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Antimony Uptake, Efflux and Speciation in the Arsenic Hyperaccumulator Pteris Vittata L.

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Title:
Antimony Uptake, Efflux and Speciation in the Arsenic Hyperaccumulator Pteris Vittata L.
Creator:
Tisarum, Rujira
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (108 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Soil and Water Science
Committee Chair:
MA,LENA Q
Committee Co-Chair:
RATHINASABAPATHI,BALASUBRAMANI
Committee Members:
TEPLITSKI,MAXIM
GUY,CHARLES L
BONZONGO,JEAN-CLAUDE J
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Anions ( jstor )
Antimony ( jstor )
Arsenic ( jstor )
Gametophytes ( jstor )
Hyperaccumulators ( jstor )
Oxidation ( jstor )
pH ( jstor )
Plant roots ( jstor )
Speciation ( jstor )
Species ( jstor )
Soil and Water Science -- Dissertations, Academic -- UF
antimony
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Soil and Water Science thesis, Ph.D.

Notes

Abstract:
Antimony (Sb) and arsenic (As) share many chemical properties including their prevalence in the environment in +3 and +5 oxidation states. In this study, Sb uptake, efflux and speciation in the arsenic hyperaccumulator Pteris vittata were investigated, and the obtained results discussed comparatively to those obtained with As. Solid phase extraction (SPE) was investigated as alternate method to HPLC-ICP-MS for Sb speciation by taking advantage of a silica-based anion exchange cartridge (SBAEC). The results showed that (SBAEC) retained SbV anions which are more polar than SbIII, and adding citric acid to form SbIII- and SbV-citrate complexes reversed the (SBAEC) retention capacity. The separation of SbIII by the cartridge was successful in plant tissues by retaining SbIII and recovered 92-104% SbV compared to HPLC-ICP-MS. The first experiment examined the uptake of Sb, its efflux and speciation in P. vittata sporophytes after 1 d exposure. The results showed that P. vittata took up more antimonite (SbIII) than antimonate (SbV) and both SbIII and SbV accumulated primarily in the roots. Sb in the roots was mostly stable with 19% of SbIII being oxidized and 2% of SbV being reduced. Additionally, 26% of the total Sb concentration was effluxed out into the growth media. After 7 d exposure, three P. vittata accessions from Florida, China, and Brazil had a similar ability to accumulate Sb. The mechanism of SbIII uptake was hypothesized to be via aquaporin transporters. The second experiment evaluated the effects of SbIII competitors (glycerol, silicic acid, glucose, and arsenite (AsIII)), and inhibitor of the aquaporin transporter (silver (Ag)) on Sb uptake by P. vittata gametophytes. Phosphate (PV) was included to determine its impact on SbIII and SbV uptake by P. vittata. P. vittata gametophytes had a higher ability to uptake SbIII than SbV after 2 h exposure without toxicity symptoms. The presence of SbIII competitors and of Ag used as aquaporin inhibitor had no impact on SbIII uptake. PV did not impact SbV uptake, but it reduced SbIII uptake. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
Bibliography:
Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: MA,LENA Q.
Local:
Co-adviser: RATHINASABAPATHI,BALASUBRAMANI.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-08-31
Statement of Responsibility:
by Rujira Tisarum.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
8/31/2015
Classification:
LD1780 2014 ( lcc )

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ANTIMONY UPTAKE, EFFLUX AND SPECIATION IN THE ARSENIC HYPERACCUMULATOR PTERIS VITTATA L. By RUJIRA TISARUM A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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© 2014 Rujira Tisarum

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To my family

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4 ACKNOWLEDGMENTS I would like to thank my advisor Dr. Lena Q. Ma for accepting me into her group and for her support and encouragement. I am grateful for the guidance she provided both in my research and professional development. I would al so like to thank my co advisor Dr. Bala Rathinasabapathi and committee members Dr. Charles Guy, Dr. Jean Claude J. Bonzongo and Dr. Max Teplitski for their valuable suggestions and the time they spent on ensuring the smooth progress of my research. My rese arch and lab training was very much influenced by the dedicated efforts of my friend s Xiaoling Dong and Dr. Jay T. Lessl. I also thank Dr. Shiny Mathews for all the help she provided. I thank my friends Hao, Piyasa, Xin, Yingjia, Ky, Letuzia, and Evandro for ongoing support in research and in personal matters. Special thanks also to Mr. Kafui Awuma for his help with my research. I thank my previous advisor s Dr. Jingtair Siriphanich , Dr. Kanogwan Seraypheap and Dr. Edward Sisler for their encouragement and help during the application process to the University of Florida. Special thanks also to Dr. Edward Sisler for Christmas shelter and Dr . Y ossapol Palapol for research assistance . Finally, I would like to thank my parents, aunt, grandma, and brother for their love, support and motivation as well as the Royal Thai Government and Dr. Ma for financial support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURE S ................................ ................................ ................................ ......................... 9 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................................ ... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 14 Antimony ................................ ................................ ................................ ................................ 14 Sb in Soil s ................................ ................................ ................................ ............................... 15 Sb in Waters ................................ ................................ ................................ ............................ 16 Sb Toxicity ................................ ................................ ................................ .............................. 17 Sb Uptake in Plants ................................ ................................ ................................ ................. 19 Sb Speciation in Plants ................................ ................................ ................................ ........... 22 Arsenic Hyperaccumulator Pteris vittata ................................ ................................ ............... 23 Sb Speciation and Analysis ................................ ................................ ................................ .... 24 Research Objectives ................................ ................................ ................................ ................ 26 2 A NEW METHOD FOR ANTIMONY SPECIATION IN PLANT BIOMASS AND NUTRIENT MEDIA USING SOLID PHASE EXTRACTION ................................ ............ 28 Introductory Remarks ................................ ................................ ................................ ............. 28 Materials and Methods ................................ ................................ ................................ ........... 30 Instrument for Sb Analysis and Speciation ................................ ................................ ..... 31 Sb Separation Using SBAEC ................................ ................................ .......................... 31 Sb Speciation in Plant Tissues and Spent Growth Media ................................ ............... 34 Results and Discussions ................................ ................................ ................................ .......... 35 Sb V Retention on Cartridge in DI Water ................................ ................................ ......... 35 Sb III Retention on Cartridge under C itric Acid ................................ ................................ 36 Sb Speciation in Plant Biomass and Growth Media ................................ ........................ 37 Conclusion ................................ ................................ ................................ .............................. 38 3 ANTIMONY ACCUMULATION IN ARSENIC HYPERACCUMUL ATOR PTERIS VITTATA L. ................................ ................................ ................................ ............................. 44 Introductory Remarks ................................ ................................ ................................ ............. 44 Material s and Methods ................................ ................................ ................................ ........... 46 Pre treatment and Sampling of P. vittata ................................ ................................ ....... 46

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6 Sb Accumulation in Three P. vittata Accessions ................................ ............................ 46 Sb Uptake, Translocation and Speciation in Excised P. vittata Fronds .......................... 47 Total Sb, Extraction and Speciation ................................ ................................ ................ 47 Statistical Analysis ................................ ................................ ................................ .......... 4 8 Results and Discussions ................................ ................................ ................................ .......... 48 Sb S peciation in P. vittata Roots ................................ ................................ ..................... 49 Sb Uptake and Translocation ................................ ................................ ........................... 50 Sb Efflux ................................ ................................ ................................ .......................... 52 Sb Translocation i n Excised P. vittata Fronds ................................ ................................ 53 Conclusion ................................ ................................ ................................ .............................. 54 4 UPTAKE OF ANTIMONITE AND ANTIMONATE BY THE ARSENIC HYPERACCUMULATOR PTERIS VITTATA L . : EFFECTS OF CHEMICAL ANALOGS AND A TRANSPORT INHIBITOR ................................ ................................ .. 61 Introductory Remarks ................................ ................................ ................................ ............. 61 Materials and Methods ................................ ................................ ................................ ........... 64 Gameto phyte Culture ................................ ................................ ................................ ....... 64 Sb Accumulation in P. vittata Gametophytes ................................ ................................ . 64 Sb III Competitors and Aquaporin Inhibitor on Sb III Uptake by P. vittata Gametophytes ................................ ................................ ................................ .............. 65 Effect of P V on Sb III and Sb V Uptake by P. vittata Gametophytes ................................ .. 65 Total Sb, Sb Speciation, Si and P Analysis ................................ ................................ ..... 66 Statistical Analysis ................................ ................................ ................................ .......... 66 Results and Discussions ................................ ................................ ................................ .......... 67 Sb Accumulation in P. vittata Gametophytes ................................ ................................ . 67 Sb III Competitors and Aquaglyceroporin Inhibitor Ag on Sb III Uptake by P. vittata Gametophytes ................................ ................................ ................................ .............. 68 Effect of P V on Sb III and Sb V Uptake by P. vittata Gametophytes ................................ .. 71 Conclusion ................................ ................................ ................................ .............................. 74 5 CONCLUSION AND D IRECTION OF FUTURE RESEARCH ................................ .......... 83 APPENDIX A ROLE OF GLUTATHIONE ON ARSENIC UPTAKE AND TRANSLOCATION ............ 86 Introductory Remarks ................................ ................................ ................................ ............. 86 Hypoth eses ................................ ................................ ................................ .............................. 86 Materials and Methods ................................ ................................ ................................ ........... 86 Results ................................ ................................ ................................ ................................ ..... 87 B EFFECT OF NANO TIT ANIUM DIOXIDE ON ARSENIC UPTAKE BY PTERIS VITTATA ................................ ................................ ................................ ................................ . 94 Introductory Remarks ................................ ................................ ................................ ............. 94 Hypothesis ................................ ................................ ................................ .............................. 94

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7 Materials and Meth ods ................................ ................................ ................................ ........... 94 Results ................................ ................................ ................................ ................................ ..... 94 C ROLE OF ARSENIC RESISTANT BACTERIA IN ENHANCING TOMATO GROWTH ................................ ................................ ................................ ............................... 96 Introductory Remarks ................................ ................................ ................................ ............. 96 Materials and Methods ................................ ................................ ................................ ........... 96 Plant Material ................................ ................................ ................................ .................. 96 Bacteria ................................ ................................ ................................ ............................ 96 Results ................................ ................................ ................................ ................................ ..... 97 LIST OF REFERENCES ................................ ................................ ................................ ............... 99 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 108

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8 LIST OF TABLES Table page 2 1 Sb retained on the cartridge preconditioned with 15 mL of 1 mg/L Sb V in 2 mM citric acid of different pH and after discarding the first 70 mL of filtrate ......................... 40 2 2 Sb retained on the cartridge from gr owth media ( 0.005 X HS and 0.005 X HS spent media) , and P. vittata roots after discarding first 60 mL of filtrate ................................ ... 40 2 3 Sb speciation in P. vittata roots extraction after exposure to 8 mg/L Sb III , or Sb V for 24 h by HPLC ICP MS before and after passing through the cartridge .......................... 41 2 4 Comparative data from recent studies on Sb speciation by different media via GF AAS detector. ................................ ................................ ................................ ..................... 41 3 1 Percentage of Sb retained on SepPak cartridge. ................................ ................................ 56 3 2 Total Sb concentration and speciation in roots and media, total Sb concentrations in frond and efflux media after exposing P. vittata plants to 8 mg/L Sb for 1 d and being efflux ed for 1 d . ................................ ................................ ................................ ........ 56 4 1 Sb concentrations in the tissues, media and the growth rate of P. vittata after exposure to 0.8, 8.0, and 80 mg/L Sb III or Sb V for 2 h . ................................ ..................... 75 A 1 GSH in P. vittata and P. ensiformis sap after exposure to As III and As V for 2 d. .............. 88 A 2 As speciation in P. vittata and P. ensiformis media after exposure to As III and As V for 2 d. ................................ ................................ ................................ ................................ 93 A 3 GHS in P. vittata and P. ensiformis media after exposure to As III and As V for 2 d, with none detected <0.5 M GSH . ................................ ................................ .................... 93 C 1 Summary of 6 treatments. ................................ ................................ ................................ .. 97

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9 LIST OF FIGURES Figure page 1 1 The structure of anion exchange cartridge containing Cl binding to quaternary ammonium group , which is covalently linked to the silica backbone. .............................. 27 2 1 Percentage of Sb III and Sb V retained on the cartridge in DI water at different pH. ........... 42 2 2 Percentage of Sb retained on the cartridge in DI water adjusted pH 6 and sampled every 50 mL ................................ ................................ ................................ ....................... 42 2 3 Percentage of Sb retained on the cartridge in DI water adjusted pH 6. The cartridge was preconditioned with 15 mL of 1 mg/L Sb V and then collected the filtrates every 10 mL after discard ing first 50 mL ................................ ................................ .................... 43 2 4 Percentage of Sb retained on the cartridge in media containing 2 mM citric acid adjust ed to pH 6, preconditioned with 15 mL of 1 mg/L Sb V and then sampled every 15 mL after discard ing the first 30 mL. ................................ ................................ ............. 43 3 1 Stability of 1.6 or 8 mg/L Sb III or Sb V in DI water (no plant) after 24 h. .......................... 57 3 2 Sb concentrations in the roots and the growth media of P. vittata after exposing to 1.6 or 8 mg/L Sb III or Sb V for 1 d ................................ ................................ ...................... 58 3 3 Sb contents in the roots and fronds of three accessions of P. vittata after exposing to 8 mg/L Sb III for 7 d . ................................ ................................ ................................ ........... 59 3 4 Sb concentration (mg/L) in the media during P. vittata uptake from 8 mg/L Sb III for 1 d and Sb concentration in the media (mg/kg roots fresh weight) during subsequent P. vittata efflux in DI water for 1 d ................................ ................................ ................... 59 3 5 Sb contents in the petioles and pinnae of excised P. vittata fronds after exposing to 8 mg/L Sb III or Sb V for 1 d ................................ ................................ ................................ .... 60 4 1 Eight week old P. vittata gametophyte clusters, each grown from 0.125 mg spore on 0.5X MS agar ................................ ................................ ................................ ..................... 76 4 2 Effect of silicic acid on Sb and Si concentrations and glucose on Sb concentrations in the tissues of P. vittata after exposure to glucose containing 65 µM Sb III for 2 4 h ........ 77 4 3 Sb and Ag concentrations in the tissues of P. vittata after pretreatment with AgNO 3 for 1 h then exposed for 2 h to ................................ ................................ .......................... 78 4 4 Effect of P V on Sb concentrations in the tissues of P. vittata after exposure to solutions containing 65 µM Sb III or Sb V for 2 4 h ................................ ............................ 79

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10 4 5 Effect of P V on P concentrations in the tissues of P. vittata after exposure to 65 µM Sb III or Sb V for 2 4 h ................................ ................................ ................................ ......... 80 4 6 Effect of Sb V and Sb III on Sb and P concentrations the tissues of 12 week P V starved P. vittata after exposure to 65 µM Sb V containing varying P V concentration or 65 µM Sb III varying P V concentration for 2 h ................................ ................................ ................ 81 4 7 Effect of P V on Sb and P concentrations the tissues of 12 week P V starved P. vittata after exposure to 0.65 mM P V containing varying Sb III concentration for 2 h . ................. 82 A 1 As speciation in P. vittata sap after exposure to As III and As V for 2 d. ............................. 87 A 2 As speciation in P. ensiformis sap after exposure to As III and As V for 2 d. ....................... 88 A 3 As in P. vittata after exposure to As III and As V for 2 d. ................................ ..................... 89 A 4 As spec i ation in P. vittata root after exposure to As III and As V for 2 d. ............................ 89 A 5 As speciation in P. vittata rhizome after exposure to As III and As V for 2 d. ..................... 90 A 6 As spec i ation in P. vittata frond after exposure to As III and As V for 2 d. .......................... 90 A 7 As in P. ensiformis after exposure to As III and As V for 2 d. ................................ .............. 90 A 8 As speciation in P. ensiformis root after exposure to As III and As V for 2 d. ..................... 91 A 9 GSH in P. vittata organs after exposure to As III and As V for 2 d. ................................ ..... 91 A 10 GSH in P. ensiformis organs after exposure to As III and As V for 2 d. ............................... 91 A 11 TBARs in P. vittata after exposure to As III and As V for 2 d. ................................ ............. 92 A 12 TBARs in P. ensiformis after exposure to As III and As V for 2 d. ................................ ...... 92 B 1 As contents in P. vittata after exposure to 2 mg As V and nano TiO 2 in DI water for 5 d. ................................ ................................ ................................ ................................ ......... 95 B 2 As contents in P. vittata after exposure to 150 mg /L AlAsO 4 or FeAsO 4 and 100 mg/L nano TiO 2 in 0.2X HS for 3 d. ................................ ................................ .................. 95 C 1 The bars represent root length and shoot height means and standard errors for 5 observations. ................................ ................................ ................................ ...................... 97 C 2 The bars represent dry weight of root s , shoot s and total means and standard errors for 5 observations. ................................ ................................ ................................ .............. 98

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11 LIST OF ABBREVIATIONS Ag Silver ARB As resistance bacteria As Arsenic As III Arsenite As V Arsenate Cl Chloride ion DI water Deionized water EDTA Ethylene diamine tetraacetic acid Fe Iron GF AAS Graphite furnace atomic absorption spectrophotomet ry HG Hydride generation HPLC H igh performance liquid chromatography HS Hoagland solution ICP MS Inductively coupled plasma mass spectrometry P Phosphorus PDC Pyrrolidine dithiocarbamate P V P hosphate Sb Antimony Sb III Antimonite Sb V Antimonate SBAEC S ilica based anion exchange cartridge Si Silicon SPE Solid phase extraction

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANTIMONY UPTAKE, EFFLUX AND SPECIATION IN THE ARSENIC HYPERACCUMULATOR PTERIS VITTATA L. By Rujira Tisarum August 2014 Chair: Lena Q. Ma Co C hair: Bala Rathinasabapathi Major: Soil and Water Science Antimony (Sb) and arsenic (As) share many chemical properties including their prevalence in the environment in +3 and +5 oxidation states. In this study, Sb uptake, efflux and speciation in the arsenic hyperaccumulator Pteris vittata w ere investigated , and the obtained results discussed comparatively to those obtained with As . S olid phase extraction (SPE) was inves tiga ted as alternate method to HPLC ICP MS for Sb speciation by taking advantage of a silica based anion exchange cartridge (SBAEC). The results showed that (SBAEC) retained Sb V anions which are more polar than Sb III , and adding citric acid to form Sb III and Sb V citrate complexes reverse d the (SBAEC) retention capacity. Th e separation of Sb III by the cartridge was successful in plant tissues by retaining Sb III and recovered 92 104% Sb V compared to HPLC ICP MS. The first experiment examined the uptake of Sb , its efflux and speciation in P. vittata sporophytes after 1 d exposure. The results showed that P. vittata took up more antimonite ( Sb III ) than antimonate ( Sb V ) and both Sb III and Sb V accumulated primarily in the roots. Sb in the roots was mostly stable with 1 9% of Sb III being oxid ized and 2% of Sb V being redu c ed . Additionally, 26% of the total Sb concentration was effluxed out into the growth media. After 7

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13 d exposure, three P. vittata accessions from Florida, China, and Brazil had a similar ability to accumulate Sb. The mechanism of Sb III uptake was hypothe sized to be via aquaporin transporters . The second experiment evaluated the effects of Sb III competitors ( glycerol, silicic acid, glucose, and arsenite (As III ) ) , and inhibitor of the aquaporin transporter ( silver (Ag) ) on Sb uptake by P. vittata gametophytes. Phosphate (P V ) was included to determine its impact on Sb III and Sb V uptake by P. vittata . P. vittata gametophytes had a higher ability to uptake Sb III than Sb V after 2 h exposure without toxicity symptoms. The presence of Sb III competitor s and of Ag used as aquaporin inhibitor had no impact on Sb III uptake. P V did not impact Sb V uptake, but it reduced Sb III uptake .

