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On the Interactions Between Single Walled Carbon Nanotubes and a Model Aquatic Organism

Permanent Link: http://ufdc.ufl.edu/UFE0042748/00001

Material Information

Title: On the Interactions Between Single Walled Carbon Nanotubes and a Model Aquatic Organism Biological Responses and Bioaccumulation Potential
Physical Description: 1 online resource (145 p.)
Language: english
Creator: Youn, Sejin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: carbon -- nanotubes
Environmental Engineering Sciences -- Dissertations, Academic -- UF
Genre: Environmental Engineering Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The rapid growth of nanoscience and nanotechnology, and the resulting widespread production and use of manufactured nanomaterials (MNs) have stimulated research on their environmental fate and potential implications. Among the most commonly used and studied MNs, carbon nanotubes (CNTs) have attracted the attention of both engineers and scientists, and this is due primarily to their unique physical, chemical, and electrical properties. The anticipated applications of CNTs are multiple and they are expected to radically improve the rate of success in biomedical, optical, electronic, and environmental research; to name a few. However, large scale production and use of CNTs would undoubtedly lead to the introduction of this new class of hazardous materials into the environment, with potential adverse effects on human health and ecosystem functions. The above concerns have led to the development of nanotoxicology, an emerging research field that focuses on the biological implications of MNs. In the past decade, most nanotoxicity studies have produced rather conflicting conclusions, due primarily to an overall failure to adequately address the diversity in the physicochemical properties of CNTs, and how such variability could be linked to the observed impacts on cells and organisms. To narrow the knowledge gap in our current understanding of the environmental implications of CNTs, this study was designed to investigate the interaction between single walled carbon nanotubes (SWNTs) and aquatic organisms, using the freshwater algae, Pseudokirchneriella subcapitata, as a model aquatic primary producer. Besides the physicochemical characterization of SWNTs and produced SWNT-suspensions, the study focused primarily on the biological responses of P. subcapitata when exposed to SWNTs under different environmental scenarios. The potential for SWNTs bioaccumulation was also assessed as a function of water chemical composition and SWNT suspension manufacturing methods. SWNTs used in this study were obtained from commercial sources and SWNT suspensions were produced in our laboratory by a non-covalent functionalization method that uses aqueous surfactant solutions and mechanical energy. The biological responses of P. subcapitata exposed to SWNTs were investigated using typical 96-hour toxicity assays and long-term exposure studies. In these experiments, changes in algal biomass were monitored using biochemical tracers (e.g. chlorophyll a); while changes in cell morphology (using transmission electron microscopy), the occurrence of oxidative stress (by measuring changes in GSH levels), and the degree of aggregation of SWNTs (using a nanospectrolyzer) were tracked over time to investigate the mechanisms of observed biological responses. Finally, phase distribution studies were conducted using a modified shake-flask method. This set of laboratory studies emphasized the role of dissolved organic matter (quantity and type) through the traditional octanol-water distribution approach to asses the bioaccumulation potential of SWNTs as a function of the aqueous chemistry of SWNT-suspensions. The major findings from the above studies can be summarized as follows. Aqueous suspensions of SWNTs stabilized with non-toxic surfactants can still be toxic to P. subcapitata. However, the magnitude of the observed toxic effects can be reduced by manipulating the suspension manufacturing process. Although this conclusion has been reached based on studies that rely on a single test organism (P. subcapitata), the results suggest that the development of a green approach which eliminates the toxicity of SWNTs during the manufacturing process is possible. Accordingly, a wide variety of model organisms (e.g. bacteria, invertebrates, and vertebrates) should be used in future studies to account for biochemical differences and SWNT-sensitivity specific to each organism type. Aqueous suspensions of SWNTs prepared in either natural river water collected from the Suwannee River (SR) or synthetic water (DI-water) spiked with different types of organic matter isolates (e.g. humic acid, fulvic acid, and tannic acid) were used in phase distribution experiments aimed at determining the octanol-water distribution coefficients (Kow) of SWNTs. The results show the following. SWNTs suspended directly into synthetic surfactant such as gum Arabic (GA) and sodium dodecyl sulfonate (SDS) do not partition between aqueous and organic phases. SWNTs become trapped at the water-octanol interface even when high energy homogenization techniques are used. While the dispersion of the highly hydrophobic SWNTs is nearly impossible in water, the presence of high levels of dissolved organic matter in tested waters produced SWNT suspensions with a dispersion degree and stability that are quite similar to those obtained with the ideal and commonly used synthetic surfactants. Our findings suggest that SWNTs introduced into organic-rich waters could remain well dispersed, and therefore, prone to long-range transport/dispersal while exhibiting an increased probability of interaction with pelagic organisms. The chemical composition of the water, namely the type and quantity of organic matter present in the water sample in which SWNTs are suspended controls the degree of SWNT suspension and stability. For instance, a complex mixture of organic compounds dominated by humic and fulvic acids (e.g. SR water) produced stable SWNT suspensions with a higher degree of dispersion than suspensions obtained with solutions dominated with tannic acid or algal and bacterial extracts. When SWNTs suspended in organic-rich waters are used in Kow experiments for determination of bioaccumulation tendency, it was found that SR water would favor the transfer of SWNTs from water to biological tissues. Dose-exposure studies using SWNT suspended in organic-rich waters showed little to no toxicity to P. subcapitata based on 96 hour toxicity studies. While the organic coating of SWNTs favors their transfer from aqueous solution to organic phase, it appears that the same coating would eliminate the bio-aggressivity of SWNTs. Overall, findings from this study point to SWNT surface modifications by different stabilizing agents as main criteria for adverse biological effects. Studies on a wide variety of organisms and the investigation of the potential for SWNT bio-magnification throughout a model food chain are significant avenues for future research.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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.
Statement of Responsibility: by Sejin Youn.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Bonzongo, Jean-Claud.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042748:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042748/00001

Material Information

Title: On the Interactions Between Single Walled Carbon Nanotubes and a Model Aquatic Organism Biological Responses and Bioaccumulation Potential
Physical Description: 1 online resource (145 p.)
Language: english
Creator: Youn, Sejin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: carbon -- nanotubes
Environmental Engineering Sciences -- Dissertations, Academic -- UF
Genre: Environmental Engineering Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The rapid growth of nanoscience and nanotechnology, and the resulting widespread production and use of manufactured nanomaterials (MNs) have stimulated research on their environmental fate and potential implications. Among the most commonly used and studied MNs, carbon nanotubes (CNTs) have attracted the attention of both engineers and scientists, and this is due primarily to their unique physical, chemical, and electrical properties. The anticipated applications of CNTs are multiple and they are expected to radically improve the rate of success in biomedical, optical, electronic, and environmental research; to name a few. However, large scale production and use of CNTs would undoubtedly lead to the introduction of this new class of hazardous materials into the environment, with potential adverse effects on human health and ecosystem functions. The above concerns have led to the development of nanotoxicology, an emerging research field that focuses on the biological implications of MNs. In the past decade, most nanotoxicity studies have produced rather conflicting conclusions, due primarily to an overall failure to adequately address the diversity in the physicochemical properties of CNTs, and how such variability could be linked to the observed impacts on cells and organisms. To narrow the knowledge gap in our current understanding of the environmental implications of CNTs, this study was designed to investigate the interaction between single walled carbon nanotubes (SWNTs) and aquatic organisms, using the freshwater algae, Pseudokirchneriella subcapitata, as a model aquatic primary producer. Besides the physicochemical characterization of SWNTs and produced SWNT-suspensions, the study focused primarily on the biological responses of P. subcapitata when exposed to SWNTs under different environmental scenarios. The potential for SWNTs bioaccumulation was also assessed as a function of water chemical composition and SWNT suspension manufacturing methods. SWNTs used in this study were obtained from commercial sources and SWNT suspensions were produced in our laboratory by a non-covalent functionalization method that uses aqueous surfactant solutions and mechanical energy. The biological responses of P. subcapitata exposed to SWNTs were investigated using typical 96-hour toxicity assays and long-term exposure studies. In these experiments, changes in algal biomass were monitored using biochemical tracers (e.g. chlorophyll a); while changes in cell morphology (using transmission electron microscopy), the occurrence of oxidative stress (by measuring changes in GSH levels), and the degree of aggregation of SWNTs (using a nanospectrolyzer) were tracked over time to investigate the mechanisms of observed biological responses. Finally, phase distribution studies were conducted using a modified shake-flask method. This set of laboratory studies emphasized the role of dissolved organic matter (quantity and type) through the traditional octanol-water distribution approach to asses the bioaccumulation potential of SWNTs as a function of the aqueous chemistry of SWNT-suspensions. The major findings from the above studies can be summarized as follows. Aqueous suspensions of SWNTs stabilized with non-toxic surfactants can still be toxic to P. subcapitata. However, the magnitude of the observed toxic effects can be reduced by manipulating the suspension manufacturing process. Although this conclusion has been reached based on studies that rely on a single test organism (P. subcapitata), the results suggest that the development of a green approach which eliminates the toxicity of SWNTs during the manufacturing process is possible. Accordingly, a wide variety of model organisms (e.g. bacteria, invertebrates, and vertebrates) should be used in future studies to account for biochemical differences and SWNT-sensitivity specific to each organism type. Aqueous suspensions of SWNTs prepared in either natural river water collected from the Suwannee River (SR) or synthetic water (DI-water) spiked with different types of organic matter isolates (e.g. humic acid, fulvic acid, and tannic acid) were used in phase distribution experiments aimed at determining the octanol-water distribution coefficients (Kow) of SWNTs. The results show the following. SWNTs suspended directly into synthetic surfactant such as gum Arabic (GA) and sodium dodecyl sulfonate (SDS) do not partition between aqueous and organic phases. SWNTs become trapped at the water-octanol interface even when high energy homogenization techniques are used. While the dispersion of the highly hydrophobic SWNTs is nearly impossible in water, the presence of high levels of dissolved organic matter in tested waters produced SWNT suspensions with a dispersion degree and stability that are quite similar to those obtained with the ideal and commonly used synthetic surfactants. Our findings suggest that SWNTs introduced into organic-rich waters could remain well dispersed, and therefore, prone to long-range transport/dispersal while exhibiting an increased probability of interaction with pelagic organisms. The chemical composition of the water, namely the type and quantity of organic matter present in the water sample in which SWNTs are suspended controls the degree of SWNT suspension and stability. For instance, a complex mixture of organic compounds dominated by humic and fulvic acids (e.g. SR water) produced stable SWNT suspensions with a higher degree of dispersion than suspensions obtained with solutions dominated with tannic acid or algal and bacterial extracts. When SWNTs suspended in organic-rich waters are used in Kow experiments for determination of bioaccumulation tendency, it was found that SR water would favor the transfer of SWNTs from water to biological tissues. Dose-exposure studies using SWNT suspended in organic-rich waters showed little to no toxicity to P. subcapitata based on 96 hour toxicity studies. While the organic coating of SWNTs favors their transfer from aqueous solution to organic phase, it appears that the same coating would eliminate the bio-aggressivity of SWNTs. Overall, findings from this study point to SWNT surface modifications by different stabilizing agents as main criteria for adverse biological effects. Studies on a wide variety of organisms and the investigation of the potential for SWNT bio-magnification throughout a model food chain are significant avenues for future research.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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.
Statement of Responsibility: by Sejin Youn.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Bonzongo, Jean-Claud.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0042748:00001


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ON THE INTERACTIONS BETWEEN SINGLE WALLED CARBON NANOTUBES AND A MODEL AQUATIC ORGANISM: BIOLOGICAL RESPONSES AND BIOACCUMULATION POTENTIAL By SE JIN YOUN 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 2011 1

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2011 Sejin Youn 2

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To my family 3

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ACKNOWLEDGMENTS I would like to thank my academic advis or and PhD committee chairman, Dr. JeanClaude J. Bonzongo, for shari ng his precious knowledge, experience and most of all, his sense of humor. I was very lucky to meet and work with him. I would like to thank my other committee members, Drs. C. Y. Wu, J. J. Delfino, and K.J. Ziegler for their help, guidance, and advice. Special thanks go to Pr ofessor Gabriel Bitton for his help with different bioassays and for mora l support. I would like also to thank all the members of my research group. Finally, I thank my dad, mom, and brother for their eternal love, support, and encouragement. I could not do it without them. 4

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TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 8LIST OF FI GURES .......................................................................................................... 9LIST OF ABBR EVIATION S ........................................................................................... 11ABSTRACT ................................................................................................................... 14 CHAPTER 1 INTRODUCTION AND PR OBLEM STAT EMENT ................................................... 182 CARBON NANOTUBES: PRODUCTION PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL IM PLICATION S ....................................................................... 222.1 Production and Us e of CNT s ............................................................................ 222.2 Potential Toxicity and Envi ronmental Behavio r of CNTs ................................... 252.2.1 Physical Characteristics (e .g. Shape, Length, Surface Area, Speciati on) .................................................................................................... 272.2.2 Toxic Im puritie s ....................................................................................... 292.2.3 Dispersion (i.e. Degree and Methods of Dispersion ) ............................... 302.2.4 Oxidative Stress by Reac tive Oxygen Spec ies (ROS)............................. 312.2.5 The Combination of Any of These Factors .............................................. 322.3 Fate and Behavior of CNT s in Aquatic Systems ............................................... 332.4 Dispersion of CNTs ........................................................................................... 372.5 Concluding Remarks ......................................................................................... 413 MITIGATION OF THE IMPACT OF SINGLE-WALLED CARBON NANOTUBES ON A FRESHWATER GREEN ALGAE: PSEUDOKIRCHNERIELLA SUBCAPITATA ....................................................................................................... 483.1 Introductory Remarks ........................................................................................ 483.2 Materials and Methods ...................................................................................... 503.2.1 Preparation and Characteriza tion of SWNT Su spensions ...................... 503.2.1.1 Gum Ar abic (GA) ........................................................................... 513.2.1.2 Sodium c holate (S C) ...................................................................... 513.2.1.3 Pluronic ac id F108 (P F) ................................................................. 523.2.2 Algal Growth in SWNT-Con taining Culture Media and SWNT Aggregation State in Algal Suspens ions. ...................................................... 533.2.2.1 Effects of increasing SWNT concentrations with fixed concentration of surfactants to P. subcapitata ....................................... 53 5

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3.2.2.2 Long-term exposure of algal cells to a pre-identified toxic combination of GA-SWN Ts and SC-S WNTs. ......................................... 543.2.3 Determination of Glutathione and Reactive Oxygen Species as Indicator Oxidat ive Stre ss ............................................................................. 543.2.4 Determination of the Aggregation State of SWNTs in Algal Culture Media ............................................................................................................ 553.2.5 Transmission Eectron Microscopy Studies .............................................. 563.3 Results and Discussions ................................................................................... 573.3.1 Characterization of Raw SWNT and Their Su spensions ......................... 573.3.2 Effect of Increasi ng SWNT Concentrations on P. subcapitata Growth .... 583.3.3 Interactions of P. subcapita ta with SWNTs Introduced into Culture Media at Different Points along the Growth Curve. ....................................... 603.3.3.1 GA -SWNT ...................................................................................... 603.3.3.2 SC-SWNT ...................................................................................... 613.3.4 Potential Mechanisms of Ob served Biological Responses of P. subcapitata Exposed to SWNT Suspens ions ................................................ 623.3.4.1 Potential ROS activity and impact on P. subcapitata growth .......... 623.3.5 State of Aggregat ion of Suspended SWNTs and Impacts on Algal Biological Re sponses .................................................................................... 663.3.5.1 GA -SWNT ...................................................................................... 663.3.5.2 SC-SWNT ...................................................................................... 693.3.5.3 PF -SWNT ....................................................................................... 703.3.6 TEM Observations of P. subcapitata Cells Grown in the Presence and Absence (control) of GA-SWN Ts .................................................................. 713.3.7 Impacts of SWNT-impur ities on Algal Growth ......................................... 723.4 Concluding Remarks ......................................................................................... 734 ROLE OF DISSOLVED ORGANIC MATTER IN THE PARTITIONING OF SINGLE WALLED CARBON NANOTUBES BETWEEN AQUEOUS AND ORGANIC PHASES: IMPLICATION S FOR BIOACCUMULATION ........................ 904.1 Introductory Remarks ........................................................................................ 904.2 Materials and Methods ...................................................................................... 944.2.1 Aqueous Organic-rich Solutions Used to Prepare SWNT Suspensions .. 944.2.2 Preparation and Characteriza tion of SWNT Su spensions ....................... 954.2.3 Octanol-water Part itioning Exper iments .................................................. 974.2.4 Algal Growth in NOM-SWNT Containing Cult ure Media .......................... 974.3 Results and Discussion ..................................................................................... 974.3.1 Characterization of SWNT Suspensions in Aqueous Solutions with Different Types of Organic Ma tter ................................................................. 974.3.2 Impact of NOM Concentrations and pH on SWNT Suspension ............. 1004.3.3 Octanol-water Partitioni ng Behaviors of SWNTs ................................... 1014.3.3.1 SWNT suspensions in synt hetic surfactants and algal organic matter and determination of octanol-water partitioning coefficients. ..... 1024.3.3.2 SWNT suspensions prepared in natural SR water and organicrich synthetic solutions and use in octanol-water partitioning experiment s .......................................................................................... 103 6

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4.3.3.3 Impacts of increasing concent ration of organic matter and pH on SWNT distribution between water and oct anol ..................................... 1044.3.4 Mechanisms of SWNT Phase Partiti oning ............................................. 1054.3.5 Relationship between SWNT Tr ansfer in Octanol Phase and Algal Growth Inhibi tion ......................................................................................... 1094.4 Concluding Remarks ....................................................................................... 1115 CONCLUSIONS AND RE COMMENDATION S ..................................................... 123LIST OF REFE RENCES ............................................................................................. 126BIOGRAPHICAL SK ETCH .......................................................................................... 145 7

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LIST OF TABLES Table page 2-1 Toxic effects of carbon nanotubes as reported in t he literat ure .......................... 443-1 Final surfactant concentrati ons in algal cu lture m edia ........................................ 893-2 Detected metal impurities in SWNT sus pensions ............................................... 894-1 Dissolved organic carbon (DOC) and spec ific UV absorpti on (SUVA) ............. 1214-2 Octanol-water partition ing experiment using both OM isolate-SWNT and SRSWNT suspensions and increased centrifugation speed ................................. 1214-3 Octanol-water distribution of SWNT as a function of dissolved organic concentrati ons. ................................................................................................. 1224-4 Octanol-water partition of SWNT from aqueous suspensions prepared in pH adjusted to 5 and 4 for TA and FA solutions, respectively. ............................... 122 8

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LIST OF FIGURES Figure page 2-1 Schematic diagram s howing how a hexagonal s heet of graphene is rolled up to single walled car bon nanotubes (S WNT). ...................................................... 422-2 Possible mechanisms of reactive oxygen species (ROS) production in the presence of single walled ca rbon nanotubes (S WNTs). ..................................... 433-1 Molecular structures of selected su rfactants (a) basic unit of gum Arabic (GA), (b) sodium cholate (SC), and (c) pluronic acid F108 (PF). ........................ 753-2 Length distributions of individual SWNTs suspended in 1 % GA determined by analysis of AFM im age (see inse t). ................................................................ 753-3 Absorption spectra of freshly prepar ed SWNTs in gum Arabic (GA-SWNT).. .... 763-4 Absorption spectra of freshly pr epared SWNTs in sodium cholate (SCSWNT) and pluronic acid F108 (PF-SW NT). ...................................................... 763-5 Effect of increasing concentrati ons of single-walled carbon nanotubes (SWNTs) on the growth of P. subcapitata in a standard 96-hour chronic algal assay. ................................................................................................................. 773-6 Effect of increasing concentra tions of SWNTs on the growth of P. subcapitata in a standard 96-hour chr onic algal a ssay. ......................................................... 783-7 Effect of increasing concentra tions of SWNTs on the growth of P. subcapitata in a standard 96-hour chr onic algal a ssay. ......................................................... 793-8 Biological response of P. subcapitata to GA-SWNT and SC-SWNT introduction to the culture medium at different algal growth phases. .................. 803-9 Effect of increasing concentrations of GA on P. subcapitata growing in culture m edia ...................................................................................................... 813-10 Diagram of the relationship between carbon nanot ubes exposure and concentration of surfactant. ................................................................................ 823-11 Near infrared fluorescence spectra of GA-SWNTs in algal suspensions with GA concentra tions .............................................................................................. 833-12 Raman spectra of GA-coated SWNTs (0.5 ppm) in algal suspensions measured on the first (t0) and fourth (tf) day of algal exposure experiments. ...... 843-13 Near infrared fluorescence spectra of single walled carbon nanotubes suspended in sodium cholate (SC-SW NTs) in algal suspensions. ..................... 85 9

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3-14 Raman spectra of single walled carbon nanotubes suspended in sodium cholate (SC-SWNTs) in algal suspens ions. ........................................................ 863-15 Near infrared fluorescence spectra of PF-SWNTs in algal suspensions ............ 873-16 Raman spectra of SWNTs suspended with PF in algal suspensions ................. 883-17 TEM images of P. subcapitata ............................................................................ 894-1 Potential behavior and fate of carbon nanotubes in natural aquatic systems as a function of key envi ronmental condi tions .................................................. 1134-2 Map of the Suwa nnee River watershed showing t he sampling loca tion. .......... 1134-3 Raman spectra of single wa lled carbon nanotubes (SWNTs) .......................... 1144-4 Concentration of single walled ca rbon nanotubes (SWNTs) suspended in synthetic organic matter c ontaining wate rs. ...................................................... 1154-5 Octanol-water partitioning of singlewalled carbon nanotubes (SWNTs). ......... 1164-6 Octanol-water partitioning behavio r of natural organic matter suspended single walled carbon nanotubes (NOM-SWN Ts) .............................................. 1174-7 Schematic diagram of a SWNT at an octanol-w ater interf ace .......................... 1184-8 Interfacial behavior of SWNTs bet ween octanol and water phases. ................. 1194-9 Effect of NOM-SWNTs on the growth of P. subcapitata in a standard 96 hour chronic algal assay. .......................................................................................... 120 10

