&EPA
United States
Environmental Protection
Agency
Proceedings of the Interagency
Workshop on the Environmental
Implications of Nanotechnology
SEPTEMBER 5 - 7, 2007
WASHINGTON, DC
/ \
Office of Research and Development
National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Table of Contents
Metals, Metal Oxides
Fate, Transformation, and Toxicity of Manufactured Nanomaterials in Drinking Water 1
Paul Westerhoff, David Capco, Yongsheng Chen, John C. Crittenden
Pulmonary and Systemic Inhalation Toxicity of Multiwalled Carbon Nanotubes 2
Jacob McDonald
Pharmacokinetics and Biodistribution of Quantum Dot Nanoparticles in Isolated
Perfused Skin 3
Nancy A. Monteiro-Riviere, Hyun A. Lee, Leshuai W. Zhang, Mudassar Imran,
Vicki L. Colvin, William Yu, Jim E. Riviere
Metal Nanoparticle Tissue Distribution Following In Vivo Exposures 4
Alison Elder, Nancy Corson, Robert Gelein, Pamela Mercer, Amber Rinderknecht,
Jacob Finkelstein, Gunter Oberdorster
The Bioavailability, Toxicity, and Trophic Transfer of Manufactured ZnO Nanoparticles:
A View From the Bottom 5
PaulM. Bertsch, Travis Glenn, Brian Jackson, Andrew Neal, Phillip Williams
Biochemical, Molecular, and Cellular Responses of Zebrafish Exposed to
Metallic Nanoparticles 6
DavidS. Barber, Nancy Denslow, Kevin Powers, David Evans
Acute and Developmental Toxicity of Metal Oxide Nanoparticles to Fish and Frogs 7
Christopher Theodorakis, Elizabeth Carraway, George Cobb
Mechanistic Dosimetry Models of Nanomaterial Deposition in the Respiratory Tract 8
Bahman Asgharian, Brian A. Wong
Nanostructured Materials for Environmental Decontamination of Chlorinated Compounds 9
Yunfeng Lu, Vijay T. John
Responses of Lung Cells to Metals in Manufactured Nanoparticles 10
John Veranth, Christopher A. Reilly, Garold S. Yost
A Toxicogenomics Approach for Assessing the Safety of Single-Walled Carbon Nanotubes
in Human Skin and Lung Cells 11
Mary Jane Cunningham, Edward R. Dougherty, Daniel E. Resasco
Microbial Impacts of Engineered Nanoparticles 12
Delina Y. Lyon, Pedro J.J. Alvarez
An Integrated Approach Toward Understanding the Inflammatory Response of Mice to
Commercially Manufactured CuO/Cu, Fe203/Fe, and Ti02 Nanoparticles 13
Vicki Grassian
Hysteretic Accumulation and Release of Nanomaterials in the Vadose Zone 14
Tohren C.G. Kibbey, David A. Sabatini
The Office of Research and Development's National Center for Environmental Research iii
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Interagency Workshop on the Environmental Implications of Nanotechnology
Carbon-Based Nanomaterials
Role of Particle Agglomeration in Nanoparticle Toxicity 15
Terry Gordon, Lung Chi Chen, Beverly S. Cohen
Chemical and Biological Behavior of Carbon Nanotubes in Estuarine Sedimentary Systems 16
P. Lee Ferguson, G. Thomas Chandler, Watty A. Scrivens
Fate and Transformation of C6o Nanoparticles in Water Treatment Processes 17
Jaehong Kim, Joseph Hughes
Cross-Media Environmental Transport, Transformation, and Fate of Manufactured
Carbonaceous Nanomaterials 18
Peter J. Vikesland, Linsey C. Marr, Joerg Jinschek, Laura K. Duncan,
Behnoush Yeganeh, Xiaojun Chang
Transport and Retention of Nanoscale Fullerene Aggregates in Water-Saturated Soils 19
Kurt D. Pennell, Joseph B. Hughes, LindaM. Abriola, Yonggang Wang, YusongLi,
John D. Partner
Repercussion of Carbon-Based Manufactured Nanoparticles on Microbial Processes
in Environmental Systems 20
Ronald' F. Turco, Bruce M. Applegate, Timothy Filley
Size Distribution and Characteristics of Aerosol Released From Unrefined Carbon
Nanotube Material 21
Judy Q. Xiong, Maire S.A. Heikkinen, Beverly S. Cohen
Physical and Chemical Determinants of Carbon Nanotube Toxicity 22
Robert Hurt, Agnes Kane
Environmental Impacts of Nanomaterials on Organisms and Ecosystems: Toxicity and
Transport of Carbon-Based Nanomaterials Across Lipid Membranes 23
Dmitry I. Kopelevich, Jean-Claude J. Bonzongo, Gabriel Bitton
Structure-Function Relationships in Engineered Nanomaterial Toxicity 25
Vicki L. Colvin
Interactions of Pure and Hybrid Polymer Nanofibers With Cells 26
Perena Gouma
Other Nanomaterials
Cellular Uptake and Toxicity of Dendritic Nanomaterials: An Integrated Physicochemical
and Toxicogenomics Study 27
Mamadou S. Diallo, William A. Goddard, Jose Luis Riechmann
Assessment of Nanoparticle Measurement Instruments 28
Patrick T. O 'Shaughnessy
Development of Nanosensors for the Detection of Paralytic Shellfish Toxins (PSTs) 29
Robert Gawley
iv The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Transformations of Biologically Conjugated CdSe Quantum Dots Released Into Water
and Biofilms 30
Patricia Holden, Jay L. Nadeau
Nanotechnology: A Novel Approach To Prevent Biocide Leaching 32
Patricia Heiden, Benjamin Dawson-Andoh, Laurent Matuana
Evaluating the Impacts of Nanomanufacturing Via Thermodynamic and Life Cycle Analysis 33
BhavikR. Bakshi, L. James Lee
Appendices
Agenda
Post-Participants List
Presentations
Executive Summary
The Office of Research and Development's National Center for Environmental Research
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Metals, Metal Oxides
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Interagency Workshop on the Environmental Implications of Nanotechnology
Fate, Transformation, and Toxicity of Manufactured Nanomaterials
in Drinking Water
Paul Westerhoff, David Capco, Yongsheng Chen, and John C. Crittenden
Arizona State University, Tempe, AZ
Objective: Although the current market for nanomaterials is small and their concentration may not be
high enough in the environment to cause human health or environmental problems, this market is increasing
rapidly, and the discharge of nanomaterials to the environment in the near future could be significant as
manufacturing costs decrease and new applications are discovered. The accumulation of nanomaterials in cells
may have significant environmental and human impacts. However, at present, very little is known about the
fate, transport, transformation, and toxicity of these man-made nanomaterials in the environment. The
objectives of this project are to: (1) characterize the fundamental properties of nanomaterials in aquatic
environments; (2) examine the interactions between nanomaterials and toxic organic pollutants and pathogens
(viruses); (3) evaluate the removal efficiency of nanomaterials by drinking water unit processes; and (4) test
the toxicity of nanomaterials in drinking water using a cell culture model system of the epithelium. This study
considers the physical, chemical, and biological implications of nanomaterial fate and toxicity in systems that
will provide insight into the potential for nanomaterials to be present and to pose health concerns in finished
drinking water.
Approach: A multidisciplinary approach is proposed that includes experiments to identify fundamental
uniqueness of nine nanomaterial properties and toxicity and quite applied experiments aimed directly at
understanding the fate and reactions involving nanomaterials in drinking water treatment plants. Advanced
nanomaterial characterization techniques will be employed to determine the size distribution, concentration,
and zeta potential of nanomaterials in buffered distilled water and model waters representative of raw drinking
water supplies (anions, cations, natural organic matter [NOM]). Adsorption of dissolved pollutants (anions,
metals, range of synthetic organic chemicals) and NOM are proposed to quantify the potential for
nanomaterials to transport such compounds and be transformed by the compounds (e.g., via aggregation,
change in zeta potential). Coagulation processes will be studied by compressing the electric double layer of
nanomaterials and exposing nanomaterials to alum coagulations, using mono- and heterodisperse solutions;
comparable filtration work also will be conducted. Adsorption of virus onto nanomaterials and subsequent
disinfectant shielding will be studied. Toxicity screening will include toxicity of nanomaterials on several cell
lines selected to mimic oral ingestion routes in drinking water.
Expected Results: This project will provide fundamental information about the fate, transport, and
transformation of nanomaterials in drinking water resources and the first evidence that such nanomaterials can
or cannot be removed by conventional drinking water treatment processes. An improved assessment will be
developed for the potential exposure risks of nanomaterials in drinking water. This research would ultimately
provide essential information that would support policy and decisionmaking regarding handling, disposal, and
management of nanoscale materials in commerce, manufacturing, and the environment.
EPA Grant Number: R831713
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Pulmonary and Systemic Inhalation Toxicity of Multiwalled
Carbon Nanotubes
Jacob McDonald
Lovelace Respiratory Research Institute, Albuquerque, NM
Inhalation of multiwalled carbon nanotubes (MWCNTs) at particle concentrations ranging 0.3-5 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, 1, or 5 mg/m3 respirable aggregates of MWCNTs for 7 or 14 days (6 hours/day). Histopathology of lungs
from exposed animals showed alveolar macrophages containing black particles; however, there was 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. MWCNT exposures
to 0.3 mg/m3 and higher particle concentrations caused nonmonotonic systemic immunosuppression after 14
days, but not after 7 days. Immunosuppression was characterized by reduced T-cell-dependent antibody
response to sheep erythrocytes, as well as by T-cell proliferative ability in the presence of the mitogen,
Concanavalin A (Con A). Assessment of nonspecific natural killer (NK) cell activity showed that animals
exposed to 1 mg/m3 MWCNTs had decreased NK cell function. Gene expression analysis of selected cytokines
and an indicator of oxidative stress were assessed in lung tissue and spleen. No changes in gene expression
were observed in lung; however, interleukin 10 (IL-10) and NAD(P)H oxidoreductase 1 (NQ01) mRNA levels
were increased in the spleen.
EPA Grant Number: R8'32527
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Pharmacokinetics and Biodistribution of Quantum Dot Nanoparticles
in Isolated Perfused Skin
Nancy A Monteiro-Riviere1', Hyun A. Lee1'2, Leshuai W. Zhang1, Mudassar Imran1'2, Vicki L. Colvin3,
William Yu3, and Tim E. Riviere1'2
Center for Chemical Toxicology Research and Pharmacokinetics, North Carolina State University,
Raleigh, NC; Biomathematics Program, North Carolina State University, Raleigh, NC; Department of
Chemistry, Center for Biological and Environmental Nanotechnology, Rice University, Houston, TX
The disposition and pharmacokinetics of nanoparticles in tissues are crucial parameters for targeting
nanotechnology-based drug delivery systems as well as for defining their toxicological profile. Quantum dots
(QD), nanomaterials that naturally fluorescence, can be synthesized with varying surface coatings that
modulate disposition and are amenable to localization in skin and other tissues due to intense fluorescence. QD
were synthesized with a 8.40 nm x 5.78 nm CdSe core and either polyethylene glycol (PEG) or COOH
coatings. These QD621 have a maximum emission wavelength of 621 nm and a hydrodynamic size (in water)
of approximately 37 nm. Flow-through diffusion cells were used to assess QD penetration through porcine
skin, along with laser scanning confocal microscopy (LSCM). The isolated perfused porcine skin flap (IPPSF)
was used to determine whether intra-arterially perfused QD would distribute to the skin. QD were mixed with
300 mL of media and were intra-arterially infused into the IPPSF (6.67 nM, 3.33 nM, 1.67 nM, or 0.83 nM)
for 4 hours (dose phase), and then QD media was replaced with fresh media and the IPPSF was perfused for an
additional 4 hours (washout phase). Upon termination of the perfusion, the IPPSF was cut into 6 segments,
flash-frozen in liquid nitrogen, cryosectioned at 25 jam, and imaged by LSCM. The arterial and venous
perfusate was sampled and the fluorescence quantitated. Flow-through diffusion cells showed penetration of
QD621 only in the upper stratum corneum layers of skin. This is in contrast to studies with QD565 and QD655
that showed slight coating-dependent epidermal penetration. In the QD621 infusion study, COOH-coated QD
had greater tissue extraction than PEG. Images indicate aggregation of infused QD in the skin vasculature.
Transmission electron microscopy localized QD621 within the capillary walls. A pharmacokinetic model of
arterial-venous extraction and tissue biodistribution of QD was developed based on a model previously used to
quantitate platinum distribution in the same experimental system. Significant arterial-venous QD extraction
was observed at all doses, with COOH QD showing greater predicted tissue deposition, an agreement in line
with the confocal studies above. A unique kinetic finding was periodicity (approximately 90 minutes) in
arterial extraction, an observation not seen after chemical infusions. These data begin to define nanomaterial
characteristics that correlate to tissue uptake and persistence. They are important for risk assessment and drug
delivery, because they suggest that QD not specifically targeted for medical applications can biodistribute to
tissues, have unique pharmacokinetic patterns of arterial extraction, and potentially may cause adverse effects.
EPA Grant Number: R831715
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Metal Nanoparticle Tissue Distribution Following In Vivo Exposures
Alison Elder , Nancy Corson , Robert Gelein , Pamela Mercer , Amber Rinderknecht,
Jacob Finkelstein , and Gunter Oberdorster
Departments of Environmental Medicine and Pediatrics, University of Rochester, Rochester, NY
Manipulation of the physicochemical properties of materials at the nanoscale has the potential to
revolutionize electronic, diagnostic, and therapeutic applications. Because of the potential large-scale use of
nanomaterials, it is important to determine if there is any unique toxicity of the nanoscale materials as
compared to the bulk. As has been hypothesized for nanosized ambient air, or ultrafine particles, nanoparticles
(NP) may evade particle clearance mechanisms at the site of exposure, thus potentially coming into contact
with epithelial and endothelial cells and translocating to sites distant from original deposition. It also is
possible that inflammation and oxidant stress will occur as a result of unique NP properties or from prolonged
retention. In the last year of this project, we have focused on the biodistribution and fate of engineered NP that
were administered via the respiratory tract or systemically. The first 2 years of the project were focused on
detailed physicochemical characterizations of the NP systems we used to test our hypotheses and on the in
vitro uptake and effects of NP, particularly nanosized Pt (flowers, multipeds, flower spheres, and pod spheres;
11-35 nm; 1-27 m2/g). Acellular reactivity assays, as well as in vitro and in vivo assessments of toxicity and
inflammatory potential, revealed that the Pt NP are relatively nontoxic, with activity similar to that of
nanosized Ti02. We quantitated the uptake of the Pt shapes by cultured endothelial cells and found that the
particles with larger surface area per mass (Pt flowers) were taken up to a greater degree than those with
smaller surface area (Pt multipods). Results from preliminary in vivo exposures in rats also showed that Pt
flowers were retained in the lungs to a greater extent than Pt multipods, although neither particle type induced
severe inflammation. We also found that a significant fraction (-80%) of the instilled dose was cleared from
the lungs within the first 24 hours following exposure. To investigate more specifically the impact of particle
surface on tissue distribution, we exposed rats to quantum dots (QDs; CdSe-ZnS core-shell crystals with
polymer cap and biomolecule coating) with three different surface functionalizations (PEG, PEGamine,
carboxylic acid) via intratracheal microspray (ITM) and intravenous (IV) exposures. We found that QDs
delivered via ITM did not induce a severe lung inflammatory response 24 hours after exposure (maximum of
3.7% neutrophils in lavage fluid, carboxyl QDs) and that they did not, for the most part, translocate out of the
lungs. The PEGamine- and carboxyl-coated QDs were found in the lung-associated lymphoid tissue (Cd
signal), but Cd was not detected in any of the other tissues we examined. Following IV exposure, the QD
surface characteristics significantly impacted tissue localization. For example, PEG-coated QDs had the
highest retention in most tissues; however, they did not accumulate in the bone marrow, whereas both the
PEGamine- and carboxyl-coated QDs did. Tissues from exposed rats are currently being examined using
fluorescence microscopy to identify the cell types that might take up QDs in tissues where significantly
elevated Cd signals were found. We are currently performing a more detailed biokinetics study (1 hour, 24
hours, 7 days postexposure) of QD tissue distribution following ITM and IV exposures and collecting excreta
so that we can more fully account for the delivered dose of material. These studies, through comparisons with
other metal NP, are helping to define the biodistribution of nanomaterials as a function of their
physicochemical characteristics and also to establish NP-related effects following in vitro and in vivo
exposures.
EPA Grant Number: R831722
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
The Bioavailability, Toxicity, and Trophic Transfer of Manufactured
ZnO Nanoparticles: A View From the Bottom
Paul M. Bertsch ' , Travis Glenn ' , Brian Jackson ' , Andrew Neal' , and Phillip Williams
Savannah River Ecology Laboratory, Athens, GA; University of Georgia, Athens, GA;
^Dartmouth College, Hanover, NH
Objectives: The overall objectives of this research project are to: (1) evaluate the bioavailability and
toxicity of manufactured nanoparticles (ZnO) as a function of particle size to the model soil bacteria,
Burkholderia cepacia, and the model detritivore, C. elegans, as referenced against aqueous Zn2+; (2) evaluate
the ability of manufactured ZnO nanoparticles to be transferred from one trophic level to the next, as assessed
in the simple food chain consisting of preexposed B. cepacia and C. elegans; and (3) evaluate the synergistic
or antagonistic effects of manufactured ZnO nanoparticles on the toxicity of Cu2+ to B. cepacia and C. elegans.
These three overall objectives will be approached in the context of the following four hypotheses:
• Hypothesis 1: The bioavailability and toxicity of manufactured ZnO nanoparticles increases with
decreasing particle size (i.e., 6 nm versus 80 nm).
• Hypothesis 2: The toxicity of ZnO nanoparticles to B. cepacia and C. elegans is lower than an
equivalent concentration of dissolved Zn +.
• Hypothesis 3: The bioavailability and toxicity of ZnO nanoparticles introduced via trophic transfer
differs from direct exposure.
• Hypothesis 4: ZnO nanoparticles alter the bioavailability and toxicity of dissolved metals.
Approach: We will study the influence of particle size of ZnO nanoparticles, (i.e., 3 nm versus 80 nm) on
bioavailability and toxicity (lethal and sublethal effects) and will compare these results with exposure to an
equivalent concentration of aqueous Zn +. Additionally, we will examine the effect of nanoparticles on the
toxicity of a dissolved constituent (Cu +). We will employ optical and fluorescent microscopy, element-
specific, synchrotron-based microspectroscopy, and hyphenated separations-ICPMS techniques to determine
the distribution of nanoparticles within each organism and potential transformations of nanoparticles.
Additionally, we will employ a transgenically modified strain of C. elegans in which we have incorporated a
metal-specific promoter (metallothionein-2 [mtl-2]) that turns on expression of green fluorescent protein (GFP)
in the presence of bioavailable metals. We expect that the nanoparticles will not switch on the GFP promoter,
but transformations (dissolution) of the nanoparticles that release the free metal will induce GFP expression.
Additionally, this transgenic strain will be used to study the effect of the bioavailability of Cu2+ in the presence
of ZnO nanoparticles and the potential that bioavailability will be lowered as indicated by lower GFP
expression. These observations will be coupled with measurements of lethal and sublethal responses for C.
elegans exposed directly and indirectly from grazing on preexposed B. cepacia, including behavior and
reproduction. We speculate that C. elegans will bioaccumulate greater quantities of ZnO nanoparticles when
feeding on preexposed B. cepacia compared to direct exposure as a result of the likelihood that intracellular
ZnO nanoparticles will be surface-modified by biocompatible molecules (e.g., peptides, proteins, other
intracellular ligands) in B. cepacia.
Expected Results: These studies will provide among the first data on the bioavailability and toxicity of a
widely used nanoparticle/nanocomposite (ZnO) to a model bacteria and detritivore and the first data available
on potential for manufactured nanoparticles to be transferred through the food chain. The general lack of
information on the bioavailability and toxicity of manufactured nanoparticles to microorganisms and higher
organisms and on the ability of manufactured nanoparticles to be transferred from prey to predators leads to a
number of very basic questions that will need to be resolved to ensure that the potential human health and
ecological risks associated with the widespread use and disposal of manufactured nanoparticles are properly
evaluated. This project will provide critical information needed to begin to bridge these knowledge gaps.
EPA Grant Number: R832530
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Biochemical, Molecular, and Cellular Responses of Zebrafish Exposed
to Metallic Nanoparticles
DavidS. Barber, Nancy Denslow, Kevin Powers, and David Evans
University of Florida, Gainesville, FL
The goals of this research 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, we have examined the behavior of metal particles in aqueous
environments 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. We, therefore, conclude that copper
nanoparticles exert a toxic effect on zebrafish gill that is not solely the result of dissolution of the particles.
NSF Grant Number: BES-0540920
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Acute and Developmental Toxicity of Metal Oxide Nanoparticles
to Fish and Frogs
Christopher Theodorakis1, Elizabeth Carraway2, and George Cobb3
Southern Illinois University, Edwardsville, IL; 2Clemson University, Clemson, SC;
Texas Tech University, Lubbock, TX
Objectives: The objectives of this research 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 will coincide with the relative toxicity of their soluble salts or oxides.
Approach: 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 postfertilization) for minnows and 10-week exposures (hatch until metamorphosis completion) for
X. laevis. Endpoints will include survival, growth, percent hatch, developmental abnormalities, and rate of
metamorphosis (for X. laevis). 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 conducted 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.
Expected Results: 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 groundwater 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 Workshop on the Environmental Implications of Nanotechnology
Mechanistic Dosimetry Models of Nanomaterial Deposition
in the Respiratory Tract
Bahman Asgharian and Brian A. Wong
CUT Centers for Health Research, Research Triangle Park, NC
Objective: Accurate health risk assessments of inhalation exposure to nanomaterials will require
dosimetry models that account for interspecies differences in dose delivered to the respiratory tract.
Mechanistic models offer the advantage to interspecies extrapolation that physicochemical properties of
particles and species differences in ventilation, airway architecture, and physiological parameters can be
incorporated explicitly to describe inhaled dose. The objective of this research is to extend existing, verified
mechanistic models of particle deposition in the respiratory tract of rats and humans both to cover the range of
size for nanoparticles and nanotubes. Deposition mechanisms are described based on first principles and semi-
empirically as required. Semi-empirical models of penetration from the upper respiratory tract (URT) also can
be used to describe regional deposition fraction in the URT and could be extended to localized modeling. The
approach includes model verification with experimental data obtained both in human and rat casts of the upper
respiratory tract as well as in vivo studies of respiratory tract deposition.
Approach: Manufactured nanoparticles and nanotubes will be obtained from manufacturers and
generated in our laboratories. Deposition of nanomaterial will be measured in nasal casts of humans and rats.
These data will allow calculation of the fraction of inhaled material that passes through the URT and enters the
lower respiratory tract (LRT). Next, existing models of LRT deposition will be extended to include
mechanisms for nanomaterial. For nanoparticles, existing models for fine and coarse particles will be extended
by accounting for the mechanisms of axial diffusion and mixing. This will address the previous inadequate
treatment of dispersive effects in the existing models that has limited their applicability to nanosized particles.
For nanotubes, deposition depends on nanotube orientation in the air. Net orientation of a cloud of nanotubes
entering each airway will be found to calculate their deposition. A software package with a graphical-user
interface will be developed to provide rapid computational capabilities to run simulations based on these
models. A series of nose-only exposure events in Long-Evans rats will be conducted to measure regional and
lobar deposition of nanoparticles in the respiratory tract. Deposition models will be verified in rats by
comparing deposition predictions against measurements from nose-only exposures, and in humans by
comparing the model predictions against available data in the literature.
Expected Results: This effort will result in mechanistic dosimetry models to predict the localized
deposition of inhaled nanomaterial in the respiratory tract of rats and humans. Specific products include:
• Deposition measurements of nanosized particles in casts of human and rat nasal URT airways
• Semi-empirical relationships to predict nanomaterial deposition in the URT airways
• Respiratory tract deposition models of nanoparticles and nanotubes in humans and rats
• Measurements of regional and lobar deposition of nanomaterial in the heads and lungs of rats
• A user-friendly software package to implement models and provide rapid simulation capability.
EPA Grant Number: R832531
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Nanostructured Materials for Environmental Decontamination
of Chlorinated Compounds
Yunfeng Lu and Vijay T. John
Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA
This research project is directed towards the development of novel mesoporous materials that act as
supports for zerovalent iron nanoparticles used in the breakdown of chlorinated compounds. Halogenated
organic compounds, such as chlorinated aromatics, chlorinated aliphatics, and polychlorinated biphenyls, are
typical of dense nonaqueous phase liquids (DNAPLs) that are prevalent at contaminant sites. In recent years,
the use of zerovalent iron has represented a promising and innovative approach to the destruction of these
compounds. Of particular interest is the number of publications recently that describe the use of nanoparticles
of iron (Fe) in remediation through hydrodechlorination. The enormous surface area of nanoparticles leads to
enhanced efficiencies. Additionally, the colloidal nature of Fe nanoparticles indicates that these materials may
be pumped to contaminated sites. Alternatively "funnel and gate" treatment systems may be devised, where
porous barriers of iron particles are constructed in the path-contaminated groundwater plumes.
Due to the high surface energy of nanoparticles, iron nanoparticles tend to aggregate, leading to larger
units that do not maintain colloidal stability. Although Fe nanoparticles that exceed 10-15 nm exhibit
ferromagnetism, this also leads to aggregation and inefficient transport. Finally, Fe is hard to functionalize
with organic compounds to attempt to maintain stability in aqueous or in organic systems. Our technical
approach combines the simplicity and affordability of the sol-gel processing techniques for ceramic synthesis
with the efficiency and spontaneity of surfactant/silica cooperative assembly to manufacture nanostructured
decontamination materials. We use a simple aerosol processing technique to encapsulate Fe nanoparticles in
silica microparticles that can be easily functionalized, leading to facile transport to trichloroethylene (TCE)
interfaces and partitioning at the TCE-water interface. Sample morphologies of such particles are shown
below. Our results indicate the following: (1) functionalized composite particles significantly adsorb TCE; (2)
composite particles are effective in TCE decontamination; (3) composite particles partition to the TCE-water
interface; and (4) composite particles have optimal size characteristics to be effective in transport through
sediments. Representative particles are shown below.
EPA Grant Number: GR832374
The Office of Research and Development's National Center for Environmental Research
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Interagency Workshop on the Environmental Implications of Nanotechnology
Responses of Lung Cells to Metals in Manufactured Nanoparticles
John Veranth, Christopher A. Reilly, and Garold S. Yost
University of Utah, Salt Lake City, UT
Objective: This research project is based on the hypothesis that transition metals in particles induce
proinflammatory signaling and cell damage through the production of reactive oxygen species. Established cell
culture models and toxicology assays will be applied to the analysis of manufactured nanomaterials. Based on
the literature and our own data, we expect that the small physical size and high surface area of nanoparticles (d
< 30 nm) will increase cellular uptake and increase induction of proinflammatory signaling, compared to larger
particles with the same elemental composition. In vitro studies with human and rat lung cells will evaluate the
effects of manufactured nanoparticles in the as-sold condition, and the same materials after the particles have
been subjected to surface modification simulating fire and wastewater treatment conditions. The emphasis will
be on lower cost nanomaterials that are sold in powder or liquid suspension form, because these materials are
expected to be produced and ultimately released in the largest amount.
Approach: A phased approach will be used to maximize useful results within the budget. In the first
phase, low-cost assays will be used to screen a wide range of samples with sufficient replicates for statistical
power. This phase will emphasize measurement of cytotoxicity, induction of the proinflammatory cytokine IL-
6, and dissolution rate in simulated lung fluid. Industrial collaborators will assist in prioritizing materials for
testing and in providing chemically similar materials of various sizes and grades. Materials selected in the
screening phase will be used for more detailed, mechanistic studies. The second phase will test selected
materials for particle uptake by the cells, for the induction of additional cytokines, and for the effect of
antioxidants. Phase two physical characterization will include electron microscopy, BET surface area, zeta
potential, and trace element analysis. In the third phase, the most inflammatory and most benign nanomaterials
will be used in hypothesis-based toxicology experiments to evaluate plausible mechanisms by which the
particles induce specific responses in cells. Cell culture toxicology studies with BEAS-2B cells, an
immortalized human lung epithelial cell line, are emphasized and consistent with the goal of refining,
reducing, and replacing animal use. However, it is necessary to establish the relevance of cell culture data to
whole animals and to human health. Experiments using normal macrophages and normal epithelial cells that
are freshly harvested from rats will be conducted to test the ability of the cell culture assays to predict the
induction of inflammation by specific nanomaterials.
Expected Results: The screening phase will provide new data on a range of commercially available
nanoparticles, using a consistent set of physical and cell culture assays to facilitate comparisons between
materials. The surface modification studies will contribute to understanding the environmental fate of
nanoparticles by evaluating whether the treatments enhance or decrease the biological effects of specific
nanomaterials. The evaluation of plausable mechanisms and the experiments with freshly isolated rat airway
cells will provide a transition between cell culture studies, inhalation studies, and extrapolation to sensitive
human populations.
EPA Grant Number: R831723
The Office of Research and Development's National Center for Environmental Research 10
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Interagency Workshop on the Environmental Implications of Nanotechnology
A Toxicogenomics Approach for Assessing the Safety of Single-Walled Carbon
Nanotubes in Human Skin and Lung Cells
Mary Jane Cunningham1, Edward R. Dougherty2, and Daniel E. Resasco3
1 The Houston Advanced Research Center, The Woodlands, TX; 2Texas A&M University,
College Station, TX; University of Oklahoma, Norman, OK
High-throughput biotechnologies were used to screen for toxicity of nanomaterials in this combined
toxicogenomics and systems biology approach. Primary human epidermal keratinocytes and primary human
bronchial epithelial cells were exposed in vitro for 24 hours to single-walled carbon nanotubes and other nano-
and low-micron-scale particulate substances. RNA isolated from the cell pellets was copied, labeled, and
hybridized onto gene expression microarrays containing between 10,000 and 20,000 human genes. A complete
comparison between these two cell systems, using a four-tiered bioinformatics approach, was performed.
Statistical analysis showed that the triplicate arrays run for each biological sample was very reproducible.
Hierarchical agglomerative clustering showed that the greatest variation between gene expression profiles was
between the two cell systems, regardless of nanomaterial exposure. Potential biomarkers were identified, and
several correlated with previous literature references. Pathway analysis showed that the active pathways in
both cellular systems were genes and proteins involved in membrane integrity and remodeling.
NSF Grant Number: 0536679
The Office of Research and Development's National Center for Environmental Research 11
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Interagency Workshop on the Environmental Implications of Nanotechnology
Microbial Impacts of Engineered Nanoparticles
Delina Y. Lyon and Pedro J.J. Alvarez
Department of Civil and Environmental Engineering, Rice University, Houston, TX
Fullerenes compose a class of nanomaterials that show potential for imminent medical, industrial, and
technological applications. One model fullerene, C6o, is insolube in water, but will form a suspension termed
nC6o upon extended exposure to water or after introduction to water via a solvent. The microbial impacts of
nC6o are analyzed using the bacteria Escherichia coli and Bacillus subtilis as indicator species of both
environmental impact and potential toxicity to higher level organisms. nC6o displayed strong antimicrobial
properties against both bacteria, with a number of factors, such as salt concentration and particle size,
mitigating toxicity. Several eukaryotic studies have implicated reactive oxygen species (ROS) as the mediators
of toxicity. However, ROS may not be the only factor responsible for killing prokaryotic cells, as the
antibacterial activity of nC6o persists in the absence of light and oxygen, challenging the feasibility of
photocatalytic ROS formation. Other research on fullerenes suggests that they exert their antibacterial effect
via direct damage to the cell membranes. This research project explores three possible mechanisms for the
antibacterial activity of nC6o. It could: (1) physically disrupt the cell membrane; (2) generate ROS; or (3) exert
ROS-independent oxidative stress. Results from flow cytometry analysis and other analytical techniques point
to nC6o acting as a direct oxidant, possibly requiring direct contact with the cell. Defining the antibacterial
mechanism allows the manipulation of the antibacterial activity for both disinfection applications and
mitigation of undesired environmental impacts.
EPA Grant Number: R832534
The Office of Research and Development's National Center for Environmental Research 12
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Interagency Workshop on the Environmental Implications of Nanotechnology
An Integrated Approach Toward Understanding the Inflammatory
Response of Mice to Commercially Manufactured CuO/Cu, Fe2O3/Fe,
and TiO2 Nanoparticles
Vicki Grassian
The University of Iowa, Iowa City, IA
As applications of nanoscience and nanotechnology in a wide-range of commercial uses continue to
expand, there is growing interest in understanding the environmental and health implications of nanomaterials.
Inhalation of manufactured nanomaterials may be one potential route for nanoparticle exposure to humans. The
implications of exposure to these airborne nanoparticles need to be determined through exposure studies of
well-characterized nanoparticles. By evaluating which materials are more likely to cause deleterious health
effects before large amounts are introduced in the environment, the risk and resources that would be devoted
toward trying to eliminate and replace the materials may be avoided. In this research, we have fully integrated
studies of the physical and chemical properties of commercially manufactured nanoparticles with inhalation
toxicological studies of these same nanoparticles to determine those properties that most significantly affect
toxicity. Using murine models for inflammation, inhalation exposures of CuO/Cu, Fe203/Fe, and Ti02
nanoparticles were investigated to determine how size and composition affects inflammatory response.
EPA Grant Number: R831717
The Office of Research and Development's National Center for Environmental Research 13
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Interagency Workshop on the Environmental Implications of Nanotechnology
Hysteretic Accumulation and Release of Nanomaterials in the Vadose Zone
Tohren C.G. Kibbey and David A. Sabatini
School of Civil Engineering and Environmental Science, University of Oklahoma, Norman, OK
Objectives: Manufactured nanomaterials are increasingly being considered for use in a wide range of
applications, and their use is projected to expand substantially during the next 10 years as costs decrease and
new applications are discovered. At present, little is known about the fate, transport, or transformation of
nanomaterials in the environment, or their inherent risks to human or environmental health. The objective of
this project is to study the vadose zone accumulation and release of a wide range of manufactured
nanomaterials, with emphasis on hysteretic interactions with air/water interfaces and specific mineral surfaces.
Nanomaterials can enter the vadose zone through infiltration of atmospheric dispersions, or from groundwater
contaminated by landfill leachate or other sources. Depending on the nature of the materials and interactions
with critical interfaces, the vadose zone may either provide a sink for nanomaterials, preventing their spread
throughout the environment, or become a long-term contaminant source.
Approach: This research project will be conducted through three primary tasks. Task 1 (batch
adsorption/adhesion experiments) is designed to assess adsorption/adhesion affinities with critical liquid/solid
and liquid/air interfaces. Task 2 (saturated deposition/dispersion transport experiments) is designed to evaluate
dynamic interactions between nanomaterials and mineral surfaces. Task 3 (dynamic hysteretic unsaturated
transport experiments) is designed to provide detailed information about the effects of wetting/drying history,
infiltration, and unsaturated soil behavior on the accumulation and release of nanomaterials. These tasks use a
range of experimental systems to study specific mechanisms influencing the dynamic accumulation and release
of manufactured nanomaterials in the vadose zone, and make extensive use of inline detectors to
simultaneously track concentration, particle size, and zeta potential distributions. A novel technique for
measurement of air/water interfacial area throughout hysteretic wetting/drying cycles will provide fundamental
experimental information about the role of wetting state history and air/water interfacial areas in the
accumulation and release of nanomaterials. Nanomaterials selected for this work cover a wide range of
structures, compositions, and physical and chemical properties, in addition to different potential applications.
The solid media selected for this work will include fully characterized whole soils and aquifer materials, as
well as critical mineral subsets of the whole materials. An unsaturated flow and transport modeling effort
conducted as a part of Task 3 will integrate the results of experimental tasks.
Expected Results: This research project will provide significant benefits to society in terms of improved
a priori assessment of manufactured nanomaterial mobility in the environment and associated risk. Outcomes
of the work will provide indications about the classes of nanomaterials most likely to accumulate in the vadose
zone, the roles of mineral surfaces, air/water interfacial areas, and wetting/drying history on accumulation.
This work will provide essential new information necessary to assess the mobility of manufactured
nanomaterials in the environment and the role of vadose zone interactions in decreasing or increasing ultimate
risk to human or environmental health.
EPA Grant Number: R832529
The Office of Research and Development's National Center for Environmental Research 14
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Carbon-Based Nanomaterials
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Interagency Workshop on the Environmental Implications of Nanotechnology
Role of Particle Agglomeration in Nanoparticle Toxicity
Terry Gordon, Lung Chi Chen, and Beverly S. Cohen
New York University School of Medicine, Tuxedo, NY
Objective: The objective of this study is to determine the biological consequences of nanoparticle
agglomeration. We 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 we 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.
Our 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. We will expand
upon these findings and identify whether realistic exposure conditions, which 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.
Approach: To test the hypothesis that there is a difference in the toxicity of fresh (predominantly singlet)
versus aged (predominantly agglomerated) nanoparticles, we 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, we also
will generate particles with different amounts of metal content, as found in carbon nanoparticles generated by
metal catalysts.
Expected Results: As determined in preliminary studies, we expect 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, our 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 will address a number of the research needs identified in this
solicitation, including toxicity and exposure assessment.
EPA Grant Number: R832528
The Office of Research and Development's National Center for Environmental Research 15
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Interagency Workshop on the Environmental Implications of Nanotechnology
Chemical and Biological Behavior of Carbon Nanotubes
in Estuarine Sedimentary Systems
P. Lee Ferguson, G. Thomas Chandler, and Watty A. Scrivens
University of South Carolina—Columbia, Columbia, SC
Objectives: The general objectives of this research project are to: (1) determine factors controlling the
fate of single-walled carbon nanotubes (SWNTs) and their synthetic byproducts in estuarine seawater,
sediment, and sediment-ingesting organisms; (2) examine the impact of SWNTs and byproducts on the
disposition of model organic contaminants in estuarine sediments; (3) determine whether the presence of
SWNTs and byproducts in estuarine sediments affects the bioavailability of model organic contaminants to
estuarine invertebrates; and (4) assess the toxicity of SWNTs and byproducts to suspension- and deposit-
feeding estuarine invertebrate models in seawater suspension alone and/or in combination with estuarine
sediments.
