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Arsenic transformation in marine macroalgae Granchinho, Sophia Catarina Reineke 2000

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ARSENIC T R A N S F O R M A T I O N IN M A R I N E M A C R O A L G A E by S O P H I A C A T A R I N A R E I N E K E G R A N C H I N H O B.Sc., The University of British Columbia, 1998 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A August 2000 © Sophia Catarina Reineke Granchinho, 2000 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t 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 a gree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u rposes may be g r a n t e d by t h e head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t 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 f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C olumbia Vancouver, Canada Date al Septemher ^ooo ABSTRACT A high performance liquid chromatography-inductively coupled plasma-mass spectrometry ( H P L C - I C P - M S ) system was utilized for the determination of arsenate (As(V)), arsenite (As(III)), monomethylarsonate ( M M A ) , dimethylarsinate ( D M A ) and arsenosugars X -XIII. In marine algae and in a marine fungus, the system was used to study pathways for the biotransformation of arsenicals. Fucus gardneri, Nereocystis luetkeana and Fusarium oxysporum melonis were grown in media enriched with arsenicals. Arsenosugars in the algae were identified by comparing the retention times with the organoarsenic compounds previously identified in oyster tissue standard reference material (NIST-1566a) and Fucus sample standard ( IAEA-140/TM) . Two H P L C columns and two mobile phase conditions were used. Arsenate, when added to Fucus gardneri under different environmental conditions, was reduced to As(III) and methylated to D M A and several different arsenosugars. The amount of arsenic species varied depending on the environmental condition. High levels of salinity and high levels of phosphate resulted in lower amounts of the arsenic species compared to low levels of salinity and phosphate. The presence or absence of antibiotics in the medium did not result in any major changes in the amount of arsenic species produced. This may indicate that the presence of more complex arsenicals in environmental algae samples is dependent on symbiotic interactions between the algae and its surroundings, rather than resulting from independent synthesis by the algae. The bull kelp Nereocystis luetkeana reduced and methylated As (V) to D M A . Significant amounts of more complex arsenic species, such as arsenosugars, were not observed in the cells i i or medium. The bioaccumulation and biomethylation of arsenic species by Nereocystis was found to be different than Fucus gardneri. The Fusarium fungus, which grows with Fucus gardneri in a parasitic relationship produces arsenite and D M A when incubated with arsenate. The amounts, however, were about 1000 times lower than produced by Fucus. Water soluble arsenic species were determined in a variety of marine algae collected from two sampling areas in British Columbia. Arsenate, M M A and arsenosugars X , XII and XIII were the predominant species extracted from the samples. The total arsenic content was determined by I C P - M S and the water content was determined by freeze-drying for all the samples collected. It was found that the kelp samples contained the highest amounts of total arsenic as well as the highest water content. The extraction efficiency varied between samples. i i i T A B L E OF CONTENTS Abstract i i Table of Contents iv List of Tables ix List o f Figures x i List o f Abbreviations x i i i Acknowledgments xiv Chapter 1. G E N E R A L I N T R O D U C T I O N 1 1.1 A R S E N I C I N T H E M A R I N E E N V I R O N M E N T 2 1.2 T O X I C I T Y O F A R S E N I C 4 1.3 M A R I N E A L G A E 6 1.3.1 General Information 6 1.3.2 Commercial Use of Marine Algae 7 1.3.3 Arsenosugars in Marine Algae 8 1.3.4 Previous Studies of Arsenic Transformation in Marine Algae 11 1.4 F U N G I 13 1.5 O B J E C T I V E OF P R E S E N T W O R K 14 Chapter 2. E X P E R I M E N T A L M E T H O D S F O R T H E A N A L Y S I S OF A R S E N I C 15 2.1 I N S T R U M E N T A T I O N 15 2.1.1 High Performance Liquid Chromatography (HPLC) 15 2.1.1.1 Apparatus 15 2.1.1.2 Conditions 16 2.1.2 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 17 iv 2.2 G E N E R A L C U L T U R E M A I N T E N A N C E 19 2.2.1 Conviron Environmental Chamber (Model C M P 3023) 19 2.2.2 Reagents and Chemicals 19 2.2.3 Medium and Antibiotics 20 2.3 S A M P L E P R E P A R A T I O N F O R A N A L Y T I C A L A N A L Y S I S 23 2.3.1 Sample Storage 23 2.3.2 Extraction 23 2.3.3 Acid Digestion of Samples 24 2.4 S P E C I A T I O N OF S T A N D A R D C O M P O U N D S B Y H P L C - I C P - M S 25 2.4.1 Standard Reference Materials for Ion-Pairing HPLC-ICP-MS Condition 25 2.4.2 Analytical Standards for Ion-Pairing HPLC-ICP-MS Condition 28 2.4.3 Analytical Standards for Anion-Exchange HPLC-ICP-MS Condition 30 2.4.4 Analytical Standards for Total Analysis by ICP-MS 32 Chapter 3. T H E B I O M E T H Y L A T I O N A N D B I O A C C U M U L A T I O N O F A R S E N I C A L S B Y M A R I N E A L G A E FUCUS GARDNERI U N D E R D I F F E R E N T E N V I R O N M E N T A L C O N D I T I O N S 33 3.1 I N T R O D U C T I O N 33 3.2 S A M P L E C O L L E C T I O N A N D T R E A T M E N T 36 3.2.1 Sample Collection 36 3.2.2 Sample Treatment 36 3.2.2.1 Treatment of Fucus gardneri before the Acclimation Period 36 3.2.2.2 Treatment of Fucus gardneri during the Acclimation and Exposure Periods ... 38 3.2.2.2.1 Fucus gardneri collected October 1998, acclimated in seawater or artificial seawater, and exposed to As(V) in seawater or artificial seawater... 38 3.2.2.2.2 Fucus gardneri collected February 1999, acclimated in seawater, and exposed to As (V) in artificial seawater 40 3.2.2.2.3 Fucus gardneri collected October 1999, acclimated in artificial seawater, and exposed to As(V) in artificial seawater at different phosphate levels 41 3.2.2.2.4 Fucus gardneri collected June 1999 and acclimated in seawater in the absence of antibiotics 42 3.2.2.2.5 Fucus gardneri collected August 1999, acclimated in seawater, and exposed to As (V) in seawater in the absence of antibiotics 43 3.2.3 Sample Preparation 44 3.2.4 Culture and Medium Conditions 44 3.3 R E S U L T S OF E X P O S U R E OF FUCUS GARDNERI T O A R S E N I C ( V ) I N D I F F E R E N T M E D I A 46 3.3.1 Introduction 46 3.3.2 Results from Fucus gardneri Acclimated in Seawater or Artificial Seawater, and Exposed to Arsenic(V) in Seawater or Artificial Seawater 47 3.3.2.1 Acclimation Period 47 3.3.2.2 Exposure Period 49 3.3.3 Results from Fucus gardneri Acclimated in Seawater and Exposed to Arsenic(V) in Artificial Seawater 56 3.3.3.1 Acclimation Period 56 3.3.3.2 Exposure Period 57 3.4 R E S U L T S OF E X P O S U R E OF FUCUS GARDNERI T O A R S E N I C ( V ) IN D I F F E R E N T P H O S P H A T E C O N C E N T R A T I O N M E D I A 62 3.4.1 Introduction 62 3.4.2 Results and Discussion 63 3.4.2.1 Acclimation Period 63 3.4.2.2 Exposure Period 65 3.5 R E S U L T S OF E X P O S U R E OF FUCUS GARDNERI T O A R S E N I C ( V ) IN T H E A B S E N C E O F A N T I B I O T I C S 71 3.5.1 Introduction 71 3.5.2 Results and Discussion 72 3.5.2.1 Acclimation Period 72 3.5.2.2 Exposure Period 73 3.6 S U M M A R Y 77 Chapter 4. B I O M E T H Y L A T I O N A N D B I O A C C U M U L A T I O N O F A R S E N I C A L S B Y M A R I N E A L G A E NEREOCYSTISLUETKEANA 79 4.1 I N T R O D U C T I O N 79 4.2 S A M P L E C O L L E C T I O N A N D T R E A T M E N T 81 4.2.1 Sample Collection 81 4.2.2 Sample Treatment 81 4.2.2.1 Treatment of Nereocystis luetkeana before the Acclimation Period 81 4.2.2.2 Treatment of Nereocystis luetkeana during the Acclimation and Exposure Periods ; 82 4.2.3 Sample Preparation 83 4.2.4 Culture and Medium Conditions 84 4.3 R E S U L T S A N D D I S C U S S I O N 86 4.3.1 Acclimation Period 86 4.3.2 Exposure Period 89 4.4 S U M M A R Y 93 Chapter 5. A R S E N I C T R A N S F O R M A T I O N B Y T H E F U N G U S FUSARIUM OXYSPORUM M E L O N I S 95 5.1 I N T R O D U C T I O N 95 5.2 S A M P L E C O L L E C T I O N A N D T R E A T M E N T 97 5.2.1 Agar Conditions and Treatment of Fusarium oxysporum melonis before the Exposure Period 97 5.2.2 Treatment of Fusarium oxysporum melonis during the Exposure Period 98 5.2.3 Sample Preparation 99 5.2.4 Culture and Medium Conditions 99 5.3 R E S U L T S A N D D I S C U S S I O N 101 5.3.1 Analysis of the Extracts of the Fusarium oxysporum melonis after the Exposure Experiments 101 5.3.1.1 Results after Exposure to As(V) 101 5.3.1.2 Results after Exposure to D M A 103 5.3.2 Analysis of the Culture Media from the Exposure Experiments 103 5.3.2.1 Results of the Medium Collected after Exposure to As (V) 103 5.3.2.2 Results of the Medium Collected after Exposure to D M A 105 5.4 S U M M A R Y 106 v i i Chapter 6. A R S E N I C S P E C I A T I O N OF D I F F E R E N T M A R I N E A L G A E F O U N D IN T H E B R I T I S H C O L U M B I A C O A S T 107 6.1 I N T R O D U C T I O N 107 6.2 W A T E R C O N T E N T 109 6.3 A R S E N I C S P E C I A T I O N 111 6.4 A S S E S S M E N T O F A C C U R A C Y OF E X T R A C T I O N M E T H O D 113 Chapter 7. S U M M A R Y A N D F U T U R E C O N S I D E R A T I O N S 116 B I B L I O G R A P H Y 119 v i i i LIST OF TABLES Table 1.1 Arsenic Species found in the Marine Environment 3 Table 1.2 Arsenosugars found in some common Marine Algae 8 Table 2.1 Summary of Experimental H P L C Conditions 16 Table 2.2 Operating Parameters of I C P - M S 18 Table 2.3 Operating Parameters for the Conviron Environmental Chamber 19 Table 2.4 Contents of Artificial Seawater, A S P 6 F2 21 Table 2.5 A 3 Antibiotics for Axenation of Bacteria and Fungus 22 Table 3.1 Medium Conditions for the Exposure Period for Fucus gardneri collected October 1998 39 Table 3.2 Medium Conditions for the Exposure Period for Fucus gardneri collected February 1999 40 Table 3.3 Medium Conditions for the Acclimation Period for Fucus gardneri collected October 1999 . 41 Table 3.4 Medium Conditions for the Exposure Period for Fucus gardneri collected October 1999 42 Table 3.5 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) following Acclimation and As(V) Exposure in Seawater or Artificial Seawater 52 Table 3.6 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) following Acclimation in Seawater and Exposure to As (V) in Artificial Seawater 59 Table 3.7 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after Acclimation in different Phosphate Concentration Media 65 Table 3.8 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after As (V) Exposure in different Phosphate Concentration Media 67 Table 3.9 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after Acclimation in Seawater with no added Antibiotics 73 Table 3.10 Arsenic Speciation of Seawater Samples (ppb, wet weight) and Fucus gardneri Extract Samples (ppm, dry weight) after As(V) Exposure in the absence of Antibiotics 75 ix Table 4.1 Medium Conditions for the Exposure Period for Nereocystis luetkeana 83 Table 4.2 Arsenic Speciation of Nereocystis luetkeana Extracts (ppm, dry weight) before and after Acclimation in Seawater 88 Table 4.3 Arsenic Speciation of Nereocystis luetkeana Extracts (ppm, dry weight) after As (V) Exposure in A S P 6 F2 Medium 91 Table 5.1 Medium Conditions for Fusarium oxysporum melonis 98 Table 5.2 Arsenic Speciation of Fusarium oxysporum melonis Extracts after As (V) Exposure (ppb, wet weight) 102 Table 6.1 Water Content of Marine Algae from British Columbia Coast 110 Table 6.2 Relative Amounts of Arsenicals found in some Marine Algae (ppm, dry weight).... 112 Table 6.3 Determination of Extraction Efficiency by using Total Digestion and Determination of Detection Efficiency by using the Extraction Method 114 x LIST OF FIGURES Figure 1.1 Proposed Pathway for the Biogenesis of Arsenosugars in Marine Algae 10 Figure 2.1 Schematic Diagram of I C P - M S ( V G Elemental, Fisons Instrument) 18 Figure 2.2 Oyster Tissue S R M Standard 26 Figure 2.3 Fucus Sample Standard 27 Figure 2.4 Kelp Powder Standard 27 Figure 2.5 Five Arsenic Standards 28 Figure 2.6 Arsenate Calibration Curve for Ion-Pairing H P L C - I C P - M S 29 Figure 2.7 Standard Solution of Four Arsenic Compounds 30 Figure 2.8 Arsenate Calibration Curve for Anion-Exchange H P L C - I C P - M S 31 Figure 2.9 Arsenate Calibration Curve for Total Digestion 32 Figure 3.1 Fucus gardneri Si lva. . . . 35 Figure 3.2 Map of Collection Sites 35 Figure 3.3 Fucus Extract before and after 14 days of Acclimation in Seawater 48 Figure 3.4 Fucus Extract before and after 14 days of Acclimation in A S P 6 F2 Medium 48 Figure 3.5 Fucus gardneri Exposed to As (V) in Seawater Medium 50 Figure 3.6 Controls for the Exposure Experiment 50 Figure 3.7 Fucus gardneri Exposed to As (V) in A S P 6 F2 Medium 51 Figure 3.8 Seawater Samples collected during the Exposure of Fucus gardneri to Arsenic(V). . 53 Figure 3.9 A S P 6 F2 Medium Samples collected during the Exposure of Fucus gardneri to Arsenic(V) 54 Figure 3.10 Fucus Extracts before and after 14 days of Acclimation in Seawater 57 Figure 3.11 Fucus gardneri Exposed to Arsenic(V) in A S P 6 F2 Medium 58 xi Figure 3.12 A S P 6 F2 Medium Samples collected during the Exposure of Fucus gardneri to Arsenic(V) 60 Figure 3.13 Arsenic Species found in Fucus gardneri Extracts before and after 14 days of Acclimation 64 Figure 3.14 Arsenic(V) Speciation of Fucus gardneri Samples following Exposure in different Phosphate Concentration Media 66 Figure 3.15 Arsenate (As(V)) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 68 Figure 3.16 Arsenite (As(III)) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 68 Figure 3.17 Dimethylarsinate ( D M A ) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 69 Figure 4.1 Nereocystis luetkeana Postels and Ruprecht 80 Figure 4.2 Nereocystis luetkeana Bulb Extracts before and after 9 days of Acclimation in Seawater 87 Figure 4.3 Nereocystis luetkeana Blade Extracts before and after 9 days of Acclimation in Seawater 87 Figure 4.4 Nereocystis luetkeana Bulb Extracts after Exposure to As(V) in A S P 6 F2 Med ium. 89 Figure 4.5 Nereocystis luetkeana Blade Extracts after Exposure to As(V) in A S P 6 F2 Medium 90 Figure 4.6 A S P 6 F2 Medium Samples collected during the Exposure of Nereocystis luetkeana to Arsenic(V) 92 Figure 5.1 Micrograph of Fusarium oxysporum melonis 96 Figure 5.2 Fusarium oxysporum melonis Extracts after Exposure to Arsenic(V) in A S P 6 F2 Medium 102 Figure 5.3 Short Term Effects of the Fusarium oxysporum melonis Exposed to As (V) over 45 days 104 Figure 5.4 Long Term Effects of the Fusarium oxysporum melonis Exposed to As(V) over 45 days 105 xii LIST OF A B B R E V I A T I O N S A D P adenosine diphosphate A s B arsenobetaine A s C arsenocholine As(III) arsenite As(V) arsenate A T P adenosine triphosphate cps counts per second D M A dimethylarsinate, also dimethylarsinic acid H P L C high performance liquid chromatography I A E A International Atomic Energy Agency ICP inductively coupled plasma M M A monomethylarsonate, also monomethylarsonic acid M S mass spectrometry m/z mass to charge ratio NIST National Institute of Standards and Technology O D S octadecylsilica P D A potato dextrose agar P D B potato dextrose broth ppb parts per bill ion, ng/g or ng/ml ppm parts per mill ion, pg/g or pg/ml ppt parts per thousand, mg/g or mg/ml P T F E polytetrafluoroethylene (Teflon) r.f. radio frequency S A M S-adenosylmethionine Sc standard deviation from calibration curve sp. species S R M standard reference material T E A H tetraethylammonium hydroxide T M trace metals T R A time resolved analysis X - X I V arseno sugars X through X I V x i i i A C K N O W L E D G M E N T S I would like to give my deepest appreciation to my research supervisor Dr. W.R. Cullen for his guidance, advice, encouragement and financial support throughout the course of this project. I would also like to thank Dr. Elena Polishchuk for her time, patience and expert advice on growing and maintaining the cultures for this project. I 'm particularly grateful to Bert Mueller for the technical assistance in I C P - M S and to Catherine Franz for preparing the fungus samples for identification. I am indebted to my past and present colleagues for their continual help and useful discussions. They include Viv ian La i , Corinne Lehr, Bianca Kiupers, Iris Koch , Paul Andrewes, L i x i a Wang, Changqing Wang, Ulr ik Norum and Sarah Maillefer. I would also like to thank all the people from Biological Services for always keeping me in a cheerful mood. Finally, I would like to thank my sister and my parents for their daily support, love and encouragement. xiv 1 G E N E R A L I N T R O D U C T I O N Arsenic is an element that is most commonly associated with poison; therefore it may be a surprise to learn that it is present in elevated levels in many types of seafood and edible marine algae. In the environment arsenic is found in many types of mineral deposits, particularly those containing sulphides. It accompanies many metals including Cu , A g , A u , Pt and Fe; hence, arsenic is a good indicator in geochemical prospecting surveys for elements of commercial importance.1 Arsenic has mainly found uses as weapons and poisons from the Middle Ages to World War I, nonetheless some arsenical compounds have been found to have therapeutic uses in treating specific types of acute leukaemia. ' Even though arsenic is now being used for various applications other than poison, it is still found in the environment from extremely toxic to relatively non-toxic forms. Therefore it is important to determine how plants and animals in the environment take up arsenic, and how the arsenic is treated and transformed by them. 1 1.1 ARSENIC IN T H E M A R I N E E N V I R O N M E N T Arsenic is an ubiquitous element. It is found in the atmosphere, in the earth's crust, in rocks, in soils, in sediments, in organisms, in freshwater and in seawater, and even in interstellar gas.4 The sources of arsenic in the marine environment are both natural and anthropogenic. Natural sources of arsenic in the marine environment include weathering, volcanic activity, and soils and sediments.5 Some of the main anthropogenic sources are mine tailings; smelting of gold, silver, copper, and lead ores; fossil fuel combustion; herbicides and pesticides. 5 ' 6 In seawater, the arsenic concentrations typically range from 1.0 ppb to 8.0 ppb. ' The predominant species that is present in the ocean is arsenate [As(V)]; however, significant amounts of arsenite [As(III)], monomethylarsonic acid [ M M A ] and dimethylarsinic acid [ D M A ] have also been observed and are believed to be associated with the biological activity of marine algae. 9 ' 1 0 Marine animals and plants contain a higher and wider range of concentration levels of arsenic (0.78 to over 100 ppm). 