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The uptake of arsenicals by the marine algae Isochrysis galbana and Dunaliella tertiolecta Tsang, Angela Ka Yen 1990

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T H E U P T A K E OF ARSENICALS BY T H E MARINE A L G A E ISOCHRYSIS  GALBANA  A N D DUNALIELLA  TERTIOLECTA  by ANGELA K A Y E N TSANG B.Sc. (Hons.), The University of Hong Kong, 1987  A THESIS SUBMITTED LN PARTIAL F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF M A S T E R O F SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES D E P A R T M E N T O F CHEMISTRY  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A OCTOBER 1990 © Angela K a Yen Tsang, 1990  I n presenting this thesis i n p a r t i a l fulfilment of the requirements for a n advanced degree at T h e U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l m a k e i t freely available for reference a n d study. I f u r t h e r agree t h a t p e r m i s s i o n for extensive copying of this thesis for scholarly purposes m a y be g r a n t e d b y t h e H e a d o f m y D e p a r t m e n t o r b y b i s o r h e r r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r financial g a i n s h a l l n o t be a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n .  DEPARTMENT OF CHEMISTRY T h e U n i v e r s i t y of B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V 6 T 1W5 D a t e : 12 O c t o b e r 1990  ABSTRACT  T w o m a r i n e a l g a e , Isochrysis galbana  a n d Dunaliella  tertiolecta,  were  g r o w n i n t h e presence o f arsenate, arsenite, monomethylarsonate ( M M A ) , a n d d i m e t h y l a r s i n a t e ( D M A ) . C e l l g r o w t h w a s m o n i t o r e d b y u s i n g in vivo f l u o r e s c e n c e a n d c e l l d e n s i t y m e a s u r e m e n t s . T h e effects o f d i f f e r e n t l e v e l s o f arsenicals o n the arsenic uptake were examined using Hydride Generation Atomic Absorption ( H G A A ) Spectrometry a n d Graphite Furnace  Atomic  Absorption ( G F A A ) Spectrometry. T h e spin-echo i H N M R spectra of these algae are reported. Significant arsenic uptake a n d incorporation occurred w h e n both algae w e r e s u b j e c t e d t o e l e v a t e d l e v e l s o f a r s e n i c a l s . / . galbana  w a s m o s t affected  by  (2 - 8 p p m ) .  arsenite  at  the range  of concentrations  tested  Total  incorporation i s also the highest i n this m e d i u m . T h e alga seems to be able to adapt to h i g h arsenate concentrations. D.  tertiolecta  w a s most  affected  (10.7 p p m ) . W a t e r s o l u b l e e x t r a c t s  b y elevated levels  of  arsenate  o f a l g a l cells g r o w n i n t h i s  medium  contain at least four arsenicals w h i c h possibly include arsenate a n d arsenite. G r o w t h of b o t h algae w a s not affected b y D M A a t the levels e x a m i n e d . Growth enhancement  i s observed i n a l l M M A treated cultures. A r s e n i c  speciation of the r e s i d u a l m e d i a showed t h a t b o t h algae c a n reduce arsenate to a r s e n i t e .  ii  TABLE OF CONTENTS  Page ABSTRACT  ii  T A B L E O FC O N T E N T S  iii  LIST O F T A B L E S  vi vii  LIST O F FIGURES  ix  LIST O F ABBREVIATIONS ACKNOWLEDGEMENTS  x  DEDICATION  xi  I.  INTRODUCTION  1.1  Arsenic Compounds and the M a r i n e Environment  1  1.1.1  Occurrence a n d Speciation of A r s e n i c a l s  2  2 2 5  1.2  Biotransformation of Arsenic i n the Environment 1.2.1 1.2.2 1.2.3 1.2.4  1.3  Seawater Marine Plants and Algae Marine Animals  Organoarsenic Compounds i n Food Chains Biomethylation of A r s e n i c Biosynthesis of Areenic-Containing Ribofuranosides Metabolism of Arsenic-Containing Ribofuranosides  Scope of w o r k  7 7 8 10 11 13  iii  Page H.  EXPERIMENTAL  2.1  Instrumentation  15  2.1.1 2.1.2  15  2.1.3 2.1.4 2.2  16  2.2.1  Culture Methods  16  17 18  Media Culture Conditions  Analytical Methods N M R Studies  18 19  Analytical Procedures  20  2.3.1 2.3.2 2.3.3  20 20  Graphite Furnace Atomic Absorption ( G F A A ) Hydride Generation Atomic Absorption ( H G A A ) W e t Digestion of Freeze-dried Algae w i t h N i t r i c A c i d , Sulfuric A c i d a n d Hydrogen Peroxide 2.3.4 E x t r a c t i o n a n d P u r i f i c a t i o n o f A r s e n i c M e t a b o l i t e s o f D. tertiolecta C u l t u r e d i n A r s e n a t e E n r i c h e d M e d i a  23  Gel Permeation Chromatography  24  HPLC-GFAA  24  RESULTS A N D DISCUSSION  3.1  Graphite Furnace Atomic Absorption Spectrometry 3.1.1  Calibration, L i m i t of Detection a n d Precision of G F A A Analysis Hydride Generation Atomic Absorption Spectrometry 3.2.1  23  m.  3.2  15 15 16  General Culture Maintenance  2.2.2 2.2.3 2.3  Atomic Absorption Spectrometry (AAS) N u c l e a r Magnetic Resonance Spectroscopy ( N M R ) and Mass Spectrometry ( M S ) H i g h Performance L i q u i d Chromatography ( H P L C ) Chemicals a n d Reagents  Calibration, l i m i t of Detection a n d Precision of H G A A Analysis  iv  26 28 29 31  Page 3.3  Determiiiation of Total Arsenic  32  3.4  Culture Experiments  34  3.4.1  Fluorescence a n d C e l l Counting Studies  34 E f f e c t o f A r s e n i c a l s o n G r o w t h o f / . galbana E f f e c t o f A r s e n i c a l s o n G r o w t h o f D. tertiolecta A r s e n i c U p t a k e b y J . galbana a n d D. tertiolecta Arsenic Speciation i n the Culture M e d i a Whole C e l l * H a n d C Nuclear Magnetic Resonance ( N M R ) Spectroscopy of M a r i n e A l g a e  35 43 51 56  3.4.2 3.4.3 3.4.4  1 3 3.4.5  Uptake of M M A and D M A b y the A l g a l Cells at Stationary Phase  Isolation a n d Identification of Arsenic Compounds i n  D. tertiolecta  Purification of Crude Extract b y G e l Permeation Chromatography Purification of C r u d e E x t r a c t b y H P L C - G F A A  57 67  71 73 76  3.5  Summary  78  TY.  BIBLIOGRAPHY  80  v  LIST OF T A B L E S  Tables  Page  1.1  Arsenic Concentrations i n Selected M a r i n e P l a n t s  4  1.2  Organoarsenicals Isolated f r o m Selected M a r i n e A n i m a l s  6  2.1  C o m p o s i t i o n (g L * ) o f t w o S t o c k S o l u t i o n s u s e d i n 1  the E n r i c h m e n t Solution  17  2.2  Graphite Furnace Operating Parameters  21  2.3  O p e r a t i n g Conditions for the H y d r i d e Generation A s s e m b l y  21  3.1  A r s e n i c Determinations i n S t a n d a r d Reference M a t e r i a l s  34  3.2  Arsenic U p t a k e a n d Relative C e l l Density of M a r i n e A l g a l  3.3  Cells Grown i n Arsenic Enriched M e d i a A r s e n i c C o n c e n t r a t i o n (ppm) of the C u l t u r e M e d i a before a n d after U V - i r r a d i a t i o n  3.4  A r s e n i c (ug) f o u n d i n E x t r a c t s o f D. tertiolecta i n Different Arsenicals  vi  52 53  Cultured 72  LIST O F FIGURES  Figures  Page  1.1  S t r u c t u r e s o f a r s e i r i c - c o n t a i n i n g isolated from algae  1.2  F o r m u l a s of organoarsenic compounds isolated f r o m  4  marine organisms  6  1.3  Challenger's m e c h a n i s m for the m e t h y l a t i o n of arsenic  9  1.4 1.5  Structure of S-adenosylmethionine ( S A M ) Proposed scheme for the f o r m a t i o n of arsenic-containing ribofuranosides from dimethylarsinic acid  9  1.6  Proposed scheme for the biosynthesis of arsenobetaine  10  from arsenic-containing ribofuranosides  12  1.7  Structure of a trimethylarsinoriboside  12  2.1  T h e C a r r - P u r c e l l - M e i b o o m - G i l l ( C P M G ) pulse sequence  19  2.2  Schematic d i a g r a m of the h y d r i d e generation system  22  2.3  Wet ashing apparatus  25  3.1  T y p i c a l c a l i b r a t i o n curve for the d e t e r m i n a t i o n o f  3.2  arsenic by G F A A T y p i c a l calibration curve for the determination of  28  arsenic by H G A A  31  3.3  E f f e c t of a r s e n a t e o n g r o w t h o f I. galbana  38  3.4  E f f e c t o f a r s e n i t e o n g r o w t h o f 7. galbana  39  3.5  E f f e c t of M M A o n g r o w t h o f I. galbana  40  3.6  E f f e c t of D M A o n g r o w t h o f I. galbana  41  3.7  E f f e c t o f a r s e n i c o n t h e r e l a t i v e c e l l d e n s i t i e s o f I. galbana  42  3.8  E f f e c t o f a r s e n a t e o n g r o w t h o f D. tertiolecta  46  vii  Figures  Page  3.9  E f f e c t o f a r s e n i t e o n g r o w t h o f D. tertiolecta  47  3.10  E f f e c t o f M M A o n g r o w t h o f D. tertiolecta  48  3.11  E f f e c t o f D M A o n g r o w t h o f D. tertiolecta  49  3.12  E f f e c t o f a r s e n i c o n t h e r e l a t i v e c e l l d e n s i t i e s o f D. tertiolecta  50  3.13  1  3.14  4 0 0 M H z * H s p i n echo N M R s p e c t r a o f / . galbana  63  3.15  4 0 0 M H z * H s p i n echo N M R s p e c t r a o f D. tertiolecta  64  3.16  7 5 M H z i C N M R s p e c t r u m o f I . galbana  65  3.17  7 5 M H z 13C N M R s p e c t r a o f D. tertiolecta  66  3.18  T h e v a r i a t i o n o f t h e * H N M R s p e c t r u m o f J . galbana  H N M R s p e c t r a o f / . galbana  washed i n D 0  62  2  3  in  the presence of M M A  69  T h e v a r i a t i o n o f t h e H N M R s p e c t r u m o f D. tertiolecta i n the presence of M M A  70  3.20  L i q u i d c h r o m a t o g r a m o f w a t e r s o l u b l e e x t r a c t o f D. tertiolecta  73  3.21  * H N M R o f w a t e r s o l u b l e a r s e n i c c o m p o u n d i s o l a t e d from D. tertiolecta c u l t u r e d i n a r s e n a t e e n r i c h e d m e d i a a n d purified by Sephadex L H - 2 0  74  H P L C - G F A A chromatogram of water soluble extract of D. tertiolecta  77  3.19  3.22  J  viii  LIST OF ABBREVIATIONS AA  Atomic Absorption  AAS  Atomic Absorption Spectrometry  FAB  Fast Atom Bombardment  GFAA  Graphite Furnace Atomic Absorption  GTA  Graphite Tube Atomizer  HGAA  Hydride Generation Atomic Absorption  HPLC  H i g h Performance L i q u i d Chromatography  i.d.  inner diameter  MS  M a s s Spectrometry  m  multiplet( N M R )  m/z  m a s s to c h a r g e r a t i o  NMR  N u c l e a r M a g n e t i c Resonance  o.d.  outer diameter  ppb  parts per b i l l i o n , also n g m L -  ppm  parts per m i l l i o n , also ug m L *  RSD  relative standard deviation  s  singlet ( N M R )  SAM  S-adenosylmethionine  T  temperature  1  1  T *  effective s p i n - s p i n r e l a x a t i o n time  UV  Ultraviolet  w/v  weight per volume  6  chemical shift ( N M R )  2  ix  ACKNOWLEDGEMENTS  I a m extremely grateful to i n y supervisor, Professor W . R . C u l l e n , for b i s expert advice a n d guidance t h r o u g h o u t the course of t h i s w o r k . I w i s h to g i v e s p e c i a l t h a n k s to M r . M i k e L e B l a n c f o r h e l p i n g m e i n a l l culture  experiments  Oceanography,  which  are  carried  out  i n .the  Department  a n d to M s . D e e p t h i H e t t i p a t h i r a n a f o r t e a c h i n g  me  of the  operations of N M R Spectrometers a n d various other i n s t r u m e n t s . T h e i r t i m e a n d patience spent d u r i n g this w o r k are v e r y m u c h appreciated. I t h a n k M r . H a o L i for providing analytical data o n the  arsenic  speciation studies a n d a l l m y l a b colleagues for t h e i r f r i e n d l y a n d h e l p f u l endeavours. F i n a l l y , I t h a n k the technical staff of this D e p a r t m e n t , i n p a r t i c u l a r t h e N M R staff, f o r t h e i r a s s i s t a n c e d u r i n g t h e c o u r s e o f t h i s w o r k .  x  T h i s w o r k i s d e d i c a t e d to m y M o m a n d D a d who have always provided me w i t h their total support, love a n d encouragement.  