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14 CHAPTER 1 INTRODUCTION Antimony Antimony (Sb), with an atomic number of 51, is a member of group 15 of the periodic table . It is in the same group as phosphorus (P) and arsenic (As), but it has a larger size with an monos (alone) ( Emsley, 2001 ) , as it is usually found with sulfur and metals such as selenium, tellurium, copper, silver (Ag), and lead ( Filella et al., 2002a ; Greenwood and Earnshaw, 1998 ) . Sb is typically found in the form of stibnite (Sb 2 S 3 ) and valentinite (Sb 2 O 3 ) and has 4 oxidation states ( 3, 3, 0, and 5), with only two oxidation states ( 3 and 5 ) commonly found in the environment ( Filella et al., 2002a ) . Sb is a silvery white element , which breaks easily but can be mixed with other metals to increase strength ( ATSDR, 1992 ) . Sb has two forms: metallic and oxide. When metallic Sb is mixed with lead and zinc, the y are called alloys ( ATSDR, 1992 ) , which are used as a hardener in batteries, antifriction alloys, small arms , tracer bullets, and cable sheathing ( Filella et al., 2002a ) . Sb oxide, a white powder produced by adding oxygen, is widely used in flame retardants in textiles, papers, adhesives, tires, brake linings, and plastics ( ATSDR, 1992 ; He et al., 2012 ) . In the past, the major use of Sb was in alloys due to its low cost but, presently, it is a major component of flame retardant chemicals ( Filella et al., 2002a ) . The United States did not mine Sb in 2013 but one company that import s Sb, plus a minor portion i s derive d from the recycling of lead acid batteries ( USGS, 2014 ) . China is the largest Sb producer in the world, followed by Russia, Bolivia, Tajikistan, and South Africa ( USGS, 2014 ) . Sb has no known biological role but exhibits toxicity at high intake doses ( Emsley, 2001 ) . Sb is generally ingested via diet in amounts from 0.002 1.3 mg daily and an average person who

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15 weighs 70 kg has 2 mg Sb in the body. At a dose of 100 mg , Sb, can cause liver damage ( Emsley, 2001 ) . Sb is considered a pollut ant of priority interest by the United States Environmental Protection Agency and the World Health Organization recommends an Sb level in drinking water of less than 20 µg/L ( He et al., 2012 ) . Sb also has medical uses , for example, Sb is used in the treatment of tropi cal diseases and HIV, but this use has been decreasing due to its toxicity ( Filella et al., 2002a ; Wilson et al., 2010 ) . Sb 2 O 3 has been assigned by the International Ag ency for Research on Cancer (IARC) as a substance suspected of being carcinogenic in humans ( Krachler et al., 2001 ) . Sb 2 O 3 and SbCl 3 are able to act as inhalative lung carcinogens in female rats by increasing the mutation rate of sist er chromatids ( Ulrich, 2005 ) . However, Sb is still used to treat several tropical protozoan diseases (e.g. , leishmaniasis, schistosomiasis, ascariasis, trypanosomasis and bilharziasis) ( Filella et al., 2002a ) . Sodium stibogluconate (C 12 H 38 Na 3 O 26 Sb 2 ) has been used for more than 75 years to treat leishmaniasis ( Tschan et al., 2009 ) . Sb in S oil s Sb is not an abundant element as it is the 63 rd most abundant of the 92 naturally occurrin g The background Sb concentration is 0.02 7.01 mg/kg with an average of ~ 1 mg/kg but can reach 80,200 mg/kg in contaminated soils ( Wilson et al., 2010 ) . Sb is released to the environment from anthropogenic and natural sources including from mining, smelting and the burning of fossil fuels, waste disposal, shooting activities, and vehicles (a fire retardant in brake linings) ( Feng et al., 2013 ; Tschan et al., 2009 ) . Natural sources are from rock weathering and soil runoff ( Filella et al., 2002a ) . The Sb concentration is higher in surface soils and decreases with soil depth ( Steely et al., 2007 ) . Sb in soil solution, contaminated soils near smelters and at shooting ranges is mainly present as a pentavalent oxyanion, Sb(OH)

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16 ( Okkenhaug et al., 2011 ) . Humic acid in the soil can induce Sb III oxidation, via electron acceptors quinone and/or disul f ide functional groups on humic acid ( Steely et al., 2007 ) . Besides humic acids, Fe and Mn oxyhydroxides, O 2 , H 2 O 2 , and iodate can also oxidize Sb III ( Fan et al., 2014 ) . In the presence of O 2 in th e pH range of 11 13, Sb III oxidation occurs with an extremely slow rate and has a half life of 170 years in the dark ( Leuz et al., 2006 ) . Oxidation of Sb III by H 2 O 2 is much faster, with a half life of 118 d ( Leuz et al., 2006 ) . In addition, Fe(III) oxyhydroxides oxidize Sb III with a half life of 40 d whereas humic acid (5 mg/L DOC) can oxidize Sb III to Sb V with a half life of 17 min in sunlight ( Leuz et al., 2006 ) . Light plays an important role in Sb III oxidation by humic acid , which increas es the reaction rate by up to 900 times ( Fan et al., 2014 ) . There are several strategies to preserve Sb speciation during storage including removing iron oxyhydroxides and microbe s by filtration, reducing Sb III oxidation rate by low temperature, and addition of chemicals such as la ctic, ascorbic, citric, tartaric acids, and e thylene diamine tetraacetic acid ( EDTA ) , which prevent Sb III from oxidation ( Filella et al., 2009 ) . Gluthathione (GSH) has been reported to promote the reduction of antim o n ate ( Sb V ) to the more toxic antimon ite ( Sb III ) in the antimonial drug meglumine antimonate at pH 5 ( Frézard et al., 2001 ) . Green rus t, layered Fe(II) Fe(III) hydroxides in sub oxic and mildly alkaline environments, also reduce Sb V to Sb III ( Wilson et al., 2010 ) . Furthermore, KI, thiourea, and L cysteine can reduce Sb V as they have been used as pre reductant agents for Sb analysis by detecti ng Sb III ( Wilson et al., 2010 ) . Sb in W aters Sb concentration in unpolluted water is typically less than 1 µg/L and the mean Sb concentration in surface marine water is 0.2 µg/L ( Filella et al., 2002b ) . Sb pollut ed water near

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17 the largest Sb mine in China has an Sb content of 0.3 11 mg/L, with Sb V being the dominant form ( Liu et al., 2010 ) . Under oxic conditions, the hydrolytic species Sb(OH) 6 is the dominant Sb aqueous species over an extended range of pH ( Krupka and Serne, 2002 ) . This anionic species is dominant at pH values > 2.5 from oxidizing to slightly reducing conditions ( Krupka and Serne, 2002 ) . At moderately reducing conditions, the speciation is dominated by the Sb III hydrolytic species Sb(OH) 2 + at pH < 2, Sb(OH) 3 at pH of 2 12, and Sb(OH) 4 at pH > 12 ( Krupka and Serne, 2002 ) . Methylated Sb species have also been detected in some marine waters and river waters, but they are only 10% of the total dissolved Sb in these waters ( Krupka and Serne, 2002 ) . Microbe and algae might be the majo r producers of methylated Sb species ( Fil ella et al., 2007 ) . Sb T oxicity The World Health Organization (WHO) sets the tolerance daily intake (TDI) for Sb at 6 g/kg of body weight and the USEPA drinking water standard has a maximum contaminant level for Sb set at 6 µg/L ( Filella et al., 2002a ; Wilson et al., 2010 ) . Sb is generally found in all human tissues ( < 1 µg/g), particularly in the lungs, lymph nodes, and hair ( Filella et al., 2002a ) . Exposure to Sb can cause irritation of the resp iratory tract and lead to pneumoconiosis ( Ulrich, 2005 ) . The toxicity of Sb depends on its speciation, with organo antimonials (methylated Sb) being less toxic than Sb V and Sb III ( Wilson et al., 2010 ) . Sb III binds to the sulfhydryl group of red blood cells whereas Sb V has less affinity for erythrocytes ( Ulrich, 2005 ) . Sb metabolism is still unclear. The Sb analog, As, is detoxified via methylation but this is not true for Sb ( Krachler et al., 2001 ) .

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18 Environmental contamination from Sb mining in China is a current concern and residents living near Sb mines have died from liver cirrhosis after long term exposure to high Sb concentrations in the environment around the Xikuangshan Sb mi ne. This exposure is also suspected to have caused dermatitis and pneumoconiosis in the local residents ( Feng et al., 2013 ) . Sb contaminated food grown in the mine ar ea i s the main source of the high Sb concentrations (16 µg/g) in the hair of the Sb mining residents, whereas non mining residents typically have only 0.5 µg/g in hair ( Feng et al., 2013 ; Liu et al., 2011 ) . Rice is a major Sb contaminated food (33% of the total Sb intake) and other Sb sources are veg etables, drinking water, and meat ( Feng et al., 2013 ) . Experiments on the effects of Sb exposure on soil fauna found 50% decrease in reproduction of earthworms, potworm s, and collembola when exposed to 70, 316, and 169 mg Sb/kg soil for 28 d ( Tschan et al., 2009 ) . Soil algae Chlorococcum infusionum had a chlorophyll density reduction (EC50) after being exposed to 257 mg Sb/kg Luvisol soil for 14 d ( Hammel et al., 1998 ) . Sb toxicity in plants is usually shown by growth reduction and barley showed 50% decrease in root elongation and l ettuce had a 50% reduction in shoot biomass after growing in soil spiked with 6,819 and 7,549 mg Sb/kg soil ( Tschan et al., 2009 ) . Paddy rice had 14% less plant biomass following exposure to 5 mg/L Sb in hydroponic media for 14 d; whereas a high Sb concentration of 50 mg/L c aused plant death ( Feng et al., 2011a ) . Maize lost 25 and 28% of root and shoot biomass, respectively, after being exposed to 50 mg/L Sb for 3 weeks ( Pan et al., 2011 ) . The n on As accumulating ferns of Cyclosorus dentatus and Microlepia hancei also had 35 38% reductions in plant biomass after exposure to 20 mg/L Sb for 14 d in hydroponic media ( Feng et al., 2009 ) .

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19 Sb U ptake in P lants Plants have two different processes to transport water and mineral nutrients across membranes: passive and active transport. Passive transport does not require energy and transports the solutes from high to low concentration s . Passive transport consists of 2 subgroups: simple and facilitated diffusion. Simple diffusion allows gas, water, and small uncharged solutes to pass through the membrane via phospholipid or membrane protein channels. The phospholipid bilayer of the pl asma membrane allows gases such as O 2 and CO 2 to pass through and block water and small uncharged solutes. Water and small uncharged solutes require membrane channels to facilitate their movement into the cell s . Aquaporin channels transport w ater whereas aquaglyceroporin channels transport glycerol and other small uncharged solutes. Facilitated diffusion conveys large uncharged solutes through the membrane by carrier proteins. The conformation of carrier proteins changes after binding to substrates, whi ch facilitates the movement of substrates across the membrane. Active transport requires energy to transport ion solutes from low to high concentration by carrier proteins and ion pumps. Carrier proteins in active transport can co transport two molecules either the same direction (called symporter proteins) or in the opposite direction (called anti porter proteins). Phosphate (P V ) is taken up by plants via the roots by symporter proteins against the concentration gradients. There are 2 mechanisms involve d in this co transport: high and low affinity transport. Hi affinity transport proteins are active under low P V concentrations, whereas low affinity transport proteins tend to be more active under high P V concentrations. Sb is not an essential nutrient f or plants, but plants can take up Sb through roots and through deposition from the atmosphere onto the surfaces of aerial plant parts ( Tschan et al.,

PAGE 20

20 2009 ) . Grasses grown in clean soil showed high Sb content in the leaves because of dust deposits of Sb on the leaves ( Tschan et al., 2009 ) . High Sb concentration in the leaves of Tussilago farfara grown in a military shooting range near Lucerne, Switzerland was also from Sb surface deposition ( Robinson et al., 2008 ) . Sb concentration in plants tends to relate to Sb concentration in soil ( Okkenhaug et al., 2011 ; Tschan et al., 2009 ) . Plant tissues have typical background Sb concentrations of 0.01 0.1 mg/kg , with levels of 5 10 mg/kg causing phytotoxicity and growth reduction ( Feng et al., 2013 ; Xue et al., 2014 ) . Sb content can reach 1,600 mg/kg in mining areas ( Tschan et al., 2009 ) . There is littl e knowledge about Sb uptake mechanisms or which Sb species are taken up and translocated by terrestrial plants. Plants have two strategies to prevent toxicity from heavy metals: avoidance and tolerance ( Dalvi and Bhalerao, 2013 ) . The first strategy occurs when plants are exposed to heavy metals and plants initially avoid uptake through immobilization by mycorrhizal asso ciations or by forming a complex with root exudates ( Dalvi and Bhalerao, 2013 ) . The latter strategy occurs after heav y metals enter into the root cells and plants detoxify heavy metals by binding to the cell wall, active efflux pumping at plasma membranes, complexation with organic acids, and inactivation through sequestering into the vacuole ( Dalvi and Bhalerao, 2013 ) . Some plants might have the ability to translocate heavy metals to the shoot s and plants accumulate heavy metals >1,00 0 mg/kg in their aboveground parts are called hyperaccumulators ( Ma et al., 2001 ) . Different plants have different Sb uptake mechanisms: some plants have limited Sb translocation whereas other plants have high Sb accumulation in the shoots ( Xue et al., 2014 ) . Sb accumulation by native herbaceous plants around an Sb mine at the Xikuangshan mining area in China can be classified into 3 groups: low and high Sb accumulation in the roots and high Sb

PAGE 21

21 accumulation in the shoots ( Xue et al., 2014 ) . Phytolacca americana , Micanthus sinensis , Artemisia argyi , and Imperata cylindrica accumulate lower Sb concentrations in the roots compared to the Sb concentration in the soil, whereas Cynodon dactylon and Amaranthus paniculatus accumulate high Sb amounts in the root s ( Xue et al., 2014 ) . Boehmeria nivea , Eleusine indica , Aster subulatus , and Equisetum ramosissimum accumulate more Sb in the shoot than in the root, but they are not an Sb hyperaccumulator because the concentration is < 1,000 mg/kg Sb in the shoot s ( Xue et al., 2014 ) . Plants generally accumulate Sb in the roots, as is found in Chinese brake fern ( Pteris vittata , ), rye, wheat, w ater couch, s mart weed, Kikuyu grass, s edge , rice and Juncus ( Mathews et al., 2011 ; M ü ller et al., 2009 ; Shtangeeva et al., 2012 ; Telford et al., 2009 ) . Other plant s such as maize, sunflower, radish, B. nivea , E. indica , A. subulatus , and E. ramosissimum accumulate Sb ma inly in the shoots ( He, 2007 ; Tschan et al., 2008 ) . Sb uptake in plants is associated with three major factors; Sb phytoavailability in the media, Sb speciation, and the plant species ( Feng et al., 2013 ) . The solubility of Sb is associated with Sb phytoavailability in soil ( Feng et al., 2013 ) . Barley and lettuce have a 50% reduction in growth after being exposed to 6,819 and 7,549 mg Sb /kg soil, which equaled to 39 and 41 mg/L Sb in soil solution ( Tschan et al., 2009 ) . Maize has an affinity for both Sb III and Sb V while rye prefer entially accumulates Sb V and wheat and rice preferentially uptake of Sb III ( Pan et al., 2011 ; Shtangeeva et al., 2012 ; Tschan et al., 2008 ) . In the Sb mining area with an Sb concentration in the soil of 443 mg/kg, Artemisia argyi accumulated low Sb concentration (12 mg/kg ) in the root s , whereas Eleusine indica accumulated 118 mg/kg in the root s ( Xue et al., 2014 ) .