1 1 The wide range of concentration levels depends on factors such as the species type, habitat and feeding habits. The most common non-toxic organoarsenic form found in marine animals is arsenobetaine.11 Arsenic-containing ribofuranosides (arsenosugars) were first identified in 1981 in the 12 13 • * brown macroalga Ecklonia radiata. ' Further investigations of other brown algae species, in addition to algae from other classes, revealed the existence of 15 arsenosugar compounds. 1 3 ' 1 4 ' 1 5 Because these compounds can occur at high concentrations (several mg As/kg wet weight) in marine organisms, including those used as human food, there is considerable interest regarding their toxicological behaviour. 2 Some common arsenic species that are found in the marine environment and in marine biological systems including marine algae are listed in Table 1.1. Table 1.1 Arsenic Species found in the Marine Environment Name Abbreviation Chemical Formula Arsenate As (V) Arsenite As(III) Monomethylarsonic acid M M A Dimethylarsinic acid D M A Trimethylarsine Me3As Arsenobetaine A s B Arsenocholine A s C Arsenosugars 3 X - X I V A s O ( O H ) 3 A s ( O H ) 3 C H 3 A s O ( O H ) 2 ( C H 3 ) 2 A s O ( O H ) A s ( C H 3 ) 3 ( C H 3 ) 3 A s + C H 2 C O O " ( C H 3 ) 3 A s + C H 2 C H 2 O H See figure below O X ( C H 3 ) — A s — C B x ^ O yP~ C H 2 C H C H 2 — Y X Y X - O H - O H X I - O H - O P 0 3 H C H 2 C H ( O H ) C H 2 O H XII - O H - S 0 3 H XIII - O H - O S 0 3 H X I V - N H 2 - S 0 3 H Numbering system (X-XIV) according to Shibata et al.15 3 1.2 T O X I C I T Y O F ARSENIC Arsenic is a Group 15 element and it is the 20 most abundant element in the earth's crust. 1 6 Arsenic is commonly found in the +5, +3 and 0 oxidation states. Arsenic is well known to be acutely toxic. It is also a carcinogen, long-term exposure to arsenic can cause skin, lung, liver and bladder cancer. 5 ' 1 7 The toxicity and carcinogenicity of arsenic depends on the chemical form of the arsenic species. Trivalent inorganic arsenic compounds are more acutely toxic than pentavalent inorganic arsenic compounds. Organometallic compounds in the +5 state are less toxic than inorganic ones and some compounds, such as arsenobetaine, are essentially non-toxic in all 5 18 systems tested. ' The dependence of toxicity on the chemical form of arsenic makes their identification necessary. The relative toxicity of arsenic compounds follows the general trend shown be low. 6 ' 1 9 ' 2 0 H3As>As2O3[As(III)]>(RAsO)n>As 2O 5[As(V)]>R nAsO(OH)3.n>R4As+>As(0) where R=alkyl or aryl n=l,2 Trivalent arsenic compounds, such as arsenite, have a high affinity for thiol groups (S-H) 21 22 23 and can interact with active sites of many enzymes. ' ' Arsenate inhibits A T P (adenosine triphosphate) synthesis by the formation of an unstable arsenate ester of A D P (adenosine diphosphate) instead of A T P . The formation of A T P may be inhibited by the formation of these 4 arsenate esters, which are not able to store energy like A T P . The energy of such an ester bond cannot be recovered metabol ica l ly . 5 ' 1 0 ' 2 1 ' 2 3 ' 2 4 ' 2 5 Organometallic compounds of arsenic (such as arsenosugars, arsenobetaine and arsenocholine) can occur in high concentration in the marine environment, including biota used for human food, but studies suggest that arsenosugars exhibit no cytotoxic or mutagenic effects.2 6 The one common feature of the organoarsenicals that are found in nature is that they contain methyl groups attached to the arsenic. It has been suggested that the formation of methylated • 77 arsenic compounds in marine organisms is a mechanism for the detoxification of arsenic. 5 1.3 M A R I N E A L G A E 1.3.1 General Information Marine algae, or seaweeds, are the oldest members of the plant kingdom, extending back many hundreds of millions of years. They have little tissue differentiation, no true vascular tissue, no roots, stems, leaves, or flowers and thus take their mineral salt directly from their surrounding medium, i.e. from the seawater. Algae range in size from microscopic individual cells to huge plants more than 100 feet (30 meters) long. Algae are commonly classified into a number of groups according to their colour: Chlorophyta are the green algae, Phaeophyta are the brown algae, Rhodophyta are the red algae and Cyanophyta (or Myxophyta) are the blue-green algae. Under the widely used five-kingdom classification system, the brown, red and green algae belong to the Protista kingdom. The blue-green algae belong to the kingdom Monera along with bacteria and viruses. Each species has its 30 31 characteristic photosynthetic pigments in the body. ' Zonation patterns within algal assemblages are dictated by tidal exposure and wave impact, as well as by species interactions such as grazing by invertebrates, and by competition for space and l ight. 2 9 The brown algae (Phaeophyta) probable represent a very old group among the plant kingdom. Almost all Phaeophyta thrive in the sea, for example, Nereocystis luetkeana (Bull kelp), Fucus gardneri (Brown algae), Laminaria groenlandica (Kombu) and Eisenia bicyclis (Arame). ' ' ' A l l brown algae (Phaeophyta) are multicellular, and most are macroscopic. 2 8 The red algae (Rhodophyta) are the seaweeds that can be seen growing along shores and cast up on beaches. Almost all red algae are multicellular and are a large group of small to 6 medium-sized plants, for example: Porphyra spp., Mastocarpus pepillatus and Gigartina 2 8 exasperata. The large and clearly defined group of the marine algae is the Chlorophyta or green algae. They comprise forms of highly diverse lines of development and phases of greatly varying habit. The green algae inhabit, above all , fresh water regions and subsist under most varying conditions. The species found mostly on the British Columbia coast is the Ulva fenestrata. This project involves experiments with Nereocystis luetkeana and Fucus gardneri. 1.3.2 Commercial Use of Marine Algae Algae have been put to a number of uses in the past, and their applications are increasing. The kelp trade developed in the seventeenth century and, until quite recently, was an important branch of the iodine industry. Kelp (Nereocystis luetkeana) has been used to weave baskets and also make great musical instruments.3 4 Brown seaweeds were collected and burned, and their ashes were used as a source of soda to make glass during the 16th and 17th century. Later it was discovered that the brown seaweed was rich in iodine and was used for iodine-extraction until after the First World W a r . 2 8 , 3 0 ' 3 1 In the modern marine algae industry, seaweeds are used in many products. ' Some examples of the use of seaweed as food are as follows: Nor i is an ingredient for 'sushi' in Japanese food. Arame is used with soups or taken with soya-sauce in Japanese food . 3 0 ' 3 1 ' 3 6 Alginates from brown algae are used as stabilizers in ice creams, bakery and confectionery products, as thickening agents in soups and sauces, as alginate strings in meat sausages and as clarifying agents in beverages.2 8 Seaweeds are also used in food products for other animals. 31 35 They are used as fertilizers and used for the production of agar. ' 7 Analyses of certain edible seaweeds also show that many contain significant amounts of protein, vitamins and minerals essential for human nutrition. ' Both Fucus gardneri and Nereocystis luetkeana are used in the production of vitamin supplements. 1.3.3 Arsenosugars in Marine Algae Marine algae are able to accumulate arsenic more efficiently than the higher members of the food web: in some algae, arsenic concentrations have been found to be 3000 times higher than those present in the surrounding seawater. Most of the arsenic in marine macroalgae exists in complex forms and a variety of arsenosugar derivatives have been isolated and characterized. 3 8 ' 3 9 ' 4 0 ' 4 1 The arsenosugars found in some of the common brown macroalgae are listed in Table 1.2. Table 1.2 Arsenosugars found in some common Marine Algae Algae Arsenosugars present Reference Brown Algae Fucus gardneri X , XII , XIII 42,43 Ecklonia radiata X , X I , XII 39,40 Undaria pinnatifida X , X I , XII 44,45 Red Algae Porphyra tenera (Nori) X , X I 45 Green Algae Codium fragile (Miru) X , X I 45 8 Edmonds and Francesconi were the first to isolate arsenosugars from the marine alga Ecklonia radiata. A total of 15 arsenosugars have been found from different species of green, brown and red algae. 2 5 The question of how and why arsenosugars are formed by the marine algae has been studied extensively. It is believed that the algae accumulate arsenate from the seawater, because arsenate is the predominant species found in seawater at a typical range of approximately 1.0 to 8.0 ppb. 7 ' 8 ' 1 0 It is believed that arsenate, being chemically similar to phosphate, is readily taken up by the algae from the water via the phosphate transport systems located in the algal cell membranes. 5 ' 1 0 ' 2 5 Although the precise mechanism involved in the formation of arsenosugars is not known, a pathway as proposed by Edmonds and Francesconi 2 5 ' 4 1 is shown in Figure 1.1. The pathway initially follows the mechanism outlined by Challenger by using S A M (S-adenosylmethionine) as the methylating agent (CFi3+ donation indicated by A in Figure 1.1). However, in the final steps the adenosyl group (indicated by B in Figure 1.1) of the methylating agent is transferred to the arsenic atom of dimethylarsinate, ultimately to form arsenosugars and arsenolipids. 8 ' 2 5 ' 4 6 9 Figure 1.1 Proposed Pathway for the Biogenesis of Arsenosugars in Marine Algae' The A represents methyl group donated by S A M , and B is the adenosyl group from S A M O II OH—As— O H I O H 1* O H — A s - O H O H C H , 4---H O O C — C H — ( C H J J J — S — C N H , S-adenosylmethionine N H , <o. ; N -Vy o II OH—As—OH I C H 3 O H — A s - O H I C H , CH, H O O C — C H — ( C H , ) , — S — C H , I ^ 2 v , y y NH, N N J O H O II H 3C—As—OH CH, -> H3C—As—OH CH, 0 II (CH^As-CH^ ^ 70H OH OH 0 (CH3)F-As-CH2v ^Ov ya OH OH NH, N N 0 OH (CHj)—As—CH^  yO-CHjCHCHj- R OH OH Arsenate can produce toxic effects in the algae, including inhibition of growth and phosphorus metabolism. Changes in the algal cell morphology are also seen. It has been suggested, that while the conversion of arsenate to arsenite creates a more toxic arsenic compound, arsenite is not released into the cell environment but it is pumped out immediately to avoid an excessive build-up of toxic arsenic concentrations inside the algal c e l l . 2 7 ' 4 7 1.3.4 Previous Studies of Arsenic Transformation in Marine Algae Although arsenosugars have been isolated from seaweeds, there are only a limited number of reports that describe the transformation of arsenicals by marine macroalgae grown in culture media. Klumpp and Peterson9 claimed that the brown macroalga, Fucus spiralis, took up the arsenic as arsenate and transformed it into water-soluble organoarsenic compounds which were then further transformed into a lipid-soluble organoarsenic compound and 12 water-soluble * 48 organoarsenic compounds. However these compounds were not positively identified as arsenosugars. Sanders and W i n d o m 1 0 used arsenate, arsenite and dimethylarsinate as substrates for cultures of marine macroalgae Valonia macrophysa. Digestion of the cells in dilute nitric acid led to an increase in methylated arsenicals, suggesting that more complex arsenic compounds were produced. Cullen et al. ' found that Polyphysa peniculus transformed arsenate into arsenite and dimethylarsinate. When the alga was treated with arsenite, dimethylarsinate was the major metabolite in the cells and in the growth medium. Trace amounts of monomethylarsonate were also detected in the cells. Significant amounts of more complex arsenic species, such as 11 arsenosugars, were not observed in the cells or medium. Cullen et al. concluded that the arsenate taken up by Polyphysa peniculus is quickly reduced to arsenite, followed by methylation to monomethylarsonate. M M A due to its low passive diffusion coefficient is not excreted to the growth medium. The compound remains in the cells, where it is more likely to be reduced and further methylated to D M A (which is the end product for Polyphysa peniculus). D M A is then diffused into the growth medium. The fast excretion of the reduced and/or methylated arsenic Q compounds to the medium decreases the need for further detoxification. Arsenic compounds found in marine animals are generally believed to come from arsenicals produced by marine algae. It has been suggested that arsenobetaine and arsenocholine are formed from arsenosugars, which are found in marine algae. 2 5 12 1.4 FUNGI The fungi (singular, fungus) are plant-like, spore-bearing organisms, which lack chlorophyll and are unable to synthesize their food. Consequently, they depend on other organisms for their nutrition. Fungi live as saprobes, which bring about the decay of organic materials, or as parasites, which attack living protoplasm, and in so doing cause diseases of plants, animals, and humans. 4 9 Fungi have a very broad definition, which encompasses the bacteria, the slime molds, and the so-called true fungi. The bacteria differ from the other two groups in that they possess primitive rather than the highly organized nuclei that are found among the slime molds and the fungi. 4 9 Fungi are often found to grow with algae in a parasitic relationship. The fungus can affect the algal cells by competing for nutrients, by changing the physical state of the medium, and by releasing substances, which inhibit the growth or k i l l the cells. Few studies have been done on these processes. 5 0 13 1.5 O B J E C T I V E O F PRESENT W O R K The study of the interaction of marine algae with arsenicals is important; because arsenic compounds produced by the algae are generally believed to be one of the sources of the arsenic compounds found in marine animals. In addition to the human toxicological aspects, arsenosugars are also of interest because of their pivotal role in the cycling and biotransformation of arsenic in marine systems. Thus the first objective of this work was to determine i f and how arsenosugars are produced by Fucus gardneri. The Fucus was exposed to As (V) under different environmental conditions to determine i f these conditions would affect the uptake of As(V) by the algae. The different environmental conditions that were examined were variations of salinity, phosphate, and antibiotic concentrations in the medium. The second objective was to duplicate one of the above experiment using Nereocystis luetkeana in order to determine i f the Nereocystis is able to accumulate As (V) from the surrounding media in a similar fashion as Fucus gardneri. The experiment that was performed was to acclimate Nereocystis luetkeana in seawater, followed by exposure to As(V) in artificial seawater The third objective was to identify a particular resilient fungus growing with Fucus gardneri and to determine i f this fungus was involved in the uptake of arsenic compounds by the brown algae. The last objective was to determine the arsenosugars present in a variety of British Columbia algae samples found to grow along with Fucus gardneri and Nereocystis luetkeana. The water content of each samples was found as well as the extraction efficiency of the method used. 14 2 E X P E R I M E N T A L M E T H O D S F O R T H E A N A L Y S I S OF A R S E N I C 2.1 I N S T R U M E N T A T I O N 2.1.1 High Performance Liquid Chromatography (HPLC) 2.1.1.1 Apparatus The H P L C system consisted of a Waters Model 510 delivery pump, a Reodyne six-port injection valve with a 20 u L sample loop, an appropriate column and its corresponding guard column. The H P L C system was connected to the inductively coupled plasma mass spectrometer ( ICP-MS) nebulizer by using P T F E tubing (2.5 cm) with the appropriate fittings. The analytical column used to analyze the medium samples, was an anion-exchange column (Hamilton, PRPxlOO, 250 mm x 4.6 mm) with a guard column of the same material. The mobile phase was 20 m M phosphoric acid, adjusted to p H 6.0 by using ammonium hydroxide. The column used to analyze the extract samples was a reversed-phase Ci8 column ( G L Sciences Inertsil O D S , 250 mm x 4.6 mm). The guard column was packed with the same material (Supelco) as in the Inertsil ODS column for the reversed-phase conditions. The mobile phase was made up of 10 m M tetraethylammonium hydroxide, 4.5 m M malonic acid, and 0.1 % M e O H , adjusted to p H 6.8 by using nitric acid. A guard column preceded the analytical column to filter out any precipitates that might form when the injection sample combined with the mobile phase, and any other large molecules that might have been present. 15 2.1.1.2 Conditions The H P L C system, connected to the I C P - M S , was used to analyze the extracted samples and medium samples collected from the exposure experiments. The conditions for the columns, mobile phases and flow rates are summarized in Table 2.1. The chromatographic columns were equilibrated with the mobile phase for about 50 minutes prior to analysis. A l l the standard samples were filtered through a 0.45 um syringe filter (Millipore) just prior to injection onto the H P L C column, while all other samples were filtered through a 0.22 pm syringe filter before injection. Filtration of the samples removed small particles that might affect the efficiency of the column or might even contaminate the column. Most importantly, removal of the small particles w i l l prevent the blocking of the injection loop, lines and column. The effluent from the H P L C column was fed to the nebulizer of the I C P - M S via the P T F E tubing and connections. Arsenic compounds in the samples were identified by matching the retention times of the peaks in the chromatograms with those of standard arsenic compounds. The mobile phases for the H P L C were filtered through a millipore 0.45 pm filter after they were made up. Table 2.1 Summary of Experimental H P L C Conditions Conditions Column Mobile Phase Flow Rate (ml/min) Anion exchange (medium samples) Ion Pairing (extract samples) Hamilton P R P X 100 Inertsil ODS ( G L Sciences, Japan) 20 m M phosphoric acid, p H 6.0 10 m M tetraethylammonium hydroxide(TEAH), 4.5 m M malonic acid, 0.1 % M e O H pH6 .8 1.5 1.0 16 2.1.2 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) A V G Plasma Quad 2 Turbo Plus inductively coupled plasma-mass spectrometer ( V G Elemental, Fisons Instrument) was used as a detector for the H P L C eluent. It was also used to measure the total arsenic, via direct injection into the system. The I C P - M S was equipped with a S X 300 quadrupole mass analyzer, a standard ICP torch, an argon plasma and a de Galan V-groove nebulizer. The mass analyzer was operated in the time resolved analysis (TRA) mode using T R Analysis software. The 75, 77, 82 and 103 mass to charge (m/z) signals were monitored. A l l signals were collected and the data were transferred to the computer ( V G data system). The data were then exported to a Microsoft Excel 7.0 program for further processing. The T R A mode operation allowed for the monitoring of mass to charge (m/z) 75, 77 and 82; and also 103 for the total digestion analysis. The m/z ratio of 75 corresponds to that of 7 5 A s (100 % isotopic abundance), but also to the possible interfering species A r CI signal. The m/z ratio of 77 corresponds to that of A r 3 7 C l + signal. The interference due to A r 3 5 C l + can thus be accounted for by subtracting the peak area at m/z 77 from the peak area at m/z 75, having corrected for the isotopic ratio of CI to CI (75.77:24.23). There may also be a peak at m/z 77 due to 7 7 Se. Se has isotopes of 77 and 82, so the contribution of the 7 7 Se peak can be accounted for by subtracting the peak area at m/z 82 from the peak are at m/z 77, having corrected for the isotopic ratio of Se to Se (isotopic ratio: 7.63:8.74). The m/z ratio of 103 corresponds to that o f R h . A schematic diagram of the I C P - M S system is displayed in Figure 2.1 and the commonly used operating parameters for the ICP-MS are shown in Table 2.2. 