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Arsenic speciation studies on some marine invertebrates of British Columbia Dodd, Matthew 1988

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ARSENIC SPECIATION STUDIES ON SOME MARINE INVERTEBRATES OF BRITISH COLUMBIA By MATTHEW DODD B.Sc, U n i v e r s i t y of Science and Technology, Ghana, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1988 © Matthew Dodd, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of O ^ M S T ^ / The University of British Columbia Vancouver, Canada Date Qc^hpbejr ? ( ^<gg DE-6 (2/88) i i ABSTRACT Graphite furnace and hydride generation atomic absorption, GFAA and HGAA, techniques have been developed and applied to the determination of arsenic concentrations i n some marine invertebrates, mainly bivalves and gastropods, of B r i t i s h Columbia. To t a l arsenic concentrations i n b i v a l v e s vary with species, ranging from 0.6-9.1 ng g"^ - (wet weight b a s i s ) . Arsenic concentrations i n the bivalve s h e l l s show a wider range of 0.1 to 26.3 ng g ' l (dry weight b a s i s ) . Gastropods show r e l a t i v e l y higher arsenic concentrations i n the s o f t t i s s u e s , 17.3-48.4 fig g"^, and concentrations i n the s h e l l s range from 1.4 to 16.3 fig g"^. There i s no c o r r e l a t i o n between arsenic l e v e l s i n the s o f t - t i s s u e s and s h e l l s . There i s also no c o r r e l a t i o n between arsenic l e v e l s i n the organisms and the surrounding sediments and sediment pore waters. HPLC-GFAA techniques have been developed and used f o r the separation and q u a n t i t a t i o n of-arsenite, arsenate, methylarsonic acid, dimethylar-s i n i c acid, arsenobetaine, arsenocholine iodide and tetramethylarsonium iodide. This technique together with TLC, NMR, FAB and thermospray LCMS were employed f o r the detection of water-soluble arsenic compounds i n 5 species of clams - Butter clam Saxidomus giganteus. Horse clam Schizothoerus n u t t a l l i . S o f t - s h e l l e d clam Mva arenaria. Native-l i t t l e n e c k clam Protothaca staminea and Manila clam Venerupis laponica. Varying amounts of arsenobetaine and tetramethylarsonium ion are found i n a l l the clams. Butter clams show the pres ence of a t h i r d compound which appears to be trimethylarsine oxide. Small amounts of unknown i i i a rsenic containing compounds are present which are yet to be character-ized . Arsenic s p e c i a t i o n i n 3 gastropods was also examined. The Northwest neptune Neptunea l v r a t a . the Thick-ribbed whelk Berinpius c r e b r i s c o t a t a and Phoenician whelk Neptunea phoenicius a l l contain arsenobetaine and at l e a s t two u n i d e n t i f i e d a r s e n i c a l s . i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES x LIST OF FIGURES x i i LIST OF ABBREVIATIONS x v i ACKNOWLEDGEMENT x v i i INTRODUCTION 1 1.1 Arsenic i n Marine Organisms 1 1.2 Biotransformation of Arsenic i n the Marine Environment . . . 3 1.2.1 Redoxtransformation Between Arsenic (III) and Arsenic (V) 3 1.2.2 The Biomethylation of Arsenic 4 1.2.3 Organoarsenic Compounds i n Marine Algae 8 1.2.4 Organoarsenic Compounds i n Marine Invertebrates and Fis h 12 1.2.4.1 The Conversion of Arsenosugars to Arsenobetaine 13 1.2.5 Organoarsenic Compounds i n Food Chain 15 1.3 Scope of Work 16 V EXPERIMENTAL 18 2.1 Instrumentation 18 2.1.1 Atomic Absorption Spectrometry . . . . 18 2.1.1.1 Graphite Furnace Atomic Absorption . . . . 18 2.1.1.2 Hydride Generation Atomic Absorption . . . 18 2.1.2 High Pressure Li q u i d Chromatography 20 2.1.3 Mass Spectrometry and Nuclear Magnetic Resonance Spectrometry 20 2.2 Chemicals and Reagents 21 2.3 Sample Site and Sample C o l l e c t i o n 22 2.3.1 Thetis Island 22 2.3.2 Yellow Point 22 2.3.3 Sherard Point 24 2.3.4 Coles Bay 24 2.3.5 P a t r i c i a Bay 24 2.3.6 Quatsino Sound-Rupert-Holberg Inlets 24 2.3.7 A l i c e Arm-Hastings Arm 27 2.3.8 Sample Storage 27 2.4 A n a l y t i c a l Procedures 29 2.4.1 Graphite Furnace Atomic Absorption (GFAA) . . . . 29 2.4.2 Hydride Generation Atomic Absorption (HGAA) . . . . 30 2.4.3 Determination of Total Arsenic 31 2.4.3.1 Wet Ashing with N i t r i c Acid, S u l f u r i c Acid and Hydrogen Peroxide . . . 31 2.4.3.2 Microwave Digestion 33 2.4.4 Extraction of Organoarsenic Compound i n the Marine Organisms 33 v i 2.4.5 Determination of Arsenic i n Extracts 34 2.4.5.1 Determination of Arsenic i n Extracts by GFAA 34 2.4.5.2 Determination of Arsenic i n Extracts by Hydride Generation AA A f t e r UV Decomposition (UV-HGAA) 34 2.4.6 HPLC-GFAA 35 2.7 P u r i f i c a t i o n Procedures f o r Arsenic i n Extracts 37 2.7.1 I s o l a t i o n of Arsenic Compounds i n NH^+OH Fra c t i o n 39 2.7.2 I s o l a t i o n of Arsenic i n HC1 Fra c t i o n 41 2.8 Determination of Arsenic i n Shells 42 RESULTS AND DISCUSSIONS - ANALYTICAL PROCEDURES 43 3.1 Determination of Arsenic 43 3.1.1 Determination of Arsenic by Using GFAA 43 3.1.1.1 C a l i b r a t i o n , Limit of Detection and P r e c i s i o n of GFAA Analysis 45 3.1.2 Determination of Arsenic by HGAA 46 3.1.2.1 Sele c t i o n of Operating Conditions f o r HGAA 48 3.1.2.2 C a l i b r a t i o n , Limit of Detection and P r e c i s i o n of HGAA Analysis 51 3.1.3 Comparison of GFAA and HGAA 52 3.2 Determination of Tot a l Arsenic 54 3.2.1 Wet Ashing with a Mixture of S u l f u r i c Acid, N i t r i c A cid and Hydrogen Peroxide 54 3.2.2 Bomb Decomposition i n a Microwave Oven 57 3.3 Determination of Arsenic i n Solution by Using HGAA following UV Decomposition 59 v i i 3.3.1 HPLC Separation of A r y l a r s e n i c a l s 61 3.3.2 Photooxidation of Arylarsonic Acids 65 3.3.3 Nature of Arsenicals A f t e r Photodecomposition . . . 67 3.3.4 HGAA and GFAA of Solutions A f t e r Photodecomposition 69 3.3.5 E f f e c t of Acid on Photodecomposition 72 3.3.6 E f f e c t of Organic Solvents on Photodecomposition . . 76 3.3.7 C a l i b r a t i o n Curves, Limit of Detection and Pr e c i s i o n of UV-HGAA 77 3.3.8 Recovery Studies 79 3.3.9 Summary of UV-HGAA 81 3.3.10 Comparison of GFAA and UV-HGAA Determination of Arsenic i n Extracts 81 3.4 Determination of Arsenic Species by HPLC 84 3.4.1 Separations on Ion-Exchange Column 85 3.4.1.1 C a l i b r a t i o n and Limit of Detection f o r Separations on Protein Pak DEAE Column . . 91 3.4.1.2 Applications of the HPLC-GFAA Separations on Protein-Pak Column to Extracts 93 3.4.2 Separations on C^g Reversed-Phase Column 94 3.4.2.1 Separations of Arsenicals with Tetrabutylammonium as Ion-pair Reagent . . 95 3.4.2.2 E f f e c t of Anion Associated with Tetrabutylammonium on the Separation of Arsenicals 99 3.4.2.3 C a l i b r a t i o n and Limit of Detection f o r Separation on Reversed Phase Column with TBAN as Ion-Pair Reagent . . . 102 3.4.2.4 Separation of Arsenobetaine, Arseno-choline and Tetramethylarsonium Iodide with Heptane Sulfonic A c i d as Ion Pair Reagent 102 v i i i RESULTS AND DISCUSSION - ARSENIC CONCENTRATIONS AND SPECIATION . . 105 4.1 T o t a l Arsenic Concentrations 105 4.1.1 Data Presentation 105 4.1.2 T o t a l Arsenic Concentrations i n Soft-Tissues . . . 105 4.1.3 T o t a l Arsenic Concentrations i n Soft-Tissues of Clams from Coles Bay and P a t r i c i a Bay . . . . 108 4.1.4 Concentration of Arsenic i n Oysters 110 4.1.5 Arsenic Concentrations i n Bivalve Tissues C o l l e c t e d from A l i c e Arm and Hastings Arm, October 1986 I l l 4.1.6 Arsenic Concentrations i n Soft-Tissues of Gastropods C o l l e c t e d from A l i c e Arm and Hastings Arm, October 1986 . 118 4.1.7 Arsenic Concentrations i n Soft-Tissues of Bivalves C o l l e c t e d from A l i c e Arm and Hastings Arm, October 1987 120 4.1.8 Arsenic Concentrations i n Crabs 122 4.1.9 D i s t r i b u t i o n of Arsenic i n Bivalves 123 4.2 Arsenic Speciation 125 4.2.1 Arsenic Speciation i n Clams 125 4.2.1.1 I d e n t i f i c a t i o n of Arsenic Compounds i n Ammonium Hydroxide Fractions 128 4.2.1.2 I d e n t i f i c a t i o n of Arsenic Compounds i n Hydrochloric Acid Fractions 136 4.2.1.3 Arsenic Compounds i n the Unadsorbed Fr a c t i o n 140 4.2.2 Arsenic Speciation i n V i s c e r a l Mass Foot and Siphon Tissues of Geoduck 140 4.2.3 Arsenic Speciation i n Gastropods 143 4.3 Ar s e n i c a l s i n Shells 149 i x SUMMARY 154 REFERENCES 157 X LIST OF TABLES Tables Page 1.1 Arsenic Concentrations i n Selected Marine Organisms 2 2.1 Furnace Operating Parameters 29 2.2 Operating Conditions of HGAA 30 3.1 Comparison of GFAA and HGAA 53 3.2 Arsenic Determinations i n Standard Reference Materials 55 3.3 Recovery of Arsenic from Dolt-1 57 3.4 Recovery of Arsenic i n Standard Reference Materials 58 3.5 HPLC Separation of a group of A r y l a r s e n i c a l s . . . . 62 3.6 Gradient Program f or the Separation of A r y l a r s e n i c a l s on Reversed Phase 65 3.7 HGAA and GFAA of A r y l a r s e n i c a l s Before and A f t e r UV Decomposition 69 3.8 HGAA and GFAA of Arsenobetaine, Arsenocholine iodide and Tetramethylarsonium Iodide Before and Af t e r UV Decomposition 71 3.9 Recovery of Arsenic from Samples of Clam Extracts 80 3.10 Analysis of Clam Extracts by UV-HGAA and GFAA . . . . 82 3.11 Arsenic Concentrations In Extracts by Using GFAA and UV-HGAA 83 4.1 Arsenic Concentrations i n Soft-Tissues of Clams Co l l e c t e d at Thetis Island, October 1985 . . . 106 4.2 Arsenic Concentrations i n Soft-Tissues of Clams C o l l e c t e d at Thetis Island, October 1986 . . . 107 4.3 Arsenic Concentrations i n Clams Co l l e c t e d from Coles Bay and P a t r i c i a Bay, May 1987 109 x i 4.4 Arsenic Concentrations i n P a c i f i c Oysters 110 4.5 Arsenic Concentrations i n Bivalve Soft-Tissues, A l i c e Arm and Hastings Arm, October 1986 I l l 4.6 Concentrations of Arsenic i n Surface Sediments Alice-Hastings Arms 114 4.7 Concentrations of Arsenic i n Sediment Pore Water 115 4.8 Arsenic Concentrations i n Some Bivalves From Selected Coastal Locations 117 4.9 Arsenic Concentrations i n Gastropods Col l e c t e d from Alice-Hastings Arm, October 1986 119 4.10 Arsenic Concentrations i n Bivalves Alice-Hastings Arms and Quatsino Sound 121 4.11 Arsenic Concentrations i n Crab Leg Muscle Tissue 122 4.12 D i s t r i b u t i o n of Arsenic i n Clams 124 4.13 Ex t r a c t i o n of Soft-Tissues of Clams 126 4.14 Fr a c t i o n a t i o n of Water-soluble Arsenic Compounds i n Clams on Dowex 50 127 4.15 Ext r a c t i o n of Three Tissues of Geoduck 142 4.16 F r a c t i o n a t i o n on Dowex 50 of Water Soluble Arsenic i n Three Tissues of Geoduck 143 4.17 Ex t r a c t i o n of Soft-Tissues of Gastropods 144 4.18 F r a c t i o n a t i o n of Water-Soluble Arsenic i n Gastropods on Dowex 50 145 4.19 Arsenic Concentrations i n Sea Shells 150 4.20 Determination of Arsenic i n Clam Shells by HGAA and UV-HGAA 151 4.21 Arsenic Concentrations i n Soft-Shelled Clam and Sediments 153 x i i LIST OF FIGURES Figures Page 1.1 Challenger's Mechanism for the B i o l o g i c a l Methylation of Arsenic 5 1.2 Structure of S-Adenosyl Methionine (SAM) 6 1.3 Structures of Arsenoribofuranosides Isolated from Algae 10 1.4 Proposed Scheme for the Formation of Arsenosugars from Dimethylarsinic Acid 11 1.5 Structures of Selected Organoarsenic Compounds Found i n Marine Invertebrates and F i s h 12 1.6 Proposed Scheme for the Biosynthesis of Arsenobetaine from Arsenic Containing Sugars . . . . 14 2.1 Schematic Diagram of Hydride Generation Assembly 19 2.2 Thetis Island and Vancouver Island Showing Sampling Locations 23 2.3 Saanich Peninsula Showing Sampling Locations . . . . 25 2.4 Rupert-Holberg I n l e t Showing Sampling Locations . . . 26 2.5 A l i c e Arm and Hastings Arm Showing Sampling Locations 28 2.6 Wet Ashing Apparatus 32 2.7 Photodecomposition Apparatus 36 2.8 E x t r a c t i o n Scheme 38 2.9 P u r i f i c a t i o n of NH4OH Fr a c t i o n 40 2.10 P u r i f i c a t i o n of HC1 Fract i o n 42 3.1 T y p i c a l C a l i b r a t i o n Curve f o r Arsenic Determination by GFAA 45 x i i i 3.2 E f f e c t of NaBH4 on HGAA 49 3.3 E f f e c t of Potentiometer Setting on HGAA 50 3.4 T y p i c a l C a l i b r a t i o n Curve f o r HGAA 51 3.5 HPLC Chromatogram of A r y l a r s e n i c a l s 64 3.6 HPLC Chromatogram of A r y l a r s e n i c a l s Before and A f t e r UV Decomposition 66 3.7 HPLC Chromatogram of Arsenite Before UV Decomposition 68 3.8 HPLC Chromatogram of Arsenite A f t e r UV Decomposition 68 3.9 E f f e c t of N i t r i c Acid on UV-HGAA . . . 72 3.10 E f f e c t of S u l f u r i c Acid on UV-HGAA 73 3.11 E f f e c t of Hydrochloric Acid on UV-HGAA 73 3.12 E f f e c t of Acetic Acid on UV-HGAA 74 3.13 Response of Arsenobetaine Solution i n various H2SO4 Solutions i n UV-HGAA, 15 min I r r a d i a t i o n 75 3.14 Response of Arsenobetaine Solution i n Various H2SO4 Solutions i n UV-HGAA, 30 min I r r a d i a t i o n 75 3.15 Response of Arsenobetaine Solution i n Various H2SO4 Solutions i n UV-HGAA, 60 min I r r a d i a t i o n 76 3.16 C a l i b r a t i o n Curve f or Arsenobetaine Using UV-HGAA 78 3.17 Scatter Diagram f o r the Determination of Arsenic i n Extracts by GFAA and UV-HGAA 84 3.18 Separation of Arsenicals on Protein Pak Column, pH 4 87 3.19 Separation of Arsenicals on Protein Pak Column pH 6.65 88 3.20 Separation of Arsenicals on Protein Pak Column pH 10 89 1 x i v 3.21 Typ i c a l C a l i b r a t i o n Curve of Arsenobetaine by HPLC-GFAA, Protein Pak Column 92 3.22 HPLC-GFAA of Manila Clam Extract 93 3.23 HPLC-GFAA Chromatogram of Arse n i c a l s , Reversed-Phase, TBAN, 95:5, pH 6.8 96 3.24 HPLC-GFAA Chromatogram of Arse n i c a l s , Reversed-Phase, TBAN, 90:10, pH 6.8 98 3.25 HPLC-GFAA Chromatogram of Arsenicals, Re versed-Phase, TBAP 100 3.26 HPLC-GFAA Chromatogram of Arse n i c a l s , using Reversed-Phase, TBAS 101 3.27 Ty p i c a l C a l i b r a t i o n Curve f or HPLC-GFAA Using Reversed Phase, TBAN 102 3.28 HPLC-GFAA Chromatogram of Arse n i c a l s , Heptane Sulfonic Acid as Counter Ion 104 4.1 Alice-Hastings Arms Showing 1986 Sampling Sites 113 4.2 *H NMR of a: Arsenobetaine from Butter Clam b: Synthetic Arsenobetaine 130 4.3 Thermospray LCMS of a: Arsenobetaine from Butter Clam and b: Standard Arsenobetaine 131 4.4 1-H NMR of Minor Arsenic Compound i n NH4OH Fracti o n 132 4.5 -^H NMR Spectrum of Synthetic Arsenocholine 133 4.6 *H NMR Spectrum of Synthetic Tetramethyl-arsonium Iodide 133 4.7 Thermospray LCMS of Minor Arsenic Compound i n Butter Clam NH4OH Fra c t i o n 135 4.8 *H NMR Spectra of Arsenic Compound i n HC1 Fractio n from Butter Clam 137 4.9 Thermospray LCMS of P u r i f i e d HC1 F r a c t i o n of Butter Clam 138 4.10 Thermospray LCMS of Standard Tetramethyl-arsonium iodide 139 XV A.11 Thermospray LCMS of P u r i f i e d Unadsorbed Fr a c t i o n of Butter Clara 141 4.12 NMR of Minor Arsenic Compound i n Whelk 146 4.13 XH NMR of P u r i f i e d HC1 Fract i o n of Whelk 147 4.14 Thermospray LCMS of HC1 Fract i o n of Whelk 148 x v i LIST OF ABBREVIATIONS AA - atomic absorption AAS - atomic absorption spectrometry FAB - f a s t atom bombardment GFAA - graphite furnace atomic absorption HGAA - hydride generation atomic absorption HPLC - high pressure l i q u i d chromatography LC - l i q u i d chromatography MS - mass spectrometry NMR - nuclear magnetic resonance TBAN - tetrabutylammonium n i t r a t e TBAP - tetrabutylammonium phosphate TBAS - tetrabutylammonium hydrogen s u l f a t e THAN - tetraheptylammonium n i t r a t e TLC - t h i n layer chromatography UV - u l t r a v i o l e t x v i i ACKNOWLEDGEMENT I want to express my utmost appreciation to my research supervisor Dr. W.R. Cullen f o r h i s continued support and valuable discussions throughout the course of t h i s work. I also thank members of my guidance committee Dr. B.C. McBride, Dr. R.C. Thompson and Dr. M.W. Blades for valuable discussions. I thank Dr. Kenneth Reimer, Royal Roads M i l i t a r y College, V i c t o r i a , B. C. for the opportunity to p a r t i c i p a t e i n sample c o l l e c t i o n cruises on C. S.S. J.P. T u l l y and C.S.S. Vector and for providing a n a l y t i c a l data on sediment and pore water. I also thank Dr. J.A.J. Thompson, I n s t i t u t e of Ocean Sciences, Sydney, B.C. for help i n obtaining samples and a l l the fr i e n d s who helped i n sample c o l l e c t i o n . I am very g r a t e f u l to the technical s t a f f of t h i s department, p a r t i c u l a r l y , Dr. G. Eigendorf and h i s s t a f f members for t h e i r assistance with the mass spectrometry experiments. I thank my lab colleagues for many h e l p f u l discussions and for the f r i e n d l y atmosphere created by them and also Mr. C. Hampton, Miss C. Reimer, my Father and Mother f o r t h e i r continuous encouragement and support. - 1 -INTRODUCTION 1.1 Arsenic i n Marine Organisms A r s e n i c a l preparations have probably been used f o r c r i m i n a l purposes more than any other poison. The destruction of human l i f e by poison has been p r a c t i c e d from very early years.^ Kings and queens i n the middle ages often c a r r i e d bezoar stones ( i n t e s t i n a l concretions found i n Persian goats) to ward o f f the e f f e c t s of arsenic.^ The f i n d i n g of r e l a t i v e l y high concentrations of arsenic (2-132 fig g~^~) i n marine plants and animals i n the 1920's^>^ was therefore viewed with much concern. This l e d to d e t a i l e d investigations of the l e v e l s and chemical forms of arsenic i n organisms and the migration and transformation of arsenic i n marine food webs.-*"^ Representative arsenic concentrations i n s e l e c t e d marine organisms are given on Table 1.1. The concentrations of arsenic found i n marine organisms are higher than i n surrounding water. Sea water contains 1-8 ng mL"^ - and because arsenic i s removed from sea water by sedimentation, i t i s present i n sediments at higher l e v e l s , i n the range 2-20 fig g " ! . ^ In contrast to the a r s e n i c a l s found i n t e r r e s t r i a l organisms which are predominantly the simple methylated species (methylarsonic and dimethylarsinic acids, trimethylarsine oxide and the methylated a r s i n e s ) , ^ the a r s e n i c a l s found i n aquatic systems seem to be dominated by a number of structu-r a l l y more complex compounds, only a few of which have been i d e n t i f i e d to date. - 2 -Table 1.1 Arsenic concentrations i n selected marine organisms Common Name Species As Hg g" 1 wet weight Ref Brown Kelp Ecklonia r a d i a t a 10 5 Edible seaweed H i z i k i a fusiforme 100 6 Clam ( g i l l ) Meretrix l u s o r i a 15-40 7 Giant clam (kidney) Tridacna maxima 2000 8 Mussel Mvtilus edilus 1 9 Shrimp Serpestes lucens 5.5 10 Pandalus b o r e a l i s 19 11 Lobster Homarus americanus 26 12 Panius cvgnus 39 13 King crab Paralithodes cantschatica 8.5 14 Snow crab Chionoecestes b a i r d i i 7.7 14 Dungeness crab Cancer magister 4.0 14 Pl a i c e Plueronectes nlatessa 3-166 15 School Whitting S i l l a g o bassensis 3.2-14.5 16 Shark Prionace glaucus 27.7-37.4 17 - 3 -1.2 Biotransformation of Arsenic i n the Marine Environment Three main types of arsenic species biotransformation have been found to occur i n the environment: (1) redoxtransformation between arsenite (As(III)) and arsenate (As(V)); (2) the biomethylation of arsenic to v o l a t i l e and h i g h l y to x i c methylarsines; and (3) the biosyn-thesis of more s t r u c t u r a l l y complex organoarsenic compounds. These three processes w i l l be discussed i n the following sections with emphasis on the marine environment. 1.2.1. Redox-transformation Between Arsenic (III) and Arsenic (V) In oxygenated water, arsenate (As(V)) dominates the s p e c i a t i o n of arsenic whereas arsenite (As(III)) i s the most common form i n the reduced oxygen-free pore waters of sediments^ and i n anoxic basins l i k e the B a l t i c Sea.^l However s i g n i f i c a n t amounts of arsenite (up to 10% of t o t a l arsenic) are found i n the surface and deep waters of oceans and conversely, some arsenate i s s t i l l present i n the anoxic waters. The redox p a i r arsenite/arsenate i s therefore not i n thermodynamic e q u i l i -brium anywhere i n the marine environment.^2 The presence of arsenite i n oxygenated sea water has been shown to be due to the reduction of arsenate to arsenite by marine b a c t e r i a or phytoplankton.22.23-25 -j^e r o i e Q f algae i n the reduction of arsenate was f i r s t e s t ablished by Blasco et al.26 who found that i t occurs i n cultures of C h l o r e l l a pyrenoidosa. a fresh water alga. The release of - 4 -arsenite by a number of cultures of marine phytoplankton species was subsequently observed by Andreae and Klumpp.23 A c o r a l species P e c i l l o p o r a v e l r u c o s a ^ and clumps of the pelagic alga Sarpassum sp.25 were shown to reduce arsenate to arsenite. This same reduction i s brought about i n sea water medium by a mixed population of bacteria.22 Pseudomonas fluorescens. a common aquatic bacterium, c a r r i e s out t h i s reduction under aerobic conditions.27 Some b a c t e r i a are responsible for the reverse process, the oxidation of arsenite to arsenate, an example being Alaligenes sp.. 28,29 however, no s i g n i f i c a n t difference was observed i n the slow oxidation of arsenite to arsenate i n s t e r i l e and n o n - s t e r i l e sea water.^0 Thus the redox s p e c i a t i o n of arsenic i n sea water appears to r e s u l t from a slow a b i o t i c oxidation of arsenite and a biogenic reduction of arsenate by a number of organisms. 1.2.2 The Biomethylation of Arsenic Interest i n t h i s subject developed as a r e s u l t of poisoning i n c i -dents i n Europe which were l i n k e d to the f a c t that victims had been l i v i n g i n rooms which e i t h e r contained r e l a t i v e l y high amounts of arsenic i n the p l a s t e r used on the walls or where the walls were covered with wall paper containing a r s e n i c a l compounds. Rooms i n which the poisoning occurred u s u a l l y had a g a r l i c odour t y p i c a l of v o l a t i l e arsenic compounds. The f i r s t systematic study of t h i s s i t u a t i o n was done by Gosio i n - 5 1891^ who i d e n t i f i e d a number of mold species which were able to produce a v o l a t i l e arsenic compound from arsenite. P e n i c i l l i u m  brevicaule (now c a l l e d Scopulariopsis b r e v i c a u l i s ) was among the producers of t h i s "Gosio gas". He suggested that the gas produced was di e t h y l a r s i n e (C2H5)2AsH, an i d e n t i f i c a t i o n which was subsequently questioned by other i n v e s t i g a t o r s . I t was not u n t i l 1932 that the true i d e n t i t y of "Gosio gas" was established by Challenger as trimethylar-sine. ^ Challenger's work further suggested that methylarsonic a c i d and dimethylarsinic a c i d were intermediates i n the production of trimethyl-arsine. He suggested the scheme f or the formation of trimethylarsine shown i n F i g . 1.1. Challenger d i d not speculate on the nature of the methylating agent then, however subsequent work on transmethylation by du Vigneaud et a l . ^ i e d h i m to study trimethylarsine production i n the presence of l a b e l l e d precursors. The precursors were studied i n bread C C H 3 + 3 2p A s ( O H ) 3 > C H 3 A s O ( O H ) 2 ( C H 3 A s ( O H ) 2 ) CCH, +3 ( C H 3 A s ( O H ) 2 ) ^ > ( C H 3 ) 2 A s O ( O H ) ^ > { ( C H . ) J U O H ) 3'2' C C H 3 + 3 2 e { ( C H 3 ) 2 A s O H ) - > ( C H 3 ) 3 A s O ~ > ( C H ^ A s F i g . 1.1: Challenger's scheme f o r the b i o l o g i c a l methylation of arsenic - 6 -cultures of S. b r e v l c a u l i s . Only CH3- C l a b e l l e d methionine was found to t r a n s f e r i t s l a b e l to arsenite to a s i g n i f i c a n t extent. This r e s u l t was taken as an i n d i c a t i o n that "active methionine" S-adenosyl methionine (SAM), F i g . 1.2, i s possibly involved i n the methylation. F i g . 1.2: Structure of S-adenosylmethionine (SAM) Cox and Alexander-^ studied the production of trimethylarsine from methylated arsenic substrates (methylarsonic and dimethylarsenic acids) by C. humicola, Gliocladium roseum and P e n i c i l l i u m sp. Only C. humicola was able to methylate inorganic substrates, however. Cullen and coworkers investigated the actions of both S. b r e v i c a u l i s and C. humicola on inorganic arsenic-^-39 with a p a r t i c u l a r view towards e s t a b l i s h i n g the d e t a i l s of the mechanism of methylation o r i g i n a l l y proposed by Challenger (Fig. 1.1). Addition of L-methionine-methyl-d_3 to the cultures of these molds, with sodium arsenite also present, r e s u l t e d i n the incorporation of the CD3 group into the a r s e n i c a l p r o d u c t s , s t r o n g l y supporting the idea that SAM i s the source of CH3+ i n F i g . 