xi  CHAPTER I INTRODUCTION  1.1  ARSENIC COMPOUNDS AND THE MARINE ENVIRONMENT A r s e n i c is r a n k e d the twentieth most a b u n d a n t element i n the earth's  crust . It is ubiquitous i n the atmosphere, i n the aquatic environment, i n 1  soils, sediments a n d i n organisms. A r s e n i c i s widely dispersed, mostly at l o w levels. F o r example, the average w o r l d w i d e concentration i n the earth's crust i s o f t e n q u o t e d a s 3 p p m ( p a r t s p e r m i l l i o n , u g g- ), a l t h o u g h i t s c o n c e n t r a t i o n 1  c a n v a r y f r o m 0.1 to s e v e r a l h u n d r e d p p m , d e p e n d i n g o n t h e g e o g r a p h i c a l location, anthropogenic i n p u t a n d other factors ' . 2  3  A r s e n i c compounds (arsenicals) have w i d e l y differing properties a n d uses " . 4  Arsenic has  6  been  recognized through the  years  for the  toxic  p r o p e r t i e s o f some o f i t s c o m p o u n d s . A r s e n i c t r i o x i d e h a s b e e n u s e d a s a 7  h o m i c i d a l poison. I n a g r i c u l t u r e , arsenicals are c o m m o n l y f o u n d as herbicides a n d pesticides. A r s e n i c compounds are used i n wood preservatives a n d i n the m i n i n g i n d u s t r y . Nonetheless, some arsenicals have therapeutic v a l u e a n d are used i n drug manufacture . 8  E n v i r o n m e n t a l a r s e n i c s t u d i e s c a n b e d a t e d b a c k to t h e w h e n b o t h Jones a n d C h a p m a n showed the presence  1920's > 9  10  of relatively h i g h  c o n c e n t r a t i o n s (2 - 132 p p m ) o f a r s e n i c i n m a r i n e p l a n t s a n d a n i m a l s . T h i s w a s v i e w e d w i t h m u c h concern because the sea has a l w a y s been the earth's richest food resource  a n d arsenic  compounds were v i e w e d as  potential  p o i s o n s * ' ' . T h e f a c t t h a t t h e t o x i c effects o f a r s e n i c d e p e n d e d o n i t s 4  6  1 1  1 2  c o n c e n t r a t i o n a n d c h e m i c a l f o r m l e d to d e t a i l e d i n v e s t i g a t i o n s o f t h e l e v e l s 1  a n d chemical structure of arsenic compounds i n m a r i n e o r g a n i s m s ' . 1 3  2 1  In  recent years, the role of biota i n arsenic speciation a n d eventually its impact on h u m a n life have been extensively studied.  1.1.1  Occurrence a n d Speciation of Arsenicals  Seawater  T y p i c a l s e a w a t e r c o n t a i n s a r s e n i c w i t h a c o n c e n t r a t i o n r a n g e of 1 8 ppb (parts per b i l l i o n , n g m L r ) . * - . Geographical distribution, depth, 1  6  1 2  2 2  2 4  biological activity, volcanic activity a n d anthropogenic  contribution  may  m o d i f y t h i s concentration. A r s e n i c occurs i n oceans i n t w o different o x i d a t i o n states. A r s e n i c i n o x i d i z e d aquatic systems i s present p r i m a r i l y as arsenate [As(V)3 a n d t h e p r e d o m i n a n t d i s s o l v e d f o r m i s H A s 0 bacteria  c a n r e d u c e A s ( V ) to a r s e n i t e  dissolved oxygen. Johnson, B r a m a n  2 5  4  2  \ Both plankton and  [As(III)] e v e n i n t h e p r e s e n c e  and others 2 6  2 7  of  showed that methylated  a r s e n i c a l s s u c h as m o n o m e t h y l a r s o n i c a c i d [ M M A ] a n d d i m e t h y l a r s i n i c a c i d [ D M A ] are present as a s m a l l percentage of t o t a l arsenic i n seawater a n d are associated w i t h the biota.  M a r i n e Plants a n d Algae  D i f f e r e n t species o f a r s e n i c h a v e v a r y i n g l e v e l s o f t o x i c i t y . I n o r d e r to 3  correctly correlate the significance of h i g h arsenic levels o n m a r i n e life, m u c h effort h a s b e e n d e v o t e d to t h e s p e c i a t i o n a n d i d e n t i f i c a t i o n o f ' n e w ' a r s e n i c c o m p o u n d s p r e s u m a b l y b i o s y n t h e s i z e d b y o r g a n i s m s a l o n g t h e food c h a i n . I n this respect, m a r i n e  autotrophs  required special attention because  2  they  comprise the base of the food web. It has been observed that some marine algae are  capable of  accumulating arsenic to concentration levels of up to several hundred p p m  28  (Table 1.1). The first successful isolation of an organoarsenical from marine algae was made by Edmonds and Francescom * . Extraction of naturally 17  29  occurring seaweed followed by isolation, purification and identification by chromatographic  and  spectroscopic  means  showed  that  arsenic  is  incorporated into two forms - water soluble and lipid soluble compounds. Three water soluble arsenic-containing ribofuranosides (ACR) (1 a - c) (Figure 1.1) were identified from the brown seaweed, Ecklonia work by others - * 15  17  29-34  radiata.  Subsequent  has shown that A C R are ubiquitous in the marine  seaweeds. A useful approach to track the speciation of trace metals i n marine organisms has been to carry out feeding experiments in the laboratory - " . 22  34  36  For example, marine algae were fed with As-labelled arsenic compounds. 74  The arsenic  metabolites  were  extracted and then isolated by using  chromatography. The distribution of the gamma-emitting label was followed by radiotracer techniques. This method had proved to be efficient in establishing the presence of known arsenic compounds and their distribution. However, the identification of 'new* arsenic compounds is not possible using only these techniques.  3  Table 1.1  Arsenic Concentrations i n Selected M a r i n e Plants  Species  Common Name  Arsenic /ppm wet weight  Arsenic Compounds found  Ref.  Ecklonia radiata  brown seaweed  10  la ,b,c  14,29  Laminaria japonica  brown seaweed  4  la  19  Hizikia fusiform  edible seaweed  10  1 a,c,d°,e inorg. As°'°  20,32  Codium fragile  green algae  1  Sargassum thunbergii  brown algae  na  a  30  lb,c c  org. A s  36  d  major arsenical found; ^ inorganic arsenic; not available; ^ ACR with tetraalkylarsonium moiety.  a  c  CH  3  O^s-CH^  ^  / ° \  / h C  H  v  V  O-CHjCHCHjR  H/ \ i/ H C  1  R  c — c 1 1 OH OH  R 1a  b c  R -OSO3H -OH 1  -OH -OH -OH  9  -0-P-0-CH CHCH OH CSH 6H 2  F i g u r e 1.1  D  -OH  e  -NH  -SO3H 2  -SO3H  Structures of arsenic-containing ribofuranosides isolated from algae  4  2  M a r i n e Animals  Marine animals accumulate arsenic to a wide range of concentrations (0.3 - 350 p p m )  28  (Table 1.2), depending on the species type, habitat, feeding  habits and other factors. The arsenic compounds found mainly occur in nontoxic organoarsenic forms, the most common being arsenobetaine (2). This was first isolated from Western rock lobster i n 1977  S 7  . Since then,  arsenobetaine has been shown to be the most abundant arsenical i n most marine animals » » > . 17  23  37  38  Besides arsenobetaine, other methylated arsenic compounds have been identified i n marine organisms. Arsenocholine (3) has been claimed to be present in shrimps ' . Tetramethylarsonium ion (4) has been found i n the 89  40  Manila clam, Venerupis  japonica  and in some other  41  gastropods ' . 42 43  Trimethylarsine oxide (5) was observed to be a common minor arsenic component in fish and some mollusks ' " . The origins of these compounds 21  43  45  (2-5), whether they are passed up the food chain from their respective food sources or are biosynthesized by the organisms is, however, not clear. It is probable that the origins vary with individual cases. The only arsenic-containing ribofuranosides (1 b, d) isolated from animals to date are from the kidney of the giant clam, Tridacna  maxima . 16  The source of arsenic is likely to be the symbiotic, unicellular green algae living in the clam tissue. There are probably many other arsenic containing compounds i n marine organisms which need to be isolated and characterized.  5  Table 1.2  Organoarsenicals Isolated from Selected M a r i n e Animals  Species  Organ Common Name (As conc/ppm)  Identified Arsenicals  Ref.  Panulirus cygnus  western rock lobster  tail (20)  2  46  Sergestes lucens  shrimp  meat (5.5)  2  47  Pandalus borealis  sririmp  meat (42)*  2,4  48,49  Tridacna maxima  giant clam  kidney (200)  lb,d  16  Venerupis japonica  m anil a clam  muscles (6)*  2,3  41  Mytilus eaulis  blue mussel  tissue (1.8)  2,3  23  Tegula pfeifferi  gastropod  muscle (3.1)  2  47  Charonia sauliae  gastropod  midgut gland (340.1)  2  49  Pleuronectes platessa  plaice  muscle (3-166)  2,5  50  0  a  wet weight basis; ^ dry weight basis  (CH ) AsCH COO3  3  (CH ) As  2  3  (CH3) AsCH CH20H  (CH ) AsO  2  3  3  (5)  (3) F i g u r e 1.2  +  (4)  (2)  3  4  Formulas of organoarsenic compounds isolated from marine organisms  6  1.2  BIOTRANSFORMATION O F ARSENIC IN T H E E N V I R O N M E N T  1.2.1  Organoarsenic Compounds i n F o o d Chains The presence of organoarsenic  animals has  led to the  compounds in marine plants and  question of whether  these compounds were  synthesized by the organism or were passed along the food chain. Selected marine  food chains  were modelled and followed using  radioarsenic,  74As 14,28,51-53  Wrench Dunaliella Lipmata  et  marina seticaudala  al  6 4  studied a  three-step food chain  (phytoplankton)  —> Artemia salina  consisting  (zooplankton)  (shrimp). They cultured the phytoplankton in  of -»  74  As  enriched seawater, fed the labelled plant material to the next higher organism in the food chain and repeated the procedure for the next trophic level. Results indicated that the organic forms of arsenic in the zooplankton and shrimps were assimilated from their respective food sources. Arsenicals were transformed along the food chain. Work by Klumpp  52  and Peterson  53  suggested  that some marine  animals were capable of taking up arsenic from the surrounding media. In the marine food chain - Fucus spiralis  (macroalgae) -> Littorina  littoralis  (herbivorous snail) - » Nucella lapillus (predatory snail), they showed that the snails could manufacture water soluble arsenicals directly from arsenate. Sanders  54  showed that other potential, indirect uptake mechanisms  are  possible. Thus, it is difficult to make generalizations about the T h sources of arsenicals even in a relatively simple ecosystem.  7  1.2.2  Biomethylation of Arsenic One common feature of the organoarsenicals found in nature is that  they contain methyl group(s) attached to arsenic. It has been proposed that the formation of methylated arsenic compounds in marine organisms is a mechanism for the detoxification of arsenic . 66  The most important work on arsenic methylation was done by Challenger in 1932 brevicaulis,  67  » . Based on the work on the mould, 68  Scopulariopsis  he identified the volatile arsenic compound that had been linked  to cases of arsenic poisoning , as trimethylarsine (11). He suggested a 69  mechanism for the conversion of arsenate (6) to trimethylarsine (Figure 1.3), with arsenite (7), monomethylarsonic acid (8) and dimethylarsinic acid (9) as intermediates.  Challenger proposed that methylation occurred by the  transfer of a carbonium ion from some already methylated compound such as betaine, methionine or a choline derivative. Subsequent to the transmethylation work by du Vigneaud and coworkers , Challenger studied trimethylarsine production by cultures of 60  S. brevicaulis  in the presence of labelled precursors . The results indicated 61  that an "active methionine", now known as S-adenosylmethionine (SAM) (Figure 1.4),  is the probable transmethylating agent. Further work by  Cullen et a l "  65  62  strongly supported this idea.  