International Perspectives on
Environmental Nanotechnology
Applications and Implications
Conference Proceedings
Volume 2 - Implications
October 7-9, 2008
Chicago, Illinois
U.S. Environmental Protection Agency
Region 5
Superfund Division
EPA905R09032
November 2009
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Disclaimer
This report does not constitute U.S. Environmnental Protection Agency policy. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
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Table of Contents
Table of Contents 3
Beth Anderson, Barbara T. Walton, and Charles G. Maurice
Chapter 6 - Introduction, Toxicity & Risk Assessment of Nanomaterials in the Environment 7
Anne Fairbrother
Managing the Risks of Nanomaterials 9
Jo Anne Shatkin
Advancing Risk Analysis for Nanomaterials and Nanotechnologies 17
I. Linkov and J. Steevens
Methods and Tools for Environmental Risk Assessment, and
Decision-Making for Nanomaterials 27
Martin Philbert
The Toxicology of Nanomaterials: Where Do We Go From Here? 33
Okkyoung Choi, Rao Y. Surampalli, and Zhiqiang Hu
Reactive Oxygen Species RelatedMicrobial Growth Inhibition by Silver Nanoparticles 35
James Lazorchak
Identification ofBiomarkers of Exposure to Metal-based Nanoparticles through Gene Expression
Profiling Using Daphnia magna Micro Arrays 41
Barbara J. Carter, Heather R. Hammers, Robert J. Griffitt, and David S. Barber
Using Microarrays to Test the Effects of Acute Exposure to Multiw ailed Carbon Nanotubes
(MWCNTs) on Gene Expression in Fathead Minnows (Pimephales promelas) 43
Stacey L. Harper, Jim Hutchison, Bettye L. S. Maddux, and Robert L.Tanguay
Integrative Strategies to Understand Nanomaterial-Biological Interactions 51
Christie M. Sayes
Nanotoxicology: Developing a Responsible Technology 57
Igor Linkov, Tommi Tervonen, Jeffery Steevens, Mark Chappell, and Jos'e Rui Figueira
Use of Multi-Criteria Decision Analysis for Classification of Nanomaterials 63
Bruce Lippy
MSDSs Fail to Communicate the Hazards ofNanotechnology to Workers 77
Q and A: Perspectives on Nanotoxicology 83
Ashley Smith, Xinyuan Liu, Kevin McNeil, Robert Hurt, and Agnes Kane
Bioavailability and Toxicity of Nickel in Metallic Nanoparticles 89
Tanapon Phenrat, Gregory Lowry, Thomas Long, and Bellina Veronesi
Partial Oxidation (Aging) and Surface Modification Decrease the Toxicity ofNano-SizedZero-
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Valent Irom 97
Alan J. Kennedy, Jacob K. Stanley, Jessica G. Coleman, David R. Johnson, and Jeffery A
Steevens
Ecotoxicological Evaluation of Carbonaceous and Metal Nanoparticles Through Bioassays
Relevant to Environmental Fate 101
Rebecca Klaper, Jordan Crago, and Devrah Arndt, and Jian Chen
Impact of Nanomaterial Structure and Composition on the Ecotoxicology ofNanomaterials on
Aquatic Species 109
Alia L. Alpatova, Pavel Babica, Syed A. Hashsham, Brad L. Upham, Susan J. Masten, and
Volodymyr V. Tarabara
Evaluation of In Vitro Toxicity ofFullerene nC60 Derivatives Formed in Conditions that
Simulate Disinfection Processes 777
F.A. Witzmann, A.D. Amos, X. Lai, and B.L. Blazer-Yost
Carbon Nanoparticle Exposure Alters Protein Expression and Cell Function in Mouse Renal
Principal Cells 773
Sylvia Chan-Remillard, Larry Kapustka, and Stephen Goudey
Can Nanoscale Particles Affect Plant Growth-Alfalfa Case Study 723
Xiaoshan Zhu, Xuezhi Zhang, Wen Zhang, Yung Chang, Hu Qiang, and Yongsheng Chen
Potential Toxicity ofNanomaterials and their Removal 725
R. Keith Esch, Li Han, David Ensor, and Karin Foarde
Endotoxin Contamination of Engineered Nanomaterials 737
Aaron P. Roberts, Leigh M. Taylor, Aaron E. Edgington, and Stephen J. Klaine
Effects of Ingested Engineered Carbon Nanomaterials on Zooplankton 737
H. P. Pace, and J. F. Ranville
Toxicity of CdSe/ZnS nanocrystals to D. magna 143
Scott Hall, Tina Bradley, Joshua T Moore, Tunishia Kuykindall, and Lauren Minella
Acute and Chronic Toxicity of TiO2 to Freshwater Aquatic Organisms 147
Joseph N. Mwangi, Ning Wang, Christopher G. Ingersoll, Doug K. Hardesty, Eric L. Brunson, Li
Hao, and Baolin Deng
Toxicity of Multi-Walled Carbon Nanotubes in Water to Sediment-Dwelling Invertebrates 757
Barbara Karn and Madeleine Nawar
Chapter 7 - Introduction, Environmental Fate & Transport ofNanomaterials 755
Jamie R Lead, Mohammed Baalousha, and Emilia Cieslak
Interactions Between Natural and Manufactured Nanoparticles 757
Vicki H. Grassian
An Integrated Approach Toward Under standing the Environmental Fate, Transport, Toxicity and
Occupational Health Hazards of Metal and Metal Oxide Nanomaterials 7 61
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Christophe J. G. Darnault, Solidea M. C. Bonina, Burcu Uyusur, and Preston T. Snee
Fate of Quantum Dots Nanomaterials in Unsaturated and Saturated Porous Media 167
Subhasis Ghoshal, Nathalie Tufenkji, and Gregory McKenna
Assessing Transport ofGoldNanoparticles and Bacteria in Porous Media Using X-ray CT
Scanning 773
Xueying Liu, Denis M. O'Carroll, Elijah Petersen, Qingguo Huang, and Lindsay Anderson
Impact of Size on Carbon Nonotube Transport in Natural Media 779
Yusong Li,Yonggang Wang, Linda M. Abriola, and Kurt D. Pennell
Evaluating the Clean-Bed Filtration Theory for Modeling Transport ofFullerene C60 Aggregates
in Saturated Porous Media 757
Paul G. Tratnyek, Vaishnavi Sarathy, James T. Nurmi, Donald R. Baer, James E. Amonette,
Chanlan Chun, R. Lee Penn, and Eric J. Reardon
Aging of Iron Nanoparticles in Water: Effects on Structure and Reactivity 793
Xuyang Liu, Mahmoud Wazne, Christos Christodoulatos, and Kristin L. Jasinkiewicz
Effects ofHumic Acid on Aggregation of Boron Nanoparticles in
Various Electrolyte Solutions 797
Marek H. Zaluski, Gilbert M. Zemansky, Adam Logar, Kenneth R. Manchester, Akshai K
Runchal, David Reichhardt, and Scott Petersen
Predictive Numerical Model of Post-Injection Distribution ofNano-Size ZVIin the Ringold
Aquifer for Mending an Existing Permeable Reactive Barrier in the 100-D Area at the Hanford
Site 205
Ann L. Miracle, Amoret L. Bunn, Jill M. Brandenberger, and Dan Gaspar
Fate and Transport of Titania Nanoparticles in Freshwater Mesocosms 273
Divina A. G. Navarro, David R Watson, Diana S. Aga, and SarbajitBanerjee
Natural Organic Matter-Mediated Phase Transfer of Quantum Dots
in the Aquatic Environment 279
Qilin Li, Bin Xie, and Steven Xu
Natural Organic Matter Enhanced C60 Fullerene Dispersion in the Aqueous Phase 22 7
Patricia Holden, Allison Horst, John Priester, and Andrea Neal
Interactions of Bacteria with Engineered Metal, Metalloid, and Metal Oxide
Nanomaterials 233
Susan Cumberland and Jamie R. Lead
Structure of Iron Oxide Nanoparticles; Influence ofpH and Natural Organic
Macromolecules 237
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Mark. A. Chappell, Aaron J. George, Katerina M. Dontsova, Beth E. Porter, Cynthia L. Price,
Pingheng Zhou, Eizi Morikawa, J. Bennett Johnston Sr, Alan J. Kennedy, and Jeffery A.
Steevens
Surfactive Stabilization of Multi-Walled Carbon Nanotube Dispersions with Dissolved Humic
Substances 243
Arianne M. Neigh, Thomas K. Darlington, Oanh Nguyen, and Steven J. Oldenburg
Evaluation of Nanoparticle and Matrix Characteristics Affecting Transport in the
Environment 253
Krishna R. Reddy, Amid P. Khodadoust, and Kenneth Darko-Kagya
Transport and Reactivity of Lactate-Modified Nanoscale Iron Particles in PCP-Contaminated
Field Sand 261
Report Backs and Panel Discussion: Implications 269
Presenter Biographies 275
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Chapter 6 - Introduction
Toxicity & Risk Assessment of Nanomaterials in the Environment
Beth Anderson, National Institute of Environmental Health Sciences
Barbara T. Walton, and Charles G. Maurice, United States Environmental Protection Agency
Integral to a discussion of nanotechnology is consideration of the implications of using these
novel materials. One critical implication, nanomaterial toxicity, was the focus of plenary,
concurrent, and poster presentations. The importance of this topic was clearly laid out in the
conference opening when Jeff Morris challenged the meeting attendees to seek to understand
which properties and characteristics of nanomaterials relate to their toxicity and to identify and
investigate doses relevant to real-world exposures.
Plenary speaker Anne Fairbrother observed that much data are needed, not only on numerous
classes of nanomaterial s but on key variables that influence toxicity. Examples of these variables
are biological species tested, toxicity tests used, test sample preparations, entry portals, dose
metrics, and modes of action. In a complementary plenary, JoAnn Shatkin focused attention on
risk analysis and placing priority on establishing what we know about toxicity and dose-response
in the context of realistic exposures.
These plenary presentations set the stage for the concurrent sessions, which provided insights on
how the field is responding to this pressing issue. One theme that emerged throughout the talks
was the importance of characterizing nanomaterials. The sheer variety of nanomaterials presents
huge obstacles for comprehensive assessments of these novel substances.
The papers demonstrated that investigations have progressed beyond evaluating the effects on
toxicity by fundamental nanomaterial characteristics such as size, shape, charge and purity.
Investigations have broadened and matured to consider variables such as aggregate size,
chlorination, coatings, dispersants, hydrophobicity, pH, reactivity, redox potential, surface area,
and reactive oxygen species (ROS) generation. A wide range of nanomaterial types are being
studied as well, including single- and multi-walled nanotubes, fullerenes, and nanoparticles
composed of silver, nickel, titanium dioxide, iron, and aluminum oxides.
To conduct these studies, investigators have used a diverse array of test species from non-
mammalian taxa such as microbes, aquatic species, and terrestrial invertebrates to mammalian
systems such as mice, rats, and human cell lines. In addition to the diversity of test species being
investigated, a multitude of endpoints are being used to assess toxicity, including oxygen uptake
rates, gene expression, mobilization and internalization of particles, oxidation, neurotoxicity, fate
and biological uptake, protein expression, and cell function.
Several general principles regarding nanomaterial toxicity emerged during the toxicity sessions:
• Nanotoxicity is often associated with ROS
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• Characterization of the nanomaterials tested is critical to data interpretation
• Aggregation typically reduces toxicity
• Aggregation and agglomeration are dynamic processes, so dissociation can occur over time
and, therefore, toxicity can also change over time
In summary, these studies demonstrate that nanomaterial toxicity is extremely complex. Despite
the vibrant and maturing field of nanotoxicology, our understanding is very limited. Because our
current understanding of the environmental implications of nanomaterials is so limited, there is
a large gulf between it and the flourishing development of new nanomaterials and applications.
A prudent approach to this disparity is to develop best practices and to be clear and transparent
about what we know, as well as what we don't know but need to know.
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Managing the Risks of Nanomaterials
Anne Fairbrother
Exponent, Bellevue, Washington, U.S.A.
My purpose in presenting this material is to provide a context within which to consider the
risks of nanomaterials, either as they are applied directly to the environment for remediation of
contaminated sites, or indirectly through manufacturing, use, and disposal. Although delegates
to this conference are no doubt well informed about particular aspects of nanotechnology and its
products, it often is useful to review basic principles. I will describe the classes of nanomaterials
and general uses of the various types of manufactured nanoparticles. This will include a
review of the properties of nanoparticles that differentiate them from macroscale products,
particularly with regard to potential human health and environmental effects. Understanding
how nanoparticles move through environmental media is fundamental to assessing risks, so I
will review what is known and where research is needed to develop methods for quantifying
nanoparticles and describing exposures. I will briefly discuss what currently is known about how
nanoparticles affect people or plants and animals. Finally, I will review the current regulatory
initiatives being considered for environment, health, and safety.
Classes and Types of Nanomaterials
Many nanoparticles occur as a result of natural processes. They are formed from sea-spray,
volcanic eruptions, lightning strikes, and forest fires. Colloidal materials in streams contain
nanosized particles, including humic and fulvic acids, proteins, and peptides. Hydrous iron
and manganese oxides occur naturally in nanosized particulates, as do clays and minerals such
as asbestos. Some nanoparticles enter the environment as combustion by-products, from fires,
various types of industrial air pollution, and from running internal combustion engines (e.g.,
automobiles). These are referred to in regulatory language as
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nanotechproject.org/inventories/consumer/), and there likely will be thousands within the next
few years. Examples include titanium dioxide and zinc oxide as photolytic and UV blockers in
paints, sunscreens, cosmetics, bottle coatings, and cement. Metal nanoparticles also are used
in mineral supplements (e.g., zinc, palladium, titanium, indium) and as combustion catalysts
(such as cerium dioxide in diesel fuels and nickel dioxide in fuel cells), thin films on solar cells,
gas sensors, refrigerants, magnets, and in medical imaging. Quantum dots are manufactured
from metals or metal oxides surrounded by a silica shell and have unique electronic, optical,
magnetic, and catalytic properties. They range in size from 1,000 to 10,000 atoms and are used,
for example, in semiconductors, electronics and computing, photovoltaic cells, and light emitting
diodes. Zerovalent iron is produced by reduction of solutions of metal salts and has been used
to remove nitrates from soils or to detoxify organochlorine pesticides and polychlorinated
biphenyls (PCBs) in contaminated soils or sediments. Nanosilver is the most well-known
metallic nanomaterial because of its antimicrobial properties. It has been used, for example, as a
deodorizer in socks, as a whitening agent in soaps, and in washing machines.
Nonmetallic nanomaterials include fullerenes. Fullerene is a generic term used to describe
carbon nanoparticles that take the form of hollow spheres or tubes. They are similar in structure
to graphite. While graphite is composed of flat sheets of carbon atoms arranged in hexagons,
fullerenes contain different arrangements of the carbon atoms that form three-dimensional
structures. The smallest and most common fullerene, C60, is a sphere of sixty carbon atoms.
Nanotubes are similar in structure to C60 but are elongated to form a tubular structure, usually
one to two nm in diameter and up to 1 mm long.
The simplest nanotubes are a single layer of carbon atoms arranged in a cylinder (single-walled
carbon nanotubes). They also can consist of multiple concentric tubes (multi-walled carbon
nanotubes) with diameters up to 20 nm and lengths greater than 1 mm. Carbon nanotubes are
synthesized from graphite using an arch discharge or laser ablation process. They can be made
more water soluble by the attachment of polyethylene glycol or phospholipids, which also
reduces the tendency of all nanoparticles to agglomerate. Carbon nanotubes are very strong
(nearly 500 times stronger than steel), very light (about one-sixth the weight of steel), and
are about 10 times more conductive than copper. They have been used in plastics and other
composites to increase strength, in flat panel displays, and in energy storage devices.
Why Are Nanomaterials Different?
Nanoscale substances behave differently than their macroscale counterparts because of their
small size. An example can be seen in the color of gold. Macroscale gold is a shiny orange-
yellow color. The same is true of a particle of gold 100 nm wide, but between 100 and about
30 nm, gold is purple, and at 30 nm in size, a gold particle is bright red. Smaller gold particles
become brownish in color. Below about 100 nm, the behavior of elements follows the rules of
quantum physics. Gravity is not important, and van der Waals forces (1) govern the attraction/
repulsion behavior between particles. Surface charge becomes very important and agglomeration
of particles occurs readily. Nanoscale particles easily penetrate cell walls and membranes without
the requirement of phagocytosis for entering cells. Phase dispersion (such as octanol / water
partitioning) is not predictable, because nanoparticles can get caught in the meniscus between
the two phases. Nanoparticles are highly chemically reactive because of their high surface to
volume ratio. They can be made efficiently and cheaply either through self-assembly of atoms
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(an inherent property of nanomaterials) or through pulverization of macroscale materials.
Transport and Fate of Nanomaterials
Nanoparticles can be highly mobile in the environment, but their mobility tends to be reduced
through agglomeration into large particles. Agglomeration increases deposition rates of
nanoparticles as a result of their propensity to attach to mineral surfaces. Although there are good
models to predict agglomeration and/or deposition of spherical particulates to form packed beds
of spheres, such models are not yet available for agglomeration and behavior of nanoparticles
that have nonspherical shapes and complex surfaces. This makes prediction of transport and
fate in environmental media (e.g., water and sediment) difficult for nanosized particles. The
field of colloid physics provides a basis for such studies, but traditionally has not studied solid
state materials or the type of multi-layered, multi-chemical particles found in manufactured
nanomaterials. However, for properly assessing risks of nanomaterials in the environment, it is
critical to know how to measure and predict the persistence, agglomeration, binding to lipids
and/or organic matter, hydrophobicity, and other properties of nanoparticles (Klaine et al. 2008).
It is likely that sediments are the final sink for nanoparticles in aquatic systems (Klaine et al.
2008). Adsorbed organic matter will stabilize charges on particles and cause fibrils to form,
which may cause particles to agglomerate via bridging mechanisms. Sediments also will
stabilize pH, and calcium ions and natural colloids (e.g., clays, fulvic and humic acids) will
retain nanoparticles. Studies on nanoparticle mobility in porous media, such as groundwater
aquifers or sand filters, have shown that mobility is a function of surface chemistry and particle
size (Wiesner et al. 2006). High ionic strength and divalent ions will increase retention of
nanoparticles in porous media. Groundwater aquifers and surface water with ionic strengths of
>10-4 M and significant concentrations of calcium or magnesium should favor nanoparticle
deposition (Wiesner et al. 2006). Even the most mobile nanoparticles should be removed in
sand filters during municipal wastewater treatment using conventional technology. However,
nanoparticles are frequently coated with polymers, polyelectrolytes, or surfactants that change
surface charges. Nanoparticles intentionally released into the environment to remediate
contaminated soils or sediments, such as zerovalent iron, have polymer coats to reduce
agglomeration and increase water solubility. This decreases the efficacy of water treatment
systems. Metal oxide quantum dots and other manufactured nanomaterials have similar coatings.
Although not intentionally released into the environment, they may be released through
wastewater or other disposal methods.
Salinity significantly increases agglomeration of nanoparticles (Niehof and Loeb 1972). This
is important in studying in vivo exposures of people and animals to nanoparticles, as blood is a
relatively saline solution. Marine organisms experience lower exposure to nanoparticles than
do their freshwater counterparts because of the salinity-induced agglomeration, which results
in lesser toxic responses (Kashiwada 2005). Additionally, currents and thermoclines in marine
environments affect the transport and movement of nanoparticles in the water column. Therefore,
predictions of transport and fate of nanoparticles in marine systems cannot be accurately
predicted from studies in freshwater systems.
