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Studies of arsenicals in some marine algae of British Columbia Ojo, Abiodun Ayodele 1994

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STUDIES OF ARSENICALS IN SOMEMARINE ALGAE OF BRITISH COLUMBIAbyABIODUN AYODELE OJOBSc (Hons), University of Ife, Nigeria, 1985A THESIS SUBMITTED IN PARTIAL FULFILJJvfENTOF THE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYINTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF CHEMISTRYWe accept this the required standard:The University of British ColumbiaDecember 1994© Abiodun Ayodele Ojo, 1994In 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 itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)_________________________________Department of CHEfVtlSiR.-YThe University of British ColumbiaVancouver, CanadaDate_________________DE-6 (2/88)STUDIES OF ARSENICALS IN SOMEMARINE ALGAE OF BRiTISH COLUMBIAABSTRACTArsenic determination in some marine algae ofBritish Columbia was carried out byusing Hydride Generation and Graphite Furnace Atomic Absorption Spectrometzy(HGAAS and GFAAS) following a two-hour wet digestion procedure. The total arsenicconcentration in Fucus distichus and other brown algae determined by using thecontinuous HGAAS was found to vary with species and the collection sites, ranging from1.8 .tg/g As to 36.9 J.Lg/g As (dry weight basis). Similarly, the arsenic concentrationsdetermined in red algae were found to vary from a low 1.3 .g/g As to a high 39.7 g/g As(also on a dry weight basis). Also, the arsenic concentrations determined in green algaewere found to vary quite widely as in the other two classes of macroalgae, ranging from0 j.g/g As (not detected) to 27.2 1gfg As. These widely varying arsenic concentrationresults suggest that the amount of arsenic accumulated by seaweed is not only related tothe class of the macroalgae as well as the particular species, but also to the samplinglocation and its soil conditions.Both the continuous HGAA and the semi-continuous HG-GC-AA techniques wereemployed for the examination of the selective reduction of four arsenic species, namelyarsenate [As(V)], arsenite [As(ffl)], monomethylarsonic acid (MMAA) anddimethylarsinic acid (DMAA). The response profiles of the four arsenic species wereplotted and the optimum arsenic analytical conditions were determined from these plots.By using a combination of extraction followed with sodium hydroxide digestion of thefreeze dried seaweed, the reducible and “hidden” arsenic species in the extracts and digestswere determined by using the semi-continuous mode HG-GC-AAS. Greater than 90% ofthe total arsenic in the seaweeds was found in the “hidden” arsenic forms.UOn-line detection techniques of High Performance Liquid ChromatographyMicrowave Oven Assisted Decomposition Hydride Generation Atomic AbsorptionSpectrometry (HPLC-Mic-HGAAS) and High Performance Liquid ChromatographyInductively Coupled Plasma Mass Spectrometry (HPLC-ICPMS) were employed for thecharacterization of arsenic species in water soluble extract ofF. distichus collected fromHead ofHastings Arm, British Columbia. Prior to the use of these techniques for arsenicspecies identification, arsenic containing fractions were isolated via the use of severalcombinations of chromatographic procedures such as gel permeation chromatography(gpc), ion exchange chromatography (WC), thin layer chromatography (tic) and reversephase high performance liquid chromatography (HPLC). The water soluble extract ofF. distichus was found to contain two major arsenosugars*, jj and 111!. as well asseveral minor, unidentified arsenic species, likely to be other arsenosugar derivatives.The biotransformation studies of “hidden” organoarsenicals in F. distichus werecarried out by using anaerobic decomposition conditions in “open” and “closed” systems.Two arsenic species, 2-dimethylarsinylethanol (DMAE) and dimethylarsiic acid (DMAA)were identified from the “open” system decomposition products by using the HPLCICPMS technique. In the “closed” system, DMAE was characterized as a majordecomposition product and DMAA was observed as another main component. Otherminor arsenic species were found in the decomposition products, however, these were notidentified.* (CH3)2As(O)CH/JCHCH(O )COU!! = (CHAs(O)CH%...,OCHCH(OH)CHOPH111TABLE OF CONTENTSTITLE PAGEABSTRACT iiTABLE OF CONTENTS ivLIST OF TABLES viiiLIST OF FIGURES xiLIST OF ABBREVIATIONS xviiACKNOWLEDGEMENT xviiiINTRODUCTION 11.1 GENERAL BACKGROUND 11.2 ARSENIC ACCUMULATION IN THE ENVIRONMENT 41.3 SPECIATION AND BIOTRANSFORMATION OF ARSENICALS IN THE MARINEENVIRONMENT 61.3.1 Speciation ofArsenicals 71.3.2 Speciation by Using CombinedMethods Fractionation Techniques Online Arsenic Speciation Techniques 101.3.3 Biotransformation ofArsenic in the Environment Arsenic Biotransformation Studies in Marine Environment Arsenicals in Marine Macroalgae Biotransformation Pathways in the Marine Environment 181.4 SCOPEOF WORK 21EXPERIMENTAL 232.1 iNSTRUMENTATION 232.1.1 AtomicAbsorption Spectrometry (AAS) Graphite Furnace Atomic Absorption (GFAA) Hydride Generation Atomic Absorption (HGAA) Hydride Generation - Gas Chromatography Atomic Absorption (HG-GC-AA) 252.1.2 High Pressure Liquid Chromatography (HPLC) HPLC-Mic-HGAAS HPLC -ICPMS 282.1.3 NuclearMagnetic Resonance (NMR) Spectrometry andMass Spectrometry (MS) 292.2 CHEMICALS AND REAGENTS 30iv2.3 SAMPLE SITE, COLLECTION AND STORAGE .312.4 ANALYTICAL PROCEDURES 362.4.1 Graphite FurnaceAtomicAbsorption (GFAA) 362.4.2 Hydride Generation AtomicAbsorption (HGAA) 382.4.3 Hydride Generation - Gas ChromatographyAtomicAbsorption (HG-GC-AA) 382.4.4 Microwave - Hydride GenerationAtomicAbsorption (Mic-HGAA) 392.4.5 High Performance Liquid Chromatography - Inductively Coupled Plasma Spectrometry(HPLC4CPMS) 402.4.6Determination ofTotalArsenic 402.4.6.1 Wet Digestion with Sulfuric Acid, Nitric Acid and Hydrogen Peroxide 422.4.6.2 Wet Digestion with Sodium Hydroxide 422.4.6.3 Wet Digestion with Nitric Acid 432.4.6.4 Methanol-Water Extraction Followed by Wet Digestion With Sodium Hydroxide 432.4.6.5 Sodium Hydroxide Extraction Followed by the Hot Base Digestion 432.4.7 Determination ofArsenic in Some Marine Algae by Neutron Activation Analysis 442.4.8 Extraction ofArsenicals in the Marine Algae 442.4.8.1 Extraction ofWater Soluble Arsenic Compounds 442.4.9 Determination ofArsenic in Extracts 452.5 PURIFICATION PROCEDURES FOR ARSENIC IN WATER SOLUBLE EXTRACTS 462.5.1 Isolation ofArsenic Containing Compounds in Water Soluble Extract ofFucus distichus 462.5.2 Preparation ofArsenosugar Standards 512.6 ANAEROBIC DECOMPOSITION OF BROWN ALGAE, F. DISTICHUS 522.6.1 Seaweed/Seawater/Sediment (SSS) 522.6.1.1 Closed System 522.6.1.2 Open System 532.6.2 Examination ofArsenicals in the Anaerobic Decomposition Products 542.7 SYNTHESIS OF 2-Dllv1ETHYLARS11,YLETHANOL 55RESULTS AND DISCUSSION - ANALYTICAL PROCEDURES 573.1 ARSENIC DETERMINATION 573.1.1 ARSENIC DETERMJNATJONBY GFAA 573.1.2 CHEMICALMODIFICATIONAND GFAA DETERA’IJNATION OFARSENIC 583.1.2.1 Nickel Modifier Optimization for Arsenic Determination by GFAA 603.1.2.2 Palladiuni Modifier Optimization for Arsenic Determination by GFAA 633.1.2.3 Determination of Some Arsenic Species Signals in 0.SM Tris buffer by GFAA Analysis 663.1.2.4 Calibration and Limit ofDetection ofGFAA Analysis 683.1.3 ARSENICDETERMINATIONBY COATINUOUS HGAA 70V3.1.4 ARSENIC SPECIESRESPONSE OPTIMIZ4 TION USINGA MINERAL ACID WITH VARYINGCONCENTRATIONS OFSODIUMBOR011YDRIDEREDUCTANT 713.1.4.1 Detemiination ofArsenic Species Response Profiles in 0.5% NaBH4Reductant Solution and VaryingConcentrations ofHydrochloric acid by Continuous HGAA 733.1.4.2 Determination ofArsenic Species Response Profiles in 1.0% NaBH4Reductant Solution and VaxyingConcentrations ofHydrochloric acid by Continuous HGAA 743.1.4.3 Determination ofArsenic Species Response Profiles in 1.5% NaBH4Reductant Solution and VaryingConcentrations ofHydrochloric acid by Continuous HGAA 753.1.4.4 Determination ofArsenic Species Response Profiles in 2.0% NaBH4Reductant Solution and VaryingConcentrations ofHydrochloric acid by Continuous HGAA 763.1.4.5 Determination ofArsenic Species Response Profiles in 2.5% NaBH4Reductant Solution and VaryingConcentrations ofHydrochloric acid by Continuous HGAA 773.1.4.6 Effect of sodium borohydride Concentration on Arsine Yield Using 0.05 iglml Arsenic Solution and4M hydrochloric acid 783.1.5ARSENIC SPECIESRESPONSE OPTIMIZATION USINGAN ORGANICACID WITHVARYING CONCENTRATIONS OFSODIUMBOROHYDRIDEREDUCTANT 803.1.5.1 Determination ofArsenic Species Response Profiles in 1% NaBH.4Reductant Solution and VaxyingConcentrations ofAcetic Acid by Continuous HGAA 813.1.5.2 Determination ofArsenic Species Response Profiles in 1.5% NaBH4Reductant Solution and VaiyingConcentrations ofAcetic Acid by Continuous HGAA 823.1.5.3 Determination ofArsenic Species Response Profiles in 2.0% NaBH4Reductant Solution and VaryingConcentrations ofAcetic Acid by Continuous HGAA 843.1.5.4 Determination ofArsenic Species Response Profiles in 2.5% NaBH4Reductant Solution and VaryingConcentrations ofAcetic Acid by Continuous HGAA 843.1.5.5 Effect of Sodium Borohydride Concentration on Arsine Yield Using 0.05 ig/ml arsenic Solutions and2M acetic acid 853.1.6 CALIBRATIONAND LIMIT OFDETECTIONS OF THE CONTINUOUSHGAA ANALYSIS 863.2 TOTAL ARSENIC DETERMINATION 883.2.1 WETDIGESTION WITHSULFURICACID, NITRJCACID AND HYDROGENPEROXIDE 893.2.1.1 Time Optimization for Wet Digestion of Seaweed Samples UsingH2S04,HNO3andH20And ArsenicDetermination by The Continuous HGAA Technique 903.2.2 Wet Digestion With Sodium Hydroxide 913.3 DETERMINATION OF ARSENIC SPECIES BY HYDRIDE GENERATION-GASCHROMATOGRAPHY-ATOMIC ABSORPTION SPECTROMETRY (HG-GC-AAS) 1013.3.1 Effect ofHydrochloric acid on the Response Profiles ofSome Arsenic Species 1013.3.2 Effect ofAcetic acid on the Response Profiles ofSome Arsenic Species 1023.3.3 Calibration and Limit ofDetection for the Semi-Continuous Mode HG-GC-AASAnalysis 1043.3.4 Investigation ofThree Wet Digestion Procedures Followed by Semi-ContinuousMode FIG-GCAASAnalysisforArsenic Speciation in the Seaweed ofBritish Columbia 1083.3.4.1 Speciation ofArsenic in the Digest of Sample Subjected to Wet Digestion UsingH2S04,HNO3andH20by the Semi-Continuous Mode HG-OC-AAS Analysis 1093.3.4.2 Speciation ofArsenic in the Digest of Sample Subjected to Wet Digestion Using 5m1 of a 2M HNO3solution by the Semi-Continuous Mode HG-GC-AAS Analysis 111vi3.3.4.3 Speciation ofArsenic in the Digest of Sample Subjected to Wet Digestion Using 5m1 of2MNaOHsoultion by the Semi-Continuous Mode HG-GC-AAS Analysis 1143.4 CHROMATOGRAPHIC SEPARATION AND ISOLATION OF ARSENICALS IN SEAWEED ... 1163.4.1 Identflcation ofArsenic Species Using High Performance Liquid Chromatography On-LineMicrowave Oven Digestion Hydride Generation AtomicAbsorption Spectrometry (HPLC-Mic-HGAAS)1183.4.2 Ident/Ication ofArsenic Species Using High Performance Liquid Chromatography On-lineInductively Coupled PlasmaMass Spectrometry (HPLC ICPMS) 121RESULTS AND DISCUSSION - ARSENIC SPECIES DETERMINATION, ISOLATION,CHARACTERIZATION AND BIOTRANSFORMATION4.1 TOTAL ARSENIC DETERMINATION 1234.1.1 TotalArsenic Concentrations in Seaweeds Collectedfrom British Columbia Coastlines in 1989,1990 and 1991 1234.2 DETERMINATION OF REDUCIBLE AND “HIDDEN” ARSENIC SPECIES iN SOME MARINEALGAE OF BRITISH COLUMBIA 1314.2.1 Concentrations ofArsenic Species in Freeze Dried Seaweed SamplesAfter the Hot BaseDigestion 1314.2.2 Speciation ofReducible and “Hidden “Arsenic Species in AqueousMethanol Extracts ofSomeMarineAlgae 1334.2.3 Speciation ofReducible and “Hidden “Arsenic Species in Dilute Sodium Hydroxide Extracts ofSomeMarineAlgae 1364.3 ISOLATION AND IDENTIFICATION OF F. DJSTJCHUSARSENICALS 1394.3.1 Purification and Isolation ofWater Soluble “Algal”Arsenic Compounds in F. distichus 1394.3.2 Identfication and Characterization ofArsenic Species in Chromatographic Isolates ofWaterSoluble Extracts ofF. distichus by using HPLC-Mic-I-1GAA and HPLC-ICPMS On Line DetectionTechniques 1454.3.2.1 Identification and Characterization ofArsenic Containing Isolates by the HPLC-Mic-HGAAAnalysis 1454.3.2.2 Characterization ofArsenic-Containing Isolates by Using HPLC-ICPMS Analysis 1474.3.3 Biotransformation Studies of “Hidden” Organoarsenic Compounds in Fucus distichus 1684.3.3.1 Anaerobic Decomposition ofF. distichus In an Open System 1694.3.3.2 Identification and Characterization ofArsenic Species in the Isolated Decomposition ProductsofF. distichus by Using HPLC-ICPMS 1724.3.4 Anaerobic Decomposition ofF. distichus In A Closed System 1814.3.4.1 Investigation of the Anaerobic Decomposition ofF. distichus In a Closed System 182SUMMARY 192195viiList of TablesTABLE 1.1 ARSENIC COMPOUNDS FOUND IN MARiNEORGSMS.2TABLE 1.2 ARSENOSUGARDERIVATIVES FOUND MAINLY IN MARINE ALGAE 3TABLE 1.3 ARSENICALS FOUND iN JAPANESEMARiNE MACROALGAE 17TABLE 2.1 OPERATING CONDITIONS FOR THE CONTINUOUS HGAA SYSTEM 24TABLE 2.2 HG-GC-AA OPERATING PARAMETERS 26TABLE 2.3 SAMPLING DATA AND SEAWEED MOISTURE CONTENT 34TABLE 2.4 FURNACE OPERATING PARAMETERS USINGNi2 SOLUTION AS AMATRIXMODIFIER 37TABLE 2.5 FURNACE OPERATING PARAMETERS USING Pd IN 2% CITRIC ACID AS A MATRIXMODIFIER 37TABLE 2.6 EXPERIMENTAL CONDITIONS FOR MICROWAVE DECOMPOSITION ANDHYDRIDE GENERATION (IvilC-HGAA) 39TABLE 2.7 (A AND B) EXPERIMENTAL AND INSTRUMENTAL CONDITIONS FOR HPLCICPMS PROCEDURE 41TABLE 3.1 ARSENIC ABSORBANCE DETERMINATIONS IN 0.5M TRIS BUFFER, pH 8.0 WITHVARYINGNi2MODIFIER CONCENTRATIONS (20 tL INJECTION OF STANDARD,BLANK AND MODIFIERRESPECTIVELY) 61TABLE 3.2 ARSENIC ABSORBANCE DETERMINATIONS IN 0.5M TRIS BUFFER, pH 8.0 WITHVARYING Pd IN 2% CITRIC ACID MODIFIER CONCENThATION (15 .tL INJECTION OFARSENIC STANDARD AND MODIFIER SOLUTIONS RESPECTIVELY) 65TABLE 3.3 ARSENIC SPECIES RESPONSE DURING GFAA 100 PPM Pd IN 2% CITRIC ACIDUSED FORMATRIX MODIFICATION OF 0.5M TRIS BUFFER 67TABLE 3.4 LODS AND RSDS OF ARSENIC DETERMINATION BY GFAA TECHNIQUE 69TABLE 3.5 REDUCTION CONDITIONS FOR SOME ARSENIC SPECIES 72TABLE 3.6 TWO HOUR WET DIGESTION CONTINUOUS HGAA DETERMINATION OF ARSENICIN STANDARD REFERENCE MATERIALS 93TABLE 3.7 COMPARISON OF RESULTS OBTAINED BY THE TWO HOUR WETDIGESTIONIHGAA PROCEDURE AND NEUTRON ACTIVATION ANALYSISTECHNIQUE 94TABLE 3.8 COMPARISON OF ARSENIC CONCENTRATIONRESULTS OBTAINED AFTER TWOHOUR WET DIGESTION PROCEDURE FOLLOWED BY GFAA AND CONTINUOUS HGAAANALYSIS AND THE INTERLABORATORY TECHNIQUE NEUTRON ACTIVATIONANALYSIS 96viiiTABLE 3.9 LODS AND RSDS OF ARSENIC DETERMINATION BY SEMI-CONTINUOUS MODEHG-GC-AAS AND IN 1MHCII2% NaBH4REDUCTANTMEDIUM. 107TABLE 3.10 LODS AND RSDS OF ARSENIC DETERMINATION BY SEMI-CONTINUOUS MODEHG-GC-AAS AND IN 1.5MCH3OO I2%NaBH4REDUCTANTMEDIUM 108TABLE 3.11 SANDERS ARSENIC SPECIATION RESULTS SELECTED FOR SEAWEEDSCOLLECTED FROM VANCOUVER ISLAND, BRITISH COLUMBIA 112TABLE 3.12 SUMMARY OF SPECIATION ANALYSIS ON DIGESTIONS OBTAINED USINGTHREE DIFFERENT WET DIGESTION PROCEDURES AN]) ARSENIC DETERMINATIONBY THE SEMI-CONTINUOUS MODE HG-GC-AASTECHNIQUE 116TABLE 4.1 ARSENIC CONCENTRATIONS IN SOME BROWN ALGAE OF BRITISH COLUMBIASAMPLED FROM 1989 TO 1991 125TABLE 4.2 ARSENIC CONCENTRATIONS IN SOME RED ALGAE OF BRITISH COLUMBIASAIvIPLED iN 1989 AND 1991 126TABLE 4.3 ARSENIC CONCENTRATIONS IN SOME GREEN ALGAE OF BRITISH COLUMBIASAMPLED iN 1989 AND 1991 127TABLE 4.4 MEAN ARSENIC CONCENTRATIONS FOR THE THREE CLASSES OF MARINEALGAE COLLECTED FROMBRiTISH COLUMBIA BETWEEN 1989 - 1991 128TABLE 4.5 ARSENIC CONCENTRATIONDATA ON SOME SEAWEEDS COLLECTED FROM THEATLANTIC PROVINCES AN]) BRITISH COLUMBIA OF CANADA 130TABLE 4.6 CONCENTRATIONS OF ARSENIC SPECIES IN HOT BASE DIGESTION OF FREEZEDRIED SEAWEED SAMPLES ANALYSED BY THE SEMI-CONTiNUOUS HG-GC-AASTECHNIQUE 132TABLE 4.7 SPECIATION OF REDUCIBLE AND “HIDDEN” ARSENIC SPECIES IN FREEZE DRIEDSEAWEED SAMPLES EXTRACTED WITH AQUEOUS METHANOL AND ANALYSED BYTHE SEMI-CONTINUOUS MODE HG-GC-AA TECHNIQUE 134TABLE 4.8 SPECIATION OF REDUCIBLE AND “HIDDEN” ARSENIC SPECIES iN FREEZE DRIEDSEAWEED SAMPLES EXTRACTED WITH 1M NaOH AND ANALYSED BY THE SEMI-CONTINUOUS MODE HG-GC-AA TECHNIQUE 137TABLE 4.9 AMOUNTS OF “HIDDEN” ARSENIC SPECIES IN FEB AN]) LEB ISOLATES USINGHOT-BASEDIGESTION AND SEMI-CONTINUOUS MODE HG-GC-AAS TECHNIQUE.. 142TABLE 4.10 REDUCIBLE AND “HIDDEN” ARSENIC SPECIES DETERMINED IN SOMECHROMATOCIRAPHIC ISOLATES OF WATER SOLUBLE EXTRACTS OF F. DISTICHUSAFTER THE MIC-HGAA ANALYSIS 145TABLE 4.11 SUMIvIARY OF ARSENIC SPECIES IN THE WATER SOLUBLE EXTRACT OF F.DJSTJCHUSBY HPLC-ICPMS ANALYSIS 165ixTABLE 4.12 ARSENIC SPECIATION IN THE ANAEROBIC DECOMPOSITION PRODUCTS OF F.DI.STICHUS BY THE SEMI-CONTINUOUS MODE HG-GC-AA ANALYSIS 169TABLE 4.13 ARSENIC DETERMINATION AND SPECIATION ANALYSIS ON THEDECOMPOSITION PRODUCTS FROMF DLSTJCHUS iNCUBATED iN A CLOSEDSYSTEM USING THE SEMI-CONTINUOUS MODE HGGCAA TECHNIQUE 184TABLE 4.14 Ef VALUES AND ARSENIC CONTENT OF THE ANAEROBIC DECOMPOSiTIONPRODUCTS OBTAINED FROMWATER SOLUBLE EXTRACTS OF F. DISTICHUS AFTERTHIN LAYER CHROMATOGRAPHY ON CELLULOSE PLATES 185xList of FiguresFIGURE 1.1 CHALLENGER’S MECHANISM FOR THE BIOLOGICAL METHYLATION OFARSENIC 14FIGURE 1.2 EDMONDS AND FRANCESCONI’S PROPOSED BIOTRANSFORMATION SCHEMEFOR ARSENIC iN THEMARINE ENVIRONMENT .. 20FIGURE 1.3 PHILLIPS AND DEPLEDGE’S PROPOSED BIOTRANSFORMATION SCHEME FORARSENIC IN THE MARINE ENVIRONMENT 21FIGURE 2.1 ASSEMBLY DIAGRAM OF THE CONTiNUOUS HYDRIDE GENERATION ATOMICABSORPTION SYSTEM 24FIGURE 2.2 HG-GC-AA ASSEMBLY 26FIGURE 2.3 SCHEMATIC DIAGRAM OF MIC - HGAAS 28FIGURE 2.4 BLOCK DIAGRAM OF A TYPICAL ICPMS INSTRUMENT COUPLED TO HPLCSYSTEM 29FIGURE 2.5 MAP 1 OF COLLECTION SITES 32FIGURE 2.6 MAP 2 OF COLLECTION SITES 33FIGURE 2.7 EXTRACTION SCHEME FOR WATER SOLUBLE ARSENIC SPECIES IN F.DISTICHUS COLLECTED FROM HEAD OF HASTINGS ARM 48FIGURE 2.8 FLOWDIAGRAM OF CBROIVLkTOGRAPHJC SEPARATION OF WATER SOLUBLEARSENIC SPECIES EXTRACTED FROMF. DISTICHUS 50FIGURE 2.9 CONTiNUED FLOW DIAGRAM OF CHROMATOGRAPHIC SEPARATION OFWATER SOLUBLE ARSENIC SPECIES EXTRACTED FROM F. DISTICHUS 51FIGURE 3.0 Ni2MODIFIER OPTIMIZATION FOR ARSENIC iN TRIS BUFFER SOLUTION BYGFAA ANALYSIS 62FIGURE 3.1 OPTIMIZATION OF ARSENIC SIGNAL WITH Pd IN 2% CITRIC ACID MODIFIER BYGFAA 66FIGURE 3.2 TYPICAL CALIBRATION CURVE FOR ARSENIC DETERMINATIONBY GFAA 68FIGURE 3.3 EFFECT OF HYDROCHLORIC ACID CONCENTRATION AND 0.5% NaBH4ASREDUCTANT ON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTiNUOUSHGAA ANALYSIS 73FIGURE 3.4 EFFEC OF HYDROCHLORIC ACID CONCENTRATION AND 1.0% NaBH4ASREDUCTANT ON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTiNUOUSHGAA ANALYSIS 74xFIGURE 3.5 EFFECT OF HYDROCHLORIC ACID CONCENTRATION AND 1.5% NaBH4ASREDUCTANT ON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTINUOUSHGAA ANALYSIS 75FIGURE 3.6 EFFECT OF HYDROCHLORIC ACID CONCENTRATION AND 2.0% NaBH4ASREDUCTANT ON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTINUOUSHGAA ANALYSIS 77FIGURE 3.7 EFFECT OF HYDROCHLORIC ACID CONCENTRATION AND2.5%NaBH4ASREDUCTANT ON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTiNUOUSHGAA ANALYSIS 78FIGURE 3.8 PLOT OF ABSORBANCE VS [NaBH4]% (wlv) 79FIGURE 3.9 EFFECT OF ACETIC ACID CONCENTRATION AND 1.0% NaBH4AS REDUCTANTON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTINUOUS HGAAANALYSIS 82FIGURE 3.10 EFFECT OF ACETIC ACID CONCENTRATION AND 1.5% NaBH4AS REDUCTANTON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTiNUOUS HGAAANALYSIS 83FIGURE 3.11 EFFECT OF ACETIC ACID CONCENTRATION AND 2.0% NaBH4AS REDUCTANTON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTINUOUS HGAAANALYSIS 84FIGURE 3.12 EFFECT OF ACETIC ACID CONCENTRATION AND 2.5% NaBH4AS REDUCTANTON THE RESPONSE OF FOUR ARSENIC SPECIES BY CONTINUOUS HGAAANALYSIS 85FIGURE 3.13 EFFECT OF SODIUM BOROHYDRIDE CONCENTRATION ON ARS1NE YIELDUSING 0.05 .tg/m1 ARSENIC SOLUTIONS AND 2M ACETIC ACID 87FIGURE 3.14 TYPICAL CALIBRATION CURVE FOR ARSENIC DETERMINATION BY ACONTINUOUS HGAA PROCEDURE 88FIGURE 3.15 BAR GRAPH SHOWING TIME OPTIMIZATION OF WET DIGESTION PROCEDUREFOR THE DETERMINATION OF ARSENIC IN SEAWEED 92FIGURE 3.16 SCATTER PLOT FOR ARSENIC DETERMINATION USING THE 2NR WETDIGESTION FOLLOWED BY HGAA ANALYSIS COMPARED WITH NEUTRONACTIVATION ANALYSIS RESULTS 97FIGURE 3.17 SCATI’ER PLOT FOR ARSENIC DETERMINATION USING THE 2HR WETDIGESTION FOLLOWED BY GFAA ANALYSIS COMPARED WITH NEUTRONACTIVATION ANALYSIS RESULTS 98FIGURE 3.18 SCATrER PLOT FOR ARSENIC DETERMINATION USING THE 21W. WETDIGESTION FOLLOWED RESPECTIVELY BY HGAA AND GFAA ANALYSIS 99xliFIGURE 3.19 EFFECT OF HYDROCHLORIC ACID CONCENTRATION ON THE RESPONSE OFAs(V), MMAA, AND DMAA DURiNG REDUCTION BY 2% NaBH4AND SEMI-CONTINUOUS HG-GC-AAS ANALYSIS 102FIGURE 3.20 EFFECT OF ACETIC ACID CONCENTRATION ON THE RESPONSE OF As(V),MMAA, AND DMAA DURING REDUCTION BY 2% NaBH4AND SEMI-CONTINUOUS HGGC-AAS ANALYSIS 103FIGURE 3.21 TYPICAL CALIBRATION GRAPHS FOR INORGANIC As(V), MMAA AND DMAA iN1M HCII2%NaBH4(wlv) REDUCTANT MEDIA BY SEMI-CONTJNL.JOUS MODE HG-GCAAS ANALYSIS 105FIGURE 3.22 TYPICAL CALIBRATION GRAPHS FOR INORGANIC AS [As(I11), As(V)], MMAAAND DMAA IN 1.5MCH3OO J2%NaBH4 (w/v) REDUCTANTMEDIA BY SEMI-CONTINUOUS MODE HG-GC-AAS ANALYSIS 106FIGURE 3.23 HG-GC-AAS CHROMATOGRAM OF As(V) - (A), MMAA - (B) ANDDMAA-(C) 110FIGURE 3.24 HG-GC-AAS CHROMATOGRAM OF DMAA DIGEST AFTER TWO HOUR WETDIGESTION IN imiH2S04, 3m1 NNO3 AND 3m1H2O 110FIGURE 3.25 HG-GC-AAS CHROMATOGRAM OF ARSENOCHOL1NE AFTER TWO HOUR WETDIGESTION IN imiH2S04,3m1 HNO3 AND 3m1H20 110FIGURE 3.26 HG-GC-AAS CHROMATOGRAM OF FREEZE DRIED F. DJSTJCHUS (HEAD OFHASTINGS ARM) AFTER TWO HOUR WET DIGESTION IN imiH2S04,3m1 HNO3 AND3m1H2O 110FIGURE 3.27 CHROMATOGRAM OF F. DLSTJCHUS (HEAD OF HASTINGS ARM) DIGEST AFTERA THREE HOUR WET DIGESTION WITH 2M HNO3 (5rnl) AT 90-95°C BY THE SEMI-CONTINUOUS MODE HG-GC-AAS ANALYSIS 113FIGURE 3.28 CHROMATOGRAM OF F. DISTICHUS (HEAD OF HASTINGS ARM) DIGEST AFTERA THREE HOURWET DIGESTION WITH 2M NaOH SOLUTION (5m1) AT 90-95°C BYTHE SEMI-CONTINUOUS MODE HG-GC-AAS ANALYSIS 115FIGURE 3.29(A): CHROMATOGRAM OF 2Ong OF ARSENATE, ARSENITE, MMAA, DMAA,ARSENOBETA1NE, ARSENOCHOLINE AND TETRAMETHYLARSONTUM ION WITHOUTOVEN DIGESTION (OVEN POWER TURNED OFF) 119FIGURE 3.29(B): CHROMATOGRAM OF 2Ong OF ARSENATE, ARSENITE, MMAA, DMAA,ARSENOBETAINE, ARSENOCHOLINE AND TETRAMETHYLARSONRJM ION WITHMICROWAVE OVEN DIGESTION IN 0. 1MK2S08ANt) 0. 1M NaOH 120FIGURE 3.30 CI-IRqMATOGRAM OF 2Ong OF ARSENATE, ARSENITE, MMAA, DMAAARSENOBETAINE AND 4Ong OF ARSENOCHOLINE AND TETRAMETHYLARSONTUMION USING THE HPLC-MIC-HGAAS ANALYSIS 120FIGURE 4.1 ISOLATION OF FEB AND LEB ON DEAF SEPHADEX A25 USING TRIS BUFFER, pH8.0 (0.05M, 0.5M) AS MOBiLE PHASE 141xliiFIGURE 4.2 CHROMATOGRAM OBTAINED FOR ARSENOSUGAR hA (TABLE 1.2) USINGHPLC-MIC-HGAA ANALYSIS WHEN MOBILE FLOW RATE WAS 1.Oml mm4 146FIGURE 4.3 CHROMATOGRAM OBTAINED FOR ARSENOSUGAR hA (TABLE 1.2) USINGHPLC-MIC-HGAA ANALYSIS WHEN MOBILE FLOW RATE WAS O.Sml mm4 146FIGURE 4.4 CHROMATOGRAM OBTAINED FROM A MIXTURE (2Ong EACH) OF ARSENITE,ARSENATE, MMAA, DMAA AND ARSENOBETAINE BY USING HPLC-ICPMS 148FIGURE 4.5 CHROMATOGRAM OBTAINED FROM THE FEBA ISOLATE OF F. DISTICHUSBYUSING HPLC-ICPMS 149FIGURE 4.6 CFIROMATOGRAM OBTAINED FROM THE LEB ISOLATE OF F. DISTICHUSBYUSING HPLC-ICPMS 149FIGURE 4.7 CHROMATOGRAMOBTAINED FOR ARSENOSUGAR 11 BY USINGCONDITION 1 151FIGURE 4.8 CFIROMATOGRAM OBTAiNED FOR ARSENOSUGAR .U BY USINGCONDiTION 2 151FIGURE 4.9 CHROMATOGRAM OBTAINED FOR SRM NBS 1566 OYSTER TISSUE BY USINGCONDITION 1 152FIGURE 4.10 CHROMATOGRAM OBTAINED FOR SRM NES 1566 OYSTER TISSUE BY USINGCONDITION 2 153FIGURE 4.11 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBA USINGCONDITION 1 154FIGURE 4.12 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBA SPIKED WITHARSENOSUGAR hA USING CONDITION 1 154FIGURE 4.13 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBA USINGCONDITION 2 155FIGURE 4.14 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBA SPIKED WITHARSENOSUGAR hA USING CONDITION 2 155FIGURE 4.15 CHROMATOGRAM OBTAINED FOR THE ISOLATE LEB USINGCONDITION 1 157FIGURE 4.16 CHROMATOGPAM OBTAINED FOR THE ISOLATE LEB SPIKED WITHARSENOSUGAR STANDARD hA USING CONDITION 1 157FIGURE 4.17 CHROMATOGRAM OBTAINED FOR THE ISOLATE LEB SPIKED WITH SRM NBS1566 OYSTER TISSUE USING CONDITION 1 158FIGURE 4.18 CHROMATOGRAM OBTAINED FOR THE ISOLATE LEB USINGCONDITION 2 158FIGURE 4.19 CHROMATOGRAM OBTAINED FOR THE ISOLATE LEB SPIKED WITHARSENOSUGAR STANDARD hA USING CONDITION 2 159xivFIGURE 4.20 CHROMATOGRAM OBTAINED FOR THE ISOLATE LEB SPIKED WITH SRM NBS1566 OYSTER TISSUE USING CONDITION 2 159FIGURE 4.21 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB1(A) USINGCONDITION 1 160FIGURE 4.22 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB1(A) SPIKED WITHARSENOSUGAR hA USING CONDITION 1 161FIGURE 4.23 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB1(A) USINGCONDITION 2 161FIGURE 4.24 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB1(B) USINGCONDITION 1 162FIGURE 4.25 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB2 (RF 0.22) 162FIGURE 4.26 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB2 (RF 0.33) 163FIGURE 4.27 CHROMATOGRAMOBTAINED FOR THE ISOLATE FEBB2 (RF 0.5 - 0.78) 163FIGURE 4.28 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB2 (RF 0.89) 164FIGURE 4.29 CHROMATOGRAM OBTAINED FOR THE ISOLATE FEBB2 (RF 0) 164FIGURE 4.30 CHROMATOGRAM OF ARSENIC SPECIES CONTAINED IN THE SEAWEEDEXTRACT FOLLOWING GEL PERMEATION CHROMATOGRAPHY ONSEPHADEXG15 170FIGURE 4.31 CHROMATOGRAM OF ARSENIC SPECIES CONTAINED IN THE FILTRATEEXTRACT FOLLOWING GEL PERMEATION CHROMATOGRAPHY ONSEPHADEXG15 171FIGURE 4.32 CHROMATOGRAM SHOWING THE FOUR ARSENIC SPECIES PRESENT IN SRMNIST 1566A OYSTER TISSUE SEPARATED ON INERTSIL ODS-2 HPLC C 18 REVERSEPHASE COLUMN 173FIGURE 4.33 DESOPRTION CHEMICAL IONIZATION MASS SPECTROMETRIC SPECTRUM OFDIMETHYLOXARSYLETHANOL (DMAE) 173FIGURE 4.34 1HNMR SPECTRUM OF DIMETHYLOXARSYLETHANOL (DMAE) IND2OSOLUTION 174FIGURE 4.35 13C NMR SPECTRUM OF DIMETHYLOXARSYLETHANOL (DMAE) IND20SOLUTION 175FIGURE 4.36 ICPMS CHROMATOGRAM OF DIMETHYLOXARSYLETHANOL (DMAE)STANDARD ON AN 1NERTSJL ODS-2 HPLC C 18 REVERSE PHASE COLUMN 176FIGURE 4.37 CHROMATOGRAM OF SEAWEED ISOLATE 1 FROM THE ANAEROBICDECOMPOSITION OF F. DISTJCHUS 177xvFIGURE 4.38 CHROMATOGRAM OBTAINED OF SEAWEED ISOLATE 2 FROM THEANAEROBIC DECOMPOSITION OF F. DJ.STJCHUS 177FIGURE 4.39 CHROMATOGRAM OBTAINED WITH A MIXTURE (1:3) OF SEAWEED ISOLATES1 AND 2 FROM THE ANAEROBIC DECOMPOSITION OFF. DJSTICHUS 178FIGURE 4.40 CHROMATOGPAM OF FILTRATE ISOLATE 2 FROM THE ANAEROBICDECOMPOSITION OF F. DISTICHUS 178FIGURE 4.41 PROPOSED BIOTRANSFORMATION OF DMAA TO ARSENOBETAINE IN MARINEORGANISM. 181FIGURE 4.42 CHROMATOGRAM OBTAINED FOR NIST 1566A OYSTER TISSUE STANDARDREFERENCEMATERIAL 186FIGURE 4.43 CHROMATOGRAM OBTAINED FORDMAE 186FIGURE 4.44 CHROMATOGRAM OBTAINED FOR SRA1 BY THE HPLC-ICPMSANALYSIS 187FIGURE 4.45 CHROMATOGRAM OBTAINED FOR SRA2 BY THE HPLC-ICPMSANALYSIS 188FIGURE 4.46 CHROMATOGRAM OBTAINED FOR SRB1 BY THE HPLC-ICPMSANALYSIS 188FIGURE 4.47 CHROMATOGRAM OBTAINED FOR SRB2 BY THE HPLC-ICPMSANALYSIS 189FIGURE 4.48 CHROMATOGRAM OBTAINED FOR SF BY THE HPLC-ICPMSANALYSIS 189FIGURE 4.49 CHROMATOGRAM OBTAINED FOR CF BY THE IIPLC-ICPMSANALYSIS 190xviList of AbbreviationsAA Atomic AbsorptionAAS Atomic Absorption SpectrometryDCIMS Desorption Chemical Ionization Mass SpectrometryDEAE DiEthylAminoEthylF. distichus Fucus distichusGC Gas ChromatographyGFAA Graphite Furnace Atomic AbsorptionGPC Gel Permeation ChromatographyHG-GC-AAS Hydride Generation Gas Chromatography Atomic AbsorptionSpectrometryI[GAA Hydride Generation Atomic AbsorptionHPLC High Performance Liquid Chromatography (or High Pressure LiquidChromatography)HPLC-ICPMS High Performance Liquid Chromatography Inductively CoupledPlasma Mass SpectrometryHPLC-Mic- High Performance Liquid Chromatography Microwave OvenHGAAS Assisted Decomposition Hydride Generation Atomic AbsorptionSpectrometryICPMS Inductively Coupled Plasma Mass SpectrometryIEC Ion Exchange ChromatographyMic Microwave Oven Assisted DecompositionNAA Neutron Activation AnalysisNBS National Bureau of Standard, USANJES National Institute ofEnvironmental StandardsNIST National Institute of Standards and TechnologyNMR Nuclear Magnetic ResonanceNRC National Research Council, CanadaSRM Standard Reference MaterialTLC Thin Layer ChromatographyTris Tris(hydroxymethyl)aminomethanexviiACKNOWLEDGMENTI hereby express my gratitude to God for the rich and invaluable experience andsupport that I received from my supervisor, Dr. W.R. Cullen. I am also grateful to Godfor providing me with an excellent guidance committee comprising of Drs. Chris Brion,Guenter Eigendorf and Alan Storr (replacing Dr. Adrian Wade, who has moved on).I wish to express appreciation to Drs. W.R. Cullen and Kenneth Reimer for theopportunity to participate in several sampling cruises on C.S.S. 3.P. Tully and C.S.S.Vector. Dr. Cullen always came back with many seaweed samples whenever he visitedany coastal areas ofBC. Many thanks.My profound appreciation goes to Dr. Xiao Chun Le (a former lab colleague) forhis expertise and assistance with the HPLC- ICPMS and HPLC-Microwave-HGAASexperiments. I am very grateful to Dr. G. Eigendorf for his friendly and expert advice. Ialso thank my friends for many uplifting discussions and my family* for their excellentsupport and abiding faith in me.Finally, I dedicate this thesis to all those seeking success through hard work anddifferent circumstances. God is with all of them, as He is with me. All things worktogether for good to those who love God (Romans 8:28).*My mom, Mrs Deborah Olu Ojo; my brothers; my wife, Trina; and my daughter,7 Toluwalase Ayomide Ojo.xviiiINTRODUCTION1.1 GENERAL BACKGROUNDArsenic has been widely studied because of its ubiquity, chemistry and usage(l-8). It is anaturally occurring element present in soil, water, air, and all living matter(9-22). As a result ofthe ubiquity of arsenic in the environment, in addition to its biological toxicity and redistribution,this twentieth most abundant element in nature has evoked a lot of public concern(l-5). This inpart, is because arsenical preparations have been misused for criminal purposes in the form ofpoison(2-6). Furthermore, another and perhaps the most significant reason for evoking the public’sconcern is the people’s ignorance about the chemistry of arsenic. The ability of arsenic to formcovalent bonds with sulfur and its similarity to phosphorus are the two fundamental reasons for thetoxicity of arsenicals. However, this (toxicity) characteristic of arsenicals is dependent on thevalency state of arsenic itselfl9-14). Penrose(9) found that the toxicity of arsenic compoundsdecreases according to the following order:R3As(R=H, Me, Cl, etc) >As203(Asifi)> (RAsO) >As205(A V)>RAsO(OH)3(n=l,2) > RAs+ > As(O).Thus, trivalent arsenicals are particularly more toxic to aquatic organisms (3, 6) than thepentavalent arsenic compounds and inorganic arsenicals exhibit greater toxicity than the organicarsenic compounds. Since arsenite is more toxic than most other forms of arsenic, its oxidation isconsidered as a form of protective mechanism in living organisms(5). In the arsenicbiogeochemical cycle, the formation of organoarsenicals via biomethylation of arsenic is consideredas well to be a detoxification process in the environment(5, 6). In the marine environment, severalarsenical species are present (2-6, 8). Some examples of those found in marine organisms aregiven in Tables 1.1 and 1.2 below.C -I —C, e B 0 g C 0 BI..’b-’———1—I)-‘0c I+•iDI+iirrrr-(•)t’J C)08c!jIc)o000r)not00i-000g——83Except for compounds 2 and Ufwhich are lipid soluble, all the other arsenicals are watersoluble. The organoarsenicals Q - ) are relatively non toxic and can accumulate in marineanimals and plants. When humans ingest marine animals containing arsenobetaine (the majorarsenical present in them), the arsenical is rapidly excreted in an unchanged form(16, 17).However, with the ingestion by humans, of arsenosugars found in brown algae a drastic differencein the urinary excretion patterns and metabolites resulted compared to when arsenicals in crab meatwere eaten(17). The ingestion of arsenosugars from the kelp nori by nine volunteers revealed thepresence of up to six unidentified arsenic species metabolites (dependent on the individual) whichare suspected to be chemically modified arsenosugars(17). (These unidentified arsenicalmetabolites were found not to be similar to DMAA, AB, AC, MMAA, arsenite, arsenate and twoor three arsenosugar standards available for their identification.) The author(17) noted thatdifferent individuals metabolize arsenosugars in different ways.The beneficial aspects for arsenicals as a paint pigment, fungicide, parasiticide, algaecide,dessicant, herbicide, pesticide, bactericide and, in the production of glass, enamels, transistors,lasers and semi-conductor devices are marred by the usage of arsenic as poison in homicidal andsuicidal events(2, 7) especially in the middle ages. The need for further understanding of arsenicand its compounds is therefore very important and necessary especially in recent times because thecommercial use and production of both inorganic and organic arsenic compounds have raised localconcentrations of this element in the environment much above the natural backgroundconcentrations(2, 3, 18-22).1.2 ARSENTC ACCUMULATION IN THE ENVIRONMENTArsenic may enter the biosphere as the result of industrial activity or through deliberateapplication, either asf a pesticide in feed additive andlor in medicine. In the natural environment,arsenic is rarely encountered as the free element. It is usually found as component of sulfidic ores,in which it occurs as metal arsenides. Soils found above sulfide ore deposits can contain arsenicconcentrations S8000 ppm(2).3The occurrence, absence, or fate of various dissolved and solid arsenic species in thenatural environment can be usefully predicted through geochemical modeling. Geochemicalmodeling is a useful means of identifying the major controls on the distribution of, and speciationof arsenic in the environment(5). It makes use of, and interprets the responses of natural systemsto pH and thermodynamic redox potential, E [defined as ER = E° - (0.059/n) log Q at StandardTemperature and Pressure (STP); E° refers to the standard electrode potential, n is the number ofelectrons gained or lost by a species, and Q is the reaction quotient; and at STP, EH = 0.059 pE,where pE is the hypothetical electron activity at equilibrium](5). The stability diagrams obtainedfrom the plots of EH versus pH are commonly used to determine the predominant soluble arsenicspecies and relevant solids(S). In the environment, the redox pair of arsenate/arsenite,H3AsO4+2H + 2e E H3AsO +H20, is not in thermodynamic equilibrium. The redox level isset by the oxygen/water couple, 02 + 4H+ + 4e 4-— 2H0. It therefore appears that therange of water soluble inorganic arsenic compounds is quite limited, and that pH is the majorfactor controlling the differences in aqueous arsenic speciation in the freshwater and the marineenvironment(S). At least on thermodynamic grounds, As(V) should strongly dominate over As(III)in oxygenated waters. For example, As(V)/As(III) ratios of i0’ - 1026 have been calculated forseawater, depending on the choice of pE (pE = 20.6 - pH, in oxygenated waters)(2-5, 23, 24).However, due to biologically mediated redox reactions, thermodynamically predicted As(V)/As(III)ratios are rarely observed(5). Consequently, in most natural waters including uncontaminatedsurface and deep ocean waters, significant amounts (-10% of total As) of arsenite have beenfound. And similarly, arsenate still exists even under anoxic (reducing) conditions. In addition, thecycle of arsenic in soil, land, water and the sea involves organic arsenicals, many of which havebeen identifled(5-7, 