&EPA
United States
Environmental Protection
Agency
Proceedings of the
Interagency Environmental
Nanotechnology Grantees Workshop
NOVEMBER 19 - 21,2008
SHERATON TAMPA RIVERWALK HOTEL
TAMPA, FL
National Center for Environmental Research
Science To Achieve Results (STAR) Research Program
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Interagency Environmental Nanotechnology Grantees Workshop
Table of Contents
Abstract Information 1
Metals, Metal Oxides: Remediation and Exposure
Novel Supported Materials for Targeted Remediation of Chlorinated Compounds 2
JingjingZhan, Tonghua Zheng, Gerhard Piringer, YunfengLu, Gary McPherson,
Vijay John
Synthesis and Application of a New Class of Stabilized Nanoscale Iron Particles for
Rapid Destruction of Chlorinated Hydrocarbons in Soil and Groundwater 3
Dongye (Don) Zhao, Christopher Roberts
Nanoparticle Stability in Natural Waters and Its Implication for Metal Toxicity to
Water Column and Benthic Organisms 4
James Ranville
Metals, Metal Oxides: Fate/Transport
The Effect of Surface Coatings on the Environmental and Microbial Fate of Nano-Iron
and Fe-Oxide Nanoparticles 5
Gregory V. Lowry
Fate and Effects of Nanosized Metal Particles Examined Along a Simulated Terrestrial
Food Chain Using Genomic and Microspectroscopic Techniques 6
Jason Unrine, Olga Tsyusko, Simona Hunyadi, Jonathan Judy, Paul Bertsch, Aaron Wilson
The Bioavailability, Toxicity, and Trophic Transfer of Manufactured ZnO
Nanoparticles: A View From the Bottom 7
Paul M. Bertsch, Brian Jackson, Andrew L. Neal, Phillip Williams, Travis Glenn,
Nadine J. Kabengi, Benjamin Neely, Hongbo Ma, Jason M. Unrine, Pamela J. Morris,
Arthur Grider
Bioavailability and Fates of CdSe and Ti02 Nanoparticles in Eukaryotes and Bacteria 9
Patricia A. Holden, Galen Stucky, Jay L. Nadeau
Metals, Metal Oxides: Toxicity
Microbial Impacts of Engineered Nanoparticles 10
ShailyMahendra, Delina Y. Lyon, Dong Li, Mark Wiesner, Pedro J.J. Alvarez
Biochemical, Molecular, and Cellular Responses of Zebrafish Exposed
to Metallic Nanoparticles 11
David S. Barber, Nancy Denslow, Kevin Powers, David Evans
Characterization of the Potential Toxicity of Metal Nanoparticles
in Marine Ecosystems Using Oysters 12
Amy H. Ringwood, Melissa McCarthy, David Carroll, Joel Berry
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Interagency Environmental Nanotechnology Grantees Workshop
Pulmonary and Systemic Inhalation Toxicity of Multi-Walled and
Single-Walled Carbon Nanotubes 13
Jacob McDonald, Leah Mitchell, Scott Burchiel, Randy Vander Wal, Andrew Giggliotti
Acute and Developmental Toxicity of Metal Oxide Nanoparticles in Fish and Frogs 14
Christopher Theodorakis, Elizabeth Carraway, George Cobb
Carbon-Based Sensors and Exposure
Conducting-Polymer Nanowire Immunosensor Arrays for Microbial Pathogens 15
AshokMulchandani, Wilfred Chen, Nosang V. Myung, Marylynn V. Yates
Carbon Nanotubes: Environmental Dispersion States, Transport, Fate, and Bioavailability 16
Walter J. Weber, Qingguo Huang
Cross-Media Environmental Transport, Transformation, and Fate of Manufactured
Carbonaceous Nanomaterials 17
Peter J. Vikesland, Linsey C. Marr, Joerg Jinschek, Laura K. Duncan,
Behnoush Yeganeh, Xiaojun Chang
Transport and Retention of Nanoscale Fullerene Aggregates in Quartz
Sands and Natural Soils 18
Kurt D. Pennell, Joseph B. Hughes, LindaM. Abriola, Yonggang Wang, YusongLi
Photochemical Fate of Manufactured Carbon Nanomaterials in the Aquatic Environment 20
Chad T. Jafvert, Wen-Che Hou
Fate and Transformation of C6o Nanoparticles in Water Treatment Processes 21
Bo Zhang, Min Cho, John D. Fortner, Jaesang Lee, Ching-Hua Huang,
Joseph B. Hughes, Jae-Hong Kim
Carbon-Based Toxicity
Role of Particle Agglomeration in Nanoparticle Toxicity 22
Terry Gordon, Lung Chi Chen, Beverly S. Cohen
Potential Environmental Implications of Manufactured Nanomaterials: Toxicity,
Mobility, and Nanowastes in Aquatic and Soil Systems 23
Jean-Claude Bonzongo, Dmitry Kopelevich, Gabriel Bitton
Structure-Function Relationships in Engineered Nanomaterial Toxicity 25
Vicki Colvin
Aquatic Toxicity of Carbon-Based Nanomaterials at Sediment-Water Interfaces 26
Joseph N. Mwangi, Ning Wang, Christopher G. Lngersoll, Doug K Hardesty,
Eric L. Brunson, Hao Li, Baolin Deng
Aquatic Toxicity of Waste Stream Nanoparticles 27
Terry Gordon, Lung Chi Chen, Isaac Wirgin
Ecotoxicology of Fullerenes (C6o) in Fish 28
Theodore B. Henry, June-Woo Park, Shaun Ard, Fu-Min Menn,
Robert N. Compton, GaryS. Sayler
The Office of Research and Development's National Center for Environmental Research iv
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Interagency Environmental Nanotechnology Grantees Workshop
Effects of Nanomaterials on Human Blood Coagulation 29
Peter L. Perrotta, Perena Gouma
Engineered Nanomaterial Ecological Effects Research Within
ORD's National Health and Environmental Effects Research Laboratory 30
Stephen A. Diamond, Christian Andersen, Amanda Brennan, Robert Burgess,
Kay Ho, Sarah Hoheisel, Mark G. Johnson, DavidR. Mount, Paul Rygiewicz
Innate Immune Response of an Aquatic Vertebrate Model to Manufactured
Nanoparticles Assessed Using Genomic Markers 31
Rebecca Klaper, Jian Chen, Frederick Goetz
Other Nanomaterials: Life Cycle Analysis and Remediation
Nanostructured Membranes for Filtration, Disinfection, and Remediation of
Aqueous and Gaseous Systems 32
Kevin Kit
Comparative Life Cycle Analysis of Nano and Bulk Materials in Photovoltaic
Energy Generation 33
V Fthenakis, S. Gualtero, H.C. Kim
The Life Cycle of Nanomanufacturing Technologies 34
Thomas L. Theis, Hatice Sengul, Allan Fluharty
Evaluating the Impacts of Nanomanufacturing Via
Thermodynamic and Life Cycle Analysis 35
Bhavik Bakshi, L. James Lee
Other Nanomaterials: Exposure
Impact of Physiochemical Properties on Skin Absorption
of Manufactured Nanomaterials 36
Xin-Rui Xia, Nancy A. Monteiro-Riviere, Jim E. Riviere
Safety/Toxicity Assessment of Ceria (A Model Engineered NP) to the Brain 37
Robert A. Yokel, Rebecca L. Florence, Jason Unrine, Michael T. Tseng,
UschiM. Graham, Rukhsana Sultana, D. Allan Butterfield, Peng Wu, Eric A. Grulke
Other Nanomaterials: Sensors and Treatment
Nanotechnology: A Novel Approach To Prevent Biocide Leaching 38
Patricia Fleiden, Benjamin Dawson-Andoh, Laurent Matuana
Other Nanomaterials: Fate/Transport
Internalization and Fate of Individual Manufactured Nanomaterial Within Living Cells 39
Galya Orr, David J. Panther, Kaylyn J. Cassens, Jaclyn L. Phillips,
Barbara J. Tarasevich, Joel G. Pounds
The Office of Research and Development's National Center for Environmental Research v
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Interagency Environmental Nanotechnology Grantees Workshop
Methodology Development for Manufactured Nanomaterial Bioaccumulation Test 40
Yongsheng Chen, Yung Chang, John C. Crittenden, Qiang Hu, C.P. Huang,
Milton Sommerfeld
Agglomeration, Retention, and Transport Behavior of Manufactured
Nanoparticles in Variably Saturated Porous Media 41
Yan Jin, John Xiao
Biological Fate and Electron Microscopy Detection of Nanoparticles
During Wastewater Treatment 42
Paul Westerhoff, Terry Alford, Bruce Rittman
Other Nanomaterials: Toxicity
Genomics-Based Determination of Nanoparticle Toxicity: Structure-Function Analysis 43
Alan T. Bakalinsky, Alex Hadduck, Vihangi Hindagolla, Mark Smith, BinXie, Qilin Li
Biological Activity of Mineral Fibers and Carbon Particulates: Implications for Nanoparticle
Toxicity and the Role of Surface Chemistry 44
Prabir K. Dutta, Amber Nagy, Brian Peebles, W. James Waldman
A Rapid In Vivo System for Determining Toxicity of Manufactured Nanomaterials 45
Robert L. Tanguay, Stacey Harper
Cellular Uptake and Toxicity of Dendritic Nanomaterials:
An Integrated Physicochemical and Toxicogenomics Study 46
Mamadou S. Diallo, William A. Goddard, Jose Luis Riechmann
Nanoparticle Toxicity in Zebrafish 47
Gregory D. Mayer, Jay L. Nadeau, Anja Nohe, V. Smorodin
Zinc Oxide Nanoparticles: It's the Contact That Kills 48
John M. Veranth, N. Shane Cutler, Philip J. Moos
Mass-Mobility Relationships for Silica Nanoparticle Agglomerates:
Implications for Transport and Morphological Properties 49
Jacob H. Scheckman, Jaimie Hamilton, Sotiris E. Pratsinis, Peter H. McMurry
Appendices
Agenda
Post-Workshop Participants List
Presentations
Executive Summary
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
Abstracts
The abstracts contained in this document are a combination of abstracts that were taken from the EPA
Web Site and submitted by presenters. The abstracts taken from the EPA Web Site may not reflect the
presentation but only the scope of the project.
Abstracts Unavailable at the Time of Print
Menachem Elimelech
Andrij Holian
Gi Soo Kang
David Pui
Tian Xia
Abstracts Taken From the EPA Web Site
Bhavik Bakshi
Yongsheng Chen
Mamadou Diallo
John Fortner
Terry Gordon
Patricia Heiden
Yan Jin
Rebecca Klaper
Gregory Mayer
Ashok Mulchandani
Elijah Petersen
Robert Tanguay
Chris Theodorakis
Paul Westerhoff
Xin-Rui Xia
Abstracts From the Meeting Last Year
David Barber
Peter Vikesland
The Office of Research and Development's National Center for Environmental Research 1
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Interagency Environmental Nanotechnology Grantees Workshop
11:00 a.m. Wednesday, November 19,2008
Novel Supported Materials for Targeted Remediation
of Chlorinated Compounds
Jingjing Zhan, Tonghua Zheng, Gerhard Piringer, Yunfeng Lu, Gary McPherson, and Vijay John
Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA
Nanoscale zero-valent iron (ZVI) particles are a preferred option for the reductive dehalogenation of
trichloroethylene (TCE). However, it is difficult to transport these particles to the source of contamination due
to aggregation. This study describes a novel approach to the preparation of ZVI nanoparticles that are
efficiently and effectively transported to contaminant sites. The technology developed involves the
encapsulation of ZVI nanoparticles in porous sub-micron silica spheres that are easily functionalized with alkyl
groups. These composite particles have the following characteristics: (1) they are in the optimal size range for
transport through sediments; (2) dissolved TCE adsorbs to the organic groups thereby bringing tremendously
increasing contaminant concentration near the ZVI sites; (3) they are reactive as access to the ZVI particles is
possible; (4) when they reach bulk TCE sites, the alkyl groups extend out to stabilize the particles in the TCE
bulk phase or at the water-TCE interface; and (5) the materials are environmentally benign. This research has
demonstrated these concepts extensively through reactivity studies and column transport, capillary, and
microcapillary transport studies. These iron/silica aerosol particles with controlled surface properties also have
the potential to be applied efficiently for in situ remediation and permeable reactive barriers construction.
In extensions of the work, the researchers have shown that these particles function effectively as reactive
adsorbents for TCE. This work will describe the synthesis of such composite nanoscale materials through an
aerosol-assisted method and through solution methods to illustrate the versatility and ease of materials
synthesis, scale up, and application. The research also will describe the development of carbon submicron
particles that serve as supports for zerovalent iron with optimal transport and reactivity characteristics.
EPA Grant Number: GR832374
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
11:20 a.m. Wednesday, November 19,2008
Synthesis and Application of a New Class of Stabilized Nanoscale Iron Particles
for Rapid Destruction of Chlorinated Hydrocarbons in Soil and Groundwater
Dongye (Don) Zhao1 and Christopher Roberts2
Departments of1 Civil Engineering and2 Chemical Engineering,
Auburn University, Auburn, AL
The overall goal of this research project is to develop an in situ remediation technology using a new class
of stabilized iron-based nanoparticles for the rapid destruction of chlorinated hydrocarbons in soil and
groundwater. The specific objectives are to: (1) synthesize a new class of stabilized iron-based nanoparticles
using low-cost and "green" stabilizers such as starch and cellulose; (2) test the stabilized nanoparticles for
dechlorination of select contaminants (tetrachloro-ethylene, trichloroethylene (TCE), and polychlorinated
biphenyls) in soil and groundwater; and (3) test the feasibility of an in situ remediation process that is based on
the nanoparticles.
Building on the researchers' prior success in synthesizing cellulose-stabilized Fe-Pd nanoparticles of
controlled size, the work in this stage focused on studying transport of the nanoparticles in porous media,
testing the effectiveness of the nanoparticles for degradation of TCE sorbed in soils, and carrying out a pilot
test at a Northern Alabama site to test the deliverability and effectiveness of stabilized Fe-Pd nanoparticles.
Results revealed that the cellulose-stabilized nanoparticles (18.1 ± 2.5 nm) are highly mobile through four
model porous media: coarse glass, fine glass, fine sand, and a loamy sand soil. The transport data can be
interpreted using both classical filtration theory and a modified convection-dispersion equation with a first-
order removal rate law. At full breakthrough, a constant concentration plateau (C/C0) is reached, ranging from
0.99 for the glass beads to 0.69 for the soil. Although Brownian diffusion is the predominant mechanism for
particle removal in all cases, gravitational sedimentation also plays an important role, accounting for 30
percent of the contact efficiency for the coarse glass beads and 6.7 percent for the soil. The attachment
efficiency for CMC-Fe was found to be 1 to 2 orders of magnitude lower than reported for other surface-
modified ZVI nanoparticles. The particle removal and travel distance are strongly dependent on interstitial
flow velocity. Simulation results indicate that once delivered, nearly all nanoparticles are removed by soil
matrix within 16 cm at a groundwater flow rate of 0.1 m/day. For the first time, this work demonstrated that
the stabilized Fe-Pd nanoparticles can in situ effectively degrade TCE in soil pores. When treated with 120 mL
(10 pore volumes) of a stabilized Fe/Pd suspension (Fe = 0.5 g/L, Pd/Fe = 0.1 wt%), greater than 38 percent of
TCE contained in a fine sand column was completely dechlorinated. The investigators also observed that
addition of surfactant may enhance or inhibit the dechlorination by the nanoparticles depending on the content
of leachable soil organic matter. Long-term pilot tests confirmed the superb soil deliverability of the stabilized
nanoparticles under field conditions. Following two consecutive injections of approximately 300 gallons (150
gallons each) of a Fe-Pd suspension (Fe = 0.5 g/L, Pd = 1% of Fe) into a heavily contaminated aquifer, the
levels of PCE and TCE in two monitoring wells were consistently lowered by less than 85 percent for nearly
600 days, and to a lesser extent, PCBS, DCE, and VC concentrations also were lowered. The results also
suggest that the injection of the stabilized nanoparticles induced and enhanced long-term biological
dechlorination of various chlorinated solvents.
EPA Grant Number: GR832373
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
11:40 a.m. Wednesday, November 19,2008
Nanoparticle Stability in Natural Waters and Its Implication for Metal Toxicity
to Water Column and Benthic Organisms
James Ranville
Colorado School of Mines, Golden, CO
The overall goal of this project is to determine the potential ecotoxicological implications of nanoparticles
(NPs), in particular metal-containing quantum dots (QDs). More specifically, this research is investigating the
stability of QDs in surface waters as well as the relative aquatic toxicity of QDs compared to their constituent
metals. The research focuses primarily on CdSe/ZnS QDs because of the high toxicity of Cd and Zn to aquatic
species.
The researchers' approach is to perform acute and chronic toxicity testing of QDs using Daphnia magna.
During these tests, the stability of the QDs was monitored using fluorescence and ICP-MS analysis of 0.02 |im
and 0.003 |im filtrates. Several novel methods for QD detection and characterization also are being examined.
QD uptake and distribution in D. Magna is being investigated by synchrotron XRF using the Brookhaven
NLS.
Toxicity of QD was found to be influenced by QD size and surface coating. Comparison of QD toxicity to
dissolved Cd and Zn found similar levels of toxicity, suggesting there is no significant enhanced toxicity of
NPs over their constituent metals. Surface coating affected the short-term (48 hour) rate of dissolution of the
QDs, with a non-ionic polymer (PEO) coated QD being more stable than an anionic (MUA) polymer-coated
QD. In long-term (3 month) stability tests, both types of QDs were observed to degrade; however, the
differences between surface coatings were still observed.
The significance of the initial results is that although toxicity due to QDs is seen, the level of the effect is
not too dissimilar to what is seen for dissolved metals. This could suggest that risk assessments for dissolved
metals could be applied to metal-containing NPs. Furthermore, under oxic conditions, the QDs appear to
dissolve on the month time scale, suggesting they will not persist in the aquatic environment.
Future work on stability will include examination of aggregation of NPs with natural colloids under
variable water chemistry conditions. Non-lethal toxicity tests (feeding and reproduction) will be performed
with D. magna. Finally, acute and chronic tests on benthic organisms will be conducted.
EPA Grant Number: R833324
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
1:20 p.m. Wednesday, November 19, 2008
The Effect of Surface Coatings on the Environmental and Microbial
Fate of Nano-lron and Fe-Oxide Nanoparticles
Gregory V. Lowry
Center for Environmental Implications of Nanotechnology, Department of Civil and Environmental
Engineering, Carnegie Mellon University, Pittsburgh, PA
Nanomaterials such as zerovalent iron (nZVI) are used for groundwater remediation. Polyelectrolyte
surface coatings are used to inhibit nZVI aggregation and enhance the transport of them in the subsurface. The
polyelectrolyte coating also may affect the interaction of the particles with soil bacteria, and hence their
potential toxicity. This study: (1) measured the rate and extent of desorption of polyelectrolyte coatings used
to stabilize nZVI, including polyaspartate, carboxymethyl cellulose, and polystyrene sulfonate; and (2)
determined the effect of polymer coatings and the oxidation of Fe° on the toxicity of nZVI to Escherichia coli
under either aerobic or anaerobic conditions. Desorption of polyelectrolyte was very slow, with less than 30 wt
percent of each polyelectrolyte desorbed after 4 months. The higher molecular weight polyelectrolyte had a
greater adsorbed mass and a slower desorption rate for PAP and CMC. The nZVI mobility in sand columns
after 8 months of desorption was similar to freshly modified nZVI, and significantly greater than unmodified
nZVI aged for the same time under identical conditions. Based on these results, polyelectrolyte-modified
nanoparticles will remain more mobile than their unmodified counterparts even after aging. This long-term
mobility indicates a potential to reach sensitive receptors in the environment. However, coatings dramatically
decreased the toxicity of nZVI to E. coli. Bare nZVI under anoxic conditions caused a log 3 inactivation of E.
coli cells within 1 hour at 100 mg/L particle concentration. Polymer-coated particles with the same Fe° content
were not toxic. Oxidized particles without Fe° also were not toxic to E. coli, indicating that redox activity
correlated with toxicity. Because the coatings do not readily desorb, the potential for surface-modified nZVI
toxicity will remain as that of coated nZVI, and the oxidation of nZVI in the subsurface by aging or by the
interaction with DNAPL will further decrease the bactericidal effect.
EPA Grant Number: R833326
The Office of Research and Development's National Center for Environmental Research
5
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Interagency Environmental Nanotechnology Grantees Workshop
1:40 p.m. Wednesday, November 19, 2008
Fate and Effects of Nanosized Metal Particles Examined Along a Simulated
Terrestrial Food Chain Using Genomic and Microspectroscopic Techniques
Jason Unrine1, Olga Tsyusko1, Simona Hunyadi1'2, Jonathan Judy1,
Paul Bertsch1, and Aaron Wilson1
1 Department of Plant and Soil Science, University of Kentucky, Lexington, KY;
2Savannah River National Laboratory, Aiken, SC
Risk from exposure to manufactured nanoparticles in terrestrial food webs depends on their propensity for
uptake and retention by detritivorous soil organisms and subsequent trophic transfer to higher trophic levels as
well as inherent particle toxicity. The overall objectives of this research are to: (1) investigate the relative roles
of particle size and chemical composition in a series of nanosized metal particles (specifically Cu, Ag, Au) in
determining soil bioavailability and oral uptake in a model soil detritivore; (2) elucidate mechanisms
governing gastrointestinal uptake, tissue distribution, retention, and trophic transfer of nano-sized Cu, Ag, and
Au along a simulated terrestrial food chain; and (3) investigate interactions among size and chemical
composition of noble metal nanoparticles in determining bioavailability and toxic mode of action. Thus far, we
have demonstrated the size-dependent uptake of Au, Ag and Cu nanoparticles in the earthworm Eisenia fetida
from simulated soils. Bioaccumulation factors differed between exposure to metals as nanoparticles and
equivalent concentrations of metal salts. The particles were absorbed from soil, taken up into internal tissues in
the earthworms, and penetrated cell membranes as demonstrated by laser ablation inductively coupled plasma
mass spectrometry (LA-ICP-MS), bulk ICP-MS analyses, synchrotron-based x-ray microanalysis, and
transmission electron microscopy (TEM). Some evidence of increased mortality and decreased reproductive
success associated with exposure to Au and Ag was observed. The research also examined changes in
expression of genes related to oxidative stress and metal homeostasis. Although no significant differences from
controls in expression of genes related to oxidative stress were observed, there were significant changes in
expression of metallothionein as a result of exposure to Cu and Ag nanoparticles. The next phase of this
research will investigate the kinetics of uptake and elimination of metal nanoparticles in earthworms as well as
trophic transfer of nanomaterials along a simulated food chain consisting of soil, earthworms, and bullfrogs.
EPA Grant Number: R833335
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
2:00 p.m. Wednesday, November 19,2008
The Bioavailability, Toxicity, and Trophic Transfer of Manufactured ZnO
Nanoparticles: A View From the Bottom
Paul M. Bertsch1, Brian Jackson2, Andrew L. Neat, Phillip Williams4, Travis Glenn4, Nadine J. Kabengi1,
Benjamin Neely5, Hongbo Ma4, Jason M. Unrine1, Pamela J. Morris5, and Arthur Grider6
1 Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY;
2 Trace Element Analysis Core Laboratory, Dartmouth College, Hanover, NH;
3Biotechnology and Biological Sciences Research Council, Rothamsted Research, Harpenden,
Hertfordshire, United Kingdom; 4Department of Environmental Health Science, University of Georgia,
Athens, GA; 5Marine Biomedicine and Environmental Sciences Center, Medical University of South
Carolina, Charleston, SC; 6College of Family and Consumer Sciences, University of Georgia, Athens, GA
Decomposers and detritivores are central players relevant to potential ecological risks associated with the
release of manufactured nanomaterials to the environment due to their intimate contact with soil and because
they are at the base of the food chain. Key processes of interest include the role of ecological receptors on the
uptake, transformation, and transfer from one trophic level to the next, as well as the lethal and sub-lethal
toxicity endpoints of metal and metal oxide nanomaterials referenced to the dissolved ionic form of the metal.
The overall objectives of this research are to evaluate: (1) the bioavailability and toxicity of manufactured
nanoparticles (ZnO-np) as a function of particle size to the model bacteria, Burkholderia vietnamiensis PRI301
and the model detritivore Caenorhabditis elegans as referenced against aqueous Zn2+; (2) the ability of
manufactured ZnO-np to be transferred from one trophic level to the next as assessed in the simple food chain
consisting of pre-exposed PR1 and C. elegans; and (3) the synergistic or antagonistic effects of manufactured
ZnO-np on the toxicity of Cu2+ to PR1 and C. elegans. These three overall objectives are being approached in
the context of the following four hypotheses:
Hypothesis 1: The bioavailability and toxicity of manufactured ZnO-np increases with decreasing particle
size (i.e., 2 nm vs. 80 nm).
Hypothesis 2: The toxicity of ZnO-np to PR1 and C. elegans is lower than an equivalent concentration of
dissolved Zn2+.
Hypothesis 3: The bioavailability and toxicity of ZnO-np introduced via trophic transfer differs from direct
exposure.
Hypothesis 4: ZnO-np alter the bioavailability and toxicity of dissolved metals.
The first 2+ years of the project have been focused on the following activities:
(1) Characterization of the ZnO-np under physicochemical conditions representative of the exposure
experiments (Kabengi et al., 2008. Electron beam interaction induces growth transformation in
manufactured ZnO nanoparticles. Microscopy and Microanalysis [in revision]).
(2) Bioavailability and toxicity of ZnO-np to B. vietnamiensis PRI301 and C. elegans as referenced to
dissolved Zn2+, including spatial analysis of Zn in tissues of C. elegans (Unrine et al., 2008.
Bioavailability, trophic transfer, and toxicity of manufactured metal and metal oxide nanoparticles in
terrestrial environments. In: Vicki H. Grassian (ed.). Nanoscience and Nanotechnology. John Wiley and
Sons; Ma et al., 2008. Bioavailability and toxicity of manufactured ZnO nanoparticles in the nematode
Caenorhabditis elegans. Environmental Toxicology and Chemistry [in press]; Neely et al., 2009.
Cytotoxicity of engineered ZnO nanoparticles to Burkholderia vietnamiensis PR130i: comparison to Zn2+
and the effects of counter-ion utilization. Environmental Science and Technology [in review]).
The Office of Research and Development's National Center for Environmental Research 7
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Interagency Environmental Nanotechnology Grantees Workshop
(3) Expanding research based on initial results to include the model earthworm Eisenia fetida and an acetate
utilizer metal sensitive bacteria Cupriavidus Necator (two manuscripts in preparation).
(4) Initiating experiments on the trophic transfer of ZnO-np from pre-exposed bacteria to nematodes.
(5) Examination of Cu2+ toxicity to C. elegans in the presence and absence of ZnO-np.
Characterization studies have revealed that acetate used in the synthesis and stabilization of the 2 nm
ZnO-np inhibits surface reactivity through the passivation of surface sites. The removal of acetate leads to
aggregation of the ZnO-np primary particles but promotes greater surface reactivity. The 80 nm particles,
which are not synthesized in a high acetate background, are far more difficult to stabilize but have greater
surface reactivity. The results of TEM characterization of the 2 nm ZnO-np has revealed that particle growth is
induced in the e-beam due to acetate degradation, leading to anomalous size estimates of primary particles (4-8
nm) compared to dynamic laser light scattering (1-2 nm). Acetate utilization (as a C source) also was
demonstrated in microbial exposure experiments and the loss of acetate resulted in the destabilization/
aggregation/agglomeration of primary particles.
In ZnO-exposure experiments, it has been demonstrated that the EC50 for lethality, behavior, and
reproduction to the nematode model C. elegans was not different from dissolved Zn2+ for the 2 nm ZnO
(s-ZnO-np) particles, whereas there was no observed toxicity for the 80 nm (1-ZnO-np) or 1.2 jim ZnO (bulk-
ZnO) particles. Although no differences in the three toxicity endpoints were observed between dissolved Zn2+
and the s-ZnO-np, there were differences in the spatial distribution of Zn and gene expression
(metallothionien-2) in exposed organisms as elucidated by micro-X-ray fluorescence spectroscopy and
epifluorescence microscopy. Likewise, the growth rate of the bacterial models B. vietnamiensis PRI301 and C.
necator displayed no difference between the s-ZnO-np and Zn2+. However, higher acetate utilization rates were
observed for C. necator in the presence of Zn2+ compared to s-ZnO-np, and there was evidence for greater
membrane damage for the s-ZnO-np exposed bacteria. This suggests greater Zn bioavailability from Zn2+
compared to s-ZnO-np and different toxicity mechanisms. Ongoing work on protein expression in C. necator
has provided evidence for differences in the up- and downregulation of specific proteins between the
s-ZnO-np and the Zn2+ exposed organisms. Identification of key proteins exhibiting differential expression is
underway.
Experiments designed to examine the synergistic/antagonistic effects of s-ZnO-np on metal toxicity have
provided evidence that s-ZnO-np reduce Cu2+ toxicity at a Zn concentration above 100 mg L"1 as compared to
Zn2+. Feeding s-ZnO-np exposed bacteria to nematodes has not provided evidence for significant trophic
transfer of the s-ZnO-np; however, this may be more related to experimental challenges using GFP expression
as the primary assessment endpoint.
The results of these studies suggest that the size of ZnO-np is a critical parameter controlling
bioavailability and observed effects using several ecologically relevant endpoints to decomposers and
detritivores, with smaller particles being more bioavailable along with concomitant observed effects. The
results also indicate that, although the observed effects of ecologically relevant endpoints (growth, behavior,
reproduction) between s-ZnO-np and Zn2+ expressed as a common total Zn concentration are not significant,
there are differences in Zn distributions within organisms (nematodes and earthworms) as well as in gene and
protein expression (nematodes and bacteria). This suggests that there may be differences in the mechanisms of
toxicity between s-ZnO-np and Zn2+.
EPA Grant Number: R832530
The Office of Research and Development's National Center for Environmental Research 8
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Interagency Environmental Nanotechnology Grantees Workshop
2:20 p.m. Wednesday, November 19,2008
Bioavailability and Fates of CdSe and Ti02 Nanoparticles
in Eukaryotes and Bacteria
Patricia A. Holden1, Galen Stucky2, and Jay L. Nadeau
1 Donald Bren School of Environmental Science and Management, University of California at
Santa Barbara, Santa Barbara, CA; 2Department of Chemistry, University of California at
Santa Barbara, Santa Barbara, CA;3 Faculty of Medicine, McGill University, Montreal, Quebec, Canada
Semiconductor nanocrystals differ in important ways from bulk semiconductor materials. Their increased
band gap means that they function as strong oxidizing and/or reducing agents, and their small size allows them
to pass into living cells. Conjugation of biomolecules to the crystal surface can alter any or all of these
properties. In preliminary experiments, we observed that only bioconjugated CdSe quantum dots are taken up
by bacteria and eukaryotic cells. Intracellular fluorescence varies, apparently by electron transfer-mediated
quenching and nanoparticle breakdown. Bare quantum dots are as toxic to growing bacteria in part due to Cd2+,
implying possible extracellular breakdown, but subsequent fates and toxicity relationships are unknown.
Particle size dependencies are implied, but insufficiently understood for use in risk analysis. A systematic
inquiry into size- and chemistry-dependent uptake and fate processes is needed. This research is focused on
quantifying cellular-scale processes that affect nanoparticle entry, stability, and toxicity for a variety of
bacterial and eukaryotic cells. This project is concentrating on two nanoparticles: CdSe whose metals are
toxic, and Ti02 whose toxicity arises solely from its size and electron transfer activity. Both short-term
labeling and longer term growth experiments are being performed to quantify particle entry into cells and
toxicity; also under study is the energy transfer between nanoparticles and energized membranes as a
mechanism. The relative importance of near-cell breakdown, whole-particle electron scavanging, and
intracellular particle reformation as fates are being quantified. This project also is addressing how
nanoparticles and cells may cooperate in transmembrane transport as well as toxicity. This research is focused
on predicting cellular-scale exposure and toxicity for bacteria and eukaryotes in soil and water.
Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured
Nanomaterials: a Joint Research Solicitation—EPA, NSF, NIOSH, NIEHS, EPA-G2006-STAR-F2.
EPA Grant Number: R833323
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
3:20 p.m. Wednesday, November 19,2008
Microbial Impacts of Engineered Nanoparticles
Shaily Mahendra, Delina Y. Lyon, Dong Li, Mark Wiesner, and Pedro J.J. Alvarez
Department of Civil and Environmental Engineering, Rice University, Houston, IX
The rapid growth in production and use of nanomaterials in commercial products has raised concerns
about their beneficial and harmful effects on the environment. An evaluation of potential environmental
impacts needs to consider how they will interact with microorganisms, which are at the foundation of all
known ecosystems and participate in primary production, nutrient cycling, and waste decomposition. They also
serve as good indicators of the potential effects on higher organisms. In this research, representatives of two
classes of nanomaterials, fullerenes, and metal-containing TiC>2, ZnO, and Fe(0), were evaluated for their
effects on bacteria and viruses.
Buckminsterfullerene water suspensions (nC6o) exerted potent antimicrobial activity similar to that of
nano-silver. The antimicrobial activity of nano-sized ZnO, TiC>2, Fe(0), and Si02 was significantly lower.
Multiple samples of nC6o prepared using various methods caused time-dependent and dose-dependent
antibacterial activity towards bacterial pure cultures. However, the effect of nC6o on soil microbial
communities was negligible. Although neither sunlight nor oxygen eliminated the long-term antibacterial
activity of nC6o, its toxicity was increased by smaller particle size in a manner disproportionate to the increase
in surface area to volume ratio. However, toxicity was significantly mitigated by salts, which promoted
coagulation and precipitation. Natural organic matter present in soil effectively sorbed nC6o and reduced its
bioavailability and, consequently, its antibacterial activity. This indicates the need to consider nC6o interactions
with common constituents in environmental matrices to obtain representative results of potential impacts.
Although eukaryotic cell damage by fullerenes has been attributed to reactive oxygen species (ROS), no
evidence of ROS-mediated damage in bacteria killed by nC6o was observed in this study. Instead, flow
cytometry studies with dyes that assess membrane potential and reductase activity suggested that nC6o acts as a
direct oxidant that interferes with energy transduction. Furthermore, the colorimetric methods used to evaluate
ROS production and damage were confounded by interactions between nC6o and the reagents that yield false
positives, revealing a need to re-evaluate previous studies that concluded that toxicity is due to ROS damage.
In contrast, polyhydroxylated fullerene (fullerol) produced ROS through UV photosensitization. Inactivation
of MS2 bacteriophage increased in the presence of fullerol-derived ROS as compared with UV-A illumination
alone. These results suggest a potential for fullerenes to impact microbial populations in both natural and
engineered systems.
In toto, this research identifies the mechanisms of antibacterial activity of nC6o and antiviral mechanisms
of fullerol, and provides a methodology by which the potential environmental impacts of other nanomaterials
can be evaluated.
EPA Grant Number: R832534
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
3:40 p.m. Wednesday, November 19,2008
Biochemical, Molecular, and Cellular Responses of Zebrafish
Exposed to Metallic Nanoparticles
David S. Barber, Nancy Denslow, Kevin Powers, and David Evans
University of Florida, Gainesville, FL
The goals of this project are to: (1) determine if metallic nanoparticles produce toxicity that is distinct
from that of soluble forms of the metal in zebrafish; and (2) determine how physical properties of particles are
related to toxicity. To this end, the behavior of metal particles in aqueous environments have been examined
over time with respect to particle aggregation, surface charge, and dissolution. All particles tested exhibited
aggregation in aqueous suspensions. Mean particle size by volume increased to 20 microns 48 hours after
addition of 50-nm copper nanoparticles to water. Despite their small volume contribution, large numbers of
small particles remained in suspension for the duration of the experiment. Under these conditions, little or no
change in zeta potential occured. Aluminum, nickel, and silver nanoparticles produced little or no lethality in
zebrafish exposed to concentrations up to 10 mg/L for 48 hours. However, exposure to aluminum
nanoparticles produced changes in gill structure and function as well as changes in gene expression. Unlike
these metals, exposure to copper nanoparticles produced lethality in zebrafish within 48 hours. Copper
nanoparticles were less acutely toxic to adult female zebrafish than copper sulfate, with a 48-hour LC50 of 1.5
mg/L for nanocopper versus 0.25 mg/L for copper sulfate. The lethal effects of copper nanoparticle exposure
appeared to be mediated at least in part by the particles and not solely by dissolution. In tanks treated with 1.5
mg/L copper particles, only 0.1 mg/L of dissolved copper was present at 48 hours, which is equivalent to a
concentration of copper sulfate producing 15 percent mortality. This conclusion also was supported by
differences in biochemical and molecular changes following exposure to the two forms of copper. Serum BUN
and ALT levels, gene expression patterns in liver, and liver histopathology showed similar minimal responses
to both forms of copper. Both forms of copper also produced injury to the gill epithelium; however, the
observed gene expression responses were markedly different in gill samples, indicating that the particles
induced a different transcriptome level response than did copper sulfate. The investigators therefore conclude
that copper nanoparticles exert a toxic effect on zebrafish gill that is not solely the result of dissolution of the
particles.
This work is supported by National Science Foundation grant BES-0540920.
The Office of Research and Development's National Center for Environmental Research
11
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Interagency Environmental Nanotechnology Grantees Workshop
4:00 p.m. Wednesday, November 19,2008
Characterization of the Potential Toxicity of Metal Nanoparticles
in Marine Ecosystems Using Oysters
Amy H. Ringwood1, Melissa McCarthy1, David Carrolf, and Joel Berry2
1 University of North Carolina-Charlotte, Charlotte, NC; 2 Center for Nanotechnology and Molecular
Materials, Wake Forest University, Winston-Salem, NC
The fate and effects of nanoparticles on aquatic organisms are important environmental concerns that must
be addressed as the production and uses of nanoparticles continue to increase. The purpose of these ongoing
studies is to characterize the toxicity of various metal nanoparticle preparations on oysters, Crassostrea
virginica, a common estuarine species. As filter-feeders, oysters are a very valuable model species for
characterizing nanoparticle bioavailability and interactions with basic cellular processes. This research project
is designed to address a number of important issues regarding metal nanoparticle toxicity in marine organisms
(e.g., morphological changes of metal nanoparticles in seawater, adverse effects on fundamental cellular
responses related to lysosomal integrity, effects on antioxidants and oxidative damage, the relative sensitivity
of different life history stages, and cellular and tissue accumulation patterns). The results of these studies will
be used to evaluate the following overall hypotheses:
Hi: Metal nanoparticle morphology and size are important determinants of toxicity.
H2: Embryonic and larval stages are more sensitive than adult forms.
H3: Oxidative damage is a common mechanism of cellular toxicity.
The results of recent studies with silver nanoparticles, approximately 15-20 nm seeds in which laboratory
exposure studies were conducted with adult and embryonic oysters, are presented here. The potential for
hepatotoxicity was evaluated using a lysosomal destabilization assay, and lipid peroxidation assays were used
to assess oxidative damage in both gill and hepatopancreas tissues. For the embryo assays, newly fertilized
oyster embryos were exposed to the nanoparticles and the percent normal development after 48 hours was
assessed. These studies were used to address issues such as the relative sensitivity of embryos compared to
adults, tissue distribution, and cellular accumulation and effects. Generally, embryos tended to be slightly less
sensitive than adults, and hepatopancreas tissues were more sensitive than gills. Atomic absorption
spectrometry was used to verify the accumulation of the nanoparticles. Significant relationships were observed
between tissue Ag levels and toxicity as well as with exposure concentrations. These kinds of basic studies are
essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as
well as aquatic organisms.
EPA Grant Number: RD833337
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
4:20 p.m. Wednesday, November 19,2008
Pulmonary and Systemic Inhalation Toxicity of Multi-Walled
and Single-Walled Carbon Nanotubes
Jacob McDonald, Leah Mitchell, Scott Burchiel,
Randy Vander Wal, and Andrew Giggliotti
Lovelace Respiratory Research Institute, Albuquerque, NM
Inhalation of multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNT)
at particle concentrations up to 1 mg/m3 did not result in significant lung inflammation or tissue damage, but
caused systemic immune function alterations. C57BL/6 adult (10-12 week) male mice were exposed by whole-
body inhalation to control air or 0.3 or 1 mg/m3 respirable aggregates of MWCNTs or SWCNTs for 14 days,
with either immediate sacrifice or sacrifice of a recovery group 30 days after the end of exposure.
Histopathology of lungs from exposed animals showed alveolar macrophages containing significant amounts
of black particles; however, there was minimal to no inflammation or tissue damage observed. Bronchial
alveolar lavage fluid also demonstrated particle-laden macrophages; however, white blood cell counts were not
increased compared to controls. Both types of carbon nanotubes caused systemic immunosuppression after 14
days and after recovery. Immunosuppression was characterized by reduced T-cell-dependent antibody
response to sheep erythrocytes as well as T-cell proliferative ability in the presence of the mitogen
Concanavalin A (Con A).
EPA Grant Number: R832527
The Office of Research and Development's National Center for Environmental Research
13
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Interagency Environmental Nanotechnology Grantees Workshop
4:40 p.m. Wednesday, November 19,2008
Acute and Developmental Toxicity of Metal Oxide Nanoparticles
in Fish and Frogs
Christopher Theodorakis1, Elizabeth Carraway2, and George Cobb3
1 Southern Illinois University—Edwardsville, Edwardsville, IL; 2Clemson University,
Clemson, SC; 3 Texas Tech University, Lubbock, IX
The objectives of this research project are to determine the environmental hazard associated with selected
metal oxide nanoparticles (Fe203, ZnO, CuO, and Ti02) in terms of acute and chronic toxicity to fathead
minnows (Pimephase promelas) and the African clawed frog (Xenopus laevis). The hypotheses are that
nanoparticle exposure will affect the survival, growth, development, egg hatchability, and metamorphosis of
these organisms in a dose-dependent fashion, and differences in relative toxicity (LC50, EC50, NOEC, LOEC)
of these nanoparticles coincide with the relative toxicity of their soluble salts or oxides.
Fathead minnows and frogs will be exposed to metal oxide nanoparticles during 96-hour acute toxicity and
developmental toxicity tests. Chronic tests will include 28-day early life stage tests (starting within 24 to post
fertilization) for minnows and 10-week exposures (hatch until metamorphosis completion) for Xenopus.
Endpoints will include survival, growth, percent hatch, developmental abnormalities, and rate of
metamorphosis (for Xenopus). Acute toxicity (growth, survival) endpoints will be reported as LC50s, and
chronic toxicity endpoints will be reported as EC50s, NOECs, and LOECs. Nanoparticles will be kept in
suspension in the water using aeration- or peristaltic pump-induced water currents (i.e., minimizing settling of
nanoparticles). Mixing of aged and fresh nanoparticles in test solutions will be minimized using flow-through
systems. Physiochemical characterization of nanoparticles before and during tests will be carried out by atomic
force and electron microscopic methods. Metal concentrations will be monitored in water and tissues by means
of atomic absorption spectrophotometry. Nanoparticles will be synthesized chemically at Clemson University.
It is expected that the nanoparticles will increase mortality and developmental abnormalities in fish and
frogs, and decrease growth rates, rates of metamorphosis, and hatchability. Calculation of LC50s and EC50s
for acute and developmental toxicity is of benefit because these chemicals have the potential for widespread
release into aquatic environments, either due to large-scale manufacture or use or to applications in
decontamination of ground water and waste streams. However, little, if anything, is known about their
potential hazard in aquatic environments. The LC50s and EC50s would allow ecological risk assessment of
these particles at an early stage in the development of this technology. It should be noted that, even if none of
these nanoparticles show any affect on minnow or frog larvae, this would still be useful information.
EPA Grant Number: R832842
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
8:40 a.m. Thursday, November 20, 2008
Conducting-Polymer Nanowire Immunosensor Arrays for Microbial Pathogens
Ashok Mulchandani, Wilfred Chen, Nosang V. Myung, and Marylynn V. Yates
University of California at Riverside, Riverside, CA
A promising approach for the direct (label-free) electrical detection of biological macromolecules uses
one-dimensional (1-D) nanostructures such as nanowires and nanotubes, configured as field-effect transistors
that change conductance upon binding of charged macromolecules to receptors linked to the device surfaces.
Combined with simple, rapid and label-free detection, these nanosensors also are attractive due to the small
size, low power requirement, and most of all, the possibility of developing high-density arrays for
simultaneous analyses of multiple species. Although current nanosensors based on carbon nanotubes and
silicon nanowires has elucidated the power of 1-D nanostructures as biosensors, they have low throughput and
limited controllability and are unattractive for fabrication of high-density sensor arrays. More importantly,
surface modifications, typically required to incorporate specific antibodies, have to be performed post-
synthesis and post-assembly, limiting our ability to address individually each nanostructured sensing element
with the desired specificity.
The overall objective of this research project is to develop a novel technique for the facile fabrication of
bioreceptor (antibody) -functionalized nanowires that are individually addressable and scalable to high-density
biosensor arrays, and to demonstrate its application for label-free, real-time, rapid, sensitive, and cost-effective
detection of multiple pathogens in water. Electropolymerization of conducting polymers between two contact
electrodes is a versatile method for fabricating nanowire biosensor arrays with the required controllability. The
benign conditions of electropolymerization enable the sequential deposition of conducting-polymer nanowires
with embedded antibodies onto a patterned electrode platform, providing a revolutionary route to create a
"truly" high-density and individually addressable nanowire biosensor arrays. The nanowire immunosensor
arrays utility will be used to simultaneously quantify three important model pathogens, poliovirus, hepatitis A
virus (HAV), and rotarvirus.
The researchers will use their recently reported (Ramanathan et al., 2004) simple yet powerful facile
technique of electrochemical polymerization of biomolecule-friendly conducting polymers, such as
polypyrrole, in prefabricated channels of tailor-made aspect ratio between two contact electrodes at site-
specific positions to synthesize nanowires of tailor-made properties for fabricating individually addressable
high-density nanowire biosensor arrays. Detection of pathogens will be achieved by the extremely sensitive
modulation of the electrical conductance of the nanowires brought about by the change in the electrostatic
charges from binding of the pathogens to the antibodies. Effects of monomer concentration, dopant type and
concentration, aspect ratio, and electrochemical polymerization mode on the sensitivity, selectivity, and
durability of poliovirus, HAV, and rotavirus antibodies-functionalized polypyrrole nanowires as label-free
bioaffinity sensors of these important model viral pathogens in water will be investigated to establish optimum
synthesis conditions of biomolecules-functionalized nanowires to successfully realize our innovation to
practice.
The lack of methods for routine rapid and sensitive detection and quantification of specific pathogens has
limited the amount of information available on their occurrence in drinking water and other environmental
samples. The nanowire biosensor arrays developed in this study would improve the ability to provide rapid and
ultrasensitive quantification of pathogens. The end results of this research will be a nanoelectronic sensor for
rapid, sensitive, selective, and reliable detection of multiple important viruses simultaneously that will be
useful not only for water and environmental monitoring but also homeland security, health care, and food
safety. Additionally, the technique of hierarchical assembly of high-density nanowire arrays developed in this
research also will find application in the rapidly advancing fields of proteomics and genomics.
EPA Grant Number: GR832375
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
9:00 a.m. Thursday, November 20, 2008
Carbon Nanotubes: Environmental Dispersion States,
Transport, Fate, and Bioavailability
Walter J. Weber and Qingguo Huang
University of Michigan, Ann Arbor, MI
The overarching goal of this research project is to evaluate factors that control the environmental
dispersion states, transport, fate, and bioavailability of carbon nanotubes, thereby providing a foundation for
human and ecological risk assessment. Specifically, single-walled and multi-walled 14C-labeled carbon
nanotubes will be synthesized, purified, and characterized using techniques previously established in our
laboratory. These radio-labeled materials will then be used to systematically investigate: (1) the dispersion
states of these nanomaterials under typical environmental conditions, (2) their transport behaviors within and
through a series of different types of soil and sediment media, and (3) their bioavailability to selected critical
aquatic and terrestrial food-chain organisms.
The researchers have developed and refined a means for producing single-walled and multi-walled 14C-
labeled carbon nanotubes by using radioactively labeled methane as a feedstock for the synthesis of carbon
nanotubes via chemical vapor deposition methods. Carbon nanotubes will be mixed with natural organic
matter and subjected to a wide range of aquatic conditions (i.e., pH, ionic strength, etc.) to elucidate their
dispersion state in natural environments. Carbon nanotube transport through a series of soil and sediment
sorbent materials having different geochemical properties will be tested in dynamic column studies, and
relationships among the breakthrough behaviors and the properties of both the nanotubes and the geosorbent
materials will be analyzed. Carbon nanotube bioavailability to a fish, an aquatic worm, and an earthworm will
be tested in lab-scale systems to examine the potentials of these nanomaterials to enter food chains in different
environments, and factors controlling ecological bioavailability will be determined.
The proposed study will: (1) provide fundamental information regarding carbon nanotube dispersion
states, transport, fate, and bioavailability in different environmental systems; (2) identify factors controlling
these environmental behaviors; and (3) establish deterministic models capable of predicting behaviors under
different environmental conditions. This information is critically needed by the U.S. EPA and the research
community for rigorous assessments of the environmental fate, transport, and ecological risks of carbon
nanotubes in various soil/water/sediment systems.
EPA Grant Number: R833321
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
9:40 a.m. Thursday, November 20, 2008
Cross-Media Environmental Transport, Transformation, and Fate
of Manufactured Carbonaceous Nanomaterials
Peter J. Vikesland, Linsey C. Marr, Joerg Jinschek, Laura K. Duncan,
Behnoush Yeganeh, and Xiaojun Chang
Department of Civil and Environmental Engineering, Virginia Polytechnic Institute, Blacksburg, VA
Despite the rapid growth in nanotechnology, very little is known about the unintended health or
environmental effects of manufactured nanomaterials. The results of several recent studies suggest that
manufactured nanomaterials may be toxic. Because experience with naturally occurring nanoscale particles
present in air has shown that they are hazardous to human health and that they can easily travel global-scale
distances in the atmosphere, such scenarios involving engineered nanoparticles must be explored. This
research project seeks to examine carbonaceous nanomaterial fate and transport in the environment. In
particular, the investigators are interested in how these particles behave when transferred from water to air or
vice versa. This presentation focuses on the characterization of aqueous aggregates of C6o fullerene.
The discovery that negatively charged aggregates of C6o are stable in aqueous environments has elicited
concerns regarding the potential environmental and health effects of these aggregates. Although many previous
studies have used aggregates synthesized using intermediate organic solvents, this study employed an
aggregate production method believed to emulate more closely the fate of fullerene on accidental release—
extended mixing in water. The aggregates formed by this method are heterogeneous in size (20 nm and larger)
and shape (angular to round), but are crystalline in structure, exhibiting a face-centered cubic (FCC) habit as
determined by electron diffraction. In addition, particle shape and surface charge changed when C6o was mixed
in the presence of electrolytes (NaCl, CaCl2) or sodium citrate at concentrations from 1 to 100 mM. These
changes in solution composition affect aggregate formation and stability and suggest that C6o fate and transport
will be a function of the composition of the solution.
NSF Award Number: 0537117
The Office of Research and Development's National Center for Environmental Research
17
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Interagency Environmental Nanotechnology Grantees Workshop
10:00 a.m. Thursday, November 20, 2008
Transport and Retention of Nanoscale Fullerene Aggregates
in Quartz Sands and Natural Soils
Kurt D. Pennell1'2, Joseph B. Hughes1, Linda M. Abriola3, Yonggang Wang1, and Yusong Li4
1 School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA;
2Department of Neurology, Center for Neurodegenerative Disease, Emory University, Atlanta, GA;
3Department of Civil and Environmental Engineering, Tufts University, Medford, MA;
4Department of Civil Engineering, University of Nebraska-Lincoln, Lincoln, NE
The goal of this research project is to advance the understanding of nanoscale fullerene (nC6o) aggregate
transport and retention in porous media through a combination of experimental and mathematical modeling
studies. The specific objectives of this research are to: (1) quantify the fate and transport of crystalline nC6o
aggregates in water-saturated soils as a function of soil properties and systems parameters; (2) investigate the
effects of C6o fullerene on soil water retention, water flow, and transport in unsaturated soils; and (3) develop
and evaluate numerical models to describe carbon nanomaterial transport, retention, and release in subsurface
systems.
Stable aqueous suspensions of nC6o aggregates were prepared by dissolving fullerene in tetrahydrofuran
(THF), which was mixed with an equal volume of water, evaporated at 75°C, and sparged with N2 gas. Batch
and column experiments were performed to assess the aggregation and transport behavior of fullerene
nanoparticles in water-saturated quartz sands and natural soils as a function of electrolyte concentration and
species. As the electrolyte concentration was increased from 1 to 100 mM, the change in nCeo particle diameter
was minimal in the presence of NaCl but increased by more than seven-fold in the presence of CaCl2. The
latter effect was attributed to the agglomeration of individual nC6o aggregates, consistent with a net attractive
force between the nanoparticles and suppression of the electrical double layer. At low ionic strength (3.05
mM), nC6o aggregates were readily transported through 40 to 50 mesh Ottawa sand, appearing in the column
effluent after introducing less than 1.5 pore volumes of an nC6o suspension, with approximately 30 percent and
less than 10 percent of injected mass retained in the presence of CaCl2 or NaCl, respectively. At higher ionic
strength (30.05 mM) and in finer Ottawa sand (100-140 mesh), greater than 95 percent of the introduced nC6o
particles were retained in column regardless of the electrolyte species. Approximately 50 percent of the
deposited nC6o particles were recovered from 100 to 140 Ottawa sand after sequential introduction of de-
ionized water adjusted to pH 10 and 12. These results indicate that nC6o transport and retention in water-
saturated quartz sands is strongly dependent on electrolyte conditions, and that release of deposited nC6o
aggregates requires substantial changes in surface charge, consistent with retention in a primary energy
minimum.
Introduction of up to 65 pore volumes of nC6o suspensions containing 1 mM CaCl2 into columns packed
with either Appling soil or Webster soil resulted in 100 percent retention of the injected nCeo mass. Retention
of nC6o aggregates occurred primarily within 6 cm of the column inlet, with solid phase concentrations
approaching 130 jag/g. The addition of Suwannee River humic acid (20 mg/L) to the nC6o suspension resulted
in slightly enhanced nC60 mobility, although effluent breakthrough was not observed. However, when nC60
suspensions were prepared with 1,000 mg/L polyethoxylate (20) sorbitan monooleate (Tween 80), nC6o
aggregates were readily transported through Appling soil, with less than 40 percent of injected mass retained.
These results clearly demonstrate that Appling soil and Webster soil possess a large retention capacity for nC6o
aggregates, but that nC6o transport can be greatly enhanced in the presence of stabilizing agents.
A mathematical model that incorporates nonequilibrium attachment kinetics and a maximum retention
capacity was utilized to simulate experimental nC6o effluent breakthrough curves and deposition profiles as a
function of quartz sand size fraction and flow rate. Fitted maximum retention capacities (Smax) ranged from
0.44 to 13.99 \xg!g, and were found to be correlated with normalized mass flux. The resulting correlation
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
provides a means to estimate Smax as a function of flow velocity, nanoparticle size, and grain size of the porous
medium. Collision efficiency factors, estimated from fitted attachment rate coefficients, were relatively
constant (ca. 0.14) over the range of conditions considered. The fitted attachment rate coefficients, however,
are more than one order of magnitude larger than the theoretical collision efficiency factor computed from the
Derjaguin-Landau-Verwey-Overbeek (DLVO) theory (0.009). Subsequent analyses suggest that neither
physical straining nor attraction to the secondary minimum was responsible for this discrepancy. Patch-wise
surface charge heterogeneity is shown to be the likely contributor to the observed deviations from classical
DLVO theory. These findings indicate that modifications to clean-bed filtration theory and consideration of
surface heterogeneity are necessary to accurately predict nC6o transport behavior in saturated porous media.
EPA Grant Number: R832535
The Office of Research and Development's National Center for Environmental Research
19
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Interagency Environmental Nanotechnology Grantees Workshop
10:40 a.m. Thursday, November 20, 2008
Photochemical Fate of Manufactured Carbon Nanomaterials
in the Aquatic Environment
Chad T. Jafvert and Wen-Che Hou
Division of Environmental and Ecological Engineering, Purdue University, West Lafayette, IN
The photochemical transformation of aqueous C6o clusters (nC6o) in sunlight (West Lafayette, IN, 86° 55'
W, 40° 26' N) and lamp light (X = 300-400 nm) has been investigated. Upon exposure to light, the brown to
yellow color of nC6o was lost gradually and the cluster size decreased as the irradiation time increased. TOC
analysis indicated that nC60 products/intermediates were soluble in the aqueous phase and C60 may have
mineralized or partially mineralized. The rate of C6o loss in sunlight was faster for smaller clusters compared to
larger clusters (i.e., /c,hs = 3.66 x 10~2 h"1 and 1.42 x 10~2 h"1 for Ceo loss from 150-nm and 500-nm nCeo
clusters, corresponding to half-lives of 18.9 h and 40.8 h, respectively, at the same initial C6o concentration).
Dark control samples showed no loss, confirming phototransformation as the underlying degradation process.
The presence of 10 mg/L fulvic acid, changes in pH, and the preparation method of nC6o clusters had
negligible effects on the reaction rate. Deoxygenation resulted in a decreased loss rate, indicating 02 played a
role in the phototransformation mechanism. These findings suggest that release of nCeo into surface waters will
result in photochemical production of currently unknown intermediate compounds.
EPA Grant Number: R833340
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
11:00 a.m. Thursday, November 20, 2008
Fate and Transformation of C6o Nanoparticles in Water Treatment Processes
Bo Zhang1, Min Cho1, John D. Fortner2, Jaesang Lee3, Ching-Hua Huang1,
Joseph B. Hughes1, and Jae-Hong Kim1
1 School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA;
2Department of Chemistry and 3Department of Civil and Environmental Engineering,
Rice University, Houston, IX
The oxidative reactivity of THF derivatives formed during THF/nC6o synthesis was evaluated with indigo
dye as a model compound. The results showed that the formation of previously undetected oxidizing agents
during THF/nC6o synthesis accounted for the degradation of indigo dye by THF/nC6o (THF/nC6o/unwashed),
while THF/nC6o after vigorous washing (THF/nC6o/washed) and nC6o prepared without the use of THF were
not reactive.
y-Butyrolactone (GBL) was detected by GC-MS in the THF/nC6o/unwashed as one of THF derivatives, but
showed no reactivity with indigo dye. An organic peroxide was detected in the THF/nC6o/unwashed by HPLC,
and was reactive with indigo dye. This compound also was found to account for the elevated antibacterial and
bactericidal activities of THF/nC6o/unwashed on Escherichia coli. Analysis by LC/(+ESI)MS and 1H NMR
showed that the detected THF peroxide was tetrahydro-2-(tetrahydrofuran-2-ylperoxy)furan. The formation of
THF peroxide during the preparation of aqueous stable C6o aggregates provides another potential explanation
for the reactivity and oxidative stress mechanisms of the THF/nC6o system reported in the literature, although
it does not exclude the potential reactivity and toxicity of nC6o itself.
EPA Grant Number: R832526
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
11:20 a.m. Thursday, November 20, 2008
Role of Particle Agglomeration in Nanoparticle Toxicity
Terry Gordon, Lung Chi Chen, and Beverly S. Cohen
New York University, Nelson Institute of Environmental Medicine-Tuxedo, Tuxedo, NY
The objective of this study is to determine the biological consequences of nanoparticle agglomeration. The
researchers hypothesize that there will be a difference in the toxicity of fresh (predominantly singlet) versus
aged (predominantly agglomerated) carbon nanoparticles, and in testing this hypothesis will: (1) measure the
agglomeration rate of several types of carbon nanoparticles; (2) identify whether agglomeration is affected by
differing exposure conditions, including humidity and particle charge; and (3) compare the toxicity of singlet
versus agglomerated particles in mice exposed via the inhalation route. A number of investigators have clearly
demonstrated in instillation studies that nanoparticle toxicity is governed, in part, by particle size. The
investigators' preliminary studies have demonstrated that freshly formed nanoparticles produce lung injury and
inflammation in mice and the extent of adverse effects is influenced by genetic host factors. The current study
will expand on these findings and identify whether realistic exposure conditions that lead to carbon
nanoparticle agglomeration alter the pulmonary response in mice. Particle agglomeration of nanoparticles is
known to be influenced by number concentration and other physical factors. Almost all particle agglomeration
data have been derived, however, under static conditions, whereas occupational exposure to nanoparticles
occurs under dynamic conditions. It is critical, therefore, that the influence of agglomeration on nanoparticle
toxicity be examined under dynamic conditions.
To test the hypothesis that there is a difference in the toxicity of fresh (predominantly > singlet =) versus
aged (predominantly agglomerated) nanoparticles, the investigators first will establish the agglomeration of
freshly generated carbon nanoparticles at various distances (i.e., aging times) downstream from particle
generation in a dynamic exposure system. After careful initial characterization of > singlet = and agglomerated
particles, inbred mice will be exposed to nanoparticles (generated in an arc furnace) at various stages of
particle agglomeration and the lungs will be examined for injury and inflammation. To ensure that pulmonary
differences in response are due to particle agglomeration, groups of mice will be exposed to > singlet = or
agglomerated particles at the same time using the same operating conditions and control of humidity and
particle charge. To determine whether initial findings for a single type of particle composition are applicable to
other nanoparticles, the researchers also will generate particles with different amounts of metal content as is
found in carbon nanoparticles generated with metal catalysts.
As determined in preliminary studies, it is expected that nanoparticle toxicity will be influenced by a
variety of exposure conditions, including particle size, number, agglomeration state, charge, and composition.
By careful characterization of particle agglomeration in a dynamic system, the inhalation toxicity data should
provide key information regarding the toxicity of emerging nanoparticle technologies. The data obtained in the
proposed animal studies can readily be used for extrapolation to occupational and ambient settings. In
summary, the results from this project address a number of research needs, including toxicity and exposure
assessment.
EPA Grant Number: R832528
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
11:40 a.m. Thursday, November 20, 2008
Potential Environmental Implications of Manufactured Nanomaterials: Toxicity,
Mobility, and Nanowastes in Aquatic and Soil Systems
Jean-Claude Bonzongo, Dmitry Kopelevich, and Gabriel Bitton
University of Florida, Gainesville, FL
The potential effects of manufactured nanomaterials (MNs) were evaluated by testing the hypothesis that:
"chemical elements used in the production of MNs could lead to environmental dysfunctions due to: (1) the
potential toxicity of these elements and their derivatives; (2) the small size-driven mobility of MNs through
heterogeneous porous media and ultimate contamination of aquifers; (3) their toxicity to microorganisms and
the resulting negative impacts on key environmental microbial-catalyzed reactions; and (4) the large surface
area which would allow MNs to act as carriers/delivers of pollutants adsorbed onto them" To address this
broad hypothesis, three well-established small-scale toxicity tests (i.e., the Ceriodaphnia dubia acute toxicity
test, the Pseudokirchneriella subcapitata chronic toxicity test, and MetPLATE™) were used. In addition,
studies at the system level were conducted using a combination of column and batch experiments to investigate
the transport behavior of MNs in heterogeneous porous media and the interactions of MNs with microbial-
catalyzed oxidation of organic matter in sediments. Finally, in addition to the above experimental work,
molecular dynamics simulations were performed to investigate the potential interactions between NMs and
cellular membrane components. The major findings of this research are briefly summarized as follows.
Carbon- (i.e., C6o, single-walled carbon nanotubes) and metal- (i.e., nanometals including oAg. oCu. oCo.
oNi. oAl and CdSe quantum dots) based nanomaterials were used in different laboratory experiments. All
tested MNs showed some degree of toxicity response to either one or more of the above three microbiotests,
with oCu and oAg being the most toxic. The use of experimental conditions that mimic likely scenarios of
MNs' introduction to aquatic systems showed that toxicity response of test model organisms to MNs under
such conditions would be affected by key water quality parameters such as organic matter content and solution
chemistry. Column studies of SWNTs transport in heterogeneous porous soils showed that soil characteristics
and the chemical composition of MN suspensions affect transport behaviors, and that the latter can be
quantitatively predicted by use of mathematical models such as the convection-dispersion equation. Finally,
the use of sediment slurries spiked with either each type of MNs or pollutant (i.e., mercury) bound to MNs
allowed the assessment of: (1) the impact of MNs on microbially catalyzed oxidation of organic matter; and
(2) the potential for Hg-bound to Si02-Ti02 nanocomposites obtained from flue gas remediation studies to
become available in sedimentary environments as a function of pH. Overall, these findings help shed light on
the potential environmental implications of MNs. However, several questions remain unanswered, as these
short-term laboratory investigations may not be able to predict the environmental fate/transport and
implications of MNs on a long-term basis. On the other hand, the use of prediction modeling tools can help
address the above concern.
These modeling studies were performed using a coarse-grained molecular dynamics (CGMD) model,
which approximates small groups of atoms as a single united atom. So far, our modeling efforts have been
limited to the interactions between carbon-based nanomaterials and cell membranes. The latter are modeled as
lipid bilayers, thereby neglecting other constituents of the membrane such as membrane proteins. This model
for cell membranes is consistent with the experimental indication that interaction of NMs with membrane
lipids plays a dominant role in mechanisms of cytotoxicity. For model carbon-based NM (i.e., C6o and carbon
nanotubes), we observed an extremely small barrier for the permeation of these NM into the hydrophobic
interior of a lipid bilayer. On the other hand, the calculated residence time of these NM within the bilayer
interior is very large, which could possibly lead to destabilizing interactions between NM and the membrane.
To assess possible mechanisms of the membrane disruption by NM, we performed computational studies of
physical properties of a membrane with embedded NMs. This analysis indicates that carbon-based
nanoparticles do not lead to changes in the membrane bending and lipid tilt moduli (i.e., these nanoparticles do
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
not affect the membrane deformations). Another possible effect of NM on a cellular membrane is a change of
the lateral pressure profile within the membrane, which may affect function of mechano-sensitive membrane
proteins. It was observed that relatively small carbon-based nanoparticles do not alter the lateral pressure
profile. This analysis is being extended to larger nanoparticles (nanotubes of larger diameter and longer length)
as well as nanoparticles containing charged and/or hydrophilic groups, which may disrupt the membrane
through interactions with lipid head groups.
EPA Grant Number: R832635
The Office of Research and Development's National Center for Environmental Research
24
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Interagency Environmental Nanotechnology Grantees Workshop
12:00 p.m. Thursday, November 20, 2008
Structure-Function Relationships in Engineered Nanomaterial Toxicity
Vicki Colvin
Rice University, Houston, IX
As nanotechnology develops into a mature industry, the environmental and health effects of its core
materials are of increasing importance. A significant challenge for this area of research is that for every class
of engineered nanoparticle (e.g., nanotubes, metal nanocrystals), there are literally thousands of possible
samples with various sizes, surfaces, and shapes. This huge parameter space cannot be narrowed by focusing
only on commercial materials, as few systems are in commerce at this point. Indeed, most nanotechnology
companies are optimizing and evaluating hundreds of material prototypes for possible commercial use. In such
a climate, all stakeholders benefit from an understanding of how fundamental nanoparticle characteristics (e.g.,
surface chemistry, size, and shape) control their biological effects.
This aim is the overarching objective of this project, which will provide the first structure-function
relationships for nanoparticle toxicology. This information benefits industry in that it will suggest material
modifications that may produce systems with minimal environmental and health impact. It benefits regulators
by not only indicating whether information on one nanoparticle type can be used to predict the properties of a
related material, but also by setting a framework for evaluating newly developed nanoparticle variants. Finally,
a correlation between biological effects and nanoparticle structure will enable the development of chemical
methods to alter more toxic nanomaterial species into less toxic materials upon disposal.
To realize these structure-function relationships requires that we develop new analytical tools as well as
evaluate material datasets with systematic changes in fundamental properties. Our specific objectives are to:
(1) expand the characterization of nanoparticle structure in biological media, and (2) characterize the effects of
nanoparticles on cell function. This data will be used to test the hypothesis that nanoparticle structure (e.g., size
and shape) directly controls cytotoxicity. A secondary hypothesis is that of the four major materials parameters
in engineered nanoparticles (size, shape, composition, and surface), surface will be the most important in
governing cellular effects. These hypotheses will be tested in several major classes of nanoparticles.
This study exploits recent advances in nanochemistry that allow for the production of highly size- and
surface-controlled nanoparticles from a variety of materials. These model systems provide the systematic
variations in nanoparticle "structure" required for structure-function relationships. Our model systems will
include engineered carbon nanoparticles, both C6o and single-walled carbon nanotubes; up to eight distinct
sizes of nanoscale iron oxides; and a wide variety of nanoscale titania with varying surface coatings. All of
these materials have been reported to generate oxygen radicals under some circumstances; thus, we expect to
correlate our "structures" with the acute cellular toxicity in three human cell lines. This overarching objective
is strongly supported by ongoing efforts to expand the characterization of nanoparticle structure directly in
biological media (objective #1). Additionally, structure-function trends are made much more general if they
can be rationalized by some basic mechanism. Thus, objective #2 aims to both characterize nanoparticle-cell
interactions as well as put forward a mechanism to explain any observed acute toxicity.
The introduction of a new class of materials into consumer products will require information about the
potential behavior and risks these systems pose to the environment and people. Risk management will be
improved with the information provided in this grant, particularly in that the investigators will establish
structure-function relationships for several major classes of nanomaterials.
EPA Grant Number: R832536
The Office of Research and Development's National Center for Environmental Research
25
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Interagency Environmental Nanotechnology Grantees Workshop
2:00 p.m. Thursday, November 20, 2008
Aquatic Toxicity of Carbon-Based Nanomaterials at Sediment-Water Interfaces
Joseph N. Mwangi1, Ning Wang3, Christopher G. lngersolf, Doug K. Hardesty3,
Eric L. Branson Hao Li2, and Baolin Deng1
1 Department of Civil and Environmental Engineering and 2Department of
Mechanical and Aerospace Engineering, University of Missouri, Columbia, MO;
2USGS, Columbia Environmental Research Center, Columbia, MO
Carbon nanotubes (CNTs) are relatively insoluble in water and are likely to accumulate in sediments if
released into the aquatic environment. The potential impacts of CNTs released into the environment are largely
unknown. The objective of this study was to evaluate the potential toxicity of commercially available or
modified CNTs to sediment-dwelling invertebrates. Short-term 14-d water-only tests were conducted by
exposing the amphipod (Hyalella azteca), the midge (Chironomus dilutus), the oligochaete (Lumbriculus
variegatus), and rainbow mussels (Villosa ins) to a thin layer of five types of CNT materials with periodic
replacement of water. A 14-d whole sediment toxicity test was conducted by exposing amphipods to CNTs
spiked into silica sand and Florissant soil (99:1 sediment to CNTs ratio on dry weight basis). In the water only
tests, the survival of the invertebrates was significantly reduced in three as-produced CNT and not in two
modified CNT samples relative to the control. The growth of some test organisms also was found significantly
reduced with exposure to CNTs. The survival and growth of the amphipods in whole sediment toxicity tests for
the two types of sediment were significantly reduced relative to the control. Light microscopy photographs and
transmission electron microscopy (TEM) images of surviving organisms at the end of the exposures
demonstrated the presence of CNTs in the gut of the amphipods, midge, and oligochaete. The CNTs appeared
to smother the organisms and may interfere with their ability to feed. Other mechanisms may exist for the
demonstrated toxicity such as by dissolution of toxic metals from the CNTs. Additional whole sediment tests
will be conducted to determine the dose-response relationships of selected nanomaterials spiked into sediment.