17 Figure 2.1 Schematic Diagram of ICP-MS (VG Elemental, Fisons Instrument) Detector Quadrupole Quadrupole RF Generator Amplifier Quadrupole RF DC Control Lenses Interface ICP Bias Voltage Supplies D O 1 ICP RF Generator • D O J -Vacuum Pumps PlasmaQuad M C S Control ^ IEEE Interlace Table 2.2 Operating Parameters of ICP-MS Feature Specific Conditions Forward r.f. power Reflected power Outer (cooling) gas flow rate Intermediate (auxiliary) gas flow rate Nebulizer gas flow rate Nebulizer type Analysis mode Quadrupole pressure Expansion pressure 1350 W <10 W 13.8 L/min 0.65 L/min 1.002 L/min de Galan T R A , 1 sec time slice 9 x 10"7 mbar 2.4 mbar 18 2.2 G E N E R A L C U L T U R E M A I N T E N A N C E 2.2.1 Conviron Environmental Chamber (Model C M P 3023) The Conviron, a commercial incubator, was used to hold the samples during the growth, acclimation and exposure periods. The Conviron can be set at specific temperatures and light conditions to obtain optimal settings for the growth of the marine algae and fungus. Table 2.3 shows the operating parameters. Table 2.3 Operating Parameters for the Conviron Environmental Chamber Sample Temperature Light Intensity Photoperiod Fucus gardneri 15 °C/ 7 °C 100 lux 12 hour day and night cycle Nereocystis luetkeana 1 8 ° C / 7 ° C 200 lux 12 hour day and night cycle Fusarium oxysporum 15 °C none none 2.2.2 Reagents and Chemicals A l l chemicals used were of at least analytical grade and obtained from commercial sources, unless otherwise stated. The chemicals and reagents used were: methanol ( H P L C grade, Fisher), tetraethylammonium hydroxide (20 wt. %, Aldrich), malonic acid (BDH), nitric acid (68-71 %, sub-boiling distilled, Seastar Chemicals), phosphoric acid (99.999 %, 85 wt. %, Aldrich) and ammonium hydroxide (30 %, Fisher). Standard solutions of arsenite (from arsenic trioxide, AS2O3, Fisher), arsenate (from sodium arsenate heptahydrate, Na2HAs04»7H20, Sigma), monomethylarsonic acid (Pfalz & 19 Bauer), dimethylarsinic acid (Aldrich), and arsenobetaine (synthesized as described by Edmonds, T O et ar) were made up in deionized water. Standard samples of oyster tissue S R M (NIST-1566a), Fucus sample ( IAEA-140/TM) and kelp powder (laboratory standard-Galloway's naturally kelp powder, Richmond, B . C . , Canada) were also available to confirm the retention times of arsenosugars obtained from the H P L C - I C P - M S . R h standard solution for total digestion was made up from RJ1CI3. Deionized water, with a resistivity better than 1 M Q , was used to make up all the standard solutions, the mobile phases for the H P L C system and the sample solutions for extraction and total digestion. Because it was necessary to avoid even trace contamination, the following precautions were taken with all the glassware and plasticware: The glassware and plasticware were cleaned by soaking in 2 % Extran solution for at least one night. They were rinsed with deionized water, and then soaked in 0.1 M HNO3 solution for at least one night. They were then rinsed with deionized water and air-dried. Any glassware used to store the standard solutions or used for the algae and fungus experiments were autoclaved. 2.2.3 Medium and Antibiotics The artificial seawater that was used in the arsenic exposure experiments was prepared according to the recipe for A S P 6 F2 (see Table 2.4), which was previously used by Fries 5 1 to culture members of the family Fucaceae. The only deviation from the recipe was the substitution of CaCl2*2H20 for C a C b ' ^ O (the formation with one water molecule is probably an error). In addition, the p H of the artificial seawater was adjusted from 9.72 to ~ 7.76 (which was the p H of the seawater that was collected with the algae) by addition of HC1. The p H reported for the 20 A S P 6 F2 artificial seawater by Fries is 8.3. The seawater was prepared without the vitamins, autoclaved and stored at room temperature. The vitamin solution and the vitamin B12 solution were sterilized by using 0.22 um sterile filters and then added to the autoclaved seawater. The artificial seawater with the vitamins was then stored in the refrigerator at 4 °C. Table 2.4 Contents of Artificial Seawater, ASP6 F2 5 1 M e d i u m A S P 6 F2 M i c r o n u t r i e n t Solut ion (per m l H?0) N a C l 24 g nitrilotriacetic acid 50 mg M g S 0 4 ' 7 H 2 0 8 g Fe (as chloride) 1 mg KC1 0.7 g Zn (as chloride) 0.25 mg CaCl 2 «2H 2 0** 0.55 g M n (as chloride) 0.5 mg N a N 0 3 0.1 g C o 5 Hg Na 2-glycerophosphate 50 mg C u 10 ug K 2 H P 0 4 2 m g B (as H3BO3) 1 mg K I 0.065 mg M o (Na-salt) 0.25 mg K B r 96.9 mg T R I S (hydroxy methyl) aminomethane l g V i t a m i n Solut ion (per ml H?0) Micronutrient solution 1 ml Thiamine HC1 0.2 mg Vi tamin solution 1 ml Nicot inic acid 0.1 mg B 1 2 1 Hg C a panthothenate 0.1 mg Dist i l led Water 1000 ml Pyridoxin HC1 0.04 mg p-aminobenzoic acid 0.01 mg Biot in 0.5 ug Thymine 0.8 mg Inositol 1 mg Orotic A c i d 0.26 mg "or ig ina l recipe has C a C l 2 * H 2 0 The antibiotics were prepared according to the recipe for A3-antibiotics as described by Xuewu (see Table 2.5). 5 3 The antibiotics were sterilized by using 0.22 um sterile filters before being used. The antibiotics were used for the axenation of the bacteria and fungi in marine algae. 21 Table 2.5 A3 Antibiotics for Axenation of Bacteria and Fungus Antibiotics A3 Streptomycin sulphate 2 g Nystatin 0.5 g Erythromycin 1.5 g Rifampicine 0.02 g Distilled Water 100 ml Antibiotics were purchased at Sigma. Stock solutions were diluted with 100 volume o f medium for treating plant materials. 2.3 S A M P L E P R E P A R A T I O N F O R A N A L Y T I C A L ANALYSIS 2.3.1 Sample Storage A l l biomass samples collected were weighed, frozen (-20 °C) immediately and then freeze-dried. Freeze-drying is a common method to preserve samples in environmental studies. It provides a convenient mode of preservation because it results in the elimination of water and the denaturation of protein. The freeze-drying procedure does not significantly alter arsenic speciation making it easier to handle the samples and also gives consistent results for quantitative analysis. A l l freeze-dried samples were kept at -20 °C until they were extracted. A l l the liquid samples taken during the experiments were frozen immediately to preserve sample integrity until the time of the analysis. The liquid samples were analyzed by anion-exchange H P L C - I C P - M S . 2.3.2 Extraction A l l the freeze-dried samples collected were extracted using a procedure similar to that described by Shibata and Mor i ta . 4 4 Kelp powder (a laboratory standard), oyster tissue S R M (NIST-1566a) and Fucus sample ( IAEA-140/TM) were similarly extracted as reference materials. Extractions were carried out by weighing accurately (-0.5 g) of the dried powders into 15 ml centrifuge tubes, adding 5 ml methanol/water (1:1), sonicating for 10 minutes, centrifuging for 10 minutes and transferring the liquid layer of the extracts into 250 ml round bottom flasks by using a Pasteur pipet. Each sample was extracted a total of 5 times. The combined extract solutions for each sample were evaporated to near dryness (~ 1-2 ml), and made up to 10.0 ml 23 using a volumetric flask. The extract solutions were frozen until just before analysis. The samples were analyzed by using the ion-pairing H P L C - I C P - M S method. 2.3.3 Acid Digestion of Samples Freeze-dried samples (-0.50-100 mg), the residue after extraction (-0.100-200 mg original for dry samples) and the extracted solution (-5 ml) were analyzed for total arsenic by using a total digestion procedure similar to that described by N0rum et al.54 i The total digestion was carried out by placing the accurately weighed freeze-dried samples into 20 ml glass test tubes (outer diameter of 16 mm). Concentrated nitric acid (2 mL, doubly distilled in quartz, Seastar, Sidney, B C ) was added to each sample together with 2-3 Teflon boiling chips. The tubes were transferred to a block heater (Standard Heatblock by V W R Scientific Products) and the temperature increased in 10 °C steps every hour, starting at 70 °C and reaching 150 °C. A t 150 °C the samples were evaporated to dryness over the next 1-2 days. Once the samples were evaporated, the test tubes were removed and allowed to cool. The samples were redissolved in 4 ml "Acid-Rhodium" solution (1% nitric acid and 5 ppb Rh). The rhodium served as an internal standard during I C P - M S analysis. The redissolved digest were vortex-mixed and filtered (0.45 um) into storage vials (2 x 1.5 ml eppendorf) using disposable syringes. The samples were stored at -20 °C until the time of analysis. The acid digest was analyzed by using I C P - M S and by monitoring m/z 75 and 103 for arsenic and rhodium, respectively. 24 2.4 SPECIATION O F STANDARD COMPOUNDS BY HPLC-ICP-MS 2.4.1 Standard Reference Materials for Ion-Pairing HPLC-ICP-MS Condition A major problem of arsenic speciation in environmental samples is the absence of pure samples with many organoarsenic compounds. Many of the compounds important in environmental and biological systems are difficult to synthesize. This problem was solved by using oyster tissue S R M (NIST-1566a), Fucus sample ( IAEA-140/TM) , and kelp powder (laboratory standard) as standard reference materials for the peak identification of arsenosugars. 3 6 ' 4 4 The ion-pairing chromatography system used here is one that has been developed for the analysis of anions, particularly arsenosugars. 5 5 ' 5 6 Ion-pairing chromatography with I C P - M S detection is a technique that combines a mobile phase containing an ion-pairing reagent with a reversed-phase column. Tetraethylammonium hydroxide ( T E A H ) is the ion-pairing reagent, and the mobile phase is adjusted to a p H of 6.8 with malonic acid and nitric acid. A small amount of methanol is added to control the chromatographic behaviour in reversed-phase systems. Figure 2.2 shows a chromatogram of the oyster tissue obtained in the present study using the ion-pairing H P L C - I C P - M S condition ( T E A H ) . The major arsenic compounds A s B , D M A , arsenosugars X and X I present in this standard reference material, were previously identified by 9 ft Shibata et al. As (V) and D M A are not baseline resolved but the individual peaks are slightly resolved making it possible to identify both peaks. The elution order shown in Figure 2.3 for the Fucus standard is D M A , As (V) and arsenosugars X , XII and XIII. This is another standard to confirm the presence and retention times of the arsenate and arsenosugars. 25 Figure 2.4 shows a chromatogram of the commercially available food product, kelp powder, under T E A H condition. The kelp powder contains four major peaks; arsenosugars X , X I , XII and XIII were identified by comparing the retention times with known samples by Shibata et al.44'45 Using the present chromatographic system, the arsenosugars are separated from the other ions. Although arsenosugars X I and XII are not baseline resolved from each other, their individual peaks are resolved, making it possible to identify both arsenosugars in unknown samples. Figure 2.4 shows that arsenosugar XII alone elutes slightly earlier, which allows this compound to be identified in the absence of arsenosugar X I . Likewise, arsenosugar X I can be identified in the absence of arsenosugar XII . The elution order for the sugars most likely indicates the increasing anionic character when going from arsenosugar X to X I I I . 5 6 Figure 2.2 Oyster Tissue S R M Standard 180000 160000 140000 120000 100000 80000 60000 40000 20000 0 AsB DMA 100 200 300 400 500 Retention time(s) 600 700 800 26 Figure 2.3 Fucus Sample Standard 450000 400000 350000 ^ 300000 250000 Q. ° 200000 150000 \ 100000 50000 0 XII D M A XIII I 100 200 300 400 500 Retention time(s) 600 700 800 Figure 2.4 Kelp Powder Standard 400 500 600 Retention time(s) 2.4.2 Analytical Standards for Ion-Pairing HPLC-ICP-MS Condition A standard solution containing 5 compounds was used to establish the retention times for A s B , As(III), As (V) , M M A and D M A under T E A H condition (Figure 2.5). The retention times for the extracts from the samples were matched with the retention times of the peaks in the standard solutions to determine the arsenic species in the extracts. In this case As(III) and A s B are not baseline resolved and M M A , D M A and As(V) are not baseline resolved, but their individual peaks are resolved, making it possible to identify As(III), A s B , M M A , D M A and As(V) in the unknown samples. Figure 2.5 Five Arsenic Standards 35000 -, As(lll) AsB 30000 j 25000 I 20000 4 in OL O 15000 \ 10000 4 5000 j 0 0 50 100 150 200 250 300 Retention time(s) 28 The standard solutions were also used to determine calibration curves for quantifying the arsenic compounds that were in the extract samples. The calibration curves for As(III), M M A , D M A and As (V) were used to determine the concentrations of each compound in the extract samples over time. The calibration curves obtained were linear for all the compounds in the range of 0 to 100 ppb. A n example for one of the calibration curves of arsenate is shown in Figure 2.6. Figure 2.6 Arsenate Calibration Curve for Ion-Pairing HPLC-ICP-MS 4.00E+06 -, 3.50E+06 4 0.00E+00 -I , , , , , , 0 20 40 60 80 100 120 Concentration (ppb) 29 2.4.3 Analytical Standards for Anion-Exchange HPLC-ICP-MS Condition The media samples collected were examined by an anion-exchange H P L C - I C P - M S system. Standard solutions of four arsenic compounds (As(III), M M A , D M A and As(V)) were used to determine the retention times. The standard solutions were also used to determine calibration curves for quantifying the arsenic compounds that were in the medium. A s can be seen from Figure 2.7, none of the peaks overlap at their baseline, making it easy to identify the peaks. As(III) is present as a neutral molecule (pK a 9.3), D M A is about 50 % mono-negative charged (pK a 6.28) and M M A , which elutes next, is predominately in singly charged anionic form (98.6 %, p K a i 3.6, p K ^ 8.2). The later elution of As (V) compared with the other compounds is most likely due to the fact that As(V) is anionic, being 90 % singly charged and 10 % double charged ( p K a I 2.2, p K ^ 6.9). 5 6 Figure 2.7 Standard Solution of Four Arsenic Compounds 50000 i 45000 j As(lll) g. 25000 -0 0 50 100 150 200 250 300 350 400 450 Retention time(s) 30 Calibration curves for As(III), M M A , D M A and As(V) were used to determine the concentrations of each compound in the medium over time. The calibration curves obtained were linear for all the compounds in the range of 0 to 500 ppb. A n example for one of the calibration curves of arsenate is shown in Figure 2.8. Figure 2.8 Arsenate Calibration Curve for Anion-Exchange HPLC-ICP-MS 2.5E+06 2.0E+06 « 1.5E+06 re £ 1.0E+06 5.0E+05 0.0E+00 y = 3805.5X + 63212 R2 = 0.9982 100 200 300 400 Concentration (ppb) 500 600 Although neither of these chromatographic systems can be used alone for the analysis of complex mixtures, such as those found in environmental samples, a combination of ion-pairing and anion-exchange chromatography can be used to obtain a satisfactory separation and identification of at least 9 arsenic species. The errors for the medium and extract samples were obtained from the standard deviation of the analytical results by using the calibration curves. Other sources of errors, such as 31 biological variations and environmental variations in the media which may be significant, were not included. 2.4.4 Analytical Standards for Total Analysis by ICP-MS The total arsenic analysis involved injecting the samples onto I C P - M S containing 10 ppb PJ1CI3 as an internal standard with nitric acid (1 %) as the carrier. A calibration curve (see Figure 2.9) was made for arsenic standard solutions ranging in concentration from 0 to 100 ppb. The standard solutions were also spiked with 10 ppb RhCl3. The ratio of arsenic detected to rhodium detected was determined for each standard and sample, and the calibration curve was used to determine the total arsenic content of the samples. Figure 2.9 Arsenate Calibration Curve for Total Digestion 1.2 -, y = 0.0101x-0.0014 R2 = 0.9998 0 20 40 60 80 100 120 •0.2 J Concentration(ppb) 32 3 T H E B I O M E T H Y L A T I O N A N D B I O A C C U M U L A T I O N OF A R S E N I C A L S B Y M A R I N E A L G A E FUCUS GARDNERI U N D E R D I F F E R E N T E N V I R O N M E N T A L CONDITIONS 3.1 INTRODUCTION Arsenic is probably one of the better known elements because of the toxic properties of some of its compounds. Against this background it is not surprising that reports, at the beginning of this century, of high levels of arsenic in marine organisms attracted much interest. Since then, concerns regarding the forms of arsenic in marine organisms, their toxicity, how they were accumulated, and what role they played in the biochemical functions of the organisms have been investigated. Marine algae have been the subject of many arsenic metabolic studies because of their ecological and nutritional importance. Such studies of the interaction of marine algae with arsenicals are relevant because arsenic compounds produced by algae are generally believed to be the source of the arsenic compounds found in marine animals, although it is not well established how and when these transformations take place. 5 ' 1 0 A s mentioned in Chapter 1, there are only a limited number of reports that discuss the biotransformation of arsenicals by marine macroalgae in culture medium. 8 ' 9 ' 1 0 ' 2 4 Complex arsenic compounds are reported to be produced by these algae under these conditions, but they have not been positively identified as arsenosugars. 33 The bioaccumulation of the arsenicals by marine algae also depends on the health of the algae and the surrounding environmental factors such as substratum, salinity, temperature, water 9 8 movements and light. To determine how each condition affects the uptake of arsenate by marine algae, several experiments were performed in which a specific condition was altered. In this case the effects of salinity, different phosphate levels and no antibiotics in the medium were looked at. Fucus gardneri Silva, also known as Fucus distichus was chosen for this investigation. It is marine algae that is abundant in the British Columbia coastal area and mainly found in the middle and lower inter-tidal zones, where it grows on rocks. The colour of the Fucus is olive green to yellow-green and it is characterized by the branching filamentous thallus (see Figure 3 .1) . 