The occurrence of MMA, D M A and trimethylarsine oxide in sediments as well as the presence of other organoarsenic compounds in marine organisms seems to support Challenger's mechanism of biomethylation. However, the final reduction product, trimethylarsine, has not yet been observed in marine systems. 8  2e*  CCHs*]  HJJABO^  >  As(OH)  (6)  > CH AsO(OH)  3  3  (7)  (8)  2e' CH ABO(OH) 3  [CH *] 3  > {CH As(OH)2}  2  2  > (CHg^OCOH)  3  (9)  2e  [CHs*]  (CH )2AsO(OH)  > {(CH^aAsOH)  3  > (CH ) AsO 3  3  (10)  (CH ) As 3  3  (11) F i g u r e 1.3  Challenger's mechanism for the methylation of arsenic T h e a r s e n i c (III) i n t e r m e d i a t e s i n b r a c e s a r e u n k n o w n a s monomers; C H A s ( O H ) i s found as ( C H A s O ) a n d ( C H ) 2 A s O H as (CH )2As-0-As(CH ) fie a  3  2  CH HO  3  3  3  x  2  3  t-CH-CHrCHr I NH  2  C  C  OH OH (12) F i g u r e 1.4  Structure of S-adenosylmethionine (SAM) 9  1.2.3  Biosynthesis of Arsenic-Containing Ribofuranosides Edmonds and Francesconi  18  have proposed a biosynthetic route for the  formation of arsenosugars (Figure 1.5). OH  i  CH-As=0 CH 3  -  OH I CH -As CH  O  SAM  3  3  3  OH OH (13)  O  (i)  9~9 OH F i g u r e 1.5  OH  (14) Proposed scheme for the formation of arseniccontaining ribofuranosides from dimethylarsinic a c i d  Dimethylarsinic acid is produced by the sequential reduction and methylation of arsenate by microorganisms as outlined in Section 1.2.2. Here, S A M is assumed to switch its role of [ C H ] source to that of a donor of the 3  +  adenosyl group . Enzymatic hydrolysis of (13) would lead to (14) which could 67  form the sugars (1 a-e) by reaction with available algal metabolites. However, the two key intermediates (13) and (14) have not yet been isolated i n the marine  ecosystem.  Thus the  exact mechanism  arsenosugars has yet to be established.  10  for the  formation  of  1.2.4  Metabolism of Arsenic-Containing Ribofuranosides It  is  still  arsenobetaine '  18 68  not  known  how  or ( C H ) A s . L u n d e 3  +  4  marine  51  animals  accumulate  proposed that algae concentrate  arsenicals, which are passed through the food web to marine animals at higher trophic levels. Since no arsenobetaine has been isolated from marine algae,  and  since  Edmonds et a l ' 1 7  2 9  seawater  is  unlikely  to  be  a  significant  source,  suggested that the A C R (1) found in algae are likely to be  the precursors of arsenobetaine. The metabolic pathway (Figure 1.6) is proposed. The facile transformation of A C R present in the brown algae Ecklonia  radiata  conditions  15  into  dimethyloxyarsylethanol  (15)  under  anaerobic  supports this view. However, if this is correct, no information  pertaining to the route to arsenobetaine (18), whether via arsenocholine (16) or dimethylarsylacetic acid (17) has yet been established. Recent work by Shibata and Morita alternative  hypothesis  trimethylarsinoriboside (19) Sargassum  thunbergii.  of  the  36  has provided the basis for an  origin  of  arsenobetaine.  has been isolated from the marine  A alga,  It seems likely that under anaerobic conditions the  trimethylarsenoriboside could be degraded to arsenocholine which could be readily converted into arsenobetaine in fish or elsewhere . Further work is 68  required as the trimethylarsenoriboside has so far been reported in one species of alga only and then only in trace amounts.  11  anaerobic decomposition  (1)  •  CH ,  3  CH  oxidation  0=As-CH COOH  0=As-CH CH OH 2  i  3  2  2  CH,  CH,  (17)  (15)  2e  2e CH  3  CH  +  (CH3)3AsCH CH OH 2  o x i d a t i  2  °  +  3  2  (18)  Proposed scheme for the biosynthesis of arsenobetaine from arsenic-containing ribofuranosides  CH  3  CH -A s-CT 3  +  2  c  \  OH  .O-CH 9HCH -S04 2  \  c — -c  H  2  OH  OH  (19) F i g u r e 1.7  +  (CH )3As CH COO-  n  (16)  F i g u r e 1.6  3  Structure of a trimethylarsinoriboside 12  1.3  S C O P E O F WORK Marine algae have been shown to concentrate arsenic to high levels  relative to their environment with concentration factors of 1000 to 10000 reported - . Lunde , Iroglic et a l » , Andreae * , Sanders ' 70  72  73  74  Windom , and Wrench et a l * 77  54  78  75  22  27  24  76  and  have investigated the bioaccumulation of  arsenic by some marine algae. A l l carried out algal growth experiments in media dosed with arsenate tagged with the gamma-emitting A s isotope and 74  studied the arsenic uptake and metabolism. Results indicate that the wide range of algae tested are able to alter the arsenic speciation in the growth media. Arsenate was taken up and subsequently released into the culture media as varying proportions of arsenite, monomethylated and dimethylated arsenic compounds. Further extraction and isolation procedures suggested that marine algae are able to biosynthesize organoarsenic. It has been proposed that arsenate is taken up by phytoplankton because of the chemical similarity between arsenate and phosphate. Arsenate may enter cells by transport mechanisms that are unable to discriminate between the two species. The phytoplankton, however, do not retain large quantities of arsenic within the cell. The degree of accumulation, the type and or number of organic arsenicals produced varies from species to species. Growth studies on freshwater algae showed similar behaviour. The present investigation involves a study of the effects of various forms of arsenic (arsenate, arsenite, M M A , DMA) on laboratory cultures of marine algae. Two single-celled species, constituting two different classes of phytoplankton Dunaliella  -  tertiolecta  Isochrysis galbana  (Class  Prymesiophyceae)  and  (Class Chlorophyceae) - are used. These were chosen 13  because they are easy to grow and are at the base of the food chain. Moreover, they have been commonly employed as commercial food for marine molluscs, crustaceans and fish. Therefore, they offer the possibility of use in further feeding experiments involving "arsenic-enriched" algae to study the transfer of arsenic along the marine food chain.  14  C H A P T E R II EXPERIMENTAL  2.1  INSTRUMENTATION  2.1.1  Atomic Absorption Spectrometry (AAS) A Varian Techtron Model A A 1275 Atomic Absorption Spectrometer  was used for arsenic determination. This was equipped with a Varian Spectra A A hollow cathode lamp operating at 9 mA. The monochromator was set at 193.7 nm, one of the resonance lines of arsenic, and the slit width was 1 nm. The A A Spectrometer was equipped with a deuterium background corrector and a Hewlett Packard 82905A printer.  2.1.2  N u c l e a r Magnetic Resonance Spectroscopy (NMR) a n d Mass Spectrometry (MS) 1H N M R spectra were obtained on Bruker WH200 and Bruker WH400  Spectrometers.  13C N M R spectra  were obtained on a Varian  XL-300  Spectrometer. Chemical shifts are quoted relative to tetramethylsilane (TMS) as an external standard. Mass spectral data were obtained by using a Kratos A E I MS9 Mass Spectrometer.  2.1.3  H i g h Performance L i q u i d Chromatography ( H P L C ) The  HPLC  system consisted of Waters M45 and M510 pumps  controlled by an Automated Gradient Controller, Waters U 6 K injector, and a  15  u-Bondapak C ^ , 3.9 mm (i.d.) x 30 cm, steel column. Graphite Furnace A A (GFAA) was used as an arsenic specific detector; fractions were collected from the H P L C  column with a Gilson Micro Fractionator  and transferred  manually to the automatic sample delivery system of the A A Spectrometer.  2.1.4  Chemicals a n d Reagents All chemicals used were of analytical grade and obtained from  commercial sources unless otherwise stated. Deionized water (Aquanetics Aqua Media System) was used for A A methods. All glassware  and plastic ware were cleaned by soaking them  overnight in 2% Extran solution, rinsing with water, and soaking in dilute Hydrochloric acid overnight. The containers were then rinsed with water and deionized water until the wash was neutral to litmus. Standard stock arsenic solution (1000 ppm) for G F A A was prepared by dissolving 1.3203 g of Arsenic (IH) oxide and 2 g of Sodium hydroxide i n 20 mL of water. The solution was diluted to 200 mL, neutralized with Hydrochloric acid, and made up to 1000 mL.  2.2  GENERAL CULTURE MAINTENANCE  2.2.1  C u l t u r e Methods Stock cultures of Isochrysis galbana  and Dunaliella  tertiolecta  were  obtained from the North East Pacific Culture Collection (The Department of Oceanography, U.B.C.).  16  Media  Enriched natural seawater was used as the culture medium. The seawater was collected well offshore i n West Vancouver and treated with activated charcoal, filtered through a 2 um pore-size glass filter, and stored in borosilicate glass bottles. For the preparation and maintenance of stock cultures, filtered seawater (10 L) was autoclaved and then enriched with a  separately  autoclaved stock of mixed nutrients (Table 2.1) . . 79  Table 2.1  80  Composition (g Lr ) of two Stock Solutions used i n the E n r i c h m e n t Solution 1  Nutrients and Trace Metals [10 mL]° NaNOo Na2Si0 .9H2P NaoglyceroPCL NaoEDTA H BO Fe(N&) (S0 ) .6H 0 FeCl .6HoO MnSO^HoO ZnS0 .7H O CoS0 .7H 0  4.667g 3.000 0.667 0.553 0.380 0.234 0.016 0.054 0.0073 0.0016  3  3  s  2  4  2  2  a  4  4  2  2  Vitamins [1 m L ] thiamine HC1 vitamin B biotin  0.1 g 0.002 0.001  1 2  a  0  Volume of stock solutions to add to 1 L of natural seawater base is in parenthesis.  17  C u l t u r e Conditions  Experimental cultures were grown i n 2 L of culture medium i n autoclaved, cotton-plugged, 2.8 L Erlenmeyer flasks. A n inoculum of 6 days old stock culture was placed in the medium. The arsenic compound (arsenite, arsenate, monomethylarsonic  acid or dimethylarsinic acid) i n aqueous  solution was added to obtain a known concentration of elemental arsenic in each culture medium. Background arsenic concentrations were less than 2ppb. All cultures were grown at 16°C and intermittent  light at an  irradiance of approximately 100 uE.nv s-i from fluorescent lamps. A 16h: 8h 2  light-dark cycle was maintained. The cultures were not continuously shaken, but were agitated once every day.  2.2.2 A n a l y t i c a l Methods Growth was estimated by measuring the in vivo fluorescence of about 8 mL of culture aliquots, at ca. 24 hours intervals, using a Turner Designs (Model 10) Fluorometer. The cultures were grown until the population reached  stationary  phase, usually 9 - 1 3 days. Cell counting was performed on a Coulter Counter Model TATJ (70 um aperture) one day before harvest. Samples were mixed by gentle inversion prior to counting. Cells were harvested by centrifugation at 3000 revolutions per minute at 4°C for 15 minutes i n a Sorvail T60000B Centrifuge and washed twice with 0.3% Sodium chloride. Cell concentrates were frozen, freeze-dried and then stored in the freezer. Total arsenic incorporated into cells was found by  18  acid digestion of samples followed by Hydride Generation Atomic Absorption (HGAA). The residual culture media were filtered through a glass membrane fibre filter (0.45 um) and stored i n the cold room at 15°C for future arsenic analysis by H G A A .  2.2.3 N M R Studies The  algal cells were harvested at the stationary phase and washed  three times with deuterium oxide to remove the excess medium and then packed into a 5 mm NMR tube. The * H spin-echo NMR spectra were recorded by using the Carr-Purcell-Meiboom-Gill pulse sequence (Figure 2.1) with a delay time (t) of 30 ms  8 1  . A small pre-saturation pulse was applied to the  water resonance prior to accumulation. A n acquisition time of 0.426 s and a spectral width of 5000 Hz were used. The free induction decay was collected in 4K of data points zero-filled to 3 2 K A 0.1 Hz line-broadening function was applied during Fourier transformation.  