )2AsO(QH) 3  2 e  ~ » (CH ) As(OH) 3  V  2  2  (CH ) AsO 3  3  (CH ) As 3  3  OH  OH  Figure 1.1a. The Challenger mechanism , showing alternating reducing and oxidative addition steps. Figure 1.1b. The structure for S-adenosylmethionine ( S A M ) ; the circled methyl group is donated during the methylation steps in the mechanism.  In marine sediments arsenic concentrations can range from 0.4-455 p p m . The majority 18  of arsenic in sediment is associated with Fe and M n oxides and some is associated with carbonates and organic material . M M A and D M A have been found in environmental marine 20  sediment samples . Laboratory experiments conducted aerobically and anaerobically with 20  cultures from marine sediments, amended with M M A and D M A , suggest that tetramethylarsonium ion may be formed and thus may be present in trace quantities . Reliable 29  evidence for the presence o f arsenobetaine (but not A C , T M A O or M e A s ) in environmental +  4  'if)  estuanne waters now exists .  9  Marine algae contain levels o f arsenic that are considerably higher than the levels in the surrounding water, ranging from 0.8 to 12.1 ppm wet weight . The major water-soluble forms 31  o f arsenic in most species o f marine algae are the arsenosugars, some o f which are shown in Table l . l ' . One exception is a Japanese edible algae, hijiki (Hizikiafusiforme), which 3 1  3 2  contains 50% o f its arsenic as arsenate . 33  Marine animals also contain higher levels o f arsenic compared to the levels in the surrounding water , although biotransformation studies via the marine food chain indicate that 31  biomagnification o f arsenic probably does not take place . In contrast to most previous studies, 34  one study has shown that biomagnification takes place in a short marine food chain (seaweed -> herbivorous marine gastropod - » carnivorous marine gastropod) . Arsenobetaine is the major 35  arsenic compound in most marine animals, although the tetramethylarsonium ion has been found in marine snails and clams, arsenocholine has been found in gastropods and dogfish muscle , 36  trimethylarsine oxide has been found in fish, and arsenosugars have been found in bivalve mollusks . 31  A pathway for the formation o f arsenosugars, and, from them, arsenobetaine, has been proposed and is shown in Figure 1.2 ' . 32 37  10  (CH )2AsO(OH)  2 e  3  SAM  ~ > (CH ) As(OH) 3  2  CH HoN (CH ) As(OH) 3  +  N  <  3  S-hCH  2  .0,  +  2  OH  OH  NH, O (CH ) AsCH v .0 ^N 3  2  2  M OH  N  N  ( 2  -  1 )  OH  O  O (CH ) AsCH . 3  2  2  OH  /O/QR  R  (CH ) AsCH .  +  3  .0  O [O]  (CH ) As. 2  (2.2)  2  OH  OH  O  3  2  (CH ) As. 3  X>H  2  (2.3)  2e~, then I CH  XOO" 2e", then CH +  +  3  3  [O]  (CH ) As3  3  *0H  Arsenocholine (AC)  (CH ) Asv^^COO" 3  3  Arsenobetaine (AB)  Figure 1.2. Pathway showing the formation o f dimethylarsinoribosides and arsenobetaine . 30  N  The pathway for the synthesis o f arsenosugars is similar to the Challenger mechanism up until the formation o f D M A (the starting molecule in Figure 1.2). After the reduction of D M A to ( C H ) A s O H , S-adenosylmethionine ( S A M ) is proposed to be the donor o f the ribosyl moiety. 3  2  The intermediate 2.1 in Figure 1.2 has been isolated from the kidney o f the clam Tridacna maxima (symbiotic algae grow in the mantle o f the clam and algal products accumulate in the kidney), providing support for this pathway . The formation o f arsenobetaine from 38  arsenosugars, as proposed in Figure 1.2, involves an intermediate compound 2.2 (dimethylarsinoylethanol, D M A E ) , which has not yet been identified as a naturally occurring compound . However, trace amounts o f dimethylarsinoylacetic acid (2.3), as well as 32  arsenosugars and arsenobetaine, have been reported in a mussel sample , which may lend 39  support to one o f the routes in the proposed pathway in Figure 1.2. Small amounts o f a trimethylarsonioriboside (Figure 1.3, 3.1) have been identified in algae leading to the proposal o f an alternate pathway for the formation o f arsenobetaine, shown 40  in Figure 3. The formation o f arsenobetaine from the trimethylarsonioriboside has been demonstrated to be a facile process in laboratory experiments ' , which may indicate that 41 42  although the levels o f the starting trimethylarsonioriboside are very l o w in algae, this pathway may, to some extent, contribute to the formation o f arsenobetaine in the marine environment . 32  12  o  (CH ) AsCrW 3  OCH CH(OH)CH OS0 ~  ,0  2  OH  2  OH  2  3  2e"  ( C H ) A s C H \ .0 3  2  2  OH CH  .OCH CH(OH)CH OS0 " 2  2  3  OH  + 3  ( C H ) A s C H \ / 0 'VOCH CH(OH)CH OS0 " +  3  3  2  2  OH  (CH ) AsV/\ 3  3  OH  On  2  3  (3-1)  JC^  (CH ) AsV COO" 3  Arsenocholine (AC)  3  v/  Arsenobetaine (AB)  Figure 1.3. Pathway showing the formation o f a trimethylarsonioriboside (3.1) followed by the formation o f arsenobetaine . 30  13  M u c h is known about arsenic species in the marine environment, and reviews by Cullen et al.  20  and Francesconi et al. address this topic in more detail than that given here. Such a 32  wealth o f knowledge is available because of the ubiquitous presence o f arsenic in marine samples, as well as sufficiently high levels o f arsenic for reliable speciation analysis. However, the information that is obtained is dependent on the analytical methods available and as a result, little reliable data exists for arsenic compounds that are not water soluble. Additionally, the presence o f trace amounts o f crucial intermediates in the formation o f organoarsenicals in environmental samples is dependent on the detection limits possible by using current analytical methods.  1.4.3. Arsenic species in the terrestrial environment  Arsenic concentrations in freshwater systems vary depending on the geological composition o f the area and the input from anthropogenic sources, such as agriculture or mining operations. Levels in rivers and lakes range from 0.1 to 75 ppb in some reports , and they can 43  be substantially elevated in hot springs (up to ppm levels) . A s ( V ) is usually the major species, 44  but high proportions (30-75% o f total arsenic) o f A s (III) have been known to occur ' , as well 21  45  as M M A , D M A , T M A O and trivalent methyl- and dimethylarsenic species ' . Methylation o f 46 47  inorganic arsenic by anaerobic microorganisms associated with lake sediments has been observed , and biomethylation is expected to take place following the pathway proposed by 48  Challenger (Figure 1.1). In soils, arsenic is associated with Fe and A l compounds . The amount o f arsenic in the 49  fraction that is soluble and hence available to plants has been estimated to be 0.07-0.2% of the estimated total, and the species o f arsenic in this fraction were found to be A s ( V ) and A s (III) . 50  Another study showed that from 9 soil samples, only one sample contained arsenite in a  proportion greater than 1.38% o f the estimated total arsenic, and A s ( V ) was assumed to constitute the remainder . However, arsenic in terrestrial soil and sediment is not limited to 51  inorganic forms only; ( C H ) A s H , ( C H ) A s H , ( C H ) A s , ( C H ) ( C H ) A s , ( C H ) ( C H ) A s and 3  2  3  2  3  3  2  5  3  2  2  5  2  3  ( C H ) A s were generated with sodium borohydride from river sediments . 52  2  5  3  A s will be discussed in Chapters 4 and 5, few studies have examined the speciation o f arsenic compounds in plants and other terrestrial biota, and details o f previous studies are given in sections 4.3 and 5.3. A Japanese study concludes that methyl species, o f unknown chemical structure, are present in a green alga, a diatom, a freshwater prawn, a marsh snail, freshwater fish and fly larvae, sampled from an area impacted by geothermal waters and containing elevated arsenic levels . Mostly inorganic species, some D M A and trace amounts of M M A and 53  arsenobetaine are present in ants living in an arsenic contaminated area . Vegetables grown in 54  arsenic amended soil were found to contain mostly arsenate, but trace amounts of M M A were present in lettuce, potato and swiss chard . 55  The findings o f arsenobetaine and arsenocholine, but not arsenosugars, in mushrooms ' ' ' 56  57  58  59  led to the postulation by some researchers that arsenosugars are not involved  in the biosynthesis o f arsenobetaine . 32  Since the discovery o f arsenosugars in the terrestrial  environment ' , however, the possibility has emerged that pathways (e.g., Figure 1.2) in the 60 61  terrestrial environment are similar to those in the marine environment. M o s t studies to date indicate that arsenobetaine and arsenosugars are not as common in the terrestrial environment as they are in the marine environment. Among mushrooms, for example, the form o f arsenic varies greatly from one species to the next. Some species contain only inorganic arsenic, and others contain only arsenobetaine. Clearly, studies to determine the arsenic compounds present in terrestrial samples are necessary to elucidate the chemical processes taking place in the terrestrial environment.  15  1.5. Environmental chemistry of antimony  1.5.1. Antimony sources, uses and exposure to humans  Antimony occurs at about one-tenth the concentration o f arsenic in the Earth's crust, and is usually found as stibnite (Sb S ), as well as in ores o f copper, silver and lead . The major use 62  2  3  of antimony is in the form o f Sb 03, as a flame retardant in textiles, paper and plastics ' . 62 63  2  Antimony compounds are used to a lesser extent in paints and ceramics; as catalysts, glass decolourizers and metal hardeners; and in the semiconductor industry ' . Antimony compounds 62 64  are also used as a treatment for tropical parasitic diseases ' . 65 66  Sources o f antimony in the environment are usually similar to those for arsenic, since antimony often occurs together with arsenic-containing ores . Hence, weathering o f rocks in 67  areas with high arsenic and antimony content, geothermal activities, mining and smelting operations, and industries utilizing antimony can all contribute to the introduction o f antimony into the natural environment. F o r example, high levels o f antimony relative to other metalloids have been observed in landfill and sewage sludge fermentation gases ' . 68  69  Humans are not frequently exposed to antimony, except in working conditions that involve antimony, such as battery charging , antimony processing, and welding . Usually, 64  66  ecological exposure to antimony is also accompanied by exposure to other toxic compounds, such as those o f lead, arsenic, cobalt or silica . Nevertheless, the U S - E P A considers antimony 66  to be a priority pollutant ' 70  71  and the threshold limit permitted for antimony and its compounds in  work room air is 0.5 mg Sb/m  37 0  . The United States Food and D r u g Administration ( U S - F D A )  tolerates a maximum o f 2 ppm o f antimony in food , and the accepted daily limits for humans 66  16  orally exposed to antimony compounds (over an extended period o f time) range from 24.5-32.5 u.g o f antimony compound per day . 62  1.5.2. Antimony species in the environment  Sb ( V ) species are thermodynamically most favourable under aerobic conditions and hence Sb(OH)6~ is predicted to be the predominant species o f inorganic antimony in oxygenated water . Sb (III) compounds are thermodynamically predicted to be oxidized to Sb ( V ) at neutral 72  p H and in an oxidizing environment. Sb 03 dissolves to a limited extent in water at neutral p H 2  (<5 ppm), forming an undissociated species, that is, H S b 0 or Sb(OFf)3 . The structure o f 72  2  organoantimony compounds in aqueous solution is unknown, except for M e S b C l , M e S b B r 3  2  3  2  and M e S b I , which hydrolyze at neutral p H to form M e S b ( O H ) , and possibly M e S b 0 ' . 7 3  3  2  3  2  7 4  3  In accordance with the thermodynamic prediction, most studies report predominantly Sb (V) with small proportions o f Sb (III), methyl- or dimethylantimony species in seawater ' , 75 76  estuarine waters ' , r i v e r s ' ' , waste waters ' , geothermal waters 75  77  75  78  79  80  81  82  and condensed water  from landfill gas . Levels o f antimony in waters are typically less than 1 ppb , although polluted 69  62  waters can contain levels o f antimony from 300-800 p p b ' . 75  81  Sb ( V ) as Sb(OH) " was the major species extracted from soils , and it was postulated 82  6  that M e S b O was present in one such extract . Compounds forming C H S b H , ( C H ) S b H , 80  3  3  2  3  2  ( C H ) S b and ( C H ) S b were found in river sediment that was reacted with sodium 3  3  2  5  3  borohydride , indicating the presence o f small, but detectable levels o f organoantimony 52  compounds in soils and sediments. Antimony was shown to be associated with iron and aluminum in sediments , but in soils it was mostly in a form not extractable by sequential 83  leaching procedures; the highest extractable amount was bound to F e - M n oxides . Antimony 84  17  concentrations o f up to 1489 ppm have been found in soil samples near an antimony smelter . In contrast, the levels o f antimony in the Earth's crust are estimated to be between 0.2 and 1 ppm . 66  Less than 1% o f antimony can be extracted from soil by using an aqueous extraction method , 82  so it is not surprising that levels in human fluids were not significantly elevated, with respect to controls, following internal exposure to antimony contaminated soil . 86  The volatile antimony compounds S b H and MesSb have been found in gases sampled 3  above hot springs, landfills and from sewage ' ' . 68  69  87  Very few studies have speciated antimony in  biota. D o d d et al. found methylated antimony species in aquatic plants collected from an area impacted by mining and hence containing elevated levels o f antimony . Kantin reported mostly 88  Sb ( V ) in extracts o f marine algae, with low levels o f Sb (III) in some samples , and similar 89  results were found in extracts o f mollusk shells, with only one sample containing Sb (III) . Total 90  levels o f antimony were found to be elevated in biota collected near an antimony smelter with respect to those collected from control areas, and the authors concluded that uptake, but not biomagnification, o f antimony takes place ' . 85  91  1.5.3. Biological and chemical transformations of antimony  The findings o f methylated antimony compounds in environmental samples, as mentioned in section 1.5.2, suggest that methylation is taking place in the environment. Recently, it was confirmed that biological methylation o f antimony takes place by anaerobic soil cultures and, to 92  a lesser extent, by aerobic cultures o f the fungus Scopulariopsis brevicaulis . 93,94  The volatile  antimony compound formed, Me3Sb, was found to oxidize rapidly in an aerobic environment ; 94  soluble dimethyl- and trimethylantimony compounds were found in liquid culture media . S. 93  brevicaulis was capable o f converting only 0.001-0.01% o f antimony in liquid culture to 18  M e S b , which is much less than its ability to convert arsenic to M e A s (~ 1%) 3  . The  3  volatilization o f antimony by S. brevicaulis from mattresses contaminated with the fungus and containing antimony as a flame retardant has been suggested to be a cause o f the sudden infant death syndrome (SIDS), or cot death ' . However, stibine, postulated to be the volatile 95 96  compound responsible for the antimony poisoning, has not yet been found as an antimony metabolite formed by S. brevicaulis cultures amended with antimony. This observation, together with the low conversion o f antimony to volatile compounds by the fungus, makes it unlikely that antimony is linked to SIDS. Additionally, reliable epidemiological evidence linking the presence of antimony in mattresses to victims o f SIDS is lacking . 97  Sb (III) is oxidized to Sb ( V ) by fungal cultures as well as by the freshwater alga 98  Chlorella vulgaris".  The antimony that was accumulated by C. vulgaris cells appears to be  bound to proteins whose molecular weight is around 4 x 10 D a . The bacterium Stibiobacter 4  9 9  senarmontii is reported to use S b 0 as a substrate for growth and may also convert this 2  3  compound to Sb ( V ) . Another bacterium Thiobacillus ferrooxidans  has been observed to  oxidize stibnite . 100  Trimethylstibine is insoluble in water, but it quickly and spontaneously forms the water soluble compound M e S b O in the presence o f oxygen. This is postulated to contribute to the 3  mobilization o f M e S b in the environment . Trimethylstibine was observed to form the water 101  3  soluble [Me4Sb ] ion in the presence o f alkyl halides (which may be present in the environment) +  in polar solvents, leading to the speculation that this may also be a process occurring in the aqueous environment . N o evidence exists yet for the presence o f the [ M e S b ] ion in the 102  +  4  environment. The lack o f analytical methodology for the trace detection o f such antimony species likely limits the range o f compounds found in the environment.  19  1.6. Objectives and scope of work  The general theme in this work is to expand the knowledge about the species of arsenic and antimony occurring in the terrestrial environment. The goal of Chapter 2 is to describe improvements and clarifications of some existing analytical methods for arsenic and antimony compounds. These include high-performance-liquidchromatography coupled to inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) and electrospray ionization-ion trap mass spectrometry (ESI-IT-MS) for the analysis of arsenic compounds, and hydride-generation gas chromatography atomic absorption spectrometry (HGGC-AAS) for antimony compounds. Two objectives are sought in Chapter 3; one is to learn more about the pathways to the formation of arsenic compounds in mushrooms, in an attempt to address the uncertainty associated with pathways to the formation of arsenosugars and arsenobetaine, described in section 1.4.2. The other objective is to increase the knowledge of antimony behaviour in the environment by studying controlled laboratory interactions of antimony with organisms. Although some studies concerning the biological transformations of antimony have been carried out (described in section 1.5.3), clearly the number is limited, and nothing is known about the transformations of antimony in biota used for human consumption. In Chapters 4 and 5, the objective is to increase the knowledge of arsenic speciation in terrestrial ecosystems, including biota such as plants and lichens, which have not previously been studied, by using modern speciation methods. This information may be used to help determine the impact of increased arsenic loading in terrestrial environments.  20  The speciation analysis o f antimony in environmental samples in Chapter 6 contributes to the understanding o f antimony in the environment and adds to the limited existing data, especially for biota, as summarized in section 1.5.2.  21  References  1. Most, K . - H . P h . D . Thesis, University o f Graz, Austria, 1939; Przyoda, G . ; Feldmann, J.; Cullen, W . R. English Translation, to be published. 2. Squibb, K . S.; Fowler, B . A . In Biological and Environmental A . , E d . ; Elsevier: Amsterdam, 1983; pp 233-269. 3. Mervis, J. Science 1996,  Effects of Arsenic; Fowler, B .  273, 578.  4. McCallum, R. I. Proc. roy. Soc. Med. 1977,  70, 756-763.  5. Valentine, B . The Triumphal Chariot of Antimony; James Elliot: London, 1893. 6. Mellor, J. W . A Comprehensive Treatise on Inorganic and Theoretical Chemistry; John Wiley: N e w Y o r k , 1960; V o l . I X , pp 339-342. 7. Mortimer, C . E . Chemistry, 6th ed.; Wadsworth: California, 1986; pg 681. 8. Birchall, T.; Delia Valle, B . Chem.Comm.,  1970, 675.  9. Shibata, Y . ; Morita, M . ; Fuwa, K . Adv. Biophys. 1992,  28, 31-80.  10. Shiomi, K . ; Chino, M . ; Kikuchi, T. Appl. Organomet. Chem. 1990, 11. Kaise, T . ; F o k u i , S.Appl.  Organomet. Chem. 1992,  4, 281-286.  6, 155-160.  12. Sakurai, T.; Kaise, T.; Matsubara, C . Appl. Organomet. Chem. 1996,  10, 727.  13. Maeda, S. 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W . - M . ; Cullen, W . R., manuscript in preparation. 88. Dodd, M . ; Pergantis, S. A . ; Cullen W . R ; L i , H . ; Eigendorf, G . K . ; Reimer, K . J. Analyst, 1996, 121, 223-228. 89. K a n t i n R . Limnol. Oceanogr. 1983, 28, 165-168. 90. Cullen, W . R.; Dodd, M . ; Nwata, B . U . ; Reimer, D . A . ; Reimer, K . J. Appl. Organomet. Chem. 1989,3,351-353. 26  91. Ainsworth, N . ; Cooke, J. A . ; Johnson, M . S. Environ. Poll. 1990, 65, 79-87. 92. Gurleyiik, H . ; V a n Fleet-Stalder, V.; Chasteen, T. G . Appl. Organomet. Chem. 1997,11, 471-483. 93. Andrewes, P.; Cullen, W . R.; Feldmann, J.; K o c h , I.; Polishchuk, E . , Appl. Organomet. Chem. 1998, in press. 94. Jenkins, R. O.; Craig, P. J.; Goessler, W . ; Miller, D. Ostah, N . ; Irgolic, K . J. Environ. Tech. 1998, 32, 882-885.  Sci.  95. Richardson, B . A . Lancet 1990, 335, 670. 96. Sprott, T. J. Chem. New Zealand 1995, May, 20-25. 97. Howatson, A . G . ; Patrick, W . J. A ; Fell, G . S.; Lyon, T. D. B . ; Gibson, A . A . M . Lancet, 1995, 345, 1044-1045. 98. Perezcorona, T.; Madrid, Y . ; Camara, C . Anal. Chim. Acta 1997, 345, 249-255. 99. Maeda, S.; Fukuyama, H . ; Yokoyama, E . ; Kuroiwa, T.; Ohki, A . ; Naka, K . Appl. Organomet. Chem. 1997, 11, 393-396. 100. Summers, A . O.; Silver, S. Ann. Rev. Microbiol.  1978, 32, 637-672.  101. Parris, G . E . ; Brinckmann, F. E . Environ.Sci. Tech. 1976, 10, 1128-1134. 102. Parris, G . E . ; Brinckmann, F. E . J.Org.Chem.  1975, 40, 3801-3803.  27  Chapter 2  METHODS FOR THE ANALYSIS OF ARSENIC AND ANTIMONY  2.1. Methods for the Analysis of Arsenic  2.1.1. Introduction Many methods have been proposed for the analysis o f total arsenic in environmental samples, but only the most commonly used ones will be summarized here. Usually samples are digested to convert the arsenic in the sample matrix into a water soluble inorganic species. D r y ashing followed by wet digestion, and wet digestion alone are the most commonly used techniques . Arsenic is then quantified in a sample by analyzing the solution. Photometric 1  analysis following arsenic complexation is known , but has been replaced with hydride generation 2  (HG) techniques preceding flame atomic absorption spectrometry ( F A A S ) , atomic absorption 3  spectrometry ( A A S ) with a quartz tube atomizer ' ' , graphite furnace atomic absorption 4 5 6  spectrometry ( G F A A S ) , inductively coupled plasma (ICP) with optical emission spectrometry 7  ( O E S ) , and I C P with mass spectrometric detection ( M S ) . Direct analysis o f solutions 8  9  containing arsenic (as well as other metals) is very common, using some o f the previously mentioned methods: G F A A S , I C P - O E S and I C P - M S ' ' . Electrochemical methods are 1 0  1 1  1 2  1 3  known as w e l l . Solid samples (subjected to little or no sample preparation, i.e., no wet 14  digestion) are frequently analyzed by using neutron activation analysis ( N A A ) ' 1 5  1 6  and X-ray  fluorescence methods ( X R F ) . 1 7  The speciation o f arsenic compounds is based almost exclusively on chromatographic methods coupled to element-specific detection. These methods include gas chromatography 28  ( G C ) and high performance liquid chromatography ( H P L C ) , as well as the less commonly used supercritical fluid chromatography (SFC) and capillary electrophoresis (CE). Arsenic compounds found in most samples are polar or ionic in character, and not volatile. Therefore they are not directly amenable to G C , so that G C methods usually involve the derivatization o f the arsenic species. Hydride generation is the most common method, using sodium borohydride as the reducing agent, as shown:  3NaBHt  ( a q )  + 2As(OH)  3 ( a q )  + 3 H 0 -> 2 A s H t + 6 H t + 3 N a H B 0 2  3  2  2  3 ( a q )  The aqueous species As(V)/(III), C H A s O ( O H ) , ( C H ) A s O ( O H ) and ( C H ) A s O can 3  2  3  2  3  3  be derivatized to the volatile hydrides A s H , C H A s H , ( C H ) A s H and ( C H ) A s , respectively, 3  3  2  3  2  3  3  with boiling points o f - 6 2 . 5 ° C , 2°C, 36°C and 52°C respectively . The inorganic species o f A s 18  (V) and (III) can be speciated by using different reaction conditions to form A s H . M o r e 3  specifically, A s (III) can be determined alone at pH>4 and A s (III) plus A s ( V ) can be determined quantitatively at pH<2, so that the amount o f A s ( V ) is calculated by difference. A n extraction step to selectively complex A s (III) compounds can be carried out to differentiate between the normally indistinguishable (by H G ) methylarsenic (III) and methylarsenic ( V ) acids, which are then reacted to form their hydrides, and separated by G C . 1 9  Methylarsenic(III)thiols, but not methylarsenic ( V ) compounds, have been selectively derivatized into hydrides by adjusting the p H to 6 during the H G reaction and they were then detected by A A S following G C separation . 20  M o s t G C separations are carried out by using a packed column, but capillary G C with flame ionization detection (FED), electron capture detection ( E C D ) , A E S and M S , has been used as w e l l ' . Derivatization is carried out by using thioglycolic acid methylester 21  22  29  ( H S C H C O O C H ) and arsenic compounds that have been analyzed include a series o f 2  3  organoarsenic halides, such as Lewisite ( ( C l H C = C H ) A s C l ) , an arsenical considered to be a 2  chemical warfare agent. The method o f H P L C can separate a range o f arsenic species and does not require derivatization. The liquid sample can be directly analyzed and the results obtained may be more representative o f the arsenic speciation in the original sample or extract. Arsenicals are most often identified and detected by using I C P - M S or I C P - O E S for the following reasons. The flow rates from H P L C columns are similar to those used in the I C P uptake and nebulization system (around 1 ml/min) allowing for easy coupling. I C P - M S and I C P - O E S are very sensitive (providing l o w detection limits o f pg levels and ng levels, respectively) and are capable o f detecting several elements simultaneously. Ion exchange chromatography is often used for the analysis o f arsenic compounds, because arsenicals typically exist as anions or cations, depending on the p H used. The technique depends on the attraction between the ions and the oppositely charged stationary phase. Gel permeation chromatography has also been used for the speciation o f arsenic compounds, including a trimethylarsonioriboside . 23  The use o f ion-pairing chromatography is very common for arsenic speciation. In this technique, an "ion-pairing reagent", possessing an alkyl chain and an ionic end, is added to the mobile phase and allows the separation to take place on a C18 column, instead o f on an ion exchange column. The current theory for ion-pairing chromatography is based on the electric double layer model. In this model, an electric double layer forms when the lipophilic end o f the ion-pairing reagent interacts with the C18 column, and a counter ion interacts with the ionic end of the reagent. The resulting layers o f charge enable mobility o f the analyte through the column  30  to be a function o f coulombic attraction, as well as interaction with the non-polar stationary phase ' . 24 25  Vesicular chromatography coupled with derivatization of the eluted analytes into their hydrides and microwave induced plasma ( M I P ) - A E S analysis has been used for arsenic speciation . In this technique, a mobile phase containing micelles, formed from a surfactant such 26  as didodecyldimethylammonium bromide ( D D A B ) in a concentration above the critical micellar concentration ( C M C ) , is used, together with a C I 8 column. The interaction of the analyte with the chromatographic system is thought to resemble anion exchange. Supercritical fluid chromatography (SFC) with supercritical C 0 as the mobile phase has 2  also been used to speciate arsenic compounds. Arsenic compounds were reacted with thioglycolic acid methylester ( T G M ) and the resulting volatile species were then extracted by using supercritical C 0 and detected by using capillary SFC with F I D . In another study, 2 7  2  coupling o f S F C to I C P - M S allowed separation of trimethylarsine ( ( C H ) A s ) , triphenylarsine 3  3  ( ( C H ) A s ) and triphenylarsenic oxide ( ( C H ) A s O ) with no derivatization . 28  6  5  3  6  5  3  Capillary electrophoresis ( C E ) has been applied to arsenic speciation. C E has been carried out with U V detection ' ' , direct injection nebulizer ( D I N ) - I C P - M S 29  30  31  32  detection, and  H G - I C P - M S detection ' . The use of I C P - M S detection in the aforementioned studies gave 33 34  better detection limits, but also required coupling devices that are not commercially available. The arsenic compounds A s ( V ) , A s (III), M M A A and D M A A were separated in all these studies. A study by Schwedt et al. incorporated a technique for HAs0 S " speciation , which allowed the 2  31  3  monothioarsenate ion to be determined at ppm levels in soil extracts. Mass spectrometry has been used as a detector for arsenic compounds, in some cases following H P L C separation. Conventional and microbore H P L C was coupled to liquid secondary ion mass spectrometry ( L S I - M S ) , continuous flow L S I - M S ( C F - L S I - M S ) , and direct 31  liquid introduction mass spectrometry ( D L I - M S ) to speciate arsenic animal feed additives . Fast atom bombardment ( F A B ) combined with M S as well as tandem M S ( M S / M S ) ' , field 3 6  3 7  desorption M S ( F D - M S ) , atmospheric pressure chemical ionization and electrospray ionization 36  combined with a triple quadrupole M S ( A P C I - M S and E S I - M S ) , desorption chemical 38  ionization M S ( D C I - M S ) and matrix-assisted laser desorption ionization/time-of-flight M S (MALDI-TOF-MS) ' 3 9  4 0  are techniques that have been used for the characterization o f arsenic  compounds as standards, and in some cases, the identification o f arsenic species in samples. The method o f electrospray ionization (ESI) with ion trap mass spectrometry (IT-MS) was used in the present study. E S I is a soft ionization technique in which ions in solution are transformed into gas phase ions, suitable for M S analysis. It is especially useful for ionic, heat labile and high molecular weight compounds and allows M S analysis to be performed on compounds present in liquids (e.g., following H P L C separation). Ions are produced when the sample solution is nebulized and the droplets formed are subjected to a high voltage, causing their surfaces to be electrically charged. The solvent evaporates from the droplet, causing the charge density at the surface to increase; the droplet becomes unstable, breaking into smaller droplets; and sample ions are ejected into the gas phase by electrostatic repulsion from the small droplets. The ions can then be analyzed by using a mass analyzer such as an ion trap . 41  The ion trap mass analyzer allows for high sensitivity, simplicity, and tandem techniques (MS"), among other features . Ions are trapped in the space created by three electrodes: a ring 42  electrode, and two end-cap electrodes. A n ac voltage o f constant frequency and variable amplitude (an r f voltage) is applied to generate an electric field and its amplitude is kept at a low value initially so as to maintain stability o f the ions in the trap. A mass scan takes place when the amplitude o f the applied r f voltage is increased, causing instability and subsequent ejection and detection o f ions o f increasing m/z. M S " experiments are performed by ejecting all ions from the  32  trap except for the selected ion, and then applying a supplementary i f voltage to the end caps to translationally excite the ions. Product ions result from collisionally induced dissociation o f the excited ions with helium buffer gas.  33  2.1.2. Experimental  Chemicals and reagents  2.1.2.1.  Arsenic standards were obtained as sodium arsenate, N a H A s 0 . 7 H 0 (Aldrich), arsenic 2  4  2  trioxide, A s 0 3 (Alfa), methanearsonic acid, C H A s O ( O H ) (Vineland Chemical), and cacodylic 2  3  2  acid, ( C H ) A s O ( O H ) ( B D H ) , and were dissolved in deionized water to make standard solutions. 3  2  Extracts o f kelp powder (Galloway's, Vancouver, B C ) and N o r i (Porphyra tenera) o f known arsenosugar content were used to identify the retention times o f arsenosugars; these were 43  verified by comparison to pure arsenosugars generously donated by K . Francesconi and T. Kaise. Arsenobetaine , arsenocholine , trimethylarsine oxide , and tetramethylarsonium iodide had 44  45  46  47  been synthesized previously according to known methods. Methanol ( H P L C grade, Fisher), tetraethylammonium hydroxide ( T E A H , 20% in water, Aldrich), malonic acid ( B D H ) , concentrated phosphoric acid (Aldrich), ammonium hydroxide ( I M , Fluka), pyridine (Fisher), and formic acid ( B D H ) were used as reagents for mobile phases and extractions.  2.1.2.2.  Instrumentation and methods of analysis  2.1.2.2.1.  HPLC-ICP-MS  The H P L C apparatus consisted o f a Waters 510 double piston pump, a Rheodyne six-port injection valve with a 20 pl loop, in-line filters, a guard column for each analytical column packed with the same stationary phase, and the analytical column. Columns and mobile phases are listed in Table 2.1. A V G Plasmaquad P Q 2 Turbo I C P - M S ( V G Elemental) was used as a detector. Parameters for the I C P - M S are given in Table 2.2. The m/z monitored were 75, along with m/z 77 and 82 in the case o f real samples, to correct for any interference from the C l - A r molecular 34  ion.  j 5  C l - A r gives a m/z o f 75; monitoring m/z 77 corresponding to 4 U  CI- A r would allow this  interference to be confirmed. However m/z 77 is also a Se isotope, and hence monitoring S e 82  allows differentiation from the interference. Chromatographic signals at m/z 77 were not present at the same retention time as those at m/z 75 when they appeared, and hence interference was never a problem. In most analyses, m/z 103 (Rh) was monitored as well, because R h was added to the mobile phase and used as an internal standard to correct for plasma instability. The H P L C was coupled to the spray chamber o f the I C P - M S by using a minimum o f P T F E tubing (10 cm x 0.5 mm i d . ) with the appropriate P T F E fittings. Data from the I C P - M S were processed by using chromatographic software , and identification o f arsenicals in samples was made by comparison 48  of retention times with those o f standards by using at least two chromatographic systems. Semiquantitative concentrations o f arsenic compounds were determined by using external calibration curves for each compound corresponding to a matching standard, or to D M A for arsenosugars.  Table 2.1.  H P L C conditions for arsenic speciation  Chromatography  Column  Mobile phase  Anion exchange  Hamilton P R P - X 1 0 0 , 150 x 4.6 or 250 x 4.6 mm  20 m M ammonium phosphate, p H 6.0  Cation exchange  Supelcosil L C - S C X or Whatman S C X Partisil 5, 250 x 4.6 mm  20 m M pyridinium formate, p H 2.7  1.0  Ion-pairing  G L Sciences O D S , 250 x 4.6 mm  10 m M T E A H , 4.5 m M malonic acid, 0.1%-0.5% MeOH, pH6.8  0.8  Flowrate (mL/min) 1.0 or 1.5  35  Table 2.2. Operation parameters for I C P - M S Feature  Specific Conditions  Forward radio-frequency power  1350 W  Reflected power  <10W  Cooling gas flow rate (Ar)  13.8 L/min  Intermediate (auxiliary) gas flow rate (Ar)  0.65 L/min  Nebulizer gas flow rate (Ar)  1.002 L/min  Nebulizer type  de Galan  Analysis mode  Time Resolved Analysis ( T R A )  Quadrupole pressure  9 x 10" mbar  Expansion pressure  2.5 mbar  7  2.1.2.2.2. ESI-IT-MS Arsenosugar standards (diluted to give solutions o f about 500 ppb) and the kelp extract were analyzed. The kelp was extracted according to published methods  43  and contained the  following approximate concentrations o f arsenosugars in the extract: X , 175 ppb; X I , 30 ppb; X I I , 70 ppb; XIII, 220 p p b  49  A Finnigan L C Q ™ including a flow injection system consisting o f a syringe pump delivering solutions in 50% M e O H at 100 pl/min was used. This instrument can utilize atmospheric pressure ionization (API) in two modes: E S I and atmospheric pressure chemical ionization (APCI), o f which the former was used for this study. The ring electrode was kept at 0.76 M H z and the i f amplitude varied from 0 to 8500 V during mass scans. Experiments were carried out in negative and positive ion modes. This was done by changing the polarity o f potentials applied to (a) the ion source including the electrospray capillary and the heated capillary tube; (b) the ion optics, specifically the interoctapole lens, situated between two  36  octapoles; and (c) the conversion dynode in the ion detection system. The partial pressure o f helium in the ion trap was kept at 0.1 P a (1 mTorr). F o r M S " experiments, the selected ions were subjected to supplementary voltages (supplied to the endcaps) ranging from 12 to 33 V and further fragmentation took place as a result o f collisions with He. Trapping o f fragments, fragmentation o f daughter ions and changing o f voltages were carried out in real time while monitoring the mass spectra.  37  2.1.3. Results and Discussion  2.1.3.1. Speciation of arsenic compounds by HPLC  2.1.3.1.1. Anion exchange chromatography The column used in these studies was a resin based column (poly(styrenedivinylbenzene)) with trimethylammonium groups providing the exchange sites (Hamilton P R P - X 1 0 0 , see Table 2.1) . The elution order o f some arsenic compounds when using 20 m M ammonium phosphate at p H 6 as the mobile phase is shown in Figure 2.1: arsenobetaine ( A B ) / A s (III)/arsenosugar X , D M A , M M A , arsenosugar X I , A s (V), arsenosugar X I I and arsenosugar XIII. The chromatogram obtained from a mixture o f A s (III), A s (V), M M A and D M A was overlayed with that from a mixture o f arsenosugars. Standard cationic compounds were not analyzed by using this system but they are expected to elute in the dead volume, prior to arsenobetaine. I C P - M S was used as a detector in all H P L C experiments. A s (III) is present as a neutral molecule ( p K 9.3), arsenobetaine is a neutral zwitterion, a  and arsenosugar X is in the neutral fully protonated form. These neutral species are separable from the cationic species, and to some extent from each other. Their retention times differ by about 10 seconds; therefore, although they are not baseline resolved, if one is present as the major species, a tentative identification can be made based on the retention time. D M A is the next eluting compound and about 50% is in the singly charged anionic form ( p K 6.28). M M A , a  which elutes next, is predominantly in the singly charged anionic form (98.6%,  pK i a  3.6, p K  a 2  8.2) . The later elution o f arsenosugar X I compared with M M A may be a result o f the anionic character o f the phosphate group in arsenosugar X I (see Figure 1.1, structures o f arsenosugars), and possibly enhanced interaction o f the sugar with the resin.  38  8000  i  0  120  240  i  i  i  r  360  480  600  720  840  time(s)  Figure 2.1. Chromatogram o f standard arsenic compounds by using Hamilton P R P - X 1 0 0 anion exchange column (15 cm) with 20 m M ammonium phosphate, p H 6.  39  A s ( V ) is anionic, being 90% singly charged and 10% doubly charged, hence its later elution. Arsenosugars X I I and XIII, containing sulfonate (XII) and sulfate (XIII) groups (see Figure 1.1, Chapter 1, for structures o f arsenic compounds), are most likely singly charged and anionic and they elute later than A s (V), when using the chromatographic system described in Figure 2.1. This later elution may be due to the increased interaction between the organic groups on these molecules with the resin. Arsenosugars have been separated on an anion exchange system in another study , where 50  2 0 m M ammonium carbonate at p H 10.3 was used to give the same elution order for the arsenosugars as that observed under the present conditions. The use o f the carbonate mobile phase resulted in co-elution o f arsenosugar X with cationic species, and co-elution o f arsenosugar X I with D M A . Hence this system using ammonium phosphate as the mobile phase offers some improvement.  2.1.3.1.2. Cation exchange chromatography Cation exchange chromatography with I C P - M S detection was used to confirm or identify the compounds that co-elute in the anion exchange system described above. The chromatogram in Figure 2.2 shows the elution order o f some arsenic species: arsenosugar X I , X I I I / M M A , D M A , arsenosugar X , arsenobetaine ( A B ) , trimethylarsine oxide ( T M A O ) , arsenocholine ( A C ) , and M e A s . Chromatographic peaks corresponding to the arsenic species A s ( V ) and A s (III) +  4  are not shown in Figure 2.2, to provide clarity o f presentation, since these compounds elute closely together with other species. Nevertheless, their retention behaviour was observed and is discussed below. The chromatograms for a mixture o f standards ( M M A , A B , T M A O , A C , and M e A s ) was overlayed with the chromatogram for kelp extract, containing arsenosugars X , X I , +  4  X I I (with unknown retention time) and XIII.  40  35000 30000 H  standard cationic species kelp extract  25000 20000 </>  15000 10000 5000 0 I  0  180  I  I  I  360  540  720  l  i  900 1080  time (s)  Figure 2.2. Chromatogram of standard arsenic compounds by using Whatman SCX cation exchange column with 20 mM pyridinium formate, pH 2.7.  