Redox transformations are very important in the degradation of organic compounds and also
affect precipitation and dissolution reactions. These are mediated by microorganisms through
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enzymatic activity and indirectly through formation of reactive oxygen species. It is not known
if nanomaterials can be transformed by redox processes. It is plausible that fullerenes could be
oxidized (e.g., hydroxylated) by soil fungal enzymes such as cytochrome P450, peroxidases, or
lactases, as they have a high propensity to accept electrons (Wiesner et al. 2006). This is an area
in need of research.
Measuring Nanomaterials in the Environment
A variety of methods are available for characterizing nanoparticles. These include high
resolution imaging techniques such as scanning and transmission electron microscopy that
can be used to measure the shape, size, and crystalline nature of the particles. Scanning probe
techniques such as atomic force microscopy also measure the size and shape of nanoparticles.
UV-vis spectroscopy, infrared spectroscopy, and x-ray photon spectroscopy are tools for
studying the surface chemistry of nanoparticles. X-ray diffraction measures the surface area
of particles. Solubility of nanoparticles can be measured by ultrafiltration, dialysis, and flow-
field fractionation coupled to ICP-MS (Klaine et al. 2008). However, none of these methods is
well-suited to quantifying the amount of nanoparticles in environmental media (soil, sediment,
or water). Further, it can be difficult to separate nanomaterials from natural colloidal matter.
Experimental methods such as near-field acoustic holography (Shekhawat and Dravid 2005)
show promise, but currently are too expensive and time-consuming to be applicable to high
throughput environmental screening. Measurement of nanoparticles in environmental media
remains an area of high research need for environmental monitoring and risk assessment.
Effects of Nanomaterials
Studies of the effects of nanomaterials on biota and people began only a few years ago, but are
increasing exponentially. This section of my presentation provides an overview of the types of
effects that may be observed and considerations for standardizing and improving toxicity tests on
different types of organisms.
Aquatic organisms are exposed to nanoparticles primarily through gut intake followed by
translocation within the body (Roberts et al. 2007; Fernandes et al. 2007). Terrestrial animals are
exposed through the lung (inhalation) and gut (diet), while plants are most likely to be exposed
via root uptake. Nanoparticles can diffuse through the cell membrane or can be taken up by
adhesion and endocytosis. They are not dependent upon the circulatory system but can move
through the body via cell-to-cell contact. This is a very important consideration in understanding
nanoparticle distribution and metabolism within organisms. Potential mechanisms of toxic
action within an organism include: disruption of membranes or membrane potential, formation
of reactive oxygen species, oxidation of proteins, interruption of energy transduction, release of
toxic constituents, and genotoxicity (Klaine et al. 2008). Antibacterial activity occurs as a direct
contact between a positively charged nanoparticle and the bacterial cell surface. This changes the
surface phosphorylation and membrane permeability, causes oxidative stress and formation of
highly reactive epoxides resulting in DNA damage, and affects the integrity of the bacterial cell
membrane (Klaine et al. 2008).
Using cell cultures, fullerenes have been shown to have antibacterial properties, to be cytotoxic
to human cell cultures, to cause DNA damage, and to inhibit protein folding. In laboratory
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rodents, single-walled carbon nanotubes have been reported to cause lung granulomas and
necrosis, pneumonitis, and activate immune cells (Wiesner et al. 2006). Metal nanoparticles have
also been shown to cause pulmonary inflammation, alveolar degeneration, and DNA damage
in rodents (Handy et al. 2008; Wiesner et al. 2006). Effects in aquatic organisms have been
summarized by Handy et al. (2008), Klaine et al. (2008) and more recently by Ziccardi et al.
(2008). These include oxidative stress and lipid peroxidation in liver, gill, and brain offish, along
with glutathione reduction in the gill reported as a result of fullerene exposure. Cytochrome
P450 enzyme expression may be downregulated and gill pathology is evident in some fish
exposed to fullerenes. Fullerenes and metal nanoparticles have also been reported to cause
delayed hatch, reduced growth, increased molting, oxidative stress, and mortality in various
aquatic invertebrates. However, evidence of effects appears to be very dependent upon test
conditions, particularly methods used to solubilize the nanoparticles and get them into solution.
Toxicity to terrestrial organisms has been studied in plants and soil microbes (summarized by
Klaine et al. 2008). Exposure to metal nanoparticles has been reported to reduce root elongation
and decrease seed germination, depending upon species, test conditions, and concentrations in
soils. Conversely, Yang et al. (2007) showed increased seed germination and photosynthesis in
spinach exposed to titanium dioxide nanoparticles. Soil microbes are not affected by exposure to
fullerenes (Johansen et al. 2008).
Results of studies of effects of nanoparticles on both aquatic and terrestrial species depend
greatly upon the test conditions. Because such tests have not yet been standardized, it is difficult
to make comparisons among species exposed to the same nanomaterials or within species using
different particle types. Questions for standardizing aquatic test systems include (but are not
limited to): how do pH, hardness, ionic strength, or organic ligands affect toxicity? Is water the
correct exposure route or is diet more appropriate? How should particles be solubilized (e.g.,
sonication, stirring, functionalization using tetrahydrofuran)? Should both water soluble forms
and agglomerates be tested (e.g., water column versus sediment organisms)? What methods are
available for measuring exposure concentrations? For terrestrial bioassays, should the particles
be mixed into soil by spraying a solution onto the soil or by mixing in dry particles? Because
particles adhere to the soil solid phase, how should exposure be measured and expressed?
For all studies, it is important to characterize the stock solution to quantify the average and
range of particle sizes used in the study. It is also important to measure particle sizes throughout
the experiment as changes may occur because of agglomeration and other factors. There also
is a question about how exposure should be quantified. Should exposure be expressed in terms
of surface area, or number of particles per unit volume (ng/L), or mass (ng/kg)? There is no
apparent reason for not using standard aquatic and terrestrial test organisms in bioassays to
assess nanoparticle toxicity, although it is not known which organisms represent the most (or
least) sensitive species.
Regulatory Concerns for Nanomaterials
While the science of nanomaterials is not new, applications of nanoparticles to consumer
products and their use in commerce is relatively recent. Recognizing the commercial potential
of this emerging technology, governments are providing billions of dollars in research funding
for nanomaterials science and commercialization. However, very little funding has been
available to study the environmental fate, human health and safety, or ecological effects of
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the manufacture and use of nanoparticles. In the U.S., only 5 percent of the 2009 $1.5 billion
budget for the Nanotechnology Initiative is directed toward environmental issues and human
health and safety. At the time of this writing, the U.S. has not proposed any new approaches to
assessing the risk or regulating nanomaterials, as existing laws provide the framework for doing
so. Products containing nanosilver used as a biocide (e.g., in washing machines or socks) are
regulated under the Federal Insecticide Fungicide and Rodenticide Act (FIFRA) in the same
manner as other biocidal products. Carbon nanotubes recently were determined to be new
substances and, as such, require registration under the Toxic Substances Control Act (TSCA).
Similarly, silica and alumina nanoparticles have been designated a "significant new use" and
are regulated under section 5(a)(2) of TSCA. EPAs Nanoscale Materials Stewardship Program
provides a framework for voluntary submission of exposure and effects information. California
has asked manufacturers of carbon nanotubes to monitor worker health (though inhalation) and
report exposures and associated effects. Canada recently instituted reporting requirements for
nanomaterials used in quantities greater than 1 kg/yr. Australia has been working to develop an
International Organization for Standardization (ISO) nanomaterial standard for occupational
health and safety, but primarily funds innovation and development in manufacturing. In Britain,
the Department of Environment, Food, and Rural Affairs (Defra) established a nanotechnology
research coordination group and commissioned several white papers on the topics of health and
safety (e.g., Crane and Handy 2007). At the time of this writing, Britain's Health and Safety
Executive advised companies or universities supplying carbon nanotubes to include health and
safety information on their materials (including "Caution: substance not yet fully tested") and
an indication of the concentration of the substance in the material. Several nongovernmental
organizations, industries, and governments have called for responsible production and use of
nanomaterials through a better understanding of potential effects and standardized approaches
to testing and regulation. Most notable are the German Chemical Industry Association (VCI)
who published several white papers on the topic (VCI 2008), and Environmental Defense in
partnership with DuPont, who published a nanorisk framework (Environmental Defense -
Dupont 2008). The Scientific Committee on Emerging and Newly Identified Health Risks in
the European Union has recognized that risk methodologies require modifications for use with
nanomaterials, stating that additional guidance is needed on how to conduct standardized toxicity
tests. The Organization for Economic Cooperation and Development (OECD) established a
Working Party in 2006 to address these and other questions for evaluating risks of manufactured
nanomaterials in the workplace and environment.
Conclusions
The science of nanomaterials and its commercial applications continues to grow exponentially,
as do concerns about possible environmental, health, and worker safety risks from exposure to
nanoparticles. Assessing potential risks is hampered by lack of information on transport and
fate of nanoparticles in the environment and standard methods for assessing effects to aquatic
or terrestrial organisms and human health. We need to know how to prepare and characterize
test materials. We need information about the full life-cycle of products, and how the associated
nanoparticles may move into and through the environment. We lack methods for measuring and
tracking nanoparticles in water, soil, or sediment and have no standard quantification metric.
Regulatory agencies continue to struggle with whether to define nanoparticles as new or existing
substances or as substantially new uses of existing substances. Appropriate values that would
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trigger requirements for additional testing or product controls are lacking. There is no doubt that
the public has been calling for an increased level of precaution with regard to nanomaterials,
as has happened with other emerging technologies (e.g., biotechnology). Although we can
learn many basic principles from the science of colloidal physics and chemistry, manufactured
nanomaterials have unique properties that affect both exposure and potential effects. Without
appropriate funding to develop test and measurement methods and to address the risk-related
questions described in this presentation, both regulators and the public are left without the
necessary tools to make informed decisions about risk management of nanomaterials.
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(1) Van der Waals forces are attractions between atoms and molecules caused by their
polarization, as the electron-rich area around one atom/molecule attracts the electron-poor region
around another.
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Advancing Risk Analysis for Nanomaterials and Nanotechnologies
Jo Anne Shatkin
CLF Ventures, Inc. U.S.A.
Introduction
Decisions about the use and acceptable levels of substances in products and in the environment
are increasingly determined using quantitative risk analysis. There are many questions and few
answers about the toxicity of nanoparticles, stemming in part from an inability to adequately
measure or predict the key characteristics of particles as they relate to toxicity.
Risk analysis as it relates to health and the environment encompasses a multi-disciplinary set
of methods to assess the levels at which substances may cause harm and to judge whether the
associated risks are acceptable. Risk analysis involves both science and judgment; the science
of characterizing materials, their toxicity, and their exposure characteristics, is weighed against
other materials and standards established as acceptable by society or within organizations.
Terminology varies across disciplines and organizations, but the assessment phase of risk
analysis generally consists of the following steps: hazard characterization, exposure assessment,
dose response analysis, and risk assessment. Underlying each of these steps is the judgment of
the available data, interpretation according to models, assessment of uncertainty, communication,
and evaluation of the acceptability of risk levels in societal context.
The rapidly expanding development and use of materials in the nanoscale range has generated
new challenges to the application of current risk analysis methods for environmental, health,
and safety concerns. The unique properties that may exist for these materials potentially have
significant implications for current approaches to the hazard identification, exposure assessment
and dose-response components of the traditional risk assessment paradigm that informs risk
management decisions, and may confound the accurate assessment of potential risks as well
as require changes to the way such risks are communicated to stakeholders and managed by
policymakers.
One of the challenges for risk assessments of nanoscale materials is that the small size of
materials conveys a much greater ratio of surface area to mass, which affects behavior in
biological and environmental systems, generally increasing activity over larger scale materials.
Currently, there is a poor understanding of the key factors that contribute to these behaviors,
which have been noted in literature and among convened experts to potentially include a breadth
of interrelated factors, including: surface area, surface charge, surface chemistry, porosity,
aggregation and agglomeration state, particle size and distribution, and level of contamination.
A further complication with assessing risks at the nanoscale is that materials produced are best
described as mixtures, that is, as produced they tend to have a breadth of sizes, impurities, and
characteristics, and they are generally combined with other materials for applications. The field
of risk analysis has been challenged by the issues raised in analyzing and assessing the risks
from mixtures, and this complexity is compounded with poorly characterized mixtures at the
nanoscale.
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The biggest issue in using risk analysis for decision making about nanomaterials is the current
lack of data to inform assessments. There is a lack of agreement on how to characterize
engineered nanoscale materials for toxicological studies, combined with limitations in the
availability of methods for measuring them. The available data are generally in systems with
limited applicability to real world exposures, such as in vitro assays that have not been validated
against in vivo data. Only a few studies have measured exposure levels, and environmental
models for estimating exposures to nanomaterials also have limitations, since the unique
properties of nanomaterials appear to also extend into the properties that dominate transport and
fate in complex biological and environmental systems.
To address these challenges, the Emerging Materials and Nanomaterials Specialty group
(EMNMS) of the Society for Risk Analysis organized a public/expert workshop, Nano Risk
Analysis: Advancing the Science for Nanoscale Material Risk Management held on September
10-11, 2008, in Washington, DC. The workshop created a multidisciplinary discussion
among experts in risk analysis, nanotechnology researchers, environmental science, other
key stakeholders and members of the public interested in risk analysis, risk communication,
and nanotechnology to identify approaches for risk analysis that assess the unique aspects of
nanotechnology and nanomaterials. To facilitate the discussion, five topical white papers, each
co-authored by a combination of nanotechnology and risk experts, were drafted and presented
on topics of: hazard identification and uncertainty; toxicology; exposure assessment; risk
characterization; and risk communication. These papers were vetted in plenary and facilitated
deliberative discussion sessions and will become a series of publications. For each topic, authors
considered the challenges posed by nanomaterials and nanotechnologies, and the opportunities to
apply the tools and methods developed for risk analysis to these current concerns.
One conclusion of the workshop was that despite current limitations, risk analysis remains a
valid approach to identifying, assessing and informing the management of engineered nanoscale
materials. This was also a conclusion of the European Food Safety Authority. Below, the issues
nanotechnology and nanomaterials raise for each step of the risk analysis process are identified,
and ideas discussed and issues raised at the Nano Risk Workshop are summarized, in the context
of other international efforts to address risks from nanomaterials and nanotechnologies.
Specific Considerations For Each Step Of Risk Assessment
Many of the uncertainties introduced by nanomaterials are not novel for risk assessment.
Radiation, respirable fibers and other particles also challenge classical toxicology models,
required defining new metrics and tolerable levels. Many of the complexities remain issues
for legacy and emerging substances in the environment - determining threshold levels versus
background (need to monitor background levels), age dependent dosimetry, body burdens from
non-engineered sources, defining internal dose -as was done for radiation, fractionation of
mixtures, and consideration of cumulative exposures. None of these sources of complexity are
specific to nanoscale materials.
Every step of the risk analysis process includes uncertainty. There are additional challenges
due to the physical dimensions of nanoparticles and the limited understanding and ability to
predict their impact on biological and environmental risks. New tools are needed to estimate and
measure real world exposures. Others have addressed the need to define and prioritize research
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needs for dose-response, exposure assessment and material characterization of engineered
nanomaterials in the short and long term. In particular, needs include fate and transport models,
biomarkers, and incorporating new scenarios for exposure (e.g. worker exposure during
recycling).
A few key points to keep in mind regarding the state of the science of nanomaterial EHS risks:
• Nanomaterials may behave like particles and have physical effects in addition to chemical
effects, which may require new measurements and assessments to determine;
• Material properties such as surface area, particle size, particle number, and aggregation state
may be important determinants of toxicity at the nanoscale;
• Existing mass- and concentration-based environmental health and safety (EHS) thresholds
may not be adequate to assess health and environmental risks of nanoscale particles to
workers and others;
• Small changes in particle size and surface properties may drastically affect toxicity;
• Nanomaterials' small size means they have the potential to "translocate" in the body (e.g.,
cross the blood/brain barrier); and
There is a poor understanding of the environmental fate and effects of nanomaterials, in part
because larger particle dynamics are not likely predictive for nanoscale particles.
Uncertainty about Material Characterization Challenges
the Identification of Hazard
Hazard characterization forms the question to be addressed in the risk assessment. Hazard
characterization questions for nanomaterials can be similar to chemical substances and mixtures;
the essential differences include the currently limited understanding of the key particle attributes
to measure, and the ability to measure them. Generally, chemicals are reported in mass quantities,
as a concentration (e.g. mass of a chemical per unit of volume in air, food, water, or blood). For
nanomaterials, the surface characteristics appear to be important parameters to characterize, and
at the moment, the measurement priorities are poorly understood. For nanomaterials, mass may
not be the best measure for characterizing risk. It may be important for risk characterization to
measure several surface properties (surface area, charge, level of contamination), but this is not
standardized, and measurement methods vary. Risk screening strategies proposed by ILSIRF,
OECD and ISO have denned over a dozen potential measures to characterize nanoscale materials
and are working toward standardizing methodologies. One proposal is to use a minimum
set of characterization parameters in all studies, to ensure comparability of data until the key
relationships between physical characteristics and toxicity are identified.
The SRANano Risk workshop addressed issues of nanomaterial characterization in the context
of uncertainty analysis, because this affects the exposure assessment, dose/response analysis
and risk characterization stages of risk assessment. Paoli and Shatkin suggested that while
traditional techniques for quantitative uncertainty analysis may be helpful for interpreting and
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estimating the uncertainty associated with nanomaterial characterization, it will be more fruitful
to conduct quantitative evaluations of uncertainty when the relationships between nanomaterial
characteristics and risk are better identified.
Currently, there is a pervasive uncertainty associated with the behavior of nanomaterials,
including which factors are associated with absorption and adverse effects, and the limits of
those factors. For example, is there a difference in effects from exposure to particles with an
average diameter of 25 nanometers versus particles with average diameter of 50 nanometers,
and if so, what if the particle size distributions overlap; can these be distinguished? The current
limitations for measurement and analysis contribute to the uncertainty, since we are both
uncertain about what to measure, and how to measure it. This "model uncertainty" contributes
as much to the current inability to conduct quantitative risk and uncertainty estimates as the
limitations in the current database. Further, it is clear from existing studies that we are not able to
generalize about these properties across different types of nanomaterials.
Nanomaterial characterization is not more uncertain than other substances, it is simply less
understood. There are plenty of examples in recent history that have required new units of
measurement for risk characterization. There are at least two possible factors that complicate
measurement: nanomaterials tend to exist as mixtures of particle sizes, purity, and aggregation
rates, and their properties may vary when incorporated into products; and some particles exhibit
dynamic behavior, that is, they associate and dissociate with each other and with biological
molecules such as proteins. Again, these concerns are not necessarily unique to nanoscale
materials. Pathogenic organisms grow, and ecosystems are dynamic. Further, mixtures have been
accommodated in risk analysis using toxicity equivalents and other measures of relative potency.
The complexity of contributing factors suggests nanoscale materials and nanotechnologies need
to be assessed under realistic exposure conditions.
There is a diversity of views on the issue of the importance of particle size. For example, there is
no evidence to suggest that 100 nanometers (nm), the generally accepted upper limit of nanoscale
particles, has any biological relevance. It is merely a scale. However, it is widely recognized that
people are investigating applications of nanoscale materials for a reason - material properties
change at the nanoscale. Some suggest that even aggregated nanoparticles, that can be several
hundred nanometers or more in diameter, still possess nanoscale properties. What is not clear is
when size matters, and how much, compared to surface and other physical properties. To date, it
has not been possible to generalize the findings from one study or type of material to another.
Exposure Assessment for Nanomaterials
Characterizing exposures to nanomaterials requires new measurement metrics be developed.