PAGE 22

22 Sb S peciation in P lants Sb V is the dominant species in plants regardless whether exposed to either Sb I II or Sb V ( M ü ller et al., 2009 ; Müller et al., 2013 ; Okkenhaug et al., 2011 ; Ren et al., 2014 ) . Plants growing in the Xikuangshan mining area in China have Sb V as the dominant species (70%), with only 30% of Sb conc entration present as Sb III ( Okkenhaug et al., 2011 ) . The smaller Sb III portion in the plants might come from preferential plant uptake of Sb V and limited Sb V reduction in the plants ( Okkenhaug et al., 2011 ) . Rice exposed to both Sb III and Sb V for 4 h in a hydroponic system (with 38% Sb III oxidation in Sb III media and no Sb V reduction in Sb V media) mainly accumulates Sb in the roots as Sb V (95 97% of total Sb), suggesting Sb II I oxidation in the roots ( Ren et al., 2014 ) . P. vittata growing in s oil and quartz substrate spiked with 16 mg/kg Sb V for 49 d accumulates mainly Sb in the roots with 74 93% Sb as Sb V ( M ü ller et al., 2009 ) . The minor amount of Sb III might come from Sb V reduction in the roots ( Müller et al., 2013 ) . Sb III oxidation and Sb V reduction also have been reported in bacteria and algae ( Filella et al., 2007 ; Hamamura et al., 2012 ; Lehr et al., 2007 ; L i et al., 2013 ; Nguyen and Lee, 2014 ; Torma and Gabra, 1977 ) . Bacteria isolated from the Xikuangshan Sb mine, China ( Acinetobacter , Comamonas , Stenotrophomonas , and Variocorax ), the Ichinokawa Sb mine, Japan ( Pseudomonas ), and from acid mine water in northern Quebec, Canada ( Thiobacillus ferrooxidans ) can oxidize Sb III ( Hamamura et al., 2012 ; Li et al., 2013 ; Torma and Gabra, 1977 ) . Most Sb III oxidizing bacteria cannot oxidize As III except Agrobacterium tumefaciens ( Lehr et al., 2007 ) . Bacteria ( Sinorhizobium ) isolated from sediments collected in the vicinity of an Sb factory has been shown to reduce Sb V to Sb III ( Nguyen and Lee, 2014 ) . Recently, a bacterium, in the order Bacillus , isolated from sediment samples from the alkaline, hyper saline Mono Lake ( pH approximately 10) , California, USA can reduce Sb V to Sb III ( Abin and Hollibaugh, 2013 ) .

PAGE 23

23 The bacterium uses Sb V as a ter minal electron acceptor for anaerobic respiration and microcrystals of Sb 2 O 3 are released as by products ( Abin and Hollibaugh, 2013 ) . The authors suggested th at Sb 2 O 3 production can be applied to nanotechnology products because Sb 2 O 3 has been used as a substrate for several Sb products such as flame retardants, ceramic pigment opacifiers, and catalysts for the production of polyethylene terephthalate ( Abin and Hollibaugh, 2013 ) . Fresh water and marine algae accumulate 0.1 0.2 m g/ k g Sb , with most Sb in the cytosol ( Filella et al., 2007 ) . Sb V is the dominant Sb species in algae and some algae have the ability to oxidize Sb III to Sb V ( Filella et al., 2007 ; Lehr et al., 2007 ) . After expos ing to Sb III , 40% Sb V was found in the excretion by algae Chlorella vulgaris , which suggest s a detoxification mechanism ( Filella et al., 2007 ) . A eukaryotic thermoacidophilic algae Cyanidiales isolated from Yellowstone National Park can oxidize Sb III and the Sb III oxidation rate increases by pre exposing to Sb III for one culture cycle ( Lehr et al., 2007 ) . Arsenic Hyperaccumulator Pteris vittata The As hyperaccumulator P. vittata (Chinese brake fern) is a vascular terrestrial fern in the division of Pteridophyta ( Lakela and Long, 1976 ) . An erect short rhizome is covered by yellow scales and the stipe and rachis are covered with hairy scales. It is a medium large fern with the short basal pinna length increasing upwards to the top and the pinna can be large up to > 1 m ( Hoshizaki and Moran, 2001 ; Wee, 1998 ) . P. vittata originates from Asia and is found from South Carolina to Louisiana, Southe rn California and in Florida. It has a wide distribution and can be found in sunny sites, in limestone rocks and in building crevices ( Hoshizaki and Moran, 2001 ; Jones, 1987 ) . It is a hardy plant and prefers sun with moist dry soil ( Hoshizaki and Moran, 2001 ) .

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24 The life cycle of P. vittata is similar to other ferns with alternating cycles of a diploid sporophyte stage and a haploid gametophyte stage ( Xie et al., 2009 ) . The abundant haploid spores are located in a line alon g the margin of the lower side of the pinna , and they are released after maturity and germinate under humid conditions ( Gumaelius et al., 2004 ; Xie et al., 2009 ) . A germinating spore forms a tiny single layered sheet of cells called a prothallium (an autotrophic haploid gametophyte) and P. vittata heart shaped prothall ium contain s egg forming archegonia and sperm forming antheridia ( Gumaelius et al., 2004 ; Xie et al., 2009 ) . The prothallium has a root like stem called a rhizoid to absorb water and nutrients for gamete production ( Xie et al., 2009 ) . When archegonia and antheridia mature, usually at different time from the same prothall ium, the sperm is released and fertilizes with an egg to generate a new diploid sporophyte ( Xie et al., 2009 ) . P. vittata gametophyte and sporophyte have been reported to accumulate high As in their tissues ( Gumaelius et al., 2004 ; Ma et al., 2001 ) . P. vittata has a dominant ability to take up and translocate As to the frond s and is the first identified As hyperaccumulator with 1,000 mg As /kg dry weigh t in its frond s ( Ma et al., 2001 ; Rascio and Navari Izzo, 2011 ) . Studies on uptake of other metals by P. vittata has been reported and P. vittata accumulates Cd, Ni, Pb, Zn, Cr, and Sb mainly in the root s ( de Oliveira et al., 2014 ; Fayiga et al., 2004 ; Mathews et al., 2011 ) . Sb S peciation and A nalysis The distribution, speciation, biogeochemistry and ecotoxicity of Sb in the environment have been poorly documented until recently due to the difficulty in analyzing Sb ( Reimann et al., 2010 ) and the low extraction efficiency. Most Sb is retained within samples and there is a lack of stability of the investigated Sb throughout the ent ire analytical process ( Krachler et al., 2001 ) . An aggressive extraction method with nitric acid can oxidize Sb III in the samples whereas

PAGE 25

25 non aggressive extractants such as water, dilute nitric acid, sodium hydroxide and enzymes extra ct low amounts of Sb without Sb III oxidation ( Miravet et al., 2005 ) . Miravet et al. ( 2005 ) compared the mild extractants among water, 9:1 methanol water and citric acid on the certified reference material Virginia tobacco leaves and found that sonication and sonication shaking increased the extraction efficiency from 3 23% to 26 45%. Sb specia tion determinations usually utilize hydride generation (HG) and high performance liquid chromatography (HPLC) techniques. The HG detects stibine (SbH 3 ) so Sb V must be reduced to Sb III prior to efficient SbH 3 formation by KI, KBr, thiourea, and L cysteine ( Nash et al., 2000 ) . HPLC has become a more popular method of Sb speciation due to its more accurate ability to detect methyl Sb species. HPLC separation of Sb species is based on different adsorption capacities of the analytes on the column with different retention times. This technique separates the mixture of components by passing pressurized samples through a solid adsorbent column. An anion exchange column has been used for Sb speciation by HPLC due to Sb III and Sb V behav ing as anion s . Solid phase extraction (SPE) is an extraction method using a solid phase and a liquid phase to isolate an analyte of interest. SPE has been used for various purposes: purification, trace enrichment, desalting, derivati z ation and class fract ionation ( Ferenc and Biziuk, 2006 ) . An anion exchange cartridge of SPE has an operating principle similar to liquid chromatog raphy by exchanging anionic compounds through chloride ion (Cl ) binding to an anionic site in the cartridge (an aliphatic quaternary ammonium group , which is bonded to the silica (Si) surface cartridge) ( Ferenc and Biziuk, 2006 ) ( Figure 1 1 ) . The pH in an anion exchange cartridge is a major key for separation as ionic analytes behave differently under varying pH ( Ferenc and Biziuk, 2006 ) .

PAGE 26

26 Research Objectives This study was divided into two sections: Sb speciation method by SBAEC and Sb uptake by P. vittata . The overall objective of the first section was to develop a new method for Sb speciation. In order to meet this objective, a strong anion exchanger cartridge was investigated for Sb speciation. The overall objective of the second section was to investigate Sb III and Sb V transport and accumulation pathways in P. vittata . In order to meet thi s objective, P. vittata sporophytes were used for Sb III and Sb V uptake study and P. vittata gametophytes were used for further study on Sb III uptake route. This dissertation was structured as follows C hapter 2 evaluated a pro posed cartridge on Sb speciation ; Chapter 3 focused on Sb accumulation, translocation, and efflux in P. vittata sporophytes ; Chapter 4 emphasized the route of Sb III uptake in P. vittata gametophytes ; Chapter 5 provided a general conclusion and future research direction .

PAGE 27

27 Figure 1 1 . The structure of anion exchange cartridge containing Cl binding to quaternary ammonium group , which is covalently linked to the silica backbone. ammonium

PAGE 28

28 CHAPTER 2 A NEW METHOD FOR ANTIMONY SPECIATION IN PLANT BIOMASS AND NUTRIENT MEDIA USING SOLID PHASE EXTRACTION Introductory Remarks Sb is a toxic element and is commonly associated with sulfur minerals ( McDermott et al., 2010 ) . The concentration of Sb ranges 0.2 to 0.3 mg/kg whereas the background in surface soil varies from 0.02 to 7.01 mg/kg ( Wilson et al., 2010 ) . Sb has been used in batteries, car brake linings and in fire retardants and it is released to the environment at a rate of 38,000 tons per year from both natural and anthropogenic sources ( Zheng et al., 2000 ) . A steady increase of Sb levels in snow and ice in the Arctic and in the aerosol s amples collected from the city of Tokyo indicate that Sb contamination is a worldwide environmental pr oblem ( Krachler et al., 2005 ) . Sb is a possible carcinogen , with no known biological function ( Krachler et al., 2001 ) . It induces keratitis, dermatitis, conjunctivi tis and gastritis and has been shown to cause lung cancer in rats ( Smichowski, 2008 ) . In plants, Sb inhibits photosynthesis and decreases utilization of essential elements ( Feng et al., 2013 ) . The toxicity of Sb largely depends on its chemical species, with inorganic species being more toxic than organic ( Nash et al., 2000 ) and the reduced inorganic species Sb III bein g 10 times more toxic than the oxidized Sb V species ( Smichowski, 2008 ) . The amount and type of Sb taken up by plants var y with species. For example, rye takes up more Sb V than Sb III whereas wheat and rice take up more Sb III than Sb V ( Huang et al., 2012 ; Shtangeeva et al., 2012 ) . Sb in plant tissues exist s as both inorganic and organic forms, with inorganic Sb being the dominant species ( Craig et al., 1999 ; Müller et al., 2009 ) . Thus, information on Sb speciation is important to understand Sb behavior in the environment as well as Sb translocation and transformation in plants.

PAGE 29

29 There are several analytical techniques that can be used to analyze total Sb, such as low cost o f equipment g raphite furnace atomic absorption spectrophotomet ry ( GF AA S ) and high cost of equipment such as the i nductively coupled plasma mass spectrometry ( ICP MS ) . In term of Sb speciation , the common ly used analytical method couple s HPLC with ICP MS ( Hansen and Pergantis, 2008 ) . Other Sb speciation methods focus on Sb separation based on the affinity for Sb III for sulfur group ( S). For example, pyrrolidine dithio carbamate (PDC) containing S group has been used for Sb III separation with non polar organic solvents such as methylisobuthylketone ( García et al., 1995 ) or xylene ( Serafimovska et al., 2011 ) . However, this technique has the disadvantage of re quiring manual injection of the organic media into the graphite furnace ( Serafimovska et al., 2011 ) . Using a column to separate Sb III PDC complexes has been developed to overcome liquid liquid extraction methods. Separating Sb III PDC complexes requires a solid phase extraction (SPE) based on Chromosorb ® self packed resin column ( Saracoglu et al., 2003 ) , which might cause inaccurate results. The non polar SPE Isolute ® silica based octyl (C 8 ) cartridge retain s Sb III PDC complex ( Yu et al., 2002 ) , but the cartridge is not globally available. In addition to PDC chelating agent, there are several SPE techniques for Sb speciation . For example, an ionic Amberlite ® IRA 910 resin for adsorb ing Sb V ( Sánchez Rojas et al., 2007 ) , Dowex ® 1x4 resin for retain ing Sb III Cl complex ( ) , and modified columns with dimercaptosuccinic acid or tetra ethylene pentamine for retain ing Sb III ( Huang et al., 2007a ; Mendil et al., 2013 ) . However, those techniques have not been successful for Sb speciation as they are limited by graphite furnace analysis, procedure complication and commercial availability. For example, an ionic Amberlite ® IRA 910 column requires self packing and the Dowex ® 1x4 column requires hydrochloric acid elution , which is impractical for

PAGE 30

30 analysis by GF AA S . Furthermore, dimercapto succinic ac id for chemically modifying mesoporous titanium dioxide micro columns or tetra ethylene pentamine for binding silica gel columns are not commercially available so they have to be synthesized in a laboratory. The reproducibility of those results obtained i n such a way remains uncertain . The pre packed strong anion exchanger cartridges proposed in this study have the ability to separate Sb III from Sb V , which can be detected by GF AA S or ICP . The cartridges are reasonably priced and ready for immediate use . In addition, they are reproducible and commercially available worldwide. This work was the first application of SPE in separating Sb speciation in plant biomass and hydroponic nutrient media. It also sheds light on Sb speciation in plant tissues and media while offering an alternative method against an expensive HPLC technique. The aim of this study was to investigate a silica based strong anion exchanger cartridge or SBAEC for Sb III and Sb V separation under pH c ontrolled conditions for Sb analysis by GF AAS. Furthermore, the results from Sb speciation in the root s of As hyperaccumulator P . vittata were confirmed by HPLC ICP MS. Materials and Methods All chemicals were analytical reagent grade. Standard stock solutions of Sb III and Sb V (1 g/L) were prepared by dissolv ing potassium antimonyl tartrate ( C 8 H 4 K 2 O 12 Sb 2 · 3H 2 O , Fisher) and potassium hexahydroxy antimonate ( KSb(OH) 6 , Sigma Aldrich) in DI water and hot DI water, respectively. The Sb III and Sb V stock solu tions were stored in a refrigerator at 4ºC. Working solutions were freshly prepared daily by further dilution from these stock solutions to the ranges needed . A citric acid solution was prepared by dissolving monohydrate citric acid (HOC(COOH)(CH 2 COOH) 2 . H 2 O, J.T. Baker) in DI water. NaOH and HCl were used for pH

PAGE 31

31 adjustments. All labware used in this study was washed and soaked in 1 M nitric acid for 24 h and rinsed several times with tap water followed by DI water before use. Instrument for Sb Analysis and Speciation GF AAS ( AA240Z Zeeman, Varian, Australia) was used for Sb analysis. The linear range was 10 for Sb was 0.15 palladium nitrate modifier solution containing 0.25% citric acid in 2.5% nitric acid were introduced with an auto sampler. HPLC ICP MS was used to verify the accuracy of the SBAEC method in 2 mM citric acid system. A Hamilton PRP X100 AEC column (250x4.1 mm, 10 µM; Hamilton, UK) with a guard column combined with a Waters 2695 HPLC system to separate Sb species. The mobile phase consisted of 20 mM EDTA (NH 4 ) 2 and 2 mM potassium hydrogen phthalate adjusted to pH 4.5 with ammonium hydroxide. The sample was sonicated and filtered (0.22 µm), injecting 50 µL wit h a flow rate at 1.2 mL/min at 40°C. A Nexion 300X Inductively Coupled Plasma Mass Spectromet ry (ICP MS) (Perkin Elmer, USA) was connected to HPLC to analyze Sb concentration. Sb V and Sb III standard solutions at 10 µg/L were run to obtain retention time. Stock solutions of Sb species were prepared from C 8 H 4 K 2 O 12 Sb 2 ·3H 2 O (Sigma Aldrich, >99%) and KSb(OH) 6 (Fluka, >99%) with DI water. The stock solution at 1 g/L was stored in the dark at 3°C until use. All standard solutions were diluted from stock solution with 2 mM citric acid on the day of analysis. Sb III and Sb V standard solutions were calibrated on ICP MS using a 100 mg/L multi element environmental calibration standard (PerkinElmer, in 5% HNO 3 ). The for Sb III and Sb V , respectively. Sb Separation Using SBAEC SPE is an extraction method common in liquid chromatography (LC) in which solid and liquid phase s are interac t ed to separate chemical com pounds of interest ( Ferenc and

PAGE 32

32 Biziuk, 2006 ) . The sorbents for SPE refer to the stationary phase us ed in LC columns ( Ferenc and Biziuk, 2006 ) . Anion exchange column such as Hamiltion PRP X100 and Synchropak Q300 are used for Sb speciation by HPLC ( Zheng et al., 2001 ; Zheng et al., 2000 ) . However , the affinity of Sb species on the columns depends on Sb III complex ( Zheng et al., 2000 ) . Sb III citrate complex has a higher affinity to the column rather than Sb V citrate complex whereas without citrate complexing agent, the affinity of Sb V is greater than Sb III on the column ( Zheng et al., 2000 ) . SBAEC used in this study was Sep Pak Accell Plus QMA Plus Short cartridges obtained from Waters ( WAT020545, Milford, MA) . The cartridge contain s a silica based, hydrophilic, strong anion exchanger with pore size of 300 Å, and sorbent particle size of 37 55 µm . The silica backbone in the cartridge contains quaternary ammonium group (R) as anion charged site and Cl as anion exchanger. Anion compounds from the sam ple were exchanged to Cl . To test the efficiency of the cartridge in Sb V retention, 45 mL of Sb III or Sb V solution was passed through the cartridge at a flow rate of 10 mL/min. Sb III or Sb V solution at 200 µg/L was prepared in DI water at pH 4, 6, or 8. The filtrate from the cartridge was collected for Sb analysis by GF AAS. The sample without cartridge filtration was used for total Sb concentration. The Sb III or Sb V concentrations were calculated from a difference between total Sb and Sb passed throug h the cartridge. To ensure better separation of Sb V from Sb III , the discard volume of filtrate was determined by applying 250 mL of 200 µg/L Sb III + Sb V mixture containing 0, 25, 50, 75 and 100% Sb III . S b s peciation was determined every 50 mL aliquots of the filtrate. The results indicated that 200 mL of filtrate needed to be discard ed before collecting filtrate for effective retention of Sb V by the cartridge .