Approach: Our research plan will address these objectives through a series of experiments designed to
provide a holistic picture of the behavior of SWNTs and their synthetic byproducts upon entry into the
estuarine environment. These experiments will include tracing the fate and phase-association of 14C-SWNTs
and byproducts under simulated estuarine conditions and through ingestion by deposit-feeding organisms;
batch sorption studies to examine the affinity of SWNTs for model hydrophobic organic contaminants (HOC)
in the estuarine environment; laboratory-scale bioaccumulation experiments designed to test modulation of
HOC bioavailability by co-occurring SWNTs in estuarine sediments; and dose-response experiments designed
to test the potential for SWNTs and byproducts to directly cause adverse effects on a sensitive estuarine
infaunal invertebrate (the harpacticoid copepod Amphisascus tenuiremus).
Expected Results: This project will, for the first time, address the physical, chemical, and biological
behavior of novel and emerging carbon nanotube materials under environmental conditions typical of
estuaries. In total, we will address not only the potential for SWNTs to be transported, accumulate, and cause
direct deleterious effects within estuarine environments, but also the potential for linked effects on the
biological and chemical behaviors of known priority pollutants common in estuarine sediments. This combined
approach represents a novel way of addressing the environmental impact of an emerging synthetic
nanomaterial, and thus will provide the U.S. Environmental Protection Agency and the scientific community
with an entirely new and highly relevant dataset for risk assessment of SWNT-derived contaminant discharge.
Further, the work will generate new scientific knowledge related to the behavior of these highly novel
nanomaterials under conditions not normally tested in the course of nanoscience research (e.g., nonmammalian
biological systems, highly saline aqueous solutions, and complex sediment media). This knowledge may
become useful in designing new nanoscale technologies in, for example, environmental engineering or "green"
manufacturing techniques.
EPA Grant Number: R831716
The Office of Research and Development's National Center for Environmental Research 16
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Interagency Workshop on the Environmental Implications of Nanotechnology
Fate and Transformation of C60 Nanoparticles in Water Treatment Processes
Jaehong Kim and Joseph Hughes
Georgia Institute of Technology, Fulton, GA
The environmental impact of carbon fullerenes is of great concern due to projections for bulk production
in the near future and the recent discovery that they form nanoscale water-stable aggregates upon release to
water. Understanding the fate and the transformations of carbon fullerenes during water treatment, currently
our first line of defense against ingestion pathways, is of particular importance. Human exposure to these
materials via water ingestion will be strongly influenced by the behavior of these aggregates in potable water
treatment systems.
Objective: The objective of this research project is to examine the response of water-stable fullerene
aggregates to processes that are used in potable water treatment, using C6o and its stable aggregate, nano-C6o,
as a model compound. More specifically, this project will test the following hypotheses:
• Nano-Ceo with an electron-rich surface will undergo chemical transformation through addition of
oxygen or chlorine atom and/or charge destabilization when subjected to oxidation by commonly used
oxidants and disinfectants such as ozone, UV light, free chlorine, and monochloramine.
• A unique, weakly negatively charged surface of nano-C6o will lead to unique electrostatic and
hydrophobic interactions with metal hydroxide-soluble complexes and precipitates, with polymeric
membrane surfaces, and with hydrophobic surfaces of activated carbon.
• The size characteristics of nano-C6o will lead to unique filtration characteristics when filtered through
nanoporous membranes and unique adsorption kinetics/equilibrium characteristics when adsorbed by
activated carbons with varying pore-size distributions.
Expected Results: The outcome of this research project will provide basic, fundamental, yet practical
knowledge in chemical and physical behavior of this nanomaterial during commonly practiced engineering
processes. New information, such as colloidal stability, chemical reaction kinetics, reaction product identity,
transport behavior, and adsorptive characteristics, will advance scientific knowledge in use, disposal, and
treatment of this growing class of materials and will trigger additional research on water treatment
technologies and facilitate appropriate toxicological studies.
EPA Grant Number: R832526
The Office of Research and Development's National Center for Environmental Research 17
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Interagency Workshop on the Environmental Implications of Nanotechnology
Cross-Media Environmental Transport, Transformation, and Fate of Manufactured
Carbonaceous Nanomaterials
Peter J. Vikesland, Linsey C. Marr, Joerg Jinschek, Laura K. Duncan,
Behnoush Yeganeh, andXiaojun Chang
Department of Civil and Environmental Engineering, Virginia Tech, 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, we are interested in how these particles behave when transferred from water to air or vice versa.
This project 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 project employed an
aggregate production method believed to emulate more closely the fate of fullerene upon 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.
EPA Grant Number: R832534
The Office of Research and Development's National Center for Environmental Research 18
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Interagency Workshop on the Environmental Implications of Nanotechnology
Transport and Retention of Nanoscale Fullerene Aggregates
in Water-Saturated Soils
Kurt D. Pennell1'2, Joseph B. Hughes1, Linda M. Abriola3, Yonggang Wang1, Yusong Li3,
and John D. Partner
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA;
Department of Neurology, Center for Neurodegenerative Disease, Emory University, Atlanta, GA;
3Department of Civil and Environmental Engineering, Tufts University, Medford, MA
The goal of this research project is to advance our understanding of nanoscale fullerene (n-C6o) 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 n-C6o
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 n-C6o 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. The
resulting suspension contained approximately 3.0 mg/L of n-C6o aggregates with an average diameter of 95
nm, as determined by dynamic light scattering (DLS). In the first set of experiments, a pulse of suspended n-
C6o solution was introduced into water-saturated columns packed with either 40-50 mesh glass beads or
Ottawa sand at a Darcy velocity of 2.8 m/d. Effluent samples were collected continuously and analyzed by
ultraviolet (UV) spectrometry to determine the C6o concentration, and by DLS to monitor changes in C6o
aggregate size. Following each experiment, the column was sectioned into 1-cm increments, extracted in water
with sonication, and analyzed by UV spectrometry to determine n-C6o retention profiles. In the presence of 1.0
mM CaCl2, n-C6o effluent concentrations gradually increased to a maximum value and then decreased sharply
upon re-introduction of the n-C6o-free solution. Retention of n-C60 in the glass bead columns ranged from 8 to
49 percent of the introduced mass, while up to 77 percent of the injected mass was retained in Ottawa sand
columns. The observed retention capacities were consistent with the delayed breakthrough of n-C6o observed in
the Ottawa sand columns and were corroborated by batch retention measurements. In the absence of
background electrolyte, effluent n-C6o concentrations coincided with those of a nonreactive tracer (Br~),
demonstrating the important role of electrostatic interactions in n-C6o transport and retention. A second set of
n-C60 transport experiments was conducted at several pore-water velocities in columns packed with various
size fractions of Ottawa sand. Decreasing flow rate and smaller grain size resulted in greater n-C6o retention,
with nearly complete retention observed with 80-100 and 100-140 mesh Ottawa sand.
The n-C6o effluent concentration and retention data were simulated using a mathematical model that
incorporated nonequilibrium attachment kinetics and a limiting retention capacity term. The numerical model
successfully captured the characteristics of both the effluent concentration and particle retention profiles.
Experimental and simulation results suggest that n-C6o aggregate attachment is strongly dependent on porous
media surface area and flow rate. Simulated attachment capacity increased with increased specific surface area,
and for a given sand size fraction, simulated n-C6o attachment rates were greater at higher flow rates. Extended
Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, which included van der Waals, electrostatic repulsion,
and hydrophobic interaction forces, was used to evaluate potential mechanisms governing n-C6o attachment.
This analysis suggests that an energy barrier of about 27 kT exists between n-C6o aggregates and Ottawa sand
surfaces, with a secondary minimum attraction region of 0.3 kT. Attachment rate coefficients derived from
secondary energy minimum theory were found to be in close agreement with those fit using the numerical
model. Additional studies are being conducted to further elucidate the mechanisms responsible for n-C6o
transport and retention as a function of ionic strength, grain size, flow rate, and the presence of stabilizing
agents.
EPA Grant Number: R832535
The Office of Research and Development's National Center for Environmental Research 19
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Interagency Workshop on the Environmental Implications of Nanotechnology
Repercussion of Carbon-Based Manufactured Nanoparticles on Microbial
Processes in Environmental Systems
Ronald F. Turco, Bruce M. Applegate, and Timothy Filley
Purdue University, West Lafayette, IN
The use of nanotechnology has tremendous potential for economic growth and is a key feature of
sustainable development. Despite the impending increase in industrial production and the certain releases of
Carbon-Based Manufactured Nanoparticles (CMNP) to the environment, almost nothing is known about their
environmental impact. To engage in a publicly transparent evaluation of risks and benefits and to develop
public policy and technology to manage potential risks, fundamental scientific environmental research must be
completed. The goal of this research project is to provide fundamental information about the impact of CMNP
on water, soil, and subsurface ecosystems.
Objective 1: We propose that there will be a shift in the structure of soil microbial populations in systems
exposed to CMNP because the nanomaterial will exert pressure on the microbial population.
Approach: The intrinsic features describing activity will be estimated in four ways. We will: (1) draw
information from the ratio of key fatty acids taken from the phospholipid fatty acids (PLFA) fraction and relate
it to a background status of the soil microbial populations; (2) use genetic approaches (e.g., density gradient
gel electrophoresis [DGGE] with both bacterial and fungal primers); (3) use enzyme assays for dehydrogenase,
urease, and cellubiase; and (4) use respiration and trapping of C02 to estimate aerobic activity in the presence
of the CMNP.
Objective 2: The long-term fate of CMNP in the environment and their entrance into soil and aquatic
biogeochemical cycles will mostly be a function of the activity of the specific oxygenase, ligninase, laccase,
and fenton systems resident in microbial populations.
Approach: Using 13C-fullerenes in soil microcosm studies outlined in Hypothesis 1, we will track CMNP
carbon to determine how the soil microbial biomass responds to CMNP. We also will assess the degree to
which CMNP carbon is assimilated into microbial biomass, or is converted to a form bound with soil carbon.
Additionally, we will inoculate various litter forms (wood and leaves) spiked with C-labeled fullerene with
aggressive decay fungi where our goal is to assess the degree to which CMNP carbon is assimilated into fungal
biomass or converted to functionalized forms (free and bound).
Objective 3: Water-borne CMNP represent an, as yet, unassessed toxicological risk to aquatic organisms
because of their capacity to physically interact with cell membranes and possibly causing harm to the cells.
Approach: We will use a lux-gfp-based assay to estimate the impact of the CMNP on the processes of
respiration and growth, allowing us to arrive at the first CMNP structure-to-microbial function model. This
objective will involve monitoring bacterial bioluminescence to evaluate the impact of CMNP (amount or
structure) on bacterial response in aqueous systems.
Expected Results: The expected results of this research are very substantial. The knowledge gained from
our research will be used by governments and industry for developing public policy and technology for the
management of any environmental risks from CMNP. The research can be integrated with educational
programs and used to disseminate knowledge about the behavior of nanomaterials.
EPA Grant Number: R831720
The Office of Research and Development's National Center for Environmental Research 20
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Interagency Workshop on the Environmental Implications of Nanotechnology
Size Distribution and Characteristics of Aerosol Released From Unrefined
Carbon Nanotube Material
Judy Q. Xiong, Maire S.A. Heikkinen, and Beverly S. Cohen
Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY
Carbon nanotubes (CNTs) are among the most dynamic and fast-growing nanomaterials due to their novel
properties. The potential of human exposure to this new type of material in the workplace, as well as in the
general environment, is rising, and its impact on human health is of great concern.
In this study, we have investigated the size distributions of airborne CNT particles that were laboratory-
generated by using a vortex agitator and dispersed with a very low flow of HEPA-filtered air. The number-
weighted particle size distributions were monitored by a 13-stage Electrical Low Pressure Impactor (ELPI) and
a 6-stage Integrating Screen Diffusion Battery (ISDB). Several industrial-grade unrefined CNT samples (raw
materials) of various types have been examined, including single-walled, double-walled, and multiwalled
nanotubes. The CNT samples were collected onto the aluminum substrates placed on each stage of the ELPI.
For ISDB sampling, the samples were collected on an array of stainless steel screens, as well as mica discs
attached on the wall between the screens. The experimental data demonstrated that all types of CNT raw
materials examined can be dispersed into the air to a significant extent. The sizes of particles generated were
widely distributed across all 13 stages of the ELPI, including the filter stage ranging from 7 nm to 10 ^m. The
ISDB results showed that the particles released from CVD-SWCNT material (HP-grade, Helix, TX) have a
solo peak under 10 nm, with a mode of 2.5 nm and GSD of 1.24 in number-weighted distributions. The
experimental data also showed that the size distributions varied with the type of CNTs and with the methods
by which they were manufactured. The image analysis results by Atomic Force Microscopy showed that the
CNTs tend to agglomerate rather than exist as single particles, physically.
These results suggest that CNTs can possibly become airborne under certain agitation conditions during
manufacturing and handling processes and can expose workers via inhalation and dermal absorption. As
deposition efficiency and sites of inhaled particles within the respiratory system largely depend on particle size
distribution, the deposition pattern of agglomerated CNT should be similar to those larger, equivalent-sized
nonagglomerated particles. Nevertheless, entrained particles depositing on/in the deep lung surfaces of the
bronchioles or alveoli will contact pulmonary surfactants in the surface hypophase and the agglomerated CNT
are likely to (ultimately) be de-agglomerated. Therefore, to investigate human exposure to airborne CNTs, the
full-size range of inhalable particles must be taken into account.
NIOSH Grant Number: R01 OH008282
The Office of Research and Development's National Center for Environmental Research 21
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Interagency Workshop on the Environmental Implications of Nanotechnology
Physical and Chemical Determinants of Carbon Nanotube Toxicity
Robert Hurt and Agnes Kane
Brown University, Providence, RI
There is real opportunity to reduce carbon nanotube health risks by understanding toxicity mechanisms
and modifying the specific material features that trigger those mechanisms. This research project considers the
role of two characteristic nanotube features: catalytic impurities and hydrophobic surface area. Electron
micrographs show that most nanotube catalyst particles are encapsulated by carbon shells, which has led to the
widespread impression that the metal is fluid inaccessible and unavailable for known biomolecular toxicity
pathways. This project describes quantitative assays for the bioavailability of CNT nickel, iron, and yttrium in
model extracellular fluids and phagolysosomal simulants. Toxicologically significant amounts of nickel and
iron are released from 12 commercial nanotubes, both as-produced and "purified," and this bioavailability
depends on material stresses (sonication, oxidation), physiological fluid properties (pH, ligands), and sample
age. We also present preliminary work on the selective removal of the bioavailable portion of the metal as a
potential detoxification strategy. Finally, amino acid and vitamin profiling is used to probe the effect of
hydrophobic surface area on cell culture media. We find that single-wall nanotubes inhibit HepG2 cell
proliferation by an indirect mechanism involving dose-dependent media depletion by physical adsorption of
small-molecule solutes, especially folate.
EPA Grant Number: R831719
The Office of Research and Development's National Center for Environmental Research 22
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Interagency Workshop on the Environmental Implications of Nanotechnology
Environmental Impacts of Nanomaterials on Organisms and Ecosystems:
Toxicity and Transport of Carbon-Based Nanomaterials Across Lipid Membranes
Dmitry I. Kopelevich1, Jean-Claude J. Bonzongo2, and Gabriel Bitton2
Departments of Chemical Engineering and2Environmental Engineering Sciences,
University of Florida, Gainesville, FL
The dramatic increase in production rates of nanomaterials (NM) and the anticipated widespread use of
engineered nanoparticles in commercial and industrial applications suggest that NM will inevitably enter the
environment, including the biosphere. These expectations stem from the high potential of nanotechnology to
substantially benefit human societies by creating new means of detecting pollutants, cleaning polluted waste
streams, recovering materials before they become wastes, and expanding the currently available resources, to
name a few. In this research, we hypothesize that NM could lead to environmental dysfunctions due to: (1) the
potential toxicity of these materials and their derivatives; (2) the nanometer-size that makes manufactured
nanomaterials prone to biouptake/bioaccumulation; and (3) the large surface area which might lead NM to act
as carriers/delivers of pollutants adsorbed onto them. Our objectives are to: (1) assess the toxicity of
nanomaterials on biota using short-term micro-biotests and investigate the impacts of NM on microbial-driven
ecological functions; (2) determine the mobility of metal-based and carbonaceous NM in porous media as well
as the toxicity of NM in soil leachates; and (3) determine possible mechanisms of toxicity of different types of
nanomaterials.
Through laboratory studies, the potential toxicity of tested NM was assessed using: (1) the Ceriodaphnia
dubia acute toxicity assay; (2) the Selenastrum capricornutum (or P. subcapitatd) chronic toxicity test; and (3)
MetPLATE™, an enzyme-based test specific to metal toxicity. The impacts of NM on selected ecosystem
functions, such as the microbial degradation of organic matter, were assessed using sediment slurries, while
ongoing studies using soil columns investigated the fate and transport of NM in porous media. In addition to
the above experimental work, a model is being developed to investigate the interactions of NM with cellular
membranes. Our modeling studies are performed using a coarse-grained molecular dynamics (CGMD) model
(molecular dynamics simulations), which approximates small groups of atoms as a single united atom.
In this study, we model cell membranes 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 NM with membrane lipids plays a dominant role in mechanisms of cytotoxicity.
Although our ongoing research deals with nanometallic particles (e.g., Ag, Cu, Co, Ni, and nano-metal
oxides), quantum dots (e.g., CdSe, CdS), and carbonaceous NM (i.e., C6o, SWNT, MWNT), this presentation
will be limited to C6o and model carbon nanotube data only.
Based on the above-mentioned toxicity tests, we first examined the toxicity of different solvents (e.g.,
THF, SDS, SDBS, PVP, Triton X-100, Triton X-15, Sodium cholate, Gum Arabic) that potentially could be
used to obtain highly dispersed fullerene suspensions. Our results show that most solvents are very toxic, even
at trace levels (e.g., 0.005% V/V). Therefore, the presence of trace levels of toxic solvents in samples used in
organism-based toxicity tests easily could lead to erroneous results. Second, using aqueous C6o suspensions
prepared by a procedure adapted from that of Degushi (by making sure that the residual THF level in control
water samples, if any, produces no toxicity), both the invertebrate- and algal-based toxicity tests showed the
negative impact of tested NM, with the algal-based test being more sensitive than the invertebrate-based test.
The prepared aqueous C6o suspension also was used to spike both lake and wetland sediments. In this latter
case, a clear negative impact on rates of acetate decomposition by microbial sedimentary processes was
observed. Third, the computational investigation of molecular mechanisms of NM toxicity focusing on
interactions of nanoparticles with cell membranes is being conducted, in parallel with the above-described
laboratory experiments. For model carbon-based NM (C6o and carbon nanotubes), we observed an extremely
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Interagency Workshop on the Environmental Implications of Nanotechnology
small barrier for the permeation of the NM into the hydrophobic interior of a lipid bilayer. Conversely, the
calculated residence time of the NM within the bilayer interior is very large, which possibly could lead to
destabilizing interactions between NM and the membrane. We have initiated theoretical studies to assess
possible physical and chemical mechanisms of the membrane disruption by NM, and our preliminary results
will be discussed.
EPA Grant Number: R832635
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Interagency Workshop on the Environmental Implications of Nanotechnology
Structure-Function Relationships in Engineered Nanomaterial Toxicity
VickiL. Colvin
Rice University, Houston, TX
Objective: 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 (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, shape) control their biological effects.
This aim is the overarching objective of this research project, which stated another way, 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 also 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.
Approach: This project exploits recent advances in nanochemistry, which 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 Ceo 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.
Expected Results: 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 we will establish
structure-function relationships for several major classes of nanomaterials.
EPA Grant Number: R832536
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Interagency Workshop on the Environmental Implications of Nanotechnology
Interactions of Pure and Hybrid Polymer Nanofibers With Cells
Perena Gouma
The State University of New York-Stony Brook, Stony Brook, NY
Nanostructured materials, such as natural polymers, are commonly used to build scaffolds that enable cell
growth and proliferation while supporting cell differentiation. This research project addresses the need for
assessing the cell-nanomaterial interactions. It is believed that the degree of cell attachment to the scaffold has
a direct influence on cell motility, proliferation rate, and control of phenotype. In this study, the nature of
osteoblast attachment to nanostructured fibers of pure cellulose acetate (CA) and cellulose acetate reinforced
with hydroxyapatite nanoparticles (CA-HA) is being reported. The fibrous mats were prepared by means of
electrospinning, a potent nanomanufacturing technique. Osteoblast cells (SaOS-2) were seeded on the
electrospun mats at a density of about 68,000 cells/well. CA-HA composite scaffolds appeared to favor cell
spreading, with hydroxyapatite nanoparticle aggregates enhancing cell attachment to the fibers by providing
anchoring sites.
EPA Grant Number: R832537
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Other Nanomaterials
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Interagency Workshop on the Environmental Implications of Nanotechnology
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
Objective: Dendrimers are relatively monodisperse and highly branched nanoparticles that can be
designed to chelate metal ions, encapsulate metal clusters, bind organic solutes or bioactive compounds, and
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 (EPA) begins its assessment of
the impact of nanotechnology on human health and the environment, there is a critical need of 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 phosphatidyl-
choline lipid bilayers as model cell membranes; and (2) endothelial and kidney cells as model human cells.
Approach: To achieve this overall objective, we 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.
Expected Results: 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
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Interagency Workshop on the Environmental Implications of Nanotechnology
Assessment of Nanoparticle Measurement Instruments
Patrick T. O'Shaughnessy
Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA
A typical industrial hygiene analysis of workplace dust exposure does not include instrumentation to
detect particles in the nanometer size range. One of the goals of this research project is to compare a suite of
aerosol measurement instruments for the purpose of demonstrating their differences and similarities to more
effectively evaluate workplaces that may have a nanoparticle aerosol. The instruments analyzed include a
scanning mobility particle sizer, portable condensation particle counter, surface area monitor, photometer, and
optical particle counter. The measurements made by these instruments were compared to mass concentration
measurements made by gravimetric analysis, and count concentration and size distribution made by
transmission electron microscopy. All instruments are connected via ports attached to a 20 L sealed chamber
acting as a plenum through which dilution air flowed at 25-L/min. Prior to this work, an assessment of various
methods for aerosolizing nanoparticles from the bulk powder were compared. These methods included both
dry powder dispersers and nebulization of a liquid suspension and involved powders consisting of titanium
dioxide, iron oxide, silicon dioxide, and single-walled carbon nanotubes. Polystyrene latex spheres with
diameters less than 100 nm also were tested as a control for particles with known geometry and size
distribution. Multiple trials of each dust type were conducted, and t-tests were used to perform pair-wise
comparisons of instrument output for instruments that were directly comparable. Conversions were made to
some measurements to compare, for example, count measurements with surface area measurements. The
results indicate a need to apply a shape factor to make direct correlations between instruments, especially when
comparing between instruments with different units—count, surface area, or mass concentrations. This
information will be useful for comparing results obtained by different instruments and for choosing an
appropriate instrument for evaluation of nanoparticles in the workplace.
NIOSH Grant Number: R01 OH008806
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Interagency Workshop on the Environmental Implications of Nanotechnology
Development of Nanosensors for the Detection of Paralytic
Shellfish Toxins (PSTs)
Robert Gawley
University of Arkansas, Fayetteville, AR
This research project will focus on progress in the development of nanosensors for detection of paralytic
shellfish toxins (PSTs), of which saxitoxin is the parent. Our recent efforts have focused on the following: (1)
determining the PST profile in blue mussels from Puget Sound in the summer of 2006; (2) correlating the
fluorescence response of our chemosensors to the blue mussel extracts in solution; and (3) incorporating our
chemosensors into a self-assembled monolayer for incorporation into a sensing device.
EPA Grant Number: GR832382
The Office of Research and Development's National Center for Environmental Research 29
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Interagency Workshop on the Environmental Implications of Nanotechnology
Transformations of Biologically Conjugated CdSe Quantum Dots Released
Into Water and Biofilms
Patricia Holden1 and Jay L. Nadeau2
University of California-Santa Barbara, Santa Barbara, CA;
McGill University, Montreal, Quebec, Canada
Objective: Semiconductor nanocrystals (quantum dots) 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 have observed that nucleobase-conjugated
CdSe quantum dots (QDs) were actively taken up by soil and aquatic bacteria (for example, Bacillus subtilis
and Escherichia coli). Effects on microbial viability attributed to the presence of the QDs included slower
doubling times, heavy metal sequestration, and "blebbing" of metals into the environment. We propose to
quantify these effects using a variety of biologically conjugated QDs and an assortment of microbial species,
monitoring the process of QD uptake and breakdown and characterizing the breakdown products that result
from bacterial metabolism of these particles. Possible hazards to microbial populations with extrapolation to
humans through contamination of soil and water with QD breakdown products will be analyzed and quantified.
Approach: Bare, core-shell, and biologically conjugated QDs will be studied. Abiotic breakdown kinetics
and products in aqueous environments will be determined by inductively coupled plasma (ICP) spectrometry
for QDs as a function of exposure to light, pH, and oxidizing or reducing conditions. In preliminary
experiments, biologically conjugated QDs are easily taken up by B. subtilis, but the process is light and pH-
dependent. Some breakdown occurs inside and outside of cells. Working with Pseudomonas aeruginosa and
Staphylococcus aureus to represent Cd-sensitive and Cd-resistant strains, we will quantify population growth
and fluorescence for pure liquid cultures previously exposed to QDs. Conventional methods (shake flask,
viable and direct counting over time) will be used to assess the effects of labeling on bacterial growth rates
under high- and low-nutrient conditions. QD fluorescence will be monitored throughout, and final results will
be adjusted for the dilution effect of growing populations. Concentrations of Cd and Se will be assessed inside
and outside cells, and membrane associations of whole QDs and breakdown products will be quantified. The
relationship of QD release and breakdown to cell viability will be assessed. DNA damage in bacteria will be
assessed by quantifying 8-oxoguanine, a product of oxidative DNA damage, by microscopy and a
commercially available fluorescent label. These experiments will provide basic insight into cellular
interactions with QDs. The potential for single-base pair damage from whole QDs and breakdown products
will be assessed using time-correlated, single-photon counting techniques. Because most bacteria exist as
biofilms in nature, we will culture mono- and dual-species bacterial biofilms under continuous flow conditions
in a commercially available flow cell. Using digital photomicroscopy and computerized image analysis, we
will assess the effects of QD labeling on biofilm growth. Unsaturated biofilms also will be cultured on
membranes to assess the effects of QD labeling on development under soil-like conditions, and as a function of
nutrient and water availability. Cryo-environmental scanning electron microscopy (ESEM), coupled with
energy dispersive spectrometry (EDS), will be used to visualize ultrastructural QD associations. Biofilms
cultured in the absence of QDs will be exposed under a range of experimental conditions and assessed over
time for viability and QD content. For all biofilm experiments, QD effects on exopolymeric substances (EPS)
can be quantified by GC-MS of derivatized glycosyl residues, and DNA and protein content determined by
standard fluorometric and colorimetric methods, respectively. Finally, column studies, using packed porous
media under saturated and unsaturated conditions, will be conducted to assess QD and Cd mobility as a
function of bacterial colonization. Because EPS is expected to chelate Cd, we will quantify whole QDs, Cd,
Se, and biopolymers in breakthrough experiments, followed by sacrificial characterization of residual analytes.
Expected Results: For a range of conditions and for a variety of environmental factors, we will discover
the fates and interactions of bare, core-shell, and conjugated CdSe QDs with bacteria. We will discover QD
effects on bacteria and DNA and differentiate effects of QDs from the effects of the independent metal species.
Both well-mixed liquid culture and biofilm modes of bacterial cultivation will be used, reflecting the full range
of planktonic to attached modes of growth in nature. Experiments also will be performed with porous media
The Office of Research and Development's National Center for Environmental Research 30
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Interagency Workshop on the Environmental Implications of Nanotechnology
columns to quantify how bacterial colonization affects the transport and fate of QDs. This research project will
provide a comprehensive investigation into bacterial QD interactions, which is imperative to understand the
impact and fates of these nanoparticles in the environment. This work is necessary for comprehending the
environmental fates and impacts of QDs, which are increasingly widespread devices in nanotechnology.
EPA Grant Number: R831712
The Office of Research and Development's National Center for Environmental Research 31
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Interagency Workshop on the Environmental Implications of Nanotechnology
Nanotechnology: A Novel Approach To Prevent Biocide Leaching
Patricia Heiden , Benjamin Dawson-Andoh , and Laurent Matuana
Michigan Technological University, Houghton, MI; West Virginia University, Morgantown, WV
Objective: 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 not only to serve as a protective reservoir for the biocide that
prevents its loss by leach or by degradation, but also to release 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.
Approach: 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.
Expected Results: 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, to 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 32
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Interagency Workshop on the Environmental Implications of Nanotechnology
Evaluating the Impacts of Nanomanufacturing Via Thermodynamic
and Life Cycle Analysis
Bhavik R. Bakshi and L. James Lee
Ohio State University, Columbus, OH
Objective: This 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 support these hypotheses. However, validating them has been challenging.
Approach: 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 statistical sound manner via several case studies.
Expected Results: 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 project 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 33
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Appendices
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Interagency Workshop on the Environmental Implications of Nanotechnology
Hotel Monaco
700 F Street, NW
Washington, DC
September 5-7, 2007
AGENDA
DAY1, Wednesday, September 5, 2007
8:00 - 8:45 a.m. Registration
8:45 - 9:00 a.m. Welcome
Gary Foley, Director, National Center for Environmental Research, Office
of Research and Development (ORD), U.S. Environmental Protection
Agency (EPA)
9:00 - 9:20 a.m. Nanotechnology Environmental and Health Implications (NEHI), National
Nanotechnology Initiative (NNI) Research Needs
Celia Merzbacher, Assistant Director for Technology Research and
Development, Office of Science and Technology Policy, Executive Director,
President's Council of Advisors on Science and Technology
9:20 - 9:40 a.m. Department of Energy (DOE) Research User Facilities
AltafH. Carim, Office of Basic Energy Sciences, DOE
9:40 - 10:00 a.m. National Science Foundation (NSF)
Cynthia J. Ekstein, Chemical, Bioengineering, Environmental, and
Transport Systems Division, NSF
10:00 - 10:20 a.m. National Institute for Occupational Safety and Health (NIOSH)
Vladimir Murashov, Office of the Director, NIOSH
10:20 - 10:50 a.m. National Institute of Environmental Health Sciences (NIEHS)
Nigel Walker, National Toxicology Program (NTP), NIEHS
10:50-11:20 a.m. BREAK
11:20 - 11:40 a.m. Office of Research and Development Introduction
George M. Gray, Assistant Administrator, ORD, EPA
11:40 - 11:50 a.m. Science To Achieve Results (STAR) Nanotechnology Program
Chris Saint, Division Director, ORD, EPA
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DAY 1, Wednesday, September 5, 2007 (continued)
11:50- 1:00 p.m. LUNCH
Metals, Metal Oxides
1:00 - 1:20 p.m. Fate, Transformation, and Toxicity of Manufactured Nanomaterials in
Drinking Water
Paul Westerhoff, Arizona State University
1:20 - 1:40 p.m. Pulmonary and Systemic Inhalation Toxicity of Multiwailed Carbon
Nanotubes
Jacob McDonald, Lovelace Respiratory Research
Institute
1:40 - 2:00 p.m. Pharmacokinetics and Biodistribution of Quantum Dot Nanoparticles
in Isolated Perfused Skin
Nancy Monteiro-Riviere, North Carolina State University
2:00 - 2:20 p.m. Metal Nanoparticle Tissue Distribution Following In Vivo Exposures
Alison Elder, University of Rochester
2:20 - 2:50 p.m. BREAK
Metals, Metal Oxides (continued)
2:50 - 3:10 p.m. The Unavailability, Toxicity, and Trophic Transfer of Manufactured
ZnO Nanoparticles: A View From the Bottom
Jason Unrine, University of Georgia
3:10 - 3:30 p.m. Uptake and Toxicity of Metallic Nanoparticles in Freshwater Fish
David Barber, University of Florida
3:30 - 3:50 p.m. Acute and Developmental Toxicity of Metal Oxide Nanoparticles to Fish
and Frogs
George Cobb, Texas Tech University
3:50 - 4:10 p.m. Mechanistic Dosimetry Models of Nanomaterial Deposition in the
Respiratory Tract
Bahman Asgharian, CUT Centers for Health Research
4:10 - 4:30 p.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
4:30 p.m. ADJOURN-DAY 1
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DAY2, Thursday, September 6, 2007
8:30 - 8:40 a.m. Welcome
Metals, Metal Oxides (continued)
8:40 - 9:00 a.m. Nanostructured Materials for Environmental Decontamination of
Chlorinated Compounds
VijayJohn, Tulane University
9:00 - 9:20 a.m. Responses of Lung Cells to Metals in Manufactured Nanoparticles
John Veranth, University of Utah
9:20 - 9:40 a.m. A Toxicogenomics Approach for Assessing the Safety of Single-Walled
Carbon Nanotubes in Human Skin and Lung Cells
Mary Jane Cunningham, Houston Advanced Research Center
9:40 - 10:00 a.m. Microbial Impacts of Engineered Nanoparticles
Delina Lyon, Rice University
10:00 - 10:20 a.m. An Integrated Approach Toward Understanding the Inflammatory
Response of Mice to Commercially Manufactured CuO/Cu, Fe2O3/Fe,
and TiO2 Nanoparticles
Vicki Grassian, The University of Iowa
10:20 - 10:50 a.m. BREAK
10:50 - 11:10 a.m. Hysteretic Accumulation and Release of Nanomaterials in the Vadose
Zone
Tohren Kibbey, University of Oklahoma
Carbon-Based Nanomaterials
11:10- 11:30 a.m. Role of Particle Agglomeration in Nanoparticle Toxicity
Terry Gordon, New York University School of Medicine
11:30- 11:50 a.m. Chemical and Biological Behavior of Carbon Nanotubes in Estuarine
Sedimentary Systems
Lee Ferguson, University of South Carolina
11:50 - 12:10 p.m. Fate and Transformation of C6o Nanoparticles in Water Treatment
Processes
Jaehong Kim, Georgia Institute of Technology
12:10- 1:20 p.m. LUNCH
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DAY2, Thursday, September 6, 2007 (continued)
Carbon-Based Nanomaterials (continued)
1:20 - 1:40 p.m. Cross-Media Environmental Transport, Transformation, and Fate of
Manufactured Carbonaceous Nanomaterials
Peter Vikesland, Virginia Tech
1:40 - 2:00 p.m. Transport and Retention of Nanoscale Fullerene Aggregates in Water-
Saturated Soils
Kurt Pennell, Georgia Institute of Technology
2:00 - 2:20 p.m. Repercussion of Carbon-Based Manufactured Nanoparticles on
Microbial Processes in Environmental Systems
Ronald Turco, Purdue University
2:20 - 2:50 p.m. BREAK
Carbon-Based Nanomaterials (continued)
2:50 - 3:10 p.m. Size Distribution and Characteristics of Aerosol Released From
Unrefined Carbon Nanotube Material
Judy Xiong, New York University School of Medicine
3:10 - 3:30 p.m. Physical and Chemical Determinants of Carbon Nanotube Toxicity
Robert Hurt, Brown University
3:30 - 3:50 p.m. Environmental Impacts of Nanomaterials on Organisms and
Ecosystems: Toxicity and Transport of Carbon-Based Nanomaterials
Across Lipid Membranes
Dmitry Kopelevich, University of Florida
3:50 - 4:10 p.m. Structure-Function Relationships in Engineered Nanomaterial Toxicity
Vicki Colvin, Rice University
4:10 - 4:30 p.m. Interactions of Pure and Hybrid Polymer Nanofibers With Cells
Perena Gouma, State University of New York-Stony Brook
Other Nanomaterials
4:30 - 4:50 p.m. Cellular Uptake and Toxicity of Dendritic Nanomaterials: An
Integrated Physicochemical and Toxicogenomics Study
Mamadou Diallo, California Institute of Technology
4:50 p.m. ADJOURN-DAY 2
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DAY3, Friday, September 7, 2007
8:30 - 8:40 a.m. Welcome
Other Nanomaterials (continued)
8:40 - 9:00 a.m. Assessment of Nanoparticle Measurement Instruments
Patrick O 'Shaughnessy, The University of Iowa
9:00 - 9:20 a.m. Development of Nanosensors for the Detection of Paralytic
Shellfish Toxins (PSTs)
Robert Gawley, University of Arkansas
9:20 - 9:40 a.m. Transformations of Biologically Conjugated CdSe Quantum Dots
Released Into Water and Biofilms
Patricia Holden, University of California-Santa Barbara
9:40 - 10:00 a.m. Nanotechnology: A Novel Approach To Prevent Biocide Leaching
Patricia Heiden, Michigan Technological University
10:00 - 10:20 a.m. Evaluating the Impacts of Nanomanufacturing Via Thermodynamic
and Life Cycle Analysis
Bhavik Bakshi, The Ohio State University
10:20 a.m. MEETING ADJOURNMENT
-------
Interagency Workshop on Environmental Implications of Nanotechnology
September 5-7, 2007
Hotel Monaco
700 F Street, NW
Washington, DC
FINAL PARTICIPANTS LIST
Anthony Andrady
RTI International
Paul Anninos
ICF International
Bahman Asgharian
The Hamner Institutes for Health Sciences
Bhavik Bakshi
The Ohio State University
Mark Baldwin
U.S. Environmental Protection Agency
David Barber
University of Florida
Thomas Barn well
U.S. Environmental Protection Agency
Raanan Bloom
U.S. Food and Drug Administration
Ann Bradley
Integral Consulting, Inc.
Lena Brunet
Rice University
Peter Canepa
U.S. Environmental Protection Agency
Altaf Carim
U.S. Department of Energy
Yung Chang
Arizona State University
Tom Chandler
University of South Carolina
Jason Chen
DuPont Company
Yongsheng Chen
Arizona State University
George Cobb
Texas Tech University
Vicki Colvin
Rice University
Mary Jane Cunningham
Houston Advanced Research Center
Tina Maragousis Conley
U.S. Environmental Protection Agency
Paul DeLeo
The Soap and Detergent Association
Mamadou Diallo
California Institute of Technology
John DiLoreto
NanoReg, Inc.