EPA Grant Number: RD833316
The Office of Research and Development's National Center for Environmental Research
26
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Interagency Environmental Nanotechnology Grantees Workshop
2:20 p.m. Thursday, November 20, 2008
Aquatic Toxicity of Waste Stream Nanoparticles
Terry Gordon, Lung Chi Chen, and Isaac Wirgin
New York University, New York, NY
The objective of this study is to determine the biological consequences of nanoparticle contamination of
the aquatic environment. The investigators hypothesize that there will be a particle-type dependent difference
in the developmental toxicity of manufactured nanoparticles in aquatic species, and in testing this hypothesis,
we will: (1) measure the differential toxicity of several types of nanoparticles in an estuarine species of fish,
Atlantic tomcod; and (2) identify whether the embryo and larval stages of development of tomcod are
particularly susceptible to carbon nanoparticle versus nanotube toxicity. A number of investigators have
clearly demonstrated that nanoparticle toxicity in the mammalian lung is governed, in part, by particle size.
The investigators' previous studies have demonstrated that freshly formed nanoparticles produce lung injury
and inflammation in mice and the extent of adverse effects is influenced by particle type as well as genetic host
factors. Little research has been published, however, on whether these physico-chemical properties of
nanoparticles influence their toxicity in aquatic species. Thus, while a considerable data base has been
established to understand the influence of physico-chemical properties of nanoparticle toxicity in a gaseous
medium, it will be critical to understand the ability of various nanoparticles to produce toxicity once they have
entered the waste stream and the aquatic environment. In the proposed studies, a group of particle toxicologists
will collaborate with a fish toxicologist to explore the toxicity of a variety of manufactured nanoparticles in an
established fish model of aquatic toxicity.
To test the hypothesis that there is a particle-type dependent difference in the aquatic toxicity of
manufactured nanomaterials, the researchers will expand their preliminary results to examine the aquatic
toxicity of a wide range of nanoparticles. The primary approach is to study the toxicity of particles present in
nanoparticle manufacturers' waste products because they have the greatest opportunity of entering the aquatic
environment. The investigators propose to study nanoparticle toxicity in tomcod fish at sensitive
developmental stages: embryo and larval stages. The proposed endpoints will include: (1) basic toxicity
endpoints (e.g., survival and time to hatching); (2) developmental morphology; (3) behavior (larval activity);
and 4) gene expression changes.
As determined in preliminary studies, we expect that nanoparticle toxicity will be influenced by a variety
of exposure conditions, including particle type (e.g., carbon toner particle vs. fullerene vs. nanotube), particle
concentration, stage of manufacturing process (e.g., raw soot precursor material vs. purified final material vs.
sludge waste product), and the natural composition of the aqueous medium. By careful analysis of the several
endpoints included in the proposed developmental toxicity experiments, this work will provide key
information regarding the toxicity of emerging nanoparticle technologies, and the data obtained in the
proposed aquatic studies can be used readily for extrapolation to ambient environments. In summary, the
results from this project address a number of research needs, including toxicity and exposure assessment.
EPA Grant Number: R833317
The Office of Research and Development's National Center for Environmental Research
27
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Interagency Environmental Nanotechnology Grantees Workshop
2:40 p.m. Thursday, November 20, 2008
Ecotoxicology of Fullerenes (C60) in Fish
TheodoreB. Henry1'2'7, June-Woo Park1, Shaun Ard\ Fu-Min Menn1,
Robert N. Compton3'4, and Gary S. Sayler1'5'6
1 Center for Environmental Biotechnology; 2Department of Forestry, Wildlife and Fisheries; 3Department of
Physics; 4Department of Chemistry; 5Department of Microbiology; 6Department of Ecology and
Evolutionary Biology, The University of Tennessee, Knoxville, TN; 7Ecotoxicology and Stress Biology
Research Center, University of Plymouth, Plymouth, Devon, United Kingdom
Establishing the toxicity of nanoparticles (NPs) is essential to protect human and environmental health and
to guide appropriately the development of nanotechnology. The researchers' investigations involve assessment
of the ecotoxicology of un-derivatized C6o in model fish species and attempt to link particle characteristics to
toxicological effects. Larval zebrafish were exposed to the following treatments: (1) C6o aggregates generated
by stirring and sonication (72 h) of C6o in water (12.5 mg C6o/500 mL water); (2) C6o aggregates generated by
established methods with tetrahydrofuran (THF) vehicle; (3) THF vehicle (i.e., method 2 without C6o added);
and (4) "fish water" control. The Affymetrix zebrafish array was used to assess changes in gene expression
(14,900 gene transcripts), and results indicated that changes in expression were related to decomposition
products of THF rather than to toxicity from C6o- Subsequently, the researchers investigated the interaction of
other contaminants with C6o aggregates and have determined that aggregate characteristics (e.g., size and
charge) can change in the presence of a co-contaminant and that C6o can alter contaminant bioavailability in
zebrafish. A separate objective was to assess dietary toxicity of C6o (500 mg/kg food) in rainbow trout exposed
for 6 weeks. Effects of dietary exposure were evaluated by organ histopathology, measurements of oxidative
stress, and effects on osmoregulation. Results of this exposure indicate minimal toxicity from C6o; however,
assessment of the actual uptake of C6o and distribution among tissues is ongoing.
EPA Grant Number: R833333
The Office of Research and Development's National Center for Environmental Research
28
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Interagency Environmental Nanotechnology Grantees Workshop
3:40 p.m. Thursday, November 20, 2008
Effects of Nanomaterials on Human Blood Coagulation
Peter L. Perrotta1 and Perena Gouma2
1 West Virginia University, Morgantown, WV;
2Stony Brook University, Stony Brook, NY
Common human diseases including myocardial infarction and stroke are caused by abnormalities of blood
coagulation that predispose to thrombosis (clots). These diseases are influenced by environmental factors, but
not all risk factors for clotting disorders are known. Because nanomaterials that enter the workplace or home
could have short- and/or long-term effects on the blood coagulation system, the researchers are studying the
effects of nanosized materials on the blood coagulation system using a variety of techniques. An important part
of these studies involved documenting adequate dispersion of nanoparticles within biological media.
Interestingly, nanoparticle (NP) size can be verified in plasma-containing solutions by dynamic light scattering
(DLS) when the nanoparticles are of uniform size and shape. Using these well-dispersed NP-plasma
suspensions for clotting studies, it appears that NPs have the effect of shortening clotting times in vitro. They
also are capable of altering the ability to generate thrombin, the most physiologically relevant clotting enzyme.
Based on the importance of thrombin in human coagulation, the investigators have explored several sensor
strategies for detecting clotting proteins like thrombin. The investigators recently have begun to study plasma
obtained from rats exposed to ultrafine and nanometer-sized particles through inhalation. Differences in
endogenous thrombin potential (ETP) and fibrinogen levels can be identified between exposed and control
animals. In addition, global proteomic profiling techniques (differential gel electrophoresis, DIGE) and more
targeted multiplexed (Luminex) panels have demonstrated significant alterations in rat proteins involved in the
coagulation and inflammatory systems.
EPA Grant Number: R832843
The Office of Research and Development's National Center for Environmental Research
29
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Interagency Environmental Nanotechnology Grantees Workshop
4:00 p.m. Thursday, November 20, 2008
Engineered Nanomaterial Ecological Effects Research Within
ORD's National Health and Environmental Effects Research Laboratory
Stephen A. Diamond1, Christian Andersen2, Amanda Brennan1, Robert Burgess3,
Kay Ho3, Sarah Hoheisel1, Mark G. Johnson2, David R. Mount1, and Paul Rygiewici?
1 Mid-Continent Ecology Division, U.S. Environmental Protection Agency, Duluth, MN; 2 Western
Ecology Division, EPA, Corvallis, OR; 3Atlantic Ecology Division, EPA, Narragansett, RI
Ecological effects of manufactured nanomaterials are being investigated at three of the EPA's ecological
research laboratories: the Atlantic, Mid-Continent, and Western Ecology Divisions. These efforts are focused
on and guided by EPA's regulatory needs. Accomplishments to date include the review of ecological effects
test guidelines to ascertain their applicability or adequacy for testing nanomaterials. Scientists from the
ecology divisions, along with scientists from seven other countries, reviewed 25 harmonized test guidelines,
five additional test guidelines, and a guidance document on testing difficult substances published by the
Organization for Economic and Cooperative Development (OECD). These efforts and additional test guideline
reviews will be summarized. Initial nanomaterials research has included development of consistent and
repeatable approaches for conducting nano-scale Ti02 toxicity assays in freshwater systems; methods that will
likely be applied to nanoscaled silver and other nanomaterials. Through collaboration with the Army Corp of
Engineers and academic researchers' studies on suspension and toxicity of Ceo fullerenes and effects of carbon
nanotubes on plant vigor are either complete, or nearing completion. The C6o research is notable for its focus
on the relationship of natural organic matter, C6o particle size and stability, as well as the effect of solar
radiation on both processes, and toxicity. Ecology division researchers also are in the planning stages of
studies that will link closely fate processes with toxicity of nanoscaled silver. These results and planned
research will be presented within a framework of EPA's regulatory needs and international collaborations
within the OECD.
EPA Grant Number: R832843
The Office of Research and Development's National Center for Environmental Research
30
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Interagency Environmental Nanotechnology Grantees Workshop
4:20 p.m. Thursday, November 20, 2008
Innate Immune Response of an Aquatic Vertebrate Model to Manufactured
Nanoparticles Assessed Using Genomic Markers
Rebecca Klaper, Jian Chen, and Frederick Goetz
University of Wisconsin-Milwaukee, Milwaukee, WI
The innate immune system is one of the first physiological systems to interact with foreign materials and
therefore will be key to understanding how organisms will be affected by exposure to nanomaterials. Recent
studies have indicated that the innate immune system of fish responds to certain pathogen patterns differently
than that of mammals. Therefore, the response of the mammalian immune system may not necessarily be
representative of the immune reaction of aquatic vertebrates such as fish. Past cellular studies have
concentrated on general cytotoxicity.
The overall objective of this research project is to assess the innate immune reaction of an aquatic model,
the rainbow trout, to manufactured nanomaterials of varying chemistries at levels not inducing cellular
toxicity. This study will create a mechanism with which to test other nanomaterials, provide data to support
ecological risk assessments, and ultimately inform decisions as to which materials will be the safest to
industrialize and use with respect to aquatic environments. Our hypothesis is: nanomaterials of dissimilar
chemical composition will stimulate different patterns of trout macrophage gene expression, and nanomaterials
of similar chemical characteristics (e.g., charge, shape, and functional group) may be grouped with respect to
their bioactivity, expressed as a particular gene response pattern. Specifically, the chemical properties of
nanomaterials will impact the genomic response of the immune system: nanomaterials of dissimilar chemical
composition will stimulate different patterns of macrophage gene expression and the response will be dose-
dependent.
A range of water-soluble C6o and carbon nanotubes with different chemical compositions and surface
chemistries will be synthesized and tested for their effects on trout macrophages. A trout primary macrophage
cell culture system will be used to determine the: (1) dose versus cell viability for each synthesized
nanomaterial type; (2) level of expression (by quantitative PCR) of marker genes associated with
inflammatory, antiviral, and anti-inflammatory responses with respect to nanomaterial dose at levels that have
no deleterious effect on cell viability; and (3) global patterns of gene expression for those materials that cause
significant changes in marker genes using custom trout immune microarrays.
Methods developed here will improve risk assessment by creating a mechanism to test other nanoparticles
prior to commercial release. The goal of this project will be to help identify nanomaterials with the least
negative environmental impact for environmentally conscious manufacturing. Risk managers will use this data
to identify particles for restricted release to limit harm to aquatic species.
EPA Grant Number: R833319
The Office of Research and Development's National Center for Environmental Research
31
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Interagency Environmental Nanotechnology Grantees Workshop
8:40 a.m. Friday, November 21, 2008
Nanostructured Membranes for Filtration, Disinfection, and Remediation
of Aqueous and Gaseous Systems
Kevin Kit
Department of Materials Science and Engineering, The University of Tennessee, Knoxville, TN
Nanofiber filtration media comprised primarily of biopolymer chitosan were produced by electrospinning.
Electrospinning of pure chitosan has proved to be difficult due to limited solubility and high degree of
intermolecular hydrogen bonding. The investigators have been able to form nanometer-sized fibers without
bead defects by electrospinning chitosan blends with synthetic polymers poly(ethylene oxide) and
poly(acrylamide) with up to 95 percent chitosan in blend fibers. The processing window was expanded by
modifying the spinning apparatus to operate at elevated temperatures. Fiber morphology was affected by
polymer molecular weight, blend ratios, polymer concentration, and spinning solution temperature.
The physical (aerosols, polymer beads), chemical (chromium IV), and microbial (Escherichia coli K-25)
filtration efficiencies of the fabricated nanofibrous filter media were characterized. Surface chemistry of these
blend fibers was characterized using X-ray Photoelectron Spectroscopy. Surface properties of blend fibers
showed a strong correlation with the structure and morphology of the fibers. Much higher chromium binding
capacities compared to similar blend ratio chitosan films were observed. Nanofibrous filter media has been
fabricated by electrospinning a layer of chitosan nanofibers onto a non-woven spun bonded polypropylene
fabric. These coated filter media have been tested for their metal binding and antimicrobial properties, and
results showed applicability towards effectively filtering heavy metals and bacteria from waste media. The
filtration performance of these nanofibrous filter media has been tested against latex polystyrene beads, and
aerosol particles and filtration efficiencies of these media were a function of pore size, fiber diameter, and size
of filtrate.
EPA Grant Number: GR832372
The Office of Research and Development's National Center for Environmental Research
32
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Interagency Environmental Nanotechnology Grantees Workshop
9:00 a.m. Friday, November 21, 2008
Comparative Life Cycle Analysis of Nano and Bulk
Materials in Photovoltaic Energy Generation
Vasilis Fthenakis, S. Gualtero, and H. C. Kim
Center for Life Cycle Analysis, Earth and Environmental Engineering Department,
Columbia University, New York, NY
Life-cycle analysis (LCA) is used to assess potential environmental impacts from the rapidly growing
implementation of photovoltaic (PV) systems. Nano materials are investigated for use in photovoltaic and
other energy generation applications. The information derived from the LCA of bulk material-based PV are
extrapolated to the processes used for their nanomaterial equivalents. For each of the life stages of PV (i.e.,
material production, cell/module manufacture, installation, operation/maintenance, recycling, and disposal),
resource utilization, process efficiencies, extra controls/steps, conversion efficiencies, recyclability, and the
environmental fate of the micro and the nonmaterial alternatives will be investigated. This way, data and
relationships will be built that will enable the quantification of the environmental effects of nanomaterials from
existing micromaterial life-cycle inventory data.
EPA Grant Number: R833334
The Office of Research and Development's National Center for Environmental Research
33
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Interagency Environmental Nanotechnology Grantees Workshop
9:20 a.m. Friday, November 21, 2008
The Life Cycle of Nanomanufacturing Technologies
Thomas L. Theis, Hatice Sengul, and Allan Fluharty
Institute for Environmental Science and Technology, University of Illinois at Chicago, Chicago, IL
A significant component of the driving force behind the evolution and acceleration of nanotechnology lies
in the prevalence of diverse manufacturing routes for nanoscale products. All nanoscale products must proceed
through various manufacturing stages to produce a material or device with nanoscale dimensions. This
research project explores manufacturing routes of nanoscale products with special attention focused on those
attributes that are likely to have significant environmental implications.
Nanomanufacturing methods are usually classified into one of two groups: "top-down," which is achieved
by carving or grinding methods (such as lithography, etching, and milling); or "bottom-up" in which matter is
assembled at atomic scale through nucleation and/or growth from liquid, solid or gas precursors by chemical
reactions or physical processes (using techniques such as sol-gel or epitaxy). "Top-down" manufacturing is the
more common approach used today to produce nanoproducts; it is generally believed that such techniques are
more waste-producing than "bottom-up" techniques. In contrast, it is often suggested that "bottom-up"
nanomanufacturing technologies should be the ultimate tools for sustainable manufacturing because they allow
for the customized design of reactions and processes at the molecular level that minimize unwanted wastes.
Regardless of the specific product or type of manufacturing process, certain general statements can be
made about the sources of relatively high waste-to-product ratios and potential environmental impacts of
manufacturing processes. Nanomanufacturing involves:
• Strict purity requirements and less tolerance for contamination during processing than more conventional
manufacturing processes;
• Low process yields or material efficiencies;
• Repeated processing, postprocessing, or reprocessing steps of a single product or batch during
manufacturing;
• Use of toxic/basic/acidic chemicals and organic solvents;
• Need for moderate to high vacuum and other specialized environments such as high heat or cryogenic
processing;
• Use or generation of greenhouse gases;
• High water consumption; and
• Chemical exposure potential in the workplace and through technological/natural disasters.
NSF Award Number: 0646336.
The Office of Research and Development's National Center for Environmental Research
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Interagency Environmental Nanotechnology Grantees Workshop
9:40 a.m. Friday, November 21, 2008
Evaluating the Impacts of Nanomanufacturing Via
Thermodynamic and Life Cycle Analysis
Bhavik Bakshi and L. James Lee
The Ohio State University, Columbus, OH
This proposed research project will develop original life cycle inventory data for the manufacture of
polymer nanocomposites, test two new hypotheses for thermodynamics-based life cycle assessment (LCA) and
impact assessment with limited information, and develop a tool for exploring economic and environmental
aspects of alternate manufacturing combinations for selected nanoproducts and conventional processes. The
following hypotheses will be tested: (1) among alternatives for making similar products, the one with a higher
life cycle thermodynamic efficiency has a smaller life cycle impact; and (2) emissions with a smaller life cycle
thermodynamic efficiency have a larger ecotoxicological impact. The second law of thermodynamics and
hierarchical systems theory supports these hypotheses. However, validating them has been challenging.
Through collaboration with leading academic groups, industry, and a national laboratory, life cycle
inventory data and modules will be developed for the synthesis and use of nanoclays and carbon nanofibers.
These modules will be combined with life cycle information at different spatial scales, ranging from equipment
to ecosystems, and used to perform multiscale or hybrid LCA of several potential products. Different scenarios
for the manufacture, use, end of life, emissions, and exposure of typical consumable and durable products,
such as automotive body panels and food wrapping film, will be analyzed along with estimates of uncertainty.
Thermodynamic LCA will treat industrial and ecological systems as networks of energy flow and combine the
features of systems ecology, LCA, and systems engineering. The proposed hypotheses will be tested in a
statistically sound manner via several case studies.
LCA of nanotechnology is essential for guiding and managing risk in research, development, and
commercialization while preventing irrational optimism or unfounded fear of this emerging field. However, it
presents formidable obstacles because data and knowledge about resource consumption, emissions, and their
impact are either unknown or not readily available. This study will lay the foundation for LCA of polymer
nanocomposites and other emerging technologies. Validation of the first hypothesis will provide useful insight
about nano versus traditional technologies, while the second hypothesis will provide a proxy for the
ecotoxicological impact of the emissions. These hypotheses will be useful for nano and other emerging
technologies before detailed emissions data and ecotoxicological studies are available. As more information
about manufacturing, emissions, and their impact becomes available, it will be incorporated in the proposed
studies and tool.
EPA Grant Number: R832532
The Office of Research and Development's National Center for Environmental Research
35
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Interagency Environmental Nanotechnology Grantees Workshop
10:20 a.m. Friday, November 21, 2008
Impact of Physiochemical Properties on Skin Absorption
of Manufactured Nanomaterials
Xin-Rui Xia, Nancy A. Monteiro-Riviere, and Jim E. Riviere
North Carolina State University, Raleigh, NC
The wide applications of manufactured nanomaterials will create enormous potential for human exposure
and environmental release. Skin, as the largest organ protecting the body from exogenous toxins and
particulates, will be a major portal of entry for nanomaterials. The investigators' preliminary study has shown
that fullerene nanoparticles can penetrate deep into the stratum corneum (the primary barrier of the skin) and
be modulated by solvents and ion-pairing agents. Currently, there is no method available for quantitative
assessment of the skin absorption of the manufactured nanomaterials.
The objective of this research project is to establish a structure-permeability relationship for skin
absorption of manufactured nanomaterials for safety evaluation and risk assessment. Four dominant
physiochemical properties (particle size, surface charge, hydrophobicity, and solvent effects) in skin absorption
will be studied. Fullerene and its derivatives will be used as model nanomaterials. The absorption and
disposition kinetics and dose-response relationships will be measured experimentally for quantitative model
development.
The novelty of this project is to study one parameter of interest (e.g., size) while keeping other parameters
(e.g., surface charges and hydrophobicity) constant, in contrast to most of the current research focusing on the
toxicological effects of the nanomaterials. Three well-developed experimental methods will be used in
consideration of throughput, cost, and biological complexity. Diffusion experiments will provide in vitro
absorption kinetic information by measuring the nanomaterial flux across the skin. Tape-stripping is designed
to provide in vitro disposition kinetic information of the nanomaterials in the stratum corneum. An isolated
perfused porcine skin flap (IPPSF) technique will provide ex vivo absorption kinetic information that has
proved to be effective for human in vivo prediction.
The ion-pairing effects, solvent effects, and the impact of particle size and hydrophobicity on skin
absorption of nanomaterials will be quantitatively measured to provide three sets of absorption kinetic data: in
vitro absorption, ex vivo absorption, and in vitro disposition kinetics. The quantitative data obtained in this
project will be used to develop quantitative structure-permeability relationships based on the physiochemical
properties of nanomaterials, which will define a general applicable approach for quantitative risk assessment
and safety evaluation of manufactured nanomaterials.
EPA Grant Number: R833328
The Office of Research and Development's National Center for Environmental Research
36
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Interagency Environmental Nanotechnology Grantees Workshop
10:40 a.m. Friday, November 21, 2008
Safety/Toxicity Assessment of Ceria (A Model Engineered NP) to the Brain
Robert A. Yokel1'2, Rebecca L. Florence1, Jason Unrine3, Michael T. Tseng8, UschiM. Graham ,
Rukhsana Sultana5, D. Allan Butterfield5'6, Peng Wu7, and Eric A. Grulke7
1 Department of Pharmaceutical Sciences; 2 Graduate Center for Toxicology; 3Department of Plant and Soil
Sciences; 4 Center for Applied Energy Research; 5Department of Chemistry; 6 Center of Membrane Sciences;
7Chemical and Materials Engineering Department, University of Kentucky, Lexington, KY; 8Departments of
Anatomical Sciences & Neurobiology, University of Louisville, Louisville, KY
Objective: The objective of this research project is to characterize the biodistribution and toxicity of
nanoscale ceria that had entered blood.
Rationale: Ceria was chosen as a model insoluble and stable metal oxide tracer with extensive engineered
nanomaterial (ENM) applications.
Material: A commercial 5 percent crystalline aqueous ceria dispersion, mean size approximately 30 nm
(by particle size determination); primary size approximately 3 to 5 nm (by high resolution transmission
electron microscopy [HR-TEM]); surface area approximately 13 m2/g.
Procedures: The effect of saline and 10 percent sucrose on ceria agglomeration was assessed. Ceria was
i.v. infused into un-anesthetized rats (0, 50, 250 or 750 mg/kg), which were terminated 1 hour or 20 hours
later. Its biodistribution was assessed by microscopy and ICP-AES/ICP-MS cerium analysis. The potential to
produce toxicity was assessed by microscopy. Neurotoxic or neuroprotective potential was assessed by 4-
hydroxy-2-nonenal (HNE), 3-nitrotyrosine (3-NT), and protein carbonyls in frontal cortex (FC), hippocampus
(HC), and cerebellum (CB). Five minutes prior to termination anesthetized rats were given i.v. Evans blue
(EB)-albumin and Na fluorescein (Na2F) as blood-brain barrier (BBB) integrity markers.
Results: Saline and 10 percent sucrose caused ceria agglomeration in vitro. Fresh blood incubated with
ceria for 1 hour showed primary and agglomerated ceria by EM and energy-dispersive X-ray spectroscopy.
Systemic ceria ty2 in the rat was less than 1 hour. Brain EB and Na2F increased somewhat in rats terminated at
20 hours, but was less consistent in 1-hour rats. Tissue [Ce] in rats terminated at 1 hour and 20 hours was dose-
dependent (spleen > liver > brain > blood serum). At 20 hours, 4-HNE increased in the HC; 3-NT changed
little in FC, HC or CB; and protein carbonyls decreased in the CB. No significant effects were seen at 1 hour.
Conclusions: Ceria was cleared by peripheral reticuloendothelial tissues. Much less ceria entered the
BBB cells or the brain. The results provide a foundation to study the impact of the physico-chemical properties
of ENMs on peripheral organ distribution, brain entry, and neurotoxic or neuroprotective potential.
EPA Grant Number: R833772
The Office of Research and Development's National Center for Environmental Research
37
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Interagency Environmental Nanotechnology Grantees Workshop
11:00 a.m. Friday, November 21, 2008
Nanotechnology: A Novel Approach To Prevent Biocide Leaching
Patricia Heiden , Benjamin Dawson-Andoh2, and Laurent Matuana
1 Michigan Technological University, Houghton, MI; 2 West Virginia University, Morgantown, WV;
3Michigan State University, East Lansing, MI
The primary objective of this research project is to develop a practical and effective approach to prepare
biocide-loaded nanoparticles (organic and copper-based biocides) that can be efficiently introduced into wood
to reduce or eliminate biocide leach into sensitive environments. Preventing biocide loss to leach also is
expected to increase the useful lifetime of wood products while using less biocide. To accomplish this
objective, the nanoparticle must be constructed to serve as a protective reservoir for the biocide that prevents
its loss by leach or by degradation, but that also releases biocide into the wood in a controlled manner at a rate
that maintains the minimal amount of biocide required within the wood for wood preservation.
A new nanoparticle preparation method is being developed to prepare hydrophobic nanoparticles that
serve as a biocide reservoir and will moderate the biocide release rate. The nanoparticles will be stabilized in
water so that they may be delivered into wood using a conventional modified full pressure-treatment method.
American Society for Testing and Materials (ASTM) and American Wood Preservers' Association (AWPA)
approved methods respectively will be used to determine the biological efficacy of treated sapwood of pine
and birch against the brown rot fungus, Gloeophyllum trabeum, and the white rot fungus, Trametes versicolor,
and the leach rates of biocide from the nanoparticle-treated wood. Wood controls will be prepared by treatment
with the same amount of biocide introduced by conventional solution or emulsion methods and evaluated in
the same tests in side-by-side studies. All results will be compared and assessed for statistically significant
differences.
This project will demonstrate the environmental benefits of introducing biocide into wood using
hydrophobic nanoparticles as a delivery vehicle and controlled release device for organic and inorganic
biocides. The primary benefits expected from use of nanoparticles as controlled release devices for biocide in
wood are an increased service life of wood and a reduction of biocide loss to leach, which is expected to allow
wood to be effectively protected with lesser amounts of biocide than is used now. These benefits are expected
to be realized by using a new and more efficient nanoparticle preparation to give a slow biocide release rate
coupled with good nanoparticle stability in aqueous suspensions. These features will allow the nanoparticles to
be delivered efficiently into wood, but once in wood maintain a slow release rate. Successful completion of
this project will benefit all ecosystems containing preserved wood. Even greater benefits are expected for
wetlands and other moist ecosystems through reduction of biocide contamination, and in forest ecosystems
harvested for wood by extending the service life of preserved wood and wood products.
EPA Grant Number: GR832371
The Office of Research and Development's National Center for Environmental Research
38
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Interagency Environmental Nanotechnology Grantees Workshop
11:20 a.m. Friday, November 21, 2008
Internalization and Fate of Individual Manufactured Nanomaterial
Within Living Cells
Galya Orr, David J. Panther, Kaylyn J. Cassens, Jaclyn L. Phillips,
Barbara J. Tarasevich, and Joel G. Pounds
Pacific Northwest National Laboratory, Richland, WA
The cellular interactions and intracellular fate of manufactured submicrometer and nanoscale materials
dictate the cellular response and ultimately determine the level of toxicity or biocompatibility. However, the
cellular interactions and pathways of particles with specific sets of properties are largely unknown. In addition,
little is known about the cellular interactions and pathways of individual or small nanoparticle aggregates, as
they are likely to be presented to cells in vivo, mostly because of their tendency to agglomerate under
experimental conditions. In this study, the researchers investigated the initial interactions and internalization
pathways of individual precipitated amorphous silica particles with specific surface properties and size by
following one particle at a time. Using time lapse fluorescence microscopy, it was found that both 100 nm and
500 nm particles can take advantage of the actin turnover machinery within microvilli to advance their way
into alveolar type II epithelial cells, an expected target cell for inhaled submicrometer and nanoscale materials.
This pathway is strictly dependent on the positive surface charge of the particles and on the integrity of the
actin filaments unraveling charge-dependent coupling of the particles with the intracellular environment across
the cell membrane. To identify the molecules that capture the particles at the cell surface, the researchers
therefore searched for a negatively charged, transmembrane molecule that could mediate the coupling of the
particles with the actin filaments. Using flow cytometry, time lapse fluorescence, and laser confocal
microscopy, it was found that syndecan I, a transmembrane heparan sulfate proteoglycan, mediates the initial
interactions of the particles at the cell surface, their coupling with the intracellular environment, and their
internalization pathway. Together, the findings reveal a new mechanism by which positive surface charge
supports particle recruitment by polarized epithelial cells bearing microvilli, and identify a critical role for
syndecan I in the cellular interactions and subsequent potential toxicity of these particles.
EPA Grant Number: R833338
The Office of Research and Development's National Center for Environmental Research
39
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Interagency Environmental Nanotechnology Grantees Workshop
11:40 a.m. Friday, November 21, 2008
Methodology Development for Manufactured Nanomaterial Bioaccumulation Test
Yongsheng Chen1, Yung Chang1, John C. Crittenden1, Qiang Hu1,
C.P. Huang2, and Milton Sommerfeld1
1 Arizona State University, Tempe, AZ; 2 University of Delaware, Newark, DE
Because of their small size and high specific surface area, manufactured nanomaterials have enhanced
mobility and, potentially, greater toxicity as they have almost unrestricted access into aquatic organisms and
the human body. However, there are no data available on whether these manufactured nanomaterials are toxic
within months or years. So, these nanomaterials could constitute a new class of non-biodegradable pollutants
and may bioaccumulate in the food chain. Consequently, it is imperative to develop a suitable methodology to
evaluate the potential risks of bioaccumulation of manufactured nanomaterials in aquatic organisms so that we
can understand their potential impacts and avoid serious environmental consequences, such as with DDT
(dichlor-diphenyl-trichloroethane) and PCBs (polychlorinated biphenyls). The objectives of this research
project are to: (1) develop suitable manufactured nanomaterial bioaccumulation testing procedures to ensure
data accuracy and precision, test replication, and the comparative value of test results; (2) evaluate how the
forms of these manufactured nanomaterials affect the potential bioavailability and bioconcentration factor
(BCF) in phytoplankton; 3) determine the potential biomagnification of manufactured nanomaterials in
zooplankton; and 4) determine the potential biomagnification of manufactured nanomaterials in fish.
This research project brings together a multidisciplinary team, which includes nanomaterial engineers and
chemists, physiologists, and molecular biologists. A hypothesis of whether manufactured nanomaterials can be
accumulated in aquatic organisms will be tested. The bioconcentration, bioaccumulation, and biomagnification
of manufactured nanomaterials will be evaluated in a simulated food chain and aquatic organisms including
algae, daphnia, and zebrafish. Advanced analysis techniques and methods, including image shape analyzing
particle counter, transmission electron microscopy (TEM), secondary ion mass spectrometer (SIMS), and
electron microscopy, will be employed for analysis of nanomaterial size, exploration of bioavailability and
dispersion pathways of nanomaterials entering into cells of an aquatic organism, and determination of the ratio
of nanomaterials dispersed in the organs of an organism.
Any risk assessment requires basic information on toxicity to biota and the likelihood of uptake into the
food chain. This study will provide essential nanomaterial bioaccumulation testing procedures and
fundamental data on the movement and transformation capabilities of nanomaterials in aquatic organisms and
the first evidence that such nanomaterials can or cannot be biologically accumulated in aquatic organisms. This
research would ultimately allow us to better understand the consequences of manufactured nanomaterials in
the environment.
EPA Grant Number: R833327
The Office of Research and Development's National Center for Environmental Research
40
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Interagency Environmental Nanotechnology Grantees Workshop
2:00 p.m. Friday, November 21,2008
Agglomeration, Retention, and Transport Behavior of Manufactured
Nanoparticles in Variably Saturated Porous Media
Yan Jin and John Xiao
University of Delaware, Newark, DE
The production of significant and increasing quantities of synthetic nanomaterials and the very limited
knowledge on their potential environmental and health effects have caused increasing public concerns. The
overall objective of this research project is to develop an understanding of the fate of nanoparticles released
into the subsurface environments. The hypothesis of this study is that nanoparticles are likely to be mobile and
have the potential to contaminate water resources either as contaminants themselves or by facilitating the
transport of other toxic substances. The investigators propose to conduct a comprehensive study to
systematically investigate the major processes that control the movement of nanoparticles in the subsurface
under environmentally relevant conditions. Our specific objectives are to: (1) determine agglomeration
behavior of nanoparticles under different solution chemistry (pH, ionic strength, and presence of dissolved
humic material); (2) measure mobility of nanoparticles in model porous media under both saturated and
unsaturated flow conditions; and (3) experimentally elucidate the attachment and retention mechanisms of
nanoparticles at various interfaces at the pore scale.
Ti02 and Fe nanoparticles will be used as models representing two major categories of nanoparticles that
have been used or have the potential to be used in large quantities commercially. Agglomeration of
nanoparticles will be evaluated in batch experiments by dynamic light scattering. Transport and potential
transformation will be studied with a series of laboratory column experiments using model sand of various
surface properties. Sorption and reaction models will be combined with transport models to describe the
transport experiments quantitatively. An innovative approach of using confocal microscopy to visualize and
analyze particle-particle and particle-interface interactions in micromodels will provide resolution high enough
to reveal detailed particle arrangement in bulk solution and at interfaces to elucidate the mechanisms involved
in particle attachment and retention at the pore scale.
This study integrates experiments across disciplines (environmental soil physics/hydrology and
physics/material science) and scales (column, batch, and pore scale). The results of this study will lead to
better understanding of particle-particle and particle-interface interactions at the microscopic level, as well as
particle agglomeration, retention, and movement in porous media under various chemical (pH, ionic strength,
presence of dissolved humic material) and physical (variable water content) conditions at the macroscopic
scale. The investigators expect to provide conclusive evidence about the conditions under which transport of
nanoparticles is expected and the quantitative magnitude of the process. Such information will contribute to the
overall understanding of how nanomaterials interact with the natural environment and provide a scientific basis
for determining exposure pathways and developing exposure guidelines, which is the first element in risk
assessment to quantify potential human health effects.
EPA Grant Number: R833318
The Office of Research and Development's National Center for Environmental Research
41
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Interagency Environmental Nanotechnology Grantees Workshop
2:20 p.m. Friday, November 21,2008
Biological Fate and Electron Microscopy Detection of Nanoparticles
During Wastewater Treatment
Paul Westerhoff, Terry Alford, and Bruce Rittman
Arizona State University, Tempe, AZ
The market for nanomaterials is increasing rapidly, and nanoparticles (NPs) present in consumer products,
industrial wastes, biomedical applications, and so on will become significant in the near future for wastewater
treatment just as nutrients, pathogens, metals, and synthetic organic chemicals have been important for the last
few decades. Waste water treatment plant (WWTP) discharges (treated effluent, biosolids, and possibly
aerosols) may become significant routes for NPs to enter the environment. Today, almost no information is
available on the fate of manufactured NPs during biological wastewater treatment.
The goal of this research project is to quantify interactions between manufactured NPs and WW biosolids.
We will model their fate with a mechanistic model that reflects and helps us gain mechanistic understanding.
We hypothesize that dense bacterial populations at WWTPs should effectively remove NPs from sewage,
concentrate NPs into biosolids, and/or possibly biotransform NPs. The relatively low NP concentrations in
sewage should have negligible impact on the WWTPs biological activity or performance.
This project involves environmental engineers and spectroscopy experts who will quantify the removal of
four classes of manufactured NPs (metal-oxide, quantum dots, C6o fullerenes, carbon nanotubes) during WW
treatment. The unique size and surface characteristics of these NPs are expected to behave differently from
greater than 1 |im sized particles currently in wastewaters. The relative importance of four NP removal
mechanisms will be quantified: (1) adsorption to the outer cell walls; (2) enmeshment into the extracellular
polymeric substances (EPS); (3) partitioning into the cytoplasm; and (4) cellular uptake and synthesis. Batch
adsorption experiments will use NPs with whole biosolids, cellular biomass only, and EPS from three types of
biological reactors (aerobic heterotrophic, aerobic heterotrophic and autotrophic nitrifying, and anaerobic
methanogenic) and from full-scale WWTP reactors. NP application to the same three types of laboratory
bioreactors operated in a semi-continuous mode will validate adsorption onto biosolids and quantify the NP
biotransformation and toxicity to the biological community/activity. Imaging techniques (environmental SEM,
TEM) will be developed to understand "where" NPs reside with biosolids. Techniques to extract NPs from
complex biological matrices also will be explored. Finally, NP removal reactions will be incorporated into
existing mechanistic WWTP models.
This research project addresses three broad questions:
(1) What mechanisms remove NPs?
(2) Can NPs be imaged within bacteria and WWTP biosolids?
(3) Do NPs affect biological WW treatment?
Data and mechanistic interpretation/modeling directly supports all four of the stated U.S. Environmental
Protection Agency interests from the Request for Proposals. Experiments will assess the toxicity and biological
effects of NPs on the three common mixed WW bacterial communities. The project quantifies the fate
(biosorption, biotransformation) of manufactured NPs in contact with complex biological matrices (i.e., WW
biosolids). This study will be among the first to apply imaging and extraction procedures for NPs in complex
biological matrices. By understanding NP removal in WWTPs, this project helps identify potential NP
exposure pathways (effluent discharge to rivers, lakes; land application of biosolids; biosolids incineration) to
the environment and provides insight for considerations during life-cycle assessments (e.g., additional
treatment requirements at WWTPs).
EPA Grant Number: R833322
The Office of Research and Development's National Center for Environmental Research
42
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Interagency Environmental Nanotechnology Grantees Workshop
2:40 p.m. Friday, November 21,2008
Genomics-Based Determination of Nanoparticle
Toxicity: Structure-Function Analysis
Alan T. Bakalinsky1, Alex Hadduck1, Vihangi Hindagolla1, Mark Smith1, Bin Xie2, and Qilin Li2
1 Department of Food Science and Technology, Oregon State University, Corvallis, OR; 2Department of
Civil and Environmental Engineering, Rice University, Houston IX
The researchers' long-term goal is to determine mechanisms by which manufactured nanomaterials may
cause cytotoxicity in realistic environments of exposure. To assess potential toxicity and to determine
mechanisms through which two such materials may elicit toxic responses, cell yield and survival of the yeast
Saccharomyces cerevisiae and Escherichia coli were determined in the presence of underivatized fullerene and
functionalized gold nanoparticles. Three independent batches of aqueous fullerene nanoparticles solubilized
initially in toluene (tol/nC6o) or THF (THF/nC60) or directly in water (aq/nC60) at about 30 |ig/mL exhibited no
observable effect on cell yield of either wild-type yeast or E. coli in minimal medium relative to control cells.
In contrast, cell yield of 3 among 48 yeast cell wall mutants tested (ecm30, ecml7, and get2) was better in the
presence of tol/nC6o than in its absence. In 27 separate exposures of wild-type yeast at different cell
concentrations to this same dose of nC6o prepared from all three lots of the three types of fullerene, yeast
survival relative to control cells was unaffected 50 percent of the time, was better 20 percent of the time, and
worse 30 percent of the time. Survival of E. coli exposed to this same dose of tol/nCeo in 0.9 percent saline was
worse or the same as that of a control about 70 or 30 percent of the time, respectively. No striking differences
were observed in either zeta potential or particle size of the one tol/nC6o lot that exhibited greater toxicity than
the other tol/nC6o lots.
Yeast cell yield was unaffected by exposure to 100 \xg!mL of functionalized Au nanoparticles (Au-TMAT)
carrying a positive charge and containing an 11 atom core 0.8 nm in diameter. In contrast, yeast survival was
reduced by exposure to Au-TMAT concentrations of less than 1 |ig/mL. A specific amount of these particles
appeared to kill a fixed number of cells rather than a fixed fraction of cells. For example, 1 |ig killed about
100,000 cells regardless of the number of cells exposed. To identify genes and mechanisms implicated in Au-
TMAT-mediated killing, a yeast gene deletion library was screened for mutants resistant to Au-TMAT relative
to the wild-type parent strain. Six resistant clones were isolated from the initial screen of 2,500 mutants, which
constitute about one-half of the library. Loss of GYL1, YMR155W, DDR48, and YGR207C was found to
result in Au-TMAT resistance, suggesting that these genes play roles in mediating Au-TMAT toxicity.
EPA Grant Number: R833325
The Office of Research and Development's National Center for Environmental Research
43
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Interagency Environmental Nanotechnology Grantees Workshop
3:00 p.m. Friday, November 21,2008
Biological Activity of Mineral Fibers and Carbon Particulates: Implications for
Nanoparticle Toxicity and the Role of Surface Chemistry
Prabir K. Dutta1, Amber Nagy2, Brian Peebles1, and W. James Waldman2
Departments of1Chemistry and 2Pathology, The Ohio State University, Columbus, OH
In this presentation, the researchers' work on the correlations between biological activity and
physicochemical characteristics of minerals and particulates, including the biological response (oxidative
burst), mutagenicity, and the chemical reactivity (Fenton reaction) of zeolite minerals and oxidative stress and
inflammatory responses of carbon particulates, will be summarized. Zeolites, with well-defined crystal
structures, serve as model systems for asbestos and other toxic minerals. For assessment of biological
response, phagocytosis as well as the oxidative burst has been studied. For determining chemical reactivity, the
researchers have focused on the ability of the iron-exchanged forms of the zeolites to produce hydroxyl
radicals from H202 (Fenton reaction). Mutagenic potential of erionite and mordenite and how this mutagenic
potential is modulated by iron has been examined. The impact of carbon-based particulate physicochemical
characteristics on their ability to induce oxidative stress and inflammatory responses will be reported.
Internalization of particulates by freshly isolated and differentiated human monocyte-derived macrophages
(MDM) is being examined. To determine the impact of particulate physicochemical characteristics on their
inflammatory potential, inflammatory endothelial adhesion molecule expression by immunofluorescence flow
cytometry is being examined. Fenton activity of particulates is being assayed by measurement of their ability
to catalyze the decomposition of hydrogen peroxide to hydroxyl radicals by spin trapping with 5,5-
dimethylpyroline-N-oxide (DMPO).
NSF Award Number: 0532250
The Office of Research and Development's National Center for Environmental Research
44
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Interagency Environmental Nanotechnology Grantees Workshop
3:20 p.m. Friday, November 21,2008
A Rapid In Vivo System for Determining Toxicity of Manufactured Nanomaterials
Robert L. Tanguay and Stacey Harper
Oregon State University, Corvallis, OR
Rapid growth of the nanotechnology industry is resulting in increased exposure of humans and the
environment to nanomaterials prior to the scientific investigation of potential risks. It is clear that there is a
need to develop rapid, relevant, and efficient testing strategies to assess these emerging materials of concern.
Here, the researchers propose an in vivo system for rapidly assessing the toxicity of nanomaterials at multiple
levels of biological organization (i.e., molecular, cellular, systems, and organismal). Early developmental life
stages often are uniquely sensitive to environmental insult, due in part to the enormous changes in cellular
differentiation, proliferation, and migration required to form the required cell types, tissues, and organs.
Molecular signaling underlies all of these processes. Most toxic responses result from disruption of proper
molecular signaling, thus, early developmental life stages are perhaps the ideal life stage to determine if
chemicals or nanomaterials are toxic. The hypothesis of this study is that the inherent properties of some
engineered nanomaterials make them potentially toxic. To test this hypothesis, we specifically propose to
(1) further develop our in vivo zebrafish toxicity assay to define the in vivo responses to nanomaterials, and
(2) begin to define structural properties of nanomaterials that lead to adverse biological consequences.
The investigators propose a three-tiered approach exploiting the advantages of the embryonic zebrafish
model to assess the toxicity of nanomaterials. Tier 1: Rapid screening experiments will be conducted to assess
the toxicity of a wide range of structurally well-characterized nanomaterials commercially available or
produced by researchers of the Oregon Nanoscience and Microtechnologies Institute (ONAMI). Nanomaterials
found to elicit significant adverse effects will proceed to Tier 2 testing. Tier 2: Potential cellular targets and
modes of action will be defined in vivo using a suite of transgenic fluorescent zebrafish and indicators of
cellular oxidative state. Nanomaterials will be grouped according to structural indices and effects.
Representative nanomaterials from each group will be selected for Tier 3 testing. Tier 3: Global gene
expression profiles will be used to define the genomic responses to nanomaterials. Data from these studies will
be used to define structure-activity relationships using a Nanomaterials Effects Database that the investigators
have created to collate, organize, and analyze data on nanomaterial effects across species and exposure
scenarios.
The successful completion of these studies will fill important gaps in our understanding of the human
health risk posed by exposure to nanomaterials. The proposed research will deliver (1) a validated in vivo
system for rapidly assessing existing and future novel nanomaterials, and (2) data on nanomaterial structure
effects relationships.
EPA Grant Number: R833320
The Office of Research and Development's National Center for Environmental Research
45
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Interagency Environmental Nanotechnology Grantees Workshop
3:40 p.m. Friday, November 21,2008
Cellular Uptake and Toxicity of Dendritic Nanomaterials: An Integrated
Physicochemical and Toxicogenomics Study
Mamadou S. Diallo, William A. Goddard, and Jose Luis Riechmann
California Institute of Technology, Pasadena, CA
Dendrimers are relatively monodisperse and highly branched nanoparticles that can be designed to:
(1) chelate metal ions; (2) encapsulate metal clusters; (3) bind organic solutes or bioactive compounds; and (4)
become soluble in appropriate media or bind onto appropriate surfaces. Because of these unique properties,
dendrimers are providing unprecedented opportunities to develop functional nanomaterials for a variety of
applications, including chemical separations and catalysis, chemical sensing, medical imaging, DNA/drug
delivery, and water purification. As the U.S. Environmental Protection Agency begins its assessment of the
impact of nanotechnology on human health and the environment, there is a critical need for data and
quantitative tools for assessing the environmental fate and toxicity of nanomaterials such as dendrimers. The
overall objective of this research project is to advance our fundamental understanding of the relationships
between the affinity of ethylene diamine (EDA) core poly(amidoamine) (PAMAM) dendrimers to cell
membranes and their vascular and ingestion toxicity using: (1) n-octanol and solid-supported
phosphatidylcholine lipid bilayers as model cell membranes; and (2) endothelial and kidney cells as model
human cells.
To achieve this overall objective, the investigators propose to implement an integrated physical-chemical
and toxicogenomics study that combines: (1) dendrimer synthesis and characterization; (2) measurements of
the octanol-water and liposomes-water partition coefficients of EDA core PAMAM dendrimers at
physiological pH; (3) AFM imaging of dendrimer interactions with liposomes at physiological pH;
(4) molecular dynamics (MD) simulations to determine the physical-chemical properties (e.g., size, shape,
internal structure, and extent of hydration, etc.) of EDA core PAMAM dendrimers in aqueous solutions at
physiological pH; and (5) experimental characterization of the vascular and ingestion toxicity of dendrimers
through in vitro measurements of cell viability and toxicogenomics studies of human endothelial and kidney
cells exposed to aqueous solutions of dendrimers at physiological pH.
The successful completion of this project is expected to provide industry with critical data and predictive
tools needed to assess the health and environmental impact of dendritic nanomaterials such as EDA core
PAMAM dendrimers.
EPA Grant Number: R832525
The Office of Research and Development's National Center for Environmental Research
46
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Interagency Environmental Nanotechnology Grantees Workshop
4:20 p.m. Friday, November 21,2008
Nanoparticle Toxicity in Zebrafish
Gregory D. Mayer1, Jay L. Nadeau2, Anja Nohe1, and V. Smorodin1
1 University of Maine, Bangor, ME; 2McGill University, Montreal, Quebec, Canada
The overlying objective of this research project is to investigate the toxicity of semiconductor
nanostructures using an in vivo developmental system (zebrafish, Danio rerio, embryos). The approach will
monitor, in real time, the effects of particle composition, size, and charge on uptake and accumulation of
nanostructures in multiple tissues. Additionally, the investigators will monitor the release of ions from the
particles using a transgenic zebrafish model that expresses green fluorescent protein (GFP) in the presence of
metal ions. These data will be correlated to altered embryo development after particle exposure, and the effects
will be extrapolated to human health. Finally, the researchers will develop a model to predict particle toxicity
that will help to evaluate potential health risks of the release of semiconductor nanoparticles into the
environment.
To effectively determine how particle composition, size, and charge affect toxicity, researchers will begin
by refining techniques of synthesis and characterization to alter one variable at a time. These well-
characterized particles then will be applied to cultured zebrafish, zebrafish embryos, or embryonic cells.
Uptake, accumulation, and ion release in cells and whole embryos will be quantitatively measured in real time
by multicolor confocal microscopy that will simultaneously detect the nanoparticles, GFP, and co-transfected
fluorescent organelle markers. Additionally, the force of adhesion of the range of particles to cell membranes
and the embryo will be investigated using laser tweezers. All obtained data will be used to develop a model for
the prediction of cellular uptake and resulting cellular toxicity based on the physical properties of the particles
and the cell membranes that they encounter.
The investigators expect the toxicity of semiconductor nanoparticles to depend on their size, charge, and
composition. However, because of the unique properties that arise from their small size and quantum
confinement, the exact dependence of toxicity on each of these factors is likely to be surprising and to be
poorly predictable from the behavior of the bulk materials. Also, it is expected that the nanoparticles will
increase mortality and developmental abnormalities in zebrafish. Calculation of LC50s, hatch success, uptake
routes, and acute and developmental toxicity endpoints will help validate the proposed model. The resulting
data are expected to be of value for prediction of risks of nanoparticle release, especially into aqueous
environments where the particles would have direct access to developing and adult organisms.
EPA Grant Number: R833339
The Office of Research and Development's National Center for Environmental Research
47
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Interagency Environmental Nanotechnology Grantees Workshop
4:40 p.m. Friday, November 21,2008
Zinc Oxide Nanoparticles: It's the Contact That Kills
John M. Veranth, N. Shane Cutler, and Philip J. Moos
Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah,
Salt Lake City, UT
Previously, the investigators evaluated the toxicity and transcriptional responses to six lower-cost, high
production-volume manufactured nanoparticles (carbon black, Si02, A1203, Ti02, ZnO, and Fe203) in colon
cell lines. These manufactured nanoparticles are used in cosmetics, dental products, sunscreen, food additives,
and dyes, making general population and occupational exposure likely. This research project has focused on a
model of bowel inflammation and uses RKO and CaCo human colon-derived cell lines with and without
activation by TNFa. The central hypothesis being tested is that ingested manufactured nanoparticles are taken
up by inflamed colon cells, translocate to the nucleus, and alter gene transcription, thereby further increasing
inflammation and leading ultimately to the development of pathological conditions including cancer.
In initial experiments, the metal oxide nanoparticles (A1203, Ti02, Si02, and Fe203) were not toxic, carbon
black showed modest toxicity, primarily at the highest concentrations, and ZnO displayed the most toxicity.
The TNFa pretreatment did not dramatically alter the sensitivity of the RKO and CaCo-2 cells to any of the
PM. In separate experiments, samples were prepared from all nanoPM and representative microarray
experiments were run. The investigators are following up with selective QPCR as a validation method. Ti02
and ZnO displayed transcriptional effects, with ZnO having the most pronounced effect. The data suggest that
multiple pathways are activated by the ZnO, including: stress response pathways, Zn metabolism and
transport genes, and genes that suggest alterations in redox pathways.
NanoZnO displayed the most toxicity and demonstrated the most pronounced transcriptional response.
This transcriptional response suggested that part of the exposure to nanoZnO was exposure to elemental Zn,
and therefore, perhaps the toxicity was merely Zn toxicity. Therefore, the investigators sought to determine if
the nanoZnO toxicity was due to the dissolution of ZnO to elemental Zn and the mechanism of the cell death
upon exposure to the nanoZnO. In addition, two size ranges of ZnO particulate matter were utilized to evaluate
the effects of size/surface area. The researchers set out to determine if: (1) cell and particulate matter contact
was required for ZnO toxicity; and (2) if ZnO dissolution to free Zn was dependent on the cells. A set of three
experimental conditions were used: (1) a dialysis device with a 10 kD cutoff was used to separate the ZnO
from cellular contact to ensure no ZnO particulate matter could interact directly with cells; (2) transwells with
0.4 micron pores that would allow greater interactions with cellular products but still separate the cells and the
particulate matter were used; and (3) ZnO particulate matter was placed in direct contact with the cells. The Zn
concentrations were measured in the media by ICP spectrometry and cell viability by PI exclusion. The ZnO
toxicity was only observed when the particles were in contact with the cells, but the Zn levels in the media
were equally high in the transwell and direct contact experiments, suggesting that contact and potentially
uptake is required for cellular toxicity. The investigators have found that ZnO induces apoptosis by inducing
superoxide production in the mitochondria and disruption of the mitochondrial potential. In addition, all of the
toxic effects are dependent on particle size, as the larger ZnO particulate matter always demonstrated reduced
toxicity compared to the smaller ZnO nanoparticles.
EPA Grant Number: R833336
The Office of Research and Development's National Center for Environmental Research
48
-------
Interagency Environmental Nanotechnology Grantees Workshop
5:00 p.m. Friday, November 21,2008
Mass-Mobility Relationships for Silica Nanoparticle Agglomerates:
Implications for Transport and Morphological Properties
Jacob H. Scheckman1, Jaimie Hamilton2, Sotiris E. Pratsinis, and Peter H. McMurry1
1 Particle Technology Laboratory, Department of Mechanical Engineering, University of Minnesota,
Minneapolis, MN; 2Loyola Mary mount University, Los Angeles, CA; 3Particle Technology Laboratory,
Department of Mechanical and Process Engineering,
ETH Zurich, Zurich, Switzerland
Transport and physical/chemical properties of nanoparticle agglomerates depend on primary particle size,
fractal dimension, and the number of primary particles in the agglomerate. Agglomerate properties were
determined by tandem measurements of mobility (Differential mobility analyzer, DMA), mass (Aerosol
particle mass analyzer, APM), and morphology (Electron microscopy, SEM/TEM). Of particular interest are
the effects of agglomerate structure on lung deposition. To investigate this, deposition of silica agglomerates
through a physical model simulating lung generation 22 was compared to that of spheres.
Nanoparticle agglomerates of silica were generated by oxidizing hexamethyldisiloxane in a methane/
oxygen diffusion flame. Particles leaving the flame were classified by electrical mobility size with a DMA, and
their mass was measured with the APM. The measured relationship between mass and mobility was used to
determine the fractal dimension. The effects of oxygen flow rate and mass production rate on single particle
mass, fractal dimension, and dynamic shape factor were characterized. Electron microscopy was used to
determine primary particle size and give qualitative information on particle morphology.
The generated particles were chain agglomerates with clearly defined primary particles. Average primary
size ranged from 12 to 93 nm. Fractal dimensions ranged from 1.76 to 2.39. Increasing the oxygen flow rate
was shown to decrease the primary particle size and the fractal dimension and increase the dynamic shape
factor. Increasing the production rate was shown to increase the primary particle size and mass of the product
particles without affecting the fractal dimension, and to decrease the dynamic shape factor. The effects of
oxygen flow rate and production rate on primary particle size were in agreement with the literature.
Deposition patterns were determined for particles passing through a capillary tube bundle with tube
diameters simulating lung generation 22 by measuring the particle concentration upstream and downstream of
the model with two identical condensation particle counters. Silica agglomerates with a fractal dimension of
2.0 and primary particle size of approximately 53 nm were compared to spheres produced by atomizing oleic
acid. When expressed in terms of electrical mobility equivalent diameter, deposition efficiency was the same
for the agglomerates and the spheres. Similar experiments measuring deposition in other regions of the lung
with additional fractal dimensions and with additional primary particle sizes are planned.
NSF Grant Number: BES-0646507
The Office of Research and Development's National Center for Environmental Research
49
-------
Appendices
-------
Interagency Environmental Nanotechnology Grantees Workshop
Sheraton Tampa Riverwalk Hotel
Tampa, FL
November 19 - 21, 2008
REVISED AGENDA
DAY 1, Wednesday, November 19, 2008
7:30 - 8:15 a.m. Registration
8:15 - 8:20 a.m. Welcome
Nora Savage, National Center for Environmental Research
(NCER), U.S. Environmental Protection Agency (EPA)
8:20 - 8:50 a.m. EPA and Nanotechnology
Christopher Zarba, Deputy Director, NCER, EPA
8:50 - 9:10 a.m. National Science Foundation (NSF)
Mihail (Mike) Roco, Senior Advisor for Nanotechnology, NSF
9:10 - 9:30 a.m. National Institute for Occupational Safety and
Health (NIOSH)
William (Allen) Robison, NIOSH
9:30 - 9:50 a.m. National Institute of Environment Health Sciences (NIEHS)
Srikanth Nadadur, Program Administrator, NIEHS
9:50 - 10:20 a.m. BREAK
10:20 - 10:40 a.m. U.S. Department of Energy (DOE) Nanotechnology
Characterization Facilities
Neal D. Shinn, Sandia National Laboratories
Metals, Metal Oxides
Remediation and Exposure
10:40 - 11:00 a.m. Engineered Nanomaterials Fate and Transport Research
Within the Office of Research and Development's (ORD)
National Exposure Research Laboratory (NERL)
Michele Conlon, EPA, NERL
-------
DAY 1, Wednesday, November 19, 2008 (continued)
Metals, Metal Oxides
Remediation and Exposure (continued)
11:00 - 11:20 a.m. Novel Nanostructured Catalysts for Environmental
Remediation of Chlorinated Compounds
Vijay John, Tulane University
Yunfeng Lu, University of California, Los Angeles
11:20 - 11:40 a.m. Synthesis and Application of a New Class of Stabilized
Nanoscale Iron Particles for Rapid Destruction of Chlorinated
Hydrocarbons in Soil and Groundwater
Dongye Zhao, Auburn University
11:40 - 12:00 p.m. Nanoparticle Stability in Natural Waters and Its Implication
for Metal Toxicity to Water Column and Benthic Organisms
James Ranville, Colorado School of Mines
12:00- 1:20 p.m. LUNCH (on your own)
Metals, Metal Oxides
Fate/Transport
1:20 - 1:40 p.m. The Effect of Surface Coatings on the Environmental and
Microbial Fate of Nano-Iron and Fe-Oxide Nanoparticles
Greg Lowry, Carnegie Mellon University
1:40 - 2:00 p.m. The Fate and Effects of Nanosized Metal Particles Along a
Simulated Terrestrial Food Chain Investigated Using Genomic
and Microscopic Techniques
Jason Unrine, University of Kentucky
2:00 - 2:20 p.m. The Bioavailability, Toxicity, and Trophic Transfer of
Manufactured ZnC>2 Nanoparticles: A View From the Bottom
Paul Bertsch, University of Georgia
2:20 - 2:40 p.m. Bioavailability and Fates of CdSe and TiC>2 Nanoparticles in
Eukaryotes and Bacteria
Patricia Holden, University of California, Santa Barbara
2:40 - 3:00 p.m. BREAK
-------
DAY 1, Wednesday, November 19, 2008 (continued)
Metals, Metal Oxides
Toxicity
3:00 - 3:20 p.m. Engineered Nanomaterial Health Effects Research Within
ORD's National Health and Environmental Effects Research
Laboratory (NHEERL)
Kevin Dreher, EPA, NHEERL
3:20 - 3:40 p.m. Engineered Nanomaterial Ecological Effects Research Within
ORD's National Health and Environmental Effects Research
Laboratory
Steve Diamond, EPA, NHEERL
3:40 - 4:00 p.m.
4:00 - 4:20 p.m.
4:20 - 4:40 p.m.
4:40 - 5:00 p.m.
5:00 p.m.
Microbial Impacts of Engineered Nanoparticles
Shaily Mahendra, Rice University
Characterization of the Potential Toxicity of Metal
Nanoparticles in Marine Ecosystems Using Oysters
Amy Ringwood, University of North Carolina at Charlotte
Acute and Developmental Toxicity of Metal Oxide
Nanoparticles to Fish and Frogs
Chris Theodorakis, Southern Illinois University
Other Nanomaterials
Sensors and Treatment
A Novel Approach To Prevent Biocide Leaching
Patricia Heiden, Michigan Technological University
ADJOURN-DAY 1
DA Y 2, Thursday, November 20, 2008
7:30 - 8:30 a.m.
8:30 - 8:40 a.m.
Registration
Welcome and Announcements
Carbon-Based
Sensors and Exposure
8:40 - 9:00 a.m.
Conducting-Polymer Nanowire Immunosensor Arrays for
Microbial Pathogens
Ashok Mulchandani, University of California, Riverside
-------
DAY2, Thursday, November 20, 2008 (continued)
Carbon-Based
Fate/Transport
9:00 - 9:20 a.m. Carbon Nanotubes: Environmental Dispersion States,
Transport, Fate, and Bioavailability
Elijah Petersen, University of Michigan
9:20 - 9:40 a.m. Aggregation and Deposition Behavior of Carbon Nanotubes in
Aquatic Environments
Menachem Elimelech, Yale University
9:40 - 10:00 a.m. Cross-Media Environmental Transport, Transformation, and
Fate of Manufactured Carbonaceous Nanomaterials
Peter Vikesland, Virginia Polytechnic Institute and State
University
10:00 - 10:20 a.m. Fate and Transport of C6o Nanomaterials in Unsaturated and
Saturated Soils
Kurt Pennell, Georgia Institute of Technology
10:20 - 10:40 a.m. BREAK
10:40 - 11:00 a.m. Photochemical Fate of Manufactured Carbon Nanomaterials
in the Aquatic Environment
Chad Jafvert, Purdue University
11:00 - 11:20 a.m. Fate and Transformation of C6o Nanoparticles in Water
Treatment Processes
Jaehong Kim, Georgia Institute of Technology
Carbon-Based
Toxicity
11:20 - 11:40 a.m. Role of Particle Agglomeration in Nanoparticle Toxicity
Terry Gordon, New York University School of Medicine
11:40 - 12:00 p.m. Assessment of the Environmental Impacts of Nanotechnology
on Organisms and Ecosystems
Jean-Claude Bonzongo, University of Florida
12:00 - 12:20 p.m. Structure-Function Relationships in Engineered
Nanomaterial Toxicity
John Fortner, Rice University
-------
DAY2, Thursday, November 20, 2008 (continued)
Carbon-Based
Toxicity (continued)
12:20- 1:40 p.m. LUNCH (on your own)
1:40 - 2:00 p.m. Long-Term Cardiovascular Effects of Inhaled Nanoparticles
Gi Soo Kang, New York University
2:00 - 2:20 p.m. Aquatic Toxicity of Carbon-Based Nanomaterials at
Sediment-Water Interfaces
Baolin Deng, University of Missouri-Columbia
2:20 - 2:40 p.m. Aquatic Toxicity of Waste Stream Nanoparticles
Judy Blatt-Nichols, New York University School of Medicine
2:40 - 3:00 p.m. Ecotoxicology of Underivatized Fullerenes (C6o) in Fish
Theodore Henry, University of Tennessee
3:00 - 3:20 p.m. BREAK
3:20 - 3:40 p.m. Development of Methods and Models for Nanoparticle
Toxicity Screening: Application to Fullerenes and
Comparative Nanoscale Particles
Tian Xia, University of California, Los Angeles
3:40 - 4:00 p.m. Effects of Nanomaterials on Human Blood Coagulation
Peter Perrotta, West Virginia University
4:00 - 4:20 p.m. Uptake and Toxicity of Metallic Nanoparticles
in FreshwaterFish
David Barber, University of Florida
4:20 - 4:40 p.m. Innate Immune Responses of an Aquatic Vertebrate
Model to Manufactured Nanoparticles Assessed Using
Genomic Markers
Rebecca Klaper, University of Wisconsin-Milwaukee
Metals, Metal Oxides
Toxicity (continued)
4:40 - 5:00 p.m. Chemical Fate, Biopersistence, and Toxicology of Inhaled
Metal Oxide Nanoscale Materials
Jacob McDonald, Lovelace Respiratory Research Institute
5:00 p.m.
ADJOURN-DAY 2
-------
DA Y 3, Friday, November 21, 2008
7:30 - 8:30 a.m.
8:30 - 8:40 a.m.
8:40 - 9:00 a.m.
9:00 - 9:20 a.m.
9:20 - 9:40 a.m.
9:40 - 10:00 a.m.
Registration
Welcome and Announcements
Other Nanomaterials
Life Cycle Analysis and Remediation
Nanostructured Membranes for Filtration, Disinfection, and
Remediation of Aqueous and Gaseous Systems
Kevin Kit, University of Tennessee
Comparative Life Cycle Analysis of Nano and Bulk Materials
in Photovoltaic Energy Generation
Vasilis Fthenakis, Columbia University
The Life Cycle of Nanomanufacturing Technologies
Thomas Theis, University of Illinois
Evaluating the Impacts of Nanomanufacturing Via
Thermodynamic and Life Cycle Analysis
Bhavik Bakshi, The Ohio State University
10:00 - 10:20 a.m.
BREAK
10:20 - 10:40 a.m.
Other Nanomaterials
Exposure
Impact of Physiochemical Properties on Skin Absorption
of Manufactured Nanomaterials
Xin-Rui Xia, North Carolina State University
10:40 - 11:00 a.m. Safety/Toxicity Assessment of Ceria (A Model Engineered NP)
to the Brain
Robert Yokel, University of Kentucky
Other Nanomaterials
Fate/Transport
11:00 - 11:20 a.m. Agglomeration, Retention, and Transport Behavior of
Manufactured Nanoparticles in Variably Saturated Porous
Media
Yan Jin, University of Delaware
-------
DAY3, Friday, November 21, 2008 (continued)
Other Nanomaterials
Fate/Transport (continued)
11:20 - 11:40 a.m. Internalization and Fate of Individual Manufactured
Nanomaterial Within Living Cells
Galya Orr, Pacific Northwest National Laboratory
11:40 - 12:00 p.m. Methodology Development for Manufactured Nanomaterial
Bioaccumulation Test
Yongsheng Chen, Arizona State University
12:00 - 12:20 p.m. Experimental and Numerical Simulation of the Fate of
Airborne Nanoparticles From a Leak in a Manufacturing
Process To Assess Worker Exposure
David Pui, University of Minnesota
12:20 - 12:40 p.m. Nanoparticle Disruption of Cell Function
Andrij Holian, University of Montana
12:40 - 2:00 p.m. LUNCH (on your own)
2:00 - 2:20 p.m. Biological Fate and Electron Microscopy Detection of NPs
During Wastewater Treatment
Paul Westerhoff Arizona State University
Other Nanomaterials
Toxicity
2:20 - 2:40 p.m. Genomics-Based Determination of Nanoparticle
Toxicity: Structure-Function Analysis
Alan Bakalinsky, Oregon State University
2:40 - 3:00 p.m. Role of Surface Chemistry in the Toxicological Properties
of Manufactured Nanoparticles
Prabir Dutta, The Ohio State University
3:00 - 3:20 p.m. A Rapid Ln Vivo System for Determining Toxicity of
Manufactured Nanomaterials
Robert Tanguay, Oregon State University
3:20 - 3:40 p.m. Cellular Uptake and Toxicity of Dendritic Nanomaterials:
An Integrated Physicochemical and Toxicogenomics Study
Mamadou Diallo, California Institute of Technology
-------
DAY3, Friday, November 21, 2008 (continued)
3:40-4:00 p.m. BREAK
4:00 - 4:20 p.m. Effects of Ingested Nanoparticles on Gene Regulation
in the Colon
John Veranth, University of Utah
4:20 - 4:40 p.m. Nanoparticle Toxicity in Zebrafish
Gregory Mayer, Texas Tech University
4:40 - 5:00 p.m. Lung Deposition of Highly Agglomerated Nanoparticles
Jacob Scheckman, University of Minnesota
5:00 p.m. ADJOURN
-------
Interagency Environmental Nanotechnology Grantees Workshop
November 19 - 21, 2008
Sheraton Tampa Riverwalk Hotel
200 North Ashley Drive
Tampa, FL
POST-WORKSHOP PARTICIPANTS LIST
Alessandro Alimonti
National Institutes of Health
Alan Bakalinsky
Oregon State University
Bhavik Bakshi
The Ohio State University
William Ball
The Johns Hopkins University
David Barber
University of Florida
Paul Bertsch
University of Kentucky
Mitch Bogle
U.S. Air Force
Jean-Claude Bonzongo
University of Florida
Denmont Bouchard
U.S. Environmental Protection Agency
Elizabeth Carraway
Clemson University
Yung Chang
Arizona State University
Yongsheng Chen
Arizona State University
George Cobb
Texas Tech University
Tina Conley
U.S. Environmental Protection Agency
Michele Conlon
U.S. Environmental Protection Agency
J. Michael Davis
U.S. Environmental Protection Agency
Baolin Deng
University of Missouri at Columbia
Nancy Denslow
University of Florida
Mamadou Diallo
California Institute of Technology
Steve Diamond
U.S. Environmental Protection Agency
Kevin Dreher
U.S. Environmental Protection Agency
Prabir Dutta
The Ohio State University
Menachem Elimelech
Yale University
Yucheng Feng
Auburn University
John Fortner
Rice University
Vasilis Fthenakis
Columbia University
-------
Terry Gordon
New York University School of Medicine
Eric Grulke
University of Kentucky
Kathy Hart
U.S. Environmental Protection Agency
Patricia Heiden
Michigan Technological University
Theodore Henry
The University of Tennessee
Jeremy Hirsz
Hillsborough County Health Department
Kay Ho
U.S. Environmental Protection Agency
Patricia Holden
University of California at Santa Barbara
Andrij Holian
University of Montana
Matt Hull
Virginia Tech and Nanosafe, Inc.
Ivo lavicoli
Catholic University of Sacred Heart
Joany Jackman
The Johns Hopkins University
Chad Jafvert
Purdue University
Yan Jin
University of Delaware
Vijay John
Tulane University
Gi Soo Kang
New York University School of Medicine
Jaehong Kim
Georgia Institute of Technology
Kevin Kit
University of Tennessee
Rebecca Klaper
University of Wisconsin - Milwaukee
Pradeep Kurup
University of Massachusetts Lowell
Ronald Landy
U.S. Environmental Protection Agency
Igor Linkov
U.S. Department of Defense
Shannon Lloyd
Concurrent Technologies Corporation
Greg Lowry
Carnegie Mellon University
Yunfeng Lu
University of California at Los Angeles
Shaily Mahendra
Rice University
Greg Malis
Pest Management Regulatory Agency
Charles Maurice
U.S. Environmental Protection Agency
Greg Mayer
Texas Tech University
Melissa McCarthy
University of North Carolina at Charlotte
Jacob McDonald
Lovelace Respiratory Research Institute
Julianne McLaughlin
University of Florida
Mike McLaughlin
University of Adelaide
Ann Miracle
Pacific Northwest National Laboratory
2
-------
Ashok Mulchandani
University of California at Riverside
Joseph Mwangi
University of Missouri
Srikanth Nadadur
National Institute of Environmental Health
Sciences
Shawna Nations
Texas Tech University
Arianne Neigh
nanoComposix
Bryant Nelson
National Institute of Standards and
Technologyy
Oanh Nguyen
nanoComposix, Inc.