PAGE 33

33 To reduce the discard ed volume, cartridge s w ere preconditioned by passing a certain volume of Sb solution. 15 mL of solutions containing 1 mg/L of Sb III or Sb V was used to precondition the cartridge and Sb V solution showed a better result (data not show n ). The conditioning was determined by applying 15 mL of 1 mg/L Sb V to the cartridge and analy zing the discard ed solution in 10 mL intervals after the first 50 mL of solution being discard ed . Precondition ing of the cartridge us i ng Sb V solution reduce d the discard ed filtrate volume from 200 to ~ 100 mL. Discarding ~ 100 mL of filtrate was still too m uch so citric acid was used to condition the cartridge. Citric acid could form a complex to both Sb III and Sb V and their products were anion complexes ( Zheng et al., 2001 ) . The cartridge with the matrix media of citric acid media retained Sb III citrate complex. Sb V [ Sb(OH) 6 ] is present as [Sb(OH) 3 (C 6 O 7 H 5 )] whereas Sb III [Sb 2 (C 4 O 6 H 2 ) 2 ] 2 is present as [Sb(C 6 O 7 H 6 ) 2 ] in citric acid solution ( Zheng et al., 2001 ) . An anion exchange cartridge would adsorb Sb from the samples according to the reactions shown in the following Eq uation . Sb III citrate complex: RCl + [Sb(C 6 O 7 H 6 ) 2 ] R Sb(C 6 O 7 H 6 ) 2 + Cl ( 2 1 ) This system contained 2 negatively charged complexes , which would compete for anion site s on the cartridge ( Zheng et al., 2001 ) . Solution o f 200 µg/L Sb III + Sb V mixture containing 0, 25, 50, 75 and 100% Sb III in 2 mM citric acid at pH 4, 6, and 8 was used to condition the cartridge. Using a cartridge that had already been preconditioned with 15 mL of 1 mg/L Sb V to reduce Sb V + Sb III anion e xchange sites, 70 mL of the Sb III + Sb V solution was passed through the cartridge. To determine optimal pH , sample in every 10 ml aliquot was collected for Sb speciation. Using the optimal pH,

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34 different Sb III proportions of 200 µg/L Sb III + Sb V mixture i n 2 mM citric acid were applied to the column. To determine discard volume, filtrate every15 mL aliquot was collected and analyzed for Sb speciation. Sb Speciation in Plant Tissues and Spent Growth Media Since the As hyperaccumulat or P. vittata was efficient in Sb accumulation in the roots, it was used to test the efficiency of Sb speciation using the cartridge ( Tisarum et al., 2014 ) . P. vittata was propagated from spores in our laboratory. Uniform plants with 3 4 fronds were selected and acclimatized hydroponically in 0.2X HS with constant aeration under cool and warm fluorescent lamps ( the total light intensity ~ 2 00 micromoles per m 2 ) with temperature of 23 28ºC and ~70% humidity for 4 weeks. After 4 weeks of acclimatization, P. vittata were transferred to 0.5 mM CaCl 2 (pH 6) for 24 h to desorb surface nutrients from the roots. Then P. vittata was divided into 3 treatments : control (DI water), 8 mg /L Sb III , and 8 mg /L Sb V , all adjusted to pH 6. After 24 h, P. vittata roots were washed to desorb apoplastic Sb in an ice cold solution containing 1 mM Na 2 HPO 4 and 0.5 mM Ca(NO 3 ) 2 , pH 6 for 10 min . The plant tissues were used for Sb extraction and speciation. P lant tissues were extracted using a modif ied method by Okkenhaug et al. ( 2012 ) with Sb extraction efficiency of 70 92% in plants ( Tisarum et al., 2014 ) . Briefly, p lants were harvested and washed thoroughly in DI water. They were separated into the roots and fronds, which were freeze dried for 2 d. Dry tissues were ground with l iquid nitrogen to fine powder in a ceramic mortar and freeze dried for an additional 2 d. Powdered tissue samples (~50 mg) were shaken at 100 rpm with 10 mL of 100 mM citric acid for 4 h and then sonicated at 42 kHz for 1 h (VWR DHT Ultrasonic Cleaner B35 00A) . The 10 mL extracts containing 100 mM citric acid were diluted 5 times to 50 mL with DI water and filtered (45 m filter) before separation of Sb

PAGE 35

35 species. The samples were further diluted 10 times with DI water. At this stage, the initial citric ac id concentration of 100 mM in the extracts was diluted down to 2 mM and the pH of the extracts adjusted to 6.0 by use of NaOH and /or HCl. In addition to plant biomass, we also tested Sb speciation in spent growth media. Spent media were obtained after gro wing P. vittata in 0.2X HS for 8 d . To reduce interference from other ions, the 0.2X HS was diluted 40 times with DI water to 0.005X HS. The diluted HS at 0.005X contained 14 µg/L H 3 BO 3 , 17µg/L MnSO 4 . H 2 O, 0.5µg/L CuSO 4 .5H 2 O, 1.1 µg/L ZnSO 4 .7H 2 O, 0.5 µg/L (NH 4 ) 6 MO 7 O 24 . 4H 2 O, 44.5 nM H 2 SO 4 , 168 µg/L Na 2 EDTA, 139.5 µg/L FeSO 4 ,4.7 mg/L Ca( NO 3 ) 2 .4H 2 O, 2.6 mg/L MgSO 4 . 7H 2 O, 3.3 mg/L KNO 3 and 0.6 mg/L NH 4 H 2 PO 4 ( Fayiga et al., 2008 ) . Sb speciation in plant roots and growth media were tested. The extracted plant samples and growth media were first passed through th e cartridge, preconditioned with Sb V , and then determined for Sb by GF AAS. To verify the accuracy of the cartridge method, the extractions from P. vittata roots treated with 8 mg/L Sb III or Sb V for 24 h were also analyzed for Sb speciation using HPLC ICP MS. Results and Discussions Sb V Retention on Cartridge in DI Water The anion exchange cartridge would exchange Cl to anion species from Sb III or Sb V and non negatively charged ions w ere not bound on the cartridge. A silica based, hydrophilic, anion exchanger cartridge had a higher affinity to Sb V as it was retained on the cartridge rather than Sb III . To assess impact of solution pH and discard filtrate volumes (filtrate volume required to condition the cartridge for Sb separation), solutions containing 100 µg/L Sb III or Sb V at pH 4, 6,

PAGE 36

36 or 8 were applied to the cartridge and Sb retained on the cartridge was determined ( Figure 2 1 ). The pH of Sb III or Sb V solutions had no impact on Sb retention on the cartridge. As ex pected, Sb V was rapidly retained on the cartridge whereas Sb III gradually released after 10 mL filtrate volume. The results showed that the cartridge had an ability to separate Sb V from Sb III . Further experiments on filtrate discard volume were studied a t pH 6. Under pH 6, the cartridge required discard volume of 200 m L filtrate to achieve good Sb V and Sb III separation ( Figure 2 2 ). The Sb V retained on the cartridge following 250 mL equaled 50 µg Sb V . Thus the cartridge had an ability to retain at least 50 µg Sb V and after discarding the first 200 m L of filtrate, the cartridge retained Sb V while pass ing Sb III ( Figure 2 2). The 200 mL discard volume was too much, thus, more effort focused on reduction in discard volume of filtrate. By preconditioning the cartridge with 15 mL of 1 mg/L Sb V , it reduce d the discard volume from 200 to 110 mL ( Figure 2 3 ). Sb III Retention on Cartridge under C itric Acid Due to its high Sb stability and strong negative charge complex, 2 mM citric acid was used to condition the sample to better separate Sb V from Sb III . Citric acid could form a complex with either Sb III or Sb V . The Sb III tartrate complex from potassium antimonyl tartrate is replaced by the Sb III citrate complex ( [Sb(C 6 O 7 H 6 ) 2 ] ) and Sb V ( Sb(OH) 6 ) is transformed to [Sb(OH) 3 (C 6 O 7 H 5 )] ) ( Zheng et al., 2001 ) . Both forms of Sb III and Sb V citrate complexes are negatively charged and Sb III citrate has a higher affinity to the cartridge , which allows it to be retained. Citric acid was not only used for separation of Sb III from Sb V , but it also stabilized Sb III in the solution ( Zheng et al., 2001 ) . Solutions containing different concentrations of Sb III and Sb V at pH 4, 6 and 8 were passed through the cartridge and analyzed for Sb. The pH impacted the Sb retention on the

PAGE 37

37 cartridge in both Sb III and Sb V solutions ( Table 2 1) . At pH 4 and 6 , the cartridge retained 96 and 99% Sb III and 3 and 1% Sb V whereas pH 8 retained 1% from both Sb III and Sb V . The data indicated that the cartridge accurately separated Sb III from Sb V at pH 4 and 6 from mixtures of Sb V and Sb III . A t pH 6, the best Sb III retention (~99%) was achieved while allowing ~99% Sb V to pass through the cartridge. We then determine d the minimum discard volume of filtrate required for accurate results ( Figure 2 4 ) . Sample mixtures of Sb III + Sb V in 2 mM citric acid at pH 6 were passed through cartridges preconditioned with Sb V . Samples with >50% Sb III in the mixture showed ~99% retention of Sb III after discarding 45 mL of filtrate. On the other hand, with Sb III at 0 and 25%, at least 60 mL of filtrate needed to be discarded to achieve optimal retention. Thus 60 mL of filtrat e was discarded for Sb speciation in 2 mM citric acid (pH 6) through cartridges preconditioned with 15 mL of 1 mg/L Sb V . Sb Speciation in Plant Biomass and Growth Media Cartridge based Sb speciation was applied to plan t biomass ( P. vittata roots) and growt h media, including spent 0.005X HS media after growing P. vittata for 8 d and 0.005 X HS spiked with Sb ( Table 2 2). Sb III was 100% retained by the cartridge , but its retention dropped slightly (4 12%) in the presence of Sb V in P. vittata roots ( Table 2 2 ). The HS samples were spiked with different concentrations of Sb III and Sb V containing 2 mM citric acid at pH 6. The cartridge was preconditioned with 15 mL 1 mg/L Sb V and the first 60 mL of filtrate was discarded. The growth media, which contained dif ferent elements of HS, organic acids from root exudates ( Tu et al., 2004 ) from spent media, and plant components from plant extraction did not impact the Sb III retention on the cartridge. Various Sb III proportion in Sb mixtures confirmed the ability of cartridge on Sb spec iation as results correlated to Sb III proportion in the samples ( Table 2 2 ).

PAGE 38

38 The cartridge retained 2 3% Sb from 0% Sb III mixtures (100% Sb V ) , 22 27% from 25% Sb III mixtures, 47 50% from 50% Sb III mixtures, and 72 76% from 75% Sb III mixtures ( Table 2 2 ). Without Sb V , the cartridge showed 100% Sb III retention from all sample media ( Table 2 2). To verify the ability of Sb III retention by the cartridge in 2 mM citric acid, Sb extraction of P. vittata roots treated with 8 mg/L Sb III or Sb V for 24 h were used. Using the optimized parameters, the cartridge showed effective retention of Sb III in P. vittata roots extracts with 92 and 104% Sb V recovery from Sb III and Sb V treatments, respectively ( Table 2 3 ). Sb speciation from HPLC ICP MS verified that no Sb III from P. vittata root extractions passed through the cartridge as there was no detection of Sb III ( Table 2 3 ). Conclusion This study outline d the first application of a solid phase extraction based on an anion exchange resin for Sb speciation in plant biom ass and hydroponic growth media. The concentration of Sb V was determined by GF AAS after retention of Sb III by the cartridge. The separation of Sb III from Sb V in the presence of citric acid was conducted at pH 6 with Sb V preconditioned cartridges after d iscard ing the first 60 mL of filtrate. Retention of Sb III on the cartridge under citric acid condition was confirmed by HPLC ICP MS, with 100% Sb III retention and 92 104% Sb V recovery from P. vittata roots treated with Sb III and Sb V . The method described here is simple and efficient because of the commercial availability of the cartridge used and the avoidance of the manual injection to a GF AAS and potential variations of self pack resin columns. The Sb speciation method from this study was comparable w ith recent studies using GF AAS as a n Sb detector ( Table 2 4 ). The detection limit from this study was higher than other methods, which focus on ultratrace Sb concentration in water. The cost

PAGE 39

39 from this method was cheaper than tetra ethylene pentamine bon ded silica gel , but higher than titanium dioxide and Pb PDC method ( Table 2 4 ).

PAGE 40

40 Table 2 1. Sb retained on the cartridge preconditioned with 15 mL of 1 mg/L Sb V in 2 mM citric acid of different pH and after discarding the first 70 mL of filtrate a . Sb added (µg/L) Sb retained (%) Sb III Sb V pH 4 pH 6 pH 8 200 0 96 ±0.5 99 ± 0.4 1±1.7 150 50 74 ± 0.1 74 ± 0.1 41 ± 0.5 100 100 48 ± 1.4 50 ±0.5 27 ± 1.2 50 150 27 ± 1.0 23 ±0.9 11 ±0.7 0 200 3 ± 0.4 1.0±0.6 1± 1.8 a Values are the mean of three replicates, with +/ standard error. Table 2 2. Sb retained on the cartridge from growth media ( 0.005 X HS and 0.005 X HS spent media) , and P. vittata roots after discarding first 60 mL of filtrate a . 2 mM citric acid was added to the samples and the cartridge was preconditioned with 15 mL of 1 mg/L Sb V . Sb added (µg/L) Sb retained (%) Sb III Sb V HS HS spent media P. vittata roots 200 0 100 ±0.0 100 ± 0.0 100 ± 0.0 150 50 76 ± 0.6 75 ± 0.2 72 ± 0.6 100 100 50 ± 0.4 47 ± 2.3 48 ± 1.0 50 150 27 ± 1.7 24 ± 2.2 22 ± 0.3 0 200 3 ± 1.5 2 ± 2.4 2 ± 2.1 a Values are the mean of three replicates, with +/ standard error. b HS = Hoagland solution .

PAGE 41

41 Table 2 3. Sb speciation in P. vittata roots extraction after exposure to 8 mg/L Sb III , or Sb V for 24 h by HPLC ICP MS before and after passing through the cartridge a . Treatment Sb concentration (mg/kg) Sb V recovery from the cartridge (%) Sb III Sb V Control Before 0.5 ± 0.1 µg/kg 0.2 ± 0.0 µg/kg After nd b 0. 2 ± 0.0 µg/kg Sb III Before 289.0 ± 87.6 456.0 ± 43.2 a After nd 421.0 ± 43.0 a 92 ± 2 Sb V Before 1.4 ± 0.4 45.6 ± 3.6 b After nd 47.6 ± 7.1 b 104 ± 5 a Values are the mean of three replicates, with +/ standard error, and columns with the same letters are not significantly different. b Sb III ). Table 2 4. Comparative data from recent studies on Sb speciation by different media via GF AAS detector. Media Sample application Preconcentration Factor Detection Limit (µg/L) Separation t ime (min) Cost ($/sample) Reference Titanium dioxide and Pb PDC River water, seawater 20 0.14 20 (total Sb) 15 ( Sb III ) 1.73 ( Zhang et al., 2007 ) Tetraethylenepentamine bonded silica gel Tap water, mineral water, spring water 50 0.02 14 (90 h for resin synthesis) 3.38 ( Mendil et al., 2013 ) A silica based, hydrophilic, strong anion exchanger cartridge Growth media, plant tissues 50 7.5 8 3.09 This study

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42 Figure 2 1. Percentage of Sb III and Sb V retained on the cartridge in DI water at different pH. Values are the mean of three replicates, bars representing standard error. Figure 2 2. Percentage of Sb retained on the cartridge in DI water adjusted pH 6 and sampled every 50 mL. The filtrates required 200 mL discard to retain all Sb III . Values are the mean of three replicates, bars representing standard error. 0 20 40 60 80 100 120 5 10 15 20 25 30 35 40 45 Sb retained (%) Discard volume (mL) SbIII pH 4 SbIII pH 6 SbIII pH 8 SbV pH 4,6,8 Sb III pH 8 Sb V pH 4,6,8 0 20 40 60 80 100 120 50 100 150 200 250 Sb retained (%) Filtrate volume (mL) SbIII:SbV=200:0 SbIII:SbV=150:50 SbIII:SbV=100:100 SbIII:SbV=50-150 SbIII:SbV=0-200

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43 Figure 2 3. Percentage of Sb reta ined on the cartridge in DI water adjusted pH 6. The cartridge was preconditioned with 15 mL of 1 mg/L Sb V and then collected the filtrates every 10 mL after discard ing first 50 mL. The filtrates required 110 mL discard to retain all Sb III . Values are t he mean of three replicates, bars representing standard error. Figure 2 4. Percentage of Sb retained on the cartridge in media containing 2 mM citric acid adjust ed to pH 6, preconditioned with 15 mL of 1 mg/L Sb V and then sampled every 15 mL after discard ing the first 30 mL. The filtrates required 60 mL discard to retain all Sb III . Values are the mean of three replicates, bars representing standard error. . 0 20 40 60 80 100 120 50 60 70 80 90 100 110 Sb retained (%) Filtrate volume (mL) SbIII:SbV=200-0 SbIII:SbV=150-50 SbIII:SbV=100-100 SbIII:SbV=50-150 SbIII:SbV=0-200 Sb III :Sb V =200:0 Sb III :Sb V =150:50 Sb III :Sb V =100:100 Sb III :Sb V =50:150 Sb III :Sb V =0:200 0 20 40 60 80 100 120 30 45 60 75 90 Sb retained (%) Filtrate volume (mL) SbIII:SbV=200-0 SbIII:SbV=150-50 SbIII:SbV=100-100 SbIII:SbV=50-150 SbIII:SbV=0-200 Sb III :Sb V =200:0 Sb III :Sb V =150:50 Sb III :Sb V =100:100 Sb III :Sb V =50:150 Sb III :Sb V =0:200