90'  180  6  Acquisition  Observation _ channel Decoupler T channel —  F i g u r e 2.1  Saturation  A  T h e Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence  19  2.3  ANALYTICAL PROCEDURES  2.3.1  Graphite F u r n a c e Atomic Absorption (GFAA) Graphite furnace atomization was achieved with a Varian Techtron  GTA-95 accessory using Varian Techtron pyrolitically coated graphite tubes and argon as the sheath gas. The optimized furnace program for the determination of arsenic is shown in Table 2.2. The autosampler delivered 20 uL aliquots of standard and sample. Palladium (20 uL of 100 ppm solution in ca. 2% citric acid) was added as a matrix modifier.  2.3.2  H y d r i d e Generation Atomic A b s o r p t i o n (HGAA) A continuous hydride generation system (Figure 2.1) was used for  generation of arsines. It consisted of a Gilson Miniplus 2 four-channel peristaltic pump, which was used to pump sample solutions, Hydrochloric acid and Sodium borohydride. Nitrogen was used as the carrier  gas.  Atomization was achieved i n an open ended "T" absorption quartz cell (8.5 cm x 1 cm (o.d.)) mounted in an air / acetylene flame of the standard Varian burner. The operating parameters established by D o d d were followed and are 42  shown in Table 2.3.  20  Table 2.2 Step No.  Graphite F u r n a c e Operating Parameters  Temperature (°C)  1 2 3 4 5 6 7 8  85 95 120 1400 1400 2400 2400 2400  Table 2.3  Time (sec)  Gas Flow (L min* )  Gas Type  3.0 3.0 3.0 3.0 0 0 0 3.0  Normal Normal Normal Normal Normal Normal Normal Normal  1  5 40 10 6 2 0.7 2 2  No No No No No Yes Yes No  Operating Conditions for the H y d r i d e Generation Assembly  Uptake tubes  sample: acid : NaBH : 4  Carrier gas  Nitrogen  2.8 rnmLD. 2.28 mm I.D. 2.29 mm I.D.  7.5 mL min" 2.0 mL min" 4.0 mL min"  100 mL min" through mixing coil TriTi min" through gas-liquid separator 1  25  Hydrochloric acid concentration  4M  N a B H concentration  2 % (w/v)  Integration  Run Mean  4  Read Command  21  1  1 1 1  QUARTZ ABSORPTION CELL  REACTION COIL  TRANSFER TUBE  *—/ V  PERISTALTIC PUMP  fA I  GAS / LIQUID SEPARATOR NITROGEN  DRAIN  F i g u r e 2J2  NEEDLE VALVES  PRESSURE REGULATOR  Schematic diagram of the h y d r i d e generation system  22  2.3.3  Wet  Digestion  of  Freeze-dried  Algae  with  Nitric  Acid,  Sulfuric A c i d a n d H y d r o g e n Peroxide Wet digestion was carried out i n a 250 m L round bottom flask fitted with specially designed stopper and air condenser containing a diffusion funnel (Figure 2.3). The stopper, funnel and plugs were made of Teflon. The dried sample (75 mg), Nitric acid (3 mL, 69%), Sulfuric acid (1 mL, 98%) and Hydrogen peroxide (3 mL, 30%) were mixed in the flask. The apparatus was placed in a 250 m L heating mantle and heated for three hours (T«250°C) in a fumehood. The flask was then cooled to ambient temperature. The digested sample was transferred to a 100 m L volumetric flask and made up to the mark with deionized water. Total arsenic of the complete digestate was determined by using H G A A and G F A A .  2.3.4  Extraction  and  Purification  of  Arsenic  Metabolites  of  D. tertiolecta C u l t u r e d i n Arsenate E n r i c h e d M e d i a Cells were recovered from the incubation medium by vacuum filtration through a glass fibre filter (0.45 um) and were washed with 20 mL of filtered seawater. The filter was extracted with 6 mL of chloroform/methanol (2:1 ,v/v) followed by 1.5 m L of deionized water. The emulsion was allowed to stand at room temperature. After separation, the lower chloroform layer (lipid soluble material) was removed and then evaporated to dryness under reduced pressure. It was stored in the cold room for further analysis by acid digestion - H G A A Spectrometry. The upper methanol/water layer was defatted by using diethyl ether (4.5 mL) and all solvents were then removed under the vacuum. The extract 23  was divided into two portions. One portion of the crude extract was subjected to gel permeation chromatography on a Sephadex LH-20 column and the other portion to H P L C - G F A A .  G e l Permeation Chromatography  Sephadex LH-20, previously swollen in water, was packed into a glass column (35cm x 1.5cm (o.d.)) using water as the eluent. The water soluble extract (~50 mg) was loaded into the column and fractions (2 mL) were collected and were monitored using G F A A . The arsenic containing fractions were concentrated and evaporated to dryness under vacuum. The solid obtained was re-crystallized in water and subsequently analyzed by * H N M R spectroscopy and mass spectrometry.  HPLC - GFAA  Water - methanol (95:5, v/v) mixtures, 5 mmole mL- with respect to 1  tetrabutylammonium nitrate (TBAN), was used as the mobile phase at a flow rate of 1 mL min* for separations on the reversed phase column. The p H of 1  the aqueous component was kept at 6.8 by using ammonium hydroxide (10%). Fractions of the eluent were collected at 0.5 minute intervals and 20 uL of each fraction was subsequently injected into the graphite tube for arsenic analysis. Standard arsenical solutions (0.5 mg i n 20 uL) were injected into the H P L C to establish retention times.  24  1.0.15  F i g u r e 2.3  Wet ashing apparatus (A) Teflon cylindrical plugs; (B) Telfon diffusion funnels; (C) Teflon stopper with capillary; (D) 250ml round-bottom flask. All joints are 14.5/23. All dimensions are in mm. Adapted from design described by Bajo et a l . 82  25  CHAPTERm RESULTS AND DISCUSSION 3.1  GRAPHITE F U R N A C E ATOMIC ABSORPTION S P E C T R O M E T R Y Atomic Absorption Spectrometry (AAS) is one of the most popular  techniques for determining arsenic i n environmental samples " . It offers 83  86  advantages in terms of simplicity, sensitivity and selectivity over other analytical methods . Conventional methods for atomizing samples use 87  flames such as air-acetylene or hydrogen-air flames and aqueous samples are aspirated directly into the flame. These methods give poor detection limits for arsenic due to the high background absorption in the spectral region of 200 nm where the most sensitive arsenic absorption lines are found. Graphite Furnace A A S (GFAA) is an alternative solution to this problem. It is becoming the method of choice because of its higher sensitivity which is 2 to 3 orders of magnitude better than that attainable i n the flame. Another advantage is that smaller sample volumes are needed in G F A A compared with the flame techniques. It is well documented that a proper heating program is essential for G F A A ' . Usually a temperature program is divided into 3 main stages. In 8 8  8 9  the first drying stage, the sample is heated so that solvents are vaporized without spattering. The second ashing stage is aimed to volatilize or decompose organic compounds which are then removed by the flow of purge gas. The third stage is the rapid vaporization and atomization of sample during which analyte absorption is simultaneously detected and recorded; The GTA-95 Graphite Tube Atomizer accessory used in this study 26  allows  the  programming  of operation  parameters  such  as  heating  temperature and duration as well as the type and rate of flow of purge gas. The  sample  solution (5 - 70 uL) is  automatically  introduced into  an  electrically heated graphite tube which is aligned in the sample beam of the A A Spectrometer. A n optimized temperature program is used to maximize the efficiency of atomization and to remove matrix interference as much as possible without loss of analyte signal. Arsenic is a relatively volatile element (boiling point 613°C) and losses will occur during ashing. However, matrix modification ' permits a higher 90 91  ashing temperature and the removal of matrix compounds which could otherwise interfere with the determination. Palladium(O) i n citric acid (2%)  92  has proved to be an efficient matrix modifier for arsenic determination and is adopted in the present study. Typically, 20 uL of a 100 ppm solution of palladium is added to the sample in the graphite tube. It is postulated that the added palladium forms a stable species with arsenic which does not decompose until high temperatures are reached.  27  3.1.1  Calibration, L i m i t of Detection a n d P r e c i s i o n of G F A A Analysis A  typical graph of absorbance against concentration for 20 uL  injections of a series of standard arsenite solutions is shown in Figure 3.1.  0.4  0.5 concentration of arsenic / p p m  F i g u r e 3.1  T y p i c a l calibration curve for the determination of arsenic by G F A A  The calibration curve constructed for the range up to 0.35 ppm arsenic showed a linear dependence of the G F A A absorbance on the amount of arsenic up to 0.2 ppm. The limit of detection, defined as the analyte concentration giving a signal equal to the blank plus three standard deviations of the blank was determined to be 10 ppb. The relative standard deviations for twenty injections of 20 uL of 10 ppb standard arsenite solution was calculated to be 8.2%.  28  3.2  HYDRIDE G E N E R A T I O N ATOMIC ABSORPTION SPECTROMETRY Hydride  Generation  Atomic  Absorption (HGAA)  involves  the  conversion of soluble compounds of a number of elements (As, Bi, Ge, Pb, Sb, Se, Sn and T e ) 93  95  into volatile covalent hydrides, followed by atomization  and detection using A A . This method is extremely useful because it separates the analyte from other potential matrix components and may also be used as a method of concentration prior to quantification. The relatively small number of components reaching the atomizer significantly reduces matrix effects compared with G F A A techniques. Sensitivity and detection limits for H G A A and G F A A techniques are reported to be quite comparable. One major disadvantage of H G A A is that relatively large sample volumes are needed for each measurement, particularly in the continuous flow system that is used in the present study. The  hydride generation  technique is most  often used for  the  determination of arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid - ' " 90  91  96  98  and  trimethylarsine  oxide  26  especially in environmental  samples. These compounds can form volatile hydrides that are reasonably stable against hydrolysis and decomposition at room temperature.  The  arsines and methylarsines have different boiling points and they can be trapped and concentrated by cryofocussing and separated sequentially by fractional volatilization or by gas chromatography. Furthermore, p H selective reduction permits speciation of inorganic As(LU) and As(V) mixtures - . 99  Two  main  reducing  systems  have  been  employed for  101  arsine  production . The first involves the use of metal-acid combination such as 83  29  Zinc-Hydrochloric acid. 4Zn +  8HC1 + As(OH)  ->  3  4ZnCl  2  + AsH  +  3  3H 0 2  + H  2  A major drawback of this reaction is that the time taken to complete the reaction is long, up to 10 minutes, and it is necessary to store the generated hydrides in some form of reservoir before introducing them into the detection system. The  use  of  Sodium  borohydride (NaBH )  and  4  acid  (usually  Hydrochloric acid) is effective for the generation of arsines. 