The current techniques available for sample analysis may not be sensitive or specific enough
to detect the low exposure levels of nanoscale materials, where relatively larger effects may
be seen from lower mass quantities. Historically, risk assessment has considered the mass or a
concentration of substances. As we've discussed, the number of particles, the total surface area,
and the reactivity of the surface area may become key exposure parameters. In occupational
environments, techniques such as particle counting and surface area measures apply, but
in toxicology experiments, and in broader scenarios, it is more difficult to measure these
physical attributes of nanoscale particles, particularly if they are not spherical. There may be
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a need to distinguish engineered from incidental nanoparticles. Many methods use established
relationships, e.g. the Brauner, Emmet and Teller (BET) method for calculating surface area, or
visual tools, such as TEM, that are less precise than may be warranted for this situation where
small subtle changes may have significant effects on particle behavior.
During the SRANano Risk workshop, participants suggested that the current situation of
exposure assessment for nanoparticles is not dire, and should try to rely on mass as the key
measure, and focus on evaluating how the environment affects nanoparticles. It will be important
to assess the relative bioavailability of nanoscale particles compared with larger particles for
specific exposure pathways. Another suggestion was to make a "metric matrix" to compare
exposures and inform dosimetry for nanomaterials. The needs for nanomaterial exposure
assessment include a host of uncertainties that have been addressed for other agents, and include:
internal dosing, body burdens, thresholds of toxicity and comparison to background levels.
Several participants called for measures to simplify exposure assessment.
"Nanotoxicology"
One widely observed effect from exposure to nanoparticles is inflammation, an immune system
response resulting from the generation of reactive oxygen species (ROS) when cells encounter
the surface-active nanoparticles. Inflammation is associated with the development of many
diseases such as asthma, cardiovascular disease, and immune system diseases. Inflammation
has been observed in whole animal studies (in vivo) and in cellular assays (in vitro) with a
diverse array of nanomaterials. It is presently unclear whether or in what ways the chemical
composition, size, shape, or surface characteristics affect the toxicity of nanoscale materials.
Existing studies are inconsistent in their findings. Differences observed between engineered
nanoparticles and their counterparts (besides the many as yet characterized) may include:
• dose metrics, absorption, distribution and excretion, as a result of size and/or external
modifications; and
• mechanisms of toxicity, as a result of increased access to cell matrices or generation of reactive
oxygen species — because of new characteristics such as increased surface area.
Consequently, traditional testing and detection methodologies may be inappropriately applied to
nanoparticles. Of the three routes of exposure relevant to humans, the majority of studies address
the inhalation route of exposure, considering occupational exposures during production. There
is relatively little evidence on the uptake of nanoparticles across the skin, with the exception of
photoactive compounds in sunscreens (nanoscale titanium and zinc oxides), which have not been
demonstrated to cross the skin barrier, and few studies assessing the toxicity of nanoparticles
once ingested. The data suggest that effects relate both to particle, as well as chemical, attributes.
A number of researchers are trying to develop predictive approaches to toxicity studies that
don't involve testing in whole animals. In vitro assays have to date not shown to be relatable
to whole animal studies for nanoparticles. This may relate in part to the difficulties of getting
nanoparticles dispersed in dosing solutions. Many nanoparticles are very sticky, and tend to
agglomerate, or aggregate. When they do, it increases the difficulty of measuring the toxic
effects, or the confidence level about the exposure levels associated with them. Some researchers
use aggressive techniques to separate the particles prior to dosing, so it then becomes a
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question of whether the findings can be related to real world exposures, where particles may be
aggregated. A recent study showed that some nanoparticles interfere with the reactive agents in
an in vitro assay, producing a false positive (Worle-Knirsch et. al, 2006). The lack of an agreed-
upon standard for material characterization, as well as for standardized types of test assays, has
led to a diversity of approaches such that the existing studies are of limited comparability. This
is problematic because the results tend to be equivocal - in one test system an effect is observed,
while in the next, no effect is observed.
Because the data are equivocal, there is no conclusive evidence that particle size is the main
driver of toxicity. This has led some organizations (e.g. FDA) to avoid denning nanoscale
materials by size, and others to acknowledge the challenges for communication and management
raised by establishing a bright line for nanoscale material (EFSA 2009). In the short term,
agencies likely will adopt a case-by-case approach to nanomaterial reviews. One suggestion from
the SRANano Risk workshop was to test the toxicity across ranges of particle sizes, with an
eye toward developing likelihood functions rather than bright lines. That is, design experiments
to define size ranges where nanoscale effects must be considered. This may involve tracking
size, rather than using size to classify materials, since size is not currently a good a predictor of
hazard. This approach would limit the effect of "lumping materials", and allow categorization to
be defined by variables as they occur. Categories of hazard may relate to uses, not sizes, which
would classify exposure types. Other participants suggested that nanomaterials may represent a
phase, not a class, of materials. The key need is to determine when mass-based doses result in
different effects.
Another significant issue for assessing toxicity is not unique to nanoscale substances, but may
be more pronounced because subtle changes have greater relative effects at the nanoscale. That
is, most nanoparticles tend to be present in a distribution of sizes and purity levels resulting
from manufacturing or processing that affect toxicity and behavior. The small scale makes
nanomaterials vulnerable to changes during handling, and the choice of media used for handling
may affect the state of aggregation and surface properties of some nanomaterials. However,
treating nanomaterials as mixtures may help determine the key parameters, relationships, and
defining criteria for variables other than mass and size.
The equivocal reports of nanomaterial toxicity beg for a standardized set of criteria and
measurements to be reported for toxicity studies, that can help to probe the key criteria, and
understand differences in study outcomes for seemingly similar materials. These criteria need to
be reported along with estimates of measurement error, or property tolerance levels, to inform
the importance to overall material toxicity and the associated variability. Many workshop
participants suggested approaches for ensuring standardization of parameter reporting, including:
broad analysis of the forthcoming data sets; adoption of a single harmonized standard for
nanomaterials reporting; research efforts to improve analytical measurements; grant funding
agencies adopting requirements for all toxicological studies; international harmonization of
data collection approaches; editorial requirements on manuscript submission, and adoption of
standards by professional organizations such as Society for Risk Analysis, Society of Toxicology,
The Society for Environmental Toxicology and Chemistry, and others.
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Risk Characterization for Nanomaterials
Considering risks from nanomaterials and nanotechnologies in the context of risk management
decisions may mean comparison to existing standards for more common chemical substances
for which there has been more discussion and debate. There are few available risk assessments
for nano materials. Those currently available are generally comparative—that is, compare risks
for nanoscale versions of substances to those from larger particles. For example, the National
Institute for Occupational Safety and Health (NIOSH) compared the risks from nanoscale
titanium dioxide to larger particles Niosh, 2005). Warheit et al. (2007) compared the properties
and effects of three types of nanoscale titanium dioxide in a breadth of in vitro and in vivo
toxicity assays.
In the workplace, exposure to substances can be more easily managed - it is a fairly
controlled environment, but in the broader environment, it may be the products that need to
be managed, not the substances, since their applications and uses will vary so greatly. Most
consumer exposure to nanomaterials is likely to occur when people use products that contain
nanomaterials. This is an added challenge for risk management and risk assessment, to determine
the potential risks associated with the use of nanomaterials in products, where materials have
different levels of bioavailability and exposure profiles.
Typically, one considers exposure to the active substance only, not the effects of the entire matrix
on exposure. Secondary pathways that release (nano)materials in the environment under poorly
controlled conditions increase the number of potential receptors and pathways. For example, a
packaging material that contains a layer of antimicrobial nanoparticles may have a protective
coating that ensures no direct contact with the user, or the packaged item. However, this material
can be recycled, introducing the nanoparticles into new matrices with indeterminate exposure
pathways. Alternatively, if the package is disposed of as solid waste, nanoparticles could be
released to ambient air by incineration, to water from landfill leachate, or to sewage sludge that is
applied as fertilizer to crops, depending on the method of disposal.
Representatives of regulatory agencies in Europe, Canada and the U.S. at the SRANano Risk
Workshop were in general agreement that existing risk models must be considered in terms
of the unique attributes of nanomaterials, and adapted, but not replaced. It will be critical to
understand exposure potential, and nuances of dose response assessments, which require an
adaptive approach to management. A case-by-case approach is the most logical and likely path
to understanding the dimensions of risk important for nanomaterials and nanotechnologies,
but there is need for an overarching framework, or a "road map" for developing data and
environmental health and safety research. There is general agreement that a life cycle
framework for risk analysis is warranted and appropriate. The importance of clear and proactive
communication about the potential risks was repeatedly highlighted.
Participants were in general agreement that the current risk characterization framework is
appropriate for nanomaterials. However, "nanotechnology is not old wine in a new bottle." Dose
metrics and chemical characteristics are novel, including the dynamism of the materials, that is,
the observation that characteristics can change with time and in response to the environment.
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The combined aspects of dynamism and novelty suggest a dynamic decision framework - so
information, characteristics, and decisions can be updated when more is understood. Further,
the desire to assess risks in real time, that is to ensure materials and technologies that are in use
today will be tested for indications of toxicity, means that early assessments may include some
uncertainties that will be updated when greater understanding of cause and effect relationships
are elucidated. These factors require an adaptive management approach to risk analysis.
Several frameworks have been proposed that are explicitly designed to address the adaptive
needs of evolving scientific understanding, and evaluate exposure across the life cycle of
nanoscale materials, in particular: Comprehensive Environmental Assessment (Davis, 2007);
NanoLCRA (Shatkin, 2008) and the Nano Risk Framework (EOF DuPont, 2007). There is a need
for public vetting of these and other frameworks. There is also a need for a coordinating entity
to prepare to assess a breadth of anticipated data sets from members of the Organization for
Economic Cooperation and Development, and from various data calls in the United Kingdom,
United States, and Canada.
Communicating About Nanotechnology and Nanomaterials
As with other aspects of risk analysis, communicating about the risks of nanomaterials
and nanotechnologies is not inherently different from communicating about other
classes of substances. However, the breadth of uncertainty and lack of agreement about
terminologyconfound clear communication. It is easier to communicate facts than uncertainty.
Workshop participants expressed concern that while the technical and scientific issues are being
addressed, with scientists and risk analysts identifying, characterizing and assessing the unique
attributes of nanomaterials, public perceptions may not recognize this progress. For example, it is
a myth that nothing is known about the risks of nanomaterials. Similarly, scientists do not agree
there is a need to change the risk analysis paradigm. One repeated myth is that nanotechnology is
present in foods everywhere; another is that regulation of nanomaterials is lacking.
The risk analysis community has addressed significant uncertainty for other sources of risk, and
generally manages risk by making conservative assumptions that are revised when data gaps are
filled. Proactive approaches for informing and educating people about the risks and benefits of
nanomaterials and nanotechnologies is a critical component of any risk management strategy, but
is not typically how things are done.
Suggestions for improving the transparency and public trust of the management of risks from
nanotechnologies include new organizations or partnerships that are privately funded but
publicly conducted, outside of traditional regulatory agencies. There is a need for independent
review and communication of data and risks in context. This independent evaluation requires
adaptations to current models. The complexity of issues raised, some of which are not unique
to nanomaterials or nanotechnologies, require new thinking about how to be proactive and
transparent, establishing new relationships, and new approaches for integrating research, problem
framing, risk assessment, management, and communications. Extensive efforts in Germany and
Japan to engage the public have led to increased public awareness, acceptance and perceived
benefits of nanotechnologies relative to risk.
One difficulty is the need to consider how to discuss risks from products, versus substances.
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Because nanotechnology is not a market, its an enabling technology, it is not possible to
define risks, or benefits, out of context. That is, the risks and benefits of medical applications
of nanotechnology are evaluated in a different context than consumer or cosmetic products,
however this is often presented under a single umbrella.
The perception of risk and of benefits will vary and is likely to influence public, regulatory, and
non-governmental activities regarding risk and benefit evaluations. If there are benefits, life-
saving benefits, these should be considered in the decision frame. This sentiment was echoed
by workshop participants who recognize the public will accept some risks more than others
depending on the benefits conveyed. Thus, risks associated with life-saving technologies may
be perceived differently than purely cosmetic applications. There is a great need to explain these
needs to risk managers.
Finally, there is a need for an independent and trusted entity to organize and filter information.
The proliferation of informal and decentralized communications via the internet is a double
edged sword. On the one hand, the internet and sources such as blogs flatten the landscape for
obtaining information, on the other, unreviewed, biased and incorrect information is easily
proliferated, and it is difficult to identify the sources and trustworthiness of much of the current
data. This means that conflicting information abounds and confuses the reader.
There is a need for an independent and trusted entity to make new findings about risk transparent
and understandable. Visual and graphical tools improve communication about complex topics.
One suggestion was for Society for Risk Analysis to bring the extensive resources, international
representation, and independence to this issue.
Acknowledgements
The workshop was supported by the National Science Foundation, the U.S. Environmental
Protection Agency, Johns Hopkins University Institute for NanoBioTechnology, CLF Ventures,
NanoGram Corporation, the American Chemistry Council, ILSI Health and Environmental
Sciences Institute, BASF Corporation, Evonik Corporation, the National Capital Area Chapter
of SRA, the U.S. Department of Defense Strategic Environmental Research and Development
Program, the National Institute for Occupational Safety and Health, and the Women's Council
for Energy and the Environment, as well affiliations of many other workshop participants.
References
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Methods and Tools for Environmental Risk Assessment, and
Decision-Making for Nanomaterials
I. Linkov, andJ. Steevens, US Army Engineer Research
and Development, Brookline, Massachusetts, U.S.A.
Abstract
Nanomaterials and their associated technologies hold promising opportunities for the
development of new materials and applications in a wide variety of disciplines, including
medicine, environmental remediation, waste treatment, and energy conservation. However,
current information regarding the environmental effects and health risks associated with
nanotechnology is limited and sometimes contradictory. This paper summarizes the
conclusions of a 2008 NATO workshop designed to evaluate the wide-scale implications of
nanotechnology on human health and the environment. A unique feature of this workshop was
its interdisciplinary nature and focus on the practical needs of policy decision makers. Workshop
presentations and discussion panels were structured along four main themes: technology and
innovation, human health risk, environmental risk, and policy implications. Four corresponding
working groups were formed to develop detailed summaries of the state-of-the-science in their
respective areas and to identify emerging gaps and research needs. Gaps between the rapid
advances in nanotechnology and the slower pace of human health and environmental risk science
were identified, along with strategies to reduce the associated uncertainties.
Introduction
Many potential questions are associated with the current state of development and use of
nanomaterials. For example, with the availability of over 600 consumer products worldwide
claiming to contain nanomaterials, what information exists that identifies their risk to human
health and the environment? What engineering and other personal and environmental protection
controls can be deployed to minimize the potential human and environmental health and safety
impacts of nanomaterials throughout the manufacturing and product lifecycles? How can the
potential environmental and health benefits of nanotechnology be realized? To discuss and
develop expert answers to questions such as these, the NATO Advanced Research Workshop
"Nanomaterials: Environmental Risks and Benefits and Emerging Consumer Products" brought
together 70 scientists and engineers from 19 different nations and multiple fields, reflecting the
global and interdisciplinary nature of nanotechnology and nanomaterials research.
State-of-the-science reviews of nanotechnology were presented during the plenary sessions
by renowned experts in the field, and over 20 poster presentations provided insight regarding
specific projects and issues of interest to the nanotechnology community. Discussion panels
were held to debate the implications of this information and to begin clarifying gaps in current
knowledge, and four working groups (WGs) were formed to detail these gaps and propose
solutions to address them. The WGs discussed methods and applications specific to the
following areas: (i) technology and benefits, (ii) human health risks, (iii) environmental risks,
27
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and (iv) policy implications. Prior to the conference, WG chairs prepared and circulated topical
white papers, providing a starting point for the detailed WG discussions during the meeting. This
summary paper was initially drafted by the workshop organizers and WG chairs and rapporteurs
during a one-day meeting immediately following the workshop. The conclusions described for
each WG are based on a prioritized list agreed upon during the post-workshop session. These
efforts highlight the significant challenges to professionals in assessing the risks associated with
nanotechnology; such assessments will almost certainly require a highly integrative and adaptive
process of decision-making for nanomaterial risk assessment. The full reports from each WG are
published in Linkov and Steevens (2009), but the concepts discussed and conclusions made are
summarized in the following pages.
Nanotechnology, its Applications, Consumer Products, and Benefits
Nanotechnologies already provide exciting new applications in materials science,
communications, electronics, medicine, energy, and the environment, to name just a few areas.
Nanotechnology represents a platform technology that utilizes the properties of matter that
arise at the nanometer scale. Many nanomaterials are currently being produced (some have
been for many years), such as carbon black, fumed silica, carbon nanotubes, fullerenes, silver
nanoparticles, polymer nanocomposites, dendrimers, metal oxides, organic and inorganic
semiconductors, and nanocatalysts. Nanomaterials are used, for example, in coatings, emulsions,
dispersions and films in automobile components, paper, cosmetics, textiles, and electronic
displays. The unique physicochemical characteristics of nanomaterials, particularly the high
surface-to-volume ratio (influencing solubility, chemical reactivity, and catalytic activity) and
quantum effects (influencing colour, magnetism, hardness, and electronic properties), make
them important drivers of innovation with the potential to benefit the world's entire population.
Nanotechnology can thus be viewed as a cross-sectional and enabling technology.
In addition to enabling a new manufacturing paradigm, another benefit of nanotechnology
would be its potential to help sustain the world's resources. At the workshop, this benefit was
discussed along with the view of Petersen and Egan (2002), who believe that nanotechnology
is a technology which, for the first time in history, holds the promise of providing inexpensive
energy, food, and clean water for everyone on the planet; it could thus also be used in innovative
ways to encourage political stability and responsibility.
Human Health Risk and Implications
The purpose of the Human Health WG was not to re-review extensive literature, but to consider
important findings in the context of a rapid reduction in the uncertainties of the risk assessment
process. Participants discussed mechanisms by which nanomaterials might pose a risk to
human health, including nanosized particles penetrating epithelial barriers at the portal of entry
and inducing oxidative stress. Both of these processes are fundamentally tied to the physical
and chemical nature of the material itself. An important point is that there is no such thing
as a generic "nanomaterial," as factors such as size, shape, chemistry, and solubility all affect
the biological interactions and consequences of exposure to a specific nanoparticle. This is
highlighted by recent reports of impacts from carbon nanotubes (Poland et al., 2008) and nano
silver (Benn and Westerhoff, 2008). The goal that should be kept in sight, similar to a recent
commentary (Hansen et al., 2008), is to facilitate actions taken by regulatory bodies that are
28
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charged with protecting human and environmental health through the reduction in uncertainties
and prioritization of health-based research.
It is neither feasible nor sensible to conduct safety evaluations for all nanomaterials in current or
future production; therefore, a risk assessment paradigm should be flexible and based on current
knowledge of similar materials (Linkov et al., 2008b). Along these lines, people are regularly
exposed to nanosized particles in ambient air (i.e., ultrafine particles) that are derived from
combustion processes. Although there are physicochemical differences between engineered
nanomaterials and ambient ultrafine particles, the large body of toxicological literature regarding
the latter provides a framework for understanding nanomaterial risks. In addition, large-volume
production of nanosized titanium dioxide and carbon black particles has been carried out for
several years, and it is possible that aspects of the risk assessment paradigms for these materials
could be applied more generally to nanomaterials. Useful predictive guidance can also be
gained from the literature regarding interactions of nanosized particles with skin, focusing on
penetration of the stratum corneum and drug delivery. Although this approach focuses mainly
on the respiratory tract and skin, such simplification is reasonable because of the ways in which
humans are likely to be exposed to nanomaterials, namely in occupational and environmental
settings and via consumer products.
Ecological Risk
This WG recognized that traditional risk assessment procedures are inadequate for predicting
the ecological risks associated with the release of nanomaterials. The WG discussed a number
of past case studies where the traditional approach to risk assessment failed to reveal unforeseen
risks. The WG emphasized their belief that the root of the problem lies in an inadequate
application of solid phase chemical principles (e.g., particle size, shape, and functionality) in the
risk assessment of nanomaterials. The group felt strongly that the "solubility" paradigm used
to evaluate the risks associated with inorganic or organic contaminants must be replaced by a
"dispersivity" paradigm for evaluating the risks associated with nanomaterials.
