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Arsenic in the terrestrial environment : an analysis of the Bridge River District Haug, Corinne 2002

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ARSENIC IN THE TERRESTRIAL ENVIRONMENT: AN ANALYSIS OF THE BRIDGE RIVER DISTRICT By C O R I N N E H A U G B . S c , The University of British Columbia, 1999 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Chemistry) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OP'BRITISH C O L U M B I A August 2002 © Corinne Haug, 2002 UBC Rare Books and Special Collections - Thesis Authorisation Form http://www.library.ubc.ca/spcoll/thesauth.html In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the Univ e r s i t y of B r i t i s h Columbia, I agree that the Li b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date 1 of 1 1/20/03 4:43 PM 11 ABSTRACT Terrestrial samples collected from the Bridge River district, a mining area in British Columbia, were analyzed for arsenic. Soils from the abandoned Pioneer mine were analyzed by neutron activation analysis (NAA) . The concentrations of these samples ranged from 35-1623 pg As/g. Total arsenic content of digested tree samples was determined by using inductively coupled plasma-mass spectrometry (ICP-MS). The results support the previous finding that Douglas-fir trees accumulate arsenic to a high degree. Comparison of arsenic content with tissues type and age reveals a trend that suggests the translocation of arsenic is from roots to needles. High-performance liquid chromatography (HPLC) coupled with ICP-MS was used for the determination of arsenic species in all tree samples. Inorganic arsenic, arsenate and arsenite, were the predominant species extracted from trees. Trace amounts of D M A and M M A were found in some samples. The interaction of bacteria and fungi with arsenic was investigated to yield information regarding the biotransformation of arsenic by microorganisms. Bacteria and fungi from the mine soils were isolated and pure cultures were obtained. Hydride generation-gas chromatography with atomic absorption spectrometric detection ( H G - G C -A A S ) was used to identify and quantify arsenic metabolites in the growth media of soil fungi amended with arsenate. Arsenate was reduced to arsenite, and methylated to D M A and T M A O by one of the isolates. iii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables vii List of Figures viii List of Abbreviations ix Acknowledgements xi Chapter 1. INTRODUCTION 1 1.1 General Introduction 1 1.2 Chemistry of Arsenic 1 1.3 Toxicity of Arsenic Compounds 3 1.4 Uses of Arsenic : 5 1.5 Arsenic in the Environment 5 1.5.1 Sources of Arsenic 5 1.5.2 The Marine Environment 6 1.5.3 The Terrestrial Environment 7 1.6 Introduction to Microorganisms and Arsenic 8 1.7 Objectives of the Present Studies 9 Chapter 2. EXPERIMENTAL METHODS FOR THE ANALYSIS OF ARSENIC 11 2.1 Instrumentation 11 IV 2.1.1 Introduction 11 2.1.2 Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) 12 Apparatus 12 Experimental Conditions and Procedure 14 2.1.3 High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry (HPLC-ICP-MS) 15 Apparatus 15 Experimental Conditions and Procedure 15 2.1.4 Hydride Generation-Gas Chromatography-Atomic Absorption Spectrometry ( H G - G C - A A S ) 16 2.2 Reagents and Chemicals 19 2.2.1 Standards and Reference Materials 19 2.2.2 Culture Media 20 2.3 Sample Preparation for Analytical Analysis 21 2.3.1 Acid Digestion 21 2.3.2 M e O H / H 2 0 Extraction 22 2.3.3 Culture Preparation 22 Chapter 3. BRIDGE RIVER TREE ANALYSIS 24 3.1 Introduction 24 3.2 Douglas-fir 26 3.3 Spring Collection 26 3.3.1 Sample Collection and Treatment 26 V 3.3.2 Sample Preparation and Analysis 29 3.3.3 Results and Discussion 32 Total Arsenic Analysis 32 Trees 32 Soils 34 Arsenic Speciation Analysis 34 Trees 34 Extraction Efficiency 39 Soils 40 3.4 Fall Collection 41 3.4.1 Sample Collection and Treatment 41 Freeze-drying Procedure 42 3.4.2 Sample Preparation and Analysis 42 3.4.3 Results and Discussion 43 Total Arsenic Analysis 43 Arsenic Speciation Analysis 46 Extraction Efficiency 52 Freeze-drying 52 3.5 Summary 53 Chapter 4. THE BIOTRANSFORMATION OF ARENICALS BY SOIL MICROORGANISMS 54 4.1 Introduction 54 vi 4.2 Analysis of the Bacterium Pseudomonas 56 4.2.1 Culture Preparation and Treatment 56 4.2.2 Results and Discussion 57 Growth Media 57 Cells 58 4.3 Analysis of Isolates from Bridge River Soils 59 4.3.1 Culture Preparation and Treatment 59 Pure Culture Isolation 59 Culture Incubation 61 4.3.2 Sample Preparation and Analysis 62 4.3.3 Results and Discussion 64 4.4 Summary 69 Chapter 5. SUMMARY AND FUTURE WORK 70 References 73 Appendix A 77 LIST OF TABLES Table 1.1 Some arsenic compounds found in the environment 2 Table 2.1 Operating parameters of ICP-MS 13 Table 2.2 Operating parameters of H G - G C - A A S 18 Table 3.1 Arsenic concentrations in tree samples from the spring collection 32 Table 3.2 Concentration of the arsenic species in the tree samples from Pioneer Mine 36 Table 3.3 Arsenic concentrations in tree samples collected from Pioneer Mine, fall collection 44 Table 3.4 Comparison of results from re-digestion ( A - l ;B-1) and re-runs (A-2;B-2) of site 1 Douglas-fir samples 45 Table 3.5 Concentration of the arsenic species in freeze-dried tree samples from Pioneer Mine, fall collection, E X P T A 47 Table 3.6 Concentration of the arsenic species in wet tree samples from Pioneer Mine, fall Collection, E X P T B 48 Table 4.1 Summary of the experimental setup of pure Pseudomonas cultures 57 Table 4.2 Summary of cultures isolated from the Pioneer mine soils 60 Table 4.3 Summary of the experimental setup for the analysis of B R fungi isolates 61 Table 4.4 Concentrations of inorganic arsenic species in the growth medium of a B R soil isolate determined by using H G - G C - A A 65 Table 4.5 Concentrations of methylated metabolites in the growth medium determined by H G - G C - A A 65 Vlll LIST OF FIGURES Figure 1.1 The Challenger Mechanism 8 Figure 2.1 Schematic diagram of ICP-MS 13 Figure 2.2 Schematic diagram of H G - G C - A A S 18 Figure 3.1 Map of the Bridge River district 25 Figure 3.2 Map of the Bridge River district showing the spring collection sampling locations 28 Figure 3.3 A typical calibration curve for total arsenic: ICP-MS analysis 29 Figure 3.4 Chromatogram of a five arsenic standard: H P L C - I C P - M S analysis 30 Figure 3.5 Calibration curve for arsenite: H P L C - I C P - M S analysis 31 Figure 3.6 Chromatogram of the extract of the standard reference material kelp 35 Figure 3.7 Chromatogram of a cone extract from a Spruce tree at Pioneer Mine 38 Figure 3.8 Relative amounts of arsenic species in tree samples from Pioneer Mine, fall collection 49 Figure 3.9 Chromatogram of a needle extract from the D F located at site 2 50 Figure 3.10 Chromatogram of a stem extract from the D F located at site 2 50 Figure 4.1 Calibration curve for M M A : H G - G C - A A analysis 63 Figure 4.2 Chromatogram of the growth media of isolate #713; H G - G C - A A analysis 64 ix LIST OF ABREVIATIONS A A S atomic absorption spectrometry A D P adenosine diphosphate A s B arsenobetaine A s C arsenocholine A S E accelerated solvent extraction As(III) arsenite As(V) arsenate A T P adenosine triphosphate B R Bridge River C C A chromated copper arsenate C L confidence level D F Douglas-fir D L detection limit D M A dimethylarsinic acid D N A deoxyribonucleic acid E E extraction efficiency F D freeze-dried FIA flow injection analysis G C gas chromatography G S H glutathione HCI hydrochloric acid H G hydride generation H P L C high performance liquid chromatography I A E A International Atomic Energy Agency iAs inorganic arsenic i.d. inner diameter ICP inductively coupled plasma L A luria agar L C - S A X liquid chromatography-silica based strong anion exchange L O Q limit of quantification M e O H methanol M M A monomethylarsonic acid M S mass spectrometry m/z mass to charge ratio N A nutrient agar N A A neutron activation analysis N B nutrient broth nd not detectable N F D non-freeze-dried NIST National Institute for Science and Technology N R C National Research Council O D S octadecylsilica X P C phytochelatin P D A potatoe dextrose agar P D B potatoe dextrose broth P L E pressurized liquid extraction ppb parts per billion ppm parts per million psi pounds per square inch P T F E polytetrafluoroethane (Teflon) Q A / Q C quality assurance/quality control rf radio frequency P v M C Royal Military College rpm revolutions per minute S Spruce S A M s-adenosylmethionine sex resin based strong cation exchange SD standard deviation S E M standard error of the mean S H thiol group SIM single ion monitoring mode T E A H tetraethylammonium hydroxide T M A O trimeythylarsinic acid T R A time resolved analysis U unclassified U B C University of British Columbia v/v volume per volume X A S X-ray absorption spectroscopy XI ACKNOWLEDGMENTS I am entirely grateful for the guidance and support I have received from my research supervisor Dr. W.R. Cullen. His knowledge and caring personality has made this project enjoyable. M y second supervisor, Dr. David Chen, has provided encouragement and advice during my studies and I thank him for this support. He has also given me the opportunity to expand my knowledge in the field of analytical chemistry. I would like to thank past and present members of my research group, Vivian Lai, Corinne Lehr, Kathy Sun, Hongsui Sun, Vicenta Devesa, Mill ion Woudneh, Sophia Granchinho, and Andrew Owen for their patience, continual assistance and especially their friendship. Thanks particularly to everyone who was involved in the field trip to Bridge River. In addition I would like to acknowledge Iris Koch for her advice and help. Elena Polishchuk's expertise and teaching ability has allowed for my skills to expand into the field of microbiology. Her assistance has been invaluable during the course of my project. I would also like to thank Olivia Lee for her help in the identification of the tree samples. I am especially grateful to Bert Mueller for his skills and help with the ICP-MS. Finally I would like to thank my family and friends for keeping me happy and healthy with their unconditional love and support. 1 Chapter 1 INTRODUCTION 1.1 General Introduction Environmental chemistry is a complex science involving the study of chemical species in water, soil and air. In order to understand the interactions that occur among these environmental compartments the contribution from many disciplines such as microbiology, geology and toxicology, along with chemistry, must be considered. Recently there has been a growing concern regarding contamination involving metals and metalloids. Much of this is focused on the metalloidal element, arsenic. 1.2 Chemistry of Arsenic Arsenic is placed in group 15 of the periodic table and it has one stable isotope occurring with 100% abundance. The element is not reactive in dry air but it will oxidize upon exposure to moisture. Arsenic has four common oxidation states, -3, 0, +3 and +5. The later two states are the most significant in the environment. The element forms strong covalent bonds to most non-metals, such as oxygen, sulfur, hydrogen and alkyl groups, thus arsenic is found as oxides, sulfides, hydrides and organometallic compounds. Some common arsenic species found in the environment are listed in Table 1.1 2 Table 1.1 Some arsenic compounds found in the environment Name Abbreviation Chemical Formula Arsenate, arsenic acid As(V) A s O ( O H ) 3 Arsenite, arsenous acid As(III) As (OH) 3 Monomethylarsonic acid M M A C H 3 A s O ( O H ) 2 Dimethylarsinic acid D M A ( C H 3 ) 2 A s O ( O H ) Trimethylarsine oxide T M A O ( C H 3 ) 3 A s O Methylarsine M e A s H 2 ( C H 3 ) A s H 2 Dimethylarsine M e 2 A s H ( C H 3 ) 2 A s H Trimethylarsine MesAs ( C H 3 ) 3 A s Arsenobetaine A s B ( C H 3 ) 3 A s + C H 2 C O O " Arsenocholine A s C ( C H 3 ) 3 A s + C H 2 C H 2 O H Arsenosugars X - X I V See figure below Sugar X Y X - O H - O H XI - O H - O P 0 3 H C H 2 C H ( O H ) C H 2 O H XII - O H - S 0 3 H XIII - O H -OSO3H X I V - N H 2 - S 0 3 H where R = ( C H 3 ) 2 As(O) 3 1.3 Toxicity of Arsenic Compounds Since the fifth century B C arsenic compounds have been used by physicians and poisoners.1 "White arsenic", or arsenic trioxide (AS2O3), was commonly utilized in the past as a homicidal poison. This species can be absorbed through the lungs and intestines.2 Chronic and acute poisoning can occur by ingestion of arsenic and it is also a human carcinogen. 