41  A silica gel based column containing benzene sulfonic acid functional groups as exchange sites (Whatman S C X or Supelcosil L C - S C X , see Table 2.1) was used with a mobile phase consisting o f 20 m M pyridinium formate at p H 2.7, based on methods described in previous studies ' . A t this p H , A s ( V ) has 74% singly charged anionic character and is unretained by the 51 52  column (not shown in Figure 4). p K ' s for the individual arsenosugars are not known but the p K a  a  for the dimethylarsinoyl moiety on the sugars is estimated to be 3.85 , indicating that the 53  arsenosugars should be somewhat cationic at this pH. However, the co-elution o f arsenosugar X I with A s ( V ) (not shown in Figure 2.2), which is unretained, indicates that arsenosugar X I may be more anionic or neutral in character. Arsenosugar XIII, on the other hand, was observed to co-elute with the neutral species As (III) and M M A (As (III) not shown in Figure 2.2). D M A is separated from the neutral species, indicating some cationic behaviour, possibly as M e A s ( O H ) +  2  5 4 2  . The partial protonation  of the oxygen in the dimethylarsinoyl moiety in arsenosugar X would give it some cationic character, allowing it to be retained on the column. Arsenobetaine has a p K o f 2.18 a  54  and is  20% cationic in nature (80% zwitterionic and neutral at this pH). It is probably more cationic than the previous two species, and hence retained longer on the column. T M A O is thought to exist as [ ( M e A s O H ) (OH)"], from the hydrolysis o f M e A s 0 , although the peak is very broad +  5 5  3  3  under these conditions, which may indicate the presence o f more than one species. Arsenocholine and M e A s are cations irrespective o f pH, and their retention behaviour +  4  under these conditions indicate that arsenocholine is less strongly retained. Previous studies have shown the same elution order for these two compounds under the same conditions, but the elution order was reversed when a bare silica column was used , and when an ion-pairing system 54  with a reversed phase column and sulfonate mobile phase was used . N o explanation was given 55  in the former study, and in the latter study arsenocholine was suggested to be more hydrophobic.  42  The organic nature o f the pyridinium mobile phase may cause the more hydrophobic arsenocholine to be eluted faster from the cation exchange column in the present study. However, other interactions, such as hydrogen bonding between silica O H groups and the O H group on arsenocholine, must be responsible for its longer retention time on the bare silica column.  2.1.3.1.3. Ion-pairing  chromatography  When the presence o f arsenosugars is indicated by one or both o f the two previously mentioned chromatographic systems, it can be confirmed by using a third chromatographic system. The system used here is one that has been developed for the analysis o f anions, particularly arsenosugars . Ion-pairing chromatography with I C P - M S detection is used, a 56  technique that combines a mobile phase containing an ion-pairing reagent with a reversed phase column. Tetraethylammonium hydroxide ( T E A H ) is the ion-pairing reagent, and the mobile phase is adjusted to a p H o f 6.8 with malonic acid and nitric acid. A small amount o f methanol is added (0.1-0.5% v/v). Methanol can be used to control chromatographic behaviour in reversed phase systems, with increased concentrations resulting in shorter retention times for the arsenosugars. Methanol has also been found to increase sensitivity o f arsenic compounds when using I C P - M S detection . 57  The elution order shown in Figure 2.3 for some arsenic species is arsenobetaine ( A B ) , M M A , D M A / A s (V), arsenosugar X , X I / X I I , and XIII. Chromatograms o f A B , M M A , D M A and A s ( V ) standards were overlayed with kelp extract and also with N o r i extract. N o r i extract is known to contain arsenosugars X and X I as the only arsenosugars . 43  25000 Nori extract standards and kelp extract  20000 -  ,  x  A B  I  MMA  I  I  XIII  X  I  15000 w  i  0  240  480  720  960  1200  time (s)  Figure 2.3. Chromatogram o f standard arsenic compounds by using ion-pairing reversed phase chromatography, with G L Sciences C I 8 column and 10 m M T E A H / 4 . 5 m M malonic acid, p H 6.8, 0.1% M e O H .  44  A s (III) elutes closely after arsenobetaine and is riot'shown on Figure 2.3 to retain clarity. Arsenobetaine and A s (III) are expected to be neutral molecules at p H 6.8, and to have short retention times. M M A and D M A elute in the opposite order compared to anion exchange; this may be due to enhanced interaction o f D M A (containing 2 methyl groups) with the C18 chains o f the stationary phase. D M A co-elutes with A s (V), which was also seen by other researchers . 58  Using the present chromatographic system, the arsenosugars are separated from the other ions, although arsenosugars X I and X I I are not baseline resolved from each other. Figure 2.3 shows that arsenosugar X I alone (dotted line) elutes slightly later, which allows this compound to be identified in the absence o f arsenosugar X I I . Likewise, arsenosugar X I I can be identified in the absence o f arsenosugar X I . The elution order for the sugars most likely indicates increasing anionic character when going from arsenosugar X to XIII. Although not one o f these chromatographic systems can be used alone for the analysis o f complex mixtures, such as those found in environmental samples, a combination o f anion, cation and ion-pairing chromatography can be used to obtain a satisfactory separation and identification of at least 11 arsenic species. Analyzing a sample by using two or three different chromatographic systems, although time consuming for routine use, is a very useful tool for strengthening the identification o f arsenic compounds.  2.1.3.2. Characterization of arsenosugars and compounds in kelp powder extract  Kelp powder has been previously analyzed and it has been suggested that this sample contains the four arsenosugars X , X I , X I I , and XIII, based on H P L C retention times ' . A s a 43  49  result o f this identification, kelp extract is often used as a reference for the retention times o f these arsenosugars, and the retention times are then used to identify arsenosugars in other 45  samples o f unknown arsenic speciation. A n attempt was made to identify the arsenosugars in kelp extract by using a mild ionization mass spectrometric technique, possible with the Finnigan L C Q ™ E S I - I T - M S instrument, as well as by analyzing pure arsenosugar standards. Such identification would validate other identifications based on retention times o f the reference sample. The positive-ion ionspray (IS) tandem mass spectra (i.e., M S - M S ) o f pure arsenosugars X , X I , X I I and X I I I have been presented by others . Positive and negative-ion fast atom 59  bombardment ( F A B ) tandem mass spectra have been produced for arsenosugars X I , X I I and XIII, as w e l l . It was o f interest to compare the fragmentation behaviour o f arsenosugars in the 60  present M S - M S experiments by using low-energy, low pressure ( l m T o r r ) C I D conditions in the ion trap, with that observed by using low-energy, higher pressure (4-18mTorr) C I D M S - M S 61  conditions (IS with a triple-quadrupole mass filter). Because Corr et al.  59  observed only positive  ions, comparisons were also made between the present results and the results obtained by Pergantis etal.  60  (high-energy C I D conditions, with F A B reverse-geometry four-sector mass  analyzer) for negative and positive-ion mass spectra. A summary o f M S and M S - M S analyses obtained from pure arsenosugars is given in Table 2.3.  46  Table 2.3. E S I - I T - M S experiments and fragments for pure arsenosugars. Arsenosugar  Precursor ion m/z  (mode)  selected  X, MW = 328  329  (positive)  XI, MW = 482 (positive)  311  52  ' 237  100 26 100  329 -> 237  209 237  30 100  219  75  391 329  65  483  481  393 391  (negative) 391 ->269  XIII, MW = 408  46  195  481 ->389  (positive) XII, MW = 392  329  311  (negative)  XII, MW = 392  Relative abundance (%)  329 -> 311  483 - » 329 XI, MW = 482  Product ion m/z  407  (negative)  100  237  13  329  100  237  55  389  100  245  27  389  100  267  20  223  49  193  33  392  100  281  53  391  52  373  100  269  48  269  100  225  90  197  17  408 '  24  389  100  285  62  171  52  407 -> 389  171  100  407 -> 285  285  21  241  100  213  35  153  75  407 ->• 389 -> 171  137  28  97  27  87  30  171  35  97  100  47  Only low concentrations o f arsenosugars were available and the mass spectrometer was contaminated by previous samples, which caused high background levels during the arsenosugar analyses. Hence, the M S " capability o f the ion trap mass analyzer in the mass spectrometer was useful because it allowed isolation o f specific ions o f the sugars and subsequent fragmentation. Only the two sugars X I and X I I could be analyzed by both negative and positive modes to give useful information. Molecular type ions were not observed in large enough abundance for trapping and subsequent M S - M S analysis for the other two sugars, X and XIII, in the negative and positive modes, respectively. A s mentioned earlier, arsenosugar X ( M W = 328) was characterized in the positive ionization mode by Corr et al.  59  by using an ionspray source and triple quadrupole mass  spectrometer with M S - M S capabilities. They found a single product ion forming at m/z 237, corresponding to the fragment containing arsenic and the ribose group. Other previous studies showed the formation o f fragments at m/z 311, 221 and 177 for this compound . In the present 47  work (Figure 2.4), the fragments at m/z 311 (loss o f H 0 ) , 237 (with the structure shown in 2  Figure 2.4) and 209 are produced when the ( M + H ) ion at m/z 329 is trapped and subsequently +  fragmented. The fragments obtained when m/z 311 ( [ ( M + H ) - H 0 ] ) was trapped and +  2  fragmented are m/z 209 and 195, which have not yet been identified. The product ion obtained when m/z 237 was trapped and fragmented is m/z 219 (resulting from loss o f water). Hence these results show similarity to both o f the previous studies, where the fragment o f m/z 237 is common with the study by Corr et al.  59  by Cullen et al.  47  and the fragment o f m/z 311 is common with the study  Although Pergantis et al.  60  did not analyze standard arsenosugar X , they were  able to isolate a precursor ion at m/z 329 in the positive-ion mode from an algal extract, and found fragments at m/z 311 and 237 (similar to the present study), as well as at m/z 208, 176, 165, 122 and 97.  48  B o t h positive and negative modes o f ionization were successful for arsenosugar X I ( M W = 482), as mentioned before. When ( M + H ) (m/z 483) was trapped and fragmented (see Figure +  2.5), m/z 391 corresponding to loss o f ( O H ) C H ( C H O H ) ( C H O H ) is observed. A fragment o f 2  2  m/z 329, corresponding to arsenosugar X is observed, as well as m/z 237, corresponding to the dimethylarsenic-ribose moiety. These fragments were also observed in the previous studies by Corr et al.  59  and Pergantis et al . 60  The higher energy C I D conditions led to additional ions in  those studies. When arsenosugar X I was analyzed in the negative ionization mode, and (M-H)" was trapped and fragmented (see Table 2.3 for these results, and Figure 2.6 for the structure o f ( M H)"), m/z 389 is observed as the negative ion analogous to the ion o f m/z 391 described above. A product ion o f m/z 245 is also observed, corresponding to the fragment remaining when the dimethylarsenic-ribose moiety is lost, with the structure [ ( C H C H ( O H ) C H O H ) P C V ] . When 2  2  2  m/z 389 was trapped and fragmented (Figure 2.6), m/z 267 is produced, having the structure shown in Figure 2.6, after loss o f the dimethylarsenic group. The fragment at m/z 223 may indicate loss o f C H = C H O H (44 amu) from the m/z 267 fragment. The fragment at m/z 193 is 2  unidentified. Pergantis et al.  60  observed m/z 389 and 245 as well, but not 267 or 223.  50  u  O)  co ro O) -  •O  o  oo  00  CO  e  r  (D  I  • co  cn O rco  OA  c ON  : o  oo m  t CO  N  O)  C  $  ~  <D  O  o (/3  ro  I  iri co  —L t  N  ug  I" .  % %  . o -co  s  5  CM  CM  p  O O  |  to CM  ; o I CM  oi ; CM -  in  CM  c/3 I-H  if) O  CM  oo O  O)  C  CM CM  (D  h8 CM CM  P,  CM  — f  t  =i-o  o> CM:  IT)  O  IT)  CD  o  © in  IT) •fl-  eouepunqv aA»e|ay  B  o  S o u a. bo  ro -  © o  X  CM  O  O)  I  ic) CM  o CM  s  o  2 &  52  Arsenosugar X I I ( M W = 392) was successfully fragmented in both the positive and negative ionization modes. In the positive mode, trapping o f m/z 393 ( M + H ) ion and M S - M S +  analysis (Figure 2.7) gives a large abundance o f m/z 392 ( M » ) and no m/z 393 is observed. A +  fragment having m/z 281, which may correspond to loss o f C H - S 0 H and O H (see Figure 2.7) 2  3  is also observed. These ions (m/z 393 and 281) were not observed when this compound was subjected to ionspray M S in the positive mode by Corr et al.  or to F A B - M S by Pergantis et  59  al.  60  Those authors observed m/z 375, 296 and 237 , and 375, 237, 165 and 122 , fragments 59  60  that were found only in very small abundance or not at all in the present study. M o r e structural information is obtained when arsenosugar X I I was analyzed in the negative mode. The [M-H]" ion o f m/z 391 was trapped and fragmented (Figure 2.8) and loss o f H 0 gives m/z 373, and the dimethylarsenic moiety (m/z 269, see Figure 2.8 for structure) is lost. 2  When m/z 269 was subsequently trapped and fragmented, loss o f C H = C H O H (269-44) followed 2  by loss o f C O (225-28) may have occurred to form the fragments at m/z 225 and 197 (structures ( C = 0 ) C H ( O H ) C H O R and ( O H ) C H = C H ( O R ) , where R is C H C H ( O H ) C H S 0 " ) . Interestingly, 2  2  3  m/z 197, but not m/z 225 was observed by Pergantis et al. ; m/z 197 was assigned a similar 60  structure to the one proposed in the present study. Arsenosugar X I I I ( M W = 408) was ionized successfully only in the negative mode, indicating greater stability o f the molecule in the anionic sulfate form compared to its stability in the cationic protonated form. This enhanced stability o f the anion corresponds well to the more anionic character o f this sugar as observed in its H P L C behaviour in the previous section. The anion (M-H)" at m/z 407 was trapped and fragmented (Figure 2.9) to produce the following ions: m/z 389, corresponding to loss o f H 0 ; m/z 285, corresponding to loss o f the dimethylarsenic 2  moiety as described above; and m/z 171, corresponding to [ H O - C H - ( C H O H ) - C H - 0 - S 0 " ] (i.e., 2  2  3  loss o f the dimethylarsenic-ribose moiety). Subsequent trapping o f m/z 389 produced the  53  aforementioned fragment at m/z 171. Trapping o f m/z 285, (Figure 2.10) produced fragments at m/z 241 and 213, which seem to follow the same pattern as the fragments at m/z 225 and 197 for the ion at m/z 269 in arsenosugar X I I , which may again indicate loss o f CH2=CHOH and then C O . Other product ions from m/z 285 are found at m/z 153 ( C H = C ( O H ) - C H - 0 - S 0 " ) , 137 2  2  3  (not easy to identify, but possibly resulting from loss o f O from m/z 153), 97 (HO-SO3") and 87 (unidentified). The fragmentation o f m/z 171 produced m/z 97 (HO-SO3"). Pergantis et al  60  observed some o f the same fragments: m/z 389, 285, 213 (but not 241), 171, 153 and 97. Useful structural information was obtained in the negative ion mode for all sugars but arsenosugar X . The loss o f the 122 amu fragment was observed for sugars X I , X I I and XIII in the negative mode and can be considered diagnostic o f this group o f compounds . In general, 60  fewer fragments were observed in the positive mode o f analysis. Characteristic arsenic containing fragments were observed in arsenosugars X , X I and X I I in the positive mode, however. The previous studies reported a greater number o f fragments compared with those in the present study, resulting from the use o f higher energy CUD conditions in those studies. M S M S - M S experiments were conducted in the present study, providing new information about the behaviour o f some o f the arsenosugars in this mode.  54  •a o  CM : co  j  e So co; co r- i O)  o CO  fco  -  CM  t  co if o r-to C M : co m : co •  >  s o  *«3 ON  cn CO r  O  •  CO  •s '% (-CM  5  1- ;  O  r-o co • co O  T  I  1 TJ C  ^>  £ o  p X  5-15  t*  X  8  s s  c3  .AT  60 3 no O C <D  1  (0  \  CM  Vi  T  ;  X  in CM  o  O  r-«D  »"  ir. CM  CO  -  CM  r  o  t-  03  m:  o CO CM  t  t/3  ;  CM  Ifi CM CM  O  o  o  m co  o oo  in CO  m m  o in  in  eouepunqv aAgeisy  T t o  00 fa  56  389.0  10095-  90  :  85^ 80^ 75  :  70  :  O H  65-  O H  285.3 60g  to  55-  T3 C  |  171.4 50-  >  1 45^ a: 403530408.0  25H 201510-  153.3  50-  137.3 129.4 | 1  213.3 199.3 200  2 4  ^-  229.3  275.3  3  271.3 >50~  286.6 314.3 345.2 363.3 300 '  m/z  l—r  350  i  408.9 i  400  Figure 2.9. Mass spectrum of arsenosugar XIII standard, MS-MS with m/z 407 selected, negative mode. 57  241.  10095  :  9085H 8075H  153.1  7065 60c ro  55^  £Z 3  J  < 50H  1  45J j  tr:  40213.1  3530- 87.1  20  97.1  137.1  285.2  : 171.1  10  :  5-  195.1 123.1  0 100  150  200  2407 241.9 250"~  302.5 m/z  j5o- i — i — r  359.4 J50  75o~  Figure 2.10. Mass spectrum of arsenosugar XIII standard, M S - M S - M S with m/z 407 selected, then m/z 285, negative mode. 58  A fUll mass scan in both the positive and negative modes o f the crude kelp powder extract did not show the arsenosugars as major ions. However, the M S " capability o f the L C Q ™ instrument proved valuable since the ions characteristic o f the individual arsenosugars (m/z 329 (positive mode) for arsenosugar X , m/z 481 (negative mode) for arsenosugar X I , m/z 391 (negative mode) for arsenosugar X I I and m/z 407 (negative mode) for arsenosugar XIII) could be selected from the complex matrix and then fragmented to ascertain i f the characteristic product ions were observed. Some o f the fragments that were produced during the M S and M S - M S experiments are summarized in Table 2.4. The fragments listed are ones that also appear in the pure arsenosugar standards.  59  Table 2.4. E S I - I T - M S experiments for kelp powder extract Arsenosugar  Precursor ion m/z  (mode)  selected  X , M W = 328  329  (positive)  X I , M W = 482  481  (negative)  X I I , M W = 392  391  (negative)  XIII, M W = 408  407  (negative)  407 -> 285  Product ion m/z  Relative abundance (%)  329  100  328  82  311  7  237  11  481  17  463  100  389  30  245  27  391  100  373  85  225  6  407  80  389  100  285  30  285  100  241  25  The fragments that might be considered characteristic occur in very low abundance and in some cases are no more abundant than the other peaks that appear in the mass spectra. This is the case for the precursor ion o f m/z 329 (arsenosugar X ) and m/z 391 (arsenosugar XII); the characteristic m/z 237 for arsenosugar X , and m/z 225 for arsenosugar X I I have abundances o f 11 and 6%, respectively (Table 2.4). Another problem is that other fragments are observed in addition to those obtained from M S - M S experiments with standard compounds and these are probably due to the impurity o f the  60  sample (i.e., precursor ions not with the structure o f the arsenosugar may be trapped). Interestingly, some o f the other fragments that occur when m/z 481 is trapped and dissociated in the negative mode (Figure 2.11) were also observed by Pergantis et al.  60  (e.g., m/z 407 and 287),  but not when the standard compound was analyzed in the present study. The ion occurring at 100% abundance, m/z 463, indicates loss o f water, but it is also not observed for the standard. Again, the characteristic ions at m/z 389 and 245 are at an abundance not significantly different from the other fragments whose structures are unknown. L o w concentrations for arsenosugars X I and X I I (about 15 and 35 ppb, respectively, after 1:1 dilution o f the extract with methanol), made further confirmation by M S - M S - M S experiments impossible. In the positive mode, only the ( M + H ) ion (m/z 329) for arsenosugar X could be isolated +  from the complex mixture, and in addition to the low abundance o f the characteristic m/z 237, the presence o f M « ion at m/z 328, a fragment that was not observed previously, can be seen +  (Table 2.4). When the (M-H)" ion for arsenosugar X I I I in the kelp extract (m/z 407) was trapped, the characteristic fragments o f m/z 389 (loss o f water) and 285 (loss o f dimethylarsenic group) are observed. When m/z 285 was subsequently trapped, the fragment o f m/z 241, observed in the same experiment with the standard, is seen (Figure 2.12). Another fragment o f appreciable abundance is present in Figure 2.12a at m/z 363, indicating either loss o f 44 amu ( C H = C H O H or 2  C 0 ) from m/z 407, or loss o f 26 amu ( C H ) from m/z 389. This fragment was not observed in 2  2  2  the mass spectra generated for the standard compound, nor in any other reports. The mechanisms are not obvious for the suggested mass losses and they may involve ring cleavage or molecular rearrangement. However, a similar mass loss is observed from m/z 481/463 (sugar X I ) and 391/373 (sugar XII). Dissociation processes are apparently taking place that are specific to these compounds in the kelp extract.  61  62  c o o  o  c 53 ! o M O ! co T-  oo  I  o_L CM i  c\i  —1  &  co i  CM CD in  i  rg  . f a  o  a •§ a  o r*o vo ! io oo l CM • LO-  CO  cn oo  Q^  —r  u a  CM I  o  Irt  •  -A  5  t>  L  O .53  asuepunqv aNjeiaa  -e CN  3  u  c  cd L_  ,  X  t »  3 ja  N  1  CM O) -  oo co  oo oo ro  eu  •s  a  C ^  ««  N  ^  11  coco ro  CD  L.  3  60  a  3  o *n <+* oo ccj <N N  CO CM  is  CM  C  c/i  to o>  LO  oo  o  LO CO  o  o CO  LO  o  LO  aouepunqv aAijeisy  fa  63  ^  fa B  The strongest argument for the presence o f arsenosugars in this extract is the evidence for arsenosugar X I I I because the M S - M S , as well as M S - M S - M S experiments, show characteristic fragments. The identification o f the other compounds, based on this data alone, is not as clear. The major reason for the problems with compound identification in the kelp extract is the low levels o f the compounds, especially in the presence o f large amounts o f matrix components. Chromatographic separation followed by these M S experiments would improve the identification of the arsenosugars. These results are very useful in characterizing the arsenosugar standards, verifying the cationic behaviour o f arsenosugar X , the similar anionic behaviours o f arsenosugars X I and X I I , and the strongly anionic character o f arsenosugar XIII, which was also observed in the H P L C behaviour o f these compounds. Moreover, this mass spectral information adds to that currently available in the literature.  64  2.2. Methods for the Analysis of Antimony  2.2.1. Introduction Methods for the analysis o f total antimony in samples are similar to those used for arsenic, which are described briefly in section 2.1.1. Speciation techniques for antimony are fewer in number. A recent review lists the known methods for speciation, including extraction techniques; electrochemical techniques; coupled techniques such as hydride generation-gas chromatography ( H G - G C ) with flame-in-tube A A S or I C P - M S detection; and high-performance liquid chromatography ( H P L C ) with I C P - O E S , I C P M S or H G - A A S detection . O f these, only H G - G C - A A S , H G - G C - M S and H P L C - I C P - M S 62  methods were used for this work. H P L C techniques are well established for the speciation o f arsenic compounds (section 2.1.1), but only recently have H P L C methods been developed to speciate antimony. In the first report o f such methods, I C P - O E S detection and a cation exchange column were used to separate Sb (III) and Sb ( V ) in electrolyte solutions . Anion exchange techniques coupled to I C P - M S or 63  I C P - O E S were explored by other authors, where mobile phases such as tartrate at p H 5.5 phthalate at p H 5 ' ' , 2 m M K O H and E D T A at p H 4 . 5 65  66  67  68  64  and  were used successfully to separate  Sb (III) and Sb ( V ) , and M e S b C l or M e S b 0 ' . These methods have been used with some 6 7  3  2  6 8  3  success to analyze real environmental samples ' ' . Some o f these methods were also 65 67 68  attempted for the separation o f dimethylantimony compounds o f unknown structure but with no 69  success . The application of H P L C to antimony speciation is limited by the lack o f standard compounds available; for example, no standard dimethyl and monomethylantimony ( V ) compounds are commercially available, or easily synthesized. A t the pHs studied for most of the 65  H P L C method development, Sb ( V ) exists as the anion Sb(OH)6 whereas Sb (III) exists as _  Sb(OH) when the oxide is dissolved, or as a complexed form such as the antimonyl tartrate, 3  [Sb (C 06H ) ] ~. The Sb ( V ) anion is not strongly retained on an anion exchange column and is 2  2  4  2  2  hence easily eluted from an anion exchange H P L C column. Uncomplexed Sb (III), however, precipitates easily as the oxide, and in the complexed anionic form is strongly retained on an anion exchange column. M e S b C l most likely exists at neutral p H as M e S b ( O H ) and is not 3  2  3  2  retained or is retained to a minor extent on an anion exchange c o l u m n ' . 67  68  A s described in section 2.1.1 for arsenic, H G - G C speciation techniques can be used to detect antimony species in samples as inorganic antimony compounds in the +3 and +5 oxidation states, which are derivatized to form S b H  3  ' ' . It is also be used for compounds that can be  derivatized to form methyl-, dimethyl- and trimethylstibines ( M e S b H , M e S b H and M e S b ) ' . 70  2  2  73  3  A problem has been noted by various researchers attempting to apply the hydride generation reaction to the generation o f methylated stibines: when trimethylantimony di chloride ( M e S b C l ) , trimethylantimony dihydroxide ( M e S b ( O H ) ) and dimethylantimony 3  2  3  2  dihyroperoxychloride ( M e S b C l ( 0 H ) ) were reacted with borohydride and acid to form their 2  2  2  corresponding stibine, four peaks corresponding to S b H , M e S b H , M e S b H , and M e S b , 3  2  2  3  appeared for each compound, rather than the anticipated ones o f M e S b or M e S b H ' 7 3  3  al  13  2  7 4  D o d d et  postulated that this "rearrangement" occurred when the reaction apparatus had not been  conditioned properly and found that the problem disappeared when the apparatus was rinsed with the reagents ( N a B I L solution and acid solution) for three minutes or more. In the following section, some problems with the hydride generation derivatization technique will be described for antimony, including examples o f rearrangement/ demethylation which can lead to misinterpretation o f analytical results.  66  2.2.2. Experimental  2.2.2A.  Chemicals and reagents Antimony ( V ) and (III) standards were obtained as potassium hexahydroxyantimonate,  K S b ( O H ) (Aldrich), and potassium antimonyl tartrate, K Sb2(C40 H2)2 (Aldrich). M e S b C l 2  6  6  3  2  was synthesized as described elsewhere . Stock solutions were made by dissolving these 75  compounds in deionized water and diluting the resulting solutions to 1000 or 100 mg L " as Sb. 1  Standard working solutions were made by diluting the stock solution with deionized water as necessary. NaBFLi (reagent grade, Aldrich) was dissolved in deionized water fresh daily to provide a concentration o f 2% w/v. Glacial acetic acid, citric acid, sodium hydroxide (for p H adjustment), maleic acid and concentrated sulfuric acid were all reagent grade and obtained from common distributors.  2.2.2.2.  Method of analysis for HG-GC-AAS Three methods were used: Methods 1, 2 and 3. The apparatus for Methods 1 and 2 was  composed o f a semi-continuous flow, hydride generation system developed for arsenic analysis , 76  coupled to an atomic absorption spectrometer (Varian AA1275) fitted with an Sb lamp (Varian) operating at a wavelength o f 217.6 nm. One modification was made to the basic apparatus in the form o f using a gas-liquid separator that resulted in less analyte carryover. The apparatus 77  consisted o f Tygon tubing for the peristaltic pump, and P T F E tubing (1/8" O D ) for the remainder. The glass gas-liquid separator was silanized with ( C H ) S i C l before use. 3  2  2  For Method 3 the semi-continuous flow and batch modes o f analysis were combined. Figure 2.13 shows a schematic diagram o f the apparatus used. Unsilanized glass batch reactors  67  of 60 ml volume were incorporated into the apparatus from Method 1. A gas-liquid separator was not used for Method 3 since the gases were separated from the liquids in the batch reactors. The A A S , peristaltic pump and tubing were identical to those used in Method 1. For all methods, data were collected from the A A S and processed directly by using an H P 3 3 90A integrator, or were analyzed with the aid o f Shimadzu E Z C h r o m software. For Method 1, a peristaltic pump was used to deliver standard or sample solution (ranging from 5 pJL to 200 u,L for standards, and from 1 m L to 5 m L for samples) to mix with the acid or buffer and then to mix with a solution of NaBEL, (2% w/v) in a reaction coil. For Method 2, standard solution was mixed with a solution of NaBIL, (2% w/v) in the mixing coil, and the gas-liquid mixture was mixed with I M H S 0 . F o r both Methods 1 and 2, the gases 2  4  evolved were separated in the gas-liquid separator and then swept by a flow o f helium through a P T F E U-tube at -78 °C (dry ice/acetone) to remove water and into a P T F E U-tube, where they were trapped at -196 °C (liquid N ) . Continuous hydride generation and trapping were carried 2  out for 3 minutes. The peristaltic pump was then stopped (making the system semi-continuous) and the second U-tube was heated to 60 °C, allowing the gases to be swept with H e at a flow rate o f 40 mL/min onto a Poropak P S column, which was then heated from 70 °C to 150 °C at a rate o f 30 °C/min, whereby the gases were separated. They were then detected by A A S .  68  69  For Method 3, the peristaltic pump delivered standard solution and 2% (w/v) NaBFL, solution to the first batch reaction vessel (A). A volume o f 30 m L was delivered to reactor A while helium gas was bubbled through the glass frit to strip the solution o f gases. The gas stream was bubbled through the contents o f reactor B (see Figure 2.13). Throughout the process, the gas stream was dried and trapped at -196 °C and the remaining procedure was identical to the one previously described for Methods 1 and 2. Reactants were added to reactor B in Method 3 from a 1 m L syringe inserted between the rubber stopper and the side o f the reactor. Measurement o f p H was carried out with an Accumet M o d e l 15 p H meter (Fisher Scientific) after the sample and acid had been mixed, but before the NaBFL, was added.  2.2.2.3. Sample preparation Mycelia o f the pink oyster mushroom Pleurotus flabellatus (Western Biologicals, Aldergrove, B C ) were grown with shaking in 400 m L potato dextrose broth (Difco) in a 1 L Erlenmeyer flask. Me3SbCl solution was added to the broth to give a concentration o f 1 ppm in 2  antimony. After a 14 day growing period, the mycelia, as spherical pellets, were harvested by centrifugation and rinsed with distilled water. They were then homogenized with an Ultraturrax T25 homogenizer (Jak & Kunkel) to give a solution o f lysed fungal cells, and analyzed by using H G - G C - A A S . A control experiment was carried out in which the fungus was grown in the same manner, only without the addition o f antimony to the potato dextrose broth. See Chapter 3 for more details about this experiment.  70  2.2.3. Results and Discussion  Demethylation of trimethylantimony species in aqueous solution during analysis  2.2.3.1.  by using G C - H G - A A S D o d d et al  73,  observed that peaks corresponding to SbH-3, M e S b H , and Me2SbH, as well 2  as the expected M e S b , appeared when the hydride generation apparatus had not been 3  preconditioned with the reagents used for analysis (2% N a B H j and 4 M acetic acid). In an attempt to replicate this observation, experiments were carried out by using the same method, but with an A A S instead o f an M S as the detector. M e S b C l was analyzed after a preconditioning 3  2  step which consisted o f rinsing all tubing with water, as well as after rinsing with 2 % NaBEL, and 4 M acetic acid. In all replicates (five for each), the results were the same when Me3SbCl was 2  reduced and analyzed: M e S b and a minor amount of M e S b H (corresponding to <2% 3  2  demethylation) were detected. The large amount o f demethylation that was observed by D o d d et al.  73  could not be replicated.  2.2.3.1.1.  The effect of acid  When HC1 was used as the acid in the reaction to adjust the p H o f the sample, increased demethylation was observed. Different concentrations of HC1 led to the appearance o f S b H , 3  M e S b H and M e S b H in differing amounts, indicating a p H dependence for the demethylation 2  2  process. Hence, demethylation of M e S b C l at different acidities was studied, and the results 3  2  obtained are illustrated in Figure 2.14. It was assumed that the reaction efficiency and detector response for each species is the same (which was observed to be the case for M e S b and SbH ), 3  3  allowing the amount o f each stibine to be determined. A minimum of three replicates were carried out at each p H , but reproducibility o f the normalized amounts was poor, with relative 71  100 B  90 80 70 15  £  10 H  5 H  T -  m  CO  r--  CO CO  co  CO  I o  T a>  IT)  T-  CM  O CO  t -  CM CN  co  •*  oi  CO  CO  CO  (6  CN  O  PH  F i g u r e 2.14. Percent amounts o f stibines generated from M e S b C l at varying p H , when using H G - G C - A A S . Amounts o f stibines may not add up to 100% because o f imprecision in blank correction. Except where indicated, unbuffered HC1 was used to adjust the p H . A = sulfuric acid, B = maleic acid, C = citric acid, D = citrate buffer, E = water. 3  2  72  standard deviations averaging 22% (0.1% up to 50% for amounts approaching the detection limit). The acid used in most o f the experiments was unbuffered HC1, but at some pHs different buffers and acids were tested to determine i f the demethylation was specific to HC1. These buffers and acids are specified in Figure 2.14. Small amounts of M e S b H appear at p H 3.22 and 2  p H 3.30, but at p H 6.10 no demethylation is seen for the concentration o f M e S b C l being 3  2  studied. Although a statistically significant dependence o f demethylation on the acid used was not observed for the acid concentrations studied (see Figure 2.14), it must be noted that certain acid systems give less demethylation than would be expected considering the p H . F o r example, reactions carried out by using 4 M acetic acid ( p H 2.2) resulted in statistically significantly less demethylation than experiments carried out at similar p H , by using other acids (see Table 2.5 and compare to results for 0.01 M HC1 and 0.1 M citric acid). However, when 0.6 M acetic acid was tested (Table 2.5), the amount o f demethylation was not statistically significantly different from other acid systems at similar p H , but the amount o f demethylation was calculated to be statistically significantly different from that observed when 4 M acetic acid was used. M o r e concentrated solutions o f citric acid (0.5 M and 1 M ) also showed amounts o f demethylation that were calculated to be statically significantly less than that observed when 0.1 M citric acid was used (Table 2.5). M o r e studies must be carried out to elucidate the mechanism o f the demethylation phenomenon and hence to explain this observed behaviour.  73  Table 2.5. Comparison o f amounts o f demethylation when using different concentrations o f acids. L o w e r values o f percent M e S b o f total Sb indicate higher amounts o f demethylation. 3  % M e S b o f total Sb (SD )  A c i d used  p H (calculated)  0.01 M H C 1  2.25  0.6 M acetic acid  2.6  94 (2)  4 M acetic acid  2.2  98 (2)  0.1 M citric acid  2.19 , 2.2  92.2 (0.7)  0.5 M citric acid  1.9  96.6 (0.6)  1 M citric acid  1.7  95.9 (0.9)  C  3  3  92.5 (0.6)  b  b  Unless otherwise stated, pH is calculated by using pH=-log(VK x [HA]/2); where K = 1.75 x 10" for acetic acid; K = 7.44 x 10" for citric acid; [HA] = concentration of acid. [HA] is divided by 2 because of dilution during mixing of the acid in the HG apparatus. pH was measured after mixing the acid solution with water, as described in section 2.2.2.2. SD = standard deviation, calculated from 3 replicate analyses. a  5  a  a  4  a  b  c  2.2.3.1.2. The effect of concentration When high levels o f M e S b C l (500 - 1000 ng) were analyzed, demethylation was 3  2  observed at neutral p H as well. Demethylation can only be seen when high levels o f M e S b C l 3  2  are analyzed, even though the amount o f demethylation is low at neutral p H (<1%), because the levels o f S b H , M e S b H and M e S b H are sufficiently high for detection. The detection limits are 3  2  2  estimated to be between 1 ng and 5 ng for all the stibines, although standards are not available for methyl- and dimethylantimony species. The demethylation pattern is not dependent on the concentration o f antimony being analyzed. Small ranges o f concentration (2 orders o f magnitude) were tested, however, so the possibility that the demethylation pattern changes at higher concentrations cannot be discounted.  74  2.2.3.1.3. The effect of sample matrix To illustrate the importance o f establishing the presence o f demethylation during the analysis o f trimethylantimony species, a sample known to contain trimethylantimony species was analyzed. W e found that the sample matrix in this example has an effect on the hydride generation o f methylstibines. The term "sample matrix" refers to any chemical components other than the analytes (antimony species) that make up the sample. Figure 2.15a shows a chromatogram o f an aqueous extract o f the fungus Pleurotus flabellatus  that had been grown in liquid culture and amended with M e S b C l . The stibines were 3  2  generated with the aid o f aqueous borohydride with no acid or buffer, conditions under which the M e S b C l standard in water described in section 2.2.3.1.1 showed minimum demethylation ( p H 3  2  5.6). Figure 2.15a shows peaks in addition to the one corresponding to the expected trimethylstibine for this sample. Figure 2.15b shows the same fungus extract analyzed while using a 0 . 0 5 M citrate buffer at p H 6.2. M o s t o f the demethylation appearing in Figure 2.15a was eliminated by using a buffer. To determine whether the demethylation observed in Figure 2.15a was caused by the sample matrix, a control sample from the mushroom culture that had not been amended with antimony, and hence with the identical matrix but found previously to contain a concentration o f antimony less than 0.05 ppm (Chapter 3), was analyzed after the addition o f 200 ng o f M e S b C l . The 3  2  sample was analyzed in the same manner as the sample in Figure 2.15a and the result is shown in Figure 2.15c. The same demethylation pattern can be seen, indicating that this specific sample matrix causes the demethylation. The efficiency o f the hydride generation reaction is decreased by the sample matrix, as shown by Figure 2.15d, which is the chromatogram that resulted when 200 ng of M e S b C l 3  2  75  100 90  2.15a  2.15c  80 70 H  8  60  e  3  60  w  40 30 20  1 Time (min)  Time (min)  100 i  100  90  90  80  60 -|  k  60 40 H 30 20 10  80-1  2.15b  c  2  2.15d  70 c  60  I"  w  30 20  h-H—r> 4 -  10 0  i 2 Time (min)  Time (min)  Figure 2.15a. Chromatogram of an aqueous fungus extract analyzed at neutral pH (water only, unbuffered). Figure 2.15b. Chromatogram of the same aqueous fungus extract analyzed at pH 6.2, citrate buffer. Figure 2.15c Chromatogram of antimony-free aqueous fungus extract spiked with 200 ng Me SbCl , analyzed as in Figure 2.15a. Figure 2.15d. Chromatogram of 200 ng Me SbCl in water analyzed as in Figure 2.15a. W = SbH , X = MeSbH , Y = Me SbH, Z = Me Sb. 3  3  3  2  2  2  2  3  76  dissolved in water was analyzed in the same manner as the sample in Figure 2.15a and Figure 2.15c. The amount o f total hydrides in Figure 2.15d is much greater than that in Figure 2.15c. Figures 2.15(a-d) show the importance o f matrix effects in this fungal extract on the hydride generation o f M e S b . 3  2.2.3.1.4. Other studies to determine causes of demethylation Additional studies were carried out to qualitatively determine causes o f demethylation, and are summarized in Table 2.6.  Table 2.6. Qualitative tudies to determine causes o f demethylation. See Figure 2.12 for locations o f reactors A and B in Method 3. Reactor B contained 20 ml o f I M H S 0 and an amount o f 100 ng (as Sb) o f Me3SbCl was reacted in each experiment. I, II and H I are sequential in time. 2  4  2  Expt .No. 1  Method 3  Reactions I. M e S b C l + N a B F L (reactor A ) EL Gaseous products from I bubbled through H S 0 (reactor B )  no  I. M e S b C l + N a B F L H. (Products from I) + H S 0  yes  3  2  2  2  Demethylation?  2  4  3  2  2  3  3  I. 2 ml NaBFL, (2% w/v) + H S 0 (reactor B ) H. M e S b C l + N a B F L (reactor A ) ITT. Gaseous products from II bubbled through liquid products from I (reactor B )  yes  I. M e S b C l + N a B F L (reactor A ) n. Gaseous products from I bubbled through H S 0 (reactor B ) HI. (Liquid products from II in reactor B ) + 2 ml N a B F L (2% w/v)  yes  2  3  4  3  4  2  3  2  4  2  4  77  M e S b C l is stable in I M DC1 in D 0 by using N M R analysis , and therefore 3  2  2  demethylation is not taking place prior to the hydride generation reaction. M e S b was generated 3  at neutral conditions and then bubbled through H S 0 , and it was found to be stable to acid 2  4  (Table 2.6, Experiment 1). However, when both borohydride and acid are present after trimethylstibine is generated, demethylation is observed (Table 2.6, Experiments 2 and 3). Trimethylstibine appears to dissolve in acid (or possibly become reoxidized to trimethylantimony oxide) and demethylates during the reaction with sodium borohydride (Table 2.6, Experiment 4). Therefore demethylation is taking place during the reaction, as a result o f a product o f the acid/borohydride reaction. A s well, trimethylstibine, once formed, is unstable to a product o f the acid/borohydride reaction.  