3 0 ' 3 3 ' 5 7 ' 5 8 Mature Fucus possesses receptacles at the ends of the branches, which are the fertile areas. Receptacles may be enlarged and swollen when they are mature and may appear yellowish. A t the sampling sites of Brockton Point, Stanley Park, Vancouver, and Bamfield, Vancouver Island, Fucus gardneri were the major algae species found. See Figure 3.2 for a map of the collection sites. 34 Figure 3.1 Fucus gardneri Silva a, habit o f dichotomously branched plant attached to substrate, showing conspicuous midrib and swollen receptacles Figure 3.2 Map of Collection Sites 35 3.2 S A M P L E C O L L E C T I O N AND T R E A T M E N T 3.2.1 Sample Collection Whole young brown algae Fucus gardneri were collected at Brockton point, Stanley Park, Vancouver, B C (on October 1998 and October 1999) and at Bamfield marine station, Vancouver Island, B C (on February 1999). Mature brown algae (Fucus gardneri), green algae (Ulva fenestrata) and red algae (Gigartina exasperata) were also collected at Stanley Park. Two red algae species (Porphyra spp. and Gigartina exasperata) and one green algae species (Ulva fenestrata) were also collected at Bamfield along with the Fucus gardneri species. Young Fucus gardneri were also collected attached to small rocks with a diameter of about 15 cm at Brockton point, Stanley Park on June 1999 and August 1999. Other species of plants and animals (barnacles, mussels, snails, small crabs, green algae and red algae) also came along with the rocks. A l l the samples were collected at low tide levels. Seawater collected at both sites was later sterilized by using the autoclave. 3.2.2 Sample Treatment 3.2.2.1 Treatment of Fucus gardneri before the Acclimation Period Whole young Fucus samples were chosen for the exposure experiments instead of the tips because, as indicated by Fries 5 1 , the tips of Fucus grow very slowly and consequently several months would be needed to see any growth. 36 A l l steps performed after the collection of the Fucus were done under sterile conditions in a laminar flow hood. The mature Fucus (separated into whole, swollen tips and remainder), the green algae, red algae, and a sample of young Fucus (separated into whole, tips and remainder) were washed with sterile seawater, weighed, freeze-dried and frozen until further analysis. These samples were further investigated which is discussed in Chapter 6. Samples of young Fucus (except for those attached to the rocks) were rinsed in a sterile beaker approximately 5 times with the sterile seawater collected at each site, followed by 4 washings with distilled water. The samples were then covered with a mixture of antibiotics and seawater (~2 ml per 100 ml seawater) for approximately 5 to 10 minutes. The mixture was removed and the Fucus washed with sterile seawater. The Fucus was separated and placed into 1-L Erlenmeyer flasks, which contained seawater or artificial seawater depending on the experiment (see Section 3.2.2.2 for details of each specific treatment). Fucus samples were collected before the washing, after the first washing with seawater and after the last washing with seawater. They were weighed, frozen and freeze-dried. Seawater samples were also collected at the same time. The Fucus samples that were collected attached to the rocks were not rinsed, but placed directly in an aquarium tank and covered with unsterile seawater after the removal of most of the barnacles and mussels from the rocks. 37 3.2.2.2 Treatment of Fucus gardneri during the Acclimation and Exposure Periods Fucus samples were used for different experiments, which are described according to their collection dates. 3.2.2.2.1 Fucus sardneri collected October 1998, acclimated in seawater or artificial seawater, and exposed to As(V) in seawater or artificial seawater The Fucus samples were treated as described in Section 3.2.2.1 and then they were divided into two batches. The first batch was separated and distributed into six-1 L flasks that contained sterile seawater (200 ml-collected from Stanley Park) and antibiotics (2 ml). The second batch was also distributed into six flasks which contained A S P 6 F2 medium (200 ml) and antibiotics (2 ml). Both sets of flasks were placed in the Conviron Environmental Chamber (incubator) under the conditions described in Section 2.2.1. The cultures were shaken once every few days to ensure adequate dissolved oxygen in the media. After 14 days of acclimation the Fucus samples were clean apart from a visible white fungal growth. Bacterial levels appeared to be minimal because the solution did not turn milky during the 14 days, and the levels were maintained that way by constant exposure to the antibiotic solution. The Fucus samples were combined from batch number 1 and then washed with sterile seawater 6 to 8 times. The last washing also contained antibiotics. The Fucus samples were weighed and transferred to sterile four-1 L flasks containing 200 ml sterile seawater, 2 ml antibiotics and 100 fig of arsenate. Batch number 2 was treated in the same way as batch number 1 after 14 days of acclimation, except that the Fucus samples were placed into artificial seawater (ASP6 F2 medium) instead of seawater. The treatment for each batch is 38 outlined in Table 3.1. The seawater and artificial seawater after acclimation and several of the washings were collected. Also , sub-samples of the Fucus were collected before and after the washings from both batch #1 and batch # 2. Table 3.1 Medium Conditions for the Exposure Period for Fucus gardneri collected October 1998 Flask #* Medium** Sample Arsenic batch #1 1 seawater algae from seawater 0.50 ppm As(V) 2 seawater algae from seawater X 3 seawater X 0.50 ppm As(V) 4 seawater X X batch #2 5 A S P 6 F2 algae from A S P 6 F2 0.50 ppm As(V) 6 A S P 6 F2 algae from A S P 6 F2 X 7 A S P 6 F 2 X 0.50 ppm As(V) 8 A S P 6 F2 X X * all flasks were duplicated ** all flasks contained 2 ml antibiotics in 200 ml medium X not present in the flasks The flasks containing the arsenate were prepared by adding 100 uL of 1000 ppm stock solution per flask. A l l treatments of the Fucus were carried out under sterile conditions and sealed with sterile cotton-plugs. The flasks were placed under the same conditions as before in the incubator. One ml samples of each flask were taken under sterile conditions twice a week for 28 days during the exposure experiment. The flasks were also swirled on the sampling days to ensure adequate dissolved oxygen in the media. The weight of Fucus used in the exposure experiment ranged from 16.45 to 24.78 g of wet weight per experimental flask. 39 3.2.2.2.2 Fucus sardneri collected February 1999, acclimated in seawater, and exposed to As(V) in artificial seawater The Fucus samples from February 1999 were treated similar to the October 1998 samples except for the following deviations. The Fucus samples were placed into sterile seawater (400 ml) collected from Bamfield, together with 4 ml antibiotics. After 14 days of acclimation, the bacterial levels were again minimal and were maintained that way by means of antibiotic solution. Only one fungus was observed growing with the Fucus. The fungus was collected and placed onto agar dishes for identification and further experimentation. The Fucus samples were collected together, washed with seawater and then placed into flasks that contained 200 ml artificial seawater (ASP6 F2 medium), 100 pg arsenate and 2 ml antibiotics. Again the flasks were placed into the incubator under the conditions described in Section 2.2.1 for the exposure experiment. See Table 3.2 for the experimental treatment. Table 3.2 Medium Conditions for the Exposure Period for Fucus gardneri collected February 1999 Flask #* Sample Arsenic** 1 algae from seawater 0.50 ppm As(V) 2 algae from seawater X 3 X 0.50 ppm As (V) 4 X X * all flasks were duplicated ** all flasks contained 2 ml antibiotics in 200 ml A S P 6 F2 medium X not present in the flasks The exposure experiment was also run for 28 days and samples of the medium were taken twice a week during that period. The weight of Fucus used in the exposure experiment ranged from 19.95 to 26.73 g of wet weight per experimental flask. 40 3.2.2.2.3 Fucus sardneri collected October 1999, acclimated in artificial seawater, and exposed to As(V) in artificial seawater at different phosphate concentrations The Fucus samples collected October 1999 were treated similar to the October 1998 samples except for the following deviations. The samples were placed into 400 ml artificial medium (ASP6 F2-without K2HPO4), 4 ml antibiotics, and an amount of K2HPO4 as detailed in Table 3.3. Table 3.3 Medium Conditions for the Acclimation Period for Fucus gardneri collected October 1999 Flask #* Medium**- K 2 H P Q 4 Algae 1 A S P 6 F 2 O.Oug/ml S 2 A S P 6 F 2 0.5'u.g/ml / 3 A S P 6 F 2 l .Oug/ml / 4 A S P 6 F 2 1.5ug/ml / 5 A S P 6 F 2 2.0ug/ml / 6 A S P 6 F 2 4.0 pg/ml • * all flasks were duplicated ** all flasks contained 4 ml antibiotics in 400 ml medium ~ without K 2 H P 0 4 After 14 days of acclimation, the bacterial levels were again minimal and were maintained that way by means of the antibiotic solution. The algae were rinsed separately 3 times with the K2HPO4 free artificial medium and placed into sterile flasks, which contained 200 ml K2HPO4 free medium, 2 ml antibiotics, 100 ug arsenate and selected levels of K2HPO4. Again the flasks were placed into the incubator at 15.0 ± 0.5 °C for the exposure experiment. See Table 3.4 for the exposure experimental treatment. 41 Table 3.4 Medium Conditions for the Exposure Period for Fucus gardneri collected October 1999 Flask #* Medium**- K 2 H P Q 4 Algae Arsenic 1 A S P 6 F 2 O.Opg/ml / 0.50 ppm As(V) 2 A S P 6 F 2 0.5pg/ml / 0.50 ppm As(V) 3 A S P 6 F 2 l .Oug/ml / 0.50 ppm As(V) 4 A S P 6 F 2 1.5ug/ml / 0.50 ppm As(V) 5 A S P 6 F 2 2.0ug/ml • 0.50 ppm As(V) 6 A S P 6 F2 4.0 pg/ml / 0.50 ppm As(V) * all flasks were duplicated ** all flasks contained 2 ml antibiotics in 200 ml medium ~ without K 2HP0 4 The exposure experiment was also run for 28 days and samples of the medium were taken twice a week during that period. The weight of Fucus used in the exposure experiment ranged from 14.90 to 26.65 g of wet weight per flask. 3.2.2.2.4 Fucus sardneri collected June 1999 and acclimated in seawater in the absence of antibiotics The Fucus samples attached to the rocks, were placed in an aquarium (size: 61 x 32 x 39 cm) divided into two sections and filled with non-sterile seawater (~ 10 L) for the acclimation period. One section contained two rocks with the young Fucus attached and other species (blue mussels, snails, barnacles, etc.) as well as sediment (sand) and one unknown clam species that was collected with the rocks. The other section only contained two rocks with young Fucus attached and no sediment. The seawater was removed in both sections every 5 to 6 days and new seawater was added to the aquarium after a 2-4 hour drying period. After 12 days of acclimation, the salinity of the seawater was changed from 6 ppt to 28 ppt by adding Instant Ocean synthetic seasalt (no 42 nitrate and phosphate). The clam in section one died after a week in the concentrated seawater, so the seawater was switched to 8 ppt for the last week of acclimation. The acclimation period was stopped after 4 weeks. There was observable bacterial contamination of the algae samples since the tips of algae were breaking apart. Samples of the seawater and Fucus were collected both before the seawater was changed and several hours after the fresh seawater was added. 3.2.2.2.5 Fucus gardneri collected August 1999. acclimated in seawater. and exposed to As("V) in seawater in the absence of antibiotics These samples collected on August 1999 attached to the rocks were grown in a similar manner as described for the June collection. After one week of acclimation, the seawater was removed from both sections and the exposure experiment started. Fresh (unsterile) seawater (~ 10 L) was added to each section, while 5 mg of arsenate was only added to section 1 of the aquarium (section 2 was the control). After one week of exposure, the seawater was removed in both sections and new seawater was added with only arsenate in section 1. The exposure experiment was repeated for two more weeks with the seawater and arsenate changed once every week. After 3 weeks of exposure, the algae in both tanks were falling apart and the seawater was milky, indicating strong bacterial growth with the algae. Samples were taken of the seawater and Fucus before the seawater was changed and several hours after new seawater was added. 43 3.2.3 Sample Preparation A l l medium samples taken during the experiments were frozen immediately to preserve sample integrity until the time of the analysis. The Fucus gardneri samples collected after both the acclimation and exposure periods were rinsed separately, weighed, frozen and then freeze-dried. The freeze-dried samples were kept at -20 °C until they were extracted using a 1:1 methanol/water solution. The extraction procedure used is described in Section 2.3.2 The medium and extract samples were filtered through 0.45 jam syringe filters (Millipore) and analyzed by using the H P L C - I C P - M S operating conditions given in Table 2.1. The data from the I C P - M S were processed by using chromatographic software and identification of arsenicals in the samples was made by comparison of retention times with those of standards. Concentrations of arsenic compounds were determined by using compound specific external calibration curves when possible. D M A was used as a standard for arsenosugars. 3.2.4 Culture and Medium Conditions The Fucus samples that were collected from the sampling sites underwent a rigorous treatment to make them as axenic as possible (except for the Fucus attached to the rocks). Antibiotics and other decontamination procedures were included in an attempt to make sure no living organisms, other than the Fucus, were involved in the exposure experiments. However, the use of antibiotics and multiple washings was not completely successful because a fungus was observed to grow with the Fucus during both the acclimation and exposure experiments. Nonetheless, the contamination was at a minimum because the media did not become cloudy or milky (a common sign of bacterial infection). It is probable that the fungus could affect the way 44 in which the Fucus took up the arsenic compounds from the medium, but as w i l l be described below arsenate is only slowly reduced by the fungus. Other more rigorous techniques for cleaning the algae could have been used, but many procedures would result in destroying the algal cells. The Fucus appeared to remain intact and healthy looking throughout most of the experiment. However, at the end of the experiment the Fucus appeared wilted and darker in colour (brownish/green) than when freshly collected. Since the media was not changed during the acclimation period and during the arsenic exposure period, the algae samples were likely experiencing starvation conditions at the end of each period. The culture media also became increasingly coloured (ranging from light orange to dark orange) over the course of the experiment. Notably, the medium containing the arsenic(V) species with the algae was of a darker orange colour indicating greater pigment loss than the control (medium with algae). The Fucus growing on the rocks rapidly became contaminated with bacteria and fungi and the Fucus did not remain intact or healthy looking until the end of the exposure experiment. The media changed colour and became milky very quickly after being added to the aquarium. 45 3.3 R E S U L T S O F E X P O S U R E O F FUCUS GARDNERI T O ARSENIC(V) IN DIFFERENT M E D I A 3.3.1 Introduction Seawater is an aqueous solution of sodium chloride and various other mineral salts such as magnesium sulphate. There are also dissolved gases (such as carbon dioxide and oxygen) and organic substances in the solution. The salinity of the ocean water amounts to approximately 36 parts per thousand (ppt). But it can range from 3 ppt in most part of the G u l f of Bothnia to approximately 60 ppt in Shark's Bay (Northwest Australia). 2 8 The varying salinity in the different parts of the ocean can be of significance for the distribution and occurrence of algal vegetation in different regions of the sea. For example, Fucus gardneri is mostly found on the shoreline where there is a lower salinity, while Nereocystis luetkeana is found in the deeper 78 oceans which has a higher salinity. Salinity not only affects the distribution of the algae, but also affects the uptake and transformation of the compounds found in the seawater. Changes in salinity affect the osmotic relationship of algal cells and the medium in which they are found. 5 9 Salinity is not the only factor that contributes to the plant response, other chemical factors such as p H , temperature and nutrient levels are also involved. The compounds found in the media have a measurable influence on the growth rate of seaweed and this affects the uptake of compounds from the surrounding environment. The following Sections describe the effects that different media had on the uptake of arsenic(V) by Fucus gardneri that was collected October 1998 and February 1999. 46 3.3.2 Results from Fucus gardneri Acclimated in Seawater or Artificial Seawater, and Exposed to Arsenic(V) in Seawater or Artificial Seawater 3.3.2.1 Acclimation Period The young algae collected from Stanley Park were treated with antibiotics and allowed to acclimate for 14 days as described in Section 3.2.2.2.1. Sub-samples were collected and processed as described in Section 2.3. The extract samples were analyzed by using ion-pairing H P L C - I C P - M S (Table 2.1). The predominant arsenicals found in the extracts of the Fucus samples, before the acclimation, were M M A , arsenosugars X , XII and XIII (Figure 3.3). During the acclimation period of the Fucus in the seawater (Table 3.1-batch #1), the total concentration of the extractable arsenic compounds decreased over the 14 days (Figure 3.3). The majority of the arsenic compounds, in the algae, were most likely lost into the medium in the form of water-soluble arsenic species. The total concentration of the extractable arsenic compounds from the Fucus in the A S P 6 F2 medium (Table 3.1-batch #2) also decreased over the 14 days, but not as much as the Fucus in the seawater (see Figure 3.4). Analysis o f the chromatographic data shows that the Fucus in the seawater medium retained ~ 26.8 % of the extractable arsenic content, while the Fucus samples in the artificial medium (ASP6 F2) retained - 3 8 . 0 % of the extractable arsenic content at the end of the 14 days of acclimation relative to the algae in the beginning of the experiment. The results from the analysis of the chromatographic data are shown in Table 3.5. 