16, 25-28).Further, trace amounts of arsenic may be present in air. In areas remote from industrialcontamination, air concentrations of arsenic generally are less than 0.02 mg 1’, whereas, in urbanareas they vary from less than 0.02 to 0.16 mg l’(29). However, the air and dust in industrial4surroundings such as in power plants, mines and smelters can have arsenic concentrations rangingfrom 0.8 to 300 ppm(2, 30).Arsenic is also present in all living organisms. The total human body content variesbetween 3 and 4 mg and tends to increase with age(2). The arsenic concentration of urine can varynormally from 0.1 to 1.0 ppm and it is generally high after consumption of seafood(2, 16, 17, 32).Some of the highest concentrations of arsenic in biota are encountered in marine organisms(4)because they have the ability to concentrate arsenic from water, food or sediment(32-39).Dissolved arsenic may be taken up by biota because arsenate is similar to phosphate in size, andgeometry and may enter cells by transport mechanisms unable to discriminate between phosphateand arsenate(37, 40, 41). Studies(39, 41-45) have shown that the accumulation of arsenicals andthe different arsenic concentrations found between species of marine organisms are related to theirfood source. In addition, it appears that the amount of ingested arsenic accumulated is dependenton the chemical form of arsenic present in the food(4 1), organic arsenic being preferentiallyretained relative to inorganic arsenic. Marine plants, particularly algae, are known to have higharsenic contents(4). Some marine macroalgae may contain an appreciable quantity of inorganicarsenic(46-49) as found in edible seaweed containing up to 50% of arsenic in the inorganic form.Recent studies(6, 7, 17, 50, 51) however, have shown the organic arsenosugars (Table 1.2) are thepredominant arsenicals found in marine macroalgae.Even though marine macroalgae are near the bottom of the food chain and contain thegreatest concentrations of arsenic(46, 47, 52), there is evidence(9) suggesting that arsenic is notbiomagnified by higher organisms in the food chain. Klumpp and Peterson(52) found that althoughthere is evidence of arsenic accumulation occurring at all trophic levels, no biomagnification on anentire animal basis,occurred in Patella vulgata (33.5-41.0 mg As kg1) and Littorina obtusata(48.5-59.8 mg As kg-’) which graze on macrophytes (59.1-189 mg As kg’). Similar determinations were found on kelp grazing crab Pugettia producta(53) and herbivorous gastropodswhich feed on marine algae containing high amounts of arsenic(42). Consequently, instead of5observing biomagnification of arsenic as one ascends the trophic levels, only accumulation andjorelimination of arsemc occurs(4, 18) in marine organisms.Finally, it has been suggested (2, 54) that high accumulation of arsenicals in soils arereduced because of the constant turnover of organic matter and the accompanying microbialactivities which result in volatilization of some arsenic species. This process helps in reducingarsenic toxicity to plant life. In consequence, the toxicity of arsenic to the higher trophic levels,including humans, is reduced as well. However, the dose makes the poison(55).1.3 SPECIATION AND BIOTRANSFORMATION OF ARSENICALS IN THEMARINE ENVIRONMENTThe possibility of long-term increase of arsenic levels(4) especially in areas near industrialor mining/mineral processing activities requires that continued measurement of arsenicconcentration in the marine environment be carried out so that anthropogenic inputs (5) into theocean can be estimated. According to some studies (14, 24, 25, 52) however, the usage of just the“total arsenic” concentrations for the assessment of an arsenic related threat to the health oforganisms was found to be insufficient and inadequate.Hence, in order to understand completely the bio-geochemical cycle of arsenicals, detailedinvestigations of the levels and chemical forms, as well as the migration and transformation ofarsenic in marine food webs(5, 6, 17) are necessary. In the following sections, the speciation ofarsenic and the various transformations of arsenicals that have been found to occur in the marinebiota will be explored.1.3.1 SPECIATION OF ARSENICALSIn the course of an investigation of the marine chemistry of arsenic, procedures wererequired for the determination of low concentrations and species of the element in seawater,sediments and marine plants. Much of the earlier work(56-58) on the abundance of arsenic inmarine environment is unreliable owing to the poor analytical techniques used, and to the difficulty6of obtaining reagents sufficiently free from this ubiquitous element. As a consequence for instance,in seawater(58) the results repozted were frequently more than an order of magnitude greater thanthe arsenic concentration value presently (5) accepted (ca. 2 pg As i’). Arsenic in earlier work(58) was detennined by using the Gutzeit method involving the reduction of arsenicals to arsenichydride and effecting a capi]laiy separation between arsenite and arsenate with silver nitratemoistened to a filter paper. Quantitative determination of arsenic with the Gutzeit method wasachieved by comparing length of unknown arsenic sampled with those of individual arsenicstandards on cross sectioned filter paper. Apart from the inconvenience and inaccurate arsenicdetemiinations that might result from such comparison, this method can only be applied in theabsence of hydrogen sulfide and hydrogen phosphide, both of which afford blackened colorationswith silver nitrate solutions, exactly like the arsenic hydrides. Furthermore, the presence ofmercury salts can either slow down and/or prevent the formation of nascent hydrogen andconsequently of AsH3 (during the reduction procedure).Another method in earlier work(57, 59-61) for studies on arsenic speciation employed theselective determination of arsenite and arsenate by using the molybdenum blue-photometrictechnique. In these procedures, arsenomolybdic acid foimed by the reaction of arsenate withacidified molybdate, is reduced to a blue complex and the absorption ( = 865 nm) is measured.Because the arsenites do not form a blue complex with the molybdate reagent, the concentration ofarsenate can be estimated in a sample. Following oxidation of arsenite to arsenate, theconcentration of arsenite can be estimated by difference from the total arsenic in the sample. Thismethod, however, suffers from severe interferences from phosphate which also affords a bluecomplex with the molybdate reagent and in addition, the method is very slow, requiring 2-4 hoursfor full color development(59-61).7Another earlier method(62, 63) employed for arsenic speciation is based on the formationof insoluble precipitates containing either arsenite or arsenate, allowing for differentiation betweentrivalent and pentavalent inorganic arsenic compounds. In this method, compounds of both tn- andpentavalent arsenic are reduced by stannous chloride to elementary arsenic in solutions stronglyacidified with hydrochloric acid. The arsenic is precipitated as a brownish precipitate. Eventhough this method is considered quite specific for arsenic, the sensitivity ofthe test is decreased bythe presence of colored salts, such as chromium, cobalt and nickel salts. Furthermore, the noblemetals and mercury are reduced to metallic state with stannous chloride under similarconditions(62-63).Overall, these earlier methods are inadequate and cannot be applied to environmentalsamples where arsenic occurs as species with distinct physical, chemical and biological propertiesthat are different from the inorganic compounds. These other arsenicals, inclusive of arsenite andarsenate, can be successfully speciated by using the combined speciation methods described below.1.3.2 SPECIATION BY USING COMBINED METHODSMore recently(7), several species of arsenic such as arsenite, arsenate, MMAA, DMAA,TMAO,Me4As ion, AC, AB, arsenosugars and a few arsenic containing lipids have been found tobe present in marine biota. In order to reveal the identity of each of these biologically distinctarsenic species, a combination of several separation and analytical techniques has been utilized fortheir speciation in the marine environment. Some of these combined methods are discussed in thefollowing sections. Fractionation TechniquesThe use of appropriate chromatographic separation procedures in combination with traceelement analytical techniques such as atomic absorption spectrometry (AAS) and atomic emissionSspectrometly (AES), have been effectively utilized in routine analysis(16, 64) for arsenicspeciation. Following the determination of the element (arsenic) content in the chromatographicfractions, the arsenic containing bands are identified offline by using ‘H- and ‘3C-NMRspectroscopy, X-ray chrystallography (in case of crystal formation in the chromatographicallypurified band), and mass spectmmetay(27, 28, 44, 46) as well as online by using HGAAS, HG-Microwave oven decomposition-AAS, HPLC-MS and HPLC-ICPMS(5, 17, 25, 27, 28, 50). Inthe offline arsenic speciation, the atomic absorption spectrometiy is the most widely used detectiontechnique for the determination of arsenic in the chromatographic fractions collected(27, 65). Themost sensitive resonance line of arsenic, 193.7 nm (the other not so sensitive line is 197.3nm) isnormally used in AAS studies(65-69). Furthennore, because of the lower sensitivity observed withthe flame AAS (69-78), the furnace AAS method particularly graphite fumace(27, 28) AAS is themost acceptable choice for arsenic determination. With the addition of modifiers (e.g. Ni2) tosamples during arsenic determination by GFAAS, loss of arsenic occurring in the charring stage (asignificant disadvantage of this method) is kept to a minimum and/or avoided completely(65, 79,80). In addition, the GFAAS technique is very suitable for offlJne arsenic analysis ofchromatographic fractions because samples or solution aliquots from fractions collected can beadded directly to the graphite furnace without any further pretreatment(81-88). After the collectionof fractions from an appropriate liquid chromatograph.ic separation procedures(81, 84, 88), severalother specirometric detectors for quantitation of arsenic such as atomic flourescencespectrometry(89, 90) and plasma mass spectrometry(91-94) have been employed for arsenicdetennination as well.Regardless of the spectrometiic arsenic detection method utilized, the final identification ofchromatographic fractions and arsenic speciation after the fractionation technique is achievedmainly by using ‘H- and ‘3C- NMR spectroscopy and mass spectroscopy. When crystals result9from purified fraction, X-ray crystallography is employed for the complete elucidation of thearsenic species(27, 28,95). Online Arsenic Speciation TechniquesThe online speciation of arsenic compounds using a combination of analytical techniquessuch as hydride generation AA spectmmetiy and high perfonnance liquid chromatographyinductively coupled plasma mass spectrometty is gaining widespread usage in environmentalanalysis(7, 17, 27, 28, 50, 91, 96-100). These online arsenic speciation techniques are discussedin this section.Providing appropriate standards are available(96, 97), the technique of High PerformanceLiquid Chromatography Inductively Coupled Plasma Mass Spectmmetiy (HPLC-ICPMS) canfacilitate the identification of arsenic metabolites present in trace amounts, as well as majorarsenicals in biological samples(94, 99, 100). The most common and convenient method of sampleanalysis with the ICPMS technique is by using the traditional pneumatic nebulization for liquidsample intmduction(17, 91) into the ICP. Consequently, the flow rates typically used for HPLCanalysis (0.5 - 2 ml min’) are compatible with the requirement for liquid introduction into the ICPunder this condition(l7). For the HPLC separation, both ion exchange and ion pairchromatography have been employed in the speciation of arsenic compounds online with theICPMS technique(17, 50, 98, 101-05). The HPLC-ICPMS technique has detection limits between20- 300 ng(17). Therefore, the technique is considered very adequate for trace arsenic specialionstudies in environmental and biological systems(50, 96-99). Even though ICP is the most popularplasma excitation source(l05-12), other plasma techniques, such as direct current plasma,DCP(l 12-14), microwave induced plasma, MIP(1 12, 115, 116) and capacitively coupled plasma,CCP(l 17, 118), have all been used for spectrochemical analysis on line with the HPLC proceduresduring arsenic speciation studies(86, 91, 119-24).10Another online speciation technique, the Hydride Generation procedure (HG), is widelyutilized for the identification and quantification of volatile arsenic compounds such as methylarsines which have been infrequently detected in the environment and other arsenicals such asinorganic (arsenate and arsenite) and methylated arsenic compounds commonly found in theenvironment(7, 125-3 1). This technique (HG), can be combined with several spectrometricdetection methods like Atomic Absorption Spectrometry (AAS), Mass Spectrometiy (MS), PlasmaAtomic Emission Spectrometzy (PAES) and Atomic Fluorescence Spectrometry (AFS)(7, 17, 27,123-35) during speciation analysis. Other analytical combinations with Gas ChromatographyAAS and HPLC-AAS(7, 17, 27) can be employed on line with the HG procedure for arsenicspeciation analysis. The hydride generation system is based on pH controlled reduction of thearsenic compounds with sodium borohydride (selective reduction of arsenite at pH >4; reduction ofmost other arsenic compounds at pH 1)(7). The arsines generated can be separated according totheir boiling point and/or by using gas chromatography(136). The major limitation in usinghydride generation for arsenic speciation is that, some environmentally and biologically importantorganoarsenicals such as arsenobetaine and arsenosugars do not form an arsine upon treatmentwith sodium borohydride. However, some workers(137, 138) have developed analytical methodsin which samples containing arsenicals such as arsenobetaine, arsenocholine, tetramethylarsoniumion and arsenosugars are pyrolyzed( 137) and/or microwave digested( 138). With these procedures,the arsenicals are converted to hydrides(137) and/or arsenate(138) which can be determined by thehydride generation technique.1.3.3 BIOTRANSFORMATION OF ARSENIC IN THE ENVIRONMENT1.3.3.1 Arsenic Biotransformation Studies in Marine EnvironmentInorganic arsenic can be accumulated by marine organism; and in the marine biota, theycan be subject to various biotransformations including reduction, oxidation and methylation(5, 6).11Biological transformation of arsenic can either be microbially mediated and/or the organism’sadaptive response to the accumulated arsenic(6). The microbial transformations of arsenic canoccur via redox transformation between arsenite and arsenate, as well as through biomethylation ofarsenic into volatile methyl arsines. The primary producers, including the marine phytoplanktonand macroalgae accumulate inorganic arsenic, which is then converted or biotransformed intocomplex organic molecules that are either water- or lipid-soluble arsenic compounds. When theseprimary producers are fed upon by the higher trophic level, the water- and lipid-solubleorganoarsenicals are further metabolized into other distinct arsenicals(6) and/or, they are likelyaccumulated unchanged(50).Arsenate dominates the speciation of arsenic in oxygenated water and arsenite is the mostcommon form in the reduced oxygen free pore waters of sediments(5, 15, 25). However, somearsenate still occur in anoxic waters and up to 10% of the total arsenic found in surface and deepwaters of oceans is arsenite(5). Even though bacteria are approximately 10 fold more resistant toarsenate than arsenite(6), the presence of arsenite in oxygenated seawater was indicated to be dueto the reduction of arsenate to arsenite by marine bacteria and also, by marine phytoplankton(5, 27,40, 139, 140). Andreae and Klumpp(40) observed that a number of cultures of marinephytoplankton species release arsenite from arsenate reduction. This result was establishedpreviously by Blasco (and coworkers)(141) who found that reduction of arsenate to arsenite occursin cultures of Chiorella pyrenoidosa, a fresh water alga. This reduction was found to occursimilarly in sea water containing a mixed population of bacteria(22). Furthermore, under aerobicconditions, a common aquatic bacterium, Pseudomonas fluorescens has been found to carry outthe arsenate reduction(142). The reverse process, the oxidation of arsenite to arsenate is performedby heterotrophic bacteria(6). This process is claimed(143) to be an important means ofdetoxification in the environment, as up to 7 8-96% conversion of arsenite to arsenate is achieved.Finally, the redox speciation of arsenic has been suggested to occur generally(144) because of a12slow oxidation of arsenite and a biogenic reduction of arsenate in seawater performed by a host ofmicrobes.The term “biological methylation” was first used by Challenger(1) to describe thereplacement of the oxy-groups of arsenic, selenium and tellurium compounds by methyl groupsthrough the action of moulds, resulting in the formation of organometalloids or organometalliccompounds(145). It has since been shown that biological methylation is a general process forliving organisms(146) and methylation of arsenic results in the formation of easily bioaccumulatedorganoarsenicals(4-7, 46-51). Methylation of arsenic by fungi and bacteria has been known forseveral decades(1, 145-58). Challenger and co-workers(1, 149) extensively investigated the abilityof Scopulariopsis brevicaulis to methylate organic and inorganic arsenic compounds. In thesestudies, the identity of “Gosio ga&’ was established as trimethylarsine and it was suggested thatmethylarsonic acid and dimethylarsinic acid were intermediates in the production oftrimethylarsine. In addition, Challenger(1) proposed a mechanism for the formation oftrimethylarsine, shown in Figure 1.1, based(5, 150) on the ability of S. brevicaulis in methylatingboth arsenate and arsenite to Me3As. Challenger did not speculate on the nature of the reducingagents. However, MMAA, DMAA and TMAO are easily reduced by a number of different thiols,e.g. cysteine, glutathione, and lipoic acid and it has been proposed that the sulphydryl group ofsuch thiols constitutes the source of electrons for the reduction steps in the biomethylation ofarsenic(S).Figure 1.1Challenger’s Mechanism for the Biological Methylation of Arsenic[CH34] 2eAs(OH)3 CH3AsO(OH)2 {CH3As(O )2}[CH3] 2e{CH3As(O )2} (CH3)2AsO(OH) {(CH3)2AsO }[CH3] 2e{(CH3)2AsO } (CH3)AsO (CH3)As13Cox and Alexander(151) also, using three fungi namely Candida humicola, Gliocladium roseumand a penicillum sp. (all isolated from raw sewage) found that arsenite, arsenate, MMAA andDMAA were respectively methylated to trimethylarsine. In this study, it was noted that these fimgiexhibited selective methylation which was previously noted by Challenger(i). The work of Cullenet al(152-55) strongly suggests that “active methionine” S-adenosylmethionine (SAM) is thesource of CH3 (see Figure 1.2 for SAM’s structure) in the Challenger scheme. By using thecultures of S. brevicaulis and C. humicola in the presence of sodium arsenite, the study(152)revealed the incorporation of CD3 group into the arsenical product when l-methionine-methyl-d3was added to the cultures. In addition, with the presence of labelled[74As](154) in the culture of C.humicola, labelled intermediates such as arsenite, methylarsonate and dimethylarsinate in thescheme were found as products in broken cell homogenates. The results were taken as indicationsthat S-adenosylmethionine (“active methionine”) is involved in the transfer of the methioninemethyl group to arsenic in mycological methylation(5).In the study done by McBride et al(150, 156) it was shown that aerobic micro organismsproduced trimethylarsine and the anaerobic methanogenic bacteria produced dimethylarsine whenincubated in the presence of pentavalent and trivalent arsenic derivatives. On account of theseobservations, McBride and coworkers(150) stated that an arsenic cycle existed in nature and thiscycle relies upon both biological and abiotic reactions. Since aerobes reduce and methylatedimethylarsinic acid (DMAA) to form trimethylarsine and the anaerobes reduce the compound todimethylarsine(150), it was indicated that DMAA is an important arsenical in thebiotransformation of arsenic in the ecosystem.In sea water, arsenate is the predominant arsenic species and is present at approximately1.0 - 2.0 ppb(157). Arsenate is assimilated due to its similarity to phosphate by marinephytoplankton( 158) as evident with uptake studies in both bacteria( 159) and marine yeast( 160).Regardless of the mechanism of arsenate uptake, primary producers must adapt to theaccumulation of cellular arsenate(6). Arsenate is detoxified by marine organisms viabiomethylation into methylated arsenic compounds. Since these methylated arsenic compounds are14less toxic, the biomethylation response to arsenate by the primary producer is considered abeneficial detoxification step for both the producer and the higher trophic level which feed uponit(6, 158). Fish and marine invertebrates retain 99% of the arsenic in an organic form(6). Theconcentration of inorganic arsenic rarely exceeds 1.0 mg kg’ in their tissues(161). Chemicalstructures have been determined among several of the predominant water soluble organic arseniccompounds in invertebrates and fish. Edmonds et al(162) isolated an organoarsenic compoundfrom the Western Rock Lobster, Panulirus cygnus by using repeated ion exchangechromatography and thin layer chromatography. X-ray analysis of the isolate confirmedunequivocally the arsenical as arsenobetaine (Arsenic compound , Table 1.1). This compound isubiquitous in marine animals. It has been found in the American Lobster, Homarusamericanus(163); the Octopus, Paroctopus dolfieini(164); the Dusky Shark, Carcharhinusobscurus( 165); the School Whining, Sillago bassensis( 166); the Lemon Sole, MicrostumusKitt(l 67); Crab, Cancer cancer(1 67); Alaskan King Crab, Paralithodes camtschatica; AlaskanSnow Crab, Chionoecetes bairdii; and Dungeness Crab, Cancer magista(168) as well as in theShrimp, Sergestes lucens(169).In shrimp arsenobetaine constitutes two thirds of the arsenic pool while the remainder hastentatively been identified as arsenocholine(170, 171) (Arsenic compound 2. Table 1.1). However,other investigators have been unable to confirm the presence of arsenocholine in other shrimpspecies(170, 172). Trimethylarsine oxide (Arsenic compound , Table 1.1,) has been found as aminor component in fish(173) and clam(27). Its concentration was found to increase withstorage(l 74). Although all of the intermediates of the metabolic pathway have not beenidentified(6), arsenobetaine is believed to be the end product which accumulates in marineanimals(5, 6, 170, 173). Arsenicals in Marine MacroalgaeAlgae are primaiy accumulators of arsenic in the marine environment(175). Knowledge ofthe chemical forms of arsenicals in the marine algae therefore is very important in understandingthe geochemical cycle of the element.Brown algae contain higher concentrations of arsenic at several tens of ppm while greenalgae and red algae contain a lesser amount of several ppm on a dry weight basis(49, 175, 176).Sanders work (49) revealed that arsenic is present in organic form at 53%, 78% and 57% in green,brown and red algae respectively, through the examination of 56 algae species. These resultsappear rather disturbing because of the large amount of inorganic arsenicals accumulated in themarine algae. Other workers(4, 40, 177-79), however, found both lipid- and water-solubleorganoarsenicals, as well as residual unextracted arsenic compounds in marine macroalgae. Therelative proportions of these arsenic compounds vaiy depending on the algae studied. The mostsignificant works on marine algae were performed, however, by the research groups in WesternAustralia and Japan(28, 48, 50, 51, 95, 97-99, 162, 163, 165, 166, 175, 180, 181).The first identification of water-soluble organic arsenic compounds in marine macroalgaewas carried out by Edmonds and Francesconi(180, 181). Methanol extraction of a brown kelp(Ecklonia radiata) sample followed by column chromatography on Sephadex LH-20 and SephadexDEAR gave three arsenic containing fractions( 180, 181). The arsenicals were characterized to bearsenic containing ribofuranosides or dimethylarsinylribosides (Arsenosugar 11 a-c, Table 1.2) onthe basis of micro analysis, ‘H and ‘3C NMR and field desorption mass spectrometry. Before thiswork on Ecklonia radiata, Edmonds and coworkers( 182) isolated and characterized Arsenosugarsfl. and jj from the giant clam Tridacna maxima. The determination of the crystal structures ofarsenosugar j confirmed that it was formulated correctly. By the use of similar purificationprocedures(175), many Japanese brown algae have been shown to bioaccumulate arsenosugars astheir main arsenicals (See Table 1.3). Analysis of a brown algae, Hizikiafusforme revealed fourarsenosugars (ii., jj, and jj) and also, 50% of its total arsenic content was found to beinorganic arsenic. Earlier works(184, 185) on Hizikia frsiforme had indicated the presence of16inorganic arsenate as a major arsenical in this macroalgae. Further, analysis on a green algae,Odium fragile (MIRU)(188) indicated that it contains arsenosugars J.h and 11h in addition to asmall portion of DMAA (5% of total arsenic). This was the first isolation( 175) of DMAA from amarine macroalgae. The presence of DMA.A in marine macroalgae had earlier beensuggested(176). For brevity, results of isolation and characterization of arsenicals reported insome Japanese marine macroalgae are given in Table 1.3.Table 1.3Arsenicals Found in Japanese Marine MacroalgaeOther ArsemcalsArsenosugars Isolated Isolated andNo. Marine Macroalgae and Characterized Characterized ReferenceBrown Alaae1 Laminariajaponica ha, 11b, lic nd 183(Makonbu)2 Undariapinnatijlda lla,llb,11c nd 175(Wakame)3 Eisenia bicyclEs Arame ha, hib, hic nd 1754 Thzilcifuszforme (Hijiki) , hic, , Inorganic As(V) 485 Sargassum thunbergii,hib, th. j nd 186(Umitoranoo)6 Shaerotircha divaricata ha, hib, hic, lid 2 unknown sp. 175(Ishimozuku)Red AIae7 Porphyra tenera , nd 187(Asakusanori)Green Alaae8 Odiumfragile (MIRU) ha, hib DMAA 188nd = not detectedLipid soluble arsenic has been studied( 175) but not as comprehensively as the water solublearsenicals. Based on the discovery of arsenosugar derivative (ilj) containing a glycerophosphoryl moiety Edmonds and Francesconi(181) and Knowles and Benson(13) proposed adiacylated form of arsenosugar lib for the structure of lipid-soluble arsenic. Biotransformation Pathways in the Marine EnvironmentThe concentration of arsenic in marine macroalgae is of the same order as in marineanimals (ca 10 ig g1) and some 4000 - 5000 times the concentration in sea water(166, 189). Inmarine organisms, arsenic occurs mainly as non-toxic organic compounds with only small amountsof the inorganic arsenic species present(4). Arsenobetaine is widely distributed in marine animalsat different trophic levels and is probably the end product of arsenic metabolism in marine foodchains. Arsenobetaine has not yet been identified in algae. However, the bulk of the arsenic inalgae appears to be in the form of dimethyiribosides (Table 1.2). The pathway for the biogenesisof arsenic-containing ribosides in algae requires reduction of absorbed oceanic arsenate and, byusing oxidative methylation, donation of the methyl group in S-adenosylmethionine (SAM) in twostages giving Dimethyl Arsinic Acid. This pathway is shown in Figure 1.2. Reduction followed bydonation of the adenosyl group in SAM by using oxidative adenosylation would give thenucleoside, (CH3)2As(O).—X which, on glycosidation with available algal metabolites would yieldthe range of dimethylarsinyiribosides that have been identified from algal sources(5, 7, 28, 51, 95).Edmonds and Francesconi(180) suggested that a microbially mediated stage, probablyoccurring within sediments, is necessary for the generation of arsenobetaine from arsenoribosides(or arsenosugars)(4). This suggestion has received some support from experimental work(1 90).When Ecklonia radiata (brown kelp) was allowed to decompose anaerobically in thepresence of sea water and beach sand, the algal arsenoribosides were claimed to have beenquantitatively converted to dimethylarsinyl ethanol, DMAE( 190). DMAE has not yet beenidentified in the natural environment, however. Regardless, Edmonds and Francesconi( 191, 192)went ahead and proposed that the dimethylribosides and their anaerobic degradation product,DMAE, are the most likely intermediates for the production of arsenobetaine from oceanic arsenatein marine food wbs. The biotransformation scheme(191, 192) for arsenic in the marineenvironment, shown in Figure 1.2, was based on this proposal. However, Phillips andDepledge( 12) have suggested an alternative pathway in which marine organisms can producearsenocholine via the breakdown of arsenophosphatidyl choline by phospholipases(4, 12).18Arsenocholine could then be oxidized to arsenobetaine(193). These authors(12) employed as theirstarting compound arsenoethanolamine (2-hydroxyethylarsine) which is an analogue ofethanolamine,HOCH2CHN3with As replacing N. The Phillips and Depledge biotransformationscheme of arsenic in the marine environment differs from that of Edmonds and Francesconi fromthe standpoint of the starting material. This scheme is shown in Figure 1.3. Phillips and Depledgescheme is not widely acceptable because the basis of their starting material, arsenoethanolamine isconsidered presumptuous(191) due to unavailable evidence that the analogue of ethanolamine,glycine or serine exist, even transiently, in living cells. However, Phillip and Depledge schemesuggest the possibility that there is an arsenic biotransformation cycle existing between thearsenoribosides and two methylated arsenic species (DMAA and TMAO), apart from the arseniccycle existing with DMAE via anaerobic decomposition. Furthermore, the scheme suggests thattrimethylarsineoxide is a likely intermediate for the production of arsenobetaine, the majorarsenical in marine animals. The present thesis took this suggestion and the identification ofDMAA as an anaerobic decomposition metabolite (present study) into consideration in proposingan alternative biotransformation pathway (Section for the formationlbioaccumulation ofarsenobetaine in the environment based on the likely toxic response of marine organisms totrimethylarsine following the Challenger mechanism.19Figure 1.2Edmonds and Francesconi’s Proposed Biotransformation Scheme forArsenic in the Marine Environment (Adapted from References 191 and 192)As(OH)3 [CH3 CH3AsO(OH)2(MMAA)CH3AsO(OH)2 2e CH3As(OH)2 [CH1 (CH3)2AsO(OH) 2 b (CH3)2AsOHDMAA)(CH3)2AsOH SAM (CH3)2As(O)—X hydrolysis (CH3)2As(O)—Y(CH3)2As(O)—Y Arsenosugar 11 Arsenic Lipids<2> Arsenosugar 11 (CH3)2As(O)CHCO (Dimethylarsinylethanol, DMAE)(CH32As(O)CHCOO (Dimethyloxarsylacetic acid)(CH3)2As(O)CHCO(DMAE)(CH3)AsCH2COH3C (Arsenocholine, AC)[CH34]<3> (CH)2As(O)CHCOO(Dimethyloxarsylacetic acid)(CH3)AsCH2COO(Arsenobetaine, AB)(CH3)AsCH2COH3C [Ol(AC)<1> HOAsO32 —.-+ HOAsO32 2e(oceanic (alga arsenate)arsenate)SAM, S - adenosylinethionineBiotransformations <1>, <2> and <3> occur respectively in marine algae, sediment and marine animals(fish, crustacea, etc).HOOCCH(NH2)CCS(3+420-3H (CH3)AsCH2COR(Arsenophosphatidylcholine)1.4 SCOPE OF WORKThe discussions in this chapter have shown that marine organisms accumulate arseniccompounds and several of these compounds have been characterized by various speciationprocedures in the marine environment. Arsenobetaine appears to be the most abundant arsenical inmarine animals wheeas arsenosugars are the main arsenicals found in marine macroalgae. Mostof the comprehensive isolation and characterization of arsenicals in the marine macroalgae havebeen restricted to Western Australia and Japan. Hence, there is a need for examination ofaccumulated arsenicals in other geographic locations in order to establish whether arsenicFigure 1.3Phillips and Depledge’s Proposed Biotransformation Schemefor Arsenic in the Marine Environment (Adapted from Reference 12)SAM-2WH3AsCH2CHO_______H3AsCH2CH0R(arsenoethanolaniine) (Arsenophospatidylethanolamine)CH(00CR’)CH(00R)0P(O)(0H)0CHAs HX(X is defined in Figure 1.2)[01Arsenosugar Anaerobic , (CH3)2As(0)CHCO Reduction! • (CH3)AsCH2COdecomposition (DMAE) methylation (AC)[0] (CH3)AsCH2COR[0](CH3)2As(0)CHCOO Reduction! (CH3)C2(YJ(Dimethyloxarsyl Methylation (AB)acetic acid) IReduction!methylation(CH3)AsO (TMAO)jReduction(CH3)As (TMA)(CH3)2AsO(OH)(DMAA)ICH3AsO(OH)2 Methylation HAsO32(MMAA)Reduction HAsO4221speciation varies according to different regions(4). In addition, the search for known and novelorganoarsenic compounds in marine organisms is still a subject of immense interest both from theanalytical method and speciation points ofview(51, 97, 99, 194).The debate about the pathway for the biotransformation of arsenic in the marineenvironment has not been completely resolved. In fact, arsenobetaine the end product of arsenicmetabolism in the marine food chain is a subject of controversy as to whether it is formed fromalgal arsenosugars via the dimethylarsinylethanol intermediate and/or, if organisms at differenttrophic levels have the ability to synthesize arsenobetaine.For a complete understanding of the bio-accumulation and biotransformation of the arsenicin the marine environment, more information is needed about the levels and forms of thesearsenicals, especially in marine macroalgae, a primary producer. The objective of this thesis wasto determine the levels and forms of the arsenicals (bioaccumulation and speciation) present in themarine macroalgae of British Columbia, as well as the examination of the biotransformationproducts of the “hidden” arsenicals present in the algae.Graphite Furnace and Hydride Generation Atomic Absorption techniques were developedand used to determine arsenic in several seaweed from various locations along the coast of BC.The extracts of some brown algae were fractionated by gel permeation chromatography and ionexchange chromatography. Speciation of the extracts and also, identification of isolated arseniccontaining fractions were carried out by using HG-GC-AA, HPLC-Mic-HGAA and HPLCICPMS. Comparison of sample chromatograms with those obtained for standard arseniccompounds, as well as arsenic species found in a standard reference material aid in arriving atpositive identification.Also, anaerobic transformations of ‘hidden” arsenicals in F distichus were investigated.The transformation products were analyzed after gel permeation chromatography with HG-GC-AAand the positive identifications of the products were established after HPLC-ICPMS analysis.22EXPERIMENTAL2.1 INSTRUMENTATION2.1.1 ATOMIC ABSORPTION SPECTROMETRY (AAS)Arsenic determinations were carried out by using a Varian Techtron Model AA1275 single beam atomic absorption spectrophotometer equipped with a Varian SpectraAA hollow cathode lamp which operated at 10 mA. The arsenic atomic absorption wasmonitored at 193.7 nm and the spectral band pass was mm. The AA Spectrometer wasfitted with a deuterium background corrector and an HP 82905A printer. Graphite Furnace Atomic Absorption (GFAA)A Varian Techtron GTA-95 accessory was used for GFAA analysis. Pyrolyticallycoated graphite tubes were obtained from Varian Techtron and argon was used as thepurge gas. Hydride Generation Atomic Absorption (HGAA)A continuous hydride generation assembly illustrated in Figure 2. 