2.2.3.1.5. Suggested reasons for  demethylation  "Molecular rearrangement" o f methylarsines has been observed in the past ' ' , mostly 79  80  81  in the form o f demethylation, but also resulting in the formation o f higher methylated compounds from lesser methylated ones. The previous studies do not give an explanation for the rearrangement, but rather link it to certain factors. For example, when 0.5 M H S 0 was used in 2  4  the derivatization reaction, demethylation o f monomethyl- and dimethylarsines resulted . The 81  present study (section 2.2.3.1.1) shows a similar trend, in that the use o f acid during the hydride generation o f M e S b C l results in demethylation products. However in another study the use o f 3  2  acid p H during the hydride generation reaction reduced the amount o f demethylation . In the 79  same study the use o f a sodium borohydride pellet rather than aqueous solution and elimination 79  of dissolved oxygen also reduced or eliminated the amount o f demethylation, and this was attributed to an increase in the rate o f the reduction reaction. In other words, these authors postulated that a faster rate o f reaction results in less demethylation.  78  Mechanistic pathways for demethylation or rearrangement have not been postulated in the literature. The mechanism o f the hydride generation reaction itself is not well known but studies with deuterium labeled sodium borohydride and arsenic compounds indicate that the hydrides result from direct FT transfer from the borohydride . 82  The present study indicates that demethylation takes place in solution during the hydride generation reaction. It may then be postulated to take place at one or both o f the following stages: during the actual reduction, or after M e S b is formed. 3  To address the possibility o f demethylation taking place during the reaction, the following reactions may be postulated to take place for the reduction of M e S b C l : 3  M e S b C l + 2FT - » M e S b H + 2C1" 3  2  3  Me SbH 3  2  Me Sb + H  2  3  2  (1) (2)  2  Because the Sb-C bond is o f similar strength to S b - H but less than that o f S b - C l , , 83  84  methyl groups may be lost during these reactions. The intermediate compound M e S b H may be 3  2  unstable to FT or to products o f the reaction between acid and borohydride. M e S b , once formed, appears to be unstable to the products o f the acid/borohydride 3  reaction (section 2.2.3.1.4). The presence o f borane, B H or diborane, B H<s, known to be 3  products from the reaction o f BFL," with H C 1  2  may be a key factor affecting the stability o f  85  M e S b . Adducts with B H may form, such as M e S b B H which decomposes at room 3  3  temperature . 86  3  3  The decomposition o f M e S b B H may follow a similar pathway as that proposed 3  3  for the reaction o f Me4Pb and B H 6 to M e B and metallic Pb, which involves stepwise loss o f 2  3  methyl groups from the lead compound . M e S b H B H has been isolated and reacts further to 87  2  form the stable compound M e S b B H 2  8 8 2  3  . The relative instability o f the M e - S b bond in M e S b is 3  79  also seen by using mass spectrometry  (see also Chapter 6) where the first methyl group is easily  lost from Me3Sb. M o r e experiments are necessary to deduce the mechanism o f both the reduction reaction and the demethylation and rearrangement phenomena. Therefore, when analyzing a sample for methylated antimony compounds by the method of hydride generation, the reaction conditions should be carefully tested with standard compounds such as M e S b C l . The issue o f "rearrangement" associated with methyl- or 3  2  dimethylantimony(V) compounds has not been addressed, since no standard compounds are available, but similar problems may exist for these species, making the synthesis and study o f these compounds imperative. A s well, the behaviour o f methylated antimony species in the +3 oxidation state is completely unknown, although their presence in the environment is not unlikely. 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CRC Handbook of Chemistry and Physics, 76th ed.; Lide, D . R , E d . ; C R C : B o c a Raton, 1995, pp F219-F223. 85. Cotton, F. A . ; Wilkinson, G . Advanced Inorganic Chemistry, 3rd ed; Interscience: N e w York, 1972; p 250. 86. Hewitt, F . ; Holliday, A . K . J. Chem. Soc. 1953,  530-534.  87. Holliday, A . K . ; Jessop, G . N . J. Organometal. Chem. 1967,  10, 291-293.  88. Greenwood, N . N . In Comprehensive Inorganic Chemistry; Bailar, J. C ; Emeleus, H . J.; Nyholm, R.; Trotman-Dickenson, A . F., Eds.; Pergamon: Oxford, 1973; V o l . 1, p 751. 89. Smith, J. D . In Comprehensive Inorganic Chemistry; Bailar, J. C ; Emeleus, H . J.; Nyholm, R.; Trotman-Dickenson, A . F., Eds.; Pergamon: Oxford, 1973; V o l . 1, pp 625.  85  Chapter 3  ARSENIC AND ANTIMONY IN MUSHROOMS  3.1. Introduction  Mushrooms are fruiting bodies which participate in the reproductive cycle o f a fungal organism. The fungus consists o f filamentous cells (hyphae) that grow through a substrate to form a mass called the mycelium. Fungi that produce mushrooms belong to one of two divisions, the Basidiomycota and Ascomycota, based on the type o f reproductive cell inside the mushroom used for spore production (basidium or asci). Spores are the reproductive cells that trigger the birth o f new fungal organisms. M o s t o f the commonly known fungi that produce mushrooms belong to the division Basidiomycota. Interest in the arsenic content in mushrooms has increased recently. Some species o f mushrooms appear to accumulate arsenic and other metals from soil , and their potential as 1  biological pollution indicators has been discussed . F o r mushrooms that accumulate arsenic, and 2  are edible, such as Laccaria  amethystina, toxicological consequences (if any) to consumers have  been o f concern . F o r these reasons, the uptake and speciation o f arsenic in mushrooms has been 3  studied. Determining the bioavailability and species o f arsenic in mushrooms, especially those containing elevated levels, helps in toxicological risk assessment. Chemical processes taking place in the terrestrial environment have not been studied to the extent to which those in the marine environment have been. The recent findings o f arsenobetaine and arsenocholine in mushrooms have led researchers to draw similarities between marine and terrestrial pathways for the formation o f arsenic compounds ' ' . The presence of 4 5 6  86  arsenobetaine in mushrooms in higher taxonomic positions (i.e., being more highly evolved) is 7  similar to the presence o f arsenobetaine in higher marine organisms, such as marine animals . 8  Although some authors believe that the fungi producing the mushrooms are responsible for the bio-synthesis o f more complex arsenic forms, such as arsenobetaine , no proof exists for 7  this hypothesis. This theory is favoured for two reasons. One is that arsenobetaine has not been found in soil . Arsenobetaine was, however, found in ant-hill material , and its presence in 4  9  estuarine waters was recently confirmed . The second reason is that similar chemical forms o f 10  arsenic have been seen in mushroom species collected from different locations . In support o f the 3  fungus biosynthesis theory, the fungi Agaricusplacomyces  and Pleurotus sp. (producing edible  mushrooms) methylate arsenic to a small extent . 11  Very little is known about the interaction o f antimony with fungi, or with terrestrial organisms in general. The chemical similarities o f antimony and arsenic has led to the hypothesis that fungi may interact with antimony in the way that they do with arsenic. F o r example, antimony was postulated to be methylated by the fungus Scopulariopsis brevicaulis, and this has, in fact, been shown to take place to a very small degree ' . The identity o f antimony species in 12 13  fungi which produce mushrooms is completely unknown. The following chapter describes work exploring three objectives. The first is to determine the arsenic species in edible mushrooms that are readily available in supermarkets. The second is to determine i f mycelia in axenic culture (i.e., containing only one organism) are responsible for the formation o f complex arsenicals by growing pure cultures o f fungi that are capable o f producing mushrooms in arsenic amended media. The third is to learn more about biological interactions with antimony, by growing fruiting bodies on antimony-containing substrate, and by culturing fungi that can produce mushrooms in antimony amended media.  87  3.2. Experimental  3.2.1. Chemicals and reagents Sodium arsenate, N a H A s 0 . 7 H 0 (Aldrich), arsenic trioxide, A s 0 2  4  2  2  3  (Alfa),  methanearsonic acid, C H A s O ( O H ) (Vineland Chemical), and cacodylic acid, ( C H ) A s O ( O H ) 3  2  3  2  (Vineland Chemical) were dissolved in deionized water to make standard solutions. Extracts o f kelp powder (Galloway's, Vancouver, B C ) and N o r i (Porphyra tenera) o f known arsenosugar content were obtained as described elsewhere and were used as laboratory standards in order to 14  establish the retention times o f arsenosugars. The identity and retention times o f the arsenosugars were verified by comparison to pure arsenosugars generously donated by K . Francesconi and T. Kaise. Arsenobetaine , arsenocholine , trimethylarsine oxide , and tetramethylarsonium 15  16  17  iodide were had been synthesized previously according to standard methods. Identification o f 18  arsenicals in samples was made by comparison o f retention times to those in standards. Antimony ( V ) and (III) standards were obtained as potassium hexahydroxyantimonate, K S b ( O H ) (Aldrich), antimony trichloride, S b C l (Aldrich), and potassium antimonyl tartrate, 6  3  K S b ( C 0 6 H ) (Aldrich). M e S b C l was synthesized as described elsewhere . Stock solutions 19  2  2  4  2  2  3  2  were made by dissolving these compounds (except SbCl ) in deionized water to 1000 or 100 ppm 3  as Sb. S b C l was dissolved in 6 M HC1 to 1000 ppm as Sb. Standard working solutions were 3  made by diluting the stock solutions with deionized water as necessary. For hydride generation analysis, NaBFL, (reagent grade, Aldrich) was dissolved in deionized water fresh daily to give a concentration o f 2% w/v. Ammonium citrate buffer at a concentration o f 0.05 M and p H 6 (1 M ammonium hydroxide, M i c r o Select, Fluka, and  88  analytical reagent grade citric acid, B D H ) and 1 M HC1 (Environmental grade, Alfa Aesar) were used. Other chemicals for the preparation o f mobile phases were analytical-reagent grade or higher in purity. They included methanol ( H P L C grade, Fisher), concentrated phosphoric acid (Aldrich), ammonium hydroxide ( I M , Fluka), pyridine (Fisher), formic acid ( B D H ) , potassium hydroxide ( K O H , Aldrich), tetraethylammonium hydroxide, ( T E A H , 20% in water, Aldrich), and malonic acid ( B D H ) . Mobile phases were filtered through 0.45 um cellulose nitrate filters (Millipore) prior to use. The arsenosugar stock solution used in the Scleroderma citrinum experiments was obtained by extracting commercially available macroalgae species (available for human consumption), which are known to contain arsenosugar X and X I , and D M A . The sample 1 4  consisting o f 28 g N o v a Scotian dulse (Palmariapalmatd),  6.5 g Taiwanese N o r i (Porphyra  tenera) and 6.7 g Japanese Y a k i N o r i (Porphyra tenera, all purchased at Vancouver supermarkets) was extracted with approximately 1.5 L o f MeOH/water (1:1) three times by sonication (2 hours), and the liquid was separated from the solid between sonications by filtration through Whatman #1 filter paper. The extracts were combined and concentrated to a final volume o f approximately 400 m L by evaporation with a constant stream o f air overnight at room temperature.  89  3.2.2. Apparatus and method of analysis  3.2.2.1. HG-GC-AAS analysis for antimony speciation The apparatus was composed o f a semi-continuous flow, hydride generation system developed for arsenic analysis, coupled to an atomic absorption spectrometer (Varian AA1275) 20  fitted with an Sb lamp (Varian) operating at a wavelength o f 217.6 nm. One modification was made to the basic apparatus in the form o f using a gas-liquid separator  21  that resulted in less  analyte carryover. The apparatus consisted o f Tygon tubing for the peristaltic pump, and P T F E tubing (1/8" O D ) for the remainder. The glass gas-liquid separator was silanized with ( C H ) S i C l before use. Data were collected from the A A S and process directly by using an H P 3  2  2  3 3 90A integrator, or were analyzed with the aid o f Shimadzu E Z C h r o m software. A peristaltic pump was used to deliver standard or sample solution (usually 0.1 m L to 3 mL) to mix with the acid or buffer and then to mix with a solution o f NaEfflU (2% w/v) in a reaction coil. The gases evolved were separated in the gas-liquid separator and then swept by a flow o f helium into a P T F E U-tube, where they were trapped at -196 °C. Continuous hydride generation and trapping were carried out for 3 minutes. The peristaltic pump was then stopped (making the system semi-continuous) and the U-tube was heated to 70 °C, allowing the gases to be swept with H e at a flow rate o f 40 mL/min onto a Poropak P S column, which was then heated from 70 °C to 150 °C at a rate o f 30 °C/min, whereby the gases were separated. They were then detected by A A S .  90  3.2.2.2. HPLC-ICP-MS analysis for antimony and arsenic speciation The H P L C apparatus consisted o f a Waters 510 double piston pump, a Rheodyne six-port injection valve with a 20 u L loop, in line filters, a guard column for each analytical column packed with the same stationary phase, and the analytical column. Columns and mobile phases are listed in Table 3.2. In some cases o f plasma instability and detector drift, 10 ppb R h was added to the mobile phase to provide a constant background signal. A V G Plasmaquad P Q 2 Turbo I C P - M S ( V G Elemental) was used as a detector. Parameters for the I C P - M S are given in Table 3.3. The m/z monitored were 121 and 123 (Sb), 75 (As), 77 82 (Se and Ar- C1) and 103 37  (Rh) where applicable. The H P L C was coupled to the spray chamber o f the I C P - M S by using a minimum o f P T F E tubing (10 cm x 0.5 mm i d . ) with the appropriate P T F E fittings. Extracts and media were diluted as necessary, filtered through 0.45 um syringe filters (Millipore) and analyzed by H P L C - I C P - M S using the conditions given in Table 3.1 (arsenic speciation), Table 3.2 (antimony speciation) and Table 3.3 ( I C P - M S detection). Data were processed and analyzed by using chomatographic software , and when semi-quantitative 22  concentrations o f arsenic and antimony compounds were determined, external calibration curves were used.  3.2.2.3. ICP-MS analysis for total arsenic and antimony concentrations The I C P - M S described above, outfitted with a peristaltic pump and injection loop for flow injection introduction, was used for the determination o f total arsenic and antimony in samples. The parameters listed in Table 3.3 were used, except that time resolved analysis was not used for these analyses. Solutions and standards were diluted with 1% (v/v) nitric acid (doubly distilled in quartz, Seastar) and R h (10 ppb) was added as an internal standard.  91  Table 3.1. H P L C conditions for arsenic speciation Chromatography  Column  Mobile phase  Flowrate (mL/min) 1.0 or 1.5  Anion exchange  Hamilton P R P - X 1 0 0 , 150 x 4.6 or 250 x 4.6 mm  20 m M ammonium phosphate, p H 6.0  Cation exchange  Supelcosil L C - S C X or Whatman S C X Partisil 5, 250 x 4.6 mm  20 m M pyridinium formate, p H 2.7  1.0  Ion-pairing  G L Sciences O D S , 250 x 4.6 mm  l O m M T E A H , 4.5 m M malonic acid, 0.1% MeOH, pH6.8  0.8  Table 3.2. H P L C conditions for antimony speciation Chromatography Anion exchange  Ion-pairing  23  Column  Mobile phase  Flowrate (mL/min)  Hamilton P R P - X 1 0 0 , 150 x 4.6 mm  2 mMKOH  10  Hamilton P R P - 1 , 150 x 4.6 mm  10 m M T E A H , 4.5 m M malonic acid, 0.1% M e O H , p H 6.8  0.8  Table 3.3. Operation parameters for I C P - M S Feature  Specific Conditions  Forward radio-frequency power  1350 W  Reflected power  <10 W  Cooling gas flow rate (Ar)  13.8 L/min  Intermediate (auxiliary) gas flow rate (Ar)  0.65 L/min  Nebulizer gas flow rate (Ar)  1.002 L/min  Nebulizer type  de Galan  Analysis mode  Time Resolved Analysis ( T R A ) for H P L C  Quadrupole pressure  9 x 10" mbar  Expansion pressure  2.5 mbar  7  3.2.3. Cultivation of Pleurotus flabellatus fruiting bodies on a solid substrate To qualitatively determine antimony uptake by mushrooms, the fungus P. flabellatus (commonly known as the pink oyster mushroom) was grown on a solid soil medium (wood chips, sawdust and bran, about 25% moisture) contained in polyethylene bags. The bags containing 4 kg substrate were sterilized and inoculated with P. flabellatus mycelium, at Western Biologicals Ltd., Aldergrove B C . The bags were opened in a biological safety cabinet at U B C and antimony solutions (10 m L o f 500 ppm as Sb) were added through a 0.2 pm syringe filter (Nalgene), dropwise, and the top 15 cm o f soil was mixed with a flame-sterilized spatula. The mixing was inadequate and hence concentrations were not homogeneous throughout the bag. Four bags were prepared, containing: (A) potassium antimonyl (III) tartrate; (B), potassium antimonate (V); (C) Me3SbCl ; and (D) no antimony. The bags were kept in a dark incubation chamber at 2  15 °C for 1 week, and then the temperature was increased to 20 °C for another week with illumination for 12 hours/day. The humidity was kept high by placing open dishes o f water  93  around the bags, and by spraying the bags daily with deionized water. After 2 weeks fruiting bodies appeared and they were harvested and frozen until analysis.  3.2.4. Preparation of pure submerged cultures of fungi P. flabellatus was grown from a culture obtained from Western Biologicals Ltd., Aldergrove, B C , in potato dextrose broth (Difco), a general liquid medium for fungus. The following procedures were used for all culture experiments. Solutions used to amend the media were sterilized by using syringe filters (0.22 um, Nalgene or Millipore, cellulose acetate), and they were added to give the appropriate starting concentration in each treatment (see Table 3.5). Seed culture containing mycelia that were free o f antimony and arsenic was added in a volume that was approximately 10% o f the volume o f the broth (see Table 3.5 for volumes). The fungi were incubated on a shaker (forming spherical mycelia for P. flabellatus, S. citrinum and S. crispa, and broken filaments o f mycelia f o r M procera) for the duration o f the experiment (Table 3.5) at 26 °C. F o r P. flabellatus, the spherical mycelia were harvested by centrifugation o f the biomass/liquid mixture and washing o f the mycelia with deionized water. F o r the other cultures, the biomass was harvested by vacuum filtration (Whatman # 1 filter paper) and rinsed a minimum o f three times with deionized water. Scleroderma citrinum, commonly known as the earthball, and resembling puffballs in shape and reproduction, was obtained from an uncontaminated w o o d chip substrate in Vancouver. A number o f fruiting body specimens were collected, cleaned manually by abrasion and with water to remove dirt and other debris, and sterilized on the outside surfaces with 30% hydrogen peroxide.  Table 3.4. Summary o f pure culture experiments. A B = arsenobetaine; A s S = arsenosugars from algae extract; control experiments contain non-living cells. Sb or A s species  P.  N o . of  Duration o f  Approximate  Culture  replicates  experiment  concentration  volume  (days)  (PP )  (mL)  m  flabellatus  Sb (III)  2  14  1  400  Sb (V)  2  14  1  400  2  14  1  400  N o Sb or A s  2  14  0  400  Sb (III) control  2  14  1  400  Sb ( V ) control  1  14  1  400  M e S b C l control  1  14  1  400  Sb (III)  2  43  10  400  Sb ( V )  2  43  10  400  2  43  10  400  As (V)  2  43  1  400  AB  2  43  1  400  AsS  3  27  0.3  100  N o Sb or A s  2  43  0  400  Sb (III) control  1  63  10  50  Sb ( V ) control  1  63  10  50  A s ( V ) control  1  63  1  50  A B control  1  63  1  50  AsS control  1  27  0.3  50  As (V)  2  35  1  400  N o Sb or A s  1  35  0  400  A s ( V ) control  1  35  1  200  As (V)  2  35  1  400  N o Sb or A s  1  35  0  400  A s ( V ) control  1  35  1  200  Me SbCl 3  3  2  2  S. citrinum  Me SbCl 3  M.  2  procera  S. crispa  A l l other culturing steps were carried out aseptically in a biological safety cabinet. The specimens were cut open and a small piece o f the fungus inside (containing spores) was used to inoculate sterile potato dextrose agar plates and potato dextrose broth. Within 2 weeks, growth was seen both on the plates (as a filamentous mycelium) and in the broth (as small white spherical mycelia, as for P. flabellatus).  These cultures were then used to seed other stock cultures and  the experimental cultures. M. procera and S. crispa were obtained from the American Culture Collection as axenic cultures, which were then used to inoculate Y M broth (see Table 3.4 for Y M broth ingredients, following the recipe recommended by D i f c o ) . The submerged cultures obtained were used to 24  seed the experimental cultures.  Table 3.5. Y M Broth ingredients and composition Ingredients per liter  Weight in grams  Yeast extract, Difco  3  Malt extract, Sigma  3  Peptone, Difco  5  Dextrose, Fisher Scientific  10  p H + 0 . 2 a t 2 5 °C  6.2  Controls were prepared, containing Sb and A s species, potato dextrose broth or Y M broth and mushroom mycelia that had been autoclaved. The controls were incubated for a minimum o f the same time period as the live cell experiments, and the liquid was collected by filtration (Whatman #1 filter paper). N o organisms appeared to be growing in any flasks containing autoclaved cells, determined by visual inspection o f the flasks, and by visual inspection o f potato dextrose agar and nutrient agar that had been streaked with the autoclaved cells.  N o contamination by other organisms was observed, either macroscopically (i.e. no cloudy solutions, which indicate bacterial infection) or microscopically, except in an experiment containing Sparassis crispa and amended with A s (V). Hence the experiments (except for the one containing live cells o f S. crispa) were assumed to axenic, meaning that only one organism was present in each experiment. A portion (1-5 m L ) o f the medium for each experiment was collected after the addition o f arsenic or antimony (referred to as "medium before") and reserved for analysis. The medium for each experiment was collected after harvesting o f the biomass (referred to as "medium after") as well. A l l biomass and medium samples were frozen (-20° C ) immediately until sample preparation and analysis.  3.2.5. Sample preparation and analysis Edible mushrooms were obtained canned or dried from Vancouver supermarkets. Canned mushrooms (oyster mushroom and straw mushroom) were homogenized in a blender with the liquid in the can, and freeze-dried. The freeze-dried material, and dried mushrooms were pulverized by using a mortar and pestle. The powders were then extracted. Extractions were carried out by weighing 2 g (± 0.5 mg) o f the dried powders into 50 m L or 15 m L centrifuge tubes, adding 10-15 m L MeOH/Water (1:1), sonicating for 20 minutes, centrifuging for 20 minutes and decanting the liquid layer into a R B F . Each sample was sonicated and centrifuged a total o f 5 times. The decanted extracts for each sample were pooled and rotovapped to near dryness (1-2 m L ) and then dissolved in 5 or 10 m L o f deionized water. P. flabellatus  fruiting bodies were freeze-dried and pulverized by using a mortar and  pestle. A soil sample consisting o f equal amounts o f soil from each bag was oven dried and weighed to calculate R (R = fresh weight/dry weight). Extractions were carried out as described 97  above, except that for mushrooms, 0.5 g (+ 0.5 mg) was weighed and extracted, and final extract volumes were 10 m L ; and for soils, 3 g (fresh weight) was extracted (± 0.01 g) and final volumes were 10 m L . Samples o f the fruiting bodies for P. flabellatus were digested with acid for determination o f total antimony content. The freeze-dried mushroom powders were weighed (0.5 g ± 0.5 mg) into a 500 m L round bottomed flask (RBF). Concentrated nitric acid (3 m L , doubly distilled in quartz, Seastar, Sidney, B C ) , hydrogen peroxide (3 m L , 30% in water, reagent grade, Fisher) and concentrated sulfuric acid (1 m L , reagent grade, Fisher) were added to each sample. The samples in the R B F s were boiled for 3 hours by using a heating mantle and a reflux apparatus . 25  After all the samples had cooled, the clear solutions remaining were diluted to 10 m L with deionized water and stored at 4 °C until analysis. Medium samples were diluted with deionized water and filtered as necessary, and analyzed with no further preparation. Fresh weight biomass samples (P. flabellatus  and <S".  citrinum) were prepared by weighing 10 g (± 0.01 g) o f filtered and washed mycelia spheres and homogenizing the spheres with an Ultraturrax T25 homogenizer (Jak & Kunkel). The samples were then centrifuged, and the resulting supernatant fraction was filtered. D r y weight samples (all species o f fungi) were obtained by freeze-drying the mycelia and pulverizing them with a mortar and pestle. The samples were then prepared by weighing 0.25 g (± 0.5 mg) o f the dry powder, extracting with 5 g (± 0.5 mg) deionized water by sonication for 2 hours, centrifuging the slurry, and collecting and filtering the resulting supernatant for analysis.  98  3.2.6. Isolation of an unknown compound containing antimony A n unknown antimony-containing compound was observed in all medium samples amended with inorganic antimony species when they were analyzed by using ion-pairing chromatography H P L C - I C P - M S . Due to the large proportions o f this compound in most samples, an attempt was made to isolate and partially characterize it. A sample o f potato dextrose broth medium containing Sb ( V ) in which P. flabellatus  had  been cultured was used. Undiluted medium (1 mL) was injected onto a PRP-1 column (4.6 cm x 15 cm) and the fraction eluting between 5 and 15 minutes was collected (a mobile phase o f 10 m M T E A H / 4 . 5 m M malonic acid at p H 6.8 was used; see Table 3.2 for chromatographic details). This was repeated 9 times so that a total o f 10 m L was injected onto the column. The fractions were pooled and rotovapped to dryness and then dissolved in about 2 m L with deionized water. Ethanol at -20 °C (4 m L ) was added and the mixture was kept at -20 °C for 24 hours. The mixture was centrifuged, and the supernatant was then applied to a 30 m L Sephedex L H 2 0 (Pharmacia) in methanol column ( 3 x 1 0 cm). Methanol (90 m L ) was applied to the column and collected, apart from the first 15 m L , and then evaporated to dryness. The solids remaining were dissolved in 1 m L o f deionized water.  99  3.3. Results and Discussion  3.3.1 Arsenic species in edible mushrooms M u c h o f the recent interest in arsenic in mushrooms stems from their potential as dietary sources o f arsenic, especially in specimens collected from areas high in arsenic. For example, because o f the presence o f D M A in the choice edible Laccaria  amethystina, it has been  recommended that the ingestion o f this mushroom grown on arsenic-contaminated soil be avoided . Therefore we were interested in analyzing some edible mushrooms commonly 3  available in Vancouver supermarkets, to determine the identity o f arsenic species present in the MeOH/water extractable portion. In past studies, the arsenic profile for the same species o f mushroom collected from different locations and containing different levels o f arsenic was found to be similar . Thus we hypothesized that knowledge o f the speciation o f arsenic in mushrooms 3  meant for human consumption might allow us to predict the speciation in the same mushrooms containing higher levels o f arsenic. The mushrooms analyzed in this study were bought both in the dried form and in cans. Dried mushrooms included wooden ears (probably Auricula auricularia),  which is a fungus used  in many Chinese dishes; shiitake mushrooms (Lentinus edodes); two samples o f porcini mushrooms, Porcini 1 (most likely Boletus sp.) and Porcini 2 (unidentified mushrooms that were most likely Agaricus sp., or portobello mushrooms); chanterelle mushrooms  (Cantherellus  cibarius); and a dried mushroom powder o f unknown composition. T w o species o f mushrooms were obtained in cans; they were oyster mushrooms (most likely Pleurotus ostreatus) and straw mushrooms (probably Volvariella  volvacea).  mushrooms by using MeOH/water (1:1)  The arsenic species extracted from these  are summarized in Table 3.6.  100  Table 3.6. Arsenic species in edible mushrooms, in ppb dry weight (SD ). "Trace" amounts are greater than the limit o f detection ( L O D ) but less than 2 x L O D . a  Mushroom  As (III)  As (V)  MMA  DMA  AB  Me As  +  4  Sum of species  b  Wooden ears  trace  17 (4)  23 (5)  trace  <3  <3  46  Shiitake  210 (30)  130 (10)  trace  18(4)  <5  <5  360  Porcini 1  56 (3)  30 (10)  trace  46 (1))  <5  10(4)  150  Porcini 2  <5  30 (10)  trace  70 (20)  54(6)  <5  160  Chanterelle  trace  22  <5  trace  <5  <5  32  Mushroom powder  <9  210 (50)  trace  230 (60)  910 (50)  <9  1360  Oyster mushrooms  30 (10)  28(1)  30  140 (10)  <5  <5  230  Straw mushrooms  <5  36  <5  trace  <5  <5  41  SD = standard deviation, obtained from analysis of extracts by using two different chromatographic systems (anion and cation exchange, see Table 3.1) with ICP-MS detection. Calculation of sum of arsenic species included trace amounts estimated at the detection limit, which was 5 ppb for all mushrooms, except for Auricula sp. (3 ppb) and mushroom powder (9 ppb). a  b  The dried mushroom mentioned earlier, referred to as Porcini 2 and tentatively identified as Agaricus sp., was packaged under the common name "porcini" mushroom, the common name for Boletus sp. However, the appearance o f the mushrooms revealed the fact that they had been identified incorrectly, since mushroom gills were observed, and Boletus sp. have pores rather than gills. Gills, pores or spines are found on the underside o f many mushrooms with caps. The observation o f a different profile o f arsenic species in the unidentified mushrooms, compared with that found for a (presumed) correctly identified sample o f porcini mushrooms (Porcini 1, Boletus sp.) supports the suggestion that the Porcini 2 mushrooms were identified incorrectly as porcini mushrooms. The most important difference in the arsenic profiles is the presence o f arsenobetaine (34% o f arsenic extracted) in the unidentified mushrooms. A number o f mushrooms belonging to the genus Agaricus were analyzed in a previous study and all contained 7  arsenobetaine, ranging in proportion from 55 to 96% o f the arsenic extracted. Therefore it is not unreasonable to suggest that the unidentified mushroom may belong to the genus Agaricus. 101  The only other mushroom sample that contains arsenobetaine is the mushroom powder o f unknown composition. The arsenic species found in the powder are similar to those found in Agaricus bisporus, both in identity and proportions (Agaricus bisporus: A s ( V ) 12%, M M A 7  6%, D M A 27%, A B 55%; see Table 3.7). Agaricus bisporus is a mushroom commonly cultivated and eaten, and is also known as the button or white mushroom. M o s t likely the mushroom powder is composed o f this species o f mushroom.  Table 3.7. Comparison o f proportions o f arsenic species in mushrooms (%) in the current study with those found in published studies (indicated by a footnote). Mushroom  A s (III)  As (V)  MMA  DMA  AB  M  Porcini 2  0  19  3  44  34  __  Mushroom powder  0  15  1  17  67  0  Agaricus  0  12  6  27  55  0  13  12  13  61  0  0  60  39  1  0  0  0  0  88  0  12  0  0  trace  4  8  78  10  0  trace  6  trace  94  trace  0  Porcini 1  37  20  3  31  0  7  Amanita caesara"  32  38  0  13  0  17  Agaricus  0  0  trace  trace  96  4  bisporus  3  Oyster mushroom Pleurotus  ostreatus  h  Straw mushroom Volvariella  volvacea l  Volvariella  volvacea T  a  b  campestef  a  Results from Slejkovec et al. (1997) . Results from Slejkovec et al. (1996) 7  11  . In general, appreciable proportions o f inorganic arsenic were extracted from most o f the mushrooms. I f these mushrooms were collected from areas contaminated with arsenic and  102  similar arsenic species were present, the presence o f higher levels o f inorganic arsenic could be toxicologically important. Extractable arsenic species have been identified for the first time in wooden ears fungus (Auricula auricularia),  shiitake mushrooms (Lentinus edodes), porcini mushrooms (Boletus sp.)  and chanterelle mushrooms (Cantharellus cibarius).  The l o w levels o f arsenic in the sample o f  chanterelle mushrooms made the speciation o f arsenic difficult. Me4As , a compound not +  observed in any other mushrooms in this study, is observed in Porcini 1 (Boletus sp.). This compound has been observed in other mushroom species, including Amanita sp. and Agaricus sp. in previous studies (see Table 3.7) . 7  The l o w levels o f arsenic in straw mushrooms made identification o f arsenic species difficult. Arsenate appears to be the dominant species in this study, but D M A was found to be the major species in the presumed same mushroom in a previous study . The species o f arsenic in 7  this mushroom differed between two different specimens in the previous study (see Table 3.7); one specimen contained inorganic arsenic, arsenobetaine and M M A , as well as D M A , and the other contained mostly D M A and a small amount o f arsenate. Therefore the speciation o f arsenic appears to be inconsistent for this mushroom species. Different arsenic profiles may arise from different microbial environments for the different specimens, i f uptake o f the arsenic compounds is taking place. Different metabolic pathways may be followed as well. When Pleurotus sp. were cultivated in soil amended with inorganic arsenic, only inorganic arsenic was observed in the mushroom, with 1% conversion to M M A (see Table 3 . 7 ) . n  In this study, D M A is the major species o f arsenic extracted from oyster mushrooms (Pleurotus sp.), with inorganic arsenic and M M A present as well. This may indicate differences in the growing environment o f the fungus, and that the arsenic species present in this mushroom are due to uptake from the environment rather than biosynthesis by the mushroom. Slejkovec et  103  al. , n  however, suggested that the time scale for the study involving cultivation ofPleurotus  sp.  was not long enough for the mycelium or fruiting body to biosynthesize other arsenic species. This hypothesis may also account for the differences in arsenic species observed.  3.3.2. The interaction of arsenic species with pure submerged cultures of fungi  3.3.2.1. Culture experiments with Scleroderma citrinum A s mentioned earlier, Scleroderma citrinum is commonly known as the earthball, and is similar to a puffball in appearance. It forms spherical fruiting bodies that can range up to 12 cm in diameter, and the spore mass inside is violet-black, becoming powdery and pale greenish or lilac gray when mature. Spores are propagated when the fruiting body is mature and opens. This mushroom was chosen because o f its availability and also because o f its resemblance to puffballs. Puffballs were found to contain arsenobetaine in previous studies . 7  Experiments were conducted in which the growth medium was amended with arsenate (As (V)), arsenobetaine ( A B ) and a mixture o f arsenosugars (AsS). Arsenate is the most likely form o f arsenic available in s o i l  26  and probably to fungi in the environment. Arsenobetaine is a  possible end product for some mushroom species and so it was o f interest to determine i f any chemical changes take place as a result o f mushroom metabolism. Arsenosugars are postulated to be intermediates in the biosynthesis o f arsenobetaine  27  and therefore their fate was o f interest.  104  Concentrations o f total arsenic in media and biomass for these experiments, as well as a bioconcentration factor ( B C F ) for the fresh weight biomass are summarized in Table 3.8. A B C F is defined as the quotient o f the concentration o f a material (in this case, arsenic) in an organism divided by the concentration o f the material in the solution in which the organism has been living . It can be calculated by using the following relation: 28  B C F = ([As]biomass/[As] ); ma = medium after. ma  Total concentrations were obtained by using flow injection I C P - M S analysis as described in section 3.2.2.3, except for the samples from the arsenosugar amended experiment. These concentrations were obtained by summing the concentrations o f arsenic species determined by H P L C - I C P - M S (as described in section 3.2.2.2). If a B C F is greater than 1, concentration o f arsenic by the organism from the medium is taking place . Concentration by S. citrinum was taking place only for arsenobetaine because a 29  B C F o f 23 (Table 3.8) is observed. In fact, only 7% o f the arsenic remains in solution, indicating that most o f the arsenic is taken up by the organism. S. citrinum does not appear to accumulate arsenate or arsenosugars.  The low levels o f arsenic in the biomass for the arsenate-amended  experiment may indicate that exclusion or fast excretion o f inorganic arsenic is taking place by S. citrinum.  105  Table 3.8. Total concentrations o f arsenic obtained by I C P - M S analysis (except where indicated) for experiments conducted with S. citrinum. A B = arsenobetaine, A s S = arsenosugar mixture Experiment/sample  Concentration o f A s (ppm)  B C F ([As]bi ma /[As] ) 0  SS  b  ma  (SD ) a  A s ( V ) amended Medium before  1.23 (0.03)  Medium after  1.34(0.05)  Biomass  0.20(0.03)  0.15  A B amended Medium before  1.30 (0.03)  Medium after  0.09 (0.03)  Biomass  2.04 (0.04)  AsS amended  23  0  Medium before  0.37  Medium after  0.30°  Biomass  0.24°  c  0.80  SD = standard deviation, calculated from duplicate biological experiments. BCF = Bioconcentration factor, calculated as [As] iomass/[As] ; where ma = medium after. These total arsenic concentrations were not obtained by flow injection ICP-MS analysis, but rather from the sum of arsenic species determined by HPLC-ICP-MS. See text for more detail. a  b  b  ma  c  The proportions o f arsenic species in samples from arsenic-amended S. citrinum experiments are summarized in Table 3.9. The speciation o f arsenic is expressed in proportions rather than absolute amounts because the analyses for most experiments were semi-quantitative.  106  Table 3.9. Proportions o f arsenic species (%) in experiments conducted with S. citrinum; control experiments contain non-living cells and arsenic; A B = arsenobetaine, A s S = arsenosugar mixture Experiment/sample  As(III)  As(V)  DMA  —  Sugar  Sugar  X  XT  AB  A s CV) amended Medium before  2.8  97.2  0  0  0  0  Medium after  80.3  19.7  0  0  0  0  Biomass  87.5  12.5  0  0  0  0  Control medium after  0.7  99.3  0  0  0  0  Medium before  0  1.0  0  0  0  99.0  Medium after  0  0  0  0  0  100  Biomass  0.6  0  0  0  0  99.4  Control medium before  0  0  0  0  0  100  Control medium after  0  0  0  0  0  100  Medium before  0  0  10.2  14.1  75.7  0  Medium after  0  0  9.9  34.1  56.0  0  Biomass  0  0  10.0  34.2  55.9  0  Control medium before  0  4.6  9.3  12.8  73.3  0  Control medium after  0  0  8.9  14.5  76.6  0  A B amended  AsS amended  For the experiments in which the growth medium was amended with arsenate, reduction to arsenite by the mycelia appears to be taking place (Table 3.9). N o reduction took place in the control containing arsenate and non-living cells, indicating that the reduction was due to the biological activity o f the mycelia, rather than chemical transformations in the medium. The speciation o f arsenic in the biomass was similar to that in the medium. M o s t likely the fungus takes up arsenate, reduces it to arsenite and excretes it as arsenite. A s shown by the concentrations o f total arsenic in media and biomass for the experiments in which arsenobetaine was added (Table 3.8), accumulation o f arsenobetaine from the medium  107  takes place, and Table 3.9 shows that arsenobetaine remains unchanged. This result may indicate that should arsenobetaine be present in the growing environment o f a mushroom-producing fungus in the wild, it will be taken up efficiently by the fungus and not be detectable in the soil. The proportions o f arsenic species in the medium at the beginning o f the experiment ("medium before") in Table 3.9 represents the composition o f the arsenosugar mix used for the experiments (extract o f dulse and N o r i , see section 3.2.1). The proportions o f arsenic species are similar for the control media at the beginning and at the end o f the experiment. This indicates that changes in proportions are caused by the biological action o f the fungus. The proportion o f D M A remains constant, but the amount o f arsenosugar X increases, while that o f arsenosugar X I decreases. The proportions in the biomass and in the medium at the end o f the experiment are similar. However, no arsenobetaine or other arsenic species are observed from the interaction o f the fungus with arsenosugars. In summary, these experiments show that accumulation o f arsenobetaine by S. citrinum takes place. A s well, the mycelium form o f this fungus in pure culture appears to be unable to biosynthesize methylarsenic species or other organoarsenic species from arsenate and arsenosugars. The fungus is responsible for the reduction o f arsenate to arsenite, and the probable transformation o f arsenosugar X I to arsenosugar X .  