1.1 Broken c e l l homogenates of C. humicola i n the presence of N H 2 HO' - 7 -l a b e l l e d [^As] arsenate gave l a b e l l e d arsenite, methylarsonate and dimethylarsinate (intermediates i n Challenger's mechanism) among the p r o d u c t s . ^ Trimethylarsine oxide, the immediate precursor to trimethylarsine, was r e a d i l y reduced to the arsine when introduced into cultures of C. h u m i c o l a . ^ The biomethylation of arsenic under anaerobic conditions by methano-genic b a c t e r i a was investigated by McBride and W o l f e . ^ The biomethyla-t i o n probably proceeds only to dimethylarsine. In experiments with lake and r i v e r sediments and with pure b a c t e r i a c u l t u r e s , Wong et a l . ^ observed the production of methylarsonic acid, dimethylarsinic a c i d and trimethylarsine oxide as well as the v o l a t i l e arsines dimethyl- and trimethylarsine. A l l experiments were conducted i n systems where arsenic l e v e l s are at l e a s t three orders of magnitude higher than those t y p i c a l of environments not p o l l u t e d by arsenic. Andreae-^ questioned the generality of these r e s u l t s i n view of the elevated arsenic l e v e l s at which the experiments were conducted, and suggests that b a c t e r i a l methylation i n these cases might be a response to extreme arsenic s t r e s s . He c i t e s the absence of methylarsenic species i n the i n t e r s t i t i a l water of N.E. P a c i f i c and C a l i f o r n i a coast, and i n marine mud culture experiments by McBride et a l . ^ on the basis of these observations, Andreae^ concluded that e x c r e t i o n by algae, or decomposition of deposited a l g a l material, were the only important processes leading to the presence of organoarsenicals i n natural waters. This conclusion, however, i s counterbalanced by the f a c t that, although the number of studies i s small, i n every other case'where sediment i n t e r s t i t i a l water has been analyzed f o r methylarsenic compounds they - 8 -have been found.^3,44 •j^g sediments sampled by Reimer and Thompson,^ fo r example, d i d not contain elevated s o l i d phase arsenic l e v e l s (average 22 ftg g'^) yet methylarsonic acid, dimethylarsinic a c i d and trimethylarsine oxide were present i n a l l cases. C l e a r l y , a d d i t i o n a l work i s required. I t seems l i k e l y that the pore water concentrations of organoarsenicals are c o n t r o l l e d by a balance between methylation and demethylation, and l i k e arsenate and arsenite, are also influenced by redox processes. While Andreae d i d not observe the methylarsines i n the porewaters,^ he d i d f i n d methylated arsenic species, methylarsonic a c i d and dimethyl-a r s i n i c a c i d to be c o n s i s t e n t l y present i n surface seawaters.^ These compounds were also observed i n coastal surface waters by Braman and Foreback.^-' No evidence was found f o r the presence of trimethylarsenic species i n sea w a t e r . ^ Thus i t seems l i k e l y that biomethylation according to the mechanism o r i g i n a l l y proposed by Challenger ( F i g . 1.1) occurs i n the marine environment with the reduction of arsenate and subsequent methylation to produce i n i t i a l l y methylarsonic a c i d and then dimethylarsinic a c i d . However, the f i n a l reduction and methylation to trimethylarsine does not always occur. 1.2.3 Organoarsenic Compounds i n Marine Algae Algae, to s a t i s f y t h e i r phosphorus requirements, take up phosphate ion from t h e i r environment. This presents algae with the p o s s i b i l i t y of taking up arsenate as well since arsenate i s i s o s t r u c t u r a l with phos-- 9 -phate and the two are often present at comparable c o n c e n t r a t i o n s . 2 0 i n seawater from the northeast P a c i f i c , f o r example, the arsenate concen-t r a t i o n i s near 2 ng mL"^, while the phosphate concentration v a r i e s from 0.3 ng mL"* to 10 ng mL"^ 2 0 w h i c h indicates an arsenate:phosphate r a t i o i n excess of 1.0 i n c e r t a i n areas. Uptake of arsenate by marine algae has been o b s e r v e d . 2 3 , 4 ^ Once the arsenate has entered the a l g a l c e l l , i t i s transformed to a v a r i e t y of organic arsenic compounds. Thus inorganic arsenic usually represents only a small f r a c t i o n of the t o t a l arsenic present i n the algae. Organic arsenic represented on the average 78% of the t o t a l arsenic i n s i x species of marine kelp studied by Andreae.^ In the case of samples of mixed marine phytoplankton, 87-96% of the arsenic i s i n organic form. Most of the arsenic i n algae i s i n an organic form, not soluble i n water; there are also a s u b s t a n t i a l number of water soluble organic forms. However, the chemical i d e n t i t y of the arsenic present has not been e l u c i d a t e d . 2 3 - 4 7 " 5 0 In a d d i t i o n to methylarsonate and dimethylarsinate, up to twelve soluble organic arsenic compounds have been observed i n algae. The methylarsonate and dimethylarsinate make up only a small f r a c t i o n of the organoarsenic compounds i n algae 2 3' 4** and appear to be intermediates i n the formation of the more complex organoarsenic forms4** or to be degradation products of these organoarsenic compounds. 4 7 The organo-arsenic compounds can be grouped into water-soluble and l i p i d - s o l u b l e f r a c t i o n s . L u n d e 4 5 • 4 7 » 4 ^ was probably the f i r s t to report the presence of the a r s e n o l i p i d s i n algae. Subsequent workers showed the production 10 -of these and s i m i l a r compounds i n algae, but the chemical i d e n t i t y could not be ascertained.23,48,50 I r g o l i c and c o w o r k e r s , a t t e m p t e d to i s o l a t e and characterize l i p i d - s o l u b l e arsenic compounds from the algae Daphnia magna and Tetraselmis chui grown i n arsenate containing media. They suggested that arsenocholine could be a part of the i s o l a t e d l i p i d s but no confirmation was obtained. The f i r s t p o s i t i v e i d e n t i f i c a t i o n of organoarsenic compounds i n marine algae was made by Edmonds and Francesconi.->• They i s o l a t e d and i d e n t i f i e d , by NMR spectroscopy and X-ray crystallography, two arsenic containing ribofuranosides, 3a and 3b (Fig. 1.3). Subsequently, the arsenosugars 3a, 3b, and 3c were i s o l a t e d from the brown seaweed Laminar i a j a p o n i c a . A l o n g with some inorganic arsenic, Me 2 As-CHa x O x X0-CH2-CH(Y)-CH 2R H SLSf H 3 a Y«0H, R=0S03H b Y=0H, R«0H C Y«0H. R=S03H dY=NH 2. R«S03H eY«0H. R = 0 - P - 0 - C H 2 OH CHOH iH20H F i g . 1.3: Structures of arsenoribofuranosides i s o l a t e d from algae. - 11 -3b. 3c, 3_d, and 3e were found i n the Japanese edible seaweed H i z i k i a  f u s i forme**. The exact mechanism for the formation of these arsenosugars has not been established. Edmonds and Francesconi 5^ proposed the scheme shown i n F i g . 1.4. NH 2 0 II Me2A*0(OH) 'MejAiOH" * ~ » H Y _ K N H J H J H 4b 0 n J H J H F i g . 1.4: Proposed scheme f o r the formation of arsenosugars from dimethylarsinic acid. Dimethylarsinate i s produced by the biomethylation mechanism i n i t i a l l y o u t l i n e d by C h a l l e n g e r 3 2 and discussed i n t h i s work i n Section 1.2.2. The adenosyl group of the methylating agent i s transferred to the arsenic atom to form 4b. Enzymatic hyd r o l y s i s of 4b would lead to 4c which i s followed by formation of the sugars 3a-3e. The two key intermediates 4b and 4c are yet to be i s o l a t e d i n the marine ecosystem. - 12 -1.2.4 Organoarsenic Compounds i n Marine Invertebrates and Fi s h Edmonds et a l . ^ i s o l a t e d an organoarsenic compound from the Western rock l o b s t e r Panulirus cvgnus by using repeated ion-exchange chromato-graphy and t h i n layer chromatography. X-ray analysis of the i s o l a t e showed that arsenic was present as arsenobetaine 5a. This was the f i r s t i s o l a t i o n and i d e n t i f i c a t i o n of an organoarsenical from a marine invertebrate. Since then the v i r t u a l ubiquity of arsenobetaine i n marine animals has been demonstrated. ( C H 3 ) 3 A s + C H 2 C 0 0 " ( C H 3 ) 4 A s + 5a 5b ( C H 3 ) 3 A s + C H 2 C H 2 0 H ( C H 3 ) 3 A s O 5c 5d F i g . 1.5: Structures of selected organoarsenic compounds found i n marine invertebrates and f i s h This compound has been found i n the American lobster Homarus  americanus.^ the octopus Paroctopus d o l f l e i n i . - ^ the dusky shark Carcharhinus obscurus.^ the school whitting S i l l a p o b a s s e n s i s . ^ the lemon sole Microstumus k i t t . ^ ^ crab Cancer cancer.^0 Alaskan king crab Paralithodes camtschatica. Alaskan snow crab Chionoecetes b a i r d i i and Dungeness crab Cancer magister.-^ as well as i n the shrimp Sergestes  lucens.^0 - 13 -Shiomi et a l . 6 ^ - investigated the spe c i a t i o n of arsenic i n the f l a t -f i s h Limanda he r z e n s t e i n i . the sea s q u i r t Halocynthia r o r e t z i and the sea cucumber Stichopus )aponicus. About 90% of arsenic i n the f l a t f i s h and 60% i n the sea cucumber was found to be arsenobetaine, whereas the sea s q u i r t contained two as yet u n i d e n t i f i e d arsenic compounds and no arsenobetaine. The same group reported arsenobetaine accounted f o r 90% and 60% of the arsenic present i n the muscle and mid-gut gland of the ivory s h e l l Buccinum s t r i a t i s s i m u m . 6 ^ Three arsenic compounds were detected i n the red crab Chionoecetes o p i l i o . the major one, 90%, being arsenobetaine, the other two could not be i d e n t i f i e d . 6 ^ As i n the case of the short neck clam, Tapes j a p o n i c a 6 ^ and s c a l l o p Patinopecten yessoensis. 6-* arsenobetaine was detected i n the clam Meretrix l u s o r i a . A strongly c a t i o n i c organoarsenical was also detected and i d e n t i f i e d to be tetramethyl arsonium ion 5b. Other organic arsenic compounds were present but not characterized. Unlike the v i r t u a l l y ubiquitous arsenobetaine, the r e l a t e d ion arsenocholine 5c has been found only i n shrimps.H> 6-* Trimethylarsine oxide 5d has been found as a minor component i n a number of f i s h s p e c i e s . 6 6 The concentration of trimethylarsine oxide i n f i s h increased with storage and i t i s suggested t h i s compound i s formed i n stored f i s h . 6 6 1.2.4.1 The Conversion of Arsenosupars to Arsenobetaine There are some in d i c a t i o n s that marine animals acquire t h e i r arsenic - 14 -burdens through the food web rather than d i r e c t l y from ambient marine animals, the arsenosugars, which have not been found i n marine animals except i n the kidneys of the giant clam Tridacna maxima (the o r i g i n of t h i s was a t t r i b u t e d to a symbiotic u n i c e l l u l a r green alga), are the general constituent of marine algae. I t i s suggested that the arsenosugars are probably converted to arsenobetaine within the food chain. The mechanism for t h i s conversion i s not known. However, fi4 Edmonds and Francesconi proposed the scheme i n F i g . 1.6 based on t h e i r observation that dimethyloxarsylethanol 6 i s a product of the anaerobic decomposition of the brown kelp Ecklonia r a d i a t a . ^ 4 water. 67,68 Whereas arsenobetaine i s the predominant form of arsenic i n 3 anaerobic decomposition CH 3 0=AsCH2CH2OH oxidation • 0= As — CH2C00H CH 3 I I CH 3 (a) 2e (b) CH 3+ (a) 2e (b) CHi* 1 (CH 3) 3As+CH 2CH 2OH 5c 1 oxidation ^ (CH 3) 3As +CH 2C00~ 5a F i g 1.6: Proposed scheme f o r the biosynthesis of arsenobetaine from arsenic containing sugars - 15 -However, I t remains to be established i f the route to arsenobetaine from dimethyloxarsylethanol proceeds v i a arsenocholine or dimethyloxar-s y l a c e t i c a c i d 7 and at what l e v e l i n the food web the biosynthesis occur. There i s also the need to e s t a b l i s h that the production of dimethyloxarsylethanol i s important i n the biosynthesis as suggested. 1.2.5 Organoarsenic Compounds i n Food Chains I t has been proposed that the formation of these organoarsenic compounds i n marine organisms i s a mechanism for the d e t o x i f i c a t i o n of a r s e n i c . ^ These organoarsenicals combine extreme low t o x i c i t y with considerable resistance to metabolic degradation. Not only are these compounds non-toxic i n the marine organisms that form them; they remain non to x i c when ingested by other organisms of the food c h a i n . 1 3 . 1 5 . 5 9 - 7 1 . 7 2 No p a r t i c u l a r t o x i c symptoms were observed following administration of 10 ng g" 1 of arsenobetaine to male mice; the arsenobetaine found i n the urine i s unchanged. Similar r e s u l t s were obtained by Cannon et a l . who also demonstrated the c o n s i s t e n t l y negative mutagenecity of arsenobetaine i n Ames' Salmonella typhimurium system f o r chemical mutagens. 1 3 When human volunteers ate witch flounder Glyptocephalus  cynoglossus. h a l f the ingested arsenic (5 mg of 10 mg per person) was excreted w i t h i n a day and there were no obvious changes i n the arsenic compound.^2 Other human volunteers who ate rock l o b s t e r containing arsenobetaine excreted the arsenic i n t h e i r u r i n e . 5 9 S i m i l a r r e s u l t s - 16 -were obtained f o r i n d i v i d u a l s who ate tissues of p l a i c e Pleuronecestes  p l a t e s s a . ^ 1.3 Scope of Work The r e s u l t s to date indicate that the water soluble organoarsenic compounds that have been i d e n t i f i e d - arsenobetaine, arsenocholine, tetramethylarsonium s a l t s and arsenosugars, are not the only ones present i n marine organisms. More organisms need to be examined i n the search f o r known and new organoarsenicals. Information about the structures and occurrence of these compounds i s important for the e l u c i d a t i o n of the pathways that are involved i n t h e i r formation and degradation and for i n d i c a t i n g possible changes to these compounds that may occur i n the food chain. Environmental factors as well as geographical locations need to be considered. This thesis i s concerned with the l e v e l s of arsenic and the nature of the water soluble arsen i c a l s present i n marine invertebrates - clams, mussels, oysters, cockles, crabs and s n a i l s - found i n B r i t i s h Columbia. Marine organisms from areas r e c e i v i n g sewage sludge, mine t a i l i n g s and pulp m i l l waste are examined and compared with organisms from r e l a t i v e l y unpolluted s i t e s . Graphite furnace atomic absorption (GFAA) and hydride generation AA methods are developed and used to determine arsenic i n the organisms. Organic arsenic compounds i n the organisms are extracted with methanol and i s o l a t e d by gel permeation and ion-exchange chromatography. HPLC - 17 -and thermospray LC-MS techniques are used to detect and i d e n t i f y the compounds present and NMR and Mass Spectrometry are employed f o r further confirmation. - 18 -EXPERIMENTAL 2.1 Instrumentation 2.1.1 Atomic Absorption Spectrometry A Varian Techtron Model AA 1275 Atomic Absorption (AA) Spectrometer was used f o r arsenic determinations. This was equipped with a Varian Spectra AA hollow cathode lamp operating at 7 mA. The 193.7 arsenic l i n e was used at a 1 nm sp e c t r a l s l i t w i d t h . The AA was equipped with a deuterium background corrector and a HP 82905A p r i n t e r . 2.1.1.1 Graphite Furnace Atomic Absorption Spectrometry Graphite furnace atomization was achieved with a Varian Techtron GTA-95 accessory using argon as the purge gas and Varian Techtron p y r o l y t i c a l l y coated graphite tubes. 2.1.1.2 Hydride Generation Atomic Absorption Spectrometry A continuous hydride generation system ( F i g . 2.1) was used f o r arsine generation. The design of the apparatus was s i m i l a r to that described by Sturman 7 3 except f o r the gas l i q u i d separator which was based on the design by Anderson et a l . 7 4 A Gi l s o n Minipuls 2 four channel p e r i s t a l t i c pump was used to pump a l l solutions and nitrogen was / QUARTZ ABSORPTI OlST CELL TRANSFER TUBE REACTION COIL PERISTALTI< PUMP e-*—^ GAS/LIQUID SEPARATOR SAMPLE 4-ACXD NaBH > N I TROGEN PRESSURE REGULATOR NEEDLE VALVES DRAIN F i g . 2.1: Schematic diagram of hydride generation assembly - 20 -used as the c a r r i e r gas. Atomization was achieved i n a conventional open end "T" shaped quartz absorption c e l l (8.5 cm x 1 cm (OD)) mounted i n the air/acetylene flame of a standard Varian burner. 2.1.2 High Pressure L i q u i d Chromatography (HPLC) The HPLC system consisted of Waters M45 and M510 pumps c o n t r o l l e d by Waters Automated Gradient C o n t r o l l e r . Samples were introduced onto the column v i a a Waters U6K i n j e c t o r . Chromophoric groups were detected using a Waters M418 v a r i a b l e wavelength detector and associated Waters QA-1 Data System. GFAA was used as the arsenic s p e c i f i c detector; f r a c t i o n s were c o l l e c t e d with a G i l s o n Micro Fractionator and tr a n s f e r r e d manually to the automatic sample d e l i v e r y system of the GTA-95. A C^g reverse-phase column (/iBondapak C^g 3.9 mm (ID) x 30 cm s t e e l , Waters) and anion exchange column (Protein Pak DEAE 5PW 7.5 mm (ID) x 75 cm steel,• Waters) were used for separations. 2.1.3 Mass Spectrometry and Nuclear Magnetic Resonance Spectrometry Mass s p e c t r a l data were obtained on a Kratos AES M50 spectrometer. A Vestec Kratos thermospray in t e r f a c e d to a Kratos MS80 RFA mass spectro meter was used f o r a l l liquid-chromatography-mass s p e c t r a l (LC-MS) an a l y s i s . -^H NMR spectra were obtained on Bruker WH400 spectrometer operating - 21 -at 400 MHz. Chemical s h i f t s are quoted r e l a t i v e to tetramethylsilane as external standard. D2O was used as the solvent i n a l l experiments. 2.2 Chemical and Reagents A l l chemicals used were of a n a l y t i c a l grade and obtained from commercial sources unless otherwise stated. Arsenobetaine, 5 7 arseno-c h o l i n e , 7 5 and tetramethylarsonium iodide 7** were prepared by using l i t e r a t u r e methods. A l l solvents used f o r HPLC were of HPLC grade and were f i l t e r e d through 0.45 /im M i l l i p o r e f i l t e r s before being used. Deionized water (Aquanetics Aqua Media system) was used f or AA methods and d e i o n i z e d - d i s t i l l e d water was used f o r HPLC a f t e r f i l t e r -a t i o n through M i l l i p o r e 0.45 pm f i l t e r s . A l l glassware and plasticware used was cleaned by soaking i t over-night i n 30% Decon solut i o n , r i n s i n g with water, and soaking i n d i l u t e hydrochloric a c i d overnight. U t e n s i l s were then r i n s e d with water and deionized water u n t i l the wash was neutral to litmus. Standard stock arsenic s o l u t i o n (1000 fig mL"^) f o r GFAA was prepared by t r e a t i n g 1.3202 g of A s 2 0 3 with 2 g NaOH and 20 mL water. A f t e r d i s s o l u t i o n , i t was d i l u t e d to 200 mL ne u t r a l i z e d with HC1 and made to 1000 mL. Arsenic reagents used were prepared by d i s s o l v i n g appropriate compounds i n 100 mL deionized d i s t i l l e d water to give solutions con-t a i n i n g 1000 fig mL"^ As. The compounds and weights used are as follows: - 22 -+ Arsenobetaine, 0.2613 g of (CH 3) 3AsCH 2C0 2-H 20 + Arsenocholine iodide 0.3892 g of (CH 3) 3AsCH 2CH 2OHI _ + Tetramethyl arsonium iodide, 0.3480 g of ( C H 3 ) 4 A s I _ Methylarsonic acid, 0.1840 g of (CH 3)AsO(ONa) 2-H 20 Dimethylarsinic acid, 0.3893 g of (CH 3) 2AsO-OH Arsenate, 0.4160 g of Na 2HAs0 4•7H 20 Arsenite, 0.1732 g of NaAs0 2 2.3 Sample S i t e and Sample C o l l e c t i o n 2.3.1 Thetis Island (Fig. 2.2) This i s a muddy beach on the secluded North Cove of Thetis Island, one of the Gulf Islands of B.C. This area has not been d i r e c t l y subject to p o l l u t a n t s . S h e l l f i s h , mainly clams, were dug at the i n t e r t i d a l zone at low t i d e . 2.3.2 Yellow Point (Fig. 2.2) This i s a a rocky beach, 20 km southwest of Nanaimo on Vancouver Island, an area not d i r e c t l y subject to p o l l u t a n t s . Oysters were c o l l e c t e d o f f the i n t e r t i d a l zone at low t i d e . F i g . 2.2: Thetis Island and Vancouver Island showing sampling locations 2.3.3 Sherard Point (Fig. 2.2) - 24 -This i s a g r a v e l l y beach southwest of Crofton, Vancouver Island. This area i s close to the Crofton Pulp and Paper M i l l waste o u t f a l l . Oysters were c o l l e c t e d o f f the i n t e r t i d a l zone at low t i d e . 2.3.4 Coles Bay (Fig. 2.3) This i s a muddy bay which receives sewage o u t f a l l on the west coast of the Saanich Peninsula, Vancouver Island, B.C. Clams were dug at the i n t e r t i d a l zone at low t i d e . 2.3.5 P a t r i c i a Bay (Fig. 2.3) This i s a large g r a v e l l y bay on the west coast of the Saanich Peninsula, Vancouver Island. This area i s close to the V i c t o r i a I nternational A i r p o r t and has a sewage o u t f a l l on the Southern side. Clams were dug at the i n t e r t i d a l zone at low t i d e . 2.3.6 Ouatsino Sound-Rupert-Holberg In l e t s ( F i g . 2.4) This area receives waste rock and ore t a i l i n g s from the Island Copper Mine located on the north shore of Rupert i n l e t . Clams were dug - 25 -F i g . 2.3: Saanich Peninsula showing sampling locations F i g . 2.4: Rupert-Holberg-Nerostos Inlets and Quatsino Sound showing sampling locations - 27 -at a g r a v e l l y beach near the copper mine. Mussels were c o l l e c t e d o f f a j e t t y close to the mine. 2.3.7 A l i c e Arm-Hastings Arm (Fig. 2.5) These are g l a c i a l l y fed f i o r d s which have been the center of numerous mining a c t i v i t i e s dating back to the 1900's. Samples from these areas were obtained during a cruise on the Canadian Survey ship J.P. T u l l y . Clams and mussels were c o l l e c t e d from the i n t e r - t i d a l zone at low t i d e . Crabs and whelks were caught i n crab pots. Other whelks and clams were caught i n trawl nets and i n grab samples. Grab sampling i s a technique which i s labor intensive and produces only a few speci-menfor the time spent. 2.3.8 Sample Storage A l l samples were kept i n fresh sea water f o r three days with p e r i o d i c change of the sea water. The samples were then frozen i n freezer bags at temperatures below -20°C u n t i l needed. - 28 -F i g . 2.5: A l i c e Arm and Hastings Arm showing sampling l o c a t i o n s - 29 -2.4 A n a l y t i c a l Procedures 2.4.1 Graphite Furnace Atomic Absorption (GFAA) The temperature-time parameters and i n e r t gas flow f o r each stage were optimized by using measurements of the absorbance s i g n a l during the atomization stage. Standard arsenic s o l u t i o n , 20 yiL of 1 ng mL"1, was in j e c t e d into the furnace by using the automatic d e l i v e r y system of the GTA-95. Ni c k e l n i t r a t e (20 /xL of 100 ng mL"1 solution) was added as a matrix modifier. The optimized furnace operating parameters f o r the determination of arsenic i s shown i n Table 2.1. Table 2.1: Furnace operating parameters Step Temperature Time Gas Gas Type Read Comment 0°C Sec. Flow Command 1 75 5.0 3.0 Normal Dry 2 90 30 3.0 Normal Dry 3 120 10 3.0 Normal Dry 4 1200 30 3.0 Normal Ash 5 1200 1.0 .0 Normal Ash 6 2300 1.0 .0 Normal * Atomize 7 2300 2.0 3.0 Normal Clean - 30 -2.4.2 Hydride Generation Atomic Absorption Spectrometry (HGAA) The absorption s i g n a l from a 0.1 ng mL'^ standard arsenic s o l u t i o n was studied i n order to e s t a b l i s h optimum operating conditions which were used f o r a l l subsequent determinations. These conditions are given i n Table 2.2 Table 2.2: Operating conditions of the hydride generation assembly Uptake tubes Sample: Acid: NaBH4: 2.80 mm I.D. 2.28 mm I.D. 2.29 mm I.D. PVC Viton PVC Uptake tubes Sample: Acid: NaBH4: 7.5 mL min'l 2.0 mL min" 1 4.0 mL min'l C a r r i e r gas Nitrogen 100 mL min'^ through mixing c o i l 25 mL min"^ through g a s - l i q u i d separator Hydrochloric a c i d concentration 4 M NaBH 4 concentration 2% ( i n 0.05% NaOH solution) Integration Run Mean - 31 -2.4.3 Determination of Tot a l Arsenic Two independent methods were used f o r sample decomposition, wet ashing with a mixture of n i t r i c acid, s u l f u r i c a c i d and hydrogen peroxide, and bomb decomposition with n i t r i c a c i d i n a microwave oven. 2.4.3.1 Wet Ashing with N i t r i c Acid. S u l f u r i c A cid and Hydrogen  Peroxide Wet ashing was c a r r i e d out i n a 250 mL round bottom f l a s k f i t t e d with a stopper having a c a p i l l a r y and an a i r condenser containing a d i f f u s i o n funnel ( F i g . 2.6). The stopper, funnel and plugs were made of T e f l o n . 