In the opinion of the working group, the pace of development of nanomaterials will exceed the
capacity to conduct adequate risk assessments using current methods and approaches. "New
generation" products will include materials with targeted nanotechnology-biology interactions,
DNA-scaffolded devices, composite materials with biological functions or photovoltaic
properties, materials for new environmental remediation technologies, self-assembling devices,
and polymer-based nanomaterials. These nanomaterials could be available in a variety of
size classes and with different surface functionalizations, probably requiring multiple risk
assessments for each material.
Considerations for Implementation of Manufactured Nanomaterial Policy and Governance
The participants in this working group agreed to focus discussions on policy frameworks, rather
than on the gaps of regulation which have been analyzed elsewhere. Further, the scope of
discussion was narrowed to focus on guidance deemed helpful for developing policies, and on
the information and tools (e.g., databases and web portals) that (i) support the development of
policies by regulators, industry, and others, and (ii) disseminate information to the public and
others.
29
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The WG agreed that while many different policy frameworks for manufactured nanomaterials
have been developed globally (Table 1), a significant lag period remains between the
development of nanotechnologies and the development and implementation of new policies.
While policy initiatives range from voluntary measures to mandatory legislative frameworks,
the WG recognized that governments and industry actually develop very few policies. The
WG agreed that developing regulatory tools is an important gap in the knowledge necessary for
manufactured nanomaterial regulation. Further, the WG agreed with that the starting point for
development of these tools is the set of policies and procedures already developed by regulatory
agencies and industry for traditional industrial materials, e.g., surfactants and other chemical
substances.
Conclusions
Workshop attendees shared basic agreements on policy and risk assessment needs across
countries. Attendees identified the need for a common, standardized taxonomy and terminology
for nanomaterials in which key aspects should include nanomaterial physical and chemical
characteristics, with the view that such a system would facilitate the development of
informational resources (e.g., publications, other documents, and databases) to provide easy
access and sharing across international borders as regulators attempt to understand and assess
the properties of these new materials. Attendees also agreed that assessments covering the
entire lifecycle would best inform and guide risk assessment for engineered nanomaterials
and related nanotechnologies, and that consumer and occupational health protection policies
needed additional development as well. Given the proprietary nature of these rapidly evolving
technologies, and current voluntary reporting requirements, a mechanism is needed for regularly
providing and updating information to scientists and policy makers regarding the safety profiles
and characteristics of these current and emerging nanomaterials. Attendees were very aware that
a serious nanomaterials-related health issue in one nation or region of the world would greatly
promote a negative public perception of nanomaterials risk in every other nation or area.
Simultaneous advances in different disciplines are necessary to advance nanomaterials risk
assessment and risk management. Risk assessment is an interdisciplinary field, but progress
in risk assessment has historically occurred due to advances in individual disciplines. For
example, toxicology has been central to human health risk assessment, and advances in exposure
assessment have been important for environmental risk assessment and risk management.
Nanotechnology, however, ideally involves the planned and coordinated development of
knowledge across fields such as biology, chemistry, materials science, and medicine.
Likewise, a risk assessment of nanomaterials and related technologies requires a lifecycle
approach, meaning a comprehensive assessment of the impact of nanomaterials at different
stages of production, use, and disposal/recycling. The current state of knowledge makes the
identification of major risk drivers challenging. This includes understanding environmental
pathways, fate and transport processes, and reasonably foreseeable exposures. An integrated,
holistic approach is needed to consider an individual's total exposure from relevant environments
expressed in different units across receptor groups. This would lead to risk characterizations
that are systematic and more inclusive, accommodating non-traditional information sources,
measures, and endpoints.
30
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Table 1: Elements of nanomaterial regulation frameworks discussed in each document (criteria are
numbered 1 to 4 under each category; for each document and criterion, • = document discussed the criterion,
• = document mentioned the criterion, and (blank) = document did not address the criterion; adapted from
Linkov and Satterstrom, 2008).
US EPA, 2007
US FDA, 2007
Davies, 2006
ED-DuPont, 2007
Quebec Commission,
2006
TIK Rnvnl Snrictv 1H04
UK DEFRA, 2006
Responsible NanoCode,
2006
EC SCEN1HR, 2007
EC Action Plan, 2005
1RGC, 2005, 2006, 2007
US NNI, 2008
REACH 2006
Science and
Research Aspects
1
•
•
•
•
•
•
•
•
•
2
•
•
•
•
•
•
•
3
•
•
•
•
•
•
•
•
•
4
•
•
•
•
•
•
•
Legal and
Regulatory
Aspects
1
•
•
•
•
•
2
•
•
•
•
•
•
3
•
•
•
4
•
•
Social Engagement
and Partnerships
1
•
•
•
•
•
•
•
•
•
2
•
•
•
•
•
•
•
•
3
•
•
•
•
•
•
•
•
4
•
•
•
•
Leadership and
Governance
1
•
•
•
•
•
2
•
•
•
•
•
•
•
3
•
•
•
•
•
•
4
•
•
•
•
Sub-criteria for the table are as follows:
• Science and Research Aspects
1.
2.
Development of methods for detection /
characterization / data collection
Assessment of environmental fate &
transport / impacts
3. Assessment of toxicology / human health
impacts
4. Assessment of health and environmental
exposure
Legal and Regulatory Aspects
1. Voluntary regulatory and best-practices
measures
2. Information-based regulatory tools (e.g.,
labeling)
3. Economic-based regulatory tools (e.g., tax
or fee for safety testing)
4. Liability-based regulatory tools (e.g.,
penalty for pollution)
Category 3: Social Engagement and
Partnerships
1. Promotion of education and distribution of
information / use of risk communication
tools
2. Use of stakeholder engagement tools
3. Development of partnerships with
academia, industry, public organizations,
provinces, and international regulators
4. Emphasis of ethical conduct
Category 4: Leadership and Governance
1. Transparency in nanotechnology-related
decisions
2. Consideration of benefits of
nanotechnology
3. Adaptive modification of existing or
development of new legislation
4. Consideration of precautionary principle
31
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The attendees agreed that while existing chemical risk assessment and risk management
frameworks may provide a starting point, the unique properties of nanomaterials adds a
significant level of complexity to this process. The goals of this workshop included the
identification of strategies and tools that could currently be implemented to reduce technical
uncertainty and prioritize research to address the immediate needs of the regulatory and
risk assessment communities. Such tools include advanced risk assessment, comprehensive
environmental assessment, risk characterization methods, decision analysis techniques, and other
approaches to help focus research and inform policymakers benefiting the world at large.
References
Benn TM, Westerhoff P (2008) Nanoparticle Silver Released into Water from Commercially
Available Sock Fabrics. Environ Sci Technol 42 (11):4133-4139
Hansen S F, Maynard A, Baun A, and Tickner J A (2008) Late Lessons from Early Warnings for
Nanotechnology. Nature Nanotechnol 3: 444-447
Linkov, I, Steevens, J., Adlakha-Hutcheon, G., Bennett, E., Chappell, M., Colvin, V, Davis,
M, Davis, T., Elder, A., Foss Hansen, S., Hakkinen, P., Hussain, S., Karkan, D., Korenstein, R.,
Lynch, I, Metcalfe, C., Ramadan, A., Satterstrom, F. K. (2008a, in press). Emerging Methods
and Tools for Environmental Risk Assessment, Decision-Making, and Policy for Nanomaterials:
Summary of NATO Advanced Research Workshop. J. of Nanoparticle Research.
Linkov, I, Satterstrom, K., Corey, L. (2008b). Nanotoxicology and Nanomedicine: Making Hard
Decisions. Nanomedicine 4:167-171
Linkov, L, Steevens, J. (2009, in press). Nanotechnology: Risks and Benefits. Springer,
Amsterdam.
Petersen, J.L., and Egan, D.M. (2002) Small Security: Nanotechnology And Future Defense,
Defense Horizons 8, 1-6.
Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee
W, Donaldson K (2008) Carbon Nanotubes Introduced into the Abdominal Cavity of Mice Show
Asbestos-like Pathogenicity in a Pilot Study. Nat Nanotechnol 3(7): 423-8
32
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The Toxicology of Nanomaterials: Where Do We Go from Here?
Martin Philbert, University of Michigan, USA
Dr. Philbert opened the lunch plenary session by stating that he was going to challenge his audi-
ence with controversial statements that would point to areas for further consideration. The first
statement, "There is no such thing as nanotechnology," was followed by a reprise of earlier and
existing technologies. Early incarnations of nanotechnology were used in the defense arena for
reinforcing at-risk joints, for example, in the enhanced soldier program. Another earlier applica-
tion was electronics, for example, a flash drive that can store 24 gigabytes of information. Cur-
rent applications of nanotechnology include sunscreen and zinc oxide coated windows. Language
implying that nanotechnology is on the horizon is inaccurate.
There are more than 800 self-identified products containing nanomaterials. Here Dr. Philbert
noted an interesting dichotomy. Some manufacturers use the term "nanomaterials" in labeling
their products, even though the products do not contain them. Other manufacturers have aban-
doned the term, even when their products contain them. They opt, instead, for terms such as "ul-
trafine" or "microfine" in an attempt to distance themselves from nanomaterials and any potential
public backlash. This raises the issue of perceived risk versus actual risk. Although concerns are
expressed for the impacts of nanotechnology on toxicology and risk assessment, greater concern
may be evident if nanotechnology is used to circumvent some protective biological processes, for
example, to modify defective sperm to enable fertilization.
The statement "There is no such thing as nanotoxicology" could be called, in legal parlance,
an excited utterance against interest. Nanotoxicology could be termed a loose constellation of
poorly coordinated activities, with a lack of coherent, meaningful experimental standards. Dos-
ing metrics are only clear for particles of regular geometry. However, zinc oxide particles have
been shown to have widely disparate geometries, and each geometry may have a different effect.
CAS RNs are not useful for materials with many different geometries. An evolutionary taxono-
my is required for nanomaterials.
Much current work in the field may be considered preliminary, and more work is required on the
metrics of exposure: duration, frequency, route, and magnitude. It is not known whether absorp-
tion, distribution, metabolism, and excretion are the same for all species. Complicating this is the
difficulty of labeling nanomaterials for identification without changing their surface characteris-
tics. How then does one track nanomaterials in vitro and in vivo?
The current high level doses used for mechanistic studies should be avoided, as these swamp
any effects that might be seen at concentrations that could be encountered in the environment.
Critical information can be missed, and undue emphasis could be given to effects that would not
be seen at environmental concentrations. Acute studies have a place, but time will be critical for
nanotoxicity studies, and even two-year rodent bioassays may be insufficient for some materials.
Positive controls are essential and should be reported for all studies in addition to new findings.
33
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An issue that requires attention is the establishment of de minimus standards for toxicological
studies that can be used in risk assessment. It should be recognized that not all biological change
is harmful, and between a normal state and pathology there is a gap that can be regarded as adap-
tation. A high quality database for all negative data could provide considerable cost savings.
Risk management is required for nanomaterials, and, while risk cannot be eliminated, the con-
cept that risk can be managed needs to be communicated.
Conference Questions and Answers
Question:
There may be no such thing as nanotechnology, but what about nano-epidemiology? An exposure
registry is required for people exposed to nanomaterials in the workplace to establish risks.
Answer:
The prefix "nano" gives these materials unwarranted importance, and the issue needs to be
placed in contextDmore are killed by cars than nanomaterials. Remember that nanoscale materi-
als are ambient and are present in filtered drinking water.
Comment:
We cannot make the statement that nobody has been killed by nanomaterials; we do not know
enough.
34
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Reactive Oxygen Species Related Microbial Growth Inhibition
By Silver Nanoparticles
Okkyoung Choi, Department of Civil and Environmental Engineering, University of Missouri,
Columbia, Missouri, U.S.A.
Rao Y. Surampalli, U.S. Environmental Protection Agency, Kansas City, Kansas, U.S.A.
ZhiqiangHu, Department of Civil and Environmental Engineering, University of Missouri,
Columbia, Missouri, U.S.A.
Abstract
Nanotechnology enhanced consumer products containing silver nanoparticles are emerging
but the fate and effect of silver nanoparticles in the environment remain largely unknown. To
evaluate the toxicity of silver nanoparticles to microorganisms involved in wastewater treatment,
we measured and compared specific oxygen uptake rates of nitrifying bacteria before and after
their exposure to silver nanoparticles. The active oxygen species in the presence or absence of
silver was determined in parallel by using ROS-sensitive fluorescence dyes. Silver nanoparticles
significantly inhibited the growth of nitrifying bacteria at silver concentrations less than 1 mg/L.
The inhibition was well correlated with the intracellular ROS concentrations and more ROS
was generated when it was exposed to silver nanoparticles, suggesting that the microbial growth
inhibition by silver nanoparticles is related to ROS generation in the cell.
Introduction
Silver nanoparticles are used in many consumer products because of their strong antimicrobial
activity (Benn and Westerhoff 2008; Mueller and Nowack 2008). We recently showed that
at Img/L Ag, silver nanoparticles (average size, 14 ± 6 nm) significantly inhibited nitrifying
bacterial growth (Choi et al. 2008). Although the mode of antimicrobial activity is still not clear,
it is believed that silver species may induce to generate intracellular reactive oxygen species
that can damage protein, DNA and membrane (Sondi and Salopek-Sondi 2004; Hussain et al.
2005; Lok et al. 2006). The reactive oxygen species including singlet oxygen (:O2), superoxide
(O2~), hydrogen peroxide (H2O2), and hydroxyl radical (OH-) are generated under oxygen-
limited conditions or in the presence of environmental toxicants. For instance, semiconductive
nanoparticles such as TiO2 produce photocatalytic ROS at near UV. Silver ion was also reported
to induce intracellular ROS (Inoue et al. 2002). The accumulation of high level intracellular
ROS can damage cellular components and disrupt cell functions. To help elucidate the inhibition
mechanism, we measured nitrification inhibition by various forms of silver including silver
nanoparticles, silver ions, and silver chloride colloids to evaluate the relationship between
silver concentrations and ROS production. Nitrification involving ammonia oxidation and
nitrite oxidation by typically nitrifying bacteria is important in wastewater treatment and global
nitrogen cycling. Nitrifying bacteria were chosen as model microbes because of sensitivity to
environmental change like as pH, temperature, and several toxicants (Blum and Speece 1991).
The quantitative description of the relationship between ROS and nanosilver toxicity will
35
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therefore help better understand the antimicrobial mechanism of silver nanoparticles.
Methods
Autotrophic nitrifying bacteria were cultivated in a continuously stirred tank reactor (14L)
operated at solids retention time (SRT) of 20d and hydraulic retention time (HRT) of Id. Silver
nanoparticles were made from reduction of silver ion (silver nitrate) with sodium borohydride
in 0.06 % (wt) PVA (polyvinyl alcohol) solution used to control silver particle size (Choi et al.
2008). The average size of the synthesized silver nanoparticles was 15 nm.
The toxicity of silver nanoparticles to nitrification was investigated by measuring specific oxygen
uptake rate (SOUR) after ammonium (10 mg-N/L) injection to aliquots of nitrifying cultures
(60 mL) in the presence or absence of silver nanoparticles in a closed respirometric vessel. The
degree of inhibition (%) was calculated based on the relatively decrease of SOUR in the presence
silver (Equation 1).
r> fj i -Z.V fo/\ mn wthoutnA - SOURwithnA
Degree of Inhibition (%) = 1 00 x - - - — (Equation 1)
SOURwithoutnAg
To determine intracellular ROS concentrations, aliquots of nitrifying biomass suspensions
were removed from the nitrifying bioreactor, centrifuged and resuspended in a loading buffer
solution containing 10 ^M H2DCFDA (dichlorodihydrofluorescein diacetate, Invitrogen, OR,
USA) for 30 minutes. After the centrifugation, the pellet cells were inoculated with prewarmed
growth medium, amended with nanosilver (average size: 15 nm) or silver bulk species (for
comparison) at predetermined concentrations, and plated into 96-well plates. The fluorescence
of the cells from each well was measured with 485 nm excitation and 535 nm emission filters
using a microreader (VICTOR3, Perkin Elmer, Shelton, USA). Fluorescence data were taken
automatically after 30 min incubation. Hydrogen peroxide (30%, Fisher Scientific) was used as a
standard for ROS measurements and intracellular ROS concentrations were normalized in H2O2
unit.
To determine photocatalytic ROS concentrations, APF (3'-(p-aminophenyl) fluorescein,
Invitrogen, OR, USA) was used in cell free condition to measure ROS generated by nanosilver
itself and compared ROS generation before and after exposure to fluorescent lab light for 30
minutes. The APF was added at a final concentration of 5 ^M and the photocatalytic ROS that is
mainly related to OH production was determined in mole units of OC1" in the solution.
Results
All forms of silver tested inhibited nitrification. At the same silver concentrations, silver
nanoparticles presented the highest degree of inhibition (Figure 1). As the silver concentrations
increased, the inhibition appeared to follow a saturation curve with R2 range from 0.91 to 0.97.
The concentrations of silver nanoparticle, silver chloride, and silver ion causing 50% inhibition
were determined to be 0.14 mg/L, 0.25 mg/L, and 0.27 mg/L.
36
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ioo -
\o
0s
^ 80 -
o
1 60-
^G
1 4°
O
'£ 20 -
£
/-v —
I
1
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1
nano silver
Ag ion
AgCl
Figure 1. The toxicity of silver to nitrifying bacteria at Img/L Ag. Error bars indicate one
standard deviation.
The ROS concentrations (normalized in H2O2 concentration) increased when the nitrifying
biomass suspensions were exposed to silver nanoparticles (Figure 2). Inhibition by Ag
nanoparticles as well as other forms of silver (AgCl colloids and Ag+ ions) correlated well
with the intracellular ROS concentrations (Figure 3) by using a saturation-type model. Poor
correlation, however, was noticed between the observed inhibition and the photocatalytic ROS
concentrations. Therefore, photocatalytic ROS concentrations were not a good predictor of
inhibition by Ag nanoparticles.
Discussion
Nanosilver toxicity was well correlated with intracellular ROS concentrations. All forms of silver
including Ag+ion, silver chloride and nanosilver induced intracellular ROS but the patterns of
their correlations with inhibition were different. Ag nanoparticles appeared to be more toxic than
Ag+ ions at the same level of intracellular ROS or the same total Ag concentrations, suggesting
that factors other than ROS are also important in determining nanosilver toxicity.
Nanoparticles are very mobile and active because of their small size. Recently, it has been
shown that gold nanoparticles coated with negative charged and hydrophobic ligands could
penetrate into the cell membrane without disruption (Verma et al. 2008). It was also suggested
that nanoparticles could be more toxic via a Trojan-horse type mechanism (Limbach et al.
2007). Silver nanoparticles induced more ROS production and higher toxicity at the same
Ag concentration than that of silver bulk species. It is therefore possible that these nano-size
particles may have a different transport mechanism from that of silver ion to enter or interact
with the cell.
37
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30 1
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.2 25
| 0 20 -
(U >yS
§ 5 ^ "
£ ^ 10 -
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D Ag ion D AgCl • nanosilver
Tl
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l~l~i!"!!l 1
Ti 1
J!i!i T T
l
E
hi
I
f
J_
i
1
fl
±1
E
i
iiii
Illl
0 0.05 0.1 0.2 0.4 0.6 0.8 1
Silver concentration (mg/L)
Figure 2. Changes of intracellular ROS concentrations in nitrifying bacteria exposed to different
forms of silver. ROS concentration was measured in H2O2 units. Error bars indicate one standard
deviation.