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LIST OF ABBREVIATIONS AFM Atomic force microscopy BDL Below detection limit CCC Critical coagulation concentration CNT Carbon nanotubes Co Cobalt Cu Copper CVD Chemical vapor deposition D Defect band DCF 2,7dichlorofluorescein DCFH-DA 2,7dichlorofluoresceindiacetate DDT Dichlorodipheny ltrichloroethane DI Deionized DLVO Derjaguin, Landau, Verwey, and Overbeek DNA Deoxyribonucleic acid DOC Dissoloved organic carbon DOM Dissoloved organic matter E.coli Escherichia coli EDL Electron double layer EPA United States environ mental protection agency EU European united FA Fulvic acid Fe Iron G Graphite band GA Gum Arabic 11

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GSH Glutathione HA Humic acid HiPCO High pressure carbon monoxide ICP-AES Inductively coupled plasma atomic emission spectroscopy Kow Octanol water partition coefficient MN Manufactured nanomaterial Mo Molybdenum MWNT Multi walled carbon nanotube Ni Nickel NIR Near infrared NOM Natural organic matter OM Organic matter P.subcapitata Pseudokirchneriella subcapitata PAAP Preliminary algal assay procedure PBS Phosphate buffered saline PF Pluronic acid F108 PL Photoluminescence PVP Polyvinyl pyrrolidone RBM Radio breathing mode REACH Registration, evaluation, authorization and restriction of chemical substances ROS Reactive oxygen species RPM Rotation per minute SC Sodium cholate SDBS Sodium dodecylbenzene sulfonate SDS Sodium dodecyl sulfate 12

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SR Suwannee river SUVA Specific ultra violet absorbance SWNT Single walled carbon nanotubes TA Tannic acid TEM Transmission electron microscopy THF Tetrahydrofuran TSCA Toxic substances control act UV Ulatra violet 13

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy ON THE INTERACTIONS BETWEEN SINGLE WALLED CARBON NANOTUBES AND A MODEL AQUATIC ORGANISM: BIOLOGICAL RESPONSES AND BIOACCUMULATION POTENTIAL By Sejin Youn December 2011 Chair: Jean-Claude J. Bonzongo Major: Environmental Engineering Sciences The rapid growth of nanoscience and nanotechnology, and the resulting widespread production and use of manufactu red nanomaterials (MNs) have stimulated research on their environmental fate and potential implicati ons. Among the most commonly used and studied MNs, carbon nanotubes (CNTs) have attracted the attention of both engineers and scientists, and this is due primarily to their unique physical, chemical, and electrical properti es. The anticipated applic ations of CNTs are multiple and they are expected to radically improve the rate of success in biomedical, optical, electronic, and environmental research ; to name a few. However, large scale production and use of CNTs would undoubtedly lead to the introduction of this new class of hazardous materials into the env ironment, with potential adverse effects on human health and ecosystem functions. The above concerns have led to the development of nanotoxicology, an emerging research field that focuses on the biological implic ations of MNs. In the past decade, most nanotoxicity studies have pr oduced rather conflicting conclusions, due primarily to an overall failure to adequately addr ess the diversity in the physicochemical 14

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properties of CNTs, and how such variability could be linked to the observed impacts on cells and organisms. To narrow the knowledge gap in our current understanding of the environmental implications of CNTs, this study was desi gned to investigate the interaction between single walled carbon nanotubes (SWNTs) and aquat ic organisms, using the freshwater algae, Pseudokirchneriella subcapitata as a model aquatic primary producer. Besides the physicochemical characterization of SWNTs and produced SWNT-suspensions, the study focused primarily on the biological responses of P. subcapitata when exposed to SWNTs under different environmental scenarios. The potential for SWNTs bioaccumulation was also assessed as a func tion of water chemical composition and SWNT suspension manufacturing methods. SWNTs used in this study were obtai ned from commercial sources and SWNT suspensions were produced in our laboratory by a non-covalent functionalization method that uses aqueous surfactant soluti ons and mechanical energy. The biological responses of P. subcapitata exposed to SWNTs were invest igated using typical 96-hour toxicity assays and long-term exposure studi es. In these experiments, changes in algal biomass were monitored using bioc hemical tracers (e.g. chlorophyll a); while changes in cell morphology (using transmi ssion electron microscopy), the occurrence of oxidative stress (by measuring changes in GSH levels ), and the degree of aggregation of SWNTs (using a nanospectrolyzer) were tracked over time to investigate the mechanisms of observed biological responses. Finally, phase distribution studies were conducted using a modified shake-flask method. This set of laboratory studies emphasized the role of dissolved organic matter (quantity and type) through the traditional octanol-water 15

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distribution approach to asses the bioaccumulati on potential of SWNTs as a function of the aqueous chemistry of SWNT-suspensions The major findings from the above studies can be summarized as follows. Aqueous suspensions of SWNTs stabiliz ed with non-toxic surfactants can still be toxic to P. subcapitata However, the magnitude of t he observed toxic effects can be reduced by manipulating the suspension m anufacturing process. Although this conclusion has been reached based on studies t hat rely on a single test organism ( P. subcapitata ), the results suggest that the development of a green approach which eliminates the toxicity of SWNTs duri ng the manufacturing process is possible. Accordingly, a wide variety of model organ isms (e.g. bacteria, invertebrates, and vertebrates) should be used in future studies to account for biochemical differences and SWNT-sensitivity specific to each organism type. Aqueous suspensions of SW NTs prepared in either natur al river water collected from the Suwannee River (SR) or synthetic water (DI-water) spiked with different types of organic matter isolates (e.g. humic acid, fulvic acid, and tannic acid) were used in phase distribution experiments aimed at determining the octanol-water distribution coefficients (Kow) of SWNTs. The results show the following. SWNTs suspended directly into synthetic su rfactant such as gum Arabic (GA) and sodium dodecyl sulfonate (SDS) do not partition between aqueous and organic phases. SWNTs become trapped at the water-oct anol interface even when high energy homogenization techniques are used. While the dispersion of the highly hydr ophobic SWNTs is nearly impossible in water, the presence of high levels of disso lved organic matter in tested waters produced 16

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17 SWNT suspensions with a dispersion degree and stability that are qui te similar to those obtained with the ideal and commonly used synthet ic surfactants. Our findings suggest that SWNTs introduced into organic-rich waters could remain well dispersed, and therefore, prone to long-range transport/d ispersal while exhibiting an increased probability of interaction with pelagic or ganisms. The chemical composition of the water, namely the type and quantity of organic matter present in the water sample in whic h SWNTs are suspended c ontrols the degree of SWNT suspension and stability. For in stance, a complex mixture of organic compounds dominated by humic and fulvic acids (e.g. SR water) produced stable SWNT suspensions with a higher degree of dispersion than suspensions obtained with solutions dominated with tannic acid or algal and bacterial extracts. When SWNTs suspended in organic-rich waters are used in Kow experiments for determination of bioaccumulation tendency, it was found that SR water would favor the transfer of SWNTs from water to biological tissues. Dose-exposure studies using SWNT sus pended in organic-rich waters showed little to no toxicity to P. subcapitata based on 96 hour toxicity studies. While the organic coating of SWNTs favors their transfer from aqueous solution to organic phase, it appears that the same coati ng would eliminate the bioaggressivity of SWNTs. Overall, findings from this study point to SWNT surface modifications by different stabilizing agents as main criteria for adver se biological effects. Studies on a wide variety of organisms and the investigation of the potential for SWNT bio-magnification throughout a model food chain are signifi cant avenues for future research.

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CHAPTER 1 INTRODUCTION AND PROBLEM STATEMENT Manufactured nanoparticles (MN) find use in a wide variety of human activities, from medicine and electronics, to environmental/agricultural research and applications. Accordingly, nanoscience and nanotec hnologies are currently generating an extraordinary amount of inte rest and are anticipated to have a substantial impact on several anthropogenic activities (Gwinn and Vallyathan 2006). According to the National Institute for Occupational Safety and Health the U.S. governm ent invested between $432 million and $1,240 million pe r year from 1997 to 2005 and on the global scale, the investment is expected to reach $1 trillion by 2015 (Roco 2003). Carbon nanotubes (CNT), consisting of carbon atoms arranged into the shape of a hollow tube, may be one of t he most useful MNs owing to its many unique properties such as high surface area, superior mechani cal strength to mass ra tio, and excellent electronic properties. Since the discovery of these distinctive materials, researchers have investigated many potential uses incl uding bioand environmental applications. Therefore, methods for their synthesis and industrial scale production have been extensively studied. Thus, in the near future a significant amount of multiple (MWNT) and single (SWNT) walled carbon nanotubes will likely be introduced into aquatic systems either through intentional (e.g. envir onmental remediation) or non-intentional (e.g. accidental spillage) discharges. With t he recognition of their future application in the medical and environmental fields, their to xicity and fate in the environment must be investigated. Research on the toxicity of CNTs is st ill in its infancy and published data are often controversial and difficult to compare because various competing factors (e.g. 18

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production methods, physical properties, su rface chemistry, dispersion states and impurities) collectively determi ne the reactivity and toxicity of CNTs. However, published data indicate that CNTs, once inside living or ganisms, could be difficult to remove and could induce significant damage because of their physical resemblance to asbestos (Donaldson and Tran 2002). For example, similar to the biological effects of asbestos, Mutlu et al. reported significant granuloma formations in lung airways of mice (Mutlu et al. 2010). In addition, CNT toxicity has been demonstrated in several in-vivo (Blaise et al. 2008; Cheng et al. 2009; Lam et al. 2004) and in-vitro studies (Jia et al. 2005; Manna et al. 2005; Shvedova et al. 2003). There is now plenty of evidence exhi biting the potential threats of CNTs. Toxicological investigations focusing on CNTs begun just a few years ago, but a significant number of toxicological studies have already been published in a rather short period. So far, several of the studies have resulted in controversial results due primarily to lack of understanding of the physical properties of SWNTs. In spite of the current progress, a knowledge gap persi sts, mostly with regard to the potential mechanisms of toxicity of CNTs. For instance, while t he hydrophobicity of CNTs tends to suggest a great potential for CNT lipo-solubility, and th erefore bioaccumulation, research on bioaccumulation and bio-magnification potentials remain very limited and inconclusive. Overall, further studies are required to el ucidate (i) the fate and behavior of CNTs in natural aquatic systems, and ( ii) the potential for CNT bioaccumulation and transfer through different trophic levels. Questions are al so being raised about the suitability of current research methods, which often fa il to adequately characterize CNTs used in environmental studies. 19

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Researchers are facing challenges in studying the environmental impact of SWNTs. The hydrophobic nature of SWNT s and the van der Waals forces that dominate their interactions lead to aggregation, which is followed by sedimentation of bundled SWNTs (Oberdorster et al. 2005). But certain anticipated uses of CNTs may require specifically processed CNTs su ch as surface functionalized nanotubes (Moore et al. 2003). In addition to such surface modifications, natural organic matter (NOM) present in aquatic systems could stabilize CNTs suspensions (Hyung et al. 2007). These surface modifications would enhance the dispersion of CNTs in aqueous solutions and as a result, extend their residence time in aquatic systems. Clearly, surface modifications of CNTs would impact t heir interactions with both living organisms and dissolved organic matter. However, not enough studies have been conducted to examine the stability of CNTs and their transport and fate in aquatic environments. This dissertation focuses on the intera ctions between single walled carbon nanotubes (SWNTs) and a model aquatic or ganism, the freshwater green algae Pseudokirchneriella subcapitata The study emphasizes the biological response of the above primary producer to exposure to SWNT s, and investigates the bioaccumulation potential of SWNTs in aquatic systems. To achieve these objectives, a research approach was developed as outlined below. Following an introduction section which articulates the research problem statement (Chapter 1), a review of relevant literature on SWNTs in the environment is provided. This second chapter points out the gaps and w eaknesses in our current understanding of the physiscochem ical properties of CNTs and their implications on the 20

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environment. The review also addresses the i ssue of conflicting toxicity results when CNTs are used as toxicants in bioassays. The impacts of SWNTs suspended in sele cted surfactants, namely gum Arabic (GA), sodium cholate (SC) and pluronic acid F108 (PF), to wards a model organism are investigated and the results reported in C hapter 3. The above syn thetic surfactants were selected for their biocompatib ility (no observable toxicity to P. subcapitata even for concentrations in excess of 1000mg/L) and their ability to produce stable and well dispersed SWNT suspensions. Additionally, small scale bioassays are used for the determination of potential biologica l effects on aquatic organisms. Chapter 4 presents the results of laboratory studies focusing on the distribution of SWNTs between aqueous and organic phases as a proxy for determination of bioaccumulation capability under environmen tally relevant conditions. The study compares the ability of SWNTs to transfer from aqueous solutions stabilized with synthetic (GA, SC. and PF) or natural (humic acid (HA), fulvic acid (FA), tannic acid (TA), and a non-identified mixture of organic compounds present in a natural water sample) surfactants to an octanol phase. T he bioaccumulation potential is determined by assessing the distribution of SWNTs between octanol and water phases in the traditional shake-flask method. The effects of some key environmental factors (pH, ionic strength, and organic matter concentra tion) are also investigated. Finally, Chapter 5 is a gener al conclusion summarizing the key findings of the study and stating how such findings lay t he ground work for future research avenues. 21

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CHAPTER 2 CARBON NANOTUBES: PRODUCTION, PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL IMPLICATIONS 2.1 Production and Use of CNTs Carbon nanotubes (CNT) are single or mult iple layers of graphene sheets rolled up into hollow tubes with diameters of around 1nanometer (nm) and lengths that can extend to a few micrometers ( m). Their first appearance in peer reviewed literature dates back to 1952 (Radushkevich and Lukyanovic h 1952). However, a true recognition of the importance of CNTs was not achiev ed until 1991 (Iijima 1991) The structure of CNTs is described by a chiral vector, which is the direction of the rolling, and the chiral angle (Figure 2-1). SWNTs can be classifi ed as either metallic or semi-conducting based on their electronic properties, which may be predicted from the ch iral vector (n,m) (O'Connell et al. 2002). The synthesis of CNTs has been the subjec t of several studies, and the following are the three primary synthesis methods: (1) electric arc, (2) laser ablation, and (3) chemical vapor deposition (CVD) (Lam et al. 2007). These methods have been extensively reviewed (Baddour and Briens 2005; Chen and Zhang 2009; Dresselhaus et al. 2008; Lam et al. 2006; Oncel and Yuru m 2006), and can be briefly summarized as follows. In the electric arc method, a carbon el ectrode is sublimated in the reactor by arc discharge which increases t he temperature to 6000C. Ca rbon atoms are vaporized from the first electrode and then deposited on the opposing carb on electrode. The laser ablation method is similar to the electric arc method, but carbon sources are vaporized using powerful laser in the furnace (1200C) Ejected carbon atoms are carried by inert gas flow and then deposited on the water cool ed collector at the end of the furnace. These two synthesis methods, developed in earlier stages of CNT research, have been 22

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studied under various conditions with modifications in carbon sources, choice of inert gas, metal catalysts, power of arc and lase r, temperature, and pressure. There are some reports which conflict in terms of the purity and structural integrity of the CNTs produced using these methods, but the general disadvantages are costly equipment, additional sorting procedures required due to tangled CNTs, and low production yield. In CVD methods, carbon sources are appli ed in either liquid or gas phases as opposed to the electric arc and laser ablat ion methods. Carbon atoms are vaporized from a gaseous or volatile compounds of carbon in the reactor (600~1000C), and then absorbed onto the metal catalysts anchored on t he substrate. The metal catalysts serve as nucleation sites for the growth of CNTs. It has also been reported that CVD methods provide more controls over structure due to the support of the substrates. Additionally, CVD methods are easily scaled up to mass production and are thus the most important commercial methods for SWNT production (Cassell et al. 1999). Among several different types of CVD methods, the high pr essure catalytic decomposition of carbon monoxide (HiPCO) process developed by the Sm alley research group is a method for the preparation of SWNTs in a high pressu re environment using carbon monooxide as the carbon source. It is the only practica l process capable of pr oducing SWNTs at the kilogram scale. Indeed, CNTs used in many recent toxicological studies have been produced by HiPCO process (Cherukuri et al 2004; Kagan et al. 2006; Shvedova et al. 2003). The CNT production methods mentioned abov e are similar in that carbon atoms are thermally generated from carbon sources in a furnace or reacto r filled with an inert gas under controlled temperature and pressure conditions. Additionally, the presence of 23

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metal catalysts is required to produce SW NTs in all methods. Commonly used metal catalysts are iron, nickel, cobalt, molybdenum, yttrium, and alloys of these metals, and they play a critical role in controlli ng physical properties and yield of SWNTs (Dresselhaus et al. 2008). Many aspects of the growth of SWNTs growth on metal catalysts remain in question and it is be lieved that more than one mechanism may be responsible for their development (Harris 2009) Briefly, it is believed that carbon and metal atoms are vaporized and then condensed in to a liquid metal carbide. As more carbon atoms are fed into th is mixture, they become s upersaturated and begin to grow on the surface. It is obvious that metal cata lysts are integrated into the structure of SWNTs and therefore difficult to remove. Such metal impurities in SWNTs could be either intrinsically toxic or involv ed in reactions inducing toxicity. It did not take a long time for researcher s to discover that these materials have highly desirable mechanical, thermal, photochemical, and electrical properties. SWNTs are not only strong and stiff, but also light and flexible (Baughman et al. 2002). They are the best known electrical conductors with twic e the conductivity of copper (Lam et al. 2007). It is obvious that CNTs would attr act the attention of both engineers and scientists because of these unique properties and their anticipated widespread commercial and industrial applications. It is es timated that the global CNT market will be between 900 billion and one trillion dollars in 2011(Roco and Bainbridge 2005). SWNT production on the global scale is estimated to exceed 1000 tones by 2011 (Lekas 2005). CNTs and their derivatives are used in plastics, catalysts, batteries and fuel cells, super capacitors, conductive coatings, adhesives a nd nano-composites, sensor s, air craft, and car industries (Klaine et al 2008). In addition, CNTs have properties which make them 24

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good candidates for medical and environmental applications. CNTs have been studied for drug delivery due to their ability to penetrate through cell membranes, which will maximize the bioavailability of a drug to target organs. Near-inf rared photoluminescence of semi-conducting SWNTs makes them potential cellular imaging devices (Cherukuri et al. 2006). CNTs have received a lot of attenti on for their possible use in environmental clean-up for organic compounds (Chen et al. 2007) and CNT are considered excellent sorbents for environmental contaminants compared to conventional carbon based materials. This is primarily due to their large surface area and extremely hydrophobic nature. A number of studies have reported antimicrobial properties in the environment (Kang et al. 2008; Liu et al. 2009) and they may also be applicable for water treatment and distribution systems. While the novel properties of CNTs make them useful for so many exciting applications, like a double edged sword, it is uncertain how these properties will affect human health and ecosystem functions. Some of the application of CNTs in the medical, pharmaceutical and environmental fiel ds involve direct introduction into the human body and the environment, and theref ore a precautionary approach with an assessment of the risk to human health and implications for the environment must be adopted. Recent studies indicate that some of the toxicological properties of CNTs might not be benign to biological targets (Alpatova et al. 2010; Mutlu et al. 2010; SimonDecker et al. 2009) (Alpatova et al. 2010; Mutl u et al. 2010; Simon-Deckers et al. 2009). The uncertainties and potential threats of CNTs are of serious concern. 2.2 Potential Toxicity and Environmental Behavior of CNTs The discovery of CNTs and their properti es has contributed immensely to the advance of nanotechnology. Numerous st udies have been conducted concerning the 25

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development of economical large scale production methods of CNTs. Increased production of CNTs, however, will result in an increased potential for either deliberate or accidental release into the environment. Therefore, CNTs are al so the subject of concerns owing to potential environmental hazards and human health risks. In addition to the toxicity issues common to nanosized materials, the very properties of SWNTs that make them attractive have lead to curr ent concerns. For instance, the physical character of needle shaped SWNTs brings about potential applications in medical areas, such as for nano-syringes (Rivera and Starr 2010) or antimicrobials (Kang et al. 2008). But this specific property c an be a potential threat to human health due to the similarity with asbestos (Kostarelos 2008; Sargent et al. 2009). Accordingly, SWNTs pose new challenges for engineers and scientists and collaborative studies are needed to address the currently unknown aspects of this novel material. EPA reports that CNTs regulated under Toxic Substances Control Act (TSC A) may be new chemicals which have significantly different properties from the traditional graphite or other carbon substances already listed under the above mentioned TSCA (EPA 2008). The literature is now abundant with papers deal ing with both the sy nthesis and use of CNTs, but current knowledge on the envir onmental fate and impact of MNs including CNTs remains very limited despite of the increasing interest on the impact of nanoscience and nanotechnology. In an effort to fill this gap, the federal research and development funding for investigations on the environmental implication of MNs has been significantly increased si nce 2004 (Guzman et al. 2006), and experimental data on environmental fate and toxicity of SWNTs ar e being published at a rapidly increasing 26