Travis Earles
Office of Science and Technology Policy
Robert Eganhouse
U.S. Geological Survey
Cynthia Ekstein
National Science Foundation
Alison Elder
University of Rochester
Lee Ferguson
University of South Carolina
-------
Colin Finan
Inside Washington Publishers
Gary Foley
U.S. Environmental Protection Agency
Jie Gao
University of Florida
Robert Gawley
University of Arkansas
Sarah Gerould
U.S. Geological Survey
Patricia Gillespie
New York University School of Medicine
Ernest Hotze
Duke University
Robert Hurt
Brown University
Matthew Jaffe
Crowell & Moring, LLP
Joseph Jarvis
U.S. Environmental Protection Agency
Vijay John
Tulane University
Krithika Kalyanasundaram
State University of New York-Stony Brook
Terry Gordon
New York University School of Medicine
Perena Gouma
State University of New York-Stony Brook
Vicki Grassian
The University of Iowa
Gi Soo Kang
New York University School of Medicine
Barabara Karn
U.S. Environmental Protection Agency
Tohren Kibbey
University of Oklahoma
George Gray
U.S. Environmental Protection Agency
Elizabeth Grossman
Lewis-Burke Associates, LLC
Maureen Gwinn
U.S. Environmental Protection Agency
Patricia Heiden
Michigan Technological University
Ed Heithmar
U.S. Environmental Protection Agency
Zachary Hendren
Duke University
Theodore Henry
The University of Tennessee
Dave Holbrook
National Institute of Standards and Technology
Patricia Holden
University of California-Santa Barbara
Jaehong Kim
Georgia Institute of Technology
Steve Klaine
Clemson University
Barbara Klieforth
U.S. Environmental Protection Agency
Dmitry Kopelevich
University of Florida
John Kramar
National Institute of Standards and Technology
Mitch Lasat
U.S. Environmental Protection Agency
Warren Layne
U.S. Environmental Protection Agency
Dong Li
Rice University
Zheng Li
U.S. Environmental Protection Agency
-------
Igor Linkov
Intertox, Inc.
Philip Lippel
National Nanotechnology Initiative
Greg Lowry
Carnegie Mellon University
Delina Lyon
Rice University
Subhas Malghan
U.S. Food and Drug Administration
Greg Mayer
University of Maine
Jacob McDonald
Lovelace Respiratory Research Institute
Fu-Min Menn
University of Tennessee
Celia Merzbacher
Executive Office of the President
Greg Miller
U.S. Environmental Protection Agency
Nancy Monteiro-Riviere
North Carolina State University
Vladimir Murashov
Centers for Disease Control and Prevention
Neil Naraine
U.S. Environmental Protection Agency
Madeleine Nawar
U.S. Environmental Protection Agency
Onyemaechi Nweke
U.S. Environmental Protection Agency
Galya Orr
Pacific Northwest National Laboratory
Jason Ortego
Woodrow Wilson International Center for Scholars
Patrick O'Shaughnessy
The University of Iowa
Marti Otto
U.S. Environmental Protection Agency
Kurt Pennell
Georgia Institute of Technology
Jenny Phillips
U.S. Environmental Protection Agency
James Ranville
Colorado School of Mines
Amy Ringwood
University of North Carolina-Charlotte
Jim Riviere
North Carolina State University
Christine Robichaud
Duke University
Christopher Saint
U.S. Environmental Protection Agency
Zubair Saleem
U.S. Environmental Protection Agency
Navid Saleh
Yale University
Nora Savage
U.S. Environmental Protection Agency
Robert Schoon
Woodrow Wilson International Center for Scholars
Smita Siddhanti
EnDyna, Inc.
Holly Stallworth
U.S. Environmental Protection Agency
Tzu-Yuan Su
Washington CORE
Yee San Su
AAAS Fellow
U.S. Environmental Protection Agency
Patricia Sullivan
U.S. Environmental Protection Agency
-------
Treye Thomas
U.S. Consumer Product Safety Commission
Ronald Turco
Purdue University
Anita Ullagaddi
U.S. Environmental Protection Agency
Jason Unrine
University of Georgia
Dennis Utterback
U.S. Environmental Protection Agency
John Veranth
University of Utah
Bellina Veronesi
U.S. Environmental Protection Agency
Peter Vikesland
Virginia Tech
Nigel Walker
National Institute of Environmental Health
Sciences
Barb Walton
U.S. Environmental Protection Agency
Jiangxin Wang
Arizona State University
Pat Weggel
U.S. Environmental Protection Agency
Randy Wentsel
U.S. Environmental Protection Agency
Paul Westerhoff
Arizona State University
Katrina White
U.S. Food and Drug Administration
Richard Wiggins
U.S. Environmental Protection Agency
John Wilkens
DuPont Company
Richard Williams
Pfizer Global Research and Development
Erik Winchester
U.S. Environmental Protection Agency
Betty Wonkovich
U.S. Environmental Protection Agency
Judy Xiong
New York University School of Medicine
Don Zhao
Auburn University
Katherine Zodrow
Rice University
Contractor Support
Sara Cohen
The Scientific Consulting Group, Inc.
Maria Smith
The Scientific Consulting Group, Inc.
Elizabeth Stallman
The Scientific Consulting Group, Inc.
-------
How the National Nanotechnology
Initiative is Addressing
Environmental, Health & Safety
Research Needs
Celia Merzbacher, Ph.D.
Assistant Director for Technology R&D
Office of Science and Technology Policy
Works/top on Ewtmnmenfet I
Ofts of Msno
n DC
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p NEHI Working Group
o Subgroup of the NSTC Nanoscale Science,
Engineering, and Technology Subcommittee
9 Established in 2003
o Co-chaired by FDA & EPA/ORD
» Members include research and regulatory
agencies
o Provides for information exchange
9 Aims to identify and address EHS research
needed to support regulatory decision making
|i EHS Research Categories
• Instrumentation, metrology &
analytical methods
• Nanomaterials and human health
• Nanomaterials and the environment
• Health and environmental exposure
assessment
« Risk management methods
I Principles for prioritizing research
a Maximize value of information to be gained.
» How much will uncertainty be reduced?
* How broadly applicable will the information be?
• What is the expected level of exposure?
o Seek to leverage investment against that of
other stakeholders (e.g., industry, other
countries)
o Be aware of the state of the art.
-------
Next Steps
Get public input on priorities
Compare priorities with current
research to identify any gaps
and overlaps
Develop research strategy to
address unmet research needs.
See interim
document at
www.nano.gov
|i Some important points
® EHS research is a shared responsibility
a Research absolute effects, but also net
risks
& Integrate risk research with development
o Research exposure as well as toxicity
o Develop standards
& Understand risk communication
-------
Office of Science
U.S. Department of Energi
Department of Energy (DOE) User Facilities
for Nanoscale Science:
National Resources for Researchers
Dr. AttalH. Carim
Scientific User Facilities Division
Office of Basic Energy Sciences
Interagency Workshop on the
Environmental Implications of Nanotechnology
Hotel Monaco, Washington, DC
September!, 2007
The National Nanotechnology Initiative, and DOE's role
• The National Nanotechnology Initiative (NNI) is an
interagency program, started in 2001, that coordinates
Federal nanoscate research and development activities
and related efforts among 26 participating entities
• Planned federal NNI expenditures are over $1.4 billion
in FY 2008
> SCIENCE
1 The Department of Energy is one of the original
participants in the NNI, and provides major
funding for nanoscale science, engineering, and
technology. The FY 2008 budget request
includes over $285 million for nanotechnology
in DOE's Office of Science, which supports both
fundamental research and facilities.
A. H. Carim
Basic Energy Sciences
Research community needs drive DOE activities
• Energy & environmental grand challenge areas
identified from the start of the National
Nanotechnology Initiative in FY 2001
1 DOE-SC-BES workshops cited nanoscience as a cross-cutting theme.
Basic Research Needs To Assure A Secure Energy Future (2002)
Basic Research Needs for the Hydrogen Economy (2003)
Basic Research Needs for Solar Energy Utilization (2005)
1 Major NNI- and DOE-sponsored workshop in 2004 identified key research
targets and foundational themes for energy-related nanoscience.
(All DOE-BES reports: see http://wwvi.sc.doe.gov/bes/repoits/listhtmf)
SCIENCE
A. H. Carim
Basic Energy Sciences
DEPARTMENT OF ENERGY
Tite mission of tfw Office of Basic Energy Sciences
Foster and support fundamental
research to provide the basis for
new, improved, environmentally
conscientious energy
technologies
Plan, construct, and operate
major scientific user facilities for
"materials sciences and related
disciplines" to serve
researchers from academia,
federal laboratories, and
industry
'A A.M. Carim
I? Basic Energy Sciences
All BES Scientific User Facilities
-------
Five Nanoscale Science Research Centers (NSRCs)
Brookhaven
National Laboratory
3P SCIENCE
A. H. Caiim
Basic Energy Sciences
Nanoscale Science Research Centers: Basic Info
Research facilities for synthesis, processing, analysis, and
characterization ofnanoscale materials
Provide specialized equipment, unique tools, and dedicated
scientific and support staff that are difficult for individual
institutions to put in place and maintain
Operated as user facilities and available to all. Access
determined by peer review of proposals. No cost for
precompetitive, non-proprietary work leading to publication;
cost recovery for proprietary work.
Co-located at DOE National Laboratories with existing major
user facilities (synchrotron radiation light sources, neutron
scattering facilities, other specialized facilities) to provide
characterization and analysis capabilities
A. H. Carim
Basic Energy Sciences
The five NSRCs are open for business and serving users!
Center for Functional N
(Brookhaven National Laboratory)
in process of completing installatit
commissioning ofinitialequipmer
Unique tools: x-ray synchrotron beamlines
with nanoscafe resolution
Unique instruments to study individual nanostructures
Quantitative structure, strain, orientation imaging
Sensitive trace element and chemical state analysis
Joint nanoprobe effort
between CNM and the
Advanced Photon
Source at Argonne
National Laboratory
Similar efforts underway
via CFN and the National
Synchrotron Light
Source at Brookhaven
National Laboratory
%> SCIENCE
A. H. Carim
Basic Energy Sciences
Calculating Resistance in the Smallest Possible Junctions
In the ultimate limit, one can imagine a junction across a single molecule.
Measurements have been made in recent years on the electron transport
through a single molecule of hydrogen, positioned between two metal
point contacts via nanofabrication techniques. Now novel calculations
have been done to understand the junction resistance. The resistance of
the hydrogen molecule is extremely sensitive to the choice of contact
material, a trait not seen in macroscopic junctions, and model
calculations accurately reproduce these material dependences.
y *
•M U^^" A _ "
I
Electron flow
A density plot of the conducting electrons reveals
significant build up of electron density (red) behind
thetip Pd atom, leading to a high junction resistance
and consistent with recent experiments.
•„'•' Basic Energy Sciences
Charge Transport in Low-Dimensional Structures:
2D and Quasi-1D Nanocrystal Arrayi
Experiments on low-dimensional artificial
solids made of nanocrystals have yielded new
insights:
• I-V behavior is highly nonlinear
• Threshold voltage scales linearly with
array width.
• Both structural disorder and quenched
charge disorder affect tunneling
X.-M. Lin, K. Elteto, et al.,
CNM and U. Chicago
SCIENCE
I£BJ& A. H. Carim
j,: Basic Energy Sciences
-------
Producing Defined Protein Nanotube
Hcp1 forms a hexameric ring
with a large internal diameter
Hcp1 rings stack to form
tubes in the crystal lattice
introduction of site-specific
modifications stabilizes
free-standing tubes
excess of end subunits added
can be selectively modified
length controlled by time,
concentration and specificity
::
• • ~~
K** !
Novel approaches for rapid, reproducible
measurements and .<
New Tools; Discovery Platforms
Standardized modular, micro-laboratories—designed iind batch labricate-d lor:
* Integrating nano and micro lenfllh scsiles
• Studying ttie physical i cJiemical pfop-erlies of nanDsca.lB materials and devices.
* Directly accessing Aide range ol CINT external diagnostic and characterization tools
Cantllc«f Array Platlwm
Electrical Transport * Optical
Spcctroscopy Pi.itror m
c Synthesis Platlurrrj *.
?? SCIENCE
A. H. Carim
Basic Energy Sciences
Seeing atoms:
Providing national user facilities for probing materials at the atomic scale
Neutron scattering Electron J
High Tc supercondua
microscope image showing
an abrupt interface and low
defect density for the
ferroelectric SrTiO,on Si.
A. H. Carim
Basic Energy Sciences
The (existing) BES Light Sources
LCLS atSLAC- The World's First X-ray PEL
SCIENCE
A. H. Carim
Basic Energy Sciences
The OOE-BiFS Neutron Scattering Ce^ers
Intense Pulsed
Neutron Source
\ SCIENCE
{Kifkl A. H. Carim
Basic Energy Sciences
-------
The Spallation Neutron Source
The DOE-BES Electron Scattering User Facilities
National Center for Electron Microscopy
(NCEM) at Lawrence Berkeley National
Laboratory: atomic resolution imaging
Electron Microscopy Center
(EMC) at Argonne National
Laboratory: in-situ studies,
including irradiation effects
Shared Research Equipment
(SHaRE) Program at Oak Ridge
National Laboratory:
microanalysis and
spectroscopy
A. H. Carim
Basic Energy Sciences
For more information:
1 On DOE's Office of
Basic Energy Sciences (BES):
h ftp://Vvww.se/ence. doe.gov/bes/
1 On DOE nanoscience:
http://nano.energy.gov
On the DOE-BES Scientific User Facilities:
http://www.sc.doe.gov/bes/BESfacilities.htm
%> SCIENCE
A. H. Carim
Basic Energy Sciences :
-------
NIOSH Nanotechnology Program
Vladimir Murashov
Special Assistant to the Director
National Institute for Occupational Safety and Health
Washington, D.C.
2007 Interagency Workshop on the Environmental Implications of Nanotechnobgy,
September 5, 2007
'The findings and conclusions in this presentation have not been formally
disseminated by the National Institute for Occupational Safety and Health
and should not be construed to represent any agency determination or policy."
T/OSH
About NIOSH
The National Institute for Occupational Safety and
Health is:
the U.S. Federal agency responsible for conducting
research and making recommendations for the
prevention of work-related injury and illness
NIOSH & Emerging Technologies
OSH Act directs NIOSH to "conduct special research,
experiments, and demonstrations relating to occupational
safety and health as are necessary to explore new
problems, including those created by new technology in
occupational safety and health."
29 USC 669 Sec. 20(a)(4)
Concerns Over Nanotechnology Implications
Namrtedinolog) Regulation Needed, Lribcs Say
Woodrow Wilson Center 2006
NGO Coalition 2007
NIOSH Goals Involving Nanotechnology
Understand and prevent work-related injuries and illnesses
potentially caused by nanoparticles and nanomaterials
Promote healthy workplaces through interventions,
recommendations, and capacity building
Enhance global workplace safety and health through national and
international collaboration on nanotechnology
Conduct research to prevent work-related injuries by applying
nanotechnology products
Understand and prevent work-related injuries and illnesses
potentially caused by nanoparticles and nanomaterials
• Toxicology Research
• Pulmonary effects in mice
• Nanoparticles enter blood stream
• Dermal effects
• Nanoparticle generation system
• Metrology Research
• Control Technology Research
• Exposure Assessment
• Medical Surveillance and Guidance
• Safety Research
-------
Progress Toward Safe
Nanotechnology in the
Workplace
TfOSH
• Research progress in 10 key
areas
• Continuing project plans
' Opportunities for collaboration
www.cdc.gov/niosh/topicsAianotech
Promote healthy work places through interventions,
recommendations, and capacity building
NIOSH Field Team
Approaches to safe nanotechnology:
An information exchange with NIOSH
NIOSH Topic Page
Nanoparticle Information Library
National and International Conference
www.cdc.gov/niosh/topics/nanotech
Recommendations from
NIOSH
•Summary of issues
•Approaches to consider
•Basic Guidance
•Updated as new information
comes on-line
•Input requested
www.cdc.gov/niosh/topics/nanotech
Nanoparticle Information Library (NIL
ibrary(NIL) „„«_.„
*• l"~l—."•! — ^—«*", ,
www.cdc.gov/nioshAopics/nanotech/NIL.html
NIOSH-sponsored conferences
August 29- September 1, 2007 nano-taiwan.sinica.edu.tw/EHS2007/
European NanOSH Conference -
NanotBdmlogtas:
A CnHca) Area In
OctupatKxiAl Safety and
HttMl
www. ttl.fi/EuroNanOSH
Enhance global workplace safety and health through national
and International collaborations on nanotechnology
• Collaborations with various companies (e.g. DuPont, Altairnano, Luna
Technologies)
Participation in inter-agency working groups (NEHI, GIN)
Participation in ISO TC 229 Nanotechnology Working Group on
Health, Safety and Environment
Collaboration with OECD
Collaboration with WHO
-------
Conduct research to prevent work related injuries by applying
nanotechnology products
Examine applications for filters, sensors, and protective clothing
E/ectmspun nanafibers, NIOSH
NIOSH Nanotechnology Program Funding
NIOSH Nanotechnology Program
Activities In Nanotechnology Research
I. Intramural
i. National Occupational Research Agenda: Nanotechnology Safety
and Health Research Program (2004-2008)
N. NIOSH Nanotechnology Research Center (2005-)
IN. Nanotechnology Research Supplement (2006-2010)
iv. Nano-Related Division Projects
II. Extramural
i. Research Grants
N. Joint RFAs
IN. Contracts
www. cdc.gov/niosh/topics/nanotech/strat_plan. html
Extramural Program
http://www.cdc.gov/niosh/oep/
fTlOSH
Office of Extramural Programs
Funding Opportunities
EPA-led Joint Research Solicitation
Joint Request For Applications with EPA/NCER, NSF and
NIH/NIEHS in FY 2004 through FY2006:
. Nanotechnology Research Grants: Investigating Environmental and
Human Health Issues.
Up to $8 million to support 15-25 research grants and
exploratory grants (per year): up to $1 million from NIOSH.
Focus:
• research to meet NIOSH mission of providing leadership in preventing
work-related illnesses and injuries.
NIH-led Joint Research Solicitation
Joint Request For Applications with NIH, and EPA/NCER in
FY2007:
• Manufactured Nanomaterials: Physico-chemical Principles of
Biocompatibility and Toxicity (R01)
Up to $4.1 million to support 10-15 research grants and
exploratory grants: up to $0.5 million from NIOSH.
Focus:
• to identify and investigate the relationships between hazardous working
conditions and associated occupational diseases and injuries; to develop
more sensitive means of evaluating hazards at work sites, as well as
methods for measuring early markers of adverse health effects and
injuries; to develop new protective equipment, engineering control
technology, and work practices to reduce the risks of occupational
hazards; and to evaluate the technical feasibility or application of a new or
improved occupational safety and health procedure, method, technique, or
system.
-------
Thank you!
Vladimir.Murashov@cdc.hhs.gov
-------
A NIEHS
NIEHS activities on Nanotechnology:
Nanoscale Science and Toxicology
Nigel Walker Ph.D.
National Toxicology Program
National Institute of Environmental Health Sciences
National Institutes of Health, RTP, NC
Interagency Workshop on the Environmental Implications of Nanotechnology,
Washington DC, September 5th 2007
Nano at NIEHS
• Funded by NIEHS
- Division of Extramural Research and
Training (DERT)
• Grants
Training
• Research at NIEHS
- Division of Intramural Research (DIR)
- National Toxicology Program (NTP)
• Contract based research and testing
- DIR Investigator Initiated
Application of nanotechnologyin EHS
Dept of Health
and Human Services (DHHS)
Areas of emphasis for NIEHS and NTP
• Exposure and dose metrics
- How do we measure exposure?
• Internal dose-Pharmacokinetics in biological systems
- What physiochemical properties determine the absorption, distribution and
elimination of nanomaterials?
Early biological effects and altered structure function
- What physiochemical properties determine biocompatibility?
Adverse effects
- What are the critical determinants of toxicity for those that are toxic?
Sources
Exposure
Internal
Dose
Early
biological
effect
Altered
structure/
function
Adverse
Effect
Experimental Strategies
Several workshops/reports with common
issues/recommendations
- NTP workshop on Experimental strategies
• University of Florida-Nov 2004
• http://ntp.niehs.nih.gov/go/100
- ILSI-RSI report
• Oberdorster et al 2005, Particle Fibre Toxicol 2:8
Current models able to detect manifestations of
novel mechanisms of action
- Use of both in vivo and in vitro approaches
Need comprehensive physical/chemical
characterizations
Biological levels and hazard evaluation strategies
100,000'sVday
Mechanisms
Immediate Human Relevance
Intramural - NTP Nanotechnology Safety Initiative
http://ntp.niehs.nih.gov/go/nanotech
• Scientific Focus
- Identify key physical-chemical features that govern nanomaterial safety
• Current materials under evaluation
-- Quantum dots
- Titanium dioxide
- Carbon fullerenes
- Nanoscale silver
- Multi-walled carbon nanotubes
- Nanoscale gold
- Dendrimers
• Contact: Nigel Walker, walker3@niehs.nih.gov
-------
Extramural - Enabling technologies
• Environmental Sensors
- Deployable sensor devices for a broad range of environmental exposures
• Biological Sensors
- Develop and apply technologies to link exposure with disease etiology
• Intervention devices
- Drug delivery devices and therapeutic nanoscale materials
• Remediation devices
- Primary disease prevention through the elimination of exposure
Catalysis or chelation
• Contact: David Balshaw: balshaw@niehs.nih.gov
Extramural - Fundamentals of Biological Response
• FY06-Human Health Effects of Manufactured Nanomaterials
- Joint solicitation between EPA, NSF, NIOSH, NIEHS/NIH
- Funded three applications $400K /year for 3 years
• Transmembrane transport, cardiovascular toxicity and oxidative stress
• FY07-Manufactured Nanomaterials: Physico-chemical Principles of
Biocompatibility and Toxicity
• NIEHS lead with additional partners
- NCI.NEI, NHGRI, NIDCR, NIGMS, and EPA NIOSH
• Review process completed and approx 10 grants may be funded
• Contact: Sri Nadadur, nadadurs@niehs.nih.gov
Taking the Next Step:
• Building on the NIEHS investment and core
competencies
• Partnering for integrated research success
• Consistent with US goals for safe commercialization
and innovation
NanoHealth Initiative
Scope
- Examine the fundamental physicochemical interactions of ENM with biological systems at
the molecular, cellular, and organ level, as well as associated pathophysiologic
- New knowledge of molecular, cellular, and organ system biology and identify clinically
relevant properties of ENM
- critical for design of ENM with r
safety
Contact: Sally Tinkle, stinkle@niehs.nih.gov
jnmental biocompatibility and
Research products
Biologically and clinically
relevant design principles
Curated data sharing
framework
More vigorous debate of
controversial issues
Standards setting
Network of research partners
Publications:
High impact journal articles
Technical reports
Scientific Foundation of an Emerging Science
-------
Pulmonary and Systemic
Biocompatability of Inhaled Carbon
Nanotubes
Jake McDonald
Lovelace Respiratory Research Institute
Albuquerque, NM
Instillation of CNT Resulted in Significant Lung Tissue
Damage
I
I ,,,
t
I I,
Hydn«yim.liKk..w.-..
• , l
J.-.^.
LanrAII CNT showed lesions/granulomas after 2- 5 mg/kg dose
Warheit: No dose or time dependence to pathology or inflammation
Hypothesis: Inhaled Carbon Nanotubes
will Not Cause Pulmonary Injury or
Inflammation after High Dose Exposures
LRRI Inhalation Exposure System for
CNT
Obtained Aligned MWCNT from NTP (Shenzhen, China)
- Purity >95%
- Diameter 10-20 nm
Length 5-10 urn
- Amorphous carbon < 3%
Ash (catalyst residue) < 0.2%
Special surface area 40-300 m2/g (actual 100 m2/g)
Aerosolized with jet-o-mizer followed by cyclone
Characterization of Bulk and Aerosol Composition Key
Component of Work
TEM, HR-TEM
SEM
XRD
X-Ray Photoemission Spectroscopy
Raman
Impactors (aerosol only)
Differential Mobility (aerosol only)
Mass Spectrometry
BET (gas adsorption) for Surface Area
Aerosolized MWCNT
-------
Particle Mass and Number Size
Distribution
CASCADE IMPACTOR MASS DISTRIBUTE J
0.01 0.1 OS
Aerodynamic Diameter ||lm)
Median mass = 0.7 |jm
Study Design
Male C57BI/6 mice
- 6 hr/day, 7 days/week
- Sham, 300, 1000, 5000 |jg/m3 (0.2-10 |jg
deposited/day)
- Sacrifice at 7 and 14 days
• Lavage cell counts and
biomarkers
• Lung biomarkers
• Lung pathology
• Immune Function-Spleen
• Gene upregulation: lung, spleen
CNTs in Macrophages
64W-2
Total cell number not increased
No increase in neutrophils, lymphocytes, etc
Control
^
7/v^'ii
:t«$*x .t-/.^
C'£\f
C 'J^_J '
5ma/m3
Clean Air 40 X Showing Alveoli and
Macrophages
'
" ' fr*«
Summary and Future Directions
Inhaled MWCNT showed unremarkable pulmonary
inflammation and pathology at high doses and by
inhalation.
- Contradicts initial hypothesis, which was
based on marked pulmonary effects
reported by others after instillation or
aspiration
• Dose?
• Route of Administration?
• SWCNT vs MWCNT vs ?
• Time? *
-------
Instilled Diesel Soot at ~2 mg/kg Results In Granulama
formation, Inflammatory cell Infiltration, change In
Inflammatory mediators, etc
V\s'">f'* I" ' -• •'•§••
F^b^H *tr •
.*. V ••V*TH.B4 •
V'>«-
-------
Gene Expression in Spleen
| 3-
O
T3
£ 2-
1 -
I
in nil
i ^
n
• IL-6
• IL-10
1 N001
I
T
I
control 0.3mg/m3 1 mg/m3 5mg/m3
Prostaglandin Associated
Enzymes
I
Lymph
node
Important Considerations
• We have observed systemic
immunosuppression with inhalation
exposures in the past (Diesel, woodsmoke,
coal)
• Often times no pulmonary effects
accompanied these systemic immune
function changes
• The immune responses shown here are likely
NOT unique to MWCNT, but this has not been
proven
Thanks
Collaborators
- Randy Vander Wai (USRA-NASA)
- Scott Burchiel
- JeanClare Seagrave
- Leah Mitchell
- And rew G igg liotti
- Chemistry/Exposure Staff
Necropsy Staff
Funding
- EPA NIEHS
-------
Metal Nanoparticle Tissue Distribution
following In Vivo Exposures
EPA Nanograntees Meeting, Sept. 5-7, 2007
Interagency Workshop on the Environmental
Implications of Nano techno logy
Alison Elder
Department of Environmental Medicine
University of Rochester
Introduction
Studies with ultrafme particles have demonstrated
extrapulmonary translocation.
What properties affect the tissue distribution of
nanosized particles?
Hypothesis
The tissue distribution of nanomaterials
following respiratory tract or systemic
exposure is a function of their surface
properties.
Methods:
Nanoparticle Characteristics
Characteristics of the QDots:
- CdSe/ZnS core-shell particles coated with polymer, ~5 nm core-
shell diameter (Invitrogen) — 565 nm emitters
- PEG, PEG-amine, or carboxyl conjugated surfaces
- Hydrodynamic radii:
> 14 (carboxyl), 15 (PEG-amine), 35 (PEG) nm - reported (Ryman-
Rasmussen etal, 2006)
> 13 (carboxyl), 17 (PEG-amine), 23 (PEG) nm - in saline
— Zeta potentials (in 0.9% saline, pH 7.4):
>-40.0 (carboxyl), -0.3 (PEG-amine), -1.5 mV (PEG)
Methods:
Nanoparticle Characteristics
Characteristics of colloidal Au particles:
- 5 nm primary particle size (Ted Pella, Inc.)
- Coated with albumin, 5 kDa PEG, or 20 kDa PEG
i- J
Size (dl
Surface Coating
Citrated Au
RSA-Au
PEG (5K)-Au
PEG (20K)-Au
Methods:
Nanoparticle Exposures
Exposures to QDotS (dose expressed as Cd content):
— Intratracheal microspray (5 j^g Cd/150 jjl saline)
- Intravenous injection (1.7 j^g Cd/200 \d saline)
Exposures to colloidal Au:
— Intratracheal microspray (50 j^g/150 jjl saline)
— Intravenous injection (15 j^g/200 jjl saline)
-------
Lung Inflammation following NP
Exposure?
Inflammation (as determined by percentage of lavage
fluid PMNs) can significantly alter the translocation
of nanoparticles from the lung to the blood and vice
Versa (e.g. Heckeletal, 2004)\
— QDots: no significant increases from controls;
— Colloidal Au: PEGylated particles caused significant
increases in PMNs (10-13%) when delivered via ITM; not
affected by other coatings, route of exposure.
Tissue Cadmium Content 24 hrs following Intratracheal
Microspray Exposure of Surface-Modified QDots
(Rats, 5 jig Cd spr
Lung Lavage Lavage Trachea Lymph Spleen Bone Heart Liver Kidney
Cells Sup. nodes Marrow
Cadmium Content 24 hrs following Intravenous
Exposure to Surf ace-Modified QDots
(Rats, 1.7 jog Cd injected in 200 jjl)
Lung Lavage Lavage Lymph Spleen Bone Heart Blood Liver Kidney
Cells Sup. nodes Marrow
Retention of Cd in Olfactory Bulb following
Intranasal Instillation of QDots (~4 ng) into Left
(right olfactory bulb background-corrected total tissue Cd)
Tissue Au Content 24 hrs following Intratracheal Microspray Exposure to
Colloidal Au Nanoparticles with Different Surface Coatings
IL
LALN Spleen Bone Marrow Heart
Blood Kidney
Tissue Au Content 24 hrs following Intratracheal Microspray Exposure to
Colloidal Au Nanoparticles with Different Surface Coatings
05'
S 0.3-
I
! o,
i
• Citrate
D PEG5k
D PEG 20k
L
i
In
LALN Spleen Bone Marrow Heart Blood Kidney
-------
Tissue Au Content 24 hrs following Intravenous Exposure to Colloidal Au
Nanoparticles with Different Surface Coatings
00.
LALN Spleen Bone Marrow Heart Blood Kidney
Translocation of Nanogold to the Brain
5nm Gold, surface modified:
Au in brain after ITM Exposure
Auin brain afterlV Exposure
2.
Summary of Results
Nanoparticles delivered via the lower respiratory
tract are translocated to extrapulmonary tissues
• Dependent on particle physicochemical
characteristics.
Nanoparticles can be retained in small amounts
by the brain following a single exposure
• Dependent on particle physicochemical
characteristics and portal of entry.
Remaining Questions
Short-term:
• More thoroughly evaluate kinetics of nanoparticle
translocation;
• Where are the nanoparticles localized (which cells,
what subcellular structures?)?
Long-term:
• Characterize translocation to the CNS as a function of
the particle surface and its interactions with
endogenous proteins;
• Characterize elimination of nanoparticles from the
CNS.
Acknowledgements
UofR:
Giinter Oberdorster
Jacob Finkelstein
Amber Rinderknecht
Nancy Corson
Bob Gelein
Pamela Wade-Mercer
WUStL:
Jingkun Jiang
Pratim Biswas
Grant Support:
EPA
DoD, NSF
Hypothesis
The tissue distribution of nanomaterials following
respiratory tract or systemic exposure is a function of
their surface properties.
CdSe-ZnS Quantum Dots in 0.9% Saline
PEGylated
23 nm
PEGamliie
17 nm
-------
Carboxylated PEGylated
Qdots Qdots
Carboxylated QDots in Rat Lung Tissue
-------
Bioavailability, Toxicity, and Trophic
Transfer of Manufactured ZnO
Nanoparticles: A view from the bottom
Ph Paul M. Bertsch
Co-Pis; Travis Glenn, Andrew L. Neal, Phillip Williams,
Brian P. Jackson-Dartmouth College
Collaborator; Jason M. Unrine, Pamela J. Morris -MUSC
Post doc; Nadine J. Kabengi
Ph.D. students; Hongbo Ma & Benjamin A. Neely-MUSC
-------
Nanoparticle-Bacteria interaction
>Higher OAc utilization rates with Zn2+aq con
to ZnO-np
> Evidence for bio avail ability of Zn ion, but not
ZnO-np
>Epifluorescence microscopy indicates an
increased number of cells with compromised
membranes associated with ZnO-np vs. free ion
Nanoparticle-detritivore interactions
959 eel
sequenced
Feeds on bacteria and
other particles < 5 |jm i
diameter
Behavior & Lethality data for ZnCl2 and ZnO-np
•' ZnCI2: EC50= 8.54mM (95% Cl: 6.82-10.69mM)
LC50=83.1(+19.2)mM
> ZnO-np: EC50= 9.42mM (95% Cl: 8.09-
10.97mM)
LC50=79.1(+16.8)mM
found between ZnCI2 and ZnO-np in buffered
medium (acetate buffered, pH=6.0).
-------
Uptake of nanosc^.
size deoendence
-------
Future Work
Results from year 1 and 2 provide a framework for directing the third year of
the project
> Characterization work of 80 nm size ZnO nanopatticles
> Under various chemical conditions
ith 80 nm size ZnO nanoparticles
• Continue exposure experiments with Cu2+
BioavailabilrtyToxicrty studies
> Differences in toxicity mechanisms of ZnCI2 and ZnO-np to B.
ensis, C. necator, and C. elegans
as opposed to direct exposure.
> Identify chemical speciation of Zn in concentrated regions in
'issues
"mine potential transformation of ingested ZnO-np
Manuscripts in preparation
A.L. Neal, N. J. Kabengi, and P.M.Bertsch. Toxicity of Zinc Oxides Nanoparticles
and Aqueous Zinc to the Soil bacterium Cupriavidus necator.
N.J. Kabengi, J.M. Unrine, A.L. Neal, and P.M. Bert'*h r^r*r-tar\-,*t\nn ^H
surf ace- re activity of commercially manufactured .
importance of the acetate counter-anion.
N.J. Kabengi, S. Wu, J. Shields, P.M. Bertsch. 2007. In situ investigation of Zinc
oxide nanoparticle growth by transmission electron microscopy: implications for
size determination.
H. Ma, J.M. Unrine, A.L Neal, P.L Williams, P.M Bertsch. Bioavailability and toxicity
of nano-sized ZnO in the nematode Caenorhabditis elegans.
B.A Neely, A. G. Slitter, D. W. Bearden, P. M. Bertsch, and P. J.
Morris. Microbial Growth Affects Zinc Oxide Nanoparticle Structure and
-------
"
• -^^^^p!'8'-.
rJ'!| - David Barber
University of Florida
Center for Environmental and Human
Toxicology
particle composition and
in gill toxicity
^plftnine role of particle surface charge
jliftiptake and retention of nanomaterials
in aquatic organisms
-------
silver and
KfrMOEC concentrations for
|ff(100ug/L for Cu, 1000 ug/L for
'concentration of soluble metal
*;rileased by particles
•Evaluated gill metal uptake, histology and
transcriptional changes at 24 and 48 hours
-------
Filament
72 slides. 350 picture of filaments. 1750 measurements
of half the width of a filament in micrometers (example
yellow line)
at equi-
^llgpcoriceritrations
-rafe:markedly different
between particles
JpifSfare different from soluble forms of
js|-4li»iais at equal concentrations of soluble
"f'sSftiaterial
~ *T\O2 produces some unique responses, though
does not cause overt toxicity
•Particle responses are time dependent
-------
te-.
^Pini'retention of PEG,
''quantum dots in daphnia
Cond
_pjp|HjSiS:!6f nanosized particles are
flpjiff Hiewater column for long periods of
rflKr
jsijfijtiqle exposure with time makes dosimetry
pBPtf"'
fe£tif 'Some nannmptak arp inrnmnlprplv pxnlainpd
'•"^SCTeelS'appearto depend on particle composition and are not
" . generic responses
Charge influences uptake of nanomaterials in daphnia
Future work will focus on mechanisms
j|fpflg:-Rfisearch Center
iiliis1*'
ffle-Zoology
£Y|S' Evans
-------
Acute and Developmental Toxicity
of Metal Oxide Nanoparticles
in Fish and Frogs
George Cobb and Shawna Nations
Texas Tech University
Christopher Theodorakis
Southern Illinois University
Elizabeth Carraway and Xin Xu
Clemson University
Metal Oxide Nanoparticles
•Catalysts
•UV protectants (ZnC
•Wood preservation
•Marine antifoulants
•Deodorants
•Polishing agents
•Glass
•Dental
• Semiconductors
•Antimicrobial
•Textiles
•Foot powder
•Coatinars
Objectives
•Determine the environmental hazard of
Fe2O3, ZnO, CuO, and TiO2
•Evaluate acute and chronic toxicity
•Fathead minnows (PimephasepromeJas) 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
3: % \\ itch, uio\vlli, % il.'touniti
-:c.r.oi-:b.3..tvivaur.