100
80
60
40
20
0
o
x>
c
o
00
(L)
Q
0 5 10 15 20
Intracellular ROS, (DM in H2O2)
Figure 3. ROS-related nanosilver toxicity to nitrifying bacteria. The ROS concentrations were
measured in H2O2 units. Error bars indicate one standard deviation. R square value of 0.86 was
calculated using a saturated model.
Conclusions
Silver nanoparticle was more toxic to nitrifying bacteria than silver ion or silver chloride colloid.
At the same silver concentrations, silver nanoparticles tended to generate more ROS than the
bulk silver species. The toxicity of silver nanoparticles was correlated with the intracellular ROS
concentrations.
38
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References
Benn, T. M. and P. Westerhoff(2008). "Nanoparticle Silver Released into Water from
Commercially Available Sock Fabrics." Environ. Sci. Technol. 42(11): 4133-4139.
Blum, D. J. W. and R. E. Speece (1991). "A database of chemical toxicity to environmental
bacteria and its use in interspecies comparisons and correlations." Journal of Water Pollution
Control Federation 63: 198-207.
Choi, O., K. K. Deng, N.-J. Kim, L. Ross Jr, R. Y. Surampalli and Z. Hu (2008). "The inhibitory
effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth."
Water Res. 42(12): 3066-3074.
Hussain, S. M., K. L. Hess, J. M. Gearhart, K. T. Geiss and J. J. Schlager (2005). "In vitro
toxicity of nanoparticles in BRL 3Arat liver cells." Toxicol. Vitro. 19(7): 975-983.
Inoue, Y, M. Hoshino, H. Takahashi, T. Noguchi, T. Murata, Y. Kanzaki, H. Hamashima and M.
Sasatsu (2002). "Bactericidal activity of Ag-zeolite mediated by reactive oxygen species under
aerated conditions." J. Inorg. Biochem. 92(1): 37-42.
Limbach, L. K., P. Wick, P. Manser, R. N. Grass, A. Bruinink and W. J. Stark (2007).
"Exposure of Engineered Nanoparticles to Human Lung Epithelial Cells: Influence of Chemical
Composition and Catalytic Activity on Oxidative Stress." Environ. Sci. Technol. 41(11): 4158-
4163.
Lok, C. N., C. M. Ho, R. Chen, Q. Y. He, W. Y. Yu, H. Z. Sun, P. K. H. Tarn, J. F. Chiu and C. M.
Che (2006). "Proteomic analysis of the mode of antibacterial action of silver nanoparticles." J.
ProteomeRes. 5(4): 916-924.
Mueller, N. C. and B. Nowack (2008). "Exposure modeling of engineered nanoparticles in the
environment." Environ. Sci. Technol.
Sondi, I. and B. Salopek-Sondi (2004). "Silver nanoparticles as antimicrobial agent: a case study
on E. coli as a model for Gram-negative bacteria." J. Colloid Interface Sci. 275(1): 177-182.
Verma, A., O. Uzun, Y. Hu, Y. Hu, H.-S. Han, N. Watson, S. Chen, D. J. Irvine and F. Stellacci
(2008). "Surface-structure-regulated cell-membrane penetration by monolayer-protected
nanoparticles." Nat Mater 7(7): 588-595.
Conference Questions and Answers
Question:
Do you have evidence that silver enters bacteria?
Answer:
We did not do that experiment, but previous publications of TM (transverse magnetic) studies
indicate that particle size <10 nm enters the cell.
39
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Question:
How did you make sure that the toxicity effect was due to the nanoparticles rather than silver
ions left in solution?
Answer:
After preparing the nanoparticles, we used an ion-specific electrode to determine the
concentration of remaining silver ions, and the concentration was negligible.
Question:
If there is an uptake difference in the rate nanosilver and silver ions cross the cell membrane, the
toxicity of nanosilver could be underestimated if it moves more slowly into the interior of the
cell. Did you perform any enzyme inhibition studies to look at this?
Answer:
Yes. Nanosilver inhibited AMO (ammonia monooxygenase) located on the cell membrane more
than HAO (hydroxylamine oxidoreductase) located between the periplasma and cell membrane.
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Identification of Biomarkers of Exposure to Metal-based Nanoparticles
through Gene Expression Profiling Using Daphnia magna MicroArrays
James Lazorchak
U.S. EPA, National Exposure Research Laboratory, U.S.A.
As new emerging contaminants are developed and gradually replace past environmental
pollutants, we are faced with an unprecedented challenge. We are currently able to confront
the issue of emerging contaminants before they become an environmental problem and develop
strategies to mitigate the risk that may be associated with their release into the environment.
One area where attention should be directed is the rapidly growing field of nanotechnology.
The emergence of genomic techniques has presented many exciting new possibilities in
ecotoxicology including the ability to classify chemicals based on their expression pattern or
fingerprint. Previous research focused on three well-characterized metal pollutants: copper,
cadmium, and zinc, and the invertebrate indicator species, Daphnia magna. Using a custom
D. magna cDNA microarray containing approximately 5000 cDNA clones, which identified
distinct expression fingerprints in response to sublethal copper, cadmium, and zinc exposures
and validated several genes as biomarkers of exposure (Poynton et al., 2007). The goal of this
current study was aimed at developing biomarkers of exposure that can be applied to study the
bioavailability and environmental exposure of metal based nanoparticles. The questions we
wanted to answer were: 1. Can we identify biological indicators of exposure to nanoparticles
using a similar approach as Poynton et al. 2007? 2. Can we distinguish between the particle
induced effects and chemical composition effects through comparative gene expression
profiling? 3. What exposure time produces the most robust and specific gene expression pattern?
Conference Questions and Answers
Question:
What were the genes shown that were different, and what were they?
Answer:
We do not know; they were just coded with in-house descriptors, and we have not identified their
particular functions. However, a Daphnia genome chip is now under development.
Question:
Can you speculate how genes being switched on could be used in a regulatory framework?
Answer:
Some literature suggests a "No Observable Transcription Expression Level" (NOTEL). The
NOTEL could be used as a regulatory driver, or a "No Observable Protein Expression Level"
(NOPEL). Once proteins are turned on there could be a cascade effect with adverse effects.
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Using Microarrays to Test the Effects of Acute Exposure to Multiwalled
Carbon Nanotubes (MWCNTs) on Gene Expression in Fathead Minnows
(Pimephales promelas)
Barbara J. Carter, and Heather R. Hammers
EcoArray, Inc., Gainesville, Florida, U.S.A.
Robert J. Griffitt, and David S. Barber
Center for Environmental and Human Toxicology, University of Florida
Gainesville, Florida, U.S.A.
Abstract
There are increasing concerns regarding the release of nanomaterials, in general, and nanotubes,
in particular, into the environment and their potential effects on fish and wildlife. Researchers
are only recently evaluating the potential for exposure and adverse effects of nanoparticles on
fish and wildlife through toxicological testing, and data are extremely limited. Under an EPA
Phase 1 SBIR grant, we have been given the opportunity to use state-of-the-art oligonucleotide
microarrays to examine the toxicity and gene expression patterns in fathead minnows
(Pimephalespromelas) exposed to two different sizes (< 8 nm and 50-80 nm outer diameter) of
multiwalled carbon nanotubes (MWCNTs).
We exposed adult female fathead minnows for 48-hours to three concentrations of each nanotube
(0.1 mg/L, 0.3 mg/L, and 1.0 mg/L ), a (water) control, and a carrier control (1.2 mg/LNaDDBS
for the 8 nm MWCNT, 2.2 mg/L for the 80 nm) under aerated static renewal conditions. After
exposure, we harvested gill, gonad and liver, and then measured gene expression using a fully
annotated 15,208 gene oligonucleotide microarray. We used GeneSpring (version 9.0.3) to
analyze the data, looking for differentially regulated genes (p> 0.05 and fold change >2).
At these concentrations, we found no toxicity and observed no gross organ abnormalities. From
the microarray analysis, we did observe a substantial transcriptional response to exposure in each
of the tissues, with over 400 genes exhibiting altered expression in each of the tissues. However,
there was very little commonality in the transcriptional response of these three tissues to a given
MWCNT. The majority of the differentially expressed genes from each tissue are involved in the
biological process category 0006xxx, regulating transport, transcription and protein functions.
In addition, the response of a single tissue (gill) to both sizes of nanotubes showed as many
differences as similarities.
Introduction
Nanoparticles have increased in manufacturing, industry, and commercial products over the past
two decades. However, assessing the potential effects of nanoparticles on human health is not
an easy task, as the properties of nanoparticles depend not only on the size of the particle, but
also on the structure, microstructure, and surface properties (coating) (Moore 2006, Yin et al.
2005, Burleson et al. 2004). Invariably, industrial products and wastes, including some aerosols,
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tend to end up in waterways despite safeguards; it is inevitable that nanoscale products and by-
products will also enter aquatic environments as nanotechnology industries scale up production
(Moore 2006, Borm et al. 2006). Thus, uptake of nanoparticles into the aquatic biota is a major
concern.
However, the evaluation of the potential for exposure and adverse effects of nanoparticles on
fish and wildlife has begun to be addressed only recently through toxicological testing, and
data are extremely limited. Concern about environmental contaminants that adversely affect
health, development and reproduction of exposed wildlife has led to the development of both
specific in vitro and in vivo assays to test for these effects. Gene microarrays integrate in vivo
exposures with mechanistic outcomes. Using this technology, we can test thousands of genes at
one time with mRNAs isolated from tissues of exposed animals. Under an EPA Phase 1 SBIR
grant (EP-D-08-026), we have been given the opportunity to use state-of-the-art oligonucleotide
microarrays to examine gene expression patterns in female fathead minnows (FHM, Pimephales
promelas) exposed to two different sizes of multiwalled carbon nanotubes (MWCNTs).
Materials and Methods
Nanotube suspension. We purchased two dry nanotube powder samples, <8 nm and 50-80 nm
outer diameter (O.D.), from Cheaptubes (www.cheaptubes.com, Brattleboro, VT). We suspended
each sample in water containing lOmg/ml sodium dodecylbenzene sulfonate (NaDDBS), at
a nominal concentration of 10 mg/ml. We bath-sonicated the nanotubes suspensions for four
hours, and then centrifuged at 600 rcf for 30 minutes. We retained the supernatant, and then
calculated that between 35-50% of the nanotubes remained in suspension, approximately what
was expected according to the method described by Attal et al. (2006).
Nanotube exposures/tissue collection. We performed all exposures as aerated 48 hour static
bioassays in 2L beakers, with 4 replicate beakers per concentration, and 3 adult female fathead
minnows per beaker. For each nanotube, we used five different exposure conditions: (water)
control, carrier control (1.2 mg/L NaDDBS for the 8 nm MWCNT, 2.2 mg/L for the 80 nm), 0.1
mg/L, 0.3 mg/L, and 1.0 mg/L MWCNTs. After 48 hours, we euthanized two fish from each
beaker by immersing them in 100 mg/L MS-222 (Tricaine) buffered with 10 mg/L NaHCO3
for five minutes. We opened the carcass by ventral incision, removed the liver (partial), ovary
(left horn), and gill (left), and immediately placed the tissues in 1 ml RNALater (Ambion, Inc.,
Austin, TX), storing samples at -20°C.
Hybridization of microarrays. We isolated total RNA using the RNEasy Plus Mini Kit (Qiagen,
Valencia, CA) following the manufacturer's protocol. We determined the quality of the RNA
by running a 1.0 uL aliquot on a 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara,
CA). As this was a reference design experiment, we labeled the exposed samples with cyanine
(Cy) 5-CTP (Perkin Elmer, Wellesley, MA, USA) and the reference sample with Cy-3-CTP We
labeled, hybridized, and washed the arrays according to Agilent's Two-Color Microarray-Based
Gene Expression Analysis (Quick Amp labeling) Protocol (version 5.7, March 2008). The FHM
microarrays used in this experiment were developed by EcoArray and manufactured by Agilent
Technologies, Inc. Each array contains probes for 15,208 annotated gene sequences; there are
8 arrays per glass slide. We scanned the slides with an Agilent DNA microarray scanner, which
processes the raw images and converts the data into .txt files using Agilent's Feature Extraction
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Software, Version 9.5.3.
Normalization of microarray data and statistical analysis. The resultant data was analyzed using
Gene Spring version 9.0.3 (Agilent Technologies, Inc., Santa Clara, CA). For this project we
considered the two sizes of nanotubes as different experiments, and we analyzed the tissues,
gill, liver, and ovary, within each size category independent of each other. We accepted as
differentially regulated all genes with a P-value of 0.05 or lower (statistically significant) and a
fold change >2.0.
Results
We observed no mortality in adult female fathead minnows exposed for 48 hours to dispersed
suspensions of two sizes of MWCNT at concentrations up to 1 mg/L. At necropsy, we did not
note any gross pathology in any of the organs examined.
We used gene expression analysis to investigate the response of gill, liver, and ovary to each
nanotube. We observed a substantial transcriptional response to exposure in each of the tissues,
with over 400 genes exhibiting altered expression in each of the tissues (Figures 1 and 2).
Figure 1. Differential gene expression in female
fathead minnows exposed to 1.0 mg/L<8 nm O.D.
MWCNTs compared to controls.
Liver
Ovary
Figure 2, Differential gene expression in female
fathead minnows exposed to 1.0 mg/L>50 nm O.D.
MWCNTs compared to controls.
Liver
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However, there was very little commonality in the transcript!onal response of these three tissues
to a given MWCNT. The majority of the differentially expressed genes from each tissue are
involved in the biological process category 0006xxx, regulating transport, transcription and
protein functions (Figure 3 and Table 1).
To determine whether MWCNT with different diameters produced different responses, we
compared the transcriptional response in the gill to MWCNT of <8nm O.D. and 50-80 nm O.D.
Our analysis found 60 genes whose expression was significantly altered that were common to
both exposures. Analysis of this subset of genes reveals that some genes show very similar
regulation, however there are a number which reveal dramatically different responses between
the two treatments (e.g.,, AF236669 which is active in transport, AL929504 and AC132256).
Genes which show similar responses between the two treatments are involved in, among other
things, transcription, cell adhesion and protein transport.
Discussion
The results of this study demonstrate that MWCNT which are well dispersed in water are not
acutely lethal to fathead minnows at concentrations up to 1 mg/L. Toxicity studies for MWCNTs
Figure 3. GO (Biological Process) pathways in gill after
exposure to 1.0 mg/L >50 nm MWCNTs
GO:001xxxx
GO:0007xxx
GO: 0000004 (UNK)
GO:0005xxx
GO:0006xxx
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Table 1. Biological processes represented in the gill under GO category 0006xxx. While this
specific list is from gill tissue exposed to >50 nm OD MWCNTs, genes differentially expressed
in the liver and ovary are also predominantly from GO category 0006xxx. Gill = 34%, liver =
35%, ovary = 26%. The second highest category is GO: 0000004, biological process unknown.
GO: Biological process
GO:0006071; glycerol metabolism
GO:0006118; electron transport
GO:0006122; mitochondrial electron transport, ubiquinol to cytochrome c
GO:0006139; nucleobase, nucleoside, nucleotide and nucleic acid metabolism
GO:0006259; DNA metabolism
GO:0006268; DNA unwinding during replication
GO:0006281; DNA repair
GO:0006289; nucleotide-excision repair
GO:0006306; DNA methylation
GO:0006334; nucleosome assembly
GO:0006350; transcription
GO:0006355; regulation of transcription, DNA-dependent
GO:0006366; transcription from RNA polymerase II promoter
GO:0006398; histone mRNA 3'-end processing
GO:0006405; RNA export from nucleus
GO:0006412; protein biosynthesis
GO:0006429; leucyl-tRNA aminoacylation
GO:0006457; protein folding
GO:0006464; protein modification
GO:0006468; protein amino acid phosphorylation
GO:0006508; proteolysis
GO:0006605; protein targeting
GO:0006629; lipid metabolism
GO:0006810; transport
GO:0006811; ion transport
GO:0006836; neurotransmitter transport
GO:0006865; amino acid transport
GO:0006915; apoptosis
GO:0006916; anti-apoptosis
GO:0006928; cell motility
GO:0006936; muscle contraction
GO:0006937; regulation of muscle contraction
GO:0006955; immune response
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20
Figure 4. Gill: 60 genes common to both exposures
D<8nm •>50nm
in aqueous environments are scarce, yet we found our lack of toxicity consistent with previous
work on other nanoparticles (e.g., Zhu et al., 2008; Blaise et al., 2008). It is important to note
that without use of a dispersing agent, suspensions of MWCNT rapidly aggregated and settled
out of the water column, resulting in little or no exposure of pelagic organisms. The dispersal
agent itself can cause toxicity. This highlights the importance of testing the form of the
nanomaterials that will actually be released into the environment in order to accurately assess
risk.
Exposure to MWCNT produced significant transcriptional effects on gill, liver, and ovary of
adult females. The responses in these organs were quite different, suggesting that the tissues are
responding differently, though it is unclear whether MWCNT were absorbed and reached internal
organs or if responses of liver and ovary are secondary to physiological stress due to effects on
gill. We are currently performing histopathological analysis of several tissues to help answer
these questions.
Comparing the response of the gill to different size MWCNT suggests that nanotubes of different
diameters can cause different responses. The biggest differences between the two types of tubes
were in genes involved in transport of calcium ions or cations; genes integral to transmembrane
movement of substances. The differences may be due to size, but the properties of nanoparticles
depend not only on the size of the particle, but also on the structure, microstructure, and surface
properties (coatings) (Moore 2006, Yin et al. 2005, Burleson et al. 2004).
Conclusions
The results of this study suggest that while acute exposure to MWCNTs is not toxic to fathead
minnows at concentrations up to 1.0 mg/L, such exposure does result in differential gene
expression in gill, liver and ovary tissue. Many of the genes that do change are involved
in regulating transport, transcription and protein functions. However, this is a preliminary
examination of the data, and we will be undertaking a much more detailed analysis. We intend to
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analyze the effects of the NaDDBS solvent, the 0.3 mg/L concentration, on the differential gene
expression. In addition, we are analyzing stained tissue sections for histopathologies, and will
correlate those findings with the expression data across tissues.
References
Attal, S. R. Thiruvengadathan, and O. Regev. (2006). "Determination of the concentration
of single-walled carbon nanotubes in aqueous dispersions using UV-visible absorption
spectroscopy." Anal Che. 78 (23) 8098-8104.
Blaise, C., F. Gagne, J. F. Ferard, and P. Eullaffroy . (2008). "Ecotoxicity of selected nano-
materials to aquatic organisms." Environ Toxicol. Jun 4. 23(5), 591-8.
Borm, P J.A., D. Robbins, S. Haubold, T. Kuhlbusch, H. Fissan, K. Donaldson, R. Schins, V.
Stone, W. Kreyling, J. Lademann, J. Krutmann, D. Warheit and E. Oberdorster. (2006). "The
potential risks of nanomaterials: a review carried out for the ECETOC." Particle and Fibre
Toxicology 3 (35 pages).
Burleson, D.J., M.D. Driessen, and R.L. Penn. (2004). "On the characterization of
environmental nanoparticles." Environ Sci Heath A Tox Hazard Subst Environ Eng 39 (10),
2707-53.
Moore, M. N. (2006). "Do nanoparticles present ecotoxicological risks for health of the aquatic
environment?" Environment International 32, 487-976.
Yin, H., H.P Too, and G. M. Chow. (2005). "The effects of particle size and surface coating on
the cytotoxicity of nickel ferrite." Biomaterials 26 (29), 5818-26.
Zhu, X., L.Zhu, Z. Duan, R. Qi, Y. Li, and Y Lang. ( 2008). "Comparative toxicity of several
metal oxide nanoparticle aqueous suspensions to Zebrafish (Danio rerio) early developmental
stage." J Environ Sci Health A Tox Hazard Subst Environ Eng. 43 (3), 278-84.