2 , 3 Even so, arsenic trioxide has been ingested daily in the past by people who believed it increased their strength and longevity. In the early 1900's organoarsenicals were used in medicine to treat syphilis and sleeping sickness. 1' 4 Use of organoarsenic compounds in medicine has decreased due to the availability of less toxic and more selective drugs, but they are still found to be useful in treating some types of leukemia. 5 The toxicity of arsenic varies with the chemical form it is found in. Toxicity can range from essentially nontoxic to extremely toxic depending on the oxidation state of arsenic and the groups it is bound to. Trivalent inorganic arsenic compounds are considered to be more toxic than pentavalent inorganic compounds, and organoarsenic compounds in the pentavalent state are less toxic than inorganic arsenic. Studies recently have suggested that methylated As(III) species may be more toxic than inorganic species.6 They are certainly more toxic than As(V) organoarsenicals.1 Some species, such as arsenosugars and arsenobetaine, have not shown a toxic effect on biological systems and are thus thought to be non-toxic.4 The hydride, arsine (AsHb), is considered to be the most toxic arsenic species. Thus variability of toxicity makes the detection and 4 identification of individual arsenic species a necessity. The detection and quantification of individual forms of an element is called chemical speciation and in order to understand the impact arsenic has on the environment speciation studies are essential. The mechanism of toxicity differs with the valence state of arsenic. Arsenite has a high affinity for thiols, which are sulfur analogs of alcohols. 4' 7 It is believed the arsenical binds to these sulfur groups present in enzyme active centers, disrupting the function of the enzyme. 2' 4 The arsenical Lewisite (Cl2AsCH=CHCl) was developed in the early 1900's by W . Lee Lewis to be used as a poisonous gas. It is thought to interact with thiol groups in exerting its blistering action. The antidote to Lewisite, British antilewisite (HSCH2CHSHCH2OH), and other arsenic poisons contain dithiol groups. The toxicity o f arsenate is less understood and a number of mechanisms have been proposed. Arsenate is structurally similar to phosphate, thus it can replace a phosphate group in A T P forming an easily hydrolyzed arsenate ester of A D P . This leads to uncoupling of phosphorylation and the breakdown of essential metabolic processes.2'4 Another theory is that arsenate is reduced to arsenite which then interacts with thiols as described previously. Methylated As(III) compounds have been shown to be more reactive than the toxic inorganic As(III) species, having greater cytotoxicity6 and O Q damaging D N A ' . Up until this discovery, methylation was thought to be a detoxification process but it is now suggested that it may be an activation process. 5 1.4 Uses of Arsenic The use of small amounts of arsenic in alloys with lead and copper improves the property of storage batteries.1 GaAs and InAs are used for optical equipment such as light-emitting diodes and infrared emitters. One main use of arsenicals in the past was as a pesticide in agriculture. Restrictions have decreased the use of arsenic in this way but the preservation of wood with C C A (chromated copper arsenate) is still common, although this use is being phased out. 1 0 1.5 Arsenic in the Environment Arsenic is a trace element, meaning it naturally occurs at levels of a few parts per million or less. It is a ubiquitous element found in all environmental compartments. In the earths crustal rocks it is naturally found at an average level of 2-5 ppm. Arsenic is often associated with sulfur and many other metals in mineral deposits. Common arsenic minerals are realgar (AS4S4), orpiment (AS2S3) and arsenopyrite (FeAsS). 1 1.5.1 Sources of Arsenic Both natural and anthropogenic sources are responsible for the presence of arsenic in the immediate environment. Most anthropogenic input comes from smelting operations and fossil-fuel combustion. 1 1 Arsenic is also a by-product from mining operations and accumulates as waste material. Arsenicals used as pesticides and 6 herbicides are another major anthropogenic source. Natural weathering and biological activity can result in the emission of arsenic into the atmosphere. It can then be redistributed by natural events, such as rain and wind. Arsenic can also be mobilized by bacteria and fungi, often via methylation and volatilization. Interactions of microorganisms with arsenic will be discussed in more detail in Section 1.6 and in Chapter 4. 1.5.2 The Marine Environment Trace amounts of arsenic, mainly as arsenate, are found in seawater (~2ppb). Arsenite, M M A and D M A have also been identified in ocean waters and are certainly the products of biological activity. 1 3 Arsenic species in marine organisms are found at levels much higher (up to 100 ppm) than that of the surrounding oceanic waters.11 The implications of this, with respect to the food chain, have been studied to a great extent. The forms of arsenic found in marine animals differ from those found in seawater. Arsenobetaine (AsB), which was first isolated from a rock lobster in 1977 1 4, has been found abundantly in most marine animals."' 1 5 ' 1 6 The origin of this compound is still not clear. A s B is excreted rapidly in urine by humans 1 7, therefore ingestion of these marine animals may not be a toxic hazard. Arsenosugars 1 5' 1 6 and arsenocholine1 8 have also been identified in marine animals. Marine algae contain levels of arsenic ranging from 0.8 to 12.1 ppm wet weight, most of which is found in the form of arsenosugars. It is thought that marine organisms metabolize inorganic arsenic found in the water column to species such as A s B and arsenosugars, although the precise pathways have not yet been elucidated. 7 1.5.3 The Terrestrial Environment Less is known about arsenic in the terrestrial environment as compared to the marine environment. Arsenic concentrations in fresh water ranges more than that in seawater.'1 Normally it is found in the low ppb range but in contaminated areas it can be present in ppm. The concentration usually depends on the geological composition of the area but it can be influenced by anthropogenic input. Pollution of ground water by arsenic has been a major cause of concern recently. Mass poisonings have been identified in areas where the main source of drinking water comes from wells contaminated with arsenic.9 There are two main proposals to try and account for this contamination. One such explanation is that upon drilling the irrigation well, oxygen is introduced into the anoxic, pyrite containing soil below the water table. Pyrite, which usually contains ~ 1 % arsenic, will be oxidized, releasing the arsenic to the ground water. The other theory is that bacteria reduce the arsenic-rich iron oxyhydroxides in anoxic groundwater thus releasing the arsenic to the surrounding. 1 9 Arsenate is commonly the major form found in soils but only a small amount of the arsenic present has been shown to be bioavailable to plants. Studies regarding the speciation of arsenicals in terrestrial plants are limited and the topic has only recently been attracting attention. Inorganic arsenic is usually the major form found in plants but M M A and D M A are often seen in trace amounts.2 1"2 4 Arsenosugars were once believed to be found only in marine algae until the development of more sensitive analytical methods allowed for the identification of these compounds in some terrestrial 25 26 21 organisms. Arsenobetaine has also been identified in mushrooms , plants and 8 lichens . A more detailed discussion of the interaction of arsenic with terrestrial plants and soils will be given in Chapter 4. 1.6 Introduction to Microorganisms and Arsenic The first report of the volatilization of arsenic by microorganisms was made by Gosio in the late 19 t h century. In the early 1930's Challenger et a l 2 8 found that by growing the fungi Scopulariopsis brevicaulis in the presence of arsenic trioxide a volatile arsenic compound, which he identified as trimethylarsine, was produced. This was presumably the same gas detected earlier by Gosio and has become known as Gosio gas. Challengers' studies led to a proposed metabolic pathway for the biomethylation of arsenicals (Figure 1.1). It was later suggested that S-adenosylmethionine (SAM) is the 29 source of the carbonium ion and that a thiol may be the reducing agent in this scheme. 2e- C H 3 + 2e" A s O ( O H ) 3 • As (OH) 3 • C H 3 A s O ( O H ) 2 • C H 3 A s ( O H ) 2 C H 3 + 2e~ C H 3 + C H 3 A s ( O H ) 2 • ( C H 3 ) 2 A s O ( O H ) • (CH 3 ) 2 As(OH) • ( C H 3 ) 3 A s O 2e" ( C H 3 ) 3 A s O • ( C H 3 ) 3 A s Figure 1.1 The Challenger Mechanism 9 Bacteria and fungi have been isolated that show tolerance to inorganic arsenic. Oxidation of arsenite is one possible mechanism of resistance to arsenite that has been seen in bacteria such as Pseudomonas and Alcaligenes. The reduction of arsenate to A S H 3 has also been reported for these microbes. 1 1 Many trace elements in the environment can undergo conversions to different species via microbial action. Indeed, arsenic speciation and distribution in the terrestrial and marine environments are affected by this activity. Thus it is important to probe the influence of microorganisms when investigating the cycling of arsenic in the environment. 1.7 Objectives of the Present Studies Humans are exposed to arsenic via inhalation, food and drinking water. In order to understand how these exposure routes affect humans it is necessary to analyze the species present in all areas of the environment. The increase in the number of contaminated sites around the world has necessitated studies of the less understood terrestrial environment. In particular little is known about arsenic speciation in terrestrial biota. Recently, biota have been discovered that have the ability to hyperaccumulate arsenic. 2 3 ' 3 0 These plants have been suggested to be used in the phytoremediation of contaminated sites. Before this can be applied it is necessary to understand the mechanisms behind the anomaly. The work presented here probes this topic. One objective of this thesis was to determine the speciation and total arsenic concentrations of trees growing in an area contaminated with arsenic. In the 1960's Warren et al discovered that Douglas-fir trees have a remarkable 10 affinity for arsenic and thus it was suggested these trees could be used as biogeochemical indicators in the search for precious metal deposits. Douglas-fir trees were investigated in the present study in an attempt to understand the interactions and processes occurring in arsenic accumulating biota. To our knowledge, the speciation of Douglas-fir trees has not been reported previously. The second objective was to isolate bacteria and fungi from surface soils found at the contaminated site. Once isolated the interactions of these microorganisms with arsenic was analyzed in an attempt to further understand the cycling of arsenic in the terrestrial environment. Undoubtedly, the first two objectives are linked in a very significant way. The microbial environment can potentially affect the uptake of arsenic by biota. In the present work an attempt is made to understand the interactions between microbes and terrestrial trees. The information from these results can help determine the impact of arsenic contaminated sites on the environment. Another objective was to investigate methods used in the analysis of arsenic. Preparation procedures are important in maintaining the integrity of the original samples. It is a common practice to freeze-dry material before analysis. Studies regarding the effect this process has on the speciation of the original sample are limited. In the present work the arsenic content of fresh and freeze-dried tree samples were compared to explore this matter. 