5As(OH) + 6 N a B H 3  4  + 3HC1 + 3 H 0  -»  2  5AsH + N a B 0 3  3  3  + 5H B0 3  3  + 3NaCl + 9 H  2  This system was found to be superior to the metal-acid system with respect to reduction yield, reaction time and contamination of the blank. Optimum reduction conditions developed by D o d d  42  were adopted for  the present study. Samples were pumped and mixed continuously with HC1 (4M) and then N a B H solution (2% w/v). The reduction of arsenicals and 4  decomposition of N a B H produces arsines and Hydrogen respectively. A flow 4  of Nitrogen carrier gas takes the volatile analyte hydride to the liquid-gas separator and thence into a heated quartz tube atomizer where the hydride decomposes. A n air-acetylene flame is positioned directly under the atomizer which is aligned i n the optical path of the Spectrometer. The absorbance is recorded when a steady state signal is obtained.  30  3.2.1  Calibration* L i m i t of Detection a n d P r e c i s i o n of H G A A Analysis A typical graph of absorbance against concentration for standard  arsenite solutions is shown in Figure 3.2.  o.e  concentration Figure 3.2  of a r s e n i c / ppb  T y p i c a l calibration curve for the determination of arsenic b y H G A A  The curve shown is linear to 80 ppb arsenic. In practice, a linear relationship between absorbance and concentration can be obtained up to a concentration of 120 ppb. The limit of detection defined as the analyte concentration giving a signal equal to the blank, plus three standard deviations of the blank, was determined to be 0.6 ppb. The precision of determinations was estimated from twenty replicate analysis of 10 ppb arsenite standard. The relative standard deviation at this concentration was found to be 2.0%. 31  3.3  D E T E R M I N A T I O N O F T O T A L ARSENIC Arsenic determination by G F A A and H G A A requires that the sample  be presented in liquid form. Thus, it is important to have methods to solubilize the phytoplankton material, a largely organic matrix, to afford a solution  suitable  described ' ' 82  83  102  -  105  for .  analysis.  The  two  Many most  digestion widely  methods  used  have  methods  been  for  the  decomposition of organic samples are wet ashing and dry ashing. In wet ashing, the sample is treated with concentrated mineral acid(s) and or oxidizing agent(s) in solution. Most often this mixture is heated; Various reagent mixtures have been described ' 85  104  '  106  . More recently, acid  digestion in a specially designed pressurized vessel contained in a microwave oven has been employed . In dry ashing, the sample is treated at high 107  temperature in the presence of atmospheric oxygen. A n ashing aid, such as Magnesium nitrate, is usually added to hasten oxidation and reduce sample losses due to retention by the walls of the container. Hoenig and Borger  108  have obtained good recoveries of trace elements  in plant material by using a wet digestion procedure with H N 0 - H 2 S 0 - H 0 2 3  4  2  acid mixture. This acid combination, chosen in the present study, avoids the use of Perchloric acid. Work with Perchloric acid at elevated temperatures requires specially designed fume hoods which are not available to us. The wet ashing apparatus employed is shown in Figure 2.3  8 2  . It  consists of a round-bottom flask, a stopper with capillary and an air condenser containing a diffusion funnel and four cylindrical plugs. The plugs are used as a filling material. The stopper, funnel and plugs are made of Teflon which is highly resistant to chemical attack. One essential feature of 32  this apparatus is the Teflon stopper with capillary. Teflon is a material which swells slightly upon heating and provide an excellent seal. It also serves as a thermal insulator, maintaining the distillate at temperatures below boiling, thereby detaining them in the condenser. This is particularly advantageous in avoiding losses of sample material during digestion. The sample material is refluxed with a mixture of HN03-H2S04-H 0 2  acid (1:3:3 m L g *  1  2  dry material). The organic material is wet ashed at  atmospheric pressure for 3 hours (T«250°C). The apparatus is then removed from the heating source and set aside to cool. A colourless solution is obtained and made up to 100 mL in a volumetric flask for further, analysis. In order to confirm the appropriateness of the digestion methods, the same procedure was carried out with certified reference materials. Samples included National Research Council of Canada (NRCC) Marine Analytical Standards, dogfish muscle DORM-1 and National Bureau of Standards (NBS), orchard leaves S R M 1571. A summary of results obtained for these standards are shown in Table 3.1.  33  Table 3.1  A r s e n i c Determinations i n Standard Reference Materials  Sample  As certified /ugg-ia  Dogfish muscle DORM-1  17.7 ± 2 . 1  Orchard leaves S R M 1571  10 ± 2  As found /ag g1 b  16.5±1.7 9.7 ± 0.8  ° Mean and 95 % tolerance limits. b Mean and standard deviation of mean for 6 determinations.  3.4  C U L T U R E EXPERIMENTS  3.4.1  Fluorescence a n d C e l l C o u n t i n g Studies In  vivo  photosynthesis  fluorescence 109  has  been widely used to monitor  or phytoplankton biomass  110  either  in marine and freshwaters  and to indicate algal growth i n laboratory experiments ' " . The intensity 56  111  113  of fluorescence at the wavelength which the algae fluoresces ' 109  114  is  measured using a fluorometer. Since there is a close correlation between chlorophyll production and the biomass of the algae species used in this study  110  '  115  , in vivo fluorescence provides a convenient estimation of growth.  The major advantage of using in vivo fluorescence is that the measurement is easy to make and the method is non-destructive. Rapid estimation of growth can be achieved without carrying out extraction or assay procedures. However, fluorescence yield varies between species and within a species when subjected to diverse conditions. There are many environmental factors which can affect fluorescence and of which little is known on a 34  quantitative basis. Therefore, no attempt is made in this study to compare fluorescence values between cultures inoculated at different times of the study. Only the time variation of in vivo fluorescence of I. galbana and D. tertiolecta  i n media containing different concentrations of arsenic is  monitored. The growth cycle of marine algae cultures is usually composed of a short lag phase when there is no apparent cell division, then an exponential growth phase when the growth is maximum, followed by a stationary phase when the cell density is at a maximum and constant. The growth of I. galbana  and D. tertiolecta  were followed using in vivo fluorescence until  the stationary phase was reached. Cell densities of algal cells were measured using a Coulter Counter and in this work these densities are expressed relative to that of the control culture within the same batch of experiments (Figure 3.7 and 3.12). Cell densities i n duplicate flask are found to vary to less than 5%. Results are listed in Table 3.2.  Effect of Arsenicals o n G r o w t h of I.  galbana  Arsenate  The growth of algae at various levels of arsenate is plotted  against time i n Figure 3.3. Growth in the presence of 0.5, 1.1, 5.4 and 10.7 ppm arsenic is not significantly different from that of the arsenate-free control although a slightly longer lag phase (~2 days) is observed in all the arsenate enriched media. (Cell densities on day 14 are found to be similar to that of the control in all the cases tested.)  35  Arsenite  The growth of I. galbana  in media containing arsenite is shown  in Figure 3.4. In media containing 2 and 4 ppm arsenic,  /. galbana  grew  quite like the control. At 6 ppm arsenic, an enhancement in growth was observed. At 8 ppm arsenic, some reduction i n growth was recorded. (The cell densities on day 13 are about 81% and 66% of the control at arsenic concentrations of 6 and 8 ppm respectively.) In comparison with cultures grown i n arsenate, those grown in arsenite are more affected at the arsenic concentrations tested. Monomethvlarsonic Acid fMMAI  The plot of growth against incubation  time i n M M A enriched media is shown i n Figure 3.5. There is a general enhancement in growth at a concentrations of 0.5, 1, 5 and 10 ppm. (Cell densities of the culture measured on day 14 often exceeded that under conditions where no arsenic is present.) Dimethvlarsinic Acid fDMAI  Growth of /. galbana  in D M A enriched  media against time is shown in Figure 3.6. Growth up to an arsenic concentration of 20 ppm seems to be slightly affected. At concentrations of 40 and 100 ppm, most algae cells are killed. (The cell densities on day 14 at 2 and 20 ppm arsenic are 93% and 82% respectively.) These results show that at low arsenic concentrations, (e.g.< 2 ppm) growth inhibition of /. galbana  is largest in arsenite enriched media. Under  these laboratory culture conditions, the algae also show some reduction in growth when dosed with arsenate and D M A , although the effect is not as significant as arsenite. On the other hand, M M A seems to exert some positive growth effect on I. galbana  at the arsenic concentrations tested. 36  A plot of relative cell densities against arsenic concentration for the different arsenicals is shown in Figure 3.7. Arsenite, M M A and D M A all cause a decrease in relative cell densities at the stationary phase (-day 1315) as the concentration of arsenic increases. Note that the cell densities of cultures treated with M M A are more than that of the control at all arsenic concentrations tested. Cell densities in arsenate enriched media showed a 'dip' in relative cell densities showing that the algae may have some means of coping with arsenate stress at the higher concentrations  37  F i g u r e 3.3  Effect of arsenate o n growth of I. galbana The error found in the intensity measurements is ±0.2 .  38  6 ppm  incubation time / days  F i g u r e S.4  Effect of arsenite o n growth of / .  galbana  The error found in the intensity measurements is ±0.2 .  39  0  2  4  6  8  10  12  14  16  incubation time / days  F i g u r e 3.5  Effect of M M A o n growth of / . galbana The error found in the intensity measurements is ± 0 . 2 .  40  16  incubation time / days  F i g u r e 3.6  Effect of D M A o n growth of I. galbana The error found in the intensity measurements is ± 0 . 2 .  41  140  F i g u r e 3.7  Effect of arsenic o n the relative cell densities of /. galbana  42  Effect of Arsenicals o n G r o w t h of D.  Arsenate  The growth of D. tertiolecta  tertiolecta  in media containing arsenate is  shown in Figure 3.8. In media containing 2.1 ppm arsenic, growth is similar to that observed in the control culture. At a lower arsenic concentration of 0.5 ppm, growth follows closely to the control for the first five days after which growth is significantly inhibited. (The cell density on the day 9 at this arsenic concentration is found to be 48% of the control.) On the other hand, growth at 5.4 ppm arsenic behaved differently; there is a greater inhibition in growth for the first five days after incubation than that in the later stage of the growth cycle. (In fact, cell density at the time of harvest is about 65% of the control, which is more than that at  0.5 ppm.) This may imply  D. tertiolecta has some means of tolerating higher arsenic concentration. It is in agreement with the much longer lag phase observed at 10.7 ppm arsenic. Cells started to grow 8 days after inoculation at this concentration of arsenate. Arsenite  The effect of arsenite on growth of D. tertiolecta is represented  in Figure 3.9. At a low arsenic concentration of 0.5 ppm, arsenite caused a significant growth inhibition. (Cell density on day 9 is just 63% of the control.) At intermediate concentrations of 1 and 5 ppm, growth is similar to the control. At 7 ppm, a reduction in growth is observed again. The growth characteristics of D. tertiolecta at various concentrations of arsenate and arsenite  are  quite different from that in  I.  galbana.  