1 was used forthe generation of arsines in the determination of total arsenic concentrations in samples.The HGAA assembly consisted of a Gilson Minipuls 2 four channel peristaltic pump whichwas used to continuously deliver the sample (or standard) solutions, hydrochloric acid (oracetic acid), and sodium borohydride solution. By using nitrogen as the carrier gas, thearsines generated were fed into an open ended T-shaped quartz curvette[8.5 cm x 1 cm (o.d.) mounted and alligned in an air/acetylene flame of a standard Varianburner. The operating conditions of this system followed those established by Dodd(27)and are described n Table 2.1.23Figure 2.1Assembly Diagram of the Continuous Hydride GenerationAtomic Absorption System (Adapted from Reference 27)Quartz Absorption CellReaction PeristalticCoil PumpHydride “4 SampleTransfer 4, 4TubeI“4AcidGas/Liquid NaBH4Separator____NitrogenDrain Needle PressureValves RegulatorTable 2.1Operating Conditions for the Continuous HGAA System(Adapted from Reference 27)Uptake Tubes Sample: 2.8mm i.d. 7.5m1 mirr’Acid: 2.3mm i.d. 2.Oml mirr’NaBH4: 2.3mm i.d. 4.Oml mirr’Carrier Gas Flow Nitrogen: Through mixing coil lOOmi min1“ Through gas/liquid separator 25m1 min1HCI acid concentration* 4MNaBH4concentration* 1.5% (w/v)Integration Run mean* Concentration values determined in this study (Section Hydride Generation - Gas Chromatography Atomic Absorption(HG-GC-AA)The HG-GC-AA apparatus shown in Figure 2.2 was employed for the speciationof arsenicals in sample solutions obtained with or without an appropriate digestionprocedure. The system consists of a peristaltic pump for mixing known volumes ofsample (or standard) solutions with hydrochloric acid (or acetic acid) and sodiumborohydride solution. The arsines generated were led through a gas-liquid separator usinga helium carrier gas into a moisture Teflon U-trap (30 cm length x 0.8 cm id.) immersedin a dry ice-acetone slurry and subsequently swept into a hydride trap, a Teflon U-tube(30 cm length x 0.4 cm i.d.) cooled by liquid nitrogen (-196 oC). The hydride trap wasthen warmed by using a water bath (70 oC) and the arsines were liberated and carried ontoa Porapak-PS column (80-100 mesh, 50 cm length x 0.4 cm i,d., silanized with silyl-8column conditioner as described by Reimer[195J) within a Varian Vista 6000 GasChromatograph (GC). By using a pre-set temperature program, the volatile arsines wereseparated on the column, and detected by passing them into an air-hydrogen flame in aquartz curvette (8 mm i.d. x 10 cm) placed carefully in the light path of an arsenic hollowcathode lamp. Detection of the arsenic absorption was achieved by using an 810 Jarrell -Ash Atomic Absorption Spectrophotometer coupled to a Hewlett Packard 3390AIntegrator. The operating parameters established by Li(196) were followed and are shownin Table 2.2.25Table 2.2HG-GC-AA Operating Parameters (Adapted from Reference 196)Hydride Generation Gas Chromatograph Atomic Absorption Spectrometer2% NaBH4Solution Initial temperature 70 OC 810 Jarrell-Ash SpectrometerlMHydrochloric acid Ramp Rate 30 oC mm-’ X(nm): 193.7, Slit width (nm): 103 ml Sample Size Final temperature 150 oC/ 2 mm Lamp Current (mA): 10Trapping time 3 mm Helium flow rate 1:30 ml miir’ Quartz Burner: 8 mm i.d. X 10 cmPump rate 3 ml mm-’ Helium flow rate 2:20 ml mm-’ Flame: Mr/HydrogenAir flow rate: 120 ml mm-’H2 flow rate: 75 ml mm-’Integration: 3 secondsFigure 2.2HG-GC-AA Assembly (Adapted from Reference 196)Perictaitic PumpReaction Coil11 eli urnSampleAcidNa13114Ventheliumqas!Liquid Dr34lccfAcetone 1 Gas Chrornatograpl - AASepartor Waler iraP Liquid Nitrogen2. ReleasIng Ar-sinesWater bath262.1.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)The HPLC system consisted ofWaters Model 510 or Model M45 solvent deliverypumps and a Waters U6K injector for sample introduction onto an appropriate column.The columns used consisted of three commercially available analytical reversed phase Cl 8columns (Waters 10 j..i Bondapak C18, 300 mm x 3.9 mm; Phenomenex 10 ji BondcloneC18, 300 mm x 3.9 mm; and GL Sciences Intersil ODS-2, 250 mm x 4.6 mm) and aWaters RCM 25 x 10 preparative reverse phase C18 column (Prep Nova C18 HR 60A).The analytical and preparative columns were preceded by a guard column packed with therespective stationary phases. The arsenic specific detectors used for effluents from theanalytical HPLC column were either a hydride generation atomic absorption spectrometer(HGAAS) or an inductively coupled plasma mass spectrometer (ICPMS). The graphitefurnace atomic absorption spectrometer (GFAAS) was used as the arsenic specificdetector for the preparative HPLC work; separated fractions collected with a GilsonMicro Fractionator were manually transferred to the automatic sample delivery system ofthe GTA-95. The HPLC - Microwave decomposition (Mic) - HGAA and HPLC - ICPMSmethodologies are described in sections and HPLC-Mic-HGAASThe schematic diagram of the Microwave - HGAAS is shown in Figure 2.3. Theinstrumental parameters were established by Le(17). The HPLC effluent(s) are mixedwith the decomposition reagent (R) (a solution containing 0.1 M potassium persulfate and0.3 M sodium hydroxide), at a T-joint (V)(1/16”, Mandel, Canada) before the solutionmixture flows through a PTFE decomposition coil (5 m X 0.8 mm i.d.) situated in anoperating microwave oven (MO) (500 W, 2450 MHz, Sharp Electronics, Japan), wherethe decomposition takes place. The microwave oven effluents then react with thecontinuous flow of acid (A) and sodium borohydride (B) to generate arsines. The arsine isatomized in a flame heated quartz curvette: an atomic absorption spectrometer serves asan arsenic specific detector.27PFigure 2.3Schematic Diagram ofMic - HGAAS (Adapted from Reference 17) HPLC-ICPMSA block diagram illustrating the arrangement of components in a typical ICPMSinstrument and combined with an HPLC system is shown in Figure 2.4. The VG PlasmaQuad 2 Turbo Plus ICPMS used in this work was equipped with a SX300 quadrupolemass analyzer, either a Meinhard concentric nebulizer or a de Galan V-groove nebulizerand a standard RD1 torch (Fassel Configuration). The spray chamber was cooled to 4°Cusing a mini-chiller (cool flow CFT-25, Neslab). Optimum conditions were obtained forthe sampling position and ion lens voltage with respect to signal - to - noise ratio at mlz 75(Ask) by introducing a 30 ng ml-1 arsenite solution in 1% nitric acid. Single ion monitoringWS MO lBDGLS — Sample flowR— Decompositionreagent flowA— Acid flowB — NaBH4 flowP — Peristaltic pumpW— WasteV— Sample injection valveMO — Microwave ovenlB — Ice water batchT1&T2— T-jointsD — To detector (AAS)0— Carrier gas (N2)GL— Gas/liquid separatorG28mode was used with the quadrupole mass analyzer. The outlet of the HPLC analyticalcolumn was connected directly to the ICP nebulizer inlet by using PTFE tubing (200 mmX 0.4 mm i.d.) and appropriate fittings. A multi- channel analyzer was used to monitorthe arsenic signal at m/z 75 and a VG data system stored the data obtained before achromatogram of the completed chromatographic run was plotted on an Epson FX-850printer.Figure 2.4Block Diagram of a Typical ICPMS InstrumentCoupled to ITPLC System2.1.3 NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROMETRY ANDMASS SPECTROMETRY (MS)‘H NMR spectra were obtained by using Bruker WH 400 or Bruker WH 200spectrometers. A Varian XL-300 spectrometer was used to obtain ‘3C NMR spectra.Chemical shifts are quoted relative to tetramethylsilane (TMS) as an external standard.Mass spectra data were obtained by using Kratos AES M50 or Kratos AEI MS9mass spectrometers. Desorption Chemical Ionization Mass Spectrometry technique wasused except where otherwise stated.292.2 CHEMICALS AND REAGENTSAll chemicals used from BDH, Fisher Scientific, Aldrich, Eastman Kodak, Alfa andMCIB were classified as analytical grade. Aqueous stock solutions (1000 ppm of arsenic)of arsenite, arsenate, MMAA and DMAA were prepared by dissolving appropriateamounts ofAs203 (BDH),Na2HAsO4.7H0(MCIB),CH3AsO(ONa)2.60(Alfa), and(CH3)2AsO(OH) (Fisher). Unless otherwise stated, standard solutions were prepared byserial dilution of these stock solutions with deionized water. Sodium borohydride(Aldrich) solutions were made fresh daily in deionized water. The HPLC mobile phasesused in this work were: (i) 10 mM tetraethylammonium hydroxide + 4.5 mM malonic acid+ 0.05 or 0.1% methanol (HPLC grade, Fisher) at pH 6.8; (ii) 10 mMsodiumheptanesulfonate + 0.15% methanol at pH 3.5, (iii) 10 mM methanesulfonic acid +4 mM malonic acid + 1% methanol at pH 3.1 and, (iv) 25 mM tetramethyl ammoniumhydroxide + 25 mM malonic acid at pH 6.8. These were all prepared in deionized distilledwater. The pH of these HPLC mobile phases was adjusted by using diluted nitric acid andsodium hydroxide and the solutions were filtered through a 0.45 urn membrane filterbefore use. Other solvents from commercial sources were either of analytical or HPLCgrade and were used after filtration through Millipore 0.45 membrane filters. Allglassware and plasticware was cleaned by soaking them overnight in 2% Extran solution,rinsing with tap water, and soaking in dilute hydrochloric acid overnight. These glass andplastic containers were then rinsed thoroughly using tapwater and finally using deionizedwater.The stock solutions (1000 ppm of arsenic) of arsenobetaine, arsenocholine andtetramethylarsonium iodide were prepared from synthetic samples(27) by dissolvingappropriate “amounts of these compounds in 0.O1M hydrochloric acid.Dimethylarsinylethanol (DMAE) standard was synthesized as described in section 2.7(1000 ppm of arsenic stock solution of DMAE was prepared in deionized water).302.3 SAMPLE SITE COLLECTION AND STORAGEBritish Columbia occupies part of the vast belt of mountainous teffain that flanksthe western margins of the Americas(197). The sampling locations are found in thewestern terrestial system averaging some 300 km in width. Even though some sites arecomposed of virgin soil, most locations have seen the influence of various marine trafficsuch as log booms, chip scows, fish boats and pleasure seacrafts. Additionally, industrialoperations such as mining (metals and fossil fuels) and forestry (logging, pulp and paper)were or still are part of some sampling environments. These and other factors such asgeological history of the particular soil have been claimed(198-205) to influence thearsenic content of the soil on which the seaweed, a primary producer derived its nutrients.The natural arsenic content in virgin soils varies from 0.1 to 4Oppm, with an average ofabout 5 - 6ppm depending on the geographic region(199). In the continental crust ofBritish Columbia’s coastal regions, offshore marine arsenophosphites have been analysedto contain an average arsenic concentration of 28.3 ppm(203, 204). Additionally, as aresult of considerable mobility of arsenic found in sandstone, shale and coal from miningareas(205), some of the collection sites are suspect to arsenic contamination.In order to investigate the amount of arsenic accumulated, as well as the arsenicspecies in the seaweeds of British Columbia, the following locations, shown in Figures 2.5and 2.6, were sampled. At the collection site, the wet samples were packaged in plasticfreezer bags and stored under -20° C. Before analysis, the seaweed samples were freezedried (except where otherwise stated) and the moisture content determined as shown inTable 2.3. The average moisture content was found to be 82% of the wet weight. Thisvalue (82%) is in agreement with similar determinations(206, 207). The freezedehydration procedure ensured that substances of interest in the seaweed were preservedduring storage. Other seaweeds not freeze dried were packaged in plastic freezer bagsand stored under cold conditions at 4°C.31Figure2.5••••••....•••.....I______________________________________•‘:••KEYALASKAA-AliceArm•‘“jB-AnthonyIsland4.-CC-Anyox/ED-GranbyBayE-HastingsArm••F-JuanPerezSound---—-•G-KitimatArmDIXONENTRANCEP1-GilttoyeesInlet1••.•-HiltonPointI•••H-LangaraIsland54N.•I-Masettlnlet-J-RennellSound1-ClonardBayGAROIER-EllsBay-ISLANDA•..•.:.••çANAI.•••U.•e-ShieldsIsland-TartuInlet-;t:•.K-TasuSoundKFzAea13-FairfaxInlet‘.11••-MinesiteShoreB11BRITISHCOLUMBIAQUEENCHARLOTTE1’MAINLANDSOUND•:(-454N•.Figure2.6-QUEENCHARLOTTE1.BRITISHCOLUMBIASOUND:..MAINLAND.•KINGCCMKNIGHTINLET..HOPEINLETBUTEINLETENOOTKA5o’.,4+o,VANCOUVEHNORTHPACIFICOCEANASHR49°NAMPHITRITE49°N__________________________l’L144.\.•••.•KEY‘‘‘.A-JervisInletCAPEFLATTERWHIDBEYB-PendrellIslandC-PendrellSoundSAN.JUANID-QuatsinoSoundSEATTLE-KoprinoHarbour/-VarneyBay£-ReadIslandF-RupertInlet•WASHINGTONG-Vancouver,PointGrey:•.••COLtJUWA-$39’V.125W.Table 23Sampling Data and Seaweed Moisture ContentTable 2.3 cont...%H20No. Sampling Location/Site Seaweed Class Seaweed Species Content1 Alice Arm ® Kitsault Beach, Phaeophyceae Fucus distichus 8705/15/902 Alice Arm @ Alice Arm Phaeophyceae Fucus distichus 78Head, 05/13/9 13 Anthony Island © Anthony Chlorophyceae Codium sp. 91Island, 05/20/9 14 Anyox © Granby Bay Beach, Phaeophyceae Fucits distichus 8905/14/905 Anyox ©Granby Bay Beach, Phaeophyceae Fuczis distichus 8105/21/916 Hastings Arm @ Hastings Phaeophyceae Fucus distichus 88Arm Head, 05/14/907 Juan Perez Sound © Hot Phaeophyceae Fucus distichus 82Spring Island Beach, 04.17.898 Juan Perez Sound @ Hot Floridiophyceae Iridaea splendeus 93Spring Island Beach, 04/17/899 Kitimat Arm © Giltoyees Phaeophyceae Fucus distichus 70Inlet, 05/10/9110 Langara Island © Beach Phaeophyceae Fucus dislichus 78Shore, 04/13/8911 Langara Island @ Beach Phaeophyceae Alaria marginata 88Shore, 05/17/9112 Langara Island © Beach Chlorophyceae Ulva sp. 88Shore, 05/17/9 113 Langara Island © Beach Chlorophyceae Endocladia muricata 88Shore, 05/21/9114 Masset Inlet © Yakoun Phaeophyceae Fucus distichus 81Yellow River, 05/14/9015 Rennell Sound @ Clonard Bay Phaeophyceae Fucus distichus 80Beach, 04/14/8916 Rennell Sound @ Shields Phaeophyceae Fuczis distichus 89Island, 05/1’9/9017 Rennell Sound @ Tartu Inlet, Phaeophyceae Fucus distichus 8505/20/9018 Rennell Sound @ Ellis Bay, Phaeophyceae Fucus distichus 8005/18/9134%H20No. Sampling Location/Site Seaweed Class Seaweed Species Content19 Rennell Sound @ Clonard Phaeophyceae Macrocys(icpyr{fera 85Bay, 05/18/9120 Rennell Sound Ellis Bay, Floridiophyceae Callophyllis 7705/18/91 edentaota21 Rennell Sound @ Ellis Bay, Chlorophyceae Ulvafenestrala 8105/18/9122 Tasu Sound @ Fairfax Inlet Phaeophyceae Fucus distichus 73Shore, 05/17/8923 Tasu Sound Fairfax Inlet Phaeophyceae Fucus distichus 76Shore, 05/21/9024 Tasu Sound Minesite Phaeophyceae Fucus distichus 80Shore, 05/21/9025 Jervis Inlet Island Copper- Phaeophyceae Fucus distichus 79mine Beach, 04/19/8926 Pendrell Sound @ Pendrell Phaeophyceae Fucus distichus ndIsland Beach, 05/23/9 127 Quatsino Sound @ Varney Phaeophyceae Fucus distichus ndBay Beach, 04/18/8928 Quatsino Sound © Varney Phaeophyceae Fucits distichus 83Bay Beach, 05/21/9129 Quatsino Sound @ Kaprino Phaeophyceae Fucus distichus 75Harbour Prideau Pt., 05/22/9 130 Quatsino Sound © Varney Floridiophyceae Endocladia muricata 88Bay Beach, 05/21/9131 Quatsino Sound © Varney Chlorophyceae Ulva sp. 86Bay Beach, 05/21/9 132 Quatsino Sound @ Varney Chlorophyceae Unkown sp. 91Bay Beach, 05/21/9 1 (Filamentus).33 Quatsino Sound @ Varney Chlorophyceae Unkown sp. (green 76Bay Beach, 05/21/9 1 algae)34 Read Island © Oyster Lease Phaeophyceae Alaria sp. 83Farm, 05/23/9 135 Rupert Inlet © The Southern Phaeophyceae Nereocystis 93Side, 10/28/87 luetkeana.36 Vancouver © Howe Sound, Chlorophyceae Enteromopha sp. 51Furry Creek, 07/07/8937 Vancouver’@ Point Grey, Phaeophyceae Fucus distichus ndWreck Beach, 05/08/89nd = not determined; unkown sp. = species name not determined; Phaeophyceae = brownalgae; Chlorophyceae = green algae; Floridiophyceae = red algaeI352.4 ANALYTICAL PROCEDURES2.4.1 GRAPHITE FURNACE ATOMIC ABSORPTION (GFAA)A Varian Techtron GTA-95 graphite furnace coupled with a VarianTechtron Model AA 1275 atomic absorption spectrometer equipped with adeuterium background corrector and a Varian spectra AA hollow cathode lampwas used in all GFAA arsenic determination experiments. The hollow cathodelamp was used at 10 mA and all absorbances were measured at the 193.7 nmresonance line. The peak area integration (1 second transient signal averaging)mode was used for arsenic analysis after atomization in Varian Techtronpyrolytically coated graphite tubes. Argon was used as purge gas. Sample (15 p.1)and/or standard arsenic (15 j.L1) solutions were introduced into the furnace usingthe automatic delivery system of the GTA-95. During the GFAA procedures,either 20 p.1 of 100 jig mr1(27) of Ni2 solution (as nitrate or chloride)(208) or15 p1 of 100 jig mr’ palladium in 2% citric acid (see section for Pdmodifier optimization) was added as a matrix modifier. The operating furnaceprograms for the determination of arsenic in the presence of nickel and palladiummodifiers are shown in Table 2.4 and 2.5 respectively.36Table 2.4Furnace Operating Parameters UsingNi2+Solutionas a Matrix Modifier (Adapted from Reference 27)Step Temperature Time Gas Gas Read Command Comment(°C) (See) Flow Type (*)1 75 5.0 3.0 Argon Dry2 90 30.0 3.0 Argon Dry3 120 10.0 3.0 Argon Dry4 1200 30.0 3.0 Argon Ash5 1200 1.0 0.0 Argon Ash6 2300 1.0 0.0 Argon * Atomize7 2300 2.0 3.0 Argon CleanTable 2.5Furnace Operating Parameters Using Pd in 2%Citric Acid as a Matrix ModifierStep Temperature Time Gas Gas Read Command Comment(°C) (Sec Flow Type (*))1 85 5.0 3.0 Argon Dry2 95 30.0 3.0 Argon Dry3 120 10.0 3.0 Argon Dry4 1400 20.0 3.0 Argon Ash5 1400 1.0 3.0 Argon Ash6 1400 2.0 0.0 Argon Ash7 2400 1.0 0.0 Argon * Atomize8 2400 2.0 0.0 Argon * Atomize9 2400 2.0 3.0 Argon CleanI372.4.2 HYDRIDE GENERATION ATOMIC ABSORPTION (HGAA)The atomic absorption signals obtained from the reduction of 0.1 i.g mi-1 standardarsenic solutions of arsenate (AsV), arsenite (Aslil), monomethylarsonic acid (M1vIAA),dimethylarsinic acid (DMAA) mixed with different concentrations of hydrochloric acid(HCI) and acetic acid (CH3COOH) were studied in order to establish optimum reductant(sodium borohydride, % NaBH w/v) concentration. The study, discussed in Section 3.2,was used to determine that the optimum concentrations of NaBH4,HC1, andCH3OOHwere 1.5% w/v, 4M and 2M respectively. These optimum concentrations were used forall arsenic analysis by the continuous HGAA procedure. The run mean (continuous signalaveraging) integration mode was employed during continuous HGAA arsenic analysis.2.4.3 HYDRIDE GENERATION - GAS CHROMATOGRAPHY ATOMICABSORPTION (HG-GC-AA)Atomic absorption signals from the selective reductions of 0.040 tg As standardsolutions of arsenate (AsV), monomethylarsonic acid (MMAA) and 0.080 tg As solutionof dimethylarsinic acid (DMAA) with 2% NaBH4 (w/v) reductant solution were studied inorder to estimate the optimum acid effect on their response profiles. Differentconcentrations of hydrochloric acid (HC1) and acetic acid (CH3COOH) were used for thestudy.In the study described in Section 3.1.3, the optimum concentrations ofhydrochloric acid and acetic acid were determined to be 1M and 1.5M respectively. Theseacid concentrations were used for all arsenic determination and speciation carried out byusing the semi-continuous mode HG-GC-AAS procedure. The three second signalaveraging peak area integration mode was employed for arsenic absorption signaldetection. /382.4.4 MICROWAVE - HYDRIDE GENERATION ATOMIC ABSORPTION(MIC-HGAA)Methodology making use of on-line coupling of microwave oven assisteddecomposition with flow injection and hydride generation Atomic Absorption detectionwas used for the determination of reducible and “hidden” arsenicals. The schematicdiagram and experimental conditions, developed by I.e et al(209, 210), for microwavedecomposition and hydride generation procedure are shown in Figure 2.3 and Table 2.6respectively.Table 2.6Experimental Conditions forMicrowave Decomposition and Hydride Generation(Mic-HGAA) (Adapted from Reference 17)Reagents and Flow Injection HGAASConditions Without Microwave With MicrowaveDecomposition DecompositionSeaweed Samples 100 p.1 100 p.1K,S,O None 0.1M (2.7%)NaOH none 0.1MDigestion time None ContinuousHC1 Cone. 0.70M 3.OMHC1 Flow Rate 3.4 mI/mm 3.4 mI/mmNaBH4Cone. 0.65M in 0.1M NaOH 0.65M in 0.1M NaOHNaBH4Flow Rate 3.4 mI/mm 3.4 mI/mmCarrier Gas Flow Rate 160 mI/mm 160 mI/mmCysteine 1.0% NoneIn the continuous operation mode of Mic-HGAA, sample and reagents solutions arecontinuously taken up by the peristaltic pump, meeting at the T-joint (V in Figure 2.3),before flowing into the digestion coil. This continuous introduction of the sample resultedin a continuous steady state arsenic absorption signal, which is recorded on the integrator.When the microwave power is turned off, only the reducible arsenicals are observed.39Whereas, when the microwave oven power is turned on in the presence of thedecomposition reagents (Table 2.6), both reducible and “hidden” arsenicals are observedas arsines. Absorption measurement of atomized arsines in the flame heated quartz tube(aligned carefully in the light path of the arsenic hollow cathode lamp of the AA) wassubsequently obtained. In other analysis (Section the arsenicals were separatedon an HPLC column before Mic-HGAAS detection. The HPLC condition used isdescribed in Table 2.7a (1) in section High Performance Liquid Chromatography - Inductively Coupled PlasmaSpectrometry (HPLC-ICPMS)The detailed experimental and instrumental operating conditions for the HPLCICPMS analytical procedures are listed in Table 2.7 (a and b). The columns wereequilibrated with the appropriate eluant before any sample introduction for at least twohours at 1 ml nE-’. The seaweed extracts and chromatographic fractions or isolates werealso centrifuged to remove any suspended particulates prior to ion pair chromatographyon the C18 reverse phase HPLC columns. Identification of arsenic compounds present inthe seaweed samples was made by matching and comparing their retention times obtainedfrom the HPLC-ICPMS chromatograms with those of known standards [using spikedand/or unspiked arsenic compounds as well as previously(50) identified arsenic speciesfound in standard reference material, NIST 1566a Oyster Tissue].2.4.6 DETERMINATION OF TOTAL ARSENICFreeze-dried seaweed samples were wet digested prior to total arsenicdetermination by using two different conditions: (i) with a mixture of sulfuric acid, nitricacid and hydrogen’peroxide, [1 ml (98%H2S04):3 ml (69% HNO3):3 ml (30%H20)pergram freeze dried weight]; and (ii) with a solution of sodium hydroxide (5 ml of2M NaOH per gram freeze dried weight). Total arsenic was also determined in seaweed40after it had been extracted with (i) methanol-water solvent system and (ii) sodiumhydroxide solvent system. Investigation of nitric acid wet digestion for total arsenicdetermination/speciation was briefly investigated as well.Table 2.7 (a and b)Experimental and Instrumental Conditionsfor HPLC-ICPMS Procedure (Adapted from Reference 17)(a): HPLC ConditionsColumn Mobile Phase Flow Rate(ml mm-’)1) Bondclone 10 C18 (i) 10 mM Sodiumheptanesulfonate + (a) 1.0300 X 3.9 mm id 0.1% methanol, pH 3.5(Phenomenex)(ii) 10 mM Tetraethylammonium (a) 1.0hydroxide +4 mM malonic acid, pH 6.8 (b) 0.8(iii) 25 mM Tetraethylammonium (a) 1.0hydroxide +25 mM malonic acid, pH 6.8(iv) 10 mM Methanesulfonic acid + 4 mM (a) 1.0malonic acid + 1% methanol, pH 3.12) t Bondapak 10 C18 (i) 10 mM Tetraethylammonium (a) 0.8300 X 3.9 mm id hydroxide + 4.5 mM malonic acid, pH 6.8(Waters)3) Inertsil ODS-2 (i) 10 mM Tetraethylammonium (a) 0.8250 X 4.6 mm id hydroxide + 4 mM malonic acid + 0.1%(GL Sciences, Japan) methanol, pH 6.8(b): ICPMS ConditionsForward r.f. power: 1.35KWReflected power: < lowOuter gas (cooling) Flow Rate: 13.8 L mm-’Intermediate gas (auxiliary) Flow Rate: 0.70 L min’Nebulizer gas Flow Rate: 0.96 L min’Spray Chamber: Water Cooled to 4°CSampling Cone Orifice Diameter: 1.0 mmSkimmer Cone Orifice Diameter: 0.70 mmSingle Ion Monitoring Mode: m/z 75412.4.6.1 Wet Digestion with Sulfuric Acid. Nitric Acid and Hydrogen PeroxideThis method has been described and applied for the determination of total arsenicin the bivalves of BC, Canada(27, 211, 212). However, because of matrix interferenceand loss of arsenic experienced during direct application to the seaweeds of BC, Canada,the method was modified as follows: With the aid of a pestle and mortar, ground freeze-dried seaweed sample (0.25 g) was taken, and placed in a 500 nil round bottom flask setunto a 500 ml heating mantle (110 V, 500 W). The heating mantle regulator was set suchthat it supplied a temperature of 200° C during the digestion. The digestion reagentsnamely, 1 ml of 98% sulfuric acid, 3 ml of 69% nitric acid and 3 ml of 30% hydrogenperoxide were added sequentially to the flask. The components of the digestion apparatusdescribed previously(27) were then connected to the flask after which heat was applied. A2 hour digestion time, which was determined after a timed digestion study involving realseaweeds (detailed in Section, was employed for all determinations with thisprocedure. After the digestion, the digests were allowed to cool before making them upto 100 ml mark in a 100 ml volumetric flask using deionized water. The solutions of eachseaweed digestate, often with further dilutions, were analyzed for arsenic by using thecontinuous mode HGAA technique. Wet Digestion with Sodium HydroxideFor the determination of methylated arsenic compounds in the seaweed, freezedried samples (0.03-0.17 g) placed directly into separate 40 ml tall glass test tubes weredigested with 5 ml of 2M sodium hydroxide. A hot water bath heated to 90 - 95°C by analuminium heating block was employed for the heating carried out for three hours. Oncooling, the digests were neutralized with dilute hydrochloric acid and made up to 40 ml(in most cases) with deionized water. Arsenic determination and speciation were carriedout on the digestates, which in most cases were diluted prior to analysis by the HG-GCAAS technique.422.4.6.3 Wet Digestion with Nitric AcidWhen nitric acid (5 ml of 2M HNO3)served as the digestion medium, freeze driedseaweed samples ( 1 g) were placed in separate 40 ml tall test tubes and digested forthree hours at water bath temperature 90-95° C. On being cooled, the digestates weremade up to 50 ml in 50 ml volumetric flasks using deionized water. Arsenic analysis wascarried out on the digestates by using the semi-continuous mode HG-GC-AAS technique. Methanol-Water Extraction Followed by Wet Digestion With Sodium HydroxideThis procedure was used to investigate arsenic species in extracts obtained fromsome seaweed samples. Known quantities of freeze dried seaweed samples (0.10 - 0.32 g)were placed in separate 40 ml tall glass test tubes and were extracted for 36 hours in anultrasonic bath using 1:4 mixture of water:methanol. The extracts obtained were filtered,washed with some aliquots of aqueous methanol which were combined, and the extractsample was made up to the 50 ml mark in a 50 ml volumetric flask by using more aqueousmethanol. A 25 ml aliquot was hot base digested and, after appropriate neutralizationwith dilute hydrochloric acid, the digestate was made up to 25 ml in a volumetric flaskusing deionized water. The seaweed residue recovered from the aqueous methanolextraction was hot base digested as well. The undigested aqueous methanol extract, aswell as the digested extract and seaweed residue were analyzed for arsenic by using thesemi-continuous mode HG-GC-AAS technique. Sodium Hydroxide Extraction Followed by the Hot Base DigestionThe sodiuni hydroxide (1M) extraction was utilized., for investigating arsenicspecies in extracts after the hot base digestion (described previously in Section amounts of freeze dried samples (0.08 - 0.45 g) were placed in separate 40 ml tall43glass test tubes, and were extracted with 1M sodium hydroxide solution for 36 hours in anultrasonic bath. Each sample extract obtained was filtered, washed with small aliquots ofdeionized water which were combined and the extract made up to 100 ml in a volumetricflask using deionized water. Each extract was divided into two equal portions and aportion of the extract was then subjected to a hot base digestion. The seaweed residueobtained after the NaOH extraction was similarly treated with the hot base digestion. Theundigested extract portion, as well as the digested extract and seaweed residue wereexamined for arsenic species by using the semi-continuous mode HG-GC-AAS technique.2.4.7 DETERMINATION OF ARSENIC IN SOME MARINE ALGAE BYNEUTRON ACTIVATION ANALYSISNeutron Activation Analysis (NAA) was performed on some powdered freeze-dried seaweed samples, as an Initial, independent and confirmatory technique. The NAAwas carried out at Queen’s University, Ontario, Canada.2.4.8 EXTRACTION OF ARSENICALS IN THE MARINE ALGAE2.4.8.1 Extraction ofWater Soluble Arsenic CompoundsThe frozen seaweed was thawed and dried with paper towels prior to beinghomogenized in an aluminium blender (4 L) using 1-3 ml of methanol per gram seaweed(wet weight). The homogenized samples were either placed in a large plastic containerand/or a large glass column prior to being extracted with methanol. Steeping the seaweedin methanol in a large plastic container for 1-2 days usually resulted in a dark brownish-green methanol extract following careful decantation and filtration. After repeated (3-6times) extraction ‘with methanol, all extracts were pooled together. In the glass columnmethod, methanol was passed continuously through the sample and the eluate wascollected. Up to 23 litres of methanol extract was normally collected. Most extractionswere regarded as complete when the extracted seaweed became greenish white in color.44The combined methanol extracts were filtered and evaporated by using a rotaryevaporator. Processing of up to 23 L seaweed extract at 400 C to minimize thepossibility of arsenical decomposition was tedious and time consuming. The dark oily (reextracted) syrup that was obtained after evaporation was then partitioned between water(1 L) and cliethylether (3 x 200 ml). The dark green ether layers, often after arsenicdetermination, were not further examined. The water layer was evaporated to yield a darkbrown syrup which was dissolved/suspended in methanol (0.2 L). The mixture was thenpoured into acetone (1.2 L) and allowed to stand for 1 hour. The supernatant, whichcontained less polar lipid materials, was decanted and discarded. The residue wasweighed and dissolved with deionized water (500 ml). The water soluble extract was keptin Nalgene plastic bottles in a freezer pending further analysis.2.4.9 DETERMINATION OF ARSENIC IN EXTRACTSThe concentrations of arsenic in extracts were determined by using GraphiteFurnace Atomic Absorption (GFAA) and Hydride Generation Gas ChromatographyAtomic Absorption (HG-GC-AA) techniques. Because of the working concentrationrange (0.01 - 0.20 jig m11 As) of the GFAA, extracts with higher arsenic concentrationswere diluted with deionized water prior to analysis. By injecting 15 p.1 of the extract intothe graphite tube, arsenic concentration was determined directly without any furthersample treatment. In all determinations, aqueous arsenic standards were used, exceptwhen stated otherwise. Normal calibration procedure was used with triplicatedeterminations carried out on each sample. The one second integration area absorbancemode was used in most GFAA analysis.Speciation of arsenic in the extracts was carried out by using the HG-GC-AAtechnique. The extracts were usually divided into two equal portions and one of them wassubjected to the hot base digestion (Section The seaweed residues recoveredfrom the extraction procedures (sections and were hot base digested as45well. The undigested extract portion, as well as the digested extract and residue wereanalyzed for arsenic species. Arsenic compounds of known concentrations [10 ppbAs(Jll), As(V), MMAA and 20 ppb DMAA] were used as arsenic standards and theirchromatograms were compared with those of the extracts. Normal calibration procedurewas used with duplicate determinations carried out on each sample (in most cases,otherwise triplicate analysis was used). The peak area absorbance mode of the AAS wasnormally utilized with the HG-GC-AAS technique.2.5 PURIFICATION PROCEDURES FOR ARSEMC IN WATER SOLUBLEEXTRACTS2.5.1 ISOLATION OF ARSENIC CONTAINING COMPOUNDS IN WATERSOLUBLE EXTRACT OF FUCUS DISTICHUSThe F distichus collected from Head of Hastings Arm (6.77 kg wet weight,5.89 mg As) was extracted according to the extraction procedure described inSection and schematically shown in Figure 2.7. After the initial methanolextractions/filtrations, the insoluble salty materials were removed and the methanol extractobtained was evaporated to an oily residue (90.01 g syrup, 5.40 mg As). The residue waspartitioned between water (1 L) and diethylether (3 x 200 ml). The ether layer which wasanalyzed by GFAA to contain 0.28 mg As was discarded and not further investigated.The aqueous layer was evaporated, dissolved in methanol (200 ml), and the mixturepoured into 1.2 L acetone. This mixture was left undisturbed for one hour. Thesupernatant solution, analyzed to contain 1.29 mg As after decantation was discarded offwithout further analysis. The precipitate (58.70 g, 3.83 mg As), containing mainly polarsubstances, was dissolved in water and made up. to 500 ml. Further removal of anyremaining less polar lipid like materials was attempted by using water-methanol-chloroform (1:1:1) extraction. The pale green chloroform/methanol layer was analyzed tocontain 0.16 mg As and was not further investigated. The water layer was evaporated and46the residue (57.0 g) obtained was re-extracted twice in methanol to obtain an insolublematerial (29.6 g, 20 .Lg As) which was discarded off. The methanol soluble material wasevaporated and dissolved in 100 ml deionized water (31.1 g syrup, 3.65 mg As). Thewater soluble extract obtained thus, was divided into two equal portions and each wasseparately subjected to chromatographic clean up on a gel permeation Sephadex LH2Ocolumn (2.5 cm x 90 cm) using water as the mobile phase. Fractions, 8.5 ml averagevolume, were manually collected and arsenic determined in each by using GFAA.47I F. distichusISyrup r(3.65mg As)Evaporation, dissolved in lOOml WaterI (3.65mg As)Solution 1(1.29mg As)DiscardedI (3.67mg As)Evaporation, methanol re-extractionFigure 2.7Extraction Scheme for Water Soluble Arsenic speciesin F. distichus Collected From Head ofHastings Arm(Adapted/Modified from Reference 95)____________ ____________(6.77kg, 5.89mg As)Methanol extractions, filtration and evaporation(90.Olg, 5.40mg As)Ether/water partitioningtesidueEther Layer I (0.28mg As)‘I,DiscardedI Aqueous Layer I (5.12mg As)Dissolved/Suspended in methanol,poured into acetone, decantationSolid Residue I (3.83mg As)water/methanol/chloroform (1:1:1)Chloroform Layer I (0.16mg As) I Aqueous LayerI Methanol Soluble MaterialIII Water Soluble ExtractInsoluble Materials I (20j.ig As)JrDiscarded48Arsenic containing fractions eluted as one band from each gel permeationchromatographic run between 159 ml and 743 ml. All arsenic containing fractions fromeach chromatographic run were combined and evaporated to syrup. Each separate syrupwas dissolved in methanol and was rechromatographed on a Sephadex LH2O column (2.5cm x 40 cm), using methanol as the mobile phase. The arsenic cdntaining compoundseluted as a single band between 60 ml and 277 ml in the two chromatographic runs. Thearsenic containing fractions in both runs were combined and evaporated to a syrup(26.70 g, 3.11 ml As). The syrup was dissolved using 50 ml of 0.05Mtrishydroxymethylaminomethane hydrochloride (Tris) buffer (pH 8.0) and was appliedonto a weak anion exchange DEAE Sephadex A25 column (2.5 cm x 40 cm) equilibratedwith the same buffer. By using initially the same 0.05M Tris buffer (pH 8.0) solution asthe mobile phase, a single arsenic containing band was eluted off the column between179 ml and 400 ml. When the mobile phase was changed at 1016 ml to 0.5M Tris buffer,pH 8.0, a later eluting single arsenic containing band was obtained at elutions volumes1070 ml - 1476 ml. Both the fast eluting arsenic containing band (FEB) and the latereluting arsenic containing band (LEB) were collected and evaporated to syrups (27.08 g,3.01 ml As; 12.29 g, 0.20 mg As; respectively). The FEB band was dissolved in methanoland stored in the refrigerator for four days prior to further analysis. Some white materialprecipitated (2.95 g, 7.74 jig As), which was separated and discarded. Furtherpurification was attempted by using a range of chromatographic procedures. These areoutlined in Figures 2.8 and 2.9. The identification of arsenic species in the isolates wascarried out by using a range of techniques, details of which will be given in Section 2.8Flow Diagram of Chromatographic SeparationofWater Soluble Arsenic Species Extracted from F. distichus19.49g Syrup,2.90mg As, FEBA.