Tom O'Farrell
U.S. Environmental Protection Agency
Galya Orr
Pacific Northwest National Laboratory
Manomita Patra
U.S. Environmental Protection Agency
Kurt Pennell
Georgia Institute of Technology
Peter Perrotta
West Virginia University
Elijah Petersen
University of Michigan
Helen Poynton
U.S. Environmental Protection Agency
David Pui
University of Minnesota
James Ranville
Colorado School of Mines
Amy Ringwood
University of North Carolina at Charlotte
William Robison
Centers for Disease Control and Prevention
Mihail Roco
National Science Foundation
Charlita Rosal
U.S. Environmental Protection Agency
Nora Savage
U.S. Environmental Protection Agency
Jacob Scheckman
University of Minnesota
Thomas Seager
Rochester Institute of Technology
Anne Sergeant
U.S. Environmental Protection Agency
Neal Shinn
Sandia National Laboratories
Robert Tanguay
Oregon State University
Thomas Theis
University of Illinois at Chicago
Christopher Theodorakis
Southern Illinois University Edwardsville
Olga Tsyusko
University of Kentucky
Jason Unrine
University of Kentucky
Katrina Varner
U.S. Environmental Protection Agency
John Veranth
University of Utah
Julie Vernon
University of Florida
3
-------
Bellina Veronesi
U.S. Environmental Protection Agency
Peter Vikesland
Virginia Polytechnic Institute and State
University
Randall Wentsel
U.S. Environmental Protection Agency
Paul Westerhoff
Arizona State University
Tian Xia
University of California at Los Angeles
Xin-Rui Xia
North Carolina State University
Robert Yokel
University of Kentucky College of Pharmacy
Sejin Youn
University of Florida
Jeff Zad
Schiller International University
Christopher Zarba
U.S. Environmental Protection Agency
Don Zhao
Auburn University
Contractor Support
Jen Hurlburt
The Scientific Consulting Group, Inc.
656 Quince Orchard Road, Suite 210
Gaithersburg, MD 20878
Telephone: (301) 670-4990
E-mail: jhurlburt@scgcorp.com
Maria Smith
The Scientific Consulting Group, Inc.
656 Quince Orchard Road, Suite 210
Gaithersburg, MD 20878
Telephone: (301) 670-4990
E-mail: msmith@scgcorp.com
4
-------
Nanotechnology
W. Allen Robisori, Ph.D.
National Institute for Occupational Safety
and Health
Office of Extramural Research
19 November 2008 Tampa, FL
Purpose
Increase knowledge of nanotechnology and
manufactured nanomaterials
Occupational Safety and Health
Applications/Implications
Complements intramural program
Background
2001-2008
• R01, R03, R21, R43/44 funding
mechanisms
• Program Announcements
• Joint Request for Applications
(EPA, NIEHS, NSF)
CDC]
NIOSH Extramural Funding
• 2001/2002
$850K (R43/44 NIOSH)
• 2004
$1 00K (R43 NIOSH)
• 2005
$1 ,46M (R01 NIOSH)
• 2005
$789K(RFA EPA lead)
• 2006
$1 00K (R43 NIOSH)
• 2006
$359K (RFA EPA lead)
• 2007
$500K (RFA NIEHS lead)
• 2008
$800K (joint RFA + NIOSH)
(About $5M total)
CDC|
Hw/TTH?
NIOSH Extramural Funding
Types of Research Funded
2007
Four active grants R01 extramural grants
Two end in 2008
2008
New R01, R03 and R44 grants
• 13 different projects
• Sensors for Portable Monitors
• Novel Protection Garments
• Lung Oxidative Stress/Inflammation
> (lung cells, macrophages)
-------
Types of Research Funded
Workplace Assessment Methods (air)
Monitoring Airborne Carbon Nanotube Particles
> (characterizing)
Role of Surface Chemistry in Toxicology
> (oxidative stress, inflammation)
Toxicity of Inhaled Nanoparticles
6u/7»H?
For More Information
>Pr
the Workplace
>DHHS (NIOSH) Publication No. 2007-123
>NIOSH Nanotechnology Research Center
^•Summary of extramural projects in appendix
> 'V
For More Information
• W. Allen Robison (
• 404.498.2530
• www.cdc.gov/niosh/oep
Other Web Sites
www2a.cdc.gov/niosh-nil
www.cdc.gov/niosh/r2p/
www.cdc.gov/niosh/programs/
www.cdc.gov/niosh/topics/nanotech/ultrares.html
www.cdc.gov/niosh/topics/nanotech/critical.html
6u/7»W?
-------
NIEHS
National Institute of
Environtmnttl Health Sciences
NIEHS Activities on Nanotechnology:
Applications and Implications
Sri Nadadur, Ph.D.,
Division of Extramural Research & Training
National Institute of Environmental Health Sciences
National Institutes of Health, RTP, NC
interagency Nanotechnology Grantee Meeting, Tampa, FL Nov2008
NIEHS NanoteGhiiology Health Implications
Routes of exposure and systemic
distribution
Correlate physical and chemical
characteristics of ENM with
biological response
Identify biomarkers of exposure
and biological response
Develop models to evaluate and
predict biological response
Interaction of ENM with
biomolecules
Transmembrane transport,
cellular uptake, subcellular
localization and retention
Identify cell and organ-specific
toxicity response pathway
Effect of structural and surface
modifications
•^V NIEHS
Nil Research Interests In HiMseMon
>Nano Delivery Systems
VBioimaging & Informatics
>Organ-tissue nano-engineering
>Medical Devices
> Biocompatibility and Toxicity
Environmental health & safety
Dept of Health
and Human Services (DHHS)
1
i
1
NIH
CDC
FDA
1
1
1
26 ICs
NIOSH
NCTR
•^v NIEHS
Extramural Research Program-Health Implications
Types of ENM
- Carbon (C60CS, fullerenes, SWNT, MWNT, CB), QDs, metal, silica, polystyrene,
Routes of Exposure
- Respiratory, dermal, gastric, ocular
Physico-chemical characterization
- Size, shape, structure, surface area, charge, aggregation, surface ligands,
functional groups
Metabolism, Transport
- Cell/organ-specific transport, bio-persistence, biotransformation, elimination
Molecular mechanisms of toxicity
- Interaction with macromolecules, signaling pathways, stress pathways, immune
function, xenobiotic metabolism, DNA repair, epigenetics, etc.,
Biomarkers of Exposure/response
- High throughput approaches (genomics, proteomics, metabonomics)
Aniehs
Nil- N&natMtaatosy Resetreh Funding
FY 199B FY 1929 FY2D0D
FY2QD1
FY 2002
FYZXD
FY2004
FY 2005
FY200B
SBIR/STTR
FY
2001
2002
2003
2004
2005
2006
Actual
4.4
7.9
7.3
11.8
16.3
17.2
^ NIEHS
-*?tssss
Extramural Research Program-Health Implications
>Comparative in vitro toxicity screening for oxidative stress response of
commercially available nanoparticles- -Andre Nel, UCLA
>Cardiovascular toxicity of nickel nanoparticles and particles coated with
sulfuric acid studied following inhalational exposure - Lung-chi Chen, NYU
> Comparative toxicology of CNP (C60CS, SWNT, MWNT)- alterations in macrophage
membrane function for lung inflammation and injury-AndriJ Hoilan,
University of Montana
>Understand relative influence of nanomaterial characteristics on nanomaterial
biological interactions at multiple levels of biological organization-
Rob Taugary, Oregon State Univ.
-------
"V NIEHS
Nan
• Em
- Sensor arrays - Primarily affinity based
- Functional profiling to detect unknown toxins
Biological Sensors
- Develop technologies to link exposure to disease etiology
- Mechanistic research (cellular dynamics, signal pathways)
- Biomarker detection (in vivo imaging, sensor arrays)
Nanotech arrays to recognize gasoline and diesel combustion
products—Ash ok Mulchandani, U. of California at Riverside
Polymer wire "tuning fork" to detect Volatile organics—NJ
Tao, Arizona State U.
Nanoparticle based immunoassay systems for detection of N-Fibrinogen
-YuheLin, PNNL
Contact: David Balshaw: balshaw@niehs.nih.gov
^>NIEMS
MENS NaMtaebMltn Programs
Implications Applications
Interaction of Engineered Nanomateriais with
Biological Systems
Structure
Activity
Predictive Models
Risk Assessment
•^v NIEHS
©fee
Hanoteehnology - Ramedlatian efforts
>2008 RFA- Development and Application of Nanotechnology-
based Tools to Understand Mechanisms of Bioremediation
>Sensors (for As, PCBs)
•Mechanisms of Hg adsorption from mixed pollutant streams - carbon
nanotubes - R. Hurt, Brown U
•Dechlorination of TCE by nanosized metallic systems and by chelate-modified
hydroxyl radical reaction - bi-metallic (Fe/Ni) nanoparticles - D
Bhattachatyya, U Kentucky
•Ground water As decontamination using nanoparticle and granular zero-valent
iron - nZVI-based permeable reactive barriers - D Sedlak, UC Berkeley
•Activated carbon as a multi-functional amendment treatment to treat PCBs and
mercury - nZVI impregnated AC - R Luthy Stanford U*
Contact: Heather henry, henryh@niehs.nih.gov
Aniens
NIEHS Next Step: Building thi
laioloaltli ind Safety Enterprise
> Build on the NIH investment and expertise
> Invite stakeholder participation
> Target questions within a shared research strategy
> Harmonize with US goals for commercialization and
innovation
¦A NIEHS
m
Notional
Toxicology
| Program |
w
1TP Hanoteehnology Safety Research
Nanoscale titanium dioxide (sunscreens)
Dermal penetration studies, in vivo and in vitro
Phototoxicology and photocarcinogenicity
Quantum dots
Pharmacokinetics studies
Dermal studies
Carbon fullerenes
Oral and pulmonary toxicity studies
Material formulation and characterization in progress
Dendrimers
Pharmacokinetics and biocompatibility
Collaborative efforts with NCL
Contact: Nigel Walker, waiker3@niehs.nih.gov
^ NIEHS
ManoHeslth and Safely Enterprise
• Characterizaton of Materials
• Biochemical -Interactions
¦ Pathophysiological Mechanisms
¦ Training Program
M
,F. .
1
-
-------
Aniens
•^SSSKl
Targeted Research Prtjtctt
Implications Applications
Interaction of Engineered Nanoscale Materials |
with Biological Systems
Biologically and clinically
relevant design principles
Strategic product
design and development
Curated data sharing
framework
Network of research partners 1
Shorter time from
concept to manufacture
Data for hazard
identification
.1
Standards setting
Computational Models for Safe Design
High Throughput Screening
-------
Reactive Composites
for Targeted Remediation of TCE
Tingjing Zhan, Tonghua Zheng, Bhanu
Sunkara, Gerhard Piringer, Gary McPherson, Yunfeng
Lu*, Vijay John*
Department of Chemical & Biomolecular Engineering
Tulane University. New Orleans, LA
Supported by EPA Grant GR832374 W5Z* |
Background
~ Dense nonaqueous phase liquid (DNAPL)
~ Density greater than water (plumes transport to lower sediment layers)
~ Low (but finite) solubility in water.
c 1
~ Example: trichlomethylene (TCE) — ^ =c ^
~ Density: 1.46 g'mL (heavier than water)
~ Solubility: 1100 ppm in water
~ Toxicity: Organ damage, carcinogen
Excerpted from:
http://www.dnapl.grou
p.shef.ac.uk/main.htm
2: Chemical Remediation of TCE
~ Zero valent nanoscale iron (ZVI) is an effective
reductant for remediation of TCE with the following
advantages:
> environments friendly
> high efficiency
> ld\v cost
~ Mechanism
M
el, a
C=C' + n e" + m H+ H2C=CH2,CH3—CH3 + 3C1"
C/ %H
Y.Liu, eta\ Environ. SciTechnol. 2005, 39, 1338
wsz*
~ ZVI particles have poor mobility, hence a low efficiency
in in-situ remediation.
Excerpted from: http://www.dnapl.group.shef.ac.uk/main.htm
3: Challenge
Injection of ZVI particles
Fe° exhibits
ferromagnetism
a«»iaias»»a , ,
vomj oqnia and thus
Icimj btdaeo) aggregates,
limiting its
ft iwod mobility in soil
Objective
Effective in-situ remediation of TGE requires the successful
delivery of reactive iron particles through soil. Our goal is to
engineer reactive particles that have good mobility through soils
and directly target TG:S.
Outline
~ Synthesis of ZVI containing composite particles.
~ Reaction Characteristics
~~~Transport characteristics of the particles
~~~ Partitioning characteristics between the bulk aqueous and
the DNAPL phase
I: Particle synthesis
1: What are particle design requirements?
• Particles that are reactive to TCE
• Particles that will partition to TCE or to the TCE/water interface
• Particles that are of the correct size range for optimal mobility
through sediments
2: Idea: Incorporating nano-scale iron into porous submicron
Silica particles that are f-unctionalized with alkyl groups
Characteristics:
~~~ Nanoscale zero valent iron ensures high reactivity;
~~~ Silica can be functionalized easily by varying the precursor;
~~~ Silica is environmentally friendly
1
-------
Y.Lu, Mal Milure, 1999,398,223
Precursor
solution:
Surfactant,
ethanol, tetraethyl
orthosilicate
Aerosol
Silicas
Lamellar
3: Particle preparation
Y. Lu, et al Nature
1999,398, 223
Aerosol technology: a simple, rapid method to obtain particles.
Can be easily scaled up.
KTliS
tex«
F e/Ethy 1-Silica
Precursor solution: 4.0g FeCl3*6H20,15ml H20, 3.0gTEOS and 1.2g ETES.
(TEOS: tetraethyl orthosilicate; ETES: ethyl triethoxysilane) .
4: Reason to use functionalized silicas
OH CH OCHjCHj OCH2CH3
-H,0 | I
CH3CH2—Si—CXHjCH, +CH3CH2—Si—OCHjCH, ~ CHjCHj—S— O — Si — CH2CH,
OCHjCH, OCHjCHk OCHjCH, OCH2CH,
• kM
transport _
2 • • • •!
VjV
•
contact with
through
soil
bulk TCE1
H20 phase
• TCE
Organic phase
* Hypothesis: Organic functional groups adsorb dissolved TCE facilitating
contact with ZVI and also extend in the organic phase (TCE) to help particle
stability.
BET surface area (m2/g)
Pore volume (cm3/g)
Particle density (g/cm3) 4.0
Characterization
~ Fe/Ethyl-Silica particles are spherical with nanoiron throughout the silica
matrix;
~~~ Fe/Ethyl-Silica particles are porous.
Zheng et aL, ES&T, 2008 BKsJ
GC Spectra of Reaction Products
(a) at hrO
(b) after 8 hr reaction with Fe/Ethyl-Silica
(c) after 1 hr reaction with Pd/Fe/Ethyl-Silica
Rlhmr
wru/c
1
b lnJoct
Reaction is relatively slowfor Fe/Ethyl-Silica
Almost all the TCE disappeared after 1 hr of
reaction with Pd/Fe/Ethyl-Silica
Palladized particles produce more saturated
ethane instead of ethene
Trace amounts of chlorinated hydrocarbons are
evident - disappears at long reaction times.
Effect of Surface Modification
(a) Fe/ Ethyl- Silica (b) Fe/Silica
Time(hr) Timefhrl
a) TCE disappears faster for Fe/Ethyl-Silica particles in the first few hours of reaction.
b) Product evolution rates are comparable for Fe/Silica and Fe/Ethyl-Silica particles;
~ Modification with ethyl functional groups leads to the adsorption of TCE on
Fe/Ethyl-Silica particles.
2
-------
Reasons to use functionalized silicas
MCM-41 MMS MES AC
Silica Functionalized silica Y: adsorbed / initial
• Organic functional groups adsorb dissolved
TCE, facilitating contact with ZVI and also
extend in the organic phase (TCE) to help
particle stability.
II: Transport properties
Filtration theory
PARTICLE
TRAJECTORY
STREAMLINE
1 \t
[\\ v0i ~Diffusion - influenced by interparticle
1 \\ van der Waals interactions
~~\Y\ •Sedimentation - gravitational effects
COLLECTOR -J
APj) •Interception - Particles following flow
Jyj streamlines come into contact with a
A INTERCEPTION
z// sediment grain (collector).
6 SEDIMENTATION
' /
C DIFFUSION
/ / Small Particles - Diffusion dominated
Large Particles - Dominated by sedimentation
Basic transport mechanisms in filtration and interception
Kuan-MuYao; Habibian, M.T.; O'Melia, C. R..
Environ. Sci. Techno!. 1971, J, 1105-1112.
SPSS*,
Tufenkji-Elimelech model
Tufenkji, N.; Elimelech, M ..Environ. Sci. Techno!. 2004,38, (2), 529-536.
" = +0.55AsNfXU5 + V-22K*XeUK°w
1 I I
Diffusion Interception Sedimentation
Commeicial Reactive Nanoscale Iron Particles (RNIPs)
7 = ,i.5 13 • Hi \i • I 3<)l - 10 ; ~ • 3.OS • lii t/ "
Ee/Ethyl-Silica particles
7/0 = 5.543 x 1(T9 dfm + 1.391 x lO'5^® + 1.671 1084*
• based on differences in particle densities
• 200nm-lfim is the correct size range for optimal
mobility through sediments
©fife*
Aggregation Characteristics
Bare-RNIP (Toda Kogvo Corp.) Fe/Ethyl-Silica
iLrZ\ A
J J A
v *>
r i
50.0 prn
(In accordance to the result previously
reported : Phenrat, T. et al Environ. Sci.
Tkch. 2007, 41, 284)
* RNIP aggregates significantly due to magnetic interactions.
Incorporation of ZVI into the silica matrix substantially
decreases aggregation,
W&z*
Size distribution
a
nrinnnnn nn
Diameter (nm)
• Almost all Fe/Ethyl-Silica particles are in
the size range for optimal mobility.
3
-------
Column test
1.2cm(i.d.) burette packed with Ottawa Sand
fcsiBtl Ee/Ethyl-Silfca
• Fe/Ethyl-Silica suspension transports through the soil readily, while most of
RNIP particles are retained at the top of the column.
Effluent analysis
CO to
d o
• Fe/Ethyl-Silica
—O— RNIP i
ifl
if 0.4
S
0.2
L/
7
0 10 20 30 40 50 60
Eluted volume (mL)
• -66% of F#Ethyl-Silica particles are eluted through
the sediment, while RNIP does not elute.
Capillary experiment
1.5 mm (i.d.) Capillary packed with Ottawa Sand
S*ci
-------
Summary
> Synthesis of adsorptive-reactive Fe/Ethyl-Silica composite
particles.
P The Fe/Ethyl-Silica particles are in the correct size range for
optimal mobility through model soils.
^ Fe/Ethyl-Silica particles may preferentially accumulate and
localize at the TCE/water interface, making dechlorination more
efficient.
^•Adsorption of TCE on the particles leads to a dramatic reduction
in solution TCE concentration.
>The composite particles can be used in in-situ remediation and in
the development of reactive barriers.
oteb*
5
-------
Synthesis and Application of
Polysaccharide-Stabilized Fe-Pd
Nanoparticles for in situ Dechlorination
in Soil and Groundwater
Progress Report III: Nov 19, 2008
Don Zhao, Chris Roberts1, F. He and J.C. Liu1
Department of Civil/Environmental Engineering
1 Department of Chemical Engineering
Auburn University, Auburn, AL 36849
IB? S
Primary Accomplishments in Year 3
Conducted batch and column tests for
degradation of TCE sorbed/trapped in
soils using CMC-stabilized ZVI
nanoparticles
Tested and modeled transport behaviors
of CMC-stabilized ZVI nanoparticles in
porous media
Pilot-tested in situ dechlorination in soils
using CMC-stabilized ZVI nanoparticles
•particles
C) as a
e e
I0 e e
eCP
e q o I
pH, IS,
T, Cations
Step 1. Solution Step 2. Fe3+ or Fe2+ Step 3. Formation of
with 0-1% (w/w) complexes with Fe(°> clusters coated
of a stabilizer. stabilizer w'**1 stabilizers
Add a
Secondary
metal, Pd
(0.1% of Fe)
Step 4. Formation of stabilized Fe-
Pd bimetallic nanoparticles.
He eta/,, Industrial & Engineering Chem. Res. 2007, 46(1), 29-34.
Stabilized vs Non-Stabilized ZVI Nanoparticles
Starch-Stabilized
Non-Stabilized
Commercially Available Iron
"Nanoparticles", RNIP (Toda America Inc.)
Toda claims: "RNIP are zero valent iron solids with an average particle
size of 70 nm."
"RNIP are available as a water-based slurry."
http://www.todaamerica.com/products/eco/rnip/rn
ip_01.html
-------
CMC can Facilitate Synthesis of Nearly Mono-
disperse Pd Nanoparticles that can Catalyze TCE
Degradation
fe:
V H :
h 'tV ' >,r''
Pd = 0.2 mM; CMC = 0.15 wt°/o, Temp. = 95 °C
Column Set-up for Studying Transport of
CMC-ZVI Nanoparticles in Four Porous Media
Breakthrough Curves of Br and CMC-Stabilized
ZVI Nanoparticles through Four Porous Media
1 2 3 4 5 6 7
23456789
Br = 50 mg/L; Fe = 0.2 g/L; Empty Bed Contact
Time (EBCT) was 28 min. Lines are model
simulation.
Breakthrough Curv es of CMC-Stabilized ZVI
Nanoparticles through Sand at Various Velocities
1.0 ¦
0.8 ¦
a= o.6.
U
0.4 •
0.2 ¦
O q El
if* : .•o"'
ii
1}9 ~ V=0.0706 cm/s
| f t V=0.0353 cm/s
|| O V=0.0176 cm/s
!| Model, 0.0706 cm/s
Model, 0.0353 cm/s
Jj Model, 0.0176 cm/s
01234567?
Pore Volume
Fe =
9.2 g/L; Lines are model simulations.
Predicted Maximum Travel Distance of CMC-
Fe Nanoparticles in Sand as a Function of
Pore Liquid Velocity
2
-------
Breakthrough and Elution Curves of CMC-Fe
Nanoparticles through a Sand Bed
Elution starts here
Model, 0.2g/L Fi
Model, 0.2g/L Fi
Pore Volume
Influent Fe = 0.2 g/L; EBCT = 28 min; Pore liquid velocity = 0.0353 em/s;
Ca = 40 mM.
Transport in a
I
• Tracer transport
2-D Sand Box (Kanel et al.
i&fiT, 2008)
•
J
r r
l: i
Aft'! I>iteu«a TfalB" i!
O
ifT-
[Time " •• nun
J
• ZVI Nanoparticle (Fe = 0.2 g/L, CMC=
=0.16%)
r j
mi
¦P"
L '"J
mm
A.irr irrTlinri i tnir- ij
O
Tim* ¦ 9 iO min
In situ Degradation of TCE Spiked in a Sand
Column
Degradation of TCE and generation of CI" in a sand column
during the nanoparticle treatment
(TCE = 15 mg, Fe = 0.5 g/L, Pd/Fe = 0.1 wt%, Cellulose = 0.4 wt.%,
suspension pore velocity = 0.0118 cm/s, EBCT = 84 min)
Column Set-up for in-situ Degradation of
TCE in a Sand Column
TCE was spiked in the sand bed and 0.5 g/L ZVI nanoparticle
suspension was pumped through the column
(a) (b)
(a) Column setup for in-situ TCE degradation; (b) A close-up of
the column as Fe/Pd nanoparticle suspension was introduced.
Surfactant-Enhanced TCE Desorption
from an Organic Soil
5CMC SDS
SCMC SDBS
SCMCTwe
SCMC HDTMA
100 120 140
-All surfactants enhanced TCE desorption, with SDS being most
effective, resulting in a TCE mass desorption of ~18% in 120 h at
1xCMC surfactant dosage
-TCE desorbed was increased to 22% at 5xCMC SDS
-TCE0 in Soil = 0.52 mg/g
-------
Effects of Surfactants on TCE Degradation
in Aqueous Phase
without Surfactant
10CMC SDS
5CMC SDS
1CMC SDS
0.5CMC SDS
- SDS enhances TCE degradation: 1xCMC >0.5CMC= 5xCMC >10xCMC.
At 1xCMC SDS, the rate constant was increased by a factor of -1.7 than
without surfactant
Test conditions: TCE=10mg/L, Fe=0.1g/L, Pd/Fe=0.1wt%, Ceilulose=0.2
wt%
Effect of SDS on Degradation of TCE Sorbed
in an Organics-Rich Soil
Effect of DOM on TCE Degradation in Water
1.0 •
0.8 •
¦ TCE degradation with DOM
¦ TCE degradation without DOM
C0 = 100 mg/L
Fe=0.3 g/L
TOC= 348 mg/L
Field Assessment of CMC-Stabilized Fe-Pd
Nanoparticles at an Alabama Site
A sectional view of the aquifer and location of injection well
and monitoring wells. (K=7xl0 5 m/s or 20 ft/d)
The Nanoparticle Tank and Injection Setup
Suspension of CMC-Stabilized Fe-Pd
Nanoparticles before Injection
4
-------
Concentration Histories of Iron Nanoparticles in MW-1 Concentration Histories of Iron Nanoparticles in MW-2
Compared to Tracers Compared to Tracers
Concentration Evolution of PCE, TCE and PCB 1242 in
Groundwater from MW-1 Following Injection of 150
G (0.2 g/L) Fe-Pd Nanoparticle Suspension
o
MW-1 TCE C0=2 ppm
§
~
MW-1 PCE C0=1.5 ppm
A
A
MW-1 PCB1242 C0=17ppb
V
MW-1 1 - C,(Br)/C0(Br)
o
16 A
o
wvvv w vvv
I
A
;
O
Time, Days
Note: Little degradation was detected in the up-gradient well
5
-------
Concentration Evolution of PCE, TCE and PCB 1242 in
MW-1 following two Injections of (150 G 0.2 g/L
+ 150 GO.5 g/L) Fe-Pd Nanoparticle Suspension
Initial concentrations: PCB= 17.0
|jg/L, PCE = 1500 |jg/L,
TCE = 2000 |jg/U cis-DCE=8500 |jg/L,
VC=1100 |jg/L
1 PCB 1242
1 VC
PCB 1242
-O- PCE
—TCE
L^tce
—7- cis-DCE
-M— VC
PCE "
200
Tin
300 400
|ej_da^s__
Concentration Evolution of PCE, TCE and PCB 1242 in
MW-2 Following two Injections of (150 G 0.2 g/L
+150 GO.5 g/L) Fe-Pd Nanoparticle Suspension
Initial concentrations : PCB= 60.0
|jg/L, PCE=5000 |jg/L,
TCE=4200 |jg/L,cis-DCE = 13000
|jg/L, VC = 2200 |jg/L
Summary
* CMC can facilitate size-controlled
synthesis of ZVI nanoparticles
* Transport of CMC-stabilized Fe
nanoparticles are controllable and can be
modeled by CDE & filtration theory
* CMC-stabilized ZVI can degrade TCE in
soil, but must overcome mass transfer
and sorption limitation and DOM
inhibition
Acknowledgements
• USEPA STAR Grant (GR832373)
• Dr. Nora Savage - EPA Project Manager
* Golder Consultants, Atlanta
* Dr. Gupta in Chemical Engineering
Department for DLS analysis
-------
8/11/2009
Characteristics, Stability, and Aquatic
Toxicity of CdSe/ZnS Quantum Dots
Dr. James Ranville
Department of Chemistry & Geochemistry
Colorado School of Mines
Golden, CO 80401
CdSe/ZnS Quantum Dots
Bright, photostable fluorophores
Basic Structure
- Metalloid core, CdSe
* Wurtzite crystal structure, 1:1 Cd/Se mole ratio
- Protective shell, ZnS
Environmental Fate of Metals in Quantum Dots
Quantum dots
CdSe/ZnS Direct Uptake
Benthic Aquatic species
Research Approach
• Characterization
•Core
•UV-Vis absorption, Fluorescence
•Core/Shell
•TEM
•Core/Shell/Polymer (Hydrodynamic)
•Light scattering, Metal ratios (ICP-AES/MS),
Metal size distributions (FFF-ICP-MS)
•Stability
•Aggregation, dissolution
•Short-term (48 hours)
•Long-term
•Toxicity/Uptake
• Acute (48-hr) D. Magna
prookhaven National Light Source: (.i-XRF
Materials
Four types of QDs used in experiments
•Two core sizes, two coatings (PEO, MUA)
Optical properties depend on core size
•UV-Vis Absorbance used to size core
C
-------
8/11/2009
TEM
•Red EviTag PEO coating
•Polymer coating not observable
•Core/shell size about 5-7nm
•UV wavelength max of 609nm gives
about 5 nm core
•ZnS coating computed from
chemical analysis to be about 1.2 nm
ICP-AES/MS: Metal Ratios
Core
Red
Green
Metal Mole Ratio
Cd/Se
Zn/Cd
Cd/Se
Zn/Cd
a
2.1
1.4
1.3
5.6
PEO
b
3.2
1.4
ND
5.6
c
1.6
1.5
2.1
5.8
a
23
0.14
ND
0.23
MUA
b
22
0.14
11
0.23
c
9.1
0.16
7.1
0.26
a. ICP-AES: QD in hard water
b. ICP-AES: QD in DI water
c. Integrated signal from FFF-ICP-MS
• Cd:Se ratio not 1:1
• Excess Cd, especially for MUAQDs
• Zn/Cd higher for smaller (green) QD
Elemental Size Distribution by Fl FFF-ICP-MS
Possible explanation:
Cd associated with
polymer due to poor
washing during
synthesis
Element Ratios
Void QD Peak
Zn/Cd 0.34 0.16
Cd/Se 8.4 9.1
Elemental Size Distribution by Fl FFF-ICP-MS
Possible explanation:
No dissolved Zn detected
in a 3K Dalton filtrate
Zn associated with free
polymer
Very little Fl in void peak
Element Ratios
Void QD Peak
Zn/Cd 8.1 1.2
Cd/Se 1.6
Elemental Size Distribution by Fl FFF-ICP-MS
Green EviTags PEO coated
300 600 900 1200 1500
Possible explanation:
No dissolved Zn detected
in a 3K Dalton filtrate
Zn associated with free
polymer or ZnS
Some Fl in void peak
Element Ratios
Void QD Peak
Zn/Cd 40.3 4.3
Cd/Se 2.1
Hydrodynamic Size (Core/Shell/Polymer)
Fl FFF and DLS Characterization
FI-FFF-ICP-MS
Dynamic light scattering
0 20 40 60 SO 100 120 140 160
Size (nm)
DLS requires higher concentration than FFF
Fluorescence comes from CdSe Core
2
-------
8/11/2009
FFF-ICP-MS Discussion
Large excess of Cd associated with QD
- Possibly associated with polymer coating
Zn in Void peak
- Unlikely to be dissolved
- Low fluorescence: Zn possibly associated with
unattached polymer
- High fluorescence: Zn possibly present as ZnS
What are the implications for stability and toxicity?
Long Term Stability: Loss of fluorescence (PEO QDs)
2.E+07
48-hr acute toxicity
2.E+07
—T=0 his
¦ ~ •
6nM HR ~->. —T= 196 his
A"""
tT
1
500 550 600 650 700
Wavelength (nm)
t
i +
*•*+ +
~
+
£
i i 4
200 400 6(
Time (Hours)
800 1000
Acute Toxicity: Methods
48 hr acute toxicity tests
- USEPA Standard Test Protocol
- Mortality (ie immobile) end point
- EPA hard water
Daphnia magna obtained from cultures maintained in
our lab at 20°C and a 16:8 hr day:night cycle
PEO and MUA coated CdSe/ZnS quantum dots
Exposure Concentration Range
- 0.1 -30 x 10 9 mol QDs/L
Two d iffe re nt s ize dots we re te sted
- Green Dots - 2.5 nm core diameter
- Red Dots- 5 nm core diameter
Stability of QDs monitored throughout 48hrtest
- Fluorescence: 0, 24 and 48 hrs
- ICP-AES metals analysis of solutions (total metals in solution):
0 and 48 hrs
- 3kDa filtrations w/ ICP-AES metals analysis (dissolved
metals): 0 and 48hrs
Acute Toxicity:
Fluorescence intensity (CPS)
throughout acute toxicity test
18
ID
i ie
i
~
k
° 14
¦ Green MUA
"J 12
¦ Red MUA
escenc
CPS)
OD o
¦
A Green PEO
A Red PEO
o 6
IL 4
re 0
d) 2. -
Q_
~
t
h.
¦
¦
0 hrs
24hrs
48hrs
Acute Toxicity: Percent dissolved metals at beginning
(0 hrs) and end (48hrs) of acute toxicity tests for a 7.5 nmol/L
QD solution
_ 100
re
| 80
¦g 60
1 40
w
Q 20
86
61
27
0 hrs
34
15
Cd Zn
Green MUA
Cd Zn
Red MUA
Cd Zn
Green PEO
Cd Zn
Red PEO
0 hrs (mg/L)
BDL
0.014
BDL
0.006
BDL
BDL
BDL
0.013
48 hrs (mg/L)
0.222
0.044
2.3
0.083
BDL
BDL
BDL
0.045
Acute Toxicity: MUA
QDs
0.8
¦e
0.6
m
0.4
&
0.2
0
5 10 15 2D 25
QD Concentration (nmol/L)
-
~ .10 nm
2 3 4 5
QD Concentration (mg dots/L)*
~
Equivalent Cd Concentration (mg/L)
Equivalent Zn Concentration (mg/L)
3
-------
8/11/2009
MUA Toxicity Discussion
Toxicity seems to be a mass based
phenomenon
Dissolved metals present at 48 hrs
(i.e. MUA QDs release metals)
There is enough Cd to cause
observed death (not enough total Zn)
Rate of metal release is important
Acute Toxicity: PEO QDs » .
0.5 1 1.5 2
Equivalent Cd Concentration (mg/L)
0.5 1 1.5 2
Equivalent Zn Concentration (mg/L)
PEO Toxicity Discussion
Toxicity seems to be a particle number phenomenon
Distinct differences in toxicity are observed when
toxicity curves are plotted on a mass basis (smaller
QDs are more toxic)
No detectable dissolved metals found in solution at
48 hrs, yet toxicity is observed
- If metal toxicity, metals must be released in the
daphnid gut
Cd is not completely bioavailable, as dissolved Cd is
more toxic than both PEO QDs on an equivalent Cd
basis
Dissolved Zn is potentially the toxic agent for the
Red PEO QDs, as the two dose-response curves
overlap
Acute Toxicity: Conclusions
1 Stability has a strong influence on QD toxicity
- The stability & toxicity may be related to impurities more
than the actual QD core/shell
1 Dissolved Cd can explain observed toxicity for MUA
QDs
1 However, no dissolved metals in PEO QDs at 48hrs
suggests an alternate pathway
- Metals are released after QDs are ingested
- Toxicity due to the particle
- Impurities in the QD stock solution
Uptake & Metal
Availability: |_i-XRF
Brookhaven National
Light Source
Red: Ca Blue: Zn Cyan: Se
Future Work
Stability Experiments
- Aggregation experiments with model environmental colloids
- Further dissolution rate experiments with more well-defined
QDs
Toxicity Experiments
- Sub-lethal tests with D. Magna at low QD concentrations
• Feeding and fecundity endpoints
- Tests with benthic species (H. azteca)
Characterization
- Further application of FFF-ICP-MS to QD synthesis through
collaborations with QD researchers
4
-------
8/11/2009
Funding Acknowledgements
DOE ERSP Grant: ER64419
Edna Bailey Sussman Fellowship
Student Investigators
FFF work
Sungyun Lee
Emily Lesher
Tox & Stability
Jesse Forth
Heather Pace
5
-------
Effect of surface coatings on the
fate of NZVI and Fe-oxide NPs
Gregory V. Lowry
Associate Professor
Carnegie Mellon University, Pittsburgh, PA
Deputy Director
Center for Environmental Implications
of N anotechnology
EPA STAR Grantees Meeting
November 19-21, 2008
Collaborative Effort
Robert Tilton-CHE/BME
Ned Minkley-Microbiologist
Pedro Alvarez-Env. Eng. and microbiologist
Chris Kim-geochemist
Ph.D. students
Nanomaterial Sources
Finished Products
Agriculture
Manufacturing
Nanoparticle-based
Groundwater Treatment
Polyaspartate coating Decreases
ROS and Cytotoxicity in Glial
Cells (BV2) and Neurons (N27)
Fluorescent and chemiluminescent probes to measure OS-specific endpoints
OxyBURST® H2HFF Green BSA—-H202
Lucigenin and MitoSOX™ Red—O2"
MitoTracker® Red-membrane permeability/mitochondrial respiration
ENLITEN-intracellular ATP
o ° NPs:l-120ppm
DMEM and RPMI
¦ Intracellular Probes
Long et al. (2006). ES&T 40 (14) 4346; Phenrat et al„ ES&7(in press)
1
-------
Coatings Decrease OS response by Microglia
(BV2) and Cytotoxicity to Neurons (N27)
N27 Cytotoxicity : fresh nZV! > SM-RNIP > "aged" nZVI « magnetite
ROS : fresh nZVI > "aged" nZVI38 magnetite > SM-RNIP
Fate of NZVI and effect of coatings on
mobility and interaction with bacteria
Partially oxidized Fully oxidized
Surface Modified NZVI
Phenrat et al., ES&T(in press)
Key Questions
What is the oxidation rate of NZVI in the
environment?
- Geochemical effects
- Microbial effects
What is the fate of the coatings?
- Resistance to desorption
- Effect on mobility
Do aging and coatings affect bactericidal
properties?
Is there synergy between NZVI, coatings, and
bacteria that enhance remediation?
- Coatings as a carbon source
Functionalized Reactive Nanoiron (NZVI)
Nanoiron (RNIP)
Surface modifiers
~
TCE + Fe° HC Products + CI + Fe2+/Fe3+
Liu, Y., Lowiy, G.V. et al, (2005) ES&T 39,1338
Liu and Lowiy (2006) ES&T 40,6085
Saleh et al., 2005 Nano Lett. 5 (12) 2489.
RNIP
Modifier
C, potential
(mV)
Average Dia
(nm)
RNIP (none)
-29.6±2.8
146±4
PAP (MW=2.5k)
-51.7±0.4
32.6±18.6
PSS (MW=70k)
-48.9±1.5
31.1±16.6
Slow desorption of
polymeric surface modifiers
Objective: investigate the rate and extent
of desorption of adsorbed polyelectrolyte
from NZVI over a 4-month period
¦ Effect of molecular weight
¦ Effect of the type of surface interaction
(specific vs. non-specific)
Kim et al., 2009 ES&T 43 (10) 3824.
Methods
¦ PAP (2.5K, 10K), PSS (70K, 1M) and CMC (90K, 700K)
were adsorbed to RNIP for 5 days in an end over end
rotator at 30rpm.
¦ Adsorption and desorption :
Analyzed by UV-Vis absorbance (PSS) or total organic
carbon (CMC, PAP) to determine the adsorbed mass (mg
adsorbed polymer/m2 of NZVI) and desorbed mass over 4
months.
¦ Transport of aged materials:
- 12.5-cm saturated silica sand column using 1 g/L NZVI
(6=0.33, vave =1.08 x 103 m/s)
- Compared mobility of freshly modified particles and after
8 months of aging
2
-------
Desorption of Polyelectrolytes from NZVI
Coating
Adsorbed
mass
(mg/m2)a
2 weeks a
(% remaining)
4-6 weeks
(% remaining)
8 weeks a
(% remaining)
16 weeks®
(% remaining)
\
PAP2.5K
0.85+0.2
90.9 + 3.0
86 + 4.7
81.7 + 5.5
73.9 ±8.1
PAP10K
1.47+0.1
94.5 + 2.3
91.5+2.5
ESEBBbIII
87.2 ± 2.8
Kg2£|^jf|
PSS70K
93.5 ± 0.6
PSS1M
2.55+0.5
95.1 ± 4.7
CMC90K
2.09+0.0
87.6 + 2.1
83.1 + 2.9
81.1 + 3.1
79.7 ± 3.3
CMC700K
3.71+0.4
igJCt:g^83Ei
igjMg»a
90.4 + 0.9
89.6 ± 1.2
Kim etal., 2009 ES&T 43 (10) 3824.
Rate of Desorption
Kim et al., 2009 ES&T 43 (10) 3824.
PAP
40 60 80 100 12(
Time (day)
PSS
0.00 1
Lrapid
0.05
0.10
. PAP10K
__ slow
0.15
0.20
PAP2.5K
0.25
0.30
0.35
40 60 80 100 120
Time (day)
Coatings Affect Bactericidal
Properties of NZVI
Objectives: Determine how the following
conditions affect the toxicity of NZVI
¦ Polymer and NOM coatings
¦ Oxidation state of NZVI
¦ Environmental conditions
- (aerobic or anaerobic)
Summary of Findings Thank You
¦ High MW coatings do not readily desorb from
NZVI ¦ Questions?
- <30% desorbed after 4 months
- Rate is a function of MW and interaction with surface
- NZVI remains potentially mobile after 8 months
compared to bare NZVI
¦ Coatings and 02 decrease bactericidal effects
- Coatings inhibit NZVI contact with cells
- Presence of DO has greater effect on toxicity than
oxidation of particles (Fe° content)
¦ possibly due to surface passivation of the particles
3
-------
Center for Environmental Implications
of NanoTechnology
® 4 Core Institutions: Duke, CMU, Va Tech, Howard
® U Kentucky, Stanford, Rice University, NC State,
Colorado School of Mines, Clemson
® 5 years-$14.4 M from NSF + EPA
® 25 faculty currently funded
® 17 International partners on 3 continents
® Collaborators with 5 US government entities
4
-------
Bioavailability and Toxicity of Nanosized Metal
Particles Along a Simulated Terrestrial Food Chain
Fate, transport, and implications of trophic transfer of
manufactured nanoparticles in the environment
Pis: Jason Unrine1, OlgaTsyusko1, Paul Bertsch1, Andrew Neal2
Postdocs: W. Aaron ShouIts-Wilson1, Simona Hunyadi1-3
Graduate Student: Jonathan Judy1
Undergraduate Student: Alison Willis4
1.University of Kentucky, Department of Plant and Soil Sciences, Lexington, KY
2.Rothamsted Research Center, Harpenden, UK.
3.Savannah River National Laboratory, Aiken, SC
4.ToxicoIogy Excellence for Risk Assessment, Cincinnati, OH; Antioch College,
Yellow Springs, OH.
Grant No. 9732138.
Research Experiences for
Undergraduates
HIGHER TROPHIC LEVELS
insec+ivores omnivores herbivores carnivores
s
Overall project objectives
® Determine interactions between particle size
and particle composition in determining ADME
and toxicity in earthworms and amphibians.
® Investigate the plausibility of nanomaterial
trophic transfer along a simulated laboratory
food chain.
® Determine if simulated environmental and
biological modifications influence
bioavailability and toxicity^-ggJB
Hypotheses
® Nanomaterials have relatively low
bioavailability in soils.
® Uptake from soils, toxicity and
distribution of nanomaterials within
organisms is size and material
dependent.
® Biological responses are related to
release of metal ions.
Approach
® Focus on both mechanistic
and ecologically meaningful
endpoints.
® Focus on exposure scenarios
that are more environmentally
relevant.
COMMUNITY
POPULATION
INDIVIDUAL
® Systematically address
structure activity relationships.
ORGAN SYSTEM
Tissue
NANOPARTICLE
CELL
MOl LOU 1
Structure Activity Relationships
Cu° +0.34
Ag° +0.74
Group 11
"Noble Metals"
-------
Test Materials
Test material characterization
® Au (4, 18, 20 and 55 nm colloidal
spheres; HAuCI4; citrate capped).
® Ag (20 and 55 nm colloidal spheres;
AgN03; citrate capped).
® Cu (20-40 nm and <100 nm powder;
CuS04; Sigma).
DJ
4 nm 18 nm
(! AF4-UVA/IS-DLS-ICP-MS
« Primary particle and agglomerate size.
(! TEM
« Primary particle shape and size
(! ICP-MS/XRF
« Particle purity and concentration
a Ion Chromatography
« Surfactants
(! XANES
« Oxidation state
$ PALS
Electrophoretic mobility
Phase 1 - Small Au particles
Artificial soil mesocosms
Eisenia fetida
68 % silica sand
10 % peat moss
20% kaolin
2 % limestone
35% moisture content
|| l|S llfi ||S
"xio HH9 "xn "US
Laser Ablation-ICPMS
300 g of soil
10 animals per
Exposure concentration
g 3.5 mg kg-1
Laser Ablation Cell
Spot Size ~ 5-400 pm
Uptake of nanoscale Au in Eisenia fetida
3.5 nm 10 ppm Au, Au 197
500 1000 1500 2000 |
Distance X (urr
18 nm 10 ppm Au Au 197
0 200 400 600 800 10001200 14001600 1800
Phase 2 - earthworm subchronic
toxicity and reproduction
3-way completely
randomized design xio
Particle size - 2 size classes (20 and 55 nm or 20-40 and <100)
Particle concentration - 3 nominal concentrations (5, 20 or 62 mg kg-1)
Particle composition - 3 compositions (Cu, Ag or Au)
Controls - citrate, H20, HAuCI4, CuS04, AgN03
Replicates - 3 mesocosms, 10 animals per mesocosm
Total mesocosms = 87
Total treatments = 29
Total animals - 870
-------
Earthworm Mortality (28 d)
28 days
(Sacrifice adults)
28 more days-
Count offspring
Exposure
Mortality Reproduction
characterization
Growth
Gene expression
Accumulation
In situ characterization
Soil concentration mg kg-1
Soil concentration mg kg-1
Gene expression - Q-RT-PCR
<• Metal homeostasis
< Metallothionein
<• Oxidative stress
< Catalase
< Superoxide dismutase
< Glutathione peroxidase
<• Molecular chaperones
< Heat shock proteins 60 and 70
< Ubiquitin
<• Housekeeping gene
• Beta-actin ¦¦¦¦¦¦¦
3
-------
mtl expression -Ag
1
- Ag 20 nm
Ag 55 nm
Ag ion
0.5
"It 11
T
0
¦0.5
Tlnl
ht
¦1.5
- I
1
-2
¦2.5
-3
1
|
*5ppm
¦ 20ppm
¦3.5
-4
*62ppm
mtl expression - Cu
20-40 nm Cu
<100 nm Cu
jm
bslv-- FF— §F—
Copper K-edge XANES
20-40 nm Cu powder oxidation
T = 10 months
4
-------
Cu oxidation process
~
~
~
<1
>10 i
min
20-40 nm Cu
@ 10 months
<100 nm Cu @
10 months
<100 nm Cu powder oxidation
T = 1 month
T = 10 months
LCF weights
-<=—=J
Cu I = 0.488
Cu II =0.151
Cu 0 = 0.360
LCF weights
Cu I =0.476
Cu II =0.456
Cu 0 = 0.068
<100 nm Cu in blood vessel
Fluorescence
20-40 nm Cu exposed worm gut
Cu Ka
Fluorescence
LCF weights
Cu I =0.656
Cu II = 0.256
Cu 0 = 0.088
LCF weights
Cu I
Cu I
Cu 0 = 0.000
0.000
1.000
Distribution of 55 nm Au in the
earthworm cross-section
Au La Zn Ka
-------
6
Outflow
(to detector)
nflow Inflow
Exploded View
Channel
Flow
Membrane
Crossflow
FI-FFF-UV/VIS-DLS-ICPMS
Wyatt Technology
Corp.
- Upper Platel
-------
'20 nm Au
exposed
¦20 nm Au
standard
2000 3000 4000
Retention time (s)
Abundance
Ion 197.OO (196.TO t
O 1,07.70): DATA2.D
35000
Ion 197.OO (196.TO t
O lj|t7.TO): DATA3.D
30000
20 nm Au
/ I lT \ 20 nm Au
25000
20000
1 \ 1 \ + BSA
10000
5000
5.00 10
OO 15.OO 20.00 25
OO 30.00 35.OO 40.00 45. OO
Future directions
c< Determine uptake and elimination rates
in earthworms.
c< Toxicity of smaller particles at higher
concentrations.
c< Further develop methods for in situ
characterization of particles/metals in
soils and tissues.
c< Add another trophic level (amphibians).
PUBLICATION
Jason Unrine, Paul Bertsch and Simona Hunyadi. 2008. Bioavailability, trophic
transfer and toxicity of manufactured metal and metal oxide nanoparticles in
terrestrial environments. In Nanoscience and Nanotechnology: Environmental and
Health Impacts. Vicki H. Grassian, Ed. John Wiley & Sons, Hoboken, NJ. pp 345-
360.
Acknowledgements
ft
•William Rao
•Antonio Lanzirotti
•Diane Addis
•Phillip Williams
•Travis Glenn
-U£tPA.Sc»nc«T«A
Atiulu ffTAJtyProgni
Grant - ESSS
• Portions of this work were performed at BeamlineX26A, National Synchrotron Light Source (NSLS), Brookhaven
National Laboratory. X26A is supported by the Department of Energy (DOE) - Geosciences (DE-FG02-92ER14244 :
The University of Chicago - CARS) and DOE - Office of Biological and Environmental Research, Environmental
Remediation Sciences Div. (DE-FC09-96-SR18546to the University of Kentucky). Use of the NSLS was supported
by DOE under Contract No. DE-AC02-98CH10886.
•This material is based upon work supported by the National Science Foundation under Grant No. 9732138. Any
opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and dc
not necessarily reflect the views of the National Science Foundation (NSF).
-------
PI: Paul M. Bertsch
Co-PIs: T. Glenn, A. L. Neal, P. Williams-UGA,
B. P. Jackson-Dartmouth College
Collaborator^ J.M. Unrine-UK, P. J. Morris -MUSC, J. Grider-UGA
Post doc; N. J. Kabengi- UK
Ph.D. students: H. Ma-UGA & B. A. Neely-MUSC
Fate, transport, and, effects of manufactured
nanoparticles in the environment
HIGHER TROPHIC LEVELS
insec+ivores omnivores herbivores carnivores
s
DETRITIVORES
^^MICROBIOLOGY {¦§
I I
SEDIMENT GEOCHEMISTRY
sorption
and aging
effects
OBJECTIVES to evaluate:
One: the bioavailability and toxicity of manufactured nanoparticles (ZnO-np as a
function of particle size to model soil bacteria (Burkholderia vietnamiensis and
Cupriavidus necator) & the model detritivores Caenorhabditis elegans and Eisenia
fetida referenced against aqueous Zn2+ ions and ZnO-bulk
Two: the ability of manufactured ZnO to be transferred from one trophic level to
the next as assessed in the simple food chain consisting of pre-exposed B.
vietnamiensis & C. elegans
Three: the synergistic or antagonistic effects of manufactured ZnO-np on the
toxicity of Cu2+ to C. elegans.
HYPOTHESES
1: The bioavailability and toxicity of manufactured NPs increases with
decreasing particle size (i.e. 2 nm vs. 80 nm)
2s The toxicity of ZnO-np to model soil bacteria and C. elegans is lower than an
equivalent concentration of dissolved Zn2+
3: The bioavailability and toxicity of NPs introduced via trophic transfer differs
from direct exposure
4: ZnO-np alter the bioavailability and toxicity of dissolved metals
Characterization - the critical first phase
BET SA=5.7 m2/g
ZnO nanoparticles
• Versatile nanomaterial
• inexpensive to produce
• Found in pigments,
rubber additives,
sunscreens, personal
care products,
biological and chemical
sensors, varistors,
transducers,
photoelectrodes, and
catalysts
Crystalline model of ZnO with acetate
coordinating a Zn atom (adapted from Wang
2004)
HR-TGA-Air dried ZnO-np
4X3 ax
Inrrmrim£Q—
175.44 °C 1st Acetate population
4
600 *000
lataBBBttMH
1
-------
TGA-Air dried ZnO-np
Characterization; Acetate Importance
189.72^°C 2nd
Acetate population
4
I 50 rim I
9
~ I
\
I— 50 n
4
0
£ I
2
o I
a I
4
8
> I
3 I
if,
o I
1500 mg L*1 Zn
Methylene
Blue assay for
ZnO surface
reactivity
Recent results:
Toxicity of ZnO-np
to C. elegans under
UV irradiation can
be predicted from
MB assay
2
Characterization: Changing nature - artifact of TEM observation
Kabengi , Bertsch, Shields and Wu
O min 9 min 14min
-------
Nanoparticle-Bacterium Interactions -
B. vietnamiensis PR1
> Acetate concentrations
were normalized to 10.7
mM across conditions.
> PR1 was unable to grown
with 10.7 mM acetate at pH
5
> Growth unaffected by:
100 mg L1Zn at pH 6
25 mg L 1 Zn at pH 7
Aquooui Zjn'-
163 >96 ntg L '
188 6 * 8 !>mg L '
Aqiwouv 2n'J
96 6 * 14 1 mo L
104 4 * 13.5 log L '
y Calculated EC50 based on OD610 at 24
hours, using 0 mg L1 metal as a
reference
> EC50 of Zn2* and ZnO-np values are
not sig. different at pH 6 or 7
Acetate and Lactate Utilization
10.7 mM acetate
pH 6
10.7 mM acetate
PH 7
/ -5
_o_ lactate
X
. acetate
-> OD610
\ . .
10 15
Time (h)
1.6
1.4
1.2
1 ?
0.8 cT
0.6 0
0.4
0.2
0
Floe formation in
bacterial culture
A V At 250 my L < ZnO-np (pH6),
significant flocculation of primary
particles at 24 hours associated with
cell growth
> Floes do not form at 250 mg L1 ZnO-
np in the absence of PR1 suggestive of
biological induction
> Floes could result from acetate
degradation and/or exopolymer
secretion
3
• Acetate and lactate are 95% depleted by 12 h and had
similar utilization patterns
• Degradation of acetate (NP counter-ion) may affect NP
stability
-------
Nanoparticle-
Bacteria interactign
Cupriavidus necator-metal sensitive
> No significant difference between Zn2+aq and ZnO-
np growth rates
> Higher OAc utilization rates with Znz+aq compared
to ZnO-np
> Evidence for bioavailability ofZn ion, but not
ZnO-np
>Epifluorescence microscopy indicates an
increased number of cells with compromised
membranes associated with ZnO-np vs. free ion
PKUTJJUMICS Z-D gels- Arthur Grider - UGA
0.1 uM Zn Ac versus 0.1 uM ZnO np
Acidic Side Basic Side
Acidic Side Basic Side
i i
I I
I 11 s
n
I If \
o
n
i i
O
i i
0.1 i M Zn Ac
0.1 |xM ZnO np
Behavior & Lethality C. elegans exposed to ZnClj
and ZnO-np
Concentration-response relationships for reproduction
of C. elegans on exposure to ZnO-np (~) or ZnCI2 (~)
(error bar denotes standard error, n=3): EC50(ZnO-
np)= 53 mg/L Zn (0.8 mM); EC50(ZnCI2)=60 mg/L Zn
(0.91 mM).
Genome-enabled environmental analysis-bioavailable metals
Caenorhabditis elegans
transgenic mtl2::GFP
H. Ma, T. Glenn, and P. Williams
GFP reporter fused to
mtl2 promoter
Simple organism (approx.
959 cells), genome
sequence complete
Feeds on bacteria and
other particles < 5 pm in
diameter
4
-------
ZnO-np vs. Zn2+ exposed nematodes
>
>
Effects of ZnO-np and ZnCI2 on Cu toxicity
Effects of ZnO-np orZnCI2 on Cu toxicity: mtl-2
expression in transgenic organisms
Toxicity of larger ZnO-NP (40-1 OOnm): 4h lethality
Initial experiments on trophic transfer of ZnO-np
3500
3000
2500
Direct Exposure
1
1—| D ZnO-np
¦ Cd
1500
1000
£ 500
n "1 1
n n :
oT 0
LL
o
Blank Control 25 50
100 100 250 500
3500
3000
2500
Exposure through PR1
C3 ZnO-np
¦ Cd
2000
1500
1000
500
n n n i n 1 n n n
Blank Control ^ ^
00 100 250 500
mm
[Metal] in fj.M
Earthworm exposure to ZnO-np in artificial soil
> Bioaccumulation of Zn in Eisenia fetida after
14 days of exposure to artificial soil
containing 1000 mg Kg1 Zn
> No difference in Zn concentration between
treatments
XRF-
Maximum Zn intensities are
independent of exposure
concentration
Areas of maximum intensity
are more evenly distributed as
exposure concentration
increases and at lower
concentrations for ZnO
nanoparticle exposed worms
The maximum intensities in
ZnCI2 exposed nematodes are
approximately twice as high
as in ZnO-np exposed
nematodes and there is more
even GFP expression for
ZnCI2, suggesting differential
bioavailability or tissue
dependent reactivity, e.g.,
dissolution.
-------
Summary and Major Conclusions
Characterization
> Size determination and
surface chemistry is a critical
issue
> TEM may not be the best
method for size determination
for small metal oxide
nanomatierals
> Acetate controls 1-2 nm ZnO-
np reactivity, passivates
surface sites; not so for bulk
(1.2 pm) intermediate for
larger (~80nm) particles
> Removal of acetate leads to
flocculation/ aggregation of 1-2
nm ZnO-np primary particles
but promotes surface
reactivity
> No difference in growth rate
between ZnO-np & Zn^aq)
for C. necator and B.
vietnamiensis PR1301
> Higher OAc utilisation rates
with Zn^compared to
ZnO-np in Cupriavidus
necator
> Evidence for Zn
bioavailabilityfrom Zn ion,
but not ZnO-np
>Cells with compromised
membranes associated with
ZnO-np compared to free ion
> Different mechanism(s) of
toxicity ?
Trophic transfer in bacterial-nematode model challenging
ZnO-np are bioavailabie from soils as demonstrated
in earthworm exposures
Manuscripts
H. Ma, P.M. Bertsch, T. C. Glenn, N.J. Kabengi, P. L. Williams. In press. 2009.
Toxicity of manufactured zinc oxide nanoparticles in the nematode
Caenorhabditis Elegans. Environmental Toxicology & Chemistry.
DOI: 10.1897/08-262)
H. Ma, N.J. Kabengi, P.M. Bertsch, J.M. Unrine, T. C. Glenn, P. L. Williams. In
Review. Phototoxicity of nanoparticulate ZnO under natural sunlight
irradiation in the nematode Caenorhabditis Elegans. Environmental
Science and Technology
B. A. Neely, D. W. Bearden, A. J. Sutter, N. J. Kabengi, P. M. Bertsch, and P. J.
Morris. 2008. Toxicity of engineered ZnO-NP to Burkholderia
vietnamiensis PR1301: Comparison to Zn2+ and the affects of counter-
ion utilization. Environmental Toxicology and Chemistry /In Review)
J. Unrine, P.M. Bertsch, and S. Hunyadi. 2008. Bioavailability, trophic
transfer, and toxicity of manufactured metal and metal oxide
nanoparticles in terrestrial environments. Pp. 343-364 inV.
Grassian (editor) Nanoscience and Nanotechnology, John
Wiley & Sons, Inc.
Manuscripts in preparation on:
The spatial distribution of Zn and metallothionein expression in C. elegans
exposed to dissolved Zn2+ and ZnO nanoparticles
Toxicity of ZnO-NP and Aqueous Zntothe Soil bacterium Cupriavidus
necator.: A Protoemics approach.
6
-------
Bioavailability and Fates of
CdSe and Ti02 Nanoparticles
in Eukaryotes and Bacteria
P. A. Holden
Bren School of Environ. Sci. & Mgmt., University of CA, Santa Barbara
J. L. Nadeau
Dept. Biomedical Engineering, McGill University
G. D. Stucky
Dept. Chem. & Biochem., Materials Research Laboratory, University of CA, Santa Barbara
UCCEilN
Cprets* for fcnvlrorvi*^ if
-------
Why Bacteri
i
rid ant:
diverse
Abundai
Biodi
Catalysts
m
Report of 2 Subprojects
1. Effects and fates of Cd(ll) vs.
CdSe QDs in P. aeruginosa
2. J\02 interactions w/ P. putida:
aggregate stability.
1. CdSe QDs: Cell labeling
Mammalian A9 cells w/ B. subtiliswl yellow Adenine auxotrophic E.
green QD-dopamine. QD-adenine. coli w/ green QD-adenine.
—~ Photoactivated uptake and fluorescence
—»¦ Conjugate and receptor mediated
—~ External binding prerequisite
—~Transient membrane damage
—~Cellular processing
—~Toxicity from Cd(ll)
(Kloepfer et al., 2003; Kloepfer et al., 2005; Clarke et al., 2006)
Labeling-inspired questions:
• Is light necessary?
• Are bare QDs internalized?
• Is external binding prerequisite?
• What are the quantitative fates of QDs?
• How are they toxic?
P. aeruginosa growth depends on dose.
Cd(ll)
QDs
" 1 O 20
to mQiL
• Control
O lOmgA.
O J* i mild.
H i"
o» • * 0 a,a
0 0 0.0
* a&* *
looflSSi^ .
0 S 10 1$ JO » » M
Time pi]
10 20 10 40
Tims [h]
QDs dissolve quickly,
1*
1*
i»
£»tf.Sqitt*0.1Q
J
8 M
5
•
•
i:l
....but incompletely.
» « It H It) 050- ^
TtawN
f«
5
I"
S 034
3
ae ojo
•
03$
Dissolution was greater in sterile con to Is.
» «e 60 SO 100 120 140
TeUI CO Cortcomritton [«»!]
2
-------
QDs add to Cd(ll) toxicity, above a threshold
Cd(ll| at E h ImgfL)
Above the threshold;
membrane damage in QD-grown cells.
Control Cd acetate -treated CdSe QD -treated
A B
500 nm 500 nm
500 nm
Above the threshold:
intracellular ROS in QD-grown cells.
O Cd »c»tAte - tfoated
«
z
a
• CdS* QO - ir**t*d
T
a
c 60
» «?
gS
1 .
1 • , o
40
1 |
=
5 s
e?
£i~20
• 1 1
1 t I
i
ijl
£
, s - i'
20 40 60 eo 100 1J0 140
Total Cd Concentration [mg/L]
(Method: DCFH-DAto dichlorofluorescein, DCF).
Above the threshold: metals in QD-grown cells.
Cd, Se and CdSe rich-regions
Se oxidation state: "tracer" for QD integrity
1.6
XANES A
A '''
Cellular selenium appears to be
IV, se (0)
mostly Se(0), with some organo-
*8
11
< complexes
If \ X _ ^
However:
1
f \/~
—» STEM suggests intact QDs
0,
J
—»XRD supports (wurtzite)
„ ™ ™ ™ ™ ™ „
Metal / loid fates w/ cells
0.15 pg cadmium/cell (dissolved, mostly)
- 4% of administered
- 4600X enrichment
0.0083 pg QD-cadmium / cell
- Based on 200,000 QDs / cell
- 30X enrichment
3
-------
1. Summary
• QDs appear more toxic than Cd(ll)
- Above a threshold
- Related to ROS
- Sorption to membrane not a prerequisite
• Pseudomonas alters fate of QDs
- Intracellular: QDs appear mostly broken down
- Extracellular: QDs are relatively stabilized
2. Titanium Dioxide NPs
P25 (25 nm) LST (16 nm)
i\ \
100 nm
1 ¦
•
2. How do agglomerates disperse?
HI: Blosurfactant mediated
H2: Metabolism of chemical linkers
H3: Preferential binding to cells
4
-------
Ongoing and future
• Mechanisms: what's behind the
observations of bacteria and QDs?
• High Throughput Screening (HTS): where
are there transferable paradigms?
• Scaling up: how are soil ecosystem
processes and soil biota affected?
ucceiN
holden@bren.ucsb.edu
Acknowledgements
People
- John Priester
- Peter Stoimenov
- JP Zhang
- Chris Ehrhardt
- Randy Mielke / J PL
- Sam Webb/SSRL
- Allison Horst
Funding
- U.S. EPA
- U.S. DOE (DE-FG02-06ER64250)
- UC Toxic Substances Research & Teaching, Nanotoxicology Lead
Campus Program
- NSF/EPA UCCEIN
5
-------
Office of Research and Development (ORD)
National Health and Environmental Effects
Research Laboratory (NHEERL)
Manufactured-Engineered Nanoma ferial
Health Effects Research Program
Kevin Dreher, Ph.D.
Science Lead, Nanomaterials/Nanotechnology Health Effects
U.S. Environmental Protection Agency
National Health and Environmental Effects Laboratory
Research Triangle Park, NC
d re her. kevi n@epa. gov \ ^
Interagency Environmental
Nanotechnology Grantees
Workshop
November 19 - 21, 2008
Tampa, FL
ORD, NHEERL Manufactured-Engineered
Nanomateria! Health Effects Research Program
ORD Research Themes:
> Sources, Fate, Transport, and Exposure
Risk Assessment Methods and Case Studies
Preventing and Mitigating Risks
?/C/
ORD, NHEERL Manufactured-Engineered
Nanomateria! Health Effects Research Program
Categories of Nanoparticles
Anthropogenic
I 1
I Envii
Natural;
Biological
Environmental
Occupational |
\
Engineered
~it
fijM
111
Laboratory
R&D
ffiEflanm
Manufactured
ORD, NHEERL Manufactured-Engineered
Nanomateria/ Health Effects Research Program
Research Implementation: "Nano" Health Effects Team
¦ Team's Vision: Research for the Responsible
Development and Application of Nanomaterials
Leading to a Sustainable Technology
Team's Long Term Goals: 1) determine the health
effects of manufactured-engineered nanomaterials
and their applications; 2) establish
approaches/models/methods to quantify and predict
these effects/risks.
Team's Composition: 10 projects with 15
investigators from each NHEERL. Health Division
with expertise in the following areas of toxicology:
pulmonary, cardiovascular, neurological,
developmental, mutagenesis, cancer, reproductive,
and ocular.
Draft Development
"Safety for Success"
ORD, NHEERL Manufactured-Engineered
Nanomateria! Health Effects Research Program
"Mufti- Tiered Approach" for Nanomaterials
Health Effects Research
Tier 1
Physicochemical Characterization
Collaborations
-ORD, NCCT "ToxCast"
-NTP
Vim
Tier 2
In Vitro Toxicology ^
(Non-Cellular and Cellular Based Assays)
Common Set of Well
Characterized Manufactured-
Engineered Nanomaterials
Identify
and Validate
Alternative
In Vitro Tests
-Predictive Toxicity
-Design In Vivo
Testing ^
^ Tier 3
In Vivo Toxicology
1
-------
OR D, NHEERL Manufactured-Engineered
Nanomaterial Health Effects Research Program
Tier 1; Physicochemical Characterization
Supplier Contractor
Surface Surface
Commercial Area Area
Nanomaterial Source (m2/gr) (m2/gr)
Supplier Contractor
% Purity %Pufity
Supplier
Crystalline
Form
Contractor
Crystalline
Form
Ti02(25nm) DegussaP25 50+/-15 50.9
>99.5 >99
anatase
rutile
86% anatase
14% rutile
52.9
99.9
86% anatase
4% rutile
Nanostructured
Ti02 & Amorphous
(30-40nm) Materials Inc. >30 22.2
95 99.9
¦\T
rutile
Ti02 (10nm) AlfaAesar 100-130 118
| 99 98.8
anatase anatase
100 -130 101
| 99 98.8
| anatase anatase
100 -130
99 97.3
anatase
anatase
Ti02(32nm) AlfaAesar 45 49.8
99.9 97.9
anatase
M
45 41.5
99.9 99.1
anatase
anatase
Ti02
(200-400nm) Mknano 6.8
99.97 98.7
rutile
rutile
not
Ti02 (200nm) Acros provided 6.99
not
provided 99.9
anatase
anatase
ORD, NHEERL Manufactured-Engineered
Nanomaterial Health Effects Research Program
Current List of Monufac tired- Engineered
Nanomateriais For Health Effects Research
NHEERL
EPA and
OECD
NMs
ORD NRS
List of
Nanomaterial
Health |
Ecology |
Federal Offices
List
NMs
SWCNT
MWCNT
X X
X X
OSW;
OPPTS;
Yes
Yes
TiOz
X
X
OSW; OW;
OPPTS; OAR;
NT?; NIOSH
FDA
Yes
Yes
CeOz
X
OTAQ; OAR;
ORD/NCEA;
Yes
Yes
Zero Valent
Iron
X
X
OSWER;
OW
Yes
Yes
Ag
I
J
OPP; OW,;
CPSC;
FDA
Yes
Yes
ORb, NHEERL Manufactured-Engineered
Nanomaterial Health Effects Research Program
V
Non-Cellulcr
Assays
Tier 2: In Vitro Toxicology
Non-Cellular Assays: Biochemical Interactions - antioxidant depletion
(£SH; Vit. C); protein binding; Surface Properties - reactivity
(TBARS; ESR; DCFH); charge (zeta charge); aggregation (DLS;
SEM; TEM).
Cellular Models: Pulmonary toxicity (airway epithelial cells; alveolar:
macrophages and epithelial cells); Cardiovascular toxicity
(cardiomyocytes; endothelial cells); Liver toxicity (HepG2 cells);
Gastrointestinal toxicity (Caco-2; NCM460 cells); Neurotoxicity
(microglia and neuronal cells; astrocytes); Ocular toxicity (lens and
retinal pigment epithelial cells).
Cellular Endpoints: Growth; Cytotoxicity; Cellular Uptake; Oxidative
Stress; Altered Function (phagocytosis; cytokine production; barrier
permeability; etc.).
Strategic Information: 1) Ranking/Design - LC50 concentrations for
cellular and non-cellular endpoints to prioritize and design in vivo
testing. 2) Provide mechanistic and biokinetic data at the cellular
level; 3) Provide an iterative model to identify alternative testing
approaches.
ORD, NHEERL Manufactured-Engineered
Nanomaterial Health Effects Research Program
Summary
1. ORD Nanotechnology Research Strategy has been developed to addresses
the impact and research needs which nanotechnology has on the Agency (US
EPA Nanotechnology White Paper, 2007).
2. To address some of the challenges associated with assessing the health
effects of manufactured-engineered nanomateriais ORD's strategy
incorporates:
--the National Academy of Sciences vision for "Toxicity
Testing in the 21st Century";
--a multi-tired approach for the screening of Agency
relevant nanomateriais to prioritize them for subsequent
in vivo testing, assist in their design as well as establish
collaborations (NCCT; EPA's 1st Nano Health Effects
CRADA; NTP).
3. The multi-tiered approach will be employed in a comparative and iterative
manner to identify in vitro assays that correlate with in vivo responses in
order to identify and develop validated alternative toxicity testing methods
for nanomateriais.
2
-------
Engineered Nanomaterial Ecological Effects
Research Within ORD's National Health and
Environmental Effects Laboratory
Steve Diamond
USEPA/Mid-Continent Ecology Division
for
USEPA/ Mid-Continent Ecology Division
USEPA / Western Ecology Division
USEPA / Western Ecology Division
USEPA / Western Ecology Division
USEPA/Western Ecology Division
USEPA/Atlantic Ecology Division
USEPA / Atlantic Ecology Division
David R Mount
Christian Andersen..
Mark G. Johnson ....
Paul Rygiewicz
David Olszyk
Robert Burgess
Kay Ho
RESEARCH * DEVELOPMENT
nullfflnp fj svimificfmm(iotUmfnr*(nmt emirmmcntal^kcti
United States Environmental Protection Agency
Office of Research and Development (ORD)
National Health and Environmental
Effects Research Laboratory
Human Health Ecology Divisions
Divisions MBHBi
RESEARCH ^DEVELOPMENT, -Jr
Hitllillrrg Evaluate current methods for assessing hazard
> Assess hazard for nanomaterials
y Identify nanomaterial characteristics that predict toxicity
>- Identify mechanisms of action, ADME (accumulation,
distribution, metabolism, and elimination)
>- To incorporate knowledge of production volume and potential
pathways of exposure, and to do so within a product
life-cycle framework
Interagency Environmental Nanotechnology Grantees Workshop
Tampa, FL, November19-21, 200S
RESEARCH K. DEVELOPMENT
RuildfijZSi scientificfoimttatlnwfartmnvl
EPA Mission: Protect human health and the environment.