7 7 The dried sample, 0.25 g, or wet sample, 1.0 g, was trans-f e r r e d into the f l a s k . N i t r i c acid, 3 mL of 69% , s u l f u r i c acid, 1 mL of 98% and hydrogen peroxide, 3 mL of 30% were added. The apparatus was placed i n a 250 mL heating mantle and heated f o r 3 h at 250°C i n a fume hood. The heat was switched o f f and the f l a s k allowed to cool. The digestate was tra n s f e r r e d to a 100 mL volumetric f l a s k and made to the mark with deionized water. Arsenic was determined i n the digestate by HGAA using the conditions l i s t e d i n Table 2.2. Where the sample s i z e was greater than 1.0 g, the solutions added were i n the r a t i o 1:3:3 mL H2S04:HN03:H202 per g. - 32 -F i g . 2.6: Wet-ashing apparatus. (A) Teflon c y l i n d r i c a l plugs; (B) Teflon d i f f u s i o n funnel; (C) Teflon stopper with c a p i l l a r y . (D) 250 mL round-bottomed f l a s k . A l l j o i n t s are 14/23. A l l dimensions are i n mm. Adapted from the design described by Bajo et a l . 7 2 - 33 -2.4.3.2 Microwave digestion The d r i e d material, 0.25 g, or wet sample, 1.0 g, was placed i n the T e f l o n pressure decomposition vessel (Parr Instrument Company, 45 mL). N i t r i c acid, 6 mL of 69% s o l u t i o n , was added. The v e s s e l was closed with i t s screw cap and heated f o r 2 mins at f u l l power (500 W) i n the microwave oven (Toshiba Model No. ERX 5610C). A f t e r cooling to ambient temperature, the s o l u t i o n was made to 100 mL i n a volumetric f l a s k and arsenic determined by GFAA and HGAA. Standard arsenic solutions were made i n n i t r i c a c i d blanks prepared by heating 6 mL concentrated n i t r i c a c i d i n the bomb for 2 min and d i l u t i n g to 100 ml. For GFAA, 20 (iL was i n j e c t e d into the furnace. Normal c a l i b r a t i o n s and standard additions were used. 2.4.4 Extraction of Organoarsenic Compound i n the Marine Organisms Frozen specimens were thawed and shucked. The s o f t tissues were bulked and homogenized i n a blender. A 10 g p o r t i o n of the homogenate was reserved f o r the determination of t o t a l arsenic by a c i d digestion. The homogenate was weighed and transferred into an Erlenmeyer f l a s k . Methanol, 2.5 mL per g of tissue was added. The f l a s k was stoppered with a rubber plug and l e f t on a mechanical shaker for 2 days. The methanol extract was f i l t e r e d o f f and the residue re-extracted with methanol f o r another 2 days. The extracts were combined and arsenic was determined by HGAA and GFAA. The residue was a i r d r i e d and arsenic was - 34 -determined by HGAA a f t e r a c i d digestion. 2.4.5 Determination of Arsenic i n Extracts The concentration of arsenic i n the extracts was determined by GFAA and by HGAA a f t e r UV decomposition. 2.4.5.1 Determination of Arsenic i n Extracts by GFAA Arsenic was determined by i n j e c t i n g 20 fjiL of the extract into the graphite tube d i r e c t l y without any sample pretreatment. However, i n most cases, i t was found necessary to d i l u t e aliquots of the extracts with deionized water to give a working concentration range of 10-100 ng mL"l As. Aqueous arsenic standards were used with normal or standard a d d i t i o n c a l i b r a t i o n . Determinations were done i n t r i p l i c a t e s and peak area absorbance was used i n most cases. 2.4.5.2 Determination of Arsenic i n Extracts by Hvdride Generation AA  A f t e r UV Decomposition (UV-HGAA) Aliquots of the extracts were made to 50 mL with water to give working concentrations between 10-100 ng mL'^ As. The solutions were placed i n quartz containers, 2.5 cm O.D. by 28 cm. The tubes, maximum - 35 -24, were arranged around a 1200 W medium pressure lamp (Hanovia) i n a fan cooled carousel (Fig. 2.7).78 (79 ^ h e solutions were i r r a d i a t e d f o r a s p e c i f i e d period of time (1-2 h). The solutions were allowed to cool, r e c o n s t i t u t e d to volume and analyzed by HGAA. In standard a d d i t i o n c a l i b r a t i o n s , As was added as arsenobetaine p r i o r to i r r a d i a t i o n . For normal c a l i b r a t i o n s aqueous arsenate standards were used. 2.4.6 High Performance L i q u i d Chromatography - Graphite Furnace Atomic  Absorption (HPLC-GFAA) Arsenate, arsenite, methylarsonic acid, dimethylarsinic acid, arseno-betaine and arsenocholine were used as standard arsenic compounds to develop the HPLC-GFAA system which was subsequently used to analyze the extracts. For separations on the Protein Pak column, 5 mM ammonium acetate s o l u t i o n was used as the mobile phase. The pH of the mobile phase was adjusted with a c e t i c a c i d or ammonium hydroxide to a working range of 4-10. The flow rate was maintained at 1 ml min" 1. Fractions were c o l l e c t e d at 30 second i n t e r v a l s by using the f r a c t i o n c o l l e c t o r and t r a n s f e r r e d to the GTA-95 sample cups; 20 ixL of the f r a c t i o n s were i n j e c t e d f o r GFAA. Water-methanol mixtures 5 mM with respect to tetrabutylammonium ( n i t r a t e , s u l f a t e or phosphate) or water-methanol mixtures 5 mM with respect to heptanesulfonic acid, served as a mobile phase f o r separa-tions on the reverse phase /jBondapak C^g column. The working pH range - 36 -F i g . 2.7: Apparatus f o r the photodecomposition of organoarsenicals i n s o l u t i o n : A, aluminum c y l i n d e r ; B, UV lamp; C, quartz sample tube; D, sample carousel; E, c o o l i n g fan; F, t e f l o n support - 37 -was kept between 4-7. Flow rate was 1 mL min'^ and f r a c t i o n s were c o l l e c t e d and analyzed as with the Protein Pak column. Varying concentrations of the standard arsenic solutions, 20 ^L volumes, were in j e c t e d to e s t a b l i s h r e t e n t i o n times and c a l i b r a t i o n curves. The p u r i f i e d extracts were then analyzed against the standard chromatograms. The residue obtained a f t e r the p u r i f i c a t i o n of the extract was dissolved i n 1 mL of water and passed through a 0.45 /im Durapore, membrane f i l t e r (Millex HV, M i l l i p o r e Corporation). The f i l t r a t e was determined f o r arsenic and 20 fth a l i q u o t s were in j e c t e d into the HPLC. 2.7 P u r i f i c a t i o n Procedures f o r Arsenic Compounds i n Extracts The procedures used for the ex t r a c t i o n of the s o f t - t i s s u e s of the organisms and the p u r i f i c a t i o n of the extracts are summarized i n F i g . 2.8. The combined methanol extract was evaporated to dryness on the Rotovapor to remove methanol. The residue was suspended i n a small volume of water. Repeated extractions with d i e t h y l ether were performed u n t i l the ether phase was c o l o r l e s s . The aqueous phase, which was regarded as a water soluble arsenic f r a c t i o n was evaporated to dryness on the Rotovapor and dissolved i n a small volume of water (10-20 mL). This s o l u t i o n was t r i t u r a t e d with methanol and the s o l u t i o n was f i l t e r e d from the insoluble residue which was discarded. The methanol s o l u t i o n was evaporated to about 10 mL. The s o l u t i o n was subjected to gel - 38 -Mollusk Ho™g«"fe« „ MeOH extract Ether/H20 partition * Y H20 layer Concentrate • H20 Sephadex LH 20 |Dowex 50W-X8 NH4 F i g . 2.8: Schematic representation of the procedures f o r the extraction of arsenic compounds from organisms and f o r p u r i f i c a t i o n of the extracts permeation chromatography on Sephadex LH-20, 2.5 cm x 60 cm column, e l u t i o n with methanol. Fractions, 12.5 mL volume, were c o l l e c t e d and As determined i n each by GFAA. The arsenic compounds eluted within one band, re t e n t i o n volumes of the s p e c i f i c extracts are given i n the r e s u l t s and discussion (Section 4.2). The As containing f r a c t i o n s were combined and evaporated to dryness on the Rotovapor. The residue was - 39 -diss o l v e d i n 10 mL of water and put onto a Dowex 50 W-X8 (H +) 200-400 mesh column. (1.5 cm x 20 cm or 2.0 cm x 30 cm). (The r e s i n was prepared by f i r s t e x tracting i t with acetone on a sintered-glass f i l t e r u n t i l the f i l t r a t e was c o l o r l e s s . I t was then washed with 2 M NaOH (3 times), then with d i s t i l l e d water u n t i l a l l the a l k a l i was eliminated. I t was washed with 2 M HC1 (3 times) and f i n a l l y with d i s t i l l e d water u n t i l n e u t r a l . The r e s i n was converted to the H + form by t h i s procedure). For the e l u t i o n of the arsenic compounds, the column was f i r s t washed with water, 100-200 mL (unadsorbed f r a c t i o n ) and then with 5% ammonium hydroxide sol u t i o n , 200-300 mL, (NH4OH f r a c t i o n ) , water 50 mL (water f r a c t i o n ) and 2 M hydrochloric a c i d (HC1 f r a c t i o n ) . Frac-tions , 12.5 mL were c o l l e c t e d and each f r a c t i o n analyzed f o r arsenic. The arsenic containing f r a c t i o n s i n the unadsorbed, NH4OH and HC1 fr a c t i o n s were bulked separately and concentrated on the Rotovapor. 2.7.1 I s o l a t i o n of Arsenic Compounds i n NH/,0H f r a c t i o n A schematic representation of the p u r i f i c a t i o n and i d e n t i f i c a t i o n of the arsenic compounds i n the NH4OH f r a c t i o n i s shown i n F i g . 2.9. The s o l i d obtained from the NH4OH f r a c t i o n was dissolv e d i n a minimum amount of water and subjected to gel permeation chromatography on a Sephadex LH-20 column (2.5 cm x 30 cm). E l u t i o n was c a r r i e d out with 500 mL water. Fractions of 12.5 mL were c o l l e c t e d . The arsenic containing f r a c t i o n s ( s p e c i f i c r e t e ntion volumes f o r the d i f f e r e n t organisms are given i n Section 4.2) were combined, evaporated to - AO -^ 1"LH20 |Dowex 2-xT (0) H2O (b) 0.2* HCI ^» |Dowex 50W-X8 thermospray L C M S NMR * NH4+ HPLC TLC Proteln-Pak silica F i g . 2.9: P u r i f i c a t i o n and i d e n t i f i c a t i o n of arsenic compounds i n NH4OH f r a c t i o n dryness, d i s s o l v e d i n water and chromatographed on a Dowex 2 x 8 column (1.5 cm x 50 cm, OH" form). The column was f i r s t washed with water, 100 mL, and then with 0.2 M HCI (200 mL). Fractions of 12.5 mL were c o l l e c t e d . The arsenic containing f r a c t i o n were concentrated to 10 mL and chromatographed on Dowex 50 W-X8 (1.5 cm x 20 cm, 200-400 mesh, H + form) with 0.2 M aqueous ammonium hydroxide, 200 mL, as the eluent. Fractions of 5 mL were c o l l e c t e d . The arsenic containing f r a c t i o n s were combined and evaporated to dryness. The residue obtained was dissolved i n a minimum amount of water. A small a l i q u o t of t h i s s o l u t i o n was examined by TLC on s i l i c a gel, ethanol-acetic acid-water (65:1:34, v-v-v) as eluent against standards of arsenobetaine, arsenocholine and tetramethylarsonium iodide. The spots were v i s u a l i z e d by exposure to iodine vapour. The Rf's of the arsenic containing spots were obtained -arsenobetaine 0.52, arsenocholine 0.32 and tetramethylarsonium iodide 0.38. The remaining s o l u t i o n was then s i m i l a r l y processed by TLC. The - 41 -area with Rf corresponding to the arsenic compound i d e n t i f i e d i n the preliminary t i c experiment was scraped,:homogenized i n 10 mL methanol and centrifuged. The methanolic extract from the scrapings was concentrated. F i n a l p u r i f i c a t i o n was achieved by HPLC on a Protein Pak DEAE column (7.5 mm x 7.5 cm) with 5 mM ammonium acetate s o l u t i o n , pH 6.65. The flow was maintained at 1 mL min" 1 and 0.5 mL f r a c t i o n s were c o l l e c t e d . The arsenic containing f r a c t i o n s ( f r a c t i o n numbers 5-7) were combined and evaporated to dryness. The residue obtained was analyzed further by NMR and MS. 2.7.2 I s o l a t i o n of Arsenic Compounds i n the HC1 F r a c t i o n The s o l i d obtained from the HC1 f r a c t i o n was d i s s o l v e d i n 0.2 M ammonium hydroxide, 20 mL, and subjected to gel permeation chromatogra-phy on a Sephadex LH-20 column (2.5 cm x 30 cm); water, 500 mL, was the eluent. Fractions, 12.5 mL, were c o l l e c t e d and analyzed f o r arsenic. The arsenic containing f r a c t i o n s (retention volumes between 150-200 mL) were combined and concentrated on the rotovapor to about 10 mL. The concentrate was chromatographed on a Biorex 70 column (1.5 cm by 50 cm) e l u t i o n with water, 100 mL, followed by 0.1 M HC1, 200 mL. Fractions, 12.5 mL, were c o l l e c t e d and analyzed. The arsenic containing f r a c t i o n s were bulked, concentrated and rechromatographed on Dowex 50 W-X8, e l u t i o n with 200 mL of 0.2 M ammonium hydroxide s o l u t i o n ; 5 mL fr a c t i o n s were c o l l e c t e d . The arsenic containing f r a c t i o n s were concentrated and p u r i f i e d by TLC and HPLC using the same procedures as f o r the NH4OH - 42 -f r a c t i o n , s e c t i o n 2.7.1. The f i n a l p u r i f i e d residue was analyzed by NMR and MS. A summary of the experimental scheme i s shown i n Fi g . 2.10. HCI 1 IH20 | »~ | Biorex 70 thermospray L C M S NMR TLC silica HPLC Protein-Pak F i g . 2.10: P u r i f i c a t i o n and i d e n t i f i c a t i o n of arsenic compounds i n HCI f r a c t i o n 2.8 Determination of Arsenic i n Shells A f t e r removal of s o f t t i s s u e s , the s h e l l s were washed with deionized water and a i r dried. The dr i e d s h e l l s were then crushed and ground to a fi n e powder i n a mortar. The ground s h e l l s , 1.0 g, was placed i n a 250 mL Erlenmeyer f l a s k and 15 mL of 2 M HCI was added. A f t e r d i s s o l u t i o n , the s o l u t i o n was f i l t e r e d to remove any insolub l e residue. The f i l t r a t e was made to 50 mL and arsenic determined by HGAA. Aliquots of the s o l u t i o n were then analyzed by HPLC-GFAA to determine the form of arsenic species present. - 43 -RESULTS AND DISCUSSION ANALYTICAL PROCEDURES 3.1 Determination of Arsenic Arsenic was determined by using graphite furnace atomic absorption (GFAA) and hydride generation atomic absorption spectrometry (HGAA). 3.1.1 Determination of Arsenic by using Graphite Furnace Atom  Absorption Spectrometry Atomic absorption spectrometry (AAS) i s the most widely used technique f o r the determination of arsenic. Of the 363 papers published on the various a n a l y t i c a l techniques used f o r the determination of arsenic between 1977 and 1981, 31% used AAS, 22% neutron a c t i v a t i o n , 16% molecular absorption spectrophotometry, 11% atomic emission spectro-metry, 10% electrochemistry, 7% X-ray emission spectrometry and 2% atomic fluorescence spectrometry.^ 0 In p a r t i c u l a r , graphite furnace atomizer AAS i s becoming the method of choice since flame AA systems are subject to s e n s i t i v i t y l i m i t a t i o n s . Graphite furnace methods are t y p i c a l l y 1000 times more s e n s i t i v e than flame methods f o r arsenic and determinations can be c a r r i e d out with only 5 piL of sample whereas flame methods require about 5 mL. In graphite furnace atomic absorption (GFAA) spectrometry, the sample s o l u t i o n (5-70 fiL) i s placed on the inner tube w a l l of an - 44 -e l e c t r i c a l l y heated tubular furnace, a graphite tube, placed i n the sample beam of an AA u n i t . The tube i s then heated i n three or more stages. The "dry" conditions are chosen to evaporate the solvent as quickly as possible without spattering. The second "char" stage (350-1250°C) removes v o l a t i l e sample components at a temperature as high as p r a c t i c a l without los s of analyte. The t h i r d stage involves the f a s t v a p o r i z a t i o n and atomization of the sample (2000-3000°C). The atoms r a p i d l y d i f f u s e out of the observation zone, and the r e s u l t i s a b r i e f absorption peak, the height or area of which can be r e l a t e d to the amount of analyte present. The GTA-95 used i n t h i s study has f a c i l i t i e s f o r the programming of temperature, time, gas flow and type of gas. The objective i n s e l e c t i n g operating parameters i s to s e l e c t parameters which w i l l completely desolvate the sample, remove the maximum amount of matrix material during the char stage, provide adequate a n a l y t i c a l s e n s i t i v i t y , and separate the analyte peak from non-atomic absorption peaks. This was achieved by optimizing the temperature-time parameters and gas flow rate f o r each stage by using the absorbance s i g n a l during the atomization stage. 76 The optimized program f o r the clam extracts i s shown i n Table 2.1. In the use of aqueous standards, the optimum char temperature i s 600°C, however, a higher char temperature of 1200"C i s necessary f o r the clam extracts due to the complex nature of the matrix. The a d d i t i o n of n i c k e l as a chemical modifier i s therefore e s s e n t i a l . Nickel i s added as n i c k e l c h l o r i d e or n i c k e l n i t r a t e i n a large excess. T y p i c a l l y , 20 fiL of a 100 ng mL"^ - s o l u t i o n i s added to the sample inside the graphite tube. The added n i c k e l forms a stable arsenide which atomize at a - 45 -higher temperature and permits a higher ashing temperature. Background c o r r e c t i o n i s used i n a l l cases. 3.1.1.1 C a l i b r a t i o n Limit of Detection and P r e c i s i o n of GFAA Analysis A t y p i c a l p l o t of absorbance against concentration f o r 20 /zL i n j e c t i o n s of a serie s of standard arsenite solutions i s shown i n F i g . F i g . 3.1: T y p i c a l c a l i b r a t i o n curve f o r the determination of arsenic by GFAA - 46 -3.1. A l i n e a r r e l a t i o n s h i p i s obtained f o r arsenic concentrations up to 0.2 /ig mL. Beyond t h i s concentration, curvature increases. The l i m i t of detection defined as the analyte concentration g i v i n g a si g n a l equal to the blank plus three standard deviations of the blank was determined to be 1 ng mL"1. The r e l a t i v e standard deviation f o r twenty i n j e c t i o n s of 20 (iL of 10 ng mL'1 standard arsenite s o l u t i o n was ca l c u l a t e d to be 6.2%. 3.1.2 Determination of Arsenic by Hydride Generation Atomic Absorption  Spectrometry Hydride generation atomic absorption (HGAA) spectrometry involves the production of v o l a t i l e covalent hydrides of a number of elements (As, B i , Ge, Te, Se, Sb, Sn and Pb) f o r determination by AA. This proves extremely u s e f u l because I t serves to separate the metal from other p o t e n t i a l l y i n t e r f e r i n g matrix component i n the sample. Matrix e f f e c t s are therefore less serious compared with GFAA techniques. S e n s i t i v i t y and detection l i m i t s of HGAA and GFAA techniques are quite comparable, however, the r e l a t i v e l y large sample volumes required f o r each HGAA measurement i s a major disadvantage f o r the analysis of some, e s p e c i a l l y b i o l o g i c a l , materials. The hydride generation technique i s most often used f o r the deter-mination of arsenite, arsenate, methylarsonic acid, dimethylarsinic a c i d 7 3 ' 7 4 , 8 2 " 8 7 and trimethylarsine o x i d e . 8 3 The reduction i s pH dependent which allows some speci a t i o n of m i x t u r e s . 8 ^ ' 8 6 Further - 47 -Q C s p e c i a t i o n can be accomplished by trapping (cryofocussing) the arsine mixture and making use of the r e l a t i v e v o l a t i l i t y of the warmed sample to e f f e c t separation. A l t e r n a t i v e l y , gas chromatography can be used to separate the arsines a f t e r c r y o f o c u s s i n g . 8 3 Two main reactions have been used f o r the generation of arsine. The f i r s t involves the reduction of arsenic by a metal-acid system such as zinc-hydrochloric a c i d . Zn + 2H + - Z n 2 + + 2H; 3H + A s 3 + - AsH 3 8 0 The r e a c t i o n however i s slow and r e s u l t s i n broad analyte response peaks and exh i b i t s poor r e p r o d u c i b i l i t y . In order to sharpen absorbance peaks, use can be made of b a l o o n s 8 3 to store the arsine and release i t i n a more reproducible manner. Zinc i s added i n the form of t a b l e t s 8 8 or powder. 8^ In another method, an a c i d i c s o l u t i o n of arsenic i s passed through a zinc column. 8 2 Apart from the slow nature of the reduction, i t i s often necessary to pre-reduce As(V) to As(III) and t h i s can be a lengthy process. A more e f f e c t i v e method for the production of arsines involves the sodium borohydride a c i d system, NaBH4" + 3H20 + H + - H3BO3 + 8H; 3H + A s 3 + - AsH 3 80 f i r s t used f o r t h i s a p p l i c a t i o n i n 1972,^ and since then has v i r t u a l l y replaced the metal-acid system for hydride generation. I n i t i a l e f f o r t s involved u t i l i z a t i o n of NaBH4 p e l l e t s dropped e i t h e r manually or by means of dosing f i t t i n g i nto a reac t i o n v e s s e l containing an ac i d s o l u t i o n of a r s e n i c . ^ I t i s now customary to use aqueous solutions of - 48 -NaBH4 from 0.5-8% w/v to generate the arsines, and t h i s procedure has f a c i l i t a t e d the development of automated systems: a review i s a v a i l -a b l e . 9 2 A f t e r many unsuccessful attempts, during the present i n v e s t i g a t i o n , the use of the metal-acid system (zinc column) was discarded and the sodium borohydride-acid system was adopted. The design of the hydride generator ( F i g . 2.1) i s s i m i l a r to that described by Sturman. 7 3 The te s t s o l u t i o n , hydrochloric a c i d and the sodium borohydride s o l u t i o n are pumped from t h e i r respective containers by the p e r i s t a l l i c pump and mixed. Simultaneously, the reduction of arsenic and the decomposition of sodium borohydride occurs producing arsine and hydrogen r e s p e c t i v e l y . The hydrogen evolved and the nitrogen added degasses the r e s u l t i n g s o l u t i o n during i t s passage through the mixing c o i l , following which the g a s - l i q u i d mixture passes into the separating c e l l . From here the gas i s swept by nitrogen into the heated quartz atomizer tube where i t i s detected. 3.1.2.1 Se l e c t i o n of Operating Conditions f o r HGAA The e f f e c t of hydrochloric a c i d concentrations between 0.5-8 M on the response of standard arsenite s o l u t i o n (0.1 fig mL"1) was determined by using 5% sodium borohydride s o l u t i o n at a f i x e d flow-rate. At concentrations between 2 and 8 M the response was almost in v a r i a n t , but i t decreases at lower concentrations. With standard arsenate however, the response peaked between 4 and 8 M. This behavior i s s i m i l a r to that - 49 -reported e l s e w h e r e . 0 0 ' 0 ' An a c i d concentration of 4 M was subsequently used f o r a l l determinations. The e f f e c t of varying the concentration of sodium borohydride s o l u t i o n on the response of 0.1 ppm Arsenite s o l u t i o n i s shown i n F i g . 3.2. The response i s at a maximum at concentrations between 1.5 and 2.0%, f a l l i n g o f f when the concentration i s reduced. At concentrations greater than 2.5% the response f a l l s r a p i d l y , probably owing to the interference e f f e c t of more hydrogen produced. A concentration of 2% w/v was subsequently adopted. 0.45-, F i g . 3.2: E f f e c t of NaBIfy concentration on the response of arsenite to HGAA 50 -The e f f e c t of the flow rates of sample so l u t i o n , a c i d s o l u t i o n and sodium borohydride s o l u t i o n on the analyte response was studied by using 0.1 /jg mL"1 standard arsenite s o l u t i o n , 4 M hydrochloric a c i d and 2% sodium borohydride s o l u t i o n . The flow rates were adjusted by changing the potentiometer s e t t i n g on the p e r i s t a l t i c pump. The e f f e c t of v a r i a t i o n s of the potentiometer s e t t i n g on the analyte response i s shown i n F i g . 3.3. The response increased from a s e t t i n g of 100 and l e v e l l e d o f f a f t e r a s e t t i n g of 500. The potentiometer was set at 500 for a l l subsequent determinations; the flow rates of the three solutions were 0.34 -| POTENTIOMETER SETTING F i g . 3.3: E f f e c t of potentiometer s e t t i n g on the response of arsenite to HGAA - 51 -then measured with a flow meter. Values of 7.5 mL min" 1, 2.0 mL min" , and 4.0 mL min"-'- were obtained r e s p e c t i v e l y f o r the sample s o l u t i o n , a c i d s o l u t i o n and sodium borohydride s o l u t i o n . The generating conditions selected f o r the operation of the hydride generator are shown i n Table 2.2. 3.1.2.2 C a l i b r a t i o n . Limit of Detection and P r e c i s i o n A t y p i c a l c a l i b r a t i o n graph for standard arsenite, obtained under the selected conditions, i s shown i n F i g . 3.4. A l i n e a r r e l a t i o n s h i p 0.4-| F i g . 