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44 CHAPTER 3 ANTIMONY ACCUMU LATION IN ARSENIC HYPERACCUMU LATOR PTERIS VITTATA L. Introduct ory Remarks Sb is a toxic metalloid widely distributed in the lithosphere, with average soil background concentrations being 0.3 8.6 mg/kg ( Tschan et al., 2009 ) . Sb is a chalcophile, commonly associated with sulfur rich minerals ( Anawar et al., 2011 ; Liu et al., 2010 ) . Recently, global Sb concentrations have been increasing at an alarming rate . F or example, Sb accumulation in arctic snow and ice has increased 50% during the last 30 years ( Krachler et al., 20 05 ) and 6.3 tons of Sb are annually dispersed in the aerosols of Tokyo ( Iijima et al., 2007 ) . Since the Industrial Revolution, the use of Sb has drastically increased due to its application in car brake linings and fire retardants ( Maher, 2009 ) , and as a hardening agent in bullet alloy (2 5% Sb) ( Steely et al., 2007 ) . Sb has no known essential biological function , but displays carcinogenic properties ( Krachler et al., 2001 ) . Its inorganic form is more toxic than the organic and Sb III is 10 times more toxic than Sb V ( Smichowski, 2008 ) . Dusts and ashes containing Sb can induce keratitis, dermatit is, conjunctivitis and gastritis and Sb III oxide has been shown to cause lung cancer in rats ( Smichowski, 2008 ) . Though Sb and As are analogs, their mechanisms of uptake and translocation in pl ants differ . F or example, the As hyperaccumulator P. vittata translocates As to the fronds while Sb remains in the roots ( Müller et al., 2013 ) . Rice and tomato reduce As V to As III in the root s and rapidly efflux As III out of the root s ( Xu et al., 2007 ) . On the other hand, maize and the As hyperaccumulator P teris cretica translocate Sb to the shoots regardless of whether they are treated with Sb III or Sb V ( Feng et al., 2011b ; Pan et al., 2011 ; Tschan et al., 2008 ) . The

PAGE 45

45 interactions between As and Sb in P. cretica are similar to P. vittata by increasing Sb V uptake with the addition of As V ( Feng et al., 2011b ; Müller et al., 2 013 ) . However, the presence of Sb does not effect As uptake by P. vittata ( Müller et al., 2013 ; Nagarajan and Ebbs, 2007 ) . Sb III has been reported to enter Arabidopsis via nodulin 26 like intrinsic proteins (NIPs) ( Kamiya and Fujiwara, 2009 ) . As V enter s via P V transporters, al though adding P V has no e ffect on Sb V uptake by maize and sunflower ( Tschan et al., 2008 ) . Plants usually take up metals an d accumulate them in the roots. Metal translocation to the shoots is rare and is hypothesized to play a role in defense against herbivores and pathogens ( Rascio and Navari Izzo , 2011 ) . P. vittata is unique because it has high ability to load As in the xylem and translocate it to the frond s . Translocation of other metals has not been observed in P. vittata , as they mostly accumulate in the roots including Sb ( Cai et al., 2004 ; Ma thews et al., 2011 ) . For example, P. vittata accumulates 49 mg/kg Sb in the roots when cultivated in quartz substrate with a concentration 5 mg/kg Sb V for 7 weeks ( Müller et al., 2 013 ) . In addition to Sb V , it is know that P. vittata can accumulate As V in the roots where it is then reduced to As III and translocated to the fronds ( Wang et al., 2002 ) . However, little is known about the fate of Sb V . The objectives of this study were to investigate 1) Sb uptake, efflux and speciation in As hyperaccumulator P. vittata during short term exposure, 2) Sb uptake by P. vittata accessions from Florida, China, and Brazil during long term exposure , and 3) Sb up take, translocation and Sb III oxidation of excised P. vittata fronds. Knowledge of how P. vittata takes up, transports, and metabolizes Sb will be helpful to better understand As uptake and metabolism in P. vittata .

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46 Materials and Methods P. vittata sporophytes were acquired from the greenhouse at University of Florida, USA . They were 8 months of age, 20 25 cm in height, 3 4 fronds, and uniform in size. The ferns were acclimatized hydroponically in 0.2 X Hoagland solution (HS) for 4 weeks with constant aeration with a 12 h photoperiod with photo flux of 350 µmol/m 2 s using cool and warm fluorescent lamps with temperature at 23 28ºC and 70% humidity. Pre treatment and S ampling of P. vittata After acclimatization in HS, the ferns were acclimatized in a solution of 0.5 mM CaCl 2 (pH 6) for 1 d before being transferred to opaque containers containing 1 L of deionized (DI) water with 1.6 and 8 mg/L of Sb III (potassium antimonyl tart r ate (Fisher)) or Sb V (potassium hexahydroxy antimonate (Sigma Aldrich)) (pH 6, 3 replicates). DI water was used to avoid Sb III oxidation by Fe and Mn in HS ( Belzile et al., 2001 ) . After 1 d exposure to Sb , the pla nts were rinsed with tap and DI water. Their r oots were separated and dried at 55ºC for 72 h for total Sb analysis. In addition, another set of plants exposed to 8 mg/L Sb III was used for efflux experiment . P lant roots were rinsed in DI water and placed in a solution containing 1 mM Na 2 HPO 4 and 0.5 mM Ca(NO 3 ) 2 (pH 6) for 10 min to desorb apoplastic Sb. Plants were then transferred to opaque containers containing 1 L of DI water, and 10 mL aliquots were sampled after 1, 2, 4, 8, and 24 h and analyzed for Sb . At the end, plant tissues were collected for Sb analysis. Sb A ccumulation in T hree P. vittata A ccessions The P. vittata spores from each accession were collected from the greenhouse at University of Florida, USA; Beijing, China; and Central Park in M inas Gerais state, Brazil . Spores from each accession were sprinkled separately onto moist potting mix (Miracle Gro

PAGE 47

47 potting soil mix) in 3 inch pots covered with plastic to maintain moisture and humidity. After 8 months, uniform plants were selected and acclimatized in hydroponics as mentioned above followed by transferring to 0.5 mM CaCl 2 (pH 6) for 1 d. Plants were then transferred to opaque containers containing 1 L of 0.2 X HS with 0 or 8 mg/L Sb III (pH 6, 3 replicates). After 7 d, the roots were rin sed with tap and DI water and dried at 55ºC for 72 h prior to total Sb analysis. Sb U ptake, T ranslocation and S peciation in E xcised P. vittata F ronds Excised young P. vittata fronds (no sori development ) , cut 5 cm above the rhizomes of a 1 year old plant , were washed with tap water followed by DI water and placed in a 250 mL Erlenmeyer flask with 100 mL solution containing 0 or 8 mg/L Sb III or Sb V (pH 6, 3 replicates), allowing only the stem to touch the solution. After 1 d, all plant materials were washe d with tap and DI water, and Sb concentration in the petioles and pinnae were determined. In addition to P. vittata fronds, pinnae were taken from young fronds prior to sori development. Eight incisions were made on the abaxial side using a scalpel blade. For each sample, eight pinnae (240 460 mg f resh weight ) were floated on 30 mL of DI water (pH 6) or 100 mg/L Sb III in sterile petri dishes with a 12 h photoperiod with photo flux of 350 µmol/m 2 s under cool and warm fluorescent lamps for 2 d (pH 6, 3 replicates). Tissues were rinsed in DI water and frozen in liquid nitrogen for Sb speciation. Total Sb, E xtraction and S peciation For total Sb analysis, oven dried ( 65 ºC for 2 d ) tissue was ground (20 mesh) and digested with 1: 1 v/v HNO 3 and water and 30% H 2 O 2 (USEPA Method 3050A). Sb concentration in the growth media and plant tissues were determined by a graphite furnace atomic absorption spectrophotomet ry (GF AAS, Varian 240Z, Walnut Creek, CA). In addition,

PAGE 48

48 appropriate reagent blanks and spikes we re used as quality checks , which were within expected values. Sb in plant tissues was extracted with a modif ied method of Okkenhaug et al. ( 2012 ) , with Sb extraction efficiency of 70 92% in plants ( Table 3 2 ) . Briefly, p lants were harvested and washed thoroughly in DI water before being separated into roots and fronds and then freeze dried for 2 d. They were then ground in liquid nitrogen to fine powder in a ceramic mortar and freeze dried for an additional 2 d. Samples of 50 mg of the powdered tissues were shaken at 100 rpm with 10 mL of 0.1 M citric acid for 4 h and then sonicated at 42 kHz for 1 h (VWR DHT Ultrasonic Cleaner B3500A) . Extracts were diluted to 50 mL with DI water and filtered (45 m filter) before separation of Sb species. Samples were further diluted until Sb concentrations were < 200 mg/L in 2 mM citric acid. The Solutions were then passed through Sep Pak Accell Plus QMA Plus Short cartridges ( WAT020545) from Waters ® , which retain s Sb III , but not SbV ( Table 3 1 ) . Statistical A nalysis Data were presented as the mean of three replicates, and error bars represent one standard error either side of the means. Signi fi cant di ff erences were established by using one way at p < 0.05 (v 9.3 SAS Institute Inc., Cary, NC, 2002 2010). Resu lts and Discussions As an As hyperaccumulator, P. vittata efficiently accumulates and translocates high concentrations of As with out displaying symptoms of toxicity ( Mathews et al., 2011 ) . Despite being an As analog, P. vittata does not hyperaccumulate Sb. Because Sb treatment does not

PAGE 49

49 induce visual symptoms of injury to P. vittata , we hypothesized that P. vittata detoxified Sb by either keeping it in the root s or effluxing it out to the media. Sb Speciation in P. vittata R oots To compare Sb III and Sb V uptake by P. vittata , it is important to determine their stability in the media. Both forms of Sb were stable within 24 h , showing either no oxidation or reduction ( Figure 3 1 ) . After exposure to 8 mg/L Sb for 1 d, almost all Sb inside P. vittata was concentrated in roots (179 to 4,192 mg/kg; Figure 3 2A ). To better understand Sb uptake and translocation in P. vittata , Sb speciation in the roots was determined. C i tric acid based extraction recovered 70 92% of the total Sb. When exposed to 8 mg/L Sb III , 81% Sb was present as Sb III in P. vittata roots ( Table 3 2) . Since Sb III was stable in DI water with the plant for 1 d ( Figure 3 2B ) , the Sb V in P. vittata roots was likely from Sb III oxidation to Sb V in the roots, which warrants further investigation. When exposed to 8 mg/L Sb V , 98% Sb was present as Sb V in the roots. The stability of Sb III and Sb V in the media within 24 h was consiste nt with the literature where Sb III and Sb V remain stable in DI water for up to 3 months at 25°C (de la Calle Guntiñas et al., 1992 ) . The stability of Sb III and Sb V in DI water is also established in Fe/Mn free solutions ( Belzile et al., 2001 ) . The mixture of 50 g/L Sb III and Sb V is stable for at least 5 d in 0.1 M oxalate at pH 2.2. However, in the presence of Fe and Mnoxyhydroxides, Sb III is oxidized to Sb V under light conditions ( Belzile et al., 2001 ) . In the current stud y, to minimize Sb III oxidation in the media, DI water was used for Sb uptake by P. vittata . Similar data were obtained in separate studies of P. vittata grown in soil spiked with 16 mg/kg Sb V for 49 d, of the Sb in the roots, ~93% was Sb V ( M ü ller et al., 2009 ; Müller et al., 2013 ) . This suggests some low level of reduction of Sb V in the roots. However, they reported

PAGE 50

50 low extraction efficiency of 5 20% using DI water under gaseous N 2 for 2 h ( M ü ller et al., 2009 ) , meaning the speciation data may not be accurate . Sb Uptake and Translocation F ollowing 1 d in media containing 8 mg/L Sb III , P. vittata rapidly removed 45% of the Sb III treatment ( Figure 3 2 B ), accumulating 4,192 mg/kg Sb in the roots with only 5 mg/kg Sb in the fronds (Table 3 2 ). Conversely, in 8 mg/L Sb V , P. vittata roots accumulated significantly lower Sb at 179 mg/kg ( p <0.05) ( Table 3 2 ). Similar data were obtained when P. vittata were exposed to lower Sb concentration (1.6 mg/L Sb III or Sb V ), with Sb concentrations in the roots being 957 and 30 mg/kg respectively ( Figur e 3 2 A ). To test the impact of exposure time on Sb uptake and translocation, we repeated the experiment by exposing P. vittata accessions from Brazil, China and Florida to 8 mg/L Sb III for 7 d. During th is period , there was no visual symptom of Sb toxicit y in P. vittata . Similar to short term experiment (1 d exposure) ( Table 3 2 ), Florida P. vittata accumulated almost all Sb in the roots ( 12,928 mg/kg ), with only 13 mg/kg in the fronds ( Figure 3 3 ) . T he additional 6 d resulted in an additional 8,736 mg/kg in P. vittata roots. Compared to Florida P. vittata , the other two P. vittata accessions accumulated similar concentration of Sb in the roots at higher than 99% of Sb remaining in the plant ( Figure 3 3 ). P. vittata takes up 5 times more As III than As V ( Wang et al., 2010 ) , however, the difference was not as striking with Sb . Besides P. vittata , rice also prefers to take up Sb III over Sb V ( Huang et al., 2012 ) . Three rice cultivars accumulated 1.3 2.3 times more Sb in the roots after exposed to 20 µM Sb III than Sb V for 3 d ( Huang et al., 2012 ) . Thus, P. vittata effectively accumulate d Sb in its roots with a clear preference to Sb III , which was 23 32 times more than

PAGE 51

51 Sb V ( Figure 3 2 A ). Though P. vittata was effective in taking up Sb III , Sb translocation to the fronds was limited ( Table 3 2) . Sb accumulation in P. vittata roots (95% of Sb in the plant) was also observed by Feng et al. ( 2009 ) . However, this was not the case with As hyperaccumulat or P. cretica ( Feng et al., 2011b ) . Following 2 w eeks of exposure to 20 mg /L Sb V ( p otassium pyroantimonate) in a hydroponic system, P . cretica accumulated 800 mg/kg Sb in the fronds, with only 23% of Sb in the plant being in the roots. The difference in Sb accumulation between P. cretica and P. vittata could be due to the difference in exposure time (1 d vs. 14 d). Unlike P . cretica , P. vittata showed little ability in Sb translocation even after 7 d. Our data indicated that P. vitta ta had a different Sb translocation from P . cretica ( Feng et al., 2011b ) . However, a similar pattern of P . vittata Sb accumulation in the roots was noticed in P . cretica grown in 20 mg/L Sb III ( potassium antimonyl tart r ate) for 2 w eeks ( Feng et al., 2009 ) . Different Sb accumulation pattern in P . cretica came from different Sb sources, Sb III from p otassium antimonyl tart r ate ( Feng et al., 2009 ) and Sb V from p otassium pyroantimonate ( Feng et al., 2011b ) . It s uggests P . cretica translocates Sb V to the fronds and accumulates Sb III in the roots. Different species of plants have been shown to differ in their Sb uptake capacities and tissue storage targets. Many plants including P. vittata accumulate Sb in the roots ( Mathews et al., 2011 ; M ü ller et al., 2009 ; Shtangeeva et al., 2012 ; Telford et al., 2009 ) , while maize and radish accumulate more Sb in the shoots ( He, 2007 ; Tschan et al., 2008 ) . Maize can take up both Sb III and Sb V ( Pan et al., 2011 ; Tschan et al., 2008 ) whereas rye take s up more Sb V than Sb III ( Shtangeeva et al., 2012 ) . Wheat and rice take up more Sb III than Sb V ( Huang et al., 2012 ; Shtangeeva et al., 2012 ) , similar to P. vittata in this study .