3.3.2.2. Culture experiments with Macrolepiota procera and Sparassis crispa Macrolepiota  procera is commonly known as the parasol mushroom and it is a choice  edible, growing up to 25 cm in diameter. It possesses a stem and cap with gills underneath, and the surface o f the cap is scaly. Sparassis crispa is also a choice edible and its appearance is described as resembling that o f a cauliflower, ranging from 10 to 60 c m . B o t h these 30  mushrooms were chosen for biological experiments because o f the interesting arsenic speciation 108  results found for specimens collected from the wild. These published results from Slejkovec et al  1  are summarized in Table 3.10. The major compound found in a wild specimen o f M procera  was arsenobetaine and it seems reasonable that this fungus might be capable o f bio synthesizing arsenobetaine. Arsenocholine, an arsenic compound found in minor amounts in the marine environment , was found in large amounts in wild specimens o f S. crispa, in addition to an 8  appreciable amount o f unknown arsenic species . 7  Table 3.10. Arsenic species (% o f arsenic extracted) found in wild specimens o f M procera and S. crispa (from Slejkovec et al. ); A B = arsenobetaine, A C = arsenocholine 1  Mushroom  AB  As(III), M M A ,  AC  Unknowns  3  D M A , A s (V) M. procera  100  trace  0  0  S. crispa 1  31  3 (As(V))  66  0  S. crispa 2  trace  trace  45  55  a  Unknown compound on cation exchange HPLC system.  The present arsenic speciation results for experiments conducted with the two species o f fungus, using arsenate to amend the growth medium, are summarized in Table 3.11.  109  Table 3.11. Concentrations o f arsenic species in experiments conducted w i t h M procera and S. crispa in ppm (biomass is dry weight except where indicated); control experiments contain nonliving cells and arsenic. Experiment/Sample  A s (III)  As (V)  DMA  TMAO  Sum o f  BCF  C  species  M. procera Medium before  <0.004  1 01  <0.004  <0.004  1 01  Medium after  0.062  0 67  <0.004  <0.004  0 73  Biomass (dry weight)  1.33  0 67  <0.02  <0.02  2 00  0.11  0 06  <0.002  <0.002  0 17  <0.004  0 75  <0.004  <0.004  0 75  Medium before  <0.004  1 08  <0.004  <0.004  1 08  Medium after  <0.004  0 85  0.043  0.26  1 15  Biomass (dry weight)  2.56  2 62  0.29  1.8  7 27  0.14  0 14  0.02  0.1  0 40  Control medium before  <0.004  1 05  <0.004  <0.004  1 05  Control medium after  <0.004  0 95  <0.004  <0.004  0 95  Biomass (fresh weight)  3  Control medium after  S. crispa  Biomass (fresh weight)  b  Fresh weight concentration is calculated as [biomass concentration (dry weight)]/R, where R = fresh weight of biomass/dry weight of biomass; R f o r M procera is 11.9. Fresh weight concentration is determined as forM procera, R for S. crispa is 18.2. BCF = bioconcentration factor; see Table 3.8 for calculation. a  b  0  Because the B C F values are less than 1 (Table 3.11), no accumulation o f arsenate is taking place by these fungi. The cultures o f S. crispa were not axenic and therefore the conclusion cannot be drawn that the methylated compounds ( D M A and T M A O ) are present due to metabolism by the fungus, rather than by other organisms (e.g., bacteria) present. M. procera biomass contains arsenic mostly as arsenite but excretes very little o f it, which may indicate that arsenite is sequestered by the organism after it is formed, or that the excretion process is slower 110  than the time scale o f these experiments. Neither arsenobetaine nor arsenocholine are present in the biomass or in the media, which may reflect the inability o f these species to biosynthesize these compounds.  3.3.2.3. Summary of the interaction of arsenic with fungi that can produce mushrooms Mycelia o f mushroom-producing fungi grown in pure culture appear to be unable to synthesize arsenobetaine or arsenocholine, and reduction o f arsenate to arsenite appears to be the only chemical process taking place. Arsenobetaine is efficiently taken up by S. citrinum which suggests that i f any arsenobetaine is present in the growing environment o f wild fungi, it may be accumulated by the mushroom. Similar results were observed by Slejkovec et al. . 11  That is, only  a small amount o f methylation by Pleurotus sp. fruiting bodies and Agaricusplacomyces  mycelia  in pure culture was observed. Additionally, when the growth medium for pure cultures o f Agaricus placomyces was amended with arsenobetaine and tetramethylarsonium ion, these two compounds were taken up to a high extent . The authors suggested that the biosynthesis o f 11  arsenobetaine may not be observed because o f the short time scale o f the laboratory experiments , which is probably the case for S. citrinum, M. procera and S. crispa as well. 11  Ill  3.3.3. The interaction of antimony species with fungi  3.3.3.1. Cultivation of Pleurotus flabellatus fruiting bodies Although antimony has been listed as a U S - E P A priority pollutant, very little is known about its interaction with living organisms. Mushrooms were grown in soil that had been amended with antimony in experiments designed to contribute information about the interaction o f antimony with fungi. It was o f interest to determine, qualitatively, i f antimony is taken up under these conditions and to obtain speciation information. The strawberry oyster mushroom Pleurotus flabellatus  was chosen because o f its availability as a pure culture (Western  Biologicals L t d . , Aldergrove, B C ) as well as its fast growth rate. A s mentioned in section 3.2.3, the fruiting bodies were cultivated on a solid substrate contained in polyethylene bags, a common method used by home mushroom growers. When the conditions are ideal, the fruiting bodies push through holes in the polyethylene to appear on the outside o f the bag. Oyster mushrooms have short lateral stems and they are gilled on the underside o f the cap. Pleurotus flabellatus  is  pink in colour and produces fruit in 2 weeks from the time o f substrate inoculation. Four bags were prepared, containing: ( A ) potassium antimonyl (III) tartrate; (B), potassium antimonate (V); (C) M e S b C l ; and (D) no antimony. The concentrations o f total 3  2  antimony in mushrooms from bags A , B and C were elevated compared to D, the mushroom that had been exposed to substrate not amended with antimony (see Table 3.12). Prior to acid digestion, Sb(OH) ", SbCl ", K S b 2 ( C 4 0 H ) and M e S b C l (0.125 pg each, to give a total o f 0.5 6  4  2  6  2  2  3  2  ug o f Sb) were spiked into the round bottom flask ( R B F ) containing the mushroom D, and an average recovery o f 95% was obtained (see Table 3.12). These results indicate that the acid digestion procedure was adequate to dissolve antimony in these forms and probably that bound to the mushroom matrix.  112  Table 3.12. Antimony in mushrooms after acid digestion, analyzed by hydride generation-GCAAS. Experiment  Sb species  [Sb] (ppm dry weight) in  3  mushrooms A  Sb (III)  6.5  A (following extraction )  Sb (III)  5.1  B  Sb(V)  1.6  C  Me3SbCI2  1.0  C (following extraction)  Me3SbC12  0.86  D  noSb  0.04  D (spiked, replicate 1)  no Sb  0.42  c  D (spiked, replicate 2)  no Sb  0.53  c  b  b  Sb species in this column are those added to amend the mushroom growing soil. ' Extraction Extraction was was carried carried out out by by using using MeOH/water (1:1), as detailed in section 3.2.5. Concentration of spike = 0.50 ppm  Very little antimony in the mushrooms is in a form extractable by M e O H / H 0 (1:1) for 2  mushrooms A and C , leaving the remainder bound up with the residue (Table 3.12 and Table 3.13). Semi-quantitative amounts o f antimony species found in mushroom and soil extracts are summarized in Table 3.13. Sb (III) is oxidized to Sb ( V ) , but the possibility o f oxidation by the mushrooms is unverified since no biomass-free control experiment (soil, bag and Sb (III) only) was carried out; thus, the oxidation o f Sb (III) by the soil or by the air cannot ruled out. The oxidation state o f antimony remains the same (i.e., Sb (V)) in both the soil and mushroom for experiment B . In experiment C , M e S b C l is taken up by the mushroom unchanged (i.e., no 3  2  methyl groups were lost) and it also remains unchanged in the soil. Recoveries o f soil spiked with all three antimony species, followed by extraction, are very l o w (<20%), indicating that the antimony species are strongly absorbed or adsorbed to the soil. 113  Table 3.13. Antimony extracted from mushrooms and soils (ppm dry weight), H G - G C - A A S analysis. "Trace" amounts are greater than the limit o f detection ( L O D ) but less than 3 x L O D . When summing species, trace amounts were given a value o f the L O D . 3  Sample  Sb (III)  Sb ( V )  b  Me SbCl 3  2  Sum o f Sb species  Mushroom A  <0.02  0.087  <0.02  0.087  Soil A  <0.003  1.0  <0.003  1.0  Mushroom B  <0.02  0.062  <0.02  0.062  SoilB  <0.003  0.46  <0.003  0.046  Mushroom C  <0.0002  0.001  0.0033  0.0043  SoilC  <0.001  0.016  2.3  2.32  Mushroom D  <0.02  trace  <0.02  0.01  SoilD  <0.003  trace  <0.003  0.003  Spiked soil D  0.016  0.29  0.25  0.556  % recovery  0.1%  16%  14%  0  Dry weight concentrations for soils were obtained by multiplying fresh weight concentrations by R = 1.85. Limit of detection (LOD) for Sb (V) was 0.01 ppm for mushrooms, except for mushroom C, for which a LOD of 0.0002 ppm was estimated, and 0.003 ppm for soils. Concentration of spike =1.84 ppm each a  b  0  3.3.3.2. Culture experiments with Pleurotus flabellatus  3.3.3.2.1. ICP-MS and HG-GC-AAS analysis of biomass extracts and media M o r e experiments with P. flabellatus were carried out, with the following points in mind. The problems o f antimony absorption or adsorption to soil was avoided by growing the fungus in submerged culture to form mycelial spheres. W e wished to confirm the oxidation o f Sb (III) to Sb ( V ) by comparison with controls containing antimony and non-living cells. Finally, higher concentrations o f antimony were used to allow the possible observation o f new antimony species by H P L C - I C P - M S . A summary o f concentrations in media at the beginning and the end o f the experiments is given in Table 3.14. T w o analysis techniques were used: hydride generation-GC-AAS, to obtain  114  speciation information; and I C P - M S , to determine the total amount o f Sb. The concentrations o f antimony acquired by using H G - G C - A A S differed from those obtained by using I C P - M S for the experiments in which inorganic antimony was used (Sb (III) and Sb (V)). This may indicate the presence o f an antimony complex or compound that cannot be derivatized to a hydride under the analysis conditions. The concentrations o f inorganic antimony species were determined either by using the method o f standard additions, or by using an external calibration curve based on standards in a matching matrix. Both these methods o f quantification minimize the likelihood o f inhibition o f hydride formation from inorganic antimony because o f a matrix effect. N o such difference is seen for the experiment in which MesSbCU is used, except for the media at the end of the experiment, which show a decrease in the amount o f hydride formed from M e S b C l . N o 3  2  appreciable differences are seen in the concentrations o f total Sb (determined by using I C P - M S ) in the media at the beginning and the end o f the experiment.  115  Table 3.14. Antimony in media and biomass extracts of Pleurotus flabellatus grown in submerged culture (ppm in solution, ppm fresh weight for biomass) (SD ). H G - G C - A A S analysis was used for speciation, I C P - M S was used for total Sb; control experiments contain non-living cells; na = not analyzed. a  Experiment/sample  Sb (III)  Sb ( V )  Me Sb-  b  3  Total (ICPMS)  Sb am  amended  Medium before  0.40 (0.01)  na  <0.05  1.2 (0.2)  Medium after  0.001  0.35 (0.04)  < 0.001  1.09 (0.04)  Biomass extract  0.004  0.47  < 0.002  na  Control medium before  0.39 (0.08)  na  <0.05  1.14(0.01)  Control medium after  0.37 (0.06)  na  <0.05  1.108 (0.003)  Medium before  na  0.4 (0.1)  <0.03  1.2 (0.2)  Medium after  na  0.4 (0.3)  <0.05  1.2 (0.2)  Biomass extract  < 0.002  0.57  < 0.002  na  Control medium before  na  na  na  1.27  Control medium after  na  na  na  1.36  Medium before  <0.05  na  1.11 (0.05)  1.2 (0.1)  Medium after  <0.01  na  0.45 (0.03)  1.1(0.1)  Biomass extract  < 0.0008  < 0.0008  0.42  na  Control medium before  <0.05  na  1.29  1.1  Control medium after  <0.05  na  1.22  1.0  Sb ( V I amended  M e S b C l ? amended 3  SD = standard deviation, calculated for replicate experiments. Me Sb- is a compound containing Me Sb, determined by HG-GC-AAS, meaning that the exact structure of the species prior to derivatization is unknown.  a  b  3  3  The concentrations o f antimony in the fresh biomass after homogenization were determined to be about 0.5 ppm fresh weight, by H G - G C - A A S , for all three treatments (Table 116  3.14). This concentration is similar to the concentrations obtained by H G - G C - A A S for the media samples at the end o f the experiment (0.35 to 0.45, see Table 3.14), which may indicate that no bioconcentration o f antimony is taking place by the mushroom mycelia. The antimony species found by using the method o f H G - G C - A A S in the mushrooms treated with Sb (III) and Sb ( V ) is Sb (V). The species o f antimony found in the mushrooms treated with M e S b C l is a 3  2  compound containing M e S b - . 3  3.3.3.2.2. HPLC-ICP-MS  analysis of biomass extracts and media  Two H P L C systems were used with I C P - M S as a detector: anion exchange chromatography with a mobile phase o f 2 m M K O H  2 3  (Method A ) , and ion-pairing  chromatography, with a mobile phase o f 10 m M T E A H / 4 . 5 m M malonic acid at p H 6.8 and a polymeric reversed phase column (Method B ) ; see Table 3.3 for details. The separation o f M e S b C l and Sb ( V ) (as Sb(OH) ~) by using these two chromatographic systems with I C P - M S 3  2  6  detection is presented in Figure 3.1. Sb (III) standards are not eluted regardless o f the method used. In Figure 3.1a, Me3SbCl elutes unretained on the column in the anion exchange system 2  (Method A ) and Sb(OH) " is retained, affording a separation between the two compounds; this 6  was observed previously as w e l l . When using the ion-pairing chromatography system (Method 23  B ) , both Sb(OH) " and M e S b C l are retained and separated (Figure 3.1b). 6  3  2  117  F i g u r e 3.1. Chromatograms o f standard antimony compounds (100 ppb each) on two H P L C - I C P - M S systems. 3.1a. 2 m M K O H , P R P - X 1 0 0 antion exchange column. 3.1b. l O m M T E A H , 4.5 m M malonic acid, p H 6.8, 0.1% M e O H , PRP-1 reversed phase column.  118  When Method A was used for H P L C analysis, several unknown antimony-containing compounds, labeled A l , A 2 and A 3 , were observed. A chromatogram is shown in Figure 3.2 for a sample (medium after for the experiment amended with Sb (V)) in which the unknown compounds A l , A 2 and A 3 are present. The relative amounts o f antimony compounds when some of the samples were analyzed are summarized in Table 3.15. The H P L C analysis was qualitative only, hence absolute amounts are not given, and the assumption was made that the ionization in the plasma and response was similar for all antimony compounds.  Table 3.15. Relative amounts o f antimony compounds (%) in some P. flabellatus samples analyzed by using Method A . Control experiments contain non-living cells and antimony; A l A3= unknown compounds. Experiment/sample  Me Sb-  Sb(OH) "  Al  A2  A3  Biomass extract  0  57  43  0  0  Medium before  0  66  18  14  0  Medium after  0  46  15  16  23  Control medium after  0  44  25  31  0  Biomass extract  0  56  44  0  0  Medium after  0  73  0  11  15  Biomass extract  95  5  0  0  0  Medium after  99  1  0  0  0  3  6  SUV) amended  Sbdin  amended  Me SbCl? amended 3  119  7e+3  120  240  360  480  600  720  time (s)  Figure 3.2. Chromatogram (Method A ) o f medium after 14 days o f growth for P. flabellatus amended with Sb ( V ) .  U p o n examining Table 3.15, it can be observed that a large amount o f A l (43 and 44%) is present in biomass extracts for the experiments treated with inorganic antimony. A 3 is seen only in media sampled at the end o f the experiments amended with Sb (III) and Sb (V). F o r the experiment amended with Sb (V), A 3 is not present in the medium at the beginning o f the experiment (medium before), nor in the control medium at the end o f the growing period (control medium after). Thus these results suggest that a metabolite is formed from inorganic antimony starting compounds by P. flabellatus,  and excreted into the medium. This metabolite might  contain antimony, or it may bind to antimony already present in the medium. N o new species are formed when M e S b C l is used as the starting compound. 3  2  When Method B was used for FIPLC analysis, unknown antimony-containing compounds, labeled B l , B 2 , B 3 and B 4 , were observed as well. Three chromatograms obtained by using Method B are shown in Figure 3.3. The first chromatogram (Figure 3.3a) is for the medium at the beginning o f the experiment amended with Sb ( V ) (medium before), the second chromatogram (Figure 3.3b) was obtained from the medium at the end o f the same experiment (medium after) and the third chromatogram (Figure 3.3 c) represents a fresh weight extract of the biomass collected from this experiment. Unknown B 4 is a major antimony-containing compound in the medium samples, but it is not present in the biomass extract. Chromatograms for samples from the experiment in which Sb (III) was used to amend the medium are similar to those in Figure 3.3 and are hence not shown. Due to its presence in all medium samples, including all controls, B 4 cannot be considered a metabolite. B 3 is found in small amounts in biomass extracts for experiments amended with inorganic antimony but not in medium samples; however, its presence as a metabolite was not determined.  121  5e+4  Sb(OH)  6  4e+4 -  3.3a  3e+4 B4  2e+4 1e+4 H Oe+0  1e+5 Sb(OH) 8e+4 H c CD  3.3b  6e+4 4e+4 -  J3  2e+4 -  B4  <  Oe+0 -  2e+4 Sb(OH)  3.3c  2e+4 H 1e+4 8e+3 B2 4e+3 -  B3  Oe+0  I  0  120  240  I  360 480 time (s)  600  720  F i g u r e 3.3. Chromatograms o f P. flabellatus media and biomass extracts (Method B ) for experiments amended with Sb (V). 3.3a. Medium before. 3.3b. Medium after. 3.3c. biomass extract (dry weight). B l , B 2 , B 3 and B 4 are unknown Sb-containing compounds.  122  Because o f the ubiquitous presence of B 4 , an attempt was made to characterize it. A s detailed in section 3.2.6, B 4 was isolated from a medium sample ("medium after", amended with Sb (V)) by using H P L C . Ethanol (-20° C ) was added to the isolation product to precipitate proteins according to the method published by M a et a/.  31  The sample was then applied to a  Sephedex L H 2 0 column to remove excessive salts. The final isolated compound was analyzed qualitatively. H G - G C - A A S analysis when using I M HC1 (acid pH) to adjust the p H o f the reaction afforded only S b H . When H G - G C - A A S analysis was carried out at neutral p H , stibines 3  were not produced. Hence the final isolated compound possesses the oxidation state o f +5 and does not contain methyl groups bound to the antimony. However, the presence o f antimonycontaining compounds o f unknown oxidation state that are not derivatized to hydrides cannot be discounted. This type o f compound was suggested to be present in media from these experiments in section 3.3.3.2.1 and Table 3.14. The chromatogram obtained when the isolated unknown compound was qualitatively analyzed by using Method B is shown in Figure 3.4. The first chromatogram (Figure 3.4a) was obtained when the sample used for the isolation procedure was analyzed; this was the medium at the end o f the experiment amended with Sb ( V ) (medium after), and prior to the isolation procedure. The second chromatogram (Figure 3.4b) represents the final isolated product, after ethanol precipitation and Sephedex L H 2 0 clean-up. Each o f the chromatograms also shows the relative amounts o f the compound (assuming similar detector response), and it can be seen that Sb(OH) ~ and B 4 were present in each sample. Only B 4 was the starting compound for the 6  chromatogram in Figure 3.4b, indicating that B 4 may in fact be an Sb ( V ) compound in equilibrium with Sb(OH) ". 6  123  I  3.4a  Sb(OH) *l (82%)  1.2e+5  6  8.0e+4  E 5 4.0e+4 H  B4 (18%)  <  O.Oe+Ci I  12fJ 1.6e+5 |  I  I  '240  I  360  480  I  ^  I  6CKk  720  600  720  3.4b  \Sb(OH) " \ (85%) 6  1.26+5 H  £ 8.0e+4 *•—• CO  In  <  B4 (15%)  4.0e+4 0.0e+0  —.. -  ,w120  240  r  360 Time (s)  480  F i g u r e 3.4. Chromatograms of unknown B 4 (Method B ) showing proportions o f each compound. 3.4a. Medium after for P. flabellatus amended with Sb ( V ) . 3.4b. B 4 after H P L C fraction collection, ethanol precipitation and Sephedex L H 2 0 clean-up.  124  The equilibrium may be represented by the following equation:  B4  Sb(OHV  When B 4 is isolated, the equilibrium is re-established so that Sb(OH) " is present. Other 6  attempts to characterize this compound were unsuccessful. Enhanced oxidation o f Sb (III) to Sb ( V ) appears to be taking place due to the presence o f P. flabellatus,  as suggested by the results in Table 3.14. Equal amounts o f Sb (III) were  found in the medium at the start o f the experiment (amended with Sb (III)), and in the controls treated with Sb (III) at the beginning and the end o f the experiment. R a w area counts (quantification was not carried out) are shown for Sb(OH) " present in media (Figure 3.5). 6  Although some oxidation is taking place in the medium in the absence o f living cells, enhanced oxidation is taking place by P. flabellatus  3E +7  (Figure 3.5).  T  2 E +7  --  1 E +7  -  OE+0  -I  CO  l_ CO  X)  mmmmmm medium before  mmmmm  1  m e d i u m after  1  mrnt^im  1  control medium after  F i g u r e 3.5. R a w area counts for Sb(OH) " in media for P. flabellatus culture. 6  grown in Sb (Ill)-amended  125  3.3.3.3. Culture  experiments  with Scleroderma  citrinum  The earthball, Scleroderma citrinum, was grown in pure submerged culture amended with antimony compounds to determine the interaction o f antimony with this organism. The concentrations o f total antimony in medium and biomass samples for cultures amended with antimony compounds are summarized in Table 3.16. The concentrations in media do not differ appreciably from the beginning (medium before) to the end (medium after) o f the experiments. Bioconcentration factors are similar for the three antimony compounds studied and are approximately 0.2, indicating that no accumulation o f antimony takes place by the fungus.  Table 3.16. Concentration o f total antimony (ppm) ( S D ) in media and biomass extracts o f Scleroderma citrinum grown in submerged culture (ppm for solutions, ppm fresh weight for biomass). I C P - M S analysis was used for the analysis. a  Experiment/sample  [Sb]  BCF  .([?^].^.9m?.?^I§.!?.]fn?)  Sb (III) amended Medium before  12.0 (0.5)  Medium after  10.9 (0.3)  Biomass extract  2.0 (0.7)  Sb(V)  0.18  amended  Medium before  12.7 (0.7)  Medium after  12.2 (0.3)  Biomass extract  2.5 (0.3)  0.20  Me3_SbCl amended z  Medium before  10.9 (0.7)  Medium after  10.7 (0.4)  Biomass extract  1.8 (0.4)  a b  0.17  SD = standard deviation, calculated for replicate experiments. BCF = Bioconcentration factor, calculated as [Sb] i mass/[Sb] ; where ma = medium after. b 0  ma  126  The chromatograms resulting from the analysis o f biomass extracts for the three experiments by using Method A (anion exchange H P L C - I C P - M S with 2 m M K O H ) are shown in Figure 3.6. The first two chromatograms were obtained from the analysis o f the biomass extracts for S. citrinum grown with Sb (III) (Figure 3.6a) and Sb ( V ) (Figure 3.6b). A second antimonycontaining peak o f unknown identity and with a retention time similar to that o f A l in Table 3.15, is observed in both these chromatograms. The third chromatogram (obtained by analyzing the biomass extract from the M e S b C l amended culture) shows the presence o f M e S b C l (or the 3  2  3  2  hydrolyzed form, M e S b ( O H ) ) , most likely unchanged, in the biomass (Figure 3.6c). The 3  2  medium samples analyzed by using Method A (not shown) contain only Sb ( V ) for the experiments amended with inorganic antimony, and only M e S b C l for the experiment amended 3  2  with M e S b C l . N o new antimony compounds were detected by using this method o f analysis, 3  2  contrasting with the finding o f a metabolite formed by P.  flabellatus.  127  2e+4  Oe+0 8e+4 Me SbCI 3  3.6c  2  6e+4 4e+4 2e+4 Oe+0  r~ 120  240 time (s)  360  480  F i g u r e 3.6. Chromatograms for biomass extracts (fresh weight) for S. citrinum experiments (Method A ) . 3.6a. Experiment amended with Sb (III). 3.6b. Experiment amended with Sb ( V ) . 3.6c. Experiment amended with Me3SbCl . A l is an unknown Sbcontaining compound. 2  128  When the samples were analyzed by using Method B (ion-pairing H P L C - I C P - M S with 10 m M T E A H / 4 . 5 m M malonic acid, p H 6.8), the unknown described in the previous section, B 4 , was present in all samples, including biomass extracts. The identification o f B 4 in these samples was based on the similar retention time o f the peak compared with that observed in the samples for P. flabellatus.  Considering only the area counts for Sb(OH) ", increased oxidation o f Sb (III) 6  to Sb ( V ) in the presence o f the live fungus does not take place in these experiments.  3.3.3.4. Summary of the interaction of antimony with fungi that can produce mushrooms Elevated levels o f antimony were observed in fruiting bodies o f P. flabellatus  grown on  substrate amended with antimony compounds, compared with levels found in the mushrooms grown on non-Sb containing substrate. Sb ( V ) was the only antimony species extracted from the mushrooms grown with both Sb (III) and Sb (V), and a Me Sb-containing compound was 3  extracted from mushrooms grown with Me3SbCl . 2  N o appreciable differences were observed in total antimony concentrations in culture media for both P. flabellatus  and S. citrinum, when the media were amended with antimony  compounds. Thus the mycelia do not accumulate these antimony compounds. Previous studies with other fungi have shown different results. Scopulariopsis brevicaulis appears to take up Sb (III) from solution (losses o f 30% were observed) , and Saccharomyces cerevisiae completely 13  took up Sb (III) but not Sb ( V ) . 32  When Method A was used for the analysis o f biomass extracts and media, a metabolite was detected, presumably produced from the interaction o f P. flabellatus  with inorganic  antimony compounds. N o metabolites were detected for S. citrinum by using Method A . The antimony species in biomass extracts were similar for P. flabellatus  and S. citrinum experiments,  129  showing a second antimony-containing compound eluting closely with Sb(OH) ". Further studies 6  to identify unknowns A l and A 3 are recommended. When Method B was used for analysis, the unknown compound B 4 appeared in nearly all biomass extracts and media. Thus an attempt was made to characterize it. B 4 may be in the +5 oxidation state and it appears to be in equilibrium with Sb(OH) ". W e suggest that further studies 6  address either this compound's identification or elimination (e.g., by using a growth medium with minimum carbon sources and salts). Enhanced oxidation o f Sb (III) to Sb ( V ) by P. flabellatus  takes place, although oxidation  by other factors is appreciable.  130  References  1. Slekovec, M . ; Irgolic, K . J. Chem. Spec. Bioavail. 1996,  8, 67-73.  2. Byrne, A R . ; Tusek-Znidaric, M . Appl. Organomet. Chem. 1990,  4, 43-48.  3. Larsen, E . H . ; Hansen, M . ; Goessler, W . Appl. Organomet. Chem. 1998,  72, 285-291.  4. Byrne, A . R.; Slejkovec, Z . ; Stijve, T.; Fay, L . ; Goessler, W . ; Gailer, J.; Irgolic, K . J. Appl. Organomet. Chem. 1995, 9, 305-313. 5. Kuehnelt, D . ; Goessler, W . ; Irgolic, K . J. Appl. Organomet. Chem. 1997,11, 289-296. 6. Kuehnelt, D . ; Goessler, W . ; Irgolic, K . J . Appl. Organomet. Chem. 1997,  11, 459-470.  7. Slejkovec, Z . ; Byrne, A . R.; Stijve, T.; Goessler, W . ; Irgolic, K . J. Appl. Organomet. 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A . ; Cannon, J. R.; Raston, C. L . ; Skelton, B . W . ; White, A . H . Tetrahedron Lett. 1977, 18, 1543-1546. 16. Irgolic, K . J.; Junk, T.; K o s , C ; McShane, W . S.; Pappalardo, G . C . Appl. Organomet. Chem. 1987, 1, 403-412. 131  17. Nelson, J. C . P h . D . Thesis, University o f British Columbia, 1993. 18. Cullen, W . R . ; Dodd, M . Appl. Organomet. Chem., 1989,  3, 401.  19. Morgan, G . T.; Davies, G . R. Proc. Royal Soc, Ser.A 1926,  523.  20. Cullen, W . R.; L i , H . ; Hewitt, G . ; Reimer, K . J.; Zalunardo, N . Appl. Organomet. 1994, 8, 303. 21. L e , X . C ; Cullen, W . R.; Reimer, K . J. Appl. Organomet. Chem. 1992, 22. Koelbl, G . ; Kalcher, K . ; Irgolic, K . J. J. Automat. Chem. 1993,  Chem.,  6, 161.  15, 37.  23. Lintschinger, J.; K o c h , I.; Serves, S.; Feldmann, J.; Cullen, W . R. Fresenius J. Anal. 1997, 359, 484-491.  Chem.  24. Difco Manual; Difco Laboratories: Detroit, 1984; pp 1131-1132. 25. Bajo, S.; Suter, U . ; Aeschliman, B . Analytica ChimicaActa 26. Helgesen, H ; Larsen, E . H . Analyst, 1998,  1983,149, 321-355.  123, 791-796.  27. Edmonds, J. S.; Francesconi, K . A . Appl. Organomet. Chem. 1988,  2, 297-302.  28. Aquatic Pollution, 2nd ed.; Laws, E . A , E d . ; John Wiley & Sons: N e w York, 1993; p p l 9 7 198. 29. Dushenko, W . T.; Bright, D . A ; Reimer, K . J. Aquat. Bot. 1995,  50, 141-158.  30. Pacioni, G . Simon & Schuster's Guide to Mushrooms; Lincoff, G . , E d . ; Simon & Schuster, N e w Y o r k : 1981; entries 20 and 329. 31. M a , J.; Stoter, G . ; Verweij, J. Schellens, J. H . M . Cancer Chemother. Pharmacol. 391-394. 32. Perezcorona, T.; Madrid, Y . ; Camara, C. Anal. Chim. Acta 1997,  1996,  38,  345, 249.  132  Chapter 4  ARSENIC IN THE MEAGER CREEK HOT SPRINGS ENVIRONMENT  4.1. Introduction  The Meager Creek hot springs are located north o f Pemberton, in British Columbia, Canada. H o t springs are formed when water percolates through permeable rock or fractures, is heated by the earth's crust at depth, and is then driven to the earth's surface by a combination o f artesian flow and thermal convection . H o t water is able to dissolve minerals over time, so that 1  elevated levels o f metals and metalloids are often associated with hot springs. F o r example, arsenic concentrations in water from hot springs in Yellowstone National Park have been documented to reach 1.6 ppm . Levels o f total arsenic reported for a number o f Japanese hot 2  springs range from non-detectable to 25 ppm . The presence o f arsenic in geothermal waters is 3  due to the dissolution o f arsenic containing minerals such as arsenopyrite, niccolite, enargite, orpiment, realgar, proustite and other pyrites , and the most probable form o f arsenic in 4  hydrothermal solutions is thought to be arsenite ' . Pyrite is the most likely host for arsenic at 5 6  Meager Creek . 7  The hot springs at Meager Creek provide an opportunity for studying the environmental chemistry o f metal(loid)s such as arsenic. Arsenic is a known poison and carcinogen, and its toxicity is dependent on the chemical form, or species, that it takes. F o r example arsenobetaine ((CH3) As CH COO") can be found in marine animals and mushrooms, and is much less toxic +  3  2  than arsenous acid (As(OH) ). Some arsenic compounds found in the environment are listed in 3  Table 1.1 (Chapter 1). 133  Very little is known about arsenic speciation in a hot springs environment specifically, although limited knowledge is available about non-marine ecosystems. F o r example, arsenic speciation was determined in a river in Japan receiving drainage from hot springs , but samples 8  were not collected from the immediate hot springs environment. Samples analyzed included a green alga, a diatom, freshwater fish, a freshwater prawn, a marsh snail and fly larvae, and all samples contained D M A and T M A when they were analyzed by H G - G C - A A S following alkaline digestion o f extracts. These results are inconclusive because the methodology used involves a digestion procedure, which may change the species o f arsenic, and an analytical tool that utilizes derivatization and hence does not permit identification o f the arsenic species in solution. A series of studies on freshwater and terrestrial plant uptake o f radioactive arsenic has been published ' ' 9  10  11  and arsenolipids were found to be formed in aquatic plants, although no  conclusive structural elucidation was carried out. In another study five species o f halophytes from an estuarine environment were analyzed for arsenic species and content . The authors 12  claim that compounds such as arsenobetaine, arsenocholine, tetramethylarsonium, T M A O , D M A and an arsenosugar were found in plant extracts and that metabolic synthesis o f these compounds is taking place within the plants. The present study was undertaken to determine, semi-quantitatively, arsenic levels and speciation in water and biota at Meager Creek hot springs to extend knowledge about the chemistry o f arsenic in freshwater/terrestrial environments " . 7  10  134  4.2. Experimental  4.2.1. Chemicals and reagents Arsenic standards were obtained as sodium arsenate, Na2HAs04.7H 0 (Aldrich), arsenic 2  trioxide,  AS2O3  (Alfa), methanearsonic acid, C H A s O ( O H ) (Vineland Chemical), and cacodylic 3  2  acid, ( C H ) A s O ( O H ) ( B D H ) , and were dissolved in deionized water to make standard solutions. 3  2  Extracts o f kelp powder (Galloway's, Vancouver, B C ) and N o r i (Porphyra tenerd) o f known arsenosugar content  13  were used to identify the retention times o f arsenosugars; these were  verified by comparison to pure arsenosugars generously donated by K . Francesconi and T. Kaise. Arsenobetaine , arsenocholine , trimethylarsine oxide , and tetramethylarsonium iodide had 14  15  16  17  been synthesized previously according to standard methods. Methanol ( H P L C grade, Fisher), tetraethylammonium hydroxide ( T E A H , 20% in water, Aldrich), malonic acid ( B D H ) , concentrated phosphoric acid (Aldrich), ammonium hydroxide ( I M , Fluka), pyridine (Fisher), and formic acid ( B D H ) were used as reagents for mobile phases and extractions.  4.2.2. Sampling Sampling was carried out in November, 1996 and July, 1997. Sample locations are shown in Figure 4.1. Water was sampled by hand into polypropylene bottles that had been acid washed previously. Biota were sampled by hand, stored in Ziploc® bags and kept cool until processing in the lab. There, they were washed thoroughly with tap water to remove soil and other particles, rinsed with deionized (1 Mohm) water, and frozen. Microbial mats were rinsed with only a minimum o f deionized water before freezing, to prevent loss o f mat composition. The samples were then freeze-dried and pulverized to a fine powder for analysis.  135  Figure 4.1. Map (not to scale) of Meager Creek Hot Springs area showing sampling locations 136  Identification o f plants and mushrooms (including bracket fungus) was carried out by using field guide b o o k s ' ' . Assistance from M s . O. Lee and D r . W . B . Schofield (Botany 18  19  20  Department, U B C ) , and from M r . James Black (Vancouver Mycological Society) is greatly appreciated in the identification o f moss, lichens and mushrooms.  4.2.3. Sample preparation and analysis For the determination o f total arsenic content by using I C P - M S , all samples were analyzed in duplicate resulting in <2% standard deviation. Water samples were analyzed directly by I C P - M S ( V G PlasmaQuad, V G Elemental) using R h (10 ppb) as an internal standard. A c i d digestions o f biota samples were carried out after weighing (0.3 g ± 0.5 mg) the freeze-dried powders into either a 40 m L glass vial or a 500 m L round bottomed flask ( R B F ) . Concentrated nitric acid (3 m L , doubly distilled in quartz, Seastar, Sidney, B C ) was added to each sample, and additionally, hydrogen peroxide (3 m L , 30% in water, reagent grade, Fisher) was added to each sample in glass vials. The samples in glass vials were heated directly on a hot plate and boiled for 3 hours. The contents o f the R B F s were boiled for 2 hours by using a heating mantle and a reflux apparatus  21  and then cooled. Hydrogen peroxide (3 m L ) was added to the R B F s and the  solutions were heated for another hour. After all the samples had cooled, the clear solutions remaining were diluted to 25 m L with deionized water and stored until analysis. The acid digests were analyzed by using I C P - M S , with R h (10 ppb) as an internal standard, and by monitoring m/z 75 and 103 for arsenic and rhodium, respectively. I C P - M S parameters are given in Table 4.1. Extractions were carried out by weighing 0.5 to 1 g (+ 0.5 mg) o f the freeze-dried powders into 50 m L or 15 m L centrifuge tubes, adding 10-15 m L M e O H / W a t e r (1:1), sonicating for 20 minutes, centrifuging for 20 minutes and decanting the liquid layer into a R B F . Each  137  sample was sonicated and centrifuged a total o f 5 times. The decanted extracts for each sample were pooled and rotovapped to near dryness (1-2 m L ) and then dissolved in 5 or 10 m L o f deionized water. The extracts were filtered through 0.45 um syringe filters (Millipore) and analyzed by H P L C - I C P - M S using the conditions given in Tables 4.1 and 4.2. Data from the I C P - M S were processed by using chromatographic software , and identification o f arsenicals in 22  samples was made by comparison o f retention times with those o f standards by using at least two chromatographic systems. Semi-quantitative concentrations o f arsenic compounds were determined by using external calibration curves for each compound corresponding to a matching standard, or to D M A for arsenosugars.  Table 4.1. Operation parameters for I C P - M S Feature  Specific Conditions  Forward radio-frequency power  1350 W  Reflected power  <10 w  Cooling gas flow rate (Ar)  13.8L/min  Intermediate (auxiliary) gas flow rate (Ar)  0.65 L/min  Nebulizer gas flow rate (Ar)  1.002 L/min  Nebulizer type  de Galan  Analysis mode  Time Resolved Analysis ( T R A ) for H P L C  Quadrupole pressure  9 x 10" mbar  Expansion pressure  2.5 mbar  7  138  Table 4.2. H P L C conditions for arsenic speciation Chromatography  Column  Mobile phase  Anion exchange  Hamilton P R P - X 1 0 0 , 150 x 4.6 or 250 x 4.6 mm  20 m M ammonium phosphate, p H 6.0  Supelcosil L C - S C X or Whatman S C X Partisil 5, 250 x 4.6 mm  20 m M pyridinium formate, p H 2.7  1.0  G L Sciences O D S , 250 x 4.6 mm  10 m M T E A H , 4.5 m M malonic acid, 0.1% M e O H , p H 6.8  0.8  Cation exchange  Ion-pairing  Flowrate (mL/min)  4.3. Results and Discussion  4.3.1. Total concentrations of arsenic in water samples The concentrations o f arsenic and sampling locations (Figure 4.1) for water samples taken in November 1996 and July 1997 are shown in Table 4.3. These results show no seasonal variations from midsummer to autumn sampling, with the arsenic levels being very similar with respect to both the locations sampled and the sampling date (range o f 237-303 ppb). The concentration o f arsenic in the hot springs water (average concentration o f 280 ppb) is two orders o f magnitude higher than in the cold Meager Creek water (5.4 ppb), reflecting the action of hot water on arsenic containing minerals. The cooler temperature observed at location 1 in November was probably a result o f cooler air temperatures (-5 to 0 °C) and precipitation in the form o f snow. The oxygen concentration  ([0 ]) results show that the water was well oxygenated 2  at the source, at outlet 1+2, and at the top o f the microbial mats, but more reducing conditions exist under the microbial mats and in the sediments. H P L C - I C P - M S analysis o f waters  23  showed  only the presence o f arsenate, except for trace levels o f arsenite at location 2. This indicates that if the arsenic was dissolved initially from minerals as arsenite, oxidation to arsenate took place before expulsion o f the water from the source. Arsenate is the species o f arsenic expected in most waters , although others have found up to 80% o f total arsenic as arsenite in Hot Creek, 24  California . 25  140  Table 4.3. Some physical and chemical characteristics of Meager Creek waters (see Figure 4.1 for sample locations), na = not analyzed. Location  Date  [As] (ppb)  [ 0 ] (ppm)  (SD)  %  a  Source 2  N o v . 1996  237 (8)  1  N o v . 1996  303 (2)  2  na b  3.04, 56.7%  c  1.08, 20.2%  d  T(°C)  pH  na  na  44  6.7  4 (geyser)  N o v . 1996  288  na  na  na  8 (Meager  N o v . 1996  5.4  na  3  8.3  Source 2  July 1997  na  2.70, 38.4%  56  6.4  1  July 1997  286  b  0.60, 8.5%  52  6.3  2  July 1997  277  b  0.75, 10.0%  41  6.8  1 + 2 outlet  July 1997  289  b  4.20, 59.7%  49  6.3  Creek)  a b 0 d e  d  e  SD = standard deviation from analysis of duplicate samples; other concentrations are for single samples only. Also in Feldmann et at Measurement taken at surface of microbial mat. Measurement taken below microbial mat. Measurement taken in sediment below microbial mat. 23  4.3.2. Total concentrations of arsenic in biota The concentrations o f arsenic in acid digested samples o f biota sampled at two different times, in November 1996 and in July 1997 are shown in Table 4.4. Sampling locations correspond to numbers on the map o f the sampling area in Figure 4.1.  141  Table 4.4. Samples, sampling location (see Figure 4.1), sampling times and arsenic levels (ppm dry weight) ( S D for duplicate digestions) in biota samples, B = background Arsenic  Sampling  Sampling  (ppm)  time  location  Top layer, microbial mat  290 (20)  N o v 1996  1  B r o w n microbial mat,  82 (8)  N o v 1996  5  Algae 1 (deep green algae)  249  N o v 1996  2  Sedge, Scirpus sp.  7.1 (0.4)  N o v 1996  1  Cedar, Thuja  0.96  N o v 1996  3  237 (2)  N o v 1996  4  14(2)  N o v 1996  1,2  0.30  N o v 1996  6  0.12  N o v 1996  6  Orange microbial mat, bottom  59  July 1997  5  Orange microbial mat, top  108  July 1997  5  Algae 2 (green algae)  56  July 1997  5  Sedge, Scirpus sp.  4.5  July 1997  1  Sample  plicata  Moss at geyser, Fumaria Fleabane, Erigeron  hygrometrica  sp.  