11 Chapter 2 EXPERIMENTAL METHODS FOR THE ANALYSIS OF ARSENIC 2.1 Instrumentation 2.1.1 Introduction Many methods have been used to analyze the total arsenic content of environmental samples. Usually the arsenic in the sample is first converted into water soluble inorganic species by using, for example, acid digestion. Hydride generation techniques coupled to atomic absorption spectrometry (HG-AAS)31 or inductively coupled plasma with mass spectrometric detection (ICP-MS) ' are commonly used to analyze the sample solution. The speciation of arsenic in environmental samples is accomplished by extraction of the species from the sample matrix and then the resultant solution is analyzed by using methods involving a chromatographic technique coupled to an element-specific detection system. Gas chromatography and high performance liquid chromatography are often used. In gas chromatography the arsenic compounds need to be derivatized to more volatile forms before they can be injected onto a GC column. This is commonly accomplished by the hydride generation technique using sodium borohydride as a reducing agent. The following equations summarize the reaction: (3-n)BH4" + (3-n)H+ + RnAs(OH)3.n + 2(3-n)H20 -> (3-n)B(OH)3 + R nAsH 3 . n + 3(3-n)H2 where n = 0-3 12 HPLC is a well established separation technique for speciation of arsenic compounds. 2 0 ' 3 4 Derivatization is not necessary as it is for GC. Different types of chromatography have been used but it is common to employ ion-exchange or ion-pairing methods. 2.1.2 Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) Apparatus A V G Plasma Quad 2 Turbo Plus inductively coupled plasma-mass spectrometer (VG Elemental, Fisons Instrument) was used to measure total arsenic. A standard ICP torch served as an atomizer and ionizer, and sample introduction into the argon plasma of the torch was accomplished by using a de Galan V-groove nebulizer. The ICP-MS was equipped with a SX 300 quadrupole mass analyzer which was operated in the single ion monitoring mode (SIM). The operating parameters for the ICP-MS are summarized in Table 2.1 and a schematic of the ICP-MS instrumentation is shown in Figure 2.1. 13 Table 2.1 Operating parameters of ICP-MS Feature Specific Conditions Forward r.f. power Reflected power Outer (cooling) gas flow rate Intermediate (auxiliary) gas flow rate Nebulizer gas flow rate Nebulizer type Analysis mode Quadrupole pressure Expansion pressure Scan Mode 1350 W <10 W 13.8 L/min 0.65 L/min 1.002 L/min De Galan SIM 9x 10"7mbar 2.4 mbar Peak jump Detector Quadrupole Quadrupole R F Generator i Amplifier Quadrupole R F DC Control Lenses Interface TCP Bias Voltage Supplies ml. C 1 f i n Vacuum Pumps Plasma Quad Control IEEE Interface' t A A A A , 3 Computer ICP R F Generator Gas Control Sample Figure 2.1 Schematic diagram of ICP-MS 14 Experimental Conditions and Procedure Flow injection introduction (FIA) at a rate of 0.8 mL/min was used for the analysis of digested samples (lOOpL) by using ICP-MS. The carrier solution used was a 1% nitric acid solution made from deionized water filtered through a 0.45pm Millipore filter. The solution was stored at 4 ° C until it was needed for analysis when it was then warmed to room temperature before being used. A low acidity is compatible with the ICP-MS instrumentation and is needed to preserve the speciation in the sample. Mass to charge (m/z) signals at 75, 77, 82 and 103 were simultaneously monitored. Signals at a m/z ratio of 75 correspond to the ion of interest, A s + , which has 100% isotopic abundance at this mass. It is necessary to monitor the other m/z signals in order to identify and correct for possible interfering species originating from the plasma or matrix. Thus, the m/z signal at 75 can also be due to the possible interfering species A r 3 5 C l + which arises from the argon gas used to carry the sample into the torch. Chlorine has isotopes of 35 and 37 in an isotopic ratio of 75.77:24.23 respectively. Monitoring the m/z ratio 77, corresponding to that of A r 3 7 C l + , allows for the correction of interference due to A r 3 5 C l + . However the signal at 77 can also be due to selenium which has isotopes of 77 and 82 in an isotopic ratio of 7.63:8.74. Contribution of the 77 peak from selenium can be determined in the same manner as for A r C l interference. Signals at 103 corresponding to Rh were also monitored. Rhodium is used as an internal standard to correct for plasma instability and detector drift. A l l signals were collected and the data was sent to the V G data system and then exported for further data processing using Microsoft Excel. 15 2.1.3 High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry (HPLC-ICP-MS) Apparatus The chromatography system consisted of a Waters Model 510 delivery pump, a Reodyne six-port injection valve with a 20ul sample loop and a reverse phase Cig column ( G L Sciences Inertsil O D S , 250 mm x 4.6 mm). A guard column, packed with the same material as the column (Supelco), preceded the analytical column. The ICP-MS instrumentation discussed previously was employed as a detector for the H P L C eluent in the analysis of extracted samples for speciation studies. The H P L C column was interfaced to the ICP nebulizer with P T F E tubing (2.5cm) and appropriate fittings. ICP-MS conditions were the same except that the mass analyzer was operated in time resolved analysis mode (TRA) instead of SIM. Experimental Conditions and Procedure A mobile phase consisting of 10 m M of the ion-pairing reagent tetraethylammonium hydroxide ( T E A H ) , 4.5mM malonic acid, 0.1% methanol and 5ppb Rh was used during H P L C - I C P - M S analysis. The mobile phase was filtered through a 0.45 pm cellulose nitrate filter (Millipore) and the pH was adjusted to 6.8 using 3 M nitric acid (Seastar). The analytical column was equilibrated with the mobile phase for approximately one hour prior to analysis. The flow rate was 0.8ml/min and the delivery pump's pressure was between 800 and 900 psi throughout the analysis. 16 Before injection into the H P L C system all extracts were diluted as necessary and filtered through a 0.45 um filter by using a disposable 1 ml syringe. This was done to prevent contamination of the column and/or blockage within the system. Arsenic compounds in the samples were identified by matching the retention times of the peaks in the chromatograms with those of standard arsenic compounds. Quantitative analysis was done using external calibration curves for each compound corresponding to a matching standard. 2.1.4 Hydride Generation-Gas Chromatography- Atomic Absorption Spectrometry (HG-GC-AAS) A Varian atomic absorption spectrometer (AA-1275) equipped with a heated quartz cell was used in the analysis of arsenic in microbiological samples. A semi-continuous hydride generation system was coupled to the A A S (Figure 2.2). Hydride generation (HG) is a common method used to introduce arsenic species into an atomizer as a gas. The generation of volatile hydrides was brought about by first acidifying the sample and then mixing it with a 2% aqueous solution of sodium borohydride in a reaction coil. A peristaltic pump (Gilson Minipuls 2) was used to deliver the sample solution or standard, acid and NaBH4 into the system. Helium was used to carry the hydrides evolved into a gas/liquid separator consisting of a glass Buchner funnel within a glass cylinder with a side arm for waste. The hydrides were then swept through a P T F E U-tube at ~ "78°C (liquid nitrogen/acetone) to trap any water present. The tubing following the trap was 17 disassembled and cleaned with a flow of air after every ten runs to prevent water accumulation from clogging the tubing. The hydrides where then swept through a U-tube where they were trapped at ~ "196°C (liquid nitrogen). One minute after the peripump was stopped the liquid nitrogen bath was replaced by a warm water bath ( ~ 6 0 ° C ) . The heat allowed the gasses to flow with the He carrier onto a G C column (10% SP2100 on 80/100mesh, Chromsorb) housed in a G C oven (Hewlett Packard, 5890A) where a temperature ramping program separated the gases. The hydrides were then carried through the side arm of an open-ended T-shaped quartz absorption tube (optical path: 9.5 cm x 0.8 cm i.d.) mounted in the optical path of an A A S instrument. Hydrogen gas was used as a fuel for producing a flame inside the T-tube and air was used as an oxidant. The sample was carried into the flame where atomization occurred. The source used for A A measurements was an arsenic hollow cathode lamp (Varian) operating at a wavelength of 193.7 nm. The light emitted was precisely aligned with the quartz tube to provide maximum absorption. Signals from the A A S were sent to a Dell 466/M where data was recorded and processed using Shimadzu E Z Chrom software. Table 2.2 summarizes the operating parameters for the H G - G C - A A S system and Figure 2.2 shows a schematic diagram of the H G - G C - A A S instrumentation. 18 Table 2.2 Operating parameters of H G - G C - A A S Feature Specific Conditions Wavelength 193.7 ran Slitwidth 1 ran Lamp Current 10 m A Hydrogen gas flow (flame) 75 mL/min Air flow (flame) 120 mL/min Helium gas flow (purge) 30 mL/min Helium gas flow (carrier) 3 mL/min NaBH4 2% Buffer 4 M acetic acid Temp program 5 0 - 1 5 0 Sample, Acid , NaBH4 5 mL/min Reaction Coil Peristaltic Pump II Gas/Liquid Separator 5 Helium Carrier gas Sample Acid NaBH4 • Waste Computer Atomic absorption spectrometer Ha/Air Acetone slurry water trap Liquid N2 Cryotrap GC column in GC oven Waste Figure 2.2 Schematic diagram of H G - G C - A A S 19 2.2 Reagents and Chemicals A l l chemicals used were of analytical grade and obtained from commercial sources unless otherwise stated. The chemicals and reagents used included methanol ( H P L C grade, Fisher), tetraethylammonium hydroxide ( T E A H , 20 wt. %, Aldrich), malonic acid (BDH), nitric acid (68-71%, sub-boiling distilled, Seastar Chemicals), sodium borohydride (Aldrich), acetic acid, hydrochloric acid, and hydrogen peroxide (30%, Fisher). A 3 M HNO3 (Seastar Chemicals) solution was used to adjust the p H of the T E A H mobile phase used in H P L C analysis. The water, with resistivity better than 18 M Q c m , used to make up all standard solutions, mobile phases, sample solutions for extraction and digestion, and media was deionized using an Elga deionizer. 2.2.1 Standards and Reference Materials Standard working solutions of arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, T M A O and arsenobetaine were made by diluting previously prepared stock solutions with deionized water as necessary. A n arsenate stock solution was prepared from sodium arsenate (Na2HAs04 • 7H2O, Sigma). A rhodium stock solution was made from RJ1CI3 for use as an internal standard in total digests and in the T E A H mobile phase used in H P L C analysis. 20 The following standard reference materials were used for Q A / Q C ; fucus ( I A E A -140, Monaco), oyster tissue (NIST-1566a), and D O R M - 2 (NRC, Canada). Also, kelp powder (Galloway's naturally kelp powder, Richmond, B . C . , Canada) which has been previously analyzed and shown to contain the four arsenosugars (X, XI , XII, XIII) was used as a laboratory standard.3 6 The presence of arsenosugars in samples can be confirmed by comparison of retention times with those of standards. A pine needle Standard Reference Material (NIST-1575) was also extracted because terrestrial samples were investigated. References are not only used to help identify compounds but also to reveal matrix effects. Therefore it is beneficial to use references with a similar matrix to the samples being analyzed. 2.2.2 Culture Media In the microbiological work, bacteria and fungi were first grown on either nutrient agar (NA), luria agar (LA) or potato dextrose agar (PDA). The microbes were then transferred to the liquid media, nutrient broth, luria broth or potato dextrose broth respectively. A l l media were prepared as directed by the manufacturers. The only deviation from recipes was the addition of arsenic in some cases. 21 2.3 Sample Preparation for Analytical Analysis 2.3.1 Acid Digestion Acid digestion was performed on freeze-dried and wet samples for total arsenic 37 analysis by ICP-MS. The method used was similar to that described by N 0 r u m et al. A sub-sample (-0.25-0.5g) of homogenized material was accurately weighed into a 20 ml glass test tube (outer diameter of 16 mm). Teflon boiling chips and 2 ml of concentrated nitric acid (doubly distilled in quartz, Seastar, Sidney, B.C.) were added to each test tube before being placed on a block heater (Standard Heatblock by V W R Scientific Products). The temperature of the heater which was initially set at 80°C was increased 10°C approximately every hour to a maximum temperature of ~ 1 5 0 ° C . At this time the heater was turned off and the samples were allowed to cool over night. The following day 1 ml of concentrated nitric acid and 2 ml of hydrogen peroxide were added to each test tube. The hydrogen peroxide was needed to break up the organic constituents of the samples and to ensure complete digestion. The samples were slowly heated to 1 5 0 ° C and allowed to evaporate to dryness. The samples evaporation times ranged widely from 1 to 7 days. After evaporation the samples were removed from the heating block, allowed to cool and redissolved in a solution containing 1%HNC>3 and 5ppb Rh. The digest was accurately made up to 5ml, vortex-mixed and then filtered through a 0.45 filter into a sterile 15 ml centrifuge tube (Falcon). The digests were then stored at 4°C until ready to be analyzed by using ICP-MS. 22 2.3.2 MeOH/H20 Extraction Freeze-dried and wet tree samples were extracted using a method similar to that described by Shibata and Morita. 