-------
Methods and Materials
Xenopus laevis SEM Preparation
Rinsed 3x with Sorenson's phosphate buffer
• Chemical dehydration Six EtOH exchanges: 10%-
100%, ~20min. each
• Critical point drying with Balzars CP 030 unit to
replaces ethanol with CO2
Mount on SEM stub with double sided
conductive tape
« Sputter Coat with Hummer V unit to deposit
~10nm of gold-palladium alloy
Method for Range Finding Test
Method for Definitive Test
FETAX assay
« Follow ASTM E1439-98
« 2 replicates of 8 concentrations including a
control (total exposures = 16)
• 1000, 100,10, 1, 0.1, 0.01, and O.OOlmg/L
• Control: FETAX solution
» FETAX solution: NaCl, NaHCO,, CaCl,, CaSO4'2H,O,
15 embryos per exposure
FETAX assay
• Follow ASTM E1439-98
• 3 replicates of 5 concentrations including a
control (total exposures = 18)
• 1000, 100, 10, 1, and 0.1
• Control: FETAX solution
m FETAX solution: NaCl, NaHCO3, CaC^, CaSO4'2H3O,
. 10 embryos per exposure
Behavior of nano-iron oxide in water
-// i%4f^|Wcf.,'"'"v ^"-'--'"
-------
Effects of
nano-copper oxide
to Xenopus
s Bdema/Bfistering
Eye malformation
Edema produced by Zinc Oxide
Zinc oxide effects
on the Xenopus
GI tract
Accumulation on Surface
.'*•'
Irregular
GUl C°il
'""-••' Accumulation in « EonUJgW
A!,. Gat Coil '; ( • '
Se\ erely
Malformed
head and
Irregular Gut
Coil
-------
Effects of
Zinc Oxide
on Xenopus
spinal column
f • ,' I' »'!>'' <^
{(j''' '""SSL,t"*^"f Ji * u t't '* i' ciLwiL'1
v& -:>:ij^l^iA'%!rxP
^4^,,;|J
Significantly Malrornied
--^•••' y^' " •'' ! "' * '%'fr'f'
Effects of Metal Oxides
on Xenopus Development
Range Finding
-------
Chronic Toxicity Exposure Apparatus
Delivery tube
Polypropylene
Acknowledgements
3 A Grant Number RD-8328420
Jniversity Imaging Center
Dr. EE Smith, Dr. M Crimson
Progress to date
Funding in place 1 July 2006
Assessed acute toxicity for Xenopus
Established zebrafish breeding
colonies
Nanoparticles synthesized and
commercial nanoparticles obtained
Range-finding tests in preparation
Chronic tests for Xenopus
« Acute tests for zebrafish
-------
Mechanistic Dosimetry Models of
Nanomaterial Deposition in the
Respiratory Tract
Bahman Asgharian, Brian A. Wong, O.T. Price,
David Nash, Earl Tewkbury
Division of Computational Biology
The Hamner Institutes for Health Sciences
Research Triangle Park, NC
Study Objectives
1. Deposition measurements of nanosized particle in casts of
human and rat nasal URT airways
2. Semi-empirical relationships to predict nanomaterial
deposition in the URT airways
3. Respiratory tract deposition models of nanoparticles and
nanotubes in humans and rats
4. Measurements of regional and lobar deposition of
nanomaterial in the heads and lungs of rats
5. A user-friendly software package to implement models and
provide rapid simulation capability
1. Deposition measurements of nanosized particle
in casts of human and rat nasal URT airways
Nasal Replicas and Models:
• Human nasal replicas:
- MRI scans of human nasal passages
- Input scans into computer to create wire mesh
— Use stereolithography to create plastic replicas
1. Deposition measurements of nanosized particle
in casts of human and rat nasal URT airways
• Rat nasal mold:
- Low melting point alloy to fill air spaces in cadaver
- Cast mold in plastic
Deposition of Nanoparticles
in Nasal Replicas
Generate monodisperse nanoparticles
- Electrospray generator: 5-30 nm
- Nebulizer and classifier: 30-100 nm
Measure particle counts and size at inlet and
outlet to nasal mold using Scanning Mobility
Particle Sizer (SMPS)
Calculate Deposition efficiency
C
^ inlet
500
400
300
200
100
0
Pressure Drop
Subject 12
-Subject 14
0 10 20 30 40 50 60 70
Flow Rate, 1pm
-------
(1.6
0.5
(1,4
Hiimans
10 11)0
Particle Diameler, ran
I"
Rats
*. «, * s
3 10
Particle Diameter, nm
100
2. Semi-empirical relationships to predict nanomateria
deposition in the URT airways
Conveotive diffusion equation:
2Sc.u'^ + u'^ = 4^lr
dr dA r dr ( dr'
d2c"
Sc= —
D
A = -
4 x Volume
Surface Area
_ (Surface Area)'
4 xjtx Volume
=> r| = f(A,Sc)
Semi-empiri(
Furichoria!
Relations h.sp
rj - axScb/f
Species
Human
Rat
Human
Rat
a
5.005
3.896
24.61
7.351
:al
Coeffscsen,s
b o
-0.5126
-0.1582
-0.2975
-0.2438
0.06998
0.2438
0.58
0.402
Correlation
coefficient.
(r2)
0.76
0.76
0.97
0.97
Comparison of Fitted curves with data
Model Comparison
1'jnii.fcltaiKla.niB
-------
3. Respiratory tract deposition models of nanoparticles
and nanotubes in humans and rats
I. Extrathoracic airway deposition
- From measurements: semi-empirical models
II. Lung geometry
- Symmetric: Yeh et al. (1979)
- Asymmetric: Koblinger & Hofmann (1990)
III. Airway flow architecture within the lung
- Uniform lung expansion and contraction
- Uniform velocity equal to average parabolic velocity
IV. Mathematical formulation to calculate deposition in the
lung during a breathing cycle
- Deposition by diffusion
HI. Lung Ventilation
Uniform Airway expansion:
lt+5t-mt :
Equation:
_
dx
DV(x)
TLV
A: Cross-sectional area
DV: Distal volume
TLV: Total Lung volume
Calculate flow splitting at bifurcations
I vr 1
p\~ \ DV\P\~^ Vp: Airway volume
iQ(x)
IV. Mathematical Model for Particle
deposition
Particle mass balance per airway:
mt+5t-mt =minSt-rhoutSt-mdl
dt dx dx(, dx
mass lost per unit
time per unit volume
Solution:
C(x,t),C,e-t''l'-'"->erJ
Tidal Reserve
air air
Calculation Steps:
1. Calculate deposition efficiencies per airway.
2. Particle concentrations at the inlet and exit of all airways.
3. Airflow rates at the inlet and exit of airways.
4. The time it takes for the aerosol front to pass the inlet and
exit of each airway.
5. Calculate Losses per airway:
time Distance
Losses =
.CAdxdt
Concentration at the end of inhalation
0.8
I o,
0.2
5 10 15 20
Generation Number
25
1
.1 a8
1 0-6
•r
:| 0.4
£.
£ -i
0
'- •-,.., ^ 'oin'eotu^!
Convection - diilw-ion
\;ra
PIT.
1 10 100
Particle diameter, nm
-------
Regional deposition
I
c (.1.8
£ 0.6
:| 0.4
I ID loo
Particle diameter, nm
Lobar Deposition
Model Verification
I""
4. Measurements of regional and lobar deposition of
nanomaterial in the heads and lungs of rats
> Generation system: TSI Electrospray aerosol generator
Particles: 59FeCl3 (73 mCi/mg of Fe, 44 days half life)
Particle si/.e: 5 nm to 100 nm
^ Exposure system: Cannon nose-only tower
Animals: Long-Evans rats
Kxposure duration: 30 minutes
> Detection system: SMPS for size measurements
Gamma counter for activity (mass)
Experimental procedure
^ Prior to exposure (10 minutes)
- Clean air through the nose-only tower
- Baseline measurement of breathing rates for each animal (Buxco system)
- Particle size distribution measurements using SMPS
^ During exposure (30 minutes)
- Breathing rate measurements
- Filter sample collected as a port of the nose-only tower
S Post exposure
- Particle size distribution measurement
- Animals asphyxiated by a direct flow of CO2 into the nose-only tower
- Tissue samples collected in a gamma counter and activities measured
o o o o
-------
ar Deposition
Lung Deposition
H 4 Ii, i 2-1
Particle Diameter, mn
Deposition fractions in the nasal airways of humans and rats
were measured for particles sizes between 5 nm to 100 nm
A semi-empirical deposition efficiency formula was obtained
as a function of Sc and A.
Model of particle deposition in the lung was extended to
ultrafine (nano) size range by including axial diffusion and
convective mixing (dispersion)
Lobar and regional deposition of nanoparticles were measured
in Long-Evans rats
-------
Preparation and Application of Stabilized
Fe-Pd Nanoparticles for in situ
Dechlorination in Soils and Groundwater:
Factors Affecting Particle Transport and
Reactivity
Progress Report II: Septembers, 2007
Don Zhao. Chris Roberts1, F. He and J.C. Liu1
Department of Civil Engineering
1 Department of Chemical Engineering
Auburn University, Auburn, AL 36849 /f&s
Primary Accomplishments in Year 2
Prepared nanoparticles of various size using
CMC (carboxymethyl cellulose) as a stabilizer
Tested effects of particle stabilization on
reactivity
Tested transport behaviors of ZVI
nanoparticles in porous media
Tested degradation of TCE in soils
Pilot tested in situ dechlorination in soils
using stabilized ZVI nanoparticles
Size-Controlled Synthesis of ZVI Nanoparticles
Using CMC as a Stabilizer
Step 1. Solution
Step 2. Fe^ or Fe24
complexes with
stabilizer
Step 3. Formation of
Fe(0) clusters coated
with stabilizers
Step 4. Formation of stabilized Fe
Pd bimetallic nanoparticles.
He et a/., I&EC Res. 2007, 46(1), 29-34.
Size distribution of ZVI nanoparticles synthesized
with CMC of Various M.W.
80
60
>200nm 15.6±2.6nm 16.8±0.8nm
^
18
6i
1.7nm
ESS™
I 1 D < 50nm
\KiMA 50nm < D < 200nm
ma D > 200nm
HP-7A CMC90K CMC250K EP-ML
Stabilizer
Fe2+ = 0.1g/L;CMC = 0.2% w/w, temp = 22° C
He and Zhao, Environ, Sci, Techno/. 2007, 41, 6216-6221,
-------
-------
Transport of Stabilized ZVI Nanoparticles
Media Properties
sity porosity velocity
1.49 0.421 0.0302 26.2 0.993
1.57 0.388 0.0327 21.3 0.993
1.73 0.360 0.0353 18.1 0.990
1.74 0.355 0.0358 25.9 0.97
Breakthrough Curves of ZVI through a Sand at
Various Pore velocity: (0.017, 0.035, 0.071 cm/s)
1.0
0.8
0.6
0.2
0.0
° „ „ : 0 °.
/ nlr ^ T. ' ^
ji*iC ^ -o- -i- -»-
n ° ' °
u
I D 1.2 rrUrrin
T 0.6 rrUrrin
0 0.3 nUnin
Model, 1.2 nUnin
li| Model, a6 nUnin
M Model, a3nUnin
0123456
Pore volume
7 I
-------
Concentration Histories of Tracers and
ZVI in MW 2
o°
Q
0.25 •
0.20 •
0.15 •
0.10 •
0.05 •
0.00 •
* MW-2 Br"
• MW-2 SO/'
O MW-2 Fe
^A
L-3st-$r-n-*r-6-<)r-^-^-(>-^-i
Time, days
-------
Summary
1 Developed a method for synthesizing ZVI
nanoparticles of controllable size and soil
mobility and reactivity
Factors such as CMC M.W., CMC/Fe ratio, pH,
and T can greatly affect transport and
reactivity of nanoparticles
1 The stabilized ZVI nanoparticles can be
delivered and distributed in soils
1 The nanoparticles can effectively degrade
NAPLs in soils and groundwater, and may
boost biodegradation
Publications
• 8 journal papers published
• 5 more under review
• 1 U.S. patent
• ~20 presentations
• 2 Pilot tests
Acknowledgements
USEPA STAR Grant (GR832373)
Geomatrix Consultant, Oakland, CA
Colder Associates, Atlanta, GA
Dr. Gupta in Chemical Engineering
Department for DLS analysis
-------
im. 'SrfW
UNIVERSITY
of to In
EPA Project 2004-STAR-1-A1
Grant Number RD 8317230
Co-Investigators / John M. Veranth, Chris A. Reffly, Gary S. Yost
Faculty Collaborators
N. Shane Cutler, Philip Moos, Agnes Ostafin
Students & Staff
Cassandra Deering, Mike Koch, Erin Kaser, Diane Lanza
Department of Pharmacology & Toxicology
University of Utah
Interagency Workshop on the Environmental Implications of Nanotechnology
Washington, DC
September 5-7, 2007
Where we are - Where we are going
Paper comparing lung epithelial cell responses to micron-
and nanosized oxide powder pairs published in Particle &
Fibre Toxicology (2007)
Continuing evidence that nano-sized metal oxides have
moderate potency to epithelial cells when compared to
other environmental and occupational agonists.
Studying other cell types in lung - vascular endothelial
cells appear to be sensitive to particles.
Studying activation of cell signaling pathways and changes
in gene transcription.
Ongoing testing for artifacts that may confound results.
UNIVERSITY
Hypothesis: Transition metals in particles induce pro-
inflammatory cytokine production via reactive oxygen species
production.
- Assumption: Due to their high surface area nanoparticles
are like to induce larger responses in cells than their micron-
sized counterparts.
Approach:
- Commercially available particles of metal oxides.
- Physical characterization of particles.
— In vitro cell culture screening assays & in vivo confirmation.
— Followup studies based on new evidence.
l\rvERsrrv
'UTAH
Panicle Types
Si02
— Thermally generated nanoparticles
- Fluorescent (aqueous process) nanoparticles
- Lab synthesized & surface modified nanoparticles
— Comparison to micron-sized: amorphous and Min-U-Sil
Other Oxides
— Supermicron- and nano-sized. Manufactured powders.
- TiO2, Fe2O3, A12O3, NiO, CeO2, ZnO
Comparisons to environmental PM
- Soil-derived dust
- Diesel PM
iNimsrrv
vitro Panicle loxicolo&v
Small peptide extracellular
signaling molecules that are
important for regulating cell
giowth, tissue differentiation,
inflammation, and other
* piocesses. Interleukin-6 (IL-
6) is a marker of inflammation.
IL-6 is increased in humans
exposed to high levels of air
pollution and in persons with
lung disease.
, 1T| I'I-ISA
/, '* Enzyme-linked
=,j£ V immunosorbent assay. Widely
"*"*"•*,* used for measurement of trace
^ pioteins in biological media.
Quantitative Real Time .PC 7?
jor changes in g^ne expression
^_ Isolate total RNA » Revise Transcribe to
Real-Time PCR
University of Utah
-------
BEAS-2B immortalized
lung epithelial cells in
KGM media.
Soil Dusts
- PM2.5-enriched material
representative of unpaved
roads at three sites.
Positive Controls:
- TNF-o, a macrophage-
generated cytokine known
to induce IL-6.
- V (Soluble vanadium),
the potent component of
residual oil fly ash.
UNIVERSITY Two 7V'-/""''/'-?.v on the Same Data
iii.T
Panel A shows a single experiment that found statistically
significant responses to nanoparticles
Panel B includes additional biological replicates and compares
results to positive controls. Note break in Y-axis scale.
INtvERsrrv Other studies have reported similar results.
fjm*^
Hrf^M
rkwnw
-------
Arrhythmia
e Rupturel 4 ^ Thr
Myocardial Infarction
tcRP
jm
UNIVERSITY
"UTAH
' . •
t< ! t
,' ,'
, " ',
.
' ' /
.
IL-6 EL IS A HAECs
300
250
I w
S~
_ 1S(I
3 100
so
0
• lOnmSICH
* WOnm S)O2
.ri
0 0.1
ll
0,316 1
ug/cmZ
•I
J
3.1S .
. '.' '
' J J -
Human aortic endothelial
cells show statistically
significant IL-6 ^
response at much lower
concentrations than
BEAS-2B epithelial cells.
100 nm silica appears
more potent than 10 nm
amorphous silica.
Followup work will focus
on surface-modified SiO2
particles, (amine and
carboxyl).
UNIVERSITY
"UTAH
! 20
7Vo endolhelicil cell ivpcs
Two natwpctrticle types
u HUVEC •HAEC
J J
oHUVKC BHAEC
Quantitative PCR for
Changes in Regulation of
Inflammation-Related
Genes
Both umbilical vein and
aortic endothelial cells are
responsive.
Nano-zinc is comparable
in potency to the silica.
Work in progress.
8(w • 10DnmS*O2 4hr- 1
""UTAH
sooc
I
1 "
Comparisons lo other particle tvpes.
§1-6
1
fi
i
IDHAEC •
r
h
i
UT DD ZnO DEP
1 ',.1
I 'it
_|m_ P
UT
M
DC
t
tr
t a
^
• Treatments
UT = untreated
DD = desert dust PM2. 5
ZnO=10nm
• Endothelial cells are more
responsive to nano-Zn
than to diesel soot (an
incidential nanomaterial).
• Future work on cellular
1 response mechanisms.
.
INlVERSITY
"UTAH
Particle Dosimetrv Issues
Important issue with in vitro particle studies.
Responses are often seen only at doses much
higher concentration than are plausible for lung
from inhalation exposure.
However, this may reflect the artifacts of cell
culture.
Lung surfactant is 0.05-0.2 fun thick compared to several mm of cell culture
media.
Seagrave reports much higher response with cells grown at air-liquid interface.
2-4 mm
fluid
cells Imicron thick
(not to scale)
Our doses are within range of similar studies.
Ongoing Work
Continued comparisons between lung epithelial
and endothelial cells in vitro.
Use specific inhibitors to study cell signaling
pathways activated by the more potent types of
nanoparticles.
Animal exposure (intratrachael aspiration) to
validate in vitro results.
University of Utah
-------
A Toxicogenomics Approach
for
Assessing the Safety of
Single-Walled Carbon Nanotubes in
Human Skin and Lung Cells
Presenter: Mary Jane Cunningham, Ph.D.,
Alternative Method for Predicting Toxicity
Objectives:
• Obtain expression profiles (EP) of:
-Known nanomaterials
-Unknown nanomaterials
-Compare EPs
to ID toxic effects
Use "systems biology" approach to:
-Perturb the biological system
-Reiteratively sample over time or dose
-"Data-driven" approach +"Reverse engineer"
cellular pathways
Manufacturing and Analysis of
Single-Walled Carbon Nanotubes
(SWNT)
SWNT: Manufacture and Characterization
Manufactured by a modified chemical vapor
deposition method (OU, SWeNT)
-Less than 1 % heavy metal contamination
-Two predominant species: (6,5) and (7,5)
-Avg diameter=2,76nm
Electron micrographs: SEM, TEM, STM
-------
Previous Results with
Primary
Human Epidermal Keratinocytes
(Dermal Exposure Route)
Experimental Design
Substance Harvest cells an time points
isolate total RNA
Make and fabe! cRNAs
Hybridize, scan and image anslyz
gene expression micfoarrays
Screen for 10,000s of gene activities simultaneously,
galihcare, atie-color, 30 bp otigos embedded in g&\ matrix
Profile Similarities-Noncvtotoxic Dose
Profile Similarities-Cytotoxic Dose
ci
and
SWNT
SWNT!
Summary of Results with Skin Cells
• Noncytotoxicdose:
-EP of SWNT is more similar to EP of CI (nontoxic
control)
-EP of SiO2 is the most active
• Cytotoxic dose:
-EP of SWNT is more similar to EP of SiO2 (toxic control)
-EP of CI is most active
• Significantly-expressed genes with Si02 correlated with
previous literature
-genes involved in membrane restoration/remodeling,
inflammation and irritation responses
Studies with
Primary
Human Bronchial Epithelial Cells
(Inhalation Exposure Route)
Dr. Mrinal Shah
-------
Phase Contrast Photomicrographs
Human Epidermal Keratinocytes Human Bronchia! Epithelial Celts
•HEK culture is
•HBE culture co
ciliated ceils (Goblet ceJls) simple c
rounds
-------
miRNA Expression Profiling
2006 Nobel Prize in Medicine for discoverers
microRNA or miRNAs:
-Smaii Single-stranded RNA molecules
{21-23 t>p)
-Non-coding RNAs tftat regulate
ne-thtrd of ait human qenes
-Comptemenfary t
-Csli proliferation, differentiation.
Summary from Proteomics and miRNA
Protein Expression:
• Only 8 proteins significantly expressed at 24hr.
• Need more time points
• Switch to large format gels or LFQMS
miRNA Expression:
• CVs are <20% between array replicates
• Most miRNA expression seen with SiO2
• 71 miRNAs significantly expressed
• Pathway analysis and interpretation ongoing
-no databases for pathways of rniRNAs
-relationships will need to be done manually with comparison of
data mostly from plants and microorganisms
Posiiwm: ',:• ..>•• Dr. Carolina Lema
Acknowledgements
HARC's Toxicogenomics Team
• Carolina Lema, Postdoctoral Fellow
• Mrinal Shah, Postdoctoral Fellow
Daniel Resasco (OU), Learidro Balzano (SWeNT)
Ed Dougherty, Ulisses Braga-Neto, Amin Zollanvari, Yufei Xiao
(Texas ASM)
Scott Magnuson, Michael Falduto (GenUs BioSysterns)
Bo Curry and team (Agilent Technologies)
John Wlktorowicz and team (UTMB Galveston)
National Science Foundation (BES-0436388 and BES-053667
HARC and She George P, Mitchell family
-------
of
Nanoparticles
Pedro Alvarez (Delina Lyon*) & Mark Wiesner
Interagency Workshop on the Environmental Implications
of Nanotechnology 2007
RICE
of
on
PRODUCERS
CONSUMERS
+.'K
—- /co, \
DECOMPOSERS 2<
Implications
• Disposal/accidental discharge
can effect microbial ecology
and disrupt biogeochemical
cycles
• Antibacterial activity
indicative of toxicity to higher
level organisms
Applications
• Use for water treatment?
• Other disinfection
in
Wilson Center inventory: >475 consumer products claim to
have nanomaterials
100
1 60
a.
•| 40
3
z
20
0
Major Materials
i Analysis: May IS 206?
i
J
, *
24
« 18 I
!2
Sliver CB*OO Zinc Silica TSlansHn DetxMte GoM
http://www.nanotechproiect.o
rq/44
of
nanomaterials
I
of
0.2mm
filter
Qn in
THF
CMinTHF +
H,0
C60 powder
nC6r
60
is
Standardized Microtox Assay
III
Vibrio fischeri (luminescent bacteria) with
increasing concentrations of nC60
Respiration in
E. co//
decrease/ceases
after exposure to
nCKn
Compound
nC,,,,,
Benzene
Sodium azide
EC50
(mg/L)
1.6
2.0
43-66
-------
nCgo is more toxic to bacteria than
many other common nanomaterials
Antibacterial activities have been
linked to ROS production
In some cases, cytotoxicity is linked to
photochemical ROS production
- Ti02
- nC60
nC60 is antibacterial in dark, under anaerobic
conditions
Production of ROS by fullerenes
Donor Donor+'
K'
UV Light
02 02-
02
OH orH20
Detection of superoxide ion
I In Ml)
I In WHIT
loparticles (5 |jM)
No superoxide production by THF/nC60
in the bacterial growth medium
5 =
Detection of all types of ROS
(O21,OHandO2-)
No ROS production by THF/nC60
in the bacterial growth medium
Evidence of nC60-mediated ROS
damage in other organisms
Damage due to peroxidation of cell membrane by nC60-
mediated ROS production
Fish (Oberdorster et al., Environ Health Persp, 2004)
- Some lipid peroxidation, no protein oxidation
Human Cell lines (Sayes et al., Biomater, 2005)
- Lipid peroxidation, increase in glutathione production, ascorbic
acid afforded protection
Mammalian cell lines (isakovk et ai., Toxkoisd, 2006)
- Intracellular ROS detected, lipid peroxidation, protection by n-
acetylcysteine
-------
Does nC60 produce ROS in
bacteria?
HjDCFDA s^-
Activated by ZQg
esterases, [
Oxidized by ROS \
to fluoresce ^^
^
Hydroethidine
Oxidized by
superoxide to fluoresce
•~TI HJCF 1 .X''^^^
ijPf 1
i5r ill" J
V V_-^
==:=^=^==:::^
Looking for lipid peroxidation
as evidence of ROS damage
Hallmark of lipid peroxidation is malonedialdehyde
(MDA)
MDA forms colored adducts with thiobarbituric acid
Assessing MDA-TBA adducts in cells Exposed to nC
No Evidence of Oxidative Damage
of Cytoplasmic Proteins
• An immunoassay was used to detect carbonyl
groups (evidence of ROS damage) in
cytoplasmic proteins.
• nC60 did not cause oxidative damage as
compared to the control.
control
-
11 Internal standard]
No conclusive evidence of ROS production
or ROS-mediated damage
If not ROS, then
how does nC60 exert its
antibacterial effect?
is an oxidant
Substance
ORP
value (niV)
water
221-
297
10 mg/L
nC60
483
1 M malic
acid
276
1 M ferric
chloride
690
1 M ferrous
sulfate
291
Could nC60 oxidize
cell components or
act as an uncoupler?
Membrane potential changes
Why membrane
potential changes in
Gram positive
B. subtilis but not
Gram negative E. colil
Assay monitors DiOC2
- Red fluorescence indicates higher
membrane potential
Higher red/green ratio means higher
membrane potential
CCCP is an ionophore
-------
Reverse Elecirou reimport (RET)
red by nC60
nC60 is
membrane integrity, or oxidizes proteins
What is the antibacterial
mechanism of nC60?
No conclusive evidence of ROS
production or ROS damage
- Re-evaluate previous results based on the
ability of nC^ to interfere with assays
nC60 oxidizes bacteria
- uncoupler
- oxidize respiratory proteins
Does nC60 puncture cells?
Propidium iodide enters permeablized cells and
stains nucleic acids
cannot enter
intact cells
Acknowledgements
Lena Brunei, Laura Adams,
David Brown, George Hinkal
This research was funded through Ihe EPA STAR
program (91650901-0)and Ihe Nanoscale
Science and Engineering Inilialive of Ihe NSF
(#EEC-0647452).
CBEN
-------
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
Objectives
1. Measure the agglomeration rate of carbon
nanoparticles
!. Identify whether agglomeration is affected by
altering exposure conditions such as humidity
and particle charge
3. Compare the toxicity of singlet vs.
agglomerated particles in mice exposed via the
inhalation route
Experimental Approach
Establish the agglomeration of freshly generated carbon
nanoparticles at various distances (i.e., aging times)
downstream from particle generation in a dynamic
exposure system
- Generated with a spark furnace
Expose mice to nanoparticles at different stages of
particle agglomeration
Expose to singlet and agglomerated at same time
Lungs will be examined for injury and inflammation
Are findings for carbon nanoparticles applicable to other
nanoparticles?
- Generate zinc and copper nanoparticles
Methods
Generate nanoparticles with Palas generator
using Argon
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
-------
Aged vs. Fresh Carbon
Nanoparticles
Data Presented Last Year
Low, middle, and high concentrations =
1, 2.5, and 5 mg/m3
Fresh = 1.5 sec downstream of the
particle generator (« 11 to 90 nm)
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
Effect of Other Nanoparticles?
Copper
Zinc
Effect of Copper Nanoparticles on
PMNs in Lavage Fluid
Copper concentraton (mg/
-------
Fresh Copper Nanoparticles Effect on
Protein
Same general dose-response as for PMNs
Air 0.15 0.2 .3 0.7 1.0
Exposure Concentration (mg/ni )
Exposure Concentration (mg/ni
Do All Humans Respond the Same?
I
Is*
'i
I
QUANTITATIVE GENETICS
3
-------
MINI
Illliiiii
(Hi)'
Genetic homology
of human and
mouse genomes
• Colors and corresponding
numbers on the mouse
chromosomes indicate
the human chromosomes
containing homologous
segments
. From D.O.E. Human Genome
Program Report, 1997.
Conclusions
Dose-response relationship between expo
carbon and metal nanoparticles and lung
inflammation
- Fresh » Aged effec
not for others (coppe
Humidity and cha
carbon nanoparticle
type of particle (carbon) but
~t on the toxicity of
HsE
elusions (cont....)
anoparticles
carbon nanoparticles
- Unlike with carbon nanoparticles, copper had only a small
difference between fresh and aged nanoparticles
- Copper nanoparticles were more toxic than zinc
nanoparticles
Strain differences in response suggest that genetic
susceptibility could be involved in the response to
nanoparticles
-------
This research is funded by
US. EPA -Science To Achieve
Results (STAR)Program
Grant#
RD-8325280
Nick Halzack, Karen Galdanes, Maire Heikkinen,
and Judy Xiong, Lung Chi Chen, Beverly Cohen,
Martin Blaustein
-------
• - • •
Transport and Retention of Nanoscale
C-60 Fullerene Aggregates in Water-
Saturated Soils
Kurt D. Pennell1'2, Linda M. Abriola3,
Joseph B. Hughes1, Yonggang Wang1,
Yusong Li3 and John D. Fortner1
'Georgia Institute of Technology, 2Emory
University and 3Tufts University
©
Background
• Fullerene-based nanomaterial production ^*,
is rapidly expanding ***
• Potential Toxicity: Lipid peroxidation, oxidative
stress, reactive oxygen species (ROS)
• C60 forms stable nanoscale aggregates in water:
» Aggregate diameter: 95-200 nm
» Size and stability is dependent upon ion strength
• Limited research on n-C60 transport in porous media;
high velocities, small columns, no retention profiles
• Classical filtration theory used to describe n-C60
transport and retention
Video of n-C60 Aggregate Suspension
(dia. -95 nm, 1.0 mM CaCI2, -0.3 mg/L)
Research Objectives
1. Investigate the transport and retention of n-C60
aggregates in water-saturated soils as a
function of soil properties and systems
parameters.
2. Assess the effects of n-C60 aggregates on soil
water retention, water flow and transport in
unsaturated soils.
3. Develop and evaluate a numerical simulator(s)
to describe n-C60 aggregate transport, retention
and detachment in subsurface systems.
Experimental Methods: Column Studies
•:• n-C60 suspensions: THF+H2O; 95-120 nm dia., ~ 3.0 mg/L
•:• Aqueous phase: 1 .0 mM CaCI2 + 0.065 mM NaHCO3 or D
•:• Pulse width: 3-10 dimensionless pore volumes
•:• At least two replicates per experiment (repacked column)
HPLCPump |
n^
/^ o\
Syringe Pump
Electrolyte
3-way Valve
C;i>
??•*.
1 Mean Pore
|~l Ottawa Diameter Velocity
I I 1 I |— |— | Sand (mm) (n^d)
~°',' n n n n n n n 2°-30 a71 ~ 8'°
"0lUmn|||||||IHIIII ^sh -1.0
Fraction collector Mesh
-8.0
-1.0
n~~l 80-100 0.16 ,",^°, , ,
T Mesh -1.0
*Qy?A
40-50 mesh glass beads (GB) or Ottawa Sand (OS),
c/5(j=0.33 mm
n-C60 suspensions: 95 nm dia., 1.3 or 2.3 mg/L
Aqueous phase: 1.0 mM CaCI2 + 0.065 mM NaHCO3 or Dl
n-C60zetapotential (mV): -29.2 (1 mM CaCI2); -63.98 (0 IS)
Pore-water velocity (v^ ~ 8.0 m/d; Flow rate, (Q) ~ 1.0 mL/min.
Experimental Sequence:
-------
0.8
13 0.6
Q
0.4
0.2
C
» =
n-C60 Effluent BTCs
40-50 mesh Glass Beads
Co °/o
OOocPOOOOOOOcCOOOo m 23 9;
0 _^^»^^% A 1.3 10
• A****^ • L3 w
« A*A ^.«* o Tracer
« A «•* .
// :
OH A •
*.*
ama3>" \
) 2 4 6 J
Pore\£>lumes
0.38' IS =1.0 rnM CaCl • PVV = 3 pv Q = 1 .0 mL/min
j^>
HB °/oRet "\
S 8.6
D.5 33.8
£ 483
5
-j
0.8
gO.6
0.4
0.2
0
(
8 =
n-C60 Effluent BTCs
40-50 mesh Ottawa Sand
Co %M
cpnOrCOOOCOOO OCOO A 23 98'
° B 2.3 97/
o H^4^*4*" • L3 102
•**A**^ o Tracer
• A^« A
0 p«AA'" *
• AB • A
3246!
PoreVolLmes
0 3^' IS =10 rnM CiCl ' PVV = 3 or c< DV o =10 r
B %Eet !it**\
77
48.1
2 59.7
3
L/rnin
1.5
a
0.5
0
8
n-C60 Retention Profiles
40-50 mesh Ottawa Sand *?$
Co °/i>MB °/oRet ^*1^
A 2.3 98.4 77
B 2.3 97.6 48.1
H • 1.3 102.2 59.7
*"•••••.•.
D 5 1C 15
Distance from inlet (cm)
= 0.36' IS = 1 .0 rnM CaCl • PVV = 3 or 5 pv Q =1.0 mL/min
Did n-C60 Aggregate Size Change?
•
p Effect of Ionic Strength (IS) ^
40-50 mesh Ottawa Sand *<&£
1
0.8
0.6
0.4
0.2
^55^5^
3&x>
o
O Tracer
a OS4
. A
/
0 2
\
\
4
3 12
JOB
04
\ /^^
^T 0 5 10 15
PoreV°llmS n-«.ft«n,rt«(cn|
Zeta
C
OS1 2
•.'•SI '
Potential:
0 IS
3 ImM
PW
3
3.1'
%MB %Ret
97.6 48.1
1"".: 1.8
-29.2 mV (1mM CaCI2) or -64.0 mV (Dl)
Experimental Study 2
n-C60 suspension: ~120nm, ~3.0mg/L (5 or 10 pore volumes)
Four size fractions of Ottawa sand:
20-30 (0.71mm), 40-50 (0.35mm), 80-100 (0.16mm),
100-140 (0.13mm)
Two Electrolyte Conditions: 1mM CaCI2 + 0.065mM NaHCO3
or Dl water
Total of 22 column experiments
-------
Selected n-C60 Effluent BTCs:
Effect of grain size and flow
1.2
1.0
0.8
§ 0.6
O
0.4
0.2
0.0
C
Fast Flow Rate (v ~ 8m/d)
• d
"f/^L i d
.*/•>. A*
*.* f
Jl/ *^_
rate t~^
Slow Flow Rate (v ~ 1m/d)
c = 0.71 mm
c = 0.35 mm
c = 0.16 mm
c = 0.13 mm
•
;
r~'
o
©
j\
2468 10 02468 10
Pore volumes
Pore volumes
Selected n-C60 Retention Profiles:
Effect of grain size and flow rate
Fast Flow Rate (v~8m/d)
Slow Flow Rate(v~1m/d)
. A>
A
A
^ v -v v v v
, , , , . 4
• dc = 0.71mm
• dc = 0.35mm
v dc = 0.16mm
A dc = 0.13mm
V 7 V V
© © Q ©
* f f {
A
A
8 „•••*.
• • • v •
0 2 4 6 8 10 12 14 16
Distance from Inlet (cm)
0 2 4 6 8 10 12 14 16
Distance from Inlet (cm)
Mathematical Modeling
1 11llation Model
vn \
8C a dS 82C 8C
—+—— = A—--,— v —
Bt 6 dt h dx1 p 8x
&
f"^-\
Dispersion is often ignored:
Yields exponential
retention profiles
Po
e^
V
--K,
dt
smgx -
s™x
"c-lr*'
s
,s
Flat retention
profile
(Johnson andElimelech, 1995)
Simulation Results
Fast Flow Rate (v - 8m/d) slow Flow Rate (v - 1m/d)
F
45
40
35
30
? 25
J 20-
15
10
5
0
0
ritted Parameters k£
-•- Fast
| & Slow
I
i
A «
&
ttand
25 -.
20 -
J 10 -
5 -
Q ^SQ^
°max ^*/
-o- Fast
Slow
ED
\
\l
\
0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8
dc(mm) dc(mm)
Low Ionic
• When IS -
identical &•
1 -
0.8 -
g 0.6
O
0.4 -
0.2 -
0 -
C
Strength Experiments
-> 0, n-C60 and tracer BTCs are
/en in 100-1 40 mesh OS
ri D Dl
., n 1mMCaCI2
D
"a:4
2 4 6 8 10
Pore Volumes
C0 = 3 mg/L; PW = 3.8 or
5 pv; V =8 m/d ' Retention is electrostatic in origin
-------
Secondary Minimum
•—-»O
-10 -
40 60 80 1(
0n_c60= - 29.2mV; 0sand = - 53mV; Ionic Strength = 1mM
Secondary Minimum
• Compare with Hydrodynamic Drag Force
Interaction force
due to secondary
minimum
F, = 2.4E-14N
Hydrodynamic
drag force
Fn = 2.43E-16~1.12E-15 N
> Theoretical Attachment Rate katt
3(1-eV
*„, = , an,
2ac
• Collision efficiency - the fraction of n-C60 particles that have
energy less than the secondary minimum
a = J :"f"m f(Ehyi )dE
• Maxwell distribution for frequencies of the kinetic energy
Comparison of Theoretical and Fitted \^{{ -^
'<•>
gO -^
50 -
„ 40 -
*T 3° -
^?
20 -
10 -
I
__^
^•''''
!•' — r\
4k\ dt=°-i6mm J
,''* \J at high flow rate
0 10 20 30 40 50 60
K,tt theory (1/h)
Retention Capacity Sn
• Previous Work:
» Smax = f (water chemistry, surface potential)
(Adnmzyk et af., 299f)
» Smax is influenced by "Shadow Effect" created by shear
component of the fluid flow around collector grains.
(KvaKdElvMledt,20CO)
* Difficult to quantify Smax a priori (e.g., batch experiments)
• Our Observations (wltntlxed water chemistry):
* Diffusion is the dominating mechanism for n-C60
nanoparticle transport
*7 ~ ^diffusion
* Fitted Smax= f (flow intensity, particle size)
Influence of diffusional
boundary layer?
Mass Flux in Diffusional Boundary Layer
• Flow in a Pore Tube
Diffusional Boundary
Layer Thickness:
Mass flux to the surface :
Normalized Mass Flux:
4,
*"-li
"f?\
R [M/T]
Correlation between SltBX
and
Normalized mass flux
Fitted S
max (ug/g sane
^ c
D C
1
0.1 -
>max and Normalized Mass Flux ^
""""•s. R*=0.96
"^^
0 10.0 ""* 100.0
Normalized Mass Flux Pe"1 (dc/d,,)
-------
Conclusions
• n-C60 aggregrate transport decreases and retention J
increases as grain size or flow rate are decreased.
• Detachment rate coefficient approached 0, and did not
change with grain size or flow rate.
• A mathematical model that includes non-equilibrium,
non-linear retention captured n-C60 transport and
retention behavior in Ottawa Sands.
• n-C60 aggregate transport and retention is strongly
influenced by IS; importance of electrostatic
interactions.
• Secondary minimum plays an important role in n-C60
attachment.
• n-C60 retention capacity was correlated with mass flux
in diffusional boundary layer.
Confocal Images of Neuronal Cells Treated
with Rhodamine-labeled Iron Particles i
40X
100X
Future Work
Measure and simulate n-C60 transport and retention in
a water-saturated "natural" soil(s) (e.g., Appling soil).