47 Figure 3.3 Fucus Extract before and after 14 days of Acclimation in Seawater 30000 25000 20000 Q. 15000 o 10000 5000 XII 200 400 600 800 Retention time(s) 1000 young algae before acclimation algae after 14 days of acclimation in seawater Figure 3.4 Fucus Extract before and after 14 days of Acclimation in ASP6 F2 Medium 30000 25000 20000 (A g. 15000 10000 5000 , XII 200 400 600 Retention time(s) • young algae before acclimation algae after 14 days of acclimation in ASP6 F2 medium 800 1000 48 3.3.2.2 Exposure Period After 14 days of acclimation, the Fucus samples were washed with sterile seawater and antibiotics. The samples were then incubated with 500 ppb of As (V) in seawater or artificial seawater (see Section 3.2.2.2.1 and Table 3.1). The Fucus samples were collected at the end of 28 days and processed as described in Section 2.3. , The extract of the samples from the seawater medium (Table 3.1-batch # 1), obtained at the end of the arsenate exposure experiment, showed an increase in the amount of arsenite, dimethylarsinate and/or arsenate, and arsenosugar X , with a decrease in M M A and arsenosugars XII and XIII relative to the Fucus samples at the beginning of the exposure experiment (see Figure 3.5 and Table 3.5). The controls consisted of Fucus samples that were treated under the same conditions as the above samples, except they were not exposed to As(V) . The extracts of the controls showed a decrease in M M A and arsenosugars XII and XIII (see Table 3.5 and Figure 3.6). These results are similar to that of the exposed Fucus. 49 Figure 3.5 Fucus gardneri Exposed to As(V) in Seawater Medium 40000 35000 30000 25000 g. 20000 15000 10000 5000 0 As(V) or DMA 200 400 600 800 Retention time(s) • algae after 14 days of acclimation in seawater algae after exposure to As(V) in seawater 1000 Figure 3.6 Controls for the Exposure Experiment 12000 10000 8000 Q. 6000 o 4000 2000 MMA algae after acclimation algae after exposure period with no As(V) 100 200 300 400 500 600 700 800 900 1000 Retention time(s) The extracts of the Fucus samples from the exposed experiment in A S P 6 F2 medium (Table 3.1-batch #2), showed an increase in dimethylarsinate and/or arsenate and arsenosugar X with a decrease in arsenosugars XII and XIII relative to the Fucus samples from the beginning of the exposure experiment (see Figure 3.7). Again, the controls showed a decrease in M M A , arsenosugars XII and XIII as in the seawater experiment (Table 3.5). Figure 3.7 Fucus gardneri Exposed to As(V) in ASP6 F2 Medium 30000 25000 20000 (A g. 15000 10000 5000 DMA or As(V) 200 400 600 Retent ion time(s) 800 1000 • algae after 14 days of acclimation in ASP6 F2 medium algae after exposure to As(V) in ASP6 F2 medium Analysis of the chromatographic data was performed by using the calibration curves for arsenic standards (Section 2.4.2). The results obtained are shown in Table 3.5. 5 1 Table 3.5 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) following Acclimation and As(V) Exposure in Seawater or Artificial Seawater Algae Sample Arsen ic species found As(III) M M A D M A & A s ( V ) X X I I XIII before acclimation 0 0.83 ± 0.2 a seawater medium after 14 days o f acclimation 0 0.68 ± 0.2 control-no A s ( V ) exposure 0 trace after exposure to A s ( V ) 0.97 ± 0.2 trace ASP6 F2 medium after 14 days o f acclimation 0 0.20 ± 0.02 control-no A s ( V ) exposure 0 trace after exposure to A s ( V ) 0 trace 0 0.32 ± 0 . 0 8 2.85 ± 0 . 7 1.56 ± 0 . 4 0 0.04 ± 0 . 0 1 0.40 ± 0 . 1 0.29 ± 0 . 0 7 0 0.08 ± 0 . 0 2 0.21 ± 0 . 0 5 0.11 ± 0 . 0 3 1.38 ± 0.3 0.36 ± 0 . 0 9 0.21 ± 0 . 0 5 0.13 ± 0 . 0 3 0 0.15 ± 0 . 0 4 0.84 ± 0 . 2 0.91 ± 0 . 2 0 trace trace 0.10 ± 0 . 0 2 0.75 ± 0.2 0.26 ± 0.06 0.38 ± 0 . 0 9 0.12 ± 0 . 0 3 a s c = standard deviation from analytical results obtained with the calibration curve 6 0 "Trace" amounts are greater than or equal to the detection limit The medium samples collected over the period of the exposure experiment were examined by using anion-exchange H P L C - I C P - M S (Table 2.1) and the results from the chromatographic data are shown in Figures 3.8 and 3.9. A s can be seen from Figure 3.8, the seawater samples showed a rapid decrease in As (V) while the As(III) increased accordingly. After 15 days of exposure, the As(V) increased again, accompanied by a decrease in As(III). D M A showed a slow overall increase. The algae may have taken up the As (V) into the algal cells.by an active transport system and reduced it into As(III). This would account for the decrease in As(V) and increase in As(III) that was seen in the medium. However, after 15 days the active transport system may have shut down due to insufficient nutrients in the medium, thus no more As (V) would be taken into the algal cells. The increase of As (V) in the medium at this point is most likely due to oxidation of As(III) to As(V) . 52 Figure 3.8 Seawater Samples collected during the Exposure of Fucus gardneri to Arsenic(V) 600 -100 J Time(days) The arsenic speciation in artificial seawater showed a completely different trend. The arsenate in the seawater slowly decreased, while D M A and As(III) slowly increased over the term of the experiment (see Figure 3.9). 53 Figure 3.9 ASP6 F2 Medium Samples collected during the Exposure of Fucus gardneri to Arsenic(V) 600 -100 J Time(days) The marked difference between the two experiments is probably due to the difference in the salinity of the two media. A diminished salinity w i l l most probably cause a reduction in the 9 8 photosynthetic activity and an increase in the rate of respiration. The salinity of the seawater was measured to be ~8 ppt while the artificial seawater had a salinity of ~ 28 ppt. The lower level of salinity in the seawater most likely caused the algal cells to take up and reduce more As(V) than the algal cells in the artificial seawater. This might also explain the higher concentration of A s ( V ) / D M A and As(III) that was seen in the algae from the seawater extracts compared to the A S P 6 F2 medium extracts (Table 3.5). In addition, numerous examples have shown that periods of rapid growth of seaweed occur during times when the external nutrient concentrations are relatively low. Uptake in higher plants (and possibly Fucus) is dependent on growth rates. That is, the greater the growth 54 rate, the greater the uptake rate wi l l be . 3 5 , 6 1 In the case of the seawater exposure experiment, the seawater had a lower nutrient concentration compared to the artificial seawater (level of salinity), which might have caused the algal cells to grow more rapidly in the seawater, causing it to take up more arsenate from the surrounding medium. The arsenate in the medium is most likely taken up by the algae by the same active transport mechanism as phosphate and reduced to As(III). The arsenite might then be further transformed into other compounds, such as M M A and D M A . In the absence of an active transport system these compounds are then excreted into the medium by passive diffusion. A t low levels of phosphate in the medium, more arsenate is taken up into the algae because the uptake is probably competative. 5 ' 1 0 ' 2 5 The low levels of salinity, low levels of external nutrient concentration, or possible low levels of phosphate in the seawater, might have resulted in enhanced arsenic uptake. A n experiment designed to probe the effects of phosphate on arsenate uptake is discussed in Section 3.4. 55 3.3.3 Results from Fucus gardneri Acclimated in Seawater and Exposed to Arsenic(V) in Artificial Seawater 3.3.3.1 Acclimation Period The algae collected from Bamfield Marine Station were acclimated in seawater for 14 days and samples collected as described in Section 3.2.2.2.2. The total concentration of the arsenic compounds in the Fucus extracts decreased during the acclimation period (see Figure 3.10). Presumably, they were lost in the form of water-soluble arsenic species. The predominant arsenicals found in the extracts of the Fucus samples, before and after acclimation, were arsenosugars X , XII and XIII. The absence of M M A , seen in the Stanley Park samples, should be noted. 56 Figure 3.10 Fucus Extracts before and after 14 days of Acclimation in Seawater 400000 350000 300000 250000 g- 200000 150000 100000 50000 0 XII 200 400 600 Retention time(s) 800 - young algae before acclimation algae after 14 days of acclimation in seawater 1000 Analysis of the chromatographic data shows that the Fucus extracts at the end of 14 days of acclimation retained ~ 27.5 % of the extractable arsenic content compared to the young algae in the beginning of the experiment. The data are shown in Table 3.6. 3.3.3.2 Exposure Period The Fucus samples were washed with sterile seawater and antibiotics after 14 days of acclimation and then were incubated with 500 ppb of As(V) in ASP6 F2 media (see Section 3.2.2.2.2 and Table 3.2). The extracts of the samples obtained at the end of the arsenate exposure experiment, showed an increase in the amount of arsenite, dimethylarsinate, arsenate and arsenosugar X , with 57 a decrease in arsenosugars XII and XIII relative to the Fucus from the beginning of the exposure experiment (see Figure 3.11 and Table 3.6). The controls for this experiment showed a similar decrease in arsenosugars XII and XIII as compared with the Fucus samples that were exposed to arsenate (Table 3.6). Figure 3.11 Fucus gardneri Exposed to Arsenic(V) in ASP6 F2 Medium 300000 i 250000 200000 g. 150000 100000 50000 0 As(lll) 200 400 600 Retention time(s) • algae after 14 days of acclimation in seawater algae after exposure to As(V) in ASP6 F2 medium A t 800 1000 The analytical data obtained by using the calibration curves for arsenic standards (Section 2.4.2) are summarized in Table 3.6. The results compare relative amounts of each arsenic compound found in the Fucus samples before and after the exposure experiment. 58 Table 3.6 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) following Acclimation in Seawater and Exposure to As(V) in Artificial Seawater Algae Sample Arsen ic species found As(III) D M A A s ( V ) X X I I XIII before acclimation 0 0 0 0.41 ± 0 . 0 5 4.62 ± 0 . 6 0.51 ± 0 . 0 7 after 14 days o f acclimation 0 0 0 0.06 ± 0 . 0 1 1.24 ± 0 . 2 0.16 ± 0 . 0 2 after exposure period control-no A s ( V ) exposure 0 0 0 0.04 ± 0 . 0 1 0.45 ± 0.06 0.13 ± 0 . 0 2 after exposure to A s ( V ) 2.59 ± 0 . 3 a 0.75 ± 0 . 1 2.65 ± 0 . 3 0.37 ± 0 . 0 5 0.50 ± 0 . 0 6 0.15 ± 0 . 0 2 'sc= standard deviation from analytical results obtained with the calibration curve The medium samples collected over the period of the exposure experiment were tested using the anion-exchange H P L C - I C P - M S condition (Table 2.1) and are shown in Figure 3.12. The medium samples showed a rapid reduction in As(V) in the first twelve days of exposure, while D M A and As(III) increased continuously. After the twelve days, the concentration of the arsenic species in the medium stayed relatively constant. 5 9 Figure 3.12 ASP6 F2 Medium Samples collected during the Exposure of Fucus gardneri to Arsenic(V) The biotransformation of As(V) by the algae in this experiment is different from both the seawater/seawater and artificial seawater/artificial seawater experiments (see Figure 3.8 and Figure 3.9). In all 3 experiments the organoarsenic compounds found in the algae were lost considerably during the acclimation period in the particular medium. The algae from the seawater/seawater experiment lost ~73.2 % of the original organoarsenic compounds during the acclimation period. The algae then took up the arsenate quite rapidly from the medium in the first 15 days. A n almost equivalent amount of As(III) was found in the medium and some was also found in the algae extracts. D M A increased slowly in the medium and was also found in the extract sample. 60 The algae from the artificial seawater/artificial seawater experiment lost - 6 2 . 0 % of the organoarsenic compounds during the acclimation period. Arsenate was then taken up much slower from the medium. Both D M A and As(III) were found in the medium, however, D M A was higher in concentration than As(III) in the medium and it was the only species found in the extract sample. The algae from the seawater/artificial seawater experiment lost - 72.5 % of the original organoarsenic compounds during the acclimation period. As (V) was taken up rapidly by the algae from the medium, as was true for the seawater/seawater experiment, however As(III) increased continuously and slowly, while D M A increased rapidly in the medium to achieve a higher concentration than As(III). Both D M A and As(III) were observed in the extract samples. Arsenosugars X , XII and XIII were found in the algae extracts in all 3 experiments. The concentration of these organoarsenic compounds decreased during the acclimation period. During the exposure period, arsenosugar X was the only one that increased, while the other two decreased in all 3 experiments in the algae extracts. The reason for the differences between the three experiments might be due to the different media used, different conditions of the media (salinity levels) or because of the fact that the algae were collected in different places at different times in the season. 61 3.4 R E S U L T S O F E X P O S U R E O F FUCUS GARDNERI T O ARSENIC(V) IN D I F F E R E N T P H O S P H A T E C O N C E N T R A T I O N MEDIA 3.4.1 Introduction As mentioned before arsenic is present in the natural environment and living organisms in different chemical forms.43 In the marine environment, high concentrations of arsenic occur in macroalgae with concentration factors of 1000 to 10,000 reported.62 Few studies have been reported, however, on either the mechanism of arsenic accumulation, or on the pathways through which the arsenic is accumulated. It has been suggested that arsenate enters living cells via an active phosphate transport system that is not able to distinguish between the arsenate and phosphate species.5'10 In oxygenated seawater, H2PO4" and H2ASO4" would be the predominant species of phosphorus and arsenic. However, the situation with regard to algal uptake is not clear, with several apparently 9 S conflicting reports in the literature. For example, although arsenate and phosphate were shown to compete for uptake by unicellular algae, a related study with two species of brown macroalgae could provide no evidence for a common uptake mechanism. ' ' At low phosphate levels, arsenate uptake by unicellular algae increased with increasing phosphate concentrations, presumably as a consequence of increase algae metabolism.2 5'6 3 In a study of several natural algal populations, the algae readily assimilated arsenate even though phosphate concentrations 25 remained high, indicating that arsenate uptake was not contingent upon phosphate depletion. Thus it was of interest to determine the affect of phosphate on arsenate uptake by Fucus gardneri. 62 3.4.2 Results and Discussion 3.4.2.1 Acclimation Period The young algae samples collected from Stanley Park were cleaned, treated with antibiotics, and placed in medium with different phosphate concentrations for the acclimation period as described in Section 3.2.2.2.3. Sub-samples were collected and processed as described in Section 2.3. The extract samples were analyzed by using ion-pairing H P L C - I C P - M S (Table 2.1). The major arsenicals found in the Fucus extract samples before the acclimation period were arsenosugar XII and XIII (Figure 3.13 and Table 3.7). Other arsenic compounds that were detected were As(III), D M A , As(V) and arsenosugars X and X I . After acclimation, the algal extracts showed a decrease in D M A and a slight decrease in arsenosugars XII and XIII. A l l other arsenic species in the algae extracts stayed relatively the same compared to the algae from before the acclimation period independent of the phosphate concentrations (see Figure 3.13 and Table 3.7). 63 Figure 3.13 Arsenic Species found in Fucus gardneri Extracts before and after 14 days of Acclimation DMA, Arsenate and Arsenite 0.18 0.16 0.14 0.12 •o E a Q. C | £ 0 1 0 c •f 0.08 fl) > o > c o u M 8 o § Q. (0 0.06 0.04 0.02 0.00 0.02 I DMA lAs(V) E3 As(lll) Phosphate concentration (ppm) . 1.8 i. 1.6 Q. ~ 1.4 •j: C-1.2 £ f 1-0 3 * 0 . 8 c ^ 8 -O0.6 8 0.4 0.2 0.0 4-J Arsenosugars IXIII IXII • XI • X Phosphate concentration (ppm) 64 Table 3.7 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after Acclimation in different Phosphate Concentration Media Algae sample Arsen ic species found As(III) D M A A s ( V ) X X I X I I XIII before acclimation 0.01 0.14 0.02 0.10 0.21 1.36 1.64 algae + 0.0 ug/ml K 2 H P 0 4 trace 0.09 0.01 0.13 0.19 0.67 1.20 algae + 0.5 ug/ml K 2 H P 0 4 0.01 0.10 0.03 0.11 0.21 1.21 1.36 algae + 1.5 ug/ml K 2 H P 0 4 trace 0.11 0.03 0.10 0.13 0.78 0.84 algae + 2.0 ug/ml K 2 H P 0 4 trace 0.10 0.02 0.15 0.25 1.04 1.23 algae + 4.0 ug/ml K 2 H P 0 4 0.01 0.11 0.02 0.10 0.00 1.56 1.25 "Trace" amounts are greater than or equal to the detection limit 3.4.2.2 Exposure Period After 14 days of acclimation, the Fucus samples were washed separately with K2HPO4 free artificial medium. The samples were then placed in flasks containing K2HPO4 free media, 2 ml antibiotics, 500 ppb of As(V) and selected levels of K2HPO4 (see Section 3.2.2.2.3 and Table 3.4). The Fucus samples were collected at the end of 28 days and processed as described in Section 2.3. The algae collected after the exposure period showed an increase in all arsenic compounds (As(III), M M A , D M A , As(V) and arsenosugars X through XII), except for arsenosugar XIII which decreased slightly. It can be seen that as the concentration of phosphate increased, the amount of arsenosugar XII produced in the algae decreased. Similar results were also seen for arsenosugars X , and X I . The inorganic compounds found in the algae extracts vary widely as the phosphate concentration increased and does not seem to follow a trend (see Figure 3.14 and Table 3.8). 65 Figure 3.14 Arsenic(V) Speciation of Fucus gardneri Samples following Exposure in different Phosphate Concentration Media DMA, MMA, Arsenate and Arsenite 12.0 I DMA I MMA SAs(V) • As(lll) 0.5 1.5 2 Phosphate concentration (ppm) Arsenosugars 0.5 1.5 2 Phosphate concentration (ppm) Table 3.8 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after As(V) Exposure in different Phosphate Concentration Media Algae sample Arsen ic species found As(III) M M A D M A As (V ) X X I XI I XIII algae + 0.0 ug/ml K 2 H P 0 4 9.59 2.24 0.95 0.93 0.48 1.19 2.67 0.62 algae + 0.5 ug/ml K 2 H P 0 4 3.37 0.95 0.59 0.80 0.36 0.75 2.31 0.58 algae + 1.5 ug/ml K 2 H P 0 4 6.77 2.01 6.65 1.22 0.36 0.49 2.31 1.05 algae + 2.0 ug/ml K 2 H P 0 4 6.49 1.99 2.59 0.94 0.26 0.42 1.56 0.54 algae + 4.0 ug/ml K 2 H P 0 4 1.77 0.65 0.69 1.71 0.21 0.23 0.76 0.29 "Trace" amounts are greater than or equal to the detection limit Samples of the media collected were analyzed using the anion-exchange H P L C - I C P - M S condition (Table 2.1). A s can be seen from Figure 3.15, the rate of arsenate uptake depended on the concentration of phosphate present in the medium. A t low phosphate concentrations (0.0 and 0.5 pg/ml), more arsenate was taken up by the algae and at high concentrations less arsenate was taken up by the algae. The two species that were formed and excreted by the algae were As(III) and D M A (see Figure 3.16 and Figure 3.17 respectively). The amount excreted depended on the amount of phosphate present in the medium. A t high concentrations,' lower amounts of As(III) and D M A were seen in the medium, while at lower concentrations, higher amounts of As(III) and D M A were seen. This might be because of the fact that at low phosphate concentrations more arsenate is taken up by the algae, which wi l l convert more arsenate into As(III) and D M A . 