1 6 A subsample (-0.25 - 1.0 g) of homogenized material was accurately weighed into sterile 15 ml centrifuge tubes (Falcon). For every gram of sample, approximately 10 ml of M e O H / H 2 0 solution was added to each tube and then sonicated for - lOmin . The samples were then centrifuged for ~10min and the supernatant was decanted into a round bottom flask (100 or 250ml) or 50 ml Falcon centrifuge tube via a glass Pasteur pipette. The procedure was repeated four more times for a total of five extractions. The extracts were evaporated by using a rotary evaporator (Biichi) or a SpeedVac® Plus (Savant) to near dryness (1-2 ml) and then made up to -10 ml with deionized water. The extracts were then stored at - 2 0 ° C to preserve sample integrity until ready to be analyzed by using ion-pairing H P L C - I C P - M S (Section 2.1.3). 2.3.3 Culture Preparation Microbes grown in liquid media were harvested by centrifugation of the biomass/liquid mixture at 5000-7500 rpm for 20-30 minutes (Sorvall® R C - 5 B Refrigerated Superspeed Centrifuge, Dupont instruments). The supernatant was transferred via pipet to sterile centrifuge tubes (Falcon) in a biological safety hood (NUAIRE) and then stored at "20°C prior to analysis by using H G - G C - A A S (Section 2.1.4). The cell pellets were freeze-dried (Flexi-dry, Kinetics), ground and extracted by using a procedure modified from that described in Section M e O H / H a O (1 ml, 1:1 v/v) was added to centrifuge tubes containing the cells, sonicated for ~10min and then centrifuged at 5000 rpm for 20min. The supernatant was transferred via Pasteur pipette into 15 ml sterile centrifuge tubes (Falcon). The extraction procedure was repeated four more times for a total of five extractions. The combined extracts were evaporated to near dryness by using a SpeedVac® Plus (Savant). Extracts were then redissolved in 1 ml of deionized water, vortex mixed and then sonicated for ~10min. Extracts were kept frozen prior to analysis by using H G - G C - A A S . The cell extracts were filtered (0.2 pm) before introduction into the analysis system. This was necessary because of the inevitable presence of cells in the extract. 24 Chapter 3 BRIDGE RIVER TREE ANALYSIS 3.1 Introduction The Bridge River District is located north of Pemberton in British Columbia, Canada (Figure 3.1). In the first half of the 1900's many quartz gold veins were found in this area and some of the more profitable ones were located close to the small town of Bralorne 3 8 . The Bralorne-Pioneer gold camp included the Bralorne, King and Pioneer mines and was abandoned in 1971 because of low gold prices. 3 9 The ore of these mines consist of gold in quartz veins containing small amounts of sulphides. Most of the sulphide is present as pyrite (FeS2) and arsenopyrite (FeAsS) which are usually associated with the gold in the mined ore . 3 8 ' 4 0 Arsenopyrite is an ore mineral that is naturally decomposed, slowly releasing arsenic into the environment.4 1 Mining activity results in the acceleration of this process. Thus, arsenic is a waste product from the mining of metals and results in the contamination of many areas. The association of arsenic with gold has proven useful. Arsenic can be used as a pathfinder element in the search for precious metal deposits.4 2 Soil samples were originally analyzed in attempts to locate gold-rich veins but biogeochemical techniques were introduced in the 50's 4 3 In an attempt to solve problems associated with soil sampling Warren et a l 4 2 analyzed plants for arsenic as indicators for gold mineralization. They unexpectedly discovered that Douglas-fir (Pseudotsuga menziesii) had a remarkable 25 affinity for arsenic. In this study the arsenic content of the ash of Douglas-firs collected from the Bralorne-Pioneer mining area ranged from 8 to 1550 ppm. The following chapter describes research to support the results of Warren et al and to determine present arsenic levels and speciation in samples within the Bridge River area. The objective is to further the understanding of the biochemical behavior of arsenicals in terrestrial plants. Figure 3.1 Map of the Bridge River district 26 3.2 Douglas-fir The Douglas-fir is a native conifer tree of British Columbia, Canada. It was named after a Scottish botanist, David Douglas, who introduced many indigenous British Columbia trees to Europe. Douglas-fir, genus pseudotsuga meaning "false hemlock", is not a true fir thus its name is hyphenated. There are six species but only two are native to North America, the coastal {menziesii) and interior (glaucd) Douglas-firs. The coastal species reaches greater heights (up to 85 m) and can be found on the south coast of the mainland and across Vancouver Island. The interior species occurs throughout southern B C and grows up to 42 m in height. The needles of Douglas-firs are flat and the trees contain woody cones 5-11 cm long. The wood is hard, stiff and durable and covered with thick grey-brown bark. 4 4 3.3 Spring Collection 3.3.1 Sample Collection and Treatment Soil and tree samples were collected in May 2001 from three sites in the Bridge River district; Moha(l), Mission Dam(2) and Pioneer Mine(3). Figure 3.2 shows a map of the sampling sites. Four Douglas-fir, one Amabilis fir (abies amabilis) and three Engelman spruce (picea engelmanii) where sampled from these locations. Surface soil was sampled by hand into polypropylene bottles that had been acid washed previously. Biota was sampled by hand and stored in Z i p l o c ® or black plastic bags. A l l samples 27 were kept cool following collection and upon return to the lab kept at 4°C until processing. The identification of trees was carried out by reference to a guide book 4 5 and with help from a botanist, Olivia Lee. The soil samples were sieved (2 mm mesh) then sub-sampled into 50 ml sterile centrifuge tubes (Falcon) prior to freeze-drying (Flexi-dry, Kinetics). These samples were further investigated for microbiological activity which will be discussed in Chapter 4. The needles and cones of each tree sample were separated from the stems and then finely ground. The samples were then stored in 50 ml centrifuge tubes at 4°C until ready for analysis. 29 3.3.2 Sample Preparation and Analysis A l l homogenized tree samples and reference materials were digested in duplicate for total arsenic analysis as described in Section 2.3.1. Dilutions of the sample and reference digest solutions were done as appropriate. Standard solutions ranging from 0 to 100 ppb were prepared from a stock solution of As(V) diluted to the appropriate concentrations by using a solution of 1% HNO3 spiked with 5ppb Rh as an internal standard. A l l samples and standards were analyzed in triplicate by using ICP-MS (Section 2.1.2). A calibration curve was made for the arsenic standard solutions and subsequently used to determine total arsenic content of the sample digests (Figure 3.3). Calibration curves obtained were linear in the range of 0 to 100 ppb As. Samples and standards were stored at 4°C prior to analysis. 120 Concentration (ppb) Figure 3.3 A typical calibration curve for total arsenic: ICP-MS analysis 30 The homogenized tree samples and reference materials were extracted as described in Section 2.3.2. Combined extracts were collected in a 50 ml centrifuge tube and evaporated by using a SpeedVac (Savant) and then redissolved in deionized water. A stock solution containing five arsenic compounds, A s B , As(III), As(V), M M A and D M A was prepared and diluted with deionized water to make standard solutions ranging from 10 to 100 ppb. On the day of analysis the extract solutions were thawed and then analyzed by using H P L C - I C P - M S (Section 2.1.3). The sample extracts were diluted if necessary. Arsenic compounds were identified by matching the retention times of the peaks in the sample chromatograms with those of standards (Figure 3.4). 70000 60000 50000 40000 8 30000 c 20000 o o 10000 0 AsB As(lll) 0 100 200 300 400 retention time (sec) 500 600 Figure 3.4 Chromatogram of a five arsenic standard: H P L C - I C P - M S analysis 31 The standard solutions were also used to construct calibration curves for quantifying the arsenic compounds that were in the extract samples. The curves were linear for all compounds within the concentration range of the standards. A n example of a calibration curve is shown in Figure 3.5. Extract samples and standards were kept at - 2 0 ° C prior to analysis. 700000 600000 -500000 400000 -£ 300000 -< JC ra S. 200000 100000 -0 -I , n , r r -j 0 20 40 60 80 100 120 Concentration (ppb) Figure 3.5 Calibration curve for arsenite: H P L C - I C P - M S analysis 32 3.3.3 Results and Discussion Total Arsenic Analysis Trees The concentrations of arsenic in acid digested tree samples collected from the three different Bridge River sites are shown in Table 3.1. The site numbers correspond to the sampling locations indicated on the map of the area (Figure 3.2). Table 3.1 Arsenic concentrations in tree samples from the spring collection (error reported as S E M of duplicate digestions at a 95% C L a ) Sample Localization Treeb Tissue Type Total As (trip#-site#) (site) (fig/g wet weight) 1-1 Moha DF-1 Stem 1.7 ± 0 . 3 Needle 1.0 ± 0 . 2 Cone 0.81 ± 0 . 0 8 1-2 Dam F-1 Stem 6.9 ± 0.9 Needle 1.50 ± 0 . 0 1 1-2 Dam DF-2 Stem 3.1 ± 0 . 7 Needle 2.1 ± 0 . 1 Cone 1.4 ± 0 . 2 l-3a Pioneer Mine S-2 Stem 1.5 ± 0 . 4 Needle 1.19 ± 0.01 l-3a Pioneer Mine DF-3 Stem 43 ± 6 Needle 116 ± 2 Cone 1.5 ± 0 . 8 l-3b Pioneer Mine DF-4 Stem 1 4 ± 1 Needle 40 ± 4 l-3c Pioneer Mine S-3 Stem 2.2 ± 0.5 Needle 1.4 ± 0 . 4 Cone 2.8 ± 0 . 4 l-3c Pioneer Mine S-4 Stem 1.8 ± 0 . 1 Needle 1.6 ± 0 . 3 Cone 5 ± 1 asee appendix A for the equation for S E M b D F = Douglas-fir; F=Fir; S= Spruce 33 The Moha and Mission dam sampling sites are areas that may be indirectly subjected to arsenic contamination. These two sites are located downstream from areas well mineralized with arsenic. 4 0 Table 3.1 shows that tree samples from the two sites have less than 7 ppm of arsenic in the samples, although the concentrations are higher than those usually reported for terrestrial plants from uncontaminated areas (<1 ppm). 1 1 The data for samples from the Pioneer mine indicate that the Douglas-fir trees do seem to concentrate arsenic to a higher extent than other tree species growing in the same area. A range of arsenic levels is seen with differences depending on tree species and tissue type. In both Douglas-fir trees sampled at the mine a greater arsenic content is observed in the needles. Warren et a l 4 0 concluded that first-year needles and all older growth contain the lowest arsenic content and the first-year stems appear to have the highest concentrations. Because the age of the present samples are unknown it is difficult to make any conclusions with respect to this topic. In the next section this will be investigated further. Indeed the results presented in Table 3.1 support the previous finding that Douglas-fir trees have an affinity for arsenic. Nevertheless the present results do not indicate they are hyperaccumulators, a term that has been defined as plants that can accumulate metal concentrations >1000 |ig/g. 2 3 However, King et a l 4 6 found that Douglas-fir trees grown in soil rich with arsenopyrite had arsenic concentrations >5000 pg/g in all parts of the tree. 34 Soils Analysis of the soil samples ( N A A ; R M C , Kingston) from the Bridge River district showed that the area around the Pioneer mine was highly contaminated with arsenic. Soil concentrations are in the range of 35-1623 ug/g which is comparable to mine soil data from other areas. 