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52 Sb III might enter into plant roots via aquaglyceroporin channels due to its neutral charge. P. vittata likely uses separat e channels for Sb III and As III uptake as up to 100 mM Sb III had no impact on P. vittata uptake of 0.1 mM As III ( Mathews et al., 2011 ) . Up until now, there has been no report on Sb III pathway in P. vittata . P V , As V , and Sb V are chemical analogs and As V enters the cells via P V channels ( Tamás and Wysocki, 2001 ; Wang et al., 2002 ) . Sb V pathway into plants is still unknown ( Maciaszczy k Dziubinska et al., 2012 ) . Maize reportedly ha s a different Sb V pathway from P V because its Sb V uptake is independent of P V uptake ( Tschan et al., 2008 ) . Sb V is octahedral with larger size than As V and P V whereas the latter two are tetrahedral ( Tschan et al., 2009 ) . The results from Müller et al. ( 2013 ) support the idea that Sb V is taken up by a different pathway from As V in P. vittata . Similarly, P. vittata has different As III uptake channel from Sb III while rice and Arabidopsis use the same channels for both As III and Sb III ( Bhattacharjee et al., 2008 ) . Further study on Sb V P V interactions is necessary to examine the Sb V channel in P. vittata . Sb Efflux Sb has no known biological function and can be toxic, so upon uptake, Sb needs to be detoxified. In term of As, once taken up, it is reduced to As III in the rhizomes and then efficiently translocated to P. vittata fronds (Mathews et al., 2011). This was not the case with Sb as almost all Sb was accumulated in P. vittata roots ( Figure 3 2A, Table 3 2 ). With high Sb concentrations in the roots, in lieu of translocation, P. vittata may reduce Sb exposure by effluxin g it out to the media. To test this hypothesis, P. vittata was first exposed to 8 mg/L Sb III for 1 d, which was chosen because Sb III was stable in the presence of P. vittata for up to 1 d ( Figure 3 2B ). At the end of 1 d, P. vittata roots accumulated 4,192 mg/kg Sb with 81% being Sb III ( Table 3 2 ). After

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53 the roots were rinsed, it was transferred to Sb free DI water for 1 d to measure Sb efflux from the roots ( Figure 3 4 ). Sb efflux in P. vittata roots increased rapidly during the f irst 4 h , reach ing a plateau at 92 mg/kg (f resh weight ) at 8 h ( Figure 3 4 ). The Sb remaining in the roots was Sb III ( 74 % ), after 1 d, 26 % of the total Sb in the roots was efflux ed out by P. vittata as Sb III ( Table 3 2 ). Sb III concentration in the roots decreased from 3,119 to 2,113 mg/kg while Sb V concentration remained unchanged. Both Sb speciation in the growth media and reduction in Sb III concentration in the roots indicated Sb III was effluxed out of P. vittata roots. Sin ce P. vittata was more efficient in taking up Sb III than Sb V , it was possible that Sb III was also easier to be efflu xed out of P. vittata roots than Sb V . Efflux is a general detoxification mechanism in plants, C . vulgaris effluxed 60% as Sb III and another 40% as Sb V after the cells were transferred to Sb free medium ( Maeda et al., 1997 ) . Maeda et al. ( 1997 ) suggested the Sb V efflux in the Sb free medium came from detoxification of C. vulgaris by converting Sb III to the less toxic form Sb V . In comparison, P. vittata effluxed 18% of the total As in the roots after exposure to As V for 1 d ( Huang et al., 2011 ; Xu et al., 2007 ) . The less efflux of As comparing to Sb by P. vittata might due to high As translocation efficiency. Since P. vittata was inefficien t in translocating Sb to the fronds, effluxing Sb out of the roots may help P. vittata to minimize toxicity. Sb Translocation in Excised P. vittata F ronds P. vittata efficiently took up Sb III , but was unable to translocate Sb from the roots to the fronds ( Table 3 2 ). Thus, we wanted to demonstrate if Sb translocation could occur in excised fronds after exposure to 8 mg/L Sb III or Sb V for 1 d ( Figure 3 5 ). P. vittata fronds took up Sb III and distributed it evenly in the petioles and pinnae (99 and 114 mg/kg) whereas Sb V was mainly translocated to the pinnae (12 and 158 mg/kg) . Again, P. vittata fronds were more effective in

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54 taking up Sb III than Sb V . However, under these artificially induced conditions, P. vittata fronds were much more effi cient in translocating Sb V from the petioles to the pinnae with translocation factor of 13 ( Figure 3 5 ). It suggested a n Sb translocation barrier in the root or rhizome and further study is required for a location of Sb translocation barrier. To test whether excised pinnae were capable of oxidizing Sb III , P. vittata pinnae were incubated in 100 mg/L Sb III solution with a 12 h photoperiod with photo flux of 350 µmol/m 2 s under cool and warm fluorescent lamps for 2 d , which was long enough to detect Sb speciation. After 2 d, the pinnae took up 814 ± 148 mg/kg total Sb and 599 ± 114 mg/kg was classified as Sb III . All of which remained as Sb III , indicating no oxidation processes were occurring in excised P. vittata pinnae. It was possible that Sb III w as not as mobile with equal concentration in the petioles and pinnae . On the other hand, P. vittata efficiently loaded Sb V from the petioles to pinnae , or pinnae contain ed a strong Sb V sink , which allow ed higher Sb content in the pinnae compared to the petioles. Together these results ( Figure 3 3 and Figure 3 5 ) suggest that when the roots were exposed to Sb III , there is a constraint to Sb III reaching from the roots to the petiole. Conclusion P. vittata ro ots w ere more effective taking up Sb III than Sb V , but w ere inefficient in Sb translocation and transformation regardless of speciation . Almost all Sb taken up by P. vittata was retained in the roots, with Sb species in the roots being closely related to Sb species supplied. P. vittata roots were efficient in Sb III efflux (26%) , but apparently not for Sb V . Based on Sb speciation, we have shown that the uptake and translocat ion mechanisms of Sb in P. vittata were different from As . These results will be valuable for probing and understanding the

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55 nature of Sb uptake, translocation and metabolic processes in the As hyperaccumulator P. vittata .

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56 Table 3 1 . Percentage of Sb retained on SepPak cartridge. Sb added (µg/L) Sb retained (%)* Sb III Sb V 200 0 100 ± 0.0 150 50 74 ± 0.4 50 150 24 ± 4.1 0 200 1 ± 0.5 * The cartridge retains Sb III with Sb V being in solution. Table 3 2 . Total Sb concentration and speciation in roots and media, total Sb concentrations in frond and efflux media after exposing P. vittata plants to 8 mg/L Sb for 1 d and being efflux ed for 1 d . Data represent the mean of three replicates with standard error. Percent of the total is given in parentheses . Treatment Root Sb (mg/kg) Extraction efficency (%) Frond Sb (mg/kg) Growth media (mg/L) Efflux media (mg/L) Speciation extraction Total Sb* Sb III Sb V Sb III + Sb V Sb III Uptake 3,119 ± 57 (81%) 736 ± 91 (19%) 3,855±71 4,192±131 92 4.51 ± 1.05 4.4 ± 0.8 Sb III Sb III Efflux 2,113 ± 216 (74%) 748 ± 71 (26%) 2,862±147 3,671±289 78 6.64 ± 0.59 4.5 ± 0.2 Sb III 0.72 ± 0.09 Sb V Uptake 2.15 ± 2.64 (2%) 125 ± 24 (98%) 127 ± 23 179 ± 29 70 *Total Sb from digestion with HNO 3 /H 2 O 2 .

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57 Figure 3 1 . Stability of 1.6 or 8 mg/L Sb III or Sb V in DI water (no plant) after 24 h. Data represents two replicates with standard error. 0 1 2 3 4 5 6 7 8 9 1.6 SbIII 8.0 SbIII 1.6 SbV 8.0 SbV Sb concentration (mg/L) Treatment (mg/L) SbIII Total

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58 Figure 3 2 . Sb concentrations in A) the roots and B) the growth media of P. vittata after exposing to 1.6 or 8 mg/L Sb III or Sb V for 1 d. Data represent the mean of three replicates with standard error, and bars with the same letters are not significantly different. Sb III

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59 Figure 3 3 . Sb contents in the roots and fronds of three accessions of P. vittata after exposing to 8 mg/L Sb II I for 7 d. Data represent the mean of three replicates with standard error, and bars with the same letters are not significantly different. Figure 3 4 . Sb concentration (mg/L) in the media during P. vittata uptake from 8 mg/L Sb III for 1 d and Sb concentration in the media (mg/kg roots f resh weight ) during subsequent P. vittata efflux in DI water for 1 d . Sb was present as Sb III in both loading media and efflux media as there was no Sb III oxidation during treatment. Data represent the mean of three replicates with standard error.

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60 Figure 3 5 . Sb contents in the petioles and pinnae of excised P. vittata fronds after exposing to 8 mg/L Sb III or Sb V for 1 d. Data represent the mean of three replicates with standard error, and bars with the same letters are not significantly different.

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61 CHAPTER 4 UPTAKE OF ANTIMONITE AND ANTIMONATE BY THE ARSENIC HYPERACCUMULATOR PTERIS VITTATA L . : EFFECTS OF CHEMICAL ANALOGS AND A TRANSPORT INHIBITOR Introductory Remarks Sb is a toxic metalloid in the same elemental group as P and As and can cause human diseases such as cancer and respiratory syndromes ( Feng et al., 2013 ) . Being chemical analogs, Sb and As share many chemical properties and both have 3 and 5 oxidation state form s. The 3 oxidation state of both elements is found under anoxic conditions, whereas the 5 oxidation state occurs in relatively oxic conditions. Sb is used in the manufacture of semiconductors, batteries, bullets, and flame retardants ( Abin and Hollibaugh, 2013 ) . As the ninth of most mined metal worldwide, Sb is an emerging element of concern to human health and the environment ( Abin and Hollibaugh, 2013 ; Feng et al., 2013 ) . Crops growing in Sb contaminated areas are the major source for dietary Sb intake. However, there is limited knowledge about plant Sb uptake and accumulation (Feng et al., 2013). Sb concentrations of 0.01 0.1 mg/kg dry weight are typical background concentrations found in p lant tissue, with levels of 5 10 mg/kg causing phytotoxicity and growth reduction ( Feng et al., 2013 ; Xue et al., 2014 ) . However, rice exhibits increased tolerance to Sb by accumulating 65.5 mg/kg Sb in the leaves without growth reduction ( Feng et al., 2013 ) . A similar neutral charge compound and a slightly smaller volume than glycerol should be the advantages of Sb III to transport via glycerol channel (Porquet and Filella, 2007). The aquaglyceroporin channels transport glycerol and metalloids including Sb III , As III , boric acid, and silicic acid, which are structurally similar to glycerol ( Bhattacharjee et al., 2008 ) . Arabidopsis thaliana takes up neutral species Sb III and As III via aquaglyceroporin channels with different

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62 affinities. For example, the transport protein AtNIP5;1 and AtNIP6;1 favor uptake of As III over Sb III and AtNIP7;1 favors uptake of Sb III over As III ( Bhattacharjee et al., 2008 ) . Similarly , in rice plants, As III and Sb III are taken up via glycerol channels and glycerol inhibits As III uptake ( Meharg and Jardine, 2003 ) . For example, presence of 0.5 mM Sb III reduced As III uptake by 50% in rice growing in distilled water containing 0.1 mM As III for 20 min ( Meharg and Jardine, 2003 ) . Glucose has been reported to share the pathway with As III in yeast ( Liu et al., 2004 ) , but it ha s no impact on As III uptake by rice ( Meharg and Jardine, 2003 ) . Although Sb III and As III utilize aquaglyceroporin channels as shown in rice and Arabidopsis ( Kamiya and F ujiwara, 2009 ; Meharg and Jardine, 2003 ) , our research suggested that P. vittata may have an alternate pathway ( Mathews et al., 2011 ; Meharg and Jardine, 2003 ) . Sb III does no t impact As III uptake by P. vittata and once absorbed, As i s rapidly translocated to the fro nds; whereas Sb i s primarily accumulated in the roots ( Mathews et al., 2011 ; Tisarum et al., 2014 ) . Uptake of As III by P. vittata is not inhibited by other neutral species such as silicic acid, boric acid, Sb III , and glycerol ( Mathews et al., 2011 ; Wang et al., 2010 ) , suggesting P. vittata might take up As III via different aquaporin channels. Wang et al. (2010 ) showed that 0.5 mM silicic acid and 0.3 mM boric acid have no impact on P. vittata uptake of As III at 15 µM for 1 d in 0.2X Hoagland solution ( HS ) . Mathews et al. ( 2011 ) demonstrated that 1 h uptake of 0.1 mM As III in deionized (DI) water i s not inhibited by glycerol and Sb III for up to 100 mM. They used a 1 h treatment to prevent As III oxidation in the media, which may have been too short to demonstrate the competition. Mathews et al. ( 2011 ) observed uptake inhibition of 0.1 mM As III in P. vittata by 0.01 mM AgNO 3 . However, increasing AgNO 3 concentration to 0.1 mM d oes not show stronger inhibition. They suggested that P. vittata takes up As III via a Ag sensitive

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63 As III channel ( Mathews et al., 2011 ) . These data suggested P. vittata might have an unusual Sb/ As III channel(s), which are different from other plants. There are several reports on Sb III u ptake via aquaglyceroporins, but to date, no Sb V specific transporters have been identified. As V is known to enter into cells via P V transporters ( Wang et al., 2002 ) , but Sb V does not use this transporter ( Tschan et al., 2008 ) . In experiments using maize and sunflower, there w as no interaction between Sb V and P V , likely due to the distinct differences in structure and size between P V and Sb V . The structure of P V and As V are tetrahedral, while Sb V is octahedral in aqueous solution ( Tschan et al., 2008 ) . There is no evidence of Sb V impact on P V uptake in P. vittata but it has been reported that As V can impact Sb V uptake ( Müller et al., 2013 ) . Adding 5 mg/kg As V to P. vittata growing in quartz substrate containing 5 mg/kg Sb V increased Sb V uptake from 49 to 84 mg/kg Sb , but did not enhance Sb translocation to the fronds ( Müller et al., 2013 ) . The authors suggested As V change s membrane integrity , allowing Sb V to enter indirectly ( Müller et al., 2013 ) . Because the oxidation state of Sb and As could potentially b e influenced by rhizosphere microflora, there is a need to re evaluate Sb uptake in P. vittata under sterile conditions. In this work, we used P. vittata gametophytes cultured in vitro . G ametophytes are known to have As hyperaccumulation abilities (Gumaelius et al., 2004), we expected that gametophyte cultures would take up the elements similar to sporophyte tissues. Thus the goals of this study was to use rapidly growing uniform gametophyte cultures to study 1) uptake of Sb III and Sb V and their ef fect on the growth of P. vittata gametophytes, 2) effects of Sb III analogs including glycerol, silicic acid, glucose, As III , and aquaporin inhibitor silver (Ag) on Sb III uptake by P. vittata gametophytes, and 3) effects of P V on Sb III and Sb V uptake by P. vittata gametophytes.

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64 Materials and Methods Gametophyte C ulture P. vittata spores of ~20 mg were surface sterilized in a 2.0 mL tube by mixing with 1.6 mL solution containing 50% (v/v) sodium hypochlorite for 10 min and centrifuged at 16,162 g for 3 min (B eckman Coulter Microfuge ® 16 Centrifuge). The spores were washed two times in sterile water and then diluted with 1.6 mL sterile water ( Gumaelius et al., 2004 ) . The sterile spore solution was pipetted in 10 µL increments (0.125 mg spore per 10 µL) onto 100 x 15 mm sterile petri dish containing 0.5X Murashige & Skoog (MS) agar, pH 6 containing 20 g/L sucrose and incubated with a 12 h photoperiod with photo flux of 350 µmol/m 2 s using cool and warm fluorescent lamps with temperature at 23 28ºC and humidity of 70%. After 8 weeks, spores germinated and formed fully developed gametophyte clusters, which were used in the experiment ( Figure 4 1A ). Sb A ccumulation in P. vittata G ametophytes P. vittata gametophyte clusters (8 per replicate; ~1.5 2 g fresh weight) were placed in 100 x 15 mm sterile petri dish containing 6.5, 65, and 650 µM Sb III (0.8, 8.0, and 80 mg/L) ( potassium antimonyl tartrate (from Fisher, USA) or Sb V ( potassium hexahydroxy antimonate (from Sigma Aldrich, Czech Republic)) (pH 6, 3 replicates) under sterile conditions. After 2 h exposure (to avoid Sb III oxidation), the medium was collected and the gametophytes were washed with sterile DI water 6 times to remove Sb on the surface, and they were analyzed for total Sb as well as Sb speciation ( Tisarum et al., 2014 ; Yang et al., 2007 ) . Seven gametophyte clusters were collected for total Sb analysis. One cluster from each replicate was weighed and placed onto fresh 0.5X MS agar for 4 weeks to assess toxicity from Sb exposure.

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65 Sb III C ompetitors and A quaporin I nhibitor on Sb III U ptake by P. vittata G ametophytes Glycerol (C 3 H 5 (OH) 3 , ACROS, USA), silicic acid (Na 2 SiO 3 .5H 2 O; sodium silicate pentahydrate, Fisher, USA), As III (NaAsO 2 , sodium arsenite, Fisher, USA), glucose (CH 2 OH(CHOH) 4 HCO, Fisher, USA) at concentrations of 0, 0.065, 0.65, and 6.5 mM were mixed with 65 µM Sb III . For t he Ag treatment , AgNO 3 ( Fisher, USA) at concentrations 1, 10, and 100 µM w as pretreated 1 h to gametophytes before adding 65 µM of Sb III or Sb V to allow prior aquaglyceroporin inhibition ( Mathews et al., 2011 ) . Five mM 2 (N morpholino)ethanesulfonic acid (MES) was added in silicic acid treatments to avoid polymerization from silicic acid ( Wang et al., 2010 ) . NaOH and HCl were used to adjust pH of the media to 6.0. All experiments were performed in triplicate with 1.5 2.0 g gametophyte clusters per replicate. Effect of P V on Sb III and Sb V U ptake by P. vittata G ametophytes Gametophytes grown in MS medium were placed in solutions containing P V (KH 2 PO 4 , Fisher, USA) at concentrations of 0, 0.065, 0.65, and 6.5 mM with 65 µM Sb III or Sb V (pH 6.0, 3 replicates). After 2 h treatment, the media were collected for Sb and P V analy sis. After washing to remove surface Sb and P V , gametophytes were determined for total Sb and P. Since P V showed impacts on both Sb III and Sb V uptake, the experiment was repeated with Sb exposure increased from 2 h to 4 h. To better understand the impact of P V on Sb uptake, gametophytes of 8 week s of age were P V starved by growing in MS agar media without P V for 9 13 weeks. The impact of P V concentrations on Sb III and Sb V uptake were repeated using P V starved gametophytes for 2 h. Since P V showed impact on Sb III uptake by P. vittata , the impact of Sb III on P V uptake was also

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66 assessed. P V starved gametophytes were placed in 0.65 mM P V with 0.65 and 6.5 mM Sb III for 2 h and total Sb and P V in the tissues were determined. Total Sb, Sb S peciati on, S i and P Analysis For total Sb analysis, gametophytes were freeze dried for 2 d, then ground in liquid nitrogen to a fine powder using a ceramic mortar and pestle. The powdered tissues were freeze dried for an additional 2 d, digesting ~50 mg samples with 1:1 v/v HNO 3 and water and 30% H 2 O 2 (USEPA Method 3050A). Sb concentration s in the growth media and plant tissues were determined by GF AAS ( Graphite furnace atomic absorption spectrophotomet ry ) ( Varian 240Z, Walnut Creek, CA). Sb speciation in the media was determined by Sep Pak Accell Plus QMA Plus Short cartridges ( WAT020545) from Waters . The anion exchanger resins in the cartridges retained Sb III citrate complex from Sb V citrate complex ( Tisarum et al., 2014 ) . Si was determined by ICP MS ( Perkin Elmer Corp., Norwalk, CT). P V was measured spectrophotometrically at 875 nm using the molybdenum blue reaction at a fi xed time (20 min) following addition of the color reagent as P concentration ( Carvalho et al., 1998 ) . Statistical A nalysis Data were presented as the mean of three replicates with error bars representing one standard error. Signi fi cant di ff erences were established by using on e way analysis of variance p < 0.05 (v 9.3 SAS Institute Inc., Cary, NC, 2002 2010).