B r o w n lichen, Bryoria  sp.  Yellow lichen, Alectoria  sp.  Moss at geyser, Fumaria  hygrometrica  350  July 1997  4  Moss at stream, Fumaria  hygrometrica  91  July 1997  3  3.9  July 1997  1,2  8.7  July 1997  1,2  Fleabane, Erigeron  sp.  Monkey flower, Mimulus  sp.  B r o w n lichen, Bryoria  sp.  0.30  July 1997  6  B r o w n lichen, Bryoria  sp. (B)  4.8  July 1997  7  Yellow lichen, Alectoria  sp.  0.16  July 1997  6  Y e l l o w lichen, Alectoria  sp. (B)  0.55  July 1997  7  Cup mushroom, Tarzetta cupularis  0.09  July 1997  7  Fawn mushroom, Pluteus cervinus  0.10  July 1997  7  Bracket fungus, Fomitopsis  <0.07  July 1997  7  pinicola  142  Microbial mats are found covering the rocks over which the hot water flows from the source into the nearby river. These mats are 1-6 thick and consist o f a variety o f organisms, usually predominantly bacteria and cyanobacteria but also including fungi and algae (their microbiological makeup is discussed in a later section) . Their appearance can be described as 26  alternating layers o f green, brown and orange with pockets o f purple. Strings o f deep green material can be found on rocks in areas o f faster water flow and these were labeled Algae 1. " B r o w n microbial mat" refers to brown slime covering the bottom o f a cooler stream (about 30° C), and Algae 2 refers to a sample o f stringy, absorbent algae collected near location 4. Finally, a thick mat o f bright orange was found in July in a stagnant pool o f cooler water, and this mat was also composed o f layers, with orange on top, brown and green layers, and a white crust on the bottom. A l l algae and microbial mat samples contain high levels o f arsenic, ranging from 56-290 ppm (dry weight), and the microbial mat from location 1 contains the highest amount. The lowest amounts o f arsenic in these samples are observed in the orange microbial mat (59 and 108 ppm), the brown microbial mat (82 ppm) and Algae 2 (56 ppm), all taken from locations furthest away from the hot springs source, with the exception o f Algae 2. Dilution (i.e., snow in November), precipitation (as, for example, Ca (As04)2), absorption by biota, or adsorption o f the 3  dissolved arsenic may result in lower levels o f arsenic in water farther away from the source, and these lower arsenic levels in the water would result in lower levels in the microbial mat samples. N o seasonal changes were studied for these samples. M o s s (Fumaria hygrometrica) was sampled at location 4, a geyser source, the result o f drilling by B C Hydro in 1974, and also at location 3, a cooler stream about 200 m away from sources 1 and 2 (See Figure 4.1). A greater arsenic content is observed for the moss from location 4 in July (350 ppm) with respect to that in November (237 ppm), and both these 143  amounts are higher compared with the arsenic content in moss from location 3 (91 ppm). Although the differences have not been statistically validated by taking replicate samples from each location and time, they may be due to the following possibilities. The arsenic content in samples taken from the same location in midsummer and late autumn may reflect seasonal differences in arsenic uptake rates and accumulation. L o w e r levels o f arsenic may have been present in the water at the stream location for the reasons mentioned above, leading to lower levels in the moss taken from the stream. Finally, different levels o f submergence o f the mosses in the arsenic containing water might cause differences in arsenic concentration. Sedge {Scirpus sp., most likely Olney's bulrush, a species known to grow at Meager Creek H o t springs ) and fleabane (Erigeron sp.) samples contain higher levels o f arsenic in the 1  late autumn (7.1 ppm for Scirpus sp. and 14 ppm for Erigeron sp.) compared with the samples taken in summer (4.5 ppm for Scirpus sp. and 3.9 ppm for Erigeron sp.), but again these differences have not been statistically validated. Nevertheless, because the levels o f arsenic in the water are the same at both these times, the higher arsenic levels observed in the plant samples from late autumn may indicate that the rate o f arsenic accumulation exceeds the rate o f arsenic depuration through the growing season. Studies on accumulation o f arsenic in plants growing on mine dumps showed a similar trend, where accumulation rates were associated with physiological growth o f the plants . 27  Bracket fungus and mushrooms (see Table 4.4 for Latin names), sampled from the forested area nearby (i.e., not directly adjacent to the hot springs) contain l o w levels o f arsenic (0.10 ppm and lower, Table 4.3). This may indicate that the amount o f arsenic being transported via the atmosphere from the hot springs is negligible or very low. Lichens (Bryoria sp. and Alectoria  sp.) collected from a tree directly adjacent to a steam vent (location 6) do not contain  levels o f arsenic that are elevated (0.12-0.30 ppm) with respect to samples from the forest, 144  location 7 (0.55-4.8 ppm), indicating that the lichens are not being impacted by the steam vent. Cedar leaves (Thuja plicata) were sampled directly above a warm stream that drained from the hot springs source into the river. The source o f arsenic in the cedar was probably the soil in which the tree was growing, rather than the steam generated by the stream.  4.3.3. Arsenic speciation in biota samples Semi-quantitative amounts o f arsenic species in samples from Meager Creek have been estimated and are listed in Table 4.5. The structures, names and abbreviations for arsenic compounds can be found in Table 1.1, Chapter 1.  145  <*. s  o  2  E o  s  1 os o V  V  o o V  8  <U cN  «  5 o • o CO o o  o o  i—i  o  V  o o o o o o  o o o o o  co  (N  o d  o d o  CO  CN  (S  V  V  H  H  V  V  T-<  r-1  i—1  o o o o o o  a c 3 O OO  CD  s  o o © d  on  V  o  g  o d V  J3  i2  8  00  CN  d  o  d  O  CN CN O  rn © d d  CN ©  i—i ©  i—i ©  V  V  V  d  d  V  P CN 2 d P a v o a O> S ©  00  i  Q  OH  y  on  ui  J3 <D  oo 1 3  ©  d  3  CN  O  00  CD  d CO  OO  oo  _J rt  55 ^  o  CN  CN CN  o  o d V  H  H  o o © © O O O d d o d d d o V V V V V V V  ^  O  d  oo cn co  d  ^  o d d  ^  CN  CN u->  ©  n-  d d  CN CN  I-M'  U  CN  s -  ©  CN  -  o  r  rn  r- o d -H  d  -  co  ^  CN  P  CO  O  o V  ® >T)  CN  © CO  i—•  p  cd  *w  co  d ©  CD  i  ^  CO  O  d  d  s  <*>  O  y  CO CN  d  CN  0\  o d dv O  m  CO  O  d  vo  d  V  vo  ~- -§ r . -  O  d ~T o co  v  O  p  o  a,  ii  o  s s c3 00  CN O  S oo  o  co CN O  g  d  d  v  1 s s  a. 3  > o  CD  ON ON  z  ca  Si  00  S  S  •a u  p3 s S § •8  iri  S c  7)  p  1  n VO ON ON  >  §3 NO ON  z  S  >  2  £  2  •5 .a  ON  a\  J5  ON  3  ON  VO O ^ ON ON  vo °^ RT  >  2 a •a 3 i  c  S 'C  5  o  w 00  §  3  ON ON  ON ON  9  3  in  VO  4-T  ,  ca S  00  3  •a  •2  oo  Ia 0)  s CN  'S  "3 X  'S  ON  3  oo oo  •S  ON  >> >, l—  •si  ^  op cu ca S  §5  >  ON  O  § 2 &  3  9"  r  c  S  on h, k, 5  -  T3  i—>  Oil  o o <  ca S  3 S.  3 VO  5  &  V5  © 2 o\ ON  §5  "3 "3 —  ~3.8 .5 5.  o. a.  « a « a C C C <=> o 5 o CJ ^ ^ ^5 eq ^  «  31  ON ON  j 1  ON  s 00  i  VO  o\  >  > o 2  o  ON ON  VO Ov Ov  vo  s e  r-  CN  S  Mjj  p  §1  a  d  4>  Q.  o «  s S g ~  S  "S3  ^  d  O  Q O  o o  H  co CN  o o o v o V  3  c o g •a C o CD  3  d  <u  O  Q ca  00  o  c  3  V  oo  £  &  V  CN CN  x  CD  <D  i-H O  O  2  c  d  CN  'o  a  O  a> g  C/3  .3  H  d P v o P o d  B  o  m  1  ON ON  r-l  I  3  u  o  00  H  "3 i—> 2 C  5 —>  It  a.  146  4.3.3.1. Algae and microbial mat samples A l l algae and microbial mat samples contain arsenosugars (Table 5), with the orange microbial mat containing the highest amount proportionally (44% o f sum o f extracted arsenic species). Only arsenosugars X and X I are observed in samples and in most cases the major arsenosugar is arsenosugar X . Sample chromatograms for Algae 1 and extracts used as laboratory standards (from kelp and Nori) are shown in Figure 4.2, and they show the selectivity o f the different chromatographic techniques. Figure 4.2a (ion-pairing chromatography) shows the presence o f arsenosugar X I and the absence o f arsenite. The absence o f arsenite allows arsenosugar X to be seen in Figure 4.2c (anion exchange chromatography), and the presence o f arsenosugar X I is confirmed in this chromatogram as well. Figure 4.2b (cation exchange chromatography) corroborates the presence o f arsenosugar X , well separated from the other arsenic compounds. Another sample chromatogram is illustrated in Figure 4.3, showing the matching retention times for arsenosugar X I when a sample (top layer, microbial mat) is spiked with an extract containing arsenosugar X I (Nori).  147  240  bitrary units)  120  360  480  I  600  720  4.2b Algae 1 Nori 1  As(V)/XI  -CO0)  cz  X  o o  0  0  l  I  i  i  i  i  i  i  i  60  120  180  240  300  360  420  480  540  60  120  180  240  300  600  360  T i m e (s)  Figure 4.2. Chromatograms for Algae 1 and laboratory standards showing the presence o f arsenosugars X and X I . Extracts o f N o r i labeled N o r i 1 and N o r i 2 contain predominantly arsenosugar X and X I , respectively. 4.2a. C18 column, 10 m M T E A H / 4 . 5 m M malonic acid/pH 6.8 mobile phase. 4.2b. Cation exchange column, 20 m M pyridinium formate/pH 2.7 mobile phase. 4.2c. Anion exchange column, 20 m M ammonium phosphate/pH 6.0 mobile phase.  148  As(lll)  DMA/As(V)  Top layer, microbial mat Top layer, microbial mat, spiked with XI  (0  co  in tf)  8  120  240  360  480  600  720  time (s)  Figure 4.3. Chromatograms of a microbial mat extract (top layer, microbial mat) and extract spiked with arsenosugar XI, showing co-elution of arsenosugar XI by using ionpairing HPLC-ICP-MS (C18 column, 10 mM TEAH/4.5 mM malonic acid/pH 6.8 mobile phase).  149  Arsenosugars have been found in the environment previously in marine algae, which contain, among others, arsenosugars X through XIII, often as the only species o f water soluble *  13 28 29 30  *  arsenic ' ' ' . In marine phytoplankton Heterosigma akashiwo and Skelotonema costatum (a diatom), arsenosugars X I and X I I I were found to be the major compounds in each organism, respectively . Arsenosugar X I I I was the major metabolite produced when another marine 31  diatom, Chaetoceros concavicornis, was exposed to arsenate . This previous work may 32  indicate that arsenosugars are formed de novo by these lower trophic level marine organisms. The de novo synthesis o f arsenosugars by the marine macroalga Fucus spiralis has been postulated to take place, based on the transformation o f radiolabeled arsenate in seawater to water soluble organoarsenic compounds within the alga, and subsequent speculation that the compounds were arsenosugars X and X I ' . Other marine green algae, Dunaliella 3 0  sp. and  3 3  Polyphysa peniculus do not form arsenosugars in pure culture; the former reduces arsenate to arsenite  34  and the latter methylates arsenic species . 35  Arsenosugars appear to be not as common in terrestrial and freshwater environments. The freshwater algae, Chlorella  sp. have been studied extensively and the extractable arsenic  species after exposure to arsenate is predominantly arsenate, or inorganic arsenic ' ' . Goessler 36  37  38  etal. , however, found that in addition to arsenate, Chlorella Bohm forms small amounts o f an 3S  unidentified arsenical, arsenite, M M A and D M A . Comparing the H P L C retention time o f the unknown compound to the retention time for arsenosugar X I in our studies, and noting that a similar chromatographic system to ours was used, may allow the unknown compound to be tentatively identified as arsenosugar X I . A commercial sample o f the terrestrial soil/freshwater cyanobacterium, Nostoc sp. contains arsenosugar X , making up 32% o f the total arsenic, but in 1 3  pure culture Nostoc sp. takes up arsenate as inorganic arsenic, with less than 1% D M A formed . 39  Likewise, another freshwater cyanobacterium Phormidium sp. contains mostly inorganic arsenic 150  that is not free, but N a O H digestable . A freshwater diatom was found to contain D M A (81%) by using an inconclusive method o f speciation , allowing for the possibility that the D M A found 8  is actually derived from arsenosugars in the diatom. Hence arsenosugars are possibly formed de novo by certain species o f marine algae (micro and macro), freshwater cyanobacteria and freshwater algae. Microbial mats have been described as "a heterotrophic and autotrophic community, dominated by cyanobacteria...annealed tightly together by slimy secretions from various microbial components" . The microbial compositions o f mats from Yellowstone National Park 41  and elsewhere have been found to depend on the p H , the temperature, and the H S concentration 2  in the water . Based on those factors, microbial mats at locations 1 and 2 at Meager Creek 26  possibly contain cyanobacteria species such as Synechococcus  lividus, the photosynthetic green  nonsulfur bacteria Chlorflexus sp. and the purple bacterium Chromatium tepidum . 26  However,  the mats are almost certainly not homogeneous with respect to time or area and many other bacteria are present, as mentioned earlier. F o r example, 22 species o f bacteria and 1 fungus have been cultured from the microbial mat at location l . 4 2  Therefore, the arsenosugars found in the mats are probably synthesized by cyanobacteria or other bacteria. The high proportion o f inorganic species, especially arsenate, in most samples, may imply that most o f the arsenate in the water is not metabolized by the microbes making up the mat. Similar behaviour has been observed with bacteria cultured from a marsh area in Yellowknife, N W T . Arsenite was extracted from the microbial mat sample taken from location 4 j  1, which is not surprising considering the reducing conditions imposed by the mat (Table 4.3). This suggests that arsenate-reducing bacteria are present in this mat. The microbial mat from location 1 contains M M A , and the orange microbial mat from location 5 contains D M A (Table 4.5), indicating that methylation is taking place by organisms in these mats.  151  Microbial mats were present in the algae sampling locations, and hence the arsenosugars and other arsenic compounds extracted from algae may be due to uptake from their environment. Therefore although the possibility exists that these algae form arsenosugars, no conclusions can be made about their capability to do so. A difference to note between freshwater and marine arsenosugar formation is the finding of only arsenosugars X and X I in Meager Creek and other terrestrial samples ' , whereas 13 38  arsenosugars X I I and XIII, as well as arsenosugars X and XI, are common in marine algae. Among marine algae, differences have also been noted; arsenosugars X and X I predominate in Rhodophyta and Chlorophyta, and arsenosugars X I I and X I I I are the major compounds in Phaeophyta . The phylum Phaeophyta is almost exclusively marine and hence the lack o f sugars 30  XII  and X I I I in the freshwater environment is not surprising, if these sugars are restricted to  Phaeophyta. Microbial mats appear to follow the same chemotaxonomic trend as Rhodophyta and Chlorophyta. However, the reasons for the trends are not clear, since the aglycones ( R  +  groups in the pathway shown in Figure 1.2., Chapter 1) are found as common metabolites in all organisms . 30  4.3.3.2.  Vascular The  plants  (sedge,  cedar, fleabane,  monkey  flower)  identities and amounts o f arsenic species associated with an organism probably  reflect at least four things: (a) the presence o f arsenicals outside the organism, in its food source; (b) the ability o f these compounds to enter the organism; (c) the ability, i f any, of the organism to synthesize arsenic compounds and (d) the presence o f arsenicals adsorbed onto the outside surface. A t this point it is unclear how much these factors influence the speciation o f arsenic found in plants. In previous studies, Nissen et al  9  and de Bettencourt et al.  12  have implied that  arsenicals other than arsenate found in plants were metabolites and hence synthesized by plants, 152  but de Bettencourt et al. also allowed for the possibility that arsenic speciation in halophytes reflected the arsenic species found in the estuarine waters from which they were sampled ' . In 12  44  other studies, Catharanthus roseus (periwinkle) was conclusively shown to methylate M M A to D M A in 4 % yield in pure plant tissue culture . Other authors believe that trace amounts o f 45  methylated species in plants are due to uptake from the soil after the compounds are formed by microbial activity in the soil . This theory is supported by studies in which beans and vascular 46  aquatic plants absorbed M M A from soil and water ' ' . 47  48  49  The major extractable arsenic species in vascular plants from Meager Creek hot springs are arsenite and arsenate (Table 4.5). A l l the plants, with the exception o f cedar, were growing in wet soil that consisted o f microbial mats, at the edges o f the streams. Although the soil in which the plants were growing was not sampled, it is reasonable to suggest that there may be arsenosugars in the root environments o f the plants. Therefore it is interesting to note the lack o f arsenosugars in the plants. These results are consistent with some o f the studies mentioned above, where inorganic arsenic species are the major water soluble species o f arsenic found in plants. Differences are seen between the July and November samples for sedge and fleabane (Figure 4.4). In sedge, higher levels o f arsenite and M M A are seen in November, and in fleabane, higher levels o f arsenite are seen but slightly lower levels of D M A are seen in November. These observations may suggest that more biological activity is taking place outside the plant, and accumulation o f these compounds is taking place, leading to higher levels o f arsenite and methyl species inside the plant. However, no seasonal reduction or methylation was evident in the water sampled at any locations, although water analyzed from microbial mats incubated anaerobically over time acquired arsenite and methylarsenic species . Arsenic 23  biomethylation and reduction by plants is a possibility as well, and the metabolism taking place 153  may parallel the increased growth of the plant during the summer.  Figure 4.4. Seasonal arsenic speciation in higher plants, sedge (Scirpus sp.) and fleabane (Erigeron sp.), shown as proportions o f total arsenic extracted.  Interestingly, the major extractable arsenic species in cedar is arsenite (Table 4.5). When pine seedlings, Pinus sp., were allowed to take up radiolabeled arsenate through their roots hydroponically, arsenite was found as the major species in roots and shoots , which may indicate 10  reduction by the plant. Arsenate was found to be more toxic than arsenite to roseus . 45  Catharanthus  Hence reduction to arsenite by cedar, and by the other vascular plants collected from  Meager Creek, may in fact be a detoxification mechanism. The monkey flower Mimulus sp., in addition to containing nearly equal amounts o f extractable arsenite and arsenate (2.8-3 ppm dry weight), contains a small amount (0.014 ppm) o f tetramethylarsonium ion (Table 4.5, including footnotes to the table). This is a compound that was not found in the microbial mats, in water, or in water after fermentation o f microbial mat . 23  154  The occurrence o f Me4As may indicate the presence o f a specific microenvironment in which +  this compound is available to the plant, or an ability o f the plant to synthesize Me4As . Marine +  sediments cultured aerobically with M M A and D M A released Me4As into the culture medium in +  small amounts  50  and tetraalkylated arsenicals (but not Me4As ) were reported to have been found +  in estuarine waters ' . Nevertheless, M e A s has otherwise not been found in pure or mixed 44  51  +  4  culture experiments, in sediments, or in waters; this information together with the lack of the compound in Meager Creek waters may suggest that methylation is indeed taking place in this plant. The formation o f Me4As is thought to be the final product o f the methylation pathway +  proposed by Challenger, with methylation o f trimethylarsine by methyl donors such as Sadenosylmethionine ( S A M ) or methyl halides . However, normally the reaction sequence stops 24  at or before the formation o f M e A s O (or M e A s ) . A n alternative pathway has been suggested, 3  3  which involves the exchange o f the ribosyl group on a precursor trimethylarsonioriboside (Chapter 1, Figure 1.3, intermediate 3.1) for a methyl group . The complete absence of the 52  intermediate trimethylarsonioriboside, as well as the absence o f its precursor molecule, arsenosugar XIII, in the terrestrial environment, makes this pathway unlikely in terrestrial plants. In summary, the speciation o f water soluble arsenic in vascular plants can be generalized as follows: plants may be selective for inorganic arsenic, they may be able to biomethylate arsenic in small amounts and they may detoxify arsenate by reducing it to arsenite, which may be less toxic to them.  4.3.3.3. Moss Arsenosugar X was detected in all moss samples (Table 4.5). Microbial colonies were observed in and around the moss samples, and despite the washing procedure, most likely contaminated the samples. The sampling methods used in the present study are not capable o f 155  differentiating between the contribution from microbial mat components interspersed throughout the moss sample and the contribution from arsenosugar uptake by the moss from its environment, or o f metabolism within the moss. The normalized concentrations o f arsenic species in moss are shown in Figure 4.5. Seasonal variations (from July to November) include a greater proportion o f arsenosugar X in November, and the presence o f methyl species in July. The most likely reason for these differences is that the microbial population in the moss environment changes throughout the growing season. The same reasoning can be used to explain the differences in the moss samples taken from different locations. F o r example, the moss sampled at the geyser location (location 4) appears to contain less arsenosugar, proportionally, than the moss sampled at the cooler stream location (location 3). This larger proportion o f arsenosugar X as well as the presence o f arsenite is also seen in the orange microbial mat taken from a cooler location (Table 4.5). Therefore the microbial community in the vicinity o f the moss sample at the cooler stream location may resemble a microbial mat at a cooler location, which may consist o f more arsenic-metabolizing organisms.  156  Nov m o s s (L4)  July m o s s (L4)  July m o s s (L3)  Figure 4.5. Seasonal and spatial arsenic speciation in moss (Fumaria hygrometrica) shown as proportions o f total arsenic extracted, L = location. July and November moss are from the geyser location (L4) and the second July moss is from the stream location ( L 3 ) .  4.3.3.4. Fungi including  lichens  The levels o f arsenic compounds in all fungi samples (Table 4.5) are at or close to the limit o f detection for the H P L C - I C P - M S methods used. B r o w n and yellow lichens both contain arsenosugar X L as does the yellow lichen sampled from a location not expected to be impacted by the hot springs (location 7). Pixie cups contain trace levels o f arsenosugar X . The presence o f arsenosugars in these samples is very likely a result o f synthesis by the indigenous algae or cyanobacteria living symbiotically with the fungus, making up the lichen, since the organisms were relatively isolated, and not exposed to the microbial mats with their sugar-synthesizing organisms. The organisms associated with both pixie cups (Cladonia sp.) and yellow lichen (Alectoria sp.) are green algae o f the genus Trebouxia . 53  O f course, the possibility cannot be  discounted o f sugars being present due to uptake or adsorption from other organisms living in the immediate surroundings o f the lichens, or as a result o f metabolism by the fungus. The results for the two mushrooms sampled add to the current knowledge o f arsenic speciation in mushrooms. Inorganic species are the major species found, as well as D M A in minor amounts (Table 4.5). Arsenosugar X was detected in Tarzetta cupularis, but in trace amounts; verification o f this result, especially in specimens containing higher levels o f arsenic, would be very interesting. A small amount o f an arsenosugar was reported in another mushroom, Laccaria  amethystina, but the authors considered additional chromatographic  confirmation to be necessary . Very l o w levels o f water soluble arsenic are present in these 54  fungi.  4.3.3.5. Extraction efficiency for arsenic species The sums o f arsenic species extracted are low for all samples analyzed. The % amounts of extracted arsenic with respect to the total, obtained by dividing the sum o f species in Table 4.5 by the total arsenic in Table 4.4, are shown in Table 4.6. This calculation does not take into account the amount o f arsenic that may have been extracted but was not observable by using these chromatographic methods. In past studies, this amount has been observed to be significant in some samples, even when using the method o f standard additions for quantification . 55  Analytical problems have been reported when significant levels o f sodium and potassium were present in samples; that is, a suppressed chromatographic signal for arsenobetaine resulted from the co-elution o f sodium and potassium ions during cation exchange H P L C - I C P - M S . The 5 6  suppression o f the signal for earlier eluting arsenic species (e.g., anionic species and arsenosugar X ) is possible as well, although such an effect was not reported . 56  158  Table 4.6. Percent amounts o f arsenic extracted. Sample  % Arsenic extracted  Top layer, microbial mat, N o v 1996  31  B r o w n microbial mat, N o v 1996  2.4  Algae 1 (deep green algae), N o v 1996  1.9  Sedge, Scirpus sp., N o v 1996  13  Cedar, Thuja plicata, N o v 1996  21  Moss at geyser, Fumaria  0.6  hygrometrica, N o v 1996  Fleabane, Erigeron sp., N o v 1996  47  B r o w n lichen, Bryoria  28  sp., N o v 1996  Y e l l o w lichen, Alectoria  sp., N o v 1996  41  Orange microbial mat, bottom, July 1997  5.4  Orange microbial mat, top, July 1997  1.7  Algae 2 (green algae), July 1997  6.7  Sedge, Scirpus sp., July 1997  31  Moss at geyser, Fumaria  hygrometrica, July 1997  1.1  M o s s at stream, Fumaria hygrometrica, July 1997  1.9  Fleabane, Erigeron sp., July 1997  47  Monkey flower, Mimulus sp., July 1997  67  B r o w n lichen, Bryoria  10  sp., July 1997  Yellow lichen, Alectoria  sp., July 1997  19  Yellow lichen, Alectoria  sp., July 1997 (B)  19  Cup mushroom, Tarzetta cupularis, July 1997  70  Fawn mushroom, Pluteus cervinus, July 1997  63  Extractable amounts o f arsenic range from 0.6 % for moss to 70 % for the mushroom Tarzetta cupularis.  From vascular plants, 13-67% o f total arsenic was extracted (mean o f 38%),  from moss, 0.6-1.9% o f total arsenic was extracted (mean o f 1.2 % ) , and from fungi, 10-70% o f total arsenic was extracted (mean o f 36%). The extraction method used has been successful for 159  marine plants and animals '  and is assumed to extract water-soluble species, yet it appears to  be insufficient for the extraction o f these samples. The levels o f inorganic arsenic may be underestimated, because in other studies the highest amounts o f inorganic arsenic species from terrestrial samples were extracted with water alone . Insufficient extraction may also result 59  from the presence o f arsenic compounds that are nonpolar and not soluble in methanol/water (1:1). In other studies where the presence o f arsenolipids was postulated, an extraction technique with hot ethanol was used ' ' . Other researchers have used a sequential extraction 9  10  11  technique consisting o f Soxhlet extraction with 80% methanol, cold 5% chloroacetic acid, warm 75% ethanol and hot 5% chloroacetic acid to extract all but 20% o f arsenic from wheat seeds . 60  These extraction techniques are likely to extract compounds that are more nonpolar in nature. Additionally, prior freeze-drying o f samples has been recently found to result in lower extraction efficiencies, although the reasons for this are unclear . Arsenic that is not extracted by 55  methanol/water (1:1) might be tightly bound to lipids; to cell wall components o f plants, including insoluble cellulose, calcium or magnesium pectates, or lignin; and to chitin or other cell components o f fungal samples. L o w extraction efficiencies are associated with microbial mats and algae (1.7-31%, mean of 18%), a result also seen with Nostoc sp. (34%) and possibly with Phormidium, where the 13  arsenic in the cyanobacterium was thought to be not free and possibly bound up in a non-water soluble form . Metals (possibly including arsenic) can bind to bioflocculants secreted by 40  microbial mats , and this may limit extraction efficiencies for arsenic. M o r e specifically, arsenate 41  may coprecipitate with insoluble minerals as a result o f microbial mat biomineralization. Evidence exists for the formation o f hydrated iron and manganese oxides, and iron and aluminum silicates on bacterial cells in freshwater microbial mats ' , which are minerals known to co61  62  precipitate with arsenate. There is also evidence o f ferric arsenate precipitation as a result o f  160  bacterial action  4.3.4. Summary Arsenosugars are apparently formed by cyanobacteria/bacteria, possibly by algae in the lichens Cladonia sp., Bryoria sp. and Alectoria sp., and possibly by the fungus Tarzetta cupularis.  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Goessler, W . ; Kuehnelt, D . ; Schlagenhaufen, C ; Slejkovec, Z . Irgolic, K . J. J. Anal. At. Spectrom. 1998, 13, 183-187. 57. Shibata, Y . ; Morita, M . Appl. Organomet. Chem. 1992, 58. Alberti, J.; Rubio, R.; Rauret, G . FreseniusJ.  6, 343-349.  Anal. Chem. 1995,  351, 420-425.  59. Kuehnelt, D . ; Goessler, W . ; Schlagenhaufen, C ; Irgolic, K . J. Appl. Organomet. Chem. 1997, 11, 859-867. 60. Domir, S. C ; Woolson, E . A . ; Kearney, P. C ; Isensee, A . R. J. Agric. Food Chem. 24, 1214-1217. 61. Tazaki, K . Clays Clay Min. 1997,  1976,  45, 203-212.  62. Konhauser, K . O.; Fyfe, W . S.; Ferris, F. G . ; Beveridge, T. J. Geology 1993, 1103-1106.  December,  63. LeBlanc, M . ; Achard, B . ; Othman, D . B . ; Luck, J. M . ; Bertrand-Sarfati, J.; Personne, J.Ch. Appl. Geochem. 1996, 11, 541-554. 64. Robinson, B . ; Outred, H . ; Brooks, R.; Kirkman, J. Chem. Speciation Bioavail. 1995, 96.  7, 89-  165  Chapter 5  ARSENIC IN THE YELLOWKNIFE ENVIRONMENT  5.1. Introduction  Yellowknife is located on Great Slave Lake, in the Northwest Territories, Canada. A major industry in the city is gold mining, and two gold mines, the Royal Oak Giant Mine and the Miramar C o n Mine, are presently in operation. The gold in the mined ore is associated with arsenopyrite (FeAsS), and hence arsenic waste is generated during the smelting operation. For example, when milling began at Giant Mine in 1948, aerial emissions o f 7.3 tonnes as arsenic trioxide per day were released . Currently, aerial emissions o f only 1.7 tonnes as arsenic trioxide 1  per year are released from Giant Mine, and 0.5 tonnes o f arsenic per year are discharged with the effluent into Baker Creek. The proximity o f the two gold mines to the city o f Yellowknife has allowed researchers to study the effects o f mining and arsenic on the surrounding environment. Arsenic chemistry has been studied to some extent in Pud, Kam, M e g , K e g and Peg watershed areas, which receive the drainage from C o n Mine waste. Arsenic mobility was found to be controlled by remobilization o f historically contaminated sediments rather than by present day mining practices . Waters, sediments and porewaters were analyzed for inorganic and methyl arsenic 2  species and methylarsenic (III) compounds were discovered although they were not identified. 3  Macrophytes (aquatic plants) were analyzed for total arsenic and concentrations were found to 4  be elevated compared with specimens collected from uncontaminated areas. Toxic effects o f reduced plant health and lack o f biodiversity were observed in areas o f high arsenic content.  166  Anaerobic microbes isolated from a sediment core from K a m Lake were found to methylate arsenic to monomethylarsonic acid ( M M A ) , dimethylarsinic acid ( D M A ) , trimethylarsine oxide ( T M A O ) , methylarsenic (III) compounds, arsine and trimethylarsine . 5  A s discussed in the previous chapter, little is known about arsenic speciation in biota in the freshwater and terrestrial environments. The high levels o f arsenic in the Yellowknife area allows us to study the forms that arsenic takes in the available biota. A s well, these results can be compared with those obtained from Meager Creek.  5.2. Experimental  5.2.1 Chemicals and reagents Arsenic standards were obtained as sodium arsenate, N a 2 H A s 0 . 7 H 0 (Aldrich), arsenic 4  2  trioxide, A s 0 (Alfa), methanearsonic acid, C H A s 0 ( 0 H ) (Vineland Chemical), and cacodylic 2  3  3  2  acid, ( C H ) A s 0 ( 0 H ) ( B D H ) were dissolved in deionized water to make standard solutions. 3  2  Extracts o f kelp powder (Galloway's, Vancouver, B C ) and N o r i (Porphyra tenera) o f known arsenosugar content were used to identify the retention times o f arsenosugars; the retention 6  times were then verified by comparison to those obtained from pure arsenosugars generously donated by K . Francesconi and T. Kaise. Arsenobetaine , arsenocholine , trimethylarsine oxide , 7  8  and tetramethylarsonium iodide had been synthesized previously according to standard 10  methods. Methanol ( H P L C grade, Fisher), tetraethylammonium hydroxide ( T E A H , 20% in water, Aldrich), malonic acid ( B D H ) , concentrated phosphoric acid (Aldrich), ammonium hydroxide ( I M , Fluka), pyridine (Fisher), and formic acid ( B D H ) were used as reagents for mobile phases and extractions.  9  5.2.2. Sampling Sampling was carried out in June and August o f 1997. Sample locations are shown in Figure 5.1a and 5.1b. Water was sampled by hand into polypropylene bottles that had been previously acid washed. Some samples were split and one o f the aliquots was filtered through 1 um cellulose nitrate filters (Sartorius) before freezing. Sediment samples were collected by using a trowel and they were frozen in Ziploc® bags. They were air dried over a period o f two days, then ground in a mortar and pestle. Particles that passed through a 60 mesh sieve were subsequently extracted. M o s t biota were sampled by hand and all were stored in Ziploc® bags and kept cool until processing in the lab. There, they were washed thoroughly with tap water to remove soil and other particles, rinsed with deionized (1 Mohm) water, and frozen. Mussels were sampled by hand as well as by using an Eckman grab, and a different freshwater species was collected from Campbell River for comparison. They were shelled before freezing, as were large specimens o f snails. Smaller specimens o f snails(< 0.5 cm in length) were frozen and processed whole. Fish were caught by using a gillnet, and they were gutted before freezing. Roe was removed from one fish and this was kept and frozen for analysis. The fish were thawed at a later date and filleted and skinned to obtain muscle samples, which were refrozen. Pond scum, or algal/microbial mats o f unknown composition, sampled from a puddle beside Baker Creek (location 4) were washed with a minimum o f deionized water to minimize sample loss before freezing. A l l frozen biota samples were then freeze-dried and pulverized to a fine powder for analysis.  168  F i g u r e 5.1a. M a p of Royal Oak Giant mine property and surrounding area, with sample locations.  Figure 5.1b.  M a p of Yellowknife, showing Royal Oak Giant M i n e and Miramar C o n Mine, and  sampling locations. 170  Mussels and snails , plants 11  12  and mushrooms '  13 14  were identified by using field guide  books. Assistance from O. Lee, D r . W . B . Schofield and Julie Oliveira (Botany Department, U B C ) is greatly appreciated in the identification o f moss, lichens and algae. W e are very grateful also to Chris Ollson (Royal Military College) for fish identification, M i k e Fournier (Yellowknife, N W T ) for assistance with plant identification, and James Black (Vancouver Mycological Society) for help with mushroom identification.  5.2.3. Sample preparation and analysis For the determination o f total arsenic content by using I C P - M S , all samples except water samples were analyzed in duplicate resulting in <2% standard deviation. Water samples were analyzed only once directly by I C P - M S ( V G PlasmaQuad, V G Elemental) using R h (10 ppb) as an internal standard. A c i d digestions o f biota samples were carried out after weighing (0.3 g ± 0.5 mg) the freeze-dried powders into either a 500 m L round bottomed flask ( R B F ) . Concentrated nitric acid in a volume o f 3 m L (doubly distilled in quartz, Seastar, Sidney, B C ) was added to each sample. The samples were boiled for 2 hours by using a heating mantle and a reflux apparatus and then cooled. Hydrogen peroxide (3 mL, 30% in water, reagent grade, 15  Fisher) was added to the R B F s and the solutions were heated for another hour. After all the samples had cooled, the clear solutions remaining were diluted to 25 m L with deionized water and stored until analysis. The acid digests were analyzed by using I C P - M S , with R h (10 ppb) as an internal standard, and by monitoring m/z 75 and 103 for arsenic and rhodium, respectively. I C P - M S parameters are given in Table 5.1. Extractions were carried out by weighing 0.5 to 1 g (± 0.5 mg) o f the freeze-dried powders into 50 m L or 15 m L centrifuge tubes, adding 10-15 m L M e O H / W a t e r (1:1), sonicating for 20 minutes, centrifuging for 20 minutes and decanting the liquid layer into a R B F . Each 171  sample was sonicated and centrifuged a total o f 5 times. The decanted extracts for each sample were pooled and rotovapped to near dryness (1-2 m L ) and then diluted to 5 or 10 m L with deionized water. Fish, fish roe, mussels, snails, and oyster tissue certified reference material ( C R M ) 1566, obtained from N I S T , were digested with protease, based on methods for enzymatic digestion ' ' . A n accurately weighed 1 g (±0.5 mg) sample was combined with 0.02-0.05 g o f 16  17  18  protease (Type VIII, N o . P-5380, Sigma) in a plastic 50 m L centrifuge tube. Ammonium carbonate ( B D H ) buffer at a concentration o f 0.1 M and p H 7.2 (adjusted with nitric acid) was added and the tube was sealed and vortexed. The samples were shaken for 4 hours at 37 °C, then centrifuged, and the supernatant was diluted to 25 m L with deionized water. To determine the extent o f arsenic solubilization by the protease, oyster tissue samples were extracted with buffer alone (no protease) and using the same procedure as that described for protease digestions. Water samples, extracts and protease digestions (PDs) were filtered through 0.45 um syringe filters (Millipore) and analyzed by H P L C - I C P - M S using the conditions given in Tables 5.1 and 5.2. Data from the I C P - M S were processed by using chromatographic software , and 19  identification o f arsenicals in samples was made by comparison o f retention times with those o f standards by using at least two chromatographic systems. Semi-quantitative concentrations o f arsenic compounds were determined by using external calibration curves for each compound corresponding to a matching standard, or to D M A for arsenosugars.  172  Table 5.1. Operation parameters for I C P - M S Feature  Specific Conditions  Forward radio-frequency power  1350 W  Reflected power  <10W  Cooling gas flow rate (Ar)  13.8 L/min  Intermediate (auxiliary) gas flow rate (Ar)  0.65 L/min  Nebulizer gas flow rate (Ar)  1.002 L/min  Nebulizer type  de Galan  Analysis mode  Time Resolved Analysis ( T R A ) for FfPLC  Quadrupole pressure  9 x 10" mbar  Expansion pressure  2.5 mbar  7  Table 5.2. H P L C conditions for arsenic speciation Chromatography  Column  Mobile phase  Anion exchange  Hamilton P R P - X 1 0 0 , 150 x 4.6 or 250 x 4.6 mm  20 m M ammonium phosphate, p H 6.0  Cation exchange  Supelcosil L C - S C X or Whatman S C X Partisil 5, 250 x 4.6 mm  20 m M pyridinium formate, p H 2.7  1.0  Ion-pairing  G L Sciences O D S , 250 x 4.6 mm  l O m M T E A H , 4.5 m M malonic acid, 0.1% MeOH, pH6.8  0.8  Flowrate (mL/min) 1.0 or 1.5  5.3. Results and Discussion  Arsenic compounds were identified by comparing retention times o f arsenic compounds in samples with those o f standard compounds. I f the retention time for an arsenic compound in a sample was the same as that for a standard compound, the arsenic compound was concluded to have the same identity as the standard compound. If the presence o f arsenobetaine or cationic species (such as T M A O , arsenocholine, and Me4As ) was indicated after analysis with anion +  exchange H P L C - I C P - M S , the identities o f such species were confirmed by analysis with cation exchange H P L C - I C P - M S method. Cation exchange chromatography was also used to confirm the presence o f arsenosugar X . This analytical method separates the aforementioned peaks from other peaks that co-elute with them on other chromatographic systems. The identities o f arsenosugars were confirmed by using ion pairing chromatography. Some samples were spiked with standards to confirm that retention times o f arsenic compounds did not depend on matrix. The primary purpose o f the present study was to determine the identity o f detectable arsenic species that could be extracted by using MeOH/water (1:1) extraction. The amounts o f the arsenic species found were calculated semi-quantitatively, to determine approximate levels o f the compounds (i.e., major or minor components). Standard deviations shown in Table 5.6 and for subsequent results were calculated with quantities obtained from analysis on the different chromatographic systems, where applicable. In some cases, only one number was obtained i f the better sensitivity and selectivity o f a chromatographic system allowed the species to be seen on one system but not another. The relative standard deviation, on average, is estimated to be about 30%.  