2 1' 2 3 In contrast, the total arsenic content of the soils from the other two sites (Moha and Mission Dam) was 2.2 - 13.7 pg/g. Natural arsenic concentration in North American soils is 5-14 ug/g; thus it can be concluded the Moha and Mission dam sites are relatively uncontaminated. Pioneer mine soil samples were not taken directly at the sites where trees were sampled. Therefore it is hard to make any conclusions about the relationship between arsenic concentrations in the soil and the tree. The soil with the highest arsenic content (1623 pg/g) was taken from below the mine workings where almost no biota thrived except some grass. Soil taken from the river below the mine was found to contain 280 pg/g and soil sampled near the mine building was 228 pg/g. Tree samples were collected at a farther distance from the mine but the tree roots could still be in direct contact with the high arsenic levels in the soils near the mine. Arsenic Speciation Analysis Trees Arsenic species in the tree samples were determined by using H P L C - I C P - M S . Species were identified by comparison of peak retention times with those of the five arsenic standards as described in Section 3.3.2. A sample chromatogram for the standard reference material kelp is shown in Figure 3.6. This material was used as a standard for 35 the arsenosugars X , XII and XIII. Identification has previously been reported by Lai et 200 400 600 800 retention time (sec) 1000 1200 -75 -77 82 1400 Figure 3.6 Chromatogram of the extract of the standard reference material kelp. Signals at m/z 75 correspond to arsenic (see p. 14 for details). Speciation analysis was performed only on the samples from the Pioneer mine site because the acid digestion analysis revealed low arsenic levels in the Moha and Mission Dam samples. The distribution of arsenic species in the Pioneer mine tree samples is summarized in Table 3.2. o o O o vi a Ji. p a 1 p 00 C}%VlC)%Vl%V3?'Vl?<V. o 2 ^ o 2 ' • 2 ^ 2 ^ - 2 ^ 2 2 n , 2 2 < ^ 2 f T > 2 ^ l 2 o > CT CT CT CT is- 5 / 3 3 =8: H rt n cf cf r-t-•1 cr p P P 3 P P o CT CT Cu CT CT CT CT CT CT CT S3 0 0 *> 8 8 o cr o o CT <-> ^ CT 1 l — ' O P M * W M o o o q- cr ; _ , P P 0 0 CT CT o cf er CT CT ^ CT CT 3 Cu ON 3 3 3 3 Cu Cu Cu cu H H H >-t 1-1 P p. p CT CT CT CT CT CT O Cf oo g 2 3 * Cu 5 CT O O Cf r~i P v l ON ffi 2; 2 2 2! 2 : 3-Cu Cu Cu Cu Cu o CT o o £ CD m © ~ £ S « -ON U l w 00 ^ ^ M (yi u 0 \ w ^ 0 0 rt if rt' t/5 3 o H P g[ rt" u> k> o o 3 o CT 3 I—t-"I O 3 o •-+> r^ -3 -CT P >-« CO CT 3 0 0 X ! CT CT_ CT* CO CT CT CO CT" CO I-+5 >-p O O 3 CT CT 3 CT CT CTQ" 3 * ON 37 Inorganic arsenic is the main species present in all tree samples and arsenate predominates in the majority of samples. However, 50-78% of the total arsenic is present 91. as arsenite in the needles of the Douglas-fir. Francesconi et al found that arsenate is the major arsenical in the rhizoids and petioles of a hyperaccumulating fern, Pityrogramma calomelanos, but the fronds accumulate mostly arsenite. This hyperaccumulation of arsenic as arsenite has also been shown to occur in the fronds of the brake fern (Pteris vittata).30 The toxicity of arsenate has been attributed to its ability to uncouple oxidative phosphorylation (Section 1.3). 2 3 Thus it is possible the present results could indicate that Douglas-fir reduce and store arsenic as arsenite to protect them from interruption of this process. However, arsenite is usually considered more toxic. Most marine organisms have the ability to methylate arsenate to M M A , D M A , and A s B but this pathway does not seem to be a common mechanism employed in the terrestrial environment. Only trace amounts of the methylated arsenic species M M A and D M A were found in the pioneer mine tree samples. These species were often present in concentrations above the calculated detection limit of H P L C - I C P - M S (<1 ppb) but can not be quantified as they are below the limit of quantification (see appendix A for the equations for D L and L O Q ) . In some of the Spruce trees the methylated species content was up to 32% of the total extractable arsenic. The cone samples analyzed were from two of the Spruce trees and showed different results. D M A was determined to be a major species in the extract, second to that of arsenate. A chromatogram of one of these cone samples is shown in Figure 3.7. The sample was re-extracted and analyzed a few months later and the results were confirmed. At the retention times for arsenite, M M A and arsenobetaine, above baseline 38 responses can be observed in the chromatogram. Only D M A and arsenate are present at quantifiable concentrations, but there were indications of AsB. Similar results have been shown by Devesa et a l . 4 7 Detectable but unquantifiable amounts of AsB were found in stems of Douglas-fir and pine trees sampled from an uncontaminated site in Vancouver. It is unusual to find AsB in terrestrial organisms, however, recent studies have found this arsenical in plants.22 Arsenosugars were not detected in any of the tree samples of the present study. This is not unusual for terrestrial samples. 4000 As(V) 3500 3000 0 0 100 200 300 400 500 600 700 800 retention time (sec) Figure 3.7 Chromatogram of a cone extract from a Spruce tree at Pioneer Mine 39 Extraction Efficiency (EE) The extraction efficiency (see Table 3.2 for calculation) is higher for the Douglas-fir samples (35-86 %) than the other tree samples (< 10%). A low extraction efficiency is commonly found for terrestrial plants but this does not appear to be the case for arsenic accumulators.2 3 In methanohwater extractions it is assumed only the water soluble species will be extracted. Thus, low extraction efficiency may mean there are unknown insoluble arsenic compounds in the sample or that some arsenic species are trapped inside cells or bound to cell walls. It is possible that some extracted species are not observable using the chromatography employed. 1 5 These species would not be accounted for in the calculation of extraction efficiency and hence result in a low E E . Recently new extraction methods have been studied to provide more efficient extractions of plant material, for example pressurized liquid extraction (PLE), which is also known as accelerated solvent extraction (ASE). This technique uses high temperatures and pressures. Schmidt et al have shown that higher extraction efficiencies for inorganic arsenic can result using P L E . 4 8 Their studies suggest that there are a number of variables that affect extraction efficiency of arsenic species in plant material. Increasing the temperature when doing P L E increased E E especially for arsenate. This could indicate arsenate is more strongly bound to the matrix of plant material. Similarly, organic arsenic compounds could be underestimated i f high temperatures are not used. On this basis, the extraction method used in the present work (MeOFLF^O at 2 0 ° C ) could have resulted in underestimates of arsenate and methylated species such as M M A and D M A in the trees. Another factor that can influence extraction efficiency of individual species is the degree of grinding. Extractable arsenate and D M A was found to be 40 independent of this factor but the concentration of arsenite was dependent on the amount of grinding. Differences in the extent of grinding between tree samples and parts is likely, thus it could be possible that this would affect the speciation results in the present study. Soils The mechanism of arsenate uptake from soils and translocation in plants is well documented. 4 9 Arsenate and phosphate are chemically similar and compete for the same uptake carriers in the root of plants and trees.5 0 Arsenate, which is the major arsenical present in most soils, 2 3 ' 5 1 is taken up by the plants and transported across the plasma membrane via a high affinity phosphate transport system. Unfortunately the mechanisms of uptake and transport of all other arsenic species are not well understood. Some researchers believe that arsenic present within the plant that is not arsenate is a metabolite of the plant. Thus upon uptake of arsenic from the soils, methylation and reduction occur within the plants themselves.2 4 Alternatively it has been suggested that methylated species are produced by microorganisms in the soil and subsequently taken up by plants. It is possible that under reducing conditions arsenic can be found in some soils as arsenite. Studies have shown that some plant species uptake arsenite from soils that have been treated with sodium arsenite. 5 2' 5 3 Results from these studies also reveal the plants ability to take up arsenic as dimethylarsinate and methylarsonate. Therefore, i f these species are present in soils it is possible they can be taken up by biota directly from the soils. 41 3.4 Fall Collection 3.4.1 Sample Collection and Treatment A second visit to the abandoned Pioneer mine site was made in early September of 2001 to obtain tree samples specifically from the contaminated mining site. A l l trees sampled were Douglas-fir species except for one Spruce species which was found growing directly beside one of the Douglas-firs. Similar sample handling as for the spring collection was used. Upon return to the lab, the needles and cones (if present) of each tree sample were separated from the stems. The experiment was divided into two parts; experiment A and B. Experiment A involved taking needles and stem samples from the ends of the sampled tree branches. These were assumed to be "newer" growth. These samples were freeze-dried by a procedure described in the following section. For experiment B, "older" and "newer" sub-samples were taken from the tree samples. "Older" growth was considered that which was taken closer to the base of the tree branches. Each sample was homogenized by grinding. Experiment B samples were not freeze-dried. A sub-sample of the homogenized wet material (-1-2 g) was accurately weighed into aluminum moisture pans and oven dried (85°C) to constant weight (within 0.00 lg). The moisture content of the sample was then determined. A l l homogenized samples were kept at -20°C until analysis in order to maintain the integrity of the samples. 42 Freeze-drying Procedure Analysis of biological samples often requires the material to be dried completely. 5 4 Freeze-drying is a method commonly used for this purpose, preserving the sample by eliminating all water content. One objective of experiments A and B was to determine i f the freeze-drying process affects the measured arsenic content of the samples. The freeze drying process involved cutting the samples into pieces and then freezing them in 50 ml centrifuge tubes for a few hours at - 2 0 ° C . The samples were then freeze-dried and ground. The moisture content was determined by the difference in the sample weight before and after freeze-drying. 3.4.2 Sample Preparation and Analysis Preparation and analysis for total arsenic analysis by ICP-MS was identical to that described in Section 3.3.2 for the spring collection. Modifications to this procedure were made for speciation analysis of the fall collection samples. Samples were extracted in duplicate. Extracts were collected in round bottom flasks and evaporated to near dryness by using a rotary evaporator. 43 3.4.3 Results and Discussion Total Arsenic Analysis The results presented in Table 3.3 support the previous finding that Douglas-fir trees accumulate arsenic to a high degree. The total arsenic content of the Douglas-fir located at site one is orders of magnitude higher than that of the neighboring spruce tree. The accumulation of arsenic in trees growing on contaminated soils, such as those from the Pioneer mine, may be explained by the role of mycorrhizae, which is defined as a symbiotic association of fungi and plant roots. 5 5 This type of association is common in conifer tree roots. Mycorrhizal fungi increase the absorptive ability of root systems by forming this association. Even though Douglas-fir, Pine and Spruce share many physiological properties they have been shown to uptake arsenic to very different extents. Pine trees have short root-hair zone thus have limited element absorptivity.4 6 The interaction of microorganisms with arsenic in the soils will be discussed in Chapter 4. 