Measure and simulate n-C60 transport and retention in
unsaturated porous media.
Investigate the effect of stabilizing/dispersive agents
(e.g., NOM, surfactants) on n-C60 transport and
retention in Ottawa Sands (Hyung et al., 2007, ES&T,
41:179-184).
Determine THF and y-butyrolactone (GBL)
concentrations in purified and unpurified n-C60
suspensions (Henry etal., 2007, EHP, 115:1059-1065).
Evaluate neurotoxicity of manufactured nanomaterials.
-------
Impacts of Fullerene (nC60 or C60) on
Microbiological Functions in Soil and Biosolids
Ronald Turco
Marianne Bischoff
Zhonghua Tong
Larry Nies
Leila Nyberg
Tim Filley
Kathryn Schreiner
Bruce Applegate
Colleges of Agriculture, Science and Engineering
Center for the Environment
Birck Nanotechnology Center
V
'.-rfj
PURDUE
Our question is whether C60 is impacting the
microbiology in the soil food web
The Soil Food Web
PURDUE
The talk presents the findings from a number
of ongoing projects
Soils Work
Biosolids Work
Fungal Work
PURDUE
Soils are typical of the Midwest and chemical
C60 preparations methods are established
i -*%*,
Hame OM Sand Silt Clay
Drummer 3.6 6 1? 52 31
Tracy . t.S:"S.S: '!
Formation: Deguchi, et
al., 2001
Concentration: Fortner
et al., 2005
Size: DLS system
PURDUE
Our chosen microbiology methods are well
established and documented
Evaluate Microbial Systems
Microbial Form
PLFA
Biomass Size
PCR-DGGE
Three domain model
Functions (CO2 CH4)
Glucose Assimilation (14C-CO2)
Fungal Abilities (13C) PURDUE
C60 and nC60 had little impact on soil
functions
Soil Respiration
Biomass Size
ioil-C THF-C
nC60 1 ppm / C60 1000 ppm - Drummer Soil
PURDUE
-------
Microbial profiling showed no difference after
six months
DGEE -6 months
PLFA3or6Months
PURDUE
No impact from longer incubations - Glucose
assimilation testing method established
Test procedure
Soil Challenged
Soil Incubated
Soil Tested
Response at 6 months
«C-C02
=»&
"C-Glucose
PURDUE
Combinations of fullerenes with soil water
stress show no effects
Five water potentials
Two nano materials
(nC60, C60, C12)
Two Soils
50 Day Incubation
Respiratory response
PURDUE
Soil dive
combine
Fatty Acids
stresses
PC/
10
;rsity showed no effects from C60
;d with water potential
patterns from soils with nanomatrials and underwater
[each symbol has an associated water potential)
/ * a
\I^
^7\
N^
:;;s: :r
PC-1 -^
\ developed from FAMEsfor treated and untreated so . rURDUE
Preliminary data suggests nC60 crystal size
has no effect on soil response
nC60 formed in different
size classes (mixing
speed) added to soil
Respiratory response
after 30 day exposure
PURDUE
Biosolids — (Anaerobic Systems) are not
impacted by C60
H
P^ Biosolids system & C60 (50,000 ppm)
. „„.
I200"
U 150 -
PURDUE
-------
No impact on community structure tested with
bacterial primers
'PURDUE
No impact on community structure tested with
Archaea pr
PURDUE
Ceo type and concentration showed no effect
on anaerobic system (150 days)
Bacteria
Sample ID
CM
(nig/kg
biomass
(d/w)
Fullerene
Prep
0.321
8.6
Dissolved
MeOffiEt
OH
aq-Cg,
Plated on
dried
sludge
(toluene)
Plated on
dried
sludge (o-
xylens)
Eukarya
II
Tracking Fungal utilization of C60 requires 13C60
Fungal Species
Gleophyllum trabeum
Fomitopsis pinicola
Cadophora malorum
"C-C60 added 150 ug C60 (15 atom %)
Growth media
Wood blocks
Tracking "C in
Biomass and Headspace Gases
PURDUE
Averages of 13C/12C ratios fungi grown on wood
blocks were not all that different for the two
materials.
-24.0
-24.5
-25.0
-25.5
Brown rot
4
1
1
~i
White rot
Soft rot
E -
E
f-
3
RT CM FP
Gleophyllum trabeum
Fomitopsis pinicola
Cadophora malorum
PURDUE
C60-OH and fungi is under investigation
White rot
fungi exhibit
rapid
bleaching
Brown rot
fungi show no
change in
color
no C60-ol control no fungus
C60-ol + fungus
-------
In summary, C60 and nC60 have had limited
impact on microbiology of soil and biosoilds.
Transformation of C60 by fungi is also limited.
Soil biomass size and structure unchanged
- repeat applications and solvent effects under investigation
Biosolids biomass size and structure unchanged
-functional groups are being investigated
Fungal utilization of C60 not apparent
- work on C60-oL is on going.
PURDUE
Funding Support
Funding support from National
Science Foundation Award EEC-
0404006 & US EPA Award RD-
83172001-0
PURDUE
-------
Size Distribution and Characteristics of Aerosol
Released from Unrefined CNT Material
Judy Q. Xiong, Ph.D.
Maire S.A. Heikkinen
Beverly S. Cohen
Safety and Health Aspect
Inhalation Exposures
Carbon nanotubes (CNTs) are among the most
dynamic and fast-growing nanomaterials due to their
As production rate scaling up, the potential of human
as well as in the general environments are rising.
Their impacts on human health are of great concern
by many researchers.
To determine the overall risk to human and
environment, not only the material toxicity but also
For many conventional workplace contaminants,
airborne route is considered the most crucial for
nanoparticles, the particle concentration, size
distribution, shape characters, as well as the
agglomeration status are among the main factors.
Characteristics of CNTs
Specific Aims
High aspect ratio (typically in a scope of 102 but can
Highly agglomerated.
amorphous carbon soot, metal catalysts as well as
ambient particulate matters.
The size distributions of CNTs are hard to predict, but
presumably widely spread and source dependent.
To investigate the size distribution and characteristics
of aerosol particles released from various types of
industrial grade CNT bulk materials due to agitation.
The results will provide a foundation for developing
To develop a practical method using atomic force
microscopy image analysis that is capable to classify
CNTs from other co-existing nano-sized particles in
general environments.
To develop appropriate methods for monitoring the
potential worker exposure levels to CNTs.
-------
Sample Material Sources
the methods by which they are manufactured.
Therefore, size distributions of CNT aerosol particles
also depend on the source of the material.
Up-to-date 7 Industrial-grade bulk CNT samples from
3 manufacturers have been examined.
The sample matrix includes 3 common types of
CNTs, i.e., single-walled, double-walled and multi-
walled, and 3 primary methods of production, i.e., arc
discharge (Arc), chemical vapor deposition (CVD),
and high-pressure CO conversion (HiPco).
Calibrated Aerodynamic Cut-off Size of ELPI
Methods
Airborne Particle Sampling and Sizing:
Integrated Screen Diffusion Battery (ISDB)
Stage Number
Filter
1
2
3
-1
5
6
~
S
9
10
11
12
13
Aeiodviianue Cut-size (/im)
0.00"
0029
0,0?6
0,093
0,15"
0,26?
0.38?
0.619
0.956
1.61
2,-li
-1.03
9.99
CVD-SWCNT Number Weighted Size Distribution
dp<100 nm: -80%
-------
HiPco-SWCNT Number Weighted Size Distribution
Grade: AF
Diameter: 0.
CVD-MWCNT Number Weighted Size Distribution
f-
n fl m
-
-i
Sample: CVD-MWCNT
Grade: Short
Diameter: < 10 nm
Manufacture: Helix, TX
-------
This portable device was developed for collecting
time-integrated samples of nano-sized particles,
based on diffusional collection of particles on
filtering elements and walls of a round tube (2 cm
in diameter).
The filtering elements used in this study are
stainless steel wire screens in different mesh sizes
(60-440).
On the walls of the tube, between the filtering
elements there are recessed slots for duplicate
detectors. Mica discs were used as collectors.
The sample collected on the mica discs can be
analyzed directly by AFM.
When particles are sampled into the tube the smallest
particles, with highest diffusion coefficients, are
collected first. Increasing number of bigger particles
will be collected by the subsequent filtering elements.
The collection efficiencies of the wire screens are
(Cheng and Yeh 1980, Cheng et al. 1980, 1985).
Particle size dependent deposition efficiency on the
substrate is calculated with an equation developed by
Ingham(1975).
Particle size distributions are calculated using the
Extreme Value Estimation deconvolution program
(Paatero 1990).
-------
Counts of Particles Collected on
the Wall of Each ISDB Stage
"or d J i re disMrbution a* CVT 32!&
Stages
1
2
3
4
5
6
Particle
Counts/1 0000nm2
mean
1120
1584
1520
1000
76
46
s.d.
242
156
171
102
7
13
Summary of Experimental Results
Characterization of CNTs
Sample Collection: ELPI (on Aluminum Discs
Sample Analysis: Atomic Force Microscopy.
Sample Preparation: Deagglomeration of samples
by applying appropriate surfactant/solvent and
sonication.
All common types of unrefined CNTs including
single-walled, double-walled and multi-walled
nanotube samples can be dispersed into air to a
significant extent due to agitation.
The sizes of particles generated from all CNT types
are widely distributed across 13 stages of ELPI,
ranging from 7 nm to 10 |im. The size distributions
vary with the type and the nature of bulk materials.
For HP grade CVD-SWCNT, majority of particles are
in the nano-size region (< lOOnm) based on the ELPI
data. There is also a significant portion of particles
found in the single-nanometer range based on the data
collected by ISDB.
Airborne CNT particles are highly agglomerated; no
single tubes or simple ropes were observed by AFM
in the original samples collected by ELPI or ISDB
before treatment with surfactants.
-------
Imolications
Carbon nanotubes can become airborne and expose
workers through inhalation or dermal contact during
the processes of manufacturing and handling.
The size distributions of CNTs are wide and source
dependent.
As deposition efficiency and sites of inhaled particles
within the respiratory system largely depends on
particle size, the deposition pattern of agglomerated
nanoparticles should be similar to those larger
equivalent sized non-agglomerated particles.
Particles depositing on/in the deep lung surfaces of
the bronchioles or alveoli will contact pulmonary
surfactants in the surface hypophase and the
agglomerated CNTs are likely to (ultimately) be de-
agglomerated.
Investigations that define CNTs should take into
account the full size range of particles to which
humans are likely to be exposed.
Adequate monitoring methods need to be established
for quantification and characterization of these new
On-soins Studies
Acknowledgements
Developing a quantitative sample treatment method
for AFM analysis that can effectively deagglomerate
samples by applying appropriate surfactants, solvent,
and sonication.
Exploring other advanced AFM technologies that
may be better suited for CNT characterization, such
as, Conductive-AFM and Phase Imaging.
Developing a validated field sampling method for
airborne CNT particles in workplaces.
This study was supported by U.S. National Institute
for Occupational Safety and Health (NIOSH) under
Grant 5-R01-OH008807.
Partial support was supplied by National Institute of
Environmental Health Sciences (NIEHS) under Grant
ES-0260.
-------
UH BROWN
Physical and Chemical Determinants
of Carbon Nanotube Toxicity
Robert H. Hurt, Ph.D.
Division of Engineering
Agnes B. Kane, M.D., Ph.D.
Department of Pathology and Laboratory Medicine
Brown University, Providence, Rhode Island
1. Bioavailability of nanotube metal residues
2. Adsorption of essential micronutrients by
nanotubes and implications for toxicity testing
3. TPGS as a safe, antioxidant surfactant
for green nanotube processing
4. Targeted removal of bioavailable metal
as a nanotube detoxification strategy
on
toxicity
mechanisms
on toxicity
management,
or "green"
nanomaterials
Many Nanomaterial
Samples are Complex
1. Bioavailability of Nanotube Metal Residues
• Catalytic growth methods:
- now dominant for synthesis of multiwall nanotubes (esp. large scale)
- only route for single-wall nanotube synthesis
• Most common elements in CNT catalyst formulations are Fe, Ni, Y, Co, Mo
• Ultrafine metals pose documented inhalation
health risks depending on form,
exposure route, dose
• Do metals contribute to CNT toxicity?
How can we assay for and manage
CNT metals effects?
SWNT or aggregate
Molecular
Mechanisms
of Ni Toxicity
[ Liu, Gurei. Morris,
iVkirray, 2!hitkovich,
Kane. Hyrt Advanced
Ni ion hypothesis: Ni toxicity, carcinogenicity depend on intracellular Ni2+ pool
Nickel Mobilization from SWNTs: Effect of Media and Sample Origin
lysosomal pH
Various Ni-SWNT sample;
[ Liu, Cure!, ryferris,
Murray, Slhitkovich,
Kane. Hurt, 4dVance
Materials, 2007]
-------
Cytotoxicity and Cellular Uptake of NiCI2
Newport Green DCF
Environmental and Processing Stresses
Affect Metal Bioavailability and Toxicity
[Liuetal, 2007; Guoetal., 2007]
Ki.iiii..nmTCTi..a..^a,.,.J,i.a»
CNTs show redox activity through release of bioavailable iron
[ Guo., Morris, Liu, Vaslet, Hurt, Kane, Chemistry of Materials, 2007 ]
Fe2+ o,
Fe3+
^ 1%™' I". "V'Vi,' Fe-containing
Suparcoil*d Open Circular
m BROWN
Plasmid DMA assay
Single-strand break
Induces uncoiling event
detectable by
gel electrophoresis
Iron Bioavailability and
Redox Activity of Diverse
Carbon Nanotube Samples
From Guo etal.,
Chemistry of Materials
2007
Fe mobilization
DMA
single-strand
breaks
2. Adsorption of essential micronutrients by nanotubes
and its implications for toxicity testing
Carbon nanomaterials observed to interact with molecular probe dyes
• MTT assay for cell viability gives false indication of CNT cytotoxicity
due to interaction with MTS metabolite dye
- Worle-Knirsch et al. A/ano Lett., 6 (6): 1261 -1268, 2006
• Various indicator dyes are unsuitable for quantitative
toxicity measurement
- Casey et al. Carbon, 45: 34-40 2007
• Even carbon black (negative control) can influence biological assays
- Monteiro-Riviere etal. Carton, 44 (6):1070-1078, 2006
Dose-dependent CNT adsorption
of amino acids from cell culture media
D100ug/ml
Qlmgtal
H 10 mg/nll
I
ASP MET TYR GLU HIS PHE
-------
Correlation of amino acid adsorption with hydrophobicity
'*-'l
,-»'*•*' •'/
Val "/*'.„
Hydrophobicity index
of Black and Mould, 1991
Depletion mechanism can be studied through
single-component experiments
adsorption ' ;
Isotherms u. 'i ~ desorption
for
phenol red
0. 2 -> Sulfonated SWNT adsorption
0.1 -;
• -^^BV .
Some vitamins are depleted at CNT doses as low as 10 ug/ml
s 0.0025
§ 0.002
•iH
is 0.0015
fH
§ 0.001
o
§ 0.0005
0
Ji
-Do^ix y^
"X /
; TL
!****_ [ H**_
__
|
J5
i
^,
x
x
X
j
*
,._
***
Riboflavin Biotin Pantothenic Folic Acid
Acid
D control DO.Olmg CNT/ml SO. ling CNT/ml
3 ling
CNT/ml H lOmg CNT/ml
Biological implications
of media depletion
:gyi
3. TPGS as a safe, antioxidant surfactant for green CNT processing
[Van, Von Dem Bysscte, Kme, Hurt, CARBON, in press]
• Many synthetic surfactants show appreciable
toxicity and/or environmental health risk
• a-TocopherylPolyethyleneGlycolSuccinate (TPGS)
is a water soluble form of vitamin E used as a
dietary supplement and drug delivery vehicle
• TPGS cleaves by enzymatic hydrolysis to deliver the
lipophilic a-tocopherol (Vitamin E) to cell membranes
• TPGS is commercially available. It is not marketed as
a surfactant, but is an amphiphile based on structure
• Its interactions with nanotubes and fullerenes
have never (to our knowledge) been studied
TPGS is an effective dispersant
for MWNTs and shows a unique
co-self-assembly with C60
TPGS
TPGS is a more effective
dispersant forMWNTs than Triton
4rV 'A
*,-
sta
-------
4. Targeted removal of bioavailable metal
as a nanotube detoxification strategy
* DIH2O
* Low pH
! 2%
t% a! total m
TotaJ Mi in SWNT (wE%)
The bioavailable metal represents
only from 0.5% to 9% of the total metal
Issues to address
A. What is the origin of bioavailable metal,
especially in "purified" samples?
1. kinetic limitations on acid washing?
2. surface re-deposition (ions, salts)?
3. oxidative carbon shell attack
during or after acid wash?
B. How can this bioavailable fraction be
optimally removed without tube damage?
C. Will the non-bioavailable (encapsulated) metal
be stable in the body?
(a question of biopersistance of carbon shells)
Acknowledgements
Financial support for work on
nanotoxicology / safe nanomaterials:
- US EPA (STAR Grant RD83171901'
SBRP grant at Brown (
In the
Hun
Lab
IndrekKulaots, Ph.D.
YumingGao, Ph.D.
Lin Guo
Xinyuan Liu
Love Sarin '" *e
Daniel Morris
Aihui Yan i-3^
Charles A. Vaslet, Ph.D.
Annette Von Dem Bussche, Ph.D.
Kevin McNeil
Michelle Buechner
Vanesa Sanchez
Jodie Pietruska
Ashley Smith
Brief Summary
• All carbon nanotubes studied (as-produced and "purified") release free metal
(Fe, Ni, Y) into physiological fluid phases, which trigger known toxicity pathways.
Metal bioavaliability is influenced by processing and environmental exposure.
Metal bioava liability assays should be standard CNT characterization.
• Single-walled carbon nanotubes deplete essential micronutrientsfrom medium
by physisorption and can affect cell behavior by a new indirect mechanism.
• TPGS, a water-soluble Vitamin E formulation, is also a promising safe surfactant
for carbon nanomaterial processing, esp. MWNT. Future work will attempt to use
TPGS to actively mitigate oxidant damage associated with nanomaterial exposure.
• Bioavailable metal in nanotubes can likely be removed by selective targeting
as a simple detoxification strategy (pending future work).
-------
Environmental Impacts of Nanomaterials on
Organisms and Ecosystems:
Toxicity and Transport of Carbon-Based
Nanomaterials across Lipid Membranes
Jean-Claude J. Bonzongo**, Dmitry Kopelevich *, Gabriel Bitton * *,
J. Gao", Y-M. Ban", andR Tasseff"
Dept of Chemical Engineering* and
Dept of Environmental Engineering Sciences**
University of Florida, Gainesville, FL 32611-6450
Toxicology
- Screening NM using micro-biotests for potential toxicity
- Carbon-based, metal, and metal oxide NM and quantum dots
Biogeochemistry
- Effects on Ecosystem Functions
- Use toxic NM and a series of microbial driven reactions involved in
sedimentary cycling of organic carbon to assess the potential impact of
NM on basic ecosystem functions
Molecular Modeling & Microscopy
- Mechanisms of permeation of NM into the cell
- Assess possible damage to the cell membrane by NM
Microbiotests for Screening Studies
• Small-sized test species
• Rapid, simple, low-cost
Ceriodaphnia dubia Acute Toxicity Test
A short-term (48 hr) acute assay used to assess the toxicity of
freshwater samples
Selenastrum capricornutum Chronic Toxicity Test
May equal or even surpass that of the 48-hr Ceriodaphnia
dubia acute testing
MetPLATE™
Based on the inhibition of the enzyme p-galactosidase by
metals at toxic levels in a mutant strain of E. coli
Toxicity (L€.'5(() of Different Surfactants on
Aquatic Organism Models
Cerodaphnia dubia
B
Sodium Triton SDBS SDS Triton THF PVP Gum
cholate X-15 X-100 Arabic
Toxicity (iC'5(!) of Different Surfactants on Aquatic
Organism Model,1*
Selenastrum capricornutum
100°PPm >1000ppm
600
400-
200-
II II
^ X
C60 Toxicity
THF concentration < 0.1 ppni
Biotests
MetPlate
Ceriodaphnia dubia
Selenastrum capricornutum
Observation
Not Toxic
Toxic
Toxic
EC50
(ppm)
-
0.43±0.11
0.13±0.05
.
-------
Effect of SVVNTs on ,V. capricormititm
(Surfactant: 50 ppm Gum Arabic)
250
200
150
100
50
0.5
Mean length ~ 20 nm
1 2 3
SWNT (mg/L)
" I
Biogeochemistry
Assess Impacts of NM on Basic Ecological Functions
Organic Matter (Ox/Red) >• CO2 + H2O
ANOXIC
. O2/H2O
. N03-/N2
. MnCyMn2*
- Fe3*/Fe2*
-so.2-/s2-
- C02/CH4
• Organic matter decomposition in
sedimentary environments
• Microbial community structure
and activity as indicator of
ecosystem well being
• Measurement of predominant
terminal electron-accepting
processes in sediments
C60 concentration = 0.5 ppm
15 THF concentration < 0.1 ppm
» Control
» C ',.(1- frt'afed slurry
ln[C] = -0.42 t + 4.67
"Pristine" wetland sediments
Time (days)
Preliminary Conclusions
Toxicology & Bio»eochemistry
Microbiotests
- C60 and SWNT toxicity significantly exceeds solvent toxicity
Biogeochemistry
- C60 toxicity significantly exceeds solvent toxicity
- Slows down metabolism of bacteria
- Effect sensitive to soil composition
Open questions
- CWNT at small concentrations promote algae growth ?
- Effect of trace metals in CWNT (MetPLATE™)
- Fluorescence of CWNT
• Transport into cell/cell membranes
• Develop connection with molecuar modeling studies ,
Molecular
Task 1
Understand Mechanism (s) of Permeation of NMs into Cell
CD "= ~ -O
' NM transport through cell membranes
• Model cell membrane as lipid bilayers
• NMs can penetrate cellular membranes by
mechanism different from phagocytosis
and endocytosis (Rothen-Rutishauser et
at, ES&T. 40, 4353, 2006)
• Investigate effects of particle size and
shape on transport
'O.
(Marrinketal, J. Phys. Chem. B 2004, 108, 750)
Hydrophilic head
DPPC
lipid
HycIfoplioWe tail
Groups of atoms are mapped onto a single coarse-grained bead
• e.g., four CH2 groups-* single hydrophobia bead
Good agreement with more detailed models
Significant speed-up of simulations -> fast screening of different nanoparticles
Simulations with GROMACS MD package ( - < \ > -)
Bilayer preparation: self-assembly
-------
Fullerene-like particles
Spheres of diameters Cube
o = 0.47 nm — 1.5 nm
Tetrahedron
,1.23 nm
Rod-like particle
Chain of spherical particles
a = 1.088 nm
of
Constraint Mean Force Method
Constraint force F(z0,t) to hold particle at z= z0
F(z0,t), deterministic force:
dz
F(z0,t), instantaneous random force:
T(z,t)=F(z,t)-.
S. J. MarrinkandH. J. C. Berendsen, J. Phys. Chem. 98, 4155 (1994)
Free
Negligible energy
barrier for entry
<8K
Significant energy well
in bilayer center
Qualitative differences
between spherical and
non-spherical particles
Enerj
id
6
£ 6
S
Q *
0
s
\
0
2
-SO
-10G
j;y Profiles
_-_
/"V Hwdgreup* ^ /^
/ A ™">M* A •
/ A A \ '
.^024
n - 0 67 nm
_"!?*™
""%- , .T^ .-r—
\ " ~ *-' /
\ /
\ /
\ /
\^~*s^~ ^,
im : "
Local Lipicl Structure
(-iff1 82
-! -05 Q
Lipid bond orientation
in neighborhood of
nanoparticle
No change to
tail bond angles
Preferred orientation
•Szz= 1, perpendicular
•Szz = -0.5, parallel
•Szz = 0, no preference
Taif groups
TowiJ
ood
axis-.'Vv 8 1
<&
10
10.
= exp(-At/T)
-3.5 -3 -2.5 -2
z, (nm)
Particles first move in and then rotate
No permanent damage observed in constrained simulations
Short-scale damage/pressure profile change?
Constrained MD cannot predict dynamics correctly
Use alternative approach: Kopelevich etal., J. Chem. Phys.,2005|
-------
2
of on
• Nanoparticles are observed to cause cytotoxidty by
membrane rupture (e.g., Sayes et al., NanoLetters, 10, 1881,
2004)
• Focus on possible physical mechanisms
• Bending modulus
• Important for intracellular nutrient transport
• Pressure profile
• Affects membrane proteins
• Disruption of equilibrium between lipid rafts
Summary
Molecular Modeling
No significant energy barrier to enter bilayer
Long residence time inside bilayer
Size and shape impact nanoparticle transport rates,
dynamics, and localization within membrane
Physical effects (preliminary data)
- Role of rotation of rod-like particles
- Spherical and almost spherical particles:
• No effect on bending modulus
Future work:
- Other physical effects, nanotube rotation
- Effects of NM localization within cellular membrane
Preliminary
Bending and tilt modulus from magnitude of bilayer fluctuations
(May etal., Phys. Rev. E 2007)
10*
* Pure biSayer ]
" Sphere, n - 0 88 nm ]
* Tetrahedron
k = bending modulus
ka = tilt modulus
I Membrane
bending modulus
is not affected
Mean Transport Time
. i Cube
1 Q~' - /
• t- /
/' Tetrahedron
,,-•-*
^M ""
_.„ ^_ ...... _ - -*-«- -_ _^
0.5 07 0.9 1 1
T! lime to enter me nib nine
T2 = time to enter cell interior
„
! - ,
-------
Assessment Methods for
Nanoparticles in the Workplace
Patrick O'Shaughnessy
Overall Research Objectives
1. Identify and evaluate methods to
measure airborne nanoparticle
concentrations.
2. Characterize nanoparticles to assess
their surface and bulk physical and
chemical properties.
3. Determine the collection efficiency of
commonly-used respirator filters when
challenged with nanoparticles.
Instrument Comparison
Comparison Apparatus
Instruments Compared
Tut; LlNivt:i«UY
or IOWA
Instrument Specifications
Instrument
TSI
Handheld
CPC
TSI CPC
TSI DMA
GRIMM
OPC
Matter Inst.
SAA
Model
3007
3010
3071
1.108
LQ1-DC
Application
Count
Count
Count/Diam
Count
Surface
Area
Measured
Unit
#/cm3
#/cm3
#/cm3
#/1000cm3
jim2/cm3
Limits
0-105
10-4-104
NA
0-2x10=
0 - 2000
Particle
Size Range, nm
10-1000
10-3000
5-1000
300 - 20,000
10-80
k" Tut llwvt
of IOWA
-------
Powder Types Analyzed
Iron Oxides:
- High Concentration
- Medium Concentration
Titanium Dioxides
- High Concentration
- Medium Concentration
- Low Concentration
Single Walled Carbon Nanotubes
Sof'loi'A
TiO2 Comparison
Trial
Sets
AVGI
AVG II
AVG III
AVG IV
AVG V
Geometric
Mean (nm)
89.2
118.3
129.1
151.4
104.7
GSD
2.5
2.1
1.9
1.6
2.4
SMPS
Average
Count
13,120
17,735
35,571
34,624
38,252
GRIMM
Average
Count
(fflcm3)
915
1,500
3,754
5,480
3,925
CPC
Average
Count
(#/cm3)
11,363
13,197
30,197
26,696
32,282
LQ1
cm3)
264
450
1,205
1,556
604
Average
SMPS Surface
Area
(unWcm3)
930
1,782
3,171
3,548
3,157
E Of 'lOWA '
Count Correlations
-Tut: LI.Nivt:isi"iY
E OF IOWA
Surface Area Correlations
1000 1500 2000 2500
SMPS Surface Area, um2/cm:
Aerosol Generation
Collison Nebulizer
Added bulk powder to filtered water
Nebulized at 20 psi
in
-------
Instrument Comparison
ample
ham be r
Water Contamination
Typical Water Only Results
250000
£ 100000
Q 50000
6-jet Collison Nebulizer
Ultrapure Water from Lab System
100
Particle Diameter, nanometers
r IOWA.
Water Output over Time
Particle Diameter, ni
Nebulizer Output with Powder
/ \ 25mc,L
/ \
/
Particle Diameter, n
6-jet Collison Nebulizer
Ultrapure Water from Lab System
20-nm TiO2 Added and ultra-sonicated
-Tut; UNIVI
1 OF IOWA
Water Trials
Nebulizer HeatTube
Or Hollow Desiccant Column
Desiccant Columns
-------
Water Trials
Particle Diameter, nanometers
fe'^Mt: LlNIVCRSHV
ftf IOWA
SEM of Water
SEM-EDS
i
Tut: llMVTKSIIY
of IOWA
Instrument Issues
Surface Area Analyzer
J
->
1
Corona Wire
Higi Voltage Voltage
•Particles are charged by unipolar diffusion of ions from the corona
charger.
•A filter downstream from the charger measures the current of the particles
via an electrometer.
•Active surface area (not individual particle surface area) is calculated from
the measured current. .- .,. ..
IKE lilt: LlMV
E tit- IOWA
Areas of Degradation
f Measuring filter - lack of sensitivity. Caused by
deposition of material in the glass/metalfeedthrough
-------
SMPS and GRIMM
NaCI
/*>* \
-,:: / \ 1
1 „ / \
c / \ \
/ \\
/ \
^r \
„
Diameter (nm )
Size Distributions
TiO2
G'"°°
~ T
! "ODD \
" =00 X^V. \
^^ \_
Diameter (nm)
faT^u: LlNivtRSrtv
OF tO\VA
Microscopic Sizing
ESP
T
1'™: UNIVERSITY
or torn
TEM Imaging/Counting
Large Fe2O3 Agglomerate small TiO2 Agglomerate
Captured on TEM grid via ESP collector
TEM Imaging/Counting
—THE LIwivEKSnv
or IOWA
Particle Characterization
-------
Characterization Techniques
Technique
XPS
XRD'
SEM
TEM
BET
Raman
spectroscopy
AFM'
Information
Elemental Composition
Crystallinity
Shape, homogeneity, tube size, size
distribution, and surface morphology
Surface area
Tube diameter, conductivity, purity
Tube length, diameter
faTH^; UNIV
Of k>WA
TiO2 Analysis
Crystalline or Amorphous
Phase
Primary Particle Diameter (nm)
BET Surface Area (m2/g)
Surface Functionalization
Aerosol Aggregate Size
Crystalline
Anatase
4± 1
266 ±3
O, O-H, and H2O
100±50nm
k' TlltllNIVl
of IOWA
Carbon Nanotube Analysis
Average Diameter (nm)
Surface Area (m2/g)
Catalyst Contamination
Conductivity
SWNT
4.5 ±3
457 ± 4
Co < 0.2%
Semiconductin
g
DWNT
2.8 ±2
575 ±10
Not detectable
Semiconducting
feTnE LlNIVtlSIlY
of lo\m
Field Sampling
Nano-structured Lithium Titanate
Facility
Facility Schematic
Mill
Spray Dryer
(not operating)
Rotary
Calciner
Room
hood
Installation
of New
Equipment
Open
BaV Load
Dock
Sampling Locations
ft
;Tnt: llNiv
of IOWA
Rotary Calciner
-------
Real-time Measurements
„ 50,000 7
E
| 30,000 -
| 20,000 -
1 10,000 -
E
I
.250 -,
"E
t .200
1 .150
| .100
" .050 '
s . i
19
«
n fl
ft XX^
— ^-^"--"^-^
|
*j_«J
20 24 21
T
' ' )
R. ,,nbl.
il/ I
*^w- . _U_J^
Ji—
v
*
Conclusions
Material handling of lithium titanate
disperses large particles (>1 |jm)
Ultrafine particles likely associated with
forklifts, welding, grinding
m
Acknowledgements
Faculty
Thomas Peters
- Field Assessments
• William Heitbrink
- Filtration Expertise
• Vicki Grassian
- Particle Characterization
Students
• Linda Schmoll - PhD
- Filtration Studies
• Sherrie Elzey- PhD
- Particle Characterization
• Hyun Ju Park - MS
- Water Contamination & TEM/SEM
• Ron Johnson - MS
- Field Sampling
Staff
Jonas Baltrusaitis
- SEM &TEM Analysis
Funding
FlOSH
sTnt: LI.Nivt:]eiTV
-------
Protecting the environment from nanotechnology
Robert Gawley
Department of Chemistry and Biochemistry
University of Arkansas, Fayetteville, Arkansas
Paralytic Shellfish Toxins
HN 0 HN 0 HN n "'V"'
Y 1 T 1 Y ~| "
.HN^ff(?-W "TrUv "llV""'' TlW •".N^.'l-B^"'*
Vl aL -"'"711 '"•" /NtC %NvtC ^>OH
< *-UH < ^T-OH < -^-OH
T
H2N
T
saxitoxin, SIX
*» ,
Saxitoxin, SIX
Benchmark method for detection: Mouse Bioassay
• Mush up 15-20 clams
•BoilSmininO.lNHCl
• Adjust to pH 3
• Inject into mice, time 'til death
Legal limit: 80 (i»/UM> « shellfish
Detection limit: 40 |i»/l(» »; ~ 1 |iM
The Lawrence Method
-------
Periodate Oxidation of Shellfish Extract
Shellfish Extract 1788 was oxidized with periodate and inj ected into the HPLC
with a 50^iL sample loop.
Shellfish Extract 1788, Periodate Oxidation
Peroxide Oxidation of Shellfish Extract
• Shellfish Extract 1788 was oxidized first by peroxide and inj ected into the
HPLC with a lO^L sample loop
Fluorescence Sensing
STX + Sensor ** STX-Sensor
_ [STX-Sensor]
[STX] [Sensor]
Fluorescence
absorption X ^
HOMO-1—
fluorescence
HOMO-±-l-
Photoinduced Electron Transfer
LUMO V LUMO -
HOMO-I-I- HOMO -I —
LUMO \
PET 1 | radiationless
bHOMO-I-'-
Complexed Host
LUMO V LUMO \
1 hv 1
1 i/ »- 1 /
X # ii absorption X 1 i
HOMO-1-1- At HOMO-1 — It
-I_L Lp J_L LP
LUMO
fluorescence
m "" it"
-------
Host Fluorophores
1 2
anthracene coumarin
366,390/420 328/420
Amax absorption / emission
Selectivity: SIX vs TTX
o HN
Toxicon 2005, 45, 783; corrigendum 46, 477
Effect of Crown Size
15-crown-5 18-crown-6 21-crown-7 24-crown-8 27-crown-9
4.93 5.3 4.68 10.7 16.9
K,, x 104 M-1
Can. J. Chem., 2006, 84, 1273
BDP Crown Binding Isotherm
K = 2.9 - 8.9x105 M-1 in methanol
J. Or?. Chem. 2007, 72,2187
JackThorne
Jennifer Pharr
New Class of Crowns
olllllll
1"
'"[.TOU/5
::
J " >.,*»•
f 1
' * U]U.4 5 ' '
P^^lsl
[crown] = 20 \M
Expansion of top trace
100% Fluarescertee EErthancemenf
at 1 |.iM [Saxiloxin]
Hua Mao
JackThorne
Coumarin Sensor Anchored in
Nanoenvironment of Dendrimer
Permeable Walls'
of Dendrimer
M. Shanmugasundaram
Michael Dukh
Hua Mao
Helen Hayes
-------
Binding Isotherm (Coumarin
Fluorophore)
K,,= 1.2x108M-1
M. Shanmugasundaram
JackThorne
Factors Influencing Enhancement
Polarity of environment
Solvation (entropy)
Ion Pairing
Dilution artifacts?
Sensing in a Nanoscale SAM
Drop of SIX solution
Sensor Monolayer on Slide
Sensor on a Monolayer
15000
12000-
9000-
Chem Commun 2006 1494
350 4GO 450 500 550
Wavelength, nm
Xex = 332 nm
(with Leblanc group)
Drop of 0.1 M acetic acid on
chemosensor modified quartz
782.5 _
700.
600.
500 .
400 .
300.
200.
100.