67 Figure 3.15 Arsenate (As(V)) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 120 i -•—0.0 ppm K2HP04 •«—0.5 ppm K2HP04 — 2.0 ppm K2HP04 •—4.0 ppm K2HP04 0 5 10 15 20 Time (days) Figure 3.16 Arsenite (As(III)) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media 68 Figure 3.17 Dimethylarsinate (DMA) variation in Medium Samples collected during Uptake of Arsenic(V) in different Phosphate Concentration Media The data shows that the concentration of phosphate in the medium affects the transformation of arsenate added to the medium. If arsenate and phosphate utilize a common uptake mechanism, as was suggested, then uptake wi l l be competitive. A s the relative concentration of phosphate increases, more phosphate wi l l be taken up than arsenate and vice versa. This is supported by the inhibition of arsenate transformation in the presence of high phosphate levels (2.0 and 4.0 ppm). Also , variation of phosphate (0.0 to 4.0 ppm) concentrations with arsenate produced concentration-dependent inhibition of arsenate uptake (see Figure 3.15). The reduction of As(V) to As(III) is also most likely depended on the concentration of phosphate. The phosphate is most likely needed to drive the reduction (compare reduction of As(III) in 0.0 ppm to 0.5 ppm K2HPO4 medium), but too much wi l l inhibit it. The same can be said of the methylation of As(III) to D M A . Phosphate is most likely needed to drive the 69 metabolism but too much leads to inhibition of methylation. The results obtained for this phosphate-arsenate interaction was limited, but they indicate an associated uptake of phosphate and arsenate by marine macroalgae Fucus gardneri. 70 3.5 R E S U L T S O F E X P O S U R E O F FUCUS GARDNERI T O ARSENIC(V) IN T H E A B S E N C E O F ANTIBIOTICS 3.5.1 Introduction The establishment of unialgal and axenic cultures is an important step in the culture of protoplasts, isolated cells, and tissues from seaweeds. In the presence of enriched media, contaminated seaweed materials are often overgrown with bacteria, fungi, or epiphytes. Bacteria and fungi affect the cultures either by competing for nutrients, by changing the physical state of the medium, or by releasing substances that inhibit growth or are toxic towards the plant. 5 3 ' 6 4 Epiphytes often show rapid growth and can completely cover the cultured tissue, resulting in unsuitable subjects for experimentation. Therefore, a variety of methods can be used to obtain axenic cultures. The methods for obtaining axenic materials may be divided into three categories: treatment with disinfectants; treatment with antibiotics; or a combination of treatment with r i disinfectants and antibiotics. The method chosen for this project was treatment with antibiotics, because it was found that disinfectants are not suitable for the algae: the treated tissues are either damaged 5 1 ' 5 2 or eventually k i l l ed . 5 3 The maximal possible exposure time to antibiotics has to be slightly longer than the exposure time required to k i l l the contaminant micro-organisms and bacteria attached to the algae. Therefore the side-effects of antibiotics on the growth and development of algae must be taken into account. It has been reported that antibiotics have a special activity against certain green and blue-green algae. 6 5 In land plant tissue, cell and callus cultures, some antibiotics were 71 shown to inhibit R N A and D N A synthesis and other systems. Thus it was of interest to conduct an arsenic exposure experiment without the influence of added antibiotics. 3.5.2 Results and Discussion 3.5.2.1 Acclimation Period The young algae collected on the rocks from Stanley Park were grown for four weeks in an aquarium in seawater, but with no added antibiotics as described in Section 3.2.2.2.4. The aquarium also contained rocks, sediments, snails, mussels, barnacles, small crabs, green algae, and red algae collected at the same time. Algae sub-samples were collected before and after adding new seawater every 5 to 6 days. These samples were processes as described in Section 2.3. The total extractable arsenic compounds in the algae collected showed a general increase over the 19 days of acclimation. However, this trend was best seen when the seawater was replaced, because the total amount of the extractable arsenic compounds increased after new seawater was added to each tank and then decreased again after some time in the new seawater (Table 3.9). This might indicate that the algae are obtaining arsenic compounds from the fresh seawater. The type of arsenic species is unknown since very small amounts of arsenic or none at all were detected in the seawater collected from Stanley Park. 72 Table 3.9 Arsenic Speciation of Fucus gardneri Extracts (ppm, dry weight) after Acclimation in Seawater with no added Antibiotics Algae Sample Arsen ic species found As(III) M M A D M A As (V ) X XI I XIII day 0 0.18 0 0 0 1.68 5.80 1.05 day 2 (after new seawater) 0 0.41 0 0 5.28 16.74 2.89 day 5 0 0.40 0 0.23 3.68 12.43 2.05 day 12 0 0.38 trace 0.41 3.20 9.09 2.04 day 13 (after new seawater) 0 trace 0.20 0.18 14.76 12.08 2.65 day 18 0 0.45 trace 0.43 2.37 8.11 2.58 day 19 (after new seawater) 0 trace 1.15 0.23 0.51 25.75 5.22 "Trace" amounts are greater than or equal to the detection limit This experiment is only similar to the algae experiments with added antibiotics when the seawater was not replaced and the total amount of the extractable arsenic species decreased. Apart from that, this experiment was different since the total extractable arsenic species increased every time the seawater was replaced. 3.5.2.2 Exposure Period In a separate experiment, young algae collected on the rocks from Stanley Park were allowed to acclimate for 6 days in non-sterile seawater before exposure to arsenic(V). Fresh seawater containing 500 ppb As(V) was added to the algae, after the 6 days of acclimation (see Section 3.2.2.2.5). Algae sub-samples and seawater samples were collected before and after the seawater was changed. These samples were processed as discussed in Section 2.3. Table 3.10 shows the data obtained for this experiment. The extracts of the samples obtained after the first day of addition of seawater and As(V) (t=0) showed an increase in As(III), 73 D M A and As(V) along with an increase in arsenosugars XII and XIII compared with the algae before and after acclimation. After six more days of exposure, the extracts of the samples obtained showed a decrease in all the extractable arsenic compounds; however, the seawater at this point showed an increase in As(III) and D M A with a decrease in As(V) . This might indicate that the algae took up the arsenate from the seawater and transformed it into As(III), D M A and arsenosugars as outlined in Chapter 1, Figure l . l . 2 5 ' 4 1 These compounds might then have been released into the seawater. After the addition of new seawater and As(V) on day 6, similar results were seen in the algae and seawater as described above. That is, after one day in the fresh seawater (t=7), the algae extracts showed an increase in As(III), D M A , As (V) and arsenosugars XII and XIII compared with the algae before the fresh seawater was added (t=6). However, the increase in concentration in these compounds was not as high as before which might indicate that the algae were dying or being taken over by the bacteria and fungi (seawater was becoming milky after 6 days which is a common indication of contamination). The algae extracts after being in the seawater for another 7 days showed a decrease in As(III), D M A , As (V) and arsenosugars XII and XIII as before, except in this case an increase in M M A and arsenosugars X and X I were also seen. The seawater after the 7 days again showed an increase in As(III), M M A and D M A compared with the fresh seawater with As(V) that was added in the beginning. The seawater was replaced one final time with fresh seawater and As(V) , and similar results were again obtained as above, but the concentrations of the different arsenic species seen in the algae extract were lower than the concentrations in the algae before the fresh seawater was added. 74 X X X > on < B .2 2 a E o co in oo o ON o o ( N O O ON o q o a a S * 1 s •B « .5 is u <j a •t 1- — ( N O o O ^ ca CN r o ™ N O CN 0 0 o o o CO CB is h CN HO r o N O t N r o t I 5 "a s « § O . .5 £ ^ JL •a ^o ca D CS ' « CO ca T3 — N O S u m S U M £ 6 0 OX) CD O O — O —' CN o o <0 0 0 ±3 O <U 0 0 t N - t N ° ° ° ° VO t N 0 0 CN r o r o V") ON © © ON f-r o CN N O —I oo f^-0 0 O N CO I-,. g So is o r o CN CN NO « a •e a NO JL CO o ca JL ca ca •o -a op op " « "ca ^ ca > > ca ca S I ) I ) C3 C/5 W5 1) 1) -4-* ca ca CL> 60 60 O © o © © O © <N O r o CN CN © © CN © © ^ O 0 0 ON © t S C r o g IL C/J S; r o ca .a T3 o ca a §3 u 1) ca ca ^ IS £ Q W t/l ca co ««, 60 >, t-ca ca •O « .S c 2 o •§ I +-» TO O « CO J— The difficulty in growing algae without antibiotics under laboratory conditions is that bacteria and fungi often show rapid growth in the medium and their presence can influence the outcome of the experiments in many ways . 5 3 ' 6 4 So even though it is not quite possible to state which species were involved in the uptake and transformation of arsenate, it should be noted that similar results were seen to those obtained from the antibiotic treated systems in that As (V) was taken up by the algae, reduced to As(III) and further transformed into M M A and D M A (compare Table 3.5 to Table 3.10). 76 3.6 S U M M A R Y This investigation has shown that the marine macroalgae Fucus gardneri is able to bioaccumulate and biomethylate arsenic compounds from the surrounding medium under various environmental conditions. The variable conditions in the medium that were examined for this investigation were variations in salinity, phosphate, and antibiotic concentrations. In all cases, the algae accumulated the arsenate from the medium but the concentration depended on environmental factors. This arsenical in the algal cells then transformed into several arsenic species. The major species found in the medium and algae extracts were As(III), D M A and As (V) with some small amounts of arsenosugars. None or trace amounts of M M A was also found in the all of the experiments. The reduction of arsenate to arsenite is the major process encountered. The algal cells first take up the arsenate from the medium presumably via the phosphate transport system, reduce the arsenate to arsenite inside the cells by using thiols and/or dithiols, and excrete most of the arsenite into the growth medium by means of an active transport system. 5 ' 1 0 ' 2 5 Some arsenite retained in the cells is methylated to M M A by using S A M ; however, because of the low passive diffusion coefficient 6 6 ' 6 7 , the M M A is not excreted to the growth medium, but remains in the cell where it is more likely to be reduced and further methylated to D M A . This arsenical is lost to the medium, presumably by means of passive diffusion. The D M A that is retained in the algal cells appear to be further reduced and methylated into arsenosugars by using S A M as the methylating agent (Figure l . l ) . 2 4 ' 2 5 It is not surprising to see that little or no M M A is detected when the algae is treated with arsenate. L i 2 4 reported that only traces of M M A were produced from arsenate by whole cell 77 cultures of Apiotrichum humicola and that M M A is the least transformed arsenical substrate. It was tentatively concluded that M M A would not be found as a free intermediate. The biomobility of M M A and D M A have been studied by measuring the rate at which these arsenicals diffuse through the walls of liposomes, regarding the models for biological membranes. 6 6 , 6 7 It was found that the membranes are less permeable to M M A than D M A . The diffusion coefficient of M M A is 10 times lower than D M A . Because of the low diffusion coefficient, it is possible that the cells metabolize the M M A to D M A faster than the M M A can diffuse into the growth medium. The D M A thus produced has a higher permeability coefficient, and can be excreted by the algal cells into the growth m e d i u m . 2 4 , 6 6 , 6 7 This seem to be consistent with what was observed in the above experiments. 78 4 B I O M E T H Y L A T I O N A N D B I O A C C U M U L A T I O N OF A R S E N I C A L S B Y M A R I N E A L G A E NEREOCYSTIS LUETKEANA 4.1 INTRODUCTION Nereocystis luetkeana commonly known as bull kelp, is one of the largest brown alga and can grow up to 30 meters long (see Figure 4.1). 3 3 It is found in sub-tidal waters as deep as 30 to 60 meters and constitutes a unique ecosystem largely restricted to the west coast of the Americas, and best represented in California's coastal waters. 2 9 The colour of Nereocystis is olive green and it is characterized by a long stipe, up to 25 meters long, with two clusters of flat dichotomously divided blades attached to an enlarged hollow spherical float. There can be up to 20 blades in each cluster (see Figure 4.1). The mature Nereocystis possesses receptacles at the end of the blades, which are the fertile areas. The spherical float (bulb) is filled with carbon monoxide gas, which provides a lift to the kelp's blades allowing it to form the dense canopies I T observed off the Pacific Coast. Only a few large mature plants were found at the sampling site of Bamfield, Vancouver Island. 79 Figure 4.1 Nereocystis luetkeana Postels and Ruprecht 3 3 a, habit o f mature plant showing attachment to substrate; b, habit o f two juvenile plants; A s mentioned before, arsenate is the main arsenic compound found in the seawater and appears to be the major source of the arsenic compounds found in marine algae. A sample of the bull kelp was found to contain ~ 4 times more total arsenic per weight than Fucus gardneri. This suggests that the Nereocystis is capable of taking up four times more arsenic from the surrounding environment than Fucus gardneri. To test this hypothesis, one of the arsenate exposure experiments was repeated with Nereocystis luetkeana. 80 4.2 S A M P L E C O L L E C T I O N AND T R E A T M E N T 4.2.1 Sample Collection Nereocystis luetkeana samples were collected on February 1999, approximately 3 meters away from one of the small uninhabited islands near the Bamfield marine station. The samples were approximately 3-4 meters long. They were placed in a 60 L container with seawater for transportation back to the laboratory. The seawater that was collected with the kelp was sterilized using the autoclave. 4.2.2 Sample Treatment 4.2.2.1 Treatment of Nereocystis luetkeana before the Acclimation Period The blades of the Nereocystis was approximately 3 meters long, the bulb was approximately 10 cm in diameter and the stem was approximately 1 meter long. The stem was cut to approximately 30 cm away from the bulb. The kelp was rinsed three times with tap water to remove any dirt and weighed. A t this point the kelp changed from a brown colour to a green-olive colour. A l l steps performed from this point on were carried out under sterile conditions in a laminar flow hood. The kelp was placed in sterile seawater with Betadiene, an antibiotic powder, for ~ 5 to 10 minutes and agitated continuously, and then washed twice with distilled water, followed by one rinsing with seawater. The kelp was then placed into glass fermentors (Microferm Fermentor, N e w Brunswick Scientific Co . Inc.), which contained seawater and antibiotics. 81 Kelp samples were collected before the tap water washing, after the tap water washing, and after the last washing. The samples were weighed, frozen and freeze-dried. Seawater samples were also collected at the same time as the kelp samples. 4.2.2.2 Treatment of Nereocystis luetkeana during the Acclimation and Exposure Periods The washed Nereocystis samples were placed into the fermentors that contained about 10L of seawater and 100 ml antibiotics. The fermentors were placed into the incubator under the conditions described in Section 2.2.1. Sterile air was pumped into the fermentors to ensure adequate dissolved oxygen in the media. One of the fermentors became contaminated with bacteria after 4 days as evidenced by a milky appearance and the formation of bubbles. The kelp was removed from this fermentor and washed five times with sterile distilled water and shaken in a mixture of Betadiene and distilled water for about 10 minutes. The kelp was then washed twice with distilled water and then with seawater. The kelp was placed back into the newly sterilized fermentor that contained new seawater and antibiotics. Samples of the seawater from the fermentor, and samples of the stem and blades were collected before the kelp was washed. After 4 more days of acclimation (or 9 days in total), both fermentors were contaminated (seawater was cloudy and bubbles formed on the top of the seawater). The kelp was removed from the fermentors, placed into sterile distilled water for 1.5 hours, and then placed into a mixture of Betadiene and distilled water for 15 minutes during which time the kelp was agitated continuously. The kelp was next washed twice with distilled water and then placed back into the 82 washed and sterile fermentors containing 10 L A S P 6 F2 medium, 100 ml antibiotics and 5 mg arsenate. N o As(V) was added to the medium of the control experiment. Table 4.1 describes the medium conditions for the exposure experiment. Table 4.1 Medium Conditions for the Exposure Period for Nereocystis luetkeana Fermentor #* Medium** Kelp Arsenic 1 A S P 6 F2 from seawater 0.50 ppm As(V) 2 A S P 6 F2 from seawater X * Microferm fermentor, N e w Brunswick Scientific Co . Inc. ** both fermentors contained 10 ml antibiotics in 10 L medium X not present in the flasks The fermentor containing the arsenate was prepared by adding 5 ml of 1000 ppm stock solution of As (V) to the fermentor. Both fermentors were placed under the same conditions in the incubator. Sterile air flow into the fermentors was set at a minimum to prevent a lot of bubbles from forming. Medium samples were taken once a week during the 28 day exposure experiment. The weight of the kelp used in the exposure experiment ranged from 787 to 800 g wet weight. 4.2.3 Sample Preparation A l l medium samples taken during the experiment were frozen immediately to preserve sample integrity until the time of the analysis. After the acclimation and exposure periods, the Nereocystis luetkeana samples were separated into bulb and blade, rinsed separately, weighed, frozen and then freeze-dried. The 83 freeze-dried samples were kept at -20 °C until they were extracted using a 1:1 methanol/water solution. The extraction procedure used is described in Section 2.3.2. The medium and extract samples were filtered through 0.45 um syringe filters (Millipore) and analyzed by using the H P L C - I C P - M S operating conditions given in Table 2.1. The data from I C P - M S were processed by using chromatographic software and identification of arsenicals in the samples was made by comparison of retention times with those of standards. Concentrations of arsenic compounds were determined by using compound specific external calibration curves when possible. D M A was used as a standard for arsenosugars. 