D. tertiolecta seems to be more sensitive to arsenate and arsenite. Low levels of these arsenic species inhibit D. tertiolecta production more, as indicated by 43  lower population densities of the batch cultures relative to the control. The changes in growth patterns at higher arsenate concentrations suggest that this alga is able to adapt to some arsenic stress. Monomethvlarsonic Acid fMMAl  The graph of D. tertiolecta  against  M M A concentration is shown in Figure 3.10. Growth is slightly depressed at an arsenic concentration of 0.5 ppm. (The cell density on day 10 is very close to that of the control.) At all other arsenic concentrations tested, growth enhancement is shown. This effect is similarly observed i n I. galbana.  The  mechanism for growth enhancement has not been established. However, it is interesting to know that M M A is capable of enhancing growth in a freshwater alga, Chlorella s p .  116  .  Dimethylarsinic Acid ID MAI  The  plot  of growth  against D M A  concentration is shown in Figure 3.11. Arsenic concentrations of up to 5 ppm appear to have little or no effect on algal productivity. (Cell densities on day 9 are found to be over 90% of the control.) The algae grew well in media containing D M A at concentration as high as 10 ppm.  The plot of relative cell densities against arsenic concentrations is shown in Figure 3.12. The relative cell densities increase, level off and then decrease as the arsenic concentrations increase for three of the arsenicals tested. Cells grown in M M A enriched media show a relative cell density over 100%, as is observed in I. galbana.  Cells grown in arsenate enriched media  show a maximum at about 2 ppm possibly indicating that D. tertiolecta may have some means of coping with arsenate stress at low concentrations. 44  It is evident that arsenic speciation plays an important role in determining its toxicity to algae. Studies from in vivo fluorescence show that methylated arsenic compounds, such as M M A and D M A , have little or no effect on the growth of phytoplankton. Arsenate and arsenite, at elevated concentrations, have more drastic effects. The two different species of algae display a different tolerance towards these arsenicals. Hence, one cannot predict the possible response of natural communities of marine algae to a given stress on the basis of unialgal cultures in the laboratory. Moreover, algal response under artificial conditions of cell density, light and nutrients is quite likely to differ from that in the natural environment . Cultures of larger scales would be preferable 107  and could facilitate isolation and characterization of arsenic compounds incorporated into the cells.  45  0  2  4  6  B  incubation time / days  F i g u r e 3.8  Effect of arsenate o n growth of D. tertiolecta The error found in the intensity measurements is ± 0 . 2 .  46  10  4  6  8  incubation time / days F i g u r e 3.9  Effect of arsenite o n growth of D. tertiolecta The error found in the intensity measurements is ± 0 . 2  47  10  _____ 5.1 p p m 10.3 ppn 1 ppm r - - • control —  0  2  4  6  8  10  incubation time / days  F i g u r e 3.10  Effect of M M A o n growth of D. tertiolecta The error found in the intensity measurements is ± 0 . 2 .  48  - 0.5 p p m  12  0.5 p p m  control  10 ppm  6  4  incubation time / days  F i g u r e 3.11 Effect of D M A o n growth of D. tertiolecta The error found in the intensity measurements is ±0.2  49  10  200  (H 0  F i g u r e 3.12  •  i  1  1  •  1  •  1  «  1  2 4 6 8 10 concentration of arsenic / ppm  « 12  Effect of arsenic o n the relative cell densities of D. tertiolecta  50  3.4.2  A r s e n i c Uptake by I. galbana  a n d D.  tertiolecta  The total concentration of arsenic incorporated into the cells is found by acid digestion - H G A A of the freeze dried samples. Results are shown in Table  3.2.  Both  I. galbana  and  D. tertiolecta  incorporate  higher  concentrations of arsenic when cultured in arsenate and arsenite enriched media than in any of the methylated arsenicals. Total arsenic uptake rarely exceeds 0.2 mg g" in the latter media. For a given species of algae, the total 1  arsenic uptake is generally higher from the arsenite enriched media than from arsenate. The total concentration of arsenic in I. galbana range from 2.1-5.1mgg~ 3.6 mg g  -1  1  in arsenite enriched media and from 0.2-  i n arsenate enriched media. It is observed that  incorporates more arsenic than I. galbana. from 0.01 - 8.0 mg g  -1  is found to  D. tertiolecta  The total incorporation ranges  and from 3.2 - 7.2 mg g- in arsenite and arsenate 1  enriched media respectively. Another way of monitoring uptake of arsenicals by the marine algae is by measuring the difference between the initial arsenic concentration in the media and that remaining at the time of harvest. It is expressed as the percentage of the original arsenic taken up from the media. Care has to be taken in analyzing these data as they include losses due to adsorption to the surface of cells and do not account for adsorption by the culture flasks. Moreover, these results do not account for any arsenic taken up and then released back into the media by the algae within the time frame of the experiments.  51  Table 3.2 Species  I80chry8is galbana  Dunaliella tertiolecta  A r s e n i c U p t a k e a n d Relative C e l l Densities of M a r i n e A l g a l Cells G r o w n i n A r s e n i c E n r i c h e d M e d i a  As added /ppm 0 0.5 1.1 5.4 10.7 0 0.5 2.1 5.4 10.7  I80chry8is galbana  0 2.0 4.0 6.0 8.0 0 0.5 1.0 5.0 7.0  Dunaliella tertiolecta  I80chry8i8 galbana  Dunaliella tertiolecta  Isochrysis galbana Dunaliella tertiolecta  a  0 0.5 1.0 5.0 10.0 0 0.5 1.0 5.1 10.3 0 2.0 20.0 0 0.5 1.0 5.0 10.0  Total As ° Angg1  As Uptake %  6  As per cell /mg cell  Relative Cell Density  -1  media enriched with arsenate [As(V)J 0 0.19 30.3 7.8 x l O 0.39 13.4 1.1 x 10 3.16 20.7 6.7 x 10 3.63 14.4 8.3 x 10 0 3.24 22.2 4.7 x 10 7.25 24.3 1.2 x 10" 6.16 19.8 3.4 x 104.55 15.7 1.6 x 10" media enriched with arsenite [Astfll)] 0 2.06 22.5 2.1 x 104.12 18.8 3.7 x 10 3.42 14.7 5.2 x l O 5.08 14.1 8.1 x l O 0 0.06 43.3 9.7 x 101.92 52.5 1.4 x 106.01 57.6 7.0 x 108.02 62.2 1.6 x 10 media enriched with monomethylarsonic acid [MMA] • 0 0.03 29.8 6.7 x lO0.07 23.9 1.1 x 100.06 24.5 5.9 x 100.17 14.7 8.1x10-7 0 0.03 18.7 2.6x10-7 0.06 29.5 8.4 x 100.15 25.8 2.7 x 10" 0.27 23.5 3.8 x 10media enriched with dimethylarsinic acid [DMA] 0 0.08 75.9 6.7 x 10" 0.05 62.8 6.3 x 100 0.17 49.7 6.3 x 100.03 43.2 1.1 xlO0.02 21.3 2.8 x 100.07 25.9 8.3 x 10-8 -7 -7 -7  -7  6  6  5  7  -7  -7 -7  7  6  6  B  8  7 7  7  6  6  7  6  7  6  6  6  {2.1 x 10 } 95.3 65.4 81.0 90.7 {4.9 x 10 } 48.3 86.3 65.0 22.0 6  5  {2.1 x 106} 102.1 98.4 80.9 66.2 {3.6 x 10 } 62.7 106.0 114.5 76.9 5  {1.8 x 10 ) 127.1 120.9 118.3 103.1 {3.6 x 10 ) 98.5 135.1 180.2 155.1 6  5  {2.4 x 10 } 93.0 82.1 {4.1 xlO } 94.8 93.4 91.0 75.2 6  5  cone, in nagg dry algae, RSD = 9.4%; * * of As left in the media, RSD = 8.6% ; % of cell density of the control; cell densities of control culture in parenthesis, RSD = 2.2% -1  c  52  The residual arsenic concentration in the media was found by Hydride Generation Atomic Absorption (HGAA) as described in Section 3.2. In order to check if all arsenic species present in the media were reducible by Sodium borohydride, the residual media of some batch cultures was decomposed by UV-irradiation following the procedures developed by D o d d » 42  117  >  118  . The  arsenic concentrations before and after UV-irradiation were determined by H G A A (Table 3.3) and are found to be in good agreement with each other. Thus it is assumed that little or no non-reducible arsenic compounds are present in the culture media. Table 3.3  Arsenic Concentration (ppm) of the C u l t u r e M e d i a Before a n d After UV-Irradiation °  Species  As added /ppm  Isochrysis galbana  0 0.5 2.1 5.4 10.7 0 0.5 1.1 5.4 10.7  Dunaliella tertiolecta  Isochrysis galbana  0 6 8 0 5 7  Dunaliella tertiolecta  As left in the media / ppm before irradiation after irradiation media enriched with [As(V)J 0 0.4 ± 0.01 1.6 ± 0.08 4.3 ± 0.3 9.0 ± 0.5 0 0.4 ± 0.01 1.0 ± 0 . 1 4.3 ± 0 . 3 9.2 ± 0.5 media enriched with [As(III)] 0 5.1 ± 0.4 6.9 ± 0.5 0 2.1 ± 0 . 1 3.6 ± 0.2  0 0.4 1.7 4.6 9.4 0 0.4 1.0 4.4 9.5 0 5.5 7.2 0 2.4 2.9  ± ± ± ±  0.01 0.1 0.3 0.5  ± 0.01 ±0.1 ± 0.3 ± 0.6 ± 0.3 ± 0.3 ±0.1 ±0.1  ° The sample solutions (50 mL) in sealed quartz tubes (2.5 cm o.d.) were irradiated for 6 hours using a 450 W lamp (Hanovia). Hydrochloric acid (150 uL, 12M), Hydrogen peroxide (35 uL, 30%) and Borax (1.5 mL, 0.1M) were added to the sample solutions prior to irradiation ' . 117 118  53  The percentage of arsenic taken up from the media at different concentrations of arsenicals are shown in Table 3.2. For I. galbana,  uptake is  highest at the lowest concentration for all four arsenicals tested. In arsenate, arsenite and M M A enriched cultures, uptake ranges from 13 - 30%. The uptake in D M A cultures is much higher than all other arsenicals with 76% at 2 ppm and this may be due to high adsorption of the arsenical to the cells. Certainly, at these concentrations, D M A has little effect on cell growth. Cultures of D. tertiolecta in arsenate, M M A and D M A enriched media also show the highest uptake at the lowest concentration of arsenicals, the same trend as observed in 7. galbana.  However, when D. tertiolecta  is fed  with arsenite, the reverse behaviour is observed. There is a gradual increase in uptake from 43 to 62% when the arsenic concentration is raised from 0.5 to 7 ppm. The percentage of arsenic taken up from the medium may not give a clear indication of the actual amount of arsenic incorporated into the cells. A lower percentage  of uptake (e.g. 14% at 10.7 ppm arsenate) does not  necessarily imply a smaller amount of arsenic taken up by the cells than a higher uptake percentage  (e.g.  30%  at  0.5 ppm arsenate). A better  representation of this set of data would be in terms of weight of arsenic taken up per cell. These results could be compared with the uptake per gram of freeze dried cells found by direct acid digestion. Results from these representations show the same trend of arsenic uptake by I. galbana  except for the culture in the D M A enriched media  which showed a higher arsenic per cell at a higher concentration. For D. tertiolecta,  similar patterns of uptake are obtained for arsenite and M M A 54  enriched cultures with arsenate and D M A showing variance. Because the two representations are expected to show the same trend, any difference in uptake pattern probably reflects different sources of errors. Arsenic uptake per gram of dry cell is dependent on the dry weights which may include a large and variable contribution from salt. Arsenic uptake per cell is dependent on the cell densities which are in the order of magnitude of 10 6  small errors in the measurements will be magnified in the calculation. It has been suggested that arsenate, the predominant form of arsenic 37  in seawater, is taken up by algae because of its similarity in size and geometry to the essential phosphate. Arsenate may enter into the cells * 18  119  via the phosphate transport system. Studies of the accumulation of arsenic in marine algal cultures '  27 77  and m a c r o a l g a e » 52  120  have shown that all species of  phytoplankton are not equally sensitive to arsenicals; some are inhibited at levels just exceeding ambient concentration while others are resistant to arsenic concentrations, a few orders of magnitude higher. Two possible strategies have been proposed ' * 5  121  122  by which algal  species may cope with arsenate stress. Some algae have a higher affinity for phosphate over arsenate. The selective uptake mechanism result in the exclusion of arsenate from the cell. Alternatively, the algal species are capable of processing incorporated arsenate by transforming it either into a harmless form that can be retained by the cells or into an altered  form .  