5.3.1. Water and sediment samples Water and soil were sampled in a few locations from which biota were sampled to ascertain the arsenic speciation and concentrations in the surrounding aqueous environment o f these biota. Total arsenic concentrations and speciation results for waters, and estimated water soluble species (extracted with MeOH/water 1:1) for soils are summarized in Table 5.3.  Table 5.3. Concentrations o f total arsenic and arsenic species in water samples (ppb) and soil extracts (ppm) (SD) . Total concentrations were determined by I C P - M S as described in section 5.2.3. "Trace" amount is 1 ppb (i.e., at detection limit); nd = not determined. a  Sample (location)  As (III)  Pore water (1)  296  6.2  <1  29  Sum o f species 331  Surface water (1)  2.5  98  <1  <1  101  140 (30)+  Surface water (2)  <1  126  <1  <1  126  nd  Surface water (3)  <1  184  <1  <1  184  195 (3)J  Standing water (4)  1.4  350 (40)+  <1  trace  352  480  C o n effluent (14)  <1  5.3  <1  <1  5.3  32 (26)t  Surface water (10)  <1  20.3  2.5  <1  23  68  Surface water (11)  trace  26 (5)J  2.5  <1  30  54 (4)t  As(V)  MMA  DMA  Total _ J 2 C P J ^ M S ) _  980  (0.8)J  Sediment extract (1)  19.1  0.65  <0.05  <0.05  19.8  nd  Sediment extract (2)  14.4  2.0  <0.05  <0.05  16.4  nd  Sediment extract (3)  10.8  2.8  <0.05  <0.05  13.6  nd  SD = Standard deviation, based on duplicate analysis at two different dilutions* or analysis of duplicate samples J.  a  In all surface waters the major arsenic compound is arsenate. M i n o r amounts o f methyl species are observed in water samples from Niven Lake (locations 10 and 11) and from location  175  4, which were areas o f prolific plant and algal growth. Only small amounts o f arsenite are present in some samples, except for the pore water squeezed from a sediment in the Baker Creek marsh, which contains arsenite as the major compound. This indicates strongly reducing conditions in the sediment. N o t all o f the arsenic was accounted for in some samples by using H P L C - I C P - M S analysis; the largest discrepancies are observed for the pore water sample, C o n effluent, and Niven Lake waters (locations 10 and 11). Species such as methylated arsenic(III)thiols, o f the form ( C H ) A s ( S R ) 3 - n (n=l,2,3), as well as those involving binding o f m  3  n  arsenic to colloidal organic matter such as humic and fulvic substances, were postulated to be present in waters from Yellowknife in a previous study . The H P L C behaviour for such arsenic 3  species is unknown and they might not be detectable by using the H P L C - I C P - M S methods in this study. The concentrations o f arsenic species in Table 5.3 are in ppm dry weight o f sediment. The major extractable species from sediments is arsenite, which probably reflects the insolubility of arsenate in the sediments, and it may reflect reducing conditions as well. I f the MeOH/water technique can be used to approximate the species that are water soluble, then it appears that arsenite is the major species available to organisms growing in the sediment.  5.3.2. Freshwater fish Although arsenic speciation has been studied in some detail for marine fish, very little information is available about the forms o f arsenic in freshwater fish. Because o f this lack o f information, a study was conducted to examine arsenic in fish from Yellowknife. It was also o f interest to see i f higher levels o f arsenic (compared with published background levels) would be present in the fish as a result o f their exposure to higher levels o f arsenic in the water and sediments. The sampling area chosen was the Baker Creek outlet in Yellowknife B a y (location  176  9, Figure 5.1a). N o fish are observed in Baker Creek itself after the mine discharge period begins each summer, although the lack o f fish in Baker Creek is thought to be due to elevated levels o f ammonia rather than metals . Four different fish species were caught: whitefish (Coregonus 20  clupeaformis),  sucker (Catostomus commersoni), walleye (Stizostedion vitreum) and pike (Esox  lucius). Fish muscle was chosen as the part o f the fish to be analyzed because o f its significance to consumers.  5.3.2.1.  Total arsenic in fish Some physical characteristics, the moisture content, and the arsenic content in each fish  sampled are shown in Table 5.4. F o r most o f these and other samples, acid digestions were carried out once, and analyzed in duplicate by using I C P - M S . Relative standard deviations obtained from duplicate analyses were less than 2% in all cases. When sucker 1, pike 2 and whitefish 3 were digested in duplicate, the average relative standard deviation between digestions was less than 5%. Standard deviations for other samples may be estimated to be 5% o f the total arsenic concentration. Total arsenic concentrations have previously been found to be much higher in marine fish than in freshwater fish, and the present results confirm this observation. The highest concentration o f arsenic in this study is 3.1 ppm dry weight in a whitefish, whereas arsenic concentrations in marine fish range from 3.5 ppm dry weight for mackerel to 196 ppm dry weight for plaice collected from uncontaminated areas . Converting these arsenic concentrations to 16  fresh weight concentrations (using [As]/R where R is fresh weight/dry weight in Table 5.4) gives concentrations ranging from 0.07 ppm for whitefish 3 to 0.72 for whitefish 1.  177  Table 5.4. Total arsenic concentrations in fish from location 9. Fish  Sex  Weight (g)  Moisture content in % (R)  a  Arsenic concentrati< (ppm dry wdgjit)j^l  Whitefish 1  Male  846  """"77(433)  Whitefish 2  Female  586  76 (4.24)  0.84  Whitefish 3  Female  1453  74 (3.88)  0.28 (0.02)  Whitefish 3 roe  —  137  Sucker 1  Male  656  78 (4.65)  1.24 (0.01)  Sucker 2  Male  457  81 (5.35)  0.98  Walleye 1  Male  386  79 (4.75)  0.46  Walleye 2  Male  507  77 (4.37)  0.85  Pike 1  Male  628  79 (4.72)  1.30  Pike 2  Male  988  80 (5.08)  1.40 (0.09)  a  b  3.1  0.25  Moisture content was calculated by using Q3.-l)/R*100%; R = fresh weight/ dry weight. SD = Standard deviation, based on duplicate acid digestions.  These numbers can be compared with reported arsenic concentrations in freshwater fish sampled from pristine locations, ranging from 0.025 ppm fresh weight for pike to 0.132 ppm fresh weight for white sucker ' . The concentrations o f the fish sampled in this study are lower 21  22  than those found in rainbow trout and freshwater smelt purchased from a Japanese market (1.46 and 1.08 ppm fresh weight) . M o s t likely the levels o f arsenic in Yellowknife fish are not 23  elevated compared with fish analyzed in previous studies ' ' , but more samples should be 21  22  23  analyzed to confirm this. The fish analyzed in this study all contain levels below a maximum permissible concentration o f arsenic in fish for human consumption, set by the Australian National Health and Medical Research Council, o f 1.14 mg/kg (ppm) fresh weight . 21  The sample size from this study is too small to draw any correlations between physical characteristics and arsenic concentration. However, for sucker, walleye and pike, the arsenic 178  concentration appears to increase with increasing fish size, a trend that has been documented previously . This does not appear to be the case for whitefish. This trend may not be observed 24  for every species offish, and there may be differences based on sex. The concentration o f arsenic in the roe is approximately the same as the fish from which it was taken. The roe was very fatty and difficult to digest, and care had to be taken to keep the rate o f heating slow to prevent charring. In yellowtail flounder Pleuronectes ferruginea,  the  arsenic concentrations in spawned eggs were significantly lower than those found in developing gonads and fish muscle , an observation that contrasts with the results found in this study (i.e., 25  that the concentrations are the same). Clearly, more studies are necessary to determine i f any trends are observable for arsenic concentrations in gonads and muscle for freshwater fish.  5.3.2.2. Arsenic speciation in fish Enzymatic digestion has been used previously for fish and shellfish samples. Branch et al}  6  used trypsin, a protease, to disrupt the lipid-protein membrane in fish samples and release  the cell contents. They found that trypsin digestion gave greater recoveries for arsenic from a certified reference material, D O R M - 1 (dogfish muscle) and whiting; lower recoveries for plaice, mackerel and a lemon sole specimen; and similar results for cod, when compared with methanol/chloroform (2:1) extraction. Forsyth et a / . ' 17  18  used crude protease and lipase to digest  samples for tin and lead speciation and observed reduced matrix effects during analysis, effective release o f organotin analytes from marine food matrices, and adequate recovery o f alkyllead standards. Oyster tissue and dogfish muscle C R M s were digested with a combination o f protease and lipase to yield quantitative recovery o f selenium . 26  It was predicted in this work that if recoveries were similar for enzymatic digestion, using a non-specific protease, compared with acid digestion to determine total arsenic in a C R M , then 179  protease digestion might be a suitable method for releasing arsenic from a protein-rich sample matrix, as well as other elements such as antimony. The results obtained when total arsenic was determined in oyster tissue C R M (NIST 1566) in protease digestions, acid digestions, and aqueous (buffer) extracts are shown in Table 5.5. About 70% o f arsenic is recovered from aqueous (buffer) extraction alone, whereas 100% o f arsenic is released from the matrix using protease digestion. Higher levels o f antimony are present after using the protease digestion, although all values obtained are significantly higher than the non-certified value (0.01 ppm). The reasons for this are unclear, and more studies should be carried out to obtain a more reliable value for the acid digestion procedure to use for comparison. Protease digestion was subsequently used for fish, shellfish and snail samples in an attempt to maximize extraction o f other elements, as well as arsenic.  Table 5.5. Comparison o f arsenic concentrations ( S D ) by using protease and acid digestion methods for oyster tissue (NIST 1566). The certified concentration o f arsenic is 14.0 ± 1 . 2 ppm, and the non-certified value for Sb is 0.01 ppm. a  Digestion procedure  Concentration o f A s (ppm)  Concentration o f Sb (ppm)  A c i d digestion  14.0 (0.2)  0.3 (0.2)  Protease digestion, p H 7.2  13.8 (0.2)  0.22 (0.08)  Buffer extraction, p H 7.2  11.4 (0.3)  0.17 (0.07)  a b  b  SD = standard deviation, obtained from 3 replicate extractions. SD for this sample was obtained from 2 replicate digestions.  180  i  w  i  CN CN  CN  ON VO I oo In ©  CO  in *-H  O a> ' 3 .  i (3  ON  5  d  ••o  X  CN  in  o d  vo o VO  o r-  CN  >-  >-  d  o  oo UO  m d  o  CN CO  CN i-H  CN  •n  CN  m r©  O  d  d  >-  X  X  00  in  >n  3  vo  00  3  d  d  ><  >-  d  ^  Q  3  i © V  IX  °  jo  ?1 eej C  c > o BO o 2 o a  m  00  V-H  o  d V  — or 1  o V  o  d V  >-> ON  o  O O  s  in  CO  d  O  d  O  X  ugar  ON.  HH  o  o o \ ON d *-H V o o  o  *-H  vo <N o d  s  o o  o O ci hi V  a V  O  VO  o  O  r- o ts o d d V  CO  O.  d V  ci V  CN  vo" 0 0 o o © d  o o  o o  oo  o ci V  O  ci V  3  O  00  ; co •©  Q 3  lo  : 00  X «  Id  co O  oo CN  o  ,052 (0.00  .a  o  vo o d  in  CN  o  .2? 73 CO  o  d  d CO  • o Id 1  q  v  o d  ^  CO CN O  o . d V d  in  o  d  d  co  CN  VO CN  o  o  d  ^ ^  so  VO O  d OV  d  ©  o  ON CN  ON  d  ©  co  d  -—-  CN CN ©  CN O  VO  d  o o o" d  o d  •—-  ro d•—• VO CN ©  3 d  0) CJ  00  CS  o  i s  d  CO CO  d  00  d  2 3  co  °-^H cu  o.  c/5  a  I  g  oo  sa  x  X) o Q C  S  o U c  n H  O  co  v  o  d  2  o d v  o o V  o d v  la  a  a  jCU 1 J3 : &o 1B  T3 O  o d v  o d v  ca  o d v  CO O  a  O  d  v  O  d  v  CO O  O  d  o o odd V V  o V  d m O  hi  o d v  o d  o d  o d v  o d V  o d V  "8  o B <a  H-H  P  ca  CN O  o •a  3  d  9  § 4J  s  O  hi v  c/3  E  12 1^  cu  a.  CN  CO  V5  J3 V5  IOi S IhSi  a a.  VH  CL)  -4 CJ 3  00  <u  -4 CJ 3  on  S  a  a  a  CH  ^  CU  CU  CH  CN w  CN  H*  a)  CD  o  O  _H  3  00  3  00  a CH  CU  co  >.  cu  3 CU  3  CU  CN  CN  cu  cu  cu cu 181  The semi-quantitative amounts o f arsenic species found in fish are summarized in Table 5.6. The most notable feature in these speciation results is the presence o f arsenobetaine in all fish species and individuals. Arsenobetaine is the major arsenic compound found in most marine fish ' . 16 27  However, none (i.e., less than 5 ppb arsenobetaine) was found in a previous study o f  freshwater fish including bass, pike, carp, yellow perch, striped perch, pickerel (walleye) and whitefish, even though methods that had been developed specifically to identify arsenobetaine were employed . 22  Another remarkable result is the presence o f arsenosugar X I in suckers. Arsenosugars have been found in only one other fish, a marine fish (silver drummer) that feeds on algae . 28  M o s t o f the arsenic in the silver drummer was as T M A O (rather than arsenobetaine) with arsenosugars X I and X I I I in the muscle, as well as arsenosugars X , XT, X I I and X I I I (with the structures shown in Table 1.1, Chapter 1) in the digestive tract. The source o f the arsenosugars in the silver drummer fish was postulated to be its food source, brown and red algae. Suckers are bottom feeders and may also acquire arsenosugar X I through their food, which implies that some benthic organisms are able to synthesize arsenosugars. The results in the previous chapter imply that cyanobacteria or other bacteria are able to synthesize arsenosugars X and X I and hence it is not unreasonable to suggest that organisms in the sediment may be capable o f doing so. The major arsenic compound in pike is D M A . Pike is a pelagic carnivorous feeder, meaning that it derives its food from the water and not from the sediment (e.g., other fish). The stomach contents o f pike sampled for this study consisted o f invertebrates and small fish. Invertebrates may contain D M A , or arsenosugars. Arsenosugars may be broken down to D M A (observed in the human b o d y and in mice ). F o r example, as described in the next section, 29  30  mussels and snails contain arsenosugars X and X I , and D M A , among other arsenic species,  182  although D M A is not the major species. It might be expected that pike would accumulate more arsenobetaine, because its diet includes small fish, which probably contain arsenobetaine. However, pike appears to preferentially accumulate D M A . High levels o f D M A have been observed in other fish; mackerel contained up to 25% o f its total arsenic as D M A . 1 6  T w o types o f unknown compounds are present in these samples. Unknown X in Table 5.6 was observed in very low levels in whitefish and walleye, and had an early retention time on the cation exchange system. It co-eluted with arsenate and arsenosugar X I , but attempts to identify the unknown by using other chromatographic systems were unsuccessful. Unknown Y compounds (in Table 5.6) appeared as two late eluting peaks on the ion-pairing chromatographic system as shown in Figure 5.2 (retention times approximately 700 s and 1000 s). These compounds did not co-chromatograph with arsenosugars X , X I , X I I or X I I I in kelp extract, as illustrated in Figure 5.2. The chromatograms in Figure 5.2 are scaled individually to facilitate comparison o f peak retention times. It was surmised that the compounds should be anionic i f they are late eluting on the ion-pairing system used, but they did not appear on the anion exchange system. Possibly they were irreversibly bound to the anion exchange column. Unknown compounds eluting at similar retention times (with respect to D M A ) on the same chromatographic system have been observed as arsenosugar metabolites in human urine . 29  183  Pike 2 PD Kelp extract  4e+3  3.5e+4  o 0 k( _  Q  X CO  a.  cu  in  i2  tz  o o  o o  0.0e+0  h Oe+0  240  480  720  960  1200  time (s)  Figure 5.2. Chromatogram o f protease digest (PD) o f a Yellowknife fish (Pike 2), showing small amounts o f two unknown compounds ( U K - Y ) mentioned in Table 5.6. Ion-pairing chromatography ( C I 8 column, l O m M T E A H / 4 . 5 m M malonic acid, p H 6.8, 0 . 1 % M e O H ; see Table 5.2) with I C P - M S detection was used. Abbreviations for arsenic species are in Table 1.1.  184  Branch et al.  observed that the trypsin digest o f a single specimen o f plaice contained  more D M A than the organic extract, and they postulated that partial degradation o f arsenobetaine to D M A by trypsin was taking place. T o determine if the presence of D M A in pike, and the unusual presence o f arsenosugar in sucker was a result o f the protease digestion, rather than representative o f the arsenic content in the fish, MeOH/water (1:1) extractions were performed for comparison. N o notable differences in arsenobetaine content in sucker are seen between the protease digestion results and the MeOH/water extraction (Table 5.6), although a more significant difference is observed in the amount o f D M A extracted. The greatest difference appears for sucker 2, where not only is more D M A present, but less arsenosugar X I is observed when MeOH/water was used. The reasons for this are not clear. Possibly the arsenosugar in this matrix is bound to protein and the protease digestion is required to release it. Likewise, some D M A may be bound to a product o f the protease digestion that precipitates or binds irreversibly to the chromatographic columns, whereas it may be effectively extracted as a free ion by using MeOH/water and can then be detected. Another possibility is that arsenosugar X I breaks down to form D M A during the MeOH/water extraction process. F o r pike, no significant differences are seen in arsenobetaine amounts by using the two methods (Table 5.6). Less D M A is extracted by using MeOH/water for both fish, which is the opposite observation as that for the sucker. Less unknown compound appears after MeOH/water extraction compared with protease digestion as well. Some o f these compounds were probably released by the action o f the protease on the matrix, and were probably not available for extraction by MeOH/water. When protease digestion was used, the sum o f arsenic species observed (Table 5.6) is greater than 100% o f the total arsenic (Table 5.5) for pike 1; however, considering that standard deviations are at least 30%, the amount detected after  185  protease digestion was within this amount and hence probably not significantly different from 100%. Extraction or digestion efficiencies were determined by using the relation:  Extraction/digestion efficiency (%) = (sum o f A s species)/(total A s ) x 100%.  These values are somewhat higher for MeOH/water extraction compared to protease digestion for sucker, and lower for pike. Reasons for the non-extraction o f certain species (i.e., greater amounts o f D M A extracted from sucker, and smaller amounts o f D M A extracted from pike, when using MeOH/water) are suggested above and these probably account for these differences in the sum o f arsenic species detected. However, this comparison study is too small to draw any conclusions about the effectiveness o f the different extraction techniques, except that the extraction o f arsenic species appears to be matrix dependent and varies between different species o f fish. Extraction/digestion efficiencies for most o f the fish are lower than 100%, ranging from 19 to 78%o (except pike 1 by P D ) . Protease digestion was expected to have solubilized most o f the arsenic, although time constraints did not allow determinations o f the total arsenic in the protease digestions to be made. However, if most o f the arsenic was solubilized, a large amount o f arsenic is unaccounted for in these results. This arsenic may be bound to molecules such as peptides or incompletely hydrolyzed proteins, that may precipitate or remain irreversibly bound on the chromatographic systems used here. Arsenic is known to bind to cytosolic proteins from rabbit and rat l i v e r ' . 31  32  In a previous study, 13.6 to 68% o f arsenic remained in the residue from the trypsin digest for marine fish . Some o f the arsenic in the present study may have remained in the 16  186  residue after protease digestion, especially i f it was lipid bound. Lipid bound arsenic was postulated to be significant in mackerel, which is a fatty fish . The lowest digestion efficiency 16  observed in the present results was for whitefish 3, which is a fatty fish as well, as evidenced by an apparent lipid layer being present after the protease digestion procedure. Hence lipid bound arsenic may be significant for these samples, and experiments using lipase, to hydrolyze lipids, would be useful. To summarize, this study has shown that arsenobetaine is a major extractable arsenic species in freshwater fish. Arsenosugar X I was observed for the first time in freshwater fish, and D M A was observed to be the major arsenic species in pike. Although D M A has been linked to increased tumor growth in mice exposed to carcinogens , the levels o f D M A in these fish are 33  too low to be o f toxicological concern. Protease digestion and MeOH/water extractions gave comparable results in terms o f arsenic species observed, although quantities varied, and more experiments are necessary to verify the differences. Extraction/digestion efficiencies were generally low, and again, more experiments are necessary to determine the reasons for this.  5.3.3. Freshwater shellfish  5.3.3.1. Total arsenic in  shellfish  The arsenic concentrations in freshwater mussels and snails collected from Yellowknife and from Campbell River are shown in Table 5.7. The freshwater mussel collected from Yellowknife was identified as Anadonta grandis simpsoniana and the snails are Stagnicola sp. The mussel from Campbell River is Margaritifera  falcata.  The mussels were collected from  areas containing l o w levels o f arsenic. The snails were collected from the marsh area at the Baker Creek outlet (location 1) and also from Baker Creek outside the mill area o f Giant Mine  (location 3). The mussel concentrations should thus reflect typical background concentrations, but the snails are most likely to have been impacted by high levels o f arsenic.  Table 5.7. Moisture content and arsenic concentration in freshwater shellfish. A l l shellfish are from Yellowknife, except where specified; C R = Campbell River; nd = not determined. Arsenic concentration (ppm dry weight)  Sample, sampling time (location)  Moisture content (%) (R=fresh weight/ dry weight)  Anadonta sp., June (16)  nd  6.0  Anadonta sp., August (16)  90 (9.8)  7.4  Margaritifera  nd  3.1  Stagnicola sp., shelled (1+3)  81 (5.2)  82  Stagnicola sp., whole (1+3)  69 (3.2)  83  sp. ( C R ) b  b  a  b  a  Moisture content was calculated by using (R-l)/R*100%; R=fresh weight/ dry weight. nd = not determined. The same species of snails was analyzed, but processing differed; see section 5.2.2. 0  These concentrations obtained for arsenic in freshwater mussels (3.1-7.4 ppm dry weight) are similar to those for marine mussels, where values ranging from 8.47 to 15 ppm dry weight and 2.1 to 3.7 ppm fresh weight have been f o u n d ' ' ' . The levels o f arsenic in these 34  35  36  37  freshwater snails (Stagnicola sp.) are similar to levels found in marine gastropods, which can range from 4.2 to 233 ppm dry weight ' ' . A freshwater snail contained 0.186 ppm (fresh 38  39  40  weight) arsenic , which is an order o f magnitude lower than the comparable concentrations in 41  Stagnicola sp. in the present study, o f 1.6 to 2.6 ppm arsenic (fresh weight).  5.3.3.2. Speciation of arsenic in shellfish The arsenic species that were determined following protease digestion (PD) and MeOH/water methods are shown in Table 5.8.  188  co £  s  c  CD o •§ 1  5  £ 3  co  O,  <D  w  t *  ST g 3  -  i  5  CN  o co  .5  CO  ON CO  oo  CN CO  CO CN  CN  .—. co  Hi ON !=l  <D  ,t-i  co  o  cd  oc ca  CO  d  co  CN  V  .2? 8 ca  d  V  d  <D  o d v  o <  o d v  s d v  cu -5 ca C  CD  oo  CD  ^  •  s  -  u.  2 > H «« s c  i  x:  CD  c/3  J-J  X  X  . -  c3 £ Vi  I—J  v-i  H 3  CD CJ  U  e  II  o (* U U 00 I/}  .8  <D  H .3  V  O  d  v  cu Z  cu Z  cu Z  CN  o d v  t-; CO  oo  CO v d  cu Z  cu Z  cu Z  a. Z  ON  CO  CN  co  ^2  d  o  co  o d v  VO  o  d  V  o  d  v  I  1  d  s d  d  CU  d  o d v  d  u  ca  &  a) o  d  o  •S  Si  v  CN  ON  d  Z  d  o d v  s d  CD  I 8.  v  .a  CO  CN  ^  II  d  3  d d  d  S  o d v  vo d  d  VO  d  O  V3  CO  CD  II  %  co 73 . c  15 ca fcM  O  u  g  co d  3  g  g  °1  o o d v  o  in o o  d  S  o d v  o  CN  d  d  o  V  u-i  00  d  ©  s d  a.  v  vi  1-3  d  5 iS ca CM  ||  « Q ca  d oo  H a « o cu a ©  CN  — d  ca  vo  CN  ft"  " .33 8 CK  > CD  i  c/s  <U 3 M CO  T3  P CU  on S  1«  e Q C3  S  I  a § CD ^  •O &  S  •§  S « -8  NO  cn  o cD  O -1  CO ( 3  u o  CO CJ  oo a 1 >w*  1I  —1  ca § 8 Q  3 .a  CN  o  d  d  VO  6 n 3 8 •a & <D  o  d  3  <C o  CD CL  CO  O  Si d  2 e ^  CN  d  3  c/3  o o d v  CN  (D t60  •9 § 3 Q 5 O .3 -2 « ca S O c/3 £3  5  o d v  <L>  3  a a (*>  d  CN  o  O  CD  CN CN  O ea  ^e s« O  CN CN  o  «J  o  II  5  Q C/3  ^ 2  s o  •3 a B  CD cj  §  u189  The most striking result in Table 5.8 is the small amount, i f any, o f arsenobetaine in freshwater mussels and snails. A small amount was extracted from the Campbell River mussel (3% o f total arsenic extracted, fresh weight) but this was not reproducible when the extraction was repeated on a dry weight basis. A small amount (0.6%) is present in shelled snails. The absence o f arsenobetaine has been observed for an estuarine mussel japonica?  4  Corbicula  and was suggested to be related to the low salinity o f its environment. Glycinebetaine  is used by marine animals for osmo-regulation , and it has been suggested that arsenobetaine is 42  accumulated in marine animals because o f its similarity to glycinebetaine. Therefore since estuarine and freshwater animals live in lower salinity regions, their need for osmo-regulation is reduced, resulting in less arsenobetaine entering the body o f a freshwater animal and/or excretion of more arsenobetaine. These processes would result in less arsenobetaine being accumulated in the body o f the animal. The absence o f arsenobetaine in the animals analyzed in the present study supports the view that arsenobetaine is accumulated from the environment, i.e. by marine animals. Previous studies have also indicated that the presence o f arsenobetaine in marine mussels is a result o f bioaccumulation (i.e., from food) . 43  The major arsenic compounds in the mussels are arsenosugars X and X I (Table 5.8). The arsenic speciation in Yellowknife mussels collected in June and August differs only in the smaller amount o f A s ( V ) and a significant amount o f an unknown species detected using the ion pairing chromatographic method, in the mussel collected in August (following P D ) . The unknown arsenic species had a retention time on the ion-pairing system similar to that observed for one o f the unknown compounds in pike protease digests (780 s, cf. 700 s in pike). A s ( V ) is present in all mussel samples. MeOH/water (1:1) extracted A s (III) from the Yellowknife mussel collected in August and the Campbell River mussel. In these specimens, the levels o f inorganic arsenic found are not o f toxicological concern to humans (should the mussels  190  be ingested). However, inorganic arsenic levels may be significant in situations where exposure o f mussels to arsenic is much higher, and i f accumulation o f arsenic by mussels takes place. Some MeOH/water extractions were performed to validate the arsenic species detected after using the P D technique, as for the fish samples. The sum o f arsenic species is slightly higher by using MeOH/water extraction for the Yellowknife mussel collected in August (2.7 ppm cf. 2.2 ppm for P D ) , but a lower sum results from MeOH/water extraction o f shelled snails (23 ppm cf. 32 ppm for P D ) . L i k e the results for suckers, MeOH/water apparently extracts (or renders detectable) more D M A from Yellowknife mussels (1 ppm) than does P D (0.12 ppm). The reasons suggested for the differences in the sucker results probably apply here as well. M o r e arsenosugar X is observed in protease digests, which may suggest that arsenosugar X I is being enzymatically degraded to arsenosugar X . The decomposition o f arsenosugar X I to arsenosugar X in aqueous extracts during storage has been documented previously . However, 34  this observation was not made for arsenosugar X I in sucker and hence it appears that individual matrices affect these processes. The results for the Campbell River mussel suggest that differences between fresh weight and dry weight extraction may be significant, in terms o f extraction efficiency and species extracted or detected. Better extraction efficiencies have been observed for fresh weight compared to dry weight extractions for marine mussels . F o r the two analyses o f Campbell 44  River mussels, differences in arsenic species are seen. Arsenosugars were observed in all samples, but small amounts o f arsenite, M M A and D M A are observed following fresh weight P D of Margaritifera  sp., whereas arsenite, arsenate and an unknown species are present following  dry weight extraction (Table 5.8). The differences in arsenic species may be due to differences in the preparation technique (fresh weight or dry weight, and P D or MeOH/water extraction), or to  191  sample inhomogeneity. Sample inhomogeneity was observed by Shibata et ai.  for a freeze-  dried reference material and was considered to be quite significant. In this study, arsenosugar X I I I was observed for a single fresh weight P D o f the Campbell River mussel (results not shown), but it was not found in a duplicate fresh weight P D , nor in the dry weight MeOH/water extraction. The presence o f arsenosugar XIII in the freshwater environment, had it been reproducible, would have been very significant, since up to now, only arsenosugars X and X I have been observed in the terrestrial environment as discussed in section 4.3.3.1 (Chapter 4). In snails (Stagnicola sp), the major extractable arsenic compound, in a proportion o f 4060% o f arsenic extracted, is tetramethylarsonium ion. Inorganic species (As (III) and A s (V)) amount to 25-40% o f the total arsenic extracted, which is a major difference from marine snails. In a marine snail, Tectuspyramis,  the major compounds were found to be arsenobetaine (35-  67% o f arsenic extracted) and tetramethylarsonium ion (6-26% o f arsenic extracted) . In other 38  marine gastropods, arsenobetaine was also found to be the major arsenic compound, with inorganic arsenic, D M A , arsenocholine, the tetramethylarsonium ion or arsenosugar X as minor constituents ' ' . Uncharacterized trimethylarsenic (62% o f total arsenic) and dimethylarsenic 27 40 45  (27% o f total arsenic) species were postulated to be present in a freshwater snail . 41  The role o f tetramethylarsonium ion is not clear. It has been suggested that it may serve as a precursor to or a decomposition product o f arsenobetaine. However, its presence in snails, where the uptake or synthesis o f arsenobetaine appears not to be significant, indicates that this is probably not the case. The tetramethylarsonium ion may arise from a metabolic pathway independent o f one that would produce arsenobetaine, and is probably prevalent in freshwater gastropods. Methylation, possibly following the Challenger mechanism, might be taking place. The presence o f M M A , D M A and T M A O in the snails, which would be intermediate compounds in the mechanism, supports this hypothesis.  192  Arsenobetaine is present in shelled snails in a small amount (0.6%), so the processes that occur in marine systems may also be taking place here, but to a smaller extent. Arsenosugar X was also detected in snails as a minor component (3.5%). The different sample preparation techniques used for the shelled snails may indicate that protease digestion releases more arsenic than MeOH/water extraction, although more studies should be done to verify this. The MeOH/water extract o f snails was incompletely analyzed (by anion exchange and ion-pairing chromatography only), but the total amount o f arsenic given in Table 5.8 reflects that which would be seen i f cation exchange chromatography was used, within the estimated uncertainty o f 30%. Extraction/digestion efficiencies are lower than 50% for all samples analyzed. The same reasons as those given for the fish extraction efficiencies apply here: protease digestion and MeOH/water extraction may not solubilize all the arsenic, or the solubilized arsenic may not be detectable by using these chromatographic systems. When MeOH/water (1:1) was used to extract arsenic from marine mussels (Mytulis edulis in all cases) in other studies, only 52 to 60% of arsenic was extracted ' . In another study, 47% o f arsenic in Mytulis edulis was accounted 34 36  for by H P L C - I C P - M S analysis as water soluble species, with 17% extracted in chloroform, and 32% remaining in the solid residue . Arsenic bound to lipids or other types o f molecules such as 35  proteins appears to be significant in mussel matrix. Another extraction method (MeOH/water, 9:1, with rehydration o f dry weight samples before extraction) has proven to yield higher extraction efficiencies (95-100%) for marine mussels, although a significant amount remained unaccounted for after chromatographic analysis . 44  Chitin is a component o f snails, since the radula (similar to a tongue) and parts o f the digestive system are composed o f this material . Protease digestion is inadequate for the 46  solubilization o f chitin and more specific enzymes such as chitinase and |3-(l-3)-glucanase are  193  necessary ' . In other studies, extraction o f the marine snail T. pyramis with methanol was unable to dissolve 12 to 2 5 % o f total arsenic which are significant amounts, but not as great as 38  those unaccounted for in the present study. Mollusk shells, made o f calcium carbonate crystals within a protein framework known as conchiolin, were found to require rigorous extraction with 2 M H Q  4 9  to dissolve arsenic. Hence  it is expected that the mild sample preparation used in this study would not be capable o f extracting arsenic from shells. This reasoning explains the observation that the extraction efficiency o f whole snails is about half that o f shelled snails, since the sample was probably diluted by the presence o f shells from which arsenic could not be extracted. Arsenic is most likely present in snail shells, because it has been found in other mollusk shells . 49  To summarize, the speciation o f extractable arsenic in freshwater shellfish, containing very little or undetectable amounts o f arsenobetaine, supports the suggestion that arsenobetaine accumulation in marine animals is related to osmo-regulation. Arsenosugars X and X I are the major compounds in freshwater mussels, similar to findings in marine mussels. Tetramethylarsonium ion is the major compound in freshwater snails, although inorganic arsenic is present in significant levels as well. The high accumulation o f inorganic compounds in snails could present toxicological problems for higher trophic organisms. Large amounts o f arsenic remain unidentified and this could be lipid or chitin bound. M o r e work is necessary to elucidate the nature o f this arsenic.  5.3.4. Plants  Higher plants were collected from the Yellowknife area to determine the speciation o f arsenic in plants. Sampling areas (refer to Figures 5.1a and 5. lb) included K a m Lake which 194  received accidental mining effluent discharges in the early 1970s from the C o n Mine, and has also been influenced by raw sewage from Yellowknife  50  (location 12). Baker Creek, currently  receiving mine effluent from the Giant Mine, was sampled in different areas: the marsh (location 1) and mill (location 3) areas, as well as an intermediate location in the creek (location 2). Niven Lake was also chosen as a sampling location, because it was a former sewage pond and was expected to contain residual elevated levels o f arsenic and other metals, and because o f prolific plant and algal growth. T w o locations are specified, one on the east side o f the lake, near the site o f a former dump (location 10) and the other at the north end o f the lake where the lake drains into B a c k B a y (location 11). Cattail, Typha latifolia, was common to all the sampling locations and horsetail, Equisetum fluviatile,  was found in K a m Lake and Baker Creek. These  two plants were sampled (shoots only) from different sites to determine i f any location specific differences in arsenic speciation could be seen. The aquatic plants, burweed, augustifolium,  and pondweed, Potomogetan  Sparganium  richardsonii, were sampled from the water in  Yellowknife B a y near a former Giant Mine tailings pond (location 6).  195  5.3.4.1. Total arsenic in plants The concentrations of total arsenic in plants are summarized in Table 5.9a. Uptake o f arsenic by macrophytes was studied previously for plants collected near C o n Mine and some o f 4  these results are also shown for comparison in Table 5.9b.  Table 5.9a. Arsenic concentrations in plants from Yellowknife. Sample, Time  Location  Arsenic concentration (ppm dry weight)  Emergent plants (shoots) Horsetail Equisetumfluviatile,  June  12  30  Horsetail Equisetum fluviatile,  June  1  48  Horsetail Equisetum fluviatile,  June  3  260  Cattail Typha latifolia, June  2  5.0  Cattail Typha latifolia, June  11  0.52  Cattail Typha latifolia, June  12  3.8  Bidens cernua, August  10  100  Submergent plants Milfoil, Myriophyllum (whole plant)  sp., June  11  78  Milfoil, Myriophyllum (whole plants)  sp., June  10  39  Milfoil, Myriophyllum (whole plant)  sp., August  10  17.4  Duckweed, Lemna minor, August (whole plants)  10  28  Burweed Sparganium augustifolium, August (shoots)  6  2.5  Pondweed, Potomogetan richardsonii, August (shoots)  6  20  196  Table 5.9b. Arsenic concentrations in plants from Yellowknife, previous study Location  Arsenic concentration (ppm dry weight)  near C o n Mine  17.2; range <1.0-38  fluviatile  near C o n Mine  34; range 5.5-91  Triglochin palustre, arrow grass  near C o n Mine  40  Potomogetan pectinatus (whole plants)  near C o n Mine  1219; range 190-4990  Myriophyllum plants)  near C o n Mine  143; range 30-255  near C o n Mine  28  Sample, Time Emergent plants (shoots) Typha  latifolia  Equisetum  Submergent plants  exalbescens (whole  Sparganium sp. (shoots)  Typha sp. contains the lowest levels o f total arsenic, and this is especially obvious when the levels are compared with levels in other plants collected from the same sample location. For example, compare Typha sp. (3.8 ppm) with Equisetum sp. (30 ppm) at location 12 (Kam Lake); and Typha sp. (0.52 ppm) with Myriophyllum  sp. (78 ppm) at location 11 (Niven Lake). In a  previous study , the average concentration o f arsenic in Typha sp. shoots was found to be 17.2 4  ppm dry weight with a range o f < 1.0 to 38 ppm dry weight, which is higher than the levels found in this study (0.52 to 5.0 ppm dry weight). However, the small sample size in this study precludes any direct comparison, except that Typha sp. shoots contained the lowest levels o f total arsenic in the previous study as well as in the present one. Dushenko et al. postulated that the lower levels o f arsenic in Typha sp., as well as its abundance at all sampling sites (observed in  197  the present study as well) indicates a greater tolerance to arsenic contamination by using mechanisms for the exclusion o f arsenic . 4  Equisetum sp. contains high levels o f arsenic at all the locations from which it was sampled (Table 5.9). M o s t likely the levels in these samples of Equisetum sp. are elevated (30 to 260 ppm dry weight) compared with background levels (5.5 ppm dry weight) in Equisetum sp. (sampled from Grace Lake, a location relatively unaffected by mine waste discharge) reported in the previous study . The other emergent plant in the present study is Bidens cernua, a flowering 4  plant that grew prolifically at the edges of Niven Lake in August, and the arsenic concentration in the shoots is 100 ppm dry weight. This concentration is higher than the average concentrations of arsenic found for all o f the emergent plants sampled in the previous study (Table 5.9) . 4  The submergent plants Sparganium sp., Potomogetan richardsonii, Myriophyllum  sp.  and Lemna minor contain arsenic in concentrations ranging from 2.5 to 78 ppm dry weight. The previous study showed that submergent plants contained more arsenic than emergent plants  4  (Table 5.9) but the sample size and number o f sampling locations in the present study was too small to make a direct comparison. The concentration o f arsenic in Myriophyllum  sp. appears to decrease throughout the  summer at the same location (39 ppm dry weight in June compared to 17.4 ppm dry weight in August), although this observation needs to be statistically verified. Grasses accumulate arsenic from soil at a faster rate when physiological growth o f the plant is significant and accumulation of arsenic slows down when plant growth slows . A s described in Chapter 4, higher levels o f 51  arsenic were observed for fleabane and sedge at the end of the growing season. The results for Myriophyllum  sp. indicate that an opposite trend is taking place, (i.e., arsenic concentration is  lower at the end o f the growing season) although the reasons for this are unclear. Possibly the rate o f arsenic depuration exceeds that o f uptake while plant growth is taking place.  