44 Table 3.3 Arsenic concentrations in tree samples collected from Pioneer mine, fall collection (error reported as SEM of duplicate digestions at a 95% CL a) EXPT A - F D * EXPT B -NFD Sample Tree Tissue ug As/g dry Relative fig As/g dry Relative (Trip#-site#) Type weight age weight age 2-1 DF-1 Cone 58 ± 3 New 65 ± 11 New Needle 195 ± 8 New 380 ± 10 Old Stem 207 ± 2 New 78 ± 11 Old 2-1 DF-2 Cone 42 ± 6 New 59 ± 10 New Needle 257 ± 11 New 322 ± 16 New Stem 307 ±41 New 195 ± 4 Old 2-1 S-1 Needle 0.4 ±0.01 New 0.5 ± 0.3 New Stem 0.73 ± 0.01 New 0.9 ± 0.2 Old 2-2 DF-3 Needle 57 ± 8 New 64 ± 17 New Stem 374 ± 19 New 364 ± 70 New 2-3 DF-4 Needle 56 ±0.8 New 58 ± 0.6 New Stem 105 ± 2 New 112 ± 2 New asee appendix A for the equation for S EM FD = freeze-dried material digested; NFD = non-freeze dried, wet plant material digested Total arsenic content in Douglas-fir seems to be dependent on the tissue type and age. The results from experiment A suggest that the stems have the highest total arsenic concentration and the cones have the least. This is not the case for experiment B, where the results do not show an obvious pattern with respect to tissue type. However, when taking into account the relative age of the tissues a pattern is revealed. The samples from experiment A were all taken from "newer" parts of the tree. All stems from site 1 trees that were not freeze-dried are "older" (see Section 3.4.1 for age definition). This can account for the difference in total arsenic between experiments A and B for the Douglas-firs from site 1. Thus it may be concluded that as the tree grows the arsenic is incorporated into the needles where it accumulates with time. In order to support this finding, further experiments were carried out on the Douglas-fir samples from site 1. The 45 digests were re-analyzed (experiment 2) to determine if the results could be duplicated. Also the homogenized samples were re-digested (experiment 1) and analyzed on the same day. Table 3.4 summarizes the results. The arsenic concentrations calculated for the three experiments were very similar for all samples indicating the difference in total arsenic between experiment A and B samples may not be due to experimental or instrumental error. Table 3.4 Comparison of results from re-digestion (A-1;B-1) and re-runs (A-2;B-2) of site 1 Douglas-fir samples (error reported as S E M at 95% confidence level) FD NFD Sample A A - l A-2 B B-l B-2 DF-1 Needle 195 ± 8 202 ± 6 206 ± 2 380 ± 10 364 ± 15 357 ± 0 . 5 DF-1 Stem 207 ± 2 251 ± 18 235 + 11 78 ± 11 89 ± 11 82 ± 11 DF-2 Needle 257 ± 11 280 ± 8 277 + 13 322 ± 16 316 ± 5 315 ± 4 DF-2 Stem 307 ± 4 1 317 ± 10 316 ± 2 0 195 ± 4 194 ± 6 187 ± 3 The results from experiment A and B for trees sampled from site 2 and 3 are in good agreement (Table 3.3). The samples for both experiments were all from the same "newer" parts of the tree. The data suggest stems have a greater arsenic content than the needles in this case. Indeed this supports the conclusion that translocation of arsenic is from the roots to the needles. In newer tree tissue there has not been time to accumulate arsenic within the needles. This agreement also suggests the freeze-drying process does not affect the determination of the total arsenic concentrations in tree samples. In order to completely eliminate the possibility that freeze drying is involved in the differences reported between experiment A and B it may be necessary to freeze dry the N F D samples to see i f it changes the results. 46 As briefly stated in Section Warren et a l 4 0 found that first year needles and older growth had the lowest arsenic content whereas first year stems had the highest. The present work supports this. Arsenic Speciation Analysis The arsenic species extracted from the tree samples collected from the second trip to Pioneer mine are summarized in Table 3.5 and 3.6. Inorganic arsenic predominates in all samples as was also shown in the first set of results. Most of the extractable arsenic is present as arsenite in all parts of the tree and arsenate was found to a lesser extent. Trace amounts of the methylated species M M A and D M A were identified in the chromatograms of only a few samples. This finding is consistent with the literature. 2 3' 4 7' 5 1 Figure 3.8 compares the relative amounts of arsenic species in the tree samples. Cone samples from Douglas-fir were analyzed in an attempt to probe the anomalous results obtained from the spring collection cones. Only a trace amount of D M A could be detected in one of the three cones. Cone samples contained a higher percent of their total arsenic as arsenate compared to other tree parts. Due to small sample size it is difficult to make any conclusions with respect to these findings at this time. Typical chromatograms of fall collection sample extracts are shown in Figures 3.9 and 3.10. 3 rs. II ra oT II CT cs. O o' S ra a o CT _o C & O O r a* F£ HT to CO E? ra o CT CS o CT' 3 o tO l U J a ra 4^ tO i a tO to to C O a to O ra rt2S22s2rtCTgn>2 3 S 3 & g 3 & 3 & g 3 & CT CT CT CT CT o p o O N U J to Os to to b oo to ON U J U J U J 00 00 N O U J ON H-H- H- H- H- H- H- H- H- H-N O to U J 00 o O o » — . U J © © b I—* oo to I—. H-U l U J t o *-»• C ^ CT >—> CT O IX r i -^ I-I ^ CT O CT U J to -J I-* U J U l H- H-O U J b to u i I-I P CT CT N O t—I H-o •3-5/3 ro . p f= 3 CT CT H rt rt H H C O S rt 13 3 3 3 3 a o. a a P CT CT 3 3 3 3 3 3 3 3 3 3 3 3 3 pi . p . pi . p • p . 3 3 3 3 3 3 3 P . p . P t p . P> , p^  . P . ON 00 U i U J ON K J 00 0 0 00 NO h—. 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P N O c r CD cn CD 5. o 0 I—1 cn CD CD' cn 5' CD ft-r-h l-t CD CD cn P 3 cT o 3 y 5' 3 CD 3 CD P^ o o_ a* o i-t-o' p m X "d H W 9^ cra^  era" o-CD_ era' C f 4i-GC Relative amount of As species (% of total As) O 8 o O O o 00 o C D O c c o o CD 3 CD a CD D a £2. D o o n CD ZJ CD a CD cn «—*-CD 3 3 CD a CD CD co CD CD p . CD D T l CO O T l 3 CD CD g . CD D T l 4*. FD NFD FD NFD FD NFD FD NFD FD NFD FD NFD FD NFD FD NFD FD NFD FD NFD FD NFD FD NFD • > > cn cn ' = < > 50 -75 -77 82 200 400 600 retention time (sec) 800 1000 Figure 3.9 Chromatogram of a needle extract from the DF located at site 2 (m/z 75 = As) 80000 70000 60000 50000 u 40000 0 (A *» 30000 O 20000 10000 0 As (III) As(V) Uv -75 77 82 200 400 600 retention time (sec) 800 1000 Figure 3.10 Chromatogram of a stem extract from the DF located at site 2 (m/z 75 = As) 51 The speciation of arsenic in the terrestrial environment has not been researched as extensively as it has in the marine environment. Recent studies of phytochelatins have revealed that they may play a role in determining what forms of arsenic are found in plant tissues. 5 0 ' 5 6 Phytochelatins (PC's) are metal binding peptides rich in thiol groups (SH). These compounds tend to be produced by plants in response to intracellular metal stress, such as inorganic arsenic exposure.57 Glutathione (GSH), which is a precursor to PC, readily reduces arsenate in plant cells to arsenite.56 The presence of arsenite in the tree extracts of the present study indicates the possibility that reduction may be occurring within the trees in this way. The arsenite can then complex with the thiol groups of the PC's. Trivalent arsenic is thought to have a higher binding affinity for thiols than pentavalent species. The production of arsenic-phytochelatin complexes has been described in the literature.5 0'5 6 Methanol/water extraction is useful in arsenic speciation studies as it allows for the detection of oraganoarsencals. However this method involves extraction of species under non-physiological conditions thus possibly destroying the in-situ speciation of arsenic in plant tissues. PC-As complexes are unstable under neutral or alkaline conditions thus methanol/water extraction could result in the break up of these complexes, releasing arsenite which can subsequently be oxidized to arsenate.56 Therefore, it is possible that the inorganic arsenic identified in the extracts of the present work could have been bound to PC's within the tree. X A S techniques could be used to reveal the presence of As-S bonds. Also, extraction of the tissues under different conditions and analyzing the extracts via size exclusion chromatography would be a possible step in determining whether PC-As complexes exist in the tree samples. 52 Extraction efficiency The extraction efficiency is generally very high for the Douglas-fir samples (55-140%) with the cone samples having the lowest values. In contrast, the extractable arsenic of the Spruce samples is below 25% of the total arsenic. These results are consistent with those from the Spring collection. Average extraction efficiencies for experiment A (FD) and experiment B (NFD) samples are within 1 % of each other (82% and 81% respectively). Thus on average the age of the tree sample does not seem to have an effect on extraction efficiency. The Douglas-fir cone samples that were not freeze-dried do have a lower E E than those that were freeze-dried but it is difficult to make a reliable conclusion as only two cones were analyzed. Freeze-drying Since both freeze-drying and oven drying resulted in very similar values for the water content in samples, they can be regarded as equally reliable for moisture calculations. It has been previously shown that oven drying may not be as reliable as freeze-drying.54 Results may depend on the matrix and type of plant. For example, there is the possibility that volatile organic compounds found in the sample may be evaporating in the oven. It seems that for the present study either drying method is acceptable. Francesconi et a l 2 3 found that drying fern fronds at 50-55 °C did not alter the arsenic content or species when compared with freeze-dried samples. However it has been shown that freeze drying the samples prior to analysis resulted in lower extraction efficiencies.2 4 The results in Table 3.5 and 3.6 indicate that EE's are not lower for the F D samples as compared to the N F D . 53 From Figure 3.8 it can be concluded that the extracts of wet samples (NFD) have a lower percentage of their total arsenic content as arsenite compared to those that were freeze-dried. However, this disparity is less significant in DF-3 and DF-4. The FD and NFD samples from these tree samples are comparable in age, thus suggesting the differences in the ratio of arsenite to arsenate in samples may be largely due to age differences rather than the freeze-drying procedure. However, more samples are needed to statistically validate these suggestions. 3.5 Summary The results obtained from the analysis of the Bridge River district tree samples confirm Douglas-firs ability to accumulate arsenic. The new data shows that the total arsenic content of the tree samples is dependent on species, tissue type and age. Translocation of arsenic in the Douglas-fir seems to be from the roots to the needles. The arsenic accumulates in the needles possibly providing a route for the tree to eliminate its arsenic burden. The extractable arsenic is mainly in the form of inorganic arsenic but some methylated species were identified. It is difficult to make any definite conclusions regarding how Douglas-fir and other arsenic accumulating terrestrial plants take up and grow with such a high arsenic burden. Controlled studies can be done to limit variables and probe this topic in order to understand the mechanisms involved in these unique species. The uptake of arsenic by terrestrial biota is likely influenced by the microbial environment of the soils they are growing in. In the following chapter the link between microbial activity and arsenic in the Bridge River environment is investigated. 54 Chapter 4 THE BIOTRANSFORMATION OF ARSENICALS BY SOIL MICROORGANISMS 4.1 Introduction Microorganisms have an important role in cycling and biotransformation of arsenic in the environment. As briefly discussed in Chapter 1, microbial activity usually involves the reduction, oxidation and methylation of inorganic arsenic to afford a variety of species. It is important to study these transformations in order to understand the fate and toxicity of the arsenic species present in various environmental compartments. Since the initial studies done by Challenger et al with S. brevicaulus, other fungi and also bacteria have been isolated that methylate arsenic. It is known that some microbes can methylate and volatilize water-soluble arsenic species in soils to arsines, which can be extremely toxic gases.5 8'5 9 These species have been shown to comprise a small percent of the total arsenic in the soils and to have a short lifetime in air. 5 8 However, more recent research has suggested otherwise.60 It has been suggested that trimethylarsine is the final product in the biomethylation of arsenic in soils. This gas has been found in the headspace of some cultures incubated with arsenic substrates.59'60 However, in a study by Cheng et a l 6 1 , enriched Pseudomonas cultures under anoxic conditions produced arsine gas but no trimethylarsine was detected. These authors concluded that the primary mechanism for 55 gaseous loss of arsenicals from soil is the reduction to arsine and not the methylation to trimethylarsine. This is an unverified conclusion. Pseudomonas is a genera in the bacterial group Pseudomonads.5 5 Some species of this genus have been characterized as arsenite-oxidizing bacteria.1 1 This oxidation is considered a protective mechanism as arsenite is more toxic that most other arsenicals. However other species of Pseudomonas have been shown to grow in arsenite without oxidizing it to arsenate. Pseudomonas isolates from enriched soils to which sodium arsenate was added have shown to produce arsine under anaerobic conditions.6 1 Under the same conditions the conversion of arsenite to arsine has also been reported. In the present work the biotransformation of arsenic in culture and in soils is investigated. Phytoavailability of arsenic depends on species.5 2 Therefore microbial interaction with arsenic will most certainly affect the bioavailability and uptake of arsenic by biota. It is important to explore the interaction of bacteria and fungi with arsenic in the pursuit of a comprehensive understanding of arsenic in the terrestrial environment. 