______________Sephadex LH2O (2.5cm x 82cm; Sephadex 015 (2.5cm x 55cm;MeOH:H20, 4:1) MeOH:H20,4:1)Dissolved in 33.5m1water, Divided intoTwo PortionsI II SeeFigure2.9 I7m1FEBAJr ticCM Sephadex C25_______ __ _(2.5cm x 42cm)26.5m1 FEBA_ __ _0,1MCH3OONH4,pH6.512.58g FEBA, 2.20mg As,Not Purified Further0.05M Tris buffer, pH 8.0,Evaporation and Re-extraction inMethanol, FiltrationJEvaporation0.5M Tris buffer,pH 8.0, EvaporationSephadex G15(2.5cm x 60cm, 1120)DEAE Sephadex A25(2.5cm x 50cm,0.05M Tris buffer, pH 8.0)3.98g Syrup,0.15mg As, FEBB17.87g Syrup,2.66mg As, FEBA2.19g Syrup, 135kg As, 0.20g Syrup, 13tg As,FEBB1 FEBB2tic ticI SeeFigure2.9 I50Figure 2.9Continued Flow Diagram of Chromatographic SeparationofWater Soluble Arsenic Species Extracted from F. distichusI 3.73gFEB%1 .40g; 0.36mg As;Rt 6-10mm;FEBAI Q2O FEBB2 I2.5.2 PREPARATION OF ARSENOSUGAR STANDARDSArsenosugar standard ili (]‘able 1.2) was kindly provided by Edmonds andFrancesconi in Western Australia. The solution of arsenosugar jj was centrifuged andfiltered through 0.45 p.m membrane. This arsenosugar fl and another arsenosugar JJJ2are known to be present in NIST 1566a Oyster Tissue(17, 50). Following the procedureof Shibata and Morita(50), the NIST 1566a Oyster Tissue reference material was weighed(0.5 g) and mixed with 5-10 ml methanol:water (1:1) in a 50 nil test tube. The suspensionwas sonicated for 10-15 mins, and then centrifuged. The supernatant solution waspipetted into a round bottom flask. The extraction procedure was repeated five times.I I 2.19gFBB1 ICellulose tic (200 x 200 x 0.5mm; n-butanol:acetic acid:water,O.85g; 0.14mg As;_____________R 0.13-0.53; FEBB11.5mg; 8.12tg AsRt25-41 mm,FEBB1(b)2.32g; 0.40mg As;Rf 0.19 -0.44; FEBAPreparative HPLC, C18 Column(Prep Nova C18 HR6OA, 6pm;Water:MeOH, 4:1; 5m1 miii’)0.70g; 0.13mg AsRt 5-17 mm,FEBB1(a)Fractions, NotEvaporated51The soluble extracts were combined, evaporated, and the solid residue was dissolved in10 ml of water. The solution was centrifuged and filtered through a 0.45 I.Lm membraneprior to HPLC analysis.2.6 ANAEROBIC DECOMPOSiTION OF BROWN ALGAE F. DJSTICHUSMicrobial degradation of arsenicals present in F. distkhus collected from WreckBeach, Vancouver Point Grey was studied in a closed and open anaerobic environment.Both systems involved the use of seawater, sediment and seaweed from the same location.A control experiment, without the sediment, was carried out consecutively with the closedexperiment.2.6.1 SEAWEED/SEAWATER/SEDIMENT (SSS) Closed SystemF. distichus (715.85 g wet weight), collected at low tide on March 15, 1992 fromPoint Grey Wreck Beach Vancouver, was placed in a large plastic container together with3 liters of unfiltered seawater and dark sediment (—628 g) obtained in the same location.The container was capped closed and parafilm was used to enhance air tightness. Themixture was vigorously shaken in the dark for 11 days. During this period gas wasreleased, probably hydrogen sulfide. On day 11, the cap was opened under a continuousflow of argon to release the built up gas, and the cap replaced. Each day, for the next 30days, the cap was opened and closed under a continuous flow of argon to removeaccumulated gas due to the anaerobic decomposition process. The container contentswere decanted/filtered after 41 days to separate the seaweed/sediment residue from theliquid filtrate. The1residue (1.34 kg wet weight) was extracted (four times) into methanoland the methanol extract obtained evaporated to a dark brown residue. The brownresidue (28.58 g) was dissolved in water (150 ml). This water soluble extract waspartitioned with diethylether (3 x 150 ml) to remove fatty material. The residue from the52ether layer (0.83 g, 0.90 jig As) was discarded. The aqueous layer was extracted withphenol-ether mixture (3 times). The water layer was evaporated and the residue (27.22 g)obtained re-extracted in methanol removing 18 g of insoluble material. The methanolsoluble extract (125 ml) was kept until further analysis. As well, the dark brownsupernatant filtrate was collected and evaporated. The residue (89 g) obtained wasextracted (six times) in methanol and the insoluble salty material (52.77 g) discarded. Themethanol filtrate extract was evaporated to a syrup which was re-extracted (six times) inmethanol, ifitered and the insoluble brown residue (9.44 g, 0.95 jig As) was discarded.The methanol soluble filtrate (125 ml) was kept until further analysis.A similar experiment involving only seaweed (70.40 g) and unfiltered seawaterobtained from the same source, served as the control. The procedure outlined above wasrepeated to afford a milky brown supernatant solution (450 ml) and the seaweed residue(58.62 g). The seaweed residue was extracted (3 times) with methanol, and afterevaporation, the extract residue (3.85 g) was kept until further analysis. The filtrate fromthe control experiment was evaporated to afford a greenish brown syrup (15.49 g). Thesyrup was extracted with methanol (3 times), filtered and the insoluble material (7.90 g)was separated from the methanol soluble filtrate (9.23 g brown syrup). Open SystemThe procedure employed here has been previously(28, 190) utilized for theisolation of dimethylarsinylethanol (DMAE) from the anaerobic decomposition productsof a brown kelp, Ecklonia Radiata. F. distichus (1.03 kg) was collected fresh fromWreck Beach, Vancouver Point Grey at low tide on October 17, 1992. The seaweed wasincubated in the dark at room temperature with 2 L unfiltered seawater and dark sediment(0.26 kg) in a sealed plastic container wrapped in aluminium foil for 11 days. Gas,smelling like hydrogen sulfide, was produced during this period as well as subsequently.The cap was opened under a continuous flow of inert argon gas which was maintained for53another 30 days. At the end of 41 days, the smelly mixture was decanted/filtered in orderto separate the seaweed/sediment residue from the liquid filtrate. The filtrate afterevaporation (56.21 g) and the seaweed/sediment residue were separately extracted(3 times) with methanol and the methanol soluble extracts evaporated. The filtratemethanol soluble (33.88 g) and the seaweed/sediment methanol soluble (29.52 g) extractswere kept while the methanol insoluble material was discarded without further analysis.Each of the extracts was subjected to phenol/ether/water partitioning as in the previousexperiment. The water soluble layers were evaporated and their residues, namely filtrate(30.22 g) and seaweed (21.49 g) were dissolved in water as water soluble extracts whichwere kept until further analysis.2.6.2 EXAMINATION OF ARSENICALS IN THE ANAEROBICDECOMPOSITION PRODUCTSThe various water soluble extracts were repeatedly (twice except otherwise stated)fractionated on a gel permeation chromatographic column (either Sephadex LH2O orSephadex G15; methanol and/or water served as eluants). The fractions, water solublefiltrate and water soluble seaweed extracts obtained from the “open” experiment ( separately chromatographed on a Sephadex G15 column (2.5 cm x 60 cm) usingwater as the mobile phase. The water soluble filtrate extract separated into two arseniccontaining bands at elution volumes (i) 110-408 ml and (ii) 573-964 ml. The watersoluble seaweed extract also separated into two arsenic containing bands at elutionvolumes (i) 104-401 ml and (ii) 550-760 ml.The methanol soluble extract (123 ml) from the seaweed/sediment residue of theclosedexperiment was chromatographed using a Sephadex LH2O column (2.5 cm x 80cm) and methanol “served as the mobile phase. A brown and a green band were visuallyobserved on the column, however, only the brown, early eluting band which was analyzedto contain arsenic was kept for further analysis and the green one, without arsenic, was54discarded. The arsenic containing band after evaporation (8.63 g) was rechromatographed on Sephadex LH2O (2.5 cm X 85 cm methanol as eluant) and a singlearsenic containing band was observed whose color darkened progressively as it wascollected. This single band was arbitrarily divided into two fractions on the basis of color,namely an early eluting light brown band (1.18 g) and a later eluting dark brown band(3.82 g). Both bands were subjected to cellulose TLC chromatography on 200 x 200 x0.1 tIc plates with n-butanol:acetic acid:water, 60:15:25 serving as mobile phase. Theearly eluting light brown band revealed two arsenic containing proffles at retention flowvalues (i) Rf 0.18-0.24 and (ii) Rf 0.48-0.74. The later eluting dark brown band revealedtwo arsenic containing profiles as well at retention flow values (i) Rf 0.18-0.21 and (ii) R10.58- SYNTHESIS OF 2-DIMETHYLARSINYLETHANOLlododimethylarsine was prepared by passing sulfur dioxide into a solutioncontaining 5 g of cacodylic acid, 10 g of potassium iodide, 3.3 g of concentrated sulfuricacid and 41.1 ml of water(213, 214). The yellow liquid iododimethylarsine (9.41 g, 0.041mol) obtained was added dropwise to a stirred, cooled (2°C) solution of sodium hydroxide(4.2 ml of 1OM, 0.042 mol) under nitrogen. [This and subsequent procedures employedhere were modifications of the method of Wigren(213, 214) established byFrancesconi(28) for the synthesis of dimethylarsinylethanol.] The yellow color ofiododimethylarsine was quickly discharged. After stirring for 5 minutes,bis(dimethylarsenic)oxide, (Me2As)0was formed. Addition of 1OM sodium hydroxide(4.2 ml, 0.042 mol) and bromoethanol (5.04 g, 0.40 mol) to the bis(dimethylarsenic)oxideresulted in an heterogeneous mixture which was stirred and heated (80° C) for 2.5hours(215). The solution was cooled, neutralized with hydrochloric acid and made up to100 ml in water. The aqueous solution was subjected to phenol/ether extraction and thewater layer was separated, filtered, and evaporated (18.87 g white syrup). The syrup was55characterized as dimethylarsinylethanol by using Desorption Chemical Ionization MassSpectrometry (DCIMS), Proton (‘H) and Carbon-13 (‘3C) Nuclear Magnetic Resonance(NMR) spectrometry. Ammonia was used as reagent gas for the DCIMS analysis,scanning every one second for twenty minutes prior to production of sample’s spectrumshown later in Section The ‘H NMR spectrum was recorded on a Brukerinstrument at 200 MHz with the sample in 1)20. The UC NMR Spectrum was recordedon a Bruker instrument at 50 MHz with the sample in 1)20. The ‘H and UC spectra areshown later in Section AND DISCUSSION - ANALYTICAL PROCEDURES3.1 ARSENIC DETERMINATIONThe determination of arsenic was achieved by using a number of analyticalmethods including Graphite Furnace Atomic Absorption (GFAA), Hydride GenerationAtomic Absorption (HGAA), and Hydride Generation Gas-Chromatrography AtomicAbsorption (HGGCAA).3.1.1 ARSENIC DETERMINATIONBY GFAAGFAA is an electro-thermal atomic absorption spectrometric technique in whichatomization of samples is achieved in high temperature furnaces, typically made fromgraphite for atomic absorption measurements. When the GFAA is used, a highconcentration of atoms is generated in a small volume (graphite tube) placed inside acontrolled atmosphere provided by an inert gas, argon. As a result, a higher atomizationefficiency, greater sensitivities and element specific ultra trace analysis is achieved, withbetter improvement in detection limits for many metals, typically 10 - 100 times over theclassical flame atomic absorption spectrometry.The technique of Graphite Furnace Atomic Absorption Spectrometry has beenwidely used in the determination of arsenic in various biological and environmental samplematrices(27, 65, 69-78, 216). The sampling procedure involves deposition of microvolumes, typically 5 - 70 l sample solution commonly, on the inner wall of the graphitetube furnace that has been carefI.illy aligned in the light-path of the respective analytehollow-cathode lamp. The furnace is electrically heated in several time-and-temperatureprogranmied steps in an inert atmosphere, usually provided by argon sweeping, leading toatomization(217).’ The instrument temperature is set around the boiling point of thesolvent for the drying step during which the sample is desolvated as quickly as possible.The drying step is followed by a decomposition or ashing step which serves to remove all57organic and anionic components from the investigated arsenical. A maximumdecomposition temperature (350 - 1500°C) chosen so as not to cause a loss of atoms byvolatilization, is employed for this step. The final step is the high-temperature atomization(2000 - 3000°C), carried out by fast vaporization and dissociation of the arsines intoneutral atoms capable of absorbing the resonance radiation emitted by the correspondinghollow cathode. The atomic absorption signal is then recorded by the spectrometer as apeak height or area. The absorbance recorded is directly related to the amount of analytepresent in the sample(27, 216, 217). Finally, the graphite tube furnace is subjected to ahigh-temperature cleaning step in order to prevent memory effects during subsequentdeterminations. Facilities for the programming of both the sampler and furnaceparameters are present on the Varian GTA-95 model used in this study.3.1.2 CHEMICAL MODIFICATION AND GFAA DETERMINATION OFARSENICThe composition of a sample is often complex. Therefore, during electro-thermalatomization of arsenic by graphite furnace, the atomic absorption signal can be severelyaffected by an incomplete isolation of the analyte signal from that of the contaminants inthe sample (spectral interferences), and also by non-spectral interferences caused bychanges in the atomic concentration being measured. In dealing with non-spectralinterferences the phase at which the particular interference occurs must be determined.These include sample transportation, solute volatilization, vapor phase and spatialdistribution interferences(217-220). Automatic syringe deposition of microlitre samplevolumes is the most common method of sample introduction or transportation intographite tube furnaces (e.g. the instrument used in this study). For this reason, transportinterferences are unlikely to affect the amount of desolvated sample entering the atomizerunless the sample is deposited unevenly due to faults in the automatic syringe system andits delivery setting. Formation of carbide or intercalation compounds with graphite(2 18)58are suspected responsible for incomplete release of the analyte elements in furnaceatomization. Depending on the graphite tube used, solute volatilization interferences arenot of much importance(219) during the GFAA analysis of arsenic. On the other hand,formation ofvolatile compounds of the analyte, i.e. with arsenic, is mainly responsible forlosses during thermal pre treatment of the sample. The spatial distribution interferencesobtained due to the physicochemical properties of the graphite surface(217, 219) can alsoinfluence the time dependent sample release from the wall of the pulse-oriented graphitefurnace atomizer(217) and this can in turn affect the resulting transient atomic population.However, pulse-operated atomizers are featured with programmable heating capabilitieswhich enable optimization of drying, ashing and atomization parameters unlike in constanttemperature thrnaces(218) resulting in shorter residence times experienced by theatomized species along the length of the absorption zone. At the atomization temperatureof the sample, its volatilization should be selective and not perturbed by matrixconstituents leading to better sensitivities within an environmental condition virtually freefrom interferences(217). Since the efficient generation of dense atomic populations infurnaces is accompanied by equally efficient production of interfering (molecularabsorption and light scattering spectra interferences) species(2 18), atomic absorptionmeasurements in furnace atomizers are intensely plagued by background and matrixinterferences. The background interference which is caused by the alteration or absorptionof the analytical line by species other than the analyte, if uncorrected will lead to erroneousanalyte absorbances. The background correction is usually achieved with backgroundcorrectors (deuterium or zeeman) equipped with the atomic absorption instrument. TheGFAA instrument used in this study is equipped with a continuous background correctorprovided by a hollow cathode deuterium arc lamp.The matrix type spectral interferences on the other hand are due to chemicalprocesses and or reactions of matrix components with the analyte to form gaseousmolecules (halides or oxides of the analyte)(l 8) which are not detected by the AA, and59therefore, cause loss of analyte signal. To avoid matrix interferences, many prerequisitesmust be met. These include accurate and reproducible furnace heating, correct furnacetubes (usually pyrolytically coated graphite tubes), optimized heating parameters and, inmost cases, the use of chemical modifiers(221-230). Chemical modification is a term usedto describe a means of chemically altering the sample matrix in order to change thevolatility of the analyte element or the bulk matrix constituents(79). The use of effectivechemical modifiers allows for the use of higher ash temperatures and prevention of lowtemperature loss of analyte metals as molecular species by forming intermetalliccompounds(221-230) with the analyte, which remains stable until high temperatures arereached. In the use of aqueous standards the optimum ash temperature for arsenic is600°C, however, a higher ash temperature(27) of up to 1400°C is necessary for theseaweed extracts because of their complex matrices and the type of mobile phases usedduring chromatographic purification(28, 95). Nickel Modifier Optimization for Arsenic Determination by GFAAThe use of nickel for chemical modification has been well studied(208, 221-224, 228).The experimental approach(222) utilized in the present study was based on recording ofthe absorbance signals with respect to the analyte (arsenic), the modifier (nickel) and thematrix(221) in which the analyte is being investigated. The matrix being investigated fornickel modification in this work is the tris (hydroxymethyl)aminomethane hydrochloridebuffer (Tris buffer) employed as mobile phase during anion exchange chromatographicprocedures(95). It was observed that arsenic signals were suppressed(227) and/orcompletely lost from chromatographic fractions in which Tris buffer(0.5M pH 8.0)(28, 95) was used as mobile phase during GFAA analysis. During theGFAA. analysis, the optimized(27) operating conditions used are shown in Table 2.4. Thequenching of the arsenic signal with large concentrations of Tris buffer has been observedpreviously(28, 95) when the modifier used is lOOppm Ni2 solution. Even though large60excesses of most modifiers cause depression of the analyte signal and shouldbeavoided(222), other workers(28, 95) have utilized 10,000 ppm Ni2 solution to estimatearsenic in their chromatographic fractions. They claimed reasonable sensitivity for arsenicby using GFA.A in the presence of 0.5M Tris buffer and the large concentration of nickelmodifier. In the present study however, when various concentrations of Ni2 modifierwere investigated for the matrix modification of I ppm and 10 ppm arsenic in 0.5M Trisbuffer (pH 8.0), using the furnace parameters optimized(27) for arsenic in aqueoussolution, the average absorbances from triplicate determinations shown in Table 3.1 andplotted in Figure 3.0 were obtained. The poor or “quenched” arsenic signals are seen atall modifier concentrations with the lppm As standard. Higher concentrations of modifier(>2,000 ppmNi2j, resulted in a signal improvement for 10 ppm As. In this study, arsenicstandards below lppm concentration spiked into 0.5M Tris buffer produced much smokeand negative absorbance readings, probably due to over correction by the backgroundcorrector.Table 3.1Arsenic Absorbance Determinations in 0.5M Tris buffer, pH 8.0With Varying Ni2 Modifier Concentrations(20 id injection of standard, blank and modifier respectively)NiCl2Modifier Absorbance Determination for 0.5MConcentration Tris buffer spiked with As(III) standard(ppmNi2) Blank I ppm As 10 ppm As100 0.004 0.000 0.009500 0.000 0.005 0.0201000 0.003 0.000 0.0132000 0.000 0.005 0.018‘ 5000 0.000 0.009 0.04810000 0.003 0.015 0.10861I0. 3.0Ni2Modifier Optimization for Arsenicin Tris buffer Solution by GFAA AnalysisWhen these results are compared to equivalent arsenic determinations in aqueous solution(without Tris buffer as a solvent matrix for As standard concentrations), the absorbancesignals obtained are greatly suppressed for 1 ppm As solution. At large excesses of theNi2 modifier (e.g. 10,000 ppmNi2), the 1 ppm As signal is less than 10% of its normalarsenic absorbance in simple aqueous matrix, and the 10 ppm As concentration is barely25% of its normal’arsenic absorbance signal. In addition to these significantly suppressedarsenic signals, much smoke emission during atomization, as well as memory effect duringrepeated determinations, were experienced. High concentrations ofNi2 have been found0 2500 5000 7500 10000ppm Ni2+62to cause memory effects during GFAA analysis(230). Others(28, 95) have observed thatwith known amounts of arsenic standards, or high concentrations of arsenic in realsamples, significant dilutions of the matrix improved arsenic responses in thepresence ofhigher concentration of Ni modifier. However, in dealing with real samples of seaweed,its chromatographic fractions from extract with unknown (possibly smaller) concentrationsof arsenic, the tris buffer matrix is a potential problem. This is because the “quenching” ofarsenic signal by the buffer may result in a false total arsenic recovery during GFA.Aanalysis. The usefulness of dilution may be limited by the calibration detection limit.Therefore, it can be more advantageous to use the “more universal modifiers”(222) suchas Pd, Mg, noble metals, Ce, La, Ht, W, and Zr in controlling higher levels of backgroundabsorption. Palladium Modifier Optimization for Arsenic Determination by GFAAThere has been much interest in matrix modification over the past twodecades(20 1, 222, 226) with extensive literature on the application ofNi(N03)2modifierto As and Se analysis. However, in more recent years, 1980-89, increased interest inmatrix modification revealed by up to 90 papers per year(222) produced new, efficient andmore universally applicable chemical modifiers. These modifiers (e.g. “reduced palladiummodifier”) possess differing modes of action which include but are not limited to thermalstabilizer, interference depressant, acid, surfactant, and antiform, and their applications tothe GFAA analysis of troublesome matrices encountered with biological, environmentaland “complex” solvent/extracts cannot be over-emphasized.The palladium modifier, unlike the nickel modifier, is reduced during GFAA to ahighly dispersed elemental form early in the temperature pre treatment stage between400 - 800° C(222). With relatively low amounts of Pd modifier therefore, highly efficientchemical modification is accomplished. In order to accomplish chemical modification withPd salts, they must be reduced to Pd metal. In this study, a solution reducing agent, 2%63citric acid which has been found effective(79), was mixed directly with the Pd solution.The optimized fi.irnace operating conditions were obtained by modifying successiveoperating steps using three criteria(229) to judge the correctness of each step in the finaltemperature program. The first criterion is the visual observation of the droplet during theinitial stages of the temperature program and this was achieved by using a 45° mirrorequipped with GTA-95 employed in the present study. This procedure has been claimedto be very useful(229) for checking whether the droplet evaporates smoothly and remainsin position during drying. The second criterion utilized for the fIrnace parameteroptimization was to observe the shape of the transient signal during the atomization step.The undistorted transmittance signal provides insight about the matrix influence and a day-to-day changes of the analyte signal. Another very important criterion was reproducibilityof the determination relative to the temperatures during the dry, ash and atomization steps.The long-term validity of the temperature program was checked by running standardarsenic samples and comparing their reproducibility. The ftirnace operating conditions(Table 2.5) obtained by the above procedures were optimized with the furnace injection of15 .il of standard sample (0.1 ppm, 1.0 ppm and 10.0 ppm As in 0.5M Tris buffer, pH 8.0)and varying concentrations (50 ppm, 100 ppm, 200 ppm and 500 ppm) of Pd in 2% citricacid (15 1.11) acting as chemical modifier during the GFAA analysis. The Pd modifier wasprepared by dissolving 0.25 g Pd powder in 8 ml concentrated nitric acid and 2 mlconcentrated hydrochloric acid, resulting in a dark brown solution. This solution wasmade to 250 ml in a volumetric flask by using deionized water to afford a 1000 ppm Pdstock solution. Serial dilution of the stock solution in 2% citric acid, freshly made, in100 ml volumetric flasks, gave the desired concentrations of Pd. The mean of triplicateabsorbance measurements were recorded by GFAA and the data are shown in Table 3.2and Figure 3.1.64Table 3.2Arsenic Absorbance Determinations in 0.5M Tris buffer, pH 8.0With Varying Pd in 2% Citric acid Modifier Concentration(15 id Injection of Arsenic Standard and Modifier Solutions Respectively)Pd in 2% Citric acid Absorbance Determination for 0.5M TrisModifier Concentration buffer Spiked with As(1II) Standard(ppmPd) 0.1 ppm As 1.OppmAs 10.0 ppm As50 0.046 0.207 0.420100 0.054 0.226 0.432200 0.024 0.180 0.358500 0.028 0.132 0.346Compared with nickel modification (Table 3.1 and Figure 3.0),the use ofPd in 2%citric acid modifier resulted in very good arsenic signals, both at low and highconcentrations of arsenic. The optimum Pd (in 2% citric acid) modifier concentration wasselected to be lOOppm and this concentration was used in all subsequent arsenicdeterminations of chromatographic factions (except where otherwise indicated) with theGFAA analysis.65Figure 3.1Optimization of Arsenic Signal with Pd in 2% Citric acid Modifier by GFAA0.60 —!. 0.300.200.10 —0.1 ppm As0.00— I I I I I0 100 200 300 400 500 600(Pd) in 2% Citric acid, pm3.1.2.3 Determination of Some Arsenic Species Signals in 0.5M Tris buffer by GFAAAnalysisSolutions (0.1 ppm) of arsenite (Asill), arsenate (AsV), mono-methylarsonic acid(MMAA), dimethylarsinic acid (DMAA), arsenocholine, and tetramethylarsonium iodidewere prepared by using serial dilution of respective stock standard solutions in 0.5M Trisbuffer, pH 8.0. Each of these arsenic standards was analyzed by using GFAA when Ni2(100 ppm) or Pd in 2 % citric acid (100 ppm) served as modifier. The ifirnace programshown in Table 2.5 was utilized. In this analysis, 15 il of standard arsenic solution, aswell as 15 of the modifier solution were injected into the graphite furnace. Meanarsenic responses obtained from triplicate determinations were recorded and the resultsobtained using Pd in 2% citric acid (100 ppm) are shown in Table 3.3.66Table 3.3Arsenic Species Response during GFAA 100 ppm Pdin 2% Citric acid Used for Matrix Modificationof 0.5M Tris buffer0.1 ppm As in Mean Peak Area Absorbance of0.5M Tris buffer As Determination by GFAAArsenic Species 100 ppm Pd in 2% Citric acid ModifierArsenite, As(III) 0.379 ± 0.027Arsenate, As(V) 0.343 ± 0.023Monomethylarsonic acid (MMAA)0.418 ± 0.024Dimethylarsinic acid(DMAA) 0.383±0.029Arsenocholine, (CH3AsCH2COHX0.265 ±_0.008Tetramethylarsonium iodide, (CH3)4Asi0.266 ±_0.003Note: Error limits are absolute error in each measurementWhen Ni2 solution (100 ppm) served as the matrix modifier during the analysis of0.1 ppm As in 0.5M Tris buffer, negative absorbances were obtained for all the arsenicspecies. This may be due to the inability of nickel to retain the analyte through the highashing temperature (1400° C) and the subsequently very high atomization temperature(2400° C). In addition, possible over correction by the deuterium background correctormay also be responsible for the negative absorbances.With Pd in 2% citric acid modifier (100 ppm), the analyte arsenic was probablyretained as stable species (“As-Pd”) through the high ashing (1400° C) temperature to thevery high (2400° C) atomization temperature. This served to isolate/remove the Trisbuffer matrix and other organic contaminants and the result is a useable arsenic signaldetermined by th GFAA for each arsenic species. The measured responses are quiteprecise when compared between nearly all the arsenic species.* 673.1.2.4 Calibration and Limit ofDetection of GFAA AnalysisA typical calibration graph for standard arsenite, shown in Figure 3.2, wasobtained using the furnace program shown in Table 2.5 and lOOppm Pd in 2% citric acidas matrix modifier.Figure 3.2Typical Calibration Curve for Arsenic Determination by GFAA0-os0.070.06• 0.05C,0.04.C,a0_os0. linear relationship is obtained for arsenic up to 0.10 .tg m1’. Beyond thisconcentration, curvature increases as is typically observed with atomic absorptionmeasurements(27).In Tris buffer matrix, the limit of detection (LOD) defined as the blank signal, YB’plus three standard deviations of the blank, SB (i.e. LODy8+3SB) was found to be36 nglml for a 15 il sample injection The absolute LOD was 0.54 ng As and the relativestandard deviati (RSD) for twenty GFAA readings of 0.01 .ig/ml standard arsenitesolution was calculated to be 8.0%. Similarly, in aqueous solution, the LOD wascalculated to be 46 ng/ml for a 15 p.1 solution. The absolute LOD was 0.69 ng and the0.00 0.05 0.3.0 0.15[AsJ68relative standard deviation, RSD, for twenty injection of 0.01 pg/mI standard arsenitesolution was found to be 12.1%.When compared to nickel modification of a similar matrix (simple aqueoussolution), Pd in 2% citric acid exhibits almost equivalent absolute detection limits forarsenic analysis. In aqueous solution using 100 ppm Ni2 solution as modifier, the LODwas calculated to be 31 ng/ml for a 20 p.1 solution. The absolute LOD was 0.62 ng As andthe estimate of precision in measurements, RSD for twenty injections of 0.01 p.g/mr’standard arsenite solution was found to be 3.4%. Compared with Pd in 2% citric acidmodification, the nickel modification showed better precision for arsenic in aqueousmatrix.. Table 3.4, shown below, lists the instrumental efficiency of the GFAA techniqueusing the optimised conditions for arsenic determinations, according to the results of thepresent study.Table 3.4LODs and RSDs of Arsenic Determination by GFAA TechniqueLOD of Arsenic by GFAA RSD (m)Modifier Type Aqueous Difficult Aqueous Difficultand Matrix Matrix Matrix MatrixArsenic Volume Injected ng/mI ng ng/ml ng % %100 ppm Ni2 modifier, 31 0.62 nd nd 3.4 nd20 p.1100 ppm Pd in 2% citric 46 0.69 36 0.54 12.1 8.0acid modifier, 15 p.1nd = not determinedm = 20 injections of0.01p.g/ml As (III)693.1.3 ARSENIC DETERMINATION BY CONTINUOUS HGAAAs a consequence of advantages such as separating and preconcentrating analytesfrom samples matrices, the Hydride Generation Atomic Absorption (HGAA) method hasbecome the most common technique of choice(133) for the determination of elements thatreadily form volatile hydrides. These elements include arsenic, bismuth, germanium, lead,antimony, selenium, tin and tellurium. Generally, this analytical procedure is performed byreducing the analyte of interest to its volatile hydride and subsequently sweeping thegenerated gas into the spectroscopic source, where quantitative spectrochemicalmeasurements can be taken. Early applications(130, 133, 231) of Hydride Generation(HG) which were relatively successful, although slow, made use of metal/acid asreductants. The ZnIHC1 reductant was most frequently utilized according to the equationgiven below for arsenic (As(III)).6Zn + 12HC1 + As(OH)3 6ZnC12+ AsH3 + 3H2 + 3H20Further development(133, 232) of the HGAA technique, however, produced theNaBH4/Acid reductant which has virtually replaced the metal/acid reaction. Two majorreasons for this wide acceptance ofNaBH4/Acid reduction are the possibility for greatercontrol over reaction conditions, and the potential for automation. The NaBH4 reaction isfaster than the metal/acid reaction resulting in rapid formation of the hydrides (typicalreaction periods range from 10 to 30 seconds for NaBH4/Acid reaction compared with5-15 mm for metal/acid). Two automated hydride generation systems have beendeveloped based on the use of aqueous solutions of NaBH4, namely, a peristaltic pumpsystem and the pressurized reagent pumping system(130). The peristaltic pump systemrequires less frequent analyst manipulation, whereas the pressurized reagent pumpingsystem appears to allow greater versatility with respect to reaction conditions. The use ofeither system results in increased precision and decreased analysis time. In this work, the70peristaltic pump system was utilized. The NaBH4I[{A(232) reduction with arsenite asanalyte is given in the following equation:2NaBH4+3H20+ 2HA + As(OH)3+2H3B0+ 2NaA + AsH3 + 5H2(HA = mineral acid, e.g. HC1 and/or organic acid, e.g.CH3OOH)In this continuous-HGAA procedure(27, 232) a peristaltic pump is used for the separatetransport of sample solution and NaBH4reductant solution to a junction where the hydrideformation takes place. The liquid and gaseous reaction products are mixed andtransported with a carrier (Nitrogen) gas stream to a gas liquid separator, and the gasmixture (AsH3 + 112 +N2) enters the heated quartz atomizer cuvette (see Figure 2.1). Asin this work, a continuous arsenic atom signal is observed(233). Sections 3.1.4 and 3.1.5give detailed studies of reduction selectivities(232, 234) achieved by using a mineral acidand an organic acid selected in combination with sodium borohydride (NaBH4) asreductant solution for arsenic species determination by the continuous-HGAA technique.3.1.4 ARSENIC SPECIES RESPONSE OPTIMIZATION USING A MINERALAC]]) WITH VARYING CONCENTRATIONS OF SODIUMBOROHYDRIDE REDUCTANTAccording to Braman et al(235), the reduction of arsenic compounds with NaBH4is pH dependent and is related to the pka of the individual arsenic acids. The pH andreduction products for some arsenic acids(235) are given in Table 3.5; these reduction pHvalues can be utilized in separating and identif,ring each arsenic species.71Table 3.5Reduction Conditions for Some Arsenic Species(Adapted from Reference 235)Reduction Reduction. Boiling PointMolecular