3.4: T y p i c a l c a l i b r a t i o n curve f o r determination of arsenic by HGAA - 52 -between absorbance and concentration i s obtained up to a concentration of 50 ng mL"^. Curvature increases at higher concentration (above 100 ng mL"l) which i s t y p i c a l of atomic absorption measurements. A working range of 0-100 ng mL"-'-, with l i n e a r c o r r e l a t i o n c o e f f i c i e n t greater than 0.9 was maintained f o r a l l subsequent determinations. The l i m i t of detection defined as the analyte concentration giving a si g n a l equal to the blank, plus three standard deviations of the blank, was determined using the intercept and the standard d e v i a t i o n of the slope of the c a l i b r a t i o n curve close to the o r i g i n . This was estimated to be 0.3 ng mL'^. This value compares well with other l i m i t s of detection reported f o r hydride generation t e c h n i q u e s . 7 3 ' 8 2 The p r e c i s i o n of determinations were estimated from twenty r e p l i c a t e analysis of 10 ng mL~^ arsenite standard using the standard operating conditions l i s t e d i n Table 2.2. The r e l a t i v e standard deviation at th i s concentration i s 1.7%. 3.1.3 Comparison of GFAA and HGAA Table 3.1 presents a comparison of the sample s i z e , l i m i t of detection and p r e c i s i o n f o r determinations using GFAA and HGAA. The lower detection and better p r e c i s i o n of the HGAA makes t h i s the method of choice over GFAA when sample siz e i s not a l i m i t a t i o n . In most experiments, the two methods were used to complement each other. The disadvantages of the HGAA method include the requirement that the arsenic compounds must be reducible to arsines, and the necessity that the arsines possess s u f f i c i e n t v o l a t i l i t y to allow t h e i r transfer - 53 -Table 3.1: Comparison of GFAA and HGAA GFAA HGAA Sample Size Limit of Detection P r e c i s i o n (RSD) RSD: Relative standard deviation of twenty determinations of 10 ng mL"1 standard arsenic solutions. L i m i t of Detection: Analyte concentration giving a s i g n a l equal to the blank plus three standard deviations of the blank. to the atomizer. Many arsenic compounds e x i s t which do not f u l f i l l these requirements and t h i s c l a s s includes the n a t u r a l l y occurring organoarsenic compounds such as arsenobetaine, arsenocholine, and tetramethylarsonium s a l t s found i n marine organisms. A l l these a r s e n i c a l s have to be converted to reducible d e r i v a t i v e s i f HGAA techniques are to be employed. The technique used i n the present work fo r t h i s conversion i s discussed i n the section on the determination of arsenic i n the extracts; Section 3.3. 5-50 A*L >5 mL 1 ng mL"1 0.3 ng mL" 6.7% 1.7% - 54 -3.2 Determination of Total Arsenic The determination of arsenic by GFAA and HGAA requires that the sample be present i n s o l u t i o n . Thus i t was necessary to digest the marine organisms i n t h i s work to provide a s o l u t i o n s u i t a b l e f o r an a l y s i s . Many dige s t i o n procedures have been described.93-95 These include wet d i g e s t i o n of samples with various combinations of acids 7>1 4> 5 7,93,94,95 a n ( j base^° and dry ashing with magnesium nitrate.I 5'° 5 Wet ashing with a mixture of n i t r i c a c i d , s u l f u r i c a c i d and hydrogen peroxide and a bomb decomposition with n i t r i c a c i d i n a microwave oven were chosen f o r t h i s work. These systems avoid the use of p e r c h l o r i c acid. Work with p e r c h l o r i c a c i d requires s p e c i a l l y designed fume hoods which were not available to us. 3.2.1 Wet Ashing with a Mixture of S u l f u r i c Acid. N i t r i c A c id and  Hydrogen Peroxide The d i r e c t determination of arsenic i n t h i s digestate by GFAA does not give accurate and precise arsenic values due to severe interferences mainly associated with s u l f u r i c a c i d which i s not used up i n the course of the digestion. The interference from the a c i d i n the HGAA procedures i s minimal and HGAA can be used f o r the determination of arsenic i n the s o l u t i o n from decomposition with a c i d and peroxide. A v a r i e t y of standard reference materials were chosen f o r analysis with a view to e s t a b l i s h i n g conditions f o r optimum recovery. These - 55 -samples include National Bureau of Standards, orchard leaves SRM 1571 and bovine l i v e r SRM 1577, National Research Council of Canada (NRCC) marine a n a l y t i c a l standards, dogfish muscle DORM-1, dogfish l i v e r DOLT-1 and marine sediment PACS-1. A summary of the r e s u l t s obtained f o r the standard reference materials are shown i n Table 3.2. Table 3.2: Arsenic Determinations i n Standard Reference Materials Sample As c e r t i f i e d As found eg g ' 1 a A*g g" 1 b Dogfish muscle . 17.7 ± 2.1 17.2 ± 0.9 NRCC-DORM 1 Dogfish l i v e r 10.111.4 8 . 6 ± 0 . 6 NRCC-DOLT 1 Marine sediment 211 ± 11 203.4 ± 9 . 6 NRCC PACS 1 Orchard leaves 1.0 ± 2 9 . 8 ± 0 . 3 NBS SRM 1571 Bovine l i v e r 0.055 1 0.005 0.053 1 0.010 NBS SRM 15 a Mean and 95% tolerance l i m i t s . D Mean and standard deviation of mean f o r 6 determinations. - 56 -The arsenic concentrations found are i n agreement with the c e r t i f i e d values. The optimum conditions used to obtain these r e s u l t s involved r e f l u x i n g 0.250 g of the dry sample with 1 mL 98% H2SO4, 3 mL 67% HNO3 and 3 mL 30% H 202 for 3 h. In the case of wet samples, a 1 g sample was used and the volume of H2SO4, HNO3 and H 20 2 was maintained at 1, 3 and 3 mL, re s p e c t i v e l y . These volumes were maintained by comparing the dige s t i o n of a 0.250 g freeze dr i e d Manila clam sample with 1.000 g of wet sample which gave i d e n t i c a l r e s u l t s . In cases where sample sizes l a r g e r than 1 g were used, the reagent r a t i o s were adjusted p r o p o r t i o n a l l y . Generally, f o r the marine organisms, the r a t i o of s u l f u r i c acid, n i t r i c a c i d and hydrogen peroxide used was 1:3:3 mL per g wet t i s s u e . As a further t e s t of the e f f i c i e n c y of the di g e s t i o n procedure, the r e s u l t s of the analysis of Manila clam ti s s u e can be compared with that obtained by Neutron A c t i v a t i o n Analysis. I d e n t i c a l r e s u l t s of 6 fig g " l are obtained (Neutron A c t i v a t i o n Analysis was performed by Novatrak Analysis Limited, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, Canada). In the course of the optimization of the procedure, i t was found that losses occur when the r e f l u x i n g i s c a r r i e d out f o r longer periods. Refluxing f o r 5 h gives arsenic concentration of 9.8 ± 1.6 fig g~^ for DORM-1, representing recovery of only 56%. A recovery of 50% i s obtained a f t e r r e f l u x i n g f o r 6 h. These r e s u l t s are probably due to v o l a t i l i z a t i o n of the arsenic on prolonged heating. Shorter r e f l u x i n g times, f o r example 1 h, give incomplete d i g e s t i o n evidenced by lower recoveries, l e s s than 40%, and the presence of residue i n the dige s t i o n f l a s k . - 57 -3.2.2 Bomb Decomposition i n a Microwave Oven Following the manufacturer's recommendations, a maximum of 0.5 g dry material or 1.0 g wet material was used f o r a l l digestion. The recovery of arsenic from 0.5 g samples of National Research Council, Canada (NRCC) standard reference material D0LT-1 by using HGAA to analyze the so l u t i o n a f t e r the digestion i s shown i n Table 3.3 Table 3.3: Recovery of arsenic from D0LT-l a Volume of Reagent Used Time of Recovery HN03 (69%) H 2S0 4 HCI H 20 2 Radiation [sec] [%] 3. .0 _ 60 6.0 2, .0 0.5 - 60 15.3 2. .0 1.0 - 60 22.6 1. .5 1.5 - 60 45.7 1. .5 1.5 - 90 56.0 1. .0 3.0 - 90 22.3 1. .0 1.0 1.0 90 45.2 a Sample s i z e - 0.5 g - 58 -Recovery i s poor i f the HGAA technique i s used to analyze the so l u t i o n . A probable explanation f o r these low r e s u l t s i s that the organoarsenicals present i n the marine reference material are not decomposed by the various a c i d combinations to a form which i s r e a d i l y reduced by NaBH 4 to allow detection by HGAA. A s i m i l a r s i t u a t i o n was observed by Raptis et a l . 9 3 who attempted to decompose f i s h solubles with n i t r i c a cid. More rigorous conditions are therefore necessary i f HGAA techniques are to be used f o r subsequent an a l y s i s . This suggestion i s supported by the increased recovery found when s u l f u r i c a c i d i s added to the mixture. The p o s s i b i l i t y of pressure b u i l d up i n the bomb leading to explosions l e d to abandoning the pursuit of using more rigorous conditions. Analysis of the digestate with GFAA shows good recovery of arsenic i n two NRCC marine standards studied - DOLT-1 and DORM-1; Table 3.4. This r e i n f o r c e s the suggestion that incomplete decomposition i s the cause of the low recoveries found by using HGAA. Table 3 . 4 : Recovery of arsenic i n standard reference material Sample As c e r t i f i e d As found Pg g" 1 Mg g' 1 DOLT-1 DORM-1 17.7 ± 2.1 10.1 ± 1.4 17.2 ± 0.7 10.2 ± 0.5 - 59 -The recoveries shown i n Table 3.4 were obtained by using 0.2 g of the dry material and 6 mL of 69% n i t r i c acid, and a microwave r a d i a t i o n time of 2 min. For routine analysis of the marine samples, 1 g of the homogenized wet material i n 6 mL of 69% n i t r i c a c i d was i r r a d i a t e d f o r 2 min. 3.3 Determination of Arsenic i n Solution by Using HGAA Following U l t r a v i o l e t (UV) Decomposition Various methods have been employed to convert a r s e n i c a l s , which are themselves not r e a d i l y reduced by NaBH4 to v o l a t i l e arsines, to solutions to which HGAA techniques can be applied. The methods include wet d i g e s t i o n of samples with various combination of acid,^-7'1^>93 base^° and dry ashing with magnesium nit r a t e . * * 5 However, these methods are d i f f i c u l t to apply d i r e c t l y i f the sample i s already i n so l u t i o n . A photooxidation procedure 7^ which can be used to decompose organoarsenic compounds i n s o l u t i o n to arsenate was therefore developed during the present studies. This procedure was applied to the analysis of methanol extracts of the marine organisms. The e s s e n t i a l feature of the UV-HGAA technique was the i r r a d i a t i o n of the sample i n s o l u t i o n f o r a s p e c i f i e d time (usually 1-2 h) with a 1200 W medium pressure lamp. Any arsenic compounds present i n s o l u t i o n are photooxidized to arsenate allowing the subsequent qu a n t i t a t i o n of the arsenic i n s o l u t i o n by HGAA methods. Beattie et a l . ^ 7 were the f i r s t group to report the photooxidation - 60 -of organic matter i n water to carbon dioxide on exposure of the samples to r a d i a t i o n from a medium-pressure mercury vapour lamp. Subsequently Armstrong et a l . 7 8 confirmed t h i s observation by i r r a d i a t i n g sea water with a 1200 W mercury arc tube; various organic compounds added to the sea water, e.g. pyridine, adenine, ethanol, methanol and glucose, were oxidized s u b s t a n t i a l l y to completion within a 2-3 h i r r a d i a t i o n period. Measures and B u r t o n 9 8 used UV i r r a d i a t i o n as part of a procedure f o r determining selenium i n sea water; the selenium i s oxidized to selenate i n the process. Tarn" adopted the UV photooxidation procedure of Armstrong et a l . 7 8 to the oxidation of a c i d i f i e d aqueous solutions of 2 - a r s a n i l i c acid, [2-NH2'C6H4AsO(OH)2] sodium cacodylate [(CH3)2AsOONa] and arsenazo [ (0H>2-(S03Na>2 C10H3"N2• CgH4-AsOOHONa] . The arsenate produced was subsequently reduced to arsenite which was extracted with diethylammo-nium diethyldithiocarbamate into carbon t e t r a c h l o r i d e and determined by using carbon rod AA spectrometry. Stringer and Attrep-^0 found that arsenic present i n waste water solutions of (Cg^^AsO, CH3AsO(ONa)2 and (CH3)2AsO(OH) could be determined by HGAA techniques following photo-oxidation with a 450 W lamp f o r 4 h. In the present i n v e s t i g a t i o n , the a r y l a r s e n i c a l s 4-NH2C5H4AsO(OH)2, 3-OHC 6H 4AsO(OH) 2, 3-NH2-40HC6H3AsO(OH)2, 4-N0 2C 6H 4AsO(OH) and (C 6H 5) 3As were chosen as model compounds f o r the i n i t i a l studies on the e f f i c a c y of the photodecomposition procedure. The presence of the aromatic rings allows the use of HPLC with UV detection to monitor the rate of photo-oxidation of these a r y l a r s e n i c a l s . In order to study mixtures i t was f i r s t necessary to e s t a b l i s h conditions f o r the HPLC separations of - 61 -these compounds. 3.3.1 HPLC Separation of A r y l a r s e n i c a l s Some a r y l a r s e n i c a l s such as 4-NH2C5H4AsO(OH)2, ( A r s a n i l i c a c i d ) , 3- N0 2-4-OHC 6H 3AsO(OH) 2 (Roxarsone), 4-N0 2C 6H 4As0(0H) 2 (Nitarsone) and 4- NH2CONHCgH4-AsO(OH)2 (Carbasone) are approved as animal feed a d d i t i v e s ^ and the parent compound CgH5AsO(OH)2 has been found i n o i l s h a l e , i n 0 n shale process water, and i n shale o i l s . 1 ^ 2 Thus, apart from the reason ou t l i n e d i n the preceeding section, there i s a need to develop separation methods for t h i s c l a s s of compounds. The present i n v e s t i g a t i o n was concerned with the separation of a number of a r y l arsonic acids on a reversed phase HPLC column. A Waters ^Bondapak C^g column was used with water-methanol as the mobile phase. The separation obtained f o r the group of compounds used i n t h i s study i s shown i n Table 3.5. Water-methanol, 80:20 i s the mobile phase at a flow rate of 1 mL min'l. Triphenylarsine shows the longest retention time of 19.07 min. This can be a t t r i b u t e d to the l i p o p h y l i c i n t e r a c t i o n of the three phenyl groups with the stationary phase. The i n t e r a c t i o n with the water: methanol mobile phase i s les s predominant. With 4-amino-2-hydroxy-phenylarsonic acid, 4-aminophenylarsonic acid, 4-hydroxyphenylarsonic a c i d and nitrophenylarsonic acid, even though there i s the l i p o p h y l i c i n t e r a c t i o n of the phenyl groups with the octadecyl groups, the solute solvent i n t e r a c t i o n s predominate presumably due to the i o n i z a t i o n - 62 -Table 3.5: HPLC separation of a group on a r y l a r s e n i c a l s Compound Retention time (min) UV detector Retention time (min) GFAA 2-NH 2-4-OH(C 6H 3)AsO(OH) 2 Aminohydroxyphenylarsonic a c i d 2.08 2.5 4-NH 2(C 6H 4)AsO(OH) 2 A r s a n i l i c a c i d 2.28 2.5 4-0H(C 6H 4)As0(0H) 2 4-Hydroxyphenylarsonic a c i d 2.37 3.0 4-N0 2(C 6H 4)As0(0H) 2 4-Nitrophenylarsonic a c i d 2.41 3.0 (C 6 H 4 ) 3 A s Triphenylarsine 19.07 20.0 - 63 -of the arsonic acids. This leads to f a s t e l u t i o n and poor r e s o l u t i o n . In order to separate ionizable compounds on reversed-phase columns, two a l t e r n a t i v e strategies can be employed - ion suppression and ion-pair chromatography. In ion suppression, the pH of the mobile phase i s selected to i n h i b i t i o n i z a t i o n of the solutes. The solutes which are thus uncharged, w i l l i n t e r a c t more with the stationary phase leading to bett e r separations. This technique requires c a r e f u l c o n t r o l of pH and i s applicable only to weak acids and bases. Ion-pair chromatography on the other hand permits the separation of strongly i o n i z e d compounds. In e f f e c t i o n - p a i r i n g w i l l convert a reverse phase column into an i n - s i t u ion exchanger by the introduction of an i o n - p a i r i n g agent into the mobile phase. The p a i r i n g agent i s usu a l l y a long chain quaternary ammonium s a l t f o r separation of anions or a long chain s u l f a t e or sulfonate f o r separating cations. An Improvement i n the separation of the compounds l i s t e d i n Table 3.5 i s achieved by suppressing the i o n i z a t i o n of the arsonic acids by the a d d i t i o n of a c e t i c a c i d to the mobile phase. The chromatogram obtained i s shown i n F i g . 3.5. I t was found necessary to use the gradient program shown i n Table 3.6 to shorten the r e t e n t i o n time of tr i p h e n y l a r s i n e . With the exception of the f i r s t two compounds a good baseline r e s o l u t i o n i s obtained. Attempts to improve the separation by varying the gradient program proved f u t i l e . However, f o r the purposes of the i r r a d i a t i o n experiments the separation proved adequate. 64 -1 I » 1 I I I I 1 1 1 0 2 4 6 8 10 12 14 16 18 20 TIME C m ± } 3.5: HPLC trace of a mixture of: a, 2-OH-4NH2C6H3AsO(OH)2; b, 4-NH 2C 6H 4As0(0H) 2; c, 3-N02-4-OH-C6H3AsO(OH)2; d, 4-N0 2C 6H 4As0(0H)4; e, (C 6H 5) 3As. Each 10 n% mL - 1 of As, C^g column, 10 ^L i n j e c t i o n , gradient program as l i s t e d i n Table 3.6. The retention time (min) are given f o r detection at 254 nm - 65 -Table 3 . 6 : Gradient program f o r the separation of a r y l a r s e n i c a l s on reverse phase Time Flow rate A c e t i c a c i d Methanol curve* (min) (mL min" 1) 5 mM (%) % 0.00 1 80 20 -4.00 1 60 40 3 6.00 1 40 60 3 10.00 1 0 100 3 Selected curve p r o f i l e from options on the automated gradient c o n t r o l l e r 3.3.2 Photooxidation of Arvlarsonic Acids As a t e s t of the e f f i c a c y of the UV i r r a d i a t i o n procedure, the same mixture whose chromatogram i s shown i n F i g . 3.6A was i r r a d i a t e d f o r 20 min and rechromatographed, F i g . 3.6B. I t i s obvious that a l l aromatic arsenic compounds have been destroyed. To a s c e r t a i n the time needed to completely remove the aromatic groups - that i s break the arsenic-carbon bonds - 50 mL solutions with concentrations of 5, 10, 15, 20 and 25 /ig g" 1 as arsenic of the compounds were i r r a d i a t e d f o r 5, 10, 15, 20, 30 and 60 min. The chromatograms obtained show complete removal of the aromatic groups i n 30 min over the concentration range studied. The solutions with - 66 -2 4 6 8 10 12 TIME (min) F i g . 3.6: (A) HPLC chromatogram of 10 fig g" 1 as As of a mixture of a, 2-OH-4NH2C6H3AsO(OH)2; b, 4-NH 2C 6H 4As0(0H) 2; c, 3-N0 2-4-0HC 6H 3As0(0H) 2; d, 4-N0 2C 6H 4AsO(OH) 2 and e, (CgH5) 3As. (B) Chromatogram of the same mixture following UV i r r a d i a t i o n (20 min) - 67 -concentration of 5 /ig mL"l, however require only 10 min whereas the 10 and 15 /ig mL'^ solutions are e s s e n t i a l l y decomposed within 20 minutes. Thus the time required f o r complete decomposition depends on the amount of analyte there i s i n s o l u t i o n . 3.3.3 Nature of Arsenicals A f t e r Photodecomposition To a s c e r t a i n the nature of the products formed a f t e r the photo-oxidation, ion p a i r chromatography on the same reverse phase column was c a r r i e d out. This was found necessary because the conditions established for the separation of the a r y l a r s e n i c a l s (Table 3.6 and F i g . 3.5) are not s u i t a b l e f o r the separation of compounds such as arsenate and arsenite, possible products of the breakdown of the arsenic-carbon bonds. Arsenite, arsenate, methylarsonic acid, dimethylarsinic a c i d and arsenobetaine a l l elute with the solvent front under the conditions l i s t e d i n Table 3.6. The separation of these compounds using the tetrabutylammonium ion as a counter ion i n ion-pair chromatography i s discussed i n Section 3.4.2. The use of ion-pair chromatography established that the product of the photodecomposition i s arsenate. F i g . 3.7 shows the chromatogram for arsenite obtained before the photodecomposition. F i g . 3.8 i s the chromatogram of t h i s s o l u t i o n a f t e r the photodecomposition. The photo-oxidation of arsenite to arsenate has previously been d e s c r i b e d . ^ 3 - 68 0.13-0.10-O z < 8 o CO < 0.03 o.oo-6 a 1 RETENTION TIME 12 minutes 14 16 18 F i g . 3.7: HPLC-GFAA chromatogram of 10 /xg ml" 1 s o l u t i o n of arsenite; /iBondapak C^g column; water:methanol 95:5 with 5 mM t e t r a -butylammonium n i t r a t e as counter ion; pH 6.65; flow-rate 1 ml min" 1 0.13-0.10 O z < CO an o to m < o.os 0.00-1 1 ' t • •—i r*-*—i r-*-1—i ' ' M '' ' — i 0 2 4 6 8 10 12 14 16 18 RETENTION TIME minutes F i g . 3.8: HPLC-GFAA chromatogram of arsenite following UV i r r a d i a t i o n f o r 1 hour. Same conditions as i n F i g . 3.7. The ret e n t i o n time corresponded to that of arsenate - 69 -3.3.4 HGAA and GFAA of Solutions A f t e r Photodecomposition Because arsenate i s the product of the photodecomposition of solutions of ar s e n i c a l s , hydride generation from these solutions should allow convenient determination of the concentration of arsenic. Some re s u l t s r e l a t i n g to t h i s proposition are given i n Table 3.7. Table 3.7: HGAA and GFAA of a r y l a r s e n i c a l s before and a f t e r UV decomposition Compound HGAA Signal GFAA Signal Before UV A f t e r UV Before UV A f t e r UV Signal Signal Signal Signal 4-NH 2C 6H 4As0(0H) 2 0. .011 0. .125 0. .010 0, .011 3-OHC 6H 4AsO(OH) 2 0. .012 0. .124 0. .010 0. .012 3-NH2-4-OHC6H3AsO(OH)2 0. .011 0. .123 0. .011 0. .010 4-N0 2C 6H 4As0(0H) 2 0, .009 0. .122 0. .012 0, .013 10 ng mL"-1- of As solutions used. 20 /iL were i n j e c t e d f o r GFAA measurements. Solutions, 40 /iL, were i r r a d i a t e d f o r 1 h p r i o r to repeat measurements by GFAA or HGAA. Average of 4 determinations - 70 -The GFAA r e s u l t s of the solutions before and a f t e r i r r a d i a t i o n show good agreement and indicate no s i g n i f i c a n t losses a f t e r the i r r a d i a t i o n . The s l i g h t differences i n absorbance units may be due to matrix e f f e c t s . Hydride generation from t h i s c l a s s of compounds does occur. However, the low v o l a t i l i t y of the compounds ArAsH2 produced makes the response f o r the s i g n a l small. R i c c i et a l . 1 ^ obtained a s i m i l a r r e s u l t where the s i g n a l generated from N^CgH^AsOCOH^ i s about 200 times lower than that from the arsenate. These authors found that ad d i t i o n of per s u l f a t e r e s u l t e d i n decomposition of the a r y l a r s e n i c a l to arsenate and increased the r e l a t i v e response to become nearly equivalent to that of arsenate. Photodecomposition of the a r y l a r s e n i c a l s greatly increases the response following hydride generation because of the formation of arsenate which i s reduced to the more v o l a t i l e ASH3. The i r r a d i a t i o n of 50 mL solutions of 10 fig mL"1 As of arseno-+ + betaine, (C^^AsC^COO", arsenocholine iodide ( C ^ ^ A s C ^ C ^ O H - I " and tetramethylarsonium iodide (CH3) 4As +.I" was c a r r i e d out f o r 1 hour. The GFAA and HGAA signals f o r the solutions were obtained before and a f t e r i r r a d i a t i o n . Table 3.8 shows the r e s u l t s obtained. As expected no hydride generation Is observed f o r the solutions p r i o r to the UV i r r a d i a t i o n . However, the arsenate produced on i r r a d i a -t i o n i s r e a d i l y reduced by NaBH4 leading to the response i n the HGAA s i g n a l . The GFAA signals before i r r a d i a t i o n were lower than that a f t e r the i r r a d i a t i o n . The GFAA program used was that optimized f o r inorganic arsenic thus the lower response f o r the solutions before I r r a d i a t i o n might be due to losses during the ashing stage. The three compounds showed differences i n response. In order to get equivalent s i g n a l s , the - 71 -Table 3 . 8 : HGAA and GFAA of arsenobetaine, arsenocholine iodide and tetramethylarsonium iodide before and a f t e r UV decomposition HGAA S i g n a l 0 GFAA S i g n a l b Compound2 Before UV A f t e r UV i r r a d i a t i o n i r r a d i a t i o n Before UV A f t e r UV i r r a d i a t i o n i r r a d i a t i o n (CH 3)3AsCH 2C02' 0.000 0.113 0.004 0.012 [(CH3)3AsCH 2CH 2OH]I _ 0.000 0.110 0.009 0.012 (CH 3) 4As]I 0.000 0.110 0.009 0.013 a 10 ng mL'l As of 50 mL solutions were i r r a d i a t e d f o r 1 hour, k Average of four determinations. program should be optimized f o r each compound. This reveals one of the advantages of the photooxidation procedure. The production of arsenate i n a l l cases eliminates the problems associated with the d i f f e r e n t responses of d i f f e r e n t compounds. A s i m i l a r e f f e c t i s noted when the GFAA s i g n a l f o r triphenylarsine before and a f t e r i r r a d i a t i o n i s compared. There i s a marked improvement i n response a f t e r i r r a d i a t i o n . - 72 -3.3.5 E f f e c t of Acid on Photodecomposition The UV-HGAA responses generated f o r a r s a n i l i c a c i d and arsenobetaine were studied i n various acid media. Solutions containing 10 ng ml" 1 of As were prepared i n O , 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 M ac i d solutions and i r r a d i a t e d f o r 1 h. Figures 3.9-3.12 show the responses obtained. Relative standard deviations from the mean of absorbance f o r each s o l u t i o n are 2.1% f o r HN03, 1.3% for HCI, 1.9% for H 2S0 4 and 2.0 for CH3COOH. Interferences from these acids solutions over the concentra-t i o n range studied i s therefore considered minimal. Similar r e s u l t s are obtained when 10 ng ml solutions of arsenobetaine are i r r a d i a t e d i n the aci d s o l u t i o n s . 