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rate as well. Some of the findings are su mmarized in Table 2-1 and briefly discussed below. Ever since oxidative damage in fish brains induced by fullerene was observed (Oberdorster 2004), interest in the toxicity of carbon based MNs has rapidly increased. Earlier studies had focused on lung or skin ti ssue because they were indicated as the main exposure routes of CNTs (Huczko and Lange 2001; Lam et al. 2004). Although certain test methods used in these earlier studies have been critisized as inappropriate (e.g. intratracheal instillation, inhom ogeneous CNT suspensions) (Lam et al. 2007), disturbing results obtained in these exper iments demonstrate t he potential risks of CNTs. Most CNT toxicity studies have fo cused on cytotoxicity and they have revealed that SWNTs can cause damage to cells (J ia et al. 2005; Shvedova et al. 2003). Additionally, primary pr oducers and secondary consum ers in aquatic systems are negatively affected by CNTs (Blaise et al. 2008). However, data have also been published on the lack of toxicity of CNTs on several model organisms (Cherukuri et al. 2004; Cherukuri et al. 2006; Pulskamp et al 2007). These conflicting findings point to gaps in our current understanding of the potential implications of SWNTs. CNTs are generally complicated mixtures and the lack of general agreement on toxicity data could be attributed to variations in the samp les. In fact, several hypotheses have been suggested to explain the toxicity of CNTs. 2.2.1 Physical Characteristics (e.g. Shape, Length, Surface Area, Speciation) As mentioned previously, CNTs are physically similar to asbestos fibers or other types of mineral fibers. This resemblance ha s raised concerns about the toxicity that would be anticipated from the in halation of long, thin non-biodegradable fibers. In earlier stages of CNT toxicity studies, CNTs contai ning soot were tested on guinea pigs using 27

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an intratracheal installation method and no significant toxicity was observed (Huczko and Lange 2001). Recent studies have shed more light on the relevance of CNTs physical properties on biolog ical targets. In a study comparing different length distribution of CNTs, CNTs su spended in bovine serum albumin were injected into mice, and it was found that granuloma lesion formations occurred more frequently with longer CNTs (Poland et al. 2008). The author s proposed that this phenomenon may be attributed to the incomplete phagocytosis of the longer CNTs. Moreover, it seems that the CNTs shorter than <10 m do not influence the viability of human lung cells (SimonDeckers et al. 2008). In a similar study, inve stigators discovered that while short (<300 nm) SWNTs presented no sign of toxicity, and were excreted outside of the body, long SWNTs (>10 m) induced granuloma formation (K olosnjaj-Tabi et al. 2010). Comparable observations have also been repor ted for multi-walled nanotubes (MWNTs) (Takagi et al. 2008). The biological responses of SWNTs and MWNTs are often compared in terms of ph ysical differences. When E.coli has been used as a test organism, stronger antibacterial effects were demonstrated from larger surface area and smaller diameter SWNTs (Kang et al. 2008). Interestingly, it was claimed that the smaller surface area was responsible for the negative impact on human fibroblast cells, with SWNTs eliciting a str onger toxic response than MWNTs (Tian et al. 2006). While larger surface area was hypothesized to pr ovide more cell-CNTs contacts and thus cause more membrane damage, others clai med that a CNT with less surface area would more easily penetrate into cells. Anot her important physicochemical property of SWNTs is electronic structure (i.e. meta llic and semi-conducing SWNTs) as mentioned above. As-produced SWNTs are mixtures of these two species consisting of 28

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approximately one third metallic and two thirds semi-conducting SWNTs (Saito et al. 1992). The difference in chemical reactivity between two SWNT species may be important in toxicological studies but it has been hampered by the lack of good separation method. Recently, Veciti s et al. (Vecitis et al. 2010) reported that the viability of E.coli was reduced significantly by increasing the concentration of metallic SWNTs. Therefore, how speciation of SW NTs impacts the toxicity has much research potential in terms of characterization of separated SWNTs. 2.2.2 Toxic Impurities CNTs synthesis involves the use of catal ysts such as Co, Fe, Ni, and Mo, and the alloys of these metals as well (Lam et al. 2006). Such metals are considered as impurities in that they are not necessary for the application of CNTs, and additionally, they are known to impact living organisms. In CNT toxicity studies, damage to tested organisms in comparison studies between pu rified and unpurified CNTs was attributed to the presence of the above metals (Cheng et al. 2009; Chlopek et al. 2006; Koyama et al. 2009; Murray et al. 2009; Porter et al. 2009; Vittorio et al. 2009; Walker et al. 2009). Purified SWNTs were found to be non-toxic to human muscle cells but slight changes in cell morphology were observed (Garibaldi et al. 2006). It has been demonstrated that significant oxidative stress in RAW 264.7 macrophages was probably caused by unpurified SWNTs (Kagan et al. 2006). Toxicity of unpurified SWNTs was also observed in living organisms such as estuari ne copepod (Templeton et al. 2006), and the purification process could impr ove the biocompatibility of CNTs in zebra fish (Cheng et al. 2009). Although removal of impurities from CNTs seemingly enhances their biocompatibility, there are still a few limitations to make definite conclusions. Various purification methods of CNTs have been dev eloped in the last decade (Chen and Zhang 29

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2009; Hou et al. 2008). The researchers usua lly adopt the developed methods or, in some cases, accept the manufacturers description about the purification process of CNTs which is often lack of necessary details In addition of the fact that different methods most likely yield different purity of CNTs, they also modify physical characteristics of CNTs. This aspect of CNTs will be discussed further later herein. 2.2.3 Dispersion (i.e. Degree and Methods of Dispersion) Because of their morphology and hydrophobic surface, as-produced CNTs tend to aggregate into bundles (Hilding et al. 2003). Ex tensive efforts have been made to obtain stable and individually separated CNT suspensions because their widespread application has been impeded by CNTs low solub ility in polar liquids and their tendency to aggregate (Tasis et al. 2006). The use of organic solvents and surfactants during dispersion processes could be toxic as shown in the case of tetrahydrofuran (THF) with fullerene (Fortner et al. 2005). Better dis persed CNTs could provide more contacts between organisms and CNTs. Ther efore, toxicity of CNTs was studied with respect to the manner and degree of disper sion. Suspended SWNTs in polyoxyethylene sorbitan monooleate were less cytotoxic to human MS TO-211H cells than bundled SWNTs and the authors emphasize the need for thorough mate rial characterizati on prior to the experiment and demonstrated the role of aggregati on in the toxicity of CNTs (Wick et al. 2007). Qu et al. tested purified CNTs su spended using carboxyl groups and CNTs with different degrees of aggregation in mice (Qu et al. 2009). Although no significant toxicity was observed, poorly dispersed CNTs rema ined in organs longer, unlike seemingly easily eliminated CNTs wit h higher degree of dispersion qua lity. In a more recent study, SWNTs suspended in Pluronic F108 (PF) were app lied to mice by intratracheal injection (Mutlu et al. 2010).The authors observed granulom a formations in the airways of mice 30

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treated with aggregated SWNTs which were abs ent when treated with well dispersed SWNTs. In terms of dispersion methods, onl y a handful of studies are available about their effects on the biocompatibility of CNTs. In vitro assessment of the toxicity of functionalized SWNTs and surfactant stabiliz ed SWNTs shows that SWNTs with more functional groups are less cytotoxic than ones with less functional groups or surfactant stabilized SWNTs (Sayes et al. 2006). SWNT s dispersed using different surfactants show differences in cytotoxicites, which are due to the toxicity of surfactant molecules attached on the nanotubes surfaces (Dong et al. 2008). 2.2.4 Oxidative Stress by Re active Oxygen Species (ROS) Due to their physical resemblances, CNTs are often compared with fibrous materials and viewed under the toxicological paradi gm of fibers. The ability of fibers to cause inflammation via oxidative stress has been extensively reviewed (Donaldson and Tran 2002), and the same mechanism can, a priori, be used to explain the adverse effect of CNTs. In general, oxidative stress is caused by the generation of reactive oxygen species (ROS) such as the super oxide anion, hydrogen peroxide, singlet oxygen, and hydroxyl radicals produced in cells (Stohs and Bagchi 1995). One of the defense mechanisms of cells toward foreign substances is the prod uction of ROS at the site of invasion (Figure 2-2A) (Apostol et al. 1989). These are neutralized by cellular defense mechanisms under usual circumstances (Blokhina et al. 2003). The intensity of the cellular response, however, depends on pr operties of the MNs digested, and the defense system can be overwhelmed as it reaches its threshold (Nel et al. 2006). Another ROS producing mechanism is the interaction between electron donor or acceptor sites on the surface of MNs and oxygen (Nel et al. 2006). Pure CNTs are supposed to be significantly inert (Zhang et al. 2002) but defective sites can be 31

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introduced to the sidewall eit her intentionally or unintentionally (Tasis et al. 2006). Superoxide radicals are produced by el ectron reduction and these can serve as precursors to additional ROS in the pr esence of transition metals through Fenton chemistry (Figure 2-2B). Several CNT st udies report the generat ion of ROS due to metal impurities such as Fe in cells (Kagan et al. 2006; Manna et al. 2005; Pulskamp et al. 2007) and in mammals (Shvedova et al. 2007) After metal impurities are removed via the purification process, CNTs can induce ox idative stress in fish (Smith et al. 2007) and human lung cells (Choi et al. 2009). Conv ersely, purified CNTs are reported to not induce oxidative stress in biological tar gets nor to generate free radicals in aqueous solutions (Fenoglio et al. 2006). 2.2.5 The Combination of Any of These Factors In some cases, the toxicological properties are difficult to address as a function of a single factor. As mentioned above, heavy metal impurities in CNTs are potential pollutants and also often involved in the gener ation of harmful ROS. In addition to the complications caused by heavy metals, physica l properties of CNTs can be significantly affected during the dispersion and purification process, generating further ambiguity. For instance, purified CNTs usually become shorter than as-produced ones due to the treatment conditions. Defective sites on the side walls and biol ogically malignant surfactants can be introduced during the disper sion process. This could have important toxicological consequences. These mechani sms collaborate to stimulate adverse effects, but one may provide resistance or reduce the negative impact of another. While most studies have concluded that purifi ed CNTs are more benign than unpurified CNTs, it has also been reported that the physical characteristics (e.g. surface area) play a more dominant role in determining toxicity than impurities (Tian et al. 2006). Moreover, 32

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the study using CNTs suspended in gum Arabi c reported that meta l impurities did not influence cytotoxicity (Simon-Deckers et al. 2008; Simon-Deckers et al. 2009). 2.3 Fate and Behavior of CNTs in Aquatic Systems As stated earlier, the uni que properties of CNTs and their anticipated multiple applications will eventually result in lar ge mass production, raising questions on the potential implications for both human health and the environment. This is because the introduction of CNTs to the environment c ould result in serious risks with adverse impacts on ecosystem functions as well as hu man health. While most research on the potential implications of CNTs has focus ed on toxicity aspects, studies on their environmental fate, with emphasis on physicochemical transformations and distribution in the environment remain very limited and highly challenging. The environmental behavior of CNTs is likely analogous to that of colloidal particles found in aquatic and terrestrial systems. This assumption is in part due to the fact CNTs are not truly soluble in water and their size distri bution range fits within the 1 to 1000nm range given for colloids (Holbr ook et al. 2010; Lead and Wilkinson 2006; Moore 2006). Therefore, CNTs could be prone to phase distribution between water, sediments, and biota (Klaine et al. 2008). Th is partition process would be controlled by certain key environmental parameters such as pH, ionic strength, and presence and types of dissolved organic matter (Handy et al. 2008). For instance, the adsorption of humic substances onto colloi dal particles can lead to changes in surface properties resulting in negatively charge surfaces and reduced aggregation of colloids (Lead and Wilkinson 2006). This behavior was observ ed in the aggregation trend of CNTs in the presence of Suwannee River humic substances (Hyung et al. 2007), resulting in a long residence time as suspended particles in aqueous solution. This could also result in an 33

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extended contact time with aquatic organisms su ch as algae. Since NOM is ubiquitous in any aquatic systems, their interaction wit h CNTs may alter the surface properties of CNTs and influence their fate and their biological effects. In the natural environment, even well st abilized colloids event ually aggregate over time and this is a key removal mechanism in colloidal systems (Buffle and Leppard 1995). Aggregation may occur when two particles transported by mechanisms such as Brownian motion come into contact and then stay attached (Handy et al. 2008). The likelihood of attachment is determined by t he competition between van der Waals (vdW) forces and electron double layer (EDL) forces according to Derjaguin, Landeau, Verwey, and Overbeek (DLVO) theory (Derjaguin an d Landau 1993; Verwey 1945). As two adjacent particles approach, the repulsive forc es of EDL increase, while the attractive forces of vdW predominate once they pass the energy barrier. Th e sum of these two forces determine whether the interaction bet ween two particles will be repulsive (i.e. stable) or attracti ve (i.e. aggregated). The DLVO theory may be used to understand the effects of various water chemistry parameters on CNT aggregation. As mentioned earlier, NOMs may introduce a negative charge on the surface of particles which result s in steric repulsion and natural river water enriched with NOMs coul d stabilize CNTs (Hyung et al. 2007). In an effort to better understand interactions bet ween NOM and CNTs, chemicals with simpler structures such as tannic acid (TA) (L in et al. 2009a; Lin and Xing 2008) and standard humic substance (Saleh et al. 2010) were used as surrogates for NOM in dispersion tests of CNTs. Dispersed MWNTs concentra tions showed a strong correlation with increasing TA and pH. The presence of ions and size of MWNTs also affected the 34

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quantity of dispersed MWNTs (Lin et al. 2009b) In addition, a higher input of organic matter impeded coagulation of MWNTs in a model water treatment facility (Holbrook et al. 2010). Conversely, an increase in ion co ncentrations may promote aggregation due to the compression of EDL. The concentration of electrolyte ions at which the transition from stable dispersion to aggregation occurs is known as the critical coagulation concentration (CCC). The CCC for various ions were evaluated using both SWNTs (Sano et al. 2001)(Na+: 37 mM) and MWNTs (Lin et al 2009b; Saleh et al. 2010)(Na+: 6.9 mM and 25 mM, respectively). These re sults cannot be directly compared due to differences in experimental conditions (i.e type of CNTs and methods of dispersion) and there are too few data to make draw any decisive conclusions. In addition, there is a need to assess the pot ential for bioaccumulation. There is no evidence that CNTs would behave as organi c pollutants such as polychlorinated biphenyl or dichlorodiphenylt richloroethane (DDT), but the lipophilicity of CNTs has been well recognized in their potential use as remediation tools along with their large surface area (Chen et al. 2007) Moreover, several reports have noted that CNTs are taken up by living cells (Smart et al. 2006) Certain CNTs can cross cell membranes and accumulate inside living organisms (Zhu et al. 2006a). This provides the basis for potential bioaccumulation in living organism s and contamination of different trophic levels through diet. There have been some criticisms about the rather higher concentration of MNs tested in the lab, co mpared with proposed MN concentrations in natural systems. However, for certain xenobio tics, the total ambient concentrations in water gives very little information of potential impacts to living organisms, particularly for compounds with strong bioaccumulation abilit ies. In addition to bioaccumulation, the 35

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concentration of such xenobiotic s could increase in tissues as they are passed to other organisms through diet (Mackay and Fraser 2000). This is usually favored by the low acute toxicity of such compounds, which allows organisms accumulating them to survive long enough and pass the contaminat ion to higher trophic level organisms. Only a few studies have been conduced to examine bioaccumulation of CNTs. The Biological uptake of CNTs and depuratio n process were observed in estuarine copepods (Templeton et al. 2006) and daphnia (Kennedy et al. 2008), but a quantitative analysis was not performed. During the in gestion and depuration processes, aquatic organisms modified the surface coatings of SWNTs and changed the stability of their dispersed state (Roberts et al. 2007). Be side qualitative studies, ingestion and depuration levels of 14C-labeled CNTs were quantitat ively determined using earth worms (Petersen et al. 2008). Peterson et al. reported that uptake of CNTs was significantly lower than that of pyrene and that they were not bioaccumulated in target organisms. In a terrestrial study, the results must be carefully interpreted because homogeneous mixing of CNTs in soil can be cumbersome (Klaine et al. 2008). The study of bioaccumulation of CNTs has been hampered due to the lack of an assessment method for xenobiotics inside ta rget organisms. Electron microscopic imaging technique and elemental composition analysis are not favorable because of the strong background signal coming from other carbon sources in organic materials. Photoluminescence of SWNTs has been applied in living or ganisms (Cherukuri et al. 2004; Cherukuri et al. 2006), but the change of aggregation state under the surrounding environment and detection failure due to the pr esence of metallic SWNTs are limitations of this method. Additionally, the attachment of tracing agents on t he CNT surface (Kam 36

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et al. 2004; Petersen et al. 2008; Singh et al. 2006) could alter their physicochemical properties, or the integrity of tracing agents could be da maged during experimental periods. Therefore, there is a plenty of research potential in establishing reliable and simple procedures for estimating bi oaccumulation potentials of CNTs. 2.4 Dispersion of CNTs SWNTs are prone to aggregatio n in aqueous suspension due to their hydrophobic nature and insolubility, and hence expected to settl e within a rather short time in aquatic systems (Oberdorster et al. 2005). It is noted that CNT in natural systems could encounter physical or chemical processes such as water waves, NOMs, or detergents, and the stability of CNTs in the environment will be affected by these environmental factors as well. Moreover, CNTs may not be released in their as-produced form because many useful properties of CNTs are onl y accessible when they are modified to be individually suspended. For example, wa ter soluble SWNTs are being developed as contrast agents in medical im aging (Ziegler 2005) and for t he delivery of therapeutically active molecules to target cells (Lacerda et al. 2006). In this modification process, SWNTs are dispersed in water with the aid of functional groups and the behavior and fate of functionalized SWNTs will be completely different from that of non-functionalized SWNTs in aquatic systems. Despite its signifi cance, the impact of the modification on SWNTs toxicity remains largely unknown. The extremely hydrophobic nature and str ong van der Waals interactions between tubes causes CNTs to bundle as they are synthes ized (Girifalco et al. 2000; Hertel et al. 1998). Therefore, one of the bi ggest challenges to achieving the promise of CNTs is separating tubes from each other, and thus, SWNT suspens ion is one of the major research areas in the study of CNTs. There are two major ways to achieve the 37

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dispersion of CNTs in solution, covalent and non-covalent functionaliz ation (Hilding et al. 2003). The covalent method involves bounding solublizing functional groups on the sidewall of SWNTs. Unfortunately, intrinsic sp2 bonds of graphene sheet consisting SWNTs sidewall are often destroyed in the process because of the introduction of sp3 hybridized carbon sites on the SWNTs side wall (Garg and Sinnott 1998). The breakage of sp2 bonds is undesirable, since it reduces the phenomenal strength of SWNTs which is important for applications (Garg and Sinnott 1998). Compared with the covalent method, non-covalent functionalization using dispersing agents has been proven to disperse and maintain individual SWNTs separated while preserving electrical properties and mechanical integrity. Furthermo re, mechanical and electrical properties of SWNT nanocomposites have been significantly improved usi ng surfactants (Tasis et al. 2006). Therefore, noncovalent functionalization is favor able for many applications. Surfactants, amphiphilic molecules havi ng both a polar and non-polar group, can accumulate on surfaces or interfaces between immiscible media, reduce surface tension, and thus provide stable dispersions. Similarly, surfactants and CNT interactions in aqueous solutions are primarily based on t he adsorption of the hydrophobic tail of the surfactant molecule on the hydrophobic surface of SWNT bundles, together with attraction of the hydrophilic head to the ambient water. In addition, efficient surfactant coating onto CNT surfaces induces electrostati c (Strano et al. 2003) or steric repulsion (Bandyopadhyaya et al. 2002) that could neutralize van der Waals forces between tubes. In a typical dispersion process, high shear homogenization and ultrasonication may follow after surfactants have been introduc ed. This mechanical exfoliation of the bundles assists the infiltration of surfactant molecules into the inner circle of CNT 38

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bundles in order to increase the efficiency of dispersion. Various surfactants have been studied due to the advantages of non-coval ent functionalization. A few well known examples are sodium dodecyl benzene sulfonate (SDBS), s odium dodecyl sulfate (SDS) cetyltrimethylammonium brom ide, Tween, and Pluronic (Moore et al. 2003). In addition, naturally existing substances, including NOMs (Hyung et al. 2007), DNA (Enyashin et al. 2007), and GA (Bandyopadhyaya et al. 2002), have been evaluated as dispersants for CNTs. It is obvious that surfactants used to dis perse CNTs will play an important role not only in determining the toxicity of CNTs, but also in deciding their fate in the environment. Living organisms could actively ingest coating substances used on SWNTs surfaces and resultantly change thei r behavior in the aquatic system (Roberts et al. 2007). Owing to possible medical applicat ions, surfactants are at the center of interest as the need to enhance the biocompat ibility of CNTs incr eases. Although the toxic effects of surfactants on various aquatic organisms have been well studied, their general effects on living organisms are often found to be ambiguous (Ostroumov 2006). For example, anionic surfactants can bind and interfere with bioactive molecule such as peptides, enzyme, and DNA. While this may c ause serious environmental pollution, it can also promote the decomposit ion of other pollutants (Cse rhati et al. 2002). Another problem is that, the amphi philic property of surfact ants may be responsible for bioaccumulation in aquatic organisms (Tolls et al. 1994). Non-ionic surfactants show antimicrobial activity by interfering with the permeability of the cell membrane and this may cause cell death or damage through the loss of ions or amino acids (Schubert et al. 1986). Therefore, using surfactant-sus pended CNTs in toxicology studies may 39

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complicate the interpretation of data. Moreover, this may raise a question as to whether the observed biological responses are not just artificial to xicity from surfactants or modifications of the surface chemistry. Fo r example, previous investigations on the toxicity of fullerenes have used THF as a dispersing agent (Oberdorster 2004). It was later suggested that residual TH F in unique fullerene structures may be responsible for elevated toxicities (Fortner et al. 2005; O berdorster et al. 2006). Stable dispersion of CNTs, however, must be achieved in studyin g their impact and fate in aquatic systems. First, only stable CNTs provide sufficient in teraction time with aquatic organisms to produce a threat of potential uptake or ch ronic toxic effects. Secondly, unexpected aggregations during toxicological experiments complicate the in terpretation of results. Thirdly, it is reported that CNTs can be st abilized through interactions with NOMs and thus stay in aquatic system longer than expe cted. Fourthly, since CNTs are expected to be separated and dispersed in suspensions for mo st of their applications, more realistic scenarios of exposure and discharge should a ccount for these modified CNTs rather than just the as-synthesized versions. The biological impact of surfactants and their effects on CNTs physicochemistry make the choice of surfactant used one of t he most important factors involved in CNT suspensions. Surfactants used in toxicity test must be as benign to targets as possible so that artificial effects are minimized. In recognition of these factors and taking into account preliminary lab studies, GA, sodium cholate (SC), and pluronic acid F 108 (PF) have been suggested. Basic information, toxici ty reviews, and CNTs suspensions using these surfactants are discussed in next chapter. 40