Emission Spectra of Modified Quartz
at Various Saxitoxin Concentrations
[L ^,
V . Fl*
45.5 pM SIX " ~J\
^r
Cuvette TopVevv
I 10 pM STX
1
\ quartz slide in buffer soln.
buffer only -~^-__^^_
405.0 420 440 460 480 500.5
NM
Ryan Farris
-------
Shellfish Extracts: Absorption
200 250 300 350 400 450 500 550 600
Wavelength (nm)
JackThorne
Jennifer Phan
Spiked Nontoxic (preliminary)
DOH Shellfish Extracts
2005 Alexandrlum bloom, Seattle
• Diluted 1:5 in MeOH
• Cleanup C18 SFE cartridge
• 10 |iL injected into 400 uL of
1 fiM anthracene crown-sensor
in 80:20 MeOH/pH 7.1 buffer
• Centrifuged
• A = 391; *,„ 425 nm (slit 5x5 nm)
0 500 1000 1500 2000 2500
PSP#
JackThorne
Jennifer Pharr
DOH-SF2
(10UL IN 400 1UM CROWN/B.MEOH)
2006 Alexandrium bloom
Extract of blue mussels
Bob Lona
JackThorne
Saxiphilin & C-Lobe
60 —
50 —
40 —
Coomassie
With Henry and Fritsch groups
Kinetics Measurements
Temp
11
17
25
31
37
*0x106(M-1s-1
1.108±2
1.83±1
2.675 ±9
2.68±1
5.47 ±7
0.980 ±3
2.1 ±1
4.1 ±1
8.35 ±4
27.2 ±3
[STX] = 50 nM
KD(nM)
0.884 ±3
1.153±4
1.536 ±5
3.12±1
4.98 ±2
Penny Lewis
-------
Thermo dynamic s
A ?0 nil.-'mol, .,4 S" 3SO fltlMHoP'k '
— Binding exothermic
— Entropy small — most binding energy from enthalpy
D ACp = -720cal/mol-K
— Probably involves burying of hydrophobia surfaces
— Possibly some burial of water molecules
Penny Lewis
Thanks to:
Dr Hua Mao
Dr Mahbubul Haque
Mr JackThorne
Ms Penny Lewis
Mr Ryan Farris
Arkansas Biosciences Institute
National Institutes of Health:
NIEHS, COBRE, BRIN
FDA: Dr Sherwood Hall (SIX)
Washington DOH: Dr Bob Lona (Shellfish)
-------
Evaluating the Impacts of
Nanomanufacturing via Thermodynamic
and Life Cycle Analysis
Bhavik R. Bakshi and L. James Lee
Vikas Khanna, Geoffrey F. Grubb, Yi Zhang
Department of Chemical and Biomolecular Engineering
The Ohio State University, Columbus, Ohio, USA
Interagency Workshop on the Environmental Implications of
Nanotechnology
September 5-7, 2007 Washington, DC
Motivation
n Discover problems with technology before it is
fully developed and adopted
n Guide development of nanotechnology to be
environmentally benign and sustainable
n Understanding environmental impact of
nanomaterials is essential but not enough
n Need to adopt a systems view with life cycle
thinking
nLife Cycle Analysis of emerging technologies
poses unique challenges
Challenges in LCA of Nanotechnology
n Inventory for nanomanufacturing is not available
n Impact of engineered nanomaterials on humans and
ecosystems is only partially known
n Predicting life cycle processes and activities is difficult
since the technology is still in its infancy
Objectives
n Life Cycle Evaluation of Nanoproducts & Processes
• Establish Life Cycle Inventory modules for Nanomaterials
• Perform LCA of Polymer Nanocomposites Products
n Explore predictive model for LCA and impact assessment
• Relationship between life cycle inputs and impact
• Relationship between properties of nanoparticles and their
impact
LCA of Carbon Nanofibers
n Extraordinary high tensile strength
• Tensile strength-12000 MPa, 10 times that of Steel
* Increases mechanical and impact strength of polyolefins
n Near term applications in Polymer Nanocomposites
• Automotive Body Panels
* Expected to replace Steel and Aluminum
n CNFs show more commercial potential compared to Carbon
Nanotubes (CNTs)
-------
System Boundary
Energy and Emissions from these T-...
steps are considered via. literature «.....':::::::'.'...I
and life cycle inventory databases
9NANOFIBERS
Khanna, V. Bakshi B. R. Lee L. J. 2007
k Emissions to Air, Soil, Water,
and other emissions
Data Sources and Assumptions
D Process data
• Journal papers and conference books
• Encyclopedias of chemical engineering and chemistry
D Inventory data
• SimaPro, (PRe Consultants); NREL
D Impact assessment
CML-IA (Leiden Univ. Institute of Environ. Sci.)
• Eco-Indicator 99
Assumptions
D Impact of Nanoparticles is not accounted
D Purification efficiency of 90%
D Catalyst life cycle is ignored (BEST Case)
D 100% efficiency for electric heating (BEST case)
®&
Results-Life Cycle Energy Analysis
25000 -,
20000
|f 15000
10000
5000
0
Effect of Cycle Time
Run time- from 1 hr to 300 (days) x 24 (hr)
CNFs- CH4 • CNFs- Significant Life Cycle
Energy Investment even for
continuous operation
CNFS- C2H4 • May lead to High Cost
1- Hindrance to use in large
volume applications
n i
rl CNFs-C6H6
1 1 Primary Poly Si
M Aluminum steel Polypropylene
Life Cycle Energy and Process Parameters
Effect of Feedstock and Carrier Gas Recycle
Assuming a 100% Recycle of unreacted HC
Feedstock and a 90% Recycle Carrier Gas
3-10% energy savings due to
material recycling
Process Energy still outweighs the
savings due to materials recycling
Life Cycle Energy Analysis- Cont'd
0 Purification
• Process Energy
D Indirect Effects _
CNFs- Methane CNFs- Ethylene CNFs- Benzene
Impact Assessment- Midpoint Indicators
Global Warming Potential
CNFs- CH4
Higher Environmental Impact in all categories compared
with traditional materials
Due to missing data:
• Emissions from CNF synthesis step are not included
• Release and impact of nanoparticles during
-------
Impact Assessment-Damage Indicators
m
Damage to Human Health- DALYs
O.O00025-
O.OOOO2-
3P
J; 0.000015 -
_
Q
0.00001 -
0.000005-
o-
DALY- Disability Adjusted Life Years
CNFs- CH4
CNFs- C,H, D Ozone Layer
• Radiation
Aluminum • Climate Change
• Resp. Inorganics
• Resp. Organics
° Carcinogens
Polypropylene
Steel
F=l I I
Impact Assessment-Damage Indicators
0.9 -
0.8-
0.7-
I 0.6 -
Damage to Ecosystems
S °-3
CNFs-CH4
CNFs- C,H4
PDF- Potentially Disappeared Fraction
• Land Use
• Acidification/ Eutrophication
Q Ecotoxicity
Polypropylene
Conclusions for First Objective
n On an equal mass basis:
• CNFs require significantly higher energy investment
compared with traditional basic materials
• CNFs do seem to have a larger life cycle environmental
impact than traditional materials
n High energy may lead to high cost thus restricting use of
CNFs only in niche applications
n Products based on Carbon Nanofibers may be greener
than alternatives for certain applications
• Quantity will be the deciding factor
1.
Objectives
n Life Cycle Evaluation of Nanoproducts & Processes
• Establish Life Cycle Inventory modules for Nanomaterials
• Perform LCA of Polymer Nanocomposites Products
n Explore predictive model for LCA and impact assessment!
• Relationship between life cycle inputs and impact
• Relationship between properties of nanoparticles and their
impact
LC Impact and Input-side Analysis
n LCA is primarily an output-side method, but input-side
information is more readily available
n Can input-side information provide an indication of life
cycle impact?
Zhang, Y., Bakshi, B. R., IEEE Symposium on Electronics and the Environment, 2007
Approach
n Identify relationship between inputs and impacts based
on LCA of common products and processes
n Extract empirical model via rigorous statistical methods
n Aggregation of inputs is crucial
• Mass
Ignores some types of energetic inputs
• Energy
Ignores non fuel inputs
• Exergy
Captures ability to do work, accounts for material and
energy inputs
With and without work done by ecosystems
n If a relationship is found, it can be used for predictive
LCA of emerging technologies
-------
Preliminary Results
n Modeled relationship
between mass,
energy, ICEC, ECEC
and human impact of
emissions
n Based on
thermodynamic
model of U.S.
economy*
n ECEC provides best
fit
n Similar results via
other studies
n Need to do more stud
•n
(jjl * Ukidwe, N. U., Bakshi, B. R., Ener
i
1 •
R!=0.1S
85 ' Mass 5 " '
R!=0.03
" "ICEC ' "
5"
|^..
R!=0.03
"Energy
05-
f'
'R!=0.40
18ECEC"
KV, 2007
Conclusions for Second Objective
n For emerging technologies, input information is easier to
obtain
n Preliminary studies indicate promising correlation
between life cycle inputs and impact
n Ecological cumulative exergy consumption seems best
for aggregating inputs for Predictive LCA
n Relationship between toxicology of nanoparticles and
thermodynamic properties is also promising
n More work is needed
1.
Second Law and Environmental Impact
n Exergy is not conserved, it can be lost
n Manufacturing involves reduction of product entropy
n This results in an increase of entropy in the surroundings,
which comes from the loss of exergy
n For same functionality, more exergy loss should mean
more increase in entropy of surroundings and larger
impact
Increase of Entropyj
Future Work
n LCA of conventional versus nanocomposite materials
n Further statistical evaluation of relationship between
inputs and impact
n Explore relationship between thermodynamic properties
of nanoparticles and their toxicity
n Risk analysis
n Acknowledgements
• Financial support from EPA (Grant No. R832532) and NSF
MSEC at Ohio State
-------
2007 Interagency Workshop on the Environmental Implications of Nanotechnology
U.S. Environmental Protection Agency
Interagency Workshop on the Environmental Implications of Nanotechnology
Hotel Monaco
Washington, DC
September 5-7, 2007
EXECUTIVE SUMMARY
SEPTEMBER 5,2007
INTRODUCTION AND OVERVIEW
The 2007 Interagency Workshop on the Environmental Implications of Nanotechnology was held
September 5-7, 2007, in Washington, DC, 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 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 engineered nanomaterials.
Welcome and Introduction
Gary Foley, U.S. EPA
Dr. Foley welcomed participants and remarked that this workshop would provide an opportunity to
examine the progress achieved by all research funding programs represented at the workshop. This effort
is a partnership among agencies, in which the Department of Energy (DOE), NSF, NIOSH, NIEHS, and
EPA work together. This coordination, one of many that have been initiated through involvement in the
National Nanotechnology Initiative (NNI), seeks to assess implications and applications of
nanotechnology. EPA currently is evaluating nanotechnology research needs across the Agency to
determine its next steps with respect to nanotechnology research. NCER administers EPA's extramural
research, including research grants and cooperative agreements, the fellowship program, and the Small
Business Innovation Research (SBIR) program. Each of these programs includes a nanotechnology topic
for proposal submissions. Overall, across all environmental topic areas, NCER makes about 300 awards
each year. NCER typically administers approximately 1,000 active grants. The investigator-initiated
research funded by these programs over the past 5-6 years is helping to pave the way for EPA's
intramural research program to examine nanotechnology applications relevant to the Agency's mission.
The Agency is developing a Nanomaterial Research Strategy (NRS). The scope of this research document
discusses broad themes and general approaches for extramural and in-house nanotechnology research.
ORD has identified four key research themes and seven key scientific questions addressing the research
themes where we can provide leadership for the federal government research program and support the
science needs of the Agency:
The Office of Research and Development's National Center for Environmental Research
-------
2007 Interagency Workshop on the Environmental Implications of Nanotechnology
(1) Sources, Fate, Transport, and Exposure
> Which nanomaterials have a high potential for release from a life cycle perspective?
> What technologies exist, can be modified, or must be developed to detect and quantify engineered
materials in environmental media and biological samples?
> What are the major processes or properties that govern the environmental fate of engineered
nanomaterials, and how are these related to the physical and chemical properties of those
materials?
> What are the exposures that will result from releases of engineered nanomaterials?
(2) Human Health and Ecological Research To Inform Risk Assessment and Test Methods
> What are the effects of engineered nanomaterials and their applications on human and ecological
receptors, and how can those effects be best quantified and predicted?
(3) Risk Assessment Methods and Case Studies
> How do Agency risk assessment and regulatory approaches need to be amended to incorporate
the special characteristics of engineered nanomaterials?
(4) Preventing and Mitigating Risks
> What technologies or practices can be applied to minimize risks of engineered nanomaterials
throughout their life cycle, and to apply nanotechnology to minimize risks posed by other
contaminants?
The purpose of the NRS is to guide the ORD program in nanomaterial research. Anticipated outcomes
from this research program will be focused research products to address risk assessment and management
needs for nanomaterials in support of the various environmental statutes for which EPA is responsible.
PROGRAM PRESENTATIONS
How the National Nanotechnology Initiative is Addressing Environmental, Health, and Safety
Research Needs
Celia Merzbacher, Office of Science and Technology Policy (OSTP), Executive Office of the
President
NNI is the multi-agency program that coordinates all federal nanoscale research and development
activities. The annual NNI supplement to the President's Budget reports investment in a number of areas
including environmental, health, and safety (EHS) research. The primary purpose of research and
development reported in this category is "to understand and address potential risks to health in the
environment posed by nanotechnology." The NNI has reported on expenditures for EHS research and
development by all agencies participating in NNI for each year since 2006. The amount being invested in
EHS research grew from $37.7 million in 2006 to a request for $58.6 million in 2008—an increase of
about 55%. Eight agencies plan to invest in EHS research in 2008. The NNI plans to spend $1.5 billion in
2008 on all aspects of nanotechnology research, including about $300 million on the nanomaterials
category. Examples of EHS research include: (1) U.S. Department of Agriculture-funded research on
reactivity, aggregation, and transport of nanocrystalline oxides in soil; (2) a U.S. Air Force-funded
The Office of Research and Development's National Center for Environmental Research
-------
2007 Interagency Workshop on the Environmental Implications of Nanotechnology
multidisciplinary university research initiative to study the relationship between physical and chemical
characteristics and toxicological properties of nanomaterials; and (3) NIOSH-funded research to develop
verified instruments and methods for accurately assessing airborne concentrations of nanoparticles and
the efficacy of respirator use for controlling exposure. EHS research, as defined by NNI for purposes of
budget reporting, does not include critical research that has other primary purposes. For example, the
National Cancer Institute (NCI) is funding a project on functionalized nanomaterials for cancer detection
and treatment. In addition, the National Institute of Standards and Technology (NIST) is developing
methods for the chemical characterization of nanoscale materials in three dimensions.
The Nanotechnology Environmental and Health Implications (NEHI) Working Group is a subgroup of the
National Science and Technology Council, Nanoscale Science, Engineering, and Technology
Subcommittee. The NEHI Working Group is co-chaired by the Food and Drug Administration and EPA's
ORD; its 19 member agencies include both research and regulatory agencies. The NEHI Working Group
provides an opportunity for information exchange and aims to identify and address EHS research needed
to support regulatory decisionmaking. Five high-level categories of EHS research, identified by the NEHI
Working Group in the report, Environmental, Health, and Safety Research Needs for Engineered
Nanoscale Materials, include: (1) instrumentation, metrology, and analytical methods; (2) nanomaterials
and human health; (3) nanomaterials and the environment; (4) health and environmental exposure
assessment; and (5) risk management methods. These research needs were prioritized according to three
overarching principles. First, NEHI prioritized research to maximize the value of information to be
gained, such as the extent to which the research findings would reduce uncertainty, how broadly
applicable the information would be, and the expected level of exposure. Second, NEHI sought to
leverage investments by other stakeholders, such as industry and other countries. Third, to maintain
awareness of the state-of-the-art, NEHI will periodically reassess these priorities. The research priorities
were released for public comment in August 2007; the interim document is available at
http://www.nano.gov. NNI will compare these priorities with current research to identify any gaps and
areas of overlap and will develop a research strategy to address unmet research needs.
Those who are managing and performing research to address EHS issues related to nanotechnology
should keep in mind the following additional points:
> Other stakeholders, in addition to the federal government, have a role in developing information
about the potential risks of nanomaterials, including manufacturers and other countries.
> In addition to understanding the absolute effects of nanomaterials, it also is important to understand
net risks. For example, the risks of certain materials or technologies may be acceptable if they are
replacing more harmful alternatives.
> Research to understand implications should be integrated with basic and application-oriented
research, both at the level of funding agencies and at the level of the individual researcher.
> Exposure must be studied in addition to toxicity.
> Further standards (e.g., standard reference materials and standard methods) are needed to ensure that
nanotechnology research findings are comparable across countries.
> It is important to understand public perception of the risks and benefits of nanotechnology, as well as
to communicate risk information that is useful to the public.
NNI and nanotechnology research are priorities of the administration. The OSTP's priorities memo for
FY 2009, which is now available at the OSTP Web Site, includes a list of priority areas, including EHS
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research related to nanotechnology. Thus, at the highest levels, the administration is directing agencies to
implement the research needs that they have identified. The work of the researchers at this meeting is vital
to the success of NNI.
Discussion
A participant asked Dr. Merzbacher why information on the budget for EHS research begins in 2006,
rather than in 2001, when NNI was established. Dr. Merzbacher explained that the Office of Management
and Budget did not begin asking NNI to break down budget information into different categories until
2006. Prior to that point, NNI collected this information more broadly, without separating it into
categories. The participant also asked why NNI spent less than 4% on EHS implications of
nanotechnology. Dr. Merzbacher said that the agencies are investing in research areas other than
implications. She suggested that it is not a question of the percentage spent on EHS implications, but
rather the expenditure necessary to address the needs identified.
The same participant asked how a consumer would know which products contain nanomaterials or are
produced using nanotechnology; she wondered whether NNI or EPA is involved in such communication.
She expressed particular interest in products containing nanomaterials that could enter the sewage system.
Dr. Merzbacher replied that, depending on one's definition of nanomaterials, many things could be said to
contain nanoparticles. Simply indicating that a product contains or may release nanoparticles is not
helpful. It is important to develop a basis for identifying the hazard of specific nanomaterials. At this
early stage, little information on the real risk is available.
Department of Energy User Facilities for Nanoscale Science: National Resources for Researchers
Altaf Carim, Office of Basic Energy Sciences (BES), DOE
The DOE, one of the original participants in NNI, provides major funding for nanoscale science,
engineering, and technology. The FY 2008 budget request includes more than $285 million for
nanotechnology through DOE's Office of Science, which supports both fundamental research and major
user facilities. The energy and environmental grand challenge areas were identified from the start of the
NNI in FY 2001, and these are mission areas for DOE. In addition, a major NNI- and DOE-sponsored
workshop in 2004 identified key research targets and foundational themes for energy-related nanoscience.
The mission of the BES is to: (1) foster and support fundamental research to provide the basis for new,
improved, environmentally conscientious energy technologies; and (2) plan, construct, and operate major
scientific user facilities for "materials sciences and related disciplines" to serve researchers from
academia, federal laboratories, and industry. BES Scientific User Facilities include five Nanoscale
Science Research Centers (NSRCs): the Center for Nanoscale Materials at Argonne National Laboratory,
the Molecular Foundry at Lawrence Berkeley National Laboratory, the Center for Functional
Nanomaterials at Brookhaven National Laboratory, the Center for Nanophase Materials Sciences at Oak
Ridge National Laboratory, and the Center for Integrated Nanotechnologies at Sandia National
Laboratories and Los Alamos National Laboratory. The NSRCs 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 precompetitive, nonproprietary work leading to publication; however, costs are 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.
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In addition to the NSRCs, other user facilities are available, including X-ray scattering, neutron scattering,
and electron scattering facilities. The BES light sources—including the Advanced Light Source,
Advanced Photon Source, National Synchrotron Light Source, and Stanford Synchrotron Radiation
Laboratory—are major user facilities that have a keen interest in nanoscale science. The BES neutron
scattering centers include the Intense Pulsed Neutron Source; the Manuel Lujan, Jr. Neutron Scattering
Center; the High-Flux Isotope Reactor; and the Spallation Neutron Source. The BES electron scattering
user facilities include the National Center for Electron Microscopy at Lawrence Berkeley National
Laboratory, the Electron Microscopy Center at Argonne National Laboratory, and the Shared Research
Equipment Program at Oak Ridge National Laboratory.
More information on DOE nanoscience is available at http://nano.energy.gov; information on the DOE-
BES scientific user facilities is available at http://www.sc.doe.gov/bes/BESfacilities.htm.
Discussion
Dr. Terry Gordon remarked that he had experienced difficulty finding information on the NNI Web Site.
He e-mailed three contacts listed on one agency's Web site and never received a response. He asked if a
central person is available who could provide advice or information to researchers. Dr. Carim suggested
that Dr. Gordon contact a nanoscale user facility directly to ask if he may submit a proposal or to
determine if a proposed activity is appropriate for that facility.
Dr. Jacob McDonald recalled an experience similar to Dr. Gordon's. Researchers need an improved
interface with the user facilities. In particular, they need someone who can show them how to integrate
the tools available at the user facilities into their research. He added that it is very difficult to develop a
proposal if one does not yet know how to integrate the tools. Dr. Carim agreed that this is a problem.
Facility staff members are eager to work with researchers on how to apply the tools to their research;
however, they may not understand all of the fields that their capabilities might serve. Similarly,
researchers working in those fields may not be sure which instruments might be useful for their research.
He agreed that this is an interface problem and welcomed any suggestions for resolving it.
The National Science Foundation—Discovery, Innovation, and Education in Nanoscale Science and
Engineering
Cynthia Ekstein, Chemical, Bioengineering, Environmental, and Transport Systems Division, NSF
Funding for nanoscale science and engineering (NSE) research has increased since FY 2000. NSF's FY
2008 request of $390 million for NSE research funding is one-quarter of the total federal request and one-
twelfth of world investment in this type of research. Of NSF's FY 2008 budget request for NSE research,
16.1% is intended to address societal dimensions of nanotechnology, and 7.4% is specifically for
nanotechnology EHS research. NSF supports fundamental research in seven program component areas;
infrastructure establishment through about 3,500 active projects, including 24 large centers, user facilities,
and multidisciplinary teams; and training and education affecting more than 10,000 students and teachers
per year. Nanotechnology research and development involves ethical, legal, and social issues. NSF
funding priorities for 2007-2008 for knowledge creation, infrastructure, and education include: (1) new
measurement methods and instrumentation to characterize nanoparticles and other nanostructured
materials and nanosystems, as well as their potential implications; (2) physical-chemical-biological
processes of nanostructures dispersed in the environment, including transport phenomena of nanoscale
aerosols and colloids from sources to exposure settings and the interaction of nanomaterials with cells and
living tissues; (3) safety in nanoscale manufacturing of materials and systems; (4) separation of
nanoparticles from fluids; (5) development of experimental and simulation user facilities; and
(6) educational programs for nano-EHS. NSF, EPA, NIOSH, NIEHS, and other agencies have jointly
supported nanotechnology research for 3 years; NSF also supports research through its Small Grants for
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Exploratory Research. Information on NSF-supported nanotechnology research is available at NSF's NNI
Web Site, http://www.nsf.gov/nano.
Discussion
A participant asked whether NSF-supported nanotechnology research is available on the NSF Web Site.
Dr. Ekstein responded that it is available on the NSF Web Site under "Awards."
The National Institute for Occupational Safety and Health Nanotechnology Program
Vladimir Murashov, Office of the Director, NIOSH
NIOSH is the federal agency responsible for conducting research and making recommendations for the
prevention of work-related injury and illness. In 2004, NIOSH created the Nanotechnology Research
Center in response to public concern over nanotechnology implications. The NIOSH Nanotechnology
Program has developed four strategic goals. The first goal is to understand and prevent work-related
injuries and illnesses potentially caused by nanoparticles and nanomaterials. NIOSH is addressing this
goal via research on risk assessment and risk management of nanotechnology in the workplace, including
toxicology, metrology, control technology, exposure assessment, medical surveillance and guidance, and
safety research. The report, Progress Toward Safe Nanotechnology in the Workplace, released in 2007,
addresses research progress in 10 key areas, research gaps, continuing project plans, and opportunities for
collaboration. The second strategic goal of the NIOSH Nanotechnology Program is to promote healthy
workplaces through interventions, recommendations, and capacity building. NIOSH is addressing this
goal in a number of ways. For example, the NIOSH field team partners with employers to assess
exposures in the workplace and the effectiveness of control technologies in the mitigation of those
exposures. In addition, NIOSH has developed best practice guidelines for the workplace in the regularly
updated report, Approaches to Safe Nanotechnology: An Information Exchange with NIOSH. The third
strategic goal is to enhance global workplace safety and health through national and international
collaboration on nanotechnology. To achieve this goal, NIOSH is engaging in a number of activities,
including: (1) collaborations with companies; (2) participation in interagency working groups;
(3) participation in the International Organization for Standardization TC 229 Nanotechnology Working
Group on Health, Safety, and Environment; (4) collaboration with the Organisation for Economic Co-
operation and Development (OECD); and (5) collaboration with the World Health Organization. The
fourth strategic goal of the NIOSH Nanotechnology Program is to conduct research to prevent work-
related injuries by applying nanotechnology products. To achieve this goal, NIOSH is examining the
application of nanotechnology and nanomaterials to the development of filters, sensors, and protective
clothing for occupational safety.
NIOSH Nanotechnology Program funding has increased to more than $6 million in 2007; this includes
funding for extramural programs, which has remained steady at approximately $1 million per year.
NIOSH engages in intramural activities related to nanotechnology, including: (1) the National
Occupational Research Agenda: Nanotechnology Safety and Health Research Program; (2) the NIOSH
Nanotechnology Research Center; (3) the Nanotechnology Research Supplement; and (4) Nano-related
Division Projects. NIOSH also funds nanotechnology research through research grants, joint RFAs, and
contracts to address specific needs. Information on NIOSH extramural programs can be found at
http://www.cdc.gov/niosh/oep/ and at http://www.grants.gov. Since 2004, NIOSH has been engaged with
EPA, NSF, and NIEHS in the joint RFA, "Nanotechnology Research Grants: Investigating Environmental
and Human Health Issues." From this RFA, up to $8 million has been spent each year to support 15-25
research grants and exploratory grants, with up to $1 million per year from NIOSH. Research funded by
NIOSH addresses the Institute's mission to provide leadership in preventing work-related illnesses and
injuries. In FY 2007, NIOSH has worked jointly with the National Institutes of Health (NIH) and EPA on
an NIH-led RFA, "Manufactured Nanomaterials: Physico-chemical Principles of Biocompatibility and
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Toxicity." From this RFA up to $4.1 million per year will support 10-15 research grants and exploratory
grants, including up to $0.5 million from NIOSH.
National Institute of Environmental Health Sciences Activities on Nanotechnology: Nanoscale Science
and Toxicology
Nigel Walker, NIEHS, NIH
Nanotechnology activities at NIEHS include research conducted or funded by NIEHS. Researchers in the
Division of Intramural Research (DIR), such as those in the National Toxicology Program (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 EHS. 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. Some
common issues and recommendations regarding experimental strategies emerged from several workshops
and reports, including the NTP Workshop on Experimental Strategies in 2004 and the International Life
Sciences Institute (ILSI), Risk Science Institute (RSI) report (Oberdorster, et al. Particle and Fibre
Toxicology 2005;2:8). In particular, it is important to traverse the continuum of human relevance and
determine how in vitro and in vivo work should be integrated and to consider whether the materials being
studied are the same materials to which humans ultimately will be exposed. 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 (TiO2), carbon fullerenes, nanoscale silver, multi-walled carbon nanotubes (MWNTs), nanoscale
gold, and dendrimers. The NTP uses an open process whereby any member of the public can nominate
nanomaterials and other environmental agents to be evaluated by NTP for toxicity. More information on
NTP's nanotechnology work can be found at http://ntp.niehs.nih.gov/go/nanotech.
Research is funded by NIEHS through the Division of Extramural Research and Training. Extramural
research regarding 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; (3) intervention devices, such as drug
delivery devices and other therapeutic nanoscale materials; and (4) remediation devices, including
primary disease prevention through the elimination of exposure. Extramural research funded in the area of
the fundamentals of biological response has included research funded under the FY 2006 joint solicitation
among EPA, NSF, NIOSH, and NIEHS, "Human Health Effects of Manufactured Nanomaterials."
NIEHS funded three applications at $400,000 per year for 3 years on transmembrane transport,
cardiovascular toxicity, and oxidative stress. In addition, NIEHS is the lead agency on the joint
solicitation with NCI, National Eye Institute, the National Human Genome Research Institute, the
National Institute of Dental and Craniofacial Research, the National Institute of General Medical
Sciences, EPA, and NIOSH in FY 2007, "Manufactured Nanomaterials: Physico-chemical Principles of
Biocompatibility and Toxicity." Approximately 10 grants will be funded from this RFA.
Through the NanoHealth Initiative, NIEHS is taking the next step by building on its investment and core
competencies and partnering for integrated research success. The scope of the NanoHealth Initiative is to
examine the fundamental physicochemical interactions of engineered nanomaterials (ENMs) with
biological systems at the molecular, cellular, and organ level, as well as associated pathophysiologic
processes. The rationale behind this initiative includes the acquisition of new knowledge of molecular,
cellular, and organ system biology and the identification of clinically relevant properties of ENMs. This
initiative is critical for the design of ENMs with maximum human and environmental biocompatibility
and safety and will establish the scientific foundation of an emerging science.
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Discussion
Ms. Patricia Weggel expressed concern about the communication of risk to researchers who work with
nanomaterials, such as those who collaborate with and obtain materials from DIR, through material safety
data sheets or other means. Manufacturers are not required to indicate whether nanomaterials are present
on material safety data sheets and university and EPA researchers sometimes do not realize how to protect
themselves. Dr. Walker agreed that communication of risk to researchers is an area of concern, but this is
not something that NTP is addressing.
Dr. Bellina Veronesi noted that she agreed with Dr. Walker about the need to physically characterize
nanoparticles because this work is necessary to link physical characteristics to a biological event.
Unfortunately, most characterizations are conducted in an environment very different from that of actual
biological exposures. Characterization of particles in environmental media can differ substantially from
that conducted in distilled water; therefore, nanomaterials should be described in more realistic
environments. Dr. Walker replied that NTP has encountered difficulty determining how to quantitate
modes of action in biological tissues. Further, agencies such as NIOSH, the Occupational Safety and
Health Administration, and EPA will have to address bulk materials. It is important to integrate
information about these materials in both kinds of environments to see how the material changes between
the raw state and a biological state. Characterization conducted only in a biological environment may not
be relevant for NIOSH, for which the occupational hazard is important. The ILSI-RSI report included a
useful table of the types of analyses needed at different levels, including dry state or bulk state and in
vitro or in vivo studies. It is important to provide characterizations at all of these levels until the key
determinants of biocompatibility are known.
Dr. Warren Layne asked whether the DIR has found that every minor modification of each nanoparticle
must be characterized to gain sufficient understanding of the material's properties. Dr. Walker replied that
each nanoparticle is different; a nanoparticle with three different surface characteristics may demonstrate
three different half-lives, even though all three might be considered to be the same nanoparticle. Dr.
Layne asked about ASTM International's efforts to develop standards related to nanoparticles. In light of
the differences among particles and the effects of minor modifications to particles, he wondered how a
particular nanoparticle under investigation could be compared to a single standard nanoparticle. Dr.
Walker said that an upcoming NIST-sponsored workshop will address development of standard materials.
Such standards would be useful in that one could compare particular characteristics of a nanoparticle to a
known standard, such as a size or colloidal reference; however, it would not be possible to develop
standards for every known particle. Dr. Layne asked whether the NTP has found that surface area is a
primary determinant of a particle's effects. Dr. Walker said that this may be the case for some types of
materials, but in general this is not yet known.
Dr. Zubair Saleem asked whether any of the agencies represented at the workshop have considered the
disposal of these materials in terms of sustainability or recycling. Dr. Walker said that this sounds like a
question about LCA and post-consumer use. Dr. Nora Savage added that this is a major component of
ORD's Nanotechnology Research Strategy, which soon will be released for Agency review, and then will
be released to the general public for comment.
Office of Research and Development Introduction
George Gray, ORD, U.S. EPA
EPA recognizes that nanotechnology may have benefits for society, such as enhanced products and
processes, reduced waste, and reduced energy use. The potential benefits of nanotechnology may make it
integral for addressing environmental challenges. To ensure that such benefits are realized, EPA is
funding research in the area of nanotechnology applications. Since 2001, EPA has awarded about 35
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grants and has spent approximately $12 million to fund research on the environmental applications of
nanotechnology. In addition, it is important to consider the potential environmental and public health
effects of the widespread use of this technology. The STAR Program first funded research on the
implications of nanotechnology in 2004; now the STAR Program has awarded about 50 grants totaling
$10 million to study implications. Implications research topics include: (1) exposure; (2) LCA; (3) risk
management; and (4) fate, transport, and transformation. EPA joins with other agencies to produce joint
RFAs on the environmental effects of manufactured nanomaterials. In addition, EPA's SBIR program,
which works primarily with the private sector, has awarded more than 32 contracts worth in excess of $3
million for small businesses to develop and bring to market nanotechnology-related products.
EPA must be proactive in identifying critical research needs. Program offices will be faced with policy
questions related to nanomaterials; as the Agency's science and technology arm, ORD must be sure that
science informs regulatory decisionmaking. For that reason, ORD released the Nanotechnology White
Paper (EPA 100/B-07/001) in February 2007. The white paper, developed by an intra-Agency team,
identifies key research needs in the area of nanotechnology and outlines a Nanotechnology Research
Strategy to prioritize these needs. The Nanotechnology Research Strategy will be released for public
comment in fall 2007. EPA will ask the scientific community if the Agency has effectively identified and
prioritized the research needs and the best means to achieve them. The Agency also will consider how to
mitigate identified problems and how to manage risks.
The extensive cooperation and communication across the federal government in the area of
nanotechnology will result in more rapid progress toward addressing nanotechnology implications. EPA
also is very active internationally, for example by leading two working groups for the OECD on the
health and safety implications of manufactured nanomaterials. In addition, EPA is working with the
Department of State, NSF, NIOSH, NIEHS, and DOE to develop an international RFA.
Nanotechnology holds a great deal of promise, but the American people expect federal agencies and the
academic community to be able to reap the benefits of this technology while minimizing risks. This
workshop will provide an opportunity to share research results, spread knowledge, find opportunities for
collaboration, and recognize the real potential of this technology.
Discussion
Dr. Richard Wiggins commented on the potential for public perception to hinder good science and good
policy. He asked whether anyone has considered looking at human behavior as these technologies are
developed and applied. The manner in which one communicates with the public is just as important as the
actual science. Dr. Gray agreed that this is a good point and said that federal agencies and researchers are
interested in science, technology, and problem-solving. It also is important, however, to effectively
communicate about nanotechnology so people understand what is and is not known. Studies of risk
perception regarding nanotechnology are beginning to appear. He asked Dr. Merzbacher whether she is
aware of work in the federal government focusing on communication. Dr. Merzbacher agreed that this is
an important issue. In addition to the NEHI Working Group, another interagency group under the same
high-level subcommittee addresses public engagement and communication.
A participant asked whether the U.S. Geological Survey (USGS)—which works with EPA on
environmental monitoring and other programs related to the occurrence of contaminants in the
environment—is conducting nanotechnology research or participating in any interagency nanotechnology
efforts. Dr. Sarah Gerould responded that the USGS has been involved in a number of interagency
working groups on nanotechnology. The USGS has a budding research program in nanotechnology and is
seeking further collaboration.
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The Science To Achieve Results Research Program in Nanotechnology: Deepening Our
Understanding of the Environmental Aspects of Engineered Nanomaterials
Chris Saint, ORD, U.S. EPA
Established in 1995, the STAR Program is the extramural funding arm of EPA's ORD. Its mission is to
include universities and nonprofits in EPA's research program to ensure that the highest quality science
supports sound decisionmaking. The STAR Program awards about $66-100 million annually and
currently is managing about 1,000 active research grants and fellowships. Each year the STAR Program
receives 3,000-3,500 grant applications and makes about 300 new STAR awards.
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 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. The STAR Program began to collaborate with other agencies to solicit
proposals on environmental and human health effects through two RFAs issued in 2005 (with NSF and
NIOSH) and in 2006 (with NSF, NIOSH, and NIEHS). In 2007, the STAR Program has collaborated with
NIEHS to solicit research proposals on the physicochemical principles of biocompatibility and toxicity;
the STAR Program also has collaborated with NSF and DOE to solicit proposals on environmental fate,
transport, transformation, and exposure research. Other programs managed by NCER include the Greater
Research Opportunities program, which has released two RFAs related to nanotechnology (Detection and
Monitoring in 2007 and Applications in 2004), and SBIR, which has solicited research on Applications
for Environmental Monitoring and Pollution Control in 2001, 2003, and 2004.
This workshop is the fourth in a series of workshops, but is the first truly interagency workshop in which
EPA has been involved. This workshop is intended to create novel interactions within the research
community and to help federal agencies begin to target research at crucial needs for EPA, other agencies,
and the public. More information about nanotechnology research funded by NCER can be obtained from
http ://www. epa. gov/ncer/nano.
Discussion
A participant asked how the STAR Program will facilitate international collaborations. Dr. Saint
explained that, unfortunately, EPA cannot legally provide grants to agencies or institutions outside the
United States. The Agency can, however, provide funding to an institution that is cooperating with
another institution or agency outside the United States; this is the kind of collaboration the STAR
Program encourages. Dr. Savage added that EPA attempted to work with the European Commission on a
joint RFA, but this was unsuccessful for logistical reasons. The Agency now is attempting to work on a
similar collaborative effort for the future. EPA understands that international cooperation is critical, but
EPA, other U.S. agencies, and the NNI must find the best means by which to achieve this kind of
cooperation.
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RESEARCH PROJECT PRESENTATIONS
Removal and Toxicity ofNanomaterials in Drinking Water
Paul Westerhoff, Arizona State University
The overall goal of this study is to understand the fate and significance of nanomaterials in drinking
water. The objectives of this research project are to: (1) characterize the fundamental properties of
nanomaterials in aquatic environments; (2) examine the interactions between nanomaterials and organic
pollutants and pathogens; (3) evaluate the removal efficiency of nanomaterials by drinking water unit
processes; and (4) test the toxicity of nanomaterials in drinking water using a cell culture model system of
the epithelium. The researchers used a multidisciplinary approach, including experiments to identify the
fundamental uniqueness of nine nanomaterial properties and toxicity, as well as applied experiments to
elucidate the fate and reactions involving nanomaterials in drinking water treatment plants. The
researchers found that most commercial metal oxide nanoparticles occurred primarily as aggregates in
water, but QDs did not aggregate in water. Ionic strength and ionic composition affected further
nanoparticle aggregation in water, but this depended on nanoparticle surface chemistry. Natural organic
matter (NOM) stabilized nanoparticles in water. During simulated water treatment, alum coagulation and
membrane filtration removed most, but not all, nanoparticle mass. The researchers also found that
nanoparticles can be toxic to Caco-2 epithelial cells, can affect epithelial layer morphology, and may
affect epithelial layer function. In addition, some nanoparticles penetrated confluent epithelial cell
monolayers. This study considers the physical, chemical, and biological implications of nanomaterial fate
and toxicity in systems and will provide insight into the potential for nanomaterials to be present and to
pose health concerns in finished drinking water.
Discussion
Dr. Navid Saleh asked whether the researchers used the same medium both in particle size
characterizations and in exposure of cells to nanoparticles. He suggested that one must use a standard
method of characterization to determine how the physical behavior of nanoparticles in aquatic
environments affects toxicology. Dr. Westerhoff replied that he and his colleagues used dynamic light
scattering (DLS) at different concentrations in distilled water and in the medium used for exposure of
cells to most of the nanoparticles studied. For QDs, researchers modified the medium by using only
phosphate buffered saline. The DLS work discussed in the first part of the presentation used distilled
water; this does not represent the particle size during the toxicity test. The researchers do have that
information; in fact, even more significant aggregation occurs in the growth medium used in the toxicity
test.