4.2.4 Culture and Medium Conditions The bull kelp samples that were collected from the sample site underwent a rigorous treatment to make them as axenic as possible. Antibiotics and other decontamination procedures were included in an attempt to make sure no other l iving organisms, other than the Nereocystis, were involved in the exposure experiments. However, the use of antibiotics and multiple washings was not completely successful in removing all other organisms. The bull kelp was quite large and difficult to handle and the fermentors became cloudy after a few days and a lot of bubbles formed at the top of the medium. The contamination of the medium could be controlled a little by varying the amount of sterile air that was pumped into the medium. That is, when the air supply into the fermentors was lowered, the contamination of the medium took longer to occur with fewer bubbles being formed. Harsher conditions (shaking and stronger antibiotics) were used with the Nereocystis compared to the Fucus but this was not successful in keeping the contamination under control. The presence of bacteria with the bull kelp formed the bull kelp 84 community and any changes attributed to the bull kelp were meant to also include the bull kelp community. Throughout most of the experiment, the Nereocystis appeared to remain intact and healthy looking. Although at the end of the experiment the Nereocystis appeared wilted and darker in colour (brownish/green) than when freshly collected. Since the media had not been changed during the acclimation period and the arsenic exposure period, the bull kelp was likely experiencing starvation conditions at the end of each period. The culture media also became increasingly coloured (dark orange) over the course of the experiment. The colour in the medium indicated that the Nereocystis was losing its pigmentation. 85 4.3 R E S U L T S AND DISCUSSION 4.3.1 Acclimation Period The bull kelp collected from Bamfield were treated with antibiotics and allowed to acclimate as described in Section 4.2.2. The sub-samples collected before and after the acclimation period were processed as described in Section 2.3. The extract samples were tested using the ion-pairing H P L C - I C P - M S condition (Table 2.1). The predominant arsenic compounds found in both the bulb and blade extracts of the Nereocystis, before the acclimation, were M M A and arsenosugars X and XII . During the acclimation period, the total concentration of the extractable arsenic compounds decreased over the 14 days as can be seen from Figure 4.2 and 4.3. These compounds were most likely lost into the medium in the form of water-soluble arsenic species. B y comparing Figure 4.2 and 4.3, it can be seen that the amounts of arsenic compounds were higher in the bulb extracts than the blade extracts throughout the acclimation period. 86 Figure 4.2 Nereocystis luetkeana Bulb Extracts before and after 9 days of Acclimation in Seawater 450000 400000 i i XII 350000 300000 „ 250000 to a. ° 2 0 0 0 0 0 150000 100000 kelp before acclimation kelp (bulb) after 4 days of acclimation in seawater kelp (bulb) after 9 days of acclimation in seawater 5 -MMA ^/j^ji I u c 200 400 600 800 1000 Retention time(s) Figure 4.3 Nereocystis luetkeana Blade Extracts before and after 9 days of Acclimation in Seawater 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 XII MMA A / 200 400 600 Retention time(s) 800 • kelp before acclimation kelp (blade) after 4 days of acclimation in seawater kelp (blade) after 9 days of acclimation in seawater 1000 87 The amounts of each arsenic compound in the Nereocystis before and after the acclimation periods are summarized in Table 4.2. The kelp lost -73.6 % of the total extractable arsenic in 9 days mostly in the form of arsenosugars X and XII. The blade lost - 92.5 % in the first 4 days, while the bulb only lost - 63.1 % in the first 4 days of the acclimation period. The M M A that was lost by the kelp might have been excreted into the medium or it might have been transformed into other arsenic compounds (for example D M A ) , which in turn were excreted into the medium. Table 4.2 Arsenic Speciation of Nereocystis luetkeana Extracts (ppm, dry weight) before and after Acclimation in Seawater B u l l kelp sample Concent ra t ion Relat ive percentage MMA X XII MMA X XII before acclimation 0.90 ± 0.09 a 4.64 ±0.2 28.37 ±0.8 100.0% 100 .0% 100 .0% bulb after 4 days o f acclimation 0.34 ±0.06 0.92 ± 0.09 11.85 ±0.5 3 7 . 5 % 19 .7% 4 1 . 8 % after 9 days o f acclimation 0.27 ±0.05 2.03 ±0.2 6.09 ±0.3 2 9 . 9 % 4 3 . 8 % 2 1 . 5 % blade after 4 days o f acclimation 0.32 ±0.06 1.23 ±0.09 2.03 ±0.1 3 5 . 8 % 2 6 . 4 % 7 . 1 % after 9 days o f acclimation 0.16 ±0.05 1.23 ±0.09 0.73 ± 0.07 18 .2% 2 6 . 4 % 2 .6% 's c= standard deviation from analytical results obtained with the calibration curve 88 4.3.2 Exposure Period A s mentioned in Section 4.2.2.2, the Nereocystis samples were incubated with 500 ppb of arsenate in A S P 6 F2 medium following 9 days of acclimation. The extracted samples obtained from the bulb of the Nereocystis incubated with arsenate showed an increase in D M A and As(V) species compared to Nereocystis sample from the beginning of the exposure experiment (see Figure 4.4). In addition, the arsenosugars X and XII from the incubated Nereocystis did not show any significant changes over the exposure period. Figure 4.4 Nereocystis luetkeana Bulb Extracts after Exposure to As(V) in ASP6 F2 Medium 450000 400000 XII 350000 \ 300000 kelp before acclimation 100000 150000 kelp (bulb) after 9 days of acclimation in seawater kelp (bulb) after exposure to As(V) in ASP6 F2 medium 50000 0 0 200 400 600 Retention time(s) 800 1000 89 The extracts of the blade samples did not show a significant increase of As (V) and only trace amounts of D M A was seen. This suggests that the blade and bulb took up the arsenate differently from each other as can be seen by comparing Figure 4.5 with Figure 4.4. Also , the concentration of arsenosugars X and XII decreased instead o f staying the same as in the case of the bulb extracts (Table 4.3). Figure 4.5 Nereocystis luetkeana Blade Extracts after Exposure to As(V) in ASP6 F2 Medium 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 XII 200 400 600 Reten t ion time(s) 800 •kelp before acclimation - kelp (blade) after 9 days of acclimation in seawater kelp (blade) after exposure of As(V) in ASP6 F2 medium 1000 The controls for this exposure experiment consisted of Nereocystis samples that were treated under the same conditions as the above samples, except they were not exposed to As(V) . The extracts of the controls for both the blade and bulb samples showed a decrease in M M A and arsenosugars X and XII (Table 4.3). These results are similar only to the blade samples that were exposed to As (V) . 90 Table 4.3 Arsenic Speciation of Nereocystis luetkeana Extracts (ppm, dry weight) after As(V) Exposure in ASP6 F2 Medium B u l l kelp sample Arsen ic species found M M A D M A As (V ) X XI I before acclimation 0.90 ± 0.09? 0 0 4.64 ±0.3 28.37 ±0.8 bulb after 9 days o f acclimation control-no As (V ) exposure after exposure to As (V ) 0.27 ± 0.05 0.19 ±0.05 0 0 0 2.65 ±0.2 0 0 1.69 ±0.1 2.03 ±0.1 trace 1.87 ± 0.1 6.09 ±0.4 0.33 ±0.05 4.52 ±0.3 blade after 9 days o f acclimation control-no As (V ) exposure after exposure to As (V ) 0.16 ±0.05 0.09 ± 0.02 0 0 0 trace 0 0 0.76 ±0.07 1.23 ±0.09 0.18 ±0.05 0.17 ±0.05 0.73 ±0.07 0.07 ± 0.02 0.06 ± 0.02 a s c = standard deviation from analytical results obtained with the calibration curve "Trace" amounts are greater than or equal to the detection limit 91 The medium samples that were collected over the period of the exposure experiment were analyzed by using anion-exchange H P L C - I C P - M S . Initially, the medium contained only the arsenate, while subsequent samples showed an overall decrease of As (V) and an overall increase of D M A (see Figure 4.6). During the time period between the 15 t h and 25 t h day, the amount of As(V) in the medium increased and then decreased again for no apparent reason. Figure 4.6 ASP6 F2 Medium Samples collected during the Exposure of Nereocystis luetkeana to Arsenic(V) 650 -| 550 | -•—As(V) • - D M A 50 -„ i m u • • • | -50 0 5 10 15 20 25 30 35 Time(days) § 250 -u c o O 150 -92 4.4 S U M M A R Y The data obtained for this experiment show that the Nereocystis luetkeana is capable of accumulating arsenate from the surrounding medium and transforming it into D M A , which is seen in both the extracts and the medium collected. The amount of D M A found in the bulb extracts was 2.65 ppm with trace amounts found in the blade. The arsenate in the medium is most likely taken up by a similar transport mechanism to phosphate and then transformed into other arsenic species as was discussed in Section 3.6. The amount of As(V) taken up by the Nereocystis is lower than the Fucus under similar experimental conditions. Extracts of Nereocystis following exposure to arsenate contained approximately 2.45 ppm As(V) , while the medium had approximately 250 ppb As(V) . The Fucus extract samples contained approximately 2.65 ppm As(V) and the medium had < 0.5 ppb As(V) left after the uptake under similar conditions (compare experiment to one in Section 3.3.3). Young samples were used in the Fucus experiment, while mature samples were used for the Nereocystis experiment. It is possible that the Nereocystis, being mature, was growing very slowly and thus affecting the uptake of arsenate. It has been seen that uptake of minerals in higher plants (and possibly Nereocystis) is dependent on the growth rates. That is, the greater the growth rate the greater the uptake rate w i l l be. 3 5 ' 6 1 The two different species of algae display a different tolerance towards arsenate compared to what was expected from natural samples. Therefore, one cannot predict the possible response of natural communities of marine algae to a given stress on the basis of algal 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. 93 N o As(III) was found in both the extract and medium samples of Nereocystis while As(IIl) was found in most extract and medium samples from the previous Fucus exposure experiments. Also , arsenosugar X stayed relatively the same in the bulb sample, while decreasing in the blade samples. In the Fucus extract samples, arsenosugar X increased for all exposure experiments. 94 5 A R S E N I C T R A N S F O R M A T I O N B Y T H E FUNGUS FUSARIUM OXYSPORUM M E L O N I S 5.1 INTRODUCTION A fungus isolated from Fucus gardneri was identified at Biological Services, U B C , Vancouver to be Fusarium oxysporum melonis. The Fusarium species are widely distributed in soil and on organic substrates and have been isolated from insects, running water and from roots, from permafrost in the arctic and from the sands of the Sahara. 6 9 ' 7 0 They abound in cultivated soil both in temperate and tropical regions and are amongst the fungi most frequently isolated by plant pathologists. 7 0 Fusarium strains are involved in diseases of animals and man, and in rotting food that produces toxins. A s with many soil fungi they are abundantly endowed with means of survival, one of the mechanisms of which is the capacity for rapid change, often morphologically as well as physiologically, to a new environment. Thus they can survive on a wide range of substrates and have been isolated from many preserved foods, from stored chemicals and from aircraft fuel 70 71 tanks. ' In the present case, the Fusarium oxysporum melonis was isolated from Fucus gardneri even though the algal samples underwent a rigorous cleaning treatment and was allowed to acclimate in sterile seawater containing antibiotics for 14 days. A micrograph o f the Fusarium collected is shown in Figure 5.1. 95 Figure 5.1 Micrograph of Fusarium oxysporum melonis Fusarium has the ability to penetrate the vascular tissue of roots and stems in plants, accounting for the difficulty experiencing in removing the fungus from the algae. 7 0 The decontamination procedure used did not k i l l the fungus and it is likely that any further decontamination might have killed the algae itself. However, it is likely that the Fusarium was present in all Fucus samples used in the acclimation/exposure experiments. Therefore, it is possible that the fungus could play an important role in the uptake of the arsenic compounds from the medium, thus an arsenic exposure experiment with only the fungus was undertaken. 96 5.2 S A M P L E C O L L E C T I O N AND T R E A T M E N T 5.2.1 Agar Conditions and Treatment of Fusarium oxysporum melonis before the Exposure Period The fungus that was growing with Fucus gardneri during a previous seawater acclimation experiment (Section 3.2.2.2.2), was removed by washing the fungus from the Fucus surface. This was done by washing the Fucus with sterile seawater and then collecting the fungus that came off into the seawater. The collected fungus was placed on agar dishes made from full strength Potato Dextrose Agar (DIFCO dehydrated) to grow on. After about 2 to 3 weeks, five round pieces from these agar dishes containing the fungus were added to 1/10 th strength Potato Dextrose Broth (DIFCO dehydrated), which contained 10 plain pieces of agar made up from Vi strength Potato Dextrose Agar (PDA) and an equal amount of bactoagar (DIFCO). The added agar pieces helped to give the fungus a surface to grow on, while the broth made it easier to remove the fungus for the exposure experiments. 1/10 th Potato Dextrose Broth (PDB) was added to this solution every time the fungus was removed or when there was no more broth for the fungus to grow in. The fungus was allowed to grow in this broth for one to two months during which time they turned from a light violet colour to a reddish-purple colour. After this growth period, some of the fungus was removed from the broth, washed with artificial seawater (ASP6 F2) and then placed into a 1 L flask containing artificial seawater. To the broth, 1/10 th P D B was added. The fungus was removed periodically from the broth and placed into the artificial seawater until enough biomass of the fungus was obtained to start the exposure experiment. A l l steps performed with the fungus were done under sterile conditions in a Biological Safety Cabinet. 97 5.2.2 Treatment of Fusarium oxysporum melonis during the Exposure Period The fungus that was grown in the artificial seawater was combined into one flask, washed with artificial seawater and then redistributed into 1 L flasks containing 100 m l artificial seawater and either As (V) or D M A . No antibiotics were added to the media because the antibiotics might have killed the fungus or destroyed its cells. A summary of the treatment is shown in Table 5.1. Table 5.1 Medium Conditions for Fusarium oxysporum melonis Flask #* ASP6 F2 medium** Fungus Arsenic 1 100 ml / 0.50 ppm As(V) 2 100 ml / 0.50 ppm D M A 3 100 ml y * all flasks were duplicated ** flasks did not contain antibiotics The flasks containing the As(V) and D M A were prepared by adding 50 p L of 1000 ppm stock solution to each flask. A l l treatments of the fungus were carried out under sterile conditions and sealed with sterile cotton-plugs. The flasks were placed in the Conviron incubator under the conditions described in Section 2.2.1. One ml samples from each flask were taken under sterile conditions in the Biological Safety Cabinet with samples being taken more frequently in the first four days. The flasks were also swirled on the sampling days to ensure adequate dissolved oxygen in the medium. The experiment lasted 45 days. 4 5 6 100 ml 100 ml 100 ml 0.50 ppm As(V) 0.50 ppm D M A 98 5.2.3 Sample Preparation A l l medium samples taken during the experiments were frozen immediately to preserve sample integrity until the time of the analysis. After the exposure experiment, the fungus samples were separated from the medium by filtering through a glass funnel and using a Whatman filter paper. The fungus samples were rinsed with sterile deionized water and then frozen at -20 °C with the filter paper. The samples were extracted along with the filter paper using the method described in Section 2.3.2. The medium and fungal extract samples were filtered through 0.45 um syringe filters (Millipore) and analyzed by using the H P L C - I C P - M S operating conditions given in Table 2.1. The data from the I C P - M S were processed by using chromatographic software and identification of arsenicals in the samples was made by comparison of retention times with those of standards. Concentrations of arsenic compounds were determined by using compound specific external calibration curves when possible. D M A was used as a standard for arsenosugars. 5.2.4 Culture and Medium Conditions During the growth of the fungus, no observable contamination was noticed. The broth solution and the artificial seawater solutions that contained the fungus did not become cloudy or milky, which are signs of bacteria contamination. It was also noticed that the colour of the fungus was depended on the type of medium that was used. The fungus was white and fluffy when collected from the Fucus gardneri exposure experiment. It turned to a purple colour with white streaks once it started growing on the petri dish with P D A . When the fungus was removed from the petridish, they turned to a yellowish colour in ' /2 t h P D B and to a red/purplish colour in 1/10 th P D B . On transfer to artificial seawater, the fungus became a light purple colour. The 99 change in colour by the Fusarium isolates indicates that they have a remarkable facility for adapting both their form and colour in response to the pressure of the culture environment. 7 0 100 5.3 R E S U L T S AND DISCUSSION 5.3.1 Analysis of the Extracts of the Fusarium oxysporum melonis after the Exposure Experiments The fungus was prepared and treated for the exposure experiment as described in Section 5.2. The extracts of the samples were analyzed by using the ion-pairing H P L C - I C P - M S condition (Table 2.1). 5.3.1.1 Results after Exposure to As(V) The predominant arsenic species found in the extracts of the fungus after arsenic exposure were As(III), D M A and As (V) (Figure 5.2). The controls were fungus samples under the same conditions as above except they were not exposed to any arsenic compounds. These samples showed a slight increase in the As(III) and D M A species (Figure 5.2). The concentration of these arsenic species is about 3 times less than what was found in the exposed fungus (Table 5.2). 101 Figure 5.2 Fusarium oxysporum melonis Extracts after Exposure to As(V) in ASP6 F2 Medium fungus control-no As(V) exposure fungus after exposure to As(V) 1000 Table 5.2 Arsenic Speciation of Fusarium oxysporum melonis Extracts after As(V) Exposure (ppb, wet weight) Fungus Sample Arsenic species found As(III) D M A A s ( V ) control-no As(V) exposure 1.08 ± 0.09 a 5.01 ± 0 . 