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2.5 Concluding Remarks Nanotoxicology is still in its infan cy, and in general, studies focusing on the environmental fate and impacts of NMs re main highly challenging. Numerous toxicological studies on the effects of NMs have been published in a rather short time, but many of these reports have been controversial due to a lack of understanding of the physicochemical properties of CNTs. A dditionally, the potentia l mechanisms of observed adverse biological effects have only been explored in a skin-deep manner, and many questions remain unanswered. In fa ct, studies on the environmental fate and behavior of CNTs in the environment are only beginning to receive due attention as the initial research efforts focused mostly on implicati ons on human health. Based on the above review, CNTs size shape, degree of dispersion, and the presence of metal impurities could all re sult in toxic responses. However, the discrepancy in reported results seems to be linked primarily to the lack of a good understanding of CNT characterization prior to use in and during bioassays. For instance, several authors rely simply on information provided by manufacturers. The evaluation of the toxicity pot ential of NMs is therefore made difficult without clear links between specific physicochemical characteristics and biological responses. Used dispersion processes may also add another leve l of complexity in that some of the phycochemical characteristics of pristine NM s as provided by the manufacturers may no longer be relevant following changes induced by surface modification techniques. CNTs are new materials with many undi scovered properties, and consequently, with unexpected impacts on or ganisms. Only with appropria tely validated analytical methods and carefully designed experiments c an we gain insight into the mechanisms of environmental fate and toxicity of CNTs. 41

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Figure 2-1. Schematic diagram showing how a hexagonal s heet of graphene is rolled up to single walled carbon nanotubes (SWN T). The various ways of rolling graphene into tubes are described by the tube chirality as defined by chiral vector = 2 1aman where the integers (n,m) are the number of steps along the unit vector ( 2 1a and a ) of the hexagonal lattice. Adopted from O'Connell et al. 2002. 42

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Figure 2-2. Possible mechanisms of reactive oxygen species (ROS) production in the presence of single walled carbon nanotubes (SWNTs). Examples illustrate that SWNT can participate in produc tion of ROS via (a) inflammation responses of cells that may cause oxyradical release, and (b) electron release from reactive sites and subs equent Fenton reaction by transition metals. 43

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Table 2-1. Toxic effects of carbon nanotubes as reported in the literature Target organism CNT type Production method Surfactants Effects and mechanisms Reference Guinea pigs Arc discharge TWEEN no (Huczko et al. 2001) Human skin cell SWNT HiPCO toxic (ROS) (Shvedova et al. 2003) Mice SWNT HiPCO Arc discharge mice serum toxic (Lam et al. 2004) Macrophage SWNT HiPCO PF no (Cherukuri et al. 2004) Human kidney cell SWNT toxic (Cui et al. 2005) Macrophage SWNT MWNT Arc discharge CVD toxic (Jia et al. 2005) Macrophage SWNT MWNT Dimethyl sulfoxide toxic (Murr et al. 2005) Human skin cell SWNT Dimethyl formamide toxic (ROS) (Manna et al. 2005) Human skin cell MWNT CVD toxic (Monteiro-Riviere et al. 2005) Macrophage SWNT MWNT Dimethyl sulfoxide toxic (Soto et al. 2005) Human muscle cell SWNT moderate (Garibaldi et al. 2006) Macrophage s SWNT HiPCO toxic (ROS) (Kagan et al. 2006) Human fibroblasts SWNT MWNT toxic(surface area) (Tian et al. 2006) Estuarine copepod SWNT Arc discharge toxic (impurity) (Templeton et al. 2006) Rabbit SWNT HiPCO (Rice) PF no (Cherukuri et al. 2006) Human fibroblasts/o steoblastic cell MWNT CVD toxic (metal impurities) (Chlopek et al. 2006) Human skin cell SWNT HiPCO (Rice) toxic (dispersion method) (Sayes et al. 2006) Rat macrophage human lung cell SWNT CVD SDS no (Pulskamp et al. 2007) human MSTO-211H SWNT Arc discharge TWEEN80 toxic (Wick et al. 2007) 44

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Table 2-1. Continued. Target organism CNT type Production method Surfactants Effects and mechanisms Reference Human skin cell SWNT HiPCO PF(127) toxic (dispersion method) (Zhang et al. 2007) Daphnia magna SWNT HiPCO lipid coating minimum (Roberts et al. 2007) Human lung cell SWNT HiPCO serum minimum (Davoren et al. 2007) Rainbow trout SWNT SDS toxic (ROS) (Smith et al. 2007) Mice SWNT HiPCO toxic (ROS) (Shvedova et al. 2007) Human leukaemia cell SWNT MWNT Arc discharge / CVD moderate (De Nicola et al. 2007) mice lung cell SWNT HiPCO toxic (DNA damage) (Kisin et al. 2007) mice lung cell SWNT HiPCO toxic (Saxena et al. 2007) E.coli SWNT HiPCO toxic (size) (Kang et al. 2007) Human osteoblast cell SWNT HiPCO Arc discharge Laser ablation biocompatible (Kalbacova et al. 2007) human lung cell SWNT HiPCO Arc discharge serum toxic (nutrient depletion by SWNT) (Casey et al. 2008) mice MWNT CVD serum toxic (size and shape) (Poland et al. 2008) mice MWNT TritonX100 toxic (size and shape) (Takagi et al. 2008) human astrocytoma cells SWNT toxic (dispersion method) (Dong et al. 2008) mice lung cell SWNT toxic (ROS) (Jacobsen et al. 2008) algae,daphn ia,fish SWNT toxic (Blaise et al. 2008) human lung cell MWNT CVD GA toxic (independent of size) (Simon-Deckers et al. 2008) E.coli SWNT MWNT HiPCO CVD toxic(size) (Kang et al. 2008) mice/macro phage SWNT PF(68) toxic(ROS) (Chou et al. 2008) zebra fish MWNT toxic (impurity and method) (Cheng et al. 2009) 45

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Table 2-1. Continued. Target organism CNT type Production method Surfactants Effects and mechanisms Reference human lung cell SWNT toxic (ROS) (Choi et al. 2009) human skin cell SWNT HiPCO toxic (ROS) (Murray et al. 2009) human lung cell SWNT toxic (surface oxidation) (Panessa-Warren et al. 2009) mouse MWNT floating reactant method toxic (impurity and ROS) (Koyama et al. 2009) human aortic endothelial cell SWNT MWNT CVD floating reactant method toxic (Walker et al. 2009) human lung cell SWNT HiPCO Arc discharge bovine serum toxic (dispersion method) (Herzog et al. 2009) human bronchial epithelial cell SWNT toxic (DNA damage) (Lindberg et al. 2009) human lung cell SWNT HiPCO Arc discharge bovine serum toxic (dispersion method) (Herzog et al. 2009) human bronchial epithelial cell SWNT toxic (DNA damage) (Lindberg et al. 2009) mice SWNT MWNT CVD mice serum toxic (allergic reaction) (Nygaard et al. 2009) mice, bone marrow dendritic cells MWNT CVD Tween 80 toxic (allergic reaction) (Inoue et al. 2009) osteoblastic cells SWNT HiPCO SDS toxic (Tutak et al. 2009) human macrophage SWNT HiPCO no (Porter et al. 2009) mice organ MWNT CVD PBS / PBS +1%Tween 80 toxic (dispersion method) (Qu et al. 2009) human mocyte MWNT Arc discharge toxic (De Nicola et al. 2009) lung cell SWNT HiPCO toxic (DNA damage) (Sargent et al. 2009) human astrocytoma cell SWNT DNA /SC/SDBS/ SDS toxic (dispersion method) (Dong et al. 2009) 46

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Table 2-1. Continued. Target organism CNT type Production method Surfactants Effects and mechanisms Reference mice fibroblast cell SWNT HiPCO SDBS/ PF(68)/ PF(127)/ Peptide toxic (dispersion method) (Bakota et al. 2009) E.coli MWNT CVD GA toxic (target species. impurity irrelevant) (Simon-Deckers et al. 2009) E.coli SWNT HiPCO Tween60 toxic (shape and dispersion method) (Liu et al. 2009) Daphnia magna SWNT + Cu lipid coating Cu uptake increased with CNTs (Kim et al. 2010) Mice SWNT HiPCO Tween60 toxic (size) (Kolosnjaj-Tabi et al. 2010) Mice SWNT HiPCO PF toxic (size /aggregation state) (Mutlu et al. 2010) human liver cancer cell MWNT CVD PF hydroxypro phylcellulos e (dispersion method) (Piret et al. 2010) P12 cell (neuron) SWNT CVD toxic (ROS) (Zhang et al. 2010) E.coli/rat liver cell SWNT CVD GA / PVP/ Triton X100/ SR NOM toxic (dispersion method) (Alpatova et al. 2010) E.coli SWNT HiPCO SDS/SC toxic (CNTs speciation) (Vecitis et al. 2010) Daphnia magna MWNT CVD commercial NOM toxic (Edgington et al. 2010) rat MWNT toxic (size and dispersion method) (Wako et al. 2010) mice MWNT no (Kobayashi et al. 2010) Daphnia magna MWNT toxic (size and dispersion method) (Petersen et al. 2011) *Blank space represents lack of description in the paper. 47

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CHAPTER 3 MITIGATION OF THE IMPACT OF SINGLE-WALLED CARBON NANOTUBES ON A FRESHWATER GREEN ALGAE: PSEUDOKIRCHNERIELLA SUBCAPITATA 3.1 Introductory Remarks Single-walled carbon nanotubes (SWNTs) have attracted the attention of both engineers and scientists because of their anticipated widespread commercial and industrial applications (Baughman et al. 2002; Dresselhaus et al. 2003). This interest stems primarily from their unique size-rela ted characteristics, such as strength, elasticity, high adsorption capacity, and contro llable conductivity, which has led to their introduction into a wide variety of commerc ial products. However, the potential danger of introducing of SWNTs into the environm ent during manufacturing stages and during both use and disposal of SWNT-containing products could grow with the economic success of manufactured nanomaterials (MN). Accordingly, SWNTs entering waste streams would likely interact with ecological systems, and ultimately impact the biosphere. In fact, aquatic systems, such as rivers and lakes, behave as primary environmental sinks by integrating pollutants from atmospheric deposition, terrestrial surface runoffs, and groundwater discharges raising concerns on the potential implications of SWNTs that might accumulate in these systems (Gao et al. 2009). Recent research on the biological imp lications of SWNTs has produced rather conflicting results, in that both toxic and nontoxic effects are reported (Chen et al. 2007; Chen et al. 2006; Cherukuri et al. 2004; Chou et al. 2008; Federici et al. 2007; Griffitt et al. 2007; Lam et al. 2004; Lecoanet et al. 2004; Shvedova et al. 2005; Smith et al. 2007; Warheit et al. 2004). In addition to studies focusing on the environm ental aspects, the anticipated use of SWNTs as biosensors and in medicine (Chen et al. 2003; Federici et al. 2007; Mattson et al. 2000; Trommer and Neuber t 2005) has also stimulated research 48

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on the investigation of the effects of MNs on humans. In the latter case, preliminary results have raised concerns about the biological effects of SWNTs at both the cell and organism levels (Fenoglio et al. 2006; Lam et al. 2004; Shvedova et al. 2005; Warheit et al. 2004). In fact, the currently observed toxic effects of SWNTs have been attributed to several parameters, including the levels an d types of metal impurities (Kagan et al. 2006; Koyama et al. 2009; Shvedova et al. 2003); the degree and kind of aggregation in produced SWNTs (Choi et al. 2009; Kostarelos et al. 2007), physical characteristics, such as shape and surface area (Flahaut et al. 2006; Kang et al. 2007), the type and role of surfactants used to disperse them (Dong et al. 2008; Monteiro-Riviere et al. 2005; Sayes et al. 2006; Simon-Deckers et al. 2008), and more likely a combination of two or more of the above parameters. The significance of the toxic role of us ed surfactants stems fr om the fact that ideal SWNT-suspensions are currently obtai ned by means of a wide variety of aqueous surfactant solutions, with different chem ical properties. Commonly used surfactants include gum Arabic (GA) (Bandyopadhyaya et al. 2002), sodium dodecyl sulfate (SDS) (Moore et al. 2003; O'Connell et al. 2002; Regev et al. 2004; Smith et al. 2007; Vigolo et al. 2000), sodium dodecylbenzene sulfonate (SD BS) (Attal et al. 2006; Cognet et al. 2007; Moore et al. 2003), and sodium cholat e (SC) (Moore et al. 2003). Some preliminary studies have also indicated that dissolved natural organic matter could help enhance nanoparticle suspension in aqueous solu tions (Hyung et al. 2007). While these surfactants are used to enhance the dispersion/suspension of SWNTs, some can also affect the inherent properties of SWNTs (Garg and Sinnott 1998; Silvera-Batista et al. 2009), and potentially the interactions between SWNTs and living organisms in toxicity 49

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studies (Dong et al. 2008). Our preliminary studi es on the effects of surfactant types and SWNTs toxicity to P. subcapitata and Ceriodaphnia dubia have shown that certain surfactants (e.g., SDBS, SD S, SC, Triton X-15 and Triton X-100) are toxic to these two model organisms (Gao 2008). Therefore, toxicity studi es based on the above model organisms and using SWNTs suspended in such surfactants could not provide an accurate assessment of the effects of pristi ne SWNTs. In contrast other surfactants, such as GA (Alpatova et al. 2010; Simon-De ckers et al. 2008), pluronic acid F108 (PF) (Cherukuri et al. 2004; Mutlu et al. 2010), have been found to be non-toxic at environmental relevant concentrations. Efforts to reduce or elimi nate the risks associated with SWNTs or any other ENMs would require an understanding of the nanoparticle characteristics responsible for toxicity, as well as the mechanisms of interactions between nanoparticles and organisms. In this study, a model aquatic organism, t he freshwater green algae Pseudokirchneriella subcapitata also known as Selenastrum capricornutum (US-EPA 2002), was used in a series of laboratory bioassays to investigate the biological effects of SWNTs as a function of dispersing agent types and algal growth conditions. Quantitative (algal biomass) and qualitative (morphology) changes associated with specific SWNT-treatments were monitored as a function of time, while the mechanisms of biological responses were investigat ed using a combination of biochemical and spectroscopic methods. 3.2 Materials and Methods 3.2.1 Preparation and Characterization of SWNT Suspensions. Surfactants used to suspend SWNTs in th is study were selected based on the results of bioassays conducted by our res earch group and published in peer reviewed 50

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literature. Using cr iteria such as biocompatibility with respect to target test organism and quality of SWNT suspensions obtained, three surfactants (i.e. GA, SC, and PF) were selected. Their properties and use in toxici ty investigations are summarized briefly below. 3.2.1.1 Gum Arabic (GA) GA is a natural polysaccharide approved for use as a food additive by the US Food and Drug Administration (USDA) and has been used as an emulsifier in the food and pharmaceutical industry (Anderson et al 1982). GA has been used successfully to disperse aggregated CNTs in aqueous susp ensions. In fact, GA compounds are basically carbohydrates that are fundamental compon ents of living organisms. This is particularly important in terms of biocompatibility and of pot ential bio-applications of MNs in general (Lin et al. 2004). In toxicity tests using the combination of GA and CNTs, GA has not expressed any adverse effects on target organisms (Alpatova et al. 2010; Simon-Deckers et al. 2008). 3.2.1.2 Sodium cholate (SC) SC (C24H39NaO5.xH2O), commonly known as bile salts, is an anionic surfactant with a molecular weight of 430.55 g/mol. It was used as early as 1932 in biomedical studies to enhance the absorption of drugs (Duchateau et al. 1986) by taking advantage of its biocompatibility and solubilzing properties (Hofmann and Roda 1984). SC has been proven to be very effective as a surfactant for the suspension of CNTs (Wenseleers et al. 2004), but it has shown cont rasting toxicity results. For instance, no adverse impacts were detected in tested org anisms in a study by Dong et al. (Dong et al. 2008). On the other hand, adverse biolog ical effects were reported on certain 51

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bacteria used as model organisms with (Treyer et al. 2002) or without carbon nanotubes (Liu et al. 2009). 3.2.1.3 Pluronic acid F108 (PF) The pluronic group of compounds have been used as drug delivery agents (Kabanov et al. 2002). PF is a nonionic surfac tant with an average mo lecular weight of 14,600 g/mol. It is a good surfactant for the preparation of aqueous suspensions of SWNTs (Moore et al. 2003). The use of PF suspended CNTs in bioassays has shown a good biocompatibility (Cher ukuri et al. 2004; Mutlu et al. 2010), but excessive use of this surfactant may lead to potential to xic impacts (Sayes et al. 2006). SWNT suspensions used in this study were prepared by suspending approximately 40 mg of raw SWNTs (obtained from Rice University. Rice HPR 145.1) into 200 mL of aqueous (1%, m/v) solution of surfactant. To facilitate the dispersion process, the mixture was homogenized usin g a high-shear IKA T-25 Ultra-Turrax mixer for about 1.5 h followed by ultrasonication using a Misonix S3000 for 10 min. The mixture was then ultra-centrifuged at 20,000 rpm (Beckman Coulter Optima L-80 K) for 4 h, and the supernatant carefully separated from the aggregated SWNTs at the bottom of the centrifuge tube. The obtained suspensions were spin-coated onto mica to be characterized using atomic force microscopy (AFM, Digital Instruments D3100; Veeco Co., Plainview, NY). Metal impurities in raw SWNTs were measured by monitoring loss of nanotubes weight up to 800C for 3.5 h using thermo-gravimetric analysis (TGA, Mettler Toledo TGA, Mettler-Toledo Internati onal Inc., Columbus, OH)). The elemental analysis of acid digested SWNT suspens ions was conducted after an overnight digestion with concentrated HCl at 100C, and then followed by inductively coupled plasma atomic emission spectr oscopy (ICP-AES) analysis. 52

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3.2.2 Algal Growth in SWNT-Containi ng Culture Media and SWNT Aggregation State in Algal Suspensions. Pure cultures of P. subcapitata were obtained from Hydrosphere Research (Alachua, FL) and cultivated under constant li ght exposure at room temperature. The algal growth procedure used in this study was adapted from Environmental Protection Agency (US-EPA)s P. subcapitata 96-h growth inhibition method 1003.0 (US-EPA 2002). In dose-exposure studies, 50 mL of a preliminary algal assay procedure (or PAAP) culture medium (US-EPA 2002) were transferred into 150 mL Erlenmeyer flasks and sterilized by autoclaving. Growth expe riments were conducted under continuous illumination (~86 Em-2s-1) and both control and SWNT-t reated samples were prepared in triplicates, with each experiment repeated at least twice. Changes in chlorophyll a concentrations were used as an indicator of general biological responses and were monitored over time using a Turner Quantech Digital-Filter Fluorometer. Algal growth rates determined from control samples were averaged, assigned a value of 100%, and used as a reference value to report the gr owth rates determined from SWNT-treated culture media. To investigate the effect s of SWNTs on algal growth, the following experiments were conducted. 3.2.2.1 Effects of increasing SWNT concentrations with fixed concentration of surfactants to P. subcapitata In this set of experiments, sterilized culture media were first spiked with aliquot volumes of a concentrated SWNT-suspensi on to produce growth media with various concentrations. The addition of different vo lumes of the SWNT-s uspension to culture media resulted in different final concentration s of surfactants, which was then corrected by adding needed volumes of a plain surfac tant-solution to obtain uniform final surfactants concentrations in all growth media. Based on this approach, algal exposure 53

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studies were conducted at two different fi nal surfactant concentrations (Table 3-1). Following the addition of SWNTs and after vigorous mixing, culture media were inoculated with 1 mL of a pure algal suspension. 3.2.2.2 Long-term exposure of algal cells to a pre-identi fied toxic combination of GA-SWNTs and SC-SWNTs. In addition to the above short-term (96 h) experiments, the pr e-identified most toxic combination of GA (0.023%)-SWNT (0 .5 ppm) and SC (0.048%)-SWNT (0.8 ppm) concentrations were used in a long-term (2 week) exposure study. In this set of experiments, aliquot volumes of SWNT s suspended in GA and SC were added to growing algal suspensions either at the st art or at different points along the algal Sgrowth curve (i.e., additions during lag, exponential, and plateau phases). Algal growth conditions (e.g. culture media, light expos ure) and analytical methods used in these long-term experiments were similar to t hose described in Effects of exposure to increasing SWNT concentrations in algal cultures with a fixed concentration of surfactants. 3.2.3 Determination of Gl utathione and Reactive Ox ygen Species as Indicator Oxidative Stress To investigate the biological responses of our model organism, concentrations of reduced glutathione (GSH) were determined us ing culture media spiked with a fixed amount of SWNTs and increas ing GA levels in 96-h growth experiments. The GSH assay was adapted from methods described by Hissin and Hilf (Hissin and Hilf 1976) and Cohn and Lyle (Cohn and Lyle 1966). Briefly, algal suspensions were first centrifuged at 4C and 2000 rpm for 15 min. The supernatant was discarded and the pellet was re-suspended in 5 mL of a s odium phosphate buffer (0.1 M sodium phosphate 0.005 M ethyl enediaminetetraacetic acid, pH 8). After 10 min of incubation 54