123 124  that can be excreted from the cells. Because these transformations require energy, cell growth may be inhibited, but the species can continue to grow at lowered cell densities.  55  3.4.3  Arsenic Speciation i n the C u l t u r e M e d i a The chemical forms of arsenic in the culture media at the stationary  phase were studied by using Hydride Generation-Gas ChromatographyAtomic Absorption (HG-GC-AA). It was found that the species of arsenic did not change greatly during the experiment. The majority of arsenic remained in the form initially inoculated. In the control media, only background amounts of arsenate were detected, indicating that speciation changes due to bacterial intervention are minimal. However, arsenite was found to be present in the residual culture media that were originally inoculated with arsenate, showing that both algal species are capable of reducing arsenate. This result is in agreement with the significant reduction of arsenate to arsenite that has been observed during phytoplankton blooms  123  in seawater.  Similarly, a positive correlation has been established between primary production in the photic zone and levels of dissolved arsenite . No speciation 22  changes were found to occur in the media of the M M A and D M A enriched cultures.  56  3.4.4  Whole C e l l  1  H and  1 3  C N u c l e a r Magnetic Resonance (NMR)  Spectroscopy of M a r i n e Algae Whole cell N M R spectroscopy has been used increasingly for the study of biological fluids and whole cell suspensions * . Compared with other 125  126  analytical methods, it is non-destructive and it allows various metabolites and delicately balanced chemical and cellular processes to be observed. Detailed metabolic information is also available from N M R studies of the sensitive and ubiquitous H nucleus. Such measurements rely on the spectral 1  simplification achieved by applying spin-echo techniques. Whole cell spin echo H N M R spectroscopy has recently been applied 1  to  cell suspension cultures  of Catharanthus  roseus, the  Madagascar  periwinkle by Cullen et a l . They report a time-course variation in proton 8 1  resonance signals when the cells are fed with monomethylarsonic acid. Furthermore, the appearance of the new resonance at 8 1.87 ppm which may be due to the presence of dimethylarsinate supports the case for in vivo methylation by the cells. Therefore, it was of interest to see if similar effects could be observed in cell cultures of /. galbana and D. tertiolecta. The whole cell * H N M R spectra of 7. galbana and D. tertiolecta at the stationary phase were measured by using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (90 °-(x-180 -T) -acquisition) (Figure 2.1). x  o  y  2  Two  approaches have been used to observe the effects of different arsenic species on the whole cell spin echo H N M R spectra of J. galbana and D. tertiolecta. X  The first approach involved the recording of spectra of cells cultured in different arsenic enriched media. Comparison was made with their respective control cultures. In the second approach, the methylated arsenical in 57  deuterium oxide was introduced directly into the N M R tube containing a control batch of cells (0.5 mg per 500 mL of cell culture). The * H NMR spectra were recorded over a period of 6 hours to see if there were any significant changes. Algal cells contain a large amount of water and this makes their study by H N M R spectroscopy difficult without the use of some kind of water 1  suppression technique. This was achieved by setting the decoupler at the water resonance prior to acquisition. The intensity of the water signal was substantially reduced, allowing components present at low concentration to be observed. A water suppressed *R N M R spectrum of I. galbana  is shown in  Figure 3.13A. The cells were washed three times with deuterium oxide and the chemical shift is referenced to TMS. The resonances in the spectrum have not been assigned. At this point, the * H NMR spectrum is still complex. There is a broad envelope  of  overlapping resonances  with  much  weaker  resonances  superimposed on it. By applying the C P M G pulse sequence ' , significant 127 128  resolution enhancement can be achieved as illustrated in Figure 3.13B. By choosing the time between the 90° pulse and acquisition of the free induction decay, there would be a selective elimination of signals from resonances having the spin-spin relaxation times less than 4x. Specifically, the shorter the spin-spin relaxation time, the faster the rate of decay. The effective spinspin relaxation time, T resonance ( T  2  2  , for a particular proton is related to the width of its  = I/TTW^, where w ^ is the width in Hertz of the resonance at  half height). Thus broad resonances can be selectively removed by delaying acquisition until their transverse magnetization has decayed to a sufficiently 58  small value. The spin-spin relaxation time depends largely on molecular size and decreases as molecular size increases; thus, resonances from proteins and other macromolecules can selectively be eliminated. The rl N M R spectra of whole cells I. galbana l  and D. tertiolecta  at  their stationary phase were recorded. It was found that the H N M R spectra 1  of /. galbana  were substantially independent of arsenical concentration or  species for arsenate, arsenite and M M A . The greatest difference was found from cells grown in D M A and this is shown in Figure 3.14. The whole cell ^-H N M R spectrum of  D. tertiolecta  is shown in  Figure 3.15A. 500 scans were required to acquire this spectrum compared with the 320 scans used for the spectrum of Figure 3.14B. It was observed that cultures grown in the arsenicals tested showed H N M R spectra similar 1  to those of the control culture. A n example is shown in Figure 3.15B. The  ^  N M R spectrum of cells grown in M M A show close resemblance to the control.The spectrum is dominated by resonances at 5 3.5 - 3.7 ppm which are probably signals from glycerol. Glycerol is known to be the osmoregulatory solute in a number of Dunaliella  major  species - . 129  131  A major difference in the * H NMR spectrum is observed when the cells are inoculated with M M A five days after transfer into a new growing media. Cells were harvested six days later when they were at the stationary phase and their H N M R was recorded i n deuterium oxide (Figure 3.15C). A new 1  resonance appears at 5 2.2 ppm. Assignment has not been made but this may be due to the proton resonances from M M A (8 1.4 ppm in D 0 ) whose 2  chemical environment changes when taken up by the cells or the production of new compounds. The biochemical effects of M M A on D. tertiolecta 59  seem to  be different at different stages of the life cycle. D M A treated cells do not show this uptake. Although results from whole cell *H. N M R spectroscopy do not indicate substantial uptake and or methylation of arsenicals, these processes have been demonstrated from other acid digestion and filtrate analysis. The concentrations of methylated arsenicals if produced or taken up at all may be too low to give a satisfactory signal in a ^  N M R spectrum. Alternatively, the  arsenicals taken up by the algal cell suspension could be associated or bonded to some large molecules i n the cells, and would not be observed under the given experimental conditions because of fast relaxation. The natural abundance normal pulse  1 3  C N M R of whole cells  I. galbana and D. tertiolecta were also studied. I. galbana gives a very noisy N M R spectrum (Figure 3.16) with very broad signals at about 8 25 ppm, 70 ppm and 129 ppm after acquiring signals for 12 hours. The poor signal to noise ratio reflects partly the inherent low abundance and sensitivity for detection of the  1 3  C nuclear spins. Better quality spectra might be obtained  by using a spectrometer operating at a higher magnetic field strength. The use of larger diameter tubes as well as larger number of scans would be advantageous. On the other hand, intact cells of D. tertiolecta yield a natural abundance  1 3  C N M R spectrum  dominated by resonances  of glycerol  (8 62.7 ppm(s), 72.3 ppm(s)) as shown i n Figure 3.17A. A good signal to noise ratio was obtained with a reasonably short accumulation time (~3 hours). This is particularly interesting because, as mentioned above, glycerol is the main osmoregulatory solute present at high concentrations in the c e l l s  130  '  131  .  Similar type of spectra has been observed from two other marine unicellular 60  algae, Dunaliella  salina  and Synechococcus  sp. > 129  132  ;  1 3  C spectrum show  principally signals from their respective major osmoregulatory solute. Although the high concentrations of osmoregulatory solutes from marine algae can facilitate their study by natural abundance will  hamper  attempts  to  study  osmoregulation unless specific natural abundance  1 8  1 3  other  metabolites  1 3  not  C NMR, they involved in  C enrichment of the latter is employed. The  C spectrum of D. tertiolecta  grown in the M M A and  D M A are shown in Figure 3.17. In addition to the glycerol resonances, multiplets are present near 25 ppm and 128 ppm. (These are not present in the spectra of D. salina .) 132  The pattern of these multiplets is different from  the spectrum of the control culture presumably as a result of the different growth conditions. However, little can be said about the origin of these effects, mterestingly, two broad signals at these positions were also present in the spectrum of  I. galbana  culture; some common cell metabolites in  unicellular algae may be responsible for these peaks.  61  A.  6.0  B.  5.0  4.0  .0  2.0  1.0  6.0 F i g u r e 3.13  / PPM  / PPM  H N M R spectra of / . galbana washed i n D 0 A. The spectrum is the normal water-suppressed spectrum of the sample; B. The spectrum is obtained with the C P M G spin echo sequence using a x of 0.030 s; number of scans = 320.  1  2  62  A.  B.  1  •  5  3.8  '  i  '  F i g u r e 3.14  J  3.4  •  1  •  J  3.0  •  1  •  1  2.6  -  1  .  ,  2.2  .  1  ,  ,  ,  1  1.8  400 M H z * H s p i n echo N M R spectra of I. galbana A. control; B. culture fed with D M A (20 ppm)  63  •  ,  1.4  ,  1  ,  / PPM  A,  B.  C.  4. C F i g u r e 3.15  2.4  0.6  / PPM  400 M H z * H spin echo N M R spectra of D. tertiolecta A. control; B. culture fed with M M A (10 ppm) on Day 0; C. culture fed with M M A (10 ppm) on Day 5  64  F i g u r e 8.16  75 M H z  1 8  C N M R spectrum of I.  65  galbana  CN  CJ IT-  c.  ™ i  F i g u r e 8.17  75 M H z C N M R spectra of D. tertiolecta A. control; B . culture fed with M M A (10 ppm); C. culture fed with D M A (20 ppm). 