NHEERL mission: help the Agency evaluate the risks that nanomaterials
pose to humans and ecosystems.
> Evaluate current methods for assessing hazard
> Assess hazard for nanomaterials
> Identify nanomaterial characteristics that predict toxicity
> Identify mechanisms of action, ADME (accumulation,
distribution, metabolism, and elimination)
> To incorporate knowledge of production volume and potential
pathways of exposure, and to do so within a product
life-cycle framework
Test guideline reviews initiated by OECD:
Organization for Economic and Cooperative Development
i
M Working Party for Manufactured Nanomaterials
Steering Group 4: Test Guidelines
^ Section 2: Biotic Effects (ecotoxicity tests)
Physical/Chemical Properties (Section 1)
Degradation and Accumulation (Section 3)
Health Effects (Section 4)
RESEARCH K DEVELOPMENT .
fii/Hding u scientific fmimlaUnn far smmt emfrf/wTtenral. i/cdiflWrff
RESEARCH K DEVELOPMENT
1
-------
Test guideline reviews initiated by EPA, Office of
Prevention, Pesticides and Toxic Substances (OPPTS):
850-Series test guidelines: Ecotoxicology
~ 50 test Guidelines reviewed
(24 are harmonized and reviewed for OECD)
~ aquatic, marine, sediment, soils, terrestrial direct contact
~ vertebrates, invertebrates, plants, microbes
~ acute and chronic exposure durations
Reviewers included all P.I.s for nanotechnology research at AED, MED, and WED
- also, ARCoE and USGS scientists
OPPTS TG Reviews Summary:
Adequate aspects
y Toxicological principles
> Endpoints
Inadequate aspects
> Media preparation
y P/Chem properties of materials
> Quantification of exposure
> Exposure m etiology
Inadequacies all related to particulate and fibrous nature ofnanomaterials
and the colloidal nature of exposure media
KESEARCfl «¦ DEVELOPMENT, _
Lzflnll3lttg,p$Cl
-------
NHEERL/Eco Nanotechnology Research
Additional efforts:
1) Involvement in OECD planning, review, and testing in
collaborations related to the Nanomaterials Sponsorship Program.
2) Continued collaboration with South Carolina University, Oregon
State Universities, ARCoE, USGS. Potential collaboration with
newly-funded nano centers.
3) Providing technical support to EPA regulatory offices.
Interagency Environmental Nanotechnology Grantees Workshop
Tampa, FL, November19-21, 2008
RESEARCH It DEVELOPMENT ¦
-ftMWiWd MitolffrcfmirnlfflMn fat'Mtnd rtnirtifmaTtalrf&tflMf
3
-------
$
RICE
CBEN
Microbial Impacts of
Engineered
|| Nanoparticies
Shaily Mahendra,
Delina Y. Lyon, Dong Li,
Mark Wiesner and Pedro J. Alvarez
EPA Project R832534
nC60: The Environmentally Relevant
Fullerene
Solid or solution nC60
* Highly stable water suspension
J Negatively charged surface
/ Sizes range, typically 30-300 nm
Important form of C60 in the aqueous environment
nC50 is a Potent Antibacterial Agent
Ct (ma/I * min) for 99% kill:
• 0.03-0.05 for free chlorine
• ~ 100 for nC60
• 95-180 for chloramines
0 10 20 30 40 50 60
Time (min)
Bacteria
Description
MIC
(mg/L)
Bacillus
subtilis
Gram +, soil
0.001 -
0.025
Streptomyces
albus
Gram +, soil
<0.05
Pseudomonas
aeruginosa
Gram soil
0.05 -
0.066
Ralstonia
pickettii
Gram
pathogen
0.025 -
0.0375
Burkholderia
cepacia
Gram
pathogen
0.0125
-0.025
Desulfovibrio
desulfuricans
Gram
anaerobe
0.1-0.2
Broad spectrum antibiotic
r. Sci. TechnoL, 2008
Bacterial Toxicity of Select
Nanomaterials
r*~~i
¦1
B
1
I
pin i-i
¦ ¦ i
TH F/n Cg0son/n Cg0 aq/nC60 PVP/nCg) Ag+
Adams et al.. Water Res, 2006
o-Ag NaOCI nZnO nZVI nT102 nSiOj
(sunlight)
Effect of nC60 Particle Size
nC60 Particle Size vs. Toxicity
B. subtilis MIC
(mg/L)
Average
Diameter (nm)
Surface
Area: Volume
"C60
0.75-1.0
100
0.06
>100 nm
particles
7.5-10
110
0.055
<100 nm
particles
0.01-0.1
50
{X2J
0.12
1
-------
Salts Promote Aggregation,
Decrease Toxicity
;E
Ionic Strength of Fresh Water
B 140 "
a>
I
Q 120
ffl
r
a. 100 ¦
sr"
c
S
80 v
20y]
/
0.00 0.02 0.04 0.06 0.08 0.10 0.1 2 0.14 0.1 6 0.1 8
Ionic Strength (M, NaCi)
NOM reduces nC60 Bioavailability
and Toxicity
0 10 20 30 40 50
Time (h)
Humic acid concentrations as low as 0.05 mg/L eliminate toxicity
Li et al. Environ. Toxicol. Chem. 2008
Bacterial Toxicity Mechanisms
Release of toxic ions
by QDs, nano-silver
Protein oxidation
by nC60
Disruption of cell
membrane by
SWNTs and
car boxyfu Here ne
I nterruption of electron transport and
protein oxidation upon contact by Ce02
Generation of
Reactive Oxygen
Species by Ti02
Does nC60 Produce ROS in Bacteria?
Hydroethidine
Oxidized by
superoxide to fluoresce
°2 O,-5^ H202^> OH- + OH-
Superoxide Hydrogen peroxide Hydroxyl radical
No Lipid Peroxidation due to ROS
o 10 •
•—
a.
o
•—
-
X
1
—1—
Control
nQo
/-BOOH
~ Chromogenic assay based on hydroperoxide reaction with ferrous
ions to make ferric ions
• no lipid perooxidation in nC60
-exposed samples (P
=0.15)
• fe/t-butylhydroperoxide is positive control
Lyon et al, A/a no. Lett., 2008.
Membrane Potential Collapse
¦'•""fes'"'"
Assay monitors DiOC2
- Red fluorescence indicates higher
membrane potential
Higher red/green ratio means higher
membrane potential
CCCP is an ionophore
DiOC2
Fluoresces green
in cells;
accumulates at
higher membrane
potential to
fluoresce red
Lyon and Alvarez, Environ. Sci. Technol. 2008.
2
-------
Changes in Reductase Activity
Redox Sensor™ Green
fluoresces after reduction
by live-cell reductases
nC60 (ORP = +483mV) hinders
normal oxidation-reduction activity,
similar to sodium azide (+ control),
which disrupts electron transport
Lyon and Alvarez, Environ. Sci. Techno!. 2008.
Healthy respiring cells
fluoresce (redox-sensor
green)
Direct Protein Oxidation
f-BOOH nCfin -Control
• Loss of thiol groups relative to control reflects oxidation
• 7erf-buty (hydroperoxide is positive (oxidizing) control
• Pure protein (bovine serum albumin) shows oxidation
Lyon et al, Nano. Lett, 2008.
Potential Application: Enhancing
UV Disinfection
• UV disinfection is
w^m
increasingly used to
yfe ro t» G—3V i
inactivate cyst-forming
protozoa such as Giardia
and Crystosporidium.
• However, UV is relatively
£ V*
ineffective to treat viruses
unless the contact time
and energy output are
significantly increa$ed
Virus inactivation by UV and Fullerol
A Fullerol in dark
\ v. y =-0.0071x
t \ R2= 0.48
* \
• \
UV alone
* \
y = -0.034x
N.R2 = 0.42
•\
~
\# Fullerol + UV \
3 x faster 4 y = -o.o967x
R2 = 0.48
0 20 40 60 80 100 120 140
Contact Time (min)
Hotze et al. Environ. Sci. Technol. 2007
Conclusions and Significance
• Ecotoxicology: nC60, ZnO,
Ti02, and nZVI can be toxic to
environmental bacteria, and
possibly higher organisms.
• Implications: Biodiversity
and food webs?
biogeochemical cycling?
mitigated by NOM and salinity.
• Applications: Water
disinfection, biofouling control
am
AitMiIng r!u Rlilu
0/Manufactured
NANOMATERIALS
3
-------
Acute and Developmental Toxicity of Metal
Oxide Nanoparticles in Fish and Frogs
Christopher Theodorakis
Southern Illinois University
George Cobb
Texas Tech University
Elizabeth Carraway
Clem son University
Metal Oxide
Nanoparticles
•Catalysts
•UV protectants (ZnO, TiO)
•Wood preservation
•Marine antifoulants
•Deodorants
•Polishing agents
•Glass
•Dental
•Semiconductors
•Antimicrobial
•Textiles
•Foot powder
•Coatings
Objectives
•determine the environmental hazard of
Fe203, ZnO, CuO, and Ti02
•acute and chronic toxicity
•fathead minnows (Pimephase promelas) and
African clawed frog {Xenopus laevis)
Hypothesis
•Nanoparticle exposure will affect the survival,
growth, development, egg hatchability, and
metamorphosis of these organisms
Approach
Flow-through exposure, nanoparticle
suspension in water
Xenopus laevis
Acute Study: FETAX Assay
¦ Xenopus laevis Definitive Test
¦ 3 replicates of 7 concentrations including a
control (total exposures = 21)
¦ 31.6,10, 3.16,1, 0.316 and 0.1 mg/L
¦ Control: FETAX solution
¦ FETAX solution: NaCl, NaHC03, CaC^, CaS04 2H20,
MgS04, and deionized or distilled water
¦ 10 embryos per exposure
Acute Study Results
¦ Growth
n Significant -Ap total body length at lOmg/L
¦ Mortality
¦ No mortality' observed
¦ Malformation
ii EC50 - 10 mg/L
¦ Developmental Dose Determination
¦ EC15 - 1.9 mg/L
¦ 2, 1, 0.5, 0.25, 0.125 and 0 mg/L
1
-------
. \ath' Z//0 Malformation Results
Typical Malformation Observed
Methods and Materials
¦ ZnO Nanoparticles
¦ AlfaAesar: NanoTek®
¦ 40-100nm APS
¦ Uses and properties
¦ UV protection
m Antimicrobial properties
¦ Maintains a high level of transparency in coatings,
polymers, caulks, adhesives and other resin systems.
Solutions were made by sonicating ZnO
nanoparticles in FETAX solution.
Flow-Thru Design
Water Chemistry
¦ pH, DO, conductivity,
ammonia, salinity
¦ Every 48 hrs
Zn Analysis
¦ Before new solution
¦ After new solution
¦ ~24hrs
Tissue Analysis
¦ End of study
Zn Analysis
¦ Thermo AA Series Spectrometer
¦ Flame Atomic Absorbance
¦ Solution
¦ Add 150 uL of concentrated HNOs to 30 mL
sample
¦ Tissue
¦ Freeze Dry for a minimum of 24 hrs
¦ Digest using EPA method 3050B
Electron Microscopy
¦ Scanning Electron Microscope (SEM)
¦ Hitachi S4300YP
¦ Size determination of nanoparticles
¦ Nanoparticle Preparation for Imaging
¦ Mount on SEM stub with conductive tape
¦ Hummer Y Sputter Coater
¦ ~5nm of gold-palladium alloy
2
-------
Endpoiiits for Developmental Study
¦ Mortality
¦ Time to Metamorphosis
¦ Growth
¦ SVL: Snout Vent Length
¦ TBL: Total Body Length
¦ HLL: Hind Limb Length
¦ NF Stage
Survival
ZhO Developmental Mortality
Control 0.125 0.25 0.5 1 2
ZhO Dose (mg/L)
: significant p-value<0.05 compared to all doses
Stage 66 Juveniles
Total Body Length and Hind Limb Length at Stage 66 for ZnO
Developmental Study
Control 0.125 0.25 0.5 1 2
Concentration (mg/L)
: significant p-value < 0.05 compared to control
3
-------
Methods
•Fathead minnow larvae (<24 hrs) were exposed to aquatios
suspensions of nanoparticles
• Fish were kept in reconstituted fresh water
•Water temperature was maintained at 21° C, photoperiod 18:6
day:night
• Static renewal design: Vi of the test solution was changed daily
• Nanoparticles were purchased from Alfa Aesar, Nanophase, Inc.,
and SunNanosystems
• Fifteen larvae were maintained in 400 ml test solution in 600 ml
beakers
• LC50s calculated using the Probit method
Copper Oxide
Metallic Copper
Table 1 - LC50 values for fathead minnows exposed
to metal or metal oxide nanoparticles for 96 h.
Nanoparticle
LC50
95 % Confidence
fmg/L)
Interval
CuO
0.662
0.492 - 0.866
Cu
0.009
0.006 - 0.013
0.0B*
-
Ti02
>1000
NA
ZnO
>1000
NA
*Estimated value, not enough range in response to calculate LC50
4
-------
Discussion - Xenopus
Discussion - Xenopus
¦ Nanop article size
¦ Individual particles varied greatly
¦ 23-190nm
¦ Aggregates
¦ 2-15 (jm
¦ Mortality
¦ 2 ppm ZnO induced a significant increase in mortality
¦ Chronic exposure resulted in a higher mortality rate
¦ Growth
¦ Total Body Length for Stage 66 juveniles
¦ Low dose ZnO juveniles were significantly longer than
controls (hormesis)
¦ Stage progression was accelerated
¦ Low dose ZnO tadpoles completed metamorphosis 5
days earlier than controls (hormesis)
¦ Stage progression was inhibited
¦ 58% stage 66 juveniles in 1 ppm ZnO
¦ NO stage 66 juveniles in 2 ppm ZnO
Discussion - Fathead minnow
¦ Titanium dioxide and zinc oxide nanoparticles
are non-toxic to fathead minnows in 96-h
exposures.
¦ Copper oxide nanoparticles are highly toxic to
fathead minnows
¦ Metallic copper and iron oxide nanoparticles are
very highly toxic to fathead minnow larvae
Continuing Work
¦ Measurement of metal concetrations and nanop article
size distributrions
¦ Determination of contribution of dissolved vs
particulate metals to toxcitiy
¦ Comparison of toxicity of metal nanoparticles to
dissolved ionic metals.
¦ Re-running LC50 with iron oxide to get more data
points between 0.06 and 0.10
¦ Toxicity of Cu to Xenopus
¦ Chronic toxicity of Cu, CuO and Fe203 to fatheads
Acknowledgements
¦ Mike Wages
¦ Shawna Nations, Gabriele Chavez, Jamie Rotter,
Zhi Mu
¦ Texas Tech Imaging Center
¦ Mark Grimson
¦ EPA for funding
¦ Star Grant USEPA Grant Number RD-83284201-0
5
-------
Single Conducting Polymer
Nanowire Immunosensors
Ashok Mulchandani. Nosang V. Myung,
Wilfred Chen and Marylynn V. Yates
University of California, Riverside, CA
20th Nov. 2008
OUTLINE
Introduction
Importance of nanowire and conducting polymer
Objective
Approaches
- In-situ electrochemical synthesis
- Magnetic aligning of multisegmented nanowire
- AC dielectrophoretic positioning and maskless
anchoring
Biological functionalization
Protein sensing
Summary
Gas sensor
Future work
Affinity-based detection
Health care
Homeland security
Environment
monitoring
Food safety & quality
Antibodies
Receptors
Binding proteins
Nucleic acid
Advantages
- High sensitivity
- High selectivity
Disadvantages
- Label required
- Not real-time
- Indirect
Major Advantages of 1 -D
Nanostructures as Sensing Materials
One-dimensional (1-D) nanostructures (e.g. nanowires, nanotubes...)
- High surface area to volume ratio
- Integrable into microelectronics
- Higher sensitivity than conventional
Film ^ 1-D
cr> V
Conducting polymers
• Exhibit electrical, electronic, magnetic and optical properties
of metals or semiconductors while retaining the attractive
mechanical properties and processing advantages
•Applied as conductometric, potentiometric, amperometric
and voltammetric transducers and as active layers of FETs
• Can be synthesized electrochemically
• Benign conditions enable the direct deposition of
conducting-polymer materials with embedded bioreceptors in
one step
• Conductivity can be modulated over 15-orders of magnitude
Conducting polymers
1
-------
Objective
Approaches
• Develop new methods for cost-effective
fabrication of single nanowire conducting
polymer affinity-based sensor arrays for
label-free, highly sensitive, selective,
precise, and accurate detection of
bioagents such as toxins, viruses and
bacteria at point-of-use.
Avidin coated quantum dot entrapment in
poiypyrrole NW
• ln-situ fabrication of conducting polymer
nanowires in e-beam lithography patterned
nanochanneis between pair of electrodes
• Magnetic aligning of template synthesized
multi-segmented nanowire on
prefabricated electrodes
• AC dielectrophoretic positioning and
maskless assembly on prefabricated
electrodes
Individually-addressable immunosensors array
fabrication
a
Nanowire bioaffinity sensor response
150-
50
_10fr
al
S 50-
0«
I
Injection
Concn. (nm)
0
AR/R (%)
-3
/
100
13
SO
b
Ik ......
(C)
Y^(B)
) 50 100 1!
Time (sec)
Electrical responses of an unmodified nanowire (A) to 100 nM biotin-DNA
(single stranded) and avidin-embedded poiypyrrole (200 nm) nanowires to 1
nM (B) and 100 nM (C) biotin-DNA. The responses were recorded on two
separate polypyrrole-avidin nanowires.
Ramanathan etal., 2004, Nano Letters, 4, 1237-
Ramanathan et. al., 2005, J. Am. Chem. Soc. (JACS), 127, 496-497
2
-------
TEMPLATE-DIRECTED SYNTHESIS AND
MAGNETIC ASSEMBLY OF MULTI-
SEGMENTED NANOWIRE
ucRiverside
Magnetic aligning arid assembly
Riverside!
Electrode preparation
Prefabricated gold
microelectrodes were
deposited with Ni
- Electrolyte: 0.91 M Ni(H2NS03)2 +
0.10 M H3BO3 + 30 ppm HCI
- Potential: -0.9 V
- Deposition time: -10 sec
To avoid surface oxide
formation of Nickel a
thin layer of gold was
electrodeposited
- Electrolyte: Technic Au
- Temp.: 60 °C
- Potential: -0.5 V
- Deposition time: 1 min
Riverside
Limitations
Magnetic (Ni) segment integration required
Multisegmented nanowire architecture results
in mechanical weakness especially at the
interfaces
Low aspect ratio can potentially result in
lower dynamic range
Due to limitation of use of NaOH for template
dissolution, over-oxidation of Ppy segment
resulted in lower conductivity and possibly in
lower sensing performance
TEMPLATE SYNTHESIS FOLLOWED BY
DIELECTROPHORETIC ALIGNMENT AND
MASKELESS ELECRODEPOSITION
ANCHORING
3
-------
Template synthesis
Riverside
Contact Resistance
AC Dielectrophoretic Alignment: 5 MHz, 1 V p-to-p.
a
Contact Resistance
I I I I I I
Device limitation: Modulation of contact resistance upon exposure to liquid
medium.
Riverside
1
Glutaraldehyde functionalization
Fer: PBS, pH 7
.0
?
~ N V
\$7
Gp
NHj
. HjO
(™)
N
fo]
• ywBi.Miii— it—>
4
-------
Carbodiimide functionalization
I
Glutaraidehyde vs. Carbodiimide
Fluorescence images and corresponding fluorescence line profile across single
nanowire functionalized with BSA-FITC with respective covalent route.
Protein detection
I Calibration plot showing detection of CA 125 cancer antigen in 10 mM phos
| buffer using Anti-CA 125 antibody immobilized Ppy nanowire biosensor.
Selectivity
10 mM Phosphate Buffer
Spiked Human Blood Plasma
if
CA 125 concentration (U/ml)
Calibration plots showing detection of CA125 cancer antigen in PB and spiked human blood|
plasma using Anti-CA 125 antibody immobilized Ppy nanowire biosensor.
Riverside
Summary
Multi -segmented
Masklessly connected
Polymer conductivity
Post-fabrication
Post-assembly
During or after fabrication
Ammonia: LDL 100 ppm,
poor recovery
CA 125:45% conductance change
at 1 U/ml. Dynamic range up to
1000 U/ml. No effect of other
proteins
Avidin-Biotin interactions:
LDL1 nM of Biotin-ssDNA
conjugate.
Multi-analyte sensing
Cost-benefits
Site-specific functional ization
Most cost-effective
Cost-benefits compromised
due to use of e-beam
Jithogragh^rJjjlB^^^^^^
-------
Carbon Nanotubes: Environmental Dispersion
States, Transport, Fate, and Bioavailability
Elijah Petersen, Walter J. Weber, Jr. — University of Michigan
Collaborators
Jack Huang — University of Georgia, Griffin
Jussi Kukkonen, Jarkko Akkanen, and Kukka Tervonen —
University of Joensuu, Finland
Dr. Denis O'Caroll — University of Western Ontario, Canada
Dr. Peter Landrum - NOAA
^ This a funded by Graham Environmental
^ Sustainability Institute-
V .H Gram University of Michigan
Presentation Outline
1. Background
2. Carbon-14 Nanotube Synthesis
3. Uptake and Depuration Behaviors for
Lumbriculus variegatus
4. Uptake and Depuration Behaviors for Daphnia
Magna
5. Additional Results
1. Background
Overview
Ecological
• Transport
ioavailability/Bioaccumulation
• Biodegradation
• Toxicity
1. Background:
Current Analytical Techniques and
Limitations
1. Spectrofluorimetry
Drawbacks: Carbon Nanotube Bundles, Metallic Nanotubes
2. External Chemical Labeling
Drawbacks: Changes Nanotube Physicochemical Properties
3. Raman Spectroscopy
Drawbacks: Only single-walled carbon nanotubes, qualitative
1. Background:
Advantages of C14 Nanotubes
1. Readily quantifiable
2. Can be used with all types of carbon nanotubes
3. Does not change the chemical or physical properties of
the carbon nanotubes
2. Carbon-14 Nanotube Synthesis
Chemical Vapor Deposition
1 3-way Vato
¦ „ . a. r
Catalyst
¦ 4 ri ' i
,! L
NiMgO
^ MgFeO
c Tub© Furnace
catalyst (MWNT)
catalyst (SWNT)
1
Petersen, E. J., Huang, Q. G., Weber, W. J., lx., Environmental Health Perspectives.
2008, 496-500.
Petersen, E. J., Huang, Q. G., Weber, W. J., Jr., Environmental Science and Technology.
2008, 3090-3095.
-------
2. Carbon Nanotube Characterization:
Transmission Electron Microscopy
B
\ • ?
rX \i;
"m
. . v: - ' -
100 rtm
Transmission electron micrographs of (A) single-walled at 250kx magnification and (B)
multi-walled carbon nanotubes at 1 OOkx magnification.
2. Carbon Nanotube Characterization:
Raman Spectroscopy on SWNTs
Spectrograph of single-walled carbon nanotubes. This spectrum is the average of nine
measurements
2. C14 Nanotube Synthesis:
Summary of Results for HC1 Purified
Nanotubes
SWNT
MWNT
Carbon Purity (Percentage)
92 ± 0.4
99 ± 1
Radioactivity (uCi/g)
1350 ±30
122 ±4
Detection Limit (nanograms)
34 ±1
380 ± 10
3. Uptake and Depuration Behaviors for
Lumbriculus variegatus
Aquatic Organism Uptake
After 1 hr of exposure
Roberts et al. 2007 - Used raman spectroscopy to qualitatively
detect lysophophatidylcholine coated SWNTs in daphnia'
magna.
Roberts et al.. Environ. Sci Tech. 2007; 41(8) pp 3025 - 3029
3. Uptake and Depuration Behaviors for
Lumbriculus variegatus
Lumbriculus variegatus has been
• used as a bioindicator for environmental pollution
• selected by the U.S. Environmental Protection
Agency as the freshwater organism for assessing
bioacciimulation
• commonly used in laboratory experiments for uptake
of a broad range of compounds
Petersen, E. J., Huang, Q. G., Weber, W. J., Jr., Environmental Health Perspectives.
2008, 496-500.
3. Uptake and Depuration Behaviors for
Lumbriculus variegatus
-------
3. Uptake and Depuration Behaviors for
Lumbriculus variegatus
BSAFs After 14d Exposure with Different
Spiking Conditions
r
Pyrene (0.05 mg/g)
MWNT #1 (0.37 mg/g)
MWNT #2 (0.37 mg/g)
¦ ,p=
MWNT (0.037 mg/g)
SWNT (0.03 mg/g)
SWNT (0.003 mg/g)
rbniftiilfcl ¦»HiU
MWNT Sediment Only (0.37 mg/g)
These results indicate that the carbon nanotubes measured after the 6 hour
depuration interval were in the gut of the organisms and not absorbed
into the tissue.
3. Uptake and Depuration Behaviors for
Lumbriculus variegatus
Aquatic Worm Depuration
-~-SWNT - Water
-—MWNT-Water
31 13%
Pyrene - Water
•X
-k-MWNT -Sediment
77%
-
Test Day
Water — indicates depuration in beakers with only water
Sediment — indicates depuration in beakers with sediment and water
3. Uptake and Depuration Behaviors with
Eisenia foetida
Bioaccumulation Factors (BAFs)
10
10 15 20
Test Day
Petersen, E. J., Huang, Q. G., Weber, W. J., Jr., Environmental Science and Technology. 2008,
3090-3095.
4. Uptake and Depuration Behaviors for
Daphnia Magna - Accumulation
I 700000 Tl^ o.04 ng/mL, 200 mLl l l
¦ 0.1 ng/mL. 200 mL T T I
^ 600000 - x 0.4 Lig/mL, 200 mL I
0.1 Lig/mL, 100 mL I
,p 500000 - X 0.1 pg/mL, 30 mL i\ C. II
.. BCF = —- ,,
¦| 400000 - O Cwater
I 300000 - x
8 200000 ^ I 1
100000 | ^
oi T T T T T I
0 10 20 30 40 50 60 ¦
Time I
4. Uptake and Depuration Behaviors with
Daphnia Magna - Depuration
0.04 Lig/mL AF
—I—0.07 Lig/mL AF
-X-0.4 |jg/mL AF
-¦-0.04 Lig/mL NOM
|jg/mL NOM
4. Uptake and Depuration Behaviors for
Daphnia Magna - Depuration
; e
I
-0.04 |jg/mL Algae
-0.4 |jg/mL Algae
-0.4 |jg/mL Algae#2
^=4
20 30 40
Time (hours)
-------
4. Uptake and Depuration Behaviors for
Daphnia Magna - Light Microscope Pictures
4. Uptake and Depuration Behaviors for
Daphnia Magna - Sediment Depuration
5. Additional Results
1. Changing the hydrophobicity of multi-walled carbon nanotubes
changes their octanol-water distribution behavior but does not
impact accumulation by earthworms or aquatic worms.
Petersen, E. J., Huang, Q., Weber, W. J., Jr. 2008. Relevance of Kow Measurements to the
Ecological Uptake of Carbon Nanotubes. Submitted.
2. Adding carbon nanotubes to soils affects the uptake of soil-borne
pyrene by earthworms in a concentration-dependent manner. Low
concentrations of nanotubes show no impact but higher
concentrations decrease pyrene accumulation and act similarly
black carbons.
Petersen, et al. 2008. Influence of Carbon Nanotubes on Pyrene Bioaccumulation from
Contaminated Soils by Earthworms. Submitted.
3. Polyethyleneimine (PEI) was covalently bonded to multi-walled
carbon nanotubes to form nanotubes with positive, negative, or
neutral surfaces charges, and the cellular toxicity of these
nanotubes was tested.
Shen, M., Wang, S.H., Shi X., Huang Q., Petersen, E.J., Pinto R.A., Baker, J.R., Jr., and
Weber, W. J., Jr. 2008. Submitted.
5. Additional Results
4. We have developed a novel method to quantify fullerenes in
ecological receptors and have found significant accumulation and
limited depuration by Daphnia magna.
Tervonen, K., Waissi, G., Petersen, E. J., Akkanen, J., Kukkonen J. V. K. 2008. Analysis
of fullerene-C60in Daphnia magna and kinetic measurements for its bioaccumulation and
depuration. In Preparation.
Uptake and Depuration Behaviors for Eisenia
foetida
Gut Contents
The BAF for a non-bioaccumulating chemical was estimated to be
0.0315 ±0.001.
Hartenstein, F.; Hartenstein, E.; Hartenstein, R., Gut Load and Transit-Time in the Earthworm
Eisenia-Foetida. Pedobiologia 1981, 22, (1), 5-20.
Similar results have also been obtained for Eisenia andrei.
Jager, T.; Fleuren, R.; Roelofs, W; de Groot, A. C., Feeding activity of the earthworm Eisenia
andrei in artificial soil. Soil Biol. Biochem. 2003, 35, (2), 313-322.
Uptake and Depuration Behaviors for Eisenia
foetida
BAFs After 14d Exposure
BAF
BAF for Non-Bioaccumulating Compound
0.0315
Pyrene Chelsea Soil (0.04 mg/g)
2.94 ± 0.25
Pyrene Ypsilanti Soil (0.04 mg/g)
14.0 ±0.9
MWNT Chelsea Soil (0.3 mg/g)
0.023 ± 0.01
MWNT Chelsea Soil (0.03 mg/g)
0.016 ±0.001
MWNT Ypsilanti Soil (0.3 mg/g)
0.014 ±0.003
SWNT Chelsea Soil (0.03 mg/g)
0.0061 ± 0.002
SWNT Chelsea Soil (0.1 mg/g)
0.0078 ± 0.005
SWNT Ypsilanti Soil (0.03 mg/g)
0.022 ± 0.003
Weight Percent Organic Carbon Content of Chelsea Soil: 5.95%
Weight Percent Organic Carbon Content of Ypsilanti Soil: 1.14%
-------
Uptake and Depuration Behaviors for Eisenia
foetida
Depuration After 14d Exposure in Chelsea Soil
-------
Aggregation and Deposition Behavior
of Carbon Nanotubes (CNTs) in
Aquatic Systems
Menachem Elimelech, Lisa Pfefferle, and Navid Saleh
Department of Chemical Engineering
Environmental Engineering Program
Yale University
Interagency Environmental Nanotechnology Grantees Workshop,
Tampa, Florida, November 19-21, 2008
Engineered Carbon-Based
Nanomaterials
ftilieiefie
Cflo
Fultersfje
C540
Carbon
Onion
sp3
Spz+7T
Mauterand Elimelech, ES&T, 42, 2008, 5843-5859.
¦ Unique properties
¦ Exponential growth in production and applications
¦ Environmental and health impacts are not known
Yale
Aggregation and Deposition Behavior
Determines Fate and Transport
Deposition/
Attachment
Influences rate
of settling and
transport
Influences
reactivity and
toxicity
Yale
Bacteria Attach to CNT Aggregates:
Significant Cell Damage
E, co// cells; single walled carbon nanotubes (SWNTs)
Kang, S., Pinauit, M., Pfefferle, L.D. and Elimelech, M. "Single-Walled Carbon v ,
Nanotubes Exhibit Strong Antimicrobial Activity", Langmuir, 23, 2007, 8670-8673 I all*
E, Coli Cell Membrane Damage in
Contact with SWNT Aggregates
Control (without SWNT) SWNT
Yale
SWNTs are Much More Toxic than
MWNTs
Control MWNT SWNT
Kang, S., Herzberg, M., Rodrigues, D.F. and Elimelech, M. "Antibacterial Effects
of Carbon Nanotubes: Size Does Matter!", Langmuir, 24, 2008, 6409-6413 Yale
1
-------
SWNTs are Much More Toxic than
MWNTs
MWNTs SWNTs
Aggregation Kinetics of
Multiwalled Carbon
Nanotubes (MWNTs)
Yale
MWNT Sample
¦ Commercial MWNTs
¦ Long, bundled tubes
¦ Sonication debundled and shortened the tubes
Yale
Electrokinetic Properties of MWNTs
0.0
E
o
-1.0
-2.0
-3.0
O Na*
~ Cff*
A Mg"*
A
¦ pH6
¦ Higher salt conc., lower
EPM
¦ Ca2+ and Mg2+ reduce
EPM more than Na+
10"4 1(r 10"' 1CT 10°
Salt Concentration (M)
Yale
ALV Light Scattering Setup
Dynamic light scattering
to derive hydrodynamic
radius
YAG laser (532 nm)
Scattered light intensity
measured at 90° from
incident beam
Yale
Attachment Efficiency
Time (s)
Attachment Efficiency:
( dRh(f)^
... _ V )t->o
V it Jt-MjSk
Yale
2
-------
Aggregation Kinetics with Monovalent
Salt (NaCI
)
« 1
>
o
c
q§>
CD
0e*3^°
Favorable ;
(Diffusion-limited)-
pH 6
£ 0.1
LU
-O
s G°°
T
D
I CCC - 25 mM NaCI
C
<1>
E 0.01
o
-------
Electrokinetic Properties of SWNTs
and Sand Grains
-1.5
-2.0
CO
V "2 5
CN
E -3.0
co
b
3^ -3.5
5
CL -4.0
LU
-4.5
0.1 1 10 100
KCI Concentration (mM)
~ Quartz Sand
O SWNT 9
*
o 4 HU ti
* <
Deposition Rate
ki = —— lnf — j
JL IcJ
Attachment Efficiency
vdjav
Jaisi et ah, Environ. Sci. Techno!. 2008, 42 (22), 8317-8323
Yale
Long SWNTs Result in Straining
c" —
^ 140
(/)
O Influent SWNTs
A Effluent SWNTs '
~o
£ 120
0
1 100
C
>*
oo0% sfe
<&cP ® o
2 80
"D
>>
0 100 200 300 400
X
Time (s)
Yale
Subsequent Column Runs with a
Single SWNT Sample (in Dl Water)
1.2
—O— 1C0=Fresh SWNT
—O-2C0=Eluted SWNT from 1C
—o— 3C0=Eluted SWNT from 2C
o co
-------
Acknowledgments
¦ National Science Foundation
-------
Cross-Media Environmental
Transport, Transformation,
™. and Fate of Carbonaceous
Nanomaterials
A.
Peter J. Vikesiarid.
Linsey C. Marr,
Joerg R. Jinschek,
Laura K. Duncan,
Behnoush Yeganeh and
Xiaojun Chang
Funding:
#¦
BES-0537117
™IGTAS
l^lrmiilil hislnmrlnj
Research Questions
How do
atmospheric
transformations of
nanoparticles
affect their fate in
water and soil?
What is the potential for
exposure to airborne
nanomaterials during
manufacturing?
Research Questions
What is the potential for
exposure to airborne
nanomaterials during
manufacturing?
How do
atmospheric
transformations of
nanoparticles
affect their fate in
water and soil?
C60 Fullerenes
Symmetry and conjugated it-bond system
of C60 leads to unique properties
-High reactivity to nucleophiles
-Electron affinity (2.7 eV)
-Photosensitization
0.7 nm
nC60 (20-200 nm)
Reported solubility in water is < 10~9 mg/L
1
Characterization of Airborne
Particles During Production of
Carbonaceous Nanomaterials
neilNCIdSH VCCMNEH.
CHRISTY M KUU. MATTHEW S HUl.l
AND UN*EY C MABIf
UflwvMmmi vf Ch41 ami tMttiimmmntiil Engt»uu^ VKryuas
TWl 418 Dvhwa Mutt Hm&mrg, Mrpmi imi
Bmron Set Ttclmol KM. 42
-------
Methods to produce nC60
Solvent exchange
~
Extended stirring
THF//7C60
toluerie//7C60
TTA/nCgo
aqu/nC60
nC 6o
Aerosolization
100 100c
Diameter (nm)
25
Upon
_ 20
tr 15
aerosolization
CD
.Q
E 10
the mean
3
z 5
particle
0
size decreases
8e+5
substantially
a. 6e+5
o
ut
¦2 4e+5
5
z
2e+5
0
100
Diameter (nm)
Does this difference suggest something about the fundamental
forces holding the nC60 aggregates together????
How representative of 'environmental' and
'physiological' systems
are these nC60 suspensions?
Solvent exchange
THF/nCS0 retains solvent THF
Typically monodisperse
Form via recrystallization (bottom-up) —
Extended stirring
Heterodisperse
Forms via weathering (top-down)
Natural water and physiological fluid
components
Electrolytes
Organic macromolecules
- Proteins
- Lipids
- Carbohydrates
- Humic and fulvic acids
Low molecular weight organics
- Nucleic acids
- Amino acids
- Carboxylic acids
Each of these components is expected to alter the mechanism(s)
responsible fornCm formation and stability...
Impacts of Organic
Materials on nC60
Terashima and Nagao (Chem Lett.
(2007), 36, 302)
Fulvic and humic acids increased
apparent solubility of nC60 by 8x and
540x, respectively
Xie et al. (Environ. Sci. Techol. (2008),
42, 2853)
Fulvic and humic acids caused
disaggregation of toluene//7C60 and
THF/nC6o
Deguchi et al. (Chem. Res. Toxicol.
(2007), 20, 854)
Human serum albumin stabilizes
SON//iC60 aggregates and inhibits
their aggregation
Xie et al. (ES&T)
~ 0 I mjml.
r05 nxjltil
fc I rnjUrL
• SmjurL
t- mgiVnL
Deguchi et al. (Chem. Res. Toxicol)
2
-------
Why carboxylic acids?
nC60 aggregate size decreases in the presence
of natural organic matter isolates (Duncan et at. 2008)
w/o NOM Zavg = 173 nm, PDI = 0.15
Carboxylic acid groups are
prevalent in many organic
compounds
Citrate is a well known
stabilizer of many
nanomaterials
Citrate stabilized gold
nanoparticles
100 nm
100 i^m
2) Mix C60 in
citrate solutions
1) Pulverize C6
¦"fc t-J
[Na3Ct]=0 0.1 1.0 10 mM
3) Settle nC60
for >24 hours
4) Characterize nC6
Effect of citrate on aggregate morphology
0.01 mM
citrate
0.1 mM
citrate
10 mM
citrate
Citrate concentration -2.ct
alters solution pH
HCt2"
ct3-
II pKa = 3.13
H2Ct"
it pKa = 4.76
HCt2"
It PKa = 6.39
ct3-
0.01
>
E
1 cz> 25 mM
i
O -5 1 • Variable citrate
6
pH
Should we worry about citrate mediated
dissolution and reprecipitation?
POSSIBLY...
>r F/nCs0 increasing Size and
f Decreasing toxicity^
Toxic concentration
•#¦0.01-0.1 mg/L
^•"0.75-1.0 mg/L
2.0-5.0 mg/L
-10 mg/L
u
Particle Size (nm)
Source: P. Alvarez (Rice)
Virginia
Questions?
3
-------
Photochemical Fate of Manufactured
Carbon Nanomaterials in the Aquatic
Environment (Emphasis on C60)
Chad T. Jafvert, Wen-Che Hou
Division of Environmental & Ecological Engineering
And School of Civil Engineering
Purdue University, West Lafayette, IN 47907
Purdue
UNIVERSITY
*
Truncated
icosahedron
symmetry
i 20 hexagons and
12 pentagons,
fully aromatic
*
~ 7 A in diameter
Previous Studies with C60*
4 "Solubility of C60 in solvent mixtures"
(Env. Sci. Techno!. 42: 845-851, 2008)
4 "C60's Kow and Aqueous Solubility"
(Env. Sci. Techno!. 42: 5945-5950, 2008)
4 "Sorption of C60 to Saturated Soils"
(in preparation)
^Funded by NSF
Excess free energy of mixtures (ATOL-ACN, o
THF-ACN, ~ TOI^THF 0 TOI^EOH). For the
TOI^THF data set, the abscissa is XTOL (not X-j^p).
Solubility in THF-ACN mixtures
Previous Results
4 Soivated crystals occur
4 Kow »106-7
4 Aqueous Solubility limit to 8 ng/L
t*RT\nr,<
Sorption to Saturated Soil from ethanol-water solutions
(Xeoh = ethanol mole fraction, soil = EPA15)
Tentative logKom° = -logS-logF -0.62195
1.6
1.2
J. 0.8
cj
0.4
0
0 0.2 0.4 0.6
Ce (mg/L)
Rationale for Current Study
« Carbon nanomaterials have many
uses. The potential widespread
use will eventually lead to the
appearance of carbon nano-
materials in the aquatic
environment.
* Although C60's aqueous solubility
of is extremely low, C60 is known
to form stable clusters (nC60) in
water (Deguchi et a!., 2001).
* Photochemistry of aqueous
clusters could be an important fate process.
potential emerging
(www.nanobama.com)
1
-------
Current EPA-funded Study
Project period: May 2007 - April 2009
"Photochemical transformation of aqueous C60
clusters (nC60) in sunlight"
(Env. Sci. Technol., In press, 2008)
Parameters examined:
A Cluster size
A Preparation method
4 pH - (3, 7, and 11 at /i = 19 mMj
A Humic substances
(10 mg/L Suwannee River fulvic acid from IHSS)
4 02 concentration
Photochemistry of C60 in Organic Solvents
(Potential Aqueous Reactions of nC60)
S
3ceo+f lo2-
-> &
•> C„
-*CW0„
H* O, ¦
Further oxidation arid
fragmentation
In water?
>Cfin +1 Oo
-60 ^ ^2
Arbogast et a I., 1991
Juha et al., 1994
Taylor et a I., 1991
\q . Further oxidation and
2 fragmentation
Potential products in the aqueous phase?
4 Polyhydroxlated C50 via acid (H2S04 and HN03) reaction
4 The product contains hemiketal moieties
4 C60(OH)14.16O7.8as a hypothetical structure
based on XPS curve fitting
Chiang et al., Multi-hydroxy additions onto C60 fullerene molecules. J. Chem. Soc., Chem.
Commun. 1992, 106,1103-1105.
Fullerene hemiketal (RO-C-OH) aqueous chemistry
Chiang etal. Evidence of hemiketals incorporated in the structure of fullerols derived
from aqueous acid chemistry. J. Am. Chem. Soc. 1993, 115, 5453-5457.
In the absence of O
Photo-polymerization of C60 in absence of 02 via the
[2+2] cycloaddition.
Giacalone et al. Fullerene polymers: Synthesis and properties. Chem. Rev. 2006,106, 5136-5190.
Rao et ai. Photoinduced polymerization of solid Cg, films. Science 1993, 259, 955-957.
nC60 Preparation
* THF/nC60-An equal volume of water added at 25 mL/min to C60-
saturated THF under mixing. Remove the THF on a rotary
evaporator. (Smaller clusters prepared with a faster addition rate
(1 L/min).
* Son/nC6Q - sonicate a water-toluene mixture containing C60 until
the toluene phase evaporates.
Analysis
* nCjo
> Add 0.1 M Mg(CI04)2 & extract w/ toluene
(Fortner et al., 2005)
HPLC on a Cosmosil 5PBB Column
Toluene as the mobile phase
Detector at X = 336 nm
* nCgQ size and morphology
> Dynamic light scattering (DLS)
> TEM
2
-------
Experimental Approach
Irradiation
Sunlight experiments were
performed from 10 am to 5 pm on
sunny or partly cloudy days on the
roof of Civil Engineering building
at Purdue ( 86° 55' W, 40° 26' N).
The solar intensity data were
obtained from a USDA UV-B
station within 5 miles from where
the irradiation occurred.
Lamp light experiments were
carried out in a merry-go-round
photo-reactor with 8 24-W lamps
(A= 350 ±50)
Sunlight irradiation
t*-
Merry-go-round
photo-reactor
Results
Photo-transformation of 65 mg/L THF/ nC60
in the lamp light (X=350 nm)
Irradiation time
(day)
0 10 30 65
[nC60] (mg/L)
65 19.5 2.6 0.47
I I I I
III
TEM image*
* • v- •
Mean diameter**
(nm)
500 350 250 160
After
Centrifugation***
1 1 1 f
*Scale bars indicate 1000 nm.
**Mean hydrodynamic diameters by DLS.
***Samples after centrifugation (13000xg, 1 h) and
filtration (nylon membrane, 0.2-pm pore size
10 20 30 40 50
Time (days)
0 nC60 dark controls
~ nC60 irradiated samples
¦ Aqueous phase OC
~ Recovered OC (Soluble Carbon + C60)
Photo-transformation of 65 mg/LTHF/nC60 in terms of
organic carbon (OC) content in the lamp light
I rradiated son/nC60 (A)
Dark control of son/nC60 (a>
Irradiated THF/nC60 (¦:
Dark control ofTHF/nC60
0 10 20 30 40 50 60 70
T ime (hrs)
Photo-transformation of THF/nC50 and son/nC50
under the mid-latitude solar exposure, May 13, 2008-June 6, 2008.
Conditions: pH = 7, ionic strength = 19 mM.
Mean cluster diameter:
235 nm ± 8% at all pH values
Irradiated nC60 at pH=3 (A)
Dark control of nC60 at pH=3 (a)
Irradiated nC60 at pH=7 (¦)
Dark control of nC60 at pH=7 (Q
Irradiated nC60 at pH=ll (~>
Dark control of nC60 at pH=ll
20 40 60 80 100 120
Irradiation time (hr)
Photo-transformation of THF/nC60 under the mid-latitude solar
exposure in September-October, 2007, at pH=3,7, and 11.
Conditions: ionic strength = 19 mM.
3
-------
0 10 20 30 40 50 60 70
Time (hrs)
Irradiated 150-nm nC60 (A)
Dark control of 150-nm nC60 (A)
Irradiated 500-nm nC60 (¦)
Dark control of 500-nm nC60 (O
Half-lives:
150 nm, 19 hrs
500 nm, 41 hrs
Photo-transformation of 150 nm and 500 nm diameter THF/nC50
mid-latitude solar exposure in October-November, 2007
Conditions: pH=7, ionic strength = 19 mM
Irradiated FFA only (A)
Irradiated THF/nC60 w/ FFA(H)
Dark control ofTHF/nC60 w/ FFA
0.8
f 0.6
E 0.6
Time (hrs)
Singlet oxygen generation by THF/nC50 (0.8 mg/L) under the
mid-latitude solar exposure in May 13, 2008-June 6, 2008.
Conditions: pH = 7, ionic strength = 19 mM
Summary
& Aqueous nC60 under lamp light (X = 300-400 nm) resulted in
losses of C60 and color, decrease in cluster size, "water-
soluble" products.
* Loss occurred more rapidly with smaller clusters.
4 pH, fulvic acid, & preparation method had minimal effect.
* The reaction rate was significantly reduced in deoxygenated
samples, indicating 02 plays a role.
Future work
« 10, measurements
4 Functional group-specific X-ray
photoelectron spectroscopy (XPS)
* NMR analysis
* Head space C02 analysis
4 Extend work to carbon nanotubes
i Alk
4
NMR spectrum of 50 mg/LTHF/nCM
(25% 13C-enriched) in 20% (v/v) D20
-------
Questions?
October 19-25,2008
5
-------
HOTEL MONACO, WASHINGTON O.C.
FATE AND TRANSFORMATION OF
CARBON NANOMATERIALS IN WATER
TREATMENT PROCESSES
JAE-HONG KIM, PH.D.
ASSISTANT PROFESSOR
SCHOOL OF CIVIL AND ENVIRONMENTAL ENGINEERING
GEORGIA INSTITUTE OF TECHNOLOGY
TOPIC I
STABILITY OF CARBON NANOMATERIALS
IN NATURAL WATERS AND REMOVAL BY
CONVENTAIONAL WATER TREATMENT PROCESSES
VISUAL EXAMINATION OF MWNT SOLUTIONS
50 mg MWNT ADDED AND AGITATED FOR ONE HOUR
100 mg-C/L
Dl 1 % SDS SRNOM
AFTER 1 DAY
AFTER 4 DAY
¦
91
sf
U-
¦
VARIOUS DISPOSAL SCENARIOS
STABILITY IN NOM SOLUTION
DEPENDS ON THE TYPE AND PHASE OF FULLERENES
PREPARATION METHODS
MXING
SONICATION
SOLVENT
EXCHANGE
BY MIXING
SOLVENT
EXCHANGE
BY SONICATION
c60
+
O
+
SWNT
O
+
o
o
MWNT
+
+
o
o
+ NOM INCREASES DISPERSION
- NOM DECREASES DISPERSION
O NO EFFECT
1
-------
REMOVAL OF nC60 AND MWNT
BY CONVENTIONAL WATER TREATMETN PROCESSES
nCfinREMOVAL
MWNT REMOVAL
ALUM DOSE #10 mg/L O 25mg/L A 50mg/L A 100 mg/L
COAGULANT: ALUM
RAPID MIXING: 2 MIN @200 RPM
SLOW MIXING: 30 MIN @ 25 RPM
SETTLING:60 MIN
1 mg/L nCeo or MWNT
2.5 mg-C/L SRNOM
100 mg/L AS CaCO,
SUMMARY
NOM ENHANCES STABILIZATION OF CARBON NANOMATERIALS (C60, SWNT
MWNT) IN NATURAL WATERS
ADSORPTIVE INTERACTION BETWEEN NOM AND NANOTUBES DEPENDS ON
WATER QUALITY PARAMETERS (FOR EXAMPLE, pH AND IONIC STRENGTH) AND
NOM CHARACTERISTICS
FULLERENESARE EXPECTED TO BE WELL REMOVED BY WATER TREATMENT
PROCESS
PUBLICATIONS
1. HYUNG, H.; FORTNER, J. D.; HUGHES, J. B.; KIM, J. H. (2007) "NATURAL ORGANIC
MATTER STABILIZES CARBON NANOTUBES IN THE AQUEOUS PHASE."
ENVIRONMENTAL SCIENCE & TECHNOLOGY, 41,179-184
2. HYUNG, H.; KIM, J. H. (2008). "NATURAL ORGANIC MATTER (NOM) ADSORPTION TO
MULTI-WALLED CARBON NANOTUBES: EFFECT OF NOM CHARACTERISTICS AND
WATER QUALITY PARAMETERS." ENVIRONIVENTAL SCIENCE & TECHNOLOGY, 42,
4416-4421
3. HYUNG, H.; KIM, J. H. (2009)."DISPERSION OF C60 IN NATURAL WATER AND
REMOVAL BY CONVENTIONAL DRINKING WATER TREATMENT PROCESSES." (IN
PREPARATION)
Goorgto.
TOPIC II
SP*I
CHEMICAL TRANSFORMATION
WATER STABLE C60 AGGREGATES
"
REACTION WITH 03 IN THE AQUEOUS PHASE
IN A SEMI-BATCH MODE
t = 0 min
CT = 0 mg-min/L
90 nm
Ifr* •
. ~
I ~ «
t = 5 min
CT = 6.6 mg-min/L
76 rim
t= 15 min
CT = 28.5 mg-min/L
40 nm
t = 30-60 min
CT = 74 -150 mg-min/L
< 5 nm
AGGREGATE SIZE DECREASES WITH REACTION TIME
PRODUCT CHARACTERIZATION: MS
720 m/z PEAK SUGGESTING THAT
CAGE STRUCTURE IS PRESERVED
ADDITIONAL 16-17 m/Z PEAKS INDICATING
MULTIPLE OXYGEN ADDITION
°X"
2
-------
PRODUCT CHARACTERIZATION: 13C NMR
Goorgtn
- T»ch
PRODUCT CHARACTERIZATION: XPS
295 290 285
C(1s) Binding Energy (eV)
INTERACTION WITH E.COLI
OZONATED C60 INACTIVATES E.COLI
ONLY IN THE PRESENCE OF 02 AND LIGHT
0.0
•0.2
-0.4
j-o.e
o -0.8
-1.0
-1.2
-1.4
0 20 40 60 80 100 120 140 160
Time (min) ph : 7.1 (10 mm of phosphate buffer)
TEMPERATURE : 20 ± 1 °C
LIGHT INTENSITY : 1.2 x 10"6 EINSTEIN/L.S
REACTOR : QUARTZ REACTOR (60 ML)
SAMPLE VOLUME: 30 ML
INITIAL CONCENTRATION OF E. COLI: 1.5 - 3.0 x 10s CFU/ML
3
-------
EFFECT OF DISSOLVED OXYGEN
DRASTIC RETARDATION OF DEGRADATION
KINETICS UNDER N,-SATURATED CONDITION.
• OXIDATIVE DEGRADATION PATHWAY
TOC WAS NOT CHANGED AFTER UV
PHOTOLYSIS
-*¦ PHOTOCHEMICAL TRANSFORMATION,
NOT MINERALIZATION
PRODUCT CHARACTERIZATION: LDI-MS
PRODUCT
720.1
J
£00 750 900 1050 1200 1350 1500 1650 1800 1950
TOXICITY OF UV PHOTOLYSIS PRODUCT
MIC TEST
The minimal inhibitory
of parent nC60 and the UV photolysis products for Ecoii
Concentration of C60 Cluster (or UV-
treated Products) (mg/L)
UV Illumination Time (hr)
0
25
50
70
90
110
0
+
+
+
+
+
+
1
+
+
+
+
+
+
2
+
+
+
+
4
+
+
+
6
+
+
+
8
+
+
10
+
(+: microbial growth,-
LONG-TERM EXPOSURE TO UVC (254 nm)
RESULTS IN nCso TOXICITY DECREASE.
4
-------
RADICAL REACTIVITY OF nCRI
PULSE RADIO LYSIS
8-MeVTITAN BETA MODEL TBS-8/16-1S LINEAR ACCELERATOR
AT NOTRE DAME RADIATION LABORATORY
UNDER N2-SATURATED CONDITION
eaq" + A * AS-
UNDER n2o-saturated condition
e, • + N20 + H20 ~ N2 + OH- + OH*
GAMMA RADIO LYSIS
USING SHEPHERD® 109-S6 COBALT 6l
WITH A DOSE RATE OF 0.0722 kGy mir
OH RADICAL-INDUCED OXIDATION
OF nCKn IN WATER
DOSES OF 20 AND 40 KGy CORRESPOND TO GENERATION OF 11 AND
22 mM OF OH RADICALS, RESPECTIVELY
~ EXCEPTIONAL STABILITY OF nC60 AGAINST OH RADICAL ATTACK
OH RADICAL-INDUCED REDUCTION
OF nC60 IN WATER
y-RADIOLYSIS (N2-SATURATED CONDITION)
Z33 20kkGy
U [C,J,
V
10 pM; pH = 5.5 :
DOSES OF 20 AND 40 KGy CORRESPOND TO GENERATION OF 5.4 AND
10.8 mM OF HYDRATED ELECTRONS, RESPECTIVELY
~ RECALCITRANT AGAINST HYDRATED ELECTRON
REACTIVITY OF nCB0 WITH OH RADICAL
C6II + OH* ~ intermediate
ke
SCN +OH* (+SCN ) 1 05x^1,^13.1 " OH +(SCN)2*'" (MONITORED AT 472 nm)
r
[(sen);], , k5[c6
[CJflSCNl
SECOND-ORDER RATE CONSTANT = 7.34 ± 0.31 x 109 MMs".
REACTIVITY OF nC60 WITH HYDRATED ELECTRONS
SECOND-ORDER RATE CONSTANT = 2.34 ± 0.02 x 1010 M"1S"1
Goor^fai
ii »<*>
5
-------
SUMMARY
OZONATION TRANSFORMS nC60 INTO WATER SOLUBLE FULLERENE OXIDE
SPECIES
OZONATED C60 APPEARS MORE TOXIC THAN nC60
IRRADIATION OF UV(254nm) TRANSFORMS nC60 INTO WATER SOLUBLE
FULLERENE OXIDE SPECIES
C60 PHOTOLYSIS PRODUCT APPEARS LESS TOXIC THAN nC60
C60 IN THE AQUEOUS PHASE REACTS WITH HYDROXYL RADICAL AND
HYDRATED ELECTRONS WITH RELATIVELY HIGH RATE CONSTANT RESULTING IN
UNSTABLE PRODUCT
PUBLICATIONS
1. FORTNER, J. D.; KIM, D. I.; BOYD, A. M.; FALKNER, J. C.; MORAN, S.; COLVIN, V. L.;
HUGHES, J. B.; KIM, J. H. (2007). "REACTION OF WATER STABLE Ceo AGGREGATES
WITH OZONE" ENVIRONMENTAL SCIENCE & TECHNOLOGY, 41, 7497-7502
2. LEE, J.; CHO, M; FORTNER, J. D.; HUGHES, J. B.; AND KIM, J. H. "UV PHOTOLYSIS
OF C60 CLUSTERS IN THE AQUEOUS PHASE." {IN PREPARATION)
3. LEE, J.; SONG, W.; JANG, S. S.; FORTNER, J. D.; ALVAREZ, P. J.; COOPER, W. J.;
KIM, J. H. "REACTION OF WATER STABLE C60 AGGREGATES WITH OH RADICAL
AND HYDRATED ELECTRONS" (INPREPARATION)
4. CHO, M.; FORTNER, J.D.; HUGHES, J.B.; KIM, J.H. "ESCHERICHIA COLI
INACTIVATION BY WATER SOLUBLE OZONATED C60: KINETICS AND MECHANISMS."
(IN PREPARATION)
TOPIC III
PHOTOCHEMICAL ACTIVITY OF C60
IN THE AQUEOUS PHASE DURING UV IRRADIATION
PHOTOACTIVITY OF Cfi(
C60 AS SINGLET OXYGEN PRECURSOR
"v ,ir
30, 1o2
ARB OGAST ET AL JPHY.CHEM. (1991)
C60 AS SUPEROXIDE RADICAL ANION PRECURSOR
ir
ED ED*
o2 or
3~
<-60 * <-60
YA[W\KOSHI ET AL J.AM.CHEM.SOC. (1991)
COMPARING PHOTOACTIVITY OF VARIOUS Cfin SAMPLES
DISPERSION STATUS OF C60 IN THE AQUEOUS PHASE DETERMINES
THE CAPABILITY OF C6Q TO TRANSFER PHOTOENERGY TO OXYGEN
nCgj (SOLVENT EXCHANGE)
• ^
son/c60
- PRISTINE-Cq) in TOLUENE " " i '
20 40 60 80 100 120
Irradiation Time (min)
DISPERSED AS AGGREGATES
t
I
MOLECULARLY DISPERSED
ELECTRON SPIN RESONANCE
FOR DETECTION OF SINGLET OXYGEN
c60/tx
(ABOVE C.M.C.)
C60/TX
(BELOW C.M.C.)
iJwJ|
MAGNETIC FIELD (mT)
MAGNETIC FIELD (mT)
MAGNETIC FIELD (mT)
Georgia
- -tech
6
-------
NANO-SECOND
LASER FLASH PHOTOLYSIS FOR 3Cfin* DECAY
c60/db
(ABOVE C.M.C.)
fewtirr1
SELF-QUENCHING/TRIPLET-TRIPLET ANNIHILATION
ARE ENHANCED IN AGGREGATED C60
REPORTED TOXICITY OF nC60 AGGREGATES
ORGANISMS
1^60
AGGREGATES
INHIBITION
CONCENTRATION
REFERENCE
B. subtilis
THF/nCgo
MIC: 0.08-0.10 mg/L
Lyon, D. Y, et al.,
ES&T, 2006
Son/nCgj
MIC: 0.4-0.6 mg/L
AQUA/nC60
MIC: 0.4-0.6 mg/L
E. coli
THF/nC60
Growth inhibited at
0.4mg/L
Fortner, J. D., et al.,
ES&T, 2005
Daphnia Magna
THF/nCg0
LDgj: 0.8mg/L
Oberddrster, E„ et
al., MER, 2006
Water
stirred/Cg0
LDg,: >35mg/L
Human dermal fibroblasts
Human liver carcinoma cells
Neuronal human astrocytes
THF/nCg0
LDcfl: 2-50ppb
Sayes, C. M„
Biomaterails, 2005
Human monocyte-derived
macrophage
THF/nCgg
Observed absorption
in the cytoplasm,
lysosomes, and cell
nuclei
Porter, A. E., et al.,
ES&T, 2007
POTENTIAL INTERFERENCE BY THF
THF/nCeo HAS BEEN CONSISTENTLY REPORTED TO BE MORE TOXIC
THAN OTHER FORMS OF nC60
WATER BATH
RESIDUAL THF REPONSIBLE
FOR TOXICOLOGICAL EFFECT?
OTHER FORMS OF THF
DERIVATIVES POSSIBLE?
e.g., TOXICOLOGICAL EFFECT
OF V-BUTYROLACTONE (GBL)
ON LARVAL ZEBRAFISH
HENRY, ENVIRON HEALTH PERSP.,2007
THF-PEROXIDE
Kl TITRATION SUGGESTS THE
PRESENCE OF PEROXIDE
THF/nCgo UNWASHED: 12 PPM
Ja,
THF/nCeo WASHED
Ja.
JUl
NOT PRESENT
THF/nCgo UNWASHED
REFLUXED THF
PRODUCT CHARACTERIZATION: LC/MSD
104-1Qo-o„
22.1 I I
J-o—OH +18
: Q-
7
.1
I
19
n +18 :
0
21
100 120 140 160 180 200 220 240
m/z
ELUENT: AMMONIUM ACETATE: 95%; METHANOL: 5%
7
-------
PRODUCT CHARACTERIZATION: 1H NMR
THF PEROXIDE COULD BE RESPONSIBLE FOR:
TOXICOLOGICAL EFFECTS
CHEMICAL REACTIV1TIY (DYE DEGRADATION)
AGING OF THF/nC6o
Goorgtn
- T»ch
MIC AND INACTIVATION KINETICS FOR E.COLI
Concentration
(mg/L)
Number of repeated washing
MIC STRONGLY DEPENDED ON THE
NUMBER OF REPEATED WASHING
E. COU INACTIVATION WAS
MOSTLY DUE TO THF PEROXIDE
50 100 150 200 250 300
Time (min)
SUMMARY
STATUS OF Ceo DISPERSION IN THE AQUEOUS PHASE AFFECTS ITS
ABILITY TO TRANSFER ABSORBED PHOTOENERGY TO OXYGEN
C60 PRESENT IN WATER AS STABLE AGGREGATES DOES NOT PRODUCE
102 AND 02- UNDER UV ILLUMINATION, IN CONTRAST TO PRISTINE Cg,
WHEN Cgo IS PRESENT AS AN AGGREGATE, THE LIFETIME OF KEY
INTERMEDIATE SPECIES FOR ENERGY TRANSFER IS DRASTICALLY
REDUCED, FUNDAMENTALLY BLOCKING THE ROS PRODUCTION
MECHANISM
THF PEROXIDE FORMS DURING PREPRATION OF nC60 WHICH IS
PARTIALLY RESPONSIBLE FOR THE REPORTED TOXICITY
Goorgto.
PUBLICATIONS
1. LEE, J.; FORTNER, J. D.; HUGHES, J. B.; KIM, J. H. (2007). "PHOTOCHEMICAL
PRODUCTION OF REACTIVE OXYGEN SPECIES BYCg, IN THE AQUEOUS PHASE
DURING UV IRRADIATION." ENVIRONMENTAL SCIENCE & TECHNOLOGY, 41, 2529-
2535
2. LEE, J.; KIM J- H. (2008). "EFFECT OF ENCAPSULATING AGENTS ON DISPERSION
STATUS AND PHOTOCHEIVICAL REACTIVITY OF C60 IN THE AQUEOUS PHASE."
ENVIRONMENTAL SCIENCE & TECHNOLOGY, 42, 1552-1557
3. LEE, J.; YAMAKOSHI Y.; HUGHES, J. B.; KIM, J. H. (2008). "MECHANISM OF Cg,
PHOTOCHEMISTRY IN THE AQUEOUS PHASE: FATE OF TRIPLET STATE AND
RADICAL ANION AND PRODUCTION OF REACTIVE OXYGEN SPECIES."
ENVIRONMENTAL SCIENCE & TECHNOLOGY, 42, 3459-3464
4. ZHANG, B.; CHO, M; FORTNER, J. D.; LEE, J.; HUANG, C. H.; HUGHES, J. B.; KIM, J.
H. "DELINEATING OXIDATIVE PROCESSES OF AQUEOUS C60 PREPARATIONS:
ROLE OF THF PEROXIDE." ENVIRONMENTAL SCIENCE & TECHNOLOGY (In Press)
KOREA RESEARCH FOUNDATION
ACKNOWELDGEMENTS
GEORGIA INSTITUTE OF TECHNOLOGY
JOHNFORTNER
JAESANG LEE
BO ZHANG
MIN CHO
DOOIL KIM
CHING-HUA HUANG
SEUNG SO 0 N JANG
JOSEPH HUGHES
RICE UNIVERSITY
ADINA BOYD
JOSHUAFALKNER
SEAN MO RAN
VICKI COLVIN
PEDRO ALVAREZ
UNIVERSITY OF CALIFORNIA, IRVINE
WEIHUA SONG
WILLIAM COOPER
8
-------
INDIGO DEGRADATION KINETICS WITH nCRI
son/nC60
THF/nCS0/washed THF/nC60/unwashed
0^^^W>OO°O0OCkA
o Dark ¥
]
a Light f
Control T
0 60 120 180 240 300 60 120 180 240 300 60 120 180 240 300
"Time (min) "Time (min) Time (min)
UNWASHED THF/Nc60IS MORE REACTIVE
[nC60] = 3 mg/L; [indigo] = 16 pM;pH = 7.0; temperature3 22°C. Control tests were performed without nCjQ.
POTENTIAL THF DERIVATIVE
V-BUTYROLACTONE (GBL)
...WAS FOUND IN THF/nC60 UNWASHED
"1
ur"
GC-MS ANALYSIS RESULT
9
INDIGO DEGRADATION WITH GBL, THF AND REFLUXED THF
30 60 90 120 150 180 210 240
Time (min)
-------
INDIGO DEGRADATION BY A
FRACTION COLLECTED FROM HPLC
Goorgtn
u Ttec*
WATER TREATMENT PROCESS
BENCH-SCALE TESTS TO EVALUTE REMOVAL OF
REPRESENTATIVE CARBON NANOMATERIALS (C60 AND MWNT)
BY CONVENTIONAL WATER TREATMENT PROCESSES
(COAGULATION/FLOCCULATION/SEDIMENTATION/FILTRATION)
DisliilfuLrju
10
-------
Role of Particle Agglomeration in
Nanoparticle Toxicity
Terry Gordon, PhD
NYU School of Medicine
Study Hypothesis
• There is a difference in the toxicity of fresh
(predominantly singlet) vs. aged (predominantly
agglomerated) carbon nanoparticles
• This difference also applies to metal
nanoparticles
2.0e+7
1.5e+'
J 5 Oe+6
1
I *T5
J 0 0e+0
Feasibility?
Inspired ZnO Distributions [dN/dlog(Dp)]
CMQ- JMB ow
mill
L
4 5e+5 -n I
3
3.0e+5 JI
o I
i.5e+5
0 0e+0 3j
10 100
lAging time of 3.3 min Particle Diameter (nm)
Beckett et al., Blue Journal, 2005
Objectives
• Measure the agglomeration rate of carbon j-
> Establish the agglomeration of freshly generated carbon nanoparticles at
various distances (i.e., aging times) downstream from particle generation in a
dynamic exposure system
• Identify whether agglomeration is affected by altering
exposure conditions such as humidity and particle charge
• Compare the toxicity of singlet vs. agglomerated particles in
mice exposed via the inhalation route
> Expose mice to nanoparticles at different stages of particle agglomeration
• Are findings for carbon nanoparticles applicable to other
nanoparticles?
> Generate zinc and copper nanoparticles
Methods
Data Presented Last 2 Years
Generate nanoparticles with Palas generator
Dilute particle stream with air (supplemented with
oxygen) and split into 2 paths: fresh and aged
Expose mice for 2 to 5 hrs to filtered air or carbon,
zinc, or copper nanoparticles
- gravimetric measurements
- particle size - WPS scanner (TSI, Inc.)
Examine lung lavage at 24 hrs after exposure
Fresh = 1.5 sec downstream (« 11 to 90 nm)
vs.
Aged = 3 minutes downstream (190 to 250 nm)
Fresh vs. Aged carbon nanoparticles
- Dose-response from 1 to 5 mg/m3
No difference in response with low or high humidity
Particle charge had no effect
Particle type had significant effect on results
1
-------
Effect of Other Nanoparticles?
Copper
Zinc
Copper Nanoparticles (0.8 mg/m3)
Effect of Copper Nanoparticles on
PMNs in Lavage Fluid
] Air
3 Copper
Fresh Copper Nanoparticles Effect on % PMNs
z
S
Oh
80-.
70-
60.
50-
40.
30-
I
20-
10.
QjtLA_
0.0
¦ Fresh
A Aged
1.0
Copper concentraton (mg/m )
Copper and Zinc Effects
Fresh Copper Nanoparticles Effect on Protein?
— Same general dose-response as for PMNs
Copper vs. Zinc Nanoparticles?
— Similar dose-response curves (PMNs and protein) for both copper and zinc
2
-------
(m?
Mill
Genetic homology
of human and
Ill
liiiii
mouse genomes
1 10
M|-
• Colors and corresponding
numbers on the mouse
chromosomes indicate the
iHll
mm
human chromosomes
containing homologous
segments
Ml
m ¦
• From D.O.E. Human Genome
Program Report, 1997.
Strain Response
• 2 hr exposure to 0.6 to 0.8 mg/m3 fresh zinc
nanoparticles
• 13 inbred strains of mice
- BALfl/c:
- BTBR
- MRL
- SJL
- AKR
- NON
- NZW
- C3H/He
- A/J
- Rill
- C57BL/6
- SWR
- DBA
Young Adult Strain Response to Zinc Nanoparticles
% PMNs
o -
It
ii L 11
/ ~' // /
Young Adult Strain Response to Zinc
Nanoparticles
Protein (ng/ml)
Conclusion
Jl
\/ sY// /
Strain-dependent difference in response
suggests genetic factors contribute to the
response
-------
Age Effect?
• In many epidemiology studies, elderly and
young (infants/children) are more
susceptible to inhaled particles
• Would older mice be more responsive to
inhaled nanoparticles?
Young vs. Old Adult Strain Response to Zinc
Nanoparticles
Old Adult Strain Response to Zinc Nanoparticles (12
Conclusions (cont....)
• Copper and zinc nanoparticles
- more toxic than carbon nanoparticles
- Copper nanoparticles were somewhat more toxic than zinc
nanoparticles
• Strain and age differences in response suggest that both
genetic and age-related factors can influence the response
to nanoparticles
This research is funded by
U.S. EPA-Science To Achieve
Results (STAR)Program
Grant#!
RD-8325280
Thanks to:
Conclusions
• Dose-response relationship between exposure to carbon
and metal nanoparticles and lung inflammation/injury
— Fresh » Aged effects for carbon but less so for copper and zinc
• Humidity and charge had no effect on the toxicity of
carbon nanoparticles
Karen Galdanes, Lung Chi Chen, Beverly Cohen, Martin
Blaustein, Nick Halzack
-------
ASSESSING THE ENVIRONMENTAL
IMPACT OF N ANOM ATE RIALS ON
BIOTA AND ECOSYSTEM
FUNCTIONS
Jean-Claude J. Bonzongo
Dept ofEnvironmental Engineering Sciences, University of Florida, Gainesville,
FL 32611-6450
fiigmoiirig
Project Overall Hypothesis
Chemical elements used in the production of NM
could lead to environmental dysfunctions due to:
(l)-The potential toxicity of these elements and their derivatives
(l)-The nanometer-size that make NM prone to bio-
uptake/bioaccumulation
(l)-The large surface area which might lead NM to act as
carriers/delivers of pollutants adsorbed onto them
Engineering
Research Approach & Methods
TOXICOLOGY
Screening of NM for potential toxicity
(Carbon-based, metal, and metal oxide NM and quantum dots)
Effects on Ecosystem Functions
* Effect ofNM on microbial-catalyzed
chemical reactions (carbon cycle).