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on ice, the mixture was transfe rred into a vial and sonicated within an ice bath with a Cell Disruptor W-375 (Heat Systems-Ultras onics INC, Farmingdale, NY). 500 L of the sonicated suspension were then vigorously mixed with 500 L of o -phthalaldehyde and 4.5 mL of the buffer solution and allowed to react at room temper ature for 20 min. The fluorescence of GSH was finally measur ed with a Turner Quant ech Digital-Filter Fluorometer (with emissi on and excitation wavelengths set at 420 and 350 nm, respectively). Assessment of reactive oxygen species (ROS) was adapted from acute toxicity assay described by Kobayashi (Koba yashi 2000) and Yoshida et al. (Yoshida et al. 2003). Briefly, 10 ml of algal su spensions were incubated with 2,7 dichlorofluoresceindiacetate DCFH-DA (20 uM in methanol) in the dark for 2 hrs and then washed 2 times with CM. The pellets, re-suspended in CM were incubated for 1hr and then the fluorescence was measured wit h the fluorometer (with emission and excitation wave lengths set at 530 and 488 nm, respectively). DCFH-DA, a cell permeable indicator of oxidati on, was oxidized in the presence of ROS to fluorescent 2,7dichlorofluorescein (DCF) in the cell. For all of the above algal growth ex periments, statistical differences (a<0.05) in observed biological responses amongst tr eatments were determined using a single factor analysis of variance. 3.2.4 Determination of the Aggregation St ate of SWNTs in Algal Culture Media The aggregation state of SWNTs within the culture m edia at both the initial time (t0) and after 96 h (tf) was probed using a combination of the following techniques. The vis-NIR (near infrared) absorbance and NIR fluorescence spectra of both controls and SWNT-treated algal suspensions were re corded using a Nanospectralyzer (Applied NanoFluorescence, Houston, TX), with ex citation from a 662 nm diode laser. The 55

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concentration of SWNTs in all suspensions was determined using Beer-Lamberts Law and the absorbance measured at 763 nm, similar to prior st udies (Moore 2005; ParraVasquez et al. 2007). Raman spectra were al so recorded using a Renishaw Invia Bio Raman with excitation from a 785 nm diode lase r. The relative inte ntisites of Raman peaks at 267 cm-1 are good indicator of CNTs aggrega tion state (Heller et al. 2004). 3.2.5 Transmission Eectr on Microscopy Studies TEM imaging was conducted at the University of Floridas Interdisciplinary Center for Biotechnology Research, using a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc. Schaumburg, IL). Samples were prepared using methods adopted from the literature (Ellis 2006), and digital images were acquired with a Veleta camera (VeletaOlympus Soft-Imaging Solutions Corp, Lakewood, CO) and iTEM software. Briefly, algal cultures were centrif uged at 1300 RPM for 10 min and then fixed with Trumps (1% glutaraldehyde and 4% formald ehyde in 0.1 M phosphate buffer), which was purchased from Electron Microscopy Sc iences, Hatfield, PA. Fixed cells were stored at 4C overnight and processed with a Pelco BioWave Microwave (Ted Pella, Redding, CA) to improve fixation. Samp les were next washed in 0.1 M sodium cacodylate (pH 7.24), post-fixed with 2% buffe red osmium tetroxide, water washed and dehydrated in a graded ethanol series of 25%, 50%, 75%, 95%, and 100%, followed by 100% acetone. Dehydrated samples were in filtrated in graded acetone/Spurrs epoxy resin and cured at 60C. Cured resin blocks were trimmed, cut into thin sections and collected on formvar copper slot grids. Samples were post-stained with 2% aqueous uranyl acetate and Reynolds lead citrate to improve contrast. Sections were then examined with TEM. Dig ital images were acquired with a Veleta camera (VeletaOlympus Soft-Imaging Solutions Corp, Lakewood, CO) and iTEM software. 56

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3.3 Results and Discussions 3.3.1 Characterization of Raw SWNT and Their Suspensions For the raw SWNTs used in this study, t he load of impurities amounted to about 13.4 weight % as measured by TGA, and th e elemental analysis by ICP-AES detected iron as the main metal impur ity (Table 3-2). An example image collected with AFM and determined nanotube length distri bution from it are shown in Figure 3-2. GA-suspended SWNTs exhibited a length distribution ranging from 300 to 1200 nm. AFM images of SC-SWNTs and PF-SWNTs were not obtained. However, the length distribution is expected to be similar to t hat of GA-SWNTs because it is mainly determined by sonication time (Simon-Deckers et al. 2008) which was identical for all suspensions prepared in this study. For suspensions pr epared in our laborat ory, stability was evaluated for the duration of the experimen ts using spectroscopic techniques. The absorption spectra of fres hly prepared GA, SC, and PF-SWNT suspensions (prepared in triplicates) were measured as a functi on of time to assess the stability of the suspension. As shown in Figures 3-3 and 34, the absorption spectra showed multiple peaks associated with the electronic structur e of different (the chirality vector n,m ; Figure 2-1) types within the SWNT suspensi on. These results show that absorbance of SWNTs remains unchanged over two weeks, the period used for long-term studies reported in this work. The lack of changes in days 1, 5, 9, and 14 in absorption trends/peaks indicates stability of GA-SWN Ts (Figure 3-3). In fact, changes due primarily to nanotubes aggregation would yiel d either red-shifted or broadened peaks due to the energy transfer betw een bundled tubes (O'Connell et al. 2002). As a colloidal suspension, SWNT suspensions may experi ence aggregation process over period of time which would impact the long term ex periments in particular. The aggregation 57

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process in nano-size systems drastically modifi es the overall physical characteristics of nanomaterials such as size and surface area. These physical parameters are critical factors determining their toxicity. The inse ts show the absorbance at 763 nm as a function of time and confirms that the aggregation state has not changed in a significant manner during the course of the experiments (Figures 33 and 3-4). Therefore, SWNT suspensions used in this study are consider ed stable for the duration of the bioassays. 3.3.2 Effect of Increasing SWNT Concentrations on P. subcapitata Growth Figure 3-5 shows the growth response of P. subcapitata to increasing concentrations of SWNTs in culture media containing fixed GA levels (0.023% and 0.046% v/v) in a series of 96-h growth experiments. The do tted horizontal lines represent the average algal growth rate s determined from control cultures. SWNTtreated media containing a final GA concentra tion of 0.023% show no growth inhibition effects for SWNT concentrations ranging fr om 0.01 to 0.05 ppm. However, a negative impact on algal growth, and therefore, toxici ty is observed for SWNT concentrations >0.05 ppm (Figure 3-5A). In contrast, when the same SWNT-concentration gradient is used with increased final GA-concentration fr om 0.023 to 0.046% v/v, SWNT-toxicity disappears and a tendency for growth bio-stim ulation is observed (Figure 3-5B). Figure 3-6 exhibits the growth response of P. subcapitata to increasing concentrations of SWNTs in culture media cont aining fixed SC concentrations (0.05% or 0.1% v/v) in a 96-h growth experiments. Additionally, a series of oxidative stress evaluation experiments were performed. T he dotted horizontal lines represent the average of algal growth rates determined from control cultures free of either SC or SWNTs. Vessel control is the culture medi um containing an amount of SC that is equivalent to other SWNTs tr eated culture media. Cultur e media treated with SWNTs 58

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having a final SC concentration of 0.05% di d not express an adverse effect on algal growth within 0.2 and 0.4 ppm, but toxicity wa s observed at SWNT concentration of 0.8 ppm. Notably, it is observed that algal grow th in culture media containing SWNTs at a final concentration of 0.8 ppm was signifi cantly reduced when final SC concentration was increased from 0.05 to 0.1%. The effects of PF-SWNTs on P. subcapitata after 96 hours are shown in Figure 37. The growth of P. subcapitata in culture media containi ng fixed PF concentrations (0.24% or 0.56%) showed si gnificant stimulation when compared to control (i.e. horizontal line). The growth of algae exposed to increasing gradient of SWNT with final PF concentration of 0.24% were significantly decreased with 5 and 7 ppm of SWNT, indicating a dose-dependent toxicity of SWNT The negative impact of SWNT on algal growth was significantly reduced upon increasi ng final PF concentration to 0.56%. With increased final PF concentration, SWNT conc entrations up to 7 ppm showed no toxicity. Overall, SWNT suspensions prepared in different surfactants (1% wt.) showed significant algal growth i nhibition for concentration rangi ng from 0.5 to 7 ppm. This concentration range is comparable to the result of Blaise et al. which reported toxic concentration from 1~10 ppm using P. subcapitata (Blaise et al. 2008). The negative impact of carbon nanotubes was significantly mitigated when the final concentration of each surfactant in culture media was increa sed about 2 times. In other words, for the tested range of SWNT concentrations, the increas e in levels of a non-toxic surfactant for the model organism alters the toxicity of SW NTs relative to this model organism. The potential mechanisms are discussed later herein. In addition, the growth stimulation was observed in culture media with SC and PF. The growth stimulation of P. subcapitata 59

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induced by surfactants had been observed in previous studies; various anionic and nonionic surfactants were shown to increas e growth of algae up to 250% as compared to control growth experiments (Nyberg 1988). Proof of biodegradation of certain types of surfactants by green algae was demonstrated in earlier literatures and this could have an effect on the biological responses of algae (Ernst et al. 1983) In other words, P. subcapitata used in this study may be able to ut ilize surfactants in culture media and this may explain the increased growth effects observed. 3.3.3 Interactions of P. subcapitata with SWNTs Introduced into Culture Media at Different Points along the Growth Curve. In this set of experiments, aliquot volu mes of SWNT-suspensions were added to algal growth media eitherprior to the inoc ulation with pure algal suspension (Figure 38A) or subsequently after an initial growth per iod (Figures 3-8B, 3-8C, and 3-8D). These experiments focused on the effect of the above determined toxic combination of 0.5 ppm of SWNTs and 0.023% of GA (Figure 3-5A) and 0.8 ppm of SWNTs and 0.05% of SC (Figure 3-6A) on different growth stages of P. subcapitata 3.3.3.1 GA-SWNT The results show that when SWNT is added prior to inoculation of the culture medium with algal cells, the gr owth inhibition is pronounced in the first 150 h (% growth decreased below ~60% of growth observed in control samples). However, this inhibition appears to be reversible as the algal growth recovers over time, reaching growth rate levels similar to that of the control a fter 200 h (Figure 3-8A). The addition of SWNTs several hours after the inoculation of cult ure media with algal cells showed different behaviors. First, SWNT addition to culture medi a 24 h after the initiation of the algal growth (Figure 3-8B) had a less inhibition effect than SWNT introduction during the 60

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exponential growth phase (96 h, Figure 3-8C). For the fo rmer (Figure 3-8B), algal growth rebounds from the initial inhibition following the introduction of SWNTs, and increases to reach levels that exceed growth rates in control sample s. In contrast, the addition of SWNTs after 96h had a pronounced i nhibition effect, and although a general trend towards recovery is observed over time growth rates in these culture media did not return to levels measured in control sa mples (Figure 3-8C). Finally, the addition of SWNTs at the end of the exp onential growth phase or plat eau (Figure 3-8D) leads to a drop in growth without recovery due to t he limited nutrients in these batch cultures. Overall, in these long-term growth experim ents, the tested toxic mixture of GA-SWNT would delay algal growth, but would not resu lt in a complete growth inhibition of P. subcapitata. Our preliminary experiments us ing SWNTs suspended in SDS as surfactant (data not shown) resulted in total algal growth inhibition. 3.3.3.2 SC-SWNT The results show that the introduction of SW NTs prior to inoculation of the culture medium with algal cells exhibi ted the most profound impact (F igure 3-8E). Algal growth was inhibited below 60 % of t hat of control for first 3 da ys and then sharply increased up to nearly 90% from 4th day. However, the grow ths were never recovered to the level of control during the experiment unlike reversible inhibition observed previously in certain culture media impacted with GA-SWNTs. Clearly, the change of the surfactant leads to different growth behaviors. Fi rst, SC-SWNT addition to cu lture media 1 day after the initiation of the algal growth (Figure 3-8F ) had a more inhibitory effect than SC-SWNT introduction during the exponential growth phas e (4th day, Figure 3-8G). In both cases, algal growth have been stabilized to the end point once they are impacted SC-SWNTs. Magnitude of the impa ct during the exponential growth phase (Figure 3-8G) are less 61

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pronounce than that of GA-SWNTs. The Expo sure of SC-SWNTs at the plateau decrease growth of algae but subsequent recovery was observed. In case of GASWNTs, drop of the growth at the plateau phase did not lead to recovery due to the depletion of nutrients. Ut ilization of the surfac tants may be responsible for this observed difference and it will be discussed more in ne xt experiment. In general, the tested toxic concentration of SC-SWNTs may adversely impacted algal growth, but the impact would be compensated up to certain level. 3.3.4 Potential Mechanisms of Observed Biological Responses of P. subcapitata Exposed to SWNT Suspensions Several physicochemical characteristics of SWNTs can be linked to the biological responses of P. subcapitata Such parameters would in clude (i) the generation of reactive oxygen species (ROS) and associ ated oxidative stress (Kagan et al. 2006; Shvedova et al. 2003), (ii) direct contact between cell and SWNTs resulting in cell membrane deformation, piercing, or uptake through passive or assisted transport (Kang et al. 2007; Liu et al. 2009), (iii) the levels and types of impurities associated with SWNT matrices (Kagan et al. 2006; Koyama et al. 2009; Shvedova et al. 2003), and (iv) the degree of SWNT aggregation (Kostarelos et al. 2007). The roles of some of these parameters in the biological responses observed in this study are discussed below. 3.3.4.1 Potential ROS activity and impact on P. subcapitata growth In this study, GSH, which is an impor tant biomolecule for defense against heavy metals and oxidative stress in plants, was used as a specific toxicity indicator (Noctor and Foyer 1998). GSH can be considered as a representative of nuc leophilic target biomolecules in algal cells. Since the enzymes glutathione-S-transferase and glutathione-synthetase are inducible by el ectrophilic compounds, such as SWNTs, the 62

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cellular GSH concentration would then be modulated by GSH-induction as well as by GSH-depletion caused by the presence of reactive toxicants. Therefore, disturbance of GSH metabolism is considered a toxicity pa rameter indicating cell damage as well as providing information about molecu lar modes of toxic action. GA. Levels of GSH were investigated in 96-h algal standard growth exposure assays (Figure 3-9). The final GA concentrations were varied from 0.04 to 0.17% in two sets of algal culture media containing eit her 0.5 or 1 ppm of SWNTs. The results showed that the increase of GA concentrations above the initially tested toxic combination of 0.023% GA and 0.5 ppm of SWNTs resu lted in biomass production similar or in excess to that measured in c ontrol cultures (Figure 3-9A). Although, the algal growth in culture media containing 1 ppm of SWNTs showed a slight inhibition for GA concentrations up to 0.1%, this toxicity is eliminated and the algal growth rate catches up with levels in control media as GA concentration reaches 0.17%. Measurements of GSH (Figure 3-9B) in these culture media showed an accumulation of GSH levels, regardless of SWNT concentrations and an overall increasing trend with increasing GA concentrations. Elevated levels of GSH in SWNTtreated algal media as compared to contro l samples indicate the induction of GSH stimulated by oxidative stress (Lei et al. 2006), caused by SWNTs in this case. In fact, the toxicity of SWNTs has been attributed pr imarily to the formati on of ROS and the resulting oxidative stress to living cells (Chou et al. 2008; Kagan et al. 2006; Manna et al. 2005; Shvedova et al. 2003). In the experim ents using GA-SWNTs in this study, the determination of ROS was omitt ed but toxicological studies focusing on oxidative stress have used exclusively the glutathione assay by either assessing the concentration of 63

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glutathione (Niederer et al. 2004; Vecitis et al. 2010), or observing the mitigation of oxidative damage through the introduction of an antioxidant-glutathione (Zhu et al. 2007). Moreover, the observed progressive build up in GSH levels with increasing concentrations of GA, which is considered a sign of oxidative stress induction (Gupta et al. 1991; Madamanchi et al. 1994), is also su ggestive of the fact that GSH synthesis exceeds GSH depletion as GA concentrati ons increase in the culture media. These results suggest that the presence of SWNTs stimulates the algal defense mechanisms as indicated by increased GSH levels. At the same time, the increasing GA levels enhance GSH-synthesis with produc tion rates that are higher than rates of GSH depletion through reaction with generated ROS. The overall result is a protective effect of the algal cells, and mechanisms s pecific to this proc ess would include the previously reported ant ioxidant potential of GA in pharma ceutical studies (Abd-Allah et al. 2002; Trommer and Neubert 2005). In this case, GA in much higher concentration than GSH in culture media would behave as the primary antioxidant, allowing the accumulation of GSH produced by algal cells. SC-SWNT. Levels of GSH and the formation of intracellular ROS were determined using final SC c oncentration of 0.05 and 0.1% in algal culture media containing varied concentrations of SWNTs (Figure 3-6). The results showed that production of ROS was increased with SWNTs concentration in culture media. Levels of GSH in algae treated with the combination of SWNTs (i.e 0.2 and 0.4 ppm) and 0.05% SC were maintained near c ontrol but the depletion of GS H was observed at the toxic concentration of 0.8 ppm showing the maximum ROS production (Figure 3-6B). Meanwhile, increasing trends of ROS was al so observed in culture media treated with 64

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SWNTs and 0.1% of SC but GSH level was still above that of control (i.e. above the horizontal line). It shows that oxidative stress exceeded t he anti-oxidant defense system and subsequently caused negative biological im pacts on algal growth when they were exposed to 0.8 ppm of SWNT with 0.05% of SC. On the contrary, the cellular antioxidative system of algae in culture media treated with 0.8 ppm of SWNT with 0.1% SC was still in tact. PF-SWNT. Evaluations of oxidative stre ss induced by PF-SWNTs were conducted using ROS and GSH analysis (Figur e 3-7). Overall trend of ROS and GSH levels in algae was identical between two di fferent final concentrations of PF. ROS levels were slightly increased as SWNTs were introduced into the culture media and concentration of GSH was neither accumula ted nor depleted in ent ire treated culture media. This implies that cellular defensiv e mechanisms of algae were not overwhelmed the ROS production induced by SWNTs when they were exposed to PF-SWNTs. Although oxidative stress induced by PF-SWNTs might partially contribute to growth inhibition, it seemed that di fferent mechanisms were involved with growth inhibition observed in culture media treated with PF-S WNTs (Figure 3-7A) as compared with GA and SC-SWNTs. Other than oxidative st ress, aggregation of state and physical characteristics of carbon nanotubes hav ebeen regarded as some of the major mechanisms of toxicity (Kang et al. 2008; Li u et al. 2009). Specifically, strong antimicrobial activities of carbon nanotubes have been explored: physical frictions and piercing by individually s eparated nanotubes can induce cell death. It is already shown that carbon nanotubes suspended in PF we re well separated and their dispersion qualities had not been changed during algal bioa ssays (discussed later in State of 65

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aggregation of suspended SWNTs and impacts on algal biological responses). Therefore, direct physical c ontacts between algal cells and PF-SWNTs are the plausible mechanism for negative impacts. The toxic impact of PF-SWNTs was significantly reduced in culture media wit h increased PF concentration of 0.56%. Increase of surfactant concentration can dr astically modify adsorption of surfactant on sidewall of nanotubes (Blanch et al. 2010), and subsequent ly impact the aggregation state of SWNT suspensions (Angelikopoulos et al. 2010). Most of the nanotubes are exposed to the surrounding environment at a low concentration of surfactant while the absorption of surfactant on the sidewall of carbon nanotubes is rapidl y increased with increasing concentration of surfactant (Figure 3-10). This coating formed by increased surfactant concentration can prevent direct contacts between nanotubes and living organism and exposure of harmful catalyst metals. In fact, the coating of MNs is a common technique in order to obtain desirable properties such as biocompatibility and solubility (Hardman 2006). 3.3.5 State of Aggregati on of Suspended SWNTs and Impacts on Algal Biological Responses The analysis of the physical state of SW NTs in algal suspensions was conducted at the initial (t0) and final (tf) times of the algal growth exper iments to gain insight into the potential mechanisms of observed cell-SWNT interactions. The sensitivity of NIRfluorescence and Raman spectra to environm ental effects can be used to assess changes due to SWNT aggregation and quenching mechanisms during algal growth. 3.3.5.1 GA-SWNT Figure 3-11 shows different emission s pectra obtained for sub-samples of algal suspensions containing different concentra tions of GA and SWNTs collected at times t0 66

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and tf. Since only individual and well-dis persed semiconducting SWNTs emit fluorescence (O'Connell et al. 2002), these s pectra are evidence t hat most of the SWNTs remain suspended and well-dispersed in the culture media throughout these algal exposure experiments. Howe ver, the intensity of the s pectra decreased slightly for each of the tested concentrations between times t0 and tf. These changes correlate to the relative ratio of surfactant to SWNTs; photoluminescence (PL) intensity changes are more significant as this ratio decreases. The observed decrease in emission intensities suggest that one or more of t he following could occur to SW NTs during algal growth: (1) covalent or non-covalent sidewall reacti ons that quench SWNT-fluorescence; (2) aggregation in the suspension; or (3) removal of SWNTs by sedimentation of either SWNTs or algal cells interacting with SWNTs. Decreases in fluorescence intensity can occur when the environment surrounding the SWNTs changes due to the replacement of surfactant molecules (GA in this case) by polymers, such as proteins (Cheruk uri et al. 2004; Cherukuri et al. 2006) and polysaccharides released in the culture media by growing algal cells. Therefore, it is possible that the production of dissolved organic matter (DOM) or organic exudates during algal growth could result in the formation of DOM-coat ed SWNTs. However, surfactant replacement is ty pically accompanied by characteristic solvatochromic peak shifts associated with the new environment surrounding the nanotubes (Cherukuri et al. 2004; Cherukuri et al. 2006; Moore et al. 2003). In this study, only a 1 to 2 nm solvatochromic shift in peak position was obser ved. This small difference indicates that only a very small amount of algal produced DOM could possibly replace GA molecules surrounding the SWNTs. These changes could indicate some changes to the surface 67