0.10 Ld CJ -z. < m Cn 0 . 05 -o IT) CD < 0 .00 T 0 1 2 3 M O L A R I T Y O F ACID F i g . 3.9: E f f e c t of n i t r i c a c i d on the response of 10 ng mL" a r s a n i l i c a c i d to UV-HGAA - 73 -0.10-1 Ld o < g 0 .05 o m < 0.00 0 1 2 3 MOLARITY OF ACID F i g . 3.10: E f f e c t of s u l f u r i c a c i d on the response of 10 ng mL"*-a r s a n i l i c a c i d to UV-HGAA 0.10 o -z. < §2 0 . 0 5 -o m < 0 .00 0 1 2 3 M O L A R I T Y O F ACID F i g . 3.11: E f f e c t of hydrochloric a c i d on the response of 10 ng mL"l a r s a n i l i c a c i d to UV-HGAA 74 -0.10-. L d o < CD £ 0.05 o CD < 0.00 0 1 2 3 M O L A R I T Y O F ACID F i g . 3.12: E f f e c t of a c e t i c a c i d on the response of 10 ng mL"l a r s a n i l i c a c i d to UV-HGAA The signals generated from 1 ng mL"1 As arsenobetaine solutions i r r a d i a t e d f o r 15, 30 and 60 minutes res p e c t i v e l y , were obtained i n the a c i d media. Figs. 3.13, 3.15 and 3.15 show the signals f o r the s o l u t i o n i n H2SO4. The r e l a t i v e s i g n a l i n t e n s i t i e s i n d i c a t e that higher acid concentrations require longer i r r a d i a t i o n times to generate signals i n d i c a t i v e of complete oxidation. The 3 M a c i d s o l u t i o n required about 1 hour to generate a s i g n a l comparable to that of the 0.5 M s o l u t i o n i r r a d i a t i o n f o r 30 minutes. The solutions of n i t r i c acid, hydrochloric a c i d and a c e t i c a c i d show s i m i l a r behaviour. The longer i r r a d i a t i o n times required f o r higher acid concentrations may be a t t r i b u t e d to the 75 -1 - i Ld o < CD C£ O 00 CD < 0.5-1 ' , — 0 1 2 3 MOLARITY OF ACID F i g . 3.13: Response of 1 pg ml"! arsenobetaine s o l u t i o n i n various H2SO4 solutions to UV-HGAA, 15 min i r r a d i a t i o n < CD 0. CC O 00 CD < 5-T 0 1 2 3 MOLARITY OF ACID F i g . 3.14: Response of 1 ng ml"* arsenobetaine s o l u t i o n i n various H2SO4 solutions to UV-HGAA, 30 min i r r a d i a t i o n - 76 -1 - i 0.5-0 1 2 3 4 MOLARITY OF ACID F i g . 3.15: Response of 1 fig m l - 1 arsenobetaine s o l u t i o n i n various H2SO4 solutions to UV-HGAA, 60 min i r r a d i a t i o n quenching e f f e c t of the increase i n number of species i n s o l u t i o n . However, t h i s i s not considered to be a major problem when applying the photodecomposition to extracts of clam ti s s u e , because the solvent i s usu a l l y water or methanol. 3.3.6 E f f e c t of Organic Solvents on Photodecomposition The e f f e c t of three organic solvents on the response of arsenobe-taine and a r s a n i l i c a c i d was investigated. The i r r a d i a t i o n s were c a r r i e d out i n water-methanol, water-ethano1, and w a t e r - a c e t o n i t r i l e - 77 -solutions of both arsenobetaine and arsenocholine. The breakdown of the arsenic carbon bonds i n 10 ng mL"1 As solutions i s e s s e n t i a l l y complete with i n 30 minutes of i r r a d i a t i o n time over the concentration range of 0-100-% of the organic solvents. Relative standard deviations of 1.0% are obtained. Interference or quenching e f f e c t s from organic solvents seems to be much les s than from a c i d solutions which show r e l a t i v e standard deviations of 1.5-2.3%. 3.3.7 C a l i b r a t i o n Curves. Limit of Detection and P r e c i s i o n of UV-HGAA In order to study the concentration range over which the UV HGAA technique can be used, c a l i b r a t i o n curves were obtained f o r the com-pounds i n Table 3.7 and 3.8. Solutions, 50 mL, were i r r a d i a t e d f o r 1 hour and analyzed by HGAA. A t y p i c a l p l o t f o r arsenobetaine i s shown i n Fi g . 3.16. The curve i s l i n e a r at low arsenic concentrations (less than 100 ng mL"1) but the curvature increases at higher l e v e l s . The curves f o r the ar y l a r s o n i c acids follow the same pattern, however the curves f o r arsenocholine iodide and tetramethylarsonium iodide show a greater d e v i a t i o n from l i n e a r i t y at higher arsenic concentrations (greater than 1 ng mL" 1). This behaviour might be due to a quenching e f f e c t of the iodide ion on the i r r a d i a t i o n . This was not investigated f u r t h e r since a working range of 0-200 ng mL"1 (the l i n e a r portion of the c a l i b r a t i o n curve) was adopted f o r a l l subsequent a n a l y s i s . By using the c a l i b r a t i o n curve close to the o r i g i n , F i g . 3.16 ( i n s e r t ) , the l i m i t of detection defined as the analyte concentration F i g . 3.16: T y p i c a l c a l i b r a t i o n curve f o r arsenobetaine by using UV-HGAA - 79 -gi v i n g a s i g n a l equal to the blank plus three standard deviations of the blank can be estimated. This value i s 0.3 ng mL'^. Ten solutions of arsenobetaine with concentration of 1 ng mLT^ were i r r a d i a t e d f o r 30 min and analyzed by HGAA. The r e l a t i v e standard de v i a t i o n of the ten measurements i s ca l c u l a t e d to be 0.9%. 3.3.8 Recovery Studies The p o t e n t i a l a p p l i c a b i l i t y of the UV-HGAA technique to methanol extracts of the marine organisms was investigated by recovery studies of arsenobetaine, a common constituent i n extracts of marine organisms. Varying amounts of arsenobetaine were added to a se r i e s of clam extracts and the solutions i r r a d i a t e d with the UV lamp. I n i t i a l l y , 1 mL arseno-betaine solutions of varying concentrations were added to 49 mL of the extracts and i r r a d i a t e d f o r 1 hour. Analysis by HGAA shows very poor recoveries, l e s s than 10%. The recoveries increase to about 50% when the solutions are i r r a d i a t e d f o r 4 h. However, when the extracts are d i l u t e d 20-50 times with water and i r r a d i a t e d f o r 1 h better recoveries of over 80% are obtained. Generally, the greater the amount of the extract used, the longer the i r r a d i a t i o n time necessary to achieve complete decomposition. The recoveries obtained f o r two clam extracts are shown i n Table 3.9. Varying amounts of arsenobetaine, 0, 10, 20, 30, 40 and 50 ng mL"l As, were added to 1 mL extract and made to 50 mL. The solutions were then i r r a d i a t e d f o r 2 h and arsenic determined by HGAA. S a t i s f a c t o r y recoveries are obtained, with a mean of 97.3% and a - 80 -Table 3.9: Recovery of Arsenic from Samples of Clam Extracts Sample Arsenic Added a (ng mL"1) Arsenic Found c (ng mL"1) Recovered (%) Manila Clam Extract 0 10.0 20.0 30.0 40.0 10.6 20.5 30.8 40.4 49.5 99.1 101.8 98.1 89.6 Horse Clam Extract 0 10.0 20.0 30.0 40.0 50.0 7.8 17.9 27.1 38.1 47.4 57.5 101.2 91.0] 103.8 94.8 96.1 a as arsenobetaine. k arsenic determined by HGAA following UV i r r a d i a t i o n f o r 2 h. standard d e v i a t i o n of 4.7. An i r r a d i a t i o n time of 2 h was used i n order to ensure complete decomposition of the organoarsenicals i n s o l u t i o n . - 81 -3.3.9 Summary of UV-HGAA In view of the r e s u l t s discussed so f a r , good r e p r o d u c i b i l i t y (0.9-2.7% r e l a t i v e standard deviations), low detection l i m i t (around 0.3 ng mL"1) and good recoveries (97.1 ± 4.7%), one may conclude that the UV-HGAA technique o f f e r s a convenient, s e n s i t i v e and r e l i a b l e method for the determination of arsenic i n extracts of marine organisms. The method eliminates the use of acids and bases f o r sample preparation and therefore eliminates interferences from these acids and bases. I t i s r a p i d and up to 24 samples can be i r r a d i a t e d at a time. 3.3.10 Comparison of GFAA and UV-HGAA Determinations of Arsenic i n  Extracts Extracts, 1 or 2 mL, were placed i n 50 mL volumetric f l a s k s . Arsenobetaine solutions were added and the solutions made to volume to give concentration of added As as 0, 20, 40, 60, 80 and 100 ng mL"1. The solutions were placed i n the quartz tubes and i r r a d i a t e d f o r 2 h. Arsenic was determined by HGAA. For comparative purposes, aliquots of the solutions prepared i n a s i m i l a r manner were analyzed by GFAA. For the GFAA ana l y s i s , 20 fiL of the solutions were inje c t e d ; matrix modifi-c a t i o n and background c o r r e c t i o n was employed. The use of background c o r r e c t i o n i s c r i t i c a l i n the analysis; as erroneously high values are obtained without i t . D i l u t i o n of the extracts i s also e s s e n t i a l . Undiluted solutions produced smoke i n the ashing and atomization stages - 82 -r e s u l t i n g i n negative absorbance readings because of over c o r r e c t i o n and s c a t t e r i n g of the analyte s i g n a l . Table 3.10 l i s t the r e s u l t s f o r the analysis of two clam extracts. The amount of arsenic found i n both samples using the two techniques i s i d e n t i c a l , 124.8 and 123.9 ng mL"1 f o r the Manila clam extract and 28.9 and 27.1 ng mL"1 f o r the horse clam extract. The r e l a t i v e standard deviations of the slope and intercepts of the UV-HGAA are bett e r than that of GFAA. The photooxidation followed by HGAA minimizes the interference from the organic matrix which a f f e c t s the GFAA determina-tions . Table 3.10: Analysis of clam extracts by UV-HGAA and GFAA Manila clam Horse clam GFAA UV/HGAA GFAA UV/HGAA r 0.983 0.992 0.984 0.974 b (RSD) 0.233 (2. .23) 0.104 (0.67) 0.0314 (2. .44) 0.0102 (0. .09) a x 10 4 (RSD) 18.9 (0. 14) 8.4 (0.04) 10.8 (5. .16) 3.76 (0. .1) [As] ng mL"1 123.9 124.8 27.1 28.9 r — c o e f f i c i e n t of l i n e a r regression of y - ax + b. RSD - r e l a t i v e standard deviation. - 83 -The arsenic concentration i n a selected number of extracts obtained using the two methods i s given i n Table 3.11. The scatte r p l o t i s shown i n F i g . 3.17. A good c o r r e l a t i o n i s obtained f o r the two techniques. A Table 3.11: Arsenic concentrations i n extracts by using GFAA and UV-HGAA Arsenic concentration /ig mL'1 Sample (source) UV-HGAA GFAA Manila clam 1.24 1.24 (Thetis Island) Horse clam 0.28 0.27 (Thetis Island) Manila clam 1.11 1.18 ( P a t r i c i a Bay, f i r s t extract) Manila clam 0.55 0.41 ( P a t r i c i a Bay, second extract) Native l i t t l e n e c k clam 0.70 0.67 ( P a t r i c i a Bay) Geoduck 0.49 0.46 (Chinatown, f i r s t extract) Geoduck 0.20 0.24 (Chinatown, second extract) Geoduck Siphon 0.35 0.30 (Chinatown, f i r s t extract) Geoduck Siphon 0.22 0.25 (Chinatown, second extract) Geoduck Foot 0.37 0.40 (Chinatown, f i r s t extract) Geoduck Foot 0.20 0.18 (Chinatown, second extract) - 84 -1.4-1 1.2 < H O O 0.8 H y 0.6-00 or < 0.4 H 0.2 o 4 i 1 1 1 1 1 1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 ARSENIC CONC. UV-HGAA F i g . 3.17: Scatter diagram f o r the determination of arsenic i n extracts by GFAA and UV-HGAA l i n e a r regression c o e f f i c i e n t of 0.988 i s obtained. Thus f or routine analysis, the two methods are interchangeable. 3.4 Determination of Arsenic Species by High Pressure L i q u i d Chromato-graphy (HPLC) The arsenic compounds present i n marine organisms are neither v o l a t i l e nor e a s i l y converted to v o l a t i l e d e r i v a t i v e s . HPLC i s the - 85 -method of choice f o r the separation of such non v o l a t i l e compounds. However, i n order to e s t a b l i s h the number and concentrations of organo-ar s e n i c a l s i n the sample, the commercially a v a i l a b l e non s p e c i f i c detectors ( u l t r a v i o l e t , r e f r a c t i v e index, fluorescence, conductivity) are of l i m i t e d use. Several reports demonstrate that element s p e c i f i c detection can be achieved by i n t e r f a c i n g HPLC with a graphite furnace atomic absorption spectrometer, HPLC-GFAA.105-108 i n d u c t i v e l y coupled plasma (ICP) spectrometry has also been used f o r arsenic s p e c i f i c detection following chromatographic separations.^09-111 HPLC-GFAA procedure involves the l i q u i d chromatographic separation of the com-pounds under i n v e s t i g a t i o n ; the chromatographic e f f l u e n t i s then p e r i o d i c a l l y examined by using the GFAA spectrometer. The sampling can be done v i a a s p e c i a l l y designed sampling cup^6 o r f r a c t i o n s can be c o l l e c t e d and transferred manually to the GFAA spectrometer.^ 5 The l a t t e r technique was employed i n t h i s work. Fractions, usually 0.5 mL were c o l l e c t e d with a G i l s o n f r a c t i o n c o l l e c t o r and transferred to the automatic sample d e l i v e r y system of the GTA-95. This avoided the hardware problems associated with the automated system and allowed f l e x i b i l i t y i n the use of the AA spectrometer. 3.4.1 Separations on Ion-exchange Column Various ion-exchange r e s i n s 1 ^ 8 « 1 1 ^ > 1 1 2 have been used f o r the separation and determination of arsenite, arsenate, methylarsonate, dimethylarsinate, arsenobetaine, arsenocholine and tetramethylarsonium - 86 -s a l t s . Morita et a l . 1 1 * ^ used ion-exchange columns to separate arsenate, arsenite, dimethylarsinate, methylarsinate and arsenobetaine and s u c c e s s f u l l y applied the method to the analysis of a seaweed sample. Their system was subsequently used to i d e n t i f y arsenobetaine i n the muscle and l i v e r of a shark Prionace g l a u c u s . 1 7 Brinkman et a l . d e s c r i b e d the separation and determination of arsenate, arsenite, dimethylarsinate and methylarsonate by using the HPLC-GFAA system with strong c a t i o n and anion-exchange resins as well as reversed-phase columns. These a n a l y t i c a l procedures were then applied to the determination of arsenic compounds i n drinking water and s o i l e xtracts. The separation of arsenite, arsenate, methylarsonate, dimethylarsi-nate , arsenobetaine, arsenocholine and tetramethylarsonium iodide on a weak ion exchanger i s investigated i n t h i s work. A 7.5 mmx 7.5 cm Waters Protein-Pak DEAE s t e e l column was used. The column packing i s a f u l l y porous h y d r o p h i l i c polymeric r e s i n onto which i s bound dimethyl-aminoethyl f u n c t i o n a l groups. This packing can be used over a wide pH range, from 2-12, which allows considerable f l e x i b i l i t y i n optimizing separations. The eluents used with t h i s column are d i l u t e , u s u a l l y m i l l i m o l a r , solutions of s a l t s , acids, bases or any combination of the three because the column has very low loading of exchange s i t e s . The present study u t i l i z e s 5 mM solutions of sodium acetate, pH adjusted to 4 with acetic a c i d , ammonium acetate pH 6.65 and ammonium acetate, pH adjusted to 10 with ammonium hydroxide. F i g . 3.18 indicates a t y p i c a l separation for a mixture of arsenobe-- 87 -taine, arsenite, arsenate, methylarsonate, and dimethylarsonate on the Protein-Pak column; the pH of the eluent, 5 mM sodium acetate, i s adjusted to 4 with a c e t i c acid. Arsenobetaine, arsenite and dimethylarsinate coeluted immediately a f t e r the solvent front; methylarsonate and arsenate are well resolved; however, arsenate has a long r e t e n t i o n time of over 40 min. 0.15-1 0.10-U 0 z < K 0 W 0.05 ffl < 0.00-if o a be w / -1 0 1 5 4 0 4 5 ION TIME C min ) F i g . 3.18: Separation by HPLC-GFAA of: a, arsenobetaine; b, arsenite; c, dimethylarsinate; d, methylarsonate; e, arsenate; Waters Protein-Pak DEAE column; 20 j i L of sample containing 500 ng As of each As compound placed on column; mobile phase 5 mM sodium acetate pH adjusted to 4 with a c e t i c a c i d ; rate 1 mL min - 1; f r a c t i o n s c o l l e c t e d every 30 sec; 20 juL flow of f r a c t i o n s i n j e c t e d i n t o graphite tube. - 88 -In order to resolve the three compounds coeluting together, the eluent was changed to ammonium acetate with a pH of 6.65. The chromato-gram obtained i s shown i n F i g . 3.19. Dimethylarsinate i s resolved from arsenobetaine and arsenite. The retention time of methylarsonate increases to over 60 min and arsenate i s not eluted from the column even a f t e r 80 min. 0.15-1 0 t < K 0 W < 0.10-0.05-0.00 ab 10 1 5 60 65 ION TIME C Bin > F i g . 3.19: Separation by HPLC-GFAA of: a, arsenobetaine; b, arsenite; c, dimethylarsinate; d, methylarsonate; Waters Protein Pak DEAE column; 20 /iL sample containing 500 ng As of each As compound placed on column; mobile phase 5 mM ammonium acetate, pH 6.65; flow rate 1 mL min" 1; f r a c t i o n s c o l l e c t e d every 30 sec; 20 /xL of f r a c t i o n s i n j e c t e d into graphite tube. - 89 -On changing the eluent to 5 mM ammonium acetate, the pH adjusted to 10 with ammonium hydroxide, arsenobetaine and arsenite are resolved, F i g . 3.20. Arsenate and methylarsonate are not eluted from the column a f t e r 80 min. Thus by varying the composition of the eluent, a separa-t i o n of the 5 compounds i s achieved. 0.15n W 0 z < 0 (/) EC < 0.10-0.05-0.00 T 1 5 xL LL. 10 1 5 20 RETENTION TIME < «in > F i g . 3 . 2 0 : Separation by HPLC-GFAA of: a, arsenobetaine; b, arsenite; c, dimethylarsinate; Waters Protein Pak DEAE column; 2 0 / J L sample containing 5 0 0 ng As of each As compound placed on column; mobile phase 5 mM ammonium acetate, pH adjusted to 1 0 with ammonium hydroxide; flow rate 1 mL min" 1; f r a c t i o n s c o l l e c t e d every 3 0 sec; 2 0 fiL of f r a c t i o n s i n j e c t e d Into graphite tube. - 90 -The long r e t e n t i o n times of arsenate and methylarsonate at pH 10 are not s u i t a b l e f o r routine a p p l i c a t i o n f or detection and quantitation. Generally, the column was run at pH 4 for the detection and q u a n t i s a -t i o n of arsenate and then at pH 10 f o r work with arsenobetaine, arsenite and dimethylarsinate. As seen from the three chromatograms, Figs. 3.18, 3.19 and 3.20, the a c i d i t y of the eluent has a considerable e f f e c t on the separation. This i s r e l a t e d to the pKa of the solute. The pKa i s the pH at which the ion i s h a l f i n the protonated form and h a l f i n the deprotonated form. Generally at one pH u n i t above the pKa, the predominant form of the ion i s the le s s protonated form; at one pH u n i t below pKa, the more proto-nated form predominates. Arsenic a c i d d i s s o c i a t e s as follows: H 3As0 4 H + + H 2As0 4" pKa x H 2As0 4* H + + HAs0 4 2" pKa 2 HAs0 4 2" H + + A s 0 4 3 " pKa 3 Arsenate ions, i r r e s p e c t i v e of t h e i r form on i n j e c t i o n w i l l tend to be chromatographed as the form favored by the pH of the eluent. At the eluent pH of 4, arsenate should be mainly i n the H 2As0 4" form; t h i s form should have les s i n t e r a c t i o n with the anion exchanger compared to the form present at pH 10; which would be mostly HAs0 4 2~. The retention time f o r arsenate at pH 10 i s such that i t i s not eluted even a f t e r 80 min, i n d i c a t i n g the strong i n t e r a c t i o n with the anion exchanger. The r e t e n t i o n of methylarsonate shows the e f f e c t of the a c i d i t y as w e l l . (CH 3)AsO(OH) 2 with pKa x 4.58 and pKa 2 7.82 1 1 4 would have the p a i r - 2.251" 6.67 - 11.60 - 91 -(CH 3)AsO(OH)0"/CH 3AsO(OH) 2 present at pH 4. However at pH 10, i t would mostly be i n the form (CH 3)As0 3 2" which would i n t e r a c t g r eatly with the anion exchanger leading to long retention time. The r e t e n t i o n of dimethylarsinate pKa 6.19 1 1 4 and arsenite, pKa^ 9.23 1 1 4 follows the same pattern as arsenate and methylarsonate. + Arsenobetaine, a Zwitterion (CH3)3AsCH2COO" i s not a f f e c t e d much by the changes i n the a c i d i t y . Arsenobetaine, arsenocholine and tetramethylarsonium iodide eluted together ( f r a c t i o n s 5-8) on the Protein Pak column. Further attempts to separate the three on the Protein Pak column proved unsuccessful; t h i s i s not s u r p r i s i n g since the three compounds e x i s t mostly as cations over the pH range studied. 3.4.1.1 C a l i b r a t i o n and Limit of Detection for Separations on  Protein Pak DEAE Column In order to obtain a c a l i b r a t i o n curve for the HPLC-GFAA analysis with the Protein Pak DEAE column, 20 mL of 5, 10, 20, 25 and 30 fig mL"1 As solutions of arsenobetaine were i n j e c t e d onto the column. Ammonium acetate, 5 mM pH 10, was used as the eluent at a flow rate of 1 mL min" 1. Fractions were c o l l e c t e d every 30 sec. The GFAA response was obtained f o r each f r a c t i o n as peak area absorbance. Arsenobetaine i s eluted between f r a c t i o n s 5 and 7, the sum of a l l three i n d i v i d u a l peak areas i s obtained and i s p l o t t e d against the amount of arsenic injected. The p l o t obtained i s shown i n F i g . 3.21. The curve i s l i n e a r over the - 92 -concentration range 100-600 ng As. The l i m i t of detection (defined i n Section 3.1.1.1) i s determined to be 20 ng. This i s higher than the 0.30 600 AMOUNT OF ARSENIC INJECTED (ng) F i g . 3.21: The c a l i b r a t i o n curve f o r the determination of arsenobetaine with the HPLC-GFAA system. (Waters Protein Pak, DEAE, 5 mM ammonium acetate, pH 10, flow rate 1 mL m i n - 1 ) . l i m i t measured f o r the d i r e c t determination of As by the GFAA. The higher detection l i m i t i s due to the d i l u t i o n e f f e c t of the HPLC eluent. The c a l i b r a t i o n curves f o r the other compounds showed a s i m i l a r trend. - 93 -3.4.1.2 A p p l i c a t i o n of the HPLC-GFAA Separation on Protein Pak Column  to Extracts For routine a p p l i c a t i o n of the HPLC-GFAA system to the extracts of marine organisms, the column i s f i r s t run at pH 4 to detect any methyl-arsonate and arsenate present. The eluent i s then switched to ammonium acetate, pH 10 to detect and quantitate any arsenobetaine, arsenate or dimethylarsonate present. In most cases only arsenobetaine i s detected. A t y p i c a l HPLC-GFAA chromatogram of p u r i f i e d Manila clam extract i s shown i n F i g . 3.22. 0.15-1 U U 0.10 I < K 0 (/) 0.05-0.00 5 1 1 r~ 1 0 1 5 2 0 ION TIME C a i n > F i g . 3.22: HPLC-GFAA chromatogram of 20 /<L of p u r i f i e d Manila clam extract; conditions are the same as used i n F i g . 3.20. a, corresponds to arsenobetaine. - 94 -The Protein Pak column i s used as a clean-up step i n preparing samples f o r NMR and mass s p e c t r a l analysis. 3.4.2 Separations on C^g Reversed Phase Column Reversed phase chromatography involves the use of a non-polar stationary phase and a polar immiscible mobile phase. Compounds are separated by t h e i r r e l a t i v e hydrophobicity; i . e . , the most polar compounds are eluted f i r s t while the non-polar solutes are retained longer. A Waters ^Bondapak C^g reversed phase column i s used i n t h i s study. The stationary phase consists of a layer of octadecyl-chains bonded to 10 /im s i l i c a g e l . The a r s e n i c a l s used i n t h i s study, arsenite, arsenate, methylarso-nate, dimethylarsinate, arsenobetaine and arsenocholine elute together immediately a f t e r the solvent (water-methanol, 80:20) front on the reversed phase column. This i s a t t r i b u t e d to the i o n i c nature of the compounds. The i n t e r a c t i o n of the solutes with the water methanol mobile phase predominates to the point of minimizing the solute-s t a t i o n a r y phase i n t e r a c t i o n s . The separation of i o n i c species on reversed phase can be improved by the a d d i t i o n of a s u i t a b l e l i p o p h y l i c counter-ion to produce a hydroph-obic ion-pair. The ion p a i r s are consequently retained more strongly by the C^g stationary phase. The ion p a i r i n g reagent i s usually a quarternary ammonium s a l t f o r anions or e i t h e r a l k y l s u l f a t e s or a l k y l - 95 -sulfonates f o r cations. 3.4.2.1 Separations of Arsenicals with Tetrabutylammonium as Ion-pair  Reagent In the present study, the separation af arsenite, arsenate, methyl-arsonate and dimethylarsinate on the reverse phase column i s achieved by the a d d i t i o n of tetrabutylammonium n i t r a t e (TBAN) to the water-methanol eluent. A t y p i c a l chromatogram i s shown i n F i g . 