Transport in porous media
* Soil column studies
NMs as carrier/deliver of other
pollutants
Toxicity and ToxicityMechanism(s)
Molecular modeling simulations
* permeation of NM into the cell
* damage to the cell membrane by NM
Lab investigation of toxicity and
toxicity mechanisms
T. Toxicity and toxicity mechanisms of
C60 and carbon nanotubes
Predicting Toxicity Mechanisms by use of Molecular
Dynamics Simulations (MPS)
J Task I:
~~~Understand the mechanisms of NM permeation of cell
membranes
~Task 2:
~~~Assess the potential damages to the cell membrane and
cell toxicity
Disruption of Cell Membranes and Toxicity
Physical Mechanisms
/ \
Morphological Formation of
Changes Holes
Noguchi and Takasu,
Biophys. J. , 2002
Leroueil etal.,Acc. Chem
Res, 2007
Chemical Mechanisms
e.g. Lipid peroxidation
Panessa-Warren, J. Phys.
Condens. Matter, 2006
1
-------
~Negligible energy barrier for
entry
~~~Significant energy well in the
bilayer center
~Qualitative differences
between spherical and non-
spherical particles
Lateral Pressure
Profiles Associated
with CNTs
UF i hwiii.'
Toxicity Screening Methods
Cell Membrane and Potential CNT Toxicity
i*l
Carbon Nanotube Induced Change in Lateral Pressure Profile May
Affect Membrane Proteins
^ HoiiibAi
Ceriod
Selen
Chronic
MetPLATE™
Sensitivity to
toxicants, particularly
metals. May equal or
even surpass that of
the 48-hr
A short-term (48 hr)
acute assay used to
assess the toxicity of
freshwater samples.
ite Toxicity Assay
•ubcapiata)
Based on the inhibition of the
enzyme P-galactosidase by
metals at toxic levels in a
mutant strain of E. coli.
Toxicity of THF-C60 suspensions
Biotests
ec50
(ppm)
MetPLATE
-
C. dubia
0.43±0.11
S. capricornutum
0.13±0.05
MetPLATE C. Mia S. Capricomtwn
Toxicity tests
Effect of C60 oil Microbial Degradation of Acetate in
Sediment Slurries (THF-C60)
~ Control • C60-treated sluriy
2
-------
2. Natural River Water as Solvent for NM Suspensions:
Effects of DOC, ionic strength, and pH
[see Gao et aL, Environ. Sci TechnoL 200% 43,3322-3328]
(icorjiin
Florida
* SRI
pH 4.7
Gulf of Mexico V' pH7M
1
— 1M
IlF;FLOR'ir&
Concentrations of Suspended C(
in River Waters of Different
Chemical Compositions
iDI-water ~ SR-1 HSR 2 QSR-3
Water sample types
EnguieenJig
Toxicity results of C60 Suspended in
River Water of Different Chemical Composition on Test
Model organisms
Ceriodaphnia dubia assay: NO TOXICTY detected
MetPLATE (e. coll based enzyme assay): NO TOXICITY detected
Conclusions
NANOMETALS
Toxicity of nCu arid nAg as a function of river
water DOC and Ionic Strength levels
1. FACILITATED CARON-BASED NMs / ORGANISM
INTERATIONS
Easy penetration of the cell membrane
Retention time within the membrane is a function of size and shape
Carbon nanotube accumulation within cell membranes ^ change in
lateral pressure profile
Important factor in activity of membrane proteins
Longer CNTs cause larger change of pressure profile
—-.,,-Ar\V
" Toxicity observed in lab experiments that favor cell-NM contact
3
-------
Conclusions (Cont'd)
2. TOXICITY OF CARBON-BASED NMs SUSPENDED IN
NATURAL RIVER MATRICES
Solution chemistry vs hydrophobicity
Toxicity reduction/elimination
Significant differences between Carbon and metal based NMs with
regard to aqueous suspension/solubility and toxicity
EngjhmiM
Contributors/Acknowledgement
J. Gao, Ph.D. candidate (EES, IIF) S. Youn, Ph.D. candidate (EES) Y-R
Dr. Kopelevich (CHE, UF)
Results (STAR)Program
Grant#EISEa
4
-------
Long-Term Effects of
Inhaled Nickel Nanoparticles on
Progression of Atherosclerosis
November 20,2008
Gi Soo Kang/ Dr. Lung Chi Chen
Dept. of Environmental Medicine
New York University School of Medicine
Inhaled NPs and their effects
on the cardiovascular system
• Inhalation as a major route of exposure to NPs
I
• Well-established association between ambient particles and
cardiovascular disease
• Strong potential to induce oxidative stress and
inflammatory responses
=> major mechanisms for cardiovascular disease
• Possible direct interaction with cardiovascular tissue after
translocation
Hypothesis
Why Nickel?
Inhaled NPs
Inflammatory
Responses
Progression of Atherosclerosis
~ Commonly found in environment
~ Widely used in industry
~ Potential to generate oxidative stress
~ Indications of potential cardiovascular effects by inhaled Ni
Nickel hydroxide (Ni(OH)2)
widely used as a positive electrode in alkaline battery
=>great interest in nanotechnology for various application
Few toxicological data
Study Design
• Ni NPs: spark-generated (PALAS) from metallic Ni electrodes
• Dose: -80 ^ig Ni/m3 (PEL: lmg/m3)
for 5h/d, 5d/w, for either lw or 5m
• Exposure route: whole body inhalation
5m-old male Apoe'1' mice
(N=6/group for lw study,
N=16/group for 5m study)
mt-DNA damage
Gene expression
Elemental analysis
Histology
1
-------
Deposition and Translocation
• Relatively rapid clearance from the lung
• Significant deposition/accumulation in the lung
• No significant accumulation in the blood
IMipl
Total Ni contents in the lung at Total Ni contents in the lung at 24h
indicated hrs after 1 d-exposure after the designated exposures
Oxidative Stress
Inhaled Ni NPs can induce oxidative stress
not only in the lung but also in the cardiovascular system.
• Ho-1 mRNA expression
(lung, spleen, heart, aorta)
" • mtDNA damage (aorta)
Oxidative Stress
1) Ho-1 mRNA expression
Ho-l (heme-oxygenase 1): sensitive marker for oxidative stress
=> Indication of increased oxidative stress in various organs
Oxidative Stress
2) mtDNA damage
• mtDNA: highly susceptible to oxidative stress
• mtDNA damage in aorta: association with CVD
• Determined by semi-quantitative long PCR
¦ Control B Nickel
Inflammatory Responses
Inhaled Ni NPs can induce pulmonary
and also systemic inflammatory responses.
• Pulmonary inflammation
- bronchoalveolar lavage fluid (BALF) analyses
- mRNA expression in the lung
- histopathological analysis
• Systemic inflammation
- mRNA expression in the spleen, heart, liver, aorta
- inflammatory markers in serum
Pulmonary Inflammation
1) BALF analyses
Neutrophil Influx in BALF
•kie
persisting effects by inhaled Ni NPs
-------
Pulmonary Inflammation
2) mRNA expression
mRNA expression - Lung
Lv
~ Ccl2 B Il-la ~ 11-6 ~ Tnf-a
~ Significant pulmonary inflammation at both time points
=> persisting effects by inhaled Ni NPs
Systemic Inflammation
1) Acute phase proteins
mRNA expression - Liver
~ Crp ¦ Sap
Crp: C-reactive protein
Sap: Serum amyloid p component
Systemic Inflammation
2) Gene expression in extra-pulmonary organs
mRNA expression Spleen
mRNA expression - Heart
^5:
~ Ccl2 ~ 11-6 ~ Tnf-a
~ Ccl2 ~ 11-6 ~ Tnf-a
• Indication of systemic inflammation in the long-term
Atherosclerosis
A long-term exposure to inhaled Ni NPs can enhance
progression of atherosclerosis in a sensitive animal model.
Plaque quantification
- H&E staining
Atherogenesis
- mRNA expression
http: //www3.imperial. ac.uk/pls/portallive/
docs/1/27647698. JPG
Control
NiNPs
% Luminal Area
54.3 (±10.6)
41.1 (±6.4)*
Atherosclerosis
2) Gene expression
mRNA expression in Aorta
Indication of macrophage infiltration, monocyte-adhesion
-------
Supplemental Studies
Ni NPs Toxicity: Particle Effects?
Study 1
Particle effects?
Same-size NPs of different
chemical composition
Study 2
Dissolved Ni?
Same-size NPs of
Ni(OH)2 vs NiS04
Id (4h) exposure to C57 mice
Endpoint
Pulmonary Inflammation
Carbon
Ti
Cu
Ni
CMD (nm) 49.02
34.77
35.49
38.79
Geo.STD 1.54
1.66
1.44
1.49
# cone (#/cm3) 1. 8E+07
2.5E+07
1.6E+07
1.8E+07
Total mass cone (ug/m3) 558
560
530
550
Particle generated by the same method
(PALAS spark-generator)
Comparable in size, number and mass
concentration of the particles
Ni was most potent, followed by Cu
=> role of chemical composition
Ni NPs Toxicity: Dissolved Nickel?
Nl(OH)2
NiS04-6H20
CMD (mil)
38.98
37.75
Geo. STD
1.47
1.87
# cone (#/cm3)
2.23E+07
3.94E+06
Total mass cone (ug/m3)
1200
3600
Nickel mass cone (ug/m3)
761
792
NiS04-6H20 particle generated from 0.15% solution using nebulizer
Comparable in size and nickel mass concentration of the particles
Ni(OH)2 was significantly more potent
Conclusion
Inhaled Ni NPs, at occupationally realistic levels, can induce
oxidative stress not only in the lung but also in the
cardiovascular system.
Inhaled Ni NPs can induce pulmonary and also systemic
inflammatory responses.
Long-term exposure to Ni NPs could exacerbate plaque
formation in hyperlipidemic mice.
Observed toxicity of Ni(OH)2 NPs may not be explained
solely by particle effects or dissolved Ni effect.
Significance
• The first sub-chronic inhalation study to investigate
cardiovascular effects of NPs
• Exposure below the current occupational guidelines
4
To further investigate potential toxicity of Ni(OH)2 NPs
To provide a database to establish size-specific
regulations in occupational and environmental settings
Acknowledgement
• Dr. Lung Chi Chen
• Patricia Gillespie
• Dr. Terry Gordon and his lab
• Dr. Albert Gunnison
• Dr. Jeff Koberstein (Columbia University - XPS analysis)
• Dr. Lu Chen (Columbia University - XPS analysis)
This work is supported by a NIH grant (R01-ES015495)
4
-------
Thank you !!!
Questions ???
5
-------
m
2008 Interagency Environmental Nanotechnology Grantees
Workshop, Nov 19-21 Tampa, FL
iUSGS
Aquatic Toxicity of Carbon-Based
Nanomaterials at Sediment-Water Interfaces
(April 2007-March,2010)
Joseph Mwangi, Bin Hua, Hao Li, and Baolin Deng
University of Missouri, Columbia, MO
Chris Ingersoll and Ning Wang
USGS-Columbia Environmental Research Center
Columbia, MO
m
Acknowledgments
•Collaborators
- Andrew Ritts (MU)
- James L. Kunz (USGS)
- Doug K. Hardesty (USGS)
- Eric L. Brunson (USGS)
- Jianzhong Zheng (Nanjing University)
- Electron Microscopy Core Facility (MU)
•Funding Sources
- Environmental Protection Agency STAR program
(RD-833316)
Drs. Nora Savage and Dr. Barbara Karn
[USGS
Two sides of Nanotech
Water filtration/desalination systems
Atmospheric carbon mitigation
Environmental remediation
Advanced microelectronics
Global communications
Cure for cancers
Harvesting solar energy
Microbial fuel cells
Gas separation
Hydrogen storage
Sensors
Transport and fate
Detection in the environment
Toxicity to various life forms
Molecular mechanisms of nano-bio
interactions
One of the biggest challenges facing firms commercializing
nanotechnaHgy innovations today is managing environmental,
he^lm and safety (EHS) risks - Lux Research (2006)
m
Objectives
[USGS
~ Adapt a proper method for water and sediment toxicity
testing 1-D nanomaterials (CNTs, SiC)
~ Assess toxicity of representative 1-D nanomaterials in water
or in sediment to representative sediment-dwelling
organisms:
~ Amphipods (Hyalella azteca)
~ Midge (Chironomns dilutus)
~ Oligochaetes {Lumbriculus variegatus)
~ Freshwater mussels (Villosa iris)
~ Identify factors controlling the toxicity towards the
sediment-dwelling organisms.
m
FOSGS
1-D Nanomaterials used for toxicity testing
Single-walled CNT (SWCNTs: Shenzhen Nanotech Port, Inc.
China):
Over 90% puritv, average tube diameter of 2 nm, average tube length
5-15 pm, specific surface area > 400 m2/g, < 2% ash, < 5%
amorphous carbon.
Multi-willed CNT (MWCNTs, Shenzhen Nanotech Port
Inc, China):
>95% purity, < 0.2% ash, < 3% amorphous carbon, tube diameters 10-
20 nm, tube lengths 5-15 pm, specific surface area 40- 400 m2 per g.
MWCNTs (Helix Material Solutions Inc., TX USA):
purity > 95%, total impurities <0.2 %W, pH 6-7.
Silicon Carbide (SiC, Manufactured at the University
of Missouri)
-40-200 nm diameter, 10-50 |am length
m
Nanomaterials with Well-Controlled Atomic
and Meso Structures for Toxicity Testing
^U5G3
Metal Nanoparticles
Graphitic Layer Orientation
CVD Carbon Nanotubes CVD-Template Liquid Crystal-Tempi ate Hybrid Method*
(Single-walled & Multi-walled) (Carbon Nanotubes) (Carbon Nanotubes & Nanofibers) (Carbon Nanofibers)
Metal Nanoparticles
V
I I
SiC Nanowire
wI Catalyst
~n
Conversion with SiO Vapor
-------
m
SEM micrograph of a MWCNTs sample
[USGS
Variable tube diameters and the
rope like entangled morphology
&
iOSGS
Transmission Electron Microscopy of a MWCNTs
1 Arrows show carbon
nanotubes with open
ends.
1 Dark spots are metal
clusters in the
nanotubes
Image (xl0,000) taken at lOOkvwithTEM FEI Quanta 600F
&
Testing organisms
icmk* to> * cJunyuff wwli
i
%
Amphipod, Hyaleila azteca
Midge Chironomus dilutus
Mussels, Lampsiiissiliquoidea
01 igoc hates
Lumbriculus variegates
m
Standar
musgs
Operating Procedure for HandliH§>«><*>M ¦
This Standard Operating Procedure (SOP) outlines procedures for the
safe handling, storage and use of the nanomaterials in the laboratory
and to avoid contaminating waste water. The SOP should be used in
conjunction with the Materials) Safety Data Sheets (MSDS) from the
suppliers of the nanomaterials.
1. Storage
2. Handling
3. Weighing and mixing with water or sediment
4. Replacement of water during an exposure
5. Decontamination of nanomaterial contaminated
items
6. Disposal of material
m
Test Conditions
(ASTM, 2007b, USEPA, 2000)
[uses
Test type:
Static renewal
Test Duration:
14 d
Test chamber:
300-ml beaker
Water volume:
200 ml
Water renewal:
100 ml on Monday, Wednesday, Friday
Feeding:
Monday, Wednesday, Friday.
Aeration:
air bubbling through mixture
Test water:
Hardness of 100 mg/L as CaC03
Test concentrations:
200 mg CNTs in 200 ml water
Mixing conditions:
Sonication and non sonication
Chemical residues:
dissolved metals in overlying water
Water quality:
DO, pH, conductivity, hardness, alkalinity, ammonia
Endpoints:
Survival and growth
Test acceptability:
(1) >80% survival in controls for amphipods and mussels;
(2). >70% survival in control for midge;
(3). 14-d biomass >0-d biomass for oligochaetes
2
-------
Survival of amphipods, midge, mussels, and biomass of oligochaetes after
14 d exposure in as produced or modified carbon nanotubes in water
only toxicity screening tests (Phase 1)
Sample Treatment
Mean survival (%, SD)
Mean dry biomass (mg, SD)
Amphipods
Midge
Mussels
Oligochaetes
1.
Control(MW)
86
(5)
30 (8) 98 (5)
16(2)
Non-sonicated
5(10)
60 (8)
23(17)
14(1)
Sonicated
3(5)
43(10)
43(19)
13(1)
2a.
Control(MW)
100 (0)
63(15)
80 (28)
3.3 (2)
Non-sonicated
8(10)
55 (6)
35(25)
0.8 (0.2)
Sonicated
5(10)
8(10)
5(5)
2.8.(1.3)
2b.
Control(MW)
100 (0)
75(19)
97 (5)
3.7(1)
Non-sonicated
95 (6)
60(14)
100 (0)
1 (0-3)
3.
Control (SW)
100 (0)
83 (5)
Not Tested
3.7 (0.8)
Non-sonicated
20(12)
0(0)
Not Tested
1.4(0.5)
Sonicated
0(0)
10(8)
Not Tested
2.8 (0.2)
1 - as produced MWCNTs from Helix material solutions, TX USA
2a- as produced MWCNTs from Shenzhen Nanotech Port, China
2b- nitric acid modified as produced MWCNTs frcm Shenzhen Nanotech Port, China
3. As produced SWCNTs from Shenzhen Nanotech Port, China
R] Midge (Chironomus dilutus)
6 d exposure
in control
treatment
6 d exposures in non-sonicated MWCNTs treatment
*' r^rrw^k'
m
Oligochaetes (Lumbriculus variegatus)
after 6 d exposures to MWCNTs treatment
RJSG5
t
Rainbow mussels (Villosa iris)
after 14 d exposures to
non-sonicated (top) and
Sonicated (left) MWCNTs
treatment
&
"pUSGS
A) gut after 6-d exposures with MWCNTs deposits
B) CNTs trapped between and around some of the microvilli
m
iOSGS
TEM images of gut of midges
A) MWCNTs deposits in gut after 6 d exposures
B) Control with some food deposits only
. - .
f ¦%
A
C\,»' . w v ^
..."
TEM images taken at 100 KV(JEOL JEM 1400, JEOL, Tokyo, Japan)
m
Amphipods exposures
5 ¦ J , - / ^
t a j Mr J m I
f r '
• f u ¦
[DSGa
6-d exposures to SWCNTs
¦
S ' ¦.
6-d in control
CNTs deposits
-------
m
Scanning electron micrograph imagSS"
[USGS
of SiC nanowires
as-fabricated SiC nanowires
(average diameter = 100 nm;
length = 10-50 fim)
SiC nanowires after sonication
m
TTJSG3
(»»4 Supdnti dmiM w» ttous m p
ASTMlul
Kmibai
"%'m qmliry to ociy a
«#»
»HM>
t1<»4
"tliil
U»JI
stent)
11 I;
Hp
««»u
lift
fp&r,
mu-
¦r
Kdxl*s3n3!i|o?tbe«xpDic»
rami)
8&
laoti
»0")
»0ti)
WO* J)
AS.TM lUfc-.
ASTMH»d
AVTUHmI
ASTMHwt
An^nd OitaMWril!
-ftnr:—
mat a
100(0)
XO*i:
«ttO>
nqur
100 ID"
&
Conclusions
Sonicated or non-sonicated as-produced single-walled and
multi-walled QNXs are toxic to amphipods, midge,
oligochates and mussels in water .
Sediment can reduce, but not totally eliminate, the toxicity
of as-produced MWCNTsto amphipods.
Sonication significantly increases the toxicity of SiC
nanowires to amphipods.
[USES
&
~^USG5
HlHM A* » ctmafwg M
-------
Toxicity of Nanoparticles in
an Environmentally Relevant
Fish Model
Judi Blatt Nichols
Department of Environmental Medicine
New York University School of Medicine
November 20, 2008
Interactions between the environment and
nanoparticles due to their physico-chemical
properties may influence bio-availability and
toxicity in aquatic organisms
• Particles:
* Size
• Density
* Surface functional groups
Affect particle
• Hydrophobicity
agglomeration and
• Environment:
settling or suspension
> Water hardness
in water column
Salinity
> Natural organic matter
Why early-life stages of fish?
• Very sensitive to a wide range of environmental
contaminants
• Easy to acquire large numbers allowing for robust
statistical analysis
Relatively inexpensive compared to mammals
• Treatments can mimic environmental conditions to
determine likely occurrence in wild populations
Why Atlantic tomcod?
Common fish found in Atlantic coastal estuaries from
Hudson River to Labrador
Wintertime spawners. Juveniles are dominant prey for
predatory fish during summer months. Occupy critical
node in food web - valuable indicator species
Bottom dwellers with lipid-rich livers. Exposed to and
accumulate extraordinary high levels of hydrophobic
contaminants associated with sediments (i.e., dioxins,
PCBs)
Long embryonic developmental period (30+ days)
Focal species for almost 20 years of research on toxic effects
of contaminants on ecosystems in Dr. Wirgin's lab
Hypothesis:
There are particle-type dependent differences in
early life stage toxicity
• Embryonic exposure
> Mortality
> Hatching success and rate
> Larval stage morphology
Larval exposure
> Mortality
1
-------
rypes of particles1use(
• Fullerenes
• Functionalized single-wall nanotubes:
• Polyethylene glycol (P7-SWNT)
• m-polyaminobenzene sulfonic acid
(P8-SWNT)
• Carbon black
• Metal nanoparticles:
• Ag, Cu, Fe, Ni, Zn
• Manufactured nanoparticles: 3 atoms of
metal (erbium, yttrium) within a C8o cage.
• Soot - raw material
Experimental design:
• Tomcod production: 6 mating pairs from Shinnecock Bay, Long
Island, NY used to produce embryos
• Stock suspensions of nanoparticles in 5 ppt sea water (except
fullerene-DMSO), sonicated for 1 hr, graded dilutions prepared
in 5 ppt sea water.
• Embryos exposed at 14 dp£ 30 embryos per replicate, 3 replicates
per dose, 5 doses
• Static renewal design, every 48 hours, particle suspensions
removed and replaced until embryos hatched or died (-1.5
months)
• Toxic endpoints evaluated:
• Mortality
• Hatching success
• Time to hatch
• Morphological abnormalities
Mortality and hatching with fullerene exposure
100
90
80
>1
70
50
s
40
0"-
30
20
10
0
1
1
1
1A
1 i PI
o 0.8 4 20 100 500
Fullerene concentration (pg/1)
Mean ± SD, * p
-------
Mortality with metal nanoparticle exposure
particle conc.
(US/ml)
Ag
Cu
Fe
Ni
Zn
0
14.7 ±7.7
13.8 ±8.8
14.7 ±7.7
13.8 ±8.8
14.7 ±7.7
0.016
10.9 ±2.7
8.8 ±4.1
2.8 ±4.8
12.2 ±4.3
7.9 ±6.5
0.08
14.5 ±5.5
4.0 ±3.7
11.4 ± 1.5
7.1 ±6.8
4.0 ±4.0
0.4
17.5 ± 11.5
83.3 ±17.4*
8.4 ±7.3
10.2 ±2.0
6.5 ±2.1
2
16.5 ±2.3
100*
11.1 ± 1.0
7.5 ± 6.8
17.8 ±2.4
10
7.2 ±9.1
o
o
o
o
19.1 ±3.0
100*
Mean ± SD, p
-------
• Ecotoxicology of Fullerenes
(CJ in Fish
Theodore B. Henry1'2, June-Woo Park 1, Shaun Ard1, Fu-
Min Menn1, Robert N. Compton1, Gary S. Sayler1
1. Center for Environmental Biotechnology, University of
Tennessee, Knoxville, TN USA
2. Ecotoxicology and Stress Biology Research Centre,
University of Plymouth, Plymouth UK
Acknowledgments/ Recogni
tion _
Ecotoxicology of Fullerenes (C60) in Fish
Henry, Menn, Compton, Sayler: Project P.I.s
Jun e-Wo o Park: Po s tdo c
Tze Ping Heah:
PhD student
Research Assist
A This researci
USEPA-Sc
Results (ST,
V Grant#
This research is funded by
US.EPA-Science To Achieve
Results (STAR)Program
Project duration: 2007-2010
%
m
Project Objectives *
Progress Report: Year 1 *
• Investigate physicochemical properties of
• Changes in global gene expression in
aqueous Cm aggregates
zebrafish exposed to aqueous C60
• Influence of dissolved organic material
• Evaluation of vehicle effects
• Investigate bioavailability of C6g in fish
• Aggregate characteristics
• Aqueous and dietary exposure
• Toxicity
• Investigate the toxicity of in fish
• Influence of C60 aggregates on bioavailability of
• Zebrafish, channel catfish
other toxicants
> Tissue accumulation and distnbution of C60
• Example: 17a-ethinylestradiol (EE2)
• Changes in gene expression
• Dietary exposure to C60
• Histopathology
• Experiments with rainbow trout
Background on C6o
• First manufactured carbon NP
¦ Nobel prize in Chemistry 1996
• Soccer ball shape
• Diameter = 0.7 nm
• Partially delocalized n electrons
• Structure facilitates energy transfer
• Absorption of light
• Light energy transferred to form '024
• Potential formation of free radicals
• Oxidative injury in organisms?
C6o in Consumer Products
(More than 100 fullerene patents)
Cosmetic products
• Radical Sponge
(Vitamin C60 B jl
BioResearch, I 1
Tokyo) - J
• Zelens Fullerene
C60 Day Cream
(Zelens, London)
Toxicity?
Environmental fate?
-------
Previous Research ot L6o #
Toxicity
*
m Little or no toxicity found for C60
¦ C60 applied to mouse skin (Nelson etal 1993)
¦ Mice IP administration of C60 (HouSSaetaii996)
¦ Lung cell cultures and Cqq (Baierl et al 1996)
Previous Research ot L6o #
Toxicity «t
*
¦ Little or no toxicity found for C60
¦ C60 applied to mouse skin (Nelson etal 1993)
¦ Mice IP administration of C60 (MouSSaetan996)
¦ Lung cell cultures and Cqq (Baierl et al 1996)
¦ Toxicity reported in fish and in vitro
¦ Oxidative injury in fish brains (Oberdorster 2004)
¦ Toxicity in aquatic species (Oberdorster et al 2004)
¦ Toxicity in human skin cell lines (Sayes et al 2004)
Challenges of Assessing Aquatic *
Toxicology of C6o
m
• Water solubility (< 10"9 mg/L)
• Vehicle: Tetrahydrofuran (THF)
• Dissolve C60 into THF
• Add C60-THF mixture to water
• Evaporate off THF
• Vehicle effects?
Assessment of THF Vehicle Effects
Henry, T.B., Menn, F., Fleming, J. T., Compton, R. Sayler, G. 2007.
Environmental Health Perspectives 115:1059-1065.
Experimental treatments:
Water control (synthetic soft water)
C60- water
THF-Cfin water
3 replicates
per treatment
THF- vehicle control
Larval zebrafish- age 72 hpf
Exposure duration 75 h
Exposure in 400 mL glass beakers
Endpoint: changes in gene expression
Affymetrix zebrafish array (=15,000 gene transcripts)
Larval zebrafish
(3-4 mm length)
Differentially Expressed Genes in C6o-
water Relative to Control
Number of gene (rank based on fold change)
Conclusion: little or no effect of C6o-water on zebrafish
gene expression
-------
Expression of Common Genes (182) in $
THF-Vehicle and THF-C6o »
thf-c6o
THF-vehicle control
• = C "a 40 60 80 100 120 140 160 180 200
&
rt "10"
X
U -15J
Number of gene (rank based on fold change)
==73% of genes have > change in expression in THF~C6o
treatment
Common Genes of Interest in THF-C6o
and THF-vehicle Compared to Control
Affymetrix
Probe ID
thf-c60
Control
fold
THF-water
Control
fold
Description/function
Dr. 10624
7.00
7.32
Peroxidase activity
Dr. 23788
5.39
6.05
Glutathione-S-transferase
Dr. 9492
3.69
3.43
Oxidoreductase activity
What was Causing Toxicity of THF-
• THF not detected by GC-MS
• LC50 THF = 1.73%
• THF degradation products (low ppm)
• Biologically active
• Butyrolactone
• yButanoic acid
• Furanone
• Butyrolactone tested
• LC50 butyrolactone = 47 mg/L
O-Os
THF
Butyrolactone 1
(y* Cr
Furanone
yButanoic acid
Effect of C6o Aggregates on ^
Bioavailability of EE2 *
«
• Aqueous C60 stock: 666 mg/L in pure water (stirred*
4 months)
• Experimental treatments: 3 replicates
1) 0 day
— Solvent control (0.01% EtOH)
— 17a~ehtinyIestradiol (EE2) (1 ug/L)
— C60 only: 16 mg/L, 40 mg/L, 65 mg/L
— C60 (each concentration) + EE2 (1 ug/L)
2) 28 day aged
— Repeated exposure with aged solutions
— Fresh EE2 solution (1 ug/L)
#
Effect of C6o Aggregates on #
Bioavailability of EE2 *
*
• Larval zebrafish (72 hpf) exposed for 75 hrs
• Endpoint: EE2 induced Vtg expression (qRT PCR)
• EE2 synthetic estrogen
• Vitellogenin genes (Vtg) induced by EE2
• C60 particle analyses: ZetaPALs
• Evaluate aggregate size
• Evaluate aggregate charge
3
-------
m
%
C6o Aggregate Characteristics *
• Each treatment prepared stirred then solution
allowed to settle for 1 hour
• Sample collected from mid water
• Particle size and charge assessed (ZetaPALs)
• Total C60 determined by evaporation, toluene
extraction, and UV-vis spectroscopy
C60 Aggregate Characteristics
T=0
Number Weighted Intensity
1
—C60 50% only 2 zeta= -35.82 +-1.31
—¦—C60 50% w/ ee2 2 zeta= -36.68 -M.71
; in
I .
|l 500 1000 1500 2000 2500 3(
Diameter(nm)
Most particles are near 200 nm diameter, few larger particles
Aggregate Size Distributions for 40 mg/L C60 Treatments
oo X
• C60 only
o C60W/EE2
Primary
distribution
Secondary
distribution
T=0
Primary
distribution
Secondary
distribution
T=4 weeks
Aggregate Size Distributions for 40 mg/L C60 Treatments
•Jf
• C60 only
o C60w/EE2
C60 only total = 9.4 mg/L
C60 + EE2 total = 11 mg/L
5
s
Primary
distribution
Secondary
distribution
T=0
Primary
distribution
Secondary
distribution
T=4 weeks
#
Conclusions *
•
• Presence of EE2 altered characteristics of C6o *
aggregates
Zeta potential decreased, more tendency to
aggregate
Particles were smaller; however, larger particles may
have sedimented out of aqueous phase
• C6o reduced bioavailability of EE2 (reduced
expression of Vtg)
Perhaps EE2 is absorbed within C6o aggregates
• Aging appeared to increase association of C6o with
EE2 and reduced bioavailability of EE2
Q
Q
a
i2
• EE2 bioavailability (assessed by Vtg expression) reduced by C6o
• EE2 became less bioavailable over time in presence of C6o
4
-------
Development of Methods and Models for Nanoparticle Toxicity Screening:
Application to Fullerenes and Comparative Nanoscale Particles
Two basic questions
Question 1: Are there any human diseases caused by nanomaterials?
Answer: No!
Question 2: Are there any human diseases caused by materials
such as particles or fibers?
Answer: Yes, what can we learn from it?
History of particle exposure
Lessons from Quartz (surface area, surface reactivity)
Healthy Lung
Lung Fibrosis
Quartz (Crystal Si02)
Widely used
for electronics
Lung fibrosis could be fatal (Seaton 1995), also
cause inflammation, cell death, and cancer.
"miracle mineral"
because of its soft and
pliant properties, as
well as its ability to
withstand fire and heat.
asbestos cancer: mesothelioma
Other lung disease: asbestosis
ilcome to Los Angeles!
Lessons from air pollution particle studies
-------
TEM of Ultrafine Particle
I Main sources are
I emissions or condensation
I of vapors
Carbonaceous core coated
with organic chemicals
with organic chemicals
and metals
Not EPA regulated
Nel A. Biomolecular effects of particles. Science 2005.
Several lessons from history
Oxidative stress plays a major role!
Toxicity is related to particle physical characteristics
Freshly cut,
Defective surface,
Surface reactivity,
Frustrated
phagocytosis,
Air particles:
High organic
chemicals and
metal coating,
ROS
Organic
. chemicals,
transition
'V metals
Carbon
The Hierarchical Oxidative Stress Model
High
GSH/GSSG
Ratio
« I
Low
GSH/GSSG
Ratio
Level of
oxidative sires
Tier 3
Response pathways:
Normal
Anti-
oxidant
defense
Inflammation
Cytotoxicity
Signaling pathway:
Nrf-2
MAP Kinase
NF-kB cascade
Mitochondrial
PT pore
Genetic response:
Anti-oxidant
response
element
AP-1
NF-kB
N/A
Outcome:
Phase II
enzymes
Cytokines
Chemoklnes
Apoptosis
Nel et al. Science, 311, 622-627, 2006
Examples tested in our mammalian
cell system:
Fullerenes: Polystyrene NP: Metal oxides:
Fullerol Plain, 60 nm ZnO
Aqueous/nC60 Cationic, 60 nm TI02
THF/11C6O Anionic, 60 nm Ce02
Cationic, 600 nm
Methods:
Test oxidative stress markers in mammalian cell system
Extensive physicochemical characterization
This model has to consider a wide range of nanomaterial
physicochemical characteristics
Material composition
Different sizes/shapes/aspect ratios
Different states of agglomeration
Different surface functional groups
& catalytic activities
Different stabilities/bioavailabilities
etc etc
u
Contact/interaction cell membrane
Contact/interaction proteins
Contact/ interaction DNA
Cell uptake (endocytosis/phagocytosis etc)
Subcellular localization/organellar interactions
Mitochondrial functions/ATP production
Bio-accumulation/biopersistance
etc etc
2
-------
H202 production in cells (RAW 264.7) in presence of aq/nC60,
THF/nC60 and fullerols
Comparative toxicity (PI) of the supernatant (liquid phase
containing the dissolved residue from the synthesis) and the
solid phase containing C60 aggregates.
Phase separation via centnfugalion
Phase
separation via
(16.000 rpoi)
dialysis (cut oPI of 4 nm)
*
*
LS before phase separation
L liquid pftase (supernatant)
S sow phase ToUctone (iigfl.)
I
Control rOutyctoctone Formic odd
3
-------
THF/nC60
^ — A„
THF
y-butyrolactone formic acid
THF 1> y-Butyrolactone > Dihydro-5-hydroxy-2(3H)-furanone —
C02 + H20 <—¦ Formic acid < Succinic acid <— Oxobutanoic acid <-
• The degradation product, formic acid and y-
butyrolactone can induce toxicity
• It is not the THF itself, only at high dose
• It is not clear whether fullerene speed up the
degradation process.
Table 1. Physical Charaererizaliou of Nanopaitieles"
av
electrophoretic
zfta
diameter
mobility
potential
MATH
particle
frnnk
PDI
U CmftV »))
'C (mVi
m
in Aqueous media
UFP
1034
1.0
-2-28
-29.1
8.2
PS
68
0.041
-2.85
-36.4
2.7
NH»-PS«n«
65
0.055
3.15
40.3
5.3
NHj-PS**,^
648
0.096
3.58
45,8
4.2
COOH-PS
f>6
0.063
—2.15
-27.6
0.0
TiC2
364
0.466
-1.28
-16.4
1.6
carbon black
245
0.251
-4 26
-54,6
7.1
fullerol
218
0.388
-1.76
-22.6
0.6
1
n Celt Cu
Iture Medium
UFP
1778
0.379
—0.S6
-11.0
PS
90
0.200
-1.00
-12.7
Nlla-PS^nm
527
0.339
-0.87
-11.1
NH2-PSeo»n«»
1913
1.0
-0.96
-12 2
COOH-PS
82
0.191
-0.85
-10.9
TiOa
175
0.877
-0.97
-12.4
carbon black
154
0.278
-1.06
-13.5
fullerol
106
0,700
-0.97
-12.1
a The reported
mean particle sure (average diaiueler) is calculated bawd
on an intensity v,
eighted average: PDI = polydispersify index; MATH —
microbial adhesion to hydrocarbon tc-si-
Cell toxicity to macrophages determined by PI uptake
€
18
M
Xia T, Nano Letters, 2006
Macrophage cells take up cationic NH2-PS nanoparticles
Xv# * # •.
# 1 •
The role of cellular uptake mechanisms of nanoparticles
Pinocytosis (cell drinking) Phagocytosis (cell eating)
•
1
: . • •
o-
Receptor mediated endocytosis
•, • • •
*
4
-------
Bafilomycin A1 inhibit NH2-PS induced cell death
O CI- ? v-ATPase +#¦ Cationic
¦ ++ polymer
• H+ (J) CFTR Jy Enzyme
The Proton Sponge
Hypothesis
Apoptosis
Nel et al. Nature Materials. 2008
Very much like Ardystil syndrome
The paint workers in Spain and Algeria suffered from many
complaints including nose bleeding, coughing, general
disorders of the upper airway, and bronchial hyper-reactivity.
Epidemiological and toxicological studies have suggested
Acramin, a polycationic paint component in the paint to be
responsible for this disease.
Toxicity induced by intratracheal injection of PS in mouse lung
~ Total
~ Macrophage
~Neutrophil
H Epithelial
jSl
Xia T, ACS Nano, 2008
Comparison of ZnO cytotoxicity with 2 other metal oxides
& j* Jm
100 nm J* 100 nn
5 10 15 20
Time (h)
Xia et al. ACS Nano. 2008
Cellular ROS production by Flow cytometry
RAW 264.7 B
fa
U 6
3 '
s 3
fa 2
. T h2o2
1 JvZnO
8 loo-
's 80 ¦
Oh
i Hl
!::
Superoxide *
ZnO /
f CeOj TiOj
0 5 10 15 20 n 5 in 15
Tta«(h) TimetT.)
0
BEAS-2B
b 2-
Q
h2o2
O 75-
Superoxide
.5 is-
l:
Ce02 TiOj
% MitoSOX Re
CeOj TiOj
Xia et al. ACS Nano. 2008
Time (h)
5 10 15 20
Time (h)
5
-------
ZnO has extensive dissolution in cell culture medium
0ZnO | _ _ 40. |azn0 jr
¦ZnS04 II i ¦ ZnS04 jH
_ill iji
0 12.5 25 50 Mg'ml 0 125 26 gg (jgfml
150 300 600 |JM 150 300 600 |JM
Total Zn concentration
CDMEM
—T i
BEGM
. * HjO
California MANoSystems Institute BOH
Injury through dissolution
~
Xia et al. ACS Nano.2008
Metal Fume Fever
Welders exposed to ZnO, other
metal oxides: Cu, Mg, Sn, or Cd
3-10 hrs post-exposure: flu-like
illness.fever, general malaise, chills,
dry cough, metallic taste, muscle
aches, shortness of breath
TNFa levels elevated at 3 hr,
IL-8 levels peaks at 8 hr, and
IL-6 values peaks at 22 hr
Pathophysiology: marked increases in lung PMLs 20-24 hr after
exposure
Resolves 24-48 hr after onset
Short-term tolerance: asymptomatic with repeated exposure
Acknowledgements
Collaborators:
Mark Wiesner at Duke
Lutz Maedler at Bremen
r ^ L a J Joanne I. Yeh at Pittsburgh
Andre Nel
Jeff Zink
Eric Hoek
Mike Kovochich
Monty Liong
Huan Meng
Saji George
Ning Li
Support: I S( Costas Sioutas at USC
Use mechanisms of nanomaterial cytotoxicity to
mitigate by adding safety design features
1. For toxicity, check the NP and the suspending solution
2. For fullerenes, be careful of the residual solvents; for
carbon nanotubes, decrease the impurities and rigidity
and/or functionalize the surface to increase solubility
3. For cationic particles, decrease the charge density or
replacing cationic head groups with amphiphillic head
groups
4. For ZnO, NiO, Ag, Cu, capping with surfactants,
polymers or complexing ligands to decrease dissolution
-------
Effects of Nanomaterials on
Blood Coagulation
Interagency Environmental
Nanotechnology Grantees Workshop
November 2008
Tampa, FL
Peter L. Perrotta, MD
West Virginia University
Nanomaterials & Coagulation
Rationale for Toxicology Assessment
1) Common human diseases including myocardial
infarction & stroke are related to clot formation
(thrombosis)
2) These diseases are influenced by environmental factors,
but not all risk factors are known
3) Nanomaterials entering workplace or home could have
short and/or long-term effects on the blood coagulation
system
4) Targets of nanoparticles related to toxicity are proteins
(clotting proteins)
Revised November 2008
Modern Coaaulation Cascade
Blood Nanomaterial Interactions
Nanomaterial Suspension Surface-Fixed Nanomaterials
Exposure of Nanomaterials to
Coagulation Proteins or Platelets
Nature Medicine 9, 991 - 992 (2003)
Issues in Blood Coaaulation Testin
• Blood sampling: Limit activation of clotting
proteins with blood drawing, limit protein
degradation, etc.
• Plasma: More difficult to work with than serum
• Macro vs. nano testing: Adapt assays to small
volumes
• Variability of assays: Higher than many other
assays
Standardizing Coagulation Assays In
Nanotoxicoloav Trials
• Few studies on coagulation
• •
• Most studies on biomaterial interactions
with surface fixed materials (prevent
clotting at surface)
•
~ No standardized assays for coagulation
(Nanotechnology Characterization Lab)
• Initial studies on SWCNT in animal
models and clotting systems difficult
due to dispersion problems
• • • •*,
• •
Citrate-stabilized gold NPs
(10,30,60 nm)
colloidal/H20 suspension
• NIST Reference materials
http://ts.nist.gov/measurementservices/referencematerials
1
-------
Particle Dispersion in Bioloqical Systems
Documenting dispersion before in vitro assays
- AFM: Dry vs. wet
- SEM, TEM, Cryo EM
- Dynamic light scattering & zeta potential
60 nM Au Nanooarticles
Global Clottinq Times (aPTT
11 13 15 17 19 21 23 25 27 29
Time (sees)
Activated partial thromboplastin time (aPTT)
Clotting time in seconds (max. rate of clot formation)
"Intrinsic" system: misnomer
Contact activation - biomaterials research
• "Nanoparticle-protein corona": NPs coated with proteins
• DLS limited with complex samples
• Appears useful for rapid documentation of particle size (with uniform
nanomaterials), but technique requires refinement for other particle types
• Increased time to clot formation (with 90 nm)
• Decreased amplitude: Reduced amount of clot or clot stability
• Mechanisms: Interference with clot formation in vitro through
interaction with clotting proteins?
Standardizina Coaaulation Assavs
False Negative
Results
False Positive
Results
•/ Controls
•/ Standards
•/ Matrix effects
Dobnovolskaia et al. Molec Pharmaceutics, 2008
2
-------
Endogenous Thrombin Potential (ETP
Lag Time: Time to thrombin generation
Clinical applications for determining who is at risk to form clots
Thrombin is "bottom line" in clot formation by converting
soluble fibrinogen to fibrin clots
Nanoparticle Effect on Thrombin Generation
Increased total thrombin generation (90 nm particle)
Nucleation effect in vitro?: Particles provide surface for
assembly of clotting factors to facilitate thrombin
generation
Dissemination of coagulation & inflammatory mediators
• Wary translating in vitro findings to in vivo effects
• Particle exposures most likely through lungs
• Explore in vivo studies (animal inhalation models, inflammation)
A) Inhalation exposure system schematic. B) Inhalation exposure system. C)
TEM image of ultrafine Ti02. D) SEM image of fine Ti02. E) Ultrafine Ti02
aerosol size distribution. F) Ultrafine Ti02 aerosol generation.
Nurkiewicz et al. 2008
Proposed pathway for lymphatic dissemination of coagulation and inflammatory
mediators in immune responses. Niessen et. al. Nature 2008
~
O 2x104-
IE
10 100 1000 10000
Geometric Diameter Dg (nm)
10 100 1000 10000
Geometric Diameter Dg (nm)
Particle size/distribution generated by the aerosol exposure system. A)
Ultrafine TiQ2. B) Fine TiQ2.
Nurkiewicz et ai. 2008
3
-------
Luminex Technolo
Measure multiple analytes
simultaneously in single reaction well
(instead of multiple ELISAs)
Capture analyte (ILs, cytokines, etc.) on
microspheres distinguished by
fluorescent intensity
Add fluorescently labeled reporter tag
Inject into instrument that can
distinguish which microspheres (e.g. IL1
bead) and how much fluorescence is on
the surface
r > A
> >
*
m
d
1
-¦^3 O
5*
lum inexcorp.com
Fibrinoa
en bv Luminex
• Rationale: Fibrinogen is
independent risk factor for
cardiovascular disease
• Findings: Variable
increases in fibrinogen
seen in most exposed rats
• Limited by variability of
fibrinogen assays
Confirm Findings with Increased Number of
Exposures & Alternate Assay
von Willebrand Factor (vWF)
Rationale: Risk factor
for thrombotic events
(not CV risk factor)
Inflammatory marker or
acute-phase reactant
Findings: Variable
increase in vWF with
pulmonary Ti02
exposures
Fibrinogen appears to acutely increase with short-term
exposure to fine & ultrafine Ti02
Troponins
Rationale: Marker of acute
myocardial injury
Finding: No significant
differences between control &
Ti02 exposed animals
Cannot extrapolate findings to
human exposures
2) 0.15 mg/m3 UF
Ti02
3) 0.03 mg/m3 UF
Ti02
4) 0.10 mg/m3 fine
TiO,
Wmfcrv* c>5 670t*n Cy» S86nr
-------
Protein Chanaes Detected bv DIGE
• 428 distinct protein "spots" identified by two-dimensional gel
electrophoresis
• 72 spots were quantitatively different between the test groups
and controls by DIGE (p < 0.05)
Spot 488: Hemopexin, showing
| a 1.2 fold change (p<0.005)
Spot 569: Fibrinogen, showing
a 1.5 fold change (p<0.04)
Up-regulation of Hemopexin Up-regulation of Fibrinogen
Significant differences in 45 distinct proteins
by MALDI & LC/MS/MS
Coagulation Proteins (generally upregulated]
- Fibrinogen (a, 0, y chains): major clotting protein
- Plasminogen: degrades fibrin clots
- Antithrombin: anticoagulant
- Kininogen: absorbs to materials
- Other serine-protease inhibitors (serpins): control
blood clotting proteins
Other Proteins of Interest
Inflammatory Proteins
- C-reactive protein: major inflammatory marker
- Complement C3: acute phase protein
- Complement C9: later phase complement system
- Pyrroline 5 carboxylate synthetase: stress protein
- Fetub: acute phase recovery protein
Miscellaneous Proteins
- Apolipoproteins (A1, E): lipid binding
- Desmoplakin: structural protein
- Angiotensinogen: increased by stress
- Ankyrin repeat domain
- Other poorly understood proteins not previously implicated
in inflammatory responses
Proteomic Study Conclusions
Exposure to fine and ultrafine Ti02 through
inhalation causes significant changes in the rat
plasma proteome, many related to coagulation &
inflammation
These changes may be directly involved in the
potential adverse effects of particle exposure, or
may serve as markers (biomarkers) of toxicity
Additional studies are needed to determine the
specific protein "pathways" involved in the adverse
health effects of small particle exposure (i.e.
interactome)
How can human health be protected against
hemostatic toxicity of nanomaterials?
• Minimize exposure in "zero-risk" society
• Identify synergistic risk factors for thrombotic disease
• Use model to predict potentially harmful effects of new
and/or functionalized nanomaterials
• Decrease exposure through increasing aggregation &
decreasing durability
• Develop biological sensors that can detect sub-clinical
effects on hemostasis -™1-
"Every generalization is dangerous, especially this one"
Mark Twain
5
-------
Nanotechnoloa
v Team
Nanoparticle characterization
NickWu-Mech. Eng. WVU
Darren Cairns - Mech Eng. WVU
mvrano
Coaqulation & Luminex
¦ \ (TA
Syamala Jagannathan -WVU Pathology
Jeff Frisbee - CIRCS WVU
Nanomaterial Interactions
Perena Gouma, Stony Brook University
\fTtOSH
Rat inhalation
Tim Nurkiewicz, CIRCS, WVU
Dale Porter, NIOSH
Vince Castranova, NIOSH
Proteomics
LindaCorum, WVU Pathology
Steve Wolfe, WVU Pathology
Andrew White, Univ. Charleston WV, INBRE student
Supported by Environmental Protection Agency (EPA #R832843)
6
-------
Physical characteristics of
nanoparticles affects interactions
with aquatic organisms
Feswick, A.1; J. Griffitt2; J, Luo1; D. S. Barber1
1 Center for Environmental and Human Toxicology, University of
Florida, Gainesville, FL, USA.
2- Department of Coastal Sciences, University of Southern
Mississippi, Ocean Springs, MS, USA.
Importance of particle properties on
toxicity
o Studies demonstrate that toxicity of
nanoparticles can be affected by:
Size
Surface area
Hydrophobicity
Charge
o Developing an understanding of how these
factors affect interactions with biological
systems is critical to be able to predict
toxicity
U 48 hour toxicity of metallic
^ nanoparticles
Nanoparticulate
Soluble
D. rerio
D. pulex
D. rerio
D. pulex
Nanocopper
0.94 ing/L
60 ug/L
0.13 mg/L
8.68 ug/L
Nanosilver
7.1 mg/L
40 ug/L
22.5 ug/L
0.85 ug/L
Nanoaluminum
> 10 mg/L
> 10 mg/L
7.92 mg/L
> 10 mg/L
Nano-Ti02
> 10 mg/L
> 10 mg/L
> 10 mg/L
> 10 mg/L
Nanonickel
> 10 mg/L
3.8 mg/L
> 10 mg/L
1.48 mg/L
Nanocobalt
> 10 mg/L
> 10 mg/L
> 10 mg/L
9.7 mg/L
Griffitt et al., 2008
Gill metal content after exposure
3 Ctrl mean
¦ SoUWM**
¦ Nuno mean
J
Copper Silver
1
-------
Nanosilver adheres to zebrafish gills
4) Nanoeopper
i
Uptake of two different dye-doped silica
nanoparticles
h
i;l
n ill ill
| 0.4 -
8
S 0.3 •
i
1 lii iJ li Ii 1
i" ~ mm MHM#"in""""" """
Uptake of Rubpy doped silica by gill cells
2
-------
^ Uptake of RuBpy Silica requires active
A transport
Fluorescence (510-555nm)
>00000000
¦^1
cont
RuPby
~
cont
RuPby
Treatment
RuBpy Silica treated RT-gill cells (40u
Dnc) at 4C for 1
hour
^ Uptake of nanoparticles by gills cells is
^ mediated by endocytosis
Fluorescence (nm)
3 0 0 0 0 0
40ug/ml RuBpy silica
—V— Cytochalasin-B _..j i
^
— 1
0 5 10 15 20
Time (hours)
Genistein (caveolar-like inhibitor) does not
reduce Rubpy silica uptake
—•— Control
¦ 40ug/ml RuBpy Silica
—V— Genistein
if
%
3.
]
-
Genistein reduces COOH functionalized Q-
dot uptake
—•— Control
COOHQ-dot
—V— Genistein
s.
_ 2 24 48 2 24 48 2 24 48 _
CC nanocopper nanosilver
Time w ithin T reatment
3
-------
COMET assay
Acknowledgements
o Dr. Joe Griffitt, April Feswick, Jing Luo
o Dr. Kevin Powers, Gil Brubaker
o Dr. David Julian
Conclusions
Intact nanoparticles are taken up by gill
cells and daphnia
Physical properties of nanoparticles have
significant impacts on their interaction
with biological systems.
Charge is an important determinant of
nanoparticle uptake
Effect of charge varies among models
Mechanisms of particle uptake for
particles with similar properties can differ
Oxidative injury appears to play a role in
nanosilver induced toxicity
o Funding Sources:
National Science Foundation (BES054920)
-------
Interagency Environmental Nanotechnology Grantees Workshop
November 19-21, 2008
Tampa, FL
Nanostructured Membranes for
Filtration, Disinfection, and
Remediation of Aqueous and
Gaseous Systems
Grant Number: GR832372
8/1/05 - 7/31/08
Kevin Kit (PI),
Svetlana Zivanovic and
P. Michael Davidson
miUNIVERSlTYofTENNESSEE Ur
Objectives
+ Develop electrospun nanofiber chitosan
membranes to treat aqueous and gaseous
environments by actions of filtration, disinfection,
and metal binding
+ Understand electrospinning process for chitosan in
order to control membrane structure
+ Investigate effect of membrane structure on
filtration, disinfection, and metal binding
•+• Optimize performance/efficiency of chitosan
membrane
Introduction - Chitosan
Chitosan is a carbohydrate polymer obtained from Ghitin
which is found in the shells of crustaceans, crab, shrimp etc
Decalcification in dilute HCI solution
Deproteination in dilute NaOH solution
Decolorization in sunshine or Oxalic acid
c«"
Deactylation in conc. NaOH (40-50%)
—./ Chitosan
Chitosan - Properties & Applications
-f Insoluble in water, soluble only in aq. acids
-f Amide group protonated at pH < 6.5
+Anti-microbial properties
~ Metal Binding Properties
-f Non-toxic biodegradable natural polymer
Biomedical
Dietary
Supplement
Chromatography
& Waste water
treatment
Food
Packaging
Chitosan Surface Properties
Surface properties of chitosan fibers is due to
the protonated amine sites on fiber surface.
+ Degree of protonation is a function of:
A Degree of deacetylation
A Solution pH
A % Chitosan in fiber
4 Molecular weight
k Crvstallinity
CHjOH
+ H+
CM OH
1
H( -
Electrospinning
Spinning Distance
(~ 10 cm)
Polymer
Solution
Syringe Pump
Collector
Plate
Desai - KCC Technical Talk July 9 2008
-------
Experimental Set-Up
Modified
electrospinnirig set-up
which allows us to
heat solution while
being ejected .
Enables spinning of
solutions at higher
temperatures, by
blowing hot air at
different flowrates (25
ft3/hr,75 ft3/hr)
Temperature
controlled by variac
Polymer Solution
being fed using syringe pump
Electrospinning - Key Parameters
+ Polymer Solution
^ Solution Viscosity/Entanglement
density
~Molecular Weight
~Solution temperature
~Concentration
~Solubility
^ Surface tension
~ Polymer/Solvent *
+Applied electric field e
x Voltage
A Tip-target distance
+ Solution flow-rate c
~ Solution conductivity
For electrospinning of PMMA
(c/c*)< 1, dilute region, formation of
droplets
1<(c/c*)<3, semi dilute un entangled
region, formation of droplets along
with few beaded fibers
3<(c/c*)<4, semidilute entangled
region,formation of beaded fibers
(c/c*)>6, formation of uniform fibers
without bead defects
(Ref.P Gupta et.al, Polymer 2005, 46, 4799-4810)
Fabrication of Nanofibers
- Electrospinning
Experimental Procedure
Polymers
HMWChitosan 80% DDA (Mv ~
1400kDa) from Primex
LMW Chitosan 83% DDA (Mw -100 kda)
from Sigma
HMWPEO (900 kDa) from Scientific
Polymer
PAAm (5000) kDa from Scientific Polymer
Electrospun @
+ Polymer blend ratios
^Solution temperatures
i.e 25°C, 41°C,70 °C
4
Solvents
+aq.acetic acid
Solution flow rate,
spinning voltage, tip-
target distance kept
constant (0.08m l/m in,
30kV,10cm)
Solutions prepared with optimum concentration of polymer
dissolved in optimum strength of acid solvent so as to form
bead less fiber mats
Electrospinning of Chitosan
1.4 wt % Himw Chitosan in 50 % Acetic 1.2 wt % HMW Chitosan +1.5 wt% Urea in
Acid Solution
Air Flowrate 25 ft3/hr
Air Temperature 70 °C
90 % Acetic Acid Solution
Air Flowrate 25 ft3/hr
Air Temperature 70 °C
Wm
5 wt % LMW Chitosan in 90 %
Acetic Acid Solution
Spun at room temperature
6wt% hydrolvzed chitosan (Mv~20
kDa) in 90 % Acetic Acid Solution
Spun at room temperature
Electrospinning of Chitosan blends
Chitosan/PEO blends
PEO widely electrospun
hydrophilic synthetic
polymer
Used as for non-fouling
surfaces, packaging
material for foods, binder
and thickening agent for
paints etc.
Chitosari/PAAm bjends
+ PAAm hydrophilic synthetic
polymer, having amide
groups like chitosan
+ Used as a flocculent in waste
water treatment as can bind
heavy metal ions by forming
coordination bonds
+ Cationic polyacrylamide has
been used for anti-microbial
applications
Blend solutions prepared and fiber formation
optimized to form, headless fiber mats at highest
chitosan content in blend solution
-ch2—ch-
c
/\
o nh2
Desai - KCC Technical Talk July 9 2008
-------
Chitosan/PEO - Effect of Blend ratios
Chitosan/PEO blends - Spinning at Higher Temperatures
1.33 wt% HMW Ghitosan PEO (95:05) fibers obtained at
different spinning solution temperatures
25°C HHBfBHIHi 40 C i
Chitosan/PAAm blends - FD and bead density
II ll ll
Sptfiinoa Air |*C)
Error bars represent std.dev (n=S0. letters indicate
significant difference at p<0.05
I
¦ 'I ¦
Pure 80 % DDA Chitosan
4.5 wt % LMW
Chitosan:HMW PEO
^9^10]
4.5 wt% LMW
Chitosan.HMW PEO
_J75i25]
2.0 wt% HMW
Chitosan:HMW PEO
1.33 wt% HMW
Chitosan:HMW PEO
(90:10)
1.6 wt% HMW
Chitosan:HMW PEO
75:25
1.4 wt% HMW Chitosan:PAAm blend fibers obtained at different spinning
solution temasali alt flam cats 25BMii:
Chitosan/PAAm blends - Effect of Blend ratios and
spinning temperature
Spinning solution temperature
90:10
Surface characterization
of fibers - XPS
Desai - KCC Technical Talk July 9 2008
-------
Pure PAAm
to:
¦s -m
-i\ih2 **
A
EC
vfl
f to:
fi
-NH.i* /
7m
maJiA \
tix
^——JLv.—
flOWUiM «« Wi -tEttl tU M « S/ Jfc a »1 Si
HntntnwW
Surface Composition - Pure Polymers
Sample
Atom %
"C/N"
ratio
C1s
N1s
Ols
CI2p
Al
80% DDA
HMW
chitosan
theoretical
56.14
8.77
35.08
6.4
from XPS
(film)
61.11
5.6
28.18
5.11
10.92
Pure
PEO
theoretical
66.67
33.33
CO
from XPS
(film)
66.77
32.39
0.11
c°
from XPS
(fiber)
96.26
3.74
CO
Pure
PAAm
theoretical
60
20
20
3
from XPS
(film)
67.17
13.73
18.56
0.54
4.89
from XPS
(fiber)
61.24
12.48
22.68
0.45
3.14
4.91
Effect of % chitosan in blend
10 " -»-HMWCh(tosan: PEO blends
fJ . -*-LMWCWK»sarc PbO Mentis.
Wis atom% for pore 00% DDA chitosan f
70 ao
% Chitosan in solution
Effect of % chitosan in blend
100 -
90
80
70
€0
50 -
40
30
20
10
0
i-1 imw Chitosan: PLO blends - theoretical atom%
t- LMUV Chitosan PEO blends - theoretical atom%
HMW ChSwsin.PEO btetKfa - XPS alum %
-LMWChitosan:PEO blends - XPS atom % , -
65 70 75 80 85
% Chitosan in solution
90 95 100
Effect of % chitosan in blend
140
120
#
¦!- —M
c ^CO
CO
1
o 80
cu
0
t:
3 60
w
- ¦ Sm 2 1
^ * | |
40
20
6
(pun at RT - tfveorehcal atom % b*tt»
-•-ffcert; spun at 40 C - IhootetKal atom % bass
-•-Ffcerj ipun al 70 4m C - theoretical atom% bam
Filers spun at RT • XPS atom % bam
-*-Fib#f s tpun M 40
-------
Test Surface Properties -
Electrospun chitosan
fibers
Metal Binding- Chitosan/PEO blends
Effect of % chitosan in blend solution and chitosan molecular weight
=-
¦ HMWChitosaniHMWPEO
20 -
c
i*
6 '5
10
1
O
a
j
¦ 1 MWGhltOtfUMVHMWHI O
b b
. 1 .
55
% c:r
75 50
Hasan in isientj Smution
Error bars represent std.dev (n=3, letters indicate significant difference at p<0.05
Surface Properties - Antimicrobial
Electrospun fibers
immersed in known
concentration (9 log) of
Escherichia E-coli K12
bacteria in phosphate
buffer solution for 6
hours.
NH3+ binds with
negative components
of cell wall like lipids
etc.
Survival rate of E-coli
measured after 6
hours using pour-
plate method using
Trypticase Soy Agar
(TSA) media
http://en.wikipedia.org/wiki/Escherichia_coli
2 log reduction is equivalent to 99% reduction in bacteria, 3 log is 99.9%
Anti-microbial - Chitosan/PEO blends
Effect of % chitosan in blend solution and chitosan molecular weight
hei
¦ HMW CMtosan PE0 blends
LMW ChitosarvPEO blends
mm
<&> ¦ ab
im
wt% PEO
Error bars represent std.dev (n=3, letters indicate significant difference at p<0.05numbers in paranthesis
is log reduction
Anti-Microbial Chitosan/PAAm Blends
Fiber
Diameter
(nm)
Log
reduction
(cfu/ml)
Std.Dev
cfu/g
chitosan
1.4 wt% HMWChitosan:PAAm
(75:25)@RT
132
3.11
0.35
2.61E13
1.4 wt% HMWChitosan:PAAm
(75:25)@70 °C
328.03
3.17
0.19
2.47E13
1.4 wt% HMWChitosan:PAAm
(90:10)@70°C
304.94
3.34
0.12
2.14E13
2.85 wt% LMWChitosan:PAAm
(75:25)@RT
421.75
3.15
0.04
1.96E13
Fabrication arid filtration
performance -
Nanofibrous filter media
Desai - KCC Technical Talk July 9 2008
-------
Filtration
Fabrication of a composite filtration membrane by electrospinning
chitosan blend fibers on spunbonded PP webs
Spun bond PP
Chitosan/PEO blends - Varying FD
hi
1 »«W%CWffl«nP€0 1 J)W%CMP«n PK) i Ctstewn.PfcO
:Aad ¦
JMIB435
Filtration - Metal Binding
Filter Mat
Chromium solution
? i! •" ^
Filtrate
+ 100 ml of 5 mg/l K2Cr04 solution passed through filter
+ Cr(VI) reduction measured after 5 and 10 passes using UV-Vis
+ Filtration time around 2 mins
+ 1 mm Hg vacuum applied
Effect of fiber diameter - chitosan/PEO
10
5
0
~-Binding capacity (mg chromium/gchitosan)
surface NIs conrai (atom %)
100 125
litxs diameter (rim)
f-
a fc
6 |
4 I
2
0
Effect of fiber diameter - chitosan/PAAm
70 -^-Binding capacity {mg chrorrounvg chitosan)
surface Nlscorvcn. {atom %)
50
40
30
20
10
4 8
0 0
75 100 125 150 175 200 225 250
fiber diameter (nm)
Effect of gsm/basis weight
fio
11
HMWchitosanrPEO (90:10)
Spunbond layer
100 [jm
7 ,/4»l
%
JfijiO Mm
HMWchitosan:PAAm (90:10)
I ii
Desai - KCC Technical Talk July 9 2008
-------
Surface structure & composition-After MB
HMWchitosarr.PAAm (90:10)
Surface structure & composition-After MB
Effect of film formation
0.5 gsm
1.0 gsm
1.5 gsm
Formation of film layer could affect liquid flow through
the fiber membrane possibly forming channels and
affecting the wettability of the entire mat with increased
gsm
Effect of % DDA
40
After pass no 5
¦ After pass no 10
ab
b b
b b i
. 3
67 71 80
%DDA
Filtration - Antimicrobial
E~Coli solution
1
r?
s- Filter Mat
fir
Filtrate
+ 100 ml of 4 log E-coli solution passed through filter
+ E-coli reduction measured after 1 pass using pour plate method
+ Filtration time around 2 mins
+ 1 mm Hg vacuum applied
Desai - KCC Technical Talk July 9 2008
-------
Chitosan/PEO Filter - Antimicrobial
0.4
0.35
0.3
0.2'j
-i 0.2
Sj 0.15
o
0.1
0.05
0
/
lllii
if f jf „/!- * ,f &
• . ¦¦¦ ,
•...SiSjSJj : - -
HWHsinp
B.-W SS.S, —
Conclusions
+ Demonstrated ability to form beadless chitosan based
nanofibers of controllable size and chitosan content
x Chitosan/PEO blends - 95% chitosan in blend (FD 80-
315 nm)
x Chitosan/PAAm blends - 90% chitosan in blend spun @
70°C (FD 130-350 nm)
x Heating polymer solution helps expand processing
window (% chitosan & fiber diameter)
+ Developed a model to predict Cr(VI) binding properties of
chitosan nanofibers
A For fiber diameter < 400 nm binding capacity decreased
exponentially
For S0
-------
Conclusions
•fNanofibrous filter media made using chitosan
nanofibers showed:
A 0.5 gsm chitosan.PEO (90:10) nanofibrous filter media
showed 35 mg chromium/g chitosan binding capacity
x After binding expts formation of film rich in chitosan
seen on filter media
A Poor anti-microbial properties under dynamic testing
•+PS beads and aerosol filtration efficiencies
increased with decreasing fiber size and increasing
fiber gsm
A Desired filtration efficiency can be achieved by
optimizing electrospinning process parameters to
control fiber size and porosity of filter media
Acknowledgements
+ Prof. Jochen Weiss + Keyur Desai
-f Prof. Gajanan Bhat + Christina Kriegel
-f Prof. Roberto Benson -fJiajie Li
+ Dr. Harry Meyer -fDoug Fielden
+ Dr. Peter Tsai
1
Desai - KCC Technical Talk July 9 2008
This poster indudes research
funded by
r U.S. EPA-Science To Achieve
Res ults (STAR) Program
Grant
-------
Comparative Life Cycle Analysis of Nano and Bulk
Materials in Photovoltaic Energy Generation
V. Fthenakis, S. Gualtero and H.C. Kim
Center for Life Cycle Analysis
Columbia University
Email: vmf5@colombia.edu
Web: www.clca.columbia.edu
Project Objectives
¦ Assess the life cycle mass arid energy inventories of
two main candidate nanomaterials for thin-film
photovoltaic applications
¦ Make comparisons with the materials and solar cell
structures that may replace, based on process-data
¦ Investigate the applicability of the results to other
nanomaterial-based thin-film technologies
Comparative Life-cycle Analysis Framework
IVicro PV
Production
—
Cell/PV
manufacturing
-
.Man
Operation/
Recycling/
Disposal
-Purity/amount
size/distribution
-Amounts of
auxiliary
-Conversion
-Recyclability
Parameters
/process
efficiency
-Extra
control/process
Balance of
efficienc
-Environmental fate
Nano PV i
Production
manufacturing
4
MM"
—*j
Operation/
Recycling/
Disposal
PV Paradigms for Comparison
a micro-crystalline CdTe vs. nanoscale CdTe (long-
term research -3rd generation PV technology
• Vapor Deposition vs. Solution Growth techniques
a amorphous Si vs. nano-crystalline Si (technology
near commercialization)
• Vapor Deposition techniques
Current CdTe PV
First Solar, Perrysburg, Ohio
Energy Payback Times (EPBT)
Insolation: 1700 kwh/m2-yr
3.0
2.5
« 20
a)
i 1.5
I 1.0
HI
0.5
0.0
Based on data from 13 U.S. and European PV manufacturers
Collaborative work with U. Utrecht 8 Energy Research Center Netherlands
IEA PV Task 12
Fthenakis et al., Environmental Science and Technology 42, 2168-74, 2008
6
~ BOS
Frame
~ Frameless
Module
Ribbon-Si
11.5%
Multi-Si
13.2%
Mono-Si
14.0%
1
-------
Life Cycle GHG Emissions -European and U.S. Cases
Insolation: 1700 kwh/m2-yr
00
55
40
30
20
10 -|—
o
~BOS
¦Frame
DMndi.il*
Ribbon Multi-Si
Mo r>o-3l
Rbbcn
MlM-Si Mano-SI
Cdt«
Rtobon Multl-S Mono-Sl
Cdfa
115% im
14.0%
116%
112% 14.C*
11.8% 112* 14.0%
Ca»l
Cut 2
Caw 3
Fthenakis etal., Environmental Science and Technology .42. 2168-74, 2008
Life Cycle SQ2 Emissions -European and U.S. Cases
i —
i
¦-
—
-n-
n
ft "
=1=
i
Rfabcn
115%
MJli-Si f>fero-S
132% 14.0%
COBC 1
Rbncn Mib-Si WbroS, CdTe Ribbon Mili-Si Mxio-Si
11.5% 112% 1*0% 9% 11.5% 13.2% 14.0%
Caw 2 Cased
CflTe
9%
Fihenake et al., Environmental Scierce and Technology 42, 2168-74, 2008
Life-Cycle Cd Emissions from Electricity Use
14.3
3.7
0.5 0.7 0.6 0.3 | 1 0.3
0.9
/ f S / * f
Fthenakis et at. Environmental Scierce and Technology 42, 2168-74, 2008
IMano CdTe PV Process
1. Synthesis of CdSe and CdTe nanorods
Preliminary Mass Balance
¦ Material Utilization in Nano-rods Synthesis (CdO and
Te/Se used per mass of nano-rods produced):
• Synthesis of CdTe rods: 77%
• Synthesis of CdSe rods: 73%
¦ Material Utilization in Device Fabrication is very low: <1%
Mass of CdO, TeandSe used per m2
1.5
kgfm2
Total mass of materials used per m2
610
kgfm2
(glass substrate excluded^
12
2
-------
EHS Implications
Solvent
L-D5„ (rat)
(mg/kg)
Hexane
28700
Isopropanol
5045
Toluene
5000
Pyridine
891
• Hexane is classified
as HAP by the EPA
- It can probably be
replaced by heptane
• Pyridine is an animal
carcinogen
- Its replacement is
difficult
13
Material Use: Lab vs. Commercial scales
Effect of Material Purity on Energy Use
Energy Breakdown to produce
5N (99.999%) Te
15
Major C02 emissions in CdTe manufacturing
16
2nd Paradigm: amorphous-Si PV modules
United Solar, Auburn Hills, Ml
17
Comparison of amorphous- and
nanostructured-
silicon PV
Grid Grid
n n
n n
IT©
ITO
P
a-Si alloy-250 nm
'a-Si alloy—250 nm
n
n
P
P
a-SiGe alloy -250 nm
nc-Sj alloy — 1500nm
n
a-SiGe alloy-250 nrn
n
n
P
ZnO
ric-Si alloy- 1500nm
Ag
Stainless Steel
n
ZnO
Ag/AI
Stainless Steel
18
3
-------
Comparison of amorphous- and
nanostructured- silicon PV
Add or replace layer (s) with nc-Si in a-Si
module
¦ Typically top layer a-Si (200-300 nm) and
middle or bottom layers nc-Si (1000-2000
nm)
¦ Change in deposition process (Increase
H2 dilution & deposition times)
Life cycle implications:
¦ Improved spectral response (thus
efficiency)
¦ Increase energy and (upstream) material
requirements
¦ Increase GHG emissions from module
production
Typical thin-film PV energy breakdown
£neigy
100%
f
&
Tec15/3.2 mm 6Hss(P0IS)
Flat glass, Rout glass
uncoated coated
11% 11%
decticity
34%
1
r
Electricity
J|L
Nat. efts into
1
20
¦.lectnc ty
1
dectricity
[
1
l*itu>algas
Comparisons a-Si with nano-c-Si options
Module types
a-Si
Tandem
a-Si/nano-c-Si
Thickness 1st layer a-Si (nm)
300
300
Thickness 2nd layer nc-Si (nm)
NA
1350
Deposition rate a-Si (nm/s)
0.3
0.3
Deposition rate nc-Si (nm/s)
NA
0.5
Reactor cleaning cycles
1
2
Silane input (g/m2)
2.8
12.6
Hydrogen input (g/m2)
17
213
Amorphous vs. 'Micromorph' Si Cells
Comparisons a-Si with nano-c-Si options
Cell Types
a-Si
Tandem
a-Si/nano-c-Si
Small area cell efficiency
(%)
13
15.4
Module efficiency (%)
7.6
8.7
Energy Ratio
EPBT (yr)
2.3
2.7
C02 emissions (kg
C02/m2)
59
74
Total GHG emissions*
(kg C02eq. /m2)
94
141
* A cleaning cycle with SF6 = 116g/m2 SF6
(Assuming 99% SF6 abatement efficiency)
Amorphous vs. 'Micromorph' Si Cells
Comparisons a-Si with nano-c-Si options
Forecast for 2013-2015
Cell Types
a-Si
Tandem
a-Si/nano-c-Si
Module efficiency (%)
9
12
Energy Ratio
EPBT (yr)
2
2
C02 emissions (kg
C02/m2)
20
20
Total GHG emissions*
(kg C02eq. /m2)
31
38
'A cleaning cycle with SF6 = 116 g/m2 SF6
(Assuming 99% SF6 abatement efficiency)
What did we learn?