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coverage of the surfactant, which reduces the ability of the layer to prevent fluorescence quenching mechanisms (Silvera-Batista et al. 2009; Wang et al. 2008b). However, any changes to the surfactant structure must be minimal since exposure to higher dielectric environments are expected to dec rease the PL intensity dramat ically (Silvera-Batista et al. 2010). It is also possible that the sidewalls of the nanotubes become functionalized during algal growth, reducing the PL intensity. However, Raman spectra would indicate structural changes to the carbon atoms on the sidewalls of SWNTs associated with covalent functionalization. The D-peak (at about 1300 cm-1) and G-peak (at about 1600 cm-1) on such spectra correspond to phonon modes associated with sp3 and sp2 carbon atoms of SWNTs, respectively. In this study, spectra obtained for different GA concentrations (0.023% and 0.046% with 0.5 ppm SWNTs) at both t0 and tf exhibit nearly identical D/G peak ratios (Figure 3-12), suggesting that nanotubes do not undergo significant chemical changes, such as oxidation or sidewa ll functionalization. This is because high D/G ratios are obtai ned only when covalent bonds are formed on SWNT surfaces causing the phonon modes of sp2 carbon atoms to shift to sp3 phonon modes (Dresselhaus et al. 2003). The decrease in the intensity of th e PL (Figure 3-11) and G-peak between t0 and tf (Figure 3-12) indicate that some SWNTs co uld be removed from t he algal suspensions. Such sedimentation or remo val of SWNTs could occur following the aggregation of a small fraction of SWNTs present in the suspension. This is important as previous exposure studies investigating the effe cts of the degree and kind of aggregation of SWNTs on cells have linked these parameters to biological responses in human MSTO68

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21 cells (Kostarelos et al. 2007). Although t he above discussion suggests that complete exchange of the surfactant with DOM on SWNT surfaces is unlikely, the adsorption of even a small amount of DOM could alter t he surfactant structure around the nanotubes (Wang et al. 2008b) or change the surface ch arges (Myklestad 1995), disrupting the mechanism that disperses SWNTs. The induced progressive aggregation of SWNTs in such cases would promote removal from so lution by sedimentation and possibly alter the biological response of the tested model organisms as well. However, considerable aggregation of SWNTs would be ex pected to have significant decreases in PL intensity, which is not observed. One may therefore conclude from the s pectroscopy data suggest that the SWNTs in the suspension are similar in regards to surfactant cover age and aggregation state throughout algal growth. However, the fact that the PL in tensity changes are correlated to the relative ratio of surf actant to SWNTs indicates that both of these factors could occur to a limited extent during algal growth. Although aggregat ion and surfactant changes cannot be ruled out, it is clear that these effects have minimal influence on the SWNTs during algal growth at all SWNT conc entrations. Therefore, our study suggests that well-dispersed SWNTs in aqueous suspens ions can result in negative biological effects at concentrations >1 ppm, which is in c ontrast to previous studies that found that aggregation was responsible for toxi city (Kostarelos et al. 2007). 3.3.5.2 SC-SWNT The dispersion state of SC-SWNTs used in this study was monitored using NIRfluorescence spectra and Raman spectro scopy before and after the 96 hours algal growth experiments. Photoluminescence sp ectra obtained from culture media treated with SC-SWNTs are shown in Figure 3-13. Fluorescence emitted from SWNTs at the 69

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beginning (i.e. black line) was significantly reduced at the end of the experiment (i.e. dotted line) for SWNTs concentrations of both 0.05% and 0.1% SC. As discussed earlier, decreases in fluorescence intensity ar e mainly caused by sidewall modifications, changes in the interaction between surf actant and nanotubes, or aggregation in the suspension and subsequent removal of SWNTs. Lack of decreases in absorbance and identical Raman D/G-peak ratio between before and after the experime nt indicate that removal of SWNTs and side wall functionalizat ions are unlikely the cause of changes in PL intensity (data not shown). Changes in intensity of Raman aggregation peak show signs of nanotubes aggregation in final SC concentration of 0.05%, but no significant changes are observed in 0.1% (Figure 3-14). Biological modification of SWNTs that target living organisms consumed coating ma terials of carbon nanotubes was reported (Roberts et al. 2007). If surfactants us ed to disperse SWNTs were degraded and utilized by algae in the culture media, it would disturb stability of the suspension and then result in aggregation of SWNTs. Th is aggregation induced by algae was not observed in higher SC concentration because there is enough SC in culture media to replace exposed sidewalls of carbon nanotubes. 3.3.5.3 PF-SWNT Fluorescence spectra changes were also monitored in the experiment using PFSWNTs (Figure 3-15) Emissi on spectra observed in this experiment shows that quenching of fluorescence does occur but in re latively small magnitude. This suggests that most of the SWNTs remained suspended and well dispersed in culture media. The existence of stable nanotubes suspensions duri ng algal growth are also confirmed in monitoring the aggregation peak in Raman spec tra (insets of Figure 3-16). Additional modifications that nanotubes may under go dur ing algal growth are the surfactant 70

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replacement and functionalization of the sidewalls. PF-SWNTs in culture media did not undergo significant shifts of peak position in fluorescence and Raman spectra (Figures 3-15 and 3-16), implying that surfactant r eplacement is unlikely to occur during the experiment. Finally, functionalization of si dewalls could occur by introducing defected sites on the sidewalls of nanotubes. The degr ee of the functionaliz ation has been shown to influence toxic response of living or ganisms (Cheng et al. 2009; Choi et al. 2009; Sayes et al. 2006). The degree of functionalizat ion can be assessed from two distinctive peaks of Raman spectra; the D-band (i.e. defectinduced) and Gband (i.e. graphiterelated) (Dresselhaus et al. 2008). D and G peak obtained for different SWNT concentration before and after exhibit insignifi cant changes (Figure 3-16), indicating that sidewall structures have been intact without chemical changes such as oxidation. 3.3.6 TEM Observations of P. subcapitata Cells Grown in the Presence and Absence (control) of GA-SWNTs A previous study on SWNT-organism inte ractions showed that cell death could be attributed to the physical piercing of ce ll membranes by individual SWNTs (Kang et al. 2007). TEM observations of cells from th e different treatments used in this study (Figure 3-17) showed no detectable physical ce ll-SWNT interactions. This could be due to the lack of sufficient c ontrast to distinguish betw een carbon-based background cell components and the carbon nanotubes. However, a look at cells exposed to the previously identified toxic combination of 0.023% GA and 0.5 ppm SWNTs (Figure 317B) in comparison with cells from contro l samples (Figure 3-17A) showed significant changes in the morphology of cell membranes In these dividing cells, SWNT-treated cells have reduced size and deformed cell me mbranes (Figure 3-17B). These impacts could be attributable to the destructive effe cts of ROS due to the fact that membrane 71

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integrity is a primary target (Cabiscol et al. 2000), but one may not exclude the potential physical interaction between cell and SWNT par ticles, even though our TEM images do not show such occurrences. Finally, the lack of physical impacts on either controls (e.g., Figure 3-17A) or other samples obtained from culture media treated with GA concentrations >0.023% (data not shown) supports the finding t hat GA does mitigate the toxicity of SWNTs to P. subcapitata 3.3.7 Impacts of SWNT-impurities on Algal Growth The effective concentration of SWNTs impacting algal growth was different between GA, SC, and PF-SWNT in this study. Approximately 50% growth inhibitions were observed in culture media with SWNT c oncentration of 0.5, 0.8, and 5 ppm for GA, SC, and PF-SWNT, respectively (Figures 3-5A, 3-6A, and 3-7A). Analysis of oxidative stress showed that GA and SC-SWNTs exhi bited more oxidative stress than PFSWNTs used in this study. This biological response of organisms exposed to SWNTs can also be affected by the load of im purities common to carbon nanotubes (Kagan et al. 2006; Koyama et al. 2009; Shvedova et al. 2003; Smart et al. 2006). For raw SWNTs used in this study, the load of impurities amounted to 13.4 weight % as measured by TGA, and ICP-AES analyses det ected only iron in levels above the instruments detection limits (ca. 2.8 ppm) in SWNT suspensions. Previous research has linked Feimpurities to toxicity due to its role in the formation of free radica ls and ROS (Nel et al. 2006; Shvedova et al. 2003), and the activity of ROS tends to be proportional to the concentration of Fe impurities in SWNT suspension (Kagan et al. 2006). Therefore, higher Fe concentrations were found in GA and SC-SWNTs and they yielded more severe oxidative stress than PF-SWNTs. Mo reover, the continuous exposure of algal cultures to light certainly favors different photochemical reactions, such as the Fenton 72

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reaction. With regard to the experimental resu lts obtained in this study, ROS production induced by Fe-impurities is possible, and if so, the previously reported antioxidant potential of GA (Abd-Allah et al. 2002; Trom mer and Neubert 2005) could play a role in the observed decreasing toxicity trend with in creasing GA concentrations in culture media. 3.4 Concluding Remarks As aquatic systems behave as terminal si nks for most environmental pollutants including waste NMs, aquatic organisms would likely become exposed to these emerging pollutants. This is not only in their pristine forms, but also as surface modified materials due to surfactant coatings as well as to surface transformations induced by the interaction with water chemistry parameter s. In this study, t he biological responses of the freshwater green algae, P. subcatpitata exposed to SWNTs suspended in aqueous surfactant solutions (GA, SC, and PF) of different chemical composition were investigated. GA, SC and PF-SWNT suspended in aqueous solutions resulted in stable suspensions for the duration of the experiments. In growth inhibition experiments, the adverse biological impact was dependent on c oncentrations of SWNTs and both type and amount of surfactant in al gal culture media. Mechanistic studies point to oxidative stress as the major cause of algal growth inhibition. P. subcapitata appeared to stand higher concentration of SWNTs in cultur e media when nanotubes were suspended in PF as compared to either GA or SC. Increasing the concentrations of used su rfactants in culture media reduced the toxic effects of SWNTs. In the case of GA and SC-SWNT suspensions, P. subcapitata showed less oxidative stress in culture medi a with higher concentrations of surfactant. The following mechanisms were responsible for the observed mitigation of SWNT 73

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toxicity: (i) the antioxidant activity of GA neutralizes the formation of ROS and this is supported by the measured levels of GSH which showed an accumulation over time, and (ii) higher concentration of SC would reduce the expos ure of SWNT sidewalls due to biodegradation of surfactant as observed in culture media with low concentration of SC, which resulted in increased oxidative st ress. In the case of PF-SWNTs, increased concentrations of the surf actant provided a more complete coating of nanotubes, preventing physical contacts bet ween the nanotubes and the cells. In terms of the toxicity of SWNTs, the dispersion state, the average diameter, and surface area should be known as they are key factors controlling the negative impacts of the naotubes. A significant finding of this study is the potential for mitigation of the toxic impact of carbon nanotubes. This coul d be beneficial toward biocompatibility research. Non-covalent functionalization using surfactants is simple method, nondestructive for intrinsic structure of nanotubes, and optimizable by simply switching appropriate surfactants for s pecific target organisms. 74

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Figure 3-1. Molecular structures of select ed surfactants (a) basic unit of gum Arabic (GA), (b) sodium cholate (SC), and (c) pluronic acid F108 (PF). Figure 3-2. Length distributions of indi vidual SWNTs suspended in 1 % GA determined by analysis of AFM image (inset). 75

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Figure 3-3. Absorption spectra of freshly prepared SWNTs in gum Arabic (GA-SWNT). The inserted figure shows measurements of absorbance of GA-SWNT at 763 nm for 14 days. Figure 3-4. Absorption spec tra of freshly prepared SWNT s in sodium cholate (SCSWNT) and pluronic acid F108 (PF-SWNT). The inserted figure shows measurements of absorbance of SC-S WNT and PF-SWNT at 763 nm for 14 days. 76

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Figure 3-5. Effect of increasing concent rations of single-walled carbon nanotubes (SWNTs) on the growth of P. subcapitata in a standard 96-hour chronic algal assay. The final concentration of gum Arabic (GA) used as surfactant to suspend SWNTs was adjusted in all cultur e media to a final concentration of 0.023% (a), and 0.046% (b). The horizontal line repres ents the growth of P. subcapitata in control culture media (i.e. plain culture media containing neither GA nor SWNTs). Growth rates obtained from controls were assigned a value of 100% and biological respons es measured in treated samples normalized to the control. (*) indicates a significant difference as compared to controls (<0.05) 77

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Figure 3-6. Effect of increasing concentrations of SWNTs on the growth of P. subcapitata in a standard 96-hour chronic algal assay. The final concentration of sodium cholate (SC) used as surf actant to suspend SWNTs was adjusted in all culture media to a final conc entration of 0.05% (a), and 0.1% (b). Changes in biomass measured as chlorophyll-a are shown as gray bars, and trends of glutathione (GSH) and reactive oxygen species (ROS) are illustrated as black () and open dots ( ), respectively. The dotted horizontal line represents the growth of P. subcapitata GSH, and ROS in control culture media (i.e. plain culture media) and vesse l control contains only SC. Growth rates obtained from controls were assigned a value of 100% and biological responses measured in treated samples normalized to the control. 78

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Figure 3-7. Effect of increasing concentrations of SWNTs on the growth of P. subcapitata in a standard 96-hour chronic algal assay. The final concentration of pluronic acid F108 (PF) used as surfactant to suspend SWNTs was adjusted in all culture media to a final concentration of 0.24% (a), and 0.56% (b). Changes in biomass measured as chlorophylla are shown as gray bars, and GSH and ROS are illustrated as black ( ) and open dots ( ), respectively. The dotted horizontal line represents the growth of P. subcapitata in control culture media (i.e. plain culture media) and vessel control contains only PF. Growth rates obtained from controls were assigned a value of 100% and biological respons es measured in treated samples normalized to the control. 79

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Figure 3-8. Biological response of P. subcapitata to GA-SWNT and SC-SWNT introduction to the culture medium at di fferent algal growth phases. (A and E) SWNTs added prior to inoculating with the algal cells time t0; (b and f) SWNT addition after 24 hours of growth; (C and G) SWNT addition after 96-hours and during the exponential growth phase, and (D and H) SWNT addition after 216 hours of growth (near plateau). The horizontal lines represent the percentage of algal growth in contro l culture media (i .e. culture media containing only surfactants but no SWNTs), which have been assigned values of 100%, and the observed effects in treated samples are normalized to the 80

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control. Vertical dotted lines indicate t he point of SWNT addition to the culture media. 0.040.070.10.17 chlorophylla (ppb) 0 200 400 600 800 1000 SWNT 0.5ppm SWNT 1ppm Concentration of GA (%) 0.040.070.10.17 GSH (ppb) 0 200 400 600 800 1000 (a) (b) Figure 3-9. Effect of increas ing concentrations of GA on P. subcapitata growing in culture media (standard 96hour chronic algal assay) containing fixed SWNT concentrations of 0.5 and 1 ppm. Figur e 9A shows changes in biomass measured as chlorophylla, and Figure 9B illustrates the trends of GSH in culture media. The horizontal line represents the growth of P. subcapitata in control culture media (i.e. culture m edia containing neither GA nor SWNTs). Growth rates obtained from controls were assigned a value of 100% and biological responses measur ed in treated samples normalized to the control. (*) indicates the lack of significant di fferences as compared to controls ( a>0.05) 81

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Figure 3-10. Diagram of the relati onship bet ween carbon nanotubes expos ure and concentration of surfactant. Adopt ed from Angelikopoulos. et al. (Angelikopoulos et al. 2010) 82

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Figure 3-11. Near infrared fluorescence spectra of GA-SWNTs in algal suspensions with GA concentrations of (a) 0.023% with 0.5 mg SWNTs/L, (b) 0.046% with 0.5 mg SWNTs/L, and (c) 0.046% with 1mg SWNTs/L. Spectra were measured on the first (initial) and fourth (final) day of al gal exposure experiments. 83

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Raman Shift (cm-1) 130014001600 Intensity 0 5000 10000 15000 20000 t0 tf 130014001600 Intensity 0 5000 10000 15000 20000 (a) (b) Figure 3-12. Raman spectra of GA-coated SWNTs (0.5 ppm) in algal suspensions measured on the first (t0) and fourth (tf) day of algal exposure experiments. (a) 0.023% GA; (b) 0.046% GA. 84

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Figure 3-13. Near infrared fluorescence spectra of single walled carbon nanotubes suspended in sodium cholate (SC-SWNTs) in algal suspensions with SC concentrations of 0.05% with 0.2, 0. 4, and 0.8 mg SWNTs/L (A, B, and C) and 0.1% with 0.2, 0.4, and 0.8 mg SWNTs/L (D, E, and F). Spectra were measured on the first (initial) and four th (final) day of algal exposure experiments. 85

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Figure 3-14. Raman spectra of single wa lled carbon nanotubes suspended in sodium cholate (SC-SWNTs) in algal suspensio ns with SC concentrations of 0.05% with 0.2, 0.4, and 0.8 mg SW NTs/L (A, B, and C) and 0.1% with 0.2, 0.4, and 0.8 mg SWNTs/L (D, E, and F). Spectra were measured on the first (initial) and fourth (final) day of algal exposure experiments. 86

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Figure 3-15. Near infrared fluorescence spectra of PF-SWNTs in algal suspensions with PF concentrations of 0.24% with 3, 5, and 7 mg SWNTs/L (A, B, and C) and 0.56% with 3, 5, and 7 mg SWNTs/L (D, E, and F). Spectra were measured on the first (initial) and fourth (final) day of al gal exposure experiments. 87

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Figure 3-16. Raman spectra of SWNTs suspended with PF in algal suspensions with PF concentrations of 0.24% with 3, 5, and 7 mg SWNTs/L (A, B, and C) and 0.56% with 3, 5, and 7 mg SWNTs/L (D E, and F). Inserted figures shows radial breathing mode (RBM) and aggregation peak at 267 cm-1. Spectra were measured on the first (initial) and f ourth (final) day of algal exposure experiments 88

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Figure 3-17. TEM images of P. subcapitata from a control sample (a) and from a culture medium containing final concentrations of 0.5 ppm and 0.023% (v/v) for SWNTs and GA (b). Table 3-1. Final surfactant conc entrations in algal cultur e media Final surfactant concentration in culture media (%, v/v) Low High GA 0.023 0.048 SC 0.050 0.100 PF 0.240 0.560 Table 3-2. Detected metal impurities in SWNT suspensions Suspens ion types Metal concentrations (ppm) Fe Cu Mg GA-SWNT 2.8 BDL* BDL SC-SWNT 3.6 0.2 0.2 PF-SWNT 1.2 0.2 0.4 *BDL=Below detection limit. 89

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CHAPTER 4 ROLE OF DISSOLVED ORGANIC MATTER IN THE PARTITIONING OF SINGLE WALLED CARBON NANOTUBES BETWEEN AQUEOUS AND ORGANIC PHASES: IMPLICATIONS FOR BIOACCUMULATION 4.1 Introductory Remarks The ongoing mass production and antic ipated widespread use of carbon nanotubes (CNT) in various industrial activiti es, including biomedical and environmental fields, are raising concerns about the potential risks for both the environment and human health (US-EPA 2007). The toxicity of CNTs has been studied quite extensively in the last few years and several of these studies have reported adverse biological effects on cells and test model organisms (B laise et al. 2008; Jia et al. 2005; Shvedova et al. 2003). From these pioneering studies, t he physical properties (e.g. size, surface area and speciation) (Kolosnjaj-Tabi et al. 2010; Poland et al. 2008; Simon-Deckers et al. 2008; Tian et al. 2006), the presence of metal impurities (Cheng et al. 2009; Chlopek et al. 2006; Koyama et al. 2009; Murray et al 2009; Porter et al. 2009; Vittorio et al. 2009; Walker et al. 2009) as well as t he degree of dispersion of CNTs in prepared suspensions have been linked to observed adve rse biological impact s (Mutlu et al. 2010; Qu et al. 2009; Wick et al. 2007). The toxic effects of CNTs have been associated mainly with the direct contact between ce ll membranes and the nanotubes and to the interactions of CNT-generated reactive oxygen species (ROS) with cell components (Kang et al. 2007; Rivera and Starr 2010; Shvedova et al. 2003). Under natural conditions, the magnitude of any potential bi ological response to CNT exposure would likely be due not only to the effects of the intrinsic physicochemical properties of the nanotubes, but also to the secondary charac teristics acquired through environmental transformations. 90

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One of the most important properties affecting CNTs ability to interact with microorganisms is its state of dispersion. For industrial applications, the dispersion state of CNTs can be altered via processes such as wall-functionalization, and in natural aquatic systems, interactions with dissolved organic matter (e.g. humic substances) could also play a role (Hyung et al. 2007). To better understand these interactions, the aggregation behavior of CNTs has been modeled based on rather well established principles of colloidal behavior, such as Derajaguin, Landau, Verwey, and Overbeek (DLVO) theory (Holbrook et al. 2010; Lead and Wilkinson 2006; Moore 2006). The comparison to colloids stems primarily fr om the overlapping size range of engineered nanomaterials (at least one dimension bet ween 1 and 100 nm) and colloidal particles (up to 10 m). According to DLVO theory, for two adjacent particles separated by a given distance, either repulsive forces of the electrical d ouble layer (EDL) or attractive forces due to van der Waals (vdW) interacti ons will dominate (Israelachvili 1985). While vdW interactions are fairly universal forces, EDL forces are easily affected by solutions chemistry (Handy et al. 2008; Israelachvili 19 85). For instance, increased ionic strength caused by addition of salts induces compre ssion of the EDL via screening of the surface charges, leading to dominance of attractive forces. This, in turn, brings particles together, which is the beginning of t he aggregation process. Therefore, key environmental factors such as pH, ionic strength, and quantity and quality of dissolved organic matter affect colloidal stability (Cosgrove 2005). One might naturally conclude that the same would occur for CNTs, resulting in the predictions illust rated in Figure 4-1. The effect of different electrolytes on the stability of SWNT suspensions prepared by the strong acid oxidization process has been studied in (Sano et al. 2001). As the 91