3 0 after exposure to As (V) 3.26 ± 0 . 2 14.70 ± 0 . 7 16.14 ± 0 . 8 a s c = standard deviation from analytical results obtained with the calibration curve The mass of the fungus in the exposure and controls was approximately the same but it was not determined. The volumes of extracts were the same in both cases. 102 60000 50000 40000 Q. 30000 u 20000 10000 DMA As(lll) As(V) 200 400 600 Reten t ion time(s) 800 5.3.1.2 Results after Exposure to D M A The Fusarium extracts after exposure to D M A showed no observable changes. The results obtained were similar to the controls (Table 5.2). 5.3.2 Analysis of the Culture Media from the Exposure Experiments The medium samples from the Fusarium exposure experiments were collected as described in Section 5.2 and analyzed without further treatment. The analysis were done by using the anion-exchange H P L C - I C P - M S conditions described in Table 2.1 5.3.2.1 Results of the Medium Collected after Exposure to As(V) In order to examine the interaction between the fungus and As(V) during the first few days and after the last few days of incubation, an exposure experiment was performed that was run for 45 days, with samples being taken more frequently in the first four days. The results for the short term and long term effects are shown in Figure 5.3 and Figure 5.4 respectively. During the first few hours As (V) was taken up slowly by the fungus and reduced to As(III) which in turn was released into the medium (Figure 5.3). It is of interest to notice that the As(V) first increased before it decreased as it was taken up by the fungus (Figure 5.4). This might indicate that there was an external source of As(V) , which might have come from the culture medium. It was determined from the total digestion analysis that P D A contained ~ 0.333 ppm arsenic (the M e O H / H 2 0 extract contained ~ 0.89 ppb as As(V) and ~ 1.89 ppb as As(III)), while P D B contained ~ 0.258 ppm of arsenic species per dry weight (the M e O H / F k O extract contained ~ 7.67 ppb as As(V) and ~ 1.55 ppb as As(III)). The fungus which were first grown on the P D A and then in the P D B might have absorbed or taken up the arsenic compounds from their 103 environment and when they were placed in the artificial seawater some of these compounds might have been transferred along with the fungus causing the increase in the arsenate species that was seen. Figure 5.3 Short Term Effects of the Fusarium oxysporum melonis Exposed to As(V) over 45 days 104 Figure 5.4 Long Term Effects of the Fusarium oxysporum melonis Exposed to As(V) over 45 days The medium samples collected during the long term experiment showed an overall decrease in As(V) , while As(III) increased during the first 300 hours or first 13 days (Figure 5.4). After this time, the As(III) levelled out and stayed reasonably constant (~ 40 ppb) for the remainder of the experiment. This seem to indicate that the As(V) was taken up and reduced by the fungus into As(III) over the first few days and after about 13 days of uptake the fungus produced no more As(III). 5.3.2.2 Results of the Medium Collected after Exposure to D M A The chromatograms of the medium incubated with 500 ppb D M A did not show any observable changes. N o arsenic compounds were lost or formed during the incubation period. 105 5.4 S U M M A R Y Fusarium, as with Fucus gardneri, is capable of accumulating As (V) from the surrounding medium and transforming it into As(III) and D M A , but it is not capable of accumulating D M A from the medium. D M A was found in the fungus extracts while As(III) was found in both the fungus extracts and the medium following exposure to 500 ppb As(V) . The arsenate concentration in the medium initially increased by a small amount and then decreased as it was taken up by the fungus. It is possible that the arsenate in the culture medium is responsible for this increase. The amount of As(III) produced by the Fucus in the As (V) exposure experiments (see Section 3.3.3) is much greater than that produced by the fungus. In the algae experiment, it was difficult to remove all of the fungus from the algae, therefore i f any As(III) was produced by Fucus gardneri during the acclimation/exposure experiment it may not have been produced exclusively from the Fucus itself, but some could have come from the fungus, that was growing with the Fucus. The amount of As(III) produced by the fungus wi l l most likely depend on how much fungus is present in the culture. This of course wi l l be related to the size of the Fucus samples and how much As(V) w i l l be taken up from the medium by the Fucus. A t the moment there is no way of saying which proportion of the reduction is associated with either the algae or the fungus. The relationship between the fungus and algae is probably not a true symbiotic relationship and this makes it more difficult to determine when and how much As (V) was metabolized by either the fungus or the Fucus. It seems the Fusarium species acts more like a parasitic fungus than a symbiotic fungus. 106 6 A R S E N I C SPECIATION OF D I F F E R E N T M A R I N E A L G A E FOUND IN T H E BRITISH C O L U M B I A C O A S T 6.1 INTRODUCTION Many different marine algae grow along the coast of British Columbia. For example, Fucus gardneri (brown algae), Nereocystis luetkeana (brown algae), Macrocystis sp. (brown algae), Gigartina exasperata (red algae), Porphyra sp. (red algae), Mastocarpus sp. (red algae) and Ulva fenestrata (green algae). The zonation pattern within algal assemblages are dictated by tidal exposure and wave impact, as well as by species interaction such as grazing by invertebrates and by competition for space and light. Samples of all the marine algae enumerated above were collected along the shoreline with Fucus gardneri except for Nereocystis and Macrocystis which were collected together several meters away from the shore. Previous studies of arsenic speciation in marine algae showed that arsenosugars X - X V were present in a number of the algal species. 4 6 It has been suggested that these various arsenic species found may possible be related to the algal taxonomy. 4 6 The major arsenic compound found in two brown algae from the order Laminariales (Ecklonia radiata12 and Laminaria japonica46) was shown to be arsenosugar XII , while arsenosugar XIII was not detected. On the other hand, three species of brown algae examined from the order Fucales (Hizikia fusiforme14, Sargassum thunbergii15 and Sargassum lacerifolium46) all contained arsenosugar XIII as the major arsenic compound. The relative occurrence of the different arsenic containing ribosides found in algae may reflect the presence of different enzyme systems capable of carrying out the glycosidation reaction. 4 6 107 The present study was concerned with determining the different arsenosugars present in the British Columbia algae samples and to investigate the extraction efficiency. The water content of each sample wi l l also be discussed. 108 6.2 W A T E R C O N T E N T A l l the algae samples were freeze-dried prior to extraction. Freeze-drying is a common method to preserve samples in environmental studies. It provides a convenient way of preservation because it results in the elimination of water and denaturation of protein. 3 6 Several studies have shown that the freeze-drying procedure does not affect or alter the arsenic species found in marine plants. These studies were done by comparing the arsenic species present in marine plants before and after the freeze-drying process. 3 6 The water content in the marine algae was determined by the weight before and after freeze-drying and is shown in Table 6.1. It was found that the water content in the marine algae varies from species to species. The red algae seemed to have the lowest amount of water compared to the other algae samples, while the brown algae had the highest (especially the kelp species). 109 Table 6.1 Water Content of Marine Algae from British Columbia Coast Algae Sample Water Content (%)* brown algae Fucus gardneri(m&\ux€) 84.3 ± 0.3 Fucus gardneri(yo\ing) 81.1 ± 0 . 3 Nereocystis luetkeana(bu\b) 94.1 ± 0.4 Nereocystis luetkeana(b\ade) 91.8 ± 0.4 Macrocystis sp. 92.5 ± 0.4 red algae Gigartina exasperata Porphyra sp. Mastocarpus sp. green algae Ulvafenestrata 82.8 ± 0.3 a 61.3 ± 0.2 92.9 ± 0.4 60.8 ± 0.2 Weight content = [(weight before - weight after)/weight before freeze-drying] a s c = standard deviation from analytical results obtained with the calibration curve 6.3 ARSENIC SPECIATION The marine algae samples collected from the coast of British Columbia were weighed, frozen, freeze-dried and extracted using the method described in Section 2.3.2. The extract samples were analyzed by using ion-pairing H P L C - I C P - M S (Table 2.1). The predominant arsenic compounds found in the extracts of the brown marine algae were M M A , and arsenosugars X , XI /XII and XIII. It is of interest to note that the kelp group (Nereocystis and Macrocystis) contained very high concentrations of arsenosugar XI /XI I compared to the Fucus and other algae species. This might be due to the fact that both species were found to grow in sub-tidal waters, while Fucus grew on the rocks along the middle and lower inter-tidal zones (see Table 6.2). Also the concentration of arsenic species varied between the two Fucus samples collected. A previous study done by L a i 3 6 indicates that the amount of arsenic species varied between the mature and young samples. It was suggested that the relative amount of water-soluble arsenic compounds present in Fucus might change as the plant matures. 3 6 In the red algae, the predominant arsenic compounds that were found were arsenate, and arsenosugars X and XI /XII . The Porphyra sp. also contained arsenosugar XIII, while Gigartina exasperata contained a small amount of M M A . For the green algae, Ulva fenestrata, M M A , As(V) and arsenosugars X and XIII were found. B y comparing the data to each other it can be seen that the amount of arsenic species found vary from one algal species to another. This suggests that the arsenic cycle might depend on the relationships between the different algal species and the different geographical locations. I l l Table 6.2 Relative Amounts of Arsenicals found in some Marine Algae (ppm, dry weight) Algae samples Arsenic Species M M A As(V) X XI /XI I XIII brown algae Fucus gardneri(mature) 2.31 0 3.03 2.75 1.09 Fucus gardneri(young) 0.08 0 0.26 2.32 1.27 Nereocystis luetkeana 1.34 1.01 1.76 52.67 0 Macrocystis sp. 0.96 0.27 0.76 18.40 0.43 red algae Gigartina exasperata 0.05 0.26 1.99 11.88 0 Porphyra sp. 0 0.07 0.08 4.21 0.65 green algae Ulva fenestrata 0.36 0.73 3.04 0 0.46 112 6.4 ASSESSMENT O F A C C U R A C Y O F E X T R A C T I O N M E T H O D Because of the biological nature of the samples in these experiments, considerable variations in the results are to be expected. For this reason, total digestion was done on the samples before and after the extraction method to determine the extraction efficiency. A l l the samples were digested as described in Section 2.3.3. The total digestion samples were analyzed by using R h (10 ppb) as an internal standard and by monitoring m/z 75 and 103 for arsenic and rhodium, respectively. The I C P - M S parameters are given in Table 2.2. Table 6.3 shows the total arsenic concentration of samples before and after extraction as well as the extraction efficiency. It also includes the total arsenic concentration obtained from 1:1 MeOH /H20 extractions as well as the detection efficiency. The certified arsenic concentration in oyster tissue S R M is 14.0 ± 1.2 ppm. Comparing this with the experimental result obtained by total digestion (13.7 ± 0.7 ppm), one can see that this method is very efficient in removing most of the arsenic compounds from the samples. The results obtained for the Fucus sample ( IAEA-140/TM) is also close within error to the certified value. However, the total concentration obtained by extraction is not close within error to the certified values. The arsenic concentration in the brown algae before extraction ranged from 15.9 ± 0.8 ppm to 63 ± 3 ppm. Again the arsenic concentration is the highest in the kelp samples. The arsenic concentration in the red algae ranged from 11.9 ± 0.6 to 21 ± 1 ppm, while the arsenic concentration found in the one green algae species was 11.1 ± 0 . 6 ppm. These results show that the brown algae generally contained higher amounts of arsenic compared to the red and green algae. 113 u S _4> I P B O a IP a i . a a. 3-a B 41 o — >_ S o < 3 s •3 i O a> H o B . a 'S i s IW >-~ s E -2 t a B 2 I- ofl es 1M o H o H •2* "a S D, 41 B w I5D 4 ) B C _g Q "5 "« « u o •S 3 H 41 OX) O x r o CN O O >—' r o r~ os r o no (N CN OS no OS <N d ° -H "H ly-, Os d <=> « OS oo SO o U O CN 4) 1 %J K K a "a "a .3 6 ? ~ « o ~ 5 o i n Z 2 - *f o < 2 «o ^ a a » s b o b o f ? S3 a o p 3 3 o o c y £ £ £ £ I I a « 3 e o £ ^ OS O o x o x (N r o '—I t - ; OS SO OS SO so I/O d OS OS OS 0 0 OS p- ro so CN so p CN o d d d d d d d -H -H -H -H +1 M -H +1 p oo "0 OS d 0 0 r o 0 0 i r i p cn SO OS 0 0 OS SO d CN d d cn cn d d d -H -H -H -H •H -H -H -H -H -H SO OS OS cn i n OS .—i cn OS so 0 0 CN l—< i — i a Si ^ s S a. D. CN -H m s ai S o -4) o < S ^ w ^ 1 -a 4 . M o H « S u s 4) 3 O 1 .9 .3 a o c 4) O c o o a o a 4) O C o o o -5 s i fl g „ 4> io 4> " H i l l C S 4) o 8 8 » § P ^ The extraction efficiency in the B . C . marine algae ranged from 37.1 % to 94.9 %. The results indicate that only the arsenic compounds extracted exist as water-soluble forms, while the rest might exist as water-insoluble forms. It has been suggested that the relative proportion of water-soluble arsenic compounds and lipid arsenic compounds in marine organisms varies 62 considerable between species. This study shows that the red algae and kelp have most of the arsenic in water-soluble forms, while only a small percentage was found for the Fucus and green algae. It has also been suggested that the relative proportion of water-soluble arsenic compounds and lipid-soluble compounds in the same algal species might vary from season to season. 3 6 However, the actual pattern of the inter-conversion between water-soluble and lipid-soluble arsenic compounds in the algae remains unknown. The detection efficiency ranged from 21.3 % to 109% for the extracts of the samples. These samples detected by H P L C - I C P - M S showed that in some cases, even i f al l the water-soluble arsenic compounds were extracted, they were not necessarily detected by the instrument. The results obtained from freeze-drying, extraction and total digestion showed that the arsenic speciation of algae is very much dependent on the species involved. Small variations in relative amounts of arsenic could result from environmental variations. The major arsenic species found in the marine algae were arsenosugars X , XII and XIII. It might be suggested that all marine algae use similar processes to take up the arsenic species that are found in the marine environment and transform them into arsenosugars using a similar transformation mechanism as was suggested for the Fucus gardneri (Figure 1 . l ) . 2 5 ' 4 1 115 7 S U M M A R Y A N D F U T U R E CONSIDERATIONS The H P L C - I C P - M S technique is a very useful tool for the determination of arsenic species in algae. Two H P L C methods with ICP-MS detection allowed separation and identification of at least 9 arsenic species. The use of two different conditions strengthen assignments of peak identification based on retention time data. Extracts of Fucus gardneri exposed to arsenate under different environmental conditions showed significant increase in the concentration of As(III), D M A and lesser increase in the arsenosugars. Different media (different salinity levels) produced different outcomes. The medium that contained a higher level of salinity, resulted in lower amounts of As (V) , As(III) and D M A in the algal cells compared with the Fucus grown in low salinity levels. The uptake of arsenate by the Fucus gardneri is also very much dependent on the concentration of phosphate in the medium. At higher concentrations of phosphate, lower amounts of arsenate are taken up by the algae and transformed into As(III) and D M A . The results obtained indicate competitive uptake of phosphate and arsenate by marine macroalgae Fucus gardneri. When Fucus gardneri is grown without antibiotics in the presence of As(V) , the speciation in the media and algae is not much different from the results obtained from Fucus grown in media containing antibiotics. The difficulty, however, when growing the algae without antibiotics is that we are dealing with a community instead of the Fucus alone, making it difficult to determine exactly what species in the community is actually involved in the arsenate uptake. The presence of more complex arsenicals in environmental algae samples may well be dependent 116 on symbiotic interactions between the algae and its surroundings, rather than resulting from independent synthesis by the algae. The arsenic accumulation and biotransformation in Nereocystis luetkeana was found to be different compared with the Fucus gardneri. Under natural conditions, the bull kelp is able to take up 4 times more arsenic compounds from the surrounding environment than Fucus, however this difference in uptake was not reflected in the exposure experiments. D M A is the only species found in the extracts of Nereocystis. The kelp does not seem to produce any arsenosugars. Future experiments should include a study of the uptake of arsenate by young Nereocystis. The experiments should be set up by growing Nereocystis under laboratory conditions such that potential environmental variations can be controlled. The marine fungus, which grows with the Fucus during the uptake experiment, was identified as Fusarium oxysporum melonis. The fungus is capable of taking up arsenate from the surrounding medium and transforming it into arsenite and dimethylarsinate, but only the arsenite is observed in the medium. The arsenic concentration produced by the fungus was about 1000 times lower than the Fucus. Future experiments might involve the identification of more marine fungi that are parasitic to marine algae and determining i f they are also able to accumulate and transform arsenic compounds from the media. The arsenic speciation of algae is very much dependent on the species involved. Small variations in relative amounts of arsenic could result from environmental variations. These might be geographical differences, food processing procedures and storage. It w i l l be of interest to study the uptake of arsenic compounds by various algae species that are found to grow with Fucus gardneri and Nereocystis luetkeana. 117 The major arsenic species extracted from the marine algae are arsenosugars. A certain percentage of the arsenic (depending on the algae species) remained unextracted and undetected. The nature of this arsenic is unknown and hence more studies are needed to determine its chemical and toxicological significance. 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