¦ We can project the mass and energy flows in future nanotechnology-
enabled PV, guided by changes in material utilization, purity, deposition
rates, film thickness and electric conversion efficiency.
¦ Solution grown nanostructured CdTe solar cells requires more extrinsic
materials than micro-CdTe solar cells, but less volume and lower purity
semiconductor precursors.
¦ Plasma-enhanced CVD of nc-Si requires materials for reactor cleaning
that are GHG.
¦ Adding nc-Si layers to a-Si solar cells increases energy and GHG
emissions that can be counterbalanced by cell efficiency increases.
24
4
-------
Next Steps
Detailed investigation of solvent use & recycling
efficiency
Detailed investigation of energy use in solution-
grown materials & in inkjet printing
Investigation of CIGS PV production by inkjet
printing
Investigation of nanoparticle inks replacing
screen-printed silver-glass-frit pastes for Si cell
contact metallization
25
Acknowledgment
¦ Ilan Gut, UC-Berkeley
¦ Sergio Pace a, U. Michigan
¦ Ulrich Kroll, Oerlikon Solar-Lab
¦ Christophe Ballif, Institute of MicroTechnology
¦ Andrea Feltrin, Institute of MicroTechnology
26
-------
Life Cycle of Nanostructured Materials
Thomas L. Theis
Hatice Sengul
Institute for Environmental Science and Policy
Siddhartha Ghosh
Department of Electrical and Computer Engineering
University of Illinois at Chicago
m
Ran Material |
Production ||
Why Life Cycle?
# l\«9
Consumer 1
Msiufacsixing 1
..-I- ' : |
Recycle j -j- 1
1 ^ K ffi
LarwfJs Incinerators
LU #
Humar Population and Ecological Exposure
Why nano?
Small amounts can have large effects
Different physical properties as size decreases
High specific surface areas
Function can often be "tuned" by altering composition,
size, shape, temperature, pressure
Rich basis for new designs and applications
Projected to generate $1.1 trillion in economic activity by
2016 (NNI, 2001)
Production rates >105 tonnes/yr by 2020 (Royal Society
2004)
An "enabling" technology with implications for energy,
manufacturing, electronics, transportation, healthcare,
pharmaceuticals, environmental control and purification,
sensors and national security, chemical processing, and
sustainable development
Nano-based publications
}
l 1 I
1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Nanomanufacturing
Top-down
Definition: The fabrication of nanostructures,
or the use of nano-based methods to
manufacture a product
Two types: "Top-down" and "Bottom-up"
(Royal Society, 2004)
Journal of Industrial Ecology 12(3):329-359
Etching/milling
Etching
Wet etching (chemical etching)
Dry etching
reactive ion etching
plasma Etching
sputtering
Milling
Mechanical milling
Mechanical alloying
Cryomilling
Mechanochemical bonding
Electrospinning
Lithography
•Conventional lithography
•Photolithography
•E-beam lithography
Next-gen era tion lithogra phy
Immersion lithography
Lithography with lower wavelengths than
photolithography
Extreme ultraviolet (soft X-ray) lithography
X-ray lithography
Lithography with particles
e-beam lithography
Focused ion-beam lithography
Nanoimprint lithography
Soft lithography
1
-------
Bottom-up
Vapor-phase deposition
• Vapor phase epitaxy
• Metal organic chemical vapor
deposition
• Molecular beam epitaxy
• Plasma enhanced chemical
vapor deposition
• Sputtering
• Evaporation
Nanoparticle synthesis
• Evaporation
• Laser ablation
• Flame synthesis
• Arc discharge
Liquid phase
• Precipitation
• Sol-gel
• Solvothermal synthesis
• Sonochemical synthesis
• Microwave irradiation
• Reverse micelle
Molecular beam epitaxy
Sources of nanomanufacturing
impacts
• Strict purity requirements and less tolerance
for contamination during processing than
more conventional manufacturing processes
(up to "nine nines").
• Low process yields or material efficiencies
• Repeated processing, postprocessing, or
reprocessing steps of a single product or
batch during manufacturing
• Use of toxic/basic/acidic chemicals and
organic solvents (eg. As, Ga, In, Cd, Zn, Sn,
Sb, Hg, solvents, chlorinated and perfluorinated
compounds, etc.)
Numbers of Mfg steps per wafer
100
2 20
P-
\° 0^
Unit operation
Sources of nanomanufacturing
impacts
• Need for moderate to high vacuum and other
specialized environments such as high heat
or cryogenic processing
• Use of or generation of greenhouse gases
(directly or through energy consumption)
• High water consumption
• Chemical exposure potential in the
workplace and through technological/natural
disasters
Cumulative energy requirement of
nanomaterials
100000
Carbon-containing nanomaterials nanoparticles
^ a ,
10000
f-
N
—(—
V
O)
s 10
1 '
0.1
¦
CNF-B CNF-M CNF-E CNT- CNT- CNT- cdSe |T0 TOania
SWNT-AA SWNT-CVD SWNT- ndot
-------
Some semiconductor materials
(>600)
• Elemental: Si, Ge
• lll-V binary: AlAs, GaAs, BN, GaN
• lll-V ternary: ALGa-^As, AllnAs, InAsSb
• lll-V quaternary: AIGaAsP, InGaAsN
• ll-VI binary: CdSe, CdS, CdTe, ZnO, HgTe
• IV-VI binary: PbSe, PbS, PbTe, SnS
• ll-V compound: Cd3P2, Cd3As2, Zn3Sb2
• Other: ln203:Sn02 (ITO)
• Organic: Anthracene, polymers
• Magnetic: GaMnAs
Energy requirements of materials
1
9
I
Ol ^
2
¦
-
EAF Sled Alumlnini
Poly 31
Walters
lanoluta
Gutowski et al. A Thermodynamic Characterization of Manufacturing Processes IEEE (2007)
Quantum dots
Quantum Dot Applications
« Datastorage
• Single electron
devices
• Uglht emitting
UiiKhra
• Security
application/ Bar
coding
¦ Quantum
computing
¦ Anli-counlei foiling
• Chomlcunblologlcal
¦ Lighting
* Biological
Kiwilng
¦ Slo-lmaging
¦ Displays
- Solar eels
Current 1 - S years 6-10 years 10 - 30 years
applications
O'Brien, P. "Making and Using Nanoparticlesthe Marketplace Potential", 2006
Expert Lecture Series Leaders in Nanotechnology, The Manchester Materials
Science Centre.
Floe relation
methanol (xJ)
* eiitrifi^gation(x2
Pf~
Dropwile addition
ethanol (xN)
rifu|ation (x
SYNTHESIS
ISOLATION
&
PUREFICATIO
SIZE SELECTIVE
\ PRECIPITATION
Liquid phase synthesis of CdSe quantum dots (Murray et al. 1993 J. Am Chem. Soc. 1993, 115, 8706-8715)
3
-------
Raw material use for CdSe qdots
Cumulative energy demand CdSe
q-dots
S
Q 100 ¦
111
o
10 -
1
Din-
cad
ethyl TOP Sele
nium Met
anol 1-Bu
tH 0 ~ b
tanol Transport, TOPO Argon Electricity D
sposal
Fibroblast embedded with CdSe
QD
(Wang et al. 2005. Bioapplications of
Nan osemiconductors.
Materials Today 8(5): 20-31)
DNA damage of CdSe
0 mins 60 m ins
~
A B C D
Damage'
band
-~
Undam,
band
DMA DNAAJV DNAjUV DN A/Darts
+ UWDark + damaging agent +QD's + QDs
V* Damage <5 >90 56 29
Green, M. and E. Howman
"Semiconductor quantum dots and free radical induced DNA nicking"
Chem. Commuri., 2005, 121 - 123
Aquatic reactions
Solubility: AxBy (s) —~ A+y + B~x
(log Ks0 = log [A+v] + log [B*])
Protolysis: HB(aq) H+ + B~
(pH = -log Ka + log [B ]/[HB])
Oxidation half-cell: Bx ~ B<-x+1) + e-
(ps = -log K0 + log [B(-*+1)]/[B-*])
Uptake and depuration of QDs by T. pyriformis
~
-------
Aqueous solubility search
Sulfides, most oxides: abundant
Binary selenides, tellurides: some
Nitrides, phosphides, arsenides,
stibnides, tertiary, quaternary, doped,
magnetic: none
Solubility of CdSe in water
pc-pH diagram for CdSe
1
1
HSe04 [
! Se042,C
-------
Other considerations.
Concluding remarks
• Since many semiconductors are comprised of
electron poor and electron rich components,
solubility results for CdSe may be generally true
for many compounds
• But favorable thermodynamics doesn't always
mean fast reaction rates
• Kinetics depend on many factors: temperature,
ionic strength, presents of catalysts (or
inhibitors), particle size, external oxidizing
agents, light, etc.
• And, the impact of nanostructured materials on
human and ecosystem function will depend on
other systemic factors (loading, exposure,
interdependence of components, mode of
toxicity or uptake...)
• The ability to make and control very small
structured materials has very large implications
for human health, comfort and convenience,
and economic well-being
• In comparison to basic nanoscience and the
fabricaton of nanostructures, our
understanding of environmental and life cycle
behaviors of nanomanufacturing,
nanomaterials, and nano-containing products
exhibit exceptional lags
• Even so, it is clear that there will be a suite of
significant waste management problems
6
-------
Evaluating the Impacts of
Nanomanufacturing via Thermodynamic
and Life Cycle Analysis
Bhavik R. Bakshi and L. James Lee
Vikas Khanna, Geoffrey F. Grubb
Department of Chemical and Biomolecular Engineering
The Ohio State University, Columbus, Ohio, USA
Interagency Workshop on the Environmental Implications of
N anotechnology _
5si
November 20-21, 2008, Tampa, Florida
Motivation
~ Discover problems with technology before it is
fully developed and adopted
~ Guide development of nanotechnology to be
environmentally benign and sustainable
~ Understanding environmental impact of
nanomaterials is essential but not enough
~ Need to adopt a systems view with life cycle
thinking
~ Life Cycle Analysis of emerging technologies
_ poses unique challenges
|
Life Cycle Analysis
~ Need data for
each stage of
life cycle
¦ Energy
¦ Materials
¦ Emissions
¦ Impact
~ Difficult to find
for emerging
technologies
Environment
Extraction
& Processing
Reuse or
recycle
Disposal
Challenges in LCA of Nanotechnology
~ Inventory for nanomanufacturing is not available
~ Impact of engineered nanomaterials on humans and
ecosystems is only partially known
~ Predicting life cycle processes and activities is difficult
since the technology is still in its infancy
| M
Objectives
~ Life Cycle Evaluation of Nanoproducts & Processes
¦ Establish Life Cycle Inventory modules for Nanomaterials
¦ Carbon Nanofibers
¦ Polymer Nanocomposites Products
¦ Titanium Dioxide nanoparticles
~ Develop methods to identify opportunities for improving
the life cycle
i
~ Explore predictive model for LCA and impact assessment
¦ Relationship between life cycle inputs and impact
¦ Relationship between properties of nanoparticles and their
impact
£
5
LCA of Carbon Nanofibers
~ Extraordinarily high tensile strength
¦ Tensile strength-12000 MPa, 10 times that of Steel
m Increases mechanical and impact strength of polyolefins
~ Life cycle energy consumption is at least 100 times
larger than conventional materials on a mass basis
~ Greenness of
CNF nano-
products will
depend on
quantity used
and resulting
benefit
~ Polymer
nanocomposites
Khanna, Bakshi, Lee,
J. Ind. Ecol, 2008
Poly Si
1
-------
Polymer Nanocomposites
Polymer
Packaging
~ Attractive features of Polymer Nanocomposites
¦ Enhanced mechanical properties
¦ High strength to weight ratios
| ¦ Other functionalities- electrical conductivity
_
Life Cycle- Polymer Nanocomposites
I Material I I Energy I
w
Carbon Nanofiber
(CNF) Production
Polymer Resin
Production
dr
II
Glass Fiber (GF)
Production
CNF/GF
Dispersion
] | Energy |
Composite
Manufacture
|^Energ^
Fuel Gasoline
Production
IE
^JEnergyJ
^nerg^^
Polymer Material Investigated I Energy I
Thermoplastic: Polypropylene
Thermoset: Unsaturated Polyester
Use Phase
3E
End-of-life
Recycle/
other uses
Landfill / Incineration
Functional Unit & Theory
~ Functional Unit- Different material components with
equal stiffness or strength
~ For equal stiffness design, the material property to be
maximized is:
= ASHBY'S MATERIAL INDEX
9
~ Structural elements perform a physical function
o Performance is defined as:
p= f(F, G, M) where p=mass, volume, cost, etc.
F= functional requirements; G= geometric requirements
M= material requirements; p= f1(F).f2(G).f3(M)
Optimum Design- selection of material and geometry that
maximize or minimize 'p' according to desirability
I Ashby, M., Mails. Selection in Mech. Design: Peigamon Press
!_
Energy Analysis for Equal Stiffness
•-CNI
>P-CNI
I
¦ Glass Fiber (GF)
Q Unsaturated Polyester Resin (UPR)
a Polypropylene (PP)
° Carbon Nanofiber (CNF)
~ CNF reinforced PNCs are energy
intensive compared with steel
» Product use phase will govern if net
energy savings can be realized
PP-GF-CNF <2'0'
UPR-CNF
PP-GF-CNF
CNF CNF CNF CNF CNF CNF CNF CNF
3 Vol. % 9 Vol. % 15 Vol. % 0.6 Vol. % 2.3 Vol. % 1.02 Vol. % 3.4 Vol. % 2.4 Vol. °/<
Cradle-to-Gate: Life Cycle Comparison of PNCs vs. Steel
JOL
Auto Panel Case Study — Assumptions
~ Midsize Car (3300 lbs) with polymer nanocomposite
body panels vs. steel body panels
~ Car lifetime: 150,000 Vehicle miles traveled (VMT)
~ Constant fuel economy over the vehicle life time
~ Body panels constitute 10% of the vehicle weight
~ Sedan equivalent for fuel economy calculation
~ Steel mix - 30 (virgin): 70 (recycled)
~ Energy requirements for nanoparticle dispersion are
ignored
_
Savings in Life Cycle Fossil Energy
Net Savings in lifetime fossil energy, GJ/Car (relative to steel)
T PP-CNF
I T
» Automobile use phase dominates
» Higher upstream energy is offset by savings during the use
phase
~ Savings of 1.4 - 10%
~ Use of glass fibres with CNF may be more promising in the
short run
~ End-of-life issues specific to CNF are not included and can be
significant
~ Steel might be easier to recycle/ reuse/dispose compared to
CNF reinforced nanocomposites
-10 -
¦20 -
Automotive body panels- Effect of secondary weight reduction on lifetime fossil energy^
I savinns
_
2
-------
LCA of Ti00 Nanoparticles
~ Altair hydrochloride process
¦ llmenite feed
1
¦ Tailored for nanoparticle production
¦ Near complete recycle of HCI
¦ Claims of energy savings
¦ Currently at the pilot stage (10,000 kg/yr)
~ Life cycle inventory is needed
~ Opportunity to identify improvements at early stages of
development
~ Some applications of nano Titania
¦ Sun screens and cosmetics
¦ Photocatalysts, etc.
m
L-r
UJ
Life Cycle Energy Consumption
~ Nano Ti02
consumes
much less
energy per
ton than CNF
~ However, total
quantity of
nano Ti02
used globally
may be
much larger
Gross Energy Requirement
Identifying Improvement Opportunities
~ LCA does have an improvement analysis step
¦ Focus on modifications to reduce emissions with largest
impact
¦ Often receives little attention
~ Consumption of resources has not received adequate
attention in LCA
~ This work explores the use of thermodynamic methods
for identifying improvement opportunities
¦ Energy and Exergy analysis
Improvements — Emissions vs. Exergy
Exergy
llmenite
IH
Iron
Powder
Emissions
Electricity
Ti02
Manufacture
Ti02
Nanoparticles
Summary
~ Developed life cycle inventories for polymer
nanocomposites and nano Ti02
~ LCA of polymer nanocomposites for automotive use
¦ 4-10% life cycle energy savings, mainly due to fuel savings
in use phase
~ LCA of nano Ti02
¦ Significantly less energy use and impact as compared to
carbon nanofibres
~ Completed life cycle exergy analysis of nano Ti02
¦ Complements emissions based LCA
¦ Identifies improvement opportunities
[•-
1 Ei
3
-------
Future Work
~ Focus on other nanoproducts based on CNF or nano Ti02
~ Explore statistical relation between resource use and
impact for predictive LCA
~ Risk analysis
~ Acknowledgements
¦ Financial support from EPA (Grant No. R832532) and NSF
NSEC at Ohio State
l'H O
SPOT: I
4
-------
NC STATE UNIVERSITY
"Properties on Skin Absorption o
Manufactured Nanomaterials
u
Xin-Rui Xia (PI), Nancy A. Monteiro-Riviere, Jim E. Riviere
Center for Chemical Toxicology Research &
Pharmacokinetics (CCTRP)
North Carolina State University, Raleigh, NC
Project Significance
Skin is the largest organ protecting our body from
exogenous toxins and particulates.
Skin confronts nanomaterials from occupational and
environmental exposures.
Hundreds of consumer products are already on the market.
Sunscreens made of nanomaterials (Ti02, ZnO) show
superior UV protection performance. Fullerenes is used as
radical sponge for facial moisturizer, anti-aging and anti-
oxidant additives in skin care products
Skin absorption of nanomaterials is critical in safety
evaluation and risk assessment of the nanomaterials.
Stratum corneum (uppermost
layer of skin, ca. 15 ^m) is the
primary barrier for small
molecules or particulates.
What happens if skin is
exposed to nanoparticles?
¦ Which factors affect their
absorption?
¦ How the physicochemical
properties of the
nanomaterials dictate their
skin permeability ?
Could a predictive model
be established via
structure-permeability
relationship?
The objective of this project is to establish a
structure-permeability relationship for skin
absorption of manufactured nanomaterials for
safety evaluation and risk assessment.
Four dominant physicochemical properties
(particle size, surface charge, hydrophobicity and
solvent effects) in skin absorption will be studied.
Fullerene and its derivatives will be used as
model nanomaterials.
KMXae
*30000
1 sooas
ZaUfel
P-
Im—lBolrtMdmi
1
100MC
TT
-4— /,
.100
0
Dynamic size distribution of nCfin
nanoparticles_ H
Zeta-potential of nC60 after 14-
day dialysis
We have developed a novel method to prepare nC60 nanoparticle with a narrow size
distribution. This method does not use TFA while provide nC60 concentration in
water 100 times higher than the TFA method. The nC60 nanoparticles are formed in
a SDS aqueous solution, then SDS is removed via dialysis. After exhaustive
dialysis, the nC60 nanoparticles were stable in water for years.
1
-------
Impact of nCBn Colloidal Stability on
Most nanoparticles in aqueous solutions are charged
colloidal particles.
It is hypothesized that an IP agent can neutralize the
charges on nanoparticles, while not destabilizing the
nanoparticles; so that the neutralized nanoparticles could
penetrate into the SC (knowing the fact that charged chemicals are
difficult to permeated through SC).
The effects of 5 IP agents on skin absorption of nC60 will be
studied with three techniques:
- Diffusion cell experiment,
- Tape-stripping method in vitro
- and in vivo Tape-stripping method.
¦ nC60 and most of the unprotected
nanomaterials have a very
narrow window in their colloidal
stability (even though they are
stable in pure water).
~ Ion-pairing agents (e.g. >
0.05%TFA) will cause their
aggregation.
• Biological electrolytes will
cause their aggregation.
Ion-pairing effects on particle size of * Once the nanoparticles
nC60 in aqueous solutions aggregate, they can not get
through the skin.
3 anions: TFA(trifluoroacetic acid), HFBA(heptafluorobutyric acid), and PA (phosphoric
acid); 2 ca TBAC (tetrabutylammonium chloride), TEA (triethylamine)
Ion-pairing effects on particle size of
nC60 in aqueous solutions
Ion-pairing effects on particle size of
ANnCfi0
We have modified an
industrial available dispersion
agent (insoluble in water) to a
water soluble polymer.
When mixed with the polymer
solution, nC60 particle size
increased about 10 nm.
The polymer wrapped nC60
nanoparticles (ANnC60) will
not aggregate in a strong
electrolyte (e.g., 2M KCI),
extreme pH (1 to 13), any ion-
pairing agent.
Ion-pairing effects on the skin
absorption of ANnC60 will be
studied.
Ion-Pairing Effects (TFA) on Particle Size and Zeta-
Potential of ANnC60
Ion-Pairing Concentration (°/tj
Ion-pairing effects (TFA) on particle size and Zeta-potential of ANnC6(
Correlation of SC absorption/adsorption of ANhC60 with Zeta-potential of
—~ the nanoparticles
SC absorption of ANnC60 was measured by submerging SC in a nanoparticle
solution containing different concentrations of ion-pairing agent and equilibrated at
37°C for 24 hrs. Then the SC was separated from the solution, washed, dried with
paper and digested for quantitative analysis.
-------
jnomaterial transport through the SC The original data from tape-stripping method
generally is assumed to follow Fick's are (mn-n, t) and t). The
second law of diffusion through a simple, concentration of nariOmatefial on nth strip
homogeneous membrane (Crank 1975): (cn);
Under the boundary conditions of the
experiments performed (at x = 0, C=Cx=
0 =KCv, t> 0; at 0 < x < L, C= 0, t= 0; and
at x=L, C= 0, t > 0), the concentration
profile of the nanomaterial, i.e., Cas a
function of position x and time f, is given
by the well known solution to Fick's
second law:
The location of Cn as a function of depth in the
SC(*n);
Kinetics information could be obtained and tape-stripping method
from the depth distribution of
na no materia Is
Tape-Strip Number
SC amount on each tape-strip
Nanomaterial amount on each tape-
measured with Lowry total protein
strip (doses were made daily for 4
method
days)
Skin permeability (kp);
kp=KD/L
Fullerenes detected in the skin after 4 day multiple exposures
in vivo
The animals (n =3) were dosed (500|jL) daily for 4 days, then
tape-stripping was performed on the 5th day. The biopsies were
collected after 26 tape-strips.
3
Larger dose area for tape-stripping analysis
Directly measures the quantity of nanomaterials absorbed into SG
Kinetics information could be obtained from the depth distribution of nanomaterials
No time-limit for study occupational exposure (weeks, or months)
The morphology of pig skin is similar to human skin
-------
Nanoparticles in aqueous solutions can be classified into "OmV Zeta-
potential" stable or unstable nanomaterials.
¦ Ion-pairing agents cause the "OmV Zeta-potential" unstable nanoparticles
to aggregate (ag., nC60). Thus ion-paring agents will not aid in their skin
penetration.
¦ Ion-pairing agents can be used to control the surface charge of "OmV
Zeta" stable nanoparticles (e.g., ANnC60). The SC absorption (in vitro) is
linearly correlated with Zeta-potential after a transition point.
¦ Skin permeation of nanomaterials is a slow process. No nanomaterial was
detected in the receptor solutions in 8-hr or 24-hr diffusion experiments.
" Nanomaterials could be absorbed though the skin from aqueous solutions
in long term exposures.
¦ Tape-stripping methods can be used to study the absorption kinetics of the
slow skin permeation of nanomaterials.
¦ Solvents are among the most commonly used chemicals in
workplaces. Many kinds of solvents will be used in manufacturing,
processing, application and handling of nanomaterials.
It is hypothesized that skin absorption of nanomaterials is altered
significantly by the solvent effects.
The solvent effects on the skin absorption of fullerene nanomaterials
will be studied in 6 industrial solvents (toluene, cyclohexane,
chloroform, ethanol, acetone and propylene glycol) using the diffusion,
tape-stripping and in vivo methods.
The skin permeability and partition coefficient of the nanomaterials
between SC and solvents (logKsc/s) will be measured, which can be
used for safety evaluation and risk assessment of the nanomaterials in
the solvents.
Data from in Vivo Tape-Strip after
Depth Profile of SC by Lowry Total Protein Method
Nanomaterial amount on each tape- SC amount on each tape-strip measured
strip (mn-n, t) with Lowry total protein method (mscn- n, t)
\
00 01 OS 03 04 05 06 07 08 09
A-Std Error A- CV%
SC thickness (L): 16.2 um
Partition coefficient (K): 0.855
Skin permeability (k ) 0.185 um/hr
Regression analysis of tape-stripping data
The animals were dosed for 2hr, then tape-stripping was performed
on the animals 8-hr after dose. The skin was tape-stripped for 26
times. After 10,h strip, two strips were combined into one digestion
solution for quantitative analysis.
Solvent effects on skin absorption of C60 in
Solvent effect on skin absorption of ANnCSO in
¦ i if
C6Qnblume C60Cydohexaie C6Q!Chloroform C60/Mner
NnC80/EOH ANnC60/PropylG
Solvent effects on skin absorption of fullerene nanomaterials after 4-day
multiple exposure in viyo Lm,
The animals {n =3) were dosed daily for 4 days, then tape-
stripping was performed on the animals under anesthesia within
1 hr. The skin tissue biopsies were collected after 26 tape-strips.
-------
Solvent Effects on SC Absorption/Adsorption of
nC60/ANnC60
Solvent Effects on SC Absorption of C60
nC60FD14l/EOH
nC60FD14l/Ace
C60/Chloroform
Solvent effects on SC absorption of C6Q in vitro
SC was weighed into a given nanoparticle solution of solvent. The SC
was submerged in the nanoparticle solution and equilibrated at 37°C for
24 hrs. Then the SC was separated from the solution and washed
vigorously in a flow-through washing tube with deionized water. Finally,
the SC was dried with paper and digested for quantitative analysis.
Solvent effects on SC absorption/adsorption pf nC60/ANnC60 in vitro —
SC was weighed into a given nanoparticle solution of solvent. The SC
was submerged in the nanoparticle solution and equilibrated at 37°C for
24 hrs. Then the SC was separated from the solution and washed
vigorously in a flow-through washing tube with deionized water. Finally, the
SC was dried with paper and digested for quantitative analysis.
Fullerenes exist as molecular C60 or nC60 in different
solvents which affect their skin absorption mechanism.
nC60/ANnC60 were readily absorbed into the SC in
vitro/in vivo; acetone gives higher adsorption comparing
to ethanol and propylene glycol
¦ C60 was readily absorbed into SC in vitro/in vivo;
chloroform gives higher absorption compared to toluene
and cyclohexane.
Tape-stripping methods can be used to study solvent
effects on skin absorption of nanomaterials and to
provide partition coefficients and skin permeability for
predictive model development.
Supported by the US EPA-Science to Achieve Results
(STAR) Program
[
US. EPA -Science To Achieve
Results (STAR)Program
H Grant
.
5
-------
Safety/toxicity assessment of
ceria (a model engineered NP)
to the brain
Tftft research a funded by
EPA - Science To Achieve
Resulis (STAR)Prooram
Grant #1
The research team
Robert A. Yokel & Rebecca L. Florence
- Department of Pharmaceutical Sciences, College of
Pharmacy & (RAY) Graduate Center for Toxicology,
University of Kentucky, Lexington, KY
Jason Unrine
- Department of Plant and Soil Sciences, U KY
Michael T. Tseng
- Departments of Anatomical Sciences &
Neurobiology, University of Louisville, Louisville, KY
The research team - continued
• Uschi M. Graham
-Center for Applied Energy Research, U KY
• Rukhsana Sultana & D. Allan Butterfield
- Department of Chemistry, U KY & (DAB)
Center of Membrane Sciences, U KY
• Peng Wu & Eric A. Grulke
-Chemical & Materials Engineering
Department, U KY
Objective of this research
• It is known that some physico-chemical properties of
engineered nanomaterials (ENMs) can influence
their fate (ADME), including distribution across the
blood-brain barrier (BBB).
• But the affects of various physico-chemical
properties on the entry of ENMs into the BBB and
brain cells and their beneficial and/or hazardous
effects are not well studied:
- Size
- Shape
- Surface chemistry
• Objective: Characterize the biodistribution and
effects of nanoscale ceria that had entered blood.
Rationale for selection of material
to be studied
• Ceria (CAS Reg #1306-38-3; Ce02, cerium dioxide,
cerium oxide) was selected because:
- it is an insoluble metal oxide that can be readily observed in
tissue (electron microscopy, elemental analysis), making it a
useful tracer.
- it is redox reactive.
- it is available and can be manufactured in many sizes and
shapes in the nanoscale range (up to 100 nm).
- it can be functionalized (surface chemistry altered).
- it has current commercial applications (catalyst and abrasive).
- it has been reported to be cytotoxic as well as
neuroprotective, representing the controversy about
nanoscale materials.
Ceria ENM studied in our initial work
• A 5% dispersion of ceria ENMs in water
(Aldrich cat #639648, produced by
NanoProducts, Corp.) characterized by laser
light scattering (Brookhaven 90Plus Particle
Size Analyzer).
- After 6 min probe sonication @ 50 W nearly 100%
of the ENM were ~ 30 (range 21 to 39) nm (94%
of the surface area; 77% of the volume), by
multimodal size distribution analysis.
- The remaining volume was ~ 90 to 200 nm.
- Primary size ~ 3 to 5 nm (by high resolution
transmission electron microscopy [HR-TEM])
- Surface area was ~ 13 m2/g.
- Osmotic strength was 28 mOsm.
-------
High resolution transmission electron
microscopy (HRTEM) showed the
material to be individual ceria crystals as
part of a ceria nanocomposite
Search for an iso-osmotic vehicle
for this ceria ENM
• The effects of saline and 10% sucrose on ceria
ENM agglomeration were assessed by their
addition and repeated particle size determination.
- Saline caused agglomeration.
• After 5 min: particles were 260 to 430 nm.
• After 40 min: - 98% 300 to 480 nm and 2% 2960 to 3320 nm.
- 10% sucrose caused agglomeration.
• Within 1 hour -89% were 110-140 nm and -11% 350-441 nm.
• Problem: How to administer a ceria ENM
dispersion i.v. to rats and avoid significant
erythrocyte lysis?
Studies to predict in vivo agglomeration
• Freshly drawn whole rat blood was incubated with
ceria ENM (0.14, 0.7 and 3.56 mg ceria/ml) for 1 hr,
allowed to clot, fixed in formalin, and processed for
high resolution transmission electron microscopy,
scanning TEM, and energy-dispersive x-ray
spectroscopy (HRTEM/STEM/EDS).
• Agglomerated ceria was seen in the extracellular
space between erythrocytes. EDS verified the
presence of cerium in the agglomeration.
Distribution and brain effects of
intravenously administered ceria
• Objective: Assess the ability of ceria ENM to
enter the BBB and brain cells, compared to
peripheral organs, and to produce
neuroprotection or neurotoxicity.
• Rationale for i.v. administration: Absorption of
an ENM by any route will introduce it into
systemic circulation, from which it may
distribute to the brain.
Methods
• Un-anesthetized male Fisher 344 rats, implanted
with two venous cannulae (femoral vein access,
terminating in the vena cava) were infused i.v. with:
- 0, 50, 250 or 750 mg ceria/kg in water.
- concurrent equal volume and rate of infusion of 1.8%
saline in a 2nd cannula.
• Blood was repeatedly drawn from some rats up to 4
hr for Ce analysis by inductively coupled plasma
atomic emission spectroscopy & mass
spectrometry (ICP-AES/ICP-MS).
• Rats were terminated either 1 or 20 hr after
completion of the infusion.
Methods - continued
• Five minutes before termination the rat was
anesthetized and given Na fluorescein (334
Da) and an Evans blue (EB)-albumin complex
(~ 68,400 Da) in saline i.v. as BBB integrity
markers.
-------
Methods - continued
• After termination samples were obtained of:
- brain, liver, spleen and blood to determine Ce by
ICP-AES/ICP-MS.
- brain, liver, spleen, and kidney for histological
assessment and EM localization ofceria.
- brain to determine fluorescein and EB.
- brain to determine oxidative stress markers:
• protein-bound 4-hydroxy-2-nonenal (HNE)
• 3-nitrotyrosine (3-NT)
• protein carbonyls
Results - Clinical toxicity
• Clinical toxicity was only seen in rats
receiving 750 mg ceria/kg:
-slight tachypnea
- dyspnea
- abnormal behavior
Results - Ce was rapidly cleared
from blood after completion of i.v.
ceria infusion
The half-life of cerium clearance after
termination of ceria infusion was well
under 1 hr.
Cerium concentration in plasma was
much less than whole blood, but this
was an artifact of centrifugation to
generate the plasma.
Results - Intracellular ceria was
seen in the spleen red pulp
• The ceria was seen as agglomerates.
• No histopathology was observed.
Results - Intracellular ceria was
seen in the liver
• Ceria agglomerations were seen in
Kupffer cells and hepatocytes.
• Cellular degeneration was observed in
some hepatocytes.
Ceria induced Kupffer cell
activation
• An increase of the number of Kupffer
cells was seen as a function of ceria
dose and time.
-------
Results - Intracellular ceria ENM
was seen in the kidney
• Ceria agglomerates (verified by EDS)
were seen in the vascular space and in
mesangial cells of rats terminated 20 hr
after ceria infusion.
• Abnormal tubular epithelial
proteinacious accumulation was
observed in rats terminated 20 hr after
ceria infusion.
Results -There was a near
absence of ceria ENM in the brain
• Ceria was seen in the vascular lumen in
the brain but only occasionally seen in
astrocytes or neurons.
• No visual evidence of BBB breakdown
was seen.
Results - Tissue Ce concentration was
ceria dose-dependent
• Very similar distribution of cerium was
seen 1 and 20 hr after completion of the
ceria infusion.
• Ceria concentration in the spleen was
slightly greater than in the liver, which was
greater than in the brain and serum by 2
to 3 orders of magnitude.
Results - No great changes in
oxidative stress indicators were
seen in the brain
• 1 hr after ceria infusion there were no
significant changes in protein-bound 4-
hydroxy-2-nonenal, 3-nitrotyrosine, or
protein carbonyls
• 20 hr after ceria infusion HNE increased
in the hippocampus and protein
carbonyls decreased in the cerebellum
Results - There was a small
increase in blood-brain barrier
permeability 20, but not 1, hr after
ceria infusion
• Brain fluorescein and Evans blue were
not significantly changed 1 hr after ceria
infusion.
• Brain fluorescein was elevated 20 hr
after ceria infusion.
• But there was considerable variability in
the results, especially with Evans blue.
Relating these ceria doses to its
use as a diesel fuel additive
• This ~ 30 nm ceria ENM nanocomposite
was quite non-toxic when introduced i.v.
-The 50, 250 and 750 mg ceria/kg i.v. doses
in these ~ 0.3 kg rats would equal all of the
5 ppm ceria in 3, 15 and 45 liters of diesel
fuel.
-------
Conclusions
• Ceria was rapidly cleared from the blood by
peripheral reticuloendothelial tissues.
• Much less ceria entered the BBB cells or the brain.
• Ceria ENM agglomerates in vivo.
• This ceria induced mild oxidative stress and stress
response in the brain.
• This ceria provides an inert core ENM enabling the
study of the effects of size, shape and surface
chemistry on biodistribution, biotransformation and
neurotoxic or neuroprotective potential.
-------
Internalization and Fate of Individual Manufactured
Nanomaterial Within Living Cells
Galya Orr
Systems Toxicology of Nanomaterials
Pacific Northwest National Laboratory
galya. o rr @ pnl .gov
http://biomarkers.pnI.gov/staff/orr.asp
Pacific Northwest
Manufactured amorphous silica nanoparticles are used
extensively in a wide range of industrial applications
The wide use of synthetic amorphous silica results, in part, from the
relative ease of controlling their size and purity
462 ± 20.5 nm
1 pm
92.2 ± 13.2 nm
,#>
Surface charge: Unmodified (C= -40 mV)
Surface aminated (C= +20 mV)
Pacific Northwest
Rationale
1) The cellular interactions and intracellular fate of nanomaterials
dictate the cellular response and ultimately the level of toxicity.
If we understand mechanisms that underlie the cellular interactions and
internalization pathways of well-defined nanoparticles we could
delineate relationships between particle properties, cellular response, and
mechanisms of toxicity or biocompatibility.
2) Nanomaterials are likely to be presented to cells in vivo as individual
particles or small nanoscale aggregates (<100 nm).
If we study one particle at a time we are more likely to delineate
mechanisms that occur in vivo.
Pacific Northwest
Alveolar type II epithelial cells are important target:
Air born particles ranging from 5 nm to 1 pm that enter the respiratory
tract are likely to be deposited in the alveolar region.
Type II cells play critical roles in the function of the alveoli by
secreting pulmonary surfactants, and by differentiating into type I
epithelial cells when these are damaged.
Importantly, type II cells participate in the immune response to certain
particles and pathogens by releasing chemokines.
- • » o
•fc.fr* •Nr-ri A
* '
Alveolar type II epithelial cells carry apical microvilli:
CIO: a Non-tumorigenic cell line, derived from a normal lung of an adult mouse
and preserves its phenotype, including lamellar bodies and surface microvilli:
Positively charged 500 nm particles are propelled along microvilli in a
retrograde motion, unraveling the coupling of the particle with the
intracellular environment across the cell membrane:
1
-------
Positively charged 100 nm particles travel along microvilli in a more
complex, anterograde and retrograde motions:
Pacific Northwest
The retrograde motion of the particles and the retrograde flow of
actin clusters depend on the integrity of actin filaments:
The retrograde motion of the particles and the retrograde flow of
actin clusters occur at the same rate:
12
Particles
Actin clusters
Is
s 3 5;
,-ai>
Q
cn
200 400 600 800 100
100 200 300 00 500 600
i=
y
Q
C/D
yS
V' 12.3 nm/s
yr 12.4 nm/s
time (sT
Time (sf
The retrograde motion unravels charge-dependent coupling of the
particles with the intracellular environment across the cell membrane:
docking
protein
w ^
Positively charged particles bind a negatively charged transmembrane
molecule that, in turn, interacts directly or indirectly with the actin
filaments within microvilli.
As actin monomers are added to the distal tip of the filaments, a
retrograde motion is generated, leading to the retrograde motion of the
membrane molecule and its bound particle.
Orr et alACS Nemo, 2007 1(5):463-475^
Heparan sulfate proteoglycans play a critical role in the attachment and
internalization of positively charged 500 nm particles:
Cells exposed to particles + Trypan Blue
Cells exposed to particles
Log (fluorescence intensity)
1
t exposed to particles
dJ Cells exposed to particles
Log (fluorescence intensity)
Cells treated with heparinase I
"l exposed to particles + Trypan
Log (fluorescence intensity)
Chondroitin sulfate proteoglycans play a smaller role in the attachment
and internalization of positively charged 500 particles:
Cells treated with chondroitinase &
exposed to particles
Cells treated with chondroitinase S
exposed to particles + Trypan
Cells exposed to
particles + Trypan
Log (fluorescence counts)
Log (fluorescence counts)
2
-------
Syndecan-1, a trans membrane heparan sulfate proteoglycan, engages
positively charged 500 nm particles in the movement along microvilli:
Syndecan-l is green
Particles are red
Pacific Northwest
Syndecan-1 and positively charged 500 nm particles are co-
localized in small and discrete cellular structures:
Positively charged 500 nm particles are also co-localized
with 70 kD dextran, a tracer for macropinocytosis:
The internalization of the particles is blocked by amiloride,
an inhibitor of macropinocytosis:
Syndecan-lmediates the initial interactions of the particles at the cell
surface, their coupling with actin filaments across the cell membrane,
and their subsequent internalization via macropinocytosis.
Summary
A new retrograde pathway is described, unraveling the coupling of
positively charged submicro- and nanoscale inorganic particle with the
intracellular environment across the cell membrane.
This pathway brings a new mechanism by which positive surface
charge supports particle recruitment, and potential subsequent toxicity,
in polarized epithelial cells bearing microvilli.
Heparan sulfate proteoglycans are identified as critical players in the
attachment and internalization of positively charged submicrmeter
inorganic particles.
Syndecan-1, a transmembrane haparan sulfate proteoglycan, is found to
mediate the cellular interactions and fate of the particles and therefore
govern their cellular response.
3
-------
****•#•
Pacific Northwest
This work has been supported by:
Pacific Northwest
r US CPA -Sckbc# To A£Kt«V«
Rasults (STAR) Prejiam
The Environmental Biomarkers
Initiative at the Pacific Northwest
National Laboratory (PNNL)
http ://b iomarkers.pnl.gov
The Air Force Research Laboratory
grant through ON AMI - SNNI.
ONAMI
4
-------
Methodology Development for
Manufactured Nanomaterial
Bioaccumulation Test
Pis: Yongsheng Chen, Qiang Hu, Milton Sommerfeid, Yung
Chang, John Crittenden, and CP. Huang*'
Arizona State University
"University of Delaware
Nov. 21, 2008
Outlines
Assess toxicity of manufactured
nanomateriais in several aquatic
modei organisms
Determine bioconcentration of
manufactured nanomateriais in
aquatic organisms
Evaluate biomagnification of
manufactured nanomateriais in food
chain
Test Nanomateriais
Particles
Particle Size
Purity (%)
C60
< 200 nm
99.5
SWCNTs
D < 2 nm
L = 5 - 15 (Jin
CNTs > 90
SWCNTs > 60
MWCNTs
D = 10 - 20 nm
L = 5 - 15 jjm
> 98.0
nZnO
20 nm
> 99.6
nTi02
< 20 nm
> 99.5
nAl203
80 nm
> 99.9
Model Organisms
• Algae
• Daphnia
Zebrafish Embryos
and Zebrafish
Reasons:
1. They are at the lower
level of the food chain;
2. Toxicity indicators and
their genetic database
have been well-
established
Toxicity of Nanoparticles on Green Algae
NPs
Regression Equation
Correlation
Coefficient
ECS0 (mg/L)
nZnO Suspension
y = 38.862x +49.194
High
R2 = 0.9542 i
toxicity
„ 1.049 + 0.565
C60 Suspension
y= 26.42x+ 20.456
R2 = 0.8988
13.122 + 4.182
nTi02 Suspension
y = 39.902x+ 2.7719
R2 = 0.9275
15.262 + 6.968
MWCNTs Suspension
y= 38.468x+ 4.3117
R2 = 0.9964
15.488 + 7.108
SWCNTs Suspension
y = 27.978x+ 12.097
R2 = 0.8434
22.633 + 9.605
nAl203 Suspension
y = 14.204x- 10.044
J?2 = 0.5471
>1000
Zhu X., L. Zhu, Y. Chen, Y. Lang
J. Nanoparticle Research. 2008 DO110.1007/s11051-008-9426-8
Green Algae Aggregation and Growth Inhibition
P 1 US rmMh. Algae growth inhibition
1
-------
Lipid Peroxidation and Gene Expressions
Lipid peroxidation (MDA)
• • control
^ m
C -1 mfl'l.
T
-. -
1 0-05
| 0.04
•& 0.03
1 002
Time (h)
For the first 12 h, A dose-dependent increase in
the maximum malondialdehyde (MDA) content
was clearly indicative of cellular lipid peroxidation
induced by Ti02 NPs.
J. Wang, X. Zhang, Y. Chen, et al. Chemosphere,
73(2008): 1121-1128
Gene expressions: Catalase
• - control
O -1 mg/L
~-10 mg/L
a -100 mg/Lof Ti02 NPs
Time (h)
After 1.5 h treatment, the maximum
transcripts of catalase occurred; However,
the catalase gene expression up-regulation
was transient.
JSU Fulton
Toxicity of NPs on Daphnia magna
High toxicity
Material j
(particle size)
^ ec50
(mg/L)
95% CI
lc50
(mg/L)
95% CI
nZnO (20 nm)
0.62
0.41-0.81
1.51
1.12-2.11
SWCNTs (<2nm)
1.31
0.82-1.99
2.43
1.64-3.55
C60 (<200nm)
9.34
7.76-11.26
10.52
8.66-12.76
MWCNTs (10-20nm)
8.72
6.28-12.13
22.75
15.68-34.39
nTi02 (< 20nm)
35.31
25.63-48.99
143.39
106.47-202.82
nAl203 (80 nm)
114.36
111.23-191.10
162.39
124.33-214.80
Low toxicity
(48 h)
Zhu X., L. Zhu, Y. Chen, Y. Lang
J. Nanoparticle Research. 2008 DOI 10.1007/s11051-008-9426-8
The Morphology of Daphnia magna
Control
A: nAI(Oj(10#mg/L^' ^
w
B: nTiO^Wmg/L)
C: nZnOlS mgrL)^
Treated with
different type of
NPs for 48 h
V
SWC.VTi
%
E: (lOmgAX-Z-
F: AlWCyTs (10 mg/L)
Impacts on Zebrafish Embryo Hatching
140
120
¦ 100
40
20
~ 10 mg/L nZnO ~ 1 mg/L Zn2+
¦ water control _ a
- de b| I
r bd b T
Mm
72 84 96
Hours post fertilization (hpf)
At 10 mg/L nZnO
concentration, the
released Zn2+ is
around 1 mg/L (20
nm filter was used)
Zhu X., L. Zhu, Y. Chen, Y. Lang
J. Nanoparticle Research. 2008 DO110.1007/s11051-008-9426-8
Toxicity of ZnO NPs on Zebrafish Embryo
Hatching
One did Hatch also display some abnormality
Pericardial edema (PE) and yolk sac edema (YE) induced by aggregates of
ZnO nanoparticle
ROS
Assessment
Fluorogenic dye was
used to reveal the
ROS. Specifically,
The embryos from 4-
day treatment of NPs
and Zn2+ were
prepared into single =
cell suspension, and c
then incubated with ]
fluorogenic dye, j
DCFDA, The cells 1
were then analyzed *
by flow cytometry.
The data indicates that the
nZnO NPs, not Zn2' cause
higher level intracellular ROS,
which may contribute to the
developmental toxicity
A
sA.,
Intensity of Florescence
Control PC=90
lmg/L Zn2+ PC=77
lmg/LnZnO PC=257
lOmg/LnZnO PC=429
100 mg/L nZnO P C= 60 9
PSU Fulton.
-------
Quantitative RT-PCR for Oxidant-Associated Genes
Given the higher level of ROS in nZnO-treated groups, we expect to see
increased expression of two genes (gstp2, Nqo1) coded for anti-oxidant enzyme.
S24hpf LJ48hpf D96hp
i
m
¦ 24hpf ~
48hpf ~ 96hpf
¦fi
r
n
1
-r
10mg/L nZnO 1 mg/L Zn2+
Gstp2
10mg/L nZnO 1 mg/L Zn2+
Nqo1
It is possible that nZnO-treated groups fail to up-reguiate their anti-oxidant genes, which
may explain the higher level of ROS shewn in the previous slide.
Sediments Impacts on
Toxicity of Zebrafish
Embryos
200
'150 -
p 100 -
; 50-
0 -
~ nZnO Aggregates
El Sediments + nZnO Aggregates
h a a
Mil
10 5 1 control
nZnO Concentration (mg/L)
Sediment could be a mitigating agent to
reduce the toxicity caused by nZnO NPs.
& technology in preparation
9 24hpf ~48hpf ~ 96hpf
0
it
S+IOmg'LnZnO 10mg/LnZnO
Gstp2
~ 300 n
3*
^ 250-
s 200-
•I 150-
£ 100-
z 50 -
24hpf ~ 48hpf ~ 96hpf
rih
Q
S+10mg/L nZnO 10m g/L nZn O
Nqo1
PSll Fulton
Summary Remarks
From the general toxicity tests:
• The toxicity rank order of carbon-based NPs is: SWCNTs >
C60 > MWCNTs; metal oxide NPs is: nZnO > nTi02 >
nAI203.
• nZnO caused oxidative stress on aquatic organisms
~ Toxicity is not solely caused by Zn 2+.
~ Toxicity is correlated with a higher level of ROS .
~ Toxicity appears to be inversely correlated with the
expression of two anti-oxidant genes.
• Sediment could reverse the toxicity induced by the ZnO
NPs.
Scheme for Experiments on nTi02 Bioconcentration by
Daphnia
Daphnia Nanoparticie
Clear
Water
•'Daphnia
' with NPs
Uptake for 24 h; sampling
at 0. 3, 6, 12 and 24 h
Depuration for 72 h; sampling
at 6, 12, 24, 48 and 72 h
12 24 36 48 60 72
Bioconcentration Factors (BCFs) for nTi02 in Daphnia
magna
Michaelis-Menten kinetics
Dose
Exact dose1
Whole body
/ BCFs \
^M=
-------
Biomagnification Tests by Feeding Daphnia to Zebrafish
Daphnia (8-10 days old): exposed to
0.1 mg/L nTi02 for 24 hours
Fed Iwo times each
day (about 8% wet
weight daily ratio)
Zebrafish (Danio rerio) (5-8 months old)
Experimental time (d)
Biomagnification factor (BMF) = 0.0259. From this preliminary data, it can be
speculated that there is no biomagnification of nTi02 from Daphnia to
zebrafish.
Future works
• Determine the bioaccumulation behavior of NPs under
different exposure conditions, such as static, semi-static
and flow-through system.
• Determine the distribution (or fate) of NPs in different parts
of exposure system, including water, organism body and
the excretion, based on the mass balance profile or using
a stable isotopic tracer approach.
Long-term experiments on biomagnification and toxicity
(e.g. in reproductive system) will be conducted.
Achievements
Journal articles related to this project
1. Sun H., Zhang X., Chen, Y., et ai. Enhanced accumulation of arsenate in Carp in the
presence of titanium dioxide nanoparticles. Water, Air & Soil Pollution. 2007, (178):245-
254.
2. Zhang X., Sun H., Chen Y., et al. Enhanced bioaccumulation of Cd in carp in the presence of
titanium dioxide nanoparticles. Chemosphere 2007, (67):160-166.
3. Zhu X., Zhu L., Lang Y., Chen Y. Oxidative stress and growth inhibition in the freshwater fish
Carassius auratus induced by chronic exposure to sublethal fullerene aggregates.
Environmental Toxicology and Chemistry, 2008, 27(9): 1979-1985.
4. Wang J., Zhang X., Chen Y., Sommerfeld M., Hu Q. Toxicity assessment of manufactured
nanomateriats using the unicellular green alga Chlamydomonas reinhardtii.
Chemosphere, 2008, 73: 1121 -1128.
5. Zhu X., Zhu L., Chen Y., Tian S. Acute Toxicities of Six Manufactured Nanomaterials Water
Suspensions on Daphnia magna. Journal of nanopartide research, 2009. Article in press
Presentations
6. Zhang X., Chen Y., Sun H., Crittenden J. Adsorption/Desorption of Cd by titanium dioxide
nanoparticles and sediment particles as well as their facilitated bioaccumulation of Cd into
Carp. NSTI (Nano Science and Technology Institute,) Nanotech 2007 Conference,
Santa Clara, California, USA. May 20-24, 2007
7. Zhu X., Zhang X., Chang Y., Chen Y. Toxicity of ZnO nanopartide sedimentation on the
embryo development of zebrafish (Danio rerio). NSTl (Nano Science and Technology
Institute) Nanotech 2008 Conference, Boston, Massachusetts, USA. June 1-5, 2008.
8. Zhang W., Zhu X., Zhang X., Chang Y., Chen Y., Rittman B., Crittenden J. Potential toxicity
of nanomateriais and their removal. International Environmental Nanotechnology
conference. Chicago, Michigan, October, 2008. (Orah:
ACkMWMgNMOtS
1) People from my group:
Wen Zhang, Ph.D. Student
Xiaoshan Zhu, Post-docs
Xuezhi Zhang, Post-docs
Jiangxin Wang, Post-docs
Pis: Yongsheng Chen, Yung Chang, Qiang Hu, Milton Sommerfeld, John
Crittenden, and CP Huang from U of Delaware
4
-------
[Nanoparticle Fate Project
Progress Report
David Y. H. Pui. Nick Stanley, and T.H. Kuehn
University of Minnesota; Minneapolis, Minnesota
C. Asbach, T. Kuhlbusch, H. Fissan - Institute of
Energy and Environmental Technology (IUTA);
Duisburg, Germany
Presentation Outline
Objectives and Background
Filtration Study
Wind Tunnel Testing
o Particle Dispersion
o Velocity Profiles
Burner Particle Characterization
Conclusions and Future Work
Overall Project Objectives
Measure and model the fate of
nanoparticles as they are emitted through a
leak from a nanoparticle production process
into a workplace environment.
Observe changes in particle and aerosol
properties, such as number and surface
area concentrations, morphology, and
chemical composition.
77>
to
J
wXV\
Background and Setup
SMPS
NSAM
SMPS
NSAM
xn
77
mto
J
Worker
E>posure
> *
I
Simpn n
SMPS
NSAM
TEOM
SEM/TEM
ED*
:_k_c
>
-------
New NIST Nanoparticle Standards:
60 nm and 100 nm SRM
Mant/re/inul of 100 tint atttl 60 tint Particle
Standards fry Differential Mobility Analysis
Journal of Research
National Institute of
Standards and Technology
Sas
—?.— rrr. r."zm
Percent of Neutrophils in BAL 24 hrs after Instillation of Ti02 in Rats
Correlation with Particle Surface Area
Particle Surface Area, cm2
Particle Deposition in Healthy Adult Subjects
0.01 0.1 1
particle diameter (|jm)
ICRP66 (1994); MPPDep (2000)
particle density: 1 g cnr*
respiratory flow rate: 300 cm3 s-1
breathing at rest cycle period : 5 s
Electrical Aerosol Detector
Nanoparticle Surface Area Monitor
Model 3550
^Filtration Study (filtration, submitted a/osj
Objectives: To determine compatibility of instruments and measure
overall filter efficiency based on number, SA, and size distributions.
Aerosol: DOS -10 ppm and 1 ppm (spherical particles)
Flow Rate at Flowmeter: 20 L/min and 10 L/min
Constant Output Atomizer (COA) Pressure: 25-30 psi
COA Flow Rate: 5 L/min
Make-up
iuloi
COA with
neutralizer -
and dryer
Upstream Downstream
! Filter !
Flowmeter
2
-------
LF iltration Study (FILTRATION, submitted 8/08) J
10 ppm DOS 1 ppm DOS
l= h
77s
.ulo,
NSAM and SMPS correlate very well (max discrepancy ~10%)
When concerned with an aerosol mainly composed of
nanoparticles, the surface-area filter efficiency represents:
o A more health relevant filter evaluation. m
o A better characterization of the filter.
4
Particle Dispersion Study - Setup
"C.
hm
¦»'. OM | |,
lutaj
Flow rates: 200 & 500 cfm (Face Velocities: 0.25 &
0.64 m/s)
6-Jet Atomizer (TSI Model 9306A) with 0,1 % KCI
solution at 12 l/min flow rate
Same sampling probe for SIVIPS and NSAM
Simultaneous measurements
4
NSAM/SMPS Dispersion Study
77s
ni
J
Idtoj
0.25 m/s
0.64 m/s
ArbiM IMa
Wi
~
C
Smptogtacalieo 1
IU i :
* t
4
NSAM/SMPS Dispersion Study
LDSA(SMPS) = 0.787*LDSA(NSAM)
• L ncarinn 3
L f>C.lT
inn
SwiqiUikg Location 1
Dixlaitc v fioui liffnttoii j j
CPC used to determine
amount of dispersion
within wind tunnel.
Flow is uniformly
dispersed 342 cm
downstream at 0.25 m/s
face velocity.
-V—1-t-
-AA-
LDispersion Study Comparison J
SMPS Dispersion Study CPC Dispersion Study
iuto.
/\
-—.
T
- -7^
it In] el
\
hm
i
SantplktR Location 1
:
i
Distance from Infection 3 j
3
-------
| Flow Profile at 20 cm Downstream
iutoj.
4.00e-0
3.84 e-0
3.72 e-0
3.60 e-0
3.48 e-0
3.36 e-0
3.24 e-0
3.12e-0
3.00 e-0
2.88 e-0
2.76 e-0
2.64 e-0
2.52 e-0
2.40 e-0
2.28 e-0
2.166-0
2.04 e-0
1.92 e-0
1.80 e-0
1.68 e-0
1.5 6 e-0
1.44 e-0
1.32 e-0
1.20 e-0
FLUENT model
)eriment
. J8e-0"
9.60 e-02
8.40 e-02
7.20 e-02
_ 6.00e-02
I"—1 4.80 e-02
3.60 e-02 Y
2.40 e-02
1.20 e-02—X
0.00 e+0(T
¦ Results are quantitatively similar
(max velocity = 0.40 m/s)
¦ Model shows a more distinguished
effect of the injection probe
I
1
Burner Housing Temperature
¦ Air-Fuel ratio: 28 (62% excess air)
Buriier Housing Wall Outer Temperatu re
lUtoJ
J
Burner Particle Injection System
iuIq
J
Diffusion burner will produce
re
I ftM
&
(soot) narioparticles.
Use short and narrow tube to
limit residence time.
HEPA Filter ensures only
injected particles will be
the sample aerosol. ———•
Different orifice insert diameters will be used to
simulate different leak sizes.
Assume the pressure inside of pipe/reactor is not
affected by the leak.
.uloj
Nanoparticle Fate Future Work
Experimentally and numerically investigate fate of
nanoparticles upon release into wind tunnel, using
burner setup.
o Use burner with housing to produce each test aerosol (Ti02,
Si02, and soot),
o Conduct burner produced aerosol tests in wind tunnel with
injection system.
Develop dilution set to possibly be used at sampling
location.
Study the effect of background particles on
nanoparticle fate.
Numerically model the fate of nanoparticles at IUTA
for a more complete understanding of the coagulation
and dispersion processes with high spatial resolution.
Acknowledgements
The Nanoparticle Fate project is sponsored by NSF
(NSF G2006-Star-F2 Fate and Transport)
.ulo,
4
iBurner Particle Characterization
Distribution and TEM images of soot particles
-------
Summary of Dispersion Studies
CNP toxicity may be dependent on size, size
distribution,aggregation, shape, surface chemistry,
surface area and surface charge
All of these properties could be affected by suspension
media
Can not predict optimal media for any one particle since
chemistry will be a factor
Variations in literature can in part be explained by
sources and dispersion
Overall: presence of lipids or proteins resulted in smaller
aggregates
Buford MC et al Particle Fibre Toxicol 2007
Andrij Holian, Raymond Hamilton, Nick Wu2, Dale
Porter3, Krishnan Sriram3 and Mary Buford
The University of Montana
Department of Biomedical and Pharmaeeutical Sciences
Center for Environmental Health Sciences
2The University of West Virginia
3NIOSH
NIH-ES015497
NSF-CBET-0834233
Fun with Carbon and Ti02
Nanoparticles
ifor fur
aental
-HfALTII SCIENCE?
Cytotoxicity of CNP: AM from Balb/c
A) MTS cell viability/
proliferation assay @48 hr
(N=10)
¦ Only MWNT had effects at
high cone, no effects at 4
or 24 hr
BMDM observed
proliferation
B) TUN EL assay for
apoptosis @24 hr (N=5)
¦ 200 |jg of each
E|
i
E
* P < 0.05
Hamilton RF et al., J Nanotox 2007
Balb/c mouse lung histology (H&E) following
instillation of 250 |jg SWNT (SES)
A-F 24 hrs, G&H 7 days
A, C, E, G PBS
B, D, F, H 100% FCS
A&B vehicle control
10Ox except E&F 200x
More effective dispersion
resulted in more distinct
areas of inflammation
A PC Assay
• 1X105 macrophages C57BI/6 or Balb/c
¦ 1hr@37° C mixing with particles
¦ 3hr in 96-well plate with OVA (10 mg/ml)
• 4x105 CD4+ T cells OT-II or D011.10
¦ Supernatants and/or T cells collected at 48hr
• Mac supernatants collected at 24hr
• Supernatants frozen until assayed by ELISA or Luminex
1
-------
CNP (200 ng ml) effects on AM cytokines in
presence of OVA antigen stimulation (24 lirs)
r*n fi
i-1
No effect w/o OVA Hamilton RF et al., J Nanotox 2007
SEM of Ti02 Nanospheres and
Nanowires
Nanoparticles generated by Dr. Nick Wu
Anatase crystal structure
~K$ry ki*
Hamilton RF et al.. J Nanotox 2007
Toxicity of Ti02 Nanowires (4 hr)
C57B/6 AM
A po ptosis
£
7 100-
100 150 200 250
Concentration (pg/ml)
50 100 150 200 250
Concentration (Mg/ml)
) nm X 22 microns
Comparison of Various Ti02 NP
small TiOj MS (20 nm|
small TiOj NT (20 nm)
large TiOjNS (80 nm)
- snort Ti02 NW(<5pra|
150 200 250
concentration (yg/mf)
A po ptosis
SO 100 150 200 250
concentration (ygiml)
Effect of Ti02 NP on APC activity
IL-13 Production
TtOj NS TtOj WW-1 TiQ, HW 1
Particle (100
T
rp"n"
Pa mete (100|io*mJj
2
-------
Role of scavenger receptors
^ 04
i
i
2
~
I
> Scavenger receptors (SR) - eight classes A-H
> SR-A family- SRAI, SRAII, SRAIII , SRCL, SCARA5 and MARCO
> SRA (l/ll) and MARCO are implicated in binding of environmental particles
(negatively charged) and subsequent signaling
> MARCO primary SR in murine models (Hamilton RF et al J Biol Chem 2006)
and SRCR region primary binding site (Thakur SA et al Toxicol Sci 2008)
Murphy, J. E et al, Atherosclerosis, 2005
Role of MARCO in Ti02 NW Toxicity
s
a
I"
0 25-
s
u
*
I IC57 wild typ*
czimarco j
B
control TiOjNS TiOjNW
Panicle (100 porml)
T
'Ol TiOjNS TIOjWV
Particle (100 iiff'ml)
Control
3
-------
Crocidolite
MARCO-/;
Toxicity of NP in Macrophage Cell
Lines?
MM-S (MTS ««*«y • 24hr»)
RAW I MTS aisiy - 24hr*>
THP-1 (MTS ituy ¦ 24hr%)
Uillll mini Jlllill
/////// /////.'/ /////-'¦'
None of the macrophage cell lines expressed MARCO
Conducting 2D-Gel/MS analysis of membrane proteins
Membrane oxidation by Ti02 NP
AM incubated with BODIPY
581/591
Nonpolarand electrically
neutral, inserts into membrane
Shifts from red to green
fluorescence up peroxidation
All forms of Ti02 NP were
effective
Therefore, peroxidation not
central to toxicity
Summary
conventional in vitro assays
Dispersion medium affects outcome for CNP
~
~
Shape of Ti02 NP important determinant of toxicity
~
¦ Long NW > Short NW:a> Nanospheres (In vivo identical)
MARCO important receptor for NP
~
~
MARCO not involved in long NW toxicity
RedOx probably not involved in mechanism of NW
toxicity
~
No unique changes in intracellular ROS
4
-------
flSU
Biological Fate & Electron
Microscopy Detection of NPs
During Wastewater Treatment
Paul Westerhoff
Bruce Rittmann
Terry Alford
Ayla Kiser, Yifei Wang, Troy Benn
November 2008
ASH
Project Goal
Goal: to quantify interactions between
manufactured NPs and WW biosolids:
Develop mechanistic models for NP removal in
WWTPs
We hypothesize that dense bacterial populations
at WWTPs should effectively remove NPs from
sewage, concentrate NPs into biosolids and/or
possibly biotransform NPs.
The relatively low NP concentrations in sewage
should have negligible impact on the WWTPs
biological activity or performance.
HSU
What is Wastewater Biomass
Active bacteria
*
Inert or residual :.[r..1i:
biomass ^
Extracellular ] Jf'
' Tmn— Nuciear a"3
-------
Activated Sludge Wastewater ^
Treatment Process
Consider Ti02 which is already in ^
widespread use in products
Ti02 particulates (200 nm) are used in foods and
have no toxicological or adverse health effects
(Lomer et al., 2000)
Ti02 is insoluble and chemically inert
TiOjIevel range in foods is up to 0.782% (<1 to >200
mg Ti02 per portion size):
Marshmallows, salad dressing, white chocolate, candies,
non-dairy creamer, icing
Average daily intake of TiO, estimated at 5.4 mg/day
(Ministry of Agriculture, Fisneries and Food, 1993)
Where does all the Ti02 in these products used in
society end up?
Ti02 may be a good SENTINEL nanomateriai
PSu
Wastewater Treatment Plant
Sampling of liquids & biosolids
(Mesa, Arizona)
Titanium removal in full-scale Particulate removal
dominates over removal of < 0.7 um titanium
Norifiltered Samples and Filtrate of
11:XX WWTP Effluent
135
1
¦ Nr.ii! UP'*) |
| i Fi trate |
I
fj
22 20 17
IftOhMilks m.-iy FHIii-iiI
Smniitey Ttrli«/£ir«enl
tffluent
Most Titanium ends up in
biosolids
ASU
WWTP
ID
Metals in biosolids
(ug/mg TSS)
Ag(x10-3)
71
Fe
A
12
2.04
36.5
B
11.7
2.94
69.5
C
36.4
3.27
19.3
D
3.6+/-1.4
1.78+/-0.02
4.9
E
38
2.42
35.0
F
14.7
2.72
16.8
G
31
6.39
130
Ti in Biosolids
Primary Aeration
Solids Basin
Micro-Scale TiO?
Oxidized away biosolids in organics with hydrogen peroxide
HoOo does not affect titanium dioxide
Na no-Scale Ti02
2
-------
ASU
Ti02 in commercial products are similar to
Ti02 extracted from biosolids
V
Ti02 in Toothpaste
Ti02 in Biosolids
Titanium in soil particles are not
pure Ti02
ASH
ASU
nC60 Fullerene and Biosolids
Full-scale WWTP Survey:
Biosolids contain < 50
(jg- C60/g-dry biosolids
Liquid effluent contains <
700 ng-C60/L
C60 partitions to
wastewater biosolids in
laboratory tests
Similar experiments
conducted for other NPs
with / without NOM
100 ,
90 -
CD
80 j
/u ¦¦
o
60
o
c
50
I Mass (ug) ir
I Mass (ug) ir
40
30 i I
20 f |
10
0
95 190 380 475 950 9500
Dry Biomass Added (mg/L)
ASU
Sequencing Batch Reactors
Experiments with
heterotrophs (ongoing with
nitrifiers)
SBR operation:
Aerate for 8 hours
Settle for 2 hour
Manage HRT 2x/day
Manage SRT 1x/6 day
3 reactors for HombiKat Ti02
3 reactors for carboxylated
nano-Ag
NPs fully characterized
Feed solution contained
salts, glutamic acid and
glucose
Measure fate of NPs &
performance of reactor
ASH
COD Removal in each SBR Reactor
7/21 7/26 7/31 8/5 8/10 8/15
Date
Reactors containing biomass & NPs showed no loss of performance due to NPs
COD removal occurred even in NP controls (not initially seeded with biomass)
Mass Balances During
Experiments
3
-------
Functionalized NPs
Poorly Removed
ASU
Exchange Number (2 per day)
Exchange Number (2 per day)
ASU
Summary of Key Points
1. Nanomaterials are present in commercial products and
will be released into sewage systems
2. Biosorption of engineered NMs onto wastewater
biomass will occur
3. Nano-Ag & Ti02 had no effect on heterotrophic activity
in Sequencing Batch Reactors
4. 100% removal of engineered NMs will never occur
5. Functionalized NMs are removed less well than metal
oxides
6. Engineered NMs will be present in wastewater effluents
at < 100 |ig/L levels - but this constitutes ~ 108 NM/mL
that will join the >1010 #/mL of natural nanomaterials
already in our rivers
7. Ti02 may serve as a SENTINEL NM in the environment
that indicates where other NMs will eventually occur.
4
-------
Genomics-based determination of
nanoparticle toxicity:
structure-function analysis
Alan T. Bakalinsky, Oregon State University
collaborators:
Qilin Li, Rice University
Jim Hutchison, University of Oregon
Interagency Environmental Nanotechnology Grantee Workshop
21 Nov 08 Tampa, FL
Overall project goals
• Discover genes that mediate toxicity as a first step
towards elucidating mechanisms of action
• Correlate toxicity with physical/chemical structure
Genetic approach:
makes no assumptions about mechanisms
Principle:
• A mutant with greater sensitivity or resistance
to a nanomaterial is likely to be mutated in a
gene relevant to the biological response to the
material
• Identifying the mutated genes can identify
processes central to toxicity
The mutant screen:
• Choose model organism
• Choose toxicity endpoint
• Determine wild-type response
• Screen for mutants with altered response
• Identify mutated genes
• Rationalize how gene loss leads to altered
response
How can gene loss lead to resistance?
• Impaired uptake
• Lack of activation
• Improper localization
The yeast model
Because so many cellular functions are shared across vast
taxonomic distances, what is true in Saccharomyces
cerevisiae is often true in other species.
Best understood eukaryote, experimentally tractable
>80% of its 6,000 genes characterized
>31% have human homologs
Comprehensive "deletion libraries" available
1
-------
Is nC60 toxic?
Endpoint/ nC^j conc.
Organism
nCgQ prep/size, zeta p
Referen ce
Oxidative damage in
brain tissue
0.5 ppm
Juvenile
large mouth
bass
THF/30-100 nm
Oberdorster et al.,
2004
DNA damage
2.2 ppb for aq; 4.2 ppb
for EtOH
Human
lymphocytes
Aq/178 nm, -13.5 mV
EtOH/122 nm, -31.6
mV
Dhawan et al., 2006
Mortality
1 ppm
Daphnia magna
(crustacean)
Aq
Oberdorster et al.,
2006
No mortality
0.5 ppm or 1 ppm
Fathead
minnow or
Medaka
Aqu
Oberdorster et al.,
2006
No effect in many tests
1 ppm
Soil microbial
community
THF/85 nm
Tong et al., 2007
Mortality
200 ppb
Embryonic
zebrafish
DM SO/300-1100 nm
Usenko et al., 2007
No mortality (post-wash)
24 ppm
D. magna
THF/192 nm, -31.1 mV
Aq/448, -17.8 mV
Spohn et al., 2007
Endpoint/nC60 conc.