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ionic strength of the solution increased, a disturbance of the suspensions occurred, resulting in CNT aggregation. This trend was observed in studies using SWNT (Saleh et al. 2010) and MWNT (Lin et al. 2009b; Smith et al. 2009) suspensions prepared by use of extensive sonication followed by centrif ugation. In addition to ionic strength, pH has also been found to influence the dispersion state of CNTs, in that the decrease of pH leads to aggregation of CNTs (Lin et al. 2009b; Shieh et al. 2007; Smith et al. 2009; Zhao et al. 2002). Besides pH and ionic strength, NOM is another factor affecting aquatic stability of CNTs. Presence of NOM is known to fac ilitate both the transport and sorption of different types of contaminants (Chiou et al. 1986; Yamamoto et al. 2003). Recent studies showed that NOM could lead to a ra ther uniform functionalization of CNTs, (Domingos et al. 2009; Hyung et al. 2007), wit h implications on the fate of CNTs in aquatic systems. The chemical composition of NOM is highly complex and variable owing to its diverse source s, and therefore, the interactions between CNTs and NOM are simulated by use of simple surrogates such as fulvic acid (FA) (Yang and Xing 2009), tannic acid (TA) (Lin et al. 2009b), and humic acid (HA) (Saleh et al. 2010) isolates. The adsorption of NOM onto CNTs is believed to be the result of interactions or stackings of aromatic fractions of NOM on gr aphene surfaces of CNTs (Hyung and Kim 2008; Lin and Xing 2008). Obviously, the magnitude and stability of CNT-NOM complexes are dependent on the chemical composit ion of NOM, as well as the physicochemical parameters of natur al waters. Due to the above mentioned complexity and uncertainty (e.g. structure of NOM), understanding the stability of CNTs suspended in NOM-rich solutions remains a challenge. 92

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CNTs can enter aquatic systems through ei ther intentional or unintentional discharges. Although, the behavior of CNTs in natural aquatic systems is still poorly understood, one can hypothesize based on current knowledge that CNTs stabilized by NOM in aquatic systems are more susceptible to enter the food chain. Aggregated and subsequently sedimented CNTs may also re-enter the water column through bioturbation, or they may be consumed by bottom dwellers. For instance, Daphnia magna a low level consumer, was capable of accumulating and transferring SWNTs via absorption onto its carapace (L am et al. 2007), or consumption of lipid-coated SWNTs (Roberts et al. 2007). However, this as pect of SWNT research has been hampered mainly because of a lack of adequate analytic al methods for detecting and quantifying CNTs in complex biological systems. Therefor e, there is a need to establish reliable, and ideally simple, procedures for estimating the bioaccumulation potential of CNTs. One common metric is the octanol-water coefficient (Kow), which has been used widely to predict the distribution of or ganic contaminants between aqueous and organic phases and to estimate the bioaccumulation potentials (Mackay and Fraser 2000; Neely et al. 1974). The principle of Kow mimics the interaction bet ween xenobiotics and fatty tissues of cell membranes having chemical si milarities with octanol. Previous studies have shown that experimentally determined values of Kow were positively correlated with actual values of bioconcentration for a diverse range of hydrophobic compounds (Neely et al. 1974) and several regression equations have been developed since the 1970s as predictive tools based on Kow values (Mackay 1982). In fact, regulatory programs such as the EUs registration, evaluation, and authorization of chemicals or REACH for short, have used the above approach to identif y the bioaccumulation potential through 93

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bioconcentration factor predictions (E uropean-Commission 2007). In the US, the EPA includes Kow as one of the most important chemical properties that needs to be determined for emerging MNs (US-EPA 2007). Nevertheless, Kow remains a rather primitive tool, as research on fate and potential bioaccumulation of SWNTs in natural wa ters is still in its infancy. SWNTs, once exposed to natural aquatic systems, will undergo physicochemical alterations controlled by water chemistry parameters. In such ca ses, the study of oc tanol water partitioning behavior of SWNTs suspended by typical disper sion method (e.g. synthetic surfactants) may not be the appropriate scenario because SWNTs in natural aquatic systems could be significantly modified before contacting aquatic organisms. Therefore, one may only adequately assess the potential of SWNT bi oaccumulation by use of experimental conditions that closely mi mic natural conditions. In this chapter, SWNTs suspensions we re prepared using natural and synthetic waters to help gain insight into how water chemistry controls the fate of SWNTs. In addition, the octanol-water phase distributi on percentages of SWNT were determined using waters with different chemical co mpositions to assess the impact on the bioaccumulation potential of SWNTs. 4.2 Materials and Methods 4.2.1 Aqueous Organic-rich Solutions Used to Prepare SWNT Suspensions In order to simulate the interaction between SWNTs and NOM, natural water and synthetic solutions spiked with commercially available organic compounds were used. NOM is a mixture of various compounds with diverse molecular sizes and chemical compositions (Leenheer and Cr oue 2003). Due to the complex nature of NOM, the use of simple and well characterized surrogates for NOM has provided an understanding of 94

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the interaction between hydrophobic pollutant s and NOM (Chin et al. 1997; Chiou et al. 1987; Yamamoto et al. 2003). Natural water samples used in this study were collected from the upper portion of the Suwannee River (SR) watershed (Fig. 2). Water samples were collected in prewashed polyethylene containers after rinsing with site water. Collected samples were placed in a cooler packed with ice and transported back to the laboratory. In the laboratory, the samples were immediately filtered (0.45 m) and analyzed for major ion concentrations by ion chromatography and pH. Organic-rich waters containing various well-characterized species of dissolved organic matter were prepared by use of comm ercially available HA, FA, and TA. HA was purchased from Sigma-Aldric h, FA from international humic substances society, and TA from Alfa Aesar. Organic-rich aqueous solutions were prepared by dissolving a pre-weighed amount of any one of t he above listed compounds into Nanopure water, followed by filtration (0.45 m). The concentration of dissolved organic carbon (DOC) in filtered natural water and l aboratory prepared solutions were determined by a Tekmar Dohrmann-Apollo-9000. For the experiments conducted in this study, solutions with DOC concentration gradients were prepared from mo ther solutions, and the effe ct of the DOC gradient on solutions pH was corrected by use of either 0.1M NaOH or 0.1M HCl. 4.2.2 Preparation and Character ization of SWNT Suspensions SWNT Suspensions used in this study were prepared using three different dispersing agents. First, SWNT suspensions were prepared by suspending approximately 10 mg of raw SWNTs (Rice HPR 145.1) into 100mL of DI-water 95

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containing either SR, HA, TA, or FA. T hese mixtures were homogenized using a highshear IKA T-25 Ultra-Turrax mixer for about 1.5h followed by ultrasonication using a Misonix S3000 for 10 min. T he mixtures were then ultr a-centrifuged at 14,000 rpm (Beckman Coulter Optima L-80 K) for 4h, af ter which, the supernatant was carefully separated from the aggregated SWNTs at the bottom of the centrifuge tube, and then filtered through a 20 m membrane. The obtained suspensions were characterized using Bio-Raman with excitation from a 785 nm diode laser while visible absorbance spectra and fluorescence were recorded with a Nano-Fluorescence Nanospectralyzer (Applied NanoFluorescence, Houston, TX). Second, SWNT suspensions were prepared using conventional surfactants (i.e. sodium cholate (SC), gum Arabic (GA), pluronic acid F108 (PF), and sodium dodecyl sulfate (S DS). Preparation and characterization methods have been published elsewhere (Y oun et al. 2011). Third, organic matter generated by a primary producer, Psuedokirchneriella subcatpiata was harvested and used to prepare SWNT suspensions. A culture of P. subcapitata was cultivated as described in the US-EPAs P. subcapitata 96-h growth inhibition method 1003.0. After 7 days from the time of the inoc ulation of the culture media with seed algal cells, 1L of algal culture media was c entrifuged at 1200 RPM for 10 min and then the supernatant was discarded. The pellet was next resuspended in Nanopure water and centrifuged as described above. This washing process was repeated twice. The pellet was then resuspended in 500 mL of Nanopure water and s onicated within an ice bath with a Cell disuptor W-375 (Heat Systems-Ulatrasonics INC, Farmingdale, NY.). The obtained mixture was analyzed for organic carbon content and used to prepare SWNT suspensions as described by Youn et al (Youn et al. 2011). 96

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4.2.3 Octanol-water Part itioning Experiments Experiments on phase distribution of SWNT s between 1-octanol and water were conducted using a modified shake-flask me thod. Briefly, for each of the SWNT suspension types, 5 mL were combined with an equal volume of 1-octanol in a 20 mL scintillation vial and then mixed at 300 RPM for 24h using a New Brunswick G24 horizontal shaker (Edison, NJ). The mixtur es were next stabilized at room temperature until complete separation of the two phases wa s achieved. Aliquots of water and octanol phases were removed and SWNT concentrations in each phase determined by absorbance measurements at 763 nm using a Nano-Fluorescence Nanospectralyzer. 4.2.4 Algal Growth in NOM-S WNT Containing Culture Media The 96hr P. subcapitata chronic toxicity assay was used (US-EPA method 1003.0). Briefly, 50 mL of t he preliminary algal assay pr ocedure (PAAP) culture medium were transferred into pre-autoclaved 150 mL Erlenmeyer flasks and then the effects of NOM suspended SWNTs were investigated by injecting a pre-determined toxic concentration of SWNTs into the P. subcapitata (e.g. 0.5 ppm) culture medium. Control experiments consisted of reactors containing an equivalent amount of the tested NOM solution, but without SWNT addition. All al gal growth experiments were conducted under continuous illumination and all treat ments were prepared in triplicates. 4.3 Results and Discussion 4.3.1 Characterization of SWNT Suspensions in Aqueous Solutions with Different Types of Organic Matter Table 4-1 presents the physicochemical char acteristics of the various organic rich solutions as well as the concentrations of SWNTs suspended in each of such solutions. For comparison purposes, the concentration of DOC of each organic-rich solution was 97

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adjusted to match the level of DOC naturally occurring in the SR water sample used in this study. The results show that the degr ee of SWNT dispersion is dependent on the type or chemical makeup of organic matter present in the water. Earlier studies on CNTorganic matter interactions showed that the adsorption of hydrophobic organic contaminants onto CNTs was influenced by the physicochemical properties of NOM present in water (Chin et al. 1997; Chiou et al. 1987; Yamamoto et al. 2003). Also, the presence of benzene rings in surfactants used to suspend CNTs produces highly dispersed and stable CNT suspensions (Islam et al. 2003). Overall, a strong correlation is observed between adsorption coefficient and both the molecular weight and the aromaticity of organic compound s present in water. Aromaticity can be determined by specific UV absorbance at 254 nm (SUVA254) of organic-rich solutions divided by the DOC concentration of the solution; and SUVA254 is strongly correlated with the degree of aromaticity as determined by 13C-nuclear magnetic resonance (Weishaar et al. 2003). Since the dispersion of SWNTs in aqueous so lutions is affected by the adsorption efficiency of NOM on the sidewalls of CNT s, the degree of arom aticity of organic compounds present in solutions used in this study can explain the observed differences in CNTs dispersion behaviors. In HA and TA containing solutions, the ability of dissolved organic matter to disperse SWNTs is related to the degree of aromaticity. The aromaticity of used HA (i.e. 0.076 Lmg-1m-1) was found to be the highest, and the amount of SWNTs suspended in HA-containing aqueous solution was also the highest. In contrast, TA suspensions, with a low degree of aromaticity, exhibited to the lowest concentration of suspended SWNTs. This is primarily due to interactions between aromatic moities of organic compounds on CN T surfaces (Hyung and Kim 2008; Lin and 98

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Xing 2008). SR water samples and FA-contai ning solutions showed a similar degree of aromaticity, but the SR water exhibited a higher ability to suspend SWNTs, thus resulting in a higher concentra tion of suspended CNTs. In fact the aromatic content of SR is nearly half that of HA, yet the concentra tion of SWNTs in SR water is close to that determined in HA-containing solutions. It shou ld be noted that SR water sample is obtained directly from the Su wannee River, and therefore, it s molecular characteristics are different from the comme rcially purchased HA, FA, and TA isolates (Malcolm and Maccarthy 1986). In Figure 4-3, the four different NOM-SWNT suspensio n types are compared by use of spectra obtained by sample excitation at = 785nm, and using radial breathing mode and D/G peak ratio mode for characteriza tion. Previous work revealed that the intensity of the 267 cm-1 peak increased as CNTs were aggregated (Heller et al. 2004). Therefore, Raman spectra in this range can be used to ex plore the aggregation state of SWNTs in aqueous suspensions. Variability of the peak intensity shown in the inserted figure implies that different organic com pounds impacted the separation process by different amounts, resulting in different degrees of SWNT aggregation in prepared suspensions. As seen in Table 4-1, the D/ G peak ratio changes with the type of organic matter used as surfactant, with FA being th e least aggregated suspension, followed by TA, SR, and then HA. In addition, the Ram an spectra of the D-peak (at about 1300 cm-1) and G-peak (at about 1600 cm-1) can provide insight into t he structure of carbon atoms on the side walls of SWNTs. The values of the D/G peak ratios obtained in this study are not significantly different when any two tested organic-rich aqueous solutions are compared (Table 4-1). 99

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4.3.2 Impact of NOM Concentrat ions and pH on SWNT Suspension Concentrations of SWNTs suspended in aq ueous solutions containing the different commercially available organic compounds are shown in Figure 4-4. SWNT concentrations varied with the type of or ganic compound and concentration range (50 to 800 mg/L). The obtained SWNT suspensions were stable for at least 7 days as determined by the lack of changes in absorbance measurements over time. The concentrations of SWNTs in HA-containi ng solutions increased with increasing HA concentrations. However, the concentrati ons of SWNTs suspended in TA and FA containing solutions increased only in solu tions with TA and FA concentrations below 200 and 400 ppm, respectively. This increase was then followed by a decrease in SWNT suspension ability (Figure 4-4). The decrease in the concentration of SWNTs suspended in TA and FA containing solutions could be due to decreasing pH. The impact of pH on the stability of CNT sus pensions has been investigated and it was shown that decreased pH leads to the decreased stability due to decreased electrophoretic mobility of CNTs (Lin et al. 2009b; Shieh et al. 2007; Smith et al. 2009; Zhao et al. 2002). In order to investigate the e ffect of pH on the ability of tested waters to suspend SWNTs, organic-rich aqueous solu tions were prepared with the pH of the solutions adjusted to 5 and 4 for TA and FA, re spectively (Figures 4-4B and 4-4C). Each pH is the one measured from TA and FA final concentration of 50 mg/L. The results showed that the increase in SWNT sus pension induced by pH adjustment was not significant and the concentrations of sus pended SWNTs were even reduced in the case of FA-containing water samples. The decrease in SWNT suspension ability at higher dissolved organic compound concentrations could be due to attractive depletion interactions, which is a re-aggregation phenomenon of SWNTs caused by micelle 100

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formations (Blanch et al. 2010). Specifically the pressure from surfactant micelles builds up as surfactant concentration increases and then begins to push SWNTs together, thus inducing aggregation. Overall, it is obvious that SWNTs remai ned in the supernatant after centrifugation, and the degree of aggregation an d non-covalent functionalization of SWNTs obtained with the tested organic compounds could be signi ficantly altered. These findings are particularly relevant in regards to the pot ential toxicity and fate of SWNTs, because these factors have been proposed as toxicity me chanisms in previous studies (Sayes et al. 2006; Wick et al. 2007) 4.3.3 Octanol-water Partiti oning Behaviors of SWNTs Based on the hydrophobic nature of carbon nanotubes, it is expected that CNTs would easily transfer across the boundary between octanol and water phases. For instance, fullerene, another carbon based nanomaterial, exhi bits a high octanol-water partition coefficient that is close to thos e of well known hydrophobic pollutants such as DDT (Jafvert and Kulkarni 2008). Currently, there are only two published papers in peer reviewed literature containing experimental data on CNT par titioning behavior (Petersen et al. 2010; Wang et al. 2008a). This lack of experimental data could be due to analytical difficulties in assessing CNT concen trations in complex organic matrices. In addition, the transfer of CNTs to octanol from a water phase does not occur as easily as one would expect from the trends of other hydrophobic compounds. Experiments conducted in this study using several organic solvents (e.g. toluene, methylene chloride, hexane, and olive oil) failed to yield cl ear extractions of SWNTs when initially suspended into SR water. Other researcher s have claimed a poor partition of SWNTs between aqueous and organic liquid phases (W ang et al. 2008a), yet they reported 101

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octanol-water partitioning coefficient of 105.18; a value that is close to that of the well known hydrophobic substances such as DDT (i.e. 105.75 (Mackay 1982)). This inconsistency raises questions about the accuracy of the reported Kow number. Additionally, multi-walled carbon nanotubes (MWNTs) suspended in water by acid functionalization were unable to transfer to octanol phase (Petersen et al. 2010). 4.3.3.1 SWNT suspensions in synthetic surfactants and algal organic matter and determination of octanol-water partitioning coefficients. As shown in the previous chapter, GA, SC, and PF have proven to yield stable suspensions, and SDS has been widely used as an ideal surfactant to prepare CNT suspensions. In this set of experiments, SWNT suspensions were prepared in GA, SC, PF, SDS, and algal organic matter. The obt ained suspensions were then used in octanol-water partitioning experiments. The results are present ed in Figure 4-5. The results of octanol-water partitioning experiments indicate that traditional compound transfer between two immiscible liquids based methods may not be appropriate for SWNTs suspended in surf actants such as GA, SC, PF, and SDS (Figures 4-5A, 4-5B, 4-5C and 4-5D). In the case of GA-SWNTs, t he majority of SWNTs were trapped in micelles between octanol and water phases (Figure 4-5A). These micelles were extremely robust and were stabl e for at least 4 weeks. The other SWNT suspensions prepared in synthetic surfact ants mixed with octanol phase could not be completely separated between the two phas es, making the determination of the Kow partition coefficient impossible (Figures 4-5B, 4-5C, and 4-5D). Only SWNT suspensions prepared in solution containing algal extracted organic matter showed CNT transfer to the octanol phase. Unfortunately, CNT concentrations in both aqueous and organic phases could not be determined by the analytical technique used in this study 102

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due to high degrees of aggregation. Overall, surfactants tested in this study did not lead to a clear transfer of SWNTs from aqueous solutions to organic phase. It would be reasonable to assume that t he aggregation of CNTs observed in this study suggests that the introduction of surfac tant coated CNTs to natural waters would likely lead to sedimentation and deposition onto surface sedi ments. Therefore, such aggregated CNTs could enter t he food chain through ingesti on by bottom feeders. With regard to algal organic matter, a previous study showed that algal exudates could impact the stability of colloidal suspensio ns (Koukal et al. 2007). Additionally, the presence of biochemical compounds such as polysaccharides in the algal organic matter mixture (Buffle and Leppard 1995), could point to some similarly with GA, and therefore a potential to produce relatively stable SWNT suspensions. However, algal organic matter was not able to successfully suspend SW NTs to the extent of suspension quality obtained with GA (Figure 4-5E). The DOC concentration of algal extract used in this study (i.e. 52 ppm) was cl osed to that of SR water (i.e. 62 ppm), but the measured SUVA254 of 1.92 -4 Lmg-1m-1 was much lower than that of the SR water (0.041 Lmg-1m-1). This is an indication of clear differences in the chemical makeup of compounds present in these aqueous solutions. 4.3.3.2 SWNT suspensions prepared in natural SR water and organic-rich synthetic solutions and use in octa nol-water partitioning experiments In this experiment, migration of SWNT s into the octanol phase was observed for SWNTs initially suspended in both SR water and synthetic solutions (Figure 4-6). Synthetic solutions were prepared in such a way that the final DOC concentrations were similar to that naturally occurring in SR wate r samples. At the end of the experiment, the % of SWNTs transferred from water to oct anol were 38.3, 31.5, 32.1 and 17.2 for SR, 103

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HA, TA and FA-SWNT, respectively (Tabl e 4-2). The concentrations of SWNTs in aqueous phases (i.e. SR, HA, TA, and FA so lutions) were below the instruments analytical detection limit (<0.5 ppm). Unlik e the results obtained from SWNT-surfactant suspensions discussed earlier, SWNTs suspended in organic rich waters appear to be prone to transfer to the oc tanol phase, and therefore, pot entially able to cross cell membrane and bioaccumulate. An additional set of experiments was conducted and in which SWNTs suspended in SR waters were first c entrifuged at different speeds (from 0 to 14,000 RPM for 30 minutes). Second, following t he separation of the supernatant from the pellet, the former was used in phase partitioning experiments. These experiments were designed to investigate the impact of the size of SWNTs on phase-part itioning behavior. The results showed increased SWNT concentrations in octanol phases with increased centrifugation speed (Table 4-2), indicating t hat well-dispersed and small size SWNTs transfer better to the octanol phase than the larger SWNT bundles. 4.3.3.3 Impacts of increasing concentrat ion of organic matter and pH on SWNT distribution between water and octanol In addition to SWNTs suspended in waters containing environmentally relevant concentration of DOC, the octanol-water partitioning behavior of SWNTs with different concentrations of commercially available or ganic matter isolates are shown in Table 43. The transfer of SWNTs occurred in sus pensions containing 50 and 100 ppm of HA and 50 ppm of TA. The transfer of SWNTs using FA containing solutions was observed for FA concentrations starting from 100 mg /L and the amount of transferred nanotubes decreased gradually with increasing FA concentrations. The amount of SWNTs 104