Form pH ProductAs (III), arsenous acid, HAsO2 4 AsH1 -55As (V), arsenic acid,H1AsO4 1-2 AsH1 -55Methylarsonic acid,CHAsO(OH)2 1-2 CHAs2 2Dimethylarsinic acid, (CH1)AsO(OH) 1-2 (CH1)As 35.6Trimethylarsineoxide, (CHAsO 1-4 (CHAs 70Phenylarsonic acid,6H5AsO(OH)2 1.2 6H5AsH2 148The relationship between acid concentration and sensitivity has been studied(232-3 8) for avariety of acids. The mineral acid of choice was hydrochloric acid(17, 27, 237).Similarly, fixed(232, 236) and varied(228) concentrations ofNaBH4 reductant have beeninvestigated to determine optimum arsenic species signal responses. The arsenic speciesresponse profiles obtained in some of these studies(232, 234, 238) differ significantly. Thereasons for the differences are not known(232), although kinetic factors or mixingdynamics were suggested as possibly responsible.In this study, response profiles were generated for four arsenic species, namelyarsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA) anddimethylarsinic acid (DMAA) during reduction by fixed concentration (%) of NaBH4(w/v) and varying hydrochloric acid concentrations. Solutions of arsenic speciescontaining a constant concentration of arsenic (0.05 .tg/ml As) were prepared. Each7 arsenic test solution was analysed by using continuous HGAA. Reduction with a fixedamount of NaBH4 reductant (0.5, 1.0, 1.5, 2.0 and 2.5%) and varying concentrations(2M - 6M) ofhydrochloric acid was studied.723.1.4.1 Determination of Arsenic Species Response Profiles in 0.5% NaBH4 ReductantSolution and Varying Concentrations ofHydrochloric acid by Continuous HGAAThe responses generated from each 0.05 tg/ml As solution of As(III), As(V),MMAA and DMAA in hydrochloric acid using 0.5% NaBH4 as reductant are shown as afbnction of acid concentration in Figure 3.3Figure 3.3Effect of Hydrochloric Acid Concentration and 0.5% NaBH4as Reductant on the Response of Four Arsenic Speciesby Continuous HGAA Analysis0.03 -As(Ill)0.020.01 —___ _____°MAA— T I J I I1 2 3 4 5 6 7HC1 (M)Arsenite [As(III)], arsenate [As(V)], MMAA signal responses increased rapidly and havemaximum in the range of 3M - SM HCI acid. The response of DMAA is significantlylower compared to the other arsenic species. At concentrations greater than 3M HCI acid,the DMAA response declined gradually toward zero absorbance. Similar behavioursexhibited by these arsenic species have been observed by others(232, 238).733.1.4.2 Determination of Arsenic Species Response Profiles in 1.0% NaBH4 Reductant•— 0.02010).0Solution and Varying Concentrations ofHydrochloric acid by Continuous HGAAThe response profiles of 0.05 As solutions of As (III), As (V), MMAA andDMAA in hydrochloric acid using 1.0% NaBH4 as reductant are shown as a function ofthe acid concentration in Figure 3.4.Figure 3.4Effect of Hydrochloric Acid Concentration and 1.0% NaBH4as Reductant on the Response of Four Arsenic Speciesby Continuous HGAA Analysis0.08 —M4AAsffl)0.01 —0.001 2 3 4 5 6 7HCI (M)With 1.0% NaBH4 as reductant, the responses of all arsenic species increasedcompared to the 1profile obtained in 0.5% NaBH4. Beyond 3M acid concentration, agradual decrease (except for DMAA with a sharp decline) in response which appeared to74level off from 5M acid concentration was observed. This is also consistent with similarobservation by other workers(232, 234). Determination of Arsenic Species Response Profiles in 1.5% NaBH4 ReductanjSolution and Varying Concentrations ofHydrochloric acid by Continuous HGAAResponse profiles of 0.05 ig/ml As solutions of As(III), As(V), MIvIAA andDMAA in HCI acid solutions with 1.5% NaBH4 reductant solution are shown inFigure 3.5.Figure 3.5Effect of hydrochloric Acid Concentration and 1.5% NaBH4as Reductaiit n the Response of Four Arsenic Speciesby Continuous HGAA Analysis0.06As(V)0.04As(JI1)MMAA-4—0.00I I I.’1 2 8 4 6 6 7I [IIG1] MThe reductant solution of 1.5% NaBH4 resulted in a significantly higher arsenicresponse for all species. Beyond 2M acid concentration, DMAA response initially75decreased, followed by a relatively constant response between 3M and SM acidconcentrations and, finally decreased toward zero absorbance after 5M acid. The otherarsenic species [As(III), As(V), MMAA], exhibited increased signal responses from 3Macid, which levelled off at higher acid concentrations. These results are in agreement withthe responses of arsenic species determined in the presence of a reaction or mixingcoil(232, 234, 238) which suggests the kinetic relevance(232) for arsine formation duringHGAA analysis. The 4M hydrochloric acid concentration was observed to be an optimumacid medium for the HGAA analysis of the combined arsenic species. Determination of Arsenic Species Response Profiles in 2.0% NaBH4 ReductantSolution and Varying Concentrations ofHydrochloric acid by Continuous HGAAFigure 3.6 gives the response profiles obtained for the four arsenic species undersimilar conditions described in the above sections./76[II1] MThe response profiles of all arsenic species are as good in 2.0% NaBH4/IIC1 acidmedia as in the 1.5% NaBH4/acid media in The response of DMAA decreasedrapidly after 3M acid, levelling off beyond 5M acid concentration. The other arsenicspecies showed increases in their responses, reaching an optimum at 4M acidconcentration, followed by a gradual relative decrease.. Determination of Arsenic Species Response Profiles in 2.5% NaBH4 ReductantSolution and Varying Concentrations ofHydrochloric acid by Continuous HGAAThe reduction of the four arsenic species in 2.5%NaBH4IHC1 media generated theresponse profiles shown in Figure 3.7 below.Figure 3.6Effect ofHydrochloric Acid Concentration and 2.0% NaBH4as Reductant on the Response ofFour Arsenic Speciesby Continuous HGAA Analysis0.0.4 -0.08 —0.02 —0.01 —0.00-frn--A. (III)A.(V)MAAIMAA1 2 8 4 5 6 .7I77Figure 3.7Effect ofHydrochloric Acid Concentration and 2.5% NaBH4as Reductant on the Response of Four Arsenic Speciesby Continuous HGAA Analysis0.05 —• A.(III)- • A.(V)MMAAI DMAA0.04 —0.08 -0.02 -0.01 —0.00— I I I I1 2 3 4 5 6 7[HC1] M:The sensitivity of all the arsenic species in this medium was relatively good.Similar response profiles as earlier discussed ( -, were observed. However,there was no observed general decrease in absorbances at higher acid concentrationsexcept for DMAA. Effect of sodium borohydride Concentration on Arsine Yield Using 0.05 uWmiArsenic Solution and 4M hydrochloric acidThe absorbance responses due to 0.05 j.ig/ml arsenic solutions of As(III), As(V),MMAA and DMAA. were determined and plotted as a function of sodium borohydrideconcentration as shown in Figure 3.8.781.5% NaBH4 reductant concentration was determined as optimum concentrationfor the selective reduction of arsenic species in 4M hydrochloric acid.Figure 3.8Plot ofAbsorbance Vs [NaBH4]% (wlv)0.05 —___________________________________—-- A•—— B—.-— C—0-— D0.04 —0.03 —C-,- I I I I0.0 0.5 1.0 1.5 2.0 2.5 3.0WaBI14 % (W/V)In conclusion, the selective reduction of four arsenic species has been described atvarying acid concentrations and with specific amounts of the NaBH4 reductant. Since aperistaltic pumping system combined with a mixing coil (see Figure 2.1) was employed forthis work, the results obtained as described were compared with other studies(232, 234,236, 238). Except for slight differences as noted therein, the present results are inagreement with these other workers. The optimum reductant concentrations (1.5%NaBH4 and 4M’ HC1 acid) were determined for the present study after the selectivereduction analysis.79Whereas Figure 3.3 gave response profiles which appeared to suggest that all thearsenic species signals gradually decreased, probably to zero absorbance (cf also DMAAresponse profile in Figure 3.5); the others, Figures 3.4 - 3.7, suggested response profileswith arsenic responses levelling off at increased acid concentrations. The optimum sodiumborohydride and hydrochloric acid concentrations selected for this work, 1.5% and 4Mrespectively (see Figure 3.8), took into consideration relatively good sensitivity for eacharsenic species analyzed and the fact that real samples, especially seaweed extractsanalysed in this work, contain competing interfering metal ions which can suppress(232)arsenic signals when they are selectively reduced (as well) by the reductant media. Theuse of 1.5% NaBH4 and 4M HCI solutions with HGAA for arsenic determination wasconsidered metal-ions interference free optimum conditions, as observed by otherworkers(232).3.1.5 ARSENIC SPECIES RESPONSE OPTIMIZATION USING ANORGANIC ACID WITH VARYING CONCENTRATIONS OFSODIUM BOROHYDRIDE REDUCTANTAn organic acid can also provide a (complimentary) pH reaction matrix for thedetermination of arsenite, As(III); arsenate, As(V); MMAA and DMAA using continuoushydride generation with sodium borohydride as reductant and atomic absorptionspectrometry for detection(232). As noted in the previous section (3.1.4), DMA.Aresponse was not determined at optimum sensitivity. This is common for DMAAdetermination when using any mineral acid media(232) for selective reduction. However,it has been shown(232, 238) that the response of DMAA in an organic acid/NaBH4reductant matrix when analysed by the continuous HGAA is efficiently and/or optimallydetermined. Anderson, et al(232), using 1% NaBH4 (w/v) in 0.1 M sodium hydroxidesolution, and varying concentrations of organic acids (oxalic acid, citric acid, tartaric acid,acetic acid and mercaptoacetic acid) determined the response profiles of As(III), As(V),80MMAA and DMAA arsenic (0.1 tg/ml) solutions by continuous HGAA. Both arsenite[As(ffl)] and DMAA produced large signals in each of oxalic, citric and acetic acid mediawhereas, arsenate [As(V)J and MMAA responded poorly. Furthermore, all arsenicspecies analysed produced similar peak responses in mercaptoacetic acid, which suggeststhat this organic acid is a potential valuable acid medium for total arsenic determination.The only drawback though, is that the procedure cannot be used to separate any of thearsenic species when present in the same sample. The study(232) also found that aceticacid media allowed the generation of arsines from both DMA.A and As(llI) to almostidentical extents over the entire acid concentration range studied. Furthermore, theresponse for As(V) was found to be a low and broad plateau whereas MMAA signal eventhough not significantly good, showed a steady increase over the entire acetic acidconcentration range. Therefore, acetic acid media provides an excellent complimentarypH matrix to hydrochloric acid media in effecting the total estimation and separation of allfour arsenic species using selective reduction by the continuous HGAA and NaBH4serving as reductant. Determination of Arsenic Species Response Profiles in 1% NaBH4 ReductantSolution and Varying Concentrations ofAcetic Acid by Continuous HGAAThe response profiles of 0.05 tg/ml As solutions of As(ffl), As(V), MMAA andDMAA in acetic acid using 1.0% NaBH4 as reductant are shown in Figure 3.9 as afunction of the acetic acid concentration (2M - 6M).81Figure 3.9Effect ofAcetic Acid Concentration and 1.0% NaBII4as Reductant on the Response of FourArsenic Species by Continuous HGAA Analysis‘ I I I1 2 3 4‘ I I5 8 7[Ac] MIn this reductant medium (1.0%NaBH4/acetic acid) a gradual increase in responsefor all the arsenic species was observed up to 5M acid, at which there was a sharp droptoward zero absorbance. Determination of Arsenic Species Response Profiles in 1.5% NaBH4 ReductantSolution and Varying Concentrations ofAcetic Acid by Continuous HGAAThe effect of acetic acid concentration and 1.5% NaBH4 (w/v) reductant media onthe responses of ech 0.05 jig/mi As solutions of As(III), As(V), MN{AA and DMAA areshown in Figure 3.10 as a function of acid concentrations.— AS(III)— AS(V)—.-— MMAA—4— DMAA-a4,C..I.. —-0.0182Figure 3.10Effect of Acetic Acid Concentration and 15% NaBH4as Reductant on the Response of FourArsenic Species by Continuous HGAA Analysis0.03 ——t----— A(III)—-—— As(V)—-— MMAA—4— DMAA0.00•— I I I1 2 3 4 5 6 7[HAd MIn this reductant medium (1.5% NaBH4/acetic acid), a gradual increase in the responsewas observed for DMAA and As(III) up to 3M and 4M respectively; beyond this, a steadydecline was recorded. Both arsenate and MMAA showed marginal responses, with theformer being the poorest.-833.1.5.3 Determination of Arsenic Species Response Profiles in 2.0% NaBH4 ReductantC)Solution and Varying Concentrations ofAcetic Acid by Continuous HGAAThe response profiles of 0.05 jig/mi As solutions of As(JII), As(V), MMAA andDMAA using 2.0%NaBH4/acetic acid media are shown in Figure 3.11, also as a fbnctionof acid concentration.Figure 3.11Effect of Acetic Acid Concentration and 2.0% NaBH4as Reductant on the Response of FourArsenic Species by Continuous HGAA Analysis——— AS(III)—---- MMAA—4---- DMAA0.040.03 —0.02 —0.01 —0.005 61. 2 3 4 7[IA.ej MThis reductant medium (2.0% NaBH4/acetic acid), shows a relatively bettersensitivity in response for all the arsenic species, particularly at 2M acid. Beyond this7 concentration, a general decline in response was observed.843.1.5.4 Determination of Arsenic Species Response Profiles in 2.5% NaBH4 ReductantSolution and Varying Concentrations ofAcetic Acid by Continuous HGAAFinally, the response profiles of the four arsenic solutions described in previoussections were determined in 2.5% NaBH4/acetic acid media as shown in Figure 3.12plotted similarly as a function of the acetic acid concentrations.Figure 3.12Effect ofAcetic Acid Concentration and 2.5% NaBH4as Reductant on the Response of FourArsenic Species by Continuous HGAA Analysis0. reductant medium (2.5% NaBH4/acetic acid),shows a relatively bettersinsitivity in response for all the arsenic species at 2M acid. And a relative general declinein response was oberved at other acid concentrations.In conclusion, the 2M acetic acidfNaBH4reductant media were observed to give inmost cases, optimum arsenic responses for the arsenic species analyzed by the HGAAprocedure. This conclusion is consistent with the results of Anderson et al(232).2 3 4 5 6[MAc] M85However, even though these workers(232) obtained good responses for DMAA andarsenite and poor responses for MtvIAA and arsenate in the concentration range of 0 - 2Macetic acid, fbrther investigations to show the effect of this acid on the arsenic speciessignals were not carried out beyond 2M acid. The workers(232), in addition, observedand concluded that the good responses ofDMAA and arsenite reached a constant plateaubetween the concentration range 0.5 -2. OM acetic acid. In the present study, it wasobserved that the arsenic responses fall rapidly at higher acetic concentrations. The 2Macetic acid, in agreement with the previous work(232), appears to be the highest optimumacid concentration when working with the acetic acid/NaBH4reductant media for hydride(arsine) generation prior to analysis by using the continuous HGAA technique. Effect of Sodium Borohydride Concentration on Arsine Yield Using 0.05 mg/mF1arsenic Solutions and 2M acetic acidThe response profiles due to 0.05 jig/mi As solutions of As(III), As(V), MMAAand DMAA were plotted as a function of sodium borohydride concentration inFigure,a.0—— AB—G-— C—0— DFigure 3.13Effect of Sodium Borohydride Concentration on ArsineYield Using 0.05 .tg/m1 Arsenic Solutions and 2M Acetic Acid0.06 -__________________________________0.05 -0.04 - -0.00- I0.5 1.5NaBH4 % (w/v)According to Figure 3.13, 2.5% NaBH4 reductant concentration was determined asoptimum concentration for the selective reduction of the arsenic species in 2.OM aceticacid medium.In summary, the selective reductions of four arsenic species have been studied atvarying acetic acid concentrations (2M - 6M), and specific concentration of the NaBH4reductant between’ 1.0 - 2.5% (w/v). The optimum reduction concentration was found tobe 2.5% NaBH4 (wlv) and 2.OM acetic acid for the arsenic species analyzed by thecontinuous HGAA.2.58731.6 CALIBRATION AND LIMIT OF DETECTIONS OF THE CONTINUOUSHGAA ANALYSISA typical calibration graph for standard arsenite, obtained with 1.5% NaBH4reductant and 4M hydrochloric acid medium is given in Figure 3.14Typical Calibration Curve for Arsenic Determination bya Continuous HGAA Procedure0.010 0.020 0.030As Coxicentratioa (ug/mi) 0.040A linear relationship between absorbance and concentration is obtained up to aconcentration of. ‘0.0 15 .tg/mI. Curvature increases at higher concentrations above0.020 pg/ml, which is typical of atomic absorption measurements.The limit of detection, LOD, defined as the analyte concentration giving a responseequal to the blank plus three standard devations of the blank was estimated by using the88section of the plot close to the origin. It was determined as 0.54 ng/ml As; a value farbetter than that determined by the GFAA technique reported in this study. Finally, therelative standard deviation, RSD, (an indicator of precision in measurement of the analyte)was determined by using data from 20 replicate analysis of a 1.0 ng/ml arsenite standard.It was determined to be 4.7% at this concentration.3.2 TOTAL ARSENIC DETERMINATIONIn order to determine arsenic by GFAA and HGAA, samples are required to be insolution form(239, 240) . This requirement has necessitated investigation of differentdigestion procedures(239, 241-243) and study was carried out in the present work inorder to achieve optimum arsenic determination for the seaweed digests.The decomposition of biological and environmental samples, specifically for therelease of arsenic into solution, can be achieved by wet digestion or dry ashing. Diyashing involves the decomposition of the material by combustion in air or oxygen. Bothquantitative(27, 242, 244) recoveries and high losses(27, 242, 245) of arsenic have beenreported with the use of dry ashing in biological materials. Dry ashing for arsenicdissolution is not widely recommended because, without an ashing aid(242) (e.g. amagnesium oxide/nitric acid mixture), the high temperature utilized in normal asbingprocedures will lead to loss of arsenic. Second dry ashing procedure involves lowtemperature, but a drawback is that it involves the production of an atmosphere of highlyreactive ionized oxygen produced by means of microwave induction and this apparatusrequires extra working care, expertize, and expense.Wet digestion however, has been widely utilized(27, 239-243, 246-250) and is wellrecommended(242) for arsenic analysis. Wet digestion is usually carried out by treatmentof the sample with mineral acids, peroxides, or perchlorate(27, 242) and/or base(5, 27, 40,246-250) with the application ofheat.89Three conditions(242) have been noted as important with these digestionprocedures if losses of arsenic are to be prevented during subsequent analyticaldeterminations by using GFA.A and/or HGA.A techniques. First, oxidising conditionsnecessary to prevent arsenic (III) forms from forming volatile compounds with any of F,Cl and or Br, (which can result in arsenic losses during charring) should be employed.Also, the digestion procedure must be sufficiently oxidising to break down anyorganoarsenic compounds into forms that can be analysed for total arsenic and/orspeciated (depending on the determination desired). And finally, recovery studies basedon the addition of inorganic arsenic to assess the efficiency of an ashing method, do notnecessarily establish that all arsenic species, particularly organic forms, are converted tothe inorganic form without loss. Therefore, it has been suggested(242, 251, 252) thatstandard reference materials with known arsenic concentrations and speciation can be usedto evaluate digestion methods. Furthermore, to increase confidence in the data, twodissimilar analytical procedures(25 1) could be applied to the same set of samples and theresults compared. Some or all of these alternatives should be employed to test rigorouslyany original analytical methods developed during preliminary studies.3.2.1 WET DIGESTION WITH SULFURIC ACID, NITRIC ACID ANDHYDROGEN PEROXIDEThe wet digestion procedure using sulfuric acid, nitric acid and hydrogen peroxidehas been effectively utilized for the dissolution of biological (invertebrates bivalves andgastropods) samples collected from British Columbia coastal areas(27). The wet digestionmethod using 1 ml 98%H2S04,3 ml 67% HN03 and 3 ml 30%H20was adopted in thepresent experiment with some modifications. The differences in the sample matrices aswell as the different arsenicals(4-7, 27, 28, 95) present in seaweed compared to thebivalves and gastropod tissues were taken into consideration in order to minimize orprevent arsenic losses. Arsenic losses have been previously observed(27) with longer90digestion times (> 3 hours). In the present work, initial digestion with the previous 3 hourdigestion time(27) showed some arsenic losses when the results of arsenic determinationin some seaweeds were compared to those determined on the same samples by usingNeutron Activation Analysis (NAA). [NAA is the second most widely used techniqueafter AAS for arsenic determination(27).] It is possible that different heating rates areneeded in order to release the different arsenicals present in these biological materials intosolution.To establish conditions for optimum recovery in the total arsenic determinations,two alternative methodologies mentioned in Section 3.2 above were employed. As will bediscussed in subsequent sections, the use of two dissimilar analytical procedures(25 1) inconjunction with standard reference materials(242, 251, 252) of known arsenicconcentration, enabled evaluation of the timed acid digestion dissolution proceduredeveloped for the present work. Both GFA.A and continuous HGAA, with theirrespective optimized conditions previously described, were applied to the arsenic analysisfollowing the wet digestion of real seaweed samples. The data were compared with theresults obtained by Neutron Activation Analysis on the same set of samples. Time Optimization for Wet Digestion of Seaweed Samples Using H2SO4. I-1N03and H202 And Arsenic Determination by The Continuous HGAA TechniqueTimed (1 hour, 2 hour, 3 hour) digestion experiments using 1 ml 98%H2S04,3 ml67% HNO3 and 3 ml 30%H20were performed on 0.25 g freeze dried seaweed samplesusing the modified wet digestion procedure described previously in Section the method established by Dodd(27). After the digestions, each digestate was7 made up to 100 ml by using deionized water. The solutions of each digestate wereanalyzed by using HGAA. The reductant media used consisted of 1.5% NaBH4 (wlv) and4M HCI acid. A bar graph, Figure 3.15, of the mean arsenic absorbance responsesdetermined in triplicate for each seaweed solution analyzed at specified times was plotted9)as a function of the digestion time. Optimum arsenic responses were achieved with thetwo hour timed wet digestion procedures for all the seaweed samples.Figure 3.15Bar Graph Showing Time Optimization ofWet Digestion Procedure for the DeterminationofArsenic in Seaweed40 —38 H32 —:: 28 —24-H 20 ——0• 8—01 2 3 4 5 6 7 8Analyzed Seaweed Sample NumberJ 1 hourm 2 hours3 hoursAnalyzed Seaweed Sample Numbers1 = FSP 104-89 (F. distichus, Shore Party) 5 = FLI 103-89 (F. distichus, Langara Island)2= FWB 8-89 (F. distichus, Wreck Beach) 6= RA HSI 17-89 (Red algae, Hot Spring Island)3 = FTS 15-89 (F. distichus, Tasu Sound) 7 = MSSdW (Green algae, Howe Sound)4 = BA(I) HSI 17-89 (F. distichus, Hot Spring Island) 8 = SWF 1CM 109-89 (F. distichus, Varney Bay)92A variety of standard reference materials (SRMs) were chosen for evaluating the two hourwet digestion dissolution procedure using HGAA for their arsenic determinations. TheseSRM samples include National Bureau of Standards (NBS), Orchard Leaves SRM 1571,National Research Council of Canada (NRCC) marine analytical standards, DORM-iDogfish Muscle and PACS-1 Marine Sediment.A summary of the results obtained for the standard reference materials are shownin Table 3.6 and are in reproducible agreement with the certified values.Table 3.6Two Hour Wet Digestion Continuous HGAA Determination ofArsenic in Standard Reference MaterialsSRM Sample As Certified Value As Experimental Valuej.tgfg (a) (Acid Digestion/Continuous HGAA)pg/g 0’)PACS-1 Marine 211±11 211.9±3.6Sediment (NRCC)DORM-i Dogfish 17.7±2.1 16.4±0.4Muscle (NRCC)SRM 1571 Orchard 10.0±2.0 9.2±0.9Leaves (NBS)(a) = mean and 95% tolerance limit(b) = mean and standard deviation of the mean of triplicate determinationsAs a further test of the efficacy of the two hour wet digestion procedure followedby the continuous HGAA analysis for arsenic determination, the results of the seaweedsamples analysed above (Figure 3.15) are compared in Table 3.7 with the data obtained byNeutron Activation Analysis.93Table 3.7Comparison of Results Obtained by the Two HourWet DigestionfllGAA Procedureand Neutron Activation Analysis Technique2hr Wet Digestion! Neutron Activation# Freeze Dried Seaweed Sample Continuous Analysis (NAA),HGAA, jig/g As(a) ig/g As1 F. distichus, Rennel Sound, 23.2 ± 0.6 24Clonard Bay Beach, 04/14/892 F. distichus, Vancouver Point 11.8 ± 0.6 15Grey, Wreck Beach, 05/08/893 F. distichus, Tasu Sound 31.2 ± 1.1 38Fairfax Inlet Shore, 04/17/894 F. distichus, Hot Spring Island 19.6 ± 1.1 25Beach, Juan Perez Sound,04/17/895 Iridaea Splendeus, Hot Spring 4.4 ± 0.1 4.3Island Beach, Juan PerezSound, 04/17/896 F. distichus, Langara Island 24.3 ± 1.1 25Beach Shore, 04/13/897 Enteromorpha Sp., Furiy 3.4 ± 0.3 3.4Creek, Howe Sound, 07/07/898 F distichus, Varney Bay, 25.0 ± 1.4 23Quatsino Sound, 04/18/89(a) = mean and standard deviation of mean of triplicate analysisThe Neutron Activation Analysis was performed at Queen’s University, Ontario.The results obtained by the two dissimilar techniques were found to be similar for all butthree samples (#2, #3 and #4). These differences are probably due to sampling variability,because different batches of the same seaweed samples were used for the two analyses.To study these effects further, three batches, but same seaweed samples, were prepared,and a set of sampls were sent for NAA analysis. The other two sets were subjected tothe two hour timed wet digestion procedure. Triplicate arsenic measurements werecarried out on each seaweed digestate by using GFAA and HGAA techniques on either ofthe two sets of digested samples. The arsenic determination results are shown in94Table 3.8. The effect of sampling using three different batches of the same seaweed couldbe observed in the arsenic data (Table 3.8) after using a different analytical technique foreach batch of samples. However, the concentrations of arsenic as determined using eachof the three dissimilar techniques (HGAA, GFAA and NAA) are reproducible for almostall the samples analyzed. By plotting the data in Table 3.8 as scatter graphs shown inFigures 3.16 - 3.18, the coefficients of linear regression which indicates the correlationsbetween the techniques and consequently their determinations were obtained. The resultsof arsenic determination obtained by using HGAA after the 2 hour digestion werecompared with results obtained by using the NAA technique according to the scatter plotshown in Figure 3.16, and th coefficient of linear regression was calculated to be 0.948.The scatter plot of the 2 hour wet digestion followed by GFAA analysis and the NAAtechnique (Figure 3.17) revealed a coefficient of linear regression of 0.915. And, thecoefficient of linear regression obtained for the two techniques of GFAA and HGAA(according to Figure 3.18), was found to be 0.943. Because these coefficients of linearregression are greater than 0.90, the methods and the results of their determinations areconsidered valid. Furthermore, the reproducible agreement between the experimentalarsenic results determined by both HGAA and GFAA techniques (after the 2 hour wetdigestion) and the certified concentration value of the NBS Orchard Leaves, SRM 1571reference material (Table 3.8) further validates the two methods and, consequently, theresults of their determinations.95Table3.8ComparisonofArsenicConcentrationResultsObtainedAfterTwoHourWetDigestionProcedureFollowedbyGFAAandContinuousHGAAAnalysisandtheInterlaboratoryTechniqueNeutronActivationAnalysis2hrWetDigestionProcedureNeutronActivationFreezeDried.w/gAs(a)Analysisig/gAs,#FdistichusSampleGFAAContinuousHGAANAA1TasuSound,FairfaxInlet,05/21/9035.4±1.726.5±2.429.12TasuSound,Minesite,05/21/9021.9±1.915.6±1.319.63RennellSound,ShieldsIsland,05/19/9028.2±1.722.3±1.926.64MassettInlet,YakounYellowRiver,05/14/9025.0±1.821.0±1.721.55Anyox,GranbyBayBeach,05/14/9022.9±1.819.7±1.724.76HastingsArm,05/14/9028.2±1.719.4±1.624.77ChathamSound,DundasIsland,05/13/9028.2±1.723.2±1.925.08RennelSound,TartuInlet,05/20/9030.1±1.723.9±2.029.49AliceArm,KitsaultBeach,05/15/9026.7±1.717.5±1.520.210NBSOrchardLeaves*SRM157111.3±2.39.6±1.1lo.o±2.o(b)(a)=meanandstandarddeviationofthemeanoftriplicatedetermination(b)=CertifiedArsenicValue,determinedbyNAA=StandardReferenceMaterial,notF.distichussampleFigure 3.16Scatter Plot for Arsenic DeterminationUsing the 2Hr Wet Digestion Followed by HGAA AnalysisCompared with Neutron Activation Analysis Results30.00 -:25.00 -%?; 20.00zwz15.00010.0025.000.00— I I I I I I I I I I I I I I I I I I I I I0.00 5.00 10.00 15.00 20.00 25.00 30.00NAA (jigfg As)97Figure 3.17Scatter Plot for Arsenic DeterminationUsing the 2Hr Wet Digestion Followed by GFAA AnalysisCompared with Neutron Activation Analysis Results40.00 -35.0030.00 /- /25.00—I20.00 —=C15.00 —— I I I I I I I I I I I I I I I I0.00 5.00 10.00 15.00 20.00 25.00 30.00NAA (gfg As)980.00 5.00 10.00 15.00 20.00 25.00 30.002 Hour Digcstion HGAA (ig/g As)Figure 3.18Scatter Plot for Arsenic DeterminationUsing the 2Hr Wet Digestion Followed Respectively byIIGAA and GFAA Analysis40.0035.00‘‘ 30.0025.0020.00C15.0010.00Thus, for rdutine investigations, the two laboratory methods ofHGAA and GFAA,can be used either separately or to compliment each other for the determination of arsenicconcentrations in marine macroalgae following the two hour digestion procedure99developed in the present study. Because of the limited sample size and the large numbersof fractions that were collected during chromatographic procedures (in the present study),the GFAA technique was preferentially employed for arsenic determination in thefractions. However, for total arsenic in samples, the HGAA technique was employedbecause the technique is experimentally simple to use, has few interferences, and it hasbetter sensitivity at low arsenic concentrations.3.2.2 WET DIGESTIONWITH SODRJM HYDROXIDEThe determination of arsenic in the seaweeds ofBritish Columbia was also studiedfollowing the use ofwet digestion with sodium hydroxide for sample dissolution. The wetdigestion with sodium hydroxide procedure is an established sample dissolutiontechnique(246, 250, 253) and has been described in Section The main advantageof the hot base digestion is that speciation analysis can be performed on the digest. Themethylated arsenic compounds are not degraded into inorganic arsenic(247-250) and, thehot base treatment cleaves arsenic bonds with intracellular compounds or groups exceptfor the methyl group. The digestion products of a sample containing inorganic-,monomethyl-, dimethyl- and trimethyl-arsenic compounds when subjected to the hot basedigestion are arsenate, methanearsonate, dimethylarsinate, and trimethylarsine oxide,respectively(247-250). On treatment of the digest products with sodium borohydride,they are converted to their respective arsines (in a hydride generation procedure). Byutilizing the boiling point differences exhibited by the arsines (Table 3.5) and furtherseparation of the hydrides by using gas chromatography, the speciation of the arsenicforms present in the samples were achieved after quantitative detection by means ofatomic absorption ‘spectrometry.1003.3 DETERMINATION OF ARSENiC SPECIES BY HYDRIDE GENERATION-GAS CHROMATOGRAPHY-ATOMIC ABSORPTION SPECTROMETRY(HG-GC-AAS)The application of the HG-GC-AAS using the semi continuous HG mode(254) foroptimum arsenic speciation analysis was investigated. This was carried out by studyingthe effect of using both hydrochloric acid and acetic acid media on the response profiles ofsome arsenic species when using 2% NaBH4 (w/v) as reductant. The semi continuousmethod used here is similar to that described by Reimer(195) and the instrumentalparameters (Table 2.2) employed were established by Li(196).3.3.1 EFFECT OF HYDROCHLORIC ACID ON THE RESPONSE PROFILESOF SOME ARSENIC SPECIESA standard solution containing 100 jig/mI of arsenic as arsenate, As(V), MMAAand 200 jig/mI of arsenic as DMAA was prepared in deionized water. This mixedstandard arsenic solution was serially diluted using deionized water to give a daily workingstandard of 10 ng/ml as arsenate, MMAA and 20 ng/ml as DMAA. The response profilesof individual arsenic species are plotted and shown in Figure 3.19 as a function of the acidconcentrations. The responses exhibited by all the arsenic species are consideredreproducibly good for their speciation with this method. The optimum acid concentrationselected in the present study is IM hydrochloric acid.101Figure 3.19Effect ofHydrochloric Acid Concentration on the Response ofAs(V), MMAA, and DMAA during Reduction by 2% NaBH4and Semi-Continuous HG-GC-AAS Analysis3E+6 —____________________________________3+6 DMAA (80 ng As)2E+60.0 MMAA(40 ngAs)Q 2E+6:40ngAsQ1E+6Q05E+5OE+O— I I I I I I I I I I I I I0.0 0.5 1.0 1.5 2.0 2.5HCI (M)3.3.2 EFFECT OF ACETIC ACID ON THE RESPONSE PROFILES OF SOMEARSENIC SPECIESA peristaltic pump was used to mix 4m1 of premixed arsenic standard solution of10 ng/ml as arsenate, MMAA and 20 ng/ml as DMAA with a continuous flow of 2%NaBH4 (w/v) and varying concentrations of acetic acid. As described in Section 3.3.1,the procedure of IG-GC-AAS produced a response profile for each arsenic species whichwas plotted and, is shown in Figure 3.20 as a function of the acid concentrations.102U1.UU4-1U4-4-Figure 3.20Effect of Acetic Acid Concentration on theResponse of As(V), MMAA, and DMAA during Reductionby 2% NaBH4and Semi-Continuous HG-GC-AAS Analysis3+6SE +62E+62E+0I.E +65E+5OK+0Unlike in the mineral acid determination (3.3.1), only the response of DMAA wassignificant in the acetic acid/NaBH4media. Both MMAA and As(V) responded poorly.At 1.5M acetic acid concentration, an optimum arsenic signal response was determined foreach of the arsenic species analyzed.In terms of total arsenic speciation, the two reduction media, HCI/NaBH4 andCH3OOHJNaBH4offer excellent complimentary advantages. If a sample is known tocontain little or no As(V) and MMAA but large concentrations of DMAA, theNaBH4lacetic acid reductant medium will be sufficient for its speciation. However, abetter and more precise determination of the three arsenic species will be expected with0.0 0.6 1.0 1.5 2.0 2.5Ilac Concentration (M)103the NaBH4/hydrochloric acid reductant medium for samples of unknown species andconcentrations.Finally, the optimum acid concentration determined for hydrochloric acid andacetic acid were 1M and 1.5M respectively, when 2% NaBH4 reductant solution andHG-GC-AAS are employed for arsenic analysis.3.3.3 CALIBRATION AND LIMIT OF DETECTION FOR THE SEMI-CONTINUOUS MODE HG-GC-AAS ANALYSISTypical calibration graphs obtained for inorganic arsenic (As[III], As[V]), MMAAand DMAA with standard arsenic solutions of 10, 20, 30, 40 ng as arsenite, arsenate,MMAA and of 20, 40, 60, 80 ng as DMAA are shown in Figures 3.21-3.22. Thecalibration graph shown in Figure 3.21 was obtained using 1M HCI acid/2% (w/v) NaBH4reductant media following arsenic determination by using the semi-continuousHG-GC-AAs. Figure 3.22 was obtained using 1.5M CH3OO /2% (wlv) NaBH4reductant media following arsenic determination by using the semi-continuous HG-GCAAS.104Figure 3.21Typical Calibration Graphsfor Inorganic As(V), MMAA and DMAA in1M 11C112%NaBH4(w/v) Reductant Mediaby Semi-Continuous Mode HG-GC-AAS Analysis3.OE+6 -_______________________________2.5E+6 -MMAA/ /DMAA2.OE+61.5E+6 —1.OE+6 —/7/7/ As(V)5.OE+5 -/—‘ I I I I I Io io 20 30 40 O 60 70 80 90Arsenic Concentration (ng As)105Figure 3.22Typical Calibration Graphsfor Inorganic As (As[T1T1, As[VJ), MMAA and DMAA in1.5MC1130011 2%NaBII4(w/v) Reductant Mediaby Semi-Continuous Mode HG-GC-AAS Analysis4.OE+6 -___________________________________3.5E+6 —- 3.OEI-6 —- 50/50 arsenate & arseniteDMAA2.5E+6 -I2.OE+6 -1.5E+6 - -1.OE+6 -MMAA5.OE+5 -0.OE+0 .:t.