Organism
nCgp prep/size
Reference
Growth inhibition at
0.4 ppm in low P min
med
No growth inhibition
in LB at 2.5 ppm
E. coli and 6.
subtilis
THF
Fortner et al., 2005
Growth inhibition at
0.4 ppm in low P min
med & at 2.5 ppm in
min. med + air.
No growth inhibition
in LB at 2.5 ppm or in
min. med without air
E. coli and 6.
subtilis
THF
Lyon et al., 2005
Growth inhibition in
low P min. med
8-10 ppb for THF; 0.1-
1 ppm for Toluene,
Aqu, PVP
B. subtilis
THF/39
Toluene/ ~2
Aq/75
PVP/~2
Lyon et al., 2006
Characterization Methods
Particle size
& Dynamic Light Scattering (DLS)
© Zetasizer Na no ZS (Malvern Instruments)
Surface zeta potential
® Eiectrophoretic measurement
& Zeta PALS (Brookhaven Instruments)
Morphology
Transmission Electron Microscopy (TEM)
©. JEOL-2010 TEM
C60 concentration
'!<$• UV absorbance
© Shimadzu UV-2550 spectrophotometer.
@ Total organic carbon (TOC)
Shimadzu TOC-V^u
m
lLl
Dynamic Light Scattering
-------
Yeast survival assay
Inoculum grown 24 h at 30° at 200 rpm in
YNB, washed 2X in water, resuspended in
water and diluted 10-, 100-, or 1,000-fold
into 100 or 250 pi aliquots of water with or
without 30 ppm nC60 in triplicate.
Cells plated on YEPD in duplicate after 24 h
incubation at 30° at 200 rpm.
E. coli survival assay
Inoculum grown 24 h at 37° at 200 rpm in
reduced phosphate MD, washed 2X in 0.9%
saline, resuspended in 0.9% saline and
diluted 10-, 100-, or 1,000-fold into 100 or
250 pi aliquots of 0.9% saline with or
without 30 ppm nC60, in triplicate.
Cells plated on LB in duplicate after 24 h
incubation at 37° at 200 rpm.
nC60 study: conclusions
• nC60 did not inhibit growth of either E. coli or yeast in
minimal media as assessed by final cell yields.
• nC50 generally had no impact on survival of yeast in water
over 24 h when >105 cells/ml were treated. Survival
decreased modestly when fewer cells were exposed.
• nC60 reduced survival of E. coli significantly over 24 h in 0.9%
saline, particularly at low cell concentration (<105 cells/ml).
• No obvious correlations between size or zeta potential and
cell survival.
Gold Nanoparticles
0.8 nm
11 Au Atoms
10 ligands
Charae
SR SR SR
? • P>* RSH M--*¦,' i?"'5"
|Au-Tl'l'|> »• aWsk
a-" • T1. rs— -sr \
'PPhs ( 5R
PhjP' ® PPh, bK SR
-SR Liaand name
Neutral:
\()H 2,2-mercaptoethoxyethoxyethanol (MEEE)
Cationic:
N.N.N trimethylammoniumethanethiol (TMAT)
Anionic:
hs' s—ONa* 2-mercaptoethanesulfonate (MES)
I
0
From gold triphenylphosphine (AuTPP) nanoparticles
AuNPs synthesized in J. Hutchinson laboratory, University of Oregon. Slide adapted courtesy of R. Tanguay.
TMAT Analogs
The toxicity of several compounds with similar structure to
the Au-TMAT functional group was assessed.
No reduction in survival was observed at functional group
concentrations 2-3X higher than that of the primary Au-
TMAT particle.
Tetramethylammonium Chloride
cr
h3c' sch.
Choline Chloride Tetramethylammonium Iodide
h3c-n-ch3 r
ch3
3
-------
Screen for Au-TMAT-resistant mutants
• 4,800 mutants screened in pools for survival
• 250* putative positive clones isolated
• 42 confirmed in initial re-test
• 12 confirmed in replicated re-test
• 5 candidate clones sequenced
• 4 genes identified: GYL1, DDR48, YMR155w
and YGR207c
*To date, 218 of these 250 have been re-tested
• GYL1
GTPase-activating protein, involved in ER-Golgi vesicle
trafficking, exocytosis, autophagy, ortholog of human RAB6A,
a RAS oncogene family member
• DDR48
DNA damage-responsive protein, has GTPase, ATPase activity,
no human orthologs
• YMR155W
uncharacterized protein, no human orthologs
• YGR207C
uncharacterized protein, no human orthologs
• Yeast gyllA/+ and YMR155wA/+
Heterozygotes exhibit similar drug sensitivities
as assessed by reduced growth fitness in rich
medium.
Hillenmeyer et al., (2008) Science 320:362
Gold NP study: conclusions
None of the three Au NPs reduced yeast cell yields in
minimal medium.
The positively-charged Au-TMAT reduced yeast survival
more than the negatively-charged or neutral Au derivatives.
The reduction in cell survival was reproducible with the
number of cells killed being proportional to mass of AuNP.
An entire yeast deletion library (~4,800 mutants) was
screened for resistance to Au-TMAT.
GYL1, DDR48, YMR155w and YGR207c cause susceptibility.
Additional resistant mutants have yet to be identified.
A hypothesis
Observations/known phenomena:
1. Stationary phase cells are sensitive to Au-TMAT-growingcells are not.
2. Autophagy is a normal and essential response to nutritional starvation in
stationary phase cells.
3. Autophagy involves the turnover of cytoplasm, proteins, organelles by
engulfment within specialized vesicles that fuse with the vacuole
(lysosome).
4. GYL1 plays a role in autophagy.
5. A gyllA mutant is relatively resistant to Au-TMAT.
Hypothesis:
Au-TMAT is toxic because it interferes with a GYL1 -dependent step in
autophagy.
Acknowledgements
• Bakalinskv laboratory, Oregon State University:
• Mark Smith
• Alex Hadduck
• Vihangi Hindagolla
• Matthew Boenzli
• Li laboratory, Rice University:
• Bin Xie
• M. Alexandra Bacalao
• Allison Harris
• James Winkler
• Steven Xu
• Hutchison laboratory. University of Oregon
• John Miller
Funding: EPA-STAR R833325
4
-------
Role of Surface Chemistry in the
Toxicology of Manufactured
Nanoparticles
Prabir K. Dutta
The Ohio State University
Goal: Evaluating how surface
structure of particles influences
their toxicity
• Aluminosilicates
• C particles
Asbestos-related lung diseases
Mineral
Composition
Toxicity
Crocidolite
Na2Fe2m (Fe11, Mg)3Si8022(0H)2
carcinogenic
Amosite
(¥#, Mg)7Sis022(OH)2
carcinogenic
Chrysotile
M83(Si2OsXQH)4
carcinogenic?
Erionite
NaK2MgCaj 5(Al8Si28072)
carcinogenic
Mordenite
Na8(Al8Si40O96)
benign
Erionite Toxicity —why?
Disease
Morphology Durability H202,02~ OH*
¦ Surface structure
¦ Surface reactivity
¦ Develop Biological Correlations
Hypothesis for toxicity : Hydroxyl Radical
Fe(II)
+ H202 -> Fe(III) + OH
* + OH
Need Fe(II) and H202
h2o2
from phagocytosis
Asbestos:
Erionite:
Source of both H202 and Fe(II, III) species.
Source of H202 but no Fe?
Erionite (zeolite): Ion exchanging material
(Erionite)-M+ + Fe(III) —» (Erionite)"Fe(III) + M+
• Acquisition of iron in the lung
• All zeolites should be toxic-this is not the case
• Mordenite is not toxic
1
-------
Zeolite fibers-mordenite and erionite
integrated cnemuuminescence intensity upon
macrophage-particle (NR8383) interaction
Median size
(micrometer)
10
micrograms
50
micrograms
250
micrograms
Mordenite
3.7
na
503±183
649±303
Fractionated
mordenite
1
946.5±139
1846±1134
8382±1855
Erionite
10
na
695±288
1166±589
Fractionated
erionite
3
na
965±361
1110±333
Fine erionite
0.8
na
1904
ha
na: not analyzed
• ROS relatively particle independent
• Smaller particles produce greater oxidative burst
~ Increase loading greater oxidative burst
What about the chemical role?
Fe2+ (surface) + H2Oz^ Fe3+ + OH*+ OH"
- Surface iron loading
- Hydroxyl radical production
Hydroxyl radical generation
7.E-04
6.E-04
5.E-04
4.E-04
3.E-04
2.E-04
1.E-04
0.E+00
mordenite HPLC
mordenite UV Vis
a erionite HPLC
A erionite UV-vis
oY HPLC
• Y UV vis
+fractionated mordenite
1. OH radicals generation increases with surface iron amounts
2. Small hydroxyl radical production compared to amounts of
surface iron (not all iron species are in the right redox state and
environment)
3. Erionite-bound iron> Mordenite-bound iron>zeolite Y-bound iron
Fe(III) + reductant -» Fe(II)
Ascorbic Acid, Glutathione
Hydroxyl Radical Production Relative to Surface iron
Erionite Mordenite
Comparison of hydroxyl radical production in the presence of reductants.
Mutagenesis Experiments
• Cell Line: AS52 cells
• Cells examined forTG resistance clones
(spontaneous mutation frequency =10.5 ± 2.7 TGr/1010 clonabie cells)
2
-------
0 2 4 6 8 10 12 14 16
|j.g fiber/cm
• Effects of mordenite insignificant as compared
to controls
Above 8 |ig fiber/cm2, erionite + Fe2+lincreased
mutation rate
• 16 |ig fiber/cm2 + 20 jiM, 3.3x increase
Erionite Mutagenicity
Surface Structures of Erionite and Mordenite
Mordenite
Erionite
J!-—-™
Sapfe***
ia r -v H-v v—"r;d **
•Coordination environment can modify the iron redox potential
•Chemical reactivity differences result in different biological reactivity
Manufactured C nanoparticles
y Carbon black production: 106 metric tons, 1999 (J. Occup.
Health 2001; 43: 118-128)
"Furnace Black"
~14nm
"Lamp Black"
-lOOnm
http://www.degussa.com/
Ternplated synthesis of carbon-
based model particulates
1 [jm aluminosilicate zeolite particles
carbon particulates
II
i
zeolite Y ~ 1 |jm
HI
i
carbon particulates ~ 1 pm
3
-------
Carbon-iron particulates
(C-Fe)
Template has ion exchange capability.
Fe incorporated into template.
SEM: C-Fe
XPS - surface elemental analysis
inset: iron region
] ICAM-1
| E-selectin
I VCAM-1
JL
L
TNFa DMEM
1
1
i
: C-Fe C-Fe/ DEP CFA
>0) (25) F-Al-Si (25) (100)
(25)
Macrophage Supernatants
Macrophage Treatment
Hydroxyl Radical Production
Fe(III)
+- h2o2
Fe(II) + H02*+ H+
Fe(II) +
H2°2
Fe (III) + OH* + OH
I ^ I
EPR active!!! (1:2:2:1 quartet)
DMPO = 5, 5-dimethylpyroline-N-oxide
1) Valvanidis et. al. Atmospheric Environment 34(2000)2379-2386
TEM
human monocyte-
derived macrophages
4
-------
Materials that release metal ions ?
Solid State
Redox
Released into
environment
Coordination
environment
**^yjn-1+ Mn+ Mn+1
What is the role of n in Mn+?
Role of n+?
• Investigated two compounds
Fe (II) Acetate (OAc) and Fe (II). Fe(lll)F5
• Both compounds form a precipitate in
presence of phosphate buffer
• For Fe (II) OAc : 50% of Fe(ll) precipitated
• For Fe (II). Fe(lll)F5: 100% of Fe(lll) i
precipitated
• So, solution species of both comparable:
Fe(ll)
TNF-a production after 12 hour exposure of
Murine Alveolar macrophages to the two Fe
sources in phosphate buffer
Fe (III) sample more inflammatory
TNF-a production for Murine Alveolar
macrophages treated with the two different
iron phosphate precipitates
Fe(OAc)2 PBS Precipitate
¦ 8 hrs
Fe2F5 PBS Precipitate 0 12 hrs
0 24 hrs
a II
ill
0.6 1.3 2.6 5.1 12.8
[jg Fe/wel!
1.0 2.1 4.4 8.3 20.7
[jg Fe/wel I
Differences arising from the precipitates
Cytotoxicity after 12 hour exposure
100
—m— Fe2F5
80
—~- Fe(OAc)2
o
o
o
60
40
20
—-—" " 3}~P= .016
-p = .0007
2.3
4.6
Fe (|jg/well)
6.9
n = 3
Significant difference between the 2 iron sources
5
-------
• Fe(lll) precipitate more cytotoxic than
Fe(ll)
• Fe(lll) precipitate more inflammatory than
Fe(ll)
Hypothesis : Redox state of the
element released is important
Acknowledgements
• NSF-EMSI
• NIH
Collaborators: W. James Waldman
Marshall Williams
John Long
Students: Estelle Fach
Robert Kristovich
Amber Nagy
Brian Peebles
6
-------
A Rapid In Vivo System for
Determining the Toxicity of
Nanomaterials
Robert Tanguay
Department of Environmental and Molecular Toxicology
Environmental Health Sciences Center
Oregon Nanoscience and Microtechnologies Institute (ONAMI)
- Safer Nanomaterials and Nanomanufacturing Initiative
* osu
{yf^Jjil 11-21-08 - --
The Opportunities
Proactively guide the development of safer
nanomaterials to reduce hazard
• Identify the physicochemical properties that
drive biological responses-take a broader
view
• Think nanoscience - not toxicology
• Develop predictive models from
experimental data.
• Feed the Nanomaterial Biological
Interactions (NBI) knowledgebase
Designing Safer Nanoparticles
Nanepariide
Cora
\ ' 1 Surface
I r\-v > Functional
Ml,ili„n, V J "i '
StKHI
Redesign
Test
Material
Properties
Structure/Property Relationships:
Physicochemical properties and biological responses
Nanoparticles have widely tunable properties - the key is
to enhance performance and safety at the same time.
Nanomaterial
synthesis
/ \
Platforms to Define Nanobiological
Interactions and Responses
• In vitro
- Continuous cell culture system
- Primary cell culture system
- Stem cells
• In vivo - High content studies
-Whole animal studies
• Rodents
• Fish
• Flies
• Worms
....The field is at the discovery phase.
Why do we chose not cultured cells?
Response
Proliferation
Cell death
y Metabolism
Gene expression
Phenotypic change
As a discovery platform ...Too many "blind spots"
Cell cultures -What blind spots?
• Different cell-cell interactions cannot be evaluated
• Indirect effects cannot be evaluated
• Cells in culture can only respond using their unique
repertoire of expressed gene products - limited
potential targets
• Tremendous potential for missed data - missed
opportunities
In vivo systems may offer significant advantages
if amenable to efficient assessments
-------
Why evaluate responses during
early embryonic development?
• Vertebrate embryonic development is the most complex
biological system.
• Processes of development are remarkably conserved
• Comparative genomics data supports overall conservation
of potential "targets"
• Generally more responsive to insult
• Most dynamic life stage...and the full signaling
repertoire is expressed and active, therefore fewer
blind spots.. Highest potential to detect interactions
• If a chemical or nanomaterial is developmentally toxic it
must influence the activity of a molecular pathway or
process., i.e. hit or influence a "Toxicity Pathway"
• Share many developmental, anatomical, and
physiological characteristics with mammals
• Genome is "completely" sequenced
• Molecular signaling is conserved
• Technical advantages of cell culture - power of in vivo
• Amenable to rapid whole animal mechanistic evaluations
• Hundreds of laboratories are exploiting this model -
shared resources
Consider startpoints - not endpoints
• Signaling pathways and molecular events are
conserved
• ..But fish are not rodents or humans
• Consequences of disrupted signaling often
species specific
• the mechanism by which a "target" is hit is
likely conserved, but the consequence of the
"hit" may be distinct
Tier 1: Toxicity Screening
• Toxicity testing whole organisms
- In vivo - zebrafish
Tier 2: Cellular Targets and
Distribution
• Defined in vivo
- Fluorescent nanomaterials
- Targeted assays
Assessing Biological - Nanomaterials
Interactions and responses
Tier 3: Molecular Expression
• Genomic Responses
- Whole animal gene expression
profiles
^ Structure Activity Relationships
Feed data back into design scheme
Tier 1 Testing
X
1
Multi-well plates
Purified well characterized
Forms actually in use
Aged- i.e. environmentally
Screening for responses 1-5 days
Assay Considerations
• The goal is to investigate
interactions and responses.
* Embryonic development serves as a
"biological sensor and amplifier"
• These are "forced" interactions!
Remove chorion "potential
barrier"
HAZARD Identification, not risk
assessment!
-------
Alternate Exposure Route
- Microinjection
1 cell
stage
A<*>)
24 hpf
-a
Development Stages of Assessments
€>_
3 min
319
. 4hr \ J
1. 25hr \ I
*
6 hr
4n
19 hr
24hr
jmLK> '""48
120 hr
hr
High Content Tier 1 Endpoints
(Assessed between 24 and 120 hpf)
Morphological
Malformations
i.e. pericardial edema, yolk sac edema, body axis
fin malformations, eye diameter
Circulation
Heart beat (rate)
Developmental progression
Embryo viability
Behavioral
spontaneous movement (18-24 hpf) onset and
frequency
touch response (27 hpf)
motility
Nanoparticles Assessed - to Date
Over 200 fully evaluated through tier 1.
C60, C60(OH)24, C70, SWCNT, DWCNT, dendrimers, metal
oxides, Q-dots, gold nanoparticles, viral derived
• Gold nanoparticles
• Fullerenes
Toxic Potential
Size and Surface Functionalization
C70 Concentration (ppb)
Toxic Potential
Size and Surface Functionalization
-------
Toxic Potential
Size and Surface Functionalization
C60(OH)24 Concentration (ppm)
C60 Exposures Increases Cellular Death
Acridine Orange - In vivo assessment
Control
i0£LH2b
W
200 ppb
Cell Death Head
0 800-.
Cffl Concentration
C60-Induced Cell Death
Total Cell Death
Apoptosis
o 000
MM J
\TtZ\ I
<8 910
O
Jf
>
/A
0; ™
a; 50
CH
C60 Concentration
Control SOppb 100ppb 200ppb
C60 Concentration
Acridine Orange
TUNEL
Light Exposure Increases C60 Toxicity
Concentration (ppto)
Cmdaifc
Oxidative Stress Response (Tier 2)
C60
\
| Oxidative Stress? |
Protein
p Gpnp
Damage/
Dysfunction
+ Depletion v
/ (i.e. GSH) X
Expression
s. Changes
Lipid peroxidation
Cell Death
GSH Precursor -NAC Offers Partial Protection
-------
The Antioxidant Ascorbic Acid
Offers Partial Protection
Chemical Depletion of Glutathione
Embryos Are More Sensitive to Ceo
100
_o- BSO * C* /
-*—DEM*Cm /
* 80
/ /
¦e 60
/ /
5
/ /I
^ 40
/ / /
20
0 ppb 50ppt> 100 ppt) 200 ppb
C,^ Concentration
Oxidative Environment Impacts
In vivo Cellular Death Response
Determining Nanomaterial Dose
• Defining Dose is challenging - regardless of the
platform
- Numerous obstacles
Agglomeration parameters in aqueous
media unknown
Uptake and distribution unknown
Few labeled materials
Must define dose for comparative studies
Ceo Dose Determination
• Goal: to develop a method for detecting and
quantifying C60 associated with biological and
aqueous samples.
• Analytical quantification of C60 using LC-MS
(Collaboration with Dr. Carl Isaacson and Dr.
Jennifer Field -OSU EMT)
• Pooled 100 embryos per replicate
• Use of 13C-labeled C60 surrogate to calculate losses
during extraction method.
Water Concentration Declines Over Time
C60 Embryo Water
0 2 4 6 8 10 12 14
Hours of Exposure
-------
C60 Dose Determination
C60 Mass in Embryos
0.5
0.4.
0.1
0.0
0 2 4 6 8 10 12 14
Hours of Exposure
The C60 LD50 in embryonic zebrafish is 0.1 ng/mg.
Global Gene Expression (Tier 3)
• Zebrafish oligo arrays used to evaluate gene
expression changes following C60 exposure
(>14,000 genes)
• 200 ppb C60 and 1% DMSO controls
• Expression evaluated at 12 & 24 hrs post
exposure
Embryonic Stress Response - Q-RT-PCR
36 hpf
Hsp70
Ferritin Heavy Chain
Conclusions
• Cannot predict biological responses without data.
• Many advantage by evaluating
interactions/responses in vivo
- multiple levels of organization
• Zebrafish: a discovery platform to define
nanomaterial/biological Interactions from
diverse sources
• Opportunities to define structure response
relationships
• Extremely well-suited for whole animal
mechanistic studies.
$5 :'.\NU ONAMI (@)
SAff ft NAMQMATEftlALS and nahqwanufacturwg MTIATYVE
Acknowledgements
• Dr. Stacey Harper
• Crystal Usenko
• Lisa Truong
• Kate Saili
• Dr. Jennifer Field
• Dr. Carl Isaacson
• Oregon State Radiation Center
•Air Force Research Laboratory, AFRL - FA8650-05-1 -5041
•NIEHS P30 Environmental Health and Sciences Center
•NIEHS T32 Toxicology Training Grant
-------
Why QDs?
Quantum Dot Toxicity in Zebrafish
Greg Mayer - Texas Tech University
Jay Nadeau - McGill University
Anja Nohe - University of Delaware
•Emission wavelength is related to the size
of the crystal
•Slow to photobleach and radiation
resistant
•Emission can be quenched/modulated by
attaching electron donors or acceptors to
the surface
•Can be suspended in aqueous and non-
aqueous environments
•Many colors obtained with a single UV
excitation source
•Surface can be conjugated to chemically
and biologically important molecules
QD Synth es is /So 1 u b i 1 izati o n
CdSe/ZnS core-shell
Synthesis via a two-step, single flask method.
- Injection of Selenium precursor into hot coordinating solvent
containing the cadmium precursor, CdO.
- Leads to nucleation and growth of particles
- Injection of Zn and S solutions arrests growth, forms cap around
particles.
Water solubilization is done by TOPO cap exchange with thiol
mercaptosuccinic acid (MSA) or mercaptoacetic acid (MAA)
- Reflux in methanol for 6 hours
- Yields water-soluble particles
Objectives of Investigation
• Compare molecular responses elicited by
organism from exposure to heavy metals and
semiconductor nanoparticles
• Determine how semiconductor nanoparticles
facilitate resulting cytotoxicity
ZM9 Strain
_ •—
Control 6dpf
50|jM Zn 6dpf
„ „ „ „ Ocean pout antifreeze
Carp Mean Mc-QFP £otein
"Insulator" terminator
"Enhancer "Enhancer
element" element
C 3
1
-------
MTF-1 Knockdown Model
Determine extent of MTF-1 knockdown
in wild type
Observe subsequent MT reduction in wild
type
Knockdown MTF-1 in transgenic model
and monitor heavy metal response
Morpholino Design
Exon 2/Intron 2
Splice-blocking
antisense morpholino
Intron 1 Exon 2 Intron 2 Exon 3
Resulting Non-Functional
Transcript
MTF-1 Target Genes
• Metallothionein (MT)
- Heavy metal and free
radical scavenger
- Well-conserved
- Increases with
elevated group I-IIB
heavy metal load
. AO
2
-------
Other Group IB and IIB Metals
mill
f noil
H nailII 1
8 8I""),
1 *
i: nnn(I
Mercuric
Chloride
Silver
Nitrate
Average
S:E.
13.058 mM
+/- 2.179
254.429 nM
+/-5.961
1.240 mM
+/-0.103
Quantum Dot Accumulation In
Zebrafish Embryo embryo uptake
~45 minutes post fertilization
40 Minutes
1 &r40 Minutes
2 hrs 40 Minutes
3
-------
embryo uptake
~2 hrs. post fertilization
Quantum Dot Interaction With
Zebrafish Liver Cells
10nM Green QD
for 24 hr
Membrane Stain
w/ BODIPY
ceramide
Cellular T oxicity
ll.
Quantum Dot Toxicity
Primary reasoning
- Heavy metal liberation
- Free radical generation -> oxidative stress
- Membrane damage/disruption
-------
Heavy Metal Chelation
Free Radical Elimination
Endocytic/Clathrin Inhibitors
Conclusions
Similar results with
amantidine and
Cytochalasin D
No significant difference
observed with inhibition
of calveolin-mediated or
clathrin-mediated uptake
No alteration of toxicity
with suppressed active
uptake mechanisms
Semiconductor nanoparticles accumulate in zebrafish
embryos
- Potentially damage hepatic system
Bind to cellular membrane
Do not enter cell through clathrin-dependent
endocytosis
Diameter correlates with overall toxicity
Toxicity not induced by heavy metal release or free
radical generation
Degrade and liberate free heavy metal ions ?
Acknowledgements
Adam Johnston
Emily Schaab
Lindsay Nadeau
Dr. Jay Nadeau
Samuel Clarke
Dr. Anja Nohe
Jeremy Boner
TTia '••••rch It funded by
r US- EPA - Sek«nc« To Achfev*
Rosults (STAR)Program
Grant GEESEES
5
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2008 Interagency Environmental Nanotechnology Grantees Workshop
U.S. Environmental Protection Agency
Interagency Environmental Nanotechnology Grantees Workshop
Sheraton Tampa Riverwalk Hotel
Tampa, FL
November 19 - 21, 2008
EXECUTIVE SUMMARY
November 19,2008
INTRODUCTION AND OVERVIEW
The 2008 Interagency Environmental Nanotechnology Grantees Workshop was held November 19-21,
2008, in Tampa, Florida, and was hosted by the U.S. Environmental Protection Agency (EPA), Office of
Research and Development (ORD), National Center for Environmental Research (NCER). The workshop
brought together research grantees funded by the EPA Science To Achieve Results (STAR) Program, the
National Science Foundation (NSF), the National Institute of Environmental Health Sciences (NIEHS),
and the National Institute for Occupational Safety and Health (NIOSH). Grantees discussed the latest
science regarding the potential effects of engineered nanomaterials (ENMs) on human health and the
environment. Additional talks were given by federal agency program officials. The goal of the workshop
was to stimulate communication and collaboration among scientists and engineers investigating the
potential implications of ENMs. Approximately 100 participants attended the workshop.
Welcome
Nora Savage, EPA, NCER
Dr. Nora Savage welcomed participants to the meeting and provided background about her job and
colleagues at NCER, within EPA's ORD. She reviewed the agenda for the meeting, noting some changes.
She explained the logistics of the meeting and introduced the contractor staff, including individuals from
The Scientific Consulting Group, Inc. (SCG). She encouraged participants to complete the meeting
evaluation form and return it to SCG staff; EPA would like input about future co-location of this meeting
with the Society of Environmental Toxicology and Chemistry (SETAC) Annual Meeting or other
professional society meetings. She introduced Mr. Christopher Zarba, the Deputy Director of NCER.
Sponsored Research at U.S. EPA NCER
Christopher Zarba, EPA, NCER
This year, as in the past 5 years, nanotechnology is the number one research priority. The area of
nanotechnology receives most of the funding, which illustrates how important this issue is to EPA. The
customers assist in writing the Requests for Applications (RFAs), and the proposals received in response
to the RFA are reviewed and ranked by an external peer review panel. Only those proposals that receive
excellent or very good scores move on to the next level. Customers select and prioritize proposals, with
approximately 10 to 20 percent of proposals funded. Scientists that receive EPA STAR funding are the
best and brightest, working on world-class environmental issues.
There are approximately 1,800 employees in ORD. ORD's budget in the 2009 President's Budget is
$54.1 million, which has not changed much in the last 12 years. There are 13 laboratories and research
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2008 Interagency Environmental Nanotechnology Grantees Workshop
facilities around the country. ORD's mission is to give their customers the scientific information they
need to write regulations and to set policies. The requests for research are about 10-fold more than the
available resources. National Program Directors (NPDs) are independent scientists who report to the
Assistant Administrator. They look at both extramural and intramural research being conducted in their
program areas. Since the creation of the NPDs, there has been an increasing emphasis on the use of STAR
grants, particularly for new and emerging programs. The Agency is developing a Nanomaterial Research
Strategy (NRS). This document covers broad themes and general approaches for extramural and in-house
nanotechnology research. ORD has identified four key research themes and seven key scientific questions
where ORD can provide leadership for the federal government research programs and support the science
needs of the Agency. The NRS should be available within 2-3 months. There is a possibility that an NPD
will be assigned for nanotechnology.
Established in 1995, the STAR Program is the extramural funding arm of EPA's ORD. There is
significant Agency and cross-agency involvement in the solicitation writing and review of proposals and
all solicitations are competitive. The STAR Program awards about $66-100 million annually and
currently is managing about 800 active research grants and fellowships. About 25 RFAs are issued each
year. Each year the STAR Program receives 3,000 grant applications and makes about 200 new STAR
awards. EPA tries to collaborate with other agencies; nanotechnology is a good example as EPA has
collaborations with the NSF, NIEHS, NIOSH, and the Department of Energy (DOE).
EPA is interested in nanoscale materials for a number of reasons, including the following: (1) the unique
chemical properties of nanoscale materials makes traditional risk management techniques and regulations
unsuitable in many situations; (2) these materials have potential environmental applications, such as
cleaning up past environmental problems, improving present processes, and preventing future
environmental problems; (3) the Agency has regulatory responsibilities because these products are in the
marketplace and may pose risks to human health, the environment, or both; and (4) opportunities exist to
maximize the environmental benefits and minimize impacts from the beginning, as new technologies are
developed. Specific areas of interest for the STAR Program in nanotechnology include research on
implications (e.g., potential toxicity; potential exposure; fate, transport, and transformation; and
bioavailability and bioaccumulation) and applications (e.g., pollution remediation and treatment, pollutant
or microbe monitoring and detection, and the development of environmentally benign processes for
pollution prevention).
The nanotechnology program was initiated in 2002 with $5 million. The STAR Program began by
funding exploratory research, primarily on applications of nanotechnology, in 2001; the program shifted
to exploratory research on the implications of nanotechnology in 2003. EPA's Small Business Innovation
Research (SBIR) Program also has solicited research on nanotechnology. The goal of the SBIR Program
is to bring new, innovative environmental technologies to market. In the STAR Program, grants can be
converted into cooperative agreements. This funding mechanism allows researchers within ORD to work
more collaboratively with STAR grantees. EPA and NSF have made awards to establish two Centers for
the Environmental Implications of Nanotechnology (CEIN). The centers, led by the University of
California, Los Angeles (UCLA) and Duke University, will study how nanomaterials interact with the
environment and with living systems, and will translate this knowledge into risk assessment and
mitigation strategies useful in the development of nanotechnology.
Discussion
A participant asked Mr. Zarba to describe EPA's customers. Mr. Zarba responded that their customers are
the EPA program offices (e.g., Office of Air, Office of Water) which write regulations, set Agency policy,
write criteria, etc., and need the research conducted to support their work.
The Office of Research and Development's National Center for Environmental Research 2
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2008 Interagency Environmental Nanotechnology Grantees Workshop
National Science Foundation (NSF)
Mihail (Mike) Roco
Since 2000, nano science and engineering has expanded to many disciplines and approximately $14
billion is spent worldwide on nanotechnology research and development. Nanotechnology is working at
the atomic, molecular, and supramolecular levels, in the length scale of approximately 1—100 nm range, to
understand and create materials, devices, and systems with fundamentally new properties and functions
because of their small structure. The definition encourages the following new contributions that were not
possible before: (1) understanding and exploitation of novel phenomena, properties, and functions at
nanoscale, which are nonscalable outside of the nanomaterial domain; (2) the ability to
measure/control/manipulate matter at the nanoscale to change those properties and functions; and (3)
integration along length scales and fields of application. A timeline was developed for the four
generations of nanotechnology products and processes by considering the beginning of industrial
prototyping and nanotechnology commercialization. The first generation products (2000-2004) were
passive nanostructures, such as nanostructured coatings, nanoparticles (NPs), nanostructured metals,
polymers, and ceramics. The second generation products (2005-2009) include active nanostructures such
as 3-D transistors, amplifiers, targeted drugs, actuators, and adaptive structures. Third generation products
(2010-2015) will be nanosystems such as guided assembly, 3-D networking, new hierarchical
architectures, and robotics. The fourth generation (after 2015) will include molecular nanosystems such as
molecular devices "by design," atomic design, and systems with emerging behavior.
NSF supports 26 large research and education centers on nanotechnology and two user facilities.
Currently, there are 4,000 active research awards, and approximately 10,000 students and teachers are
trained each year. The current year's nano budget at NSF is approximately $400 million. NSF spends
about 7 percent ($28 million) of its nanotechnology budget on environmental health and safety concerns
through single investigator projects, small groups, and centers. Collaborations and partnering are
important to NSF. NSF has had a number of program collaborations with EPA as well interactions with
the National Institutes of Health (NIH), DOE, NIOSH, and other agencies. In 2007, NSF collaborated
with EPA and DOE on a solicitation that focused on exposure from manufactured nanomaterials. The
collaborations and partnerships for the nano centers, networks, and user facilities were described.
Both immediate and continuing societal implications issues as well as long-term concerns must be
addressed earlier in research programs. An anticipatory and corrective approach that is both transforming
and responsible in addressing societal implications for each major nanotechnology research and
development program from the beginning is needed. Risk governance of nanotechnology is becoming
increasingly important at the national and international levels.
National Institute for Occupational Safety and Health
William (Allen) Robison, NIOSH
Dr. W. Allen Robison explained that NIOSH is small institute within the Centers for Disease Control and
Prevention (CDC) with an overall annual extramural budget of $82 million. The purposes of NIOSH's
Nanotechnology Program are to: (1) increase knowledge of nanotechnology and manufactured
nanomaterials, (2) examine the occupational safety and health aspects of nanotechnology, and (3)
examine application and implications of nanotechnology. The program complements the intramural
program. Since 2001, NIOSH has used R01, R03, and R43/44 funding mechanisms to fund
nanotechnology projects. NIOSH utilizes program announcements and has participated in joint RFAs
with EPA, NSF, and the National Institute of Environmental Health Sciences (NIEHS). Dr. Robison
highlighted the annual extramural funding amounts since 2001; the largest annual amount was $1.46
million in 2005. Funding for the current year is $800,000 and approximately $5 million has been granted
in external funding since 2001. In 2008, R01, R03, and R44 funding mechanisms were used to fund 13
The Office of Research and Development's National Center for Environmental Research 3
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2008 Interagency Environmental Nanotechnology Grantees Workshop
projects that deal with a variety of topics including sensors for portable monitors, lung oxidative stress
and inflammation, and toxicity of inhaled NPs. The extramural process is a competitive, peer-reviewed
process; proposals must be relevant to occupational safety and health. There is an emphasis on research to
practice (i.e., show how research can be used to improve the workplace). More information regarding
nanotechnology research can be found in the 2007 NIOSH report, Progress Toward Safe Nanotechnology
in the Workplace, on the NIOSH Web Site at http://www.cdc.gov/niosh/topics/nanotech, and in the
NIOSH online Nanoparticle Information Library at http://www2a.cdc.gov/niosh-nil/index.asp.
National Institute of Environmental Health Sciences Activities on Nanotechnology:
Applications and Implications
Srikanth Nadadur, NIEHS
Each of the NIH's 26 institutes and centers has a nanotechnology research program. NIH created an
intramural nano task force comprised of representatives from each of the 26 institutes and centers to work
with extramural experts to identify priority research areas for nanomedicine and health. Some of the
critical research areas include: nano delivery systems; bioimaging and informatics; organ-tissue
nanoengineering; medical devices; biocompatibility and toxicity; and environmental health and safety.
NIEHS is solely responsible for developing research programs to evaluate the environmental health
implications and safety of nanomaterials. The creation of the National Nanotechnology Initiative in 2001
helped spur an increase in funding for nanotechnology research. Last year, NIH spent approximately $200
million on nanotechnology research and approximately $30 million of that total was spent on nano
environmental health safety research.
For the study of health implications, NIEHS' work includes both basic and exposure research. Exposure
research is focused on determining routes of exposure and systemic distribution, correlating physical and
chemical characteristics of ENMs with biological response, identifying biomarkers of exposure and
biological response, and developing models to evaluate and predict biological response. Basic research
includes projects studying the interaction of ENMs with biomolecules; studying transmembrane transport,
cellular uptake, subcellular localization and retention; identifying cell- and organ-specific toxicity
response pathways; and studying the effects of structural and surface modifications.
There are three research programs within NIEHS: extramural, intramural, and the National Toxicology
Program (NTP). Extramural research is funded by NIEHS through the Division of Extramural Research
and Training in three areas: (1) nanotechnology-health implications; (2) nanotechnology-based
applications; and (3) remediation devices. Health implications research ranges from efforts to understand
basic interactions between nanomaterials and biological systems to organ-specific toxicity. Research in
enabling technologies addresses the applications of nanotechnology, including the development of: (1)
deployable environmental sensors for a broad range of environmental exposures; (2) biological sensors to
link exposure with disease etiology; and (3) intervention devices, such as drug delivery devices and other
therapeutic nanoscale materials. Remediation devices include nanotechnology-based devices for the
superfund research program aimed at eliminating exposure. Researchers in the Division of Intramural
Research (DIR), such as those in the NTP, investigate the applications of nanotechnology and
characterize nanomaterials. Materials characterized by the NTP are available to researchers for
collaborative efforts. DIR investigator-initiated research addresses the application of nanotechnology in
the areas of environment, health, and safety. The NTP's areas of emphasis include: (1) exposure and dose
metrics; (2) internal dose-pharmacokinetics in biological systems; (3) early biological effects and altered
structure or function; and (4) adverse effects related to exposure to nanomaterials. The scientific focus of
the NTP Nanotechnology Safety Initiative is to identify key physical-chemical features that govern
nanomaterial safety. Materials currently under evaluation by NTP include quantum dots (QDs), titanium
dioxide (Ti02), carbon fullerenes, nanoscale silver, multi-walled carbon nanotubes (MWCNTs),
nanoscale gold, and dendrimers.
The Office of Research and Development's National Center for Environmental Research 4
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2008 Interagency Environmental Nanotechnology Grantees Workshop
Discussion
A participant asked which study sections at NIH focus on the issues discussed. Dr. Nadadur said that
there is a standing study section named NANO that reviews research in the areas of nanotechnology, and
there is also a new special emphasis panel, Systemic Injury to Environmental Exposures (SIEE) that has
the required expertise to review grant proposals on nano environmental health safety.
Department of Energy Nanoscale Science Research Centers (NSRCs): User Facilities for the
Scientific Community
Neal D. Shinn, Sandia National Laboratories
Dr. Neal Shinn is affiliated with one of the DOE NSRCs and presented information on each center and
how each may benefit researchers. The five NSRCs, located across the United States and opened between
2006 and 2008, are research facilities for the synthesis, processing, analysis, and characterization of
nanoscale materials. They provide specialized equipment, unique tools, and dedicated support and
scientific staff. The NSRCs are operated as user facilities and are available to all researchers, with access
determined through peer review of proposals. There is no user fee for nonproprietary work leading to
publication; federal law, however, requires that costs be recovered for proprietary work. All NSRCs are
co-located at DOE National Laboratories with existing major user facilities, including synchrotron
radiation light sources, neutron scattering facilities, and other specialized facilities. Although most
NSRCs offer similar expertise, some have unique capabilities and expertise. The expectation for the
NSRCs is that they help foster impactful science and create a community of successful users. This is
reflected in metrics such as publications, citations, size of the user population, and so on.
The Center for Nanophase Materials Sciences is located at the Oak Ridge National Laboratory and has a
variety of research capabilities. The Laboratory has world-class capabilities in polymer synthesis,
computation and visualization, and computational nanotoxicology, which determines the environmental
impacts of nanomaterials. The Molecular Foundry is located at Lawrence Berkeley National Laboratory
and includes six facilities, with a principal scientist for each facility and a team of scientists working
within each facility. The Center for Nanoscale Materials is located at the Argonne National Laboratory
and is working on six integrated scientific themes, including "nanobio" interfaces, nanophotonics, theory
and modeling, X-ray microscopy, nanofabrication and devices, and electronic and magnetic materials and
devices. The Center for Integrated Nanotechnologies is a partnership between Sandia National
Laboratories and Los Alamos National Laboratory. It is focused on the integration of nanostructured
materials to exploit their special properties and the need to move nanosystems into real-world
applications. In its two facilities, the Center for Integrated Nanotechnologies has the capabilities for
synthesis, characterization, and integration and has four science thrusts: (1) nanophotonics and optical
nanomaterials; (2) nanoscale electronics and mechanics; (3) soft, biological, and composite
nanomaterials; and (4) theory and simulation of nanoscale phenomena. The Center for Functional
Nanomaterials is located at the Brookhaven National Laboratory and has five scientific themes: (1)
nanocatalysis; (2) electronic nanomaterials; (3) soft and biological nanomaterials; (4) electron
microscopy; and (5) theory and computation. Its focus is on energy applications (e.g., functional
nanomaterials for exploiting renewable energy sources, energy storage, and utilization).
The role of the NSRCs is to make specialized capabilities and expertise available to outside researchers,
and the DOE looks to the centers for technical input with respect to the developing area of engineered
nanomaterials safety. Dr. Shinn explained that operational policies currently are being crafted, and he
invited participants to be involved and have an impact on how DOE sets policy. The five NSRCs have
received approximately 800 to 1,000 user proposals and have had more than 1,000 researchers working at
the centers. There are semi-annual calls for proposals, with other mechanisms for brief access for time-
sensitive projects. Historically, there is a 55 to 93 percent likelihood of a proposal being accepted. If a
The Office of Research and Development's National Center for Environmental Research 5
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2008 Interagency Environmental Nanotechnology Grantees Workshop
proposal is rejected, in most cases feedback is provided and the researcher is encouraged to resubmit in
the next cycle. Proposals are first evaluated for feasibility and then peer-reviewed for scientific quality
and expected impact. Each proposal must include a statement of work that is reviewed by an external
panel that assesses what is clear and achievable. Projects can be 1 day to 1 year, but it must be clear what
the researcher would like to accomplish. The researcher's institution must sign an agreement that allows
the researcher to work at the NSRCs and publish. The centers are in place to help make researchers' work
successful.
Discussion
A participant stated that students that work at Argonne National Laboratory are required to complete
extensive safety training. What type of safety training is in place through this program? Dr. Shinn
responded that all users must complete safety training, but the specific training would depend on the
project.
A participant noted that early on, the environmental science community had a difficult time receiving
high rankings in proposals because review panels did not understand the science and asked whether the
DOE has considered how it is populating its review panels. Dr. Shinn responded that all of the centers list
their reviewers on their Web sites; if researchers find that they are lacking in expertise, they are
encouraged to provide this feedback to the individual center or the DOE. He added that peer-review
judgments are inherently qualitative, and reviewers could have trouble with proposals if they are not well
written.
Metals, Metal Oxides: Remediation and Exposure
National Exposure Research Laboratory (NERL) Nanomaterials Research Program
Michelle Conlon, EPA, NERL
Ms. Michele Conlon discussed the Agency's intramural nanomaterials research in which funds are used to
address Agency-driven problems. The goals of this research are to assess the impact to environment and
human health and research beneficial environmental applications. The key issues under these research
goals are the uniqueness of nanomaterials as contaminants, risk assessment approaches, mitigation
strategies, and the use of environmental nanomaterial technology. Nanomaterials are of interest because
they exhibit different characteristics than their larger size counterparts. With ENMs in particular, the issue
is that they have been changed from their natural state.
Nanotechnology research is driven by the Nanotechnology Environmental and Health Implications
(NEHI) Working Group, an interagency strategy for collaboration; the Organization for Economic
Cooperation and Development (OECD), an international cooperative program; the EPA Office of
Pollution Prevention and Toxics (OPPT) Nanoscale Material Stewardship Program, which involves inter-
Agency working groups; the EPA STAR grants program; and ORD's NRS. The purpose of the NRS is to
guide nanomaterials research within ORD; the final draft is under review and is expected to be finalized
within the next few months. NERL is working on sources, fate and transport, and exposure. It is
collaborating with EPA's National Health and Environmental Effects Research Laboratory (NHEERL) on
human health and ecological effects, with EPA's National Center for Environmental Assessment (NCEA)
on risk assessment and case studies, and with EPA's National Risk Management Research Laboratory
(NRMRL) on preventing and mitigating risks. EPA selected five nanomaterial classes on which to focus
its efforts: titanium dioxide (Ti02), zero-valent iron (ZVI), nanosilver, nanocarbon, and cerium oxide
(Ce02).
The Office of Research and Development's National Center for Environmental Research 6
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2008 Interagency Environmental Nanotechnology Grantees Workshop
Regarding sources, fate and transport, and exposure, NERL developed five research goals, and initial
research has been focused on identifying, characterizing, and quantifying nanomaterials in soil, water, and
biota media. The eventual goals are to model transport and exposure and characterize multimedia and
cross-media fate and transport. Within the next 2 to 3 years, NERL would like to: (1) separate and
characterize certain nanomaterials in soil and water matrices; (2) evaluate the detection of nanomaterials
by at least six physical and chemical methods; (3) understand the influence of certain environmental
factors on nanomaterials; (4) identify and prioritize the research needed for NCEA's comprehensive
environmental assessments; and (5) describe the properties of certain nanomaterials in the environment.
The long-term research goals are to: (1) model deposition of airborne nanomaterials; (2) model
nanomaterial behavior in surface water; and (3) design a nanomaterial exposure modeling approach. All
of this work is aimed at addressing the following major questions: Do nanomaterials move through the
environment? Is there exposure potential for humans and/or ecosystems? Do nanomaterials pose unique
exposure problems?
Reactive Composites for Targeted Remediation of Trichloroethylene (TCE)
Vijay John, Tulane University
This research project is attempting to devise new methods to remediate TCE. TCE is a dense nonaqueous
phase liquid (DNAPL). DNAPLs are a major problem, and TCE materials escape into groundwater and
create flumes that are difficult to clean up because they sink so far into the ground. ZVI is an effective
reductant for the remediation of TCE that is environmental friendly, highly efficient, and inexpensive.
The challenge is that ZVI particles have poor mobility because of their magnetic properties, so new
techniques are being created to disperse them. Because effective in situ remediation of TCE requires the
successful delivery of reactive nanoscale iron particles (RNIPs) through soil, the goal of the research is to
engineer reactive particles that have good mobility through soils and directly target TCE. Particles must
be synthesized that are reactive to TCE, will partition to TCE or to the TCE/water interface, and are of the
correct size range for optimal mobility through sediments. The idea is to incorporate nanoscale iron into
porous submicron silica particles that are functionalized with alkyl groups; the accompanying hypothesis
is that organic functional groups adsorb dissolved TCE facilitating contact with ZVI and also extend in
the organic phase to help particle stability. Using silica allows for the correct size range for optimal
mobility through sediments; almost all iron/ethyl-silica particles are in the size range for optimal mobility
and have optimal collector efficiency. Experiments show that: (1) the iron/ethyl-silica suspension
transports through the soil readily, whereas most of the RNIPs are retained at the top of the column; (2)
approximately two-thirds of iron/ethyl-silica particles are eluted through the sediment, whereas RNIP
does not elute; and (3) bare RNIP accumulates at the capillary inlet, whereas iron/ethyl-silica particles
move through the capillary. The researchers then examined a simpler technology and using carbons
prepared from sugars, incorporated the ZVI on the carbon surface for reaction. Following preparation,
electron microscopy showed prepared carbon as monodispersed uniform spherical particles. Pyrolysis and
activated carbons exhibited nearly 100 percent TCE adsorption. ZVI particles are dispersed on the carbon
surface, and the weight ratio between carbon and iron is controllable. The elution profiles and capillary
results of pyrolysis carbons indicate good elution of the materials. Furthermore, the researchers found
that: (1) iron/ethyl-silica particles may preferentially accumulate and localize at the TCE-water interface,
making dechlorination more efficient; (2) adsorption of TCE on the particles leads to a dramatic reduction
in solution TCE concentration; and (3) composite particles can be used in in situ remediation and the
development of reactive barriers. Currently, alternate technologies for adsorptive-reactive supported
nanoscale ZVI particles are in development.
Discussion
A participant noted that optimum size appears to be important and asked what size range is most optimal
and whether it would change based on the material used. Dr. John responded that silica particles are very
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different from carbon materials so the comparison is difficult. The participant then asked whether the
optimum size would be a function of porous media, and Dr. John replied that it would.
A participant commented that there is a group in Oklahoma performing work on groundwater and this
might be a source of collaboration.
A participant asked how sticking is controlled with sugar-based carbons and how they are mobile. Dr.
John responded that they do not appear to aggregate much.
Synthesis and Application of Polysaccharide-Stabilized Fe-Pd Nanoparticles for In Situ
Dechlorination in Soil and Groundwater
Donye Zhao and Chris Roberts, Auburn University
Contaminated plumes often are difficult to reach. The idea to deliver NPs to contaminants in situ first was
proposed in 1997, but there were no mobile NPs at that time. The primary accomplishments during Year
3 of this project were that: (1) batch and column tests for degradation of TCE sorbed and/or trapped in
soils using carboxymethyl cellulose (CMC)-stabilized ZVI NPs were conducted; (2) transport behaviors
of CMC-stabilized ZVI NPs in porous media were tested and modeled; and (3) in situ dechlorination in
soils using CMC-stabilized ZVI NPs was pilot tested. The researchers modified the traditional process by
starting nanoparticle synthesis by adding polysaccharide starch or carboxymethyl cellulose (CMC) before
the nanoparticles were formed (via the reduction of Fe2+ via the addition of electron donors). Following
Pd coating, the result was the formation of stabilized and soil-dispersible iron-palladium bimetallic NPs.
Researchers showed that CMC can facilitate the synthesis of nearly monodispersed palladium NPs that
can catalyze TCE degradation. Dr. Zhao described the experimental set up and results of several
experiments that demonstrated that: (1) CMC can facilitate size-controlled synthesis of ZVI NPs, (2)
transport of CMC-stabilized iron NPs are controllable and can be modeled by the convection-dispersion
equation and filtration theory, and (3) CMC-stabilized ZVI can degrade TCE in soil but must overcome
mass transfer and sorption limitation and dissolved organic matter inhibition.
Discussion
A participant asked what Dr. Zhao thought the reactive lifetime of particles is and whether, when
injections are performed, excess CMC is injected. To the first question, Dr. Zhao responded that the
lifetime depends on the composition, concentration, particle size, and conditions. If kept refrigerated, the
NP dispersion's reactivity can last for months, but all particles will be oxidized eventually. To the second
question, he responded that there always is some excess CMC, and the maximal CMC:iron ratio is
determined. The researchers try to use no more than is required for stabilization, which is approximately
0.2 percent per 0.2 g of iron.
Characteristics, Stability, and Aquatic Toxicity of Cadmium Selenide/Zinc Sulfide (CdSe/ZnS)
Quantum Dots (QDs)
James Ranville, Colorado School of Mines
CdSe/ZnS QDs are bright, photostable fluorophores that are used in biological imaging, optics, and other
applications. This project is examining them because cadmium, selenium, and zinc metal-containing QDs
are known to be toxic and they could escape into the environment in a variety of ways. The objective of
this research project is to characterize the environmental fate of QDs in the aquatic environment.
Characterization is key to this effort, and the research approach utilized ultraviolet and visible (UV-Vis)
absorption spectroscopy, fluorescence, transmission electron microscopy, inductively coupled plasma
(ICP)-atomic emission spectrometry (AES), and field-flow fractionation (FFF) to characterize the core,
shell, and polymer. Researchers also examined short- and long-term stability. Daphnia magna is being
used to determine acute toxicity and uptake. Four types of QDs were used in the experiments; the optical
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properties of each depend on core size. Researchers found that there is a large excess of cadmium
associated with QDs, given the assumed stoichiometry of 1:1 Cd to Se. The FFF results strongly
suggested that Cd is associated with the polymer coating. The researchers investigated the implications of
the characterization results for stability and toxicity and observed that: (1) mercapto-undecanoic acid
(MUA) toxicity appears to be a mass-based phenomenon; (2) there are dissolved metals present at 48
hours post-test; (3) there is enough dissolved cadmium to cause observed death; and (4) the rate of metal
release is important. In terms of polyethylene oxide) (PEO) toxicity, researchers observed that: (1) this
toxicity appears to be a particle number phenomenon; (2) smaller QDs are more toxic on a mass basis; (3)
although no detectable dissolved metals were found in solution at 48 hours, toxicity was observed; (4)
cadmium is not completely bioavailable as dissolved cadmium is more toxic than both PEO QDs on an
equivalent cadmium basis; and (5) dissolved zinc is potentially the toxic agent for the red PEO QDs. In
terms of acute toxicity, the researchers concluded that: (1) stability has a strong influence on QD toxicity;
(2) dissolved cadmium can explain the observed toxicity for MUA QDs; and (3) the lack of dissolved
metals found with PEO QDs suggests an alternate pathway of toxicity. The laboratory will continue its
characterization, stability, and toxicity experiments.
Discussion
A participant asked what the approach was for measuring dissolved cadmium. Dr. Ranville responded that
the researchers used filtration as a measure to dissolve cadmium.
Dr. Savage noted that EPA is attempting to establish a partnership with the United Kingdom. The RFA
will specify a joint U.S.-U.K. team and will be funded at $2 million each year for 4 years. If the
partnership does not work out, the usual amount of $600,000 will be offered.
Metals, Metal Oxides: Fate and Transport
Effect of Surface Coating on the Fate of NZVI and Fe-Oxide NPs
Greg Lowry, Carnegie Mellon University
There are releases from nanomaterial-related products into air, soil, and water. To develop NPs that can
be placed underground, it is necessary to coat the particle. Most nanomaterials are coated, and these
coatings are important because they affect the manner in which they behave in the environment. In
previous studies, researchers have shown that a polyaspartate (PAP) coating decreases reactive oxygen
species (ROS) and cytotoxicity in glial cells and neurons. Fresh particles have an effect at low
concentrations but oxidation and coating of particles can affect particle toxicity. The goal is to understand
how the coating affects the fate of these particles. The key questions are: What is the oxidation rate of
nanoscale ZVI in the environment? What is the fate of the coatings? Do aging and coatings affect
bactericidal properties? Is there synergy between nanoscale ZVI, coatings, and bacteria that enhances
remediation? The researchers investigated the rate and extent of desorption of adsorbed polyelectrolyte
from nanoscale ZVI during a 4-month period. Dr. Lowry briefly described the methods used to achieve
this. Researchers found that lower molecular weight coatings have higher rates of desorption; greater than
30 percent of the polyelectrolyte stays on the surface. Bare particles do not move; PAP, CMC, and
poly(styrene sulfonate) (PSS) were immediately mobile and remained mobile after 8 months. The
researchers also examined how polymer and natural organic matter coatings, oxidation state, and
environmental conditions affected the bactericidal effects and toxicity of nanoscale ZVI using
Escherichia coli. The findings showed that aerobic cultures were less affected than anaerobic cultures,
indicating that Fe° content is less important than the presence of oxygen. Fe° oxidizes quickly in an
aerobic environment, and it appears that under aerobic conditions a different iron oxide shell is formed on
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the outside of the particle. Results also indicated that PSS, PAP, and natural organic matter coatings
eliminated bactericidal effects, and coatings decreased contact between bacteria and nanoscale ZVI.
In summary, high molecular weight coatings do not readily desorb from nanoscale ZVI, and coatings and
aerobic conditions appear to decrease bactericidal effects. Under realistic groundwater conditions, these
NPs appear fairly immobile.
Discussion
A participant asked whether surface coatings are changing reduction-oxidation chemical properties, and if
this would be a problem in the real world. Dr. Lowry explained that a coating is being placed on the
particle that slows down but does not completely stop its reactivity. Even coated particles will oxidize
over time; the factor that is blocking electron transfer is the different iron oxide coatings.
A participant asked whether the coated particles can last 8 months in water. Dr. Lowry replied that iron
zero content plays a large role; if it is depleted, the particles are less likely to agglomerate. The desired
outcome is for the coating to come off so that the particles do not move, but this is not happening.
Therefore, the particles could continue to be mobile under the correct hydrogeochemical conditions.
A participant asked whether the degradation rate of the coating was checked. Dr. Lowry responded that an
undergraduate student currently is comparing the biodegradation rates of free coating polymers. The
participant asked whether a synergistic effect of anaerobic degradation was observed. Dr. Lowry
responded that the laboratory is working on this.
A participant asked whether the ROS were analyzed in the presence of oxygen, which could explain the
observed antimicrobial effects. Dr. Lowry responded that the laboratory has not measured this
specifically, but the results are counter to this as anaerobic conditions have greater antimicrobial
conditions.
Bioavailability and Toxicity of Nanosized Metal Particles Along a Simulated Terrestrial Food Chain
Jason Unrine, University of Kentucky
Dr. Jason Unrine explained that their laboratory is examining ecotoxicological effects of NPs in the
terrestrial system with a focus on detritivores. Detritivore food chains dominate in soil ecosystems, and
materials taken up by detritivores can move up the food chain. The overall objectives of the project are to:
(1) determine the interactions between particle size and particle composition in determining absorption,
distribution, metabolism, excretion, and toxicity in earthworms and amphibians; (2) investigate the
plausibility of nanomaterial trophic transfer along a simulated laboratory food chain; and (3) determine
whether simulated environmental and biological modifications influence bioavailability and toxicity. The
hypotheses are that: (1) nanomaterials have relatively low bioavailability in soils; (2) uptake from soils,
toxicity, and distribution of nanomaterials within organisms is size- and material-dependent; and (3)
biological responses are related to the release of metal ions. The laboratory is focusing on mechanistic
and ecologically relevant endpoints and used copper, silver, and gold as test materials. Results showed
that gold particles are delivered throughout the body of earthworms. Results of earthworm subchronic
toxicity and reproduction experiments indicated that in most cases, copper, silver, and gold do not cause
high mortality in earthworms, but silver nitrate (AgN03) at a soil concentration of less than 20 mg/kg has
a mortality rate of 100 percent in earthworms. The earthworms bioaccumulated all three types of metal
NPs in a size-dependent manner, and a decrease in reproductive success was seen; large particles showed
a trend of decreased reproductive success with increased exposure. Researchers also examined changes in
gene expression related to metal homeostasis, oxidative stress, and molecular chaperones. Results
indicated that metallothionein gene expression, a measure of metal homeostasis, was significantly altered
following exposure to copper and silver NPs. In the future, the laboratory plans to: (1) determine the
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uptake and elimination rates in earthworms, (2) determine the toxicity of smaller particles at higher
concentrations, (3) further develop methods for in situ characterization of particles/metals in soils and
tissues, and (4) investigate amphibians as another trophic level.
Discussion
A participant asked how AgN03 caused the mortality. Dr. Unrine responded that the mechanism had not
yet been determined, and there were no obvious molecular markers. His theory is that it somehow
interferes with earthworm ion regulation.
The Bioavailability, Toxicity, and Trophic Transfer of Manufactured Zn02 Nanoparticles: A View
From the Bottom
Paul Bertsch, University of Georgia
The overall objectives of this research project are to examine: (1) the bioavailability and toxicity of
manufactured NPs (i.e., nanoparticle zinc oxide [ZnO-np]), as a function of particle size to model soil
bacteria (Burkholderia vietnamiensis) and (Cupriavidus necator), and the model detritivore
Caenorhabditis elegans as referenced against aqueous zinc (i.e., Zn2+); (2) the ability of manufactured
ZnO-np to be transferred from one trophic level to the next as assessed in the simple food chain consisting
of pre-exposed B. vietnamiensis and C. elegans; and (3) the synergistic or antagonistic effects of
manufactured ZnO-np on the toxicity of copper to B. vietnamiensis and C. elegans. The researchers
hypothesize that: (1) the bioavailability and toxicity of manufactured ZnO-np increases with decreasing
particle size; (2) the toxicity of ZnO-np to B. vietnamiensis and C. elegans is lower than an equivalent
concentration of dissolved Zn2+; (3) the bioavailability and toxicity of ZnO-np introduced via trophic
transfer differs from that introduced via direct exposure; and (4) ZnO-np alters the bioavailability and
toxicity of dissolved metals. The first year of research focused on characterization of commercial ZnO-
nps and found evidence for at least three acetate populations. This is important because acetate inhibits
surface reactivity; removing acetate significantly increases surface reactivity. Additionally, there is much
greater surface reactivity of larger (80 nm) versus smaller (2 nm) nanoparticles. In terms of
characterization, the researchers found that: (1) size determination and surface chemistry are critical
issues; (2) transmission electron microscopy may not be the best method for size determination for small
metal oxide nanomaterials; (3) acetate controls smaller ZnO-np reactivity and passivates surface sites, but
this is not the case for larger particles; and (4) removal of acetate leads to flocculation/aggregation of
small ZnO-np primary particles but promotes surface reactivity. Results from bacterial exposure
experiments showed that: (1) there is no significant difference in the growth rate of C. necator and
B. vietnamiensis following exposure to ZnO-np and aqueous zinc; (2) C. necator displays higher acetate
utilization rates with aqueous zinc compared to ZnO-np, indicating a possible difference in
bioavailability; and (3) there are a greater number of compromised cell membranes associated with ZnO-
np than with the free ion. Experiments with nematodes indicated that: (1) mortality is not significantly
different between aqueous zinc and ZnO-np; and (2) at higher zinc concentrations (> 100 mg.L4), ZnO-
np decreases copper toxicity compared to aqueous zinc. Finally, there was no evidence for significant
trophic transfer in the bacterial-nematode model (although this may be more related to experimental
challenges), and ZnO-np is bioavailable from soils as demonstrated in earthworm exposures.
Discussion
Dr. Randy Wentsel (EPA) commented that, in terms of linkage between EPA intramural and extramural
research, Dr. Bertsch should consider working with EPA researchers regarding ecoeffects and ecological
risk assessment of these materials. Dr. Bertsch responded that he has had discussions with EPA
researchers at the Athens, Georgia, and Cincinnati, Ohio, facilities. His group also is fortunate to be part
of the Duke-Carnegie Mellon Center for Environmental Implications of Nanotechnology.
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Bioavailability and Fates of CdSe and Ti02 NPs in Eukaryotes and Bacteria
Patricia Holden, University of California at Santa Barbara
As nanomaterials enter the environment, a major question is whether NPs are toxic to bacteria and
eukaryotic cells. This research focuses on how NPs interact with cellular organisms, including
quantifying cellular-scale processes that affect nanoparticle entry, stability, and toxicity. Researchers are
examining two materials, CdSe QDs and Ti02 NPs. Researchers chose to work with bacteria because they
are abundant, biodiverse, and act as catalysts. Previous cell labeling experiments led researchers to ask the
following questions: Is light necessary? Are bare QDs internalized? Is external binding a prerequisite?
What are the quantitative fates of QDs? How are they toxic? Experimental results displayed a typical
dose-response relationship for Pseudomonas aeruginosa growth in response to exposure to both Cd(II)
and CdSe QDs. Additionally, bare QDs dissolve relatively quickly but not completely, and QDs add to
Cd(II) toxicity above a certain threshold. Above this threshold, researchers noted membrane damage,
increased intracellular ROS, and metal uptake in cells. Multiple evidence points to the probability that
QDs cause membrane damage, enter cells, and are highly reactive within the cells. Researchers concluded
that QDs appear to be more toxic than Cd(II) above a threshold, and sorption to the membrane is not a
prerequisite. Pseudomonas appears to alter the fate of QDs: intracellularly QDs appear mostly broken
down, whereas extracellularly QDs are relatively stabilized. Researchers also attempted to grow P. putida
in the presence of Ti02 NPs and determine whether the growth rate is affected by the particles. Initially,
in rich media, the particles are highly agglomerated, but after 12 hours they are highly dispersed. The
researchers hypothesized that this could be caused by: (1) the cells metabolizing the factor in the media
causing agglomeration, (2) bacterial biosurfactant production, or (3) specific adhesion. Further
experiments showed that the dispersion is caused by specific adhesion; the cells have a higher affinity to
the NPs than they have for each other. In the future, the researchers plan to examine the mechanisms
behind their observations, employ high-throughput methods, and scale up their research to include soil
ecosystem processes and biota.
Discussion
A participant asked whether QD fluorescence could be used to measure the concentration of intact QDs
within the cells. Dr. Holden responded that from a purist standpoint, she did not believe so. Labeling
indicates that as the QDs are being processed in the cells their fluorescence is changing.
Metals, Metal Oxides: Toxicity
OR I) NHEERL Manufactured-Engineered Nanomaterial Health Effects Research Program
Kevin Dreher, EPA, NHEERL
ORD's strategic plan for nanotechnology flows from the 2007 EPA Nanotechnology White Paper, the
National Nanotechnology Initiative (NNI), Woodrow Wilson International Center for Scholars documents
regarding the environmental health and safety implications of nanotechnology, the National Academy of
Sciences publication Toxicity Testing in the 21st Century: A Vision and a Strategy, and OECD's
nanotechnology document. EPA's health laboratories plan to develop an implementation plan for the
ORD strategy, which includes four basic themes. NHEERL nanotechnology research falls under the
theme of risk assessment and risk management, but all of the themes inform each other. NHEERL must
develop long-term goals to address the research question of determining the health effects of
manufactured-engineered nanomaterials and their applications and how these effects can be quantified
and ultimately predicted. High priority research areas include: (1) toxicology, hazard identification,
mechanisms of injury, and modes of action of nanomaterials and nanotechnology; (2) dosimetry,
biokinetics, and response modifiers of nanomaterials; and (3) the adequacy of existing test methods and
development of predictive approaches to assess toxicity of nanomaterials and nanotechnology. The long-
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term goal is to ultimately quantify and predict adverse health outcomes, and researchers initially are
examining manufactured nanomaterials in pursuit of this goal.
NHEERL has formed the "Nano" Health Effects Team, which includes 15 investigators representing each
NHEERL health division and a variety of expertise, to develop the implementation plan. The team also is
examining the systemic effects of inhaled or ingested nanoparticles. NHEERL's nanomaterials health
effects research employs an integrated multidisciplinary approach in its assessment of a common set of
well-characterized manufactured-engineered nanomaterials. Various types and sizes of Ti02, Ce02, and
carbon nanotubes have undergone independent physical and chemical characterization. This independent
characterization of commercially available nanomaterials showed significant differences from the
vendor's product information and underscores the need to conduct independent physical and chemical
characterizations of commercially available nanomaterials prior to conducting effects research. In terms
of alternative testing methods, NHEERL is involved in several projects that examine biochemical
interactions and surface properties via non-cellular and cellular-based assays that mimic pulmonary,
cardiovascular, liver, gastrointestinal, neuro, and ocular toxicities. In summary, to address some of the
challenges associated with assessing the health effects of manufactured-engineered nanomaterials, ORD
has developed a multidisciplinary strategy to screen and prioritize nanomaterials for in vivo toxicity
testing in a manner that ultimately will identify and develop validated alternative toxicity testing methods
for nanomaterials that predict in vivo toxicity.
Discussion
A participant asked why human health was considered a priority versus ecological concerns in regard to
nanosilver, because nanosilver is not as toxic to humans compared to aquatic organisms. Dr. Dreher
responded that Dr. Steve Diamond could answer this question better during his presentation. In terms of
human health, there will be a significant OECD effort regarding nanosilver, and NHEERL will fill in the
gaps. Nanosilver can be toxic to humans. NERL also is performing ecological work on nanosilver.
Microbial Impacts of Engineered NPs
Shaily Mahendra, Rice University
This research examines the effects of engineered nanoparticles on bacteria. Bacteria are important in
ecotoxicological studies because they are at the foundation of all known ecosystems, and as simpler
organisms, they can be indicative of the potential toxic effects on more complex organisms. Although C60
is insoluble in water, it can form a suspension, termed nC6o, when introduced to water via a solvent; nC6o
is an important form of C6o in the aqueous environment and is a potent, broad-spectrum antibacterial
agent that affects a variety of organisms. In comparing the bacterial toxicity of nC60 to other
nanomaterials, nC6o is among the most toxic. The researchers examined the effects of nC6o particle size
and found that particles were 100 times more toxic when particle size was reduced by one-half.
Researchers also observed that salt promotes aggregation (increase in particle size) of nC6o particles,
indicating that the particles would be more toxic in freshwater than in seawater. Natural organic matter,
however, reduces nC6o bioavailability and toxicity. Researchers also reviewed possible toxicity
mechanisms to determine how nC6o causes toxicity and tested three hypotheses involving changes in
membrane permeability, increased oxidative stress, or disruption of membrane oxidation/electron
transport phosphorylation. Results showed that nC6o did not appear to induce ROS-mediated damage in
bacteria, but nC6o did significantly collapse membrane potential, suggesting that nC6o results in oxidative
damage and can directly oxidize proteins. Researchers concluded that there is oxidative damage that is not
mediated by ROS but is most likely a result of oxidative stress on direct contact of nC6o with the cells. In
terms of potential applications, photocatalytic NPs could enhance UV disinfection of drinking water.
Fullerol, a hydroxylated form of C6o, enhanced virus removal by UV irradiation, shortening the contact
time by a factor of three. Because nC60 is bactericidal, release or improper disposal could have important
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environmental implications. Fortunately, this can be mitigated by natural organic matter and salinity.
Alternatively, nC6o's antimicrobial activity can be exploited to protect public health by preventing
microbial growth in water distribution and storage systems or enhancing UV disinfection practices.
Discussion
Dr. Lowry asked, if it is not ROS that implies a direct electron transfer, whether that means that nC60 must
be attached to the particle. Dr. Mahendra responded that this was the case, and the data support the fact
that there should be direct contact between the cell wall and the nanoparticle. Dr. Steve Diamond added
that a good deal of work conducted in the laboratory has found that the activation process must occur in
tissues.
Engineered Nanomaterial Ecological Effects Research Within ORD's NHEERL
Steve Diamond, EPA, NHEERL
The EPA's NHEERL is divided into health and ecology components and is one of the laboratories within
EPA's Office of Research and Development (ORD). Three of the four ecology divisions within NHEERL
(Atlantic [AED], Mid-Continent [MED], and Western [WED]) are involved in work with nanomaterials.
Research planning within ORD and NHEERL is based on documents prepared by NNI and NEHI, the
2007 EPA Nanotechnology White Paper, and the draft version of ORD's Nanotechnology Research
Strategy. Each of the three ecology divisions working on nanotechnology has completed a formal
research plan. MED will focus on freshwater systems, including freshwater sediments; AED will focus on
marine systems, including marine sediments; and WED will focus on terrestrial systems, including soils.
Ecological effects nanomaterials research aims to: (1) evaluate current methods for assessing hazard; (2)
assess hazard for nanomaterials; (3) identify nanomaterial characteristics that predict toxicity; (4) identify
mechanisms of action, accumulation, distribution, metabolism, and elimination; and (5) incorporate
knowledge of production volume and potential pathways of exposure within a product life cycle
framework. NHEERL scientists work in close collaboration with other ORD laboratories in these efforts.
Early efforts of scientists within NHEERL's ecology divisions included coordinating the review of
toxicity testing guidelines for both the Organization for Economic Cooperation and Development
(OECD) and EPA's Office of Pesticide Programs and Toxic Substances (OPPTS). Reviewers included all
of the nanotechnology principal investigators from the AED, MED, and WED as well as researchers from
the U.S. Army Corps of Engineers (USACE) and the U.S. Geological Survey (USGS). The OPPTS
review found that the toxicological principles and endpoint aspects of current testing guidelines were
adequate; however, media preparation, physical/chemical properties of materials, quantification of
exposure, and exposure metrology aspects of the current testing guidelines were inadequate. The
inadequacies identified were generally related to the particulate and fibrous nature of nanomaterials and
the colloidal nature of exposure media.