198  5.3.4.2. Arsenic speciation in plants The arsenic species found in the plants collected in Yellowknife are listed in Table 5.10. The most noteworthy feature is the predominance o f inorganic arsenic (As (III) and A s (V)) as the extractable species, amounting to greater than 90% for all plants except for Lemna minor, which contains 80% o f extractable arsenic as inorganic arsenic. For Equisetum sp. and Sparganium sp. inorganic arsenic forms were the only arsenic species extracted.  199  J3  O  c  co —  Tf  O  CN  Tf  r-  o  —< CN  t -  co  in  ~->  CN  Tt Tf  CO CN  VO  Tf  at  J3  3  o  CD  c3b  tu CJ  c*>  CD  CN  VO  CN  Ov  rt  Tf  m  M  VO  o  CN  vo  t-'  VO  ret CO  CN  ©  O  o  o  V  V  m o ©  - H  o d  V V  r-  „  o o d  m o  v  CJ  90 ©  o o  CN ©  w  Tf  CN  Q M  43 00  «  "Si  3  O O  © ©  V  V  CN  © ©' © H H  VV  X CN ©  CN  CN  O  © ©  © ©  V  CN  © ©  V  V  © ©'  V  _  © ©•  V  •—-  00  ro ©  F-H  ©'  V  "3  ©  V  o  l-H  ©  ©  ©  V  V  •n ©  CN  CD  ©  'o  CN © ©  © '  V  V  — cj  ? s  Tf ©  Tf ©  ?  © CO  © ©  ©  VO ©  O  S 3  CN  CN  ©  © ©  ©  V  —  u  CD  V  •a CD cn  *  C+H  O cn G  O  X  co c  3  co  Tf  CN  /  > n ©'  Tf  ©  —^  © ^  ©  Tf  ©  CN  ©  00 ©'  ^—^ CO  f-H  VO  1 © —'  '  CN CN  VO CN  Tf  ©  ©'  ©  vo  "/I  © d  ©  H H  l-H  ^—'  Tf CN  CO  Tf  Tf  IT)  nj  +n C/3 CA  3 J=  42  r-~  CN  in 00 © vo  00 CN  co  ©'  ©  Xa  « I O  3 .-a  N  © © ^  -  00  CN  ©  CN  l-H  •n  ~—' CO rH  CN  '  © ©  •a  I  ab  5 CN  u~ a a  3  at  a  I  if  &3 8 C3 60  I Si C ^  ST  5  !  I s  © *—' CO  ©  HH  60  I.  vo  rH  OH  M I  0, C  © ^—^  CN  VO,  o  C+H  ©  ©' ^—'  1  VO  ©  H H  © —'  ©  vS"  ©  ©  CN  +-»  in  Tf  y—V  CD  H  2  § a  J3  Is o  ©  f)  V  Tf  VO  CJ  © ©  © ©  © CN  M  © ©  V  '« Q 2 O c/j  V  CN  CN  V  C/l  C  ex  ©  ^1 c/3  C+H  o  V  d  CN ©  3< C+H  ©  CN CN ©  0>  O d  _ 0>  ecj  CN  © ©'  0>  CH  V  CN  O  °5 -a s  © ©  ©  CO  5a  I  c •—> ts It  C o s:  I cf  a cu y  53  co  cu  c5  c S  K  200  The proportions o f arsenic species, especially A s (III) and A s (V), are consistent between plants o f the same species, as seen for Typha sp., Equisetum sp. and Myriophyllum  sp. collected  in June (see Table 5.11). The major arsenic species extracted from Typha sp. is A s (V), for Myriophyllum  sp. (June) it is A s (III), and equal amounts o f A s ( V ) and A s (III) were extracted  from Equisetum sp. A s mentioned in Chapter 4, A s (III) is less toxic to periwinkle than A s ( V ) , and may be less toxic to higher plants from Meager Creek. Its presence in plants from 52  Yellowknife may indicate that reduction o f arsenate is taking place by the plant as a detoxification mechanism. The major species o f arsenic in all water samples was arsenate (Table 5.3). However, the major extractable species from Baker Creek sediments was arsenite (see Table 5.3) which may be taken up unchanged by plants.  Table 5.11. Percent arsenic species o f total arsenic extracted from plants (SD) . a  Plant  A s (III)  Methyl (DMA + MMA) 4.8 (4.4)  Sugars (X + XI)  Me As  0  0  50 (10)  0  0  0  70  0  0  0  A s (V) _____  Typha  latifolia  Equisetum  b  (8)  fluviatile  50(10)  h  +  4  Sparganium  augustifolium  Potomogetan  richardsonii  11.8  78 7  1.6  5.9  2.0  Myriophyllum  sp., June  71 (12)  25 (14)  3.4(2.2)  0  0.1 (0.1)  Myriophyllum  sp., August  26  67 3  5.5  0.9  0  Lemna minor  17.3  63  7.2  10.7  1.7  Bidens cernua  8  88  3.4  0  0.5  21(8)  70 (?)  3.6(3.3)  4.4 (5.0)  0.9(1.1)  d  Average for submergents collected in August  .30  6  SD = standard deviation, calculated for plants of the same species sampled from different areas and with different absolute concentrations of arsenic. Where no SD is indicated, only one sample was collected, so n=l. F o r S D , n = 3. °ForSD, n = 2.. Submergents collected in August include: Sparganium sp., Potomogetan sp., Myriophyllum sp. and Lemna a  b  d  minor, for SD, n = 4. 201  The difference between arsenate and arsenite distributions for Myriophyllum  sp. between  June and August is interesting to note. The sample collected in August contains the same proportion o f arsenate as the June sample contains o f arsenite (approximately 70%). This may indicate that oxidation is taking place throughout the summer, or it may indicate a slowing o f the microbial activity (in the plant's environment) or plant activity to produce A s (III), which may be related to decreased plant growth in August. In general, submergent plants collected in August contain predominantly A s ( V ) (see Table 5.11) and therefore the trend observed for Myriophyllum  sp. may extend to other  submergent plants. Dushenko et al , found that submergent plants accumulated more arsenic 4  than emergent plants (Table 5.9). They postulated that leaf uptake from the surrounding water column was significant, and that no arsenic exclusion mechanism exists for submergents. The presence o f arsenate as the major extractable arsenic compound in submergents after a summer of exposure may mirror the surrounding environment, and indeed, the major arsenic species in Yellowknife waters is arsenate. M i n o r amounts (less than 5%) o f methylated arsenic ( M M A and D M A ) were extracted from all samples o f Typha sp. (Table 5.10). The absence o f methylated arsenic from extracts o f other plants collected from the same location as Typha sp. (such as Equisetum sp. at K a m Lake) may indicate some specificity on the part o f Typha sp. to accumulate these species or to methylate arsenic from its surroundings. M i n o r amounts o f methyl arsenic are also observed in Potomogetan sp., Myriophyllum  sp. collected in both June and August, and in Bidens cernua. A  larger amount (7.2%) o f methyl arsenic is present in Lemna minor. In all these species except Typha sp. and Equisetum sp., Me4As was observed at some point (e.g., in a.Myriophyllum sp. +  specimen in June, but not in August) Arsenosugars are also present in Potomogetan sp.,  202  Myriophyllum  sp. and Lemna minor. Evidence exists for both the synthesis and uptake o f methyl  arsenic (as M M A and D M A ) from its environment, as discussed in Chapter 4  52>53  >  54  Me4As was +  found in a vascular plant from Meager Creek and its presence in these samples, albeit at very low levels, indicates a wider distribution than might otherwise be expected, considering the rarity o f its presence in sediments or waters, as discussed in Chapter 4 - . Halophytes apparently 55  contained T M A O , M e A s , arsenocholine and arsenobetaine +  4  57  56  and the presence o f these  compounds was attributed to the presence o f similar compounds in the surrounding water. The discovery o f arsenosugars in some o f these plants (Table 5.10), amounting to as much as 11% o f extracted arsenic (absolute concentration 0.68 ppm dry weight) in Lemna minor, represents the first finding o f arsenosugars in higher terrestrial plants. Halophytes (salt marsh plants) were proposed to contain an arsenosugar but no identification (i.e., comparison with standard arsenosugar compounds) was carried out . Interestingly, only arsenosugars X and 57  X I are observed in the present study, which has also been the case so far for Meager Creek samples, and Yellowknife fish and shellfish. The arsenosugars are found only in submergent plants. Myriophyllum  sp. and Lemna minor were growing in physical contact with algae, which  may be a possible source o f arsenosugars (mentioned in the next section). Submergent plants, in general, contain a larger proportion o f organoarsenic species (on average, 9% o f extracted arsenic, and up to 20% o f extracted arsenic for Lemna minor, see Table 5.11) compared with emergent plants (up to 4.8 %). The reasons for this trend are unclear. Due to their propensity for uptake from their environment, as discussed by Dushenko et al. , submergent plants may be acquiring organoarsenic from their environment, but the 4  possibility o f synthesis by the plants cannot be discounted. Extraction efficiencies are less than 100% in most cases, although two cases o f efficiencies greater than 100% were obtained. In both these cases (Equisetum sp. from Baker  203  Creek marsh and Typha sp. from Niven Lake) the standard deviations in the speciation results are about 30% and the error in these numbers may account for these unusual results. The lowest extraction efficiencies are observed for Potomogetan richardsonii, Bidens cernua and Lemna minor. Unextracted arsenic could be bound to lipids, or to cell wall components, including insoluble cellulose, calcium or magnesium pectates, or lignin.  5.3.5. Algae a n d microbial mats Algae and microbial mats were sampled from a few locations in Yellowknife. Microbial mats were sampled from standing water next to, but not a part of, Baker Creek (location 4, Figure 5.1a), in both June and August. In June only the microbial mat was present in the standing water, whereas in August other plants (Typha sp. and Equisetum sp.) were present, but very little o f the microbial mat was present. A microbial mat was also sampled from a small pond near a former Giant M i n e tailings pond (location 5). Location 10 in Niven Lake was sampled for two different types o f algae, which were painstakingly separated from each other and other organisms, and washed thoroughly. The exact composition o f the microbial mats from locations 4 and 5 was unknown and most likely included a combination o f bacteria (including cyanobacteria) and freshwater algae (including diatoms). The organisms possibly belong to genera such as Microcystis,  Oscillatoria,  Spirulina or Aphanizomenon, which have been found  in other freshwater microbial mats . T w o species o f green algae were sampled from Niven 58  Lake: Enteromorpha intestinalis, and Algae 1, o f unknown identification, but possibly Cladorpha sp.  204  5.3.5.1. Total arsenic in algae Arsenic concentrations in the microbial mats are high, ranging from 390 to 2500 ppm dry weight (Table 5.12). These levels are higher than those found in Meager Creek microbial mats (maximum o f 278 ppm dry weight).  Table 5.12. Arsenic concentrations in algae from Yellowknife. Sample, Time  Location  Microbial mat, June  4  Arsenic concentration (ppm dry weight) 2500  Microbial mat, August  4  1100  Microbial mat, June  5  390  Enteromorpha  10  6.6  10  30  intestinalis, August  Algae 1, August  A bioconcentration factor ( B C F )  can be calculated for these algae, by using fresh  weight concentrations and the following relations, where fw is fresh weight and dw is dry weight:  [Asftv] = [Asdw]/R;  where  R = fresh weight mass o f sample/dry weight mass o f sample BCF =  [ASfwMASwater]  Factors o f 0.86 and 0.38 are calculated for the microbial mats sampled from location 4 in June and August, respectively (R = 6). If the factors are less than 1, then no bioconcentration is taking place and this is likely the case for these samples. 4  205  Concentration factors can be calculated for the samples from location 10 as well; they are 9 (R = 10) for Enteromorpha intestinalis, and 68 (R = 6.5) for Algae 1. The water concentrations used for B C F calculations for the August samples are those determined in the June samples in Table 5.3 because concentrations o f arsenic are not expected to change by more than about 50% in a location, from June to August . The B C F s obtained indicate that 60  Enteromorpha  intestinalis and Algae 1 bioconcentrate and bioaccumulate arsenic. B C F s for  marine and freshwater algae have been reported to be as high as 200 to 3000 . 61  5.3.5.2. Arsenic speciation in algae The arsenic species in the microbial mats sampled from location 4 in June and August show very few differences (See Table 5.13). The major species in these samples are inorganic arsenic, and the proportion o f arsenite appears to increase slightly from June to August, where [arsenite]/[arsenate] was 0.47 in June and 0.70 in August. O n the other hand, the proportions o f D M A and arsenosugar X , although small in June, decrease to trace levels in August. The reasons for this are unclear and the differences are probably too small to draw conclusions.  206  s-<  a  CD  r—I  «n > on  oo  vo  ON CO  -d-  CN  CN O  .2? S PH  3  H-C  cD O Cd.  c/3 VH  Ja  <D  3  3  B  +H  *  S  o «  \E1  c/3  o  00  3  00  <"  8  o  OH  CN  o d v  d v  Tf  d  !>->  d  ro d  'cu  NO  X  —:  oo 3  00  cX  Q  ^  " ' -—-  C/3 C/3  ON  cd  a Q °  NO  5?  • ° CO CD  Tt  CO  CO  00  C  o  cu ON  %  O  3  d  00  o  IT)  d  o  IT)  CN  cd CJ  CU  l/->  X  CN  CU  I  s  o  a ^  S <^  o  cj cd  b  NO  d  o d  CN  CN  |  CN  o d v  > o o 8 aa «>  CN  o d  CN CN  V  d  o d v  CO  d  T3  CD  '3 0>  C2  B  co  ON  d  CD  C/3  ,0  .a J3 cu  g> &  1* "HI  2 o  •*H  co  CH  .a g  «3  5  -s 2 C/3  cu  >. H  *3 CD  p §  d  '-^  5  ~  * CN  co S  CD  O O  CN  ^  o  co  d  °  CN CU  i  5£ T1  Tt  CN  ci 8  ^ g  C/3 NO  C/3  a o •a  cd  t-i  Id  co -—' 00  a c CD a a o U  CU  s.  •  cd  cd  i-J  CD  £ £  _«J  1  00  d o NO  oo 3  CN  o d v  s cu  '5 ia  a o a  3 3  b x tu  00  3  cd  cd =  a  IE o  o  2  is  S  s3 "3 " o  CJ  o d v  CU  s  VH  >>  o d v  o  82  I I  < <3  00  3 <  # II  SC cu  OC  oo  207  The microbial mat sampled from the pond at location 5 contains arsenate as its major species, and 12.6% o f the arsenic extracted occurs as methylated arsenic and arsenosugars (Table 5.13). The absence o f arsenite in this sample compared with samples from location 4 probably indicates a lack o f organisms that reduce arsenate to arsenite. Arsenosugars X and X I (10% o f arsenic extracted) are present in this sample, whereas only arsenosugar X (3.4% o f arsenic extracted for the June sample) is present in samples from location 4. The finding o f arsenosugars in these samples and in microbial mats from Meager Creek in levels ranging from 0.8 to 44% o f water soluble arsenic (Chapter 4) indicates that their synthesis is not restricted to thermophilic organisms. That is, among the non-thermophilic organisms that make up the microbial mats from Yellowknife, some appear to be capable o f synthesizing arsenosugars X and X I . The algae sampled from location 10 belong to the phyllum o f Chlorophyta. Because they are macroscopic it was possible to thoroughly wash them without disrupting or losing the organisms. From Table 5.13, it can be seen that arsenate is the major water soluble arsenic species (90% for Enteromorpha  intestinalis and 33% for algae 1) in these algae. Arsenate is the  only inorganic species present, indicating that no reduction to arsenite takes place by, or in the environment o f these algae. Appreciable amounts o f arsenosugars are present, especially in Algae 1 (sum o f arsenosugars X and X I was 59% o f extracted arsenic). This represents the highest proportion o f arsenosugars found in freshwater organisms from both the Yellowknife and the Meager Creek environment. The terrestrial cyanobacterium Nostoc sp. contains the highest proportion o f arsenosugars in any terrestrial organism (arsenosugar X was the major arsenic compound extracted) . The presence o f only arsenosugars X and X I (and not arsenosugars X I I 6  and XIII) in the present study are in agreement with the findings from Meager Creek. These freshwater algae are probably synthesizing arsenosugars, although the possibility o f uptake from microscopic organisms living in close physical contact with the algae and possibly 208  excreting arsenosugars cannot be discounted based on these studies. A unicellular marine diatom is apparently capable o f synthesizing arsenosugars , and freshwater analogues o f these 62  microscopic organisms may exist. L o w extraction efficiencies are observed for algae and especially for microbial mats (Table 5.13). L o w extraction efficiencies were also found for Meager Creek microbial mats (333%) and algae (1.1-7%). Only 1% o f total arsenic was extracted from the microbial mat from location 4 that contained the highest levels o f arsenic o f all algae samples (2500 ppm dry weight). The low extraction efficiencies from microbial mats might be a result o f arsenate coprecipitatation with iron and manganese oxides onto or in the microbial mats, making the arsenic insoluble in MeOH/water (1:1). A s discussed in Chapter 4, evidence exists to suggest that biomineralization o f arsenic in microbial mats could take p l a c e ' ' . 63  64  65  The highest extraction efficiencies are observed for Enteromorpha intestinalis (49%) and Algae 1 (20%). Lipid soluble arsenic, which would have been extracted to only a very small extent by using MeOH/water (1:1) may make up some o f the unextracted arsenic in these algae samples. Arsenic may also be bound to cell wall components such as those mentioned in 5.3.4.2.  5.3.6. Mosses  5.3.6.1 Total arsenic in mosses The total arsenic concentrations found in mosses collected from Yellowknife are summarized in Table 5.14. T w o samples o f Drepanocladus sp. were found growing underwater (locations 1 and 4) and another sample of Drepanocladus sp. as well as the other mosses were terrestrial (see Table 5.14 for location numbers). The identity o f mosses 1 and 2 are not certain  209  but they are most likely the same species, Fumaria hygrometrica. Although Drepanocladus sp. was identified by genus only, all specimens o f this moss are o f the same unknown species . 66  Table 5.14. Arsenic concentrations in mosses from Yellowknife. Sample, Time  Location  Drepanocladus sp., June  1  Arsenic concentration (ppm dry weight) 1220  Drepanocladus sp., August  1  490  Drepanocladus sp., August  4  880  Moss l , June  5  1130  M o s s 2 , August  7  1900  Pohlia sp., August  15  1310  Drepanocladus sp., August  15  770  a  a  Moss 1 and Moss 2 are suggested to be the same species, namely Fumaria hygrometrica.  The terrestrial mosses contain, in general, the highest levels o f arsenic, and the sample from location 7 (Giant Mine tailings pond) contains the highest amount (1900 ppm dry weight, see Table 5.14). F o r terrestrial mosses, washing was probably not adequate to remove the soil in which they were growing, which may explain the very high levels o f arsenic found in them. It was possible to sample aquatic Drepanocladus sp. so that sediment was not included and thus the levels in these moss samples more accurately represent the actual concentrations o f arsenic in the moss. Drepanocladus sp. from location 1 appears to decrease in arsenic concentration from June to August, although the sampling locations were not exactly the same and replicate samples were not taken to verify this observation. Meager Creek samples o f Fumaria hygrometrica also showed seasonal differences, where the concentration o f arsenic in a November sample was  210  lower than that o f a July sample. A s with the Meager Creek samples, the seasonal differences in these samples may reflect differences in arsenic uptake rates and accumulation.  5.3.6.2. Arsenic speciation in mosses The arsenic species found in mosses are summarized in Table 5.15. Inorganic species o f arsenic are the major and only species extracted from all moss samples, except from Drepanocladus  sp. From all three locations, Drepanocladus  sp. (1, 4 and 15) contain more  extractable A s ( V ) than A s (III), whereas mosses 1 and 2, presumed to be the same species, show no trend. The most remarkable feature o f these results is the occurrence o f M e A s in +  4  Drepanocladus  sp. sampled from location 1 in June and August, but the lack o f it in the same  species o f moss sampled from the two different locations 4 and 15. The major arsenic species present in snails, which were living in the moss, is M e A s . In a similar way, arsenosugars are +  4  present in Fumaria  sp. from Meager Creek (Chapter 4), but not in the same or similar moss  species from Yellowknife locations. These results may indicate that the arsenic species extracted from moss reflect the surrounding environment. This supports the hypothesis that for mosses, the source o f arsenic species is uptake from the environment, rather than de novo synthesis. The arsenic species found in mosses from Meager Creek and Yellowknife, compared with the arsenic species found in some organisms (snails in Yellowknife, microbial mats in Meager Creek) that live in close physical contact with the mosses, are shown in Figure 5.3.  211  p  VO CN  o  ON  o  o  00  in in  p co  3 O  o> CN  CN  CN  d  d  v  V  CN  CN  cn  r~-  CN  CO  CO  CO  d  d  d  o  V  o  V  O  V ©  O oo — <U  § *  0\  ©  8  CN  o  © v  CN O d  d  v  V  NO  o d  V  VO O d  V  d  in  —'  VO ON d  a  p CN  CN  CN  o  d  o  8  d  v  v  d  V  8  O  VO  vo O  V  d  V  d  d  a s 3 "3 s  V  >»  I-H  y  o  — 'V  m o o  /• N  oo o.  •«—^  .  1—1  o o NO  ON  2a  ©  CN  ,—1  •S a CN  00  g o  ^ «  3  o  X> O  "B.  : " 3  © s  a _> s  oo  1  j  o  a 3  s 00  a  3  Q  a 'o  c" o • -a s 2 o •> •a . 1)  O,  4}  c3  •a _s § 3  •_  i  on  d  10.8  O  d •-—'  55(6)  24.5  CO  CO  o  4.86  ©  V3  <  <  oo  < 3  3 <  •J:  •2 O  O  •St  © a.  IIw  1 CJ  II  © s  I  u  g  II 00  3  <  ! Q  W  i on  212  As(lll)+As(V) MMA+DMA  um  x+xi Me As  +  4  o __ C  CO  o c o o  CU  >  CD  c_  YK moss  Y K snails  MC m o s s  MC algae  Figure 5.3. Relative amounts o f arsenic species in moss and associated organisms from Yellowknife ( Y K ) and Meager Creek ( M C ) , showing similarity in speciation between the moss and the organism (snails or algae) living in close physical contact with it. Abbreviations for arsenic species are found in Table 1 . 1 .  213  Extraction efficiencies for moss samples are low, ranging from 1.7% for moss 2 from location 7 to 15% for Drepanocladus  sp. sampled in August from location 1. L o w extraction  efficiencies were observed for mosses from Meager Creek as well. Limited extraction might be expected from the terrestrial mosses from which soil could not be completely removed, since arsenic in soil occurs in mostly non-water soluble forms, such as minerals, or adsorbed onto iron and manganese oxides. Arsenic appears to be bound to other plant components o f the mosses for the non-terrestrial species, which may include lipids and cell wall components (see section 5.3.4.2.)  5.3.7.  Lichens and mushrooms Pixie cup (PC) lichens (Cladonia sp., not positively identified as the same species) were  found at different locations: P C I near K a m Lake (location 12), P C 2 near M e g Lake (location 13), and P C 3 in the C o n tailings pond area (location 15). Other lichens were collected as well near M e g Lake (location 13) and one species was found on rocks close to the water line o f the Giant M i n e tailings pond (lichen 4, from location 7). Identifications are Cladina sp. for lichen 1 and Cladonia sp. for lichens 2 and 3 (lichens 2 and 3 were not the same species). The identity o f lichen 4 remains unknown but it was not Cladonia sp. Lichens 1, 2 and 3 were growing together. Mushrooms Paxillus involutus, Psathyrella candolleana and Leccinum scabrum were found near M e g Lake (location 13). M e g Lake is the first lake in the Meg-Keg-Peg drainage system, into which C o n Mine effluent flows, and therefore it is the first lake to receive C o n Mine effluent. The shaggy mane mushroom, Coprinus comatus, was found growing in the dry and sandy former Giant M i n e tailings pond (location 8). Puffballs Lycoperdon pyriforme  were  sampled at two different locations. Mature specimens were found at the edges o f the currently  used Giant M i n e tailings pond (location 7), and younger specimens were found at the edges o f the C o n M i n e tailings pond (location 15).  5.3.7.1.  Total arsenic in lichens and  mushrooms  Total arsenic concentrations in lichens and mushrooms from Yellowknife are summarized in Table 5.16. Levels o f arsenic in lichens are higher than the levels in Meager Creek samples, which contained a maximum o f 4.79 ppm dry weight o f arsenic (Chapter 4). The highest concentration o f arsenic in Cladonia sp. is observed in the sample from the C o n Mine tailings pond, location 15 (520 ppm); it was likely to have been submerged at times o f slow drainage from the tailings pond. The highest level o f arsenic in all lichens occurs in Lichen 4 from the Giant Mine tailings pond (location 7). This was also the sampling location for the puffball mushroom, Lycoperdon  sp., which contains the highest level o f arsenic among mushrooms. The  lichens that were sampled together, Lichens 1, 2 and 3, contain similar levels o f arsenic, with an average o f 47 ( S D o f 9) ppm dry weight. This may indicate that these different species o f lichens accumulate arsenic to a similar extent.  215  Table 5.16.  Arsenic concentrations in lichens and fungi from Yellowknife.  Sample, time  Location  P C I , June  12  Arsenic concentration (ppm dry weight) 14.3  P C I , residue , June  12  6.4  P C 2 , August  13  29  P C 2 , residue , A u g .  13  15.9 (0.7)  PC3, Aug.  15  520  Lichen 1, A u g .  15  38  Lichen 2, A u g .  15  49  Lichen 3, A u g .  15  55  Lichen 4, A u g .  7  2300  Paxillus involutus, A u g .  13  36  Psathyrella  13  13.6  Leccinum scabrum, A u g .  13  8.3  Coprinus comatus, A u g .  8  410  Lycoperdon pyriforme, A u g .  7  1010  3  b  candolleana, A u g .  c  Sample obtained when the residue remaining after MeOH/water extraction was acid digested. Residues following duplicate extractions, as for PCI (location 12). Standard deviation obtained from total arsenic content in residues of duplicate extractions. a  b  0  Some o f the mushrooms contain higher levels o f arsenic than mushrooms collected from uncontaminated areas. For example, the specimen o f Lycoperdon sp. analyzed in the present study contains 1010 ppm dry weight o f arsenic, whereas the published background values are 0.46 to 2.81 ppm dry weight for the same family o f mushrooms . Lycoperdon 67  sp. from the C o n  Mine tailings pond (location 15) was not analyzed for total arsenic because the sample size was sufficient only for speciation analysis. Total arsenic levels for Psyaihyrella sp. and Leccinum sp. collected from uncontaminated areas were less than 0.2 ppm dry weight '  69  involutus, they were 5.7-5.9 p p m . Arsenic levels in Paxillus involutus,  Psathyrella  68  68  and for Paxillus  216  candolleana and Leccinum scabrum in the present study are apparently elevated compared with the levels published for similar species collected from uncontaminated areas. The levels in the present study, however, are still close to the range o f background concentrations (non-detectable to 15 ppm dry weight o f arsenic) for most mushrooms ' . The Coprinus comatus mushrooms 58 67  from location 8 are appreciably elevated in arsenic concentration with respect to a published literature amount o f < 0.1 ppm for Coprinus micaceus . 61  Its arsenic concentration is also  elevated even when compared with the exceptionally high background levels (up to 130 ppm dry weight) found in Laccaria  sp.  70  5.3.7.2. Arsenic speciation in lichens andfungi The arsenic species extracted from lichens and mushrooms are summarized in Table 5.17. The major arsenic species in lichens are A s (III) and A s (V), with the sum o f these two species making up 62 to 93 % o f the total extracted arsenic for the lichen specimens analyzed. Arsenobetaine has been found for the first time in lichens, and it is present in all the lichens sampled. Chromatograms o f P C I and P C 2 extracts, obtained from cation exchange H P L C - I C P - M S analysis, are shown in Figure 5.4. Arsenobetaine was identified in P C I by spiking the sample extract with standard arsenobetaine and demonstrating co-chromatography o f the suspected component with the authentic material (Figure 5.4a). It was identified in P C 2 (and other lichen samples) by matching the retention time o f the presumed arsenobetaine peak in the sample with that o f the standard (Figure 5.4b).  217  c  5  8  I  vo  CN Tt  18 cu  CJ  00  <  1  h1 CO  © ©  CO  CO  J3  CN  CJ CO  •a  a a CU  vo  rTt  V  >  X  CN  CN  dV  V  ©  o H H  ©  i  © ©  © ©  V  OJ CJ  "3. co H>  3 §  CN  I  •a  © ©  x E ii  M  OS (—,  s °.  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Arsenobetaine ( A B ) in lichens by using cation exchange H P L C - I C P - M S (see Table 5.2 for details). 5.4a. Chromatograms o f Yellowknife lichen ( P C I ) extract and extract spiked with 50 ppb A B standard. 5.4b. Chromatograms o f Yellowknife lichen (PC2) extract and 100 ppb A B and A C standards. See Table 1.1 for abbreviations, U K = unknown compound.  220  Arsenosugar X is present in minor amounts in two lichen samples, P C 2 (5% o f extracted arsenic) and Lichen 3 (4% o f extracted arsenic) from location 13. Arsenosugars were not detected in Lichens 1 and 2, which were growing together with Lichen 3 at location 13 (Table 5.17a). Some similarities exist for all lichen species that were sampled from the C o n Mine M e g K a m Lakes drainage system (locations 12, 13 and 15), summarized in Table 5.18. The proportion o f A s (III) is similar for all samples (42-61% o f extracted arsenic, see Table 5.18), but a broader range is observed for the relative amounts o f A s ( V ) in the lichens (12-44% o f extracted arsenic). Amounts o f arsenobetaine range from 4 to 11%, and similar amounts o f T M A O were observed for the three lichen species (lichens 1, 2 and 3) that were growing together (4-6.5 % o f extracted arsenic). Similarities in arsenic speciation between these three lichens might be expected, due to their common environment. The distribution o f arsenic species found in the lichens collected near C o n Mine (locations 12, 13, and 15) differs from that in the Giant Mine lichen (location 7) as summarized in Table 5.18. The major differences observed are the predominance o f A s ( V ) extracted from Lichen 4, and the small proportion o f arsenobetaine found in Lichen 4 (0.5%) compared with the amounts found in the other lichens (4 to 11%). These differences may be caused by differences in lichen species (and hence metabolism o f arsenic), and/or differences in their environments. F o r example, the major form o f arsenic is probably as arsenate adsorbed onto ferric hydroxide in the Giant Mine tailings and the major form o f arsenic in the tailings pond water is arsenate . Unlike 60  lichen 4, the C o n M i n e lichen samples were less likely to be submerged in tailings pond water, except for one sample (PC3). The arsenic speciation might reflect the extent to which the lichen is directly impacted by the tailings water.  221  Table 5.18. Proportions o f total arsenic extracted, in % o f arsenic species for lichens and mushrooms. Sample (location)  As (III)  As (V)  Methyl + Sugars  AB  P C I (12)  44  44  1.9  11  TMAO + cations 0  P C 2 (13)  50  12  10  11  18  PC3 (15)  61  27  7.4  4.0  0  Lichen 1 (13)  55  26  8.6  5.1  5.9  Lichen 2 ( 1 3 )  60  18  5.1  10.3  6.5  Lichen 3 (13)  42  35  13  4.0  6.0  Average for C o n M i n e lichens (12, 13, 15)  52 (8)  27(11)  7.7(3.9)  7.5 (3.5)  6.1 (6.5)  Lichen 4 (7)  1.9  91  6.5  0.5  0  4.6  29  22  42  7.8  16  1.6  3.2  78  1.8  Lycoperdon  sp. (7)  Lycoperdon sp. (15)  a  Standard deviation of proportions of each compound.  The mushroom Lycoperdon  sp. from the Giant Mine tailings pond (location 7) also  contains a proportionally higher amount o f arsenate, although the major arsenic species in both specimens of Lycoperdon  sp. is arsenobetaine. Arsenobetaine was also observed to be the major  arsenic species extracted from Lycoperdon echinatum (78%), Lycoperdon perlatum (88%) and Lycoperdon pyriforme  (62%) in a previous study; minor components included A s (V), A s (III),  M M A and D M A . A n greater variety o f arsenic species is observed in the Lycoperdon sp. from 6 7  Giant Mine tailings pond (location 7) compared with that in Lycoperdon sp. from the C o n Mine tailings pond (location 15); see Figure 5.4 and Table 5.18. The specimen from location 7 was observed to be in a more mature form, and this may account for the differences in speciation  222  i  ] As(lll)+As(V)  |  MMA+DMA AB | AC+Me As +UK +  4  100  Figure 5.5. Relative amounts o f arsenic species in the puffball mushroom Lycoperdon from location 7 (Giant Mine tailings pond) and location 15 (Con Mine tailings pond). Differences in speciation are seen for Lycoperdon sp. from different locations. Abbreviations for arsenic compounds are in Table 1.1; U K = unknown compound.  sp.  223  observed. Again, the microbial environment influencing the fungus may also cause differences in arsenic speciation. The arsenic speciation has been determined for water-soluble species in the shaggy mane mushroom Coprinus comatus for the first time, and the major arsenic compound is arsenobetaine (88% o f extracted arsenic). M i n o r components include inorganic arsenic, D M A , arsenocholine and an unknown arsenic compound. Paxillus involutus grew in abundance next to the C o n Mine effluent stream. Its arsenic content is unusual because a major proportion o f arsenic extracted (36%) is in an unidentified form (unknown compound Y ) . The peak for unknown Y was broad and it eluted near the retention time o f arsenobetaine on the cation exchange system (see Figure 5.6 for chromatogram.) Neither unknown Y , nor a minor component, unknown X , could be identified as any of the arsenic compounds available to us as standards; some o f the cationic arsenic standards are chromatographed in Figure 5.6. A n unknown peak possessing a retention time similar to that for unknown Y , on almost the identical chromatographic system, was observed in mussels by Larsen et al.  35  The authors established that the compound was neither 2-  dimethylarsinylethanol ( M e A s ( 0 ) C H C H O H ) nor glycerylphosphoryl-arsenocholine 2  2  2  (Me As CH CH 0-P0 "-OCH CHOHCH OH). +  3  2  2  2  2  2  The other major arsenic compound extracted from Paxillus involutus is D M A (53% o f extracted arsenic). A s (III) and (V), M e A s , another unknown species (unknown X ) , and a +  4  small amount o f arsenosugar X I (2.6% o f arsenic extracted) occur as minor components.  224  2e+4 Paxillus sp. extract 25 ppb standard mix  DMA  0  240  480  720  960  1200  time (s)  Figure 5.6. Chromatogram o f Paxillus involutus extract (diluted lOx), showing the presence o f unknown compounds X and Y ( U K - X and U K - Y , respectively), as well as D M A , inorganic arsenic, and a small amount o f Me4As . Standards (including 25 ppb o f M M A , A B , T M A O and A C ) do not co-chromatograph with U K - X and U K - Y . Abbreviations for arsenic compounds are found in Table 1.1. +  225  The major arsenic compound found in Psaihyrella  candolleana is arsenate, making up  63% o f the total arsenic extracted. The next most abundant compound is arsenite, and minor components are M M A , D M A , arsenobetaine and M e A s . This mushroom species is classified in +  4  the same family as Coprinus sp. (Coprinaceae) . Similarities have been observed in arsenic 69  contents o f different mushroom species within the same family , and thus it is not surprising to 67  find some o f the same compounds (e.g., arsenobetaine) in Psathyrella candolleana and Coprinus comatus. B o t h mushrooms are edible but the major water-soluble arsenic species in Psathyrella candolleana, A s ( V ) and A s (III), are toxic to humans, whereas in Coprinus comatus the major species, A B , is not. Leccinum scabrum contains mostly D M A with minor amounts o f inorganic arsenic. In comparison, the three major arsenic compounds extracted from Boletus edulis (Chapter 3) were arsenite, arsenate and D M A . These two mushrooms belong to the same family (Boletaceae) , 69  and again, similarities in arsenic species can be seen. It has been suggested that in more "primitive" genera o f mushrooms, arsenobetaine occurs much less frequently than in genera that are more highly evolved, such as puffballs . On 67  this basis, Paxillus involutus, and Leccinum scabrum may be more primitive mushrooms. The presence o f arsenosugar X I in Paxillus involutus represents one o f the first reports o f arsenosugars in mushrooms. Arsenosugar X I was tentatively identified in an extract of Laccaria amethystina, but further chromatographic confirmation was considered to be necessary . 70  Small amounts (less than 1% o f extracted arsenic) o f arsenocholine were found in Coprinus comatus and Lycoperdon sp. Arsenocholine has been observed only in small amounts in marine samples, even though studies have demonstrated that this compound can be readily biotransformed into arsenobetaine by sediments . However, arsenocholine has been observed 71  226  previously in mushrooms, being one o f the major extracted arsenic species in Amanita muscaria  72  as well as in Sparassis  Other researchers '  67 73  crispa . 61  have suggested that the fungus itself is responsible for synthesizing  arsenicals, such as arsenobetaine, that had not previously been found in the terrestrial environment. The absence o f these compounds in soil (although arsenobetaine was recently found in ant hill material ) provides strong support for their hypothesis. However, experiments 74  summarized in Chapter 3 indicate that synthesis o f arsenobetaine or arsenocholine does not take place in pure culture o f fungi. In the present study, the arsenic compounds found in a mushroom species (Lycoperdon sp.) appear to depend on the location, indicating that the surrounding environment has a strong influence on the extractable arsenic species from a mushroom. A s well, it was shown in Chapter 3 that arsenobetaine in the culture medium was readily taken up by fungus. I f arsenobetaine was being synthesized by soil organisms, or organisms associated with the mycelia, it would be taken up efficiently by the fungus and not be detectable in the soil. Experiments with organisms cultured from the environment o f cultivated or wild mushrooms might help to elucidate the source o f complex arsenicals such as arsenobetaine, arsenocholine and arsenosugars. Extraction efficiencies for lichens and fungi range from 1.1% for Lichen 4 from location 7 to >80% for Paxillus involutus and Leccinum scabrum from location 13. The average extraction efficiency for lichens collected from the C o n Mine area (locations 12, 13 and 15) is 2 1 % with a standard deviation o f 12. The lowest extraction efficiencies are observed for P C 3 from location 15, and Lichen 4 from location 7, which are the lichens containing the highest levels o f total arsenic. The mushrooms Coprinus comatus and Lycoperdon sp. contain the highest levels of total arsenic and their extraction efficiencies are the lowest. Extraction efficiencies for Lycoperdon sp. 227  in a previously published report ranged from 24% to 128% , whereas 68% o f arsenic was extracted from Laccaria  amthystina  70  containing levels o f arsenic comparable to those in  Coprinus sp. and Lycoperdon sp. The present results suggest a negative correlation between extraction efficiency and arsenic concentration in lichens and mushrooms. However, the reasons for this apparent correlation remain unclear. A possibility is that some o f the arsenic contributing to high total levels o f arsenic is in a non-extractable form, such as in a mineralized form on the outside o f the specimen, or bound to chitin or other cell components o f the fungus. The distribution and metabolism o f arsenic may not be uniform for different levels o f arsenic uptake for fungi, which may explain differences in extraction efficiencies. Residues o f two extracted lichens were analyzed for total arsenic (see Table 5.16) and extraction efficiencies from these results were calculated as:  % Extraction efficiency = [(total arsenic)-(arsenic in residue)]/(total arsenic) x 100%.  Based on this calculation, 55% o f total arsenic was extracted from P C I , which differs from the extraction efficiency o f 13% in Table 5.17 (calculated by summing the arsenic species detected). A n extraction efficiency o f 45% was calculated by using the arsenic concentration in the P C 2 residue, which agrees quite well with the 42% reported in Table 5.17. The difference in extraction efficiencies for P C I indicates that some arsenic that was extracted was not observed by using the chromatographic systems available. This was suggested previously for other samples as well.  228  5.4. S u m m a r y A large amount o f information about arsenic in the terrestrial environment was obtained from the study o f Yellowknife biota. A n important observation was, as for the Meager Creek study, the low proportions o f water soluble arsenic species (evidenced by l o w extraction efficiencies) in the majority o f the biota studied. Arsenic that is not extracted is probably bound to lipids, cell components, proteins, or exists as minerals. Although the extractable portion can be approximated to represent the arsenic species that are bioavailable, the availability o f the nonextractable arsenic cannot be assumed to be negligible, since the metabolism o f such arsenic by higher trophic level organisms has not been reported. Freshwater fish were found to contain arsenobetaine, in contrast to previous reports. Sucker was found to contain arsenosugar X I , and probably obtains the arsenosugar from its food source, implying that benthic organisms are capable o f synthesizing these arsenic compounds. The major extractable arsenic compound in pike was found to be D M A , but the source is unknown. Amounts o f arsenobetaine in freshwater mussels were negligible or non-existent, supporting the idea that the uptake o f arsenobetaine in the marine environment is related to osmo-regulatory processes. Mostly inorganic arsenic species were extracted from plants, mosses, algae and lichens. Plant species appear to be consistent in their uptake and metabolism o f arsenic, which may indicate some transport mechanisms specific to the plant. Arsenosugars were found for the first time in higher plants. Freshwater green algae, as well as microbial mats, contain arsenosugars, probably indicating synthesis by the organisms. Arsenobetaine was found for the first time in lichens. Differences in arsenic speciation are seen for the mushroom Lycoperdon sp. from different locations, which may indicate that  229  complex arsenicals, such as arsenobetaine, are formed as a part o f the fungus microbial community and taken up by the fungus, rather than synthesized by the fungus itself.  References  1. 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Chem. 1997, 11, 859-867.  