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partitioned to octanol phase seems to be re lated to the concentration of dissolved organic carbon present in the suspension. In general, SWNTs do not transfer to octanol phase when concentrations of OM in aqueous phase are >300 mg/L). As the concentrations of organic matter in pr epared suspensions increased, persistent emulsion was observed between the octanol and water (Figure 4-7A), and the transfer of SWNTs to octanol phase seemed to be hamper ed by bubbles formed at the interface. The impact of adjusting the pH of SWNT suspensions on SWNT transfer between phases can vary markedly as shown in Tabl e 4-4. In comparison with the transfer experiment without pH adjustment as show n in Table 4-3, the trend of TA-SWNT transfer did not change as the concentration of organic carbon increased in the range between 200mg and 800 mg of TA/L. However, enhanced FA-SWNT transfer percentage was observed in the same range when SWNTs were suspended in pH adjusted FA solutions. 4.3.4 Mechanisms of SW NT Phase Partitioning There are 2 notable findings from the abov e octanol-water partitioning studies. First, unlike SWNTs suspended into synthetic surfactants, SWNT suspension in the natural SR water sample and water containing isolates of humic and fulvic acids transfer from aqueous solutions to the octanol phase us ing the simple shake-flask technique. Second, the size and concentration of SW NTs as well as solution pH and organic carbon concentration impact the octanol-w ater distribution behavior of SWNTs. Previous studies found no partitioni ng of CNTs between water and octanol (Petersen et al. 2010; Wang et al. 2008a); resu lts that differ from observations made in this study. This may be due to differences in physical characteristics of carbon nanotubes either inherited in production process or derived from the preparation method 105

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used in each study. For instance, Peterson et al (2010) used MWNTs which in general have bigger diameter than SWNTs and m ade them hydrophilic through strong acidderived functionalization, a process that in troduces defective sites on the sidewalls. Moreover, the ultra-centrifugation proc ess used in this study removes SWNT aggregates, resulting in well dispersed sus pensions. For most nanoparticles including CNTs, the diameter, length, and contact angle of the nanoparticles at the octanol-water interface would influence their behavior. Spec ifically, the partitioning of SWNTs at the interface between octanol and water can be ex plained by changes in free energy of the interface, and the change in energy induced by inserting the CNTs into either phase depends on both the diameter and the length of CNTs. For a SWNT particle of the radius R and the length L placed at the oc tanol-water interface with the contact angle ( ) (Figure 4-7), the ar ea of contact between the SWNT and water is 360 2 RL2 Awater-SWNT (1) The planar area of the octanol-water interface removed by the SWNT is L2Rsin ASWNT by the removed water -oil (2) If we can assume that indivi dual SWNT is small enough so the effect of gravity is negligible and the interface is planar, the energy change required to remove the SWNT from the interface into the octanol phase can be described as (Binks and Lumsdon 2000), w-o w-So-S2RLsin)( 360 2 RL2 E (3) where refers to the interfacial tension between SWNT, octanol and water shown as the subscript S, o and w, respectively. The interfacial tensions are related with the 106

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contact angle through Youngs equation (Binks and Clint 2002) (Figure 4-7A), S-o S-w = o-w cos and the equation 3 can be simplified as, sincos 180 RL2Ewo (4) The required energy for the transfer to the octanol phase is increased when the size of CNTs is bigger (equation 4) and subsequent ly they are stabilized at the interface rather than transferred. The impact of the size of SWNTs on the partitioning behavior was also confirmed with the SR-SWNTs pr epared using a different centrifugation time. As seen in Table 4-2, a higher octanol transfer of SWNTs was observed in the suspension with smaller size SWNTs. In addition to the impact of the physical characteristics of CNTs, the presence and type of surfactants are other key factors influencing CNTs behavior at the oilwater boundary (Binks 2002). The observed emulsion with SWNTs residing at the interface between the immiscible fluids was reported previously (Fi gure 4-8B) (Hobbie et al. 2005; Wang and Hobbie 2003) and the self -assembly of SWNTs on hydrophobic substances has been used to remove SWNTs bundles (Wang et al. 2008c). The emulsion occurs with the SWNTs themselves, but the addition of a surfactant yields more stable and larger volume emulsions (Hobbie et al. 2005). The observed formation of bubbles between octanol and water phases is due to octanol droplets surrounded by organic matter-SWNT complexes. The transfer of SWNTs from aqueous solution to the octanol phase was thus hampered with incr easing organic matter concentration, due primarily to the interfacial trapping phenomenon. Note that when the concentration of organic carbon in solution prepared using is olates obtained from commercial sources was equivalent to DOC concentration naturally occurring in the SR water sample used; 107

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the formation of droplets at t he octanol-water interface was significantly lower (Figure 46). This observation suggests that the c oncentration of dissolved organic matter is a crucial factor in the interfacial trapping of SWNTs. Other key factors influencing the interfacial trapping are the concentrations of SWNT and pH as illustrated by data presented in Table 4-4. Besides the physical characteristics (e .g. diameter and length) and surfactant type, the contact angle of SWNTs which SWNT s make with the octanol-water interface can influence the observed interfacial trapping. As illustrated in Figure 4-8C, the position of a nanotube in the suspension depends on the contact angle ( ) with the oil-water interface. When the contact angle is more than 90, SWNTs are transferred more easily into the organic phase. The contact angle of nanoparticles is determined by their hydrophobicity (Binks 2002) and can be manipulated through nanopar ticles surface modifications (Duan et al. 2004; Shen and Resasco 2009). Dissolved organic compounds can effectively increase the cont act angle of SWNTs at the interface of octanol-water by modifying the sidewalls of SWNTs. As a result, the complex organic matter-SWNT can be transported across th e interfacial boundaries between octanol and water. Although a significant migration of SWNTs into the octanol phase was observed, it was still very difficult to accurate ly measure the partition coefficient (Kow), which requires the concentration of a compound in both phases. In most cases, the SWNTs remaining in the water phase after the mixing process was below detection limit (<0.5 ppm), as determined by UV-vis-NIR spectr oscopy in this study. In addition, the determination of the amount of SWNTs in the octanol phase was hampered by 108

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aggregation. Therefore, it was more convenient to represent the results in terms of transfer percentage, which is based purely upon the fraction of SWNTs leaving the water phase rather than Kow. This allows one to exclude i ssues that may arise from the aggregation of SWNTS trapped at the water-octanol interface. Finally, following the transfer to the octanol phase of SWNTs previously suspended in aqueous solutions containing either HA, FA, or TA or suspende d directly in natural SR water; SWNTs became unstable and gradually aggregated and deposited near the water-octanol interface. This is probably due to the drastic changes of the coverage of the sidewalls of nanotubes by NOMs, and additionally, chan ges of the surrounding environment upon moving from water to octanol. Upon trans fer to the octanol phase, SWNTs would experience enhanced charge screening due to the lower dielectric constant of octanol (e.g. dielectric constants of 1-octanol and water are 10.3 and 80, respectively)(Equation 5). Subsequently, the repulsive force between SWNTs would be diminished. The Coulomb force F is given by 2 2 214 ezz Fr (5) where z is the ionic valency, e is the m agnitude of their electr ical charge (i.e. 1.6 -19C), is the relative permittivity or dielectr ic constant, and r is the distance between two charges. Once nanotubes begin to aggr egate each other, the depos ition process is accelerated in the less dense octanol phase compared to the water phase. 4.3.5 Relationship between SWNT Transf er in Octanol Phase and Algal Growth Inhibition Figure 4-9 shows the 96hr growth response of P. subcapitata in culture media containing fixed SWNTs levels (0.5 ppm), pre-suspended in aqueous solutions containing the above discussed organic matter is olates or in SR water. The 0.5 ppm 109

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was selected as concentration of SWNTs to be tested due to the significant adverse biological impacts it induces based on previous studies (Youn et al. 2011). The horizontal line in Figure 4-8 represents the average algal gr owth rates determined from control cultures. Culture m edia treated with FA-SWNTs showed a slight increase in growth of P. subcapitata compared to the control. A severely negative impact on algal growth was observed with TA suspended SWNTs. The negative biological responses observed in this case are mainly due to TA itself as shown by data obtained from algal growth in vessels containing only TA and no SWNTs (Figure 4-9). In fact, TA has well known anti-algal properties (Pillinger et al. 1994). In contra st, HAand SR-suspended SWNTs showed no toxicity, and significant growth stimulation was observed. The investigation of the mechanisms driving the observed biological responses of algae treated with HAand SR-suspended SWNTs was not attempted due to resource limitation. However, it is known that natur al dissolved organic matter can protect aquatic organisms from heavy metal toxicity (Koukal et al. 2007) and metal impurities do contribute to the toxicity of CNTs (Cheng et al. 2009; Chlopek et al. 2006; Kagan et al. 2006; Shvedova et al. 2003). Mor eover, the adsorption of di ssolved organic matter on the surface of the al gal cells (Campbell et al. 1997) may reduce the probability of direct contact between cell surfaces and SWNTs, hence eliminating the toxicity attributable to cell piercing. It is therefore postulated that some of the tested organic isolates and organic compounds present in SR water coul d provide protection against the adverse effects of SWNTs. In comparison with the octanol-water di stribution of NOM-SWNTs (Table 4-2), a higher algal growth stimulation was observ ed in culture media treated with HAand SR110

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suspended SWNTs. However, these two organic-rich aqueous solutions favored a higher transfer of SWNTs to the octanol ph ase. The octanol-water partition approach determines the tendency for bioaccumulation of target substances by measuring the level of their affinity to octanol. Therefore, a high trans fer of SWNTs from water to octanol implies higher affinity of SWNTs fo r the model organism. With this concept in consideration, the bio-stimulation observed in this study is opposite to the anticipated trend, likely because the higher affinity of nanotubes for the algal cells would lead to more physical contacts between them. Certainl y, several factors are involved in the biological responses observed. There are a few studies reporting positive responses such as growth stimulation of living syst ems caused by nanotubes (Leeuw et al. 2007; Roberts et al. 2007; Templeton et al. 2006; Zhu et al. 2006b). Interestingly, positive responses of target organisms were relat ed with adsorption of nutrients onto the large surface area of nanotubes and then subsequent ingestion (Rober ts et al. 2007; Zhu et al. 2006b). If similar nutrient concentrati on and delivery process occurred in this experiment, then higher affinity of SWNT toward cell surface would benefit the growth of P. subcapitata 4.4 Concluding Remarks In this chapter, SWNTs suspended in either a natural water sample collected from the Suwannee River or synthetic soluti ons containing either HA, FA, or TA were used in lab experiments for the determinati on of the octanol-water partition percentage of SWNTs. The use of organic -rich natural water resulted in well dispersed and stable SWNT suspensions, suggesting that natural ly occurring dissolved organic matter could behave as surfactant for sus pension of SWNTs in aqueous solutions. The ecological implication is that SWNTs could potentially be well disp ersed and well suspended in 111

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organic rich aquatic environments. This woul d then impact the transport and fate of SWNTs, as well as their interaction wit h biological membranes. Moreover, SWNTs suspended in SR water and in different solu tions produced by dissolving natural organic compound isolates (e.g. HA and FA) were easily transferred from aqueous solution to organic phase in experiments using the conv entional shake-flask method. In contrast, SWNTs suspended in synthetic surfactants (GA, SC, and PF) formed large aggregates that accumulated in the water-octanol inte rface. These results suggest that organic matter induced changes of SWNT surface proper ties drives the partition of these CNTs between water and octanol. There is therefore a need for further investigations focusing on characterization of SWNTs and on surfac e property changes induced by natural dissolved organic carbon. Toxicity studies using SWNTs suspended in aqueous solutions containing natural dissolved organic matter show that the chemical makeup of the dispersing agent (the different organic com pounds used in this study) play a critical role in determining the biological responses of P. subcapitata Additionally, the concentration of SWNTs found to be toxic in previous studies did not produce adverse biological impacts on P. subcapitata, except for TA, which is already toxic to P. subcapitata in the absence of SWNTs. Finally, SWNTs coated by HA, FA, and organic matter present in the SR samples stimulat ed algal growth in a significant manner, indicating a potential nutrient delivery mechanism. 112

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Figure 4-1. Potential behavior and fate of carbon nanotubes in natural aquatic systems as a function of key environmental condi tions such as pH, ionic strength, and dissolved natural organic matter. Figure 4-2. Map of the Su wannee River watershed showi ng the sampling location. 113

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Figure 4-3. Raman spectra of single wa lled carbon nanotubes (SWNTs) suspended in Suwannee River water (SR-SWNT), fulvic acid (FA-SWNT), tannic acid (TASWNT), and humic acid (HA-SWNT). The inserted figure shows radial breathing mode (RBM) and aggr egation peak at 267 cm-1. 114

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Figure 4-4. Concentration of single walle d carbon nanotubes (SWNTs) suspended in synthetic organic matter containing wate rs. (a) humic acid (HA)-SWNT (b) tannic acid (TA)-SWNT and (c) fulvic ac id (FA)SWNT. Black and grey bars represent SWNT suspensions prepar ed with as-prepared organic matter solutions and solutions whose pHs were adjusted to the level of organic matter at a concentration of 50 mg/L, which is 5 (i.e. TA) and 4 (i.e. FA), respectively. 115

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Figure 4-5. Octanol-water partitioning of single-walled carbon nanotubes (SWNTs). SWNTs were first suspended in aqueous so lutions containing either gum Arabic (GA), sodium cholate (SC), plur onic acid F108 (PF), sodium dodecyl sulfate (SDS), or algal organic matte r. Upper and lower images were taken before mixing and after 24 hours of stabilization, respectively. (a) GA, (b) SC, (c) PF, (d) SDS, and (e) algal organic matter. 116

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Figure 4-6. Octanol-water partitioning behav ior of natural organic matter suspended single walled carbon nanotubes (NOM-SWNTs ) using the conventional shak e flask method. (a) Suwannee Rive r water suspended SWNT (SR-SWNT) before. (b) SR-SWNT after. (c) H u mic acid suspended SWNT (HA-SWNT) before. (d) HA-SWNT after. Tests were run in triplicates. 117

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Figure 4-7. Schematic diagram of a SWNT at an octanol-water interface showing (a) the various inter facial energies and the contact angle and (b) the shaded area represents the planar area of the octanol-water in terface removed by the SWNT 118

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Figure 4-8. Interfacial behavior of SWNT s between octanol and water phases. (a) Formation of the emulsion between oct anol and water phases at the end of octanol-water partitioning experiment. The concentration of HA in the solution was 200 mg/L. (b) Diagram showing the emulsion of octanol droplets stabilized by HA-SWNTs. Black arrow indicated SWNTs suspended by HA. Adopted from Hobbie et al. (Hobbie et al. 2009) (c) Diagram of SWNT position at a planar octanol-water inte rface with a contact angle less than 90 (left), equal to 90 (center), and greater than 90 (right). Adopted from Binks (2002). 119

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Figure 4-9. Effect of NOM-SWNTs on the growth of P. subcapitata in a standard 96 hour chronic algal assay. The final concentration of SWNTs was 0.5 ppm. The horizontal line repres ents the growth of P. subcapitata in control culture media (i.e. plain culture media containing nei ther NOMs nor SWNTs). Vessel control contains only each NOM but no SWNTs. Growth rates obtained from controls were assigned to a value of 100% and other growth rates from treated samples normalized to the control. All dat a points show significant differences compared to controls. (a<0.05) 120

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Table 4-1. Dissolved organic carbon (DOC) and specific UV absorption (SUVA) of Suwannee River water (SR), humic acid (HA), tannic acid (TA), and fulvic acid (FA) and dispersion quality com parison of aqueous single walled carbon nanotubes (SWNTs) suspensions. Parameters SR HA TA FA DOC (mg/L) 62 58 61 65 SUVA254(Lmg1 m1 ) 0.041 0.076 0.008 0.043 SWNTs concentration (ppm) 8.0 9.7 2.8 4.9 Fluorescence intensity weak weak weak weak Absorbance features significant significant weak significant Raman aggregation rati o* 1.81 2.18 1.36 1.05 D/G peak ratio** 0.048 0.052 0.052 0.059 Stability (1 week)*** st able stable stable stable *Calculation based on intensity from Raman peaks (i.e. ratio 234 cm-1 to 267cm-1) **Calculation based on Raman spectra of D-peak (at about 1300 cm-1) and G-peak (at about 1600 cm-1) ***Monitoring absorbance changes at 763 nm over 1 week. Table 4-2. Octanol-water parti tioning experiment using both OM isolate-SWNT and SRSWNT suspensions and increased centri fugation speed. The percent of SWNTs transferred from aqueous solutions to octanol is calculated based on SWNT concentrations determined from t he octanol phase at the end of the partition experiment. Types of SWNT suspensions in solutions with different types of organic compounds % of SWNTs transferred from aqueous solution to organic phase SR-SWNT 38.3 HA-SWNT 31.5 TA-SWNT 32.1 FA-SWNT 17.2 SR-SWNT :centrifugation speed (RPM) 0 (no centrifugation) 48.5 4000 57.8 8000 62.9 14000 68.0 121

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Table 4-3. Octanol-water distribution of SWNT as a function of dissolved organic concentrations. The percentage of SWNTs transferred from water to octanol is determined based on SWNT concentrations in octanol phase at the end of the experiment. Concentration of organic carbon (mg/L) Percentage (% ) of SWNTs tr ansferred from water to octanol Humic acid Tannic acid Fulvic acid 50 58.7 60.9 *BDL 100 68.0 BDL 95.2 200 BDL BDL 22.3 400 BDL BDL 11.6 800 BDL BDL BDL *BDL = below detection limit Table 4-4. Octanol-water partition of SW NT from aqueous suspensions prepared in pH adjusted to 5 and 4 for TA and FA soluti ons, respectively. Octanol transfer percent is calculated from measured SWNT concentrations in octanol phase at the end of t he experiment. Concentration of organic carbon (mg C/L) Percentage of SWNTs transfe rred to the octanol phase TA FA 100 27.2 65.9 200 BDL 82.3 400 BDL 30.9 800 BDL 38.1 *BDL: below detection limit 122

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CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS Progress in nano-science has resulted in the rapid development of nanotechnology and production of new nanosized materials such as carbon nanotubes (CNTs), which are likely to be mass produced and introduced to the environment. Currently, the impact and fate of CNTs are lar gely unknown. This study has investigated the interaction between single walled carbon nanotubes (SWNTs) and an aquatic organism while assessing the bioaccumulati on potential of SWNTs in aquatic biota. The biological responses of Pseudokirchneriella subcapitata to exposure to SWNTs suspended in gum Arabic (GA), sodium cholate (SC), and pluronic acid F108 (PF) was investigated using typical 96hour algal bioassays and long-term growth studies. Changes in algal biomass and cell morphology associated with specific SWNTtreatments were monitored and the biological response mechanisms were studied through a combination of biochemical and s pectroscopic methods. The main findings can be summarized as follows. Results from short-term bioassays show significant growth inhibition in culture media containing SWNT concentrations of >0 .5 mg, 0.8 mg and 5 mg /L for GA, SC, and PF, respectively. However, the observed to xicity was significantly mitigated when the surfactant concentrations were nearly doubled while SWNT concentrations were maintained constant in culture media. On the other hand, long-term bioassays using toxic combinations of either SW NTs-GA or SWNT-SC show that P. subcapitata would easily recover from an initial growth inhibi tion effect but at di fferent rates depending on the surfactant. These results suggest that t he toxicity of SWNTs can be mitigated, and potentially eliminated at the manufacturing stage. However, these conclusions are 123

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obtained using a single model aquatic organism. The study should be extended to other organisms such as bacteria (Gram negative and positive), invertebra tes (e.g. daphnids), and vertebrates (e.g. fish). Oxidative stress appears to be the primar y mechanism of observed adverse biological impacts based on the detecti on of reactive oxygen species and measurements of glutathione conc entrations in culture media. This is supported by (1) the fact that used SWNTs exhibited high levels of iron impurities, high enough to induce Fenton-like reaction and ROS production; and (2) the lack of physical damages to cells as determined through transmission elec tron microscopy (T EM) observations. The mitigation of SWNTs toxicity is also likely related to the properties of used surfactants. For instance, GA has antioxidative properties as illustrated by GSH accumulation in algal culture media treated with GA only. In the case of SC, the exposure of the SWNT sidewall (which lead s to increased oxidative stress) due to the degradation of SC is reduced wh en there is ample SC in culture media to replace degraded SC. In the case of PF, increased co verage provided by higher concentrations of PF is suggested as a mechanism to help protect against physical contact between SWNTs and P. subcapitata .These findings point to the possibility of using the surfactants to mitigate the toxicity of SWNTs, making them not only ideal dispersing agents but also toxicity inhibitors, provi ded that they do not alter the performance sought from these nanotubes. Dispersion of SWNTs in natural Suwann ee River (SR) water and synthetic solutions of organic matter (OM) isolates (e.g. humic acid (HA), tannic acid (TA), and fulvic acid (FA)) demonstrate that the naturally occurring organic compounds yield 124

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nanotube suspensions which are as stable as those produced wit h synthetic surfactants. Thus, naturally occurring OM could enhanc e SWNT transport and hence, impact the fate of SWNTs beyond what is currently known. SWNTs suspended in SR water and aqueous synthetic solutions of HA, FA and TA are transferred to the organic phase in the traditional s hake-flask separation method. This phenomenon is not observed when SWNTs are dispersed first in synthetic surfactants such as GA or SC. This sugges ts that surface property modifications of SWNTs caused by naturally occurring OM are more favorable for the transfer process than those induced by syn thetic surfactants. From the environmental perspective, changes in SWNT properties produced after they are released into organic-rich wa ter systems may have a greater impact on potential interactions with biological memb ranes. Further investigation is therefore required in order to fully characterize SWNT surface property changes induced by OM. The toxicity of SWNTs is altered when susp ended directly into organic rich waters, while their transfer from the aqueous solution to the octanol phase is increased. This discrepancy calls for further studies with emphasis on bioaccumulation and biomagnifications. The use of other model organisms r epresenting different trophic levels in an aquatic system should be considered. 125

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145 BIOGRAPHICAL SKETCH Se Jin Youn was born in Seoul, Republic of Korea in 1976 as a son of Bern Soo Youn and Chun Ja Yeo. He received his bachel ors degree at Kyung Hee University in environmental science and engineering after which he served in the army of Republic of Korea. He received his masters degree in environmental engineer ing sciences at the University of Florida.