56 4.2 Analysis of the Bacterium Pseudomonas 4.2.1 Culture Preparation and Treatment In order to study microorganisms and how they interact with the environment it is often desirable to obtain pure cultures and study them under laboratory conditions. This involves growing the organism in appropriate culture media containing nutrients (Section 2.2.2). Pure cultures of the two strains of Pseudomonas, P. resinoverans ( U B C # 164) and P. strain 9816/11 ( U B C #175), were obtained from the culture collection of the chemistry department at the University of British Columbia. Initially luria agar plates were streaked with each bacteria. When colony growth was visible on the plate a single colony was transferred to cooled, autoclaved luria broth. The cultures were grown aerobically on a shaker and maintained at room temperature. Transfers and all other procedures were carried out aseptically in a biological safety cabinet. Appropriate amounts of aqueous solutions of arsenite or M M A were added to luria broth to give a final concentration of 500 ppb and then sterilized by autoclaving. Flasks were prepared in triplicate and two types of experimental controls were introduced. One flask was prepared containing arsenic and media without seed culture and the second control contained media and bacteria but no arsenic. Upon cooling, 5 ml of the actively growing cultures of Pseudomonas were added to the appropriate flasks. (Table 4.1) The bacteria were incubated on a shaker for 28 days at room temperature. Cells were then harvested and extracted as outlined in Section 2.3.3. Media was stored at 57 -20°C until ready for analysis by using HG-GC-AA. Acetic acid (4M) was used as the source of acidity for hydride derivitization. Under these conditions the signal from arsenate is greatly reduced. Table 4.1 Summary of the experimental setup of pure Pseudomonas cultures. Strain As Species #of Approx. Seed Final culture replicates TAsl (ppb) volume (ml) volume (ml) P. resinovorans MMA 3 500 5 50 As(III) 3 500 5 50 MMA 1 500 0 50 As(III) 1 500 0 50 N/A 1 0 5 50 P. 9816/11 MMA 3 500 5 50 As(III) 3 500 5 50 MMA 1 500 0 50 As(III) 1 500 0 50 N/A 1 0 5 50 4.2.2 Results and Discussion Growth Media Qualitative analysis of methylated arsenic species present in the medium of two strains of the soil microorganism Pseudomonas grown in the presence of MMA and arsenite was achieved by using HG-GC-AA. The retention times of the peaks in the samples chromatograms were compared with those obtained from standards to identify the arsenic species present. MMA was the only arsenical found in the growth media of both strains of bacteria following incubation with MMA. When P. resinovorans and P. 9816/11 were incubated with arsenite small peaks were seen at the retention time for 58 that of M M A but the metabolite was not quantifiable. The characteristic garlic-like odor of arsines was not observed in any of the incubated flasks. In the present study, the possible occurrence of monomethylated arsenic species in the media of Pseudomonas strains grown in arsenite suggests methylation may be occurring. This in turn could lead to the production of methylated arsines. However, there was no physical indication of arsines suggesting that there was no need to look for these compounds in the flask headspace. These results contradict those by Cheng et al as they found arsine gas in the headspace of pseudomonas cultures. In their work, Pseudomonas were shown to be arsine producers under anoxic conditions and to release arsine from arsenite.61 In order to completely understand the transformation of arsenic by Pseudomonas, volatilization studies should be done to determine i f arsines are present in the headspace of the cultures. Cultures could also be grown anaerobically as previous studies have shown that Pseudomonas may produce arsines only in an oxygen free atmosphere. Cells The harvested cells of the Pseudomonas strain 9816/11 were extracted according to Section 2.3.3 and the extracts were analyzed qualitatively by using H G - G C - A A . As in the analysis of media, identification of arsenic species was accomplished by comparison with external standards of known composition. When the bacteria were incubated with arsenite, methylated species were not detected in the cell extract. M M A and background levels of inorganic arsenic were only detected following incubation with M M A . The 59 amount of arsenic found in the cells is a very small fraction of the total arsenic added to the media. 4.3 Analysis of Isolates from Bridge River Soils It is important to understand the processes affecting the speciation and distribution of arsenic in soils in order to assess the risk associated with growing crops on arsenic rich soils and the mobility of arsenic species. Microbial transformation, such as volatilization is one of these processes. The presence of certain microbes in the soil may result in the loss of arsenic from soils whereas adsorption of arsenic will decrease the availability of the element by restricting its mobility. 4.3.1 Culture Preparation and Treatment Pure Culture Isolation Five soil samples were collected from the Pioneer mine and treated as described in Section 3.3.1. A small amount of each soil was added to a 1.5 ml Eppendorff tube and mixed with ~0.5ml sterilized water. Appropriate amounts of an As(III) standard were added to autoclaved potatoe dextrose agar (PDA) and nutrient agar (NA) to final concentrations of 2000 ppb and 1000 ppb for each medium. Plates were poured, cooled and then streaked with the soil mixtures. Thus each soil was grown on four different media. P D A is a general medium for fungal growth and N A for bacterial growth. The plates were stored at 4 ° C for approximately one week. The number of colonies and a 60 description of each were documented. Some of this information is summarized in Table 4.2. A single colony of each bacteria or fungi was transferred to the same agar initially grown on in an attempt to obtain pure cultures. The plates were stored in an incubator at 15°C until significant growth was observed and then they were stored at 4 ° C to slow growth. Pure cultures were transferred to appropriate storage vessels for preservation in the U B C collection. Small pieces of fungi were cut from the agar and transferred onto slants of P D A which were then covered with mineral oil. Single colonies of bacteria were transferred to 1.5 ml cryoflasks containing ~ 0.5ml of an autoclaved glycerol:NB mixture. These flasks were then stored in liquid nitrogen dewars. Bacteria and fungi were distinguished from one another by visual inspection. Table 4.2 Summary of cultures isolated from the Pioneer mine soils. Soil Locality Soil [As] Growth [As(III)] in #of Pure isolates (ug/g) Medium agar (ng/g) colonies (UBC#) Creek 279.9 P D A 2000 6 707-711,738 1000 10 712-717 N A 1000 5 718,719 Above creek 35.4 P D A 2000 9 720,721 1000 5 722 N A 2000 4 724 1000 7 723 Under workings 1622.5 P D A 2000 6 730,731,739 1000 5 736,737 N A 2000 6 725-729 1000 4 -Left of workings 227.9 P D A 2000 3 -1000 3 -N A 2000 5 732,734 1000 5 733,735 Waterfall 39.4 P D A 2000 4 -1000 5 -61 Culture Incubation A n aqueous solution of arsenate was filter-sterilized (0.2um) separately and added to autoclaved P D B medium in the appropriate flasks to give a final concentration of ~ 100 ppm. Three selected fungal isolates from the B R soil collection slants were used to inoculate the medium. Flasks were prepared in duplicate. Three types of controls were prepared; three flasks were inoculated each with a single fungal isolate without the addition of arsenic, duplicate flasks containing arsenic but not fungi, and one flask contained neither fungi nor arsenic (Table 4.3). A l l flasks were incubated on a shaker aerobically for 28 days at room temperature. Significant growth was observed in the flasks except for those that were not amended with fungi. In these cases no organisms appeared to be growing as determined by visual inspection. No contamination by other organisms was observed thus it was assumed only one organism was present in each experimental flask. Cells were harvested as outlined in Section 2.3.3. The media was filtered (0.22 pm) and stored at - 2 0 ° C until analysis by using H G - G C - A A . Table 4.3 Summary of the experimental setup for the analysis of B R fungi isolates Fungi U B C # # of replicates Approx. [As(V)] Approx. Final culture (ppm) volume (ml) 712a 2 100 50 712a 1 0 50 713 2 100 50 713 1 0 50 734 2 100 50 734 1 0 50 Control #1 2 100 50 Control #2 1 0 50 62 4.3.2 Sample Preparation and Analysis The H G - G C - A A system described in Chapter 2 was used to analyze the growth media of the fungi for hydride-forming arsenicals. Both 4M acetic acid and 1M HCI solutions were used to acidify samples in the analysis of media. In order to quantify and identify all arsenic species in the media it was necessary to cleanup the samples using anion (Supelco, LC-SAX) and cation (Strata, SCX) cartridges. The procedure used was similar to that described by Le et al. The cartridges were first conditioned with a solution of MeOH/H20 (1:1; v/v). Sample extracts were then applied to the cartridges and eluded with the appropriate solution. An acetic acid system was used to analyze the methylated arsenicals, T M A O , D M A and M M A . Total inorganic arsenic and arsenite were quantified using 1M HCI as the acid in the H G system. Dilutions were done as necessary. External calibration was used to quantify all species except for T M A O for which the standard addition technique was required. An example of a calibration curve is shown in Figure 4.1. 63 1600000 -| Amount of MMA (ng) Figure 4.1 Calibration curve for MMA: HG-GC-AA analysis 64 4.3.3 Results and Discussion Acetic acid (4M) was first used in the identification of T M A O in the original sterilized media samples. In this system both arsenite and arsenate are reduced to arsine (ASH3). The resultant ASH3 peak for the combined inorganic arsenic was very wide masking the possible presence of MeAsH.2 and M e 2 A s H . However, it was possible to quantify M e 3 A s from T M A O , which has a later retention time, by using the standard addition technique. T M A O was observed in only one out of the three B R soil isolates thus only the growth media of this fungus (UBC#713) was analyzed further. Figure 4.2 shows a chromatogram of the media of isolate #713. Tables 4.4 and 4.5 summarize the speciation results obtained from the analysis of the growth media of this fungus. Figure 4.2 Chromatogram of the growth media of isolate #713; H G - G C - A A analysis iAs CU o C C3 X> o 5.0 Retention time (min) 65 Table 4.4 Concentrations of inorganic arsenic species in the growth medium of a B R soil isolate determined by using H G - G C - A A . (error reported as SD of duplicate analysis) Sample Total iAs As(III) % of total As(V) % of total (ppm) (ppm) iAs (ppm)a iAs 713 - l b 115 ± 1 104 ± 4 91 10 ± 5 9 713 -2 109 ± 6 101 ± 13 93 8 ± 19 7 Control #1 108 ± 8 nd - 108 ± 8 100 Control #2 nd nd - nd -a As(V) concentration calculated by difference (As(V) = Total iAs-As(III)) bduplicate flasks indicated as -1 and -2 Table 4.5 Concentrations of methylated metabolites in the growth medium determined by H G - G C - A A (error reported as SD of duplicate analysis) Sample M M A (ppm) D M A (ppm) T M A O (ppm) 713 - l a nd 0.043 ± 0.004 0.8 ± 0.2 713 -2 nd 0.0317 ± 0 . 