__c—: I I I I I I I0 10 20 30 40 50 60 70 80 90V Arsenic Concentration (ng As)106Both Figure 3.21 and 3.22 show the expected linear relationships existing betweenabsorbance and individual arsenic species concentration at the concentration rangeemployed for the analysis. The following tables (3.9 and 3.10) give the calculated limits ofdetection (LODs), (defined as the blank signal plus three standard deviations of the blank)for each arsenic species analysed by the semi-continuous HG-GC-AAS and the two acid(HCIJCH3COOH)INaBH4media. The relative standard deviations (RSD) of twentyreplicate analyses of 10 ng As as arsenate, arsenite, MIVIAA, and 20 ng As as DMAA werealso calculated.Table 3.9LODs and RSDs of Arsenic Determination bySemi-Continuous Mode HG-GC-AAS and in1M 11C112% NaBH4Reductant MediumLOD of Arsenic by HG-GC-AAS (Semi-Continuous) RSD (m)Arsenic Species Area Abs (x 10) Absolute [As] ng [As1/4m1, ng/ml %Inorganic As (V) 1.3 1.6 0.4 1.6MIVIAA 3.2 3.2 0.8 3.1DMAA 3.4 8.3 2.1 4.1Area Abs = Area Absorbance; (m) = 20 replicate analysis of lOng As as Inorganic As, MMAAand 2Ong As as DMAA; nd = not determinedThe calculated RSD are 1.6%, 3.1% and 4.1% for arsenate, MMAA and DMAArespectively in the 1M HC1/2% NaBH4 reductant medium. In 1.5M CH3OO J2%NaBH4 reductant medium, the RSD calculations (Table 3.10) are 7.2%, 4.6% and 5.0%for inorganic arsenic [50/50 As(III) and (V)], MMAA and DM.AA respectively. TheseRSD determinations were determined at the specified concentration for each arsenicspecies.107Table 3.10LODs and RSDs of Arsenic Determination bySemi-Continuous Mode HG-GC-AAS and in1.5MCH3OO I2% NaBH4Reductant MediumLOD of Arsenic by HG-GC-AAS (Semi-Continuous) RSD (m)Arsenic Species Area Abs (x 10) Absolute [AsJ ng [As]/4m1, ng!ml % —Inorganic [As(III) + As(V), 4.8 5.4 1.3 7.250/50]IvIMAA 1.2 3.4 0.8 4.6DMAA 6.7 7.7 1.9 5.0Area Abs = Area Absorbance; (m) = 20 replicate analysis of lOng Asas inorganic As [50/50 As(III) ÷ (V)1, MMAA and 2Ong As as DMAA3.3.4 INVESTIGATION OF THREE WET DIGESTION PROCEDURESFOLLOWED BY SEMI-CONTINUOUS MODE HG-GC-AAS ANALYSISFOR ARSENIC SPECIATION IN THE SEAWEED OF BRITISHCOLUMBIAThe knowledge of the total concentration of an element is the main objective ofmany analytical schemes. However, the determination of the various forms or species ofthe analyte is even often more important(255). This is because the concentration of aspecies, especially if it is toxic, is more relevant than the total elemental concentration forsetting environmental and clinical standards and/or regulatory limits(256). The analyticalprocedures(s) used in the acquisition of data on toxic species must be reliable,reproducible and the data interpretation clear and accurate(255, 256). This studyexamined three analytical digestion procedures used for dissolution of environmental andbiological samples with respect to speciation analysis(27, 49, 246-250).The following digestion procedures were examined: i) wet digestion using a twohour heating cycle with 1 ml 98% H2S04, 3 ml 67% HNO3 and 3 ml 30% H20solution(27); ii) wet digestion using a three hour heating cycle with 5 ml of 2M HNO3108solution(49); and iii) wet digestion using a three hour heating cycle with 5 ml of 2MNaOH solution(247-250). Arsenic determination on the digests was acheived by using thesemi-continuous mode HG-GC-AAS technique. Speciation of Arsenic in the Digest of Sample Subjected to Wet Digestion UsingH2S04. HNO3 and H202 by the Semi-Continuous Mode HG-GC-AAS AnalysisThis wet digestion procedure has been described in Section 3.2.1 and previously byDodd(27). The speciation of the digest product(s) obtained after using the wet digestionprocedure has not been done although it is assumed that arsenate is the final arsenicspecies. In the current study, two methylated arsenic standards and a freeze-driedseaweed sample were examined for arsenic species present in their respective digestsolutions, after subjecting each sample to the dissolution procedure. After using serialdilution on stock solutions of DMAA and arsenocholine, to obtain 10 ppm of eachmethylated arsenic standard, aliquots (10 ml) were taken and digested according to theoptimized wet digestion procedure described in Section Similarly, 1.Olg F.distichus (freeze dried, Head of Hastings Arm) was subjected to the digestion procedure.The digests obtained were neutralized with dilute hydrochloric acid and made up to 50 mlin separate 50 ml volumetric flasks. Appropriate dilutions were made where necessarybefore arsenic determination using the semi-continuous HG-GC-AAS technique.The following chromatographic traces shown in Figures 3.23 - 3.26 show theresults of the the HG-GC-AAS analysis on (i) the standard arsenic solutions withoutdecomposition (Figure 3.23), (ii) DMAA after decomposition (Figure 3.24), (iii)arsenocholine after decomposition (Figure 3.35), and (iv) seaweed digestate (Figure 3.26);arsenocholine, without digestion, is considered as one of the “hidden” arsenicals during ahydride generation procedure. The y-axis represents the absorbance drawn to an arbitraryscale in the chromatograms shown in Figures 3.23 - 3.26.109Figure 3.23HG-GC-AAS Chromatogramof As(V) - (A), MMAA - (B)and DMAA - (C)Figure 3.24HG-GC-AAS Chromatogram ofDMAA Digest After Two HourWet Digestion in Imi H,S04,3m! HNO1 and 3m!H20Figure 3.25HG-GC-AAS Chromatogram ofArsenocholineAfter Two Hour Wet Digestionin imiH2S04,3m! HNO1 and 3m!H20Figure 3.26HG-GC-AAS Chromatogram ofFreeze DriedF. distichus (Head of HastingsArm) After Two Hour WetDigestion inimi 112S04,3m! 11N0 and 3m!11202 /C.,I.0.0IAL.j 1AFARetention Time (mm) —*110The results confirmed that the decomposition is complete and that the arsenicspecies in the digests is inorganic arsenate, As(V). Therefore, this wet digestionprocedure is appropriate for the routine analysis of total arsenic in environmental,biological and other analytical samples. Speciation of Arsenic in the Digest of Sample Subjected to Wet Digestion Using5m1 of a 2M UNO, solution by the Semi-Continuous Mode HG-GC-AAS AnalysisA wet digestion procedure using nitric acid was employed by Sanders(49) inanalysing a wide variety of algal species from different habitats in order to assess the rangeof arsenic concentrations, speciation and possible variations existing between differentalgal classes and/or habitats. In the procedure, the algae were weighed and digested inHNO3 under a contamination-free atmosphere and low heat (900C). The vials containingthe sample digests were removed before complete dryness of the digestates and theremaining residue dissolved in 10% Ultrex HNO3 (5ml). Following this digestionprocedure, it was claimed(49) that arsenic species were analyzed in the nitric acid digestsolutions by using a D.C.-arc-induced-plasma emission technique. The results revealedthat the three algal classes contained significantly different arsenic concentrations, with thePhaeophyceae containing on the average more arsenic than either the Rhodophyceae orthe Chlorophyceae (10.3 vs 1.43, and 1.54 jiglg As respectively). Also, the speciation ofthe incorporated arsenic was found to vary between the algal classes. The Phaeophyceaecontained 22% as inorganic arsenic whereas the Rhodophyceae and the Chiorophyceaccontained 43% and 47% inorganic arsenic respectively. These inorganic arsenic speciesresults are rather high and should have stimulated some justifiable anxieties about possiblearsenic poisoning.111-4-4-‘CDOC)————-.-Ni$.H ?•Ic -.—.—x1r:4 -.... cI-‘-U.-4.-4 o Cl) .—;% :; I rr .Cl)0),CD 00--CD4—•0’CD oCD 0CD g•00 CD—I.-JC)00CD0)‘1CD—‘-4.“)Q•1%)-‘pIL ,CDCDoJ%0 I 0 C,Cl)il0CDCD CD CDCDp.I2 ac,) CD CDot.CeCD —C1CD CD CDo1CCl)—————————————-‘——-‘—-,-U’—-4-3—)----3-.4-.4)--‘‘‘.<aOp-’,-’,-’Owwwwoo4bO003-Lz’OOO.Q%POOJW.t43O)Q’bViI..JQ%%O0ooQo0-JVi000%%0-.4WC)0t.3Wt)s)W1.)WVi.1.))Vi-31-300%0000000%00.“1)%0%O%0-40—-Z3Cwi:ti:3.•%0‘-‘-3I)-40’1-31-)0%-.4a. ‘•.•tei•e•o•,. T-3001-3.WL3.0%ow-‘uwwWViViI.3%OVi1-)-000%Vi1-)00————.-‘—-U’U’....‘—‘—-40%-4-4Vi-4Q%0O00O--3-40%.00Vi-3‘.4.W001-)‘-‘1-31-3.1-3ViVi%O%000%-3‘.000‘-aW0...j’-IO%’.O’OOOOO1-)W0030%‘1-.)ViVi001-30Vi.•Is)Vi1-3‘.3,IIIIIIIIIIIIIIIIIIIII1.03W00-4-4-4Vi-.30%‘.0‘.‘.)‘.00‘.4000%Vi‘.0Vi-3-.4.031.03-300-31.03000%1.03.-3001-3‘0..-4‘.0ViViC)————-U’————‘———0———————U’The “questionably” high inorganic arsenic concentrations prompted the present study onthe speciation ofarsenic in seaweed sample following nitric acid digestion.With nitric acid serving as the digestion medium, the procedure detailed in Section2.4.6.3 was followed and the subsequent F. distichus (Head of Hastings Arm) digestswere analyzed by the semi-continuous mode HG-GC-AAS technique. A typicalchromatogram obtained on the digest is shown in Figure 3.27,Figure 3.27Chromatogram of F. di.stickus (head of hastings Arm)Digest After a Three Hour Wet DigestionWith 2M 11N03 (Sm!) at 90-95°C bythe Semi-Continuous Mode IIG-GC-AAS AnalysisC.,C-,.t60.0C)60C)CCI..C)0Retention Time (mm) —*Variations in the percentage of the arsenic species shown in Figure 3.27 wereobserved in the replicate determinations of each of the three digests analyzed. Also, thedigestion was not considered complete because the calculated arsenic concentration in thedigestate was 40% of the arsenic content in the F. distichus.. Finally, the same seaweedwas digested with sodium hydroxide following Section procedure, and mainly(> 98%) dimethylted arsenic species were present. This result suggests that the nitricacid digestion seems to have partly decomposed these dimethylarsenic species intoinorganic and monomethylarsenic species. Therefore, the nitric acid digestion procedurecannot be considered appropriate for providing speciation information. Based on theseA = As(V)13 = MMAAC=DMAAABC113observations and the relatively ‘harsh” concentrated nitric acid solution used bySanders(49) the speciation results in Table 3. II may be unreliable. Speciation of Arsenic in the Digest of Sample Subjected to Wet Digestion Using5m1 of 2M NaOH solution by the Semi-Continuous Mode HG-GC-AAS AnalysisThe wet digestion procedure using sodium hydroxide has been describedpreviously [Section 3.2.2 of this thesis and in References (247-250)]: Excellent arsenicspeciation results were obtained after dissolution of samples in 2M NaOH solution from(a) five arsenic-tolerant freshwater algae isolated from an arsenic-pollutedenvironment(247); (b) the water soluble fractions of the muscles of carnivorousgastropods, crustaceans and fish(248), (c) the metabolic products of fresh water alga(Chiorella sp.) and the shrimp as a grazer(249), and finally, (d) the accumulation productsfrom the water phases of a three-step fresh water food chain(250). The hot base digestionensures that the methylated arsenic compounds are not degraded into inorganic arsenic,and arsenic speciation information is retained. The results generally show that thesummation of all arsenic species concentrations equals the total arsenic concentration inthe sample.In the present study, the total arsenic concentrations were determined by usingdirect arsenic speciation after hot base digestion of freeze dried seaweed samples.Undigested extracts of these seaweeds were also examined. Two extraction media wereused. The first, methanol/water is used for all extractions carried Out on seaweeds in thepresent study. The other, a sodium hydroxide solution(246) was employed for someseaweed extractions in order to compare the arsenic speciation information between thetwo extraction mehods.In order io establish that the dissolution procedure is complete and reliable forarsenic speciation, the hot base wet digestion was performed on a I disiichzis (collectedfrom Head of Hastings Arm). The chromatograrn shown in Figure 3.28 is typical of the114result obtained after arsenic speciation by the semi-continuous mode HG-GC-AASanalysis.Figure 3.28Chromatogram ofF. distichus (Head of Hastings Arm) DigestAfter a Three Hour Wet Digestion With 2M NaOH Solution(5m1) at 90-95°C by the Semi-Continuous ModeHG-GC-AAS AnalysisUI..I’UI..URetention Time (mm) —,Generally the speciation analysis revealed that the dimethylarsenic species(DMAA) is the major arsenical present in the F. distichzis sample.In summary, three wet digestion procedures have been investigated for sampledissolution. Arsenic determination and speciation were subsequently carried out using thesemi-continuous HG-GC-AAS technique. The studies revealed that one of the three wetdigestion procedures, wet digestion by using 5 ml of 2M HNO3 acid, cannot be consideredappropriate for providing speciation information. The efficacy of the other two wetdigestion procedures (1 ml 98%H2S04, 3 ml 67% HNO3, 3 ml 30%H20 and 5 ml 2MNaOH solution)1was considered completely sufficient for arsenic determination andspeciation. A summary of the speciation analysis discussed in the previous three sectionsfollowing the application of the three digestion procedures for F. distichus sampleF3=MMAAC = DMAAB4-C115dissolution and subsequent arsenic determinations by using the semi-continuous modeHG-GC-AAS are shown in Table 3.12.Table 3.12Summary of Speciation Analysis on DigestionsObtained Using Three Different Wet Digestion Proceduresand Arsenic Determination by the Semi-ContinuousMode HG-GC-AAS TechniqueMethod 1 Method 2 I Method 3Arsenic Species in % Area AbsorbanceDigest Jnorg As MMAA DMAA [ Inorg As MMAA DMAA lnoi-g As MMAA DMAADMAA 100 — — md nd nd nd nd ndArsenocholine 100 —— IL nd nd nd nd nd ndF. distichus 100 ——32.4 24.4 43.2 — 1.7 98.3—, not detected; nd, not determinedMethod 1, imIH2S04(98%): 3m1 HNO3 (67%): 3m!H20 (30%), 2hr, 200°C;Method 2, 5m1 2MHNO3,3hr, 90-95°C;Method 3, Sm! 2M NaOH, 3hr, 90-95°C3.4 CHROMATOGRAPHIC SEPARATION AND ISOLATION OFARSENICALS IN SEAWEEDThe separation and isolation of arsenic compounds in seaweeds has been achievedmainly by using chromatographic procedures involving Gel Permeation Chromatography(GPC), Ion Exchange Chromatography (IEC, anion and cation IEC), Thin LayerChromatography (TLC, silica and cellulose ThC), and High Performance LiquidChromatography (HPLC, normal and reverse phase)(28, 51, 95, 257-263). Thesechromatographic separations and isolations of the seaweed arsenicals were monitored byusing element-selective detection in either on-line or more usually, off-line modes.Arsenic species identification after chromatographic speciation using the off-line detectionmode involves maximum sample handling, long analysis time as well as the time needed tooptimize the chromatographic system(5 1, 162, 186, 264). The off-line method continuesto be used extensively, in spite of its drawbacks. More recently, however, element116selective detectors such as AES and AAS(51), ICPAES(264), ICPMS(50, 257, 265-266),electrochemical(96, 211) and radioactivity(261, 268-269) detectors, are being used tomonitor chromatographic isolation.A combination, involving chromatographic isolation followed by off-line detectionof arsenic in the fractions and a further separation and identification of the arseniccontaining fractions using the combined chromatography on-line detection mode, wassuccessfully applied to the seaweed samples investigated in the present study. The flowdiagrams (Figures 2.8 and 2.9) described previously detailed schematically thechromatographic separation of water soluble arsenic species extracted from F. distichus inwhich the off-line mode of arsenic detections were employed. The initial cleaning of theseaweed extracts was achieved by using isocratic elution with water or methanol servingas mobile phase on a size exclusion gel column, using a Sephadex LH2O or Sephadex G15chromatography. Further purifications were then achieved on a DEAE Sephadex A25weak anion exchange column using isocratic elution with either 0.05M or 0.5Mtris(hydroxymethyl)amino methane hydrochloride buffer (Tris buffer). The Tris buffersolution is normally prepared with a pH of 8.0. The separate arsenic containing isolates,obtained by combining fractions according to their elution profiles on the anion exchanger,were then further cleaned on a gel permeation column to remove the Tris buffer solventfrom the isolates. Most often, repeated chromatography involving both the GPC andanion exchange columns were utilized for further purifications prior to the next separationstep. These procedures have been previously utilized for the isolation of arsenosugarsfrom other marine macroalgae(28, 51, 95, 175, 18 1-183, 186). Following these repeatedchromatographic purifications, the arsenic containing isolates were taken and applied untocellulose tic plates, using an acidic solvent combination of n-butanol:acetic acid:water,60:15:25, as the mobile phase(95). The fractions containing arsenic are further purified ona preparative HPLC column using water:methanol (4:1) as the mobile phase(27). Thearsenic containing isolates separated according to their different elution profiles were-117taken for final chromatography and identification by using on-line detection proceduresdiscussed in the next sections, 3.4.1 and IDENTIFICATION OF ARSENIC SPECIES USING HIGHPERFORMANCE LIQUID CHROMATOGRAPHY ON-LINEMICROWAVE OVEN DiGESTION HYDRIDE GENERATION ATOMICABSORPTION SPECTROMETRY (HPLC-MIC-HGAAS)The use of an on-line detection system coupled to an HPLC column for theidentification of arsenic compounds has improved complex separation procedures withregard to reduced analysis time, separation efficiency, selectivity, ultra trace compounddetection and successfUl identification analysis. The separation and speciation of arsenite,arsenate, monomethylarsonic acid (MIvIAA), dimethylarsinic acid (DMAA) and the“hidden” organoarsenic compounds such as arsenobetaine (the major arsenical found inmarine animals), and arsenosugars (commonly found in marine algae) have beensuccessfully carried out by using two on-line detection systems coupled to an HPLC(17,52, 97, 100, 138, 210). These are the HPLC-On-line Microwave Oven AssistedDecomposition-Hydride Generation Atomic Absorption Spectrometry (HPLC-MicHGAAS) and I-IPLC-Inductively Coupled Plasma-Mass Spectrometry (I-IPLC-ICPMS).The separation and identification of arsenic species using the HPLC-ICPMS will bediscussed in Section 3.4.2. The main advantage of the HPLC-Mic-HGAAS on-linedetection system is that it is capable of differentiating arsenic species such as arsenite,arsenate, MIvIAA and DMA.A from the “hidden” organoarsenic compounds, such asarsenobetaine and arsenosugars, in environmental and biological samples.The procedure established by Le et al(138, 210) was adopted in the present workwhen using the ón-line HPLC-Mic-HGAAS. In the procedure, the I-IPLC effluentundergoes microwave assisted decomposition before hydride generation takes place. Asolution containing 0.IM potassium persulfate and 0.3M sodium hydroxide in the presenceofmicrowave energy was used efficiently to decompose organoarsenic compounds present-11$in seaweed extracts and arsenic containing chromatographic fractions of the extracts toarsenate. The microwave oven then meets the continuous flows of acid (A) andborohydride (B) (see Figure 2.3) in a hydride generation reaction. In order to reducedispersion and the amount of aerosol, an ice water cooling bath ([B) .is used. The arsinesproduced, after passing through the gas/liquid separator are introduced to the flame heatedquartz tube for atomic absorption detection. The summary of the experimental conditionsfor the microwave decomposition and hydride generation (Mic-HGAAS) is given in Table2.5. The following, Figures 3.29(a-b) and 3.30, are typical chromatograms which wereobtained when aqueous solutions of arsenobetaine, arsenocholine, tetramethylarsoniumion, arsenite, arsenate, MMAA and DMAA were analyzed by using Mic-HGAAS andHPLC-Mic-HGAAS analysis respectively. These analyses were performed and describedby Le(17).Figure 3.29(a):Figure 3.29 (a-b)Chromatograms of 2Ong of Arsenate, Arsenite, M11AADMAA, Arsenobetaine, Arsenocholine andTetramethylarsonium ionUsing the Mic-HGAAS AnalysisWithout Oven Digestion (Oven Power Turned Off) (Adaptedfrom Reference 17)IAs(IJl) As(V) MMAj DMAA119Figure 3.29(b): With Microwave Oven Digestion in O.1MK2S08and 0.1MNaOH (Adapted from Reference 17)Aft AC Me4As As(1H) As(V) MMAA DMAAChromatogram of 2Ong ofArsenate, Arsenite, MMAA, DMAAArsenobetaine and 4Ong of Arsenocholine and Tetramethylarsonium ionUsing the HPLC-Mic-HGAAS Analysis (Adapted from Reference 17)3;]HPLC Condition: Reversed Phase C18 Column Using SKP Gradient Elution withInitial Eluent Being 10mM Heptanesulfonate, pH 3.5 switchedafter 2 minutes to 4mM Malonic acid, pH 3.0; Flow Rate1.0mI/rninFigure 3.30o 4 S 12 16 20 24 2€ 32 36Retention Time, miii.120Figure 3.29(a) reveals only the reducible arsenic species when the microwaveoven was turned off. Under this condition, the “hidden” arsenicals are not detected. Withthe microwave oven turned on however, all the arsenicals, including the reducible and“hidden” arsenic species are detected (Figure 3.29(b)). When the HPLC system is coupledto the Mic-HGAAS and used for the analysis of arsenate, arsenite, MMAA, DMAA,arsenobetaine, arsenocholine and tetramethylarsonium ion, the chromatogram shown inFigure 3.30 is obtained.3.4.2 IDENTIFICATION OF ARSENIC SPECIES USING HIGHPERFORMANCE LIQUID CHROMATOGRAPHY ON-LINEINDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (IIPLCICPMS)The coupling ofHPLC to ICPMS for speciation analysis has provided an improvedseparation and identification procedure for complex environmental and biological samplesbecause of the high resolution obtained with HPLC columns and very high sensitivity ofthe ICP-MS detector. The HPLC-ICPMS technique has been successfi.illy employed forthe separation and detection of fifteen arsenic species(97). It has also been used for theidentification of arsenic lipids(99) in the digestive gland of the western rock lobster,Panulirus cygnus and the characterization of organic arsenic species including the“hidden” arsenic containing ribofhranosides, in samples of clam and mussel(50). Similarly,arsenobetaine was found as a major arsenical (84% of total arsenic) in a Dogfishmuscle(92).In order to identify naturally-occurring arsenicals in environmental and biologicalsamples by the HPLC-ICPMS technique, appropriate standard arsenic materials must beavailable(50, 98, 99). Arsenic compounds in samples can then be accurately identified bymatching retention times of their respective chromatographic peaks with those of spikedand/or unspiked reference arsenic standards chromatograms(17). Some standard arsenic121compounds that have been employed for identification purposes are aqueous arsenicsolutions of arsenite, arsenate, Mfh,{AA, DMAA, and TMAO. Other arsenicals that havebeen used include arsenocholine, arsenobetaine, tetramethylarsonium ion, synthetic andnatural arsenic containing ribosides(99) and arsenic species found in NBS (or NIST),NRC, and NIES standard reference materials(50).A combination of the routine laboratory arsenic standards, natural arseniccontaining ribosides (kindly provided by Edmonds and Francesconi, Western AustralianMarine Research Laboratories, Australia) and a Standard Reference Material NEST 1566aOyster tissue, Carssostrea gigas was employed for arsenic species identification in thepresent study. The NEST 1566a has previously been found to contain two major watersoluble arsenic containing ribosides(17, 50), arsenosugars jj and jjj, and also, DMAAand arsenobetaine. This standard reference material has been successfhlly used forcharacterization of arsenic species in some bivalves(50) and in some marine organisms, aswell as human urine(17).122RESULTS AND DISCUSSION - ARSENIC SPECIES DETERMINATIONISOLATION, CHARACTERIZATION AND BIOTRANSFORMATION4.1 TOTAL ARSENIC DETERMINATIONJn this section, data relating to the total arsenic content in several marine macroalgae(seaweeds) will be presented. The analyses were carried out on digests of the freeze driedseaweed samples after the application of an appropriate digestion method that used either amixture of nitric acid, sulfuric acid and hydrogen peroxide (the two hour wet digestion method)or a solution of sodium hydroxide (the hot base digestion method). Both digestion methodshave been described previously, in Section and All analyses were done usingtriplicate determinations on each sample, except where otherewise stated. The mean arsenicconcentration and the standard deviation of that mean are normally quoted on the basis offreeze dried weight, except where otherwise stated.4.1.1 TOTAL ARSENIC CONCENTRATIONS IN SEAWEEDS COLLECTEDFROM BRITISH COLUMBIA COASTLINES IN 1989, 1990 AND 1991The collection of the seaweed samples was achieved by using the scientific cruise boat,C.S.S. 3.?. Tully, except where otherwise stated. The following brown algae, listed inTable 4.1 were sampled between 1989 and 1991 and the total arsenic concentrations weredetermined following the two hour wet digestion method on homogenized freeze driedseaweed samples by using the continuous mode HGAA methodology. The arsenicconcentrations shown in Table 4.1 vary with species of the brown algae and the location fromwhich each species were collected. The concentrations of arsenic in the brown algae variedbetween 1.8 (sample collected at Giltoyees Inlet, Kitimat) and 36.9 jiglg (samplecollected at Langara Island). The considerable mobility of arsenic(205) in the soil couldprovide some explanation about the differences in the arsenic contents revealed in the brownalgae samples from these two given locations. The Kitimat Arm area, even though a potentialsource of arsenic pollution from discharges coming from the smelter refinery complex for1 — .lI ialuminium located at Kitimat (Figure 2.5) and other industrial activities including pulp mill andmethane production, its soils are well drained, infertile and subject to extreme leaching(197).Mobility of arsenic is expceted in this area and therefore a small amount of arsenic isaccumulated by the brown algae sampled at this site. Whereas, because Langara Island issubject to various manmade activities (mining at nearby Queen Charlotte Islands, Tasu, fishingprocessing and energy generating plant at nearby Masset; as well as tourism by land, ferry andair) which are potential arsenic pollution sources, and because the soils have very poordrainage, leading to soil saturation due to heavy rainfall in this location, arsenic mobility is notexpected to be significant. Therefore, the brown algae sample from Langara Island was foundto have accumulated a very significant amount of arsenic. The different species of brown algaesampled at Langara Island also revealed variations in the amount of arsenic accumulated. TheF. distichus sample collected at the beach shore of Langara Island was determined to haveaccumulated 24.3 p.g/g arsenic whereas, the Alaria marginata sample collected from the samesite was found to contain 36.9!g arsenic. The possible explanation for this observedvariation in arsenic accumulated between species of the same class of brown algae (at the samelocation), in this instance, might be related to the dates of collection. Furthermore, thisobserved high accumulation of arsenic in the brown algae collected at this site indicates that thelocation is suspect to arsenic pollution. Finally, the mean arsenic concentration obtained overallfor brown algae samples collected between 1989 and 1991 sampling (3 years) period, wascalculated to be 19.8 .tg/g As. The data relating to the other classes of marine macroalgaesampled in the present study are given in Tables 4.2 and 4.3.Jn Table 4.2, arsenic concentrations in some red algae collected from four differentlocations on British Columbia coastline areas between 1989 and 1991 are presented. Similarly,the arsenic concentations in some green algae collected from six locations along the BritishColumbia shores are presented in Table 4.3,124Table 4.1IArsenic Concentrations in Some Brown Algaeof British Columbia SampledFrom 1989 to 1991Freeze Dried Seaweed Sample, Two Hour Wet Digestion!Location and Continuous HGAA, .tg/g AsNo. Collection Date 1989 1990 19911 F distichus, Rennell Sound @Clonard Bay Beach, 04/14/89 23.2 ± 0.6 n n2 F distichus, Rennel SoundShields Island, 05/19/90 n 22.3 ± 1.9 n3 F distichus, Rennell Sound @ TartuInlet, 05/20/90 n 23.9±2.0 n4 F distichus, Rennell Sound @ EllisBay, 05/18/91 n n 6.9± 0.45 Macrocysticpyrijera, Rennell Sound@ClonardBay, 05/18/91 n n 32.0±0.46 F distichus, Quatsino Sound (Varney Bay, 04/18/89 25.0 ± 1.4 n7 F distichus, Quatsino Sound @VameyBay, 05/21/91 n 6.5 ±0.48 F distichus, Quatsino Sound @Kaprino Harbour Prideau Pt., n n 8.2 ± 0.405/22/9 19 F distichus, Langara Island @Beach shore, 04/13/89 24.3 ± 1.1 n n10 Alaria marginata, Langara Island @Langaralsland, 05/17/91 n n 36.9±0.511 F distichus, Tasu Sound @ FairfaxInlet shore, 04/17/89 31.2 ± 1.1 n12 F distichus, Tasu Sound @ FairfaxInlet, 05/21/90 26.5 ± 2.4 n13 F distichus, Tasu Sound @ Minesite, 05/21/90 n 15.6± 1.3 n14 F distichus, Juan Perez Sound @HotSpringlslandbeach,04/17/89 19.6±1.1 n fl15 F distichus, Vancouver Point Grey@WreckBeach, 05/08/89 11.8±0.6 n n16 F distichus, Anyox @ Granby Baybeach, 05/14/90 n 19.7± 1.717 F distichus, Anyox @ Granby Bay,05/21/91 7.6±0.4125IFreeze Dried Seaweed Sample, Two Hour Wet Digestion!Location and Continuous HGAA, .ig/g AsNo. Collection Date 1989 1990 199118 F distichus, Alice Arm ( Kitsaultbeach, 05/15/90 n 17.5 ± 1.519 F distichus, Alice Arm @ AliceArm, 05/13/91 n 8.5 ± 0.420 F distichus, Hastings AnnHastings Arm, 05/14/90 n 19.4 ± 1.6 n21 F distichus, Chatham Sound @Dundas Island, 05/13/90 n 23.2 ± 1.9 n22 F distichus, Masset Inlet @ YakounYellow River, 05/14/90 n 21.0 ± 1.7 n23 F. distichus, Kitimat GiltoyeesInlet, 05/10/91 n n 1.8 ±0.524 F distichus, Pendrell Sound @Island beach, 05/23/91 n n 25.1 ±0.425 Alaria sp., Read Island OysterLease Farm, 05/23/91 n n 24.6 ± 0.4Mean Arsenic Concentration per Sampling Year 22.5 21.0 15.8Overall Mean Arsenic Concentration for the 19.8 $.Lg/g AsThree Years Sampling Periodn = The seaweed was not sampled at this location for the applicable year;y = The seaweed was sampled at this location for the applicable yearTable 4.2Arsenic Concentrations in Some Red Algaeof British Columbia Sampled in1989 and 1991Two Hour Wet Digestion!Freeze Dried Seaweed Continuous HGAA, p.g/g AsNo. Collection Site and Date 1989 19911 Iridaea splendeus, Juan Perez Sound @Hot Spring Island beach, 04/17/89 4.4 ± 0.1 n2 Callophyllis edentaota, Rennell Sound @EllisBay, 05/18/91 n 39.7±0.53 Endocladia muricata, Quatsino Sound @VameyBay, 05/21/91 n 4.5 ±0.54 Endocladia Inuricata, Langara Island @Langara Island, 05/21/91 n 1.3 ± 0.5Mean Arsenic Concentration per Sampling Year 4.4 15.2Overall Mean Arsenic Concentration for the Two 12.5 J.Lg/g AsYears Sampling Periodn = This seaweed was not sampled at this location for the applicable year126Table 4.3Arsenic Concentrations in Some Green Algaeof British Columbia Sampled in 1989 and 1991Two Hour Wet Digestion!Freeze Dried Seaweed Continuous HGAA, .tg/g AsNo. Collection Site and Date 1989 ] 19911 Enteromorpha sp., Howe Sound @Furry Creek, 07/07/89 3.4 ± 0.3 n2 Ulva Fenestrata, Rennell Sound EllisBay,05/18/91 n 3.6±0.53 Ulva sp. (green sea lettuce), QuatsinoSound@Varneybay,05.21.91 n nd4 Codium sp., Anthony Island AnthonyIsland,05/20/91 n 14.3 ±0.45 Ulva sp. (green sea lettuce), LangaraIsland Langara Island, 05/17/91 n 4.2 ± 0.56 Green algae (filamentus type), QuatsinoSound @ Vamey Bay, 05/21/9 1 n 1.3 ± 0.57 Green algae, Quatsino Sound VarneyBay, 05/21/91 n 27.2 ± 0.4Mean Arsenic Concentration Per Sampling Year 3.4 8.4Overall Mean Arsenic Concentration for theTwo Years Sampling Period 7.7 p.g/g Asn= This seaweed was not sampled at this location for the applicable year;nd = Arsenic was not detected by the two hour wet digestion/continuous HGAA analysis andtherefore, taken to be [Asj0Arsenic accumulation was observed to vary also between the different species thatmake up a particular class of the macroalgae analyzed. Possible variation of accumulatedarsenic related to the site of collection is observable in the green algae samples collected fromQuatsino Sound. At Varney Bay, Quatsino Sound, a filamentus green algae was found tocontain 1.3 j.iglg As whereas, another species of the green algae at the same site wasdetermined to have accumulated 27.2 As. These observed variations in accumulatedarsenic within this class of algae are not related to different collection dates. The overall meanarsenic concentratin determined from all samples of red algae (Table 4.2) analysed between1989 and 1991 was found to be 12.5 j.ig/g As. The overall mean arsenic concentration127determined similarly from all samples of green algae (Table 4.3) analyzed between 1989 and1991 was calculated to be As.In summazy, the mean arsenic concentrations for the three classes of marine algaecollected from British Columbia between 1989 and 1991 are given in Table 4.4. These overallmean concentrations revealed that the brown algae accumulated higher amounts of arsenicwhen compared to the other two, the green and the red algae. However, high accumulation ofarsenic cannot be considered exclusive only to the brown algae since some species of the otherclasses also accumulate relatively very high arsenic. It was also observed that, accumulation ofarsenic to a high or low extent, can be dependent on the location as well as the particularspecies of the marine macroalgae.Table 4.4Mean Arsenic Concentrations forThe Three Classes ofMarine AlgaeCollected from British ColumbiaBetween 1989 - 1991No. -J Seaweed Class/Total Number Overall Mean Total Arsenic(and Species) Sampled Concentration, Ig/g As1 Brown algae (Phaeophyta), 25, (4 species) 19.32 Green algae (Chlorophyta), 8, (3 known,3 unknown species) 7.73 LRed algae (Rhodophyta), 4, (3 species) 12.5Other arsenic concentration data for seaweeds from most of the areas investigated inthe present study are not available for comparison. However, some workers(47, 49, 206) havestudied and determined arsenic concentrations in some marine macroalgae from the Atlanticprovinces of Canada and in British Columbia. The total arsenic concentrations obtained inthese studies are given in Table 3.11 and Table 4.5. In Sanders(49) arsenic analysis, the brownalgae accumulated on the average more arsenic than either the green algae or the red algae(10.3 vs 1.43, and l.54gAs respectively). According to the arsenic concentration data onsome seaweeds obtained from British Columbia and the Atlantic provinces of Canada(47, 206)128shown in Table 4.5, sini1ar arsenic accumulation patterns were observed between the threeclasses of seaweeds. This conclusion was also reached in the present study as discussedpreviously. Furthermore, it can be observed in these studies(47, 49, 206), also in agreementwith the present study, that arsenic accumulation varied between species of the same class ofseaweed as well as between the different classes. However, due to lack of knowledgeregarding the various sampling locations in these other studies, information cannot beascertained comparatively about the relationship of the collection site to the arsenic classes ofmacroalgae studied, as in the present work.Generally, the areas sampled in British Columbia in these other studies(47, 49) haveinfluences