Conference Questions and Answers
Question:
You showed morphological changes within a section of gill. Did you show any effect on oxygen
transport?
Answer:
We think it does have an effect, but we have not gotten that far yet. We can relate the changes in
the gill to other studies (rat inhalation) that have indicated toxic effects in the lung.
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Integrative Strategies to Understand Nanomaterial-Biological Interactions
Stacey L. Harper, Oregon State University and Oregon Nanoscience and
Microtechnologies Institute, Corvallis, Oregon, U.S.A.
Jim Hutchison, and Bettye L. S. Maddux, Oregon Nanoscience and
Microtechnologies Institute, Corvallis, Oregon, U.S.A., and
University of Oregon, Eugene, Oregon, U.S.A.
Robert L. Tanguay, Oregon State University, Oregon Nanoscience and
Microtechnologies Institute, Corvallis, Oregon, U.S.A., and Environmental
Health Sciences Center, Corvallis, Oregon, U.S.A.
Abstract
The rapid rate of discovery and development in nanotechnology will undoubtedly increase
the potential for both human and environmental exposures to novel nanomaterials. While
numerous applications promise benefit to human health or the environment, the potential
health and environmental risks associated with the unique properties of nanoscale materials
are unknown and may lead to unintended health and safety consequences. The current gap
in nanoparticle toxicological data dictates the need to develop rapid, relevant and efficient
testing strategies to assess these emerging materials of concern prior to large-scale exposures.
Here we present a novel approach that utilizes a dynamic whole animal (in vivo) assay to
reveal whether a nanomaterial produces adverse responses at multiple levels of biological
organization (i.e. molecular, cellular, systems, organismal). Early developmental life stages
are often uniquely sensitive to environmental insult, due in part to the enormous changes in
cellular differentiation, proliferation and migration required to form the required cell types,
tissues and organs. Molecular signaling underlies all of these processes. Most toxic responses
result from disruption of proper molecular signaling, thus, early developmental life stages are
perhaps the ideal life stage to determine if chemicals or nanomaterials are toxic. Therefore,
the embryonic zebrafish model was chosen to investigate nanomaterial biological activity
and toxic potential. Investigations using this model system can reveal subtle interactions at
multiple levels of biological organization, thus we have developed an EZ (embryonic zebrafish)
metric for nanomaterial toxicity (EZ-metric) that takes into account the types and frequency of
sublethal effects in addition to overt mortality. The EZ-metric was used to compare morbidity
and mortality elicited from exposure to over 100 novel engineered nanomaterials using the
Nanomaterial-Biological Interactions (NBI) knowledgebase at Oregon State University.
Introduction
Scientists and engineers, whether in industry, government or academia, have a common need
to understand how nanomaterials interact with biological systems. The importance of this
information is most obvious for applications in biomedicine such as targeted drug delivery,
novel therapies using nanomaterials as agents, prosthetics, regenerative medicine, diagnostics
and imaging. Information gained at the interface of nanomaterials with biological systems
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can be used to guide materials design (e.g., biomimicry), optimize synthesis processes (e.g.,
nanomanufacturing architecture), and integrate 'soft' and 'hard' platforms (e.g., bionics).
However, consideration is now being given to the environmental and health implications of
nanotechnology so information on how and why nanomaterials may interact with, and potentially
alter, biological processes is of critical importance. Ultimately, this foundational knowledge
can be used to direct the safe development of future nanomaterials and nanotechnologies and
provide input into the regulatory process, two strategies that could improve public perception of
nanotechnology.
Investigations to understand nanomaterial-biological interactions are fraught with complexities.
First and foremost would be the lack of relevant baseline data on nanomaterial characteristics,
biological effects, and determinants of the unique properties that are so desired. There are few
standard methodologies and materials that are specific for nanotechnology and nanomaterials.
Recent studies conducted by the Nanotechnology Characterization Laboratory (NCL)
indicate that adaptations of standard protocols in toxicology may be necessary for the correct
interpretation of test results [1]. Additional complexity is derived from the sheer diversity of
nanomaterials that are being/or will be tested in a broad array of animal systems and cell based
assays. Obtaining comprehensive knowledge of nanomaterial-biological interactions and
responses will likely require consideration and inclusion of the entire body of data produced from
global efforts in this research area[2]. Thus, the Oregon Nanoscience and Microtechnologies
Institute (ONAMI) is working jointly with Oregon State University to develop a knowledgebase
of Nanomaterial-Biological Interactions (NBI) to address such critical infrastructure needs for
nanotechnology.
The goal of ONAMI's Safer Nanomaterials and Nanomanufacturing Initiative (SNNI) is to
develop new nanomaterials and nanomanufacturing approaches that offer a high level of
performance, yet pose minimal harm to human health or the environment. The SNNI research
paradigm is a testing-redesign loop that utilizes multiple whole-animal systems (e.g., zebrafish,
water flea, fruit fly) to rapidly evaluate the biological responses to nanomaterial exposure[2].
One such rapid testing platform is the embryonic zebrafish assay. In this assay, developing
zebrafish embryos serve as an integrated sensing and amplification system that is sensitive to
perturbation. This experimental platform offers the power of whole-animal investigations (e.g.,
intact organism, functional homeostatic feedback mechanisms and intercellular signaling) with
the convenience of cell culture (e.g., cost- and time-efficient, 96-well plate exposure chambers,
minimal infrastructure, small quantities of nanomaterial solutions required). Here we present
data on a variety of nanomaterials that were testing using this novel approach. Oftentimes, we
are able to test a series of materials that differ in only one aspect, for instance, size or surface
groups. Such an approach is preferred when the aim is to develop design rules for benign
nanomaterials.
Methods
We used the embryonic zebrafish assay to perform screening-level toxicity evaluations of carbon
fullerenes, carbon nanotubes, nanoparticulate metal oxides, nanoscale polystyrene spheres, CdSe
Quantum Dots®, PbS nanoparticles, fluorescein-labeled cowpea mosaic viral nanoparticles,
tobacco mosaic viral nanoparticles, multi-functional dendrimers, gold nanoparticles, silver
nanoparticles, nanocrystalline cellulose and silicon nitride nanoparticles. Basically, zebrafish
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Danio rerio embryos were collected from group spawns [3] and staged [4]. The chorion
surrounding the embryo was removed enzymatically [5] at 6 hours post fertilization (hpf) and
embryos were placed in 96-well exposure plates, one animal per well, at 8 hpf. Embryos were
exposed to 100^1 of nanomaterial solution over a broad concentration range, typically 5-fold
serial dilutions ranging from 16 parts per billion (ppb) to 250 parts per million (ppm).
Waterborne-exposed embryos were evaluated at 24 hpf for viability, developmental progression
and spontaneous movement (earliest behavior in zebrafish). At 120 hpf, behavioral endpoints
(motility, tactile response) were thoroughly evaluated in vivo and larval morphology (body
axis, eye, snout, jaw, otic vesicle, notochord, heart, brain, somite, fin, yolk sac, trunk,
circulation, pigment, swim bladder) was evaluated and scored in a binary fashion (present or
absent). Control and nanomaterial-exposed groups are statistically compared using a standard
proportionality test, the Fisher Exact test. Based on the lethal and sublethal effects data from
our embryonic zebrafish assays, we have developed an EZ (embryonic zebrafish) metric for
nanomaterial toxicity (EZ-metric) that takes into account the types and frequency of sublethal
effects (morbidity) in addition to overt mortality elicited from exposure.
Results
The toxicity of gold nanoparticles was influenced by synthesis methods, purity, core size, and
surface functionalization (charge). The vast majority of dendrimers (12 out of 17) did not elicit
a response independent of the generation (-size). Those that elicited a significant response were
amine terminated but not of a specific generation. No response was elicited from exposure to
a series of 10 viral nanoparticles except for the tobacco mosaic viral capsid functionalized with
polyethylene glycol. Of eleven nanoparticulate metal oxides tested, approximately half were
benign to embryonic zebrafish and toxicity appeared to be related to particle shape and reactivity.
Discussion
The embryonic zebrafish model is extremely useful for rapidly assessing the potential of
nanomaterials to interact with and alter biological processes. Using this model, we determined
differential toxicity profiles for diverse groups of nanomaterials in an effort to define
relationships between nanomaterial physicochemical properties and the biological responses
they elicit. The EZ-metric was established to provide a relative comparison of nanomaterial-
elicited effects on integrated living systems. Our calculated EZ-metrics were consistent with
other statistical measures. However, for the majority of nanomaterials tested, we did not
observe significant adverse biological outcomes. There were some unique observations from
those nanomaterials that did elicit significant responses. Extremely small changes in size, such
as an increase in size from 0.8 to 1.5 nm, can significantly affect the biological response to
gold nanoparticle exposure. Dendrimers which have potential applications for drug delivery
were relatively benign to the developing embryonic zebrafish. Viral nanoparticles that were
functionalized with polyethylene glycol (PEG) elicited significant deleterious effects while
the other viral nanoparticles did not. This may be due to the increased residence time of
nanomaterials functionalized with PEG. The toxicity of nanoparticulate metal oxides appears to
be related to differences in shape of the materials but was not correlated with zeta potential.
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Conclusions
The embryonic zebrafish model system can be employed to rapidly gain information and provide
feedback to engineers designing novel nanomaterials. Incorporating toxicological evaluations
early in research and development schemes will allow us to close the testing-redesign loop and
favor the development of nanomaterials with minimal toxicity. Metrics that combine data on
morbidity and mortality are valuable for understanding overall whole organism response and
relating it to cellular and molecular level responses. Our results revealed that characteristics
such as purity, size, surface functionalization, synthesis method, particle shape, and reactivity are
important parameters governing nanomaterial toxic potential.
References
1. M.A. Dobrovolskaia, J.D. Clogston, B.W. Neun, J.B. Hall, A.K. Patri and S.E. McNeil,
Method for analysis ofnanoparticle hemolytic properties in vitro. Nano Lett, In press.
2. S.L. Harper, J.L. Dahl, B.L.S. Maddux, R.L. Tanguay and I.E. Hutchison, Proactively
designing nanomaterials to enhance performance and minimize hazard. International Journal
of Nanotechnology, 5 (2008), 124-142.
3. M. Western eld, The Zebrafish Book. University of Oregon Press, Eugene, OR (2000).
4. C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann and T.F. Schilling, Stages of
embryonic development of the zebrafish. Developmental Dynamics, 203 (1995), 253-310.
5. C.Y. Usenko, S.L. Harper and R.L. Tanguay, In vivo evaluation of carbon fullerene toxicity
using embryonic zebrafish. Carbon, 45 (2007), 1891-1898.
Conference Questions and Answers
Question:
Are you basically using Multi-criterion Decision Analysis to evaluate adverse effects on the
zebra fish screening-level assay?
Answer:
Yes, the EZ Metrics system is basically the same as Multi-criterion Decision Analysis.
Question:
How does the zebra fish assay relate to other toxicity assays that are closer to human exposure?
Answer:
Other researchers are using the zebra fish for biomedical research and believe that it correlates
well; there is about 80 percent gene homology. The zebra fish assay gives a target area for further
investigations. Having said that, it is difficult to see what an isolated hepatocyte preparation tells
the investigator about human physiology.
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Question:
Which effects seem to be the most sensitive in this assay?
Answer:
Pericardial edema (swelling of the heart) seems to be very sensitive and is commonly seen;
however, this effect may be mediated by several mechanisms. We have also seen effects on the
notochord that cause it to push out to the exterior.
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Nanotoxicology: Developing a Responsible Technology
Christie M. Sayes, Department of Veterinary Physiology & Pharmacology,
College of Veterinary Medicine, Texas A&M University, College Station, Texas, U.S.A.
Abstract
Nanotechnology is not only an emerging field of study, it is now an industry. Because of this,
we now see an abundance of nanomaterials in numerous consumer goods. Still further, well
established industries, such as food packaging, forestry and paper, plastics and paints, and
electronics are beginning to use nanotechnology's scientific and engineering-based advances
to better their products, profit, and marketability. The research presented here describes basic
concepts of nanotechnology and its potential health and safety risks, the current status of
nanomaterial-containing consumer products in the marketplace, and nanomaterial synthesis and
physico-chemical properties important to toxicological and ecotoxicological evaluations. This
material aims to prepare chemists, toxicologists, risk assessors, and policy-makers to meet the
rapidly growing need to understand and evaluate the risks that engineered nanomaterials may
pose to human health and the environment. A toxicological and risk assessment of nanomaterials
requires an understanding of the unique differences between these "new" materials and their
previously studied chemicals or larger-particle predecessors. For example, studies on the
biocompatibility of various metal oxide nanoparticles (such as titanium dioxide, aluminum
oxide, and iron oxide) in various crystalline forms exposed to whole animal and cultured
cells are compared and contrasted to the more commonly used micro-sized particles. Results
show that, depending on chemical composition, crystalline structure, and type (and degree of)
surface modifaction, nano-scale metal oxide particles may induce elevated levels of alkaline
or acid phosphatase and increase levels of lactate dehydrogenase (an indicator for "leaky"
membranes). However, larger metal oxide particles remain relatively inert in cultured cells, the
lungs of rats, and algae test systems. Current methods for, and challenges to, toxicological and
ecotoxicological testing of nanomaterials will be covered. Most importantly, this work identifies
strategies in the material design process that minimize potential human health and safety risks
when working with nano-scale materials.
Introduction
The use of nanomaterials is expected to have great potential to advances devices and procedures
in the medical field, improve consumer goods and industrial products, and tackle rising energy
requirements. This opportunity is based on the unique physical and quantum properties that
vary continuously with changes in the size of some materials produced between 1 and 100
nanometers. As with any new technology, the potential risks associated with nano-based
products is needed in order to properly assess the safety of the novel materials being developed.
The potential human and environmental health risks are determined by the hazards posed and the
potential exposures to the nanomaterials that are developed for use in products. Both hazard and
exposure potential will vary widely for different nanomaterials and for different products that
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incorporate nanomaterials. This work focuses on relating nanomaterial physical and chemical
properties to its potential hazards in human health and the environment. The properties of
nanomaterials are predominantly associated with their nanometer size scale, structure-dependent
electronic configurations, and extremely large surface area-to-volume ratios relative to larger-
sized chemicals and materials. Size falls in the region between individual atoms or molecules and
their corresponding bulk materials (Colvin 2004). Particle size and surface area are important
properties from a toxicological perspective because as the size of a particle decreases, its surface
area increases. This allows for a greater portion of the total atomic make-up to be located on
the material's surface rather than within its interior. These atoms on the surface of the material
may be chemically and biologically active, potentially contributing to the development of
adverse health effects. Other physical and chemical properties such as shape, surface coating,
aggregation potential, and solubility may also affect the reactivity and mobility of nanomaterials,
with the possibility of negating or amplifying any associated size-related effects.
Nanotoxicology. The evaluation of the safety of nanomaterials will likely require a
multidisciplinary approach between toxicologists and experts in materials science, chemistry,
physics, biotechnology, engineering, and/or other appropriate disciplines. The physical and
chemical properties of nanomaterials can modify cellular uptake, protein binding, translocation
from portal of entry to the target site, and the potential for causing tissue injury (Oberdorster et
al. 2005a). The unique chemical and physical properties of nanomaterials may present special
challenges to the toxicologist or ecotoxicologist when designing studies to accurately and
reproducibly identify adverse biological interactions or effects. Scientific experimentation in
this area is complicated by several factors including: the need to (1) characterize nanomaterials
during several stages of toxicological testing (e.g., before, during after nanomaterial
administration); (2) express and/or administer the dose of nanomaterials (e.g., mass, surface area,
or particle number; (3) confirm that the nanomaterial aggregation state at time of administration;
and (4) identify analytical difficulties in detecting and quantifying nanomaterials in biology and
the environment.
Risk Assessment Process for Nanomaterials. Risk assessment is the systematic scientific
characterization of potential adverse health effects resulting from human or environmental
exposures to hazardous agents or situations (NRC 1983, 1994). As with larger-sized chemical
substances, risk assessment will be the basis of assessing and regulating nanomaterials to
protect human health and the environment. A risk assessment consists of four components, 1)
hazard identification - qualitative evaluation of the adverse effects of a substance, 2) exposure
assessment - evaluation of the types (routes and media) and magnitude or levels of exposure, 3)
dose-response evaluation - relationship between dose and incidence (or severity) of an adverse
effect, and 4) risk characterization - quantitative estimation of the probable incidence of adverse
health effects under various conditions of exposure, including a description of the uncertainties
involved (Purchase 2000). Thus, the distinction between the hazard (an inherent toxic property
of a chemical that may or may not be manifested, depending on exposure potential) and risk (the
consequences of being exposed to a hazardous chemical at a particular exposure level) is critical
(Purchase 2000).
Characterizing Nanomaterials for Toxicological Evaluation. One obstacle in developing
safety information on nanomaterials involves technical issues associated with conducting
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reliable and reproducible toxicity assessment (Bucher et al. 2004) and among these issues is
the characterization of materials to be tested (Powers et al. 2007). Particle characterization in
solution or suspension is just as important as characterization the dry, as-received phase. In
addition, a characterization profile of the nanomaterial within the in-life test system should be
included. Characterization of nanomaterials can be divided into three categories based upon
the physical state of the nanomaterial (i.e. dry, wet, or in life) and are referred to as primary,
secondary, and tertiary. Primary characterization is performed on particles or materials as-
received in the dry native state. Secondary characterization is performed on particles or
materials in the wet phase as a solution or suspension. Tertiary characterization is performed on
particles or materials either in vivo or ex vivo.
Methods
The Nanomaterials. In this paper, we describe the characterization methods used to determine
the physical properties of five metal oxide nanomaterial systems: titanium dioxide, zinc oxide,
aluminum oxide, silicon dioxide, and iron oxide. All five materials were produced in either liquid
phase or aerosol phase. All five materials were designed to be crystalline, spherical, and -30 nm
Table 1. Techniques for evaluating physical properties of nanomaterials.
Property
Definition
Technique
Particle Size
The range of sizes of particles within
a sample; this measurements gives
an indication of the
aggregation/agglomeration state
o Specific surface area (SSA
BET)
o Dynamic light scattering (DLS)
o Electron microscopy (TEM &
SEM)
Chemical
Composition
Information on the material's
intrinsic chemical toxicity can be
attained; includes both composition
of the particle's core and its surface
o Spectroscopy: X-ray
photoelectric (XPS), Raman,
Inductively coupled plasma
atomic emission (ICP-AES),
Fourier-transform infrared
(FTIR), Differential thermal
analysis (DTA)
Morphology
Information on aspect ratio for non-
spherical particles; crystal structure
for crystalline materials; allotropic
forms for materials of similar
chemical composition
o X-ray diffraction (XRD)
o Electron diffraction (ED)
o X-ray Photoemission
Spectroscopy (XPS)
Surface
Information about the interface of
the solid particle and liquid solvent
can be attained
o Zeta potential & isoelectric
point (IEP)
o Electron spin resonance (ESR)
o Chemiluminescence
This list is intended for establishing a relative metric of the physical properties of nanomaterials.
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in diameter at the time of production. Each system was suspended in Milli-Q ultrapure water,
phosphate buffered saline solution, and cell culture media. Concentrations ranged 0.001 to 1000
mg/L (milligrams of material per liter of solvent).