Dr. Saleh then commented on the finding that increasing the TiO2 to 1,000 ppm results in fewer particles
penetrating the membrane. He asked whether particle size or concentration might have played a role in
this result. Dr. Westerhoff clarified that he presented a percentage of particles passing through the
membrane, not absolute numbers of particles. He then explained that he had primarily intended to contrast
titanium with cadmium (Cd). At a high concentration of particles (1,000 ppm), 2.2% of TiO2
nanoparticles pass through the cell monolayer, whereas, at a relatively low particle concentration (1 ppm),
34% of Cd QDs penetrated the cell monolayer.
Pulmonary and Systemic Biocompatibility of Inhaled Carbon Nanotubes
Jake McDonald, Lovelace Respiratory Research Institute
Previous research, published in 2004, showed that instillation of carbon nanotubes resulted in significant
lung tissue damage, up to and including death, in very short time scales. In the present research project,
investigators hypothesized that, in contrast to instillation, inhalation of carbon nanotubes would not cause
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pulmonary injury or inflammation after high-dose exposures. Researchers characterized MWNTs and
developed a whole-body inhalation system capable of delivering MWNTs to the air breathed by rodents.
Researchers exposed mice to control air or to respirable MWNTs for 7 or 14 days, examined lungs for
indications of inflammation, and assessed the systemic response. Mice exposed to MWNTs via inhalation
showed unremarkable pulmonary inflammation and pathology even at high doses. Inhalation of MWNTs,
however, caused systemic immunosuppression, characterized by reduced T-cell-dependent antibody
response to an antigen and suppressed T-cell proliferative ability in the presence of the mitogen,
Concanavalin A. Researchers found no change in gene expression in the lungs of MWNT-exposed mice;
however, they found large increases in IL-10 and in NQ01 mRNA levels in the spleens of exposed
animals, as well as an increase in prostaglandin-associated enzymes. In several other environmental or
occupational exposures, the researchers have found similar systemic immune function changes that are
not accompanied by pulmonary effects. The immune responses found in this study are likely not unique to
MWNTs.
Discussion
Dr. Igor Linkov asked whether the researchers have attempted to run a computational fluid dynamics
model with respect to particle size distribution. Dr. McDonald responded that they have not run such a
model but would be willing to collaborate with other investigators who are experienced in that approach.
Dr. Gayla Orr raised the issue of potential artifacts due to experimental conditions. She suggested that the
specific physical and chemical properties of nanomaterials (e.g., the large surface area-to-mass ratio)
make them more reactive; once agglomerated, these materials no longer have the same properties. She
asked, therefore, how nanoparticles might produce the same effects regardless of agglomeration. Dr.
McDonald clarified that he has not concluded that smaller particles are the most harmful; therefore, these
findings are not necessarily artifacts of the experimental conditions. Another participant added that each
aggregate has many nanofaces, so aggregation does not necessarily end the effects or reactivity of
nanomaterials.
Pharmacokinetics an d Bio distribution of Quantum Dot Nanoparticles in Isolated Perfused Skin
Nancy Monteiro-Riviere, North Carolina State University
The objective of this research project is to assess potential health effects—specifically, dermal absorption
and cutaneous toxicity—of manufactured nanomaterials in skin. The researchers asked the following
questions: (1) Do nanoparticles penetrate the skin? (2) Do such particles preferentially locate in the lipids
of the stratum corneum? (3) Can nanoparticles gain access to tissue spaces, a prerequisite for systemic
toxicity? Researchers used QDs with various surface coatings, including polyethylene glycol (PEG) and
carboxylic acids (COOH). They used flow-through diffusion cells and laser scanning confocal
microscopy to assess QD penetration through porcine skin. Flow-through diffusion cells showed
penetration of QD621 only in the upper stratum corneum layers of skin. This is in contrast to studies with
QD565 and QD655 that showed slight coating-dependent epidermal penetration. In the QD621 infusion
study, COOH-coated QDs showed greater tissue extraction than PEG-coated QDs. Images indicate
aggregation of infused QDs in the skin vasculature, and transmission electron microscopy (TEM)
localized QD621 within the capillary walls. A pharmacokinetic model of arterial-venous extraction and
tissue biodistribution of QDs was developed based on a model previously used to quantitate platinum
distribution in the same experimental system. Significant arterial-venous QD extraction was observed at
all doses, with COOH QDs showing greater predicted tissue deposition; this confirms results of the
confocal studies. Researchers found an approximately 90-minute periodicity in arterial extraction, an
observation not seen after chemical infusions. Such periodicity could lead to tissue redistribution on
chronic exposure, as has been found by other investigators. These data begin to define nanomaterial
characteristics that correlate to tissue uptake and persistence. The results are important for risk assessment
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and drug delivery because they suggest that QDs not specifically targeted for medical applications can
biodistribute to tissues, have unique pharmacokinetic patterns of arterial extraction, and may cause
adverse effects.
Discussion
Dr. Layne asked whether these findings have relevance to inhalation exposure in humans. Dr. Jim Riviere
recalled the potential systemic effect after pulmonary exposure found by Dr. McDonald and noted that
this would occur through redistribution. Although it is not yet known how the nanomaterials redistributed,
the potential for such redistribution is important for an understanding of the dose-to-effect pathway. Dr.
Monteiro-Riviere added that QDs are used in infusion for medical imaging, and this study shows that the
QDs can disperse through the capillaries.
Metal Nanoparticle Tissue Distribution Following In Vivo Exposures
Alison Elder, University of Rochester
Studies of ultrafine particles have demonstrated extrapulmonary translocation, but it is not yet known
which particle properties affect the tissue distribution of nanosized particles. In the last year of this
project, the focus is on the biodistribution and fate of engineered nanoparticles administered via the
respiratory tract or systemically. The researchers hypothesized that the tissue distribution of
nanomaterials following respiratory tract or systemic exposure is a function of the particles' surface
properties. Researchers exposed rats to QDs (with PEG-, PEG-amine-, or COOH-conjugated surfaces) or
colloidal gold particles (coated with rat serum albumin or PEG) via intratrachial microspray (ITM) or
intravenous injection. Researchers characterized the inflammatory response because inflammation (as
determined by percentage of lavage fluid neutrophils) can significantly alter the translocation of
nanoparticles between the lung and the blood. They found that inflammation in QD-exposed rats did not
differ from that of controls. In contrast, the PEGylated colloidal gold particles caused significant increases
in neutrophils when delivered via ITM. The researchers then determined Cd content and gold content in
lung and extrapulmonary tissues. They concluded that nanoparticles delivered via the lower respiratory
tract are translocated to extrapulmonary tissues, but this is highly dependent on particle physicochemical
characteristics. They also found that small amounts of nanoparticles can be retained in brain tissue
following a single exposure, but this is dependent on particle physicochemical characteristics and the
portal of entry. In the future, the researchers plan to more thoroughly evaluate the kinetics of nanoparticle
translocation and to determine in which cells and subcellular structures nanoparticles are localized. In
addition, researchers hope to characterize the translocation of particles to the central nervous system
(CNS) as a function of the particle surface and its interactions with endogenous proteins; they also plan to
characterize the elimination of nanoparticles from the CNS.
Discussion
A participant suggested that when they characterize the translocation of particles to the CNS, the
researchers should examine the basal ganglia because they are known to concentrate metals.
Another participant observed that the researchers found very different responses from QDs and gold
particles of similar size and coating (PEG). He asked whether the characteristics or amounts of surface
coatings might help explain these differences or whether there are other explanations for the differences
between PEG-coated QDs and gold particles. Dr. Elder responded that she had initially thought that these
differences could be explained by differences between QDs and gold in core chemistry. This may be too
simplistic however; the answer may have to do with protein interactions. The same participant asked if all
tissues were perfused. Dr. Elder explained that the tissues in this study were not perfused because
perfusion can increase the variability of results across animals or across tissues within one animal; this
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could interfere with the interpretation of results. The best way to assess the contribution from blood in
different tissues is to look for particles in the endothelium and in the tissues themselves to determine
where the particles are localizing.
Dr. Veronesi asked why the researchers think that the nanoparticles would leave the brain. Dr. Elder
answered that she does not know if they leave the brain. She added that she hopes to address that question
more specifically in the future by, for example, sampling cerebrospinal fluid in different regions of the
brain. This is a critical issue because of Dr. Veronesi's work showing that, in the presence of ambient
particulate matter, neurotoxicological effects may be attributable to the particulate fraction. It is necessary
to determine where the particles are going and how long they are staying there.
Bioavailability, Toxicity, and Trophic Transfer of Manufactured ZnO Nanoparticles: A View From the
Bottom
Jason Unrine, University of Georgia
The objectives of this research project are to: (1) evaluate the bioavailability and toxicity of manufactured
zinc oxide (ZnO) nanoparticles to model soil bacteria (Burkholderia vietnamiensis and Cupriavidus
necator) and the model detritivore Caenorhabditis elegans as a function of particle size, compared with
aqueous Zn2+; (2) evaluate the ability of manufactured ZnO nanoparticles 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) evaluate the additive, synergistic, or antagonistic effects of manufactured ZnO
nanoparticles on the toxicity of Cu2+ to B. vietnamiensis and C. elegans. The researchers hypothesized
that: (1) the bioavailability and toxicity of manufactured ZnO nanoparticles would increase with
decreasing particle size; (2) the toxicity of ZnO nanoparticles to model soil bacteria and C. elegans would
be lower than an equivalent concentration of dissolved Zn2+; (3) the bioavailability and toxicity of ZnO
nanoparticles introduced via trophic transfer would differ from direct exposure; and (4) ZnO
nanoparticles would alter the bioavailability and toxicity of dissolved metals. The researchers found that
size determination is a critical issue and that TEM may not be the best method for ZnO nanoparticle size
determination. Further, acetate controls ZnO nanoparticle reactivity and passivates surface sites; the
removal of acetate leads to aggregation of ZnO nanoparticles but promotes surface reactivity. Regarding
nanoparticle-bacteria interactions, researchers found no significant difference in growth rates of bacteria
in the presence of aqueous Zn2+ versus ZnO nanoparticles. Acetate use rates, however, were higher in the
presence of aqueous Zn2+ compared with ZnO nanoparticles. Researchers found some evidence for zinc
bioavailability from Zn2+ but not from ZnO nanoparticles. In addition, cells with compromised
membranes were more strongly associated with ZnO nanoparticle treatment than with Zn2+ treatment.
Therefore, even though growth rates in the presence of ZnO nanoparticles and Zn2+ do not differ, there
may be differences in the mechanisms of toxicity. Regarding nanoparticle-nematode interactions,
researchers found that the LC50 and EC50 of ZnO nanoparticles do not differ significantly from those of
aqueous Zn2+; however, the mechanisms of toxicity differ. In addition, at zinc concentrations of greater
than 100 mg/L, copper toxicity to nematodes is decreased more by ZnO nanoparticles than by aqueous
Zn2+. Finally, no significant green fluorescent protein (GFP) was induced by exposure of C. elegans to
either 100 \iM Cd or 500 \iM ZnO nanoparticles. In the future, the researchers plan to characterize 80 nm
ZnO nanoparticles under various chemical conditions; study the bioavailability, toxicity, and behavior of
80 nm zinc nanoparticles; and continue exposure experiments using Cu2+. In addition, the researchers plan
to conduct the following bioavailability and toxicity studies: (1) an investigation of the differences
between toxicity mechanisms of ZnCl2 and ZnO nanoparticles in B. vietnamiensis, Cupriavidus necator,
and C. elegans; (2) an examination of the bioavailability and toxicity of ZnO nanoparticles introduced via
trophic transfer as opposed to direct exposure; (3) identification of chemical speciation of zinc in
concentrated regions in tissues; and (4) an examination of potential transformation of ingested ZnO
nanoparticles.
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Discussion
Dr. Greg Mayer asked, regarding the metallothionein and GFP work, whether the researchers have any
evidence regarding immune function and its upregulation in C. elegans. Dr. Unrine responded that he
does not have such evidence, but the researchers are aware of experiments with ionizing radiation
exposure that might be related to this issue.
Biochemical, Molecular, and Cellular Responses ofZebrafish Exposed to Metallic Nanoparticles
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 toxicity of nanoparticles and their
soluble counterparts to aquatic organisms. To address the second goal, researchers exposed zebrafish to
TiO2, 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. Nanoparticles 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 nanomaterial into the environment. Changes in particles over time make dosimetry problematic.
Results suggest that the effects of some nanometals are not completely explained by dissolution; in
particular, the effects appear to depend on particle composition, and this is not a generic (uniform)
particle response. Finally, the researchers concluded that particle surface charge influences the uptake of
nanomaterials, at least by Daphnia. Future work will focus on mechanisms, such as whether particles are
entering the gill cell.
Discussion
Dr. Mayer commented that, in the cluster analysis conducted as part of the transcriptional work, silver and
copper clustered individually; however, the silver and copper appeared to have extensive commonality
except that the copper appeared to have an additional group of upregulated genes. He asked whether those
genes are associated with a hypoxia response. Dr. Barber responded that he does not yet know, but he and
his colleagues are attempting to address this kind of question. Unfortunately, even though the zebrafish
has been studied for a long time, the annotation of the genome is not very complete. The researchers are
conducting data-mining to come up with testable hypotheses. Some evidence does exist for a hypoxia-like
response.
Acute and Developmental Toxicity of Metal Oxide Nanoparticles in Fish and Frogs
George Cobb, Texas Tech University
The objectives of this research project are to determine the environmental hazard of metal oxide
nanoparticles (Fe2O3, ZnO, CuO, and TiO2) in terms of acute and chronic toxicity of these particles to
fathead minnows (Pimephase promelas} and African clawed frogs (Xenopus laevis}. The researchers
hypothesized that nanoparticle exposure would affect the survival, growth, development, egg hatchability,
and metamorphosis of P. promelas and X. laevis. Researchers have synthesized nanoparticles and have
obtained commercial nanoparticles. Acute (96-hour) exposure of X. laevis to metal oxide nanoparticles
demonstrated developmental effects for one of the nanometal oxides, ZnO (EC50 = 8 mg/L). Inhibited
growth was observed for Xenopus embryos exposed to CuO and ZnO suspensions of greater than 10
mg/L and 100 mg/L, respectively. Scanning electron microscopy showed metal oxide nanoparticles
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trapped in ciliated skin cells. This close proximity to the embryo skin may play a role in any observed
acute or chronic results. Acute tests for P. promelas are just beginning. Chronic tests will include 28-day
early life stage tests for P. promelas and 10-week exposures forX. laevis. Nanoparticles will be kept in
suspension in water using aeration- or peristaltic pump-induced water currents. Metal concentrations in
water and tissues are being measured via atomic absorption spectrophotometry.
Mechanistic Dosimetry Models of Nanomaterial Deposition in the Respiratory Tract
Bahman Asgharian, The Hamner Institutes for Health Sciences
Accurate health risk assessments of inhalation exposure to nanomaterials will require mechanistic
dosimetry models that account for interspecies differences in dose delivered to the respiratory tract. The
objectives of this research project are to: (1) measure deposition of nanosized particles in casts of human
and rat nasal upper respiratory tract (URT) airways; (2) develop semi-empirical relationships to predict
nanomaterial deposition in URT airways; (3) develop respiratory tract deposition models of nanoparticles
and nanotubes in humans and rats; (4) measure regional and lobar deposition of nanomaterial in the heads
and lungs of rats; and (5) develop a user-friendly software package to implement models and provide
rapid simulation capability. The researchers have measured deposition fractions in the nasal airways of
humans and rats for particle sizes from 5 to 100 nm. They also have obtained a semi-empirical deposition
efficiency formula for humans and rats. The researchers have extended a model of particle deposition in
the lung to the ultrafine (nano) size range by including axial diffusion and convective mixing (dispersion).
Finally, they have measured lobar and regional deposition of nanoparticles in Long-Evans rats.
Development of the software package is in progress.
Discussion
Dr. McDonald observed that in some studies, a 15- or 20-minute exposure will result in as many particles
in the gastrointestinal tract as in the lung. He asked whether this might be related to clearance. Dr.
Asgharian agreed that this is what his observations suggest. Dr. McDonald asked whether the researchers
took any other tissues after exposure that could be measured. Dr. Asgharian replied that they collected
lobes, trachea, and head.
Preparation and Application of Stabilized Fe-Pd Nanoparticles for In Situ Dechlorination in Soils and
Groundwater: Factors Affecting Particle Transport and Reactivity
Don Zhao, Auburn University
The overall goal of this research project is to develop a cost-effective, in-situ remediation technology
employing a new class of soil-dispersible, iron (Fe)-based nanoparticles for rapid destruction of
chlorinated hydrocarbons in soil and groundwater. In Year 2, the researchers completed the following: (1)
prepared nanoparticles of various sizes using carboxymethyl cellulose (CMC) as a stabilizer; (2) tested
effects of particle stabilization on reactivity; (3) tested transport behaviors of zerovalent iron (ZVI)
nanoparticles in porous media; (4) tested degradation of trichloroethylene (TCE) in soils; and (5) pilot-
tested in situ dechlorination in soils using stabilized ZVI nanoparticles. The researchers developed a
method for synthesizing ZVI nanoparticles of controllable size, soil mobility, and reactivity. They found
that factors such as CMC molecular weight, the CMC:Fe ratio, pH, and temperature can greatly affect
transport and reactivity of nanoparticles. The researchers also found that stabilized ZVI nanoparticles can
be delivered and distributed in soils. The nanoparticles can effectively degrade nonaqueous phase liquids
in soils and groundwater and may boost biodegradation.
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Discussion
A participant noted that one of the unfortunate intermediate products of ZVI and TCE dechlorination is
the carcinogen vinyl chloride and asked whether Dr. Zhao had examined this byproduct. Dr. Zhao
responded that TCE degradation has two pathways, one biotic and the other abiotic. Degradation by the
stabilized ZVI nanoparticles did not lead to the production of vinyl chloride, and TCE is completely
degraded to ethene and chloride. However, biological processes can result in vinyl chloride production.
Dr. Layne asked if it would be possible to return to the pilot site or the samples to measure vinyl chloride.
Dr. Zhao replied that researchers are still sampling at the pilot site so this would be possible. He predicted
that vinyl chloride would not be found there but that, in the second phase (the ZVI-boosted
biodegradation phase), vinyl chloride will probably be detectable.
SEPTEMBERS, 2007
Engineered Nanoparticles in Environmental Remediation Technology and Implications to
Nanoparticle Transport Through the Skin Barrier
Vijay John, Tulane University
The goal of this research project is to develop novel mesoporous materials that act as supports for ZVI
nanoparticles used in the breakdown of chlorinated compounds. Dense nonaqueous phase liquids
(DNAPLs) are pollutants of concern that are prevalent at contaminated sites. Widely used as a solvent by
industry, TCE is a DNAPL that resists biotic and abiotic degradation in natural environments. ZVI can
react with TCE through redox chemistry, and nanoscale Fe particles have been found to be most effective
in TCE remediation. In a new approach to the environmental remediation of TCE, the researchers are
investigating the use of functional Fe-silica submicroscopic particles prepared through an aerosol-assisted
route. They have found that: (1) functionalized composite particles significantly adsorb TCE;
(2) composite particles are effective in TCE decontamination; (3) composite particles partition to the
TCE-water interface; and (4) composite particles have the optimal size characteristics to be effective in
transport through sediments. High particle production rates are possible using the aerosol technique. In
work related to human health, the researchers have found that nanoparticle penetration pathways through
skin are highly dependent on both the initial microstructure and induced conformation changes. They also
have found that extensive hydration affects the skin barrier and may allow extremely small nanoparticles
to pass through hydration-induced defects in the stratum corneum. This work has implications for human
health challenges, such as transcutaneous vaccine delivery.
Discussion
Dr. Riviere commented that there is an extensive literature on lipid biophysics and the effects of
hydration. He added that this work is relevant for public health because people go swimming and this
hydrates the skin extensively.
Dr. Layne asked how openings in human skin, such as hair follicles and sweat glands, might be relevant
to the delivery of nanoparticles through human skin. Dr. John responded that nanoparticles do enter hair
follicles but cannot penetrate the follicle to enter the skin. He added that the drug delivery model appears
to indicate that sweat glands also are not the primary pathway for entering the skin.
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Responses of Lung Cells to Metals in Manufactured Nanoparticles
John Veranth, University of Utah
This research project is based on the hypothesis that, because of their small physical size and large surface
area, nanoparticles would increase cellular uptake and the induction of proinflammatory signaling
compared with larger particles with the same elemental composition. The researchers predicted that, in
comparison with other environmental and occupational agonists, nano-sized metal oxides would have
moderate potency in lung epithelial cells. They are using commercially available particles of metal oxides
(SiO2, TiO2, Fe2O3, A12O3, NiO, CeO2, and ZnO) and are conducting in vitro cell culture screening assays
and in vivo confirmation. The researchers have found that the oxide nanoparticles have low potency in
lung epithelial cells compared with soil-driven dusts and vanadium. They concluded that manufactured
metal oxide nanoparticles may pose a risk comparable to other ambient and occupational particle types,
such as micron-sized crystalline silica. Multiple cell types occur in the lung, however, and vascular
endothelial cells are in close proximity to the airspace. Translocation of nanoparticles from the lung is
well documented and cardiovascular effects occur in response to environmental air pollution. The
researchers currently are investigating responses of endothelial cells in vitro. They have found that some
types of nanosilica induce proinflammatory signaling in endothelial cells, and now are examining the
biochemical mechanisms linking particles to inflammation. Dosimetry is an important consideration with
in vitro particle studies because responses often are seen only at concentrations much higher than those
plausible for inhalation exposure; however, this may be a reflection of cell culture artifacts. Ongoing
work includes: (1) continued comparisons between lung epithelial and endothelial cells in vitro; (2) the
use of specific inhibitors to study cell signaling pathways activated by the more potent types of
nanoparticles; and (3) animal exposure via intratracheal aspiration to validate in vitro results.
Discussion
Dr. McDonald commented that this study reaffirms that one should not rush to judgment on the relative
toxicity of nanoparticles compared with larger particles. With respect to the conclusion that nanoparticles
may have potency similar to that of Min-U-Sil silica or silica, he suggested that this may be a bit
overstated. Researchers in the Rochester group have compared crystalline (Min-U-Sil) silica to
amorphous (nano) silica. They found that the nanosilica was much less potent than Min-U-Sil silica. He
added that crystalline silica is quite toxic and is regulated as such. Dr. Veranth agreed that he should have
cited that earlier work in addition to the more recent papers.
Dr. Elder commented on the notion of signaling pathways as a possible explanation for the cell responses
and noted that those pathways can be activated at the surface of the cell through receptor-mediated
processes. She asked whether particles are being taken up by the cells and whether uptake is required for
the response of the cells. Dr. Veranth responded that this is not yet known. Some of the receptors are
membrane receptors, so they could certainly respond to something outside the cell. He noted that he has
done some work with a membrane receptor and the inhibitor suppressed the response. He cited previous
research and suggested that particle contact with the cell may be triggering the response, but these
particles also are taken up. With soil dust, an inhibitor of phagocytosis did not affect the response, but for
nanoparticles we do not have an answer.
Dr. Greg Lowry asked whether Dr. Veranth conducts particle characterizations in the vehicles in which
the exposures are conducted. It may be important to examine the state of aggregation, surface charge,
functionalization of the surface, and what impacts those factors have on toxicity or other responses in this
study. Dr. Veranth replied that he has done some work of this type and has found aggregation. The most
relevant model might be to contact the particles first with one surfactant and let them become coated with
the lipids and proteins of the surfactant before applying the surfactant to the particles. He has attempted
this experiment and needs to do more work in that area. He added that this is one of the limitations of an
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in vitro model because of the use of a large amount of a fairly dilute nutrient medium. In contrast,
particles in the lung are in contact with a thick viscous surfactant. Dr. Lowry suggested that an
understanding of the mechanisms will require an understanding of the properties of the particles; it will be
necessary to characterize them in the vehicle and then do the exposure and relate those two conditions.
A Toxicogenomics Approach for Assessing the Safety of Single-Walled Carbon Nanotubes in Human
Skin and Lung Cells
Mary Jane Cunningham, Houston Advanced Research Center
The objectives of this research project are to: (1) obtain expression profiles (EPs) of known nanomaterials
and unknown nanomaterials, and to compare these EPs to identify toxic effects; and (2) use a systems
biology approach to perturb the biological system and reiteratively sample over time or dose. This is a
data-driven approach, rather than a hypothesis-driven approach, and involves reverse engineering of
cellular pathways. The goal is to create a virtual cell interaction network to predict adverse effects by
combining genomics, proteomics, metabonomics, and pharmacogenomics. In previous work with primary
human epidermal keratinocytes (dermal exposure route), the researchers found that, with a noncytotoxic
dose, the EP of single-walled carbon nanotubes (SWNT) is more similar to the EP of the nontoxic control
(CI), and the EP of silicon dioxide (SiO2) is the most active. With a cytotoxic dose, the EP of SWNT is
more similar to the EP of the toxic control (SiO2), and the EP of CI is most active. In accordance with
previous research, the significantly expressed genes in the SiO2 treatment included genes involved in
membrane restoration or remodeling and inflammation or irritation responses. In studies with primary
human bronchial epithelial cells (inhalation exposure route), the researchers found that lung cells were not
as robust in their long-term growth as skin cells, and it was not possible to perform the complete time
course. As with the skin cells, however, there was very low variation between array replicates. The EP of
SiO2 was the most different and the most active, and the EP of SWNT was more similar to the EP of CI.
Significantly expressed genes were similar to those found with skin cells and included genes active in
inflammation, irritation, and membrane remodeling. Any adverse effects observed with SWNT appear to
be limited to local inflammation caused by the physical presence of particulate material. In a principle
components analysis comparison of EPs from skin and lung cells, the researchers found the maximum
difference between tissue types rather than between types of compounds. The EP of SWNT in both lung
and skin cells is similar to that of untreated samples. The researchers observed about 10 times more
overall activity in skin cells than in lung cells. In protein expression work, they found that only six
proteins were significantly expressed at 24 hours. In miRNA expression work, the researchers found low
variability among array replicates. The greatest miRNA expression occurred in the SiO2 treatment and 71
miRNAs were significantly expressed. Pathway analysis and interpretation of the miRNA expression
work is ongoing.
Discussion
Dr. Saleh noted that SWNTs and MWNTs are extremely hydrophobic. He has found that, when bacterial
cells are exposed to MWNTs, they are nontoxic if they are not in direct contact with the cell. This is
because SWNTs and MWNTs agglomerate and, because the density is so low, they do not settle in any
aqueous suspension; instead, they float around and do not make contact with the cells. Therefore,
particularly with nanotubes, it is important to be sure that the cells are exposed. He asked how the
researchers conducted the exposure. Dr. Cunningham responded that she and colleagues sonicated the
nanotubes for an hour, causing a dispersion of individual tubes. A dosage on the order of mg/mL is
required to achieve any reduction in viability. In addition, the researchers used serum-free defined media
and achieved a heterogeneous suspension of media and nanotubes long enough to treat the cells. After 24
hours, all nanotubes settle on the cells, essentially suffocating them. She had not observed the oxidative
stress that others have found, but she noted that she is using a highly purified preparation of nanotubes.
Also, they are investigating the use of surfactants to help keep the nanotubes fully dispersed. The first
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surfactant in which they dispersed the nanotubes was cytotoxic, and the researchers tried many different
surfactants to find one that was nontoxic.
Dr. Subhas Malghan mentioned that Fe is a potential contaminant in the raw material; its removal requires
the use of other processes that may change the surface properties of the nanotubes completely. He asked
Dr. Cunningham to consider the medical applications of these materials. Dr. Cunningham responded that
the carbon nanotubes have been fully characterized, but purification occurs after the manufacturing steps
to remove heavy metals. In early work with carbon nanotubes, the large percentage of heavy metals
present could explain some of the oxidative stress found in those studies.
Microbial Impacts of Engineered Nanoparticles
Delina Lyon, Rice University
Fullerenes, such as C60, constitute a class of nanomaterials that show potential for medical, industrial, and
technological applications. C6o is insoluble in water but will form a suspension termed nC6o upon
extended exposure to water or after introduction to water via a solvent. The researchers examined the
effect of nanomaterials on bacteria because the disposal or accidental discharge of nanomaterials could
affect microbial ecology and disrupt biogeochemical cycles. In addition, antibacterial activity may be
indicative of toxicity to higher level organisms. From the perspective of applications, compounds with
antibacterial activity could be used in water treatment or other disinfection. nC60 is antibacterial. In
eukaryotes, reactive oxygen species (ROS) may mediate nC6o toxicity. In prokaryotes, however, the
antibacterial activity of nC6o persists in the absence of light and oxygen. In this study, researchers
explored three possible mechanisms for the antibacterial activity of nC6o: (1) physical disruption of the
cell membrane; (2) generation of ROS; or (3) production of ROS-independent oxidative stress. The
researchers found no conclusive evidence of ROS production or ROS damage in bacteria by nC6o. They
encourage researchers who previously showed evidence of ROS production or damage to reevaluate their
results, as these results may be biased by the ability of nC60 to interfere with assays. Findings suggest that
nC6o acts as an oxidant, possibly requiring direct contact with the cell.
Discussion
Dr. Lee Ferguson noted that the researchers used the tetrahydrofuran (THF) method to produce nC6o and
asked if they had determined whether residual THF was present in the nC6o after the evaporation. Ms.
Lyon replied that she and her colleagues had not addressed this issue; however, other groups have not
found residual THF. It is possible that the THF is incorporated into the nC6o suspension. The researchers
attempt to remove all THF, but they also find the same antibacterial activity with stirred nC6o (without
solvents). Dr. Ferguson continued that he has had problems with evaporating large amounts of THF and
using that for toxicity assays because most THF contains stabilizers and oxidation inhibitors that probably
would not evaporate. Ms. Lyon clarified that the THF used in this research project is not stabilized.
Dr. Veronesi noted that electron microscopy may help explain the mechanism of antibacterial activity.
Ms. Lyon answered that she and her colleagues have attempted electron microscopy but this has proven
logistically difficult. They have tried to embed the bacteria in agarose, thinly slice it, and look for nC6o
particles either inside the cell or elsewhere. They know that nC60 likes to sorb to bacteria, but whether it
penetrates the membrane is not clear. The nC6o particles are as small as 2 nm, so they could be penetrating
the membrane because work with QDs has shown that particles smaller than 5 nm can be incorporated
into bacteria.
Dr. Ted Henry said that he and colleagues have found that some of the degradation products of THF have
been responsible for toxicity in zebrafish. He asked how Ms. Lyon prepared the controls; in particular, he
wondered whether the preparation of the controls was the same as that for the THF nC60 treatment (i.e.,
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evaporation of the THF). Ms. Lyon explained that she and her colleagues did not use THF in the negative
controls, but they examined the antibacterial activity initially and did not find toxicity in that condition.
An Integrated Approach Toward Understanding the Inflammatory Response of Mice to Inhaled
Manufactured Nanoparticles
Vicki Grassian, University of Iowa
The goal of this research project is to use an integrative approach to determine which physicochemical
properties and conditions are important in nanoparticle toxicity. The specific objectives are to: (1) use
state-of-the-art techniques to fully characterize a variety of manufactured metal and metal oxide
nanomaterials in terms of their size, aggregation state, shape, and bulk and surface properties;
(2) determine if engineered nanomaterials are particularly deleterious to health compared with particles
from combustion processes that have been more extensively studied; and (3) evaluate the relative health
effects of different nanoparticle surface coatings. The researchers found that subchronic inhalation
exposure to 5-nm TiO2 nanoparticles (one of the smallest commercially available oxide nanoparticles)
caused an increase in the number of activated macrophages, but mice recovered 3 weeks after exposure.
Furthermore, they found no signs of pathological changes in bronchoalveolar lavage fluid or in lung
tissue. These particles are pure anatase and their surfaces are truncated by surface hydroxyl groups and
adsorbed water under ambient conditions. Thus, no surface coatings are present from manufacturing.
They also found that acute inhalation and instillation exposures did not show an effect of surface area for
5- versus 20-nm TiO2 nanoparticles. Agglomeration state (agglomerate size and porosity) may be the
most important factor in these experiments. Studies on metal nanoparticles are underway. Although 25-
nm Fe nanoparticles are similar to 5-nm TiO2 nanoparticles in response, copper nanoparticles show the
largest inflammatory response. Metal nanoparticles are coated with an oxide surface layer that will be
important in understanding the toxicity of these particles. Bulk and surface characterization shows that
these oxide coatings are composed of one or more crystalline phases. Current studies focus on further
understanding the chemical characteristics of these oxide layers and how they influence metal
nanoparticle toxicity.
Discussion
Dr. Elder agreed with the importance of airborne agglomeration state. She also asked if both the 5- and
20-nm TiO2 particles had the same crystal structure (i.e., pure anatase). Dr. Grassian clarified that any
commercially available particles above 20 nm will contain some rutile, even if they are labeled 100%
anatase; this is related to stability and thermodynamics. Below 20 nm one can find pure anatase.
Another participant mentioned the effect of the edges and corners of nanoparticles on their reactivity. He
asked whether the researchers had found parallels to silicosis, noting that fresh silica dust is more toxic
than stale silica dust. Dr. Grassian answered that she has not seen any findings regarding aging effects.
She and colleagues use commercially available materials and always do an independent characterization;
however, they have not addressed the aging effect.
Hysteretic Accumulation and Release of Nanomaterials in the Vadose Zone
Tohren Kibbey, University of Oklahoma
Any nanomaterial that is widely used will ultimately enter the environment. The vadose zone may either
provide a sink for nanomaterials, preventing their spread throughout the environment, or a long-term
contaminant source. The objective of this research project is to study the vadose zone accumulation and
release of a wide range of manufactured nanomaterials. The researchers are focusing on an examination
of hysteretic interactions with air-water interfaces and specific mineral surfaces. They are assessing
adsorption and adhesion affinities with critical liquid-solid and liquid-air interfaces. They also are
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evaluating dynamic interactions between nanomaterials and mineral surfaces using saturated deposition
and dispersion transport experiments. Finally, they are conducting dynamic hysteretic unsaturated
transport experiments to provide detailed information about the effects of wetting and drying history,
infiltration, and unsaturated soil behavior on the accumulation and release of nanomaterials. For the work
presented, the systems were specifically designed so that saturated transport of nanomaterials would not
be a factor. Therefore, any increase in retention seen in unsaturated experiments must be attributable to
the formation of the air-water interface. Regarding transport of TiO2, the researchers have found, using
miniature unsaturated transport experiments, that the average mass of nanomaterial retained in the soil
cell is much higher for the slowest drainage flow rate investigated than for the faster rates. When these
results are normalized for interfacial area, again, the slowest drainage flow rate has the highest retention
of nanomaterial. The mass per area of the nanomaterial was found to be approximately constant over a
large saturation range, suggesting that adsorption to air-water interfaces is an important mechanism over
much of the saturation range. Ongoing and future work using miniature dynamic unsaturated transport
experiments includes: (1) the use of multiple drainage-imbibation cycles and an examination of wetting-
drying history effects; (2) experiments with changing nanomaterial concentrations; (3) the use of different
porous media with smaller grain sizes; and (4) an investigation of more nanomaterials, including those
that interact with solid surfaces. The researchers also have begun large-scale experiments, including: (1)
unsaturated transport modeling to interpret results of the large-scale experiments; (2) the use of a new
column with higher resolution; (3) the use of heterogeneous packings that include a combination of fine
and coarse material to determine how the different water contents of the different layers influence the
movement of nanomaterials; (4) the use of different drainage and imbibation paths; and (5) the use of
more nanomaterials, including those that interact with solid surfaces.
Discussion
Dr. Saleh asked Dr. Kibbey to compare the expected retention of hydrophobic materials, such as carbon
nanotubes, to that of the hydrophilic materials used in this study (e.g., TiO2), in terms of unsaturated
transport. Dr. Kibbey explained that he would expect much more retention in the air-water interface for
hydrophobic materials. The effect would be greater at lower saturations where there is a greater air-water
interface. The challenge would be to disperse a sufficient quantity of nanotubes in water to begin with,
and other surface-active chemicals would probably be required to stabilize them.
Dr. Kurt Pennell asked whether the researchers measured surface tension or interfacial tension as another
measure of adsorption. Dr. Kibbey replied that they have done this in a few cases, and in some cases the
surface tension increases slightly, which is not consistent with adsorption for a dissolved compound.
However, it is not clear that the Gibbs adsorption equation can be applied to particle adsorption.
Dr. Saleem asked what kind of water the researchers used. Dr. Kibbey said that they used nanopure water
but added ionic strength. Dr. Saleem noted the potential for microbiological activity, but Dr. Kibbey
clarified that the time scale of the miniature experiments is on the order of minutes to hours.
The Role of Particle Agglomeration in Nanoparticle Toxicity
Terry Gordon, New York University School of Medicine
The hypothesis of this research project is that the toxicity of fresh (predominantly singlet) carbon
nanoparticles differs from that of aged (predominantly agglomerated) carbon nanoparticles. The
researchers further predicted that this difference also would apply to metal nanoparticles. The objectives
were to: (1) measure the agglomeration rate of carbon nanoparticles; (2) identify whether agglomeration
is affected by altering exposure conditions, such 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 nanoparticles at
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various distances downstream from particle generation. They then exposed mice to nanoparticles 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 nanoparticles 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 nanoparticles. They found that copper and zinc nanoparticles are more toxic than carbon
nanoparticles, and copper nanoparticles are more toxic than zinc nanoparticles. In contrast to carbon
nanoparticles, copper particles showed only a small difference between fresh and aged nanoparticles.
Differences in response among mouse strains suggest that genetic susceptibility could be involved in the
response to nanoparticles.
Discussion
A participant asked whether the carbon source was pure or whether it might have contained metals. Dr.
Gordon clarified that he and colleagues used 99.9 or 99.99% pure carbon, zinc, or copper electrodes.
Dr. Patrick O'Shaughnessy remarked that he did not see any results presented regarding the age versus
size distribution of particles and asked whether the fresh and aged particles have the same size
distribution. Dr. Gordon replied that, in general, the count median diameter of the fresh particles is 10-50
nm and the diameter of the aged particles is 190-250 nm. Dr. O'Shaughnessy asked whether Dr. Gordon
was more focused on fresh versus aged as the factor influencing toxicity, rather than differences in size
distribution. Dr. Gordon agreed that this was correct and added that he does not predict a substantial
change in surface area resulting from agglomeration.