of large manmade activities and as such, arsenic contamination could be expected.The veiy high arsenic concentrations found in the seaweeds analyzed from these locationssuggests possible potential arsenic accumulation related mainly to the collection sites. It can bereasonably inferred, therefore, that the sampling site plays a significant role in the accumulatedarsenic found in seaweeds, as was previously suggested in the present work.129Table 4.5IArsenic Concentration Data onSome Seaweeds Collected fromThe Atlantic Provinces and British Columbia of Canada(47, 206)Atlantic Province ig/g As (thy wt) British Columbia g/g As (thy wt)Seaweed Class/Species Total Arsenic Total ArsenicBrown algae (Phaeophyceae)-Agarumfimbriatum 1 na 45.6- Costaria costata 1 na 40.8- Cymathere triplicata 1 na 59.3- Hedophyllum sessile 2 na 57.3- Laminaria groenlandica 1 na 91.9- Laminaria groenlandica 2 na 89.4- Laminaria groenlandica 3 na 88.9- Laminaria saccharina 2 na 57.5- Laminaria setchellil 2 na 41.5- Laminaria longicruris 4 52 na- Laminaria digitata 4 50 na-Fucusvesicutosus4 58 na-Ascophyllum nodosum 4 38 na-Alarianana2 na 43.7- Egregia menziesii 2 na 52.6- Pterygophora caljfornica, fronds 2 na 61.5- Nereocystis luetkeana 5 na 141.2-Macrocystis integrzfolia 6 na 67.9- Lessoniopsis littoralis 2 na 45.3- Sargassum mubicum 2 na 54.8OVERALL MEAN 49.5 65.0Red algae Rhodophyceae)- Chondrus cr1spus 7 5 na- Phyllophora membranifolia 4 — na- Habsaccion ramentaceum 4 8 na-Ahnfeltic plicata 4 2 na- Phodymenia palmata 4 10 naOVERALL MEAN 5 naGreen algae (Chlorophyceae)- Ulva lactuca 4 4 na- Spongomorpha arcta 4 8 naOVERALL MEAN 6 nana, not available/not sampled,1, Albert Head, British Columbia; 2, Glacier Point, British Columbia;3, Bamfield, British Columbia; 4 Southwestern shores of Nova Scotia;5, Stanley Park, British Columbia; 6, Sooke, British Columbia;7, Paddy’s Head, Nova Scotia1304.2 DETERMINATION OF REDUCIBLE AND “HIDDEN” ARSENIC SPECIES INSOME MARiNE ALGAE OF BRITISH COLUMBIAFreeze dried seaweed samples were subjected to digestion either in the solid freezedried (homogenized using pestle and mortar) state and/or after extraction of the freeze driedseaweed sample using either water/methanol solvent system and/or sodium hydroxide solution.Overnight ultrasonication was employed for all the extraction procedures. Arsenic speciationwas performed on the extracts before and after the sodium hydroxide digestion procedure.Furthermore, the seaweed residue left over from the extraction procedure was also subjected tothe (sodium hydroxide) digestion procedure and arsenic speciation analysis was similarlycarried out on the subsequent digest obtained. The sodium hydroxide digestion procedure hasbeen described in Sections 3.2.2 and CONCENTRATIONS OF ARSENIC SPECIES IN FREEZE DRIED SEAWEEDSAMPLES AFTER THE HOT BASE DIGESTIONFreeze dried seaweed samples were digested by using the hot base digestion detailed inSection and the digestions were analyzed by using the semi-continuous mode HG-GCAAS. The following arsenic concentrations (data) shown in Table 4.6 were obtained. Thesearsenic data represent the species present in the digest, and, in the absence of any broken “AsC” bonds, represent the speciation of arsenicals contained in the macroalge.131Table 4.6Concentrations ofArsenic Species inHot Base Digestion of Freeze Dried Seaweed SamplesAnalysed by the Semi-Continuous HG-GC-AAS TechniqueAlgae Sample Analyzed Arsenic Concentration, tg/g As(See Referenced Table for Inorg. AsSample Description) IAs(V)1 MMAA DMAA Total AsTable 4.1, #2 d d 22.3 22.3Table 4.1, #3 d 0.2 26.8 26.9Table 4.1, #9 0.2 0.3 23.1 23.6Table 4.1, #11 d 0.03 39.0 39.1Table 4.1, #12 d d 26.8 26.8Table 4.1, #13 d 0.0 17.0 17.0Table 4.1, #14 d 0.4 18.4 18.8Table 4.1, #15 0.6 0.7 11.0 12.3Table 4.1, #16 0.3 0.3 19.3 19.8Table 4.1, #18 d nd 18.5 18.5Table4.1,#20 0.9 0.4 19.7 21.0Table 4.1, #21 0.3 0.3 22.6 23.2Table 4.1, #22 d nd 19.4 19.4Table4.2,#1 d 0.1 4.4 4.5Table 4.3, #1 2.7 nd 1.7 4.4na, Nereocystis Iuetkeana, d 0.1 35.9 36.0Rupert Inlet Southernside,10/28/87d, detected; nd, not detected; Inorg. As (As [V]), Inorganic Arsenic found as arsenate onlyusing acetic acid/NaBH4and hydrochloric acidfNaBH4reduction media, na, not analyzedpreviouslyThe total arsenic concentrations obtained (Table 4.6) from the sum of the individualarsenic species concentrations are in good agreement with the previous total arsenicdeterminations done on same seaweed samples (Table 4.1, 4.2 and 4.3). The use of thesodium hydroxide digestion procedure for sample dissolution therefore, enables not only acomplete digestion affording good speciation information from its digest but also, total arsenicdetermination. The major arsenic species in the digested macroalgae is the dimethylated arseniccompound (DMAA). However, a green macroalgae, Enteromorpha sp. collected from HoweSound in July, 1989 (different sampling locality compared to the others collected via access totheir locations using the cruiseship) contained about 61% of its total arsenic content asinorganic arsenate. Sanders(49) determined high accumulation of inorganic arsenic (47% oftotal arsenic content) in 16 species of green algae. However, this speciation study(49) has been132suspected as unreliable by Morita and Shibata(175), as well as in the present study. Otherstudies(48, 175) have reliably indicated an inorganic arsenate as a major (50%) arsenical inHizildafiis!forine, a brown algae.4.2.2 SPECIATION OF REDUCIBLE AND “HIDDEN” ARSENIC SPECIES INAQUEOUS METHANOL EXTRACTS OF SOME MARINE ALGAEGenerally, knowledge of the arsenic species within marine organisms is obtained bycanying out various extraction procedures(246, 271). However, when speciation informationis required, the use of strongly acidic or basic conditions is usually not recommended(271).Isolation of algal arsenic compounds is best carried out using as mild conditions as possible,such as provided in such extraction solvents like dilute acid(47, 49) or base(206, 246, 253),organic solvents(27, 28, 95, 272-274) (for example methanol, chloroform) and also water(98).“Algal Arsenic” has been generally and successfully isolated by using methanol extraction(28,95). Also, speciation information has been obtained from digests of algal obtained afterextraction in dilute sodium hydroxide(246, 253). This latter aspect, using dilute NaOHextraction is discussed with speciation results in Section 4.2.3. In this section, the investigationof arsenic forms in the digests of extracts obtained from the extraction of some freeze driedseaweeds using aqueous methanol (following the procedures detailed in Section ispresented in Table 4.7. The arsenic species in the digests were analyzed by using the semicontinuous mode HG-GC-AAS.133Cl)Cl)CCl) Cl).B-.Cl)—.0H0H0-30H0HOH0H0H04-I—I—I-’-I—%Q)LJ<-o<CDCDCDr4 HHHHHHHHH3H’000000000D)HHHHHHHHH£zg—.CDCDCD—.-l+00+0+00+0+0+0+0CD‘—Cl).CZ I-ICCCCCCCCCCCC-CCCCC;_.:D.———CD‘CCCCCC>CD—PPLLLLUCLLULUJJULLHJLHJULHLICIgII!-iL3IiIIINIIIHIIIhIIIHIIIIiIIIIIII[i‘‘v-1u1-iIiI’Iii.IIIbINII’oIIIIIcolji1—INioIiitIhotNIic’1IE.CDir-1iII-CD -4Algae Sample Samples (ME, R)Analyzed (See Analysed With %Referenced Table for (V.’) & Without (W/O) Arsenic Concentration .tgIg As TMHidden”Sample Description) Hot Base Digestion Inor. As MMAA DMAA Total As ArsenicTable 4.1 MEWIO 0.2 0.0 3.3 3.5#18 MEW d 0.2 10.2 10.4 66.3RW 0.0 0.1 7.1 7.2 —OVERALL TOTAL (MEW + RW) [As] 0.0 0.4 17.3 17.7 (59.O)aTable 4.1 MEWIO 0.1 0.0 0.1 0.1#20 MEW 0.3 0.4 17.1 17.8 94.7RW d nd 2.0 2.0OVERALL TOTAL (MEW + RW) [As] 0.3 0.4 19.1 19.8 (90.O)aTable 4.1 MEW/O 0.2 0.1 0.9 1.2#21 MEW 0.3 0.2 20.9 21.4 94.5RW d d 1.9 1.9OVERALL TOTAL (MEW + RW) [As] 0.3 0.2 22.8 23.3 (9l.g)aTable 4.1 MEW/O 0.9 0.0 0.9 1.8#22 MEW. 0.7 0.2 21.2 22.2 92.1RW 0.1 0.0 3.5 3.6OVERALL TOTAL (MEW + RW) [AsI 0.8 0.3 24.7 25.7 (86•2)aTable 4.2 MEW/O d d 0.2 0.2#1 MEW d 0.0 4.1 4.1 94.2RW d nd 0.3 0.3OVERALL TOTAL (MEW ÷ RW) [As] d 0.0 4.4 4.4 (94.1)aTable 4.3 MEW/O 0.1 nd d 0.1#1 MEW d nd 0.1 0.1 38.5RW 1.9 nd 0.9 2.8OVERALL TOTAL (MEW + RW) [As] 1.9 nd 1.0 2.9 (44)ana, Nereocystis luetkeana, MEW/O d 0.0 2.9 2.9Rupert Inlet @ the MEW 0.6 0.7 30.4 31.7 90.7southernside, 10/28/87 RW d 0.0 4.3 4.3OVERALL TOTAL (MEW +RW) [As] 0.6 0.7 34.7 35.9OVERALL MEAN Arsenic Concentration for all 0.3 0.3 22.3 22.8 92.1Brown Algae Analysed (g63)aME, methanol (aqueous) extract; R, Residue (seaweed) from aqueous methanol extraction;d, detected; nd, not detected; a, the relative amount of arsenic compared to total Arsenic (%) extracted fromseaweed sample by using aqueous methanol; MEAN, of overall total [As]; na, not analyzed previouslyThe concentrations of arsenic in the form of inorganic arsenic (arsenate and arsenite),MMAA and DMAA were obtained for the methanol extracts of all the analysed seaweedsamples before (MEWIO) and after (MEW) they were subjected to the hot base digestion. Theseaweed residue from the methanol extraction was base digested (RW) and the arsenic formsdetermined in the digest. The overall mean arsenic concentrations for the brown algaeindicated that 92% of the accumulated arsenic existed as “hidden” arsenic compounds. Also,135more than 86% of the total arsenic accumulated in brown algae can be extracted with aqueousmethanol. Exceptions are seen with the F distichus collected from Anyox and Alice Arm.Both of these sites are in the same locality (Figure 2.5); however, proximity cannot explain thelower amounts of methanol extractable arsenic when compared to another algae (HastingsArm) collected in the same locality. Also, a lower amount of methanol extractable arsenic(—39%) was determined for the green algae, Enieromorpha sp. It seems probable thatdifferences in the chemical forms of arsenic species contained in these algae(175) areresponsible for variations in the amount of extractable arsenic by using methanol.4.2.3 SPECIATION OF REDUCIBLE AND “HIDDEN” ARSENIC SPECIES INDILUTE SODIUM HYDROXIDE EXTRACTS OF SOME MARINE ALGAEDilute sodium hydroxide solution (1M3) was employed for the extraction of arsenicspecies from some of the freeze dried seaweed samples. Details of the extractions are given inSection The following arsenic speciation results, shown in Table 4.8, were obtained byusing the semi-continuous mode HG-GC-AAS.136Cl)J2—.rDOO o0oCl)rGojI---..0H00H0O0000tJP-.cr1 c F‘-F‘-F‘-F‘-F‘-F‘-F‘-F‘-Fr’rr’r-’HHHHHHHHH000000000HHHHHHHHH+O0000+0-O0‘InInInInInInInIn——--—---o‘—c-.p..p..p..p..p..p..p..p..p..p..p.p..p.p..--—---—------—---—-—---—---—---—--p..p..p..p...........p....p..p.©p....z() Ct’--;P0P..00cnP..C0000..t’300000P..‘>In—‘j—..’=‘0000000000‘00CtCt%p-CtCtIflp.p.00‘-.3p.I—.)-p..p..,Algae Sample Samples (SE, R)Analyzed (See Analysed th (W) & %Referenced Table for Without (W/O) Arsenic Concentration tg/g As “Hidden”Sample Description) Hot Base Digestion Inor. As IvIMAA DMAA Total As ArsenicTable 4.1 SEWIO d 0.0 9.7 9.8#18 SEW ud ud ud ud udRW d ml 1.1 1.1OVERALL TOTAL (SEW + RW)[AsJ ud ud ud ud udTable 4.1 1 SEW/O d nd 3.8 3.8#20 j SEW d nd 15.9 15.9 76.1JRW d nd 1.6 1.6OVERALL TOTAL J (SEW + RW)[AsJ d nd 17.5 17.5 (909)aTable 4.1 SEW/O d nd 2.0 2.0#21 SEW d d 23.2 23.2 91.5RW d nd 0.6 0.6OVERALL TOTAL (SEW + RW)[As] d d 23.8 23.8 (975)aTable 4.1 SEW/O d nd 3.9 3.9#22 SEW d 0.0 22.3 22.3 82.7RW d nd 1.4 1.4OVERALLTOTAL (SEW+RW)[As] d 0.0 23.6 23.7 (94.l)aTable 4.2 SEW/O d nd 0.6 0.6#1 SEW ud ud ud ud udRW d nd 0.2 0.2OVERALL TOTAL (SEW + RW)[As] ud ud ud ud udTable 4.3 SEW/O 1.6 nd 0.5 2.1#1 SEW ud ud ud ud udRW d nd 0.1 0.1OVERALL TOTAL (SEW + RW)[As] ud ud ud ud udna, Nereocystis luetkeana, SEW/O d 0.1 10.1 10.2Rupert Inlet @ the SEW ud ud ud ud udSouthernside, 10/28/87 RW d nd 2.3 2.3OVERALL TOTAL (SEW + RW)[AsJ ud ud ud ud udOVERALL MEAN Arsenic Concentration for all d d 21.63 T 21.69 85.5Brown Algae Analysed (93.4)aSE, sodium hydroxide (1M)extract; R, Residue (seaweed) from 1M NaOH extraction;d, detected; nd, not detected; ud, un-determined; a, the relative amount of arsenic compared to total Arsenic (%)extracted from seaweed sample by using aqueous methanol; MEAN, of overall total [As]; na, not analyzedpreviouslyIThe concentration of arsenic in the forms of inorganic arsenic (arsenate and arsenite),MMAA and DMAA was determined in the base extracts; the analysis was performed with andwithout hot base digestion. The seaweed residue was digested prior to analysis. The relativeamount of arsenic compared to the total arsenic in the seaweed extractable by using 1Msodium hydroxide was generally good for all the macroalgae. The speciation results using the138dilute NaOH extraction also revealed that greater than 85% of the extractable arsenic speciesexists as ‘hidden” arsenic forms in the brown algae (F distichus). When this extractionmethod is compared with the methanol extraction, the speciation results reveal that largerconcentrations of reducible dimethylarsinic acid were found in samples extracted in the 1MNaOH solution. This probably indicates that the major ‘hidden” arsenic species (DMA forms)present in the macroalgae samples are sensitive to the base extraction, more than whenextracted in methanol. Therefore, in the present study, the methanol extraction procedure wasemployed for the isolation of arsenic species present in F. distichus. Others(28, 97) have alsocarried out arsenic species isolation in seaweeds by using methanol extraction.4.3 ISOLATION AND IDENTIFICATION OF F. DISTICHUS ARSENICALS4.3.1 PURIFICATION AND ISOLATION OF WATER SOLUBLE “ALGAL”ARSENIC COMPOUNDS IN F. DISTICHUSIn this section, the studies carried out on the examination of the arsenic constituents ofthe brown alga, Fucus distichus collected from the Head of Hastings Arm in British Columbia(Figure 2.5) are presented. A combination of chromatographic purification procedures(Section 2.5) with both off-line and on-line detection was used for arsenic species identificationas described in Section 3.4.The F distichus sample was extracted in methanol and the extracts taken through theextraction procedures schematically detailed in Figure 2.7 (Section 2.5.1). The scheme was amodification of Edmonds(95) procedure. The total arsenic concentration in the seaweed (6.77x io g) was determined by using the 2 hour wet digestion/continuous-mode HGAAS and wasfound to be 0.87 .ig/g (wet weight). Extraction of the seaweed by using aqueous methanolremoved into solution 91.6% of the total arsenic. The residue (90.01 g) obtained afterevaporation of the methanol was dissolved in water and extracted with diethyl ether. The ethersoluble layer containing 5.2% of the methanol extractible arsenic was discarded. The contentsof the water layer, after evaporation, were dissolved/suspended in methanol and poured into139acetone. This procedure resulted in a solution containing the less polar material and aprecipitate of the polar substances which was filtered/separated and dissolved in water(58.70 g, 3.83 mg As). This water soluble extract was further re-extracted by usingmethanol/chloroform (1:1) to remove lipid soluble materials. The pale greenchloroform/methanol layer containing 4.1% of the total water-soluble arsenic concentration wasdiscarded. The top aqueous layer after evaporation, was re-extracted in methanol and theinsoluble salty material separated by filtration. Arsenic speciation of this insoluble saltymaterial carried out by using the hot-base digestion followed by the semi-continuous modeHG-GC-AAS, revealed a total arsenic amount of 20 ig As being the sum of the amounts ofinorganic arsenic (0.28%), DMAA (0.49%) and the “hidden” arsenic compounds (99.23%).This insoluble material was not further analyzed. The aqueous methanol soluble layer wasevaporated (31.14 g, 3.65 mg As) and dissolved in deionized water. The clean-ups of theextract were achieved by using repeated gel permeation chromatography on Sephadex LH2O(water and methanol served as the mobile phase). The arsenic containing fractions whicheluted between 60m1 and 277 ml (methanol as mobile phase) were combined and evaporated toasyrup(26.70g,3.11 mgAs).The arsenic containing syrup was dissolved and equilibrated by using 0.05M Trisbuffer prior to chromatography on a weak anion exchange DEAF Sephadex A25 (2.5 x 40 cm)column. The details of this purification step and others are schematically shown in Figures 2.8and 2.9 (Section 2.5.1) The retention volumes of the arsenic containing fractions which elutedwhen 0.05M Tns buffer was used as the mobile phase are from 179 ml to 400 ml. This wasthe major arsenic containing band, FEB (27.08 g, 3.01 mg As). The minor arsenic containingband, LEB, eluted on the weak anion exchanger when 0.5M Tris buffer was used as the mobilephase between 1070 ml and 1476 ml. It was evaporated as a syrup (12.29 g, 0.20 mg As).The above isolation on the weak anion exchanger is shown in Figure 4.1.140S..8U8‘SFigure 4.1Isolation of FEB and LEB on DEAE Sephadex A25Using Tris buffer, pH 8.0 (0.05M, 0.5M) asMobile Phase6548210-1100 850 600 850 1100 1850 1600Elution Volume (ml)The increase in the total syrup weights is attributed to the presence of the Tns buffersalt which was later removed on a gpc Sephadex LH2O column. The amounts of “hidden”arsenic species present in the FEB and LEB isolates, shown in Table 4.9, were investigated byusing the three hour hot-base digestion followed by the semi-continuous mode HG-GC-AAStechnique.141Table 4.9Amounts of “Hidden” Arsenic Species inFEB and LEB Isolates Using Hot-Base Digestionand Semi-Continuous Mode HG-GC-AAS TechniqueF distichus % Hidden Arsenic SpeciesIsolate on A25 Column “Hidden” ArsenicalsNo.1 FEB 98.92 LEB 99.9Both isolates were found to contain — > 99% of the total arsenic as ‘hidden” arsenicspecies. A further re-extraction of FEB in methanol removed some white precipitate(2.95 g, 7.74 .tg As) which was discarded. The methanol soluble FEB was evaporated (24.13g syrup) and was dissolved in 0.05M Tris buffer, pH 8.0 (50 ml). The Tris buffer - FEBextract solution was further chromatographed on DEAE Sephadex A25 (2.5 x 50 cm) by using0.05M Tris buffer, pH 8.0 as mobile phase. A major arsenic containing band was obtained.However, because of tailing the band was arbitrarily separated into two fractions: a fast bandwith retention volumes from 156 ml to 330 ml labelled as FEBA; and a later eluting minorband which eluted at retention volumes 331 ml to 770 ml, labelled as FEBB. The isolates,FEBA and FEBB were evaporated to syrups (19.49 g, 2.90 mg As FEBA; and 3.98 g, 0.15 mgAs FEBB) and were separately dissolved in aqueous methanol (1:4; water:methanol). Themajor arsenic containing isolate, FEBA (19.11 g syrup, 2.89 mg As) which was dissolved inaqueous methanol (35 ml), was applied unto a Sephadex LH2O column (2.5 x 82 cm, isocraticelution using 4:1 methanol:water as the mobile phase). The elution profile obtained afterGFAA analysis indicated only one arsenic containing band which eluted between 154 ml and449 ml. This was evaporated to a syrup (17.87 g, 2.66 .ig As, FEBA).142The minor arsenic containing band, FEBB, (3.84 g, 140 g As) was dissolved inaqueous methanol to give a lOmi solution which was chromatographed on a Sephadex G15column (2.5 x 55 cm, isocratic elution using 4:1 methanol:water as mobile phase). On this gpccolumn, FEBB separated into two arsenic containing bands. The early eluting major arseniccontaining band (labelled FEBB1) eluted between retention volumes 105 ml and 275 ml andafforded a syrup (2.19 g, 135 .tg As) after evaporation. The later eluting minor arseniccontaining band (labelled FEBB2) eluted between retention volumes 318 ml and 613 ml andalso afforded a syrup (0.20 g, 13 jig As) after evaporation.Similarly, the late eluting band LEB (Figures 2.8 and 4.1) was furtherchromatographed on Sephadex 015 column (2.5 x 60 cm, water used as mobile phase) inorder to remove the Tris buffer salt. The isolate, LEB (10.83 g, 0.18 mg As) was dissolved inwater to make a 30 ml solution and was reapplied onto the gpc column. Fractionation andsubsequent GFAA analysis revealed an arsenic containing band eluting between retentionvolumes 54 ml and 342 ml. The LEB arsenic band was evaporated to a creamy syrup (7.97 g,213 jig As). Because this is a minor arsenic containing band (containing only 6.6% of the totalisolated arsenic in the F. disrichus extract) it was not further purified. However, the arsenicspecies in the sample were investigated by using the on-line detection technique discussed laterin Section 4.3.2. Further chromatography with the objective of achieving additionalpurification was performed on the isolates FEBA, FEBBI and FEBB2 as will be discussedbelow.FEBA was chromatographed on a weak cation exchanger, CM Sephadex C25(25 x 42 cm, using 0. imol dm-3 ammonium acetate pH 6.5 as mobile phase) resulting in a veryfast arsenic containing band with retention volumes between 50 ml and 258 ml. The syrup(12.58 g, 2.20 mg As) obtained after evaporating the FEBA fractions from the cationexchanger was subjected to further chromatographic purification by using cellulose tic andpreparative HPLC. A 7 ml aliquot portion of FEBA in water (33.5 ml) was applied ontocellulose plates and chromatographic development was carried out by using 60:15:25143n-butanoi:acetic acid:water. Examination of 1.0cm layers of each tic plates used, (eachextracted into 5 ml deionized water) revealed a broad arsenic containing band, Rf 0.19 to Rf0.44. These fractions (from only the 7ml aliquot chromatographed) were combined andevaporated to syrup (2.32 g, 0.40 mg As).The tic FEBA isolate was next dissolved in deionized water (2.32 g, 3.5 ml) and wassubjected to preparative HPLC on a C 18 reverse phase column, flow rate 5 mI/mm, elutingwith aqueous methanol (4:1). This method has been used in separating some arsenic speciespreviously(27, 95). Arsenic containing fractions eiuted between retention times 6 mm and 10mm and afforded a syrup (1.40 g, 0.36 mg As) after evaporation. The syrup was examinedsubsequently for arsenic species by using on-line detection techniques, discussed inSection 4.3.2.Similar purification procedures were performed on the isolates FEBB I and FEBB2 byusing cellulose thin layer chromatography (with same conditions as previously discussed).FEBBI revealed one arsenic containing band having flow rates, Rf 0.13-0.53. It is probablethat this arsenic band could be related to the FEBA isolate on the basis of their overlappingflow rates. The tic fractions afforded a syrup (0.85 g, 0.14 mg As) after evaporation. TheFEBB1 syrup was dissolved in water:methanol (4:1) and injected repeatedly onto thepreparative HPLC C 18 reverse phase column described above. Two arsenic containing bandswere obtained; some tailing was observed from the earlier eluting peak which necessitated anarbitraiy separation of the two peaks. The earlier eluting peak was recovered betweenretention times 5 mm and 17 mm, which afforded a syrup [0.70 g, 0.13 mg As, FEBBI(a)].The later eluting minor arsenic containing fractions were recovered between retention times 25mm and 41 mm, and this afforded a syrup [15 mg, 8.12 .tg As, FEBB1(b)]. These isolateswere examined for arsenic species by using the on-line 1-fPLC-ICPMS analysis described inSection 4.3.2.The purification of FEBB2 isolate was carried Out in a similar manner by using thinlayer chromatographic procedures as described above. The elution profile from the cellulose tic144Irevealed a major arsenic containing band at Rf 0.50 - 0.78 (10.91 tg As). Some arseniccontaining material remained at the origin (0.33 jig As) and other minor arsenic containingfractions were recovered at Rf 0.22 (0.68 jig As), Rf 0.33 (0.41 jig As), and Rf 0.89 (0.18 jigAs). Some of these minor isolates are probably due to tailing or band broadening. The ticisolates were not further chromatographed; however, they were investigated further by usingthe on-line HPLC-ICPMS as described in Section IDENTIFICATION AND CHARACTERIZATION OF ARSENIC SPECIES INCHROMATOGRAPHIC ISOLATES OF WATER SOLUBLE EXTRACTS OFF. DISTICHUS BY USING HPLC-MIC-HGAA AND HPLC-ICPMS ON LINEDETECTION TECHNIQUES4.3.2.1 Identification and Characterization of Arsenic Containing Isolates by theHPLC-Mic-HGAA Analysis:The Mic-HGAA described in Section 3.4.1 can be used to differentiate the ‘hidden”organoarsenicals from the reducible marine organoarsenic compounds such as arsenate,arsenite, MMAA and DMAA. It can also be used as a detector for HPLC(1 7). The methodwas used to analyse some arsenic containing isolates, obtained from F distichus:FEBA, LEB,and FEBB (Figure 2.8) after a weak anion exchange and gel permeation chromatography.Table 4.10Reducible and “Hidden” arsenic SpeciesDetermined in Some Chromatographic Isolates ofWater Soluble Extracts ofF. distichusAfter the Mic-HGAA AnalysisArsenic Containing Reducible Species “Hidden” Species Total# Isolates jig As % As jig As % As jig As1 FEBA 1.74x 102 6.0 2.73x l0 94.0 2.90x 102 FEBB / 4.59 3.1 1.45 x 102 96.9 1.50 x 1023 LEB 3.89 2.2 l.76x 102 97.8 1.80x 102145IAll the isolates were found, not unexpectedly, to contain high percentages (> 94%) of‘hidden” organoarsenicals which are likely to be arsenic-containing ribofuranosides commonlyfound in water soluble extracts ofmarine macro algae(28, 95, 271).The HPLC-Mic-HGAA analysis was carried out on arsenosugar fl, and thechromatograms shown in Figures 4.2 and 4.3 were obtained by using two flow rate conditionswith the HPLC system. The HPLC system consisted of a phenomenex C 18 reverse phasecolumn and 10mM tetraethylammonium hydroxide + 4mM malonic acid, pH 6.8 used as themobile phase. The two flow rates used are 1.0 and 0.8 mi/mm. Through retention timescomparison, additional speciation information were obtained for FEBA, FEBB and LEBBisolates.Figures 4.2 and 4.3Figure 4.3Figure 4.3: Chromatogram Obtained forArsenosugar ha (Table 1.2) UsingHPLC-Mic-HGAA Analysis WhenMobile Flow Rate was O.8m1 mm’Figure 4.2y axis = absorbancekeiention Time(s)Figure 4.2: Chroniatogram Obtained forArsenosugar ha (Table 1.2) UsingHPLC-Mic-HGAA Analysis WhenMobile Flow Rate was 1.Oml mm’146When the flow rate was 1.0 ml mm4, the arsenosugar eluted at retention time 5.92 secwith some tailing. However, when the arsenosugar was eluted by using 0.8 ml min’ flow rate,it was recovered at retention time 7.92 sec without any tailing. When the FEBA isolate wassubjected to the HPLC-Mic-HGAA using 1.0 ml mind flow rate, a broad arsenic peak wasrecovered at retention time 7.58 sec. The unresolved broadness of the arsenic peak suggests thepossiblity of more than one similar arsenic species in the isolate. When the FEBB isolate wassimilarly analysed by using 1.0 ml mind flow rate, two arsenic peaks at 5.77 sec and 6.74 secwere observed. When using 0.8 ml mind, two arsenic peaks at 7.35 sec (early eluting peak)and 8.47 sec (later eluting peak) were observed; in this instance, the FEBB isolate was notspiked with the standard arsenosugar fl. When the FEBB isolate was spiked with thearsenosugar, two unresolved peaks were observed; the later eluting peak became broader andwith manifold increase in absorbance. These unresolved peaks when spiked with the standardarsenosugar probably suggests that the two peaks have structural similarity to the arsenosugarstandard. However, the later eluting peak in the FEBB isolate, due to its manifold broadnessand increased absorbance, seems to suggest similar identity with the arsenosugar standard iii!.Further, the HPLC-Mic-HGAA analysis was used to investigate the LEB isolate.When 1.0 mI/mm was used, two arsenic containing peaks were observed at retention times5.33 sec and 6.41 sec. When the LEB sample was spiked with the arsenosugar standard, threearsenic peaks were observed. The first and last eluting peaks were identical with thepreviously observed peaks in the LEB isolates. The middle, third peak, matched with theretention time of the arsenosugar standard. This result indicates that the arsenic species presentin the LEB are not the same as the arsenosugar fl. Characterization of Arsenic-Containing Isolates by Using HPLC-ICPMSAnalysisThe HPLC separations (on-line with the ICPMS system) were first carried out by usingaPhenomenex C 18 reverse phase column (300mm x 3.9mm id) and 10mM heptanesulfonate147+ 0.1% methanol at pH 3.5 as the mobile phase. Five standard arsenic compounds, arsenite,arsenate, MMAA, DMAA and arsenobetaine are separated under these conditions as shown inFigure 4.4. The chromatograms shown in Figures 4.5and 4.6 were obtained by using HPLCICPMS on the FEBA and LED isolates.Figure 4.4Cliroinatogram Obtained From a Mixture (2Ong each) ofA rscnitc, A rscna fe, MMAA, DMAA and Arscnobctaincby Using lIPLc-ICPMS9 ArsnatS-4bgArsenife90 lAO 270 3(0 450 54030 720Tinic ()148-t‘-I4-0C-)4-CCFigure 4.5Chromatogram Obtained From the FEBA Isolate ofF. di.cticl,us by Using IIPLC-ICPMS12iiI09A765432I032282420I!A490 iRo 270 3o 450 0 630 720lime ()Figure 4.6Chromafogram Obtained From the LEB Isolate ofF. distichus by Using II PLC-ICPMS..(I/ 90 ISO 270 360 450 540 630Time ()720149The HPLC condition employed did not separate the isolates peaks (Figures 4.5 and4.6) from the coeluted reducible arsenicals (Figure 4.4), inorganic arsenite and MMAA.However, because the major arsenicals present in these isolates have been determined to be thedimethylated ‘hidden” arsenic species, these arsenic containing peaks can not be arseniteand/or MrvIAA or both. The same isolates were examined under other HPLC conditions asfollows:The first, condition 1, utilized a Waters C 18 reverse phase column (300 x 3.9 mm id)and 10mM tetraethylammonium hydroxide + 4.5mM malonic acid at pH 6.8, as the mobilephase. The flow rate for this system was 0.8 ml min’. The second, condition 2, employed aPhenomenex C 18 reverse phase column (300 x 3.9 mm id), and 25mM tetramethylammoniumhydroxide + 25mM malonic acid at pH 6.8, as the mobile phase. The flow rate of this systemwas 1.0 ml mi&1. The arsenical standards run under the same conditions include thearsenosugar standard jj described previously (Section and a standard referencematerial, NBS 1566 (Oyster tissue, Crassosirea gigas) which has been found to contain notonly DMAA and arsenobetaine, but also two arsenosugars jjj and j(50) (Table 1.2).Chromatograms of arsenosugar ha run under conditions 1 and 2 are shown in Figures 4.7 and4.8 respectively.150Figure 4.7Clirornatogram Obtained for Arscnosugar iJ. by Using Condition 15550., .4540352O,15,.1o•60 120 1O 240 300 360420 4Time (s)Condition I = Waters C18 Reverse Phase Column (33 x 3.9 mmid),lOmMtctracthylammoniuni hydroxide + 4.5mM malonic acid at p’-1 6.8 as mobile.phase. flow rate of0.8 nil mini.Figure 4.8Claroniatograni Obtained for Arsemiosugar ha by Using Condition2807264. 56480j (•.0120 160 240 300 360 420 4QTime (I)Condition 2 = Phenomenex C18 Reverse Phase Column (33 x 3.9 mm id), 25mMtetraethylamnionium hydroxide + 25 mM malonic acid at p1-I 6.8 as mobile phase, flow rate ofLO nil miii1.151IThe retention times for the arsenosugar jj under conditions 1 and 2 after the NPLCICPMS analysis were estimated to be 265 - 334 sec and 226 - 302 sec, respectively. When theextract of the SRM NBS 1566 sample was run using conditions 1 and 2 the chromatogramsshown in Figures 4.9 and 4.10 were obtained respectively.Figure 4.9Chromatogram Obtained for SRM NBS 1566 Oyster Tissue by Using Condition 1161412.! 1008C.)c 6.4201 240cc€ CS)Condition 1 = Waters C18 Reverse Phase Column (33 x 3.9 mm id),lOmMtetraethylammonium hydroxide + 4.5mM malonic acid at pH 6.8 as mobile phase, flow rate of0.8 ml min’.1521614, .121-1° 8420Condition 2 = Phenomenex C18 Reverse Phase Column (33 x 3.9 mm Id), 25mMtetraethylammonium hydroxide + 25mM malonic acid at pH 6.8 as mobile phase, flow rate of1.0 ml miii’.The chromatograms in Figures 4.9 and 4.10 differ slightly, probably as a result ofsampling inhomogeneity(50), although the difference could be due to the different HPLCconditions employed. In Figure 4.9, three arsenic species are seen at retention times 200-246sec. 248-277 sec and 279-33 1 sec. These are arsenobetaine (AB), arsenosugar jj, andarsenosugar fl. The same species are seen in Figure 4.10 including DMAA(17, 50).The chromatograms shown in Figures 4.11 to 4.14 were obtained using both conditions1 and 2 on the FEBA isolate.Figure 4.10Chromatogram Obtained for SRM NBS 1566 Oyster Tissue by Using Condition 210 120 110 240 SQO O 420 4Olim. (e)/153Figure 4.11Clironiatogram Obtained for the Isolate FEBA.Using Condition 1.131211,1o 1I.). 9 iiS H4;.1II!’21o •———-•••——--120 180 24) 300 360 420400Timi (s)Figure 4.12Chroinatogram Obtained for the Isolate FEBA Spiked WithArseiiosugarfl Using Condition 128160Q ..12 I.81 a—ftr.11) 120 1 240 300 360 420 400L.Tim,(s)154Figure 4.13Chroniatogram Obtained for the Isolate FEBA Using Condition 21’.t 7 Ii‘-4. ii6 II4 ‘p.I?0C’) 12) 18’) 240 300 300 42048’)Tim• (a)Figure 4.14Chroniatogram Obtained for the Isolate FEBA Spiked WithArscnosugar jj Using Condition 2109F!4,.32 J i\I ‘iV •..,.___-___d--’/ 6’) 12’) 18’) 24’) 300 30042’) 4)Tma (a)‘55The chromatogram of the FEBA isolate (Figure 4.11) obtained by using condition 1revealed two arsenic compounds labelled F and G. When the FEBA isolate was spiked witharsenosugar jj, the peak G coeluted with the standard. Also, the retention times of peak Gmatched that of arsenosugar jj in the standard reference oyster tissue material (Figure 4.9).Therefore, on the basis of retention times as discussed, peak G in the FEBA isolate is thearsenosugar fl. The other peaks, F and G, do not have the same retention times asarsenosugar JJh in the reference material (Figure 4.9). However, it is likely that these arearsenosugar derivatives. Similar observations and conclusions were reached with resultsobtained with Condition 2 for the isolate.The HPLC-ICPMS chromatograms of the LEB isolate are shown in Figures 4.15 to4.20. Two arsenic compounds, D and E are present in the isolate. The LEB isolate was spikedwith the arsenosugarj and the NBS Oyster tissue extract, and the chromatograms shown inFigures 4.16 (condition 1, LEB spiked with arsenosugar fl.), 4.17 (condition 1, LEB spikedwith NBS 1566 Oyster tissue reference material), 4.19 (condition 2, LEB spiked with standardarsenosugar ha) and 4.20 (condition 2, LEB spiked with NBS 1566 Oyster tissue referencematerial) were obtained. The chromatograms of LEB spiked with the arsenosugar jj(Condition 2; Figure 4.19) confirmed that neither arsenic compound D nor E is identical withha. However, the chromatograms of the LEB isolate spiked with the oyster tissue referencematerial (Figures 4.17 and 4.20) show that the early eluting compound D coelute with thearsenosugar hhb. The other arsenic compound E does not coelute with any of the arsenicspecies in the SRM extract. Therefore, on the basis of retention times, as well as spikedsamples, the LEB isolate was found to contain the arsenosugar jjJ2 (peak D) and anunidentified compound E likely to be an arsenosugar derivative.156Figure 4.15Chronialograni Obtaincd for the Isolate LEB Using Condition 155504540 Di35 E30z25.20 , Ii5 li10’ 1106’) 12Q 18’1 240 3’X 60 420 480lime (I)Figure 4.16Cliroziaatograrn Obtained for the Isolate LEB Spiked WithArsenosugar Standard jj Using Condition 114 —13 11211 :11 Unrcsolvcd10 Eandjj9 Di87C.) 6IIc54 ‘12 j1 106’) 120 IU’) 24’) 3) 36’) 421) 480.Time(s)157Figure 4.17Clii oniatograna Obtaincd for the Isolate LEB Spikcd WithSRM NBS 1566 Oyster Tissue Using Condition 1645640‘*0: 40 I E32 D+fl. : \241:.