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67 Results and Discussions P. vittata Sb III or Sb V had no effect on their growth, indicating no toxicity under the studied Sb concentrations. P. vittata gametophytes had the same growth rate after 4 weeks of exposure to either Sb III or Sb V for 2 h ( Table 4 1 ). Sb A ccumulation in P. vittata G ametophytes Following 2 h of exposure to Sb, P. vittata gametophytes preferentially took up Sb III over Sb V ( Table 4 1 ). The gametophytes accumulated 27 mg/kg Sb after 2 h exposure to 0.8 mg/L Sb III whereas there was no Sb detected in 0.8 mg/L Sb V treatment . After increasing Sb concentration from 8 to 80 mg/L, Sb III uptake increased by 5 times (from 152 to 767 mg/kg) whereas Sb V uptake increased by 10 times (from 40 to 419 mg/kg) ( Table 4 1 ). Consistent with higher uptake of Sb III in P. vittata tissues, Sb III decrease in the media was larger than that from Sb V treatments except for 80 mg/L treatment. Sb concentrations in 80 mg/L of both Sb III and Sb V media after 2 h exposure were not different (76 77 mg/L). Sb in the media after 2 h exposure to P. vittata remained the same species, suggesting there was no Sb transformation ( Table 4 1 ). Since Sb in the media did not change species after 2 h treatment, this system allowed us to examine Sb uptake into the tissue without the complexity of non biological and microbe mediated Sb oxidation and reduction . P. vittata gametophytes showed preference of Sb III uptake over Sb V , similar to our previous study in P. vittata sporophytes ( Tisarum et al., 2014 ) . P. vittata sporophytes accumulated 23 times more Sb concentration in the roots after exposure t o 6.5 µM Sb III for 24 h than 6.5 µM Sb V ( Tisarum et al., 2014 ) . Similarly, P. vittata gametophytes accumulated 3.8 and 1.8 times more Sb III in the tissues after exposure to 65 and 650 µM Sb III for 2 h than Sb V . No Sb oxidation and reduction in the media was observed, similar to P. vittata sporophytes ( Tisarum et

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68 al., 2014 ) . These results showed that P. vittata gametophytes could be used as an efficient system to investigate Sb transport in P. vittata . Sb toxicity in plants is usually shown by growth reduction. For example, after growing for 14 d in the presence of 20 mg/L Sb, the biomass of fern plants Cyclosorus dentatus and Microlepia hancei was reduced by 38 and 35% respectively ( Feng et al., 2009 ) . Despite the high level of Sb accumulation observed, we did not find toxicity symptoms in P. vittata gametophytes. T his suggests that P. vittata tolerates to high Sb concentration in the tissues ( 767 mg/kg ), or it has a mechanism to detoxify the accumulated Sb . Sb III C ompetitors and Aquaglyceroporin I nhibitor Ag on Sb III U ptake by P. vittata G ametophytes Both As III and Sb III are transported via aquaglyceroporin in rice ( Meharg and Jardine , 2003 ) . Aquaglyceroporin has a selectivity to solutes by size and stereospecific recognition ( Maurel et al., 2008 ) . Aquaglyceroporins have been reported to transport glycerol and other small uncharged species into plant tissues including As III and Sb III ( Feng et al., 2013 ; Mathews et al., 2011 ) . The high ability of P. vittata in taking up Sb III might utilize existing channels for similarly structured molecules. In previous studies using P. vittata sporophytes, root uptake of As III by P. vittata was not impacted by aquaglyceroporin competitors such as glycerol, Sb III , or silicic acid ( Mathews et al., 2011 ; Wang et al., 2010 ) . Here we have tested the corollary of this by testing whether Sb III uptake i s competed by As III . Similar to the conclusions from our previous studies ( Mathews et al., 2011 ; Wang et al., 2010 ) , the current study show ed that the Sb III route wa s probably different from aquaglyceroporins , which is share d by glycerol and silicic acid. Mathews et al. (2011 ) suggest that As III and Sb III uptake by P. vittata sporophyte is probably via different channels, which is confirmed by this study using sterile P. vittata

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69 gametophytes. As III had no impact on Sb III uptake after 2 h exposure to P. vittata gametophytes without microbe mediated Sb and As oxidation and reduction ( Figure 4 1B ). This was expected as gametophytes have rhizoid tissues, which act similar to sporophyte roots for taking up nutrients ( Racusen, 2002 ) . To test the impact of glycerol on Sb III uptake by P. vittata gametophytes, they were exposed to 65 µM Sb III with increasing glycerol concentrations from 0.065 6.5 mM. Following 2 h of exposure, Sb uptake was reduced by ~18 24% regardless of the glycerol concentration ( Figure 4 1B). The range of Sb III uptake under varying glycerol concentration was 131 172 mg/kg. Upon exposure to 65 µM silicic acid for 2 h, P. vittata gametophytes significantly increased Sb III uptake from 160 to 225 mg/kg and silicic acid uptake increased after exposure to 6.5 mM silicic acid (from 69 86 to 203 mg/kg) ( Figure 4 2A ). In an attempt to further clarify this relationship, 4 h exposure was conducted but no additional effect on Sb III uptake by silicic acid was observed ( Figure 4 2A ). Silicic acid is a larger molecule than Sb III , thus it is possible for Sb III to utilize silicic acid channels. However, silicic acid channel might require specific structure rather than a similar size. P. vittata might have different pathways for Sb III and silicic acid uptake because no relationship was observed between Sb III and silicic acid accumulation after 4 h exposure ( Figure 4 2A ). Plants can use the same aquaglyceroporin channel route for differ ent solutes, although the affinity varies. For example, As III and Sb III in rice have been reported to use glycerol channels with varying degrees of affinity , i.e., As III > Sb III > glycerol ( Meharg and Jardine, 2003 ) .

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70 Glucose at 0.065 mM reduced Sb III uptake by 30% and extending exposure to 4 h showed no additional impact of glucose on Sb III uptake ( Figure 4 2B ). Unlike As III competing with glucose in yeast ( Liu et al., 2004 ) , Sb III under neutral conditions in this study might not form Sb polymerization , which is why no competitive uptake with glucose was observed. A poly merized species of Sb 4 O 7 2 and Sb 6 O 10 2 have been reported from the alkaline solution containing 0.01 0.1 M Sb III ( Shoji et al., 1974 ) . The conditio ns used in our study might not provide the polymerized Sb III form, based on our observations that there was probably no competition between glucose and Sb III . This study showed no impact of Ag on either Sb V or Sb III uptake, which indicated that Ag did not inhibit channels used by Sb and that it did not damage P. vittata gametophytes ( Figure 4 3A , 4 3B ). The lowest Ag concentration at 1 µM showed the highest impact on increasing Sb III uptake by 43 mg/kg from the cont rol whereas the highest Ag concentration at 100 µM had the lowest impact by increasing only 28 mg/kg from the control without significantly different ( p <0.05) ( Figure 4 3B ). P. vittata gametophytes took up more Ag ( 0 5 to 62 mg/kg ) after exposure to 100 µM Ag in both Sb V and Sb III treatments ( Figure 4 3A , 4 3B ). Ag has been widely used as an aquaglyceroporin inhibitor in studies examining As III uptake by plants ( Ma thews et al., 2011 ; Nagarajan and Ebbs, 2007 ) as it is less toxic and more specific in the action than other inhibitors ( Mathews et al., 2011 ) . Ag has a potential to inhibit aquaglyceroporin by binding to sulfhydryl groups of cysteine near conserved positions ( Niemietz and Tyerman, 2002 ) . In a previous study ( Mathews et al., 2011 ) , 10 µM Ag significantly reduced As III uptake by P. vittata roots. Our results are consistent with Sb III channel in P. vittata being insensitive to Ag as the permeability of different solutes in a single channel might come from different mechanisms ( Jung et al., 2012 ) . Ag in P. vittata gametophytes after exposure to

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71 100 µM AgNO 3 confirmed that Ag was insentitive for Sb III uptake. P. vittata gametophytes accumulated high Ag at 62 mg/kg without tissue damage (withou t Sb V and Sb III uptake change at high Ag accumulation) whereas Ag background in plants is 0.06 mg/kg ( Horovitz et al., 1974 ) . Effect of P V on Sb III and Sb V U ptake by P. vittata Gametophytes Adding 6.5 mM P V increased significantly Sb III uptake from the control at 132 to 176 mg/kg , but decreased significantly Sb V uptake from 46 to 37 mg/kg ( p < 0.05) ( Figure 4 4A , 4 4B ). P concentrations in the tissues after exposure to different P V concentrations containing 65 µM Sb V were not significantly different in the range of 4,158 4,556 mg/kg , but adding P V increased P concentrations in the tissues of Sb III treatments from 3,895 to 4,754 5,043 mg/kg ( Figure 4 5 ). Extending to 4 h exposure, P had no additional impact on either Sb III or Sb V uptake by P. vittata ( Figure 4 4A, 4 4B ). Increasing P in the tissues of both Sb III and Sb V treatments depended on P V dose and there was the same P increasing trend in both Sb III and Sb V treatment s ( Figure 4 5 ). P V starved gametophytes contained 651 681 mg/kg P after 12 weeks of starvation and rose to 1,057 1,112 mg/kg P after adding 6.5 m M P V for 2 h ( Figure 4 6 A , 4 6 B ). Various Sb III concentrations with 0.65 m M P was set up to investigate the impact of Sb III on P V uptake in P V starved gametophytes. After 2 h exposure, gametophytes took up 706 mg/kg Sb at 0.65 mM Sb III and Sb III uptake increased by 4 times at 6.5 mM Sb III ( Figure 4 7 ) . Sb III treatment did not impact P V uptake as there was no significant difference among P concentration in P V starved gametophytes after exposure to different Sb III concentrations ( Figure 4 7 ). This study showed P V reduced Sb V uptake after 2 h exposure but there was no impact of P V on Sb V uptake after 4 h exposure, which confirmed different routes for P V and Sb V uptake by P. vittata . Neither P V starvation nor the presence of P V affected Sb V uptake ( Figure 4 5 , 4 6 A ).

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72 An octahedral structure of Sb V (Sb(OH) 6 ) might not share a route with the tetrahedral structure of P V ( Tschan et al., 2008 ; Zangi and Filella, 2012 ) and Sb V uptake mechanism in plants is still unclear ( Feng et al., 2013 ) . A different route for Sb V and P V has been reported in maize and sunflower ( Tschan et al., 2008 ) , and As V increases Sb V uptake in P. vittata ( Müller et al., 2013 ) . It still leaves a question whether P V can increase Sb V uptake in P. vittata gametophytes. The low amounts of Sb V uptake by P. vittata might be via other anion transporters with low selectivity in the roots ( Feng et al., 2013 ) . P concentrations in the tissues after exposure to different P V concentrations containing 65 µM Sb III or Sb V were in the range of 3,895 5,043 mg/kg ( Figure 4 5 ) . The data indicated sufficient P V supply from MS agar media during 8 weeks and as a result, there w ere no increasing P concentrations in the tissues after exposure to different P V concentrations. We investigated the impact of P V on Sb III and Sb V uptake by P V starved gametophytes. The results showed P V decreased significantly Sb III uptake from 80 to 56 mg/kg after adding 6.5 mM P ( p <0.05) whereas P V had no impact on Sb V uptake ( Figure 4 6 A, 4 6 B ). Because Sb III is not structurally related to P V and P. vittata P V uptake can be expected to function under P V starvation, we infer red that P V uptake under the conditions of this study negatively influenced Sb III uptake indirectly. Unde r P V sufficient condition, PV gametophytes likely used low affinity P V transporters to take up P V whereas high affinity P V transporters are activated under P V starvation condition ( Chrispeels et al., 1999 ) . The high affinity P V system is activated under P V deficiency to increase the capacity of P V uptake by several responses such as increasing P V transporters, reducing P V efflux, mobilizing P V from vacuole to cytoplasm, and changing membrane lipid ( Prieto et al., 1997 ; Raghothama, 1999 ; Wang et al., 2002 ) . The P. vittata

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73 response to the high affinity P V system might change membrane permeability inhibiting Sb III to enter P. vittata indirectly . P V has been reported to have no impact on As III uptake by P V starved P. vittata sporophytes after 8 d with reapplied P V (as NH 4 H 2 PO 4 ) for 8 h and it was concluded that P V and As III used different uptake pathways ( Wang et al., 2002 ) . Opposite result has been reported when P. vittata sporophytes were grown in sand culture. In Wang et al. (2002 ) , the plants were applied with 0.107 mM As III or As V containing different P V concentrations (0.05, 0.30, and 1.00 mM P V ) for 15 weeks, and renewed every 4 d to prevent As III oxidation. P V reduced As III uptake more than that for As V in both the roots and fronds ( Huang et al., 2007 b ) . Our observations of reapplied P V (as KH 2 PO 4 ) significantly reducing Sb III uptake ( Figure 4 6B ) was comparable to their study. Following P V starvation, a resupply of P V can cause P V toxicity in the plants due to excess P V uptake ( Raghothama, 1999 ) . In this study, P V starved gametophytes might have suffered P V toxicity due to excess P V uptake ( 406 431 mg/kg increasing from the control ( 651 681 mg/kg ) after exposure to 6.5 mM P V , Figure 4 6 A , 4 6 B ) during treatment which impacted Sb III uptake. However, P V toxicity hypothesis might not be tenable as P V toxicity would also damage P. vittata gametophytes , which would have in turn impacted both Sb III and Sb V uptake. P V uptake reduced Sb III uptake when high affinity P V system was activated but Sb III did not impact P V uptake in either low or high affinity P V system. These results indicated a complex mechanism for P V interference of Sb III uptake.

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74 Conclusion Our study indicated that sterile P. vittata gametophyte cultures could be used to investigate the physiology of ion transport under controlled conditions. P. vittata gametophytes were tolerant to Sb and had a higher affinity for Sb III than for Sb V . Sb accumulation increased 5 times (from 152 to 767 mg/kg) when increasing Sb III concentration ten times in the medium whereas it increased 10 times (from 40 to 419 mg/kg) for a ten fold increase in Sb V . Sb III uptake was not inhibited by th e aquaglyceroporin competitors glycerol, silicic acid, and As III and its transport was not sensitive to Ag at the range from 1 100 µM. Glucose did not compete with Sb III , but P V decreased Sb III uptake of P V starved gametophytes, but did not affect Sb V uptake. Together our results suggest that P. vittata ha d unique features distinct from the characteristics known for P V transporters, hexose permeases and aquaglyceroporins in other plants.

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75 Table 4 1 . Sb concentrations in the tissues, media and the growth rate of P. vittata after exposure to 0.8, 8.0, and 80 mg/L Sb III or Sb V for 2 h. Data represent the mean of three replicates with standard error. Bars with the same lower case letters are not signifi cantly different between the same amount of Sb III and Sb V of Sb in tissues and Sb in media and the same lower case letters in growth rate are not significantly different ( p <0.05) among all treatments. Treatment (mg/L) Sb in tissues (mg/kg) Sb in media* (mg /L) Growth rate** (mg/d) Control nd a nd 29.8 ± 3.9 a 0.8 Sb III 26.7 ± 0.9 a 0.6 ± 0.0 a 24.8 ± 0.4 a 0.8 Sb V nd 0.8 ± 0.0 b 30.7 ± 4.7 a 8.0 Sb III 152.3 ± 2.9 a 6.7 ± 0.1 a 29.3 ± 2.5 a 8.0 Sb V 39.6 ± 0.8 b 7.6 ± 0.1 b 32.4 ± 4.6 a 80 Sb III 766.7 ± 12.2 a 77.3 ± 2.0 a 27.6 ± 2.9 a 80 Sb V 419.1 ± 8.8 b 75.6 ± 3.1 a 29.3 ± 4.6 a * Sb was present as total Sb as there was no Sb III oxidation and Sb V reduction during treatment. ** increasing weight per day of P. vittata after 4 weeks following the Sb treatment for 2 h . a L Sb ).

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76 Figure 4 1 . A) Eight week old P. vittata gametophyte clusters, each grown from 0.125 mg spore on 0.5X MS agar and B) Effect of As III and glycerol on Sb concentrations in the tissues of P. vittata after exposure to As III or glycerol containing 65 µM Sb III for 2 h. Data represent the mean of three replicates with standard error, and bars labeled with the same letters are not significant ly different ( p <0.05). a a a a A A A A 0 50 100 150 200 250 0 0.065 0.65 6.5 Sb in tissue (mg/kg dry weight ) Treatment (mM) AsIII glycerol B AS III Glycerol

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77 Figure 4 2 . Effect of A) silicic acid o n Sb and Si concentrations and B) glucose on Sb concentrations in the tissues of P. vittata after exposure to glucose containing 65 µM Sb III for 2 4 h. Data represent the mean of three replicates with standard error, and bars labeled with the same letters are not significantly different ( p <0.05). a b ab ab A A A A 0 50 100 150 200 250 300 350 0 0.065 0.65 6.5 Sb in tissue (mg/kg dry weight) Glucose ( m M) 2h 4h B

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78 Figure 4 3 . Sb and Ag concentrations in the tissues of P. vittata after pretreatment with AgNO 3 for 1 h then exposed for 2 h to A) 65 µM Sb V or B) Sb III solutions . Data represent the mean of three replicates with standard error, and bars labeled with the same letters are not significantly different ( p <0.05).