Characterization Techniques. Transmission electron microscopy (TEM) is one of the most
common and informative methods used to size nanoparticles in the dry state, in wet phase, and
in cultured cells. TEM pictures were taken on three different sample states using the JEOL
JEM-2010 microscope for dry state and cellular association analyses. For wet phase analyses,
the FEI Tecnai G2 F20 FE-TEM microscope for wet phase analyses; each TEM sample was
prepared by flash freezing 2 |al of 5 and 50 mg/mL nano-TiO2 suspension via FEI Vitrobot
at liquid nitrogen temperature (196°C) onto a 300-mesh copper/carbon grid (Ted Pella, Inc.,
Redding, CA). Alternative to the TEM method is dynamic light scattering (DLS). Although
traditional methodologies are limited to the particle's size (data is less reliable as the size
of the particle decreases), recent advances in the technique have improved light scattering
measurements (Powers et al. 2006). Here, we used the Malvern ZetaSizer Nano ZS to determine
size, size distribution, and surface charge of each nanomaterial system. For the specific surface
area measurements (SSA) measurements, we used the BET (Braunauer, Emmett, and Teller)
method to attain an area measurement that can then be converted (through stoichiometry) to a
primary particle size. There is an inverse squared relationship between surface area and radius
of a nanoparticle, so as the radius of the nanoparticle decreases, the surface area increases
exponentially (Brunauer et al., 1938). We utilized the Micromeritics ASAP 2020 instruments
for these studies. The materials were also characterized for morphology using X-ray diffraction
(XRD) (Otwinowski and Minor, 1997). XRD patterns were collected using a Siemens Platform-
Model General Area Detector Diffraction System with a Cu Ka source.
Surface Activity. Determining the surface activity of nanomaterial is non-trivial. The assay
used depends on the electron configuration of the material being tested. Here, we briefly
describe the use of luminal for TiO2 particles, but other dyes and techniques may be used to
determine reactivity of other metal oxide systems. The chemiluminescence of luminol was used
to qualitatively probe the production of RS over 20 min. This method, while not quantitative,
does provide an indicator of RS production and is completed in the dark (Arnhold et al., 1991;
Hadjimitova et al., 2002). Luminol (< 99%, Sigma) was prepared using Milli-Q water at 0.140
M NaCl, 10.0 mM PBS, and adjusted to pH 7.30 (Allen and Loose, 1976; Hallett and Campbell,
1983). Chemiluminescence intensities were measured with a SpectraMax M2 (Molecular
Devices, Sunnyvale, CA).
Results
Results show that, depending on chemical composition, crystalline structure, and type (and
degree of) surface modifaction, nano-scale metal oxide particles change in aggregation state,
may induce elevated levels of some enzymes, and increase levels of cytotoxicity (as measured by
cell density, viability, and oxidative stress).
Discussion
There are several challenges associated with the evaluation of the potential human and
environmental health risks from the development and use of novel nanomaterials include
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Table 2. Summary of results.
Metal Oxide
Nanomaterial
titanium
dioxide
(TiO2)
zinc oxide
(ZnO)
aluminum
oxide
(A12O3)
silicon
dioxide
(SiO2)
iron oxide
(Fe2O3)
Surface
Area
(m2/g)
51.19
32.1
54.350
31.4
48.6
Density
(g/cm3)
3.88
5.6
3.8
2.66
5.2
Calculated
Size in
Dry State
(nm)
30.2
33.3
29
71.8
23.5
Particle Size
in Dry State
from TEM
(nm)
27
39
48
30
30
Size in Wet Phase
(Milli-Q water) (nm)
1
ppm
125.0
150.6
42.1
52.0
188.3
10
ppm
280.8
176.1
110.3
50.1
237.2
100
ppm
298.5
167.5
870.1
60.3
750.7
Surface
Charge
in Wet
Phase
(mV)
+1.62
-55.0
-21.8
-
-
All five materials were designed to be crystalline, spherical, and -30 nm in diameter at the time
of production.
appropriate toxicological studies for the hazard evaluation of nanomaterials, characteristics of
nanomaterial-containing products, increased funding for EHS research and opportunities for
collaborations.
Conclusions
At this point, it appears that the research, development, and production of nanomaterials
are greatly outpacing the speed by which toxicological and exposure information is being
acquired on nanomaterials. An understanding of the mammalian and ecotoxicological profiles
of nanomaterials will be necessary to prioritize those nanomaterials that are safe for use and
to establish appropriate safety procedures for handling those nanomaterials that may pose
potential health hazards if there is sufficient exposure in the workplace, to consumers, or in
the environment. When conducting physical and chemical characterization of nanomaterial
properties, each material property should be measured using more the most appropriate
technique, and when possible, results should be confirmed with an additional analytical
technique. No single technique can accurately describe the properties of a nanomaterial.
Methodological limitations, non-trivial sample preparation, and incorporation of the appropriate
controls are all issues investigators should consider when analyzing nanomaterial samples.
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It is important that nanomaterials are developed responsibly, with optimization of benefits and
minimization of risks, with international cooperation to identify and resolve gaps in knowledge.
To maintain a high level of public health, occupational health, and environmental protection,
it will likely be necessary to conduct nanomaterial-specific risk assessments to evaluate any
potential human and environmental health effects and to ensure the development of safe
nanomaterial-containing consumer products.
Conference Questions and Answers
Question:
The characterization matrix for nanoparticles studies is very extensive, especially for their
surface chemistry, and is time consuming. Have you thought of using a hypothesis-driven
approach?
Answer:
Yes, the hypothesis-driven approach needs to be maintained and incorporated by new and
experienced researchers to avoid the unnecessary expenditure of time and materials. However,
the characterization of nanomaterials may differ with time-at time zero, 24 hours, 30 days, and
post-exposure-but this does not answer the question.
Question:
Is the difference in the persistence of the inflammatory response due to the severity of the
response and its time to resolve, or to the persistence in the lungs of the nanomaterials?
Answer:
The nanomaterials will be coated with lung surfactants; however, when they are acid washed, the
nanomaterials appear to be unchanged. They maintain their catalytic ability and do not appear
to be degraded. The coating nanomaterials receive in the lung does not apparently degrade the
particles.
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Use of Multi-Criteria Decision Analysis for Classification of Nanomaterials
Igor Linkov, US Army Research and Development Center, Vicksburg, Mississippi, U.S.A.
Tommi Tervonen, Faculty of Economics and Business, University ofGroningen,
Groningen, The Netherlands
Jeffery Steevens, US Army Research and Development Center, Vicksburg, Mississippi, U.S.A.
Mark Chappell, US Army Research and Development Center, Vicksburg, Mississippi, U.S.A.
Jos'e Rui Figueira, Centre for Management Studies, Instituto Superior T'ecnico,
Technical University of Lisbon, Porto Salvo, Portugal
Abstract
There is rapidly growing interest by regulatory agencies and stakeholders in the potential risks
associated with nanomaterials throughout the different stages of products' life cycle (e.g.,
development, production, use and disposal). Risk assessment methods and tools developed
and applied to chemical and biological agents may not be readily adaptable for nanomaterials
because of the current uncertainty in identifying the relevant physico-chemical and biological
properties that adequately describe the materials. Such uncertainty is further driven by
the substantial variations in the properties of the original material because of the variable
manufacturing processes employed in nanomaterial production. We propose a decision support
system for classifying nanomaterials into different risk categories. The classification system
is based on a set of performance metrics that measure both the toxicity and physico-chemical
characteristics of the original materials, as well as the expected environmental impacts through
the product life cycle. The stochastic multicriteria acceptability analysis (SMAA-TRI), a formal
decision analysis method, was used as the foundation for this task. This method allowed us to
cluster various nanomaterials in different risk categories based on our current knowledge of
nanomaterial's physico-chemical characteristics, variation in produced material, and expert
estimates. SMAA-TRI used Monte Carlo simulations to explore all feasible values for weights,
criteria measurements, and other model parameters to assess the robustness of nanomaterial
grouping for risk management purposes.
Abstract
There is rapidly growing interest by regulatory agencies and stakeholders in the potential toxicity
and other risks associated with nanomaterials throughout the different stages of the product's
life cycle (e.g., development, production, use and disposal). Risk assessment methods and tools
developed and applied to chemical and biological materials may not be readily adaptable for
nanomaterials because of the current uncertainty in identifying the relevant physico-chemical
and biological properties that adequately describe the materials. Such uncertainty is further
driven by the substantial variations in the properties of the original material because of the
variable manufacturing processes employed in nanomaterial production. To guide scientists
and engineers in nanomaterial research and application as well as promote the safe use/handling
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of these materials, we propose a decision support system for classifying nanomaterials into
different risk categories. The classification system is based on a set of performance metrics
that measure both the toxicity and physico-chemical characteristics of the original materials,
as well as the expected environmental impacts through the product life cycle. The stochastic
multicriteria acceptability analysis (SMAA-TRI), a formal decision analysis method, was used as
the foundation for this task. This method allowed us to cluster various nanomaterials in different
ecological risk categories based on our current knowledge of nanomaterial's physico-chemical
characteristics, variation in produced material, and best professional judgments. SMAA-TRI
uses Monte Carlo simulations to explore all feasible values for weights, criteria measurements,
and other model parameters to assess the robustness of nanomaterial grouping for risk
management purposes.
Introduction
Nanotechnology is a rapidly growing field of research that is already demonstrating a great
impact on consumer products. The field of nanotechnology can be defined as the production
and use of materials at the nano-scale, normally characterized as smaller than 100 nm in one
dimension (Oberdorster et al., 2007). Nanomaterials are formed through both natural (e.g.,
combustion by-products) and synthetic processes. For the purposes of this paper, we focus
our discussion solely on engineered nanomaterials, which are currently used in more than 600
different consumer products (Woodrow Wilson Institute, Online database, 2008). In spite of
their potential commercial benefits, some nanomaterials have been identified as toxic in in vivo
and in vitro tests. Clearly, our knowledge of the potential toxicity of these materials is far from
comprehensive (Oberdorster et al., 2005; Thomas and Sayre, 2005). The potential environmental
fate and toxicity (as well as potential for exposure and risk) of nanomaterials may be strongly
impacted by the material's physico-chemical characteristics. For example, potentially toxic
nanoparticles that tightly bind to soil surfaces may exhibit limited movement through the
environment. In this case, such materials may be deemed relatively safe for certain specific uses.
Such information is important as a lack of understanding of nanomaterial toxicity and risks may
delay full-scale industrial application of nano-enabled technologies.
Nanomaterial research and regulations could be guided by a systematic characterization of
factors leading to toxicity and risks in the absence of definitive data. In this paper we propose
a risk-based classification system for nanomaterials that takes into account several parameters
commonly associated with ecotoxicity and environmental risk of nanomaterials. These
parameters vary from nanomaterial physico-chemical characteristics to expected environmental
concentrations to fate and transport mechanisms. In this work, we focus primarily on ecological
risks although the same methodology could be applied to human health risk assessment. This
work does not attempt to draw exact conclusions about the environmental risks associated
with different nanomaterials, but rather to provide reasonable recommendations about which
nanomaterials may need more precise measurements and testing to be safely deployed in
consumer products.
MCDA Approaches to Classification
Clustering nanomaterials into ordered risk categories can be treated as a sorting problem in the
context of multi-criteria decision analysis (MCDA). MCDA refers to a group of methods used
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to impart structure to the decision-making process. Generally, the MCDA process consists of
four steps: (1) structuring the problem by identifying stakeholders and criteria (nanomaterial
properties in this case) relevant to the decision at hand, (2) eliciting the parameters of the model
(weights, thresholds, etc.), and assigning measurements for each alternative (e.g., nanomaterial
risk group), (3) executing the model through computer software, and (4) interpreting results
of the model and possibly re-iterating the process from step 1 or 2 by re-evaluating the model.
The goal of this MCDA process was not to select a single best alternative, but to rank or group
alternatives through a structured process. A detailed analysis of the theoretical foundations for
different MCDA methods and their comparative strengths and weaknesses is presented in Belton
and Stewart (2002). A review of MCDA applications to environmental management can be
found in Linkov et al., 2006.
The SMAA-TRI sorting method (Tervonen et al., 2009) is well suited for the proposed
classification system given the uncertainty of available information regarding the physico-
chemical characteristics of nanomaterials (see Figueira et al., 2005a, for a review of other
MCDA sorting methods). Many of the characteristics attributed to nanomaterials are limited to
a solely qualitative assessment. We used SMAA-TRI, an outranking model based on ELECTRE
TRI (see e.g., Figueira et al., 2005b) for the assignment procedure. If an alternative outranked
another, then the alternative was considered at least as good or better than another alternative.
We preferred SMAA-TRI as it extends the capabilities of ELECTRE TRI by allowing the use
of imprecise parameter values. ELECTRE TRI assigns the alternatives (different nanomaterials
in this study) to ordered categories (risk classes). Three types of thresholds are used to construct
the outranking relationships by defining preferences with respect to a single criterion. The
indifference threshold defines the difference in a criterion that is deemed insignificant. The
preference threshold is the smallest difference that would change the expert preference. Between
these two lay a zone of "hesitation" or indifference. The veto threshold is the smallest difference
that completely nullifies (raises a "veto" against) the outranking relation. The assignment
procedure involves comparing the properties associated with a specific nanomaterial (gl, g2,
..., gm) against a profile that includes ranges of criteria metric values corresponding to several
risk classes. Comparisons are performed with respect to each criterion, taking into account the
specified thresholds. The final classification decision is based on the profile criteria weights and
specified cutoff level (lambda). For example, Class 4 represents the highest risk while Class 1 is
the lowest risk (Figure 1).
Figure 1. Example measurements of profiles for each criterion gj (adapted from Merad et al.,
2004). Profiles are marked with horizontal lines.
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The assigned criteria weights represent the subjective importance of the criteria. For this
reason, ELECTRE TRI was particularly attractive for these classifications because the weights
represent "votes" for each criteria which are not affected by criteria scales. The lambda cutting
level represents the minimum weighted sum of criteria that have to be in concordance with
the outranking relation for it to hold: the lambda cutting level is used to transform the "fuzzy"
outranking relation into an exact one (whether an alternative outranks a profile or not). For
example, a lambda cutting level of 0.6 means that 60% of the weighted criteria have to be "at
least as good" for the outranking relation to hold.
Alternatives were compared by accounting for the three thresholds. An alternative and profile
with scores of 0.4 and 0.6 (for the same criterion) respectively, and an indifference threshold
of at least 0.2, demonstrates that this criterion fully supports the conclusion that the alternative
outranks the profile. Sometimes the support is not binary, but is further affected by linear
interpolation in the hesitation zone of both veto and preference thresholds (see e.g. Tervonen,
2007). In this case the support can have real values between 0 (no support) and 1 (full support).
All the parameters of ELECTRE TRI can be imprecise and represented by arbitrary joint
distributions in SMAA-TRI. This feature allows us to make conclusions about risks related to
different nanomaterials even though the information about their characteristics is limited. Monte
Carlo simulations were used in SMAA-TRI to compute acceptability indices for alternative
categorizations (i.e., for assigning nanomaterials in different risk classes). SMAA-TRI allows
performing automatic sensitivity analysis.
Output of SMAA-TRI comes as a set of category acceptability indices which describes the
share of feasible parameter values that assign alternatives to each category. The category
acceptability indices are measures indicating the stability of the parameters, i.e, if the parameters
are too uncertain to make informed decisions. A high index (>95%) signals a reasonably safe
assignment of the alternative into the corresponding category. With lower indices, the risk
attitude of the decision maker defines the final assignment. For example, if an alternative has a
80% acceptability for the lowest risk category, and a 20% acceptability for the second lowest risk
category, a risk-averse decision maker could assign the alternative to the higher risk category.
SMAA-TRI conducts the numerical simulation by comparing the effect of changing parameter
values and criteria evaluations on the modeling outcomes. Parameter imprecision can be
quantified by Monte Carlo simulations using different probability distributions (uniform, normal,
log-normal, etc). Gaussian or uniform distributions are typically used (for more information
about SMAA methods, see Tervonen and Figueira, 2007).
If some model parameters need to have their sensitivity assessed, they can be considered as
imprecise and defined as probability distributions.
Criteria
Recent articles, as well as the frameworks reviewed in this study, generally propose several
different characteristics in the risk assessment of nanomaterials. These characteristics are
generally based on extrinsic particle characteristics (size, agglomeration, surface reactivity,
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number of critical function groups, dissociation abilities), (Biswas and Wu, 2005; Borm and
Muller-Schulte, 2006; Borm et al., 2006; Gwinn and Vallyathan, 2006; Kreyling et al., 2006;
Medina et al., 2007; Nel et al., 2006; Oberdorster et al., 2005; Thomas and Sayre, 2005). These
various parameters are critical because they define the fate and relevant intact exposure pathways
as well as internal dose required to assess risk (Powers, 2007; Tsuji et al., 2006). Summary
descriptions of five basic extrinsic nanomaterial properties, agglomeration and aggregation,
reactivity, critical functional groups, particle size, and contaminant dissociation are presented
below:
• Agglomeration, weakly bound particles, and aggregregation, strongly bound or fused particles,
(ISO, 2008) are important criteria of risk because it provides a description of the physical state
of nanoparticles (NP) in the aquatic system (Kennedy et al., 2008); Wang et al., 2008). In
aqueous solutions, NP agglomeration generally occurs by two mechanisms: colloid settling and
flocculation. Flocculation occurs when Brownian-driven collisions bind unassociated particles
together through Van der Walls forces by dehydrating the interacting surfaces. Consequently, the
particle separates out of solution containing the mass of the previously unassociated particles.
Settling, on the other hand, occurs due to the pull of gravity, as described by Stokes law
relationships. Particles may settle but remain non-flocculated, settling at interparticle distances
with the lowest free energies. In the absence of surfactive agents, particle flocculation is fairly
predictable by particle charge. Charged functional groups give way to the development of a
surface electrostatic potential which extends out a few nanometers at the solid-liquid interface
forming a diffuse double layer or DDL (Bowden et al., 1977; Uehara and Gillman, 1981).
Classical DLVO theory predicts that repulsive forces between particles (arising from overlapping
DDLs) increase with increasing ion concentrations (or increasing ionic strength, I) because of
rising osmotic pressures at the solid-solution interface force the DDL to swell (Evangelou, 1998,
and references therein). Yet, classical Debeye-Huckel theory predicts a competing case where
increasing ion concentration decreases DDL thickness, throwing a system into flocculation.
Thus, at a fundamental level, the process of agglomeration represents the balance of these two
competing charge interactions.
• Reactivity/Charge. Charge may be expressed on NP either by design (such as through
functionalization) or by spontaneous degradative reactions. NPs may be functionalized with
various types of groups, such as COOH, NH2, and SH2 through standard organic synthesis
methods. Such functionalizations may be useful for manufacturing processes. For example,
single-walled carbon nanotubes (SWNTs) are typically carboxylated at their ends as part of the
isolation/purification processs (Anita Lewin, RTI International, personal communication). The
type of charge occurring on functionalized NPs is called variable charge, which means that the
magnitude of the surface electrostatic potential varies with solution pH (Uehara and Gillman,
1981). Variably charged groups characteristically exhibit a surface pKa. Thus, variably charged
surface groups may be speciated (e.g., protonated vs. deprotonated) by the classical Henderson-
Hasselbauch equation. Furthermore, the magnitude of the surface electrical potential may be
suppressed by increasing I, as described previously. Thus, the reactivity of variably charged
functional groups varies with the difference in solution pH from the surface pKa and the
magnitude of I.
• Critical functional groups: Related to the reactivity/charge, critical functional groups make up
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an important criterion given the fact that nanomaterial functionality and bioavailabilty is directly
related to chemical species. Basing risk criteria on elemental speciation is superior to elemental
composition alone because it identifies the unique set of reactions available to each species.
For example, suspended zero-valent Fe nanoparticles have been shown to catalyze reductive
degradations of aqueous organic contaminants (Joo et al., 2004). The same degradative ability
has been shown for structural Fe2+ (higher oxidation state than zero-valent Fe but different
speciation in terms of its complexation environment) domains at clay-edge and -interlayer nano-
sites in soil (Hofstetter et al., 1999; 2003). The Cd2+ cation in quantum dots exhibits no toxicity
to organisms as long as it remains complexed with Se (Derfus et al., 2004). Speciation also
determines solubility or potential dissociation of nanomaterials.