\_t? 12’I 1BO 240 Q0 3&0 42Q 4e3CLTime (e)Figure 4.18Chrornatograni Obtained for the Isolate LEI3 Using Condition 232El2824 D-r• 2016 .1 112 i’ii j1e 12’) IBO 240 30’) 36’) 420 430Tm• (I)158Figure 4.19Chroniatograzu Obtained for the Isolate LEB Spiked WithArsenosugar Standard jj Using Condition 211 -10 1i•i 8 III,,”.1 D ‘I• 6 4Ii • I.o 52I10 ——.124 18’ 24) OO 3cc) 4ZTime (i)Figure 4.20Cliromatograni Obtained for (lie Isolate LEB Spiked ‘WithSRM NBS 1566 Oyster Tissue Using Condition 25045 h‘-4 ‘U IiI f. 35 1 JI U‘n • 1C25201510I0 I —_____________• 120 180 240 3O 3%c) 420 &)Time (i)159The chromatograms of other isolates arc shown in Figures 4.21 to 4.29: FEBB1(a,Figure 4.21; FEBB1(a) spiked with the standard arsenosugar fl, Figure 4.22 (condition 1);FEBB 1(a); Figure 4.23 (condition 2); FE1313 1(b) analysed without spiking and with condition1, Figure 4.24; FEBB2 (R, 0.22), Figure 4.25; FEBB2 (R 0.33), Figure 4.26; FEBB2 (R1 0.5 -0.78), Figure 4.27; FEBB2 (R 0.89), Figure 4.28; and finally the arscnicals which remainedunmoved and unresolved at the origin (R 0), Figure 4.29.Figure 4.21Claromatograin Obtained for (lie Isolate FEBBI(a) Using Condition 11816 II‘ IA12 QI10 w.If Ii i’I’! :i1ij.‘‘tirP11j1T I IllLI(I(iiI(II(III120 180 240 300 360 4WTim• (I)160Figure 4.22Clarornatogrant Obtained for the Isolate FEB11 1(a) Spiked WithArscnosugarjj Using Condition 136 Unresolved32Q,Iandjj.7 28 (I.‘4 2420 II.c-) 1612 .18V:,14____06o 127 100 240 OO V 420 480Tim. (s)Figure 4.23Chroniatogram Obtained for (lie Isolate FEB11 1(a) Using Condition 264- 5611Bc3 32 i4 1 ‘;• &i18 1I0120 180 240 3o O ‘zo 480/ Time (.161Figure 4.24Cliromatograna Obtained for the Isolate FEBBI(b) Using Condition I16141 V12io 4•) [“.U4 ..,. .21t,jt: 11 (:01120 18’) 240 3 360 42’)Timi (1)Figure 4.25Claromatograni Obtained for the Isolate FEBB2 (RO.22)11 :.10..9 .177 .71’6I— ‘1 t120 1O’3 240 & 360 42(1Tim.(s)162Figure 4.26Chroniatograna Obtained for the Isolate FEBB2 (R1 0.33)1312; J1110.9.KI.o 6R 4: I, •‘60 120 180 24Q - SO 420Time (g)Figure 4.27Chroniatograni Obtained for the Isolate FEB82 (R1 0.5 - 0.78)50-45j.-r 40•_4-. .,.Ic. 25,15i•10 L51• f\Io---——-----— 240 ôOTime (I)163Figure 4.28Chroniatograrn Obtained for the Isolate FEBB2 (R 0.89)Ii16. 014I.’12’•H.’10;I..3 8j.6 N60 120 1W) 240 3b0 360 420 4LOTime C)Figure 4.29Chroniatograin Obtained for the Isolate FEBB2 (R0)6055 : i X50 jf, 45-“IWVfr 4()..35 I!15;-10:.5.120 1W) 240 3”() 3O 421) /2OTime (S)164.0\Cl)0 Cl) 0Isolates ofWater HPLC4CPMS ANALYSISSoluble Extracts of Spiked With Spiked With Retentioii Times Identical WithF. distichus fromHead Standard Extract of AUCaIS in SRM SID AS# ofHastings Arm Arsenosugar SRMNBS AB jj. Jj flija 1566L. PeakL xb PeakM xFEBB2 0.89) No Noa PeakN x xPeakO xFEBB2 (Rf 0, origin) No No.L. PeakW xb BroadPeakX x x xSTD AS = Standard Arsenosugarx = Peak retention time not identical to the standard arsenicalq = Peak retention time identical to the standard arsenicalNo = Isolate was not spiked with fl! or SRM extract, asappropriateYes = Isolate was spiked either with or SRM extract, orboth, as appropriateAB ArsenobetaineThe FEBB1(a) isolate contained three arsenic compounds (Peaks H, I and Q inFigures 4.21 and 4.23). Peak I had the same retention time as a known arsenosugar,Peaks H and Q do not have the same retention times as any of the known arsenic species.They remain unidentified but they are likely arsenosugar derivatives. Similarly, Peak V in theFEBB 1(b) isolate was identified as arsenosugar fl on the basis of retention time. The otherpeak, Peak U is probably another arsenosugar derivative. Furthermore, with due considreationto the purification procedure, it is probable that FEBB1(b) resulted from the tailing ofFEBB1fr) because their observed peaks are similar.Analysis of the various isolates separated initially from FEBB2 isolateindicated thatnone of the identified peaks in these isolates (Table 4.11) is identicial to any of the knownarsenical standards used for their characterization. Furthermore, a careful analysis of thechromatograms (Figures 4.25 to 4.28) revealed that they all have similar arsenic compounds,which are found in the major arsenic containing isolate, FEBB2 (R 0.5 - 0.78). However,166FEBB2 (Rf 0.33) isolate (Figure 4.26) contained a third arsenic compound (peak K) which isalso not identical with any of the standard arsenic species used. Therefore, it is reasonable toconclude that FEBB2 isolate contained three minor unidentified arsenic compounds, but theyare probably arsenosugar derivatives.In conclusion, the preceeding discussions have shown that there are four main arseniccontaining isolates obtained from the comprehensive chromatographic separation of the watersoluble extract of the F distichus investigated in this study. The first arsenic containing isolate,FEBA (major band) has been analysed by the HPLC-ICPMS technique and was found tocontain three arsenic species (Figure 4.13). One of the arsenic species was identified as thearsenosugar jj. and the other two arsenic compounds were characterized as unknown“hidden” arsenic compounds. These “hidden” arsenicals are probably arsenosugar derivatives.The second arsenic containing band, LEB (minor isolate), was found to contain two arsenicspecies (Figures 4.15 and 4.18). One of the two compounds was identified to be arsenosugarJj, found and identified in the SRM NBS 1566 Oyster tissue sample. The other arseniccompound did not have retention times that matched any of the available arsenic standards; itwas characterized as unknown “hidden” arsenic compound, suspected to be anotherarsenosugar derivative. The isolate FEBB1(a), a minor arsenic containing band, was found tocontain three arsenic compounds, one of which was identified as arsenosugar i.!. The othertwo were unidentified but are suspected arsenosugar derivatives. Finally, FEBB2 was found tocontain three arsenic compounds, but none of these have retention times identical with any ofthe available arsenic standards. They were therefore unidentified, although suspected asarsenosugar derivatives.Other workers(51) have isolated two major arsenosugar constituents from the extractof the brown algae, &irgassum lacenfolium; both were identified after repeated gel permeationchromatography ori Sephadex G15, anion exchange chromatography on DEAE Sephadex andmc on cellulose. However, several minor arsenic compounds were also present in the algaextract but they(51) claimed that the small quantities available precluded their identification.167Andreae and Klumpp(40) studied the biosynthesis and release of organoarsenic compounds bymarine algae in cell extracts and found up to 12 organoarsenicals, none of these was identified.In summaiy, the examination of the arsenic constituents in F distichus has beenstudied in the present work. Two arsenosugars, jj. and il!a were found to be the majorarsenic components in the water soluble extract of the seaweed. The F distichus sample wasalso found to contain several other, minor “hidden” arsenic compounds which have not beencharacterized, although they are likely to be arsenosugar derivatives.4.3.3 BIOTRANSFORMATION STUDIES OF “HIDDEN” ORGANOARSENICCOMPOUNDS IN FUCUSDISTICHUSIn this section, studies carried out on the biotransformation of arsenic species inF. distichus, when subjected to anaerobic decomposition conditions(28, 190), are presented. Ina related study(1 90) performed on Ecidonia radiata, another brown algae species, the “hidden”organoarsenic compounds, specifically arsenosugars(95) were converted intodimethyloxarsylethanol under the anaerobic decomposition conditions. This result providedimportant experimental evidence to support the biotransformation scheme given in Figure 1.2for the production of the major arsenic compound, arsenobetaine found in marineanimals(1 91, 192). Therefore, confirmation of similar results from seaweeds obtained fromother environments would add support to the scheme. The present study involved a differentspecies of brown algae, F distichus abundantly available in British Columbia, Canada. Thedecomposition was carried out by using both an open and a closed system procedure. An opensystem was used in the earlier work(190). The closed system approach introduced differentbiotransformation condition(277-279) considered likely to occur in a large beach deposit ofmarine algae and/or in an algae bed sediment(190).1684.3.3.1 ANAEROBIC DECOMPOSITION OF F. distichus IN AN OPEN SYSTEMThe present study was based on the procedure employed by Edmonds andco-workers(190) for the production and isolation of dimethyloxarsylethanol (DMAE) byanaerobic decomposition of the brown kelp Ecklonia radiata. Freshly collected F distichus(1.03 kg) was incubated with unfiltered seawater, and sediment collected from the same sitefor 41 days under a continuous flow of argon (Section The residue was thenseparated from the liquid by filtrationldecantation and the filtrate evaporated. The residue andthe filtrate were separately extracted with methanol and the methanol extracts were evaporatedand further subjected to water/phenol/diethyl ether extraction(1 65). This procedure separatedthe highly polar material into the aqueous phase while the low polarity substances wereextracted into the phenol/ether layer and was discarded. The aqueous layers afforded residues,filtrate (30.22 g) and seaweed (21.49 g) after evaporation. These were dissolved in water.The water soluble filtrate and seaweed samples were subjected to arsenic analysis,prior to and following the 3 hour hot base digestion by using the semi-continuous mode HGGC-AAS technique. The results are shown in Table 4.12.Table 4.12Arsenic Speciation in the Anaerobic Decomposition ProductsofF. distichus by the Semi-Continuous Mode HG-GC-AA AnalysisAnaerobic 3 Hour Hot Base Digestion (2M NaOH, 5m1)Decomposition Before, jig As After, jig AsNo. Extract Inorg. As IMAA DMAA Inorg. As MMAA DMAA1 Seaweed 1.88 d 3.44 3.84 d 11.342 Filtrate 1.33 d 5.55 1.50 d 9.93d = detected, Inorg. As = Inorganic ArsenicThe amount of arsenic determined for both decomposition products, seaweed andfiltrate extracts is 15.18 jig As and 11.43 jig As respectively. These arsenic amounts(summation) represents about 90% of the total arsenic in the seaweed. The filtrate extractcontains greater than 60% of its total arsenic as reducible arsenic species, mainlyI169dimethylarsinic acid (49%). The seaweed extract contains 65% of its total arsenic as ‘hidden”arsenic species. The result from the three hour 2M NaOH (5 ml) digestion on both samplessuggested that the ‘hidden” arsenicals are decomposing into inorganic arsenic species (e.g.1.88 g —* 3.84 ig As inorganic arsenic species observed for the seaweed extract). Perhapsthe ‘hidden” arsenic compounds are sensitive to the base digestion. Chromatographic analyseswere carried out on the seaweed and the filtrate extracts, and their chromatograms are shown inFigures 4.30 and 4.31.Figure 4.30Chromatogram of Arsenic Species Containedin the Seaweed ExtractFollowing Gel Permeation Chromatography on Sephadex GiS0.900.800.700.500.400.300.200.100.00-0.104 ‘. .== LOcq CO LO O CoElutioia Volume, ml=-4-4170The chromatogram shown in Figure 4.30 was carried out on a Sephadex 015 colunm(2.5 cm x 60 cm, water served as the mobile phase) and the seaweed extract revealed twoarsenic containing bands having retention volumes between 104 ml - 401 ml (I) and 550 ml -760 ml (2). The seaweed extract isolate (1) was found to contain 10.78 p.g As by using GFAAanalysis. The othr isolate, (2), contained 4.40 i.g As, also from GFAA analysis. Undersimilar chromatographic conditions, as described above, the filtrate extract revealed twoarsenic containing bands between elution volumes 110 - 408 ml (1) and 573 - 964 ml (2).Figure 4.31Chromatogram ofArsenic Species Contained inthe Filtrate ExtractFollowing Gel Permeation Chromatography on Sephadex G150.30‘ 0.204.—C0.10Cc)0.00-0.10C_. ,. -==0 —4 C’-4Elution. Volume, mlI171GFAA analysis showed that the filtrate isolate (1) contains 7.89 tg As and isolate (2) contains3.54 .tg As. Identification and Characterization of Arsenic Species in the IsolatedDecomposition Products ofF. distichus by Using HPLC-ICPMSThe HPLC separation (on line with the ICPMS) was carried out on an Inertsil ODS-2(250 x 4.6mm id, GL Sciences, Japan) HPLC column using 10mM tetraethylammoniumhydroxide + 4mM malonic acid + 0.1% methanol at pH 6.8 as the mobile phase. This HPLCsystem is different from those of conditions I and 2 described previously in Section flow rate employed was 0.8 ml min1. Two standard samples were used for retentiontimes comparison. These are, a standard dimethylarsinylethanol synthesized in the presentstudy by using established procedures (Section 2.7), and a reference material NEST 1566aOyster tissue. The chromatogram obtained using the present HPLC system for the Oystertissue extract is shown in Figure 4.32. The four arsenic species, which have been previouslydiscussed (Section were observed. The synthetic product was characterized to bedimethylarsinylethanol by using Desoprtion Chemical Ionization (DCIMS), ‘H- and ‘3C-NMR. The characteristic mass peaks at (M+l) (or protonated molecular ion) mlz (76.04%)167, base peak (M-OH) m/z (100%) 149 and mlz (trace) 106 (M+-CH3CH2O )wereobserved (Figure 4.33) in the DCIMS spectrum of the synthetic product. The ‘H NMRspectrum (Figure 4.34) showed chemical shifts 6 (ppm) at 2.27 (s) for two methyl groupsattached to arsenic, (CH3)2As; 2.97 (t), CH2As; 4.40 (t), CH2O and; 4.72(s), H20. The ‘3CNMR spectrum, shown in Figure 4.35 revealed chemical shifts 3 (ppm) at 16.74 for the twomethyl groups attached to arsenic, (CH3)2As; 35.26, CH2As; and; 57.16 ppm, CH2O . Thesynthesized product used as a standard in the present study, were correctly characterized to bedimethylarsinylethanol.172Figure 4.32Chromatogram Showing the Four Arsenic Species Present inSRM NIST 1566a Oyster Tissue Separated onInertsil ODS-2 HPLC C 18 Reverse Phase Column1 iiLQ 3O 450 5;L() (jFigure 4.33Desorption Chemical Ionization Mass SpectrometricSpectrum ofDimethyloxarsylethanol (DMAE)CL0168: Scan Avg 36—40 (0.92 — 1.02 zin)50 100 150 200 250 300 360aoo% -- 1 - - -I - ‘ 100%17so% 501 Q6u—1.. j - --- - —0 %50 100 150 200 250 300 360173Figure 4.34‘H NMR Spectrum of Dimethyloxarsylethanol (DMAE) inD20SolutionI‘ T.,, , ., .,,.,14 .,.,, 1.—I 711.0 10.0 9.0 9.0 7.09.0 5.0 4.0 3.0 2.01.0 0.0o rj 3.0(c174Figure 4.35C NMR Spectrum of Dimethyloxarsylethanol (DMAE) in D20SolutionII I20 200 180 160 140 120 100SO 80 40 20 0 —20PPM- 175IFigure 4.36ICPMS Chromatogram of Dimethyloxarsylethanol (DMAE) StandardOn An Inertsil ODS-2 HPLC C 18 Reverse Phase Column7264564032241680The HPLC-ICPMS chromatogram of DMAE (0.78ppm As) is shown in Figure 4.36and DMAE is the major peak eluted between 194 sec and 375 sec. Trace amounts of othercompounds seen to be present eluted at 47 sec (an unknown impurity) and between160 sec - 375 sec [possibly dimethyloxarsylacetic acid (CH3)2As(O)CHCOOH, aby-product(28)]The HPLC-ICPMS chromatograms shown in Figures 4.37 - 4.40 were obtained for thewater soluble seaweed extract isolates 1, 2, mixture of seaweed isolates 1 and 2 (1:3) and thewater soluble filtrate extract isolate 2, collected from the Sephadex Gi 5 column.720I 76Figure 4.37Chromatograrn ofSeawccd Isolate 1From the Anaerobic Dccompositioii ofF. disticlucs3228 ñ12I90 270 O0 4cc tc t’ oT1m3(S)Figure 4.38Chromatogram Obtained of Seaweed Isolate 2from the Anaerobic Decomposition of F. distichus12111098j :VV 29V O c ?6C Q-V 630 ?t’tm2.($)I 77Figure 4.39Chromatogram Obtained With a Mixtãrc (1:3) ofSeaweed Isolates 1 and 2front the Anacrobic Decomposition ofF. distichus141312itT I• :20 90 / SO 270 ) 45i 540 630 720Time(s)Figure 4.40Cliromatogram Of Filtrate Isolate 2from the Anaerobic Decomposition ofF. disticlius3632‘28a /40vo xo 450 540 630 720Time CS)178The chromatogram obtained for the water soluble seaweed isolate 1 (Figure 4.37)shows only one arsenic containing band whose retention time matches that ofdimethylarsinylethanol (DMAE). On the basis of this retention time comparison, seaweedisolate 1 is dimethylarsinylethanol, a compound that was isolated from water soluble seaweedfraction following anaerobic decomposition ofEcklonia radiata(190).The retention times of the arsenic containing seaweed isolate 2 (Figure 4.38) is similarto that of dimethylarsinic acid (Figure 4.32). On this basis, the water soluble seaweed isolate 2is dimethylarsinic acid. When the seaweed isolates 1 and 2 were mixed (1:3), thechromatogram shown in Figure 4.39 was obtained. The retention time of this mixture did notmatch either DMAE or DMAA completely but overlaps the profile of the two compounds.(Even though an apparent one broad peak is observed, it did not match just one, but the twoarsenic species.) This serves to confirm that these two arsenicals are separate components ofthe decomposition products ofF disrichus unlike the single product in the previous work(190).The chromatograrn of filtrate isolate 2, shown in Figure 4.40 revealed a broad peakwith its retention time similar to DMAA. The retention volume (110 ml - 408 ml) for filtrateisolate 1 on the gel permeation Sephadex Gi 5 column shows it to be similar to the seaweedisolate 1 which was identified as DMAE.The present result (open system), suggests that both DMAE and DMAA are anaerobicdecomposition products of F distichus. Following the isolation of DMAE from thedecomposition product of Ecklonia radiaia( 190), Edmonds and Francesconi( 191) suggestedthat DMAE occupies a key position in the biosynthesis of arsenobetaine. However, DMAE hasnot yet been identified in the natural environment. Nonethiess, Edmonds and Francesconi(1 92)proposed a scheme for the transformation of arsenic compounds in the marine environmentleading to the production of arsenobetaine from oceanic arsenate (Figure 1.2). Some of thecompounds in the scheme have been detected in the natural environment while some keyintermediates have not been encountered. The key experimental basis for the scheme, DMAE,has no direct experimental link to the production of arsenobetaine beyond the fascile formation179from algal dimethylarsinylribosides(190). Also, a synthetic trimethylarsonioriboside wasanaerobically decomposed to arsenocholine(28) and this was also taken as a support for theproposed arsenic transformation scheme. However, it has been claimed that even if thetrimethylarsomoribosides, which have only been isolated once from the brown alga Sargassumthumbergii(186), occur naturally in the environment, they are not likely to be present beyond1% of the amount of the dimethylarsinylribosides(275). The view of the present writer is thatthe anaerobic decomposition study(28) yielding arsenocholine seems quite insightful. This isbecause arsenocholine has been previously(170, 171) found in shrimps and was readilyconverted to arsenobetaine when administered to the juvenile yelloweye mullet(28). Therefore,other alternative pathways for the formation of arsenocholine need to be examined.Because dimethylarsinic acid has been found transformed to arsenobetaine in seawatercontaining mussels(279) and also because it has been suggested previously (without anyexperimental basis) that DMAA is converted to arsenobetaine in Figure 1.3 [proposed as analternative arsenic biotransformation scheme by Phillips and DePledge(12)], the present writerproposes that DMAA is probably the key intermediate for the production of arsenobetaine.DMAA has been found as a decomposition product ofF distichus in the present study, and itis commonly available in the natural environment(5, 192, 275). Therefore, a possible simplescheme can be proposed for the biotransformation of DMAA via arsenocholine leading to theproduction of arsenobetaine as shown in Figure 4.41. This pathway to arsenobetaine fromDMAA via arsenocholine takes into consideration the importance of choline(CH3)N’CH2CO as an essential dietary requirement of many organisms(12).Furthermore, during phospholipid biosynthesis, ethanolamine is abundantly available(12).Therefore, following Challenger mechanisms leading to the formation of trimethylarsine viaDMAA, and by a tvo electron transfer mechanism enzymatically mediated in the organismtowards the toxic effect of (CH3)As, a fascile formation of arsenocholine is achieved in thepresence of ethanolamine,HOCH2CHN.180Figure 4.41Proposed Biotransformation ofDMAAto Arsenobetaine in Marine OrganismChallenger HOCH2CHN(CH3)2AsO(OH)hM(CHAS (CH3)AiCH2COHXDMAA &zymeEdmoudsetal(CH3)AsCH2COHX . (CH3)AiCH2COOArsenocholine 011 ArsenobetaineThe toxic nature of trimethylarsine(9) probably explains why organisms respondrapidly to transform it to a less toxic material leading to the formation of arsenobetaine. Thenon-toxic nature of arsenobetaine to marine organisms must account for its highbioaccumulation at the higher trophic levels(5, 6, 271).4.3.4 ANAEROBIC DECOMPOSITION OF F. distichus IN A CLOSED SYSTEMThis present study examined all the possible metabolites occurring during theanaerobic decomposition process. Unlike in the ‘bpen” system in which all the arsenic speciesare completely transformed into DMAE(190) and/or, DMAE + DMAA (present study), thisclosed system allows examination of all metabolites including intermediate ones. Thespeciation information generated through the closed anaerobic decomposition procedure on F.disrichus will ensure a complete understanding about the anaerobic decomposition process onone hand, and on the other hand, offer possible new insights on the conversion to, andaccumulationof arsenobetaine in the higher trophic levels of the marine environment1814.3.4.1 Investigation of the Anaerobic Decomposition of F. distichus In a Closed SystemThe experimental details for this examination have been given in Section collected F distichus was incubated with unfiltered water and soil from the same siteunder argon for 41 days in a closed tightly sealed (using parafllm to ensure air tightness) plasticcontainer. The system was opened daily under a continuous flow of argon in order to releaseany build up of gas; suspected to be mainly sulfur containing. After the41 days, the contents of the reaction were decanted and filtered to separate a smelly dark brownliquid from the seaweed residue. In a separate control experiment, the seaweed was incubatedin unfiltered seawater, but without the sediment, collected from the same site. Similarly, after41 days, the contents of the reaction were decanted and filtered to separate the liquid from thedecomposed seaweed. The liquid filtrates in both experiments were separately evaporatedprior to subsequent analysis. The seaweed residues from both experiments, as well as theirrespective filtrate residues, were separately extracted in methanol.For the control experiment, the methanol soluble filtrate and that of its seaweed wereseparated by filtration from their respective residues and evaporated. The resulting filtrate(CF) and seaweed (CR) syrups, as well as the methanol insoluble filtrate (C1F) were subjectedto the three hour hot base digestion and arsenic speciation data were obtained as shown inTable 4.13 by using the semi-continuous mode HG-GC-AAS. The samples were found tocontain mainly dimethylated arsenic species (DMAA).For the main experiment, the unextractable seaweed residue (UR) was analyzed by theHG-GC-AAS and the amount of unextractable arsenic was found to be 17.04 p.g, withinorganic arsenic representing almost half (8.04 .ig As). Other workers(48, 49, 175) havefound high inorganjc arsenic in seaweed as well. Also, in Section 4.2.2 of the present study, itwas observed that some F distichus revealed large amounts of unextractable arsenic inmethanol.182The methanol soluble extracts from the main experiment were also further analyzed asfollows: The methanol soluble filtrate (SF) was examined for arsenic species by the semi-continuous mode HG-GC-AAS following the three hour digestion (Table 4.13). The SFsample was found to contain more than half of its total arsenic content (8.09 .tg As) asreducible DMAA (4.72 .tg As) with the rest (3.37 p.g As) being ‘hidden” arsenic species. Themethanol soluble extract of the seaweed residue was evaporated and the residue which wasdissolved in water was partitioned with diethyl ether. The fatty material (0.83 g, 0.90 .tg As)which was extracted into the ether layer was discarded. Further removal of less polar materialfrom the aqueous phase was achieved by using phenol/ether/water partitioning. Thephenol/ether layer was not further analyzed. The water layer was evaporated and the residuewas re-extracted in methanol which removed some insoluble material. The methanol solubleextract (SR) was subsequently chromatographed by using Sephadex LH2O with methanol asthe mobile phase. This revealed that the SR sample contained a single arsenic containing bandwhich eluted between 216 ml and 457 ml. The band was collected and rechromatographed onSephadex LH2O (2.5 cm x 85 cm, methanol as the mobile phase). A single arsenic containingband which eluted between 200 ml and 443 ml was obtained. Two fractions were taken basedon color: a leading light brown band, (SRA) between 200 ml and 303 ml, and a later elutingdark brown band (SRB) between 304 ml and 443 ml. Some arsenic speciation results fromthese samples are given in Table 4.13.183Table 4.13Arsenic Determination and Speciation Analysis onthe Decomposition Products from F. distichusIncubated in a Closed System Using theSemi-Continuous Mode HGGCAA TechniqueClosed System Hot Base Digestion (2M NàOH, 5m1)Anaerobic Decomposition Before, .tg As After, .tg As Total# Products/isolate Inorg As MMAA DMAA Inorg As MIvL4A DMAA g As1 Seaweed/Seawater/SedimentSamplesa SF nd nd 4.72 nd nd 8.09 8.09b IF d d 0.86 d d 0.95 0.95c UR — — — 8.04 0.30 8.70 17.04d SR.A 0.83 0.07 0.72 2.01 0.12 28.04 30.17e SRB 0.12 d 0.38 2.41 0.67 24.75 27.83Total Arsenic Species 0.95 0,07 6.68 12.46 1.09 70.53 84.082 Seaweed/Seawater Samplesa CF nd nd 0.48 nd nd 5.13 5.13b CIF — — — 0.70 0.74 11.68 13.12c CR 0.04 nd d 0.04 nd 9.24 9.28Total Arsenic Species 0.04 nd 0.48 0.74 0.74 26.05 27.53nd = not detected; d = detected; — = not determined; SF = methanol soluble filtrate of closed system; IF =insoluble filtrate material; UR = unextractable seaweed residue; SRA = early eluting methanol soluble seaweedextract (SR);SRB = later eluting methanol soluble seaweed extract (SR); CF = control filtrate extract;CIF = control insoluble material; CR = control seaweed extractThe total arsenic contents found in the isolates SRA and SRB are respectively,30.17 ig As and 27.83 p.g As. Greater than 94% of these total arsenic contents are in theforms of “hidden” arsenic species.The isolates SRA and SRB were further examined by thin layer chromatography oncellulose plates (200 mm x 200 mm x 0.1 mm, n-butanol:acetic acid:water, 60:15:25 as themobile phase). Two arsenic containing bands were obtained and their analysis is given inTable 4.14.184Table 4.14RValues and Arsenic Content of theAnaerobic Decomposition Products ObtainedFrom Water Soluble Extracts ofF. dislichusAfter Thin Layer Chromatography on Cellulose Plates11 Isolate on Cellulose tic Rf Value Arsenic Content ig As1 SRAI 0.18-0.24 21.50SRA2 0.48 - 0.74 7.053 SRB1 0.18 - 0.21 8.914 SRB2 0.58 - 0.73 18.42The HPLC-ICPMS chromatograms of some of these isolates are shown inFigures 4.44 - 4.49. (Because the time scales recorded for these chromatograms are from0 - 480 see, appropriate adjustments were made on the chromatograms of the DMAE standardand the NEST extract for the purpose of retention times comparison. The standard arsenicspecies chromatograms are given in Figures 4.42 and 4.43.) These HPLC-ICPMS analyseswere performed with assistance provided by Xiao Chun Le (of the Chemistry Department,UBC). The HPLC system consists of an Inertsil OD-2 C 18 reverse phase column, 10mMtetraethylammonium hydroxide + 4mM malonic acid + 0.1% methanol atpH 6.8 as the mobile phase, and the flow rate used was 0.8 ml min1.185Figure 4.42Chromatogram Obtained forNIST 1566a Oyster Tissue Standard Reference Material10 I98U ‘—I\,l \90 180 270 360 540 630 720Time (s)Figure 4.43Chromatogram Obtained for DMAE7264564841); L111180 270 360 450 540 630 720Time (s)186The chromatogram of isolate SRA1 (Figure 4.44) shows three arsenic peaks whoseretention times do not match any of the available standards and are therefore identified asunlcnown hidden arsenic species. Because a different HPLC system was utilized for theisolation of arsenic species in F distichus discussed previously (Section, theseunlcnown hidden arsenicals were not further characterized.The chromatogram of the isolate SRA2 revealed one major arsenic peak (Figure 4.45)which is probably a mixture ofDMAE and DMAA (this is because the peak retention overlapsboth arsenic compounds retention time profiles). This result appears quite similar to Figure4.39 data in which isolate 1 (DMAE) and isolate 2 (DMAA) were mixed (1:3) together.Figure 4.44Chromatogram Obtained for SRA1by the HPLC-ICPMS Analysisa60554035302520151050/rco 3°187Figure 4.45Chrornatograrn Ob(aincd for SRAZby (lie HPLC-ICPMS Ana’ysis55504540 4I ![Q 12Q i 24G 3 O CTim C )Figure 4.46Chroniatogram Obtained for SiB 1by (lie 1IPL-1CPMS Analysis109.876?; 42Tm ()188Figure 447Chromatogram Obtained for SR132by the IIPLC-ICPMS Analysis1816, 14‘.410020 (20 /O Z0 ?C 3O £2OTi m€. (5)Figure 4.48Chroinatograrn Obtained for SFby the IIPLC-ICPMS Analysis1098’‘.4H 6z“. _ji\\__0&L? (20 ,go 2 o 3eso o.TmQ LS)1896456.•:‘ 484032c-)24168.Figure 4.49Chromatogram Obtained for CFby the HPLC-ICPMS AnalysisThe chromatogram of isolate SRB 1 (Figure 4.46) reveals three arsenic peaks, whoseretention times are identical with those of isolate SRA1. The similar Rf values (Table 4.14)support the }{PLC-ICPMS results that identical arsenic species are present in these isolatesSRA1 and SRB1.The chromatogram of isolate SRB2 (Figure 4.47) revealed one arsenic containingband, which appears to overlap the retention profiles of both DMAE and DMAA. This isolate,SRB2, is therefore characterized as a mixture of DMAE and DMAA, similar to isolate SRA2.Only one aTsenic containing peak is present in the methanol soluble filtrate, SF (Figure4.48). Its retention time matches that of the standard DMAE (Figure 4.43). However, fourarsenic species (Figure 4.49) are present in the methanol soluble filtrate CF obtained from the£0 120 gV 21f0 Co 3eCS)190control experiment. On the basis of retention times comparison, the first eluting peak isDMAE, the second arsenosugar iji?.. and the third and fourth peaks are unknown arsenicspecies. The retention time of the third eluting peak in this CF sample matched the retentiontimes of the third eluting peaks (identified as unknown arsenic species as well) in thechromatographic isolates SRA1 and SRB1. The fourth arsenic peak was different from any ofthe peaks in the other chromatograms.The identification of DMAE and DMAA in the anaerobic decomposition products ofF distichus incubated in a closed system confirms these compounds as degradation productsof the “hidden” arsenic compounds present in this seaweed. The presence of only one arseniccompound, DMAE in the methanol soluble filtrate SF compared to four arsenic species(DMAE, arsenosugar and two unknown “hidden” arsenic species) in the methanol solublefiltrate CF suggests the importance of microbial activity, peculiar only to marine sediment, inthe complete conversion of “hidden” arsenicals to DMAE. Finally, the closed anaerobicdecomposition ofF distichus confirmed that the major decomposition product of the “hidden”organoarsenic compounds present in brown algae is DMAE (isolates SF, CF peak 1 and aminor portion of SRA2). In addition, DMAA (also a major arsenical - isolates SRA2 andSRB2) is a decomposition product, possibly produced in larger quantities as well, during acomplete biodegradation process which takes place in an open system (Section and HGAA methodologies were developed in combination with a two-hour wetdigestion procedure and applied to the determination of total arsenic in some marine macroalgae ofBritish Columbia. The total arsenic concentrations in the three classes ofmarine algaevaiy with species and the location from which each species was collected, ranging between0 ig g’ (not detected) and 39.70 .ig g1. The overall mean arsenic concentrations revealedthat the brown algae generally accumulates higher amounts of arsenic when compared to thered and/or green algae. However, the tendency to accumulate high or low arsenic in aparticular class/species seems to be dependent on the collection site and its associated soilconditions.Three digestion procedures were examined for the dissolution of environmental andbiological samples, particularly with respect to arsenic speciation in marine macro algae, andarsenic was determined by the semi-continuous mode HG-GC-AA technique. The methodemploying HNO3 wet digestion of the sample was found to be inadequate for both arsenicdetermination and speciation analysis, particularly when higher amounts of “hidden” arsenicspecies are present in the sample. Both the wet digestions using a combination ofH2S04,HNO3 andH20,as well as NaOH however provided complete sample decomposition for totalarsenic determination.Furthermore, only the sodium hydroxide wet digestion procedure retained a completespeciation information in which the methylated arsenic compounds present in the sample digestare not degraded unto inorganic arsenic. By employing the sodium hydroxide digestionfollowing extraction of some freeze dried seaweed samples, speciation analysis carried out byusing the semi-continuous mode HG-GC-AA revealed that more than 90% of the total arsenic192accumulated in the macro algae analysed was present as “hidden” arsenic species. This resultis similar to those obtained in previous work by others.The combination of both off-line and on-line analytical procedures making use ofcolumn chromatographies including gel permeation and ion exchange, thin layerchromatography, high performance liquid chromatography (HPLC) and instrumental detectiontechniques such as GFAA, HGAA, HPLC-Mic-HGAA, HPLC-JCPMS were used for theisolation and identification of water soluble “algal” arsenic species present in Fucus distichuscollected from the Head of Hastings Arm in British Columbia. The main arsenic containingisolates obtained were determined to contain several “hidden” arsenic compounds stronglysuspected to be arsenosugars based on comparison of their retention times with those ofavailable standard arsenic compounds. Two of these “hidden” arsenicals were positivelyidentified as the arsenosugars and jjj (Table 1.2) by using the on-line HPLC-ICPMStechnique. This positive identification result was possible because of the availability ofstandard arsenosugar th. provided by Francesconi and Edmonds in Australia, and the correctcharacterization of arsenic species including both arsenosugars jj and Jj in the standardreference material NBS 1566. The SR.M NBS 1566 oyster tissue extract has been previouslycharacterized to contain these arsenic species. Because the main identification technique, theHPLC-ICPMS, employed in this study requires the use of known standards for positivecharacterization, availability of other known and previously isolated arsenosugars is• necessaiybefore a complete (positive) identification of other “hidden” arsenicals isolated in the watersoluble extract,of F. distichus analyzed in this study can be made possible. The completecharacterization of these compounds is essential in order to understand the pathways involvedin the conversion of arsenic compounds in the marine environment.193The presence of dimethylarsinyl ethanol (DMAE) in the open system-anaerobicdecomposition products of F disrichus confirms that it is indeed a major transformationproduct of arsenic in the environment, in agreement with previous similar study by otherworkers. However, in this present study, DMAA was also characterized as a decompositionproduct in both the open- and closed-anaerobic decomposition experiments. This is a newcontribution to the understanding of arsenic biotransformation in the marine environment. Theclosed system approach also revealed other decomposition products which were notcharacterized completely in this study. 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