235  Chapter 6  ANTIMONY IN ENVIRONMENTAL SAMPLES  6.1. Introduction  Antimony can enter the environment as a result o f rock weathering, soil runoff and through effluents from mining and smelting. Its compounds are used as flame retardants in plastics and textiles, as additives in metal alloys, as doping agents in semiconductors, and as antiparasitic drugs . Because o f the toxicity o f some o f its compounds, antimony is listed by the 1  United States Environmental Protection Agency ( U S - E P A ) as a priority pollutant . The 2  determination o f antimony species in the environment is necessary in order to assess the toxicity and mobility o f antimony. Usually concentrations o f naturally occurring antimony are about 5-10% those o f arsenic  3  and the two elements are often found together in mineral deposits . Because elevated levels o f 4  arsenic are found in the Meager Creek and Yellowknife environments (Chapters 4 and 5), and because elevated antimony levels were found previously in biota from the Yellowknife environment , we were interested in increasing the range o f samples previously analyzed for 5  antimony content. Although methods for antimony speciation by using H P L C with element specific detection have been developed ' ' , determination o f methylantimony species other than 6 7 8  Me3SbCl2 by using these methods have not been successful . Therefore, we chose to use the 9  method o f hydride generation-gas chromatography ( H G - G C ) with A A S and M S detection for the analysis o f antimony in environmental samples, in spite o f the limitations and problems associated with this speciation method (see Chapter 2 and references at the end o f this chapter ' ). 510  11  236  6.2. Experimental  6.2.1. Chemicals and reagents Antimony ( V ) and (III) standards were obtained as potassium hexahydroxyantimonate, K S b ( O H ) (Aldrich), and potassium antimonyl tartrate, K Sb2(C 06H2)2 (Aldrich). M e S b C l 2  6  4  3  2  was synthesized as described elsewhere . Stock solutions were made by dissolving these 12  compounds in deionized water and diluting the resulting solutions to 1000 or 100 mg L " as Sb. 1  Standard working solutions were made by diluting the stock solution with deionized water as necessary. For hydride generation analysis, NaBFL, (reagent grade, Aldrich) was dissolved in deionized water fresh daily to provide a concentration o f 2% w/v. Ammonium citrate buffer at a concentration o f 0.05 M and p H 6 (1 M ammonium hydroxide, MicroSelect, Fluka, and analytical reagent grade citric acid, B D H ) and 1 M HC1 (Environmental grade, Alfa Aesar) were used for p H adjustment during derivatization.  6.2.2. Sampling and sample preparation Sampling was carried out at Meager Creek ( M C ) and in Yellowknife ( Y K ) , as described in Chapters 4 and 5, with sample locations shown in Figure 6. l a ( M C ) and Figure 6. l b ( Y K ) . Water was sampled by hand into polypropylene bottles that had been acid washed previously. Biota were sampled by hand, stored in Ziploc® bags and kept cool until processing. They were washed thoroughly with tap water to remove soil and other particles, rinsed with deionized (1 Mohm) water, and frozen. The samples were freeze-dried and pulverized to a fine powder for analysis. Snails from Yellowknife were dissected to remove the soft tissue prior to freezing. 237  Samples were digested with acid for the determination o f total antimony content. The freeze-dried powders were accurately weighed (0.5 g ± 0.5 mg) into a 500 m L round bottomed flask ( R B F ) . Concentrated nitric acid (3 m L , doubly distilled in quartz, Seastar, Sidney, B C ) and hydrogen peroxide (3 m L , 30% in water, reagent grade, Fisher) were added to each sample. The samples in the R B F s were boiled for 3 hours by using a heating mantle and a reflux apparatus . 13  After all the samples had cooled, the clear solutions remaining were diluted to 25 m L with deionized water and stored at 4°C until analysis. Extractions were carried out by weighing 0.5 g (± 0.5 mg) o f the dried powders into 50 m L or 15 m L centrifuge tubes, adding 10-15 m L MeOH/water (1:1), sonicating for 20 minutes, centrifuging for 20 minutes, and decanting the liquid layer into a R B F . Each sample was sonicated and centrifuged a total o f 5 times. The decanted extracts for each sample were pooled and rotovapped to near dryness (1-2 m L ) and then diluted to 5 or 10 m L with deionized water. Moss (Drepanocladus  sp.) from Yellowknife locations 1 and 4, and snails (Stagnicola sp.) from  Yellowknife locations 1+3, were extracted in a larger quantity to permit analysis by using H G G C - A A S and H G - G C - M S . Masses o f 1 or 2 g were weighed out and extracts were made up to a final volume o f 20 m L or 40 m L , respectively.  238  Figure 6.1a. Map (not to scale) of Meager Creek Hot Springs area showing sampling locations. 239  6.2.3. ICP-MS analysis for total arsenic and antimony concentrations A c i d digested samples and water samples were analyzed by I C P - M S for total antimony content. A V G Plasmaquad P Q 2 Turbo I C P - M S ( V G Elemental) outfitted with a peristaltic pump and injection loop for flow injection introduction was used. Parameters for the I C P - M S are given in Table 6.1. The m/z monitored were 121 (Sb), 123 (Sb) and 103 (Rh). A c i d digested samples were diluted with 1% nitric acid (doubly distilled in quartz, Seastar) and R h (10 ppb) was added as an internal standard to diluted samples and water samples. Quantification was carried out by using an external calibration curve derived from Sb standards made in 1% nitric acid and containing 10 ppb R h .  Table 6.1. Operation parameters for I C P - M S Feature  Parameter  Forward radio-frequency power  T350"w"  Reflected power  <10 W  Cooling gas (Ar) flow rate  13.8 L/min  Intermediate (auxiliary) gas (Ar) flow rate  0.65 L/min  Nebulizer gas (Ar) flow rate  1.002 L/min  Nebulizer type  de Galan  Quadrupole pressure  9 x 10" mbar  Expansion pressure  2.5 mbar  7  6.2.4. HG-GC-AAS analysis for antimony speciation The apparatus was composed o f a semi-continuous flow, hydride generation system developed for arsenic analysis, coupled to an atomic absorption spectrometer (Varian AA1275) 14  fitted with an Sb lamp (Varian) operating at a wavelength o f 217.6 nm, or at 231.4 nm for a few  241  samples (section 6.3.2). One modification was made to the basic apparatus in the form o f using a gas-liquid separator  15  that resulted in less analyte carryover. The apparatus consisted o f Tygon  tubing for the peristaltic pump, and P T F E tubing (1/8" O D ) for the remainder. Data were collected from the A A S and processed directly by using an FfP 3390A integrator, or were analyzed with the aid o f Shimadzu E Z C h r o m software run on a P C . A peristaltic pump was used to deliver standard or sample solution (ranging from 5 u L to 200 p L for standards, and from 1 m L to 100 m L for samples) to mix with the acid or buffer and then to mix with a solution o f NaBFL, (2% w/v) in a reaction coil. The gases evolved were separated in the gas-liquid separator and then swept by a flow o f helium into a P T F E U-tube, where they were trapped at -196 °C. Continuous hydride generation and trapping were carried out for 3 minutes. The peristaltic pump was then stopped (making the system semi-continuous) and the U-tube was heated to 70 °C, allowing the gases to be swept with H e at a flow rate o f 40 mL/min onto a Poropak P S column, which was then heated from 70 °C to 150 °C at a rate o f 30 °C/min, whereby the gases were separated. They were then detected by A A S . Semi-quantitative amounts were calculated by using external calibration curves.  6.2.5. HG-GC-MS analysis for confirmation of methylantimony species Extracts o f samples and standard solutions o f MesSbCb. were reacted with NaBFL, to form methylantimony hydrides. The reactions were performed in a 15 m L vial (sealed with a PTFE-faced silicone or neoprene septum, 16mm, Supelco) by using the appropriate volume o f sample (5 m L for moss extracts and 10 m L for snail extract) or MesSbCb. standard solution, and 1 m L deionized water (for standards) and then injecting 0.5 m L o f 6% NaBEL, solution through the septum. A l l reactions were carried out without the addition o f acid or buffer, with the exception o f one reaction in which M e S b C l standard solution was first made acidic by adding 3  2  242  an equal volume o f 1 M HC1, in order to generate M e S b H and M e S b H in addition to M e S b . 2  2  3  These methods were qualitative only. For the analysis, a G C - M S system consisting o f a Star 3400Cx gas chromatograph (Varian), equipped with a 1078 temperature programmable injector (Varian) and interfaced to a Saturn 4 D ion-trap mass spectrometer (Varian) was employed. A gas tight syringe (1.0 m L , Gastight #1001, Hamilton) was rinsed with 5 m L o f lab air and then used to inject 1 m L o f headspace generated from the samples onto a capillary column ( P T E ™ - 5 , 30m x 0.32mm, 0.25 um, Supelco 2-4143, poly (5% diphenyl / 95% dimethylsiloxane)). The injector was kept at 100 °C. The temperature program started at 40 °C, and stopped at 150 °C with a heating ramp o f 15 °C/min. The parameters used are shown in Table 6.2.  Table 6.2. G C - M S parameters. G C method Injector temperature  100 °C  Column temperature program  40 °C, 1 5 ° C / m i n t o 150 °C  Transfer line temperature  200 °C  Column  P T E ™ - 5 , 30 m x 0.32 mm, 0.25  M S method Mass range  115-180 m/z  Scan time  0.4 s  Segment length  4.5 min  Ion M o d e  Electron Impact  Multiplier  2150 V  Target  15 400  Ionization current  20 u A  Manifold temperature  260 °C  243  6.3. Results and Discussion  6.3.1. Antimony species and total antimony in environmental samples The semi-quantitative amounts o f antimony species in environmental samples detected by using the method o f H G - G C - A A S , as well as total antimony, determined by using I C P - M S , are shown in Table 6.3. The antimony contents in biota and water from Yellowknife and Meager Creek are summarized in Table 6.3a (biota) and Table 6.3b (water). Absolute detection limits o f 1 ng for Sb (III) and methyl antimony species were estimated and relative standard deviations between replicate analyses can be estimated to be 20%. The biota samples were chosen for analysis by H G - G C - A A S based on their ability to meet one o f two criteria: (a) the presence o f antimony in sample extracts was observed during H P L C - I C P - M S analysis or (b) total antimony content greater than 10 ppm dry weight was measured.  6.3.1.1. Inorganic antimony species In all biota sample extracts and water samples, inorganic Sb ( V ) is the major antimony species (Tables 6.3a and 6.3b). Very few biota samples have been speciated for antimony previously ' , but other researchers have also shown that Sb ( V ) is the major antimony 5 16  compound in water samples ' ' ' ' . Thermodynamically, Sb ( V ) is predicted to be the most 3  8  17  18  19  stable oxidation state under most environmental, oxygenated conditions ( p H 5 to 8 )  2 0  and  therefore it is not surprising to find that Sb ( V ) is the most abundant extractable species o f antimony in these samples.  244  o  S  H  O  O  •a  a  is  i—  r-H CN  ©  °°. Tf'  w w  .  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(moss) samples from Yellowknife  ( Y K ) location 1 and Yellowknife ( Y K ) location 4, as shown in Table 6.3a. Sb (III) is also present in all Meager Creek ( M C ) waters and water from Y K location 4 and location 11 (Table 6.3b). Other studies have shown that Sb (III) is produced in the photic zone o f an estuarine inlet, which may indicate that biological activity is responsible for the presence o f Sb (III) . In the 21  same study, reducing conditions, including the presence o f H S , did not result in significant 2  reduction o f Sb ( V ) to Sb (III) in water, although the formation o f Sb(III)-S compounds was postulated . In the present study, water from Meager Creek was sampled from locations near 21  microbial mats, which have been shown to exist under reducing conditions (Chapter 4) and to produce M e s S b . Reducing conditions and microbial metabolism may lead to the presence o f Sb 22  (III) in these waters.  6.3.1.2. Methylated  antimony species  Methylated antimony species were detected in a few samples by using H G - G C - A A S . Biota samples containing methylantimony species include Drepanocladus  sp. from Y K locations  1 and 4 and snails (Stagnicola sp.) from Y K locations 1+3 (Table 6.3a). A chromatogram showing stibine, dimethylstibine and trimethylstibine that were generated from a standing water sample ( Y K location 4) is shown in Figure 6.2.  247  C.14  0.14  0.12  Me SbH 2  1.9 min  o.iq  V o I t  s o.oa  0.06\  0.04  0.02  o.oq  -0.02  0  0.5  1.0  1.5  2.0  2.5  3.0  3.5  4.0  time (minutes)  Figure 6.2. Chromatogram obtained by using HG-GC-AAS (217.6 nm) showing stibines generated at neutral pH from 100 mL of a sample of standing water from location 4 in Yellowknife.  248  The moss samples from Yellowknife that contained methylantimony compounds were all identified as Drepanocladus sp. Interestingly, no detectable antimony species could be extracted from another sample o f Drepanocladus sp., at Y K location 15 (see Figure 6. lb). The inability to extract antimony in Drepanocladus sp. from Y K location 15 is probably because o f lower levels of total antimony in this sample. The discovery o f methylantimony compounds in snails represents, to our knowledge, the first finding o f methylated antimony in an animal. Methylantimony compounds in biota have been observed rarely in past studies. Dodd et al. identified predominantly a trimethylantimony compound in one sample and a dimethylantimony compound in another sample o f the same species o f macrophyte from Yellowknife . The results from the present study differ from that by D o d d et al. because the 5  5  dimethylantimony compound is possibly the same in all three moss samples. Analysis o f a soil extract by using H P L C - I C P - M S indicated the presence o f a MesSb(V) species . 7  A dimethylantimony compound is present in a water sample from Y K location 4 and a trimethylantimony compound is present in a water sample from M C location S2 (Table 6.3b). Monomethylantimony and dimethylantimony compounds, assumed to be methylstibonic acid and dimethylstibinic acid, have been observed in river, estuary and sea water samples by other researchers and their presence was attributed to biological activity ' . 21  23  The dimethylantimony compound present in the Yellowknife water sample ( Y K location 4) may be similar to the dimethyl compound in the moss sampled from the same location ( Y K location 4), and its presence in both samples may indicate uptake and/or excretion. From these results it is impossible to differentiate between the possibility o f the moss forming a dimethylantimony species as a metabolite and excreting it into the water, and the possibility o f microorganisms in the water or sediment forming the compound and its being taken up by the moss. B o t h scenarios are possible.  249  The presence o f trimethylantimony species in Meager Creek water may be a result o f the production of M e S b by the microbial mats nearby. Recent studies have indicated that M e S b 3  3  would be oxidized rapidly to M e S b 0 ' , but other studies suggest that i f the Challenger 2 4  2 5  3  mechanism is followed for the methylation o f antimony (oxidative addition o f methyl groups, followed by reduction to the stibine, see Chapter 1, Figure 1.1) the final reduction step to M e S b 3  takes place only to a very small extent . Therefore the trimethylantimony compound in the 9  Meager Creek water sample may be present as a result o f oxidation following M e S b production 3  in the mats, or o f biological antimony methylation only as far as M e S b O . 3  6.3.1.3. Extraction efficiencies for biota and percent Sb species of total Sb in waters Extraction efficiencies o f antimony from biota range from 0.7 to 95%. However, extraction efficiencies for all samples except one are below 37% (Table 6.3a). Clearly the extraction method using MeOH/water (1:1) inadequately extracts antimony from these samples. The reasons for this are likely similar to those given for low extraction efficiencies o f arsenic from biota: antimony may be strongly bound to cellular components such as lipids, cellulose, lignin or carbohydrates. For example, metals are known to bind strongly to fungal cell walls , 26  and antimony may thus be strongly bound to cellular components. Accordingly, extraction efficiencies from fungus samples (lichen and mushrooms) ranged from 0.8 to 15% (Table 6.3a). Typhus sp. (cattail) from Y K location 12 was extracted nearly quantitatively (Table 6.3a). Some o f the antimony extracted from these samples may not have been detected by using H G - G C - A A S , which may account for the differences in amounts detected in waters by H G - G C A A S compared with the levels o f total antimony in waters (Table 6.3b, last column). Arsenic species that are "hidden" to H G - G C - A A S detection, without strong digestion techniques (e.g., U V photolysis or microwave digestion), have been found in sediment pore water samples from  250  Yellowknife . This arsenic was suggested to be bound to colloidal organic matter, or in the form o f organoarsenic compounds (such as arsenocholine or arsenosugars) . In the same way, 27  antimony that is complexed strongly to organic groups may not form hydrides under the conditions used, or Sb-S compounds may exist as well. Sb(III)-S compounds have been proposed to be present in estuarine and interstitial waters, detected by acidifying and degassing samples before H G - G C - A A S analysis at p H 6 . This method was not carried out in the present 21  study and hence the possibility has not been ruled out that such compounds are present.  6.3.1.4. Total concentrations of antimony compared with arsenic The total concentrations o f arsenic (from Chapters 4 and 5) and antimony (from Table 6.3) in some Yellowknife and Meager Creek biota and water samples are summarized in Table 6.4. A n average [Sb]/[As] ratio o f 6.4% can be calculated for the biota samples. F o r water samples other than those taken from Baker Creek (locations 1 and 3) and the effluent from C o n Mine (location 13), the average ratio is 4.0%.  251  Table 6.4. Comparison o f total concentrations o f antimony and arsenic (ppm unless otherwise stated) for selected samples Sample  [Sb]  [As]  [Sb]/[As] x 100%  Y K Biota samples (location #) Moss 1, June (5)  190  1130  17  Drepanocladus sp., June (1)  12  1220  9.8  Drepanocladus sp., August (1)  60  490  12  Drepanocladus sp., August (4)  28  880  3.2  Stagnicola sp., August (1+3)  6  82  7.3  Typha sp. (12)  0.20  3.8  5.3  Bidens cernua (10)  0.7  100  0.7  Lemna minor (10)  0.39  28  1.4  Myriophyllum sp., August (10)  0.28  17.4  1.6  Potomogetan richardsonii (6)  0.8  20  4.0  Sparganium augustifolium (6)  0.26  2.5  10  Lichen 4 (7)  120  2300  5.2  Cladonia sp. (13)  1.4  29  4.8  Lycoperdon sp. (7)  60  1010  5.9  Coprinus sp. (8)  34  410  8.3  0.5  8.7  5.7  Location 10  3.7  68  5.4  Location 11  1.8  53  3.4  Location 4  50  740  6.8  Location 1  260  120  220  Location 3  380  220  170  Con effluent, location 14  31  51  61  Location 2,  5  277  1.8  Location S2  5  237  2.1  Location 4  12  288  4.2  M C Biota samples (location #) Mimulus sp. (1+2) Y K Water Samples in ppb  M C Water Samples in ppb  252  Ratios o f [Sb]/[As] in soil and rocks average about 1 0 %  M  and therefore it may appear  that uptake o f antimony by biota is slightly less than that o f arsenic, a phenomenon that has been observed before . A slight deviation o f the average ratio obtained (6.4%) from the typical crustal 4  ratio (10%) might also be observed i f soil ratios were lower than 10% in the Yellowknife environment. The ratio in waters is slightly lower (4%) than the ratio in biota. Although these slight differences are noted, the results are still within an order o f magnitude o f the typical relative levels o f antimony and arsenic in the natural environment. It is interesting to note that the concentrations o f antimony in waters from Baker Creek ( Y K locations 1,2 and 3) and in C o n Mine effluent ( Y K location 14) are similar to those o f arsenic. The probable reason for this is that arsenic is effectively removed by alkaline precipitation with ferric sulfate from the effluents, before the effluents are discharged into the environment , whereas antimony is not. Co-precipitation o f arsenic with ferric hydroxides is 28  postulated to take place during effluent treatment, and it has been observed that antimony is removed 10 times less efficiently than arsenic from solution by co-precipitation with ferric and manganese hydroxides . 17  6.3.2. The confirmation of antimony in samples containing methylantimony compounds by using HG-GC-AAS The presence o f methylantimony species in samples was confirmed by two methods: (a) by using H G - G C - A A S at a different wavelength to corroborate that peaks are due to antimony compounds and not the result o f spectral interferences, and (b) by using H G - G C - M S to confirm the structure and presence o f hydrides derived from samples (next section, 6.3.3). The three moss samples, from Y K locations 1 and 4, as well as the water sample from Y K location 4, were analyzed by using H G - G C - A A S with the A A S operating at a wavelength o f 231.4 nm, which is a  253  secondary absorption line specific to antimony. Peaks appeared at the same retention times as those found at a wavelength o f 217.6 nm, and in similar abundances, as summarized in Table 6.5. The snail extract was not analyzed because o f limited sample size.  Table 6.5. Relative amounts (% o f sum of methyl species, estimated by normalizing area counts) for methyantimony peaks in moss and water samples.  "Sample ( Y K  location^)"™"™"™ Sb (III) (RT=0.7)  M e S b - (RT=1.9) 2  Me Sb-(RT=2.3) 3  A A S at 231.4 nm Drepanocladus  sp. June ( 1 ) 1 2  88  0  Drepanocladus  sp., A u g . (1)  10  90  0  Drepanocladus  sp., A u g . (4)  5  95  0  17.5  59  23.5  Water, A u g . (4)  A A S at 217.6 nm Drepanocladus  sp., June ( 1 ) 1 7  83  0  Drepanocladus  sp., A u g . (1)  23  77  0  Drepanocladus  sp., A u g . (4)  6  94  0  17.5  59  23.5  Water, A u g . (4)  6.3.3.  The use of headspace HG-GC-MS for the speciation of antimony compounds MesSb was generated in sealed vials from standard solutions o f MesSbCb. by using  hydride generation methodology. A sample o f the headspace was injected into the G C - M S . A detection limit o f 0.08 ng Sb for M e S b was obtained, corresponding to 1 ng Sb in solution 3  before derivatization. However, the analysis o f headspace following hydride generation suffers from imprecision, since R S D values no better than 20% were observed for 5 replicate analyses.  254  4e+4  6.3a  3e+4  Me Sb 3  3e+4 2e+4 •g  2e+4 H  8 1e+4 1e+4  H  5e+3 Oe+0 H 15  30  1  I  45  60  75  90  time (s) 5e+3  151  6.3b 4e+3  Me Sb  +  2  153 3e+3 o o  Sb  +  121  2e+3  136 M e S b  +  ^  123  Oe+0  +  3  167  138  1e+3 -A  [Me Sb-H]  l l l l l i M i i i i l l l l l l i i , , !  110  120  130  140  150  160  170  180  190  m/z  Figure 6.3a. Total ion chromatogram resulting from headspace-HG-GC-MS analysis showing M e S b generated from 30 ng Me3SbCl (neutral). Figure 6.3b. Mass spectrum at 54.60 s corresponding to M e S b . 3  2  3  255  In Figure 6.3, a chromatogram and mass spectrum are shown, corresponding to standard trimethylstibine (30 ng Sb as M e S b ) generated by using 30 p L o f 1000 ppb M e S b C l solution 3  3  2  with 1 m L o f deionized water and 0.5 m L o f 6% (w/v) N a B F L solution. The dominant characteristic o f all mass spectra involving antimony compounds is the appearance o f the isotopic pattern due to masses o f 121 and 123 (naturally occurring at about 52:48) in all Sb-containing fragments. This isotopic pattern is observed in Figure 6.3 at m/z 165/167, corresponding to [Me Sb-H]7[Me m  3  1 2 3 3  S b - H ] ; at m/z 151/153 corresponding to M e +  136/138, corresponding to M e 1 2 1  Sb7  MS  2 9  1 2 3  1 2 1  Sb /Me +  1 2 3  1 2 1 2  Sb /Me +  1 2 3 2  S b ; at m/z +  S b ; and at m/z 121/123 corresponding to +  S b . This mass spectrum is similar to one obtained previously by using the same G C +  and also to spectra obtained by using a quadrupole mass spectrometer . The loss o f a 5  methyl group from methylstibines is the prevalent fragmentation pattern and is considered to be typical for methylated organometallic compounds . 30  A s discussed in Chapter 2, enhanced demethylation o f trimethylstibine is observed when the hydride generation reaction is performed at low p H . Therefore, acidic H G conditions resulting in demethylation can be used to generate mass spectra for methylstibine and dimethylstibine. In Figure 6.4, the chromatogram obtained from hydride generation of M e S b C l 3  2  (100 ng Sb) at low p H (Figure 6.4a) and the mass spectra obtained for the peaks assumed to be methylstibine (Figure 6.4b) and dimethylstibine (Figure 6.4c) are shown. The fragmentation pattern for dimethylstibine is similar to that observed for trimethylstibine, because the most abundant m/z appears as a result o f methyl loss ( M e S b from M e S b H ) . The mass spectrum for +  2  the peak at a retention time o f 39.27s most likely corresponds to the one expected for M e S b H , 2  even though the high background levels obscures a clear, characteristic fragmentation pattern. However, fragments at m/z 121/123 and 136/138 were observed, corresponding to Sb and +  M e S b , respectively. +  256  5e+4  6.4a  4e+4 3e+4  MeSbhL  v>  § o o  2e+4  Me SbH 9  AAJ  1e+4  T  T  30  15  45 time (s)  60  90  75  7e+2 - i 6e+2 5e+2 w 4e+2 t— O 3e+2 o 2e+2 1e+2 Oe+0 110  120  130  140  150  160  170  180  190  190  Figure 6.4a. Total ion chromatogram resulting from headspace-HG-GC-MS analysis showing stibines generated from 100 ng M e S b C l (1 M HC1). Figure 6.4b. Mass spectrum at 39.27 s corresponding to M e S b H . Figure 6.4c. Mass spectrum at 49.61 s corresponding to M e S b H . 3  2  2  2  257  The use of H G - G C - M S was useful in the past for the identification and confirmation o f methylated antimony species in plant samples from Yellowknife . The direct injection o f 5  fractions o f gas samples into an ion trap G C - M S resulted in the conclusive identification o f trimethylstibine, trimethylbismuthine and methyltin compounds in landfill and fermentation gases . The headspace H G - G C - M S method developed in this work was anticipated to provide 29  information about the presence o f methylstibines following H G o f Yellowknife samples. Chromatograms and mass spectra for peaks corresponding in retention time to dimethylstibine for moss samples from Yellowknife are shown in Figures 6.5, 6.6 and 6.7. The mass spectra indicate that the compound found in the headspace following hydride generation o f the sample extracts is indeed dimethylstibine, by comparison with the mass spectrum shown in Figure 6.4c. The chromatogram and mass spectra for the peaks corresponding in retention time to dimethylstibine and trimethylstibine for the snail extract are shown in Figure 6.8. Again, comparison of the mass spectra with those for standards (Figures 6.3b and 6.4c) indicates the presence o f dimethyl- and trimethylstibine following H G of the extract. Differences in m/z abundances are probably due to interfering ions causing slightly different fragmentation patterns. For example, in Figure 6.8c (the mass spectrum for the peak corresponding to trimethylstibine) m/z 166 and 168 are observed (corresponding to Me3Sb ) rather than 165 and 167 +  (corresponding to [Me3Sb-H] ), which were the m/z observed for the standard compound +  (Figure 6.3b). F o r this sample, background subtraction was necessary to isolate the major m/z o f interest, because o f low levels o f antimony in the extract and large amounts o f other matrix components.  258  2.5e+4 -j 2.0e+4 -  6.5a  1.5e+4 -  ]|  Me SbH 2  I |  \  1 .Oe+4 -  o  A \  l\  5.0e+3 O.Oe+0 i 15  0  i 30  i 45  i 60  i 75  90  time (s)  1.6e+3 1.4e+3 -  6.5b  MeSb  1.2e+3 1 .Oe+3 § o o  138  Sb  CO  8.0e+2 H  +  +  121  136  121  6.0e+2 -  Me Sb  4.0e+2 -  ^153  2.0e+2 0.0e+0  110  +  2  lilili 120  130  140  1111,1111 I I I I | M . . I H I I  150  160  170  180  190  m/z  Figure 6.5a. Total ion chromatogram resulting from headspace-HG-GC-MS analysis of 5 mL of moss extract (June, YK Location 1) showing Me SbH. Figure 6.5b. Mass spectrum at 46.90 s corresponding to Me SbH. 2  2  259  1.60+4 - i 1.4e+4 1.2e+4 LOe+4 if) r~  8.0e+3 -  O O  6.0e+3 -  u_  4.0e+3 2.0e+3 0.0e+0 45 time (s)  1.4e+3 1.2e+3 -  136  6.6b  MeSb  +  1 .Oe+3 -  Sb « c  8.0e+2 -  °  6.0e+2 -  +  i l l  .3  122 is! Me Sb  4.0e+2 -  +  2  2.0e+2  IM11111•111111111.... 11111 0.0e+0  110  120  130  140  150  160  170  180  190  m/z  Figure 6.6a. Total ion chromatogram resulting from headspace-HG-GC-MS analysis of 5 mL of moss extract (August, YK Location 1) showing Me SbH. Figure 6.6b. Mass spectrum at 46.96 s corresponding to Me SbH. 2  2  260  2e+4  1e+4 H  to c o o  1e+4  H  5e+3  Oe+0 H  F i g u r e 6.7a. Total ion chromatogram resulting from headspace-HG-GC-MS analysis of 5 m L of moss extract (August, Y K Location 4) showing Me2SbH. F i g u r e 6.7b. Mass spectrum at 46.91 s corresponding to M e S b H . 2  261  Me.SbH  6.8a  I Me Sb 3  1e+3 H  IVj Oe+0 H  1  15  30  60  45 time (s)  75  9to  136^  3e+2 H |  6.8b  MeSb Sb  2e+2 H  +  +  o o  151  ^138  1e+2  Me Sb  +  2  153  l-lll ,'• I ••'  iiu  Oe+0 110  120  130  140  150  160  ii.lnlll  170  180  190  180  190  3e+2 150.  6.8c 2e+2 H  Sb  [Me Sb-H]  +  121  MeSb  +  152  136  1e+2 H  ^138  Me Sb  n il  110  120  I.I I.  i  i  166  130  140  I  1  150  LUi  +  3  \  Oe+0  +  2  168  1  r  160  170  m/z  Figure 6.8a. Chromatogram (sum of Sb containing ions) resulting from headspace-HG-GC-MS analysis of 10 mL of snail extract (YK Location 1+3) showing Me SbH and Me Sb. Figure 6.8b. Background corrected mass spectrum at 46.96 s corresponding to Me2SbH. Figure 6.8c. Background corrected mass spectrum at 54.05 s corresponding to Me Sb. 2  3  3  262  6.4. S u m m a r y Antimony was extracted from environmental biota samples from Yellowknife and Meager Creek, with extraction efficiencies ranging from 0.7 to 37% for all samples except for Typha sp., from which 95% o f antimony was extracted. Total antimony levels in most samples, when compared with arsenic levels, reflected the relative abundance o f naturally occurring antimony (about 5-10% o f arsenic). The exceptions were water samples from Baker Creek, receiving mine effluent from Giant M i n e in Yellowknife, which contained antimony at concentrations similar to those for arsenic. This probably indicates that treatment o f effluent to remove arsenic is successful, but that antimony is inefficiently removed during the treatment process. Speciation analysis was carried out by using H G - G C - A A S . The major antimony species in all samples, including biota extracts and water, was Sb (V). Sb (III) and methylated antimony species were detected in some samples as well. The presence o f methylated antimony species in moss from Yellowknife and a water sample from Yellowknife was confirmed by using H G - G C A A S at a second absorption wavelength, increasing the likelihood that the peaks obtained are due to the presence o f antimony compounds. A headspace H G - G C - M S method was developed for the speciation o f antimony compounds and this was used to confirm methylantimony species in the headspace following H G o f extracts o f moss and snail samples from Yellowknife. Because of the abundance of Drepanocladus sp. at location 1 in Yellowknife, and the seasonal consistency in its methylantimony content, this species o f moss can be used as a laboratory standard for dimethylantimony. Future work could involve the use o f moss extracts to study H P L C behaviour of the dimethylantimony compound present in the moss by using I C P - M S detection or M S detection for structural information.  References  1. Maeda, S. In The chemistry of organic arsenic, antimony and bismuth compounds, Patai, S. E d . ; John Wiley & Sons: Chichester, 1994; pp 737-742. 2. Keith, L . H . ; Telliard, W . A . Environ. Sci. Technol. 1919,13, 416. 3. Cutter, G . A . ; Cutter, L . S. Mar. Chem. 1995,  49, 295-306.  4. Stewart, K . C ; M c K o w n , D . M . J. Geochem. Explor. 1995, 54, 19-26. 5. Dodd, M . ; Pergantis, S. A . ; Cullen W . R.; L i , H . ; Eigendorf, G . K . ; Reimer, K . J. Analyst 1996,  121, 223-228.  6. Lintschinger, J.; K o c h , I.; Serves, S.; Feldmann, J.; Cullen, W . R. Fresenius J. Anal. 1997, 359, 484-491. 7. Ulrich, N . Anal. Chim. Acta 1998,  Chem.  359, 245-253.  8. Smichowski, P.; Madrid, Y . ; D e L a Calle Guntinas, M . B . ; Camara, C . J. Anal. At. Spectrom. 1995, 10, 815-821. 9. Andrewes, P.; Cullen, W . R.; Feldmann, J.; K o c h , I.; Polishchuk, E . Appl. Organomet. Chem. 1998, in press. 10. K o c h , I.; Feldmann, J.; Lintschinger, J.; Serves, S. V . ; Cullen, W . R.; Reimer, K . J. Appl. Organomet. Chem. 1998, 12, 129-136. 11. Dodd, M . ; Grundy, S. L . ; Reimer, K . J.; Cullen, W . R. Appl. Organomet. Chem. 1992, 207. 12. Morgan, G . T.; Davies, G . R. Proc. Royal Soc, Ser. A 1926,  6,  523.  13. Bajo, S.; Suter, U . ; Aeschliman, B . Analytica Chimica Acta 1983,  149, 321-355.  14. Cullen, W . R.; L i , H . ; Hewitt, G . ; Reimer, K . J.; Zalunardo, N . Appl. Organomet.  Chem.,  1994, 8, 303 15. L e , X . C ; Cullen, W . R.; Reimer, K . J Appl. Organomet. Chem. 1992, 16. Kantin R . Limnol. Oceanogr. 1983,  6, 161.  28, 165-168.  17. M o k , W . - M . ; W a i , C . M . Environ. Sci. Technol. 1990, 24, 102-108.  264  18. Mohommad, B . ; Ure, A . M . ; Reglinski, J.; Littlejohn, D . Chem. Speciation Bioavail. 3, 117-122. 19. Yamamoto, M . ; Urata, K . ; Murashige, K . ; Yamamoto, Y . Spectrochim. Acta 1981, 671-677.  1990,  36B,  20. Pourbaix, M . Atlas of Electrochemical Equilibria in Aqueous Solutions; National Association o f Corrosion Engineers: Houston, Texas, 1974; p 524-532. 21. Bertine, K . K . ; Lee, D . S. In Trace Metals in Seawater, NATO Conference Series, Series IV: Marine Sciences; Wong, C. S.; Boyle, E . ; Bruland, K . W . ; Berton, J. D . ; Goldberg, E . D . , Eds.; Plenum: N e w Y o r k , 1983; pp 21-38. 22. Feldmann, J.; Lehr, C ; K o c h , I; Andrewes, P.; L a i , V . W . - M . ; Cullen, W . R., manuscript in preparation. 23. Andreae, M . O.; Asmode, J.-F.; Foster, P.; Van't dack, L . Anal. Chem. 1981, 1771. 24. Parris, G . E . ; Brinckmann, F. E . Environ. Sci Tech. 1976,  53, 1766-  10, 1128.  25. Jenkins, R. O.; Craig, P. J.; Goessler, W . ; Miller, D . Ostah, N . ; Irgolic, K . J. Environ. Tech. 1998, 32, 882-885.  Sci.  26. Sarret, G . ; Manceau, A . ; Spandini, L . ; Roux, J . - C ; Hazemann, J.-L.; Soldo, Y . ; EybertBerard, L . ; Menthonnex, J.-J. Environ. Sci. Technol. 1998, 32, 1648-1655. 27. Bright, D . A . ; Dodd, M . ; Reimer, K . J. Sci. Tot. Environ. 1996,  180, 165-182.  28. Halverson, G . B . ; Raponi, R. R. Water Poll. Res. J. Canada. 1987, 29. Feldmann, J.; K o c h , I.; Cullen, W . R. Analyst, 1998,  22, 570-583.  815-820.  30. Nekrasov, Y . S.; Zagorevskii, D . V . In The Chemistry of Organic Arsenic, Antimony and Bismuth Compounds; Patai, S., E d . ; John Wiley: Chichester, 1994; pp 237-264.  265  Chapter 7  CONCLUSIONS AND FUTURE WORK  Considerable knowledge was gained about arsenic and antimony species in the terrestrial environment. Existing methods for speciation analysis were adapted for this work. Three H P L C methods with I C P - M S detection allowed separation and identification o f at least 11 arsenic species. The agreement o f retention times between chromatographic peaks in samples and standards, by using more than one method, corroborates identifications based on retention times. Arsenosugar standards were analyzed by using tandem E S I - I T - M S and fragmentation patterns specific to this mass analyzer were obtained. Only partial identification o f arsenosugars in a crude kelp extract was achieved by using M S - M S and M S - M S - M S techniques (only arsenosugar X I I I could be identified with any certainty). Future work should address clean-up o f the kelp extract (e.g., the use of H P L C methods, before introduction to E S I - I T - M S ) . The demethylation o f trimethylantimony species during analysis by using H G - G C A A S was characterized as being dependent on p H ; lower p H resulted in more demethylation. Mechanistic reasons for demethylation were sought and it was established that methyl groups were lost during the H G reaction, rather than as a result o f instability of the starting compound (Me3SbCl ) or the product (Me Sb) to acid. Care is 2  3  recommended (and was taken) in the analysis o f environmental or other samples for  266  antimony by using H G - G C methods to ensure that the methylantimony species identified are not an artifact o f the method. Non-toxic and very low levels o f toxic water soluble arsenic species were identified in some edible mushrooms, and consequently these do not present a health concern to consumers. It would be interesting to determine i f arsenic speciation changes as the arsenic concentration increases for the mushrooms analyzed. In some cases the extractable arsenic was inorganic and hence higher levels might become o f greater toxicological concern. Pure culture experiments with fungi that can produce mushrooms indicated that the synthesis o f arsenobetaine, arsenocholine or arsenosugars does not take place by the mycelia during the short experiment times. Arsenobetaine is accumulated by Scleroderma citrinum which may suggest that i f any arsenobetaine is present in the growing environment o f wild mushrooms, it may be accumulated by the fungus. The interaction o f antimony with fungus was confounded by the ubiquitous presence o f an unknown antimony compound for which only partial characterization was accomplished. Pleurotus flabellatus  formed an antimony-containing metabolite (of  unknown identity, detected by using H P L C - I C P - M S ) from inorganic antimony. This fungus also oxidized Sb (III) to Sb(OH) ". Future experiments should include growth 6  media that contain a minimum amount o f salts and/or carbon sources, to simplify the matrix and hopefully eliminate the presence o f unidentified antimony-binding compounds. N o v e l results were obtained from a study o f the arsenic species in samples from two terrestrial environments: a hot springs environment (Meager Creek, B C ) and from an area impacted by smelting and mining (Yellowknife, N W T ) . Arsenosugars are apparently synthesized by cyanobacteria and other bacteria in microbial mats from both locations.  267  Thus thermophilic as well as non-thermophilic organisms have this capability. Small amounts o f arsenosugars were also found in lichens, in higher plants, in some mushrooms, in green algae (belonging to the phylum Chlorophyta), in freshwater mussels and in suckers. Because they are bottom feeders, suckers probably accumulate arsenosugars from benthic organisms. Suckers also contain arsenobetaine which was the major arsenic compound found in all other fish analyzed, except for pike. In pike, the major detectable arsenic compound was D M A . On the other hand, neither freshwater mussels nor snails contain appreciable quantities o f arsenobetaine. This is in direct contrast to findings in the marine environment where marine mussels and gastropods usually contain arsenobetaine as a major or as the only arsenic compound. Freshwater mussels contain mostly arsenosugars and snails contained mostly tetramethylarsonium ion, as well as inorganic arsenic. Arsenobetaine was found for the first time in lichens and it is present in all specimens from Yellowknife. It was also found in some mushrooms, including Coprinus comatus and Lycoperdon pyriforme. The identity o f arsenic species was determined for the first time in Paxillus involutus, Psathyrella candolleana and Leccinum scabrum. The major arsenic species extracted from higher plants, as well as lichens, mosses, algae and microbial mats were inorganic (arsenite and arsenate). A large amount o f arsenic remained unextracted or undetected in all types o f samples. The nature o f this arsenic is unknown and hence more studies are imperative to determine its chemical and toxicological significance. Arsenic that is not extracted may be bound to lipids, cell components, proteins, or it may exist in a mineral form.  268  Antimony species were determined in some samples from Meager Creek and Yellowknife, and mostly Sb ( V ) was found (although, as for arsenic, a large fraction remained unextracted). In moss samples, however, a dimethylantimony species in the form o f a compound that resulted in dimethylstibine being formed from hydride generation of the extract, is present. Derivatization o f extracts o f the same moss species from two different locations, and from the same location at different times (June and August) yielded dimethylstibine in all cases. Future work to identify this compound would be very interesting. Arsenic speciation in the terrestrial environment, in general, does not appear to be as complex as in the marine environment. F o r example, arsenosugars and arsenobetaine, which are the major arsenic compounds in marine plants and animals, respectively, occur either rarely or in very small amounts in terrestrial samples. This may indicate that different metabolic pathways are followed in the terrestrial environment, or that the processes in the marine environment are not important in the terrestrial environment. Little is known about the speciation o f antimony in the environment, and this study helps to establish a knowledge base for this element. Inorganic antimony appears to be the predominant species and there is no evidence yet for antimony analogues o f arsenosugars or arsenobetaine. However the apparent absence o f these compounds may be due to the present lack o f appropriate analytical methodology for antimony speciation.  269  

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