Preliminary research at MED has focused on approaches to producing consistent nanomaterial exposure
media for aquatic toxicity testing. The effect of ionic strength on the particle size of titanium dioxide has
been quantified, as well as settling rates and resulting stable bulk concentrations. The effect of UV
exposure on the toxicity of C6o and titanium dioxides is being studied in collaboration with USACE
scientists. MED researchers also have initiated work on nanosilver, which is increasingly being used in
consumer products. Preliminary assays have been completed, and researchers have successfully imaged
nanosilver in organisms using two-photon, scanning, and confocal microscopy. Single- and multiwall
carbon nanotubes have been obtained from Nikkiso Company, Ltd. (Japan) to be used in OECD
Sponsorship Program assays. Scientists from WED have coauthored a manuscript regarding the effects of
single-walled carbon nanotubes (SWCNTs) on root elongation of crop species in the journal
Environmental Toxicology and Chemistry. In the near term, NHEERL will continue its involvement in
OECD planning, review, and testing; its collaborations with South Carolina University, Oregon State
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University, USACE, and USGS; and its provision of assistance and technical support to EPA regulatory
offices.
Discussion
A participant asked whether collaborations were formal or informal. Dr. Diamond responded that most
collaborations currently are informal. There is one formal collaboration, which is an ongoing Cooperative
Agreement with the University of Minnesota.
Characterization of the Potential Toxicity of Metal NPs in Marine Ecosystems Using Oysters
Amy Ringwood, University of North Carolina at Charlotte
While more nanomaterials are being released into the environment, there are numerous potential
environmental risks of engineered NPs that are not well characterized or understood. This research
focuses on oysters (Crassostrea virginica), a widely-distributed estuarine bivalve species that lives in a
wide range of salinities. Filter-feeding bivalves are good models for characterizing the potential risk of
nanoparticles, because they are highly effective at removing particles, have high filtration rates, and
sample water column and surface/resuspended sediments. Additionally, there is extensive background
information regarding their toxic responses to metals and organic contaminants. The potential toxicity of
nanoparticle exposure to adult oysters is being investigated based on lysosomal destabilization, lipid
peroxidation, antioxidant responses, and cellular and tissue accumulation. The potential effects on oyster
embryos also are being investigated to compare the relative sensitivity of developmental stages and
adults. Nanoparticle exposure experiments were conducted with nanosilver seeds, which are
approximately 15 nm in diameter. Short-term (2-day) exposures were conducted in which adult oysters
were exposed to a range of Ag nanoparticle concentrations; and similarly, 48-hour embryo development
assays were conducted. The range of exposure concentrations selected for these studies was relatively
low. The results of the adult oyster exposures indicated increased rates of lysosomal destabilization
associated with Ag nanoparticle exposure. Furthermore, the levels of destabilization observed are
associated with reproductive failure. Results of lipid peroxidation studies indicated that gills did not show
oxidative damage, but hepatopancreas tissues did, and the response was more threshold-dependent than
dose-dependent. There was no evidence of depleted or altered glutathione status in either tissue. For
embryos, adverse effects were not seen until the highest dose was given, indicating a similar threshold
response. Dr. Ringwood summarized that, in terms of lysosomal destabilization in adult oysters, there are
significant adverse effects, and dose-dependent responses are based on exposure and tissue
concentrations. In regard to adult oxidative damage, there were significant increases in lipid peroxidation
with hepatopancreas tissues at the same concentrations at which adverse effects on lysosomal
destabilization were observed. There was, however, no significant oxidative damage to the gill tissues.
Next steps include characterization in seawater, investigations with other nanosilver preparations (e.g.,
rods, etc.), examination of antioxidant responses, and investigation of metallothioneins.
Discussion
A participant asked whether there was a nanosize effect. Dr. Ringwood responded that some work has
been done with the ion itself, which appeared to be less toxic than the NPs. She reminded the audience
that this is a work in progress.
Acute and Developmental Toxicity of Metal Oxide NPs in Fish and Frogs
Chris Theodorakis, Southern Illinois University
The objectives of this research project are to determine the environmental hazard of metal oxide NPs
(Fe203, ZnO, CuO, and Ti02) in terms of acute and chronic toxicity of these particles to fathead minnows
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(FMs) and African clawed frogs. The researchers hypothesized that nanoparticle exposure would affect
the survival, growth, development, egg hatchability, and metamorphosis of FM and African clawed frogs.
In experiments conducted to date, mortality was seen in the frogs at 1.0 and 2.02 mg/L (nominal
concentrations). As expected, chronic exposure resulted in a higher mortality than acute exposure did.
Frog growth was accelerated by low doses of ZnO and slowed by higher doses of ZnO. CuO and Fe203
NPs are highly toxic to FMs, while Ti02 and ZnO were not shown to be toxic in standard 96-hour tests.
Future work will include: measuring metal concentrations, characterizing nanoparticle size distribution,
determining the contribution of dissolved versus particulate metals to toxicity, comparing the toxicity of
metal NPs to dissolved ionic metals, comparing the Lethal Concentration 50 (LC50) of the metal oxide
NPs to the LC50 of the freely dissolved metal oxide, studying the toxicity of metallic copper to African
clawed frogs, and conducting chronic toxicity tests for metallic Cu, CuO, and Ti02 in FMs.
Other Nanomaterials: Sensors and Treatment
A Novel Approach to Prevent Biocide Leaching
Patricia Heiden, Michigan Technological University
With preserved wood, introduction of biocide is necessary, and leach is a potential problem. The
hypothesis is that biocide-containing NPs could penetrate the wood interior, enhance service life via a
stable and controlled release, and reduce or prevent leach. The objectives of this research are to "fix"
biocides into core-shell NPs and control biocide release by matrix hydrophobicity. Dr. Heiden highlighted
the initial nanoparticle synthesis, nanoparticle properties, and wood properties targets, comparing them to
current results. In terms of nanoparticle size, the initial target was less than 100 nm in diameter; this has
been achieved. Currently, the researchers are working on core-shell composition. A significant decrease
in leaching has been achieved; obtaining zero leach, however, is not possible at this time. The
nanoparticle size is suitable for delivery into wood if the NPs are not aggregated; sonicating before
treating wood improves efficiency. Delivery efficiency of 68 percent was achieved. Observed NPs appear
to be aggregates of much smaller core-shell NPs, which provides larger ill-defined core-shell NPs;
functionally, the NPs appear to work as intended to provide good control over the active ingredient
release rate. In terms of controlled release into water, as methyl methacrylate (MMA) is increased, there
is a decrease in the rate of release. Additionally, a background loss of mass with NPs is not seen. The
control showed significant release initially, whereas nanoparticle-treated wood showed a much smaller
initial release; ultimately, nanoparticle-treated wood had 55 percent less leach than the control. The effect
of using a polar co-monomer was similar. The biological efficacy is quite good, but researchers would
like to replace gelatin with chitosan. Researchers also decided to examine copper-containing NPs, but
discontinued their work because of the manufacturer's formulation with unknown components. The new
approach utilizes a 1:4 copper:tebuconazole complex (CTC), which has many advantages in that: (1)
inorganic/organic biocides are usually used in combination, (2) the complex may leach less than either
biocide alone, (3) the complex can be obtained in high yield via simple methods, (4) it can be delivered
into wood by various routes, and (5) the complex dissociates in water. CTC nanoparticle size appears to
be similar to that of the tebuconazole NPs, but the data need to be replicated. The delivery efficiency of
CTC into wood also appears to be similar to gel:MMA NPs with tebuconazole. The researchers plan to
optimize the formulation and measure leach, as well as carry out some studies using chitosan instead of
gelatin. There are plans to predominantly evaluate and optimize leach in the remaining studies.
Researchers also will evaluate the biological efficacy or lowest leaching samples.
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November 20, 2008
Carbon-Based Sensors and Exposure
Single Conducting Polymer Nanowire Immunosensors
Ashok Mulchandani, University of California, Riverside
Conducting polymers exhibit electrical, electronic, magnetic, and optical properties of metals or
semiconductors while retaining attractive mechanical properties and processing advantages. They can be
applied as conductometric, potentiometric, amperometric, and voltammetric transducers and as active
layers of field-effect transistors (FETs), and they can be synthesized electrochemically. Benign conditions
enable the direct deposition of conducting-polymer materials with embedded bioreceptors in one step.
Conductivity can be modulated over 15 orders of magnitude. The objective of this research project is to
develop new methods for cost-effective fabrication of single nanowire conducting polymer affinity-based
sensor arrays for label-free, highly sensitive, selective, precise, and accurate detection of bioagents such
as toxins, viruses, and bacteria at point-of-use. The approach to the research includes: (1) in situ
fabrication of conducting polymer nanowires in e-beam lithography patterned nanochannels between a
pair of electrodes; (2) magnetic alignment of template synthesized multi-segmented nanowire on
prefabricated electrodes; and (3) AC dielectrophoretic positioning and maskless assembly of template
synthesized nanowire on prefabricated electrodes. In situ fabrication has the advantage of biological
functionalization during fabrication and sequential site-specific deposition into individual channels. It is,
however, expensive due to the need for e-beam lithography. The magnetic alignment and assembly
identified the following limitations: (1) magnetic (Ni) segment integration is required; (2) the multi-
segmented nanowire architecture results in mechanical weakness, especially at the interfaces; (3) the low
aspect ratio can potentially result in lower dynamic range; and (4) the sodium hydroxide required for
template dissolution over-oxidized the polypyrrole segment, resulting in lower conductivity and possibly
in lower sensing performance. The maskless assembly is the most cost-effective method. Future work
includes: (1) demonstrating an immunosensor for viruses; (2) demonstrating a nucleic acid nanosensor;
(3) integrating micro-fluidics for improved handling and real-time sensing; and (4) demonstrating a multi-
analyte sensor array.
Carbon-Based Fate/Transport
Carbon Nanotubes (CNTs): Environmental Dispersion States, Transport, Fate, and Bioavailability
Elijah Petersen, University of Michigan
The overarching goal is to evaluate factors that control the environmental dispersion states, transport, fate,
and bioavailability of CNTs, thereby providing a foundation for human and ecological risk assessment.
Specifically, single-walled and multi-walled 14C-labeled CNTs will be synthesized, purified, and
characterized using techniques previously established in the researchers' laboratory. These radio-labeled
materials will then be used to systematically investigate: (1) the dispersion states of these nanomaterials
under typical environmental conditions; (2) their transport behaviors within and through a series of
different types of soil and sediment media; and (3) their bioavailability to selected critical aquatic and
terrestrial food-chain organisms. The researchers have developed and refined a means for producing
single-walled and multi-walled 14C-labeled CNTs by using radioactively labeled methane as a feedstock
for the synthesis of CNTs via chemical vapor deposition methods. CNT bioavailability to Daphnia
magna, an aquatic worm, and an earthworm was tested in lab-scale systems to examine the potential of
these nanomaterials to enter food chains in different environments and the factors controlling ecological
bioavailability. The uptake and depuration behaviors for these bioavailability studies were presented.
Results of the research include: (1) changing the hydrophobicity of multi-walled CNTs changes their
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octanol-water distribution behavior but does not impact accumulation by earthworms or aquatic worms;
(2) adding CNTs to soils affects the uptake of soil-borne pyrene by earthworms in a concentration-
dependent manner (low concentrations of nanotubes show no impact but higher concentrations decrease
pyrene accumulation and act similarly to black carbons); (3) polyethyleneimine was covalently bonded to
multi-walled CNTs to form nanotubes with positive, negative, or neutral surface changes, and the cellular
toxicity of these nanotubes was tested; and (4) a novel method to quantify fullerenes in ecological
receptors was developed and the test results showed significant accumulation and limited depuration by
Daphnia magna.
Aggregation and Deposition Behavior of CNTs in Aquatic Environments
Menachem Elimelech, Yale University
The use of engineered carbon-based nanomaterials has grown exponentially in recent years, but their
environmental and health impacts are not known. This research project is studying the aggregation and
deposition behavior of carbon-based nanomaterials as this will determine the fate and transport of these
nanomaterials through the environment. Experiments have shown SWCNTs to be much more toxic than
MWCNTs. The electrokinetic properties of MWCNTs were characterized to understand their aggregation
behavior and humic acid was found to stabilize MWCNTs. The deposition behavior of SWCNTs was
studied; long SWCNTs were found to be strained. Findings to date include: electrostatic interactions
control the aggregation behavior of CNTs; humic substances stabilize CNTs by electrosteric repulsion;
and CNT transport in porous media is relatively limited because of straining.
Discussion
A participant asked if a new method of measuring the surface charge of SWCNTs was needed. Dr.
Elimelech responded that his group measures size, which indicates the transfer properties of the
SWCNTs.
Cross-Media Environmental Transport, Transformation, and Fate of Carbonaceous Nanomaterials
Peter Vikesland, Virginia Polytechnic Institute and State University
Little is known about the unintended health or environmental effects of manufactured nanomaterials, but
some evidence suggests that they may be toxic. For example, nC6o produced using the tetrahydrofuran
(THF) method is suggested to cause oxidative stress in fish brain tissue and is potentially toxic to human
cell lines. The goal of this research project is to examine carbonaceous nanomaterial fate and transport in
the environment. The researchers focused on the question: How do atmospheric transformations of NPs
affect their fate in water and soil? The project focused on the characterization of the aqueous aggregates
of C6o fullerene. Due to its shape and electronic structure, C6o is highly reactive towards nucleophiles,
exhibits a sizable electron affinity, and can be photosensitized. C6o is extremely insoluble in water, but it
can form stabled water suspensions through the use of transitional solvents or long-term stirring in water;
this environmentally relevant form of fullerenes is called nC60. Natural water and physiological fluid
components are expected to alter the mechanism(s) responsible for nC6o formation and stability. These
components include: electrolytes, organic macromolecules (proteins, lipids, carbohydrates, humic and
fulvic acids), and low molecular weight organics (nucleic acids, amino acids, carboxylic acids). The nC60
aggregate size decreases in the presence of natural organic matter isolates. Carboxylic acid groups are
prevalent in many organic groups. Citrate is a well known stabilizer of many nanomaterials. Sodium
citrate increases the negative surface charge of these particles at low concentrations, but decreases the
negative surface charge at higher concentrations. The research conclusions are: (1) citrate stabilized nC6o
(cit-nC6o) is a new form of nC6o with unique properties; (2) carbonyl-7i interactions stabilize these
molecular crystals—these interactions are relatively weak and can be broken by alterations to solution
conditions, filtration, etc.; (3) molecular C60 is an important intermediate in carboxylic acid/nC60
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suspensions; and (4) aerosolization of nC6o results in a decrease in aggregate size. The implications of the
weakly stabilized molecular crystals on the fate and transport of C6o are unknown.
Transport and Retention of Fullerene NPs in Quartz Sands and Natural Soils
Kurt Pennell, Georgia Institute of Technology
The objectives of this research project are to: (1) investigate the transport and retention of nC60 aggregates
in water-saturated soils as a function of soil properties and systems parameters; (2) assess the effects of
nC6o aggregates on soil water retention, water flow, and transport in unsaturated soils; and (3) develop
and evaluate a numerical simulator^) to describe nC60 aggregate transport, retention, and detachment in
subsurface systems. The researchers found that nC6o aggregate transport decreases, and retention
increases, as grain size or flow rate is decreased. A mathematical model that includes non-equilibrium
attachment and maximum retention capacity accurately predicts nC6o transport and retention behavior in
Ottawa sands. The researchers also found that ionic strength strongly influences nC60 aggregate transport
and retention; the researchers attributed this primarily to electrostatic interactions. Future work will
include: (1) measurement and simulation of nC6o transport and retention in unsaturated porous media; (2)
investigation of nC60 transport and retention in heterogeneous 2-D aquifer cells; and (3) investigation of
technologies to image the retained nC6o aggregates on quartz sand surfaces (e.g., force-balance
microscopy). In a separate project, the researchers will evaluate the neurotoxicity of manufactured
nanomaterials in cell culture and mouse models (oxidative stress, dopamine system).
Photochemical Fate of Manufactured Carbon Nanomaterials in the Aquatic Environment
Chad Jafvert, Purdue University
For many organic chemicals, photodegradation is a significant environmental fate process, and
information regarding the rates and products of these reactions is therefore important in overall risk
assessment analysis. The overall objective of this research is to investigate photochemical transformation
of buckminsterfullerene (C6o) and SWCNTs under conditions of environmental relevance. Due to the
strong light absorbance of these materials within the solar spectrum, photochemical transformation in the
environment may lead to potentially more water soluble and easily bioaccumulative products. The three
subobjectives of this project are to: (1) measure photochemical transformation rates and products of C6o
solid films hydrated with aqueous solutions under solar irradiation; (2) measure solar photochemical
transformation of C6o in aqueous humic acid solutions and as clusters in aqueous solution; and (3) extend
these measurements to include the photochemical transformation of SWCNTs under similar conditions.
The photochemical transformation of aqueous C6o clusters (nC6o) in sunlight (West Lafayette, IN, 86° 55'
W, 40° 26' N) and lamp light (k = 300-400 nm) has been investigated. Upon exposure to light, the brown
to yellow color of nC60 was gradually lost and the cluster size decreased as the irradiation time increased.
TOC analysis indicated that nC6o products/intermediates were soluble in the aqueous phase and C6o may
have partially mineralized. The rate of C6o loss in sunlight was faster for smaller clusters compared to
larger clusters (i.e., kobs = 3.66 x 10 2 h 1 and 1.42 x 10 2 h 1 for C60 loss from 150 nm and 500 nm nC60
clusters, corresponding to half-lives of 18.9 h and 40.8 h, respectively, at the same initial C6o
concentration). Dark control samples showed no loss, confirming phototransformation as the underlying
degradation process. The presence of 10 mg/L fulvic acid, changes in pH, and the preparation method of
nC60 clusters had negligible effects on the reaction rate. Deoxygenation resulted in a decreased loss rate,
indicating that 02 played a role in the phototransformation mechanism. These findings suggest that the
release of nC6o into surface waters will result in photochemical production of currently unknown
intermediate compounds. Future work will include: (1) singlet oxygen measurement; (2) functional
group-specific X-ray photoelectron spectroscopy (XPS); (3) NMR analysis; (4) head space C02 analysis;
and (5) the extension of this work to CNTs.
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Discussion
A participant asked if the tests were done without any suspended solids or anything to which the
fullerenes could absorb. Dr. Jafvert replied that only water was used. In some cases, the researchers did
not buffer the solutions and the pH dropped, indicating that they had gotten some carboxyl groups. In
some cases, the researchers used phosphate species to buffer the pH. The ionic strength was controlled,
and no solid materials were seen other than the C60 particles.
A participant asked whether a C6o particle absorbed to a mineral surface, some bacteria, or some other
biological material would change the rate of the dissolution. Dr. Jafvert responded that it possibly could.
The researchers would like to do C6o coatings on walls and other materials to see if there are enhanced or
decreased rates of reaction.
Fate and Transformation of Carbon Nanomaterials in Water Treatment Processes
Jae-Hong Kim, Georgia Institute of Technology
The objective of this research is to examine the response of water-stable fullerene aggregates to processes
that are used in water treatment, using C6o and its stable aggregate, nano-C6o, as model compounds. The
researchers investigated the stability of carbon nanomaterials in natural waters and removal by
conventional water treatment processes. The results showed that: (1) natural organic matter (NOM)
enhances stabilization of carbon nanomaterials (C6o, SWCNT, MWCNT) in natural waters; (2) adsorptive
interaction between NOM and nanotubes depends on water quality parameters (e.g., pH and ionic
strength) and NOM characteristics; and (3) fullerenes are expected to be well removed by water treatment
processes. In the study of the chemical transformation of water stable C60 aggregates, the results showed
that: (1) ozonation transforms nC6o into water soluble fullerene oxide species; (2) ozonated C6o appears
more toxic than nC6o; (3) irradiation of UV (254 nm) transforms nC6o into water soluble fullerene oxide
species; (4) C60 photolysis product appears less toxic than nC60; (5) C60 in the aqueous phase reacts with
the hydroxyl radical and hydrated electrons with a relatively high rate constant resulting in an unstable
product. The results from the study of the photochemical activity of C6o in the aqueous phase during UV
radiation showed that: (1) the status of the C6o dispersion in the aqueous phase affects its ability to
transfer absorbed photoenergy to oxygen; (2) C6o present in water as a stable aggregate does not produce
1©2 and 02* under UV illumination, in contrast to pristine C6o; (3) when C6o is present as an aggregate, the
lifetime of key intermediate species for energy transfer is drastically reduced, fundamentally blocking the
ROS production mechanism; and (4) peroxide forms during preparation of nC60, which is partially
responsible for the reported toxicity.
Discussion
A participant asked whether the NPs entered the cell during the E. coli destruction of protein in the cell.
Dr. Kim responded that it is not possible to see it in the cell matrix.
Carbon-Based Toxicity
The Role of Particle Agglomeration in Nanoparticle Toxicity
Terry Gordon, New York University School of Medicine
The objective of this study is to determine the biological consequences of nanoparticle agglomeration.
The hypothesis of this research project is that the toxicity of fresh (predominantly singlet) carbon NPs
differs from that of aged (predominantly agglomerated) carbon NPs. The researchers further predicted
that this difference also would apply to metal NPs. The objectives were to: (1) measure the agglomeration
rate of carbon NPs; (2) identify whether agglomeration is affected by altering exposure conditions, such
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as humidity and particle charge; and (3) compare the toxicity of singlet versus agglomerated particles in
mice exposed via inhalation. The researchers used a dynamic exposure system to establish the
agglomeration of freshly generated carbon NPs at various distances (i.e., aging times) downstream from
particle generation. They then exposed mice to NPs generated in an arc furnace at different stages of
particle agglomeration and examined lungs for injury and inflammation. The researchers found a dose-
response relationship between exposure to carbon and metal NPs and lung inflammation such that the
effects of fresh particles were greater than those of aged particles for carbon particles, but not for copper
particles. Humidity and particle charge had no effect on the toxicity of carbon NPs. The researchers found
that copper and zinc NPs were more toxic than carbon NPs, and copper NPs were more toxic than zinc
NPs. In contrast to carbon NPs, copper particles showed only a small difference between fresh and aged
NPs. Differences in response among mouse strains suggest that genetic and age-related factors can
influence the response to NPs.
Discussion
A participant commented that everyone is looking for a susceptible strain. He asked whether there was
any consistent pattern with one strain being more susceptible for even a single endpoint or all endpoints.
Dr. Gordon responded that there was no consistent pattern for zinc. All of the strains responded at the
concentration that was used. In reviewing the literature, Dr. Gordon found that in comparing ozone,
nitrous oxide, and NPs, there was no consistency among strains.
Assessing the Environmental Impact of Nanomaterials on Biota and Ecosystem Functions
Jean-Claude Bonzongo, University of Florida
The hypothesis of this research project is that nanomaterials could lead to environmental dysfunction
because of their potential toxicity and the toxicity of their derivatives. Their small size makes them prone
to biouptake and bioaccumulation, while their large surface area could allow nanomaterials to act as
carriers or deliverers of pollutants that are adsorbed onto them. The objectives of this project are to:
(1) assess the toxicity of nanomaterials using short-term microbiotests and investigate the impacts of
nanomaterials on microbe-driven ecological functions; (2) determine the mobility of metal-based and
carbon-based nanomaterials in porous media, as well as the toxicity of nanomaterials in soil leachates;
and (3) identify possible mechanisms of toxicity for different types of nanomaterials. The combination of
experimental and modeling data collected so far shows that when contact is facilitated between
hydrophobic carbon-based nanomaterials (e.g., C60 and SWCNTs) and organisms by use of organic
solvents or surfactants: (1) an easy penetration of the cell membrane occurs; (2) the retention time within
the membrane varies with the nanoparticle size and shape; and (3) while C6o tends to induce toxicity
primarily by lipid peroxidation, carbon nanotube accumulation within cell membranes results in increased
pressure within the membrane with negative impacts on cell membrane functions. Additional studies on
the toxicity of carbon and metal-based nanomaterials suspended in natural river waters point to the
importance of solution chemistry as it affects both the degree of nanoparticle dispersion/suspension and
the biological response of model aquatic organisms exposed to such suspensions.
Discussion
A participant asked if toxicity experiments in this study were conducted comparatively by using both
river water-stirred nC6o (i.e., without use of THF) and suspensions produced by the THF method. Dr.
Bonzongo responded that this was the case, adding that Dl-water based suspensions were used as controls
and THF- C6o suspensions were more toxic. The participant asked if something from the THF derivative
could be causing the toxicity. Dr. Bonzongo responded that he did not have experimental evidence to
support the idea that a potential THF derivative was responsible for the observed trend in toxicity.
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ENMs in the Environment: Aggregated C6„ and Associated Impurities
John Fortner, Rice University
All stakeholders will benefit from an understanding of how fundamental characteristics of engineered
NPs control their biological effects. This research project will provide the first structure-function
relationships for nanoparticle toxicology. The guiding hypothesis of the research project is that
nanoparticle structure (e.g., size and shape) and surface chemistry directly control cytotoxicity. Within
that construct, a secondary hypothesis is that, of the four major material parameters in engineered NPs
(size, shape, composition, and surface), surface is the most important in governing cellular effects. The
specific objectives are to: (1) expand the characterization of nanoparticle structure in biological media
that can change aggregation status and surface chemistry (e.g., protein coat surfaces); and (2) characterize
the effects of NPs on cell function. The researchers found that fullerenes behave contrary to initial
estimations (i.e., there is water stable aggregate formation), and aggregates have been shown to interact
with biological systems. Before such work can be done with certainty, however, the purity of engineered
particles must be characterized and normalized: nC6o formation via THF intermediate can have impurities
that are particle associated and unassociated; THF and THF derivatives have been identified, including a
THF peroxide; and y-butyrolactone was less than 2 percent of the total of THF derivatives. The
researchers also found that the systems can be cleaned effectively; the stirred cell method provided
enhanced control and removal of greater than 99 percent of aqueous impurities. It also was found that
standard protocols for synthesis and purification are essential to compare "apples to apples."
Discussion
A participant asked whether C60 could enhance the decomposition of THF. Dr. Fortner responded that it
could not. Based on negative controls without C6o, THF decomposed to a THF peroxide in the presence of
light and oxygen regardless of C6o-
A participant commented that a number of studies have shown that these conversions do occur and that
toxic byproducts are produced. Knowing that the byproducts tend to be toxic and with all of the efforts
involved in removing THF, THF should not be used as a method. Dr. Fortner agreed. He also noted,
however, that organic impurities are nearly ubiquitous in engineered nanomaterials as they are often used
in the intermediate stages of synthesis. Therefore, this issue must be addressed for all particles with
potential impurities. Standard protocols for stating impurity levels and identification must be incorporated
into particle characterization as these issues are critical for toxicological analyses and comparison.
A participant commented that the solvate formation of THF in the clusters is similar to the solvate
formation of other molecules within precipitants of C60. Solvation is a function of temperature; as
temperature is increased, there is an increase in C6o solubility from the pure crystalline material, not the
clusters. As the temperature is further increased, the clusters are desolvated. If the clusters are formed at
higher temperatures, it may be possible to get a lot of the THF to not reside in the clusters. Dr. Fortner
agreed that this may be possible.
Long-Term Effects of Inhaled Nickel (Ni) NPs on Progression of Atherosclerosis
Gi Soo Kang, New York University
The hypothesis of this project is that inhaled Ni NPs can generate oxidative stress and inflammatory
responses not only in the lung, but also in the cardiovascular system, which in the long term can enhance
the development and progression of atherosclerosis in a sensitive animal model. An inhalation study was
conducted with 5-month-old male Apoe' mice. The dose was 80 jxg Ni/m3 for 5 hours/day, 5 days/week,
for either 1 week or 5 months. The research results showed that: (1) inhaled Ni NPs, at occupationally
realistic levels, can induce oxidative stress not only in the lung but also in the cardiovascular system;
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(2) inhaled Ni NPs can induce pulmonary and also systemic inflammatory responses; and (3) long-term
exposure to Ni NPs could exacerbate plaque formation in hyperlipidemic mice. An additional study
conducted to investigate which physicochemical properties of tested Ni NPs were responsible for the
observed toxicity revealed that toxicity may not be explained solely by particle effects or dissolved Ni
effects. This is the first long-term inhalation study to investigate cardiovascular effects of NPs, and the
results will provide a useful database to establish size-specific regulations in occupational and
environmental settings.
Discussion
A participant asked if there was any direct evidence of nickel translocation to the blood stream. Ms. Kang
replied that the researchers were not able to find any direct evidence, but pointed out that the exposure
concentration in this study was fairly low and the analytical method used might not have been sensitive
enough to detect the very low levels of nickel possibly translocated to the blood.
Aquatic Toxicity of Carbon-Based Nanomaterials at Sediment-Water Interfaces
Baolin Deng, University of Missouri-Columbia
The objectives of this research project are to: (1) adapt a proper method for water and sediment toxicity
testing of 1-D nanomaterials (CNTs, silicon carbide [SiC]); (2) assess the toxicity of representative 1-D
nanomaterials in water or in sediment to representative sediment-dwelling organisms; and (3) identify
factors controlling the toxicity toward the sediment-dwelling organisms. The approach includes three
phases: (1) 14 -day toxicity screening of CNT in water with four selected organisms; (2) 14-day sediment
tests with the CNTs identified as toxic to species in Phase 1 testing (e.g., 1% CNT spiked into sediments);
and (3) sediment tests with dilutions of sediment containing CNTs (No Observed Effect Level [NOEL])
and variations with types of sediments. The researchers found that: (1) sonicated or non-sonicated as-
produced single-walled and multi-walled CNTs are toxic to amphipods, midge, oligochates and mussels
in water; (2) the observed toxicity is partially contributed to toxic metals dissolved from the
nanomaterials such as Ni, but also is caused by purified nanomaterials (effect on growth); (3) sediment
can reduce, but not totally eliminate, the toxicity of as-produced MWCNTs to amphipods; and (4)
sonication significantly increases the toxicity of SiC nanowires to amphipods. Future studies will include:
identifying physical and chemical characteristics of the CNTs; phase 2 sediment toxicity testing; phase 3
sediment dilution testing; and mechanisms for the observed toxic effects.
Toxicity of NPs in an Environmentally Relevant Fish Model
Judi Blatt-Nichols, New York University School of Medicine
The objective of this study is to determine the biological consequences of nanoparticle contamination of
the aquatic environment. The investigators hypothesize that there will be a particle-type dependent
difference in the developmental toxicity of manufactured NPs in aquatic species, and in testing this
hypothesis, they will: (1) measure the differential toxicity of several types of NPs in an estuarine species
of fish, Atlantic tomcod; and (2) identify whether the embryo and larval stages of development of tomcod
are particularly susceptible to carbon nanoparticle or nanotube toxicity. The research results included: (1)
fullerenes cause 100 percent mortality at 500 jxg/L and hatching was delayed in all exposed doses; (2)
functionalized SWCNTs did not result in significantly more mortality to embryos than carbon black
particles, although time to hatch was significantly delayed; (3) for metal NPs, Cu was greater than Fe, Zn
was greater than Ag and Ni for mortality; (4) toxicity associated with erbium- and yttrium-containing
particles for the mix, soot, and sludge was dose-dependent and statistically significant. Future work will:
(1) determine if nanoparticle bioavailaility and toxicity is influenced by aquatic media; (2) characterize
the particles used in 5 ppt sea water and the natural waters in terms of mean diameter and zeta potential;
(3) expose a second species, Fundulus heteroclitus, to a subset of particles to determine if the effects
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found in tomcod are replicated in other species; and (4) use high-thoughput microarrays to determine
dose- and time-dependent changes in gene expression in tomcod and F. heteroclitus.
Discussion
A participant asked if the researchers had considered using the carbon materials with erbium and yttrium
atoms as tracers to look at the toxicokinetics of the carbon materials. Ms. Blatt-Nichols responded that
they would like to do that in the future.
In response to a question from a participant, Ms.. Blatt-Nichols stated that the soot was the most toxic for
erbium; the sludge was not as toxic as the soot and the finished products.
A participant asked where the soot and sludge materials were obtained. Ms. Blatt-Nichols responded that
Luna Works was the company that supplied the materials.
Ecotoxicology of Fuller en es (C60) in Fish
Theodore Henry, University of Tennessee
The research objectives are to investigate the characteristics of aqueous C6o aggregates and the impact of
dissolved organic material on the behavior of these aggregates, and to evaluate bioavailability and toxicity
of C6o (both aqueous C6o aggregates and dietary C6o) in fish by assessing changes in gene expression,
histopathology, and bioaccumulation of C6o in tissues. The hypotheses are: (1) bioavailability of aqueous
C6o aggregates is impacted by nanoparticle characteristics and presence of dissolved organic material; (2)
exposure of fish to C60 can be detected by changes in expression of biomarker genes; and (3) toxic effects
of C6o in fish can be detected only after long-term chronic exposure. Zebrafish (Danio rerio) and channel
catfish (Ictalurus punctatus) are the species that will be investigated in this research. Larval zebrafish
were exposed to the following treatments: (1) C60 aggregates generated by stirring and sonication (72 h)
of C6o in water (12.5 mg C6o/500 mL water); (2) C6o aggregates generated by established methods with
THF vehicle; (3) THF vehicle (i.e., method 2 without C6o added); and (4) "fish water" control. The
Affymetrix zebrafish array was used to assess changes in gene expression (14,900 gene transcripts), and
results indicate that changes in expression were related to decomposition products of THF rather than to
toxicity from C6o- Subsequently, the researchers investigated the interaction of other contaminants with
C6o aggregates and have determined that aggregate characteristics (e.g., size and charge) can change in the
presence of a co-contaminant and that C60 can alter contaminant bioavailability in zebrafish. The presence
of 17a-ehtinylestradiol (EE2) altered the characteristics of C6o aggregates. The Zeta potential decreased,
and there was more of a tendency to aggregate. Particles were smaller; however, larger particles may have
sedimented out of the aqueous phase. C60 reduced bioavailability of EE2 (reduced expression of
Vitellogenin genes). Aging appeared to increase the association of C6o with EE2 and reduced the
bioavailability of EE2.
Discussion
A participant asked whether the C6o aggregates were penetrating the chorion or whether de-chorionated
embryos were used. Dr. Henry responded that larvae were used; the larvae had hatched so the presence of
the chorion was not an issue for exposure.
Development of Methods and Models for Nanoparticle Toxicity Screening
Tian Xia, University of California, Los Angeles
This project aims to learn more about the health effects of nanoparticles. To date, approximately 10
particles, including fullerenes prepared by different methods, polystyrene nanoparticles with different
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surface charges, and metal oxides with different dissolution rates have been studied. Results to date
indicate that the physical characteristics of the particles and oxidative stress play key roles in particle
toxicity. Physicochemical characteristics (e.g., shape, size, surface reactivity, dissolution rate) have been
thoroughly characterized, and oxidative stress markers from cellular defense response, pro-inflammation,
and cell death, have been tested in mammalian cell systems. Tests on fullerenes prepared using THF
showed that, at the THF concentration used, the THF itself is not toxic to the cells. The degradation
products—formic acid and y-butyrolactone—were found to be very toxic and to induce cell death, but it
was not clear whether fullerenes sped up the degradation process. For the polystyrene nanoparticles,
cationic NH2-PS nanoparticles were found to be toxic, while plain and anionic nanoparticles were found
to be nontoxic. The mechanism of toxicity induced by cationic nanoparticles involves particle uptake
inside cells via specific endocytic pathways, proton sponge effects inside lysosomes, lysosomal leakage,
and mitochondrial-mediated apoptosis. For the metal oxides, ZnO was found to be toxic; the toxicity is
mainly induced by the high Zn concentration that results from ZnO dissolution. For toxicity testing, it is
important to thoroughly characterize the physicochemical properties of nanoparticles and the suspending
solutions. The lessons learned about the mechanisms of cytotoxicity from this study can be used to design
nanoparticles to mitigate toxicity. The following are some examples of the lessons learned to date: for
fullerenes, be careful of the residual solvents; for carbon nanotubes, decrease the impurities and rigidity
and/or functionalize the surface to increase solubility; for cationic particles, decrease the charge density or
replace cationic head groups with amphiphilic head groups; and for ZnO, NiO, Ag, and Cu, cap with
surfactants, polymers, or complexing ligands to decrease dissolution.
Discussion
A participant asked how cationic particles, which have a negative zeta potential in biological solutions,
could cause toxicity. Dr. Xia explained that the positive charge can reappear inside lysosomes because
particles are exposed to low pH environments and the protein coatings can come off.
Effects of Nanomaterials on Blood Coagulation
Peter Perrotta, West Virginia University
The goal of this project is to determine the effects of commercially available nanomaterials on the human
blood coagulation system. Common human diseases, such as myocardial infarction, are caused by
abnormalities of blood coagulation that predisposes a person to thrombosis (clots) and these diseases are
clearly influenced by environmental factors. Because of their large surface area and reactivity,
nanomaterials that enter the workplace or home have the potential to adversely affect blood coagulation,
which could result in clotting abnormalities. The researchers are studying the effects of nanosized
materials on the blood coagulation system using a variety of techniques. An important part of these
studies involved documenting adequate dispersion of NPs within biological media. Interestingly,
nanoparticle size can be verified in plasma-containing solutions by dynamic light scattering when the NPs
are of uniform size and shape. Using these well-dispersed nanoparticle-plasma suspensions for clotting
studies, it appears that NPs have the effect of shortening clotting times in vitro. They also are capable of
altering the ability to generate thrombin, the most physiologically relevant clotting enzyme. Based on the
importance of thrombin in human coagulation, the investigators have explored several sensor strategies
for detecting clotting proteins like thrombin. The investigators recently have begun to study plasma
obtained from rats exposed to ultrafine and nanometer-sized particles through inhalation. Differences in
endogenous thrombin potential and fibrinogen levels can be identified between exposed and control
animals. In addition, global proteomic profiling techniques (differential gel electrophoresis) and more
targeted multiplexed (Luminex) panels have demonstrated significant alterations in rat proteins involved
in the coagulation and inflammatory systems.
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Discussion
A participant asked whether the ability of citrate to complex calcium plays a role and whether citrate
would protect nanomaterials, which are intended to be introduced systemically, and make them safer. Dr.
Perrotta responded that citrate is very important; it is used to keep blood from clotting. It potentially could
be one way to make nanomaterials safer, but the short half-life of citrate may limit its usefulness.
A participant asked whether any evidence of systemic inflammation, such as c-reactive protein (CRP),
was found. Dr. Perrotta responded that CRP was definitely increased, as were other markers of an acute
inflammatory response.
Physical Characteristics of NPs Affect Interactions with Aquatic Organisms
David Barber, University of Florida
The goals of this research project are to: (1) expand the database of acute toxicity of metallic
nanomaterials in aquatic organisms; (2) evaluate the role of particle composition and dissolution in gill
toxicity; and (3) determine the role of particle surface charge in uptake and retention of nanomaterials in
aquatic organisms. To address the first goal, researchers assessed the toxicity of NPs and their soluble
counterparts to aquatic organisms. To address the second goal, researchers exposed zebrafish to Ti02,
silver, or copper particles and evaluated gill metal uptake, histology, and transcriptional changes at 24 and
48 hours. To address the third goal, researchers examined the uptake and retention of PEG, NH2, and
COOH QDs in Daphnia. The researchers found that nanometals can be acutely toxic to aquatic
organisms, but they are typically less toxic than their soluble counterparts. NPs aggregate rapidly once
they are introduced into water. Large numbers of nanosized particles, however, are likely to remain in the
water column for long periods of time; this may allow for prolonged exposure after a release of
nanomaterials into the environment. Intact NPs are taken up by gill cells and Daphnia. Physical properties
of NPs have significant impacts on their interaction with biological systems. Charge is an important
determinant of nanoparticle uptake and the effect of charge varies among models. Mechanisms of particle
uptake for particles with similar properties can differ. Oxidative injury appears to play a role in
nanosilver-induced toxicity.
Discussion
A participant commented that there was a question as to the Daphnia and whether or not what was being
seen by fluorescence after gut clearing was simple adhesion to the carapace. He suggested taking a molt
exuviate and exposing it after it has molted to find if it is strictly adhesion to the carapace. The concept of
redistribution is very important and what is seen in the gut before and after gut clearing is a critical
question. Dr. Barber responded that this was a good idea. The fact that increased fluorescence with the
PEG is seen suggests that it is not simply adhesion. (Postmeeting Note: Electron microscopy with EDS
was performed and it was confirmed that QDs are being internalized by Daphnia)
A participant asked if strand breaks were seen from silver nitrate. Dr. Barber responded that they have not
addressed that yet.
The Cellular and Gene Expression Effects of Manufactured NPs on Primary Cell Cultures of
Rainbow Trout Macrophages
Rebecca Klaper, University of Wisconsin-Milwaukee
The overall objective of this research project is to assess the innate immune reaction of an aquatic model,
the rainbow trout, to manufactured nanomaterials of varying chemistries at levels not inducing cellular
toxicity. This study will create a mechanism with which to test other nanomaterials, provide data to
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support ecological risk assessments, and ultimately inform decisions as to which materials will be the
safest to industrialize and use with respect to aquatic environments. The research hypothesis is:
nanomaterials of dissimilar chemical composition will stimulate different patterns of trout macrophage
gene expression, and nanomaterials of similar chemical characteristics (e.g., charge, shape, and functional
group) may be grouped with respect to their bioactivity, expressed as a particular gene response pattern.
Specifically, the chemical properties of nanomaterials will impact the genomic response of the immune
system: nanomaterials of dissimilar chemical composition will stimulate different patterns of macrophage
gene expression and the response will be dose-dependent. A range of water-soluble C6o and CNTs with
different chemical compositions and surface chemistries will be synthesized and tested for their effects on
trout macrophages. A trout primary macrophage cell culture system will be used to determine the: (1)
dose versus cell viability for each synthesized nanomaterial type; (2) level of expression (by quantitative
PCR) of marker genes associated with inflammatory, antiviral, and anti-inflammatory responses with
respect to nanomaterial dose at levels that have no deleterious effect on cell viability; and (3) global
patterns of gene expression for those materials that cause significant changes in marker genes using
custom trout immune microarrays. The results show that: (1) trout macrophages are a sensitive tool to
investigate the effects of NPs on gene expression; (2) side-chains attached to NPs may have just as much
of a stimulatory effect on the immune system as the NPs; (3) surfactants used to solubilize NPs may have
significant effects on gene expression—deoxycholate is a stimulator of inflammatory gene expression in
trout macrophages; and (4) C6o fullerenes and nanotubes stimulate inflammatory gene expression in trout
macrophages.
Discussion
Dr. Klaper responded to comments from others in previous talks and stated that although THF and other
surfactants were not used in these experiments, these compounds should not be banned from use in
experiments.
A participant commented that there have been a number of studies on whole fish gills showing
inflammation. He asked if there was a way to link that whole gill level response to Dr. Klaper's work. Dr.
Klaper responded that it would be interesting to see how much of the inflammatory response was due to
pure oxidative stress or other immune factors. The researchers would like to study whole organisms as
part of their next project.
A participant commented that Dr. Klaper's point about THF was a good one. This is not an academic
exercise; researchers are trying to predict what is going on in the real world. This is similar to what went
on with pesticides. Do you test the toxicity of the pure compound or what is used in the formulation that
is used industrially? Industry is using things to disperse NPs and these releases are mixtures.
A participant asked Dr. Klaper to comment on her microarray study. Dr. Klaper stated that her team used
three fish and there was a strong response for the inflammatory genes. There may be some small variation
among fish, but the tissue culture system leads to little variation among fish. In addition, the inflammatory
response was overwhelming and varied little among individual plates. The researchers would like to
review earlier time points and even lower concentrations of each particle; she thinks that they will see a
more sensitive measurement of how the treatments may affect the response.
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Metals, Metal Oxides: Toxicity
Pulmonary and Immune Effects of Inhaled Carbonaceous Materials
Jacob McDonald, Lovelace Respiratory Research Institute
The research objective is to directly compare the biological disposition, persistence, and toxicity of two
commercial nanoscale carbonaceous nanomaterials of potential wide utilization to a control material of
known toxicity. Concentration matched (by mass) inhalation exposures of CNTs and fullerenes were
compared to inhaled crystalline silica. Inhalation of MWCNTs and SWCNTs at particle concentrations up
to 1 mg/m3 did not result in significant lung inflammation or tissue damage, but caused systemic immune
function alterations. The effect appears to be regulated from a TGF-beta lung signal that manifests
through the COX-2 pathway. C6o fullerenes of median size 20 nm were produced by sublimation-
condensation. F344 rats were exposed by nose-only inhalation for 6 hours at lmg/m3, and
pulmonary/extra pulmonary disposition was monitored for 7 days. Fullerenes were measured in tissues by
LC/MS/MS. C6o fullerene inhalation showed poor lung clearance and minimal systemic translocation.
Discussion
A participant asked whether the C6o translocation could be related to dietary uptake. Dr. McDonald
responded that it would not be related; most everything that is inhaled goes into the gut. Dr. McDonald
will be conducting oral studies to answer this question.
November 21,2008
Other Nanomaterials: Life Cycle Analysis and Remediation
Nanostructured Membranes for Filtration, Disinfection, and Remediation of Aqueous and
Gaseous Systems
Kevin Kit, University of Tennessee
The objectives of this research project are to: (1) develop electrospun nanofiber chitosan membranes to
treat aqueous and gaseous environments by actions of filtration, disinfection, and metal binding; (2)
understand the electrospinning process for chitosan in order to control membrane structure; (3) investigate
the effect of membrane structure on filtration, disinfection, and metal binding; and (4) optimize
performance/efficiency of the chitosan membrane. Electrospinning of pure chitosan has proved to be
difficult due to limited solubility and a high degree of intermolecular hydrogen bonding. The researchers
were able to form nanometer-sized fibers without bead defects by electrospinning chitosan blends with
synthetic polymers polyethylene oxide) and poly (aery lamide) with up to 95 percent chitosan in blend
fibers. To date, researchers have developed a model to predict Cr(VI) binding properties of chitosan
fibers; performed a detailed surface analysis of the fiber surface, and found two highly effective chitosan
blends, one with good binding capacity and the other showing a 2-3 log reduction in E. coli K-12 with
much smaller fiber mass.
Discussion
A participant asked if the researchers ran XPS on the film. Dr. Kit responded that they did and the results
were the same for the film structure and the fiber structure.
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Comparative Life Cycle Analysis of Nano and Bulk Materials in Photovoltaic Energy Generation
Vasilis Fthenakis, Columbia University
The objectives of this research project are to: (1) assess the life cycle mass and energy inventories of two
main candidate nanomaterials for thin-film photovoltaic (PV) applications; (2) use process data to
compare the materials and solar cell structures; and (3) investigate the applicability of the results to other
nanomaterial-based thin-film technologies. Much progress has been made on the first two objectives. To
date, researchers have been able to project the mass and energy flows in future nanotechnology-enabled
PV, guided by changes in material utilization, purity, deposition rates, film thickness, and electric
conversion efficiency. Solution grown nanostructured CdTe solar cells require more extrinsic materials
than micro-CdTe solar cells, but less volume and lower purity semiconductor precursors. Plasma-
enhanced CVD of nc-Si requires materials for reactor cleaning that are greenhouse gases (GHG). Adding
nc-Si layers to a-Si solar cells increases energy and GHG emissions that can be counterbalanced by cell
efficiency increases. Future work will include a detailed investigation of solvent use and recycling
efficiency, a detailed investigation of energy use in solution-grown materials and in inkjet printing,
investigation of CIGS PV production by inkjet printing, and investigation of nanoparticle inks replacing
screen-printed silver-glass-frit pastes for Si cell contact metallization.
Discussion
A participant asked if the researchers had considered using water as a solvent in the cadmium synthesis.
Dr. Fthenakis responded that the researchers had not, but would be interested in learning more about this
potential approach.
Life Cycle of Nanostructured Materials
Thomas Theis, University of Illinois
The life cycle of a nanostructured material includes its manufacture from raw materials to its release into
the environment; each of these stages offers opportunities for exposure and efficiency. To date, most
research efforts have focused on the end of the life cycle. Bottom-up techniques (creating nanomaterials
and then assembling them) were initially thought to be less harmful to the environment, but this has
turned out not to be the case. In fact, sources of nanomanufacturing impacts include: strict purity
requirements and less tolerance for contamination during processing; low process yields or inefficiencies;
repeated processing, postprocessing, or reprocessing steps for a single product or batch; use of
toxic/basic/acidic chemicals and organic solvents; the need for moderate to high vacuum and other
specialized environments such as high heat or cryogenic processing; use of or generation of GHGs; high
water consumption; and chemical exposure potential in the workplace through technological/natural
disasters. The more complicated the structure of the nanostructured material, the more energy needed to
manufacture it. At the other end of the life cycle, this project has focused on CdSe NPs in aquatic
environments. Preliminary results show CdSe NPs to be extremely insoluble, but the expectation is that
they will dissolve after entering the environment which will have implications. Ultimately, the impact of
nanostructured materials on human and ecosystem function will depend on many factors.
Discussion
A participant asked why economic impact was not included in the life cycle assessment. Dr. Theis
responded that the economic aspect would be included later. The participant stressed the importance of
including economic impact in the assessment. Dr. Theis stated that while there is a considerable amount
of energy used in the manufacture of nanomaterials, this must be balanced with the potential energy
savings resulting from the use of these nanomaterials.
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Evaluating the Impacts of Nanomanufacturing via Thermodynamic and Life Cycle Analysis
Bhavik Bakshi, The Ohio State University
The overall goal of this research project is to help guide the development of nanotechnology to ensure
that it is environmentally benign and sustainable. Understanding the impact of nanomaterials is essential,
but not sufficient; a systems view must be adopted. Life cycle analysis (LCA) of emerging technologies
poses unique challenges. In particular, life cycle inventory data for nanomanufacturing are not available
and the impacts of ENMs on humans and ecosystems are only partially known. The first objective of this
research project is to conduct a life cycle evaluation of nanoproducts and processes. To date, the
researchers have established life cycle inventory modules for a number of nanomaterials. The second
objective is to explore a predictive model for LCA and impact assessment. Specifically, the researchers
will examine the relationship between life cycle inputs and impact and the relationship between the
properties of NPs and their impacts. The researchers have found that, from cradle to grave, polymer
nanocomposites (PNCs) are 1.6-10 times more energy intensive than steel. On a life cycle basis, the
product use phase is likely to govern if net energy savings can be realized, and the use of PNCs in
automotive body panels may result in net life cycle fossil energy savings. In addition, the life cycle
assessment of nano Ti02 shows significantly less energy use and impact as compared to carbon
nanofibers. A recently completed life cycle energy analysis of nano Ti02 has identified opportunities for
improvement. Future work will include: (1) research on other nanoproducts based on carbon nanofibers or
nano Ti02; (2) exploration of the statistical relationship between inputs and impact; and (3) risk analysis.
Discussion
A participant noted that the research did not include an impact assessment beyond energy requirements
and asked how a broader impact assessment could be built into these models. Dr. Bakshi responded that
there is a dearth of information on the environmental impacts of these NPs and that taking this type of
approach would require collaboration.
Other Nanomaterials: Exposure
Impact of Physicochemical Properties on Skin Absorption of Manufactured Nanomaterials
Xin-Rui Xia, North Carolina State University
Skin is made up of layers, with the top layer serving as the main barrier for small molecules and
particulates. The objective of this project is to establish a structure-permeability relationship for skin
absorption of manufactured nanomaterials for safety evaluation and risk assessment. Four dominant
physicochemical properties (particle size, surface charge, hydrophobicity, and solvent effects) in skin
absorption will be studied. Fullerene and its derivatives will be used as model nanomaterials. Results to
date show that fullerenes exist as molecular C60 or nC60 in different solvents and this affects their skin
absorption mechanism. In experiments, C6o, nC6o, and ANnC6o were all readily absorbed into the
uppermost layer of skin in vitro and in vivo. Tape-stripping methods can be used to study solvent effects
on skin absorption of nanomaterials and to provide partition coefficients and skin permeability for
predictive model development.
Discussion
A participant asked if the researchers had studied nC6o in dimethyl sulfoxide (DMSO). If so, how does it
behave? Dr. Xia responded that nC6o is stable in DMSO.
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Safety/Toxicity Assessment of Ceria (A Model Engineered NP) to the Brain
Robert Yokel, University of Kentucky
The long-term objectives of this project are to determine the physicochemical properties of ENMs that
influence their distribution into the cells comprising the blood-brain barrier (BBB) and the brain and to
characterize their beneficial and/or hazardous effects on the brain. The researchers are using ceria (Ce02)
as a model insoluble stable metal oxide tracer. Studies conducted to date in rats have shown that ceria is
rapidly cleared from the blood by peripheral reticuloendothelial tissues, much less ceria entered the BBB
cells or the brain than peripheral tissues, ceria ENM agglomerates in vivo, and the ceria induced mild
oxidative stress and stress response in the brain. These results provide a foundation to study the impact of
the physicochemical properties of ENMs on peripheral organ distribution, brain entry, and neurotoxic or
neuroprotective potential.
Discussion
A participant asked if the results suggested that ENMs would aggregate and coagulate quickly in blood.
Dr. Yokel responded that the ENMs could potentially aggregate after they reach the blood. He clarified
that, in the experiments discussed, the two solutions infused into the rat (the ceria ENM dispersion in
water and 1.8% saline) were not combined until they reached the blood.
Other Nanomaterials: Fate/Transport
Aggregation, Retention, and Transport Behavior of Magnetite NPs in Porous Media
Yan Jin, University of Delaware
The overall objective of this research project is to develop an understanding of the fate of NPs released
into the subsurface environments. Specific project objectives include: (1) determining the agglomeration
behavior of selected NPs under different solution chemistry (pH, ionic strength, and presence of dissolved
organic matter); (2) measuring the mobility of NPs in model porous media; and (3) elucidating retention
mechanisms of NPs at various interfaces at the pore-scale. Work to date has focused on the first two
objectives. Experiments have shown that humic acid can modify the surface charge of NPs by forming a
coating on the particle surfaces. This shifts the point of zero charge and changes the pH at which
aggregation occurs, increases the critical coagulation concentration (making it more stable), reduces
deposition, and increases mobility. The next steps will be to determine if this also will be the case with
smaller and other types of nanoparticles.
Internalization and Fate of Individual Manufactured Nanomaterials Within Living Cells
Gayla Orr, Pacific Northwest National Laboratory
Accumulating observations suggest that inhaled nanoscale particles (NSPs) are more harmful to human
health than larger particles, and these effects have been linked to the surface properties of the
nanomaterials. Current observations also suggest that NSPs might directly enter the circulatory system
through the epithelial wall. The hypothesis of this research project is that the initial interaction of NSPs
with the living cell in vivo occurs at the level of individual or small NSP aggregates (< 100 nm), and that
the physical and chemical surface properties of the individual NSPs dictate their mechanisms of
interaction with the cell, and ultimately govern their level of toxicity. Experiments conducted to date have
shown that both 100 nm and 500 nm particles can take advantage of the actin turnover machinery within
microvilli to move into alveolar type II epithelial cells, an expected target cell for inhaled submicrometer
and nanoscale materials. This pathway, however, is strictly dependent on the positive surface charge of
the particles and on the integrity of the actin filaments, unraveling charge-dependent coupling of the
particles with the intracellular environment across the cell membrane. To identify the molecules that
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capture the particles at the cell surface, the researchers searched for a negatively charged, transmembrane
molecule that could mediate the coupling of the particles with the actin filaments and found that
syndecan-1, a transmembrane heparan sulfate proteoglycan, mediates the initial interactions of the
particles at the cell surface, their coupling with the intracellular environment, and their internalization
pathway. These findings reveal a new mechanism by which positive surface charge supports particle
recruitment by polarized epithelial cells bearing microvilli, and identify a critical role for syndecan-1 in
the cellular interactions and subsequent potential toxicity of these particles.
Discussion
A participant asked how the charge is distributed. Dr. Orr responded that the distribution of the surface
charge over the particle surface was not known; the researchers measured zeta potential to approximate
the charge.
Methodology Development for Manufactured Nanomaterial Bioaccumulation Test
Yongsheng Chen, Arizona State University
The objectives of this research project are to: (1) develop suitable manufactured nanomaterial
bioaccumulation testing procedures to ensure data accuracy and precision, test replication, and the
comparative value of test results; (2) evaluate how the forms of these manufactured nanomaterials affect
the potential bioavailability and bioconcentration factor (BCF) in phytoplankton; (3) determine the
potential biomagnification of manufactured nanomaterials in zooplankton; and (4) determine the potential
biomagnification of manufactured nanomaterials in fish. The researchers tested different nanomaterials on
algae, daphnia, and adult and embryonic zebrafish to determine which were most toxic to these
organisms. For carbon-based NPs, SWCNTs were most toxic, followed by C6o and then by MWCNTs.
For metal oxides, nZnO was most toxic, followed by nTi02 and then by nAI203. nZnO was found to
cause oxidative stress in aquatic organisms and sediment could potentially be a mitigating agent to reduce
the toxicity caused by ZnO NPs. Future work includes determining the bioaccumulation behavior of NPs
under different exposure conditions, determining the distribution (or fate) of NPs in different parts of the
exposure system, and conducting long-term experiments on biomagnification and toxicity.
Discussion
A participant asked what species of green algae was studied. Dr. Chen promised to send the participant
the paper describing their work. Another participant asked if there were any physical or chemical property
changes in the nTi02 during exposure. Dr. Chen said that physical and chemical property changes did
occur, but he did not include this in his presentation because of time limitations.
Experimental and Numerical Simulation of the Fate of Airborne NPs From a Leak in a
Manufacturing Process To Assess Worker Exposure
David Pui, University of Minnesota
This project aims to determine the fate of NPs as they are emitted through a leak from a nanoparticle
production process into a workplace environment. This NP fate is determined by measuring and modeling
changes in particle and aerosol properties, such as number and surface area concentrations, morphology,
and chemical composition. To do this, the researchers simulated a leak and studied the particle changes
that occurred. A filtration study showed that results from the two types of monitors used to detect NPs
correlated very well. With an aerosol mainly composed of NPs, the surface area filter efficiency was
found to represent a more health-relevant filter evaluation and a better characterization of the filter. A
particle dispersion study showed that the nanoparticle concentration became more uniformly distributed
further out from the release location. Future plans include experimentally and numerically investigating
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the fate of NPs upon release into a wind tunnel using a burner setup, studying the effects of background
particles on nanoparticle fate, and numerically modeling the fate of NPs for a more complete
understanding of the coagulation and dispersion processes with high spatial resolution.
Discussion
A participant asked how many manufacturing facilities had been monitored with these instruments. Dr.
Pui said that one of the large chemical companies has plans to begin using these instruments for
monitoring soon.
Fun with Carbon and Ti02 NPs
Andrij Holian, University of Montana
Studies to date have shown that carbon nanoparticle toxicity may be dependent on size, size distribution,
aggregation, shape, surface chemistry, surface area, and surface charge. All of these properties could be
affected by suspension media, but predicting the optimal media for any one particle is not possible
because chemistry will be a factor. Experiments performed for this project have shown that carbon
nanoparticle toxicity is difficult to predict from conventional in vitro assays. Additionally, the dispersion
medium affects the outcome for CNTs. The researchers compared Ti02 nanospheres and nanowires and
found the shape of the nanoparticle to be an important determinant of toxicity, with long nanowires being
the most toxic and nanospheres being the least toxic. The scavenger receptor macrophage receptor with
collagenous structure was found to be an important receptor for NPs, but is not involved in long nanowire
toxicity. Redox is probably not involved in long nanowire toxicity. No unique changes in intracellular
ROS were found.
Discussion
A participant asked if the researchers observed frustrated phagocytosis. Dr. Holian stated that they did not
see this; nanowire contact with cells was enough to induce toxicity.
Biological Fate and Electron Microscopy Detection of NPs During Wastewater Treatment
Paul Westerhoff, Arizona State University
The overall goal of this project is to quantify interactions between manufactured NPs and wastewater
biosolids. This will be accomplished through the estimation of sources and loadings of nanomaterials into
wastewater treatment plants (WWTP) and through the development of mechanistic models for
nanoparticle removal in WWTPs. The researchers hypothesize that dense bacterial populations at
WWTPs should effectively remove NPs from sewage, concentrate NPs into biosolids, and/or possibly
biotransform NPs. The relatively low nanoparticle concentrations in sewage should have a negligible
impact on the WWTP's biological activity or performance. Experiments to date have shown that
functional nanomaterials are not removed as well as metal oxides. In sequencing batch reactors, Nano-Ag
and Ti02 had no effect on heterotrophic activity. Results to date suggest that Ti02 may serve as a sentinel
nanomaterial in the environment, indicating where other nanomaterials will eventually occur.
Discussion
A participant pointed out that Ti02 may not be a sentinel for another nanoparticle if the two NPs have
different point uses. Dr. Westerhoff agreed and stated that all Ti02 cannot be accounted for based solely
on what goes through the body; other sources must be considered as well.
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Other Nanomaterials: Toxicity
Genomics-BasedDetermination of Nanoparticle Toxicity: Structure-Function Analysis
Alan Bakalinsky, Oregon State University
This project aims to identify genes that mediate toxicity as a first step toward elucidating mechanisms of
action and to correlate toxicity with physical/chemical structure. Experiments showed that nC6o did not
inhibit the growth of E. coli or yeast in minimal media and had no real impact on the survival of yeast in
water over a 24-hour period although survival decreased slightly when fewer cells were exposed. Survival
of E. coli was significantly reduced over 24 hours in 0.9 percent saline, particularly at low cell
concentration. No obvious correlations were seen between size or zeta potential and cell survival. Studies
of gold NPs showed that none of the three Au NPs tested reduced yeast cell yields in minimal medium.
Positively charged Au-TMAT reduced yeast survival more than negatively charged or neutral Au
derivatives. Specific amounts of these particles appeared to kill a fixed number of cells. To identify genes
and mechanisms implicated in Au-TMAT-mediated killing, a yeast gene deletion library was screened for
mutants resistant to Au-TMAT relative to the wild-type parent strain. Six resistant clones were isolated
from the initial screen of 2,500 mutants. Loss of GYL1, YMR155W, DDR48, and YGR207C was found
to result in Au-TMAT resistance, suggesting that these genes play roles in mediating Au-TMAT toxicity.
Future work will focus on identifying additional mutant strains.
Discussion
A participant asked if the researchers had studied chromosome or DNA damage. Dr. Bakalinsky
responded that they had not, but would like to do so in the future.
Role of Surface Chemistry in the Toxicology of Manufactured NPs
Prabir Dutta and W. James Waldman, The Ohio State University
This project is working to identify correlations between biological activity and physicochemical
characteristics of minerals and particulates, including the biological response (oxidative burst),
mutagenicity, and the chemical reactivity (Fenton reaction) of zeolite minerals and oxidative stress and
inflammatory responses of carbon particulates. Zeolite minerals (aluminosilicates) and carbon particles
were chosen for study to evaluate how the surface structure of particles influences their toxicity. The
researchers found that the coordination environment can modify the iron redox potential and the chemical
reactivity differences result in different biological reactivity. Further experiments using carbon NPs of the
same size showed that it is the surface chemistry of the iron that causes the reaction. Results to date have
shown that Fe(III) precipitate is more cytotoxic and more inflammatory than Fe(II). The researchers
hypothesize that the redox state of the element released is important.
A Rapid In Vivo System for Determining the Toxicity of Nanomaterials
Robert Tanguay, Oregon State University
The hypothesis of this study is that the inherent properties of some ENMs make them potentially toxic.
To test this hypothesis, the researchers developed an in vivo zebrafish toxicity assay to define the in vivo
response to nanomaterials, and will eventually define structural properties of nanomaterials that lead to
adverse biological consequences. A wide range of nanomaterials will be tested to assess toxicity. Those
that cause significant adverse effects move on to the next stage of testing in which potential cellular
targets and modes of action are defined in vivo; nanomaterials are then grouped according to structural
indices and effects. Global gene expression profiles will be used to define the genomic responses to these
materials. A Nanomaterial Biological Interactions database will be populated with the data collected on
the properties of the nanomaterials. To date, more than 200 nanomaterials have been evaluated for
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toxicity in zebrafish. Those determined to be toxic have moved on the next stage of testing. The
researchers will continue to test nanomaterials for toxicity and ultimately, develop a database populated
with the data collected.
Discussion
A participant asked if the researchers were planning to study epigenetic responses. Dr. Tanguay replied
that they are planning to do these studies.
Cellular Uptake and Toxicity of Dendritic Nanomaterials: An Integrated Physicochemical and
Toxicogenomics Study
Mamadou Diallo, California Institute of Technology
The overall objective of this research project is to improve understanding of the cellular uptake and
toxicity of dendritic nanomaterials in aqueous solutions at physiological pH 7.4. The specific objectives
are to: (1) characterize the interactions of dendrimers with cell membranes through measurements of
physical-chemical surrogates (octanol-water partition coefficients and liposome-water partition
coefficients); (2) characterize the interactions of dendrimers with plasma proteins through measurements
of dendrimer binding to human serum albumin (HSA) protein; (3) use molecular dynamics simulations,
nuclear magnetic resonance spectroscopy, and neutron scattering to characterize the mechanisms of
interactions of dendrimers with lipid bilayers and HSA protein; (4) characterize the cytotoxicity of
dendrimers through in vitro measurements of cell viability and toxicogenomic studies; and (5) conduct
correlation analysis. Work to date shows that PAMAM dendrimers with protonated terminal NH2 groups
at pH 7.4 have a higher tendency to bind to liposomes (LogKiipw). These dendrimers also show a high
level of toxicity due to their tendency to cause membrane leakage. Other molecular mechanisms beyond
membrane leakage may be responsible for the higher toxicity of cationic dendrimers. PAMAM
dendrimers with neutral and negatively terminal groups have been found to have low to negligible
toxicity. Future work includes: quantitative internalization, live imaging 1 ms frame to track the
internalization of dendrimers, and performing correlation analysis and developing structure-activity
relationships.
Effects of Ingested NPs on Gene Regulation in the Colon
John Veranth, University of Utah
This research project focused on a model of bowel inflammation and used RKO and CaCo human colon-
derived cell lines with and without activation by TNFa. The central hypothesis being tested is that
ingested manufactured NPs are taken up by inflamed colon cells, translocate to the nucleus, and alter gene
transcription, thereby further increasing inflammation and leading ultimately to the development of
pathological conditions including cancer. In separate experiments, samples were prepared from multiple
types of metal oxide nanoPM and whole genome microarray experiments were conducted. Ti02 and ZnO
displayed transcriptional effects, with ZnO having the most pronounced effect. The data suggest that
multiple pathways are activated by the ZnO, including: stress response pathways, Zn metabolism and
transport genes, and genes that suggest alterations in redox pathways. NanoZnO displayed the most
toxicity and demonstrated the most pronounced transcriptional response. This transcriptional response
suggested that part of the exposure to nanoZnO was exposure to elemental Zn, and therefore, perhaps the
toxicity was merely Zn toxicity. Therefore, the investigators sought to determine if the nanoZnO toxicity
was due to the dissolution of ZnO to elemental Zn and the mechanism of the cell death upon exposure to
the nanoZnO. In addition, two size ranges of ZnO PM were utilized to evaluate the effects of size/surface
area. The researchers wanted to determine if: (1) cell and PM contact was required for ZnO toxicity; and
(2) ZnO dissolution to free Zn was dependent on the cells. A set of three experimental conditions were
used: (1) a dialysis device with a 10 kD cutoff was used to separate the ZnO from cellular contact to
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ensure no ZnO PM could interact directly with cells; (2) transwells with 0.4 micron pores that would
allow greater interactions with cellular products but still separate the cells and the PM were used; and (3)
ZnO PM was placed in direct contact with the cells. The Zn concentrations were measured in the media
by ICP spectrometry and cell viability by PI exclusion. The ZnO toxicity was only observed when the
particles were in contact with the cells, but the Zn levels in the media were equally high in the transwell
and direct contact experiments, suggesting that contact and potentially uptake is required for cellular
toxicity. It was also found that ZnO induces apoptosis by inducing superoxide production in the
mitochondria and disruption of the mitochondrial potential. In addition, all of the toxic effects are
dependent on particle size, as the larger ZnO PM always demonstrated reduced toxicity compared to the
smaller ZnO NPs.
Discussion
A participant asked what the molecular mechanism of zinc toxicity is. Dr. Veranth said that little is
known about mechanisms of zinc toxicity; this should be explored further.
Nanoparticle Toxicity in Zebrafish
Gregory Mayer, Texas Tech University
The objective of this research project is to investigate the toxicity of semiconductor nanocrystals using
zebrafish (Danio rerio) as an in vivo model, and zebrafish liver cells as an in vitro system. The approach
will monitor, in real-time, the effects of particle composition, size, and charge on uptake and
accumulation of nanostructures in multiple cellular compartments. Additionally, the investigators will
address the hypothesis that toxicity of metal-cored nanoparticles stems from dissoluting metal ions by
using a transgenic zebrafish model that expresses green fluorescent protein (GFP) in the presence of I-B
and II-B metal ions. These data will be correlated with embryo development after particle exposure, and
the effects will be extrapolated to human health. Finally, the researchers will develop a model to predict
particle toxicity that will help to evaluate the potential health risks of the release of differing
semiconductor NPs into the environment. Cell cultures have shown cell viability results similar to those
found by other researchers. Toxicity appears to be related to the size of the particle, with smaller particles
being more toxic. Work conducted to date suggests that nanocrystals may not be gaining entrance to the
cell through classic calveolin- or clathrin-mediated pathways. In vivo, the toxicity of quantum confined
semiconductors does not seem to be attributable to ion dissolution from the particles.
Discussion
A participant asked whether the researchers saw different effects in the different regions of the fish. Dr.
Mayer explained that it appears that the ions are moving into the gut, but because this happens before the
embryos feed, this may not be attributable to normal gut uptake. In this stage of development, it would be
difficult to discern distinct tissue patterns with this method.
Lung Deposition of Highly Agglomerated NPs
Jacob Scheckman and Peter McMurry, University of Minnesota
The objectives of this research project are to: (1) develop a stable, repeatable source of nanoparticle
agglomerates with closely controlled properties; and (2) characterize the effects of agglomerate properties
on deposition in physical models of the human lung. Transport and physical/chemical properties of
nanoparticle agglomerates depend on primary particle size, fractal dimension, and the number of primary
particles in the agglomerate. Agglomerate properties were determined by tandem measurements of
mobility (differential mobility analyzer [DMA]), mass (aerosol particle mass analyzer [APM]), and
morphology (electron microscopy [SEM/TEM]). Nanoparticle agglomerates of silica were generated by
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oxidizing hexamethyldisiloxane in a methane/oxygen diffusion flame. Particles leaving the flame were
classified by electrical mobility size using a DMA, and their mass measured with an APM. The measured
relationship between mass and mobility was used to determine the fractal dimension. The effects of
oxygen flow and mass production rates on single particle mass, fractal dimension, and dynamic shape
factor were characterized. Electron microscopy was used to determine primary particle size and to
provide qualitative information on particle morphology. The generated particles were chain agglomerates
with clearly defined primary particles. Increasing the oxygen flow rate was shown to decrease the primary
particle size and the fractal dimension and to increase the dynamic shape factor. Increasing the production
rate was shown to increase the primary particle size and mass of the product particles without affecting
the fractal dimension and to decrease the dynamic shape factor. These results represent the completion of
objective 1. Of particular interest are the effects of agglomerate structure on lung deposition. To
investigate this, deposition of silica agglomerates through a straight capillary tube model simulating lung
generation 22 was compared to that of spheres. Deposition did not depend on particle morphology in the
capillary tubes, but deposition of spheres and agglomerates differed significantly in the entrance/exit
region of the model. Future work will: investigate increased deposition in the entrance/exit region,
characterize the effects of fractal dimension, and measure deposition through more physically realistic
lung models.
Dr. Savage thanked all of the participants for their contributions and adjourned the meeting.
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