1 S  66  Uptake of M M A a n d D M A b y the A l g a l Cells at Stationary Phase  The uptake of M M A and D M A by I. galbana  is studied by following  the time-course variation of VS. N M R after addition of the arsenicals. A spectra of the control algal cell suspensions was recorded prior to the addition of the methylated arsenical into the N M R tube. Data were then accumulated (-15 minutes per spectrum) for up to 6 hours. A n example of time course study is shown in Figure 3.18. A peak at 5 1.8 ppm appeared after the addition of M M A and is accompanied by other changes especially to the intense resonances at 5 2.95 ppm and 3.25 ppm. The upfield resonance is probably associated with the arsenic-methyl group and further changes of spectra with time are small. It is hard to establish whether this peak and the others can be attributed to protons inside or outside the cells because cell lysis is bound to occur especially when the cells are subjected to stress for a relatively long period of time. Uptake studies with D M A show an additional peak at 5 1.8 ppm after the arsenical was added. Again, this peak can probably be associated with the arsenic-methyl group. The intensity of this peak does not change significantly after 6 hours. No other spectral changes were observed in this time course study. The time-course study of D. tertiolecta  after the addition of M M A is  shown in Figure 3.19. Spectral changes are seen after 1/2 hour and no further changes of any significance take place. The peak at 8 1.8 ppm can probably be assigned to the methyl-arsenic protons and changes in intensities of other peaks are apparent. Similar variations were observed after addition of DMA: 67  little can be said in the absence of specific assignments.  68  A. Control  B . after 1/2 h o u r  C . after 6 hours  —I—•— —'—r1  3.8  F i g u r e S.18  3.4  I  3.0  '  1  '  !  2.6  —I—  2.2  1 .8  T h e variation of the * H N M R spectrum of I. galbana the presence of M M A  69  / PPM  1.4  in  A . Control  B . after 1/2 h o u r  C . after 6 hours  A . 3.6 F i g u r e 3.19  3.0  -1—"  2.4  1 • 8.  1.2  / PPM  T h e variation of the *R N M R spectrum of D. tertiolecta i n the presence of M M A  70  3.4.5  Isolation D.  and  Identification  of  Arsenic  Compounds  in  tertiolecta  Previous work on Dunaliella  sp. has shown that the marine algae are  capable of accumulating arsenic . Accumulation was found to be dependent 111  on the physical  112  and chemical environment ' . Wrench and Addison 113  133  studied the metabolism of arsenate by D. tertiolecta 74  78  by using the isotope  A s . They found that complex organoarsenic compounds were rapidly formed  after addition of the labelled arsenate. Three polar arsenic metabolites were isolated by T L C and identified as arsenite, monomethylarsonic acid and dimethylarsinic acid. A large fraction of assimilated arsenic appeared in the lipid fractions but the exact chemical forms were not determined. The objective of the present study was to examine the properties of arsenic compound(s) present i n D. tertiolecta  after growing the alga in media  enriched with arsenicals. Five litre cultures of D. tertiolecta were grown in media enriched with arsenate, arsenite, M M A and D M A at an arsenic concentration of 2 ppm. Cells were recovered from the media 10 days after inoculation by filtering through a glass fibre filter. The algae were washed with filtered seawater prior to extraction  with chloroform/methanol/water  (8:4:3, v/v/v). After  separation, the chloroform layer was removed and dried under vacuum. The total arsenic present in the chloroform layer was then found by acid digestion followed by H G A A . Results (Table 3.4) showed that cells grown in arsenate contain higher proportions of arsenic in the organic extracts; 60.5% of the total arsenic incorporated are in organic forms. Cells grown in arsenite, M M A and D M A contain similar proportions of arsenic in the lipid layers and these 71  range from 31.0 to 45.5%. It seems likely that different species of arsenicals in the media affect the forms of arsenic incorporated in D.  Table 3.4  A r s e n i c (ug) found i n Extracts of D. tertiolecta C u l t u r e d i n Different Arsenicals  Arsenic Arsenic found in inoculated CHC1 layer /ug 0  Arsenate Arsenite MMA DMA 0  tertiolecta.  3  0.33 0.71 0.14 0.23  ± 0.03 ± 0.07 ±0.01 ±0.02  Arsenic found in H 0 layer /ug 2  0.22 0.85 0.32 0.36  ±0.02 ± 0.08 ± 0.03 ±0.04  % of Total Arsenic i n the organic extract 60.5 45.5 31.0 39.5  Concentration of arsenic inoculated is 2 ppm.  The above results show that the total amount of arsenic extracted and subsequently detected by H G A A is more from arsenite enriched media than from arsenate or any of the methylated species. These results can be compared with those previously found by acid digestion (Table 3.2) of cells cultured in arsenicals of the same concentration range. Cells grown in the methylated arsenicals show the same trend from both sets of experiments uptake of DMA is found to be slightly higher than that of MMA. However, the total arsenic was found to be higher in extracts of cells cultured in arsenite than in arsenate whereas higher incorporation in arsenate was found from cell digestion studies. This probably indicates that not all the arsenic present in the cells is extractable and reducible.  72  P u r i f i c a t i o n of C r u d e Extract b y G e l Permeation Chromatography  With gel permeation chromatography, separation of molecules was achieved according to size. Substances are eluted from the column of Sephadex LH-20 i n order of decreasing molecular size. This column is particularly useful to clean up marine samples because desalting can be attained at the same time, where salts are retained on top of the column. A chromatogram of the water soluble extract of cells cultured in arsenate is shown in Figure 3.20.  Only one arsenic containing band at a retention  volume of 18 mL was eluted with water as eluent. A white solid was obtained after removal of the solvent and this was re-crystallized from water. The * H N M R spectrum of the crystals is shown in Figure 3.21. 0.12  i 20  i  30  i 40  50  60  retention volume / mL F i g u r e 3.20  L i q u i d chromatogram of water soluble extract of D. tertiolecta  73  MM  • i • ' ' • i ' 10.0 9.0  1  I ' ' ' B.O  ' I 7.0  1  1  1  1  I 6.0  1  '  1  1  I ' ' 5.0  1  1  P P M  F i g u r e 8.21  I ' ' ' ' 4.0  I ' 3.0  i  I i i i i I i i i i I i i i i I 2.0 1.0 0.0  H N M R of water soluble arsenic compound isolated f r o m D. tertiolecta cultured i n arsenate e n r i c h e d media a n d purified by Sephadex LH-20  1  74  The peaks at 2 ppm(s) and 3.6 ppm(m) are peaks found in the * H N M R spectrum of the water extract of control cultures. Other peaks in the * H N M R spectrum of the purified water extract may be due to the arsenic containing metabolite(s) of the alga. Arsenic analysis of this solid by G F A A gives an arsenic content of 18.8%. However, examination of the solid under a microscope revealed the presence of two types of crystals. The F A B mass spectrum of this solid has major peaks at m/z 482,419,257, 235, 220, 205. The same purification procedures were repeated for cultures grown in arsenite and M M A enriched media. After passing the water soluble cell extracts through a Sephadex LH-20 column, an arsenic containing band was eluted. This has the same elution volume as that obtained from arsenate enriched culture. The solid isolated from arsenite enriched media was recrystallized and was found to contain 22.3% arsenic by GFAA. The H N M R X  spectrum of this solid is the same as that of the solid isolated from cells grown in arsenate enriched media. The major peaks of FAB mass spectrum are m/z 482, 419, 260, 235, 220 and 205. This probably indicates that cells grown i n arsenate and arsenite enriched media incorporate the same form of water soluble arsenic compound(s) in the cells. The amount of arsenic containing solid isolated from the M M A culture was too small for any further analysis.  75  P u r i f i c a t i o n of C r u d e Extract b y H P L C - G F A A  H P L C - G F A A has been successfully used for the separation and quantitation of arsenite, arsenate, methylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine and tetramethylarsonium salt in environmental samples . Attempts were made to purify the arsenic cOmpound(s) present in 41  the water soluble extract of D. tertiolecta by using the same methodology. A Waters u-Bondapak C  1 8  reversed phase column was used in the  H P L C sytem. This consists of a non-polar stationary phase of a layer of octadecyl-chains bonded to silica gel (10 um). Water/methanol (95:5, v/v) was used as the polar mobile phase. The elution conditions employed by Dodd  41  were followed. Separations on a reversed phase column was based on the the relative hydrophobicity of compounds where the most polar compounds are eluted first. The separation of ionic species can be improved by addition of a suitable lipophilic counterion to produce a hydrophobic ion-pair that is retained more strongly by the stationary phase . Tetrabutylammonium nitrate (TBAN) is 42  used as the ion-pairing reagent in this study. The H P L C chromatogram of the crude water extract was monitored by G F A A Spectrometry and is shown in Figure 3.22. It was observed that four peaks were eluted at retention volumes of 2, 17, 21 and 45 mL. Peaks at 17 and 45 mL correspond to the retention volumes of standard arsenite and arsenate respectively. The presence of these is not unexpected because arsenate inoculated into the media is likely to be adsorbed and reduced by D. tertiolecta  cells. The peaks at retention volumes 2 mL and 21 mL do not  correspond with any of the standard arsenic compounds available and 76  therefore remain unassigned. Further isolation of these compounds is difficult because of the presence of high concentration of the T B A counterion.  retention volume / m L  F i g u r e 3.22  H P L C - G F A A chromatogram of water soluble extract of D. tertiolecta  11  3.5  SUMMARY The uptake of arsenicals (As(V), As(IH), M M A and DMA) by the  marine algae, Isochrysis galbana  and Dunaliella  tertiolecta was monitored  by using Hydride Generation Atomic Absorption (HGAA) Spectrometry and Graphite Furnace Atomic Absorption (GFAA) Spectrometry. The effects of arsenicals on growth were monitored by in vivo fluorescence and cell counting measurements. Significant arsenic uptake and incorporation are found when both algae are subjected to elevated levels of arsenicals. I. galbana  show a higher  uptake of As(LTI) accompanied by a greater inhibition of growth. The following order of incorporation is found (the range of arsenic uptake per gram of freeze dried cells are shown in parenthesis): As(III)  >  (2-5 mgg- ) 1  I. galbana  As(V)  >  (0.3-3.6 mg g-i)  MMA  »  (0.03-0.2 mg g-i)  DMA  (0.05-0.08 mg g-i)  seems to be able to adapt to higher As(V) concentrations  (10.7 ppm). Growth of D. tertiolecta  is most affected by elevated levels of As(V)  (10.7 ppm) where a high uptake is observed. Water soluble extracts of D. tertiolecta  cultured i n As(V) contain at least four arsenicals, which  probably include As(V) and As(ni). The arsenic uptake is in the order:  As(V) > As(in) > (3.2-7.3 mgg-i) (0.06-8 mg g-i)  MMA « (0.03-0.2 mg g-i)  DMA (0.02-0.2 mg r ) 1  In all M M A treated cultures, there is a stimulation of cell densities above that of the control. In vivo fluorescence is also stimulated over the  78  course of the experiment. Methylated arsenicals such as D M A do not affect growth of both algae at the levels examined. 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