0 0 0 6 0.48 ± 0.02 Control #1 nd nd nd Control #2 nd nd nd 'duplicate flasks indicated as -1 and -2 Most of the arsenic in the growth media is present as arsenite. T M A O and D M A were also found in the media to a lesser extent but M M A was not detected. In the flasks with no arsenic, both with and without fungi, no detectible amounts of arsenic were observed. Sun et al have found a high percent of arsenite in the growth media of mine fungi incubated with arsenate. This study, and many others, have also identified the presence of extracellular methylated arsenical metabolites in the growth medium of fungi. 2 9 The soils sampled from the Pioneer Mine were found to be highly contaminated with arsenic (35-1623 pg/g) and yet many bacteria and fungi were isolated from these 66 soils. A s discussed previously, most of the arsenic in soils is present as arsenate. The results of the present experiments indicate that some microbes in the Pioneer mine soils, once isolated, can take up and metabolize arsenate to arsenite and then excrete this compound into the medium. This reduction is the first step in the biomethylation scheme proposed by Challenger et a l 2 8 (Figure 1.1). The presence of arsenite and methylated intermediates in the growth media support this mechanism. It has been suggested that a phosphate transport system is involved in the uptake of arsenate by cells and 70 subsequently thiols reduce the arsenate within the cell. In other culture experiments where arsenate was used as a substrate in the growth of fungal cells, M M A has been detected to a lesser extent or not at a l l . 2 9 The lack of M M A in the media of the present study and those of others may indicate that i f M M A is an intermediate in the methylation of arsenic by microbes it is metabolized rapidly within the cells. It has been theorized that M M A can not diffuse into the medium thus it is methylated endocellularly to D M A 70 and T M A O which are in turn able to passively diffuse into the medium. The volatilization of arsenic by isolate #713 seems an unlikely transformation pathway. The total arsenic content of the species found in the growth media is -100% of the total amount of arsenic added to the media as arsenate. Thus the arsenic added as a substrate is accounted for in the media and it is unlikely that significant amounts of arsenic would be found intracellularly or in the headspace of the flasks. Also the cultures did not exude a garlic-like odor to suggest the presence of arsines. It has been shown that the biomethylation of arsenic by the fungi 5. brevicaulis is decreased in the presence of antimony(III) compounds. 6 4 Thus, interactions with other species might be a factor in the presence and detection of metabolites by the B R isolate. Variations in biomethylation 67 could be due to exposures with other metals thus affecting the fate of the arsenic in the environment. It has been suggested that the transformation of arsenic species by microorganisms can have detrimental effects resulting from mobilization and/or release of more toxic forms into the immediate environment. However, the activity of microbes can also be used to advantage in microbial bioremediation techniques.6 5 It has been shown that genes for the biodegradation of pollutants, such as chlorinated pesticides, exist in isolates of bacteria that occur naturally in the environment.5 5 These genes have been isolated from species of the bacteria Pseudomonas. Current interest has been focused on utilizing these microbes in environmental cleanup. Speciation and mobilization of arsenicals by microbes in soils can also aid in the study of arsenic uptake and transport in plants and their use in phytoremediation. It has been proposed that the ability of some microbes, such as fungus #713, to reduce arsenate to arsenite can enhance uptake of the metal by plants. 6 5 Arsenite is sorbed less strongly than arsenate to metal oxyhydroxides in soils thus it is more mobile. Therefore, it is suggested i f conditions are optimized for microbes to reduce arsenate to arsenite, significant improvement in arsenic phytoremediation can be accomplished. Arsenate has generally assumed to be the arsenical taken up by plants from the soil, not arsenite. However, recent studies suggest that arsenite can be taken up by plants . 5 2 , 5 3 In the present work a large percentage of arsenite was found in the tree extracts and in the growth media of the B R soil fungi. These results suggest the possibility that As(V) is biotransformed to As(III) in the soil which could in turn immediately be taken up by the trees. However, these experiments 6 8 are done under laboratory conditions and can not be assumed to occur in the real environment. There are a number of factors that determine the behavior of arsenic in soils and thus need to be considered when discussing the uptake of arsenic by plants. The pH of the soil and therefore the redox state of arsenic will determine the bioavailability of it to the plants. If there is a high clay content in the soils more arsenic can be complexed and thus less is available.66 Phosphate is chemically similar to arsenate and thus if it is present in the soils there is competition for uptake by plants. Of course mechanisms for dealing with arsenic exposure will differ depending on the plant species. Indeed there are many variables that affect the mobility and availability of arsenic in the soils and often these are linked to microbial activity. The biotransformation of arsenic by microbes is very complex. The presence of nutrients, oxygen and the pH of the soil can all affect whether microbes methylate arsenic. Cox et al 5 9 suggested that acidic conditions may be needed for the production of trimethylarsine by fungi. Also, 67 phosphate has been shown to inhibit the production of trimethylarsine. 69 4.4 Summary The role of microorganisms in the speciation of arsenic within the terrestrial environment has been studied extensively. The present work probes this topic in an attempt to further understand the interactions and cycling of arsenic. Arsenic was added to pure cultures of Pseudomonas, a bacterium commonly found in soils, and to fungi isolated from the soils of a contaminated mine site. With arsenite as a substrate, mainly inorganic arsenic was identified in the growth medium of the two Pseudomonas strains analyzed. However, peaks below the detection limit were visualized at the retention time for that of M M A . When amended with M M A the medium contained only M M A . Under laboratory conditions, the highly contaminated soils sampled from the Pioneer mine were cultured in the presence of arsenic. Arsenic speciation studies were performed on the growth media of a pure fungus isolated from these cultures. The presence of the methylated metabolites, D M A and T M A O in the media imply the fungus has the ability to methylate arsenate. The major arsenical found in the media was arsenite (-90%) which suggests the fungus can reduce arsenate fairly efficiently. Such transformations may have consequences on the uptake and availability of arsenic in biota growing in this type of microbial environment. 70 Chapter 5 SUMMARY AND FUTURE WORK Novel results were obtained from the study of arsenic in samples from the mining impacted area, Bridge River. Tree samples from the district were digested and analyzed for total arsenic by using ICP-MS and speciation analysis was preformed on the tree extracts by using H P L C - I C P - M S . Douglas-fir trees were found to have a remarkable affinity for arsenic. The major arsenic species extracted from the B R trees was inorganic arsenic. However, trace amounts of methylated species were found in some samples. Some Spruce cones revealed atypical results; D M A was the second most abundant arsenical in the tissue. Less concentrated samples allowed for the identification of methylated species however they were often below the limit of quantification. Above baseline signals were seen at the retention time for that of arsenobetaine in a few tree samples. The possibility of A s B in tree tissue should be further investigated due to the lack of previous sightings of this arsenical in trees. The present work suggests that the total arsenic content and arsenic speciation of the tree samples is dependent on species, tissue type and age. However it should be noted that any discrepancy between those samples collected in the spring and those collected in the fall could be due to seasonal differences. Future work could be done to determine i f this has an affect on the arsenic content and speciation of Douglas-fir tissues. The environmental effects of having high arsenic loads in tissues of arsenic accumulating biota are relatively unknown. In homes where wood burning is a primary source of heat and fuel it would be interesting to investigate the effect of burning arsenic 71 accumulating trees such as the Douglas-fir. Ashes of these trees may also be used in gardens thus it would be useful to determine the consequences of this practice. Arsenic accumulators are being considered for use in remediation of contaminated sites. This technique is called phytoremediation and has become a topic of increasing interest recently. Plants species, such as the brake fern discovered by M a et al, have been shown to thrive on arsenic-rich soils, efficiently extracting and translocating the arsenic from the soils into its biomass. 1 1 ' 2 3 ' 3 0 These studies, and those of the present work, are therefore important in furthering the knowledge of arsenic cycling, aiding in the development of remediation techniques and environmental effects. Total arsenic analysis of soil samples from the abandoned Pioneer mine reveal the area is contaminated with arsenic. Soil concentrations range from 35 to 1623 pg As/g. Microorganisms were isolated from these soil samples and the resultant pure cultures where grown in media amended with arsenate. The major arsenical found in the media of cultures was arsenite. The methylated arsenic metabolites, D M A and T M A O , were detected in the growth media of one isolate analyzed. Assuming arsenate is the form found in these soils and is taken up by the plants, reduction is occurring within the organism. Phytochelatins may provide thiol groups for the reduction and transport of arsenic within the plant. However, it is possible that other forms of arsenic exist in the soils and arsenate may not be the only species taken up by the trees. The predominance of arsenite in both the media of the B R soil isolates and in the tree extracts suggests the possibility of arsenate reduction to arsenite in the soils followed by uptake of arsenite by the trees. The general prevalence of arsenite and the presence of methylated arsenic metabolites in soil fungi media are important findings. Definite conclusions can not be 72 drawn at this time and therefore further investigations must be performed. Speciation analysis of the soil samples would be beneficial; however this type of study is difficult to do. 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Cullen, W.R.; McBride, B . C . ; Reimer, M . Bulletin of Environmental Contamination & Toxicology 1979, 21, 157-161. 77 Appendix A EQUATIONS Standard Deviation The standard deviation (SD) is a measure of the degree of spread of data. It is defined as: SD = VS(xi-x)2/(n-l) where: n - number of measurements Xj = result i x = mean of results The standard deviation of the mean is called the standard error of the mean (SEM) and is related to SD in the following way: SEM = SD/Vn The 95% confidence limit is 2 S E M Detection Limit The detection limit (DL) is the concentration of the analyte which gives a signal significantly different from the background signal. It can be calculated using the following equation: v = 3SDR m where: y = detection limit (concentration) S D B = standard deviation of the background signal (counts/sec) m = slope of the calibration curve (counts/sec vs concentration) The limit of quantification ( L O Q ) is the lower limit for precise quantitative measurements. A suggested equation for the calculation of this value is: y = IOSDR m Reference: Miller, J.C.; Miller, J.N., eds. Statistics for Analytical Chemistry. 3rd ed. Analytical Chemistry Series, ed. Masson, M. ; Tyson, J.; Stockwell, P. Ellis Horwood Limited: Chichester, 1993, 115-117. 


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