Dr. Henry wondered whether the same kinds of strain differences would occur with other types of
toxicants. Dr. Gordon responded that this appears to be the case and offered the examples of cigarette
smoke and ozone.
Chemical and Biological Behavior of Carbon Nanotubes in Estuarine Sedimentary Systems
P. Lee Ferguson, University of South Carolina
Carbon SWNTs are hydrophobic and will likely associate strongly with sediments upon entry into the
aquatic environment. In sediments, these materials may cause toxicity to benthic, sediment-ingesting
organisms and may impact the disposition of persistent and bioaccumulative organic contaminants, such
as polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). The objectives of
this research project are to: (1) determine which factors control the fate of SWNTs in estuarine seawater,
sediment, and sediment-ingesting organisms; (2) examine the impact of SWNTs on the disposition of
model organic contaminants in estuarine sediments; (3) assess the toxicity of SWNTs to a model deposit-
feeding estuarine invertebrate in seawater; and (4) determine whether the presence of SWNTs in estuarine
sediments affects the bioavailability of model organic contaminants to suspension- and deposit-feeding
estuarine invertebrates. The researchers concluded that SWNTs entering estuaries are likely to associate
strongly with suspended particulates and concentrate in sediments during estuarine mixing. Relative to
other carbonaceous materials, SWNTs are highly sorptive to hydrophobic organic contaminants (HOCs)
and may sequester these compounds in aqueous environments. They found that purified SWNTs are
relatively nontoxic to benthic deposit-feeding organisms, but exposure of these organisms to SWNT
carbonaceous synthetic byproducts may pose a risk of adverse effects. The consequences of HOC
sorption to SWNTs in estuarine sediments for contaminant bioavailability to deposit-feeding organisms
are still unclear.
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Discussion
Dr. Robert Hurt noted that, because the researchers are studying materials with gel electrophoresis, they
are essentially studying a relatively hydrophilic form of nanotubes. He asked whether Dr. Ferguson had
considered annealing the products after separation or subjecting them to a process that would reduce them
to their more commonly used hydrophobic form. Dr. Ferguson responded that his colleague, Walter
Scrivens, has performed re-annealing after purification, but they have not done any experiments with
toxicity or sorption after re-annealing. He agreed that these materials are hydrophilic, negatively charged,
and have been oxidated. He clarified that the nanotubes themselves do not penetrate the gel; it is the short
tubular nanocarbons and the fluorescent nanocarbons that penetrate the gel. The short tubular
nanocarbons are aggregates and are much larger than the fluorescent nanocarbons. The researchers have
not been able to obtain good TEM images of the fluorescent nanocarbons, and they generally are difficult
to analyze. They are not PAHs—or at least they are not small PAHs that can be examined with gas
chromatography. He suggested that they might be nanobarrels with very short aspect ratios.
Dr. Mamadou Diallo asked if Dr. Ferguson and colleagues had assessed the effect of organic matter
modification. He noted that the effects of divalent cations could explain some of the findings. Dr.
Ferguson replied that he added DOM to the nanotubes in the presence of divalent cations and did not see
any changes in aggregation; the nanotubes aggregate in the presence of divalent cations whether or not
DOM is present. He further clarified that he also conducted the experiments without nanotubes and did
not observe a formation of large DOM flocks at the ionic strengths he was using; such an outcome would
have complicated the interpretation.
Dr. Gordon commented that he has conducted experiments with nanoparticles and cod embryo and found
similar low-dose effects, but not high-dose effects. Dr. Ferguson replied that he would like to do some of
the same experiments at lower concentrations of SWNTs. He anticipated that he would see differences in
aggregation behavior at different concentrations of the nanotubes.
Fate and Transformation of Carbon Nanomaterials in Water Treatment Processes
Jae-Hong Kim, Georgia Institute of Technology
The environmental impact of carbon fullerenes is of great concern because of projections for bulk
production in the near future and the recent discovery that they form nanoscale water-stable aggregates
upon release into the water. Understanding the fate and transformation of carbon fullerenes during water
treatment, currently the first line of defense against ingestion pathways, is of particular importance. The
objective of this research project is to examine the response of water-stable fullerene aggregates to
processes used in potable water treatment, using C60 and its stable aggregate, nano-C60, as a model
compound. In the first 2 years of the project, researchers addressed the following questions: (1) How do
carbon nanomaterials behave in a natural water matrix? (2) How does C6o react with chemicals used in
water treatment? (3) How does C6o respond to UV irradiation with respect to the production of ROS? The
researchers found that NOM enhances stabilization of carbon nanomaterials (i.e., C60, SWNTs, and
MWNTs) in natural waters. They also found that the adsorptive interaction between NOM and nanotubes
depends on water quality parameters (e.g., pH and ionic strength) and NOM characteristics. Regarding the
reaction of water-stable C60 aggregates with ozone, one of the strongest oxidants used during water
treatment, the researchers found that the reaction products were molecular fullerene oxides and that the
C6o cage structure remained intact in the product. Both mono- and di-oxygenated carbons were present,
with hydroxyl and carbonyl functional groups. Further, the products showed pH-dependent UV spectra.
Regarding the photochemical production of ROS by C6o in the aqueous phase during UV illumination, the
researchers found that the status of C6o dispersion in the aqueous phase affects its ability to transfer
absorbed photo-energy to oxygen. C6o present in water as stable aggregates did not produce singlet
oxygen under UV illumination, in contrast to pristine C60.
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Discussion
Dr. Diallo pointed out that NOM generates superoxide. Dr. Kim replied that he corrected for that.
Dr. Lowry asked whether the surfactant might play a role in ROS production. Dr. Kim explained that, for
energy transfer, the coating does not appear to have an effect. With superoxide, however, he did find an
effect of surface coating on electron transfer and recombination.
Dr. David Barber asked whether the researchers have attempted similar experiments with the oxidized
byproducts to examine ROS production. Dr. Kim answered that he has conducted such experiments and
has observed singlet oxygen production.
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, nC60 produced using the 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. In particular, the researchers determined how these particles behave when transferred
between water and air. The project focuses on the characterization of the aqueous aggregates of C6o
fullerene. The researchers found that aqueous nC6o particles (i.e., particles produced by extended mixing
in water) are irregular in size and shape, have a negative surface charge, and are crystalline in nature. In
contrast, THF nC60 particles are of regular size and shape, have a negative surface charge, and are
crystalline in nature. Sodium citrate increases the negative surface charge of these particles at low
concentrations but decreases the negative surface charge at higher concentrations. The researchers
concluded that nC60 may form either via weathering of larger particles to smaller particles or
recrystallization of nanoparticles from solution.
Discussion
Dr. Kim asked Dr. Vikesland to comment on the differences and similarities between the mechanisms of
aggregate formation in the organic phase and the aqueous phase. Dr. Vikesland answered that he does not
know how aggregates form in the organic phase or their characteristics.
Dr. Vicki Colvin asked Dr. Vikesland about the nature of the interaction with citrate or other stabilizers.
She said that it is not clear that it would necessarily be ionic, and wondered how this interaction can be
understood in a molecular sense. Dr. Vikesland responded that he would need to do Fourier transform
infrared spectroscopy and other spectroscopic methods to characterize the nature of the interaction
between citrate and the particle surfaces. He noted that researchers use acids to clean carbon nanotubes
and to break the carbon cage; it is possible that, with extended mixing, the same process could occur with
fullerenes, but this is not yet known.
Transport and Retention ofNanoscale C-60 Fullerene Aggregates in Water-Saturated Soils
Kurt Pennell, Georgia Institute of Technology
C6o forms stable nanoscale aggregates in water with aggregate diameters ranging from 95 to 200 nm
depending on the preparation method and ionic strength. Only limited data are available on transport and
retention of nC6o aggregates in porous media, and most previous studies employed high velocities and did
not determine retention profiles. In addition, classical filtration theory has been used to describe nC6o
transport and retention. The objectives of this research project are to: (1) investigate the transport and
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retention of nC6o 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 nC6o 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 are decreased. The detachment rate
coefficient approached zero and did not change with grain size or flow rate, indicating irreversible
attachment. A mathematical model that includes nonequilibrium, nonlinear retention accurately captured
nC6o transport and retention behavior in Ottawa sand. The researchers also found that ionic strength
strongly influences nC6o aggregate transport and retention; the researchers attributed this primarily to
electrostatic interactions. The secondary minimum plays an important role in nC60 attachment, and nC60
retention capacity was correlated with mass flux in the diffusional boundary layer. Future work will
include: (1) measurement and simulation of nC6o transport and retention in water-saturated "natural"
soil(s); (2) measurement and simulation of nC6o transport and retention in unsaturated porous media; (3)
investigation of the effect of stabilizing or dispersive agents (e.g., NOM or surfactants) on nC60 transport
and retention in Ottawa sands; and (4) determination of THF and y-butyrolactone concentrations in
purified and unpurified nC6o suspensions. In a separate project, the researchers will evaluate the
neurotoxicity of manufactured nanomaterials.
Discussion
Dr. Saleh asked how the researchers separated fullerenes from the column. Dr. Pennell explained that the
researchers sonicated and washed the column. They found that this worked best because they were
primarily interested in finding mass balance.
Impacts ofFullerene (nC60 or C60) on Microbiological Functions in Soil andBiosolids
Ronald Turco, Purdue University
Nanotechnology has tremendous potential for economic growth and is a key feature of sustainable
development; however, almost nothing is known about the environmental impact of carbon-based
manufactured nanoparticles. The goal of this research project is to provide fundamental information about
the impact of C6o on the soil food web. This project addresses soils, biosolids, and fungi. The researchers
found that C6o and nC6o had limited impact on the microbiology of soil and biosolids. In particular, soil
biomass size and structure were unchanged. Repeated applications and solvent effects currently are under
investigation. Biosolids biomass size and structure were unchanged by C60. The researchers are
investigating functional groups of C6o and carbon nanotubes in terms of their effects on the biomass of
anaerobic digesters. Transformation of C6o by fungi also is limited and there is no evidence of fungal use
of C60. Studies of C60-OH and fungi are ongoing and preliminary results are quite interesting.
Discussion
Ms. Lyon asked if the researchers sampled soil at time points earlier than 3 and 6 months and whether it is
possible that there is a slight initial impact on bacteria, after which they recover. Dr. Turco responded that
they sampled at 1 month and did not see any impact at that time point.
Size Distribution and Characteristics of Aerosol Released From Unrefined CNT Material
Judy Xiong, New York University School of Medicine
Particle concentration, size, distribution, shape, and agglomeration status are among the key factors for
determination of worker exposure levels to airborne nanoparticles. Carbon nanotubes (CNTs) have a high
aspect ratio, are highly agglomerated, and often coexist with other nanoparticles, such as amorphous
carbon soot, metal catalysts, and ambient particulate matter. The size distributions of CNTs are difficult to
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predict but presumably have a wide spread and are source-dependent. The specific aims of this study are
as follows: (1) Investigation of the size distribution and characteristics of aerosol particles released from
various types of industrial-grade CNT bulk materials resulting from agitation. The results will provide a
foundation for developing field and personal sampling devices for CNTs. (2) Development of a practical
method using atomic force microscopy (AFM) image analysis that is capable of classifying CNTs and
distinguishing them from coexisting nano-sized particles in general environments. (3) Development of
appropriate methods to monitor potential worker exposure levels to CNTs. The researchers found that all
common types of unrefined CNTs, including single-walled, double-walled, and multi-walled nanotube
samples, can be dispersed into the air to a significant extent from agitation. The sizes of particles
generated from all CNT types were widely distributed across 13 stages of the Electrical Low-Pressure
Impactor (ELPI), ranging from 7 nm to 10 \m\. The size distributions varied with the type and the nature
of bulk materials. For High Purity grade single-walled CNT produced by chemical vapor deposition, a
majority of particles were in the nano-size region (< 100 nm) based on the ELPI data, and a significant
proportion of particles also occurred in the single-nanometer range based on the data collected by an
Integrated Screen Diffusion Battery (ISDB). Airborne CNT particles were highly agglomerated; no single
tubes or simple ropes were observed by AFM in the original samples collected by ELPI or ISDB before
treatment with surfactants. The researchers concluded that adequate monitoring methods should be
established for quantification and characterization of these new types of materials to evaluate workers'
exposure levels and the potential health risks. Ongoing studies include: (1) the development of a
quantitative sample treatment method for AFM analysis that can effectively de-agglomerate samples by
applying appropriate surfactants, solvent, and sonication; (2) an investigation of other advanced AFM
technologies that may be better suited for CNT characterization, such as conductive-AFM and phase
imaging; and (3) the development and validation of a field sampling method for airborne CNT particles in
workplaces.
Discussion
Dr. O'Shaughnessy asked who manufactured the ISDB. Dr. Xiong answered that her research team made
the ISDB in the previous research studies. Dr. O'Shaughnessy asked if the researchers based the ISDB on
a particular source (Chang et al), and Dr. Xiong said it was based on similar principles, but the ISDB was
independently developed and designed at New York University. She added that the filtering elements of
the ISDB could use either stainless steel wire screens of different mesh sizes or porous metal filters of
different porosities. The researchers calibrated the collection efficiencies themselves.
Dr. Gordon asked whether it is common for researchers to collect data inside CNT manufacturing
facilities or whether occupational monitoring programs exist anywhere. Dr. Xiong replied that she was
uncertain about measurements conducted inside manufacturing facilities. She mentioned that some
companies have developed workplace safety programs, but they lack appropriate sampling methods for
CNTs. Dr. Savage added that NIOSH has been inside some of the CNT manufacturing facilities. She
offered to find out more about this for workshop participants.
Physical and Chemical Determinants of Carbon Nanotube Toxicity
Robert Hurt, Brown University
It may be possible to reduce CNT health risks by understanding toxicity mechanisms and modifying the
specific material features that trigger those mechanisms. The goal of this research project is to consider
two characteristic nanotube features: catalytic impurities and hydrophobic surface area. The researchers
found that all CNTs studied (both as-produced and "purified") release free metal (Fe, nickel [Ni], and
yttrium) into physiological fluid phases; this triggers known toxicity pathways. Metal bioavailability is
influenced by processing and environmental exposure. The researchers concluded that metal
bioavailability assays should be standard in CNT characterization. The researchers also found that
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SWNTs deplete essential micronutrients from the medium by physisorption and can affect cell behavior
by a new indirect mechanism. a-Tocopherylpolyethyleneglycolsuccinate (TPGS), a water-soluble
Vitamin E formulation, is a promising safe surfactant for the processing of carbon nanomaterials,
especially MWNTs. In future work, the researchers will attempt to use TPGS to actively mitigate oxidant
damage associated with nanomaterial exposure. Bioavailable metal in nanotubes probably can be
removed by selective targeting as a simple detoxification strategy; this is the basis of pending future
work.
Discussion
Dr. Saleh asked whether the researchers have systematically examined defects in CNTs and how that
might affect absorption of amino acids and vitamins. Dr. Hurt responded that he and his colleagues had
only limited data with which to address this question. They sulfonated the material; this introduces many
hydrophilic sites, which suppresses absorption. He suggested that it is not really a question of active sites
or defects, but rather the regions in between. With very defect-laden nanotubes, one can functionalize
those defects and potentially suppress this problem. The more hydrophobic materials might adsorb more
in solution.
Dr. Unrine noted that Ni metal particles can be difficult to solubulize in nitric acid even at high
temperature and pressure. Considering that the researchers found sorption of cysteine and methionine, he
asked whether they had considered how that might exacerbate metal toxicity and oxidative stress. Dr.
Hurt said that he had not followed up on the biological implications of amino acid absorption, but the
doses required to do that are high, well over 1 mg/mL. He noted that amino acid absorption may be
relevant for some nanomedicine scenarios with high nanotube density; but for most nanotoxicity assays,
vitamins will be absorbed but amino acids will not.
Environmental Impacts of Nanomaterials on Organisms and Ecosystems: Toxicity and Transport of
Carbon-Based Nanomaterials Across Lipid Membranes
Dmitry Kopelevich, University of Florida
The hypothesis of this research project is that nanomaterials could lead to environmental dysfunctions
because of the potential toxicity of these materials and their derivatives. In addition, their small size
makes nanomaterials prone to biouptake and bioaccumulation, and their large surface area might allow
them 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 on biota using short-term microbiotests and
investigate the impacts of nanomaterials on microbe-driven ecological functions; (2) determine the
mobility of metal-based and carbonaceous nanomaterials in porous media, as well as the toxicity of
nanomaterials in soil leachates; and (3) determine possible mechanisms of toxicity of different types of
nanomaterials. In microbiotests, the researchers found that both C6o and SWNT toxicity significantly
exceeded solvent toxicity. From biogeochemistry work, the researchers found that C6o toxicity
significantly exceeded solvent toxicity and slowed down bacteria metabolism; these effects were sensitive
to soil composition. It is not yet clear why SWNTs at small concentrations promoted algae growth. The
researchers plan to investigate the effect of trace metals in SWNTs as opposed to the effects of the
nanotubes themselves. They also plan to use the fluorescence of SWNTs to investigate the transport of
SWNTs into cells and cell membranes; this will allow them to develop a connection with the molecular
modeling studies. In the molecular modeling work, the researchers did not find a significant energy
barrier for carbon-based particle penetration of the cell membrane's lipid bilayer. The particles had a long
residence time inside the bilayer. Particle shape and size impacted the transport rates, dynamics, and
localization of nanoparticles within the membrane. The researchers have collected preliminary data on
physical effects on the membrane, such as the effect of particle size and shape. In future work, they will
examine other potential physical effects on the membrane.
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Discussion
Dr. Veronesi commented on the evidence for a possible physical deformation of the membrane and said
that, from a neuroscientific perspective, several types of polymodal receptors respond to deformation,
which initiates many types of inflammatory sequelae. Dr. Kopelevich suggested that the most interesting
factor to examine would be the change in pressure profiles; he said that he hopes to have data to address
this issue soon.
Dr. Hurt asked if the researchers are interested in looking at long-chain nanotubes. Dr. Kopelevich
responded that he is interested in this but it would be computationally difficult.
Structure-Function Relationships for Predicting Nano-Bio Interactions
Vicki Colvin, Rice University
All stakeholders will benefit from an understanding of how fundamental characteristics of engineered
nanoparticles control their biological effects. This research project will provide the first structure-
function relationships for nanoparticle toxicology. The hypothesis of the research project is that
nanoparticle structure (e.g., size and shape) directly controls cytotoxicity. A secondary hypothesis is that,
of the four major material parameters in engineered nanoparticles (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; and (2) characterize the effects of
nanoparticles on cell function. The researchers found that the toxicity of nanoscale TiO2 scales with
photocatalytic activity and that the phase composition is crucial. They also found that QDs demonstrate
minimal cytotoxicity at extracellular concentrations typical of most molecular imaging experiments (5-20
nm). The researchers confirmed in different QDs the previous finding that biocompatibility improves with
PEGylation. They found that biocompatible polymer coatings appear to work by preventing cell uptake of
nanoparticles.
Discussion
Dr. Hurt asked what the manufacturers of solar cell QDs would say is the potential for coatings that might
make the material safer? Dr. Colvin replied that manufacturers probably will realize that they cannot build
a business around QD systems unless they reclaim the Cd, and they are interested in packaging that
allows for recycling to reclaim the Cd for further use. Some researchers are working on systems with the
same efficiencies but without the use of heavy metals. This is promising, but the reality is that the
companies manufacturing these materials are expanding in both their funding and the market. Dr. Hurt
asked if polymer coatings are a serious option. Dr. Colvin responded that these coatings might not last in
the environment after disposal. The systems should be packaged and encapsulated and, in general, the
answer may be to recycle the materials and never allow them to enter a landfill.
A participant asked if the researchers had any information on how the QD coating might affect their
photoactivity. Dr. Colvin replied that she and her colleagues are making the QDs as they are because the
QD is already essentially stabilized by an organic substance and the chemistry that produces it. When this
coating is stripped, the photoactivity of the material is destroyed. The organic coatings must be left intact
or the material will not be effective for solar cells or other applications. She noted that her coatings do not
perturb the native coatings of the QDs; the researchers encapsulate, rather than strip, the QDs. They
examined the kinds of highly photoactive substances that researchers in biomedical engineering or in
solar cell work would want to use.
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Cellular Uptake and Toxicity of Dendritic Nanomaterials
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, neutron scattering, and neutron reflectometry 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 a correlation analysis. Work in progress includes examination of the mode of cell death, the
mechanisms of dendrimer cytotoxicity, and cellular uptake and subsequent activation of intracellular
signal transduction pathways. The successful completion of this project should provide industry with
critical data and predictive tools needed to assess the health and environmental impact of dendritic
nanomaterials, such as ethylene diamine core poly(amidoamine) dendrimers.
Discussion
Dr. Veronesi asked if surface charge affects transcytosis through cells. Dr. Diallo responded that surface
charge does affect this. He noted that he and colleagues will first use biological imaging and then will
conduct toxicogenomics work to determine the mechanism.
Interactions of Pure and Hybrid Polymer Nanofibers With Cells
Perena Gouma, State University of New York at Stony Brook
Nanostructured materials offer a high surface area-to-volume ratio and interconnected porosity. They are
used in tissue engineering to produce fibrous scaffolds that mimic the extracellular matrix (ECM). This
research project focuses on natural polymer-hydroxyapatite nanofiber interactions with osteoblasts,
which are anchorage-dependent, mononucleate cells responsible for bone formation. Cellulose acetate
(CA) is a natural polymer that has been used as a scaffold material for functional cardiac cells and
microvascular cell growth. Hydroxyapatite (HA) is a bioactive material that promotes osteoblastic
differentiation in vitro. HA is used in bone tissue engineering. Nanocrystalline HA is similar to bone
apatite. Adding nano-HA to natural polymer hybrids is expected to strengthen cell-polymer fiber
interactions. The researchers studied the interactions of human osteoblasts with fibrous nanomaterials and
their hybrids. They used electrospinning to fabricate the nanofibrous mats for bioscaffolds. The
researchers found that the osteoblasts maintained a rounded morphology on CA fibers and typically
attached to a single fiber. In contrast, osteoblasts tended to remain flat on CA-HA nanofibers and
attached to several fibers, forming an interconnected network. The osteoblasts also preferentially attached
to thinner fibers. HA nanoclusters provided anchoring sites for osteoblasts, thus enhancing cell
attachment. Further, the CA-HA hybrid mats showed enhanced cell spreading, which is known to control
cell differentiation. The researchers concluded that the size and shape of nanomaterials play important
roles in influencing cell attachment and proliferation behavior.
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SEPTEMBER 7,2007
Announcements
Warren Layne, U.S. EPA
Dr. Layne announced an international nanotechnology meeting scheduled to take place October 6-8,
2008, in Chicago. EPA currently is compiling a list of invited speakers. Dr. Layne noted that information
about this meeting will be available on the EPA Web Site soon.
Assessment Methods for Nanoparticles in the Workplace
Patrick O'Shaughnessy, The University of Iowa
A typical industrial hygiene analysis of workplace dust exposure does not include instrumentation to
detect particles in the nanometer size range. The objectives of the research project were to: (1) identify
and evaluate methods to measure airborne nanoparticle concentrations; (2) characterize nanoparticles to
assess their surface and bulk physical and chemical properties; and (3) determine the collection
efficiencies of commonly used respirator filters when challenged with nanoparticles. The researchers
compared a surface area analyzer, handheld condensation particle counter (CPC), photometer,
electrostatic precipitator, scanning mobility particle sizer, and an optical particle counter (OPC). They
analyzed Fe oxides at high and medium concentrations; TiO2 at high, medium, and low concentrations;
and carbon nanotubes. The results indicate a need to apply a shape factor to make direct correlations
between instruments, especially when comparing among instruments with different units, such as count,
surface area, or mass concentrations. This information will be useful for comparing results obtained by
different instruments and for choosing an appropriate instrument for evaluation of nanoparticles in the
workplace. In field sampling at a nanostructured lithium titanate facility, the researchers found that
material handling of lithium titanate dispersed this material as large particles (> 1 \im); any nano-sized
particles observed were mainly associated with other sources, such as diesel forklifts and welding and
grinding operations.
Discussion
Dr. Savage asked about the instruments the researchers used to do the facility measurement for the field
sampling. Dr. O'Shaughnessy replied that they primarily used the handheld CPC and the OPC. They
compared the results to gravimetric measurements using more traditional methods and the photometer.
Dr. Layne asked how uniform the agglomerates are in size. Dr. O'Shaughnessy responded that the
particles produced in his laboratory are between 90 and 150 nm, but this size distribution is partly an
artifact of the methods used to produce the particles. He referenced Dr. Gordon's work and suggested that
aging might allow more time for agglomeration to occur. In terms of results from field sampling,
however, the size distribution is still uncertain. What remains unknown is the particle sizes to which
people are actually exposed.
Mr. James Stewart noted that asbestos analysis is a parallel to the TEM particle counting process. He
asked if the researchers have looked into that kind of analysis for particle counts. Dr. O'Shaughnessy
replied that there is a NIOSH method for TEM of asbestos and the researchers will be looking into this
method. Asbestos particles, however, are larger and better defined than the agglomerates under study in
this project.
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Development of Nanosensors for the Detection of Paralytic Shellfish Toxins
Robert Gawley, University of Arkansas
The goal of this research project is to develop nanosensors for the detection of paralytic shellfish toxins
(PSTs), primarily saxitoxin. (Other shellfish toxins are chemically very similar to saxitoxin.) Currently,
the primary methods for detection of shellfish toxins are the mouse bioassay and the Lawrence High-
Performance Liquid Chromatography Method; each of these methods has limitations. The researchers
have been developing another method—fluorescence sensing, which is based on the 1:1 equilibrium
between the toxin and the sensor to produce a fluorescent complex; the sensor in the absence of the toxin
is not fluorescent. The sensors are based on crown ethers. Using a new class of crown ethers, the
researchers are achieving 100% fluorescence enhancement with a 1 \iM concentration of saxitoxin. More
recently, the researchers have investigated sensing in a nanoscale self-assembled monolayer with a long-
term aim of placing the sensor in a portable device. They have been attempting to determine which
chromophore will work best. Scientists at the Department of Health in Seattle provided the researchers
with shellfish extracts (specifically, blue mussel extracts); this helped them determine the limitations of
each of the chromophores. A method not related to fluorescence that currently is under investigation uses
the protein saxiphilin, which was isolated originally from bullfrogs. The C-lobe of saxiphilin binds
saxitoxin with nanomolar affinity. The researchers are attempting to use this protein in the development
of an electrochemical displacement assay. They hope to place the C-lobe of saxiphilin in a monolayer in a
microfluidic device for PST detection.
Discussion
Dr. Wiggins asked if the researchers are using a dichroic filter system to detect fluorescence when they
use fluorophores to create a fluorescence wavelength. Dr. Gawley confirmed that this was correct. Dr.
Wiggins asked if it would be possible to achieve a more sensitive level of detection (despite some loss of
efficiency) by doing away with the dichroic filter, polarizing the fluorescent light, and using fluorescent
depolarization to detect the fluorescence. Dr. Gawley agreed that this would be possible. He and his
colleagues primarily are working with a fluorometer rather than a microscope and have been working
with another company to optimize this kind of response.
Dr. Layne asked whether the crown ether materials are easy to synthesize. Dr. Gawley responded that the
parent crown ether can be purchased with one or two nitrogens, and one then may alkylate one or both of
the nitrogens. Synthetically, the process is trivial.
Bioavailability and Fates ofCdSe QDs and TiO2 Nanoparticles in Bacteria
Patricia Holden, University of California at Santa Barbara
The goal of this research project is to investigate the influence of bacteria on nanoparticles and the
influence of nanoparticles on bacteria. The specific objectives are to: (1) quantify planktonic bacterial
toxicity to and uptake of cadmium selenide (CdSe) QDs; (2) investigate interactions between
nanoparticles and bacterial biofilms; (3) investigate redox mechanisms with QDs; and (4) investigate size-
related toxicity of TiO2 nanoparticles. The researchers found that, in the presence of bare, 5-nm CdSe
QDs, planktonic Pseudomonas aeruginosa growth was inhibited in a dose-dependent manner as it was in
the presence of cadmium ion [Cd(II)] at equivalent concentrations. The bacteria accumulated Cd in cells
whether they were fed CdSe QDs or Cdll. They also found that planktonic P. aeruginosa broke down
CdSe QDs and this breakdown appeared to be cell-associated. It is not yet known whether one can predict
toxicity of a heavy metal-containing nanoparticle based on its heavy metal content. In biofilm P.
aeruginosa, the researchers found that the toxicity profiles of Cd were similar to those for planktonic P.
aeruginosa. Accumulation of Cd was similar for biofilm and planktonic P. aeruginosa, except that in the
biofilm bacteria, Cd also accumulated in the extracellular polymers, both in QD- and Cd(II)-exposed
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cells; it is not clear if the Cd in the extracellular polymers is in the form of CdSe or Cd(II). The
researchers observed a Cd concentration gradient in the medium, with lower concentrations between
biofilms and concentrations that were 100-fold higher in or under the biofilms. It is not yet known if the
mechanism of Cd toxicity is similar for planktonic and biofilm bacteria. The researchers investigated the
potential for electron transfer in the interactions between bacteria and nanoparticles using time-correlated
single proton counting and measuring lifetime fluorescence emission of dopamine (DA)-conjugated QDs.
The researchers found that lifetime fluorescence was enhanced for concentrated Escherichia coll
associated with QD-DA compared to either QD-DA alone or diluted E. coli associated with QD-DA. It is
not yet known if interfacial charge transfer occurs from cells to QDs. To address the fourth objective, the
researchers are working with the bacterium P. putida and several types and diameters of TiO2 particles.
They found that/1, putida growth decreased in the presence of TiO2; specifically, the larger TiO2 particles
appear to be toxic and the effects are dose-dependent. Further, they found that TiO2 aggregates appeared
to break down during bacterial growth, implying that the bacteria break down the aggregates. In the
future, the researchers will examine the greater toxicity of larger TiO2 particles. They also hope to
conduct a systematic study to determine how the bacteria are breaking down the aggregates.
Discussion
Dr. Barbara Walton asked if Dr. Holden could envision that either the aggregation or breakdown of TiO2
could be related to the differences between rutile and anatase crystal structures. Dr. Holden replied that
she thought that was possible. She and her colleagues have not yet tested this, but they would be very
interested in investigating this possibility.
Dr. Gawley asked whether the kind of de-aggregation shown with TiO2 has been done with CdSe. Dr.
Holden clarified that the researchers have not observed aggregation of the CdSe QDs with which they
work; they start with dispersed particles that remain dispersed. In the case of QDs, the nanoparticles
themselves are breaking down and liberating the Cd(II); the cells are facilitating the breakdown. She did
not know whether aggregated CdSe QDs would be broken down by bacteria but agreed that this would be
interesting to test.
Dr. Yongshen Chen asked if the bacteria must internalize the QDs to degrade them. Dr. Holden noted that
she had been very careful not to say that QDs are broken down inside cells; she can only say that the
degradation is cell-associated. It is not yet clear if the degradation is happening inside the cell or at the
surface of the cell. Similarly, with biofilms, the researchers do not yet know the form of the Cd. Dr. Chen
asked about the addition of azide and Dr. Holden clarified that this was part of the experiment with E. coli
regarding electron transfer. Dr. Chen asked if cell metabolism was inhibited (or if the cells were killed).
Dr. Holden responded that she and her colleagues are planning to investigate this issue.
Dr. Veronesi asked if QDs shift the absorbance of DA when oxidized or reduced. Dr. Holden answered
that this was correct and, in the oxidizing conditions, the emissions observed are primarily from the DA
breakdown product. In the case of reducing conditions, the emissions are from the QD itself because the
DA is lost.
A Novel Approach to Prevent Biocide Leaching
Patricia Heiden, Michigan Technological University
The objective of this research project is to develop a practical and effective approach to prepare
controlled-release, biocide-loaded nanoparticles that can be efficiently introduced into wood to reduce or
eliminate biocide leach into sensitive environments (e.g., wetlands). Preventing biocide loss to leach also
should extend the lifetime of treated wood products. The researchers have adapted a method to prepare
core-shell nanoparticles, each with a hydrophobic core that serves as a biocide reservoir and moderates
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the biocide release rate. The main research components include: (1) development of the nanoparticle
method; (2) development of efficient wood treatment; and (3) assessment of results to determine how
effectively the nanoparticles are reducing biocide leach. The researchers have been able to prepare
nanoparticles with a dry diameter of approximately 40 nm, which is smaller than the target diameter (no
more than 150 nm); however, in water the gelatin shell swells, increasing the diameter to 150-300 nm,
and it is not clear if this swelling is problematic for this application. The nanoparticle yield appears to be
sufficient, but when isolated, only 45-55% dry nanoparticles are collected; the target yield is greater than
90%. The researchers have achieved approximately 46 wt% incorporation of biocide, compared with a
target of 48 wt%. Work is ongoing to improve the method and to identify the optimal core:shell
(hydrophobic:hydrophilic) ratio to balance biological efficacy and biocide leach. In terms of wood
treatment, the researchers have found that delivery efficiency appears to be 84-97% (compared to a target
of greater than 90%) and have demonstrated biological efficacy. Preliminary results also show a
significant decrease in biocide leach in nanoparticle-treated wood.
Discussion
Dr. Wiggins asked what the hydrophobic core was in this research project. Dr. Heiden responded that it
was methyl methacrylate. Dr. Wiggins wondered if the researchers had considered the option of making
the nanoparticles by mixing the biocide with phospholipids; this would form a hydrated shell. One could
make this onsite very simply by adding water. Dr. Heiden replied that industry has used surfactants with
the organic biocides, but this method also is very susceptible to leach.
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 so it is
environmentally benign and sustainable. Understanding the impact of nanomaterials is essential, but this
is not sufficient; it is necessary to adopt a systems view with life cycle thinking. LCA of emerging
technologies poses unique challenges. In particular, life cycle inventory data for nanomanufacturing are
not available and the impacts of engineered nanomaterials on humans and ecosystems are only partially
known. Predicting life cycle processes and activities is difficult because the technology is still in its
infancy. The first objective of this research project is to conduct a life cycle evaluation of nanoproducts
and processes. In particular, the researchers will establish life cycle inventory modules for nanomaterials
and perform an LCA of polymer nanocomposite products. 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 nanoparticles and their
impacts. The researchers have found that, on an equal mass basis, carbon nanofibers require a
significantly higher energy investment, and appear to have a larger life cycle environmental impact, than
traditional basic materials. The high energy investment may lead to high costs, thus restricting the use of
carbon nanofibers to niche applications. Products based on carbon nanofibers may be greener than
alternatives for certain applications, and the quantity used will be the deciding factor. Regarding the
predictive model, the researchers found that, for emerging technologies, input information is easier to
obtain than output information. Preliminary results indicate a promising correlation between life cycle
inputs and impact. They found that ecological cumulative exergy consumption appears to be best for
aggregating inputs for a predictive LCA. The relationship between toxicology of nanoparticles and
thermodynamic properties also is promising. Future work will include: (1) an LCA of conventional versus
nanocomposite materials; (2) further statistical evaluation of the relationship between inputs and impact;
(3) an exploration of the relationship between thermodynamic properties of nanoparticles and their
toxicity; and (4) risk analysis.
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Discussion
Dr. Unrine commented on the mass-to-mass comparison and the application to automotive parts. He
suggested, for example, that if automobile body panels were made from carbon fiber, the car would be
lighter than one made with steel or aluminum and its operation would require less energy. He asked if the
model could take this into account or if the researchers had any data to address this issue. Dr. Bakshi
agreed that the mass of carbon nanofibers in automobile body panels would be small. The researchers
currently are examining nanocomposite materials. He cautioned, however, that most engineers and
scientists tend to think that if they develop a technology that uses less material, uses less energy, and is
more efficient, this will be better from an environmental perspective. Unfortunately, this does not always
happen because a technology that is more efficient also is less expensive, so it tends to be used more
extensively. Therefore, if the nanotechnology of carbon nanofibers became very good, they would likely
be used in almost everything and would be used much more extensively. In this way, any advantage may
disappear.
Dr. Mayer noticed that most of Dr. Bakshi's work is based on energy consumption and it appears that
energy consumption currently is not well understood. He asked if Dr. Bakshi believed that, in future
years, the data regarding energy consumption will be tighter, resulting in better correlations. Dr. Bakshi
explained that the researchers focused on energy consumption because this was the easiest approach, but
he clarified that he and his colleagues also had examined emissions of many other chemicals and
consumption of other materials. Regarding the large error bars for some analyses presented, he said that
he is in the process of completing a calculation of the theoretical minimum amount of energy needed to
produce carbon nanofibers; the challenge for this calculation is that many of the properties of
nanomaterials are not very well known. With this calculation, the error bars will no longer be relevant. He
added that the error bars in the analyses presented are based on the cycle time; according to manufacturers
the cycle time can be quite variable and this is why the error bars are so large.
Dr. Saleem remarked that EPA has a 2003 database for the TRACI Model and the LCA Model. Dr.
Bakshi explained that, even though the EPA data are more current, the underlying model is based on
information from the Bureau of Economic Analysis, and 1997 is the most recent database available. The
2002 model will come out next month and the researchers will incorporate the 2002 data.
Dr. Philip Lippel observed that it seems, generally, that one of the big promises of nanomaterials is the
potential to use much less material for the same purpose. He guessed that one might expect a reduction of
a factor of 10 or even a factor of 100 for most applications. This may be application-specific, but he
expected that a correction factor may be needed in the mass comparison. In addition, as these materials
become more widely used, the researchers may find that they are comparing immature production
technologies to mature ones. He asked Dr. Bakshi to what extent he expects the energy production costs
for carbon nanofibers to decrease. He added that Southwest Nanotubes is one company that may be
willing to provide relevant data. This company has been communicating very publicly recently about
scaling up through three or four generations of continued flow reactor designs and it might be willing to
share information about the associated energy costs. Such information might be helpful to the researchers'
modeling efforts. Dr. Bakshi replied that he will contact that company. He also described some of the
ways in which he and his colleagues are addressing this kind of concern. For example, the researchers
will determine theoretically the minimum amount of energy needed. In addition, they will specifically
examine the computer chip industry to determine how energy consumption has declined historically. They
then will conduct a scenario analysis based on this information. This work is ongoing and Dr. Bakshi said
he expects to have results next year.
Dr. Savage thanked all the participants for their contributions and adjourned the meeting.
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