3.23. This i s obtained using TBAN at 5 mM concentration i n water-methanol, 95:5, pH adjusted to 6.8 with ammonium hydroxide, at a flow rate of 1 mL min" 1. Arsenite i s eluted f i r s t , followed by dimethylarsinate, methylarso-nate and arsenate. The retention of these compounds i s r e l a t e d to t h e i r pKa values. Arsenite, pKa 9.23 1 1 3 remains undissociated under these conditions. I t does not p a i r up with the tetrabutylammonium c a t i o n and thus r e t a i n s i t s polar character. I t i s therefore not adsorbed onto the stationary phase leading to f a s t e l u t i o n . Dimethylarsinate pKa 6.19, 1 1^ i s present as the p a i r (CH3) 2As0 2"/(CH3) 2As0 2H whereas methylarsonate pKa^ 4.58 and pKa 2 7.82 1 1 4 probably i s present as the p a i r (CH3)As0 3 2"/ (CH3)As0 3H". Methylarsonate i s therefore more l i k e l y to form the ion-pair r e s u l t i n g i n i t s longer retention time. Arsenate, the most a c i d i c compound among the four compounds pKa^ 2.25, pKa 2 6.67 and pKa3 11.60 i s retained longer probably as the ion p a i r [TBA]2HA504^". Brinkman et a l . ^ 5 demonstrated the use of the ion p a i r reagents tetrabutylammonium phosphate (TBAP) and tetraheptylammonium n i t r a t e - 96 -w u t < 0 If) (I) < 0.2-1 0.1-0.0 i i T 0 5 1 0 1 5 RETENTION TIME 1-2 0 C min > F i g . 3 . 2 3 : HPLC-GFAA chromatogram of: a, arsenite; b, dimethylarsi-nate; c, methylarsonate; d, arsenate; Waters /iBondapak C^g column. 2 0 / i L of sample containing 5 0 0 ng As of each As compound injec t e d ; mobile phase water-methanol ( 9 5 : 5 ) 5 mM tetrabutylammonium n i t r a t e , pH adjusted to 6.8 with ammonium hydroxide; flow rate 1 mL min" 1; f r a c t i o n s c o l l e c t e d every 30 sec; 2 0 fiL of each f r a c t i o n i n j e c t e d Into graphite tube. (THAN) i n the separation of arsenite, arsenate, methylarsonate and dimethylarsinate on a C^g column. Arsenite and arsenate are completely separated with water-methanol 95:5 containing TBAP (5 mM) at pH 7 . 3 with r e t e n t i o n times of 11 min and 19 min r e s p e c t i v e l y . During that study - 97 -when a mixture of arsenite, arsenate, methylarsonate and methylarsinate was chromatographed with 5 mM TBAP i n pure water as the mobile phase ( t h i s should increase the retention of the l i p o p h i l i c i o n p a i r s ) , the peaks associated with arsenite and dimethylarsinate overlap. The methylarsonate s i g n a l i s located between those belonging to arsenite and arsenate. A l l four compounds are s a t i s f a c t o r i l y separated by using water-methanol (75:25) at pH 7.6 saturated with THAN. The retention times of arsenite, dimethylarsonate, methylarsonate and arsenate are around 6 min, 10 min, and 36 min, res p e c t i v e l y . The separation achieved i n the present study, F i g . 3.22, i s obviously much f a s t e r than that described by Brinkman et al;105 arsenate i s eluted around 15 min study versus the reported 36 min. The shorter r e t e n t i o n times obtained makes the routine a p p l i c a t i o n of the HPLC-GFAA system more f e a s i b l e . The concentration of ion-pair reagent and the composition of the mobile phase can a f f e c t the retention of the solute. Thus a change of the water-methanol composition from 95:5 to 90:10 r e s u l t s i n f a s t e r e l u t i o n of the compounds, F i g . 3.24. However, methylarsonate and arsenate are not w e l l resolved. I f the mobile phase i s saturated with TBAN, no separation i s obtained. A l l the compounds elute close to the solvent front. A s i m i l a r r e s u l t was obtained by Caruso and coworkers^- who found that no separation of arsenite and arsenate occurs when a mobile phase of water-methanol (75:25 or 95:5), pH 7.3, saturated with TBAN i s used. They a t t r i b u t e t h i s to a competition of the n i t r a t e anion and the solute anions (As02 _ and HAsO^ 2 -) i n forming ion-pairs with the tetrabutylammonium ion. The separation of the compounds obtained with - 98 -0.2-1 RETENTION TIME C min > F i g . 3 . 2 4 : HPLC-GFAA chromatogram of: a, arsenite, b, dimethylarsi-nate; c, methylarsonate; d, arsenate; Waters /xBondapak C^g column; 20 pL of sample containing 4 0 0 ng As of each As compound injected; mobile phase water-methanol ( 9 0 : 1 0 ) , 5 mM tetrabutylammonium n i t r a t e , pH adjusted to 6.8 with ammonium hydroxide; flow rate 1 mL min" 1; f r a c t i o n s c o l l e c t e d every 30 sec; 2 0 / i L of each f r a c t i o n i n j e c t e d into graphite tube. the eluent containing 5 mM TBAN ( F i g . 3.22) may therefore be due to a le s s e r competition. I t should be pointed out that arsenite with pKa 9.23 1 1 3 remains undissociated under the conditions (pH 7.3), i t does not p a i r up with the tetrabutylammonium ion and therefore i s not affe c t e d much by the concentration of the tetrabutylammonium reagent. - 99 -3.4.2.2 E f f e c t of the Anion Associated with Tetrabutvlammonium on the  Separation of Arsenicals As discussed i n the previous section, no separation of arsenite and arsenate i s achieved on the reversed phase column by using a mobile phase saturated with tetrabutylammonium n i t r a t e . The two compounds are, however, separated by using a mobile phase saturated with t e t r a b u t y l -ammonium phosphate.m The behaviour of the anion associated with the tetrabutylammonium ion was therefore investigated. The s u l f a t e , phosphate and n i t r a t e s a l t s of the tetrabutylammonium c a t i o n were used as i o n - p a i r reagents i n the separation of the a r s e n i c a l s . Water-methanol, 90:10 was used as the eluent i n a l l experiments. No u s e f u l separations are obtained with the eluent saturated with tetrabutylammonium n i t r a t e (TBAN), tetrabutylammonium s u l f a t e (TBAS) and tetrabutylammonium phosphate (TBAP). Arsenite, arsenate, dimethylarsi-nate and methylarsonate a l l elute close to the solvent f r o n t . Severe interference i s obtained i n the GFAA analysis of the f r a c t i o n s , leading to poor r e p r o d u c i b i l i t y . Much better separations are obtained, however, when a 5 mM concentration of the ion-pair reagent i s used. The best separation i s obtained with TBAN, Figs. 3.23 and 3.24, as discussed e a r l i e r i n the previous section. With TBAP, arsenite i s separated from arsenite, however methylarsonate and dimethylarsinate coelute, F i g . 3.25. I f 5 mM s o l u t i o n of TBAS i s used as the Ion-pair reagent, arse-nate, methylarsonate and dimethylarsinate coelute, F i g . 3.26. - 100 -0.13-1 u z < 0.10-0 (/] 0.05-A be 0.00-0 T 4 6 8 10 RETENTION TIME T" l - I 12 U ( min > . 3.25: HPLC-GFAA chromatogram of: a, arsenite; b, dimethylarsinate; c, methylarsonate; d, arsenate; Waters /iBondapak C^g column; 20 /xL of sample containing 400 ng As of each compound inject e d ; mobile phase water-methanol (90:10),. 5 mM t e t r a -butylammonioum phosphate, pH adjusted to 6.8 with ammonium hydroxide; flow rate 1 mL min" 1; f r a c t i o n s c o l l e c t e d every 30 sec; 20 /*L of each f r a c t i o n i n j e c t e d into graphite tube. - 101 -0.15 b e d u z < CD tc O CQ < 0 . 1 0 ' 0.05 0.00 10 RETENTION TIME (min) F i g . 3.26: HPLC-GFAA chromatogram of: a, arsenite; b, dimethylarsinate; c, methylarsonate; d, arsenate; Waters /jBondapak C^g column; 20 pi. of sample containing 400 ng As of each compound inject e d ; mobile phase water-methanol (90:10),. 5 mM t e t r a -butylammonioum s u l f a t e , pH adjusted to 6.8 with ammonium hydroxide; flow rate 1 mL min'^; f r a c t i o n s c o l l e c t e d every 30 sec; 20 (xL of each f r a c t i o n i n j e c t e d into graphite tube. 3.4.2.3 C a l i b r a t i o n and Limit of Detection f o r Separation or Reversed- Phase Column with TBAN as Ion-Pair Reagent Using a mobile phase of water-methanol (95:5) with 5 mM TBAN pH 6.8 at a flow rate of 1 mL min'^ and a procedure s i m i l a r to that described f o r the Protein Pak column (Section 3.4.1.1) the c a l i b r a t i o n curves f o r arsenate, arsenite, dimethylarsinate and methylarsonate were obtained. A t y p i c a l c a l i b r a t i o n curve f o r arsenite i s shown i n F i g . 3.27. The curve i s l i n e a r f o r the concentration range 0-500 ng As. The l i m i t of detection i s c a l c u l a t e d to be 40 ng. 102 -0.10 100 2 0 0 3 0 0 4 0 0 5 0 0 AMOUNT OF ARSENIC INJECTED(ncj) 6 0 0 F i g . 3.27: T y p i c a l c a l i b r a t i o n curve f o r the HPLC-GFAA determination of arsenite; reversed-phase, TBAN ion p a i r reagent The c a l i b r a t i o n curves f o r arsenate, methylarsonate and dimethylar-sinate follow a s i m i l a r trend and the l i m i t of detection i s 30 ng for arsenate, 40 ng for methylarsonate and 50 ng for dimethylarsonate. 3.2.2.4 Separation of Arsenobetaine. Arsenochol ine and Tetramethyl-arsonium Iodide with Heptanesulfonic Acid as Ion-Pair Reagent A mixture of arsenobetaine, arsenocholine and tetramethylarsonium iodide coelute immediately a f t e r the solvent front when chromatographed - 103 -with water-methanol 95:5 with 5 mM TBAN ion-pair reagent. The compounds e x i s t i n s o l u t i o n as cations under these conditions and are therefore not capable of forming ion-pairs with the tetrabutylammonium ions. Heptane-s u l f o n i c a c i d or dodecylbenzenesulfonic acid, however, are capable of forming ion-pairs with arsenobetaine, arsenocholine and tetramethylar-sonium ion i f they are ionized. Stockton and I r g o l i c l 0 6 separated arsenocholine, arsenobetaine and inorganic arsenic (arsenite and arsenate) on a C^g reversed phase column with the sodium s a l t s of heptanesulfonic a c i d or dodecylbenzenesulfonic a c i d as counter ions f o r the arsonium s a l t s . An e l u t i n g solvent mixture of w a t e r - a c e t o n i t r i l e - a c e t i c a c i d (95:5:6) with 5 mM heptanesulfonic acid, and a flow rate of 0.5 mL min~l e f f e c t s the separation of arsenobetaine (retention time, 12 min) from arsenocholine (retention time, 24 min). Arsenite and arsenate elute with the solvent front. This separation of arsenobetaine and arsenocholine served as a model f o r the present study. The chromatogram f o r the separation of arsenobetaine, arsenocholine and tetramethylarsonium iodide i n t h i s study i s shown i n F i g . 3.28. An e l u t i n g solvent of w a t e r - a c e t o n i t r i l e - a c e t i c a c i d (95:5:6), 5 mM i n heptanesulfonic acid, i s used, at a flow rate of 1 mL min'l. Arsenocholine iodide and tetramethylarsonium iodide are separated but not well resolved. However, t h i s separation proved adequate for the routine detection of these compounds i n the extracts of the marine organisms. - 104 -0.15-, w u 2 < K 0 (/) (I) < 0.10-0.05-0.00 0 5 10 15 RETENTION TIME < B i n > F i g . 3 . 2 8 : HPLC-GFAA chromatogram of: a, arsenobetaine; b, arsenocho-l i n e iodide; c, tetramethylarsonium iodide; /iBondapak C^g column; 2 0 J J L of sample containing 5 0 0 ng As of each compound placed on column; mobile phase w a t e r - a c e t o n i t r i l e -a c e t i c a c i d ( 9 5 : 5 : 6 ) , 5 mM heptanesulfonic a c i d , flow rate 1 mL min" 1; f r a c t i o n s c o l l e c t e d every 3 0 sec; 2 0 /*L of each f r a c t i o n i n j e c t e d into graphite tube. - 105 -RESULTS AND DISCUSSION ARSENIC CONCENTRATIONS AND SPECIATION 4.1 T o t a l Arsenic Concentration 4.1.1 Data Presentation A consistent format i s used throughout t h i s s e c t i o n f o r the presentation of data r e l a t i n g to the t o t a l arsenic content i n organisms. T o t a l arsenic analysis was c a r r i e d out by diges t i n g the homogenized sample with a mixture of n i t r i c acid, s u l f u r i c a c i d and hydrogen peroxide; the procedure i s described e a r l i e r (Section 3.2.1). Four r e p l i c a s were c a r r i e d out f o r each sample. The mean value i s quoted along with the standard deviation of the mean, e.g. 9.1 ± 0.8 indicates a mean value of 9.1 ranging from 8.3 to 9.9 over four r e p l i c a s . A l l concentrations are quoted on a wet weight basis except f o r s h e l l s where they are on a dry weight bas i s . 4.1.2 T o t a l Arsenic Concentrations i n Soft Tissues The t o t a l arsenic concentrations i n whole s o f t tissues of clams obtained from Thetis Island are presented i n Tables 4.1 and 4.2. The arsenic concentration v a r i e s with species, ranging from 1.0 ng g " l i n the Manila clam to 2.5 ng g"* i n the S o f t - s h e l l e d clam. With the exception of the So f t - s h e l l e d clam, the concentration of arsenic i n a - 106 -Table 4.1: Arsenic concentrations i n s o f t t i s s u e s of clams c o l l e c t e d at Thetis Island, October 30, 1985 Species No of Average Weight (g) As specimen with s o f t pg g~^ pooled s h e l l t i s s u e Butter clam Saxidomus giganteus 239.7 109.5 2.2 ± 0.2 S o f t - s h e l l e d clam Mya arenaria 87.0 33.1 2.5 ± 0.1 Native l i t t l e n e c k clam 59 Protothaca staminea 21.1 7.7 1.6 ± 0.1 Manila clam Venerupis japonica 15 10.5 3.5 1.0 ± 0.1 - 107 -Table 4.2: Arsenic concentrations i n s o f t t i s s u e s of clams c o l l e c t e d at Thetis Island, October 13, 1986 No of Average Weight (g) As Species specimen with s o f t /ig g" pooled s h e l l t i s s u e Horse clam 3 214.7 99.5 2.0 ± 0.1 Schizothoerus n u t t a l l i Butter clam 6 250.1 107.6 2.3 ± 0.1 Saxidomus giganteus S o f t - s h e l l e d clam 4 85.5 32.7 1 . 3 ± 0 . 1 Mya arenaria Native l i t t l e n e c k clam 21 22.2 8.3 1 . 6 ± 0 . 1 Protothaca staminea Manila clam 66 7.2 2.7 1 . 0 ± 0 . 1 Venerupis japonica - 108 -p a r t i c u l a r species does not vary much with time. The arsenic concentration i n Butter clams, for example, i s 2.2 n& g" 1 i n October 1985 and 2.3 ng g" 1 i n the same period i n 1986. Generally, the arsenic l e v e l s can be r e l a t e d to the s i z e of the species. With the exception of the S o f t - s h e l l e d clam from October 1985, the l a r g e r the s i z e of the species, the higher the arsenic content. Thus Butter clam shows the highest l e v e l and Manila clam the lowest. Correlations between f i s h weights and arsenic l e v e l s i n muscle tissues of f i s h has been o b s e r v e d . 1 6 - 1 1 5 S o f t - s h e l l e d clams from t h i s area should be examined again to e s t a b l i s h i f the trends noted above are continuing. 4.1.3 T o t a l Arsenic Concentrations i n Soft Tissues of Clams from Coles  Bay and P a t r i c i a Bay Soft tissues of clams were bulked, homogenized and analyzed. The t o t a l arsenic concentrations obtained from the two stations are pre-sented i n Table 4.3. The two species which were obtained from P a t r i c i a Bay, Native l i t t l e n e c k and Manila clams, show arsenic concentrations about two times higher than the same species obtained from Coles Bay or Thetis Island. This might be due to the nature of the sewage e f f l u e n t s discharged into P a t r i c i a Bay. However, no information about t h i s i s a v a i l a b l e . The Bent-nose clams from Coles Bay show an arsenic concentration of 4.3 n& g" 1, which i s higher than the other species analyzed, however, no data - 109 -Table 4.3: Arsenic concentrations i n clams c o l l e c t e d from Coles Bay and P a t r i c i a Bay, May 7, 1987 Name Station No. of clams As /ig g* 1 Bent-nose clam Macoma nasuta Coles Bay 75 4.3 ± 0.3 So f t - s h e l l e d clam Mya arenaria Native l i t t l e n e c k clam Protothaca staminea Manila clam Venerupis japonica Native l i t t l e n e c k clam Protothaca staminea Manila clam Venerupis japonica Coles Bay Coles Bay Coles Bay 0.6 ± 0.1 P a t r i c i a Bay 98 105 90 1.8 ± 0.1 1.3 ± 0.1 P a t r i c i a Bay >200 3.8 ± 0.1 2.2 ± 0.2 - 110 -fo r t h i s type of clam are a v a i l a b l e from the other stations f o r comparative purposes. 4.1.4 Concentration of Arsenic i n Oysters Data on arsenic analysis of oysters c o l l e c t e d from Yellow Point and Sherard Point are recorded i n Table 4.4. The arsenic concentrations i n the oysters from Sherard Point, an area near a pulp m i l l e f f l u e n t , i s e s s e n t i a l l y the same as i n oysters from Yellow Point. The arsenic l e v e l s i n the oysters are of the same magnitude as those found i n the clams. Table 4.4: Arsenic concentrations i n s o f t - t i s s u e s of P a c i f i c Oysters. Crassostrea gigas Name Statio n (Date) As Mg/g P a c i f i c oyster Sherard Point 2.1 ± 0.1 Crassostrea gigas September 18, 1987 P a c i f i c oyster Yellow Point 2.0 ± 0.1 Crassostrea gigas September 18, 1987 - I l l -4.1.5 Arsenic Concentrations In Blvalue Tissues C o l l e c t e d from A l i c e  Arm and Hastings Arm. October 1986 The arsenic concentrations i n a number of bival v e s c o l l e c t e d from A l i c e Arm and Hastings Arm i n October 1986 are shown i n Table 4.5. The statio n s are in d i c a t e d on the chart i n F i g . 4.1. The l e t t e r T indicates a trawl and the area covered by the trawl i s indi c a t e d by the s o l i d l i n e on the chart. Table 4.5: Arsenic concentrations i n bivalve s o f t t i s s u e , A l i c e Arm and Hastings Arm, October 1986 Statio n Name No. of Average Weight (g) As Date specimen with s h e l l Tissue ng g T-A3 10-27 -86 Fucan cockle (Clinocardium fucanum) 51 1. .6 0. .9 1. .8 + 0. .1 T-A3 10-27 -86 N u t t a l l ' s cockle (Clinocardium n u t t a l l i ) 11 35. .0 10. 3 1. .7 + 0. .2 T-A3 10-27 -86 Hundred-lined cockle 2 (Namocardium centifilosum) 2. .4 1. ,1 2. .5 + 0. .2 T-A3 10-27 -86 Egg cockle (Laevicardium sp.) 2 3. .9 1. .8 2. .2 + 0. .3 T-A3 10-27 -86 Pink s c a l l o p (Chlamvs h e r i c i a ) 9 9. .6 4. 1 3. .0 + 0. ,3 T-A2 10-27 -86 F i l e y o l d i a (Yoldia limatula) 4 0. .7 0. 5 2. .3 + 0. .2 Table 4.5 continued - 112 -Table 4.5 continued S t a t i o n Name No. of Average Weight (g) As Date specimen with s h e l l Tissue ng g A12 10-26-86 B a l t h i c macoma (Macoma ba l t h i c a ) 1 0. 4 0. .1 3. 9 + 0. 1 A12 10-26-86 Short y o l d i a (Yoldia s a p o t i l l a ) 1 1. 3 0. .7 3. ,9 + 0. 1 A15 10-26-86 Chalky macoma ('Macoma calcarea) 2 2. 5 0. .9 3. ,2 + 0. 4 T-Hl 10-26-86 B a l t h i c macoma (Macoma ba l t h i c a ) 3 0. .5 0. .3 3. .0 + 0. ,2 T-Hl 10-26-86 Chalky macoma (Macoma calcarea) 1 2. ,7 1. ,4 2. .1 + 0. 1 T-Hl 10-26-86 Oval y o l d i a (Yoldia mvalis) 2 2. .4 1. .0 17. .0 + 2. ,0 T-H2 10-26-86 Broad y o l d i a 1 (Yoldia thraciaeformis) 7. ,6 5. .22 9. .1 + 0. 8 T-H2 10-26-86 Short y o l d i a (Yoldia s a o o t i l l a ) 4 1. .4 0, .7 8. .0 + 0. .8 D3 10-26-86 Oval y o l d i a (Yoldia mvalis) 1 1. .8 1. .2 5, .9 + 0. .2 D3 10-26-86 Short y o l d i a (Yoldia s a o o t i l l a ) 1 1 .3 0 .6 4 .6 + 0 .2 D3 10-26-86 Chalky macoma (Macoma calcerea) 1 0 .9 0 .6 3 .4 + 0 .2 - 113 -F i g . 4.1: A l i c e - H a s t i n g Ann showing 1986 sampling s i t e s - 114 -The Arsenic concentrations i n surface sediments (0-5 cm) f o r some of the s t a t i o n s l i s t e d i n Table 4.5 are shown i n Table 4.6. The concen-t r a t i o n s were determined by neutron a c t i v a t i o n analysis on d r i e d sediments c o l l e c t e d i n 1985 by Dr. Ken Reimer, Royal Roads M i l i t a r y College, V i c t o r i a , B.C. Table 4.6: Concentrations 8 of arsenic i n surface sediments (0-5 cm); Alice-Hastings Arms Sta t i o n Type of Sediment As /jg g A- 3 T a i l i n g s ; some r i v e r sedimentation (but t h i s could be contaminated) 24. .9 + 3, .7 A- 5 E x c l u s i v e l y t a i l i n g s 26, .7 + 3, .6 A- 12 Natural sediment (reference station) 9. .4 + 3. .5 A- 15 T a i l i n g s from Anyox slag; 50 year o l d deposit 46. .5 + 5. .3 H- 1 Natural sediments 14. .9 + 1. 9 H- 2 E s s e n t i a l l y natural but could have some contamination from Anyox 36. 8 + 6. 2 Determined by neutron a c t i v a t i o n analysis on d r i e d sediments. - 115 -Table 4.7 l i s t s arsenic concentrations i n sediment pore water f or some of the stati o n s sampled. Table 4.7: Concentrations (nm) of arsenic species i n sediment (0-5 cm) pore water; Alice-Hastings Arms a Station/Depth Arsenate Arsenite Methyl- Dimethyl- Trimethyl-(cm) arsonic a r s i n i c arsine a c i d a c i d oxide A l 0-3 2-5 30.7 68.0 45.3 57.7 0.5 0.4 1.1 1.2 0.6 0.6 A5 0-3 2-5 120.0 353.3 126.7 330.7 2.7 6.3 1.1 0.8 1.1 2.7 A12 0-3 2-5 117.3 141.3 38.7 72.0 0.5 0.8 1.1 1.2 0.6 0.6 HI 0-3 2-5 26.7 49.3 10.7 58.7 0.1 0.4 1.7 1.2 <0.1 1.2 a Values obtained from Reimer and Thompson.4^ - 116 -Samples from A2, A12, A15 and D3 were obtained with a s t a i n l e s s s t e e l Smith-Mclntyre grab sampler. The samples c o l l e c t e d o f f Anyox, A15, has e s s e n t i a l l y the same arsenic content as those from the s i l l i n A l i c e Arm i n sp i t e of the very d i f f e r e n t arsenic concentrations of 46.5 fig g" 1 (A15) and 9.4 fig g'^ (A12) i n the surface sediment. Bivalves obtained by trawling i n the Hastings Arm T-Hl and T-H2 show a range of arsenic concentrations, 2.1-17.0 fig g" 1. The Oval y o l d i a has the highest concentration of 17.0 fig g" 1. The concentrations i n samples from A3 do not d i f f e r much from those i n samples from A12 and A15 despite the differences i n sediment concentrations. Thus the concen-t r a t i o n s of arsenic i n the sediment do not seem to c o r r e l a t e with the concentrations of arsenic i n the bivalv e s . The t o t a l arsenic concentrations i n the pore waters also do not co r r e l a t e with arsenic concentrations i n the bi v a l v e s . B a l t h i c macoma from A12 with t o t a l pore water arsenic concentration of 158.2 nM (0-3 cm) has 3.9 fig g " l arsenic while the same species from HI with 39.3 nM arsenic i n the pore water has 3.0 fig g" 1 arsenic. Some e a r l i e r data f o r bivalves from some of the areas studied are av a i l a b l e f o r comparison. Table 4.8 presents arsenic concentrations f o r three of the species studied; the values reported i n the present work are comparable to these l e v e l s . Information regarding arsenic concentrations i n bivalves from other locations i n B r i t i s h Columbia i s extremely l i m i t e d . - 117 -Table 4.8: Arsenic concentrations i n some bivalves from selected c o a s t a l locations Species Sample l o c a t i o n (date) As fig g'1 (Dry Weight basis) Ref Cockles Clinocardium n u t t a l i A l i c e Arm 1984 1986 0.6-1.4 1.7 a 116 Mussels Mytilus e d u l i s A l i c e Arm 1978 1982 Hastings Arm 1978 1986 9.5 7.4-9.5 7.9 2.4 a 117 118 117 Quatsino Sound 1978 1979 1986 11.5 1.3 1.5* 117 117 Anyox 1978 1986 9.4 1.8 a 117 Y o l d i a Y o l d i a thraciaeformis A l i c e Arm 1978 1981 1982 52 66 100 117 119 119 Hastings Arm 1978 1982 1986 Quatsino Sound 1978 1981 46 62 9.1 a 40 26 117 119 117 120 This work, wet weight bas i s . - 118 -4.1.6 Arsenic Concentrations i n Soft Tissues of Gastropods C o l l e c t e d  from A l i c e Arm and Hastings Arm. October. 1986 Gastropods, mostly whelks, were obtained by trawl nets. The areas covered by the trawl i s presented i n F i g . 4.1. The s o f t - t i s s u e s were separated from the s h e l l s and analyzed following wet dige s t i o n with n i t r i c acid, s u l f u r i c a c i d and hydrogen peroxide. Table 4.9 presents data on the arsenic concentrations obtained. The Ridged whelks obtained from T-A3 show a much higher arsenic concentration compared with the same species obtained from T-Hl. This seems to c o r r e l a t e with the concentration of arsenic i n the sediments. The other species, however, do not show such c o r r e l a t i o n s . Data on arsenic concentrations i n gastropods are l i m i t e d . The few reported cases include the Ivory s h e l l Buccinum striatissimum with 38 and 18 /ig g " 1 As i n the muscle and mid-gut gland r e s p e c t i v e l y . ^ The herbivorous gastropod B a t i l l u s cornutus has 5.0 and 22.7 /ig g* 1 As i n the muscle and mid-gut g l a n d . ^ Tegula p f e i f f e r i . also a herbivore, has 3.1 and 6.7 ng g" 1 As i n the muscle and mid-gut gland. ^  The carnivorous gastropods on the other hand, have much higher arsenic l e v e l s , thus Charonia sauliae has 116.5 ng g" 1 As i n the muscle and 340.1 n% g A s * n t n e mid-gut g l a n d . ^ Reisha c l a v i g e r a . also a carnivore has 53.3 pg g" 1 As i n the muscle and 39.5 ng As i n the mid-gut g l a n d . ^ With the exception of the low values found f o r Phoenician whelk the gastropods i n t h i s study have arsenic concentrations i n the range 17.3-48.4 /ig g" 1. These are generally comparable to the previously - 119 -Table 4.9: Arsenic concentrations of gastropids c o l l e c t e d from A l i c e -Hastings Arms, October 1986 Stati o n Name No. of Average Weight (g) As Date specimen with s h e l l Tissue /ig g T-A3 10-27- 86 Northern moon s n a i l ( P o l i n i c e s p a l l i d a ) 1 7. 5 2. 8 31. 9 + 1. 2 T-A3 10-27- 86 Thick-ribbed whelk 5 (Beringius crebricostata) 100. 0 37. 0 17. 4 + 0. 4 T-A3 10-27- 86 Ridged whelk (Neptunea lvr a t a ) 5 33. 9 12. 6 42. 5 + 0. 9 T-A3 10-27- 86 Phoenician whelk (Neptunea Phoenicia) 18 36. 0 11. 6 3. 9 + 0. 9 T-A3 10-27-•86 Kroyer's whelk ( P l i f i c u s k r o v e r r i ) 4 9. .1 2. .6 48. ,4 + 7. 