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79 Figure 4 4 . Effect of P V on Sb concentrations in the tissues of P. vittata after exposure to solutions containing A) 65 µM Sb III or B) Sb V for 2 4 h. Data represent the mean of three replicates with standard error, and bars labeled with the same letters are not significantly different ( p <0.05). a ab ab b A A A A 0 100 200 300 400 0 0.065 0.65 6.5 Sb in tissue (mg/kg) P V (mM) Sb 2h Sb 4h A a ab b b A A A A 0 100 200 300 400 0 0.065 0.65 6.5 Sb in tissue (mg/kg) P V (mM) Sb 2h Sb 4h B

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80 Figure 4 5 . Effect of P V on P concentrations in the tissues of P. vittata after exposure to 65 µM Sb III or Sb V for 2 4 h. Data represent the mean of three replicates with standard error, and bars labeled with the same letters are not significantly different ( p <0.05). a b b b A A A A 0 1000 2000 3000 4000 5000 6000 0 0.065 0.65 6.5 P in tissue (mg/kg dry weight) P V (mM) SbIII 2h SbV 2h Sb III 2h Sb V 2h

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81 Figure 4 6 . Effect of Sb V and Sb III on Sb and P concentrations the tissues of 12 week P V starved P. vittata after exposure to A) 65 µM Sb V containing varying P V concentration ; B) 65 µM Sb III varying P V concentration for 2 h. Data represent the mean of three replicates with standard error, and bars labeled with the same letters are not significantly different ( p <0.05).

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82 Figure 4 7 . Effect of P V on Sb and P concentrations the tissues of 12 week P V starved P. vittata after exposure to 0.65 mM P V containing varying Sb III concentration for 2 h. Data represent the mean of three replicates with standard error, and bars labeled with the same letters are not significantly different ( p <0.05).

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83 CHAPTER 5 CONCLUSION AND DIRECTION OF FUTURE RESEARCH The As uptake mechanism of the As hyperaccumulator P. vittata has been established, but there are still several gaps in understanding which need to be explored. This study provided new information about Sb uptake by P. vittata , which helps to confirm a unique mechanism for both As and Sb uptake. T he use of the SPE cartridge was proposed for an alternative Sb speciation technique in plant biomass and plant growth media. The anion exchange cartridge retained Sb V and the Sb III concentration was determined by GF AAS. In the presence of citric acid, which reduced sampl e volume and reversed Sb III retaining on the cartridge, the cartridge was preconditioned with Sb V and Sb III separation was determined after discarding the first 60 mL of filtrate. Successful Sb III separation and 92 104% Sb V recovery from P. vittata root w ere confirmed by HPLC ICP MS. This method has a lower detection limit, shorter Sb separation time, and cheaper cost than other recently used methods using GF AAS as a n Sb detector and is recommended as a simple method for a general lab without HPLC ICP MS. The cartridge is available in the commercial market and its pre pack column provided reproducible data. The need to discard 60 mL of the sample is a concern on using the SPE cartridge for Sb speciation and further study to reduce the sample volume is required. One suggestion is by increasing Sb V concentration when preconditioning the cartridge. P. vittata has different mechanisms for uptake and translocation of As III and Sb III . It is efficient in As III and As V uptake and translocation, but it was onl y efficient in taking up Sb III . P. vittata sporophyte was set up to study short (1 d) and long term (7 d) exposure to Sb III and Sb V . P. vittata accumulated most Sb in the roots and had higher affinity for Sb III than Sb V . P. vittata r oots accumulated 4,192 and 179 mg/kg after exposure to 8 mg/L Sb III and Sb V for short term

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84 exposure, respectively. Long term exposure of P. vittata accessions from Brazil, China and Florida to 8 mg/L Sb III , showed similar results to the short term exposur e with higher concentration in the roots of 12,072 13,230 mg/kg without differing Sb accumulation among three accessions. P. vittata has limited ability to transform Sb ( 1 9% Sb III oxidation and 2% Sb V reduction ) and efflux it from the roots ( 26% ). Excised P. vittata fronds translocated Sb V more efficiently from the petioles to pinnae than Sb III and were unable to oxidize Sb III . This study on Sb species in P. vittata roots indicate d the capacity of P. vittata to transform Sb into different specie s. The presence of Sb III oxidizing and Sb V reducing enzymes in P. vittata roots is a possibility , which might be investigated. To reduce the impact of Sb microbial transformation, sterile roots should be used to reduce rhizomicrobial interference. With high Sb accumulation in the roots, Sb detoxification needs to be investigated in detail as only 26% of Sb was effluxed. The rest of Sb in the roots might be bound to the cell wall or sequestered into vacuoles and subcellular localization should be explore d to answer this question. The results from experiments on Sb uptake in the P. vittata sporophytes led to a following study on how P. vittata transports Sb III and Sb V into the roots. P. vittata gametophyte was used in this study as a tool instead of a sporophyte due to its rapid growth, ease of culture, its uniformity and the advantage of eliminating microbe mediated Sb transformations . Sterile P. vittata gametophytes were treat ed with Sb III and Sb V for 2 h and showed no toxicity , indicating that P. vittata was tolerant to Sb. The same trend of higher affinity for Sb III than Sb V was also found in P. vittata gametophytes, with 5 and 10 times increase in Sb III and Sb V accumulation after exposure to a 10 fold increase in Sb concentration. Sb III is known to share uptake routes with As III , glycerol, and silicic acid. So it was expected that As III , glycerol, and silicic acid

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85 should act as Sb III uptake competitors and Ag, the aquaglyceroporin inhibitor, c ould be able to inhibit Sb III uptake into P. vittata . However, in this study, there was no impact of those Sb III competitors and the aquaglyceroporin inhibitor on Sb III uptake. Glucose, which has been reported to inhibit As III uptake in yeast, did not impact Sb III uptake in P. vittata , but P V reduced Sb III uptake under conditions that would promote an activated high affinity P V system . The results indicated a unique Sb uptake system in P. vittata different from aquaglyceroporins, hexose permeases, and P V transporters. Better understanding of Sb III and Sb V uptake routes in P. vittata is necessary for using P. vittata in phytoremediation. To further study the Sb III uptake system in P. vittata , a m olecular level analysis is required, as the Sb V uptake route might share a route with anion transporters such as nitrate and sulfate transporters. To conclude, the As hyperaccumulator P. vittata has an unique Sb III uptake system and an unknown Sb V uptake r oute. Further study is required to reveal P. vittata potential use for Sb phytoremediation.

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86 APPENDIX A ROLE OF GLUTATHIONE ON ARSENIC UPTAKE AND TRANSLOCATION Introduct ory Remarks GSH plays two roles in As transformation: reduce As V to As III and complex As III to form As( GSH ) 3 , which has been shown to be present in plants ( Pickering et al., 2006 ) . Wei et al. (20 10 ) showed that GSH increased both As uptake (77 89%) and translocation (61 85%) by P. vittata . The mechanisms of GSH induced plant As accumulation remain unclear since GSH did not change As speciation in the growth media and plant biomass. Uptake kinetics indicate that externally applied GSH is taken up by a single saturable transporter in broad bean ( Vicia faba L.) leaf tissues, with an apparent K m of 0.4 mM ( Jamai et al., 1996 ) . Hypotheses Addition of GSH reduces oxidative stress in plants ( Thiobarbituric acid reactive substances, TBARS), thereby increasing the ability to take up As by P. vittata and Pteris ensiformis and GSH will increase uptake of As III more than As V . Materials and Methods Uniform plants with 3 4 fronds were selected and acclimatized hydroponically in 0.2X HS with constant aeration under cool and warm fluorescent lamps ( the total light intensity ~ 2 00 micromoles per m 2 ) with temperature of 23 28ºC and ~70% humidit y for 4 weeks. After 4 weeks of acclimatization, P. vittata and P . ensiformis were transferred to 0.2 mM GSH in DI water containing 13 and 133 M (1 and 10 mg/L) As III or As V for 2 d. After 2 d of growth, the frond was cut 1 cm above rhizome and the sap was collected for As speciation and total GSH concentrations. T he plants were separated into the roots, rhizome and fronds and determine d 1) As speciation, 2) total GSH (GSH + GSSG) concentrations ( Wei et

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87 al. , 20 10 ) and 3) TBARS. T he growth media were collected and determine d As speciation and total GSH concentrations. Results GSH did not reduce oxidative stress as indicated in plants by the formation TBARS and did not increase the ability to take up As by P. vittata and P . ensiformis . Figure A 1 . As speciation in P. vittata sap after exposure to As III and As V for 2 d . 0 2 4 6 8 10 12 Control 10As(III) 1As(III) 10As(V) 1As(V) As (mg/L) As(III) As(V) As III As V Treatment (mg/L)

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88 Figure A 2 . As speciation in P . ensiformis sap after exposure to As III and As V for 2 d . Table A 1 . GSH in P. vittata and P . ensiformis sap after exposure to As III and As V for 2 d . Treatment GSH ( mol/mL sap ) (mg/L) P. vittata P. ensiformis Control None detected (< 0.5) None detected 10 As III 2.15 4.17 1 As III 1.30 1.17 10 As V 0.58 None detected 1 As V 2.87 None detected 0.00 0.10 0.20 0.30 0.40 Control 10AsIII 1AsIII 10AsV 1AsV As (mg/kg) AsIII AsV Treatment (mg/L)

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89 Figure A 3 . As in P. vittata after exposure to As III and As V for 2 d . Figure A 4 . As spec i ation in P. vittata root after exposure to As III and As V for 2 d . 0 50 100 150 200 Control 10AsIII 1AsIII 10AsV 1AsV As (mg/kg) Root Rhizome Frond 10As III 1As III 10As V 1As V Control 0 5 10 15 20 25 Control 10As(III) 1As(III) 10As(V) 1As(V) As (mg/kg) As(III) As(V) 10As III 1As III 10As V 1As V Control As III As V Treatment (mg/L) Treatment (mg/L)

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90 Figure A 5 . As spec i ation in P. vittata rhizome after exposure to As III and As V for 2 d . Figure A 6 . As spec i ation in P. vittata frond after exposure to As III and As V for 2 d. Figure A 7 . As in P . ensiformis after exposure to As III and As V for 2 d. 0 50 100 150 200 Control 10AsIII 1AsIII 10AsV 1AsV As (mg/kg) AsIII AsV 10As III 1As III 10As V 1As V Control As III As V 0 50 100 150 200 Control 10As(III) 1As(III) 10As(V) 1As(V) As (mg/kg) As(III) As(V) 0 50 100 150 200 Control 10As(III) 1As(III) 10As(V) 1As(V) As (mg/kg) Root Rhizome Frond 10As III 1As III 10As V 1As V Control Treatment (mg/L) Treatment (mg/L) Treatment (mg/L)

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91 Figure A 8 . As speciation in P . ensiformis root after exposure to As III and As V for 2 d. Figure A 9 . GSH in P. vittata organs after exposure to As III and As V for 2 d. Figure A 10 . GSH in P . ensiformis organs after exposure to As III and As V for 2 d. 0 5 10 15 20 Control 10As(III) 1As(III) 10As(V) 1As(V) GSH (umol/g fresh weight) Root Rhizome Frond 10As III 1As III 10As V 1As V Control 0 5 10 15 20 Control 10As(III) 1As(III) 10As(V) 1As(V) GSH ( umol /g fresh weight) Root Rhizome Frond 10As III 1As III 10As V 1As V Control Treatment (mg/L) Treatment (mg/L) Treatment (mg/L)

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92 Figure A 11 . TBARs in P. vittata after exposure to As III and As V for 2 d. Figure A 12 . TBARs in P . ensiformis after exposure to As III and As V for 2 d. 0 1 2 3 4 5 6 7 Control 10As(III) 1As(III) 10As(V) 1As(V) Root TBARs (umol/g fresh weight) Root Rhizome Frond 10As III 1As III 10As V 1As V Control 0 10 20 30 40 50 Control 10As(III) 1As(III) 10As(V) 1As(V) Frond TBARs (umol/g fresh weight) Root Rhizome Frond 10As III 1As III 10As V 1As V Control Treatment (mg/L) Treatment (mg/L)

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93 Table A 2 . As speciation in P. vittata and P . ensiformis media after exposure to As III and As V for 2 d. Treatment (mg/L) As (mg/L) As III As V P. vittata P. ensiformis P. vittata P. ensiformis Control None detected None detected None detected None detected 10 As III 0.7 5.4 10.5 6.1 1 As III None detected None detected 0.9 1.1 10 As V None detected 0.1 10.5 11.0 1 As V None detected None detected 0.9 1.1 Table A 3 . GHS in P. vittata and P . ensiformis media after exposure to As III and As V for 2 d , with none detected <0.5 M GSH . Treatment (mg/L) GSH (mM) P. vittata P. ensiformis 0 d 2 d 0 d 2 d Control 0.2 None detected 0.2 None detected 10 As III 0.2 None detected 0.2 None detected 1 As III 0.2 None detected 0.2 None detected 10 As V 0.2 None detected 0.2 None detected 1 As V 0.2 None detected 0.2 None detected

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94 APPENDIX B EFFECT OF NANO TITANIUM DIOXIDE ON ARSENIC UPTAKE BY PTERIS VITTATA Introduct ory Remarks TiO 2 has high affinity for As and it promotes both As V and As III uptake in carp. Limited research is available on plant uptake of TiO 2 thus t he impact of nano TiO 2 on As uptake by P. vittata has not been elucidated. Hypothesis Nano TiO 2 may increase As V uptake from As V ( sodium arsenate dibasic heptahydrate ) and As V minerlas : FeAsO 4 and AlAsO 4 in P. vittata . Materials and Methods Uniform P. vittata with 3 4 fronds were selected and acclimatized hydroponically in 0.2X HS with constant aeration under cool and warm fluorescent lamps ( the total light intensity ~ 2 00 micromoles per m 2 ) with temperature of 23 28ºC and ~70% humidity for 4 weeks. The plants were separated into 2 groups. The first group, P. vittata were placed in 0.5 mM CaCl 2 (pH 6) for 1 d before being transferred to opaque containers containing 1 L of deionized (DI) water with 2 mg/L As V containing 100 or 200 mg/L nano TiO 2 for 5 d. The second group, P. vittata were placed in 0.5 mM CaCl 2 (pH 6) for 1 d before being transferred to opaque containers containing 1 L of 0.2X HS with 100 mg/L nano TiO 2 containing 150 mg /L AlAsO 4 or FeAsO 4 for 3 d. After the treatments, t he plants were separated into the roots and fr onds and determine d As concentration Results Exogenous n ano TiO 2 did not promote As uptake by P. vittata .

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95 Figure B 1 . As contents in P. vittata after exposure to 2 mg As V and nano TiO 2 in DI water for 5 d. Figure B 2 . As contents in P. vittata after exposure to 150 mg /L AlAsO 4 or FeAsO 4 and 100 mg/L nano TiO 2 in 0.2 X HS for 3 d. 0 20 40 60 80 100 120 140 Control (DI) 2 AsV 100TiO2 2 AsV+ 100TiO2 200TiO2 2AsV + 200TiO2 As in tissue (mg/kg) Treatment (mg/L) Root Frond

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96 APPENDIX C ROLE OF ARSENIC RESISTANT BACTERIA IN ENHANCING TOMATO GROWTH Introduct ory Remarks In this study our objective was to use insoluble FePO 4 to test the ability of As resistance bacteria ( ARB ) in iron ( Fe ) and P solubilization and its potential benefit to the growth of tomato plants under hydroponic conditions. Materials and Methods Plant M aterial Tomatoes were grown in sterilized soil to minimize effect of bacteria from soil. Fourteen days after sowing, uniform seedlings were selected. The plants were grown in growth room from March 8 to 22 , 2011 , with average light intensity of 350 µmol / m 2 s. The soil was washed from the root system with DI water and seedlings were transferred to 1 L hydroponic containe rs with 0.5 X HS pH 6.5, aerated with an air pump. After 7 d of acclimatization to the hydroponic culture, plants were treated with ARB as described in T able C 1 . Bacteria Seven individual bacterial isolates ( Pseudomonas sp., Comamonas sp. a nd Stenotrophomonas sp.) making the ARB consortium were grown overnight separately in liquid LEB medium ( Ghosh et al., 2011 ) in a shaker incubator set at 30 C and 200 rpm to an OD 600 value of 1.5 and then mixed together to a volume of 12 m L and then 1 m L were added to 1 L of growth medium.

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97 Table C 1 . Summary of 6 treatments. Treatment Days after 1 w ee k acclimatization Day 1 3 Day 4 10 (fresh media to start condition at pH6.5) 1.Control 0.5 X HS 0.5XHS + 1 mL LEB 2.Control+ARB 0.5XHS 0.5XHS + 1 mL ARB consortium 3.P free P free 0.5XHS P free 0.5XHS + 1 mL LEB 4.P free+ARB P free 0.5XHS P free 0.5XHS + 1 mL ARB consortium 5.P free+ FePO 4 P free 0.5XHS P free 0.5XHS + 0.25 g FePO 4 + 1 mL LEB 6. P free+FePO 4 +ARB P free 0.5XHS P free 0.5XHS + 0.25 g FePO 4 + 1 mL ARB consortium Results Figure C 1 . The bars represent A) root length and B) shoot height means and standard errors for 5 observations.

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98 Figure C 2 . T he bars represent dry weight of A) root s , B) shoot s and C) total means and stand ard errors for 5 observations.

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108 BIOGRAPHICAL SKETCH Rujira Tisarum, the second of the two siblings , was born and brought up in Buriram, Thailand. She went to Chulalongkorn University to p u Following this , she switched her major to Horticulture and obtained her master degree in the Faculty of Agriculture at Kasetsart University. and Alleviation of Uneven Fruit Ripening in Mon Thong Durian ( Duriozibethinus Murray cv. Mon Thong ) In 2009, she came to the University of Florida to p u rsue a Ph . D . in Soil and Water Science supported by the Royal Thai Government . She worked on antimony uptake, speciation, and efflux by the arsenic hyperaccumulator Pteris vittata and she received her Ph.D. from the University of Florida in the summer of 2014 .