4 T-A3 10-27-•86 Lyre whelk (Buccinum plectrum) 4 12. .5 2. .8 16. .0 + 1. .7 T-A2 10-28-•86 Jordan's colus (Colus ^ordani) 4 4. .6 1 .2 20, .9 + 2. .5 T-A2 10-28-•86 Kroyer's whelk ( P l i f i c u s kroveri) 4 6 .6 2 .6 45 .3 + 4. .9 T-A2 10-28--86 Ridged whelk (Neptunea lvr a t a ) 2 38 .0 12 .6 48 .3 + 1, .2 T-A2 10-28--86 Phoenician whelk (Neptunea Phoenician) 1 13 .3 2 .8 0 .8 + 0 .1 T-Al 10-28 -86 Thick-ribbed whelk 5 (Berineius crebriscotata) 39 .1 15 .9 17 .3 + 0 .2 T-Hl 10-26 -86 Ridged whelk (Neptunea l v r a t a ) 5 35 .8 15 .0 28 .4 + 1 .2 T-Hl 10-26 -86 Jordan's colus (Colus iordani) 3 4 .8 1 .5 22 .7 + 1 .5 - 120 -reported values with the exception of the one case of the Charonia  sauliae which has very high arsenic concentrations. I t should be pointed out that the bivalves i n t h i s study which are herbivores have r e l a t i v e l y lower arsenic concentrations (0.6-6.7 fig g " 1 ) . This r a i s e s i n t e r e s t i n g questions about the nature of arsenic compounds i n these two groups (herbivores and carnivores) and possible conversions of arsenic compounds In the bivalves to those i n the gastropods, the higher members of the food chain, which feed on the b i v a l v e s . 4.1.7 Arsenic Concentrations i n Soft-Tissues of Bivalves Collected  from Alice-Hastings Arms and Quatsino Sound. October 1987 Arsenic concentrations i n three species of bivalves c o l l e c t e d from the i n t e r t i d a l zone are shown i n Table 4.10. The S o f t - s h e l l e d clam c o l l e c t e d from the Anyox sl a g heap shows the highest arsenic concentrations of 6.7 fig g" 1, which co r r e l a t e s with the arsenic concentrations i n the sediment at S t a t i o n A 15 which i s the c l o s e s t s t a t i o n to Anyox. However, the highest arsenic concentration f o r the Blue mussels are found i n samples from the head of Hastings Arm. This does not c o r r e l a t e with the arsenic i n the sediments. The arsenic concentrations i n S o f t - s h e l l e d clams from a l l stations except the north shore of Rupert I n l e t are higher, 3.5-6.7 fig g'^, than the concentrations of 1.3-2.5 fig g" 1 (Tables 4.1 and 4.2) i n clams from Thetis Island. - 121 -Table 4.10: Arsenic concentrations i n bivalves Alice-Hastings Arm and Quatsino Sound, October 1987 Name Station (Date) As fig g -1 S o f t - s h e l l e d clam Mva arenaria A l i c e Arm (Head) 4.5 ± 0.7 B a l t h i c macoma Macoma b a l t h i c a A l i c e Arm (Head) 2.6 ± 0.1 1.6 ± 0.3 So f t - s h e l l e d clam Mya arenaria Blue mussel Mvtilus edulis S o f t - s h e l l e d clam Mya arenaria Blue mussel Mvtilus e d u l i s B a l t h i c macoma Macoma b a l t h i c a Anyox (Slag heap) Anyoxy (Slag heap) Hastings Arm (Head) Hastings Arm (Head) Hastings Arm (Head) 6.7 ± 0.1 1.8 ± 0.1 5.8 ± 0.1 2.4 ± 0.1 1.8 ± 0.1 So f t - s h e l l e d clam Mya arenaria Blue mussel Mvtilus e d u l i s S o f t - s h e l l e d clam Mya arenaria Rupert I n l e t (North shore) 1.7 ± 0.1 Rupert I n l e t (North shore) 1.5 ± 0.4 Rupert I n l e t Varney Bay 3.4 ± 0.6 - 122 -4.1.8 Arsenic Concentrations In Crabs The arsenic concentrations i n the Tanner crab Chionoecetes b a i r d i i . obtained from A l i c e Arm i s given i n Table 4.11 along with previous reported data. Table 4 . 1 1 : Arsenic concentrations i n crab l eg muscle t i s s u e Species Location (Date) As fig g" 1 Ref Tanner crab A l i c e Arm Chionoecestes b a i r d i i 1986 12.1 ± 0.7 a 7.1 ± 0.2 This work This work 1980 56* 117 - 1981 79.1 ± 1.0 b 117 1983 9.0 116 1983 44. 3 b 120 Hastings Arm 1980 l l l b 125 ± 32 117 120 Heptopancrease Dry weight basis - 123 -The arsenic l e v e l s i n crabs from Hastings Arm are higher than those from A l i c e Arm. The wet weight l e v e l of 7.1 /ig g" 1 compares well with the previous data (a f a c t o r of 5-6 i s involved between wet weight-dry weight conversions). The arsenic concentration i n the l e g muscle of the crabs i n t h i s work also compares well with the arsenic concentrations reported by Francesconi et a l . (7.7 /ig g ' ^ ) . ^ 4.1 D i s t r i b u t i o n of Arsenic i n Bivalves The d i s t r i b u t i o n of arsenic i n tissues was investigated to determine i f p r e f e r e n t i a l concentration of arsenic i n any p a r t i c u l a r organ occurs. The arsenic l e v e l s found i n four tissues of clams are given i n Table 4.12. In a l l the clams studied, the highest concentration of arsenic i s found i n the v i s c e r a l mass. This observation i s i n l i n e with that made by Maher,^ 5 regarding the clam Mercenaria mercenaria. A s i m i l a r study by Shiomi et a l . , ^ revealed that the g i l l of the clam Meretrix l u s o r i a has r e l a t i v e l y higher arsenic concentrations (15-40 /ig g" 1) than the other tissues (below 10 /ig g " 1 ) . This i s i n contrast to Maher's observations.9 5 i n the present study, the g i l l was not excised from the v i s c e r a l mass, so no data are a v a i l a b l e for comparative purposes. The bivalves studied i n t h i s work were kept i n sea water a f t e r c o l l e c t i o n f o r three days to allow t h e i r gut contents to be purged. I t i s not c l e a r how t h i s a f f e c t s the arsenic concentrations i n the organ-isms. I f the gut contents contain the bulk of the arsenic, purging for - 124 -Table 4 . 1 2 : D i s t r i b u t i o n of arsenic i n clams Organism 5 As >ig g" 1 V i s c e r a l Siphon Foot Muscle mass Butter clam Saxidomus giganteus 4.3 1.0 1.0 2.7 Horse clam Schizothoerus n u t a l l i 2.7 1.9 1.1 1.4 S o f t - s h e l l e d clam Mya arenaria 4.1 1.0 1.4 1.4 Native l i t t l e n e c k clam Protothaca staminea 2.1 0.5 0.7 0.9 Manila clam Venerupis japonica 2.0 1.2 1.4 1.4 Bent-nose clam Macoma nasuta 5.9 2.5 2.8 1.6 Geoduck Panope generosa 5.6 3.2 4.1 2.5 The Geoduck was purchased from Chinatown, Vancouver, B.C. Bent-nose clam was obtained from Coles Bay, a l l other clams from Thetis Island. longer periods should r e s u l t i n lower arsenic concentrations r e l a t i v e to the i n i t i a l l e v e l s . In such a case, higher l e v e l s of arsenic In the v i s c e r a l mass may not be a true i n d i c a t i o n of a s i t e of arsenic accumu-l a t i o n . - 125 -4.2 Arsenic Speciation This s e c t i o n reports the p u r i f i c a t i o n and i d e n t i f i c a t i o n of water-soluble arsenic compounds from some bivalves and gastropods. The procedures described i n Section 2.7 were used. 4.2.1 Arsenic Speciation i n Clams Soft t i s s u e s of Manila clam (Venurupis japonica), Native l i t t l e n e c k clam (Protothaca staminea). S o f t - s h e l l e d clam (Mya arenaria). Horse clam (Schizathoerus n u t a l l i ) and Butter clam (Saxidomus giganteus) were extracted. A summary of the weights and arsenic contents of the clam s o f t - t i s s u e s and extracts i s given i n Table 4.13. E x t r a c t i o n of the s o f t - t i s s u e removes 68% of the arsenic i n Manila clam, 81% i n the Native l i t t l e n e c k clam, 79% i n the S o f t - s h e l l e d clam, 57% i n the Horse clam and 60% i n the Butter clam. When the gum obtained a f t e r evaporation of methanol i s dissolved i n water and extracted with d i e t h y l ether, 3% of the t o t a l arsenic i n the Manila clam i s ether soluble while 4% of the t o t a l arsenic i n the Native l i t t l e n e c k clam i s extracted into ether. The percentage of the t o t a l arsenic extracted into ether f o r the S o f t - s h e l l e d clam, Horse clam and Butter clam i s 1%. These percentages of ether soluble arsenic are comparable to that found by others i n clam tissue,** Western rock lobster 1-* and s h e l l f i s h ; 1 ^ 1 and higher amounts up to 17% are found i n crabs.14,95 jjo further work i s c a r r i e d out on the ether extracts. - 126 -Table 4.13: Ext r a c t i o n of s o f t - t i s s u e s of Manila, Native l i t t l e n e c k , S o f t - s h e l l e d , Horse clam and Butter clam Clam Manila Native l i t t l e neck Soft s h e l l e d Horse Butter Number of clams pooled 66 21 4 2 5 Weight of s o f t t i s s u e extracted (g) 168 165 134 198 522 As content of s o f t t i s s u e (ng) 170 248 169 396 1200 Residue a f t e r methanol ex t r a c t i o n (g) 27 25 17 42 73 As content of residue (ng) 49 47 29 168 460 As content of methanol extract (ng) 116 200 138 227 730 Percentage of t o t a l As extracted (%) 68 81 79 57 60 The e l u t i o n patterns of the water-soluble arsenic compounds i n the f i v e species of clams on Sephadex LH-20 (2.5 cm x 60 cm) are s i m i l a r , e l u t i n g at re t e n t i o n volumes between 250 and 325 mL. Standard arse-nobetaine, arsenocholine iodide and tetramethylarsonium iodide elute from the column with s i m i l a r r e t ention volumes between 225 and 325 mL, - 127 -so the ar s e n i c a l s i n the clams do not seem to be strongly associated with other molecules i n the extracts. The arsenic containing f r a c t i o n s from the LH-20 column were chromatographed on Dowex 50 WX-8; e l u t i o n with water, 5% ammonium hydroxide and 2% hydrochloric a c i d r e s u l t s i n varying concentrations of arsenic i n the f r a c t i o n s (Table 4.14). The S o f t - s h e l l e d clam and Butter clam show the highest concentration of arsenic i n the ammonium hydroxide f r a c t i o n , whereas the Horse clam, Manila clam and Native l i t t l e n e c k clam contain higher amounts of arsenic i n the HCI f r a c t i o n . The ch a r a c t e r i -Table 4.14: Fr a c t i o n a t i o n of water-soluble arsenic i n Manila, Native l i t t l e n e c k , S o f t - s h e l l e d and Butter clam on Dowex 50 W x 8 Clam As placed As i n Fractions on column (fig) Unadsorbed NH40H HCI Manila clam 100 9 22 68 Venerupis ^aponica ( 7 ) a (13) (48) Native l i t t l e n e c k clam 150 6 29 99 Protothaca staminea (3) (13) (48) S o f t - s h e l l e d clam 100 20 46 26 Mva arenaria (10) (38) (20) Horse clam 150 13 30 99 Schizothoerus n u t a l l i (6) (12) (39) Butter clam 250 7 100 88 Saxidomus giganteus (2) (24) (21) a The r a t i o (%) to the t o t a l As i s given i n parentheses - 128 -z a t i o n of the arsenic compounds In the f r a c t i o n s Is discussed i n the following sections. 4.2.1.1 I d e n t i f i c a t i o n of Arsenic Compounds i n Ammonium Hydroxide  Fractions When subjected to gel permeation chromatography on a smaller Sephadex LH-20 column (2.5 cm x 30 cm) with water as eluent, only f r a c t i o n s with r e t e n t i o n volumes 120 and 140 mL contain arsenic. Standard arsenobetaine, a compound recovered i n the ammonium hydroxide f r a c t i o n , ^ elutes with a s i m i l a r retention volume. The f r a c t i o n s , when combined and evaporated y i e l d l i g h t brown syrups. Chromatography of these syrups on Dowex 2 x 8 column; ( e l u t i o n with water followed by 0.2 M HCI) y i e l d s two sets of arsenic containing f r a c t i o n s for the Butter clam extract and one set of arsenic containing f r a c t i o n s f o r the other clams. The f r a c t i o n s with retention volume 37.5-62.5 mL contains 90% of the arsenic In the Butter clam extract placed on the column while 4% of the arsenic i s contained i n the f r a c t i o n with volume 112.5-125 mL. The f r a c t i o n s with r e t e n t i o n volume 37.5-50.0 mL contain arsenic f o r the other clams. For b r e v i t y , the i d e n t i f i c a t i o n of the arsenic compounds i n the Butter clam extract i s described as an example. The two sets of arsenic containing f r a c t i o n s are termed "major" (90%) and minor (4%). Three spots are present i n the t h i n layer chromatogram of the major f r a c t i o n . Only the spot with Rf 0.52 contains arsenic, which corres-ponds to the Rf of standard arsenobetaine under the same conditions, - 129 -( s i l i c a g el plate, ethanol-acetlc acid-water, 65:1:34). E x t r a c t i o n of the arsenic containing spot, Rf 0.52, with methanol and evaporation of the methanol y i e l d s a residue. The NMR spectrum of a D 20 s o l u t i o n of t h i s residue shows peaks at 1.86, 3.22, 3.85, 4.32 and 4.70 ppm. This spectrum corresponds to that of a mixture of arsenobetaine and betaine, the peak at 1.86 due to (CH 3) 3As~, 3.22 due to (CH 3) 3N-, 4.32 due to N-CH2- and that at 4.70 due i s HDO. Further p u r i f i c a t i o n of the residue can be achieved by HPLC on a Protein Pak column with 5 mM ammonium acetate b u f f e r as eluent i s c a r r i e d out (Section 3.4.1.2). The arsenic compound elutes with the same retention time, 2.5-3.5 min, as synthetic arsenobetaine. Evaporation of the solvent y i e l d s a residue, the NMR of a D 20 s o l u t i o n of t h i s residue shows s i n g l e t s at 1.8 (9H), 3.2 (2H), and 4.8 ppm (HOD); F i g . 4.2a. This NMR spectrum i s i d e n t i c a l with that of synthetic arsenobetaine ( F i g . 4.2b). To obtain further evidence that the arsenic compound i s I d e n t i c a l to arsenobetaine, i t was analyzed by mass spectrometry. The FAB mass spectrum indicates peaks at 105 (CH 3) 2As, 120 (CH 3) 3As, 134, 135 (CH 3) 4As, 149 (M - C0 2 + CH 3), 179 (M + H) and 357 (2M + 1) among other peaks. This corresponds to the we l l documented FAB spectrum of arsenobetaine. 1 1,60,65,77 -phe thermospray LC mass spectrum include among other peaks at 179 (M + H), 193 (M + CH 3), 214 (M+H20+MH4+) and 329 (2M+l+OCH3-CH3COO) (F i g . 4.3a). The thermospray LC mass spectrum of synthetic arsenobetaine i s shown i n F i g . 4.3b. Thus the arsenic compound i n the major f r a c t i o n i s well i d e n t i f i e d as arsenobetaine. The arsenic compound i n the other clams: Horse clam, Manila clam, Native l i t t l e n e c k clam and So f t - s h e l l e d clam are characterized as 130 -a 1 - t 1 -b 1 ppm x i 1 ppm F i g . 4.2: NMR spectra of: a, major arsenic compound i n NH4OH f r a c t i o n from butter clam; b, synthetic arsenobetaine i 0 a B O h H I/] Z w h H 100 h H (/) u h Z H n a 3 2 9 1 9 3 214 3 1 7 5 i i i 1 1 i i i I 1 1 i 1 1 1 1 1 i I i i 1 1 1 1 1 1 i i I rf i 11 i i i 11 i i M i 11 i 11 I I 3 5 3 3 8 7 "I I I I | I I I ' M I I I I | I I 460 M / Z M 7 0 2 X 7 0 2 5 X 7 r 3 0 0 3 5 0 4 0 0 329 B 0 5 TTTT TO-2 1 4 1 | I I T I I I I I I 2 0 0 2 6 0 f r - r i i i i i | r r i i I T i i i | i ' i 3 0 1 0 3 6 0 I | I I I I I I I I I | I I I I M I I I | t I 400 450 M / Z 500 F i g . 4.3: Thermospray LC mass spectra of: a, major arsenic compound from the NH^OH f r a c t i o n of Butter clam; b, synthetic arsenobetaine; eluent, water-methanol, 40:60 with 0.05 M ammonium acetate; vaporizer 201°, probe 128°, block 156°, j e t 251°. 20 /xL of 1 mg mL"1 s o l u t i o n s i n j e c t e d - 132 -arsenobetaine on the basis of s i m i l a r evidence. Arsenobetaine i s found i n a wide v a r i e t y of marine invertebrates and f i s h (see Section 1.2.4). The f i n d i n g of arsenobetaine i n the clams reported i n t h i s study goes to confirm the v i r t u a l ubiquity of t h i s organoarsenic compound i n marine invertebrates and f i s h . The t h i n - l a y e r chromatogram of the minor f r a c t i o n of the Butter clam extracts shows one arsenic containing spot, Rf = 0.68. This does not correspond to the Rf value of any of the standard arsenic compound used, (arsenocholine 0.30, tetramethylarsonium iodide 0.38 and arsenobetaine 0.52). The NMR spectrum i n D2O i s shown i n F i g . 4.4. The s i n g l e t at 1.8 ppm i s c h a r a c t e r i s t i c of a methyl attached to arsenic (based on the 5 4 3 2 ppm F i g . 4.4: 1H NMR spectrum of minor arsenic compound i n NH4OH f r a c t i o n of Butter clam - 133 -spectra of standard arsenobetaine, F i g . 4.2b, arsenocholine, F i g . 4.5 and tetramethylarsonium iodide, F i g . 4.6) i s obtained. This compound i s not a tetramethylarsonium s a l t because i t i s known, that tetramethyl-arsonium s a l t s are recovered i n the HCI f r a c t i o n a f t e r chromatography on Dowex 50.^ Trimethylarsine oxide i s recovered i n the NH^OH f r a c t i o n a f t e r chromatography on Dowex 5 0 1 1 and has an NMR spectrum with a s i n g l e t at 1.77 ppm with respect to external sodium 2,2-dimethyl-2-silapentane-5-sulfonate.-^ Thus i t i s more l i k e l y that the arsenic compound i n the minor f r a c t i o n i s trimethylarsine oxide. The thermospray LC mass spectrum of the residue F i g . 4.7, shows peaks at 137 (M + H), 155 (M + H + H 20), 177 (M + CH 3C0 2 -H20) 195 (M + CH3C0 2). Due to s c a r c i t y , further t e s t could not be performed. Trimethylarsine oxide has previously been reported i n f i s h . ^ The concentration increased with storage and i t i s suggested that trimethyl-arsine oxide i s produced i n stored f i s h . ^ Trimethylarsine oxide also occurs as a natural component i n estuary c a t f i s h 1 ^ a n d accumulates i n the tissues of f i s h as a r e s u l t of o r a l administration of inorganic arsenic. I t i s suggested that the presence of t h i s compound i s depen-dent upon b a c t e r i a l actions i n the gut t r a c t of the f i s h . - ^ 3 The presence of trimethylarsine oxide i n Butter clams i s the f i r s t reported instance of t h i s compound i n a marine bivalve and i s o l a t i o n and unequi-vocal i d e n t i f i c a t i o n i s desirable. Unlike the Butter clam, no evidence f o r the presence of trimethylarsine oxide i s found i n the other four species of clams studied. - 134 -•4_1_ A 2 ppm F i g . 4.5: NMR spectrum of synthetic arsenocholine iodide i u 2 ppm F i g . 4.6: NMR spectrum of synthetic tetramethylarsonium iodide 1 0 0 1 3 7 JIJLh 177 155 , 1 6 6 h'p'H'h'M'i 1 9 5 2 1 2 2 2 3 111 I'I .I'm i 1 i n i i I'I i i | i i i i i i i i i'| i i i i i i i i i | i i 2 4 0 2 6 0 2 8 0 I W 1 / 2 3 * * ' l l l l 1 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 l l l | I 2 2 0 F i g . 4.7: Thermospray LC mass spectra of: a, minor arsenic compound from the NH4OH f r a c t i o n of Butter clam. Eluent 1 N ammonium acetate; vaporizer 212°, probe 133°, block 171°, Jet 234". 20 fiL of 1 mg mL"1 s o l u t i o n s Injected - 136 -4.2.1.2 I d e n t i f i c a t i o n of Arsenic Compounds i n Hydrochloric A c i d  Fractions Gel permeation chromatography on Sephadex LH-20 (2.5 cm x 30 cm) affords one arsenic containing peak between the e l u t i o n volumes 120-160 mL. This i s s i m i l a r to the e l u t i o n pattern of the compounds i n the ammonium hydroxide f r a c t i o n , the main component of which i s character-i z e d as arsenobetaine (Section 4.2.1.1), suggesting that the arsenic compound i n the HCI f r a c t i o n i s almost the same s i z e as arsenobetaine. The extracts of a l l f i v e species of clams e x h i b i t the same behaviour on LH-20. Thin-layer chromatographic analyses show the p u r i f i e d HCI f r a c t i o n s from a l l f i v e species of clams contain one major arsenic compound with Rf 0.38 which i s i d e n t i c a l with the Rf of standard tetramethylarsonium iodide. Preparative TLC and HPLC clean up y i e l d s o l i d residues. The ^H NMR spectrum of the Butter clam residue i s given as an example i n F i g . 4.8. The spectrum i s i d e n t i c a l to that of standard tetramethylarsonium iodide, F i g . 4.6; with a s i n g l e t at 4.8 ppm due to (C^^As"*". The thermospray LC mass spectrum of the Butter clam residue i s shown In F i g . 4.9 with a c h a r a c t e r i s t i c peak at m/z 135 assigned to (CH3)4As +. The thermospray LC mass spectrum of standard tetramethylarsonium iodide i s shown i n F i g . 4.10. The Butter clam residue i s thus characterized as a tetramethylarso-nium s a l t . The residues from Horse clam, Manila clam, Native l i t t l e n e c k clam and S o f t - s h e l l e d clam are s i m i l a r l y characterized as tetramethyl-arsonium s a l t . - 137 -2 ppm F i g . 4 . 8 : NMR spectra of arsenic compound i n HCI f r a c t i o n from Butter clam The presence of tetramethylarsonium s a l t s i n a marine invertebrate was f i r s t reported by Shiomi et a l . ^ The presence of t h i s compound i n a l l the clams studied i n t h i s work ra i s e s questions as to the r o l e i t plays i n the biotransformation of arsenic i n the marine environment. 100 1 3 5 1 6 3 187 i i ' i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i r 1 5 0 2 0 0 2 5 0 3 0 0 I I I " I | I I I I I 3 5 0 M / Z F i g . 4.9: Thermospray LC mass spectrum of p u r i f i e d HCI f r a c t i o n of Butter clam, same conditions as i n F i g . 4.7 1 0 0 1 3 5 3 9 7 1 6 3 I II I 1 1 1 1 | I I I I I I I I I [ I 2 0 0 3 0 0 f I I' I I I I I |~)—I I I I I I I I 4 0 0 1 I I I I I I 5 0 0 | I I I I I I I 6 0 0 M / Z F i g . 4.10: Thermospray LC mass spectrum of synthetic tetramethylarsonium iodide, same conditions as in "Fig. 4.7 - 140 -4.2.1.3 Arsenic Compounds In the Unadsorbed Fractions The t h i n layer chromatogram of the concentrated unadsorbed f r a c t i o n of Manila clam shows three spots with Rf's 0.34, 0.76 and 0.96. Only the spot with Rf 0.96 contains arsenic. Native l i t t l e n e c k clam extract shows 3 spots on TLC with Rf's 0.78, 0.86 and 0.91, again only one spot Rf 0.91 contains arsenic. Butter clam extract has 2 spots on TLC with Rf 0.80 and 0.97; the spot with Rf 0.97 contains arsenic. Preparative TLC of the Butter clam extract y i e l d s about 1 mg of a l i g h t brown residue, containing less than 1% arsenic. The thermospray LC mass spectrum of t h i s residue i s shown i n F i g . 4.11. The residue appears to c o n s i s t of a mixture of compounds and a further cleanup i s necessary to a i d c h a r a c t e r i z a t i o n . Due to sample s c a r c i t y t h i s i s not pos s i b l e . None of the arsenic compounds i n the unadsorbed f r a c t i o n s i n previous s t u d i e s ^ * 6 4 have been i d e n t i f i e d to date presumbaly f o r the same reason. In order to i d e n t i f y these compounds, large quantities of clams w i l l need to be extracted because the arsenic i n the unadsorbed f r a c t i o n i s only about 2% of the t o t a l arsenic present, i . e . about 0.05 fig g" 1 of arsenic i n Butter clam. 4.2.3 Arsenic Speciation i n the V i s c e r a l Mass. Foot and Siphon Tissues  of the Geoduck (Panope generosa) Ext r a c t i o n of the Geoduck tissues with methanol removes 87% of the arsenic i n the v i s c e r a l mass, 70% of the arsenic i n the foot and 82% of 1 0 0 > h H (A z 111 h Z H 5 0 1 3 3 1 7 5 1 4 4 4li 1 5 8 1 8 9 2 0 3 2 3 5 1 2 0 1 4 0 1 6 0 1 8 0 I ' l ' i ' i'l ' l'I'i ' iTl'i ' l ' l'Iri I'I'I'I ITI'I'ITI i i'i il I'I'I i 11 i i-i'i 1111111 Ii 11 j 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 M/2 F i g . 4.11: Thermospray LC mass spectrum of p u r i f i e d unadsorbed f r a c t i o n of Butter clam, same conditions as i n F i g . 4.7 - 142 -the arsenic i n the siphon, Table 4.15. Table 4.15: Ex t r a c t i o n of three tissues of the Geoduck V i s c e r a l mass Siphon Foot Weight of tissu e extracted (g) 132 148 115 As i n tiss u e (Mg) 739 488 471 Weight of residue (g) 9 21 20 As i n residue (/ig) 94 89 152 As i n methanol extract (/ig) 635 393 330 Percentage of As extracted % 87 82 70 The pattern of d i s t r i b u t i o n of arsenic i n the unadsorbed, NH^OH and HCI f r a c t i o n s a f t e r chromatography on Dowex 50 i s also d i f f e r e n t f o r the three t i s s u e s , Table 4.16. The v i s c e r a l mass which contains the highest concentration of arsenic 5.6 /ig g " l has 61% of the t o t a l arsenic i n the HCI f r a c t i o n . Arsenic i s equally d i s t r i b u t e d between the NH4OH and HCI fr a c t i o n s i n the foot t i s s u e . The arsenic compound present i n the NH4OH i s characterized as arsenobetaine and that i n the HCI f r a c t i o n as t e t r a -methylarsonium from the HPLC, TLC and NMR behaviour. - 143 -Table 4.16: F r a c t i o n a t i o n on Dowex 50 of the water soluble arsenic i n three tissues of Geoduck Tissue As % of T o t a l As Unadsorbed NH40H HCI V i s c e r a l mass 8 17 61 Siphon 4 11 65 Foot 15 27 27 4.2.4 Arsenic Speciation i n Gastropods Phoenician Whelk (Neptunea  phoeniceus). Northwest Neptune (Neptunea l v r a t a ) . and Thick- ribbed Whelk (Beringius crebriscotata) Methanol extracts 98% of the arsenic i n the Northwest neptune and Thick-ribbed whelk and 90% of the arsenic i n Phoenician whelk (Table 4.17). These percentages are higher than those found f o r the clams (Table 4.13) which range from 57-81%. Gel permeation chromatography of the extracts on Sephadex LH-20 shows the arsenic compounds elute with the same pattern as the arsenic compounds i n the clams. Chromatography on Dowex 50, however, reveals a d i f f e r e n t trend. Unlike the clams (with - 144 -Table 4.17: Ex t r a c t i o n of s o f t - t i s s u e s of Gastropods Phoenician Northwest Thick Whelk Neptune ribbed Neptunea Neptunea Whelk phoenecius l v r a t a Beringius c r e b r i s c o t a t a Number of whelks 15 Weight of s o f t - t i s s u e s extracted (g) As content of s o f t tissues (/ig) 170 602 54 2305 175 3045 Residue a f t e r methanol ex t r a c t i o n (g) As i n residue /ig 27 60 7 29 23 75 As i n methanol extract (/ig) Percentage of t o t a l As extracted 535 90 2240 98 2950 98 the exception of the S o f t - s h e l l e d clam) which have the highest percent-age of arsenic i n the HCI f r a c t i o n , (Table 4.14), the NH4OH fr a c t i o n s of the Northwest neptune and Thick-ribbed whelk show the highest percentage of arsenic (Table 4.18). The Phoenician whelk, on the other hand, shows 50% of i t s arsenic i n the HCI f r a c t i o n and 36% i n the NH4OH f r a c t i o n . - 145 -Table 4.18: Fra c t i o n a t i o n of water-soluble arsenic i n gastropods on Dowex 50 Gastropod As (% of T o t a l As) Unadsorbed NH40H HCI Phoenician whelk 6 36 50 (Neptunea phoeniceus) Northwest neptune 16 50 30 (Neptunea ly r a t a ) Thick-ribbed whelk 11 75 11 (Beringius crebriscotata) I t should be pointed out that the arsenic l e v e l i n t h i s whelk i s much lower, 3.5 /jg g" 1 than the 42.6 and 17.4 ng g" 1 f o r the Northwest neptune and Thick-ribbed whelk re s p e c t i v e l y . Dowex 2 X 8 chromatography of the NH4OH f r a c t i o n of the Thick-ribbed whelk gives two arsenic containing f r a c t i o n s . The maj or one, 90% i s i d e n t i f i e d as arsenobetaine on the basis of HPLC, TLC, NMR, and thermo-spray LC mass spectra. The ^H NMR of the minor f r a c t i o n i s shown i n Fi g . 4.12. This i s almost i d e n t i c a l to the spectrum of the compound found i n Butter clam ( F i g . 4.4) which i s p r o v i s i o n a l l y i d e n t i f i e d as 146 -2 ppm F i g . 4.12: *H NMR of minor arsenic compound i n NH4OH f r a c t i o n of Thick-ribbed whelk trimethylarsine oxide. An attempt to remove the D 20 from the s o l u t i o n by freeze drying r e s u l t s i n a residue which does not contain arsenic; the arsenic compound i s l o s t i n the process. No furthe r t e s t could therefore be performed. The NH4OH f r a c t i o n of the Phoenicean whelk and Northwest neptune shows one arsenic containing f r a c t i o n a f t e r chromatography on Dowex 2 x 8. This i s i d e n t i f i e d as arsenobetaine based on TLC, HPLC, NMR and thermospray LC mass spectra. - 147 -Analysis by TLC reveals that the HCI f r a c t i o n of the gastropods does not contain arsenocholine (Rf 0.30) or tetramethylarsonium s a l t (Rf 0.38) but instead an arsenic containing compound with Rf 0.25 f o r the Thick-ribbed whelk and Northwest neptune and a compound with Rf 0.23 f o r the Phoenician whelk. A l l three compounds elute at the same r e t e n t i o n volume of 2.5-3.5 mL as arsenobetaine on the Protein Pak column (ammo-nium acetate b u f f e r , pH 10). On the reversed phase column with hepta-nesulfonic a c i d of the counter ion, they again elute with the same re t e n t i o n volume as arsenobetaine, 6.5-7.5 mL. F i g . 4.13 shows the ^H NMR spectrum s o l u t i o n of the Northwest neptune extract. This i s obviously d i f f e r e n t from the spectra of arsenocholine ( F i g . 4.5) or tetramethylarsonium ( F i g . 4.6) possible r 5 4 3 2 1 ppm F i g . 4.13: *H NMR spectrum of p u r i f i e d HCI f r a c t i o n of Northwest neptune - 148 -compounds i n the HCI f r a c t i o n . The thermospray LC mass spectrum ( F ig. 4.14) does not correspond to any of the known compounds. The gastropods studied to d a t e ^ ^ • ^ a r e a n known to contain arsenobetaine and small proportions of yet to be i d e n t i f i e d compounds. The presence of arsenobetaine i n the gastropods i n t h i s study i s i n l i n e with the previous reported observations. More whelks need to be examined i n order to i d e n t i f y the unknown compounds. 4.3 Arsenicals i n Shells Arsenic concentrations i n a number s h e l l s are given i n Table 4.19. Arsenic i s determined by HGAA a f t e r d i s s o l u t i o n of 1 g of the s h e l l i n 10 mL of 2 M hydrochloric a c i d (Section 2.8). I n j e c t i o n of aliquots of the hydrochloric a c i d s o l u t i o n into the graphite furnace f o r GFAA r e s u l t s i n negative absorbance readings due to s c a t t e r i n g and over-c o r r e c t i o n by the background c o r r e c t i o n system. This problem i s not solved by matrix modification. The s h e l l s are chalky materials compared mostly of calcium carbonate. Arsenic has proven p a r t i c u l a r l y d i f f i c u l t to determine by GFAA i n matrices that have large amounts of inorganic materials or elements capable of forming r e f r a c t o r y oxides such as calcium.^4 Hydrochloric a c i d also presents serious interference problems i n the analysis of arsenic by GFAA. ^ 5 I t was found necessary to use the UV-HGAA technique (Section 3.3) to determine t o t a l arsenic. The HGAA response of hydrochloric a c i d solu-tions of Manila clam s h e l l s are compared before and a f t e r UV-irradia-t i o n , Table 4.20. 1 0 0 h H (A Z s" w h Z H 100 > b H (/} W h Z H 3 8 8 i I I i | I I I I I ) I I I | I Fl 4f I I I ) I | I I I I I I I I l | I | | 1 | | | II | | | i | ) | | | ) j | ) | | | I4!3?! | I I I I I I I I I | I I I I I I I I I | I I I I I 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 VO 1 8 8 1 8 0 111M' 111' i' i11 r i l i I I'I'I'I 11 1 1 1 I ' I | 1111111 II 11111'| 11 11 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 M/ Z 3 0 0 F i g . 4.14; Thermospray LC mass spectrum of p u r i f i e d HCI f r a c t i o n of Thick-ribbed whelk, same conditions as i n F i g . 4.7 - 150 -Table 4.19: Arsenic concentrations i n sea s h e l l s S h e l l Source (Date Collected) Butter clam Thetis Island (Oct 85) 0. ,6 + 0. 02 Saxidomus giganteus S o f t - s h e l l e d clam Thetis Island (Oct 85) 0. .3 + 0. .02 Mva arenaria Coles Bay (May 87) 0. .4 + 0, .05 P a t r i c i a Bay (May 87) 0, .4 + 0, .04 Anyox Slag heap (Oct 87) 4 .4 + 0 .2 Hastings Arm (Oct 87) 26 .3 + 0 .8 Manila clam Thetis Island (Oct 86) 0 .3 + 0 .03 Venerupis ^aponica Coles Bay (May 87) 0 .5 + 0 .04 Native l i t t l e n e c k clam P a t r i c i a Bay (May 87) 0 .3 + 0 .05 Protothaca staminea Coles Bay (May 87) 0 .3 + 0 .05 Bent nose clam Coles Bay (May 87) 0 .4 + 0, .07 Macoma nasuta Blue mussel Hastings Arm (Oct 87) 0.15 + 0. .01 Mvtilus e d u l i s Rupert I n l e t (Oct 87) 0.11 + 0. .01 Lyre whelk A l i c e Arm A3 (Oct 86) 5. .1 + 0. .09 Bucinuum plectrum Phoenician whelk A l i c e Arm A3 (Oct 86) 5, .8 + 0. 8 Neptunea phoenicius Jordan's colus A l i c e Arm A3 (Oct 86) 16, .3 + 6. ,5 Colus ^ordani Northwest neptune A l i c e Arm A3 (Oct 86) 1. .4 + 0. .2 Neptunea l v r a t a A5 (Oct 86) 1 .9 + 0. .2 - 151 -Table 4.20: Determination of arsenic i n Manila clam s h e l l s by HGAA and UV-HGAA Solution Arsenic Absorbance HGAA UV-HGAA3 0.031 0.033 0.031 0.031 0.032 0.032 0.031 0.032 20 mL a l i q u o t i r r a d i a t e d f o r 30 min. No s i g n i f i c a n t increase i n arsenic response i s obtained a f t e r U V - i r r a d i a t i o n . This indicates that the arsenic i s present i n the s h e l l mostly i n inorganic or simple methylated form capable of reduction by NaBH 4 to v o l a t i l e arsines. The nature of the arsenic compound i n the s h e l l can be determined by HPLC-GFAA. When ali q u o t s , 20 fiL of the s o l u t i o n obtained from Soft-s h e l l e d clam (from Anyox) containing 4.4 ng g o f arsenic are injec t e d onto the /iBondapak C^g column, the eluent water-methanol (95:5) with 5 mM tetrabutylammonium n i t r a t e as ion p a i r reagent, only the fra c t i o n s - 152 -with r e t e n t i o n time of 15.0 and 15.5 min contain arsenic. This corres-ponds to the re t e n t i o n time of standard arsenate. No methylarsonic a c i d or dimethylarsinic a c i d i s detected. The presence of methylarsonic a c i d and dimethylarsinic a c i d i n sea s h e l l s and b i r d egg s h e l l s has been reported by Braman and Foreback^ although no d e t a i l s are given as to the type of s h e l l s and l e v e l s of arsenic. The i n a b i l i t y to detect the presence of dimethylarsinic a c i d and methylarsonic a c i d i n the s h e l l s i n the present study might probably be due to the f a c t that they are present i n quantities lower than the detection l i m i t of the HPLC-GFAA system. More s e n s i t i v e techniques such as HGAA-GC^ are required to f u l l y characterize compounds present. This suggestion i s r e i n f o r c e d by the observation that HPLC-GFAA studies of aliquots of solutions of S o f t - s h e l l clams from Hastings Arm, containing 26.3 /ig g a r s e n i c , show a f r a c t i o n with retention time 5.5 and 6.0 min, which corresponds to that of dimethylarsinic a c i d . The arsenic concentrations i n the s h e l l s do not c o r r e l a t e with the arsenic concentrations i n the s o f t tissues or arsenic concentrations i n the surface sediments. Table 4.21 shows the concentrations of arsenic i n S o f t - s h e l l e d clam s h e l l s and s o f t tissues as w e l l as arsenic concen-t r a t i o n s i n surface sediments. The highest arsenic concentration i s found i n the S o f t - s h e l l e d clam from Hastings Arm, a p r i s t i n e area. The concentrations i n the s h e l l s from Thetis Island, Coles Bay and P a t r i c i a Bay seem in v a r i a n t . With d i f f e r e n t organisms, the lowest concentrations are found i n the Blue mussels, 0.1 /ig g'^. Mussels are not i n d i r e c t contact with the sediments. The whelks show concentrations which vary from 1.4 /ig g"^ i n - 153 -Table 4.21: Arsenic concentrations In So f t - s h e l l e d clam and sediments Stati o n As (/ig g ) Comments Soft t i s s u e s 3 S h e l l b Sediment 0 Thetis Island 2. 5 0. 3 - Natural sediment Coles Bay 0. .6 0. ,4 - Natural sediment, sewage o u t f a l l P a t r i c i a Bay 2. ,0 0. .4 - Natural sediment sewage o u t f a l l Anyox 6. .7 4. .4 46.5 T a i l i n g s s l a g heap, 50 years o l d Hastings Arm 4 .5 26 .3 14.9 Natural sediment g l a c i e r fed Northwest neptune to a high of 16.3 /ig g i n Jordan's colus. I t i s possi b l e the organisms are s t o r i n g arsenic i n t h e i r s h e l l s as a form of d e t o x i f i c a t i o n , s i m i l a r to the storage of arsenic i n human hair.-'-- 154 -SUMMARY GFAA and HGAA techniques have been developed and applied to the determination of t o t a l arsenic i n some marine invertebrates of B r i t i s h Columbia. T o t a l arsenic concentrations i n bi v a l v e s o f t tissues vary with species and range from 0.6 ng g~^ to 9.1 ng g"^. The same species from d i f f e r e n t locations show d i f f e r e n t arsenic l e v e l s which do not co r r e l a t e with arsenic l e v e l s i n sediments and i n t e r s t i t i a l pore waters. The arsenic l e v e l s obtained i n t h i s work are comparable to l e v e l s obtained i n previous work by others. Arsenic concentrations i n the s h e l l s of the bival v e s show a wider range of 0.1 ng g'^ to 26.3 ng g > on a dry weight b a s i s . Again, the same species from d i f f e r e n t locations show d i f f e r e n t arsenic concentrations with no c o r r e l a t i o n with sediment arsenic l e v e l s . The arsenic concentrations i n the s o f t tissues do not co r r e l a t e with concentrations i n the s h e l l s . I t i s probable that d i f f e r e n t mechanisms are involved i n the accumulation of arsenic i n the s o f t tissues and s h e l l s . The gastropods from the same study areas (with the exception of the Phoenician whelk) show r e l a t i v e l y higher arsenic concentrations (17.3-48.4 ng gcompared to the bivalv e s . The gastropod s h e l l s show concentrations ranging from 1.4 to 16.3 ng g a n d again there i s no c o r r e l a t i o n between the arsenic l e v e l s i n the s o f t tissues and s h e l l s . D e t ailed studies are needed on the mode of arsenic uptake and accumulation i n the s o f t - t i s s u e s and s h e l l s i n the marine organisms i n order to understand the reasons f o r the differences i n arsenic l e v e l s . - 155 -HPLC-GFAA techniques were developed and used f o r the separation and qu a n t i t a t i o n of arsenite, arsenate, dimethylarsinic acid, methylarsonic acid, arsenobetaine, arsenocholine iodide and tetramethylarsonium iodide by using C^g reversed phase and Protein Pak ion exchange columns. Thermospray LC mass spectrometry was also developed f o r the detection of these compounds. These techniques along with TLC, NMR and FAB mass spectrometry can employed to the detection of water soluble arsenic compounds i n 5 species of bivalves; Butter clam Saxidomus giganteus. Horse clam Schizothoerus n u t t a l l i . S o f t - s h e l l e d clam Mya arenaria. N a t i v e - l i t t l e n e c k clam Protothaca staminea and Manila clam Venerupis  japonica. Varying amounts of arsenobetaine and tetramethylarsonium ion are detected i n a l l the clams. Butter clam shows the presence of a t h i r d compound which appears to be trimethylarsine oxide. Small amounts of unknown compounds are present i n a l l the clams which are not yet characterized. Arsenic s p e c i a t i o n i n three gastropods are also examined. The Northwest neptune Neptunea l y r a t a . the Thick-ribbed whelk Beringius  cre b i s c o t a t a and Phoenician whelk Neptunea phoenicius a l l contain arsenobetaine and at l e a s t two yet to be characterized compounds. The c h a r a c t e r i z a t i o n of these compounds i s e s s e n t i a l i n order to understand the pathways involved i n the conversion of arsenic compounds i n the marine environment. The presence of arsenobetaine i n a l l the species examined confirms the v i r t u a l ubiquity of t h i s compound i n marine invertebrates. The i d e n t i f i c a t i o n of tetramethylarsonium s a l t s i n a l l the bivalves r a i s e s questions as to the r o l e they play i n the mechanism of arsenic transfor-- 156 -mations i n marine organisms. These compounds were not considered i n the scheme proposed by Edmonds and Francesconi to account f o r the conversion of arsenate to arsenobetaine v i a arsenosugars. - 157 -REFERENCES 1. Mellor, J.W. In "A Comprehensive Treastise on Inorganic and The o r e t i c a l Chemistry", V o l . IX, Longman, 1970, p. 42. 2. Maugh, T.H. Science. 1977, 203:637. 3. Chapman, A.C. Analyst. 1926, 51:548. 4. Cox, H.E. Analyst. 1925, 50:3. 5. Edmonds, J.S. and Francesconi, K.A. J . Chem. Soc. Perkin Trans. I. 1983, 2375. 6. Edmonds, J.S., Morita, M., and Shibata, Y. J . Chem. Soc. Perkin  Trans I. 1987, 577. 7. Shiomi, K., Kakehasi, Y., Yamanaka, H., and Kikuchi, T. Appl.  Organomet. Chem.. 1987, 1:177. 8. Edmonds, J.S., Francesconi, K.A., Healy, P.C., and White, A.C. J .  Chem. Soc. Perkin Trans. I. 1982, 2989. 9. Edmonds, J.S. and Francesconi, K.A. Nature, 1977, 265:436. 10. Shiomi, K., Shinagawa.A., Igarashi, H., Yamanaka, H., and Kikuchi, T. Comp. Biochem. Physiol.. 1983, 74:393. 11. Norin, H., Ryhage, R., Christakopoulos, A., and Sandstroem, M. Chemosphere. 1983, 12:299. 12. Edmonds, J.S. and Francesconi, K.A. Chemosphere. 1981, 10:1041. 13. Cannon, J.R., Saunders, J.B., and Toia, R.F. S c i . To t a l Environ.. 1986, 31:181. 14. Francesconi, K.A., Micks, P., Stockton, R.A., and I r g o l i c , K.J. Chemosphere. 1985, 14:1443. 15. Luten, J.B., Riekwel-Booy, G., and Rauchbar, A. Environ. Health  Perspect.. 1982, 45:165. 16. Edmonds, J.S. and Francesconi, K.A. Mar. P o l l t . B u l l . . 1981, 12:92. 17. Kurosawa, S., Yasuda, K., Taguchl, M., Yamazaki, S., Toda, S., Morita, M., Uehiro, T., and Fuwa, K., Agric. B i o l . Chem.. 1980, 44:1993. - 158 -18. Penrose, W.R. CRC C r i t . Rev. Environ. Control. 1974, 4:465. 19. Andrea, M.O. In, "Organometallic Compounds i n the Environment; P r i n c i p l e s and Reactions", (ed. Craig, P.J.), Wiley-Interscience New York, 1986, 198. 20. Andreae, M.O. Limnol. Oceanogr.. 1979, 24:440. 21. Andreae, M.O. and F r o e l i c h , P.N. T e l l u s . 1984, 36B:101. 22. Johnson, D.L. Nature, 1972, 240:44. 23. Andreae, M.O. and Klump, D.W. Environ. S c i . Tech.. 1979, 13:738. 24. P i l s o n , M.E.Q., Limnol. Oceanogr.. 1974, 19:339. 25. Blake, N.J. and Johnson, D.L. Deep Sea Res.. 1976, 23:778. 26. Blasco, F., Gaudin, C. and Jeanjean, R. C.R. Hebd. Seances Acad.  S c i . Ser. D. S c i . Nat.. 1971, 273:812. 27. Myers, D.J., Heimbrook, M.E., Osteryoung, J . , and Morrison, S.M. Environ. L e t t . . 1973, 5:53. 28. P h i l l i p s , S.E. and Taylor, M.L. App. And Environ. M i c r o b i o l . . 1976, 32:392. 29. Osborne, F.H. and E h r l i c h , H.L. J . A D D I . B a c t e r i o l . . 1976, 41:295. 30. Johnson, D.L. and Pil s o n , M.E.Q. Environ. L e t t . . 1975, 8:157. 31. Gosio, B. Ber. Duet. Chem. Ges.. 1897, 30:1024. 32. Challenger, F., Chem. Reviews. 1945, 36:315. 33. du Vigneau, V., Cohn, M., Chandler, J.P., Schenck, J.R., and Simmonoss, S., J . B i o l . Chem.. 1941, 140:625. 34. Challenger, F. , L i s l e , D.B., and Dransfield, P.B. J . Chem. S o c . 1954, 1760. 35. Cox, D.P. and Alexander, M. B u l l . Environ. Contam. T o x i c o l . . 1973, 9:84. 36. Cullen, W.R., Froese, C.L., L u i , A., McBride, B.C., Patmore, D.J., and Reimer, M. J . Organomet. Chem.. 1977, 139:61. 37. Cullen, W.R., McBride, B.C., and Reimer, M. B u l l . Environ. Contam.  To x i c o l . , 1979, 21, 157. - 159 -38. Cullen, W.R., McBride, B.C., and Pick e t t , H.W. Can. J . Mi c r o b i o l . . 1979, 25:1201. 39. P i c k e t t , A.W., McBride, B.C., Cullen, W.R., andManji, H. Can. J . Mi c r o b i o l . . 1981, 27:773. 40. McBride, B.C. and Wolfe, R.S. Biochemistry. 1971, 10:4312. 41. Wong, P.T.S., Chau, Y.K., Luxon, L., and Bengert, G.A. Trace  Subst. Environ. Health. 1977, 11:100. 42. McBride, B.C., Merilees, H., Cullen, W.R., and Pick e t t , A.W. ACS Symposium Series No. 82, 1982, 94. 43. Reimer, K.J. and Thompson, J.A.J. Biogeochem.. 1988, In Press. 44. Ebdon, L., Walton, A.P., Millward, G.E., and W h i t f i e l d , M. Appl.  Organomet. Chem.. 1987, 1:427. 45. Braman, R.S. and Foreback, C.C. Science. 1973, 182:1247. 46. Klumpp, D.W. Marine B i o l . . 1980, 58:257. 47. Benson, A.A. and Nissen, P. Development i n Plant B i o l . . 1982, 8:121. 48. Klumpp, D.W. and Peterson, P.J. Marine B i o l . . 1981, 62:297. 49. Lunde, G. Acta Chem Scand.. 1973, 27:1586. 50. Wrench, J . J . and Addison, R.F. Can. J . Fis h Aquat. S c i . . 1981, 38:518. 51. Lunde, G. Acta Chem. Scand.. 1972, 26:2642. 52. Lunde, G. J . S c i . Food Agr.. 1975. 26:1257. 53. I r g o l i c , K.J., Woolson, E.A., Stockton, R.A., Newman, N.R., Zingaro, R., Kearney, P.C., Pyles, R.A., Maeda, S., McShane, W.J., and Cox, E.R. Environ. Health Perspect.. 1977, 19:61. 54. Edmonds, J.S. and Francesconi, K.A. J . Chem. Soc. Perkins Trans. I, 1983, 2375. 55. Shibata, Y., Morita, M., and Edmonds, J.S., Agr i c . B i o l . Chem.. 1987, 51:391. 56. Edmonds, J.S. and Francesconi, K.A. Experientia. 1987, 43:553. 57. Edmonds, J.S., Francesconi, K.A., Cannon, J.R., Raston, C.L., Skelton, B.W., and White, A.H. Tetrahedron L e t t . . 1977, 18:1543. - 160 -58. Shiomi, K., Shinaga, A., Yamanaka, H., and Kikuchi, T. B u l l . Jpn.  Soc. S c i . F i s h . . 1983, 49:79. 59. Cannon, J.R., Edmonds, J.S., Francesconi, K.A., Raston, C.L., Saunders, J.B., Skelton, B.W. and White, A.H. Aust. J . Chem.. 1981, 34:787. 60. Luten J.B., Riekwel-Booy, G., Van der Greeg, J . , and Ten Noever de Brauw, M.C. Chemosphere. 1983, 12:131. 61. Shiomi, K., Shinagawa, A., Hirota, K., Yamanaka, H., and Kikuchi, T. Comp. Biochem. Phvsiol.. 1983, 74:393. 62. Shiomi, K., Shinagawa, A., Hirota, K., Yamanaka, H., and Kikuchi, T. Aerie. B i o l . Chem.. 1984, 48:2863. 63. Matsuto, S., Stockton, R.A., and I r g o l i c , K.J. S c i . T o t a l  Environ.. 1986, 48:133. 64. Shiomi, K., O r i i , M. , Yamanaka, H., and Kikuchi, T. Nippon Suisan  Gakkaishi.. 1977, 53:103. 65. Lawrence, J.F., Michalik, P., Tarn, G., and Conacher, HBS, J .  Aerie. Food. Chem.. 1986, 34:315. 66. Norin, H., Christakopoulos, A., Sandstrom, M., and Ryhage, R. Chemosphere. 1985, 14:313. 67. Fowler, S.W. and Unlu, M.Y. Chemosphere.. 1978, 7:711. 68. Klumpp, D.W. Mar. B i o l . . 1980, 58:265. 69. Edmonds, J.S. and Francesconi, K.A. Experientia. 1982, 32:643. 70. Harraro-Lasso, J.M. and Benson, A.A. Environ. S c i . Res.. 1982, 23:50. 71. Kaise, T., Watanabe, S., and Itoh, K. Chemosphere. 1985, 14:1327. 72. Tarn, G.K.H., Charbonneau, S.M., Bryce, F., and Sandi, E. B u l l .  Environ. Contain. T o x i c o l . . 1982, 28:669. 73. Sturman, B.S. App. Spectroscopy.. 1985, 39:48. 74. Anderson, R.K., Thompson, M., and Culbard, E. Analyst. 1986, 111:1143. 75. I r g o l i c , K.J., Junk, T., Kos, C , McShane, W.S., and Parppalardo, C.C. App. Organomet. Chem.. 1987, 1:403. - 161 -76. Penrose, W.R., Conacher, H.B.S., Black, R., Meranger, J.C., Miles, W., Cunningham, H.M., and Squires, W.R. Environ. Health Prespect.. 1977, 19:53. 77. Bajo, S., Suter, U., and Aeschliman, B. Anal. Chim. Acta.. 1983, 149:321. 78. Armstrong, F.A.J., Williams, P.M., and St r i c k l a n d , J.D.H. Nature. 1966, 211:481. 79. Cullen, W.R. and Dodd, M. Appl. Organomet. Chem.. 1988, 2:1. 80. Brooks, R.R., Ryan, D.E., and Zhang, H. Anal. Chim. Acta.. 1981, 131:1. 81. A n a l y t i c a l Methods for Graphite Tube Atomizers. Ed. E. Rothery, Varian Techtron Pty. Ltd. Mulgrave, V i c t o r i a , A u s t r a l i a , 1982. 82. Maher, W.A. Spectroscopy L e t t . . 1983, 16:865. 83. Andreae, M.O. Anal. Chem.. 1977, 49:80. 84. Braman, R.S., Johnson, D.L., Foreback, C.C., Ammons, C.C., and Briker, J.L. Anal. Chem.. 1977, 49:621. 85. Shaikh, A.U. and Tallman, D.E. Anal. Chim. Acta. 1978, 98:251. 86. Anderson, Q.K., Thompson, M., and Culbard, E. Analyst. 1986, 111:1153. 87. Fernandez, F.J. and Manning, D.C. At. Absorpt. Newst.. 1971, 10:86. 88. Maruta, T. and Sudoh, G. Anal. Chim. Acta.. 1975, 77:37. 89. Korenga, T. Mikrochim. Acta.. 1979, 1:435. 90. Braman, R.S., Justen, J.L., and Foreback, C.C. Anal. Chem.. 1972, 44:2195. 91. Knudson, E.J. and C h r i s t i a n , G.D. Anal. L e t t s . . 1973, 6:1039. 92. Robins, W.B. and Caruso, J.A. Anal. Chem.. 1979, 51:8894. 93. Raptis, S.E., Wegscheider, W., and Knapp, G. Microchim Acta  rwienl. 1981, 1:93. 94. Van Loon, J.C. Selected Methods of Trace Analysis: B i o l o g i c a l and Environmental Samples, Wiley, New York, 1985. 95. Maher, W.A. Marine B i o l . L e t t . . 1984, 5:47. - 162 -96. Norin, H. and Christakopoulos, A. Chemosphere. 1982, 11:287. 97. Beattle, J . , Bricker, C , and Garvi, D. Anal. Chem. . 1961, 33:1890. 98. Measures, C.I. and Burton, J.D. Anal. Chim. Acta. 1980, 120:177. 99. Tarn. K.C. Environ. S c i . Technol.. 1974, 8:734. 100. Stringer, C.E. and Attrep, M. Anal. Chem.. 1979, 51:731. 101. F i s h , R.H., Walker, W., and Tannous, R.S. J . Energy Fuels. 1987, 1:243. 102. F i s h , R.H., Brinkman, F.E., and Jewett, K.L. Environ. S c i .  Technol.. 1982, 16:175. 103. Asher, C.J. and Reay, P.F. Anal. Biochem.. 1977, 78:557. 104. R i c c i , G.R., Shepard, L.S., Colovos, G., and Hester, N.E. Anal.  Chem.. 1981, 53:610. 105. Brinkman, F.E., B l a i r , W.R., Jewett, K.L., and Iverson, W.P. J .  Chromatogr. S c i . . 1977, 15:493. 106. Stocton, R.a. and I n r g o l i c , K.J. Int. J . Environ Anal Chem.. 1978, 50:1700. 107. Koizumi, H., Hadeishi, T., and Maclauglin, R. Anal. Chem.. 1978, 50:1700. 108. Woolson, E.A. and Aharonson, J . Assoc. Off. Anal. Chem.. 1980, 63:523. 109. I r g o l i c , K.J., Stocton, R.A., and Chakraborti, D. Spectrochimica  Acta B. 1983, 38:437. 110. Morita, M., Uehiro, T., and Fuwa, K. Anal. Chem.. 1980, 52:348. 111. Nisamaneepong, W., Ibrahim, M., G i l b e r t , W.T., and Caruso, J.A. J . Chromatogr. S c i . . 1984, 22:473. 112. Brinkman, F.E., Jewett, K.L., Iverson, W.P., I r g o l i c , K.J., Ehrhardt, K.C, and Stocton, R.A. J . Chromatogr.. 198, 191:31. 113. I o n i z a t i o n Constants of Inorganic Acids and Bases i n Aqueous Solutions Compiled by D.D. Perrin, 2nd Ed., Oxford, New York, Pergamon Press, 1982, IUPAC Chemical data s e r i e s , No. 29, p. 9. - 163 -114. I o n i z a t i o n Constants of Inorganic Acids and Bases i n Aqueous Solutions. Eds. E.P. Serjeant and B, Dempsey, Oxford, Neew York, Pergamon Press, 1979. IUPAC Chemical data s e r i e s , No. 23, p. 12, 115. Bohn, A. Mar. P o l l u t . B u l l . . 1975, 6:87. 116. F a r r e l l , M.A. and Nassichuk, M.D. Can. Data Rep F i s h Aquat. S c i . . 1984, No. 467. 117. Goyette, D. and C h r i s t i e , P. Environmental Protection Service, Regional Program Report 82-1. Environment Canada. 118. Amax of Canada Ltd., Annual Report f o r the K i t s a u l t Mine Environ-mental Monitoring Program. (AATDR, PE-4335), 1982, 2. 119. Goyette, D., Thomas, M., and Factor, E. Environmental Protection Service, Regional Program Report 85-04, Environment Canada. 120. Futer, P. and Nassichuk, M. Can. MS Rep. Fi s h . Aauat. S c i . . 1983, No. 1699. 121. Shiomi, K., Shinagawa, A., Igarashi, T., Hirota, K., Yamanaka, H., and Kikuchi, T. B u l l . Japan Soc. S c i . F i s h . . 1984, 50:293. 122. Edmonds, K.S. and Francesconi, K.A. S c i . T o t a l Environ.. 1987, 64:317. 123. Penrose, W.R. J . F i s h Res. Board Can.. 1975, 32:2385. 124. Sl a v i n , W. In "Graphite Furnace AAS", A Source Book, 1984, Perkin-Elmer Corporation, Norwalk, CT 06856. 

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