• Contaminant dissociation: This criterion describes risk associated with residual impurities
contained within the NP. For example, Fe oxide NP may contain S impurities depending on
whether FeC13 or Fe2(SO4)3 was used in manufacturing. Carbon nanotubes may contain Ni,
Y, or Rb metal cation impurities (Bortoleto et al., 2007; Chen et al., 2004), which may either be
entrained within or adsorbed onto the surface of the tubes. However, little is actually known
about the extent in which metallic and organic contaminants remain with the manufactured
product. Thus, the assignment of this risk criterion could change depending on better
information.
• Size: Particle size is a criterion related to the agglomeration and reactivity criteria. Obviously,
smaller particles agglomerate at slower rates. However, agglomeration is also related to the
particle size distribution or polydispersivity. For example, greater monodispersivity of particles
sizes appears to promote more stable dispersions (Chappell et al., 2008). Also, nanoparticle
reactivity is also impacted by the size of NP surface relative to the bulk of the solid. While the
surface is the reactive portion of solids, the bulk component may suppress the surface reactivity
through internal reorganizations, etc. NPs are essentially surfaces with limited bulk. Thus, the
smaller particle size, the lower bulk to potentially limit surface reactivity. Surfaces with low
accompanying bulk have been shown to possess enhanced reactivities, such as high-affinity
adsorption of metals or unique structures of assembly during agglomeration (Auffan et al., 2008;
Erbs et al., 2008). Particle size is particularly important in terms of distinguishing the unique
size-dependent chemistry of nanoparticles from classical colloid chemistry.
Processes that may influence the potential hazards of engineered nanomaterials include
bioavailability potential, bioaccumulation and translocation potential, and potential for
toxicity. These processes have been described in empirical studies and are dependent on the
characteristics of the particles as described above. It is difficult to predict the behavior of these
materials, however, in the future computational approaches are expected to provide additional
tools to estimate these processes from the physical and chemical parameters.
• Bioavailability potential: Bioavailability describes the amount of material absorbed across
cell membranes from the various exposure routes (e.g., dermal, inhalation, and oral exposures)
into system circulation in an organism (Medinsky and Valentine, 2001). This process is
controlled by the characteristics described above. For example, charge of the particles may
influence the agglomeration of the particles and hence limit the ability of the particle to cross
the gastrointestinal membranes after oral ingestion. There are however, several pathways
which nanoparticles may cross cell membranes ranging from pinocytosis, endocytosis, and
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diffusion as summarized by Unfried et al. (2007). The mechanism by which these particles are
absorbed are highly dependent on the particle composition, surface modification, size, shape, and
agglomeration.
• Bioaccumulation Potential: Bioaccumulation is the net accumulation of particles absorbed
from all sources (soil, water, air, and food) and exposure routes listed above into an organism.
Accumulation must consider the temporal aspects of exposure and include kinetic factors such
as exposure concentration, duration of exposure, clearance, biotransformation, and degradation.
Most studies to date have focused on the potential for uptake and translocation in specific tissues
(Ryman-Rasmussen et al., 2006; Gopee et al., 2007; Kashiwada, 2006) and have not addressed
the toxicokinetics of nanoparticles.
• Toxic Potential: Toxicity of engineered nanomaterials and particles in mammalian and
other animal systems has been assessed primarily through cytotoxicity screening assays;
although some in vivo studies have been completed. It is proposed that toxicological effects of
nanomaterials occurs through oxidative stress, inflammation from physical irritation, dissolution
of free metal from metal nanoparticles, and from impurities in nanomaterials (e.g., catalysts)
(Oberdorster et al. 2007). The characteristics of nanoparticles that influence toxicity include
the size, surface area, morphology, and dissolution. To date, screening studies using in vitro
approaches have observed toxicity from metal nanoparticles at lower concentrations (Bradich-
Stolle et al., 2007) than toxicity from carbon-based nanoparticles (Murr et al., 2005; Grabinski et
al., 2007).
Proposed Classification Framework
The purpose of the proposed classification system is to preliminarily group nanomaterials in risk
classes for screening level risk assessments. Such groupings should aid in prioritizing materials
for further study. In this paper, we considered five risk categories: extreme, high, medium, low,
and very low risk. In order to assign particular nanomaterials to these categories, we need to
define criteria scales, thresholds, and measurements.
The quantitative criterion, particle size, was evaluated as the mean size of the material in
units of nanometers as obtained from literature review and expert estimates. Bioavailability,
bioaccumulation, and toxic potential were measured through subjective probabilities that the
nanomaterial has significant potential in the criterion. These, as well as rest of the criteria
(agglomeration, reactivity/charge, critical function groups) were measures based on expert
judgments. The qualitative criteria, agglomeration, reactivity/charge, and critical function groups,
were measured in terms of ordinal classes: 1 was the most favorable (least risk) value class,
while 5 the least favorable (highest risk).
For the qualitative criteria, we encoded the classes with integers. The indifference thresholds
were set to 0 and the preference thresholds to 1. This choice of thresholds represented an
ordinal scale: a smaller number was preferred to a larger one, but the intervals did not carry any
information (e.g. 1 is as much preferred to 2 as 2 is to 3). If there were multiple possible classes
for an alternative, the measurement was modeled with a discrete uniform distribution, meaning
that the density function for the distribution was such that the integers corresponding to these
classes were equiprobable. Veto thresholds were not used in this phase of the framework, but will
69
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be added later when more information about the criteria becomes available. Size is a criterion
that should have some veto associated with it, so that very small materials cannot be assigned to
the safer (lower risk) categories.
Even though nanomaterial size is believed to be a factor influencing toxicity, there is little
specific information available characterizing toxic effects relative to the 1 to 100 nm size
range (Powers et al., 2007). More research is needed to define the thresholds in a more exact
manner. If a "smaller"-sized nanoparticle represents higher risk, it follows that a larger size is
"more preferable" because of its inherently lower risk. Due to these knowledge gaps, imprecise
thresholds were used for nanomaterial size with indifference threshold of 10 ± 5% and preference
threshold of 25 ±5%.
Bioavailability, bioaccumulation, and toxic potential were all measured using a cardinal but
subjective scale as described above. Because of the subjectivity of this scale, we applied
imprecise thresholds. Indifference thresholds were set to vary uniformly from 0 to 10, and
preference thresholds from 10 to 20.
The SMAA-TRI model separated the risk categories using profiles formed from measurements of
the same criteria as the alternatives. In our framework, the profile measurements were all exact
(Table 2).
Our model applied imprecise preference information in the form of weight bounds. For more
information on how these were implemented, see Tervonen and Lahdelma (2007). We judged
the toxic potential to be the most important criterion, and thus it was assigned weight bounds of
0.3-0.5. Bioavailability and bioaccumulation potentials were deemed the least important criteria,
and as a result, we were undecided on their relative importance. Both of these criteria were given
weight bounds ranging from 0.02-0.08. The rest of the criteria were assigned weight bounds of
0.05-0.15.
We used imprecise values for the lambda cutting level within the range of 0.65-0.85. Lambda
defines the minimum sum of weights for the criteria that must be in concordance with the
outranking relation to hold. The classification was performed according to the pessimistic
assignment rule, which in risk assessment applications represents a more conservative approach.
Criteria measurements. The first four criteria are measured as ordinal classes. Measurements
of reactivity/charge have associated uncertainty in that the materials can belong to either of the
indicated classes. The following three criteria have linear imprecision of 10 in both directions
from the indicated mean value. Size has uncertainty of 10% of the shown mean value.
Example
We demonstrated application of the framework by classifying five nanomaterials: nC60 (a
fullerene), MWCNT (Multi-Walled Carbon Nanotube), CdSe (quantum dot), Ag NP (Silver-
Nanoparticles), and Al NP (Aluminum Nanoparticles). Typical size ranges for these materials
were estimated based on in situ measurements from the available literature. Other properties
were assessed using authors expert judgments, taking into account the characteristics for each
criterion described in Section 3. Metrics for the five materials used in our case study (Table
2) as well other model parameters were input into the SMAA-TRI software. Even though
70
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criteria metrics used in the paper were assessed using expert judgment and its objectivity can
be questioned, the outranking algorithms used in SMAA-TRI together with the the choice of
absolute thresholds implemented in this study allows to obtain robust results (Tervonen (2007).
Category acceptability indices obtained from the simulation are presented in Figure 2. These
indices show that the data was too imprecise to make definite decisions about the risks
related to the different nanomaterials. However, there was sufficient data to make preliminary
classifications. For example, CdSe exhibited a very high index in the high risk-class. On the other
hand, Al NP may be considered relatively safe, its category acceptability indices for low and very
low risk were 34 and 34, respectively. Summing these indices gave the material an estimated
68 percent probability of being classified as "low to very low risk". C60 showed a reasonable
acceptability index (49%) for the low risk category. In terms of making risk-aware decisions for
C60 and Al NPs, we feel that further studies into expanding the potential applications of Al NP
and C60 (as opposed to CdSe) are justified.
It is important to point out that in spite of the high uncertainty of the above results, this work
represents a reasonable starting point for a more thorough follow-up analysis. And indeed, more
data is required to improve our estimates. Risk estimates based on acceptability indices below a
certain threshold (e.g. 80 %) should be viewed with caution. For example, should C60 be deemed
viable for further research and application, additional measurements will be required to further
refine the risk estimates. In spite of its limitations, the quantified risk values determined from
our simulations are helpful in characterizing the risk and uncertainty for limited and variable
data.
Extreme risk High risk Medium risk Low risk Very low risk
C60
MWCNT
CdSe
-•i fir
AINP
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34
Figure 2. Category acceptability indices of the example. A high index means, that the material
is assigned to corresponding category with a higher confidence as measured by larger share of
possible parameter values corresponding to this category.
Concluding remarks
Nanotechnology is a fast growing research field with an increasing impact on our everyday
72
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lives. Although nanomaterials are used in common consumer products, the lack of information
about human health and environmental risks may hamper the full-scale implementation of this
technology. We presented in this paper a systematic multi-criteria approach that allows for
assigning nanomaterials into ordered risk classes. Materials assigned to the highest risk class
potentially represent areas of important future toxicological studies while materials exhibiting
low risk may be recommended for targets of research aiming at commercial use. The proposed
framework takes into account measurements and expert estimates for multiple criteria that are
known to impact the toxicity of the material.
The use of SMAA-TRI approach allows for the explicit incorporation of uncertainty parameters
in the model. An appealing characteristic of the outranking model applied in SMAA-TRI is
that it allows veto effect to be modeled, meaning that a nanomaterial's poor performance in
one criterion cannot be compensated by good performance in other criteria (as is the case for
compensatory MCDA models, e.g. utility theory). This convention prevents decisions about the
risk of a particular nanomaterial being unduly based on one particular criterion (such as size vs.
surface reactivity relationships) as the material may have other physico-chemical characteristics
related to size that exhibit a greater impact on its toxicity.
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Conference Questions and Answers
No Questions.
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MSDSs Fail to Communicate the Hazards of Nanotechnology to Workers
Bruce Lippy, The Lippy Group, LLC, Baltimore, Maryland, U.S.A.
Background
In the United States, the Occupational Safety and Health Administration's Hazard
Communication Standard (29 CFR 1910.1200) requires that employers inform their workers of
the chemical hazards to which they are exposed and how they should protect themselves. The
2006 European REACH (Registration, Evaluation, Authorization and Restriction of Chemicals)
initiative on chemical hazard communication is more comprehensive and ambitious than the
OSHA requirements.
Nanomaterials are widely believed to have begun a new revolution in manufacturing that will
broadly provide improved products and capabilities in areas as diverse as sports equipment and
biomedical sensors. Engineered nanoparticles, however, have been shown in animal studies "to
reach the alveolar region; avoid macrophage engulfment; cause oxidative stress, inflammation,
and fibrosis; and translocate into the blood." (1) The National Institute for Occupational Safety
and Health (NIOSH) has raised concerns about what prevention and control actions should be
taken while toxicological research is ongoing. (2)
A more fundamental question is what should workers currently manufacturing these products
be told about the risks they face? With $88 billion worth of products containing nanomaterials
reportedly sold in 2007, there are clearly many workers potentially exposed. (3) Their numbers
have been projected by the U.S. government to grow to 2 million worldwide over the next 15
years. (4) Given the limited toxicological information that is available for most nanomaterials,
the task of effectively communicating the risks of handling these materials is daunting. Material
Safety Data Sheets (MSDSs) are required for nanomaterials that meet the definitions of
hazardous chemicals under OSHA's Hazard Communication standard. MSDSs from suppliers are
the preferred source of risk information for nanotechnology firms, according to a survey of firms
in Massachusetts. (5) The Wilson Center for Scholars website maintains the most comprehensive,
publicly-available online inventory of commercial nanoproducts. (6) As of May 31, 2008, there
were 609 materials in the Wilson Center nanoproduct database, which is growing by 3-4 products
each week.
Unfortunately, industry hasn't done a good job of communicating the hazards of standard
industrial chemicals despite the two and a half decades since the promulgation of OSHA's
Hazard Communication standard in 1983 to get it right. This author participated in an OSHA-
funded 1997 study of the peer-reviewed hazard communication literature. The results (which
are still on OSHA's website) indicated broad shortcomings with the research methods, which
generally relied on self-reported preferences rather than observations of actual behaviors and on
students as test subject, rather than workers. (7) One representative study employed an expert
panel to review the accuracy of the technical information in randomly-chosen MSDSs and found
that only 11 percent of the MSDSs were accurate in all of the following four key areas: health
effects, first aid, personal protective equipment, and exposure limits. Particularly pertinent to
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nanomaterials, the health effects data on the MSDSs frequently were incomplete and the chronic
data - the biggest unknown for nanoparticles - were often incorrect or less complete than the
acute data. (8) Of significant concern, three separate studies found that literate workers only
comprehended roughly 60 percent of the health and safety information on sample MSDSs. (9,
10, 11)
A recent review of more current literature regarding the accuracy, comprehensibility and
use of MSDSs unfortunately did not show improvements over the 1997 review. Accuracy
and completeness were found to be relatively poor: the majority of studies showed that the
MSDSs did not contain information on all the chemicals present and workers showed low
comprehensibility because of overly complex language. (12)
A key role of MSDSs is to communicate the government's regulatory requirements for specific
chemicals. The U.S. governmental efforts to research and regulate chemicals have not kept up
with industry's impressive ability to develop and produce new ones. Nanotechnology appears
to be a tsunami wave heading towards this badly leaking ship. In the U.S. there are currently
around 600 OSHA Permissible Exposure Limits (PELs) for individual chemicals, most of which
haven't been updated in 40 years, despite new research findings. The Bush administration created
only one health standard for a chemical (hexavalent chromium) in eight years and only after
receiving a court order to do so. (13) There is no definitive count of the number of chemicals
in regular use today, but an often cited estimate is around 100,000. The Chemical Abstract
Service had registered 37,966,182 organic and inorganic substances developed by industry as of
September 18, 2008. (14) Scanning Tunneling Electron Microscopy allows the manipulation of
individual atoms. Given the 118 elements available for combination, an estimate of between 10200
to 10900 distinct nanoscale particles has been posited - an unimaginable number (particularly for
regulators). (15)
Methods
The National Institute for Occupational Safety and Health (NIOSH) appears to maintain the most
complete collection of MSDSs for nanomaterials and provided the Lippy Group a copy of the 49
MSDSs collected as of September 2007. All of these documents were then individually assessed
to answer the following questions:
1. Is the actual component or components that contain nanoparticles clearly identified?
2. Is there cautionary language provided about nanomaterials?
3. What, if any, Permissible Exposure Limits or Threshold Limit Values are provided?
4. What ventilation is recommended?
5. What personal protective equipment is recommended?
6. Are explosive hazards noted where appropriate?
Results
33 percent of the MSDSs did not identify the nano-sized component in the material.
56 percent did not have any cautionary language pertaining to the nanosized component.
67 percent listed an OSHA Permissible Exposure Limit (PEL) or American Conference
of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value, but all were tied
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to the normal form of the nanomaterial (e.g. carbon black or graphite rather than carbon
nanotubes).
89 percent recommended using respiratory protection, but tied it to the normal OSHA
PELs or TLVs, which are often based on the OSHA nuisance dust standards.
79 percent recommended using local exhaust ventilation; of those that did, 25 percent
recommended a face velocity greater than 100 feet per minute even though NIOSH
has indicated that standard fume hoods operated at that rate tend to create too much
turbulence to fully contain nanoparticles, which when dry are extraordinarily buoyant.
None indicated that nanoparticles pose a much greater flammability risk even though the
minimum ignition energy decreases exponentially with particle diameter. As the British
Health and Safety Executive noted, "An increasing range of materials that are capable of
producing explosive dust clouds are being produced as nanopowders." (16)
Discussion
Manufacturers of nanomaterials have an opportunity to learn from the hazard communication
failures of the past and create informative tools that workers and employers find helpful, despite
the acknowledged gaps in our current understanding of the toxicology of nanoparticles. This
will require honestly describing what we know and don't know. At a minimum, manufacturers
must identify which components in their formulations contain engineered nanoparticles.
Listing OSHA PELs for macro-sized materials without any conditional statements may meet
regulatory requirements, but borders on the unethical. For instance, the OSHA PEL for synthetic
graphite is 15 milligrams per cubic meter, but Oberdorster reported "profound cytotoxity" for
single walled carbon nanotubes in animal instillation studies for exposures at 0.38 micrograms
per square centimeter and noted that even the low mass-based concentrations of nano-sized
materials measured in workplace air (generally less than 50 micrograms per cubic centimeter)
represent "very high particle number concentrations." (17) NIOSH has flatly stated that "...
the occupational exposure limit for graphite should not be used to allow extensive exposure
to carbon nanotubes that appear far more toxic than graphite," but this practice appears to be
common among manufacturers of carbon nanotubes. (18)
Manufacturers of nanomaterials can have an informed and safer workforce, able to respond
rationally to this remarkable new trend in manufacturing, but they must do a better job with
MSDSs and government should help.
Recommendations
1. OSHA should require that all nanoscale materials be identified on MSDSs, to answer a
question posed at a national conference by one of their nanotechnology experts. (19)
2. Given the absence of occupational exposure limits for nanomaterials, OSHA should
require conditional language be included in all MSDSs containing nanomaterials to
explain the inadvisability of using PELs derived for normal forms of the materials. One
example proffered by a hazard communication expert: "Established exposure values do
not address the small size of particles found in this product and may not provide adequate
protection against occupational exposures." (20)
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3. The Nanotechnology Environment and Health Implications Working Group of the
National Nanotechnology Initiative should consider the hazard communications needs
of workers currently creating nanoproducts, rather than wait until the toxicology data
are conclusive. Technology Safety Data Sheets, informational tools developed for the
U.S. Department of Energy to inform workers of the risks posed by new remediation
technologies, can serve as an example of an alternative, creative approach. A majority of
surveyed populations of technology developers, state environmental regulators and heavy
equipment operators found these tools "quite valuable." (21) Given the abysmal results
demonstrated thus far with the standard MSDS approach, it may be time to consider
creating Nanotechnology Safety Data Sheets.
References
1. Schulte, P., Geraci, C., Zumwalde, R., Hoover, M., and E. Kuempel. (2008).
"Occupational Risk Management of Engineered Nanoparticles." J. Occupational and
Environmental Hygiene (5) 239.
2. Ibid. 240.
3. Woodrow Wilson International Center for Scholars. (April, 2008). "New Nanotech
Products Hitting the Market at the Rate of 3-4 Per Week." http://www.nanotechproject.
org/news
4. U.S. National Nanotechnology Initiative. (April, 2008). "Frequently asked questions."
http: //www. nano. gov/html/res/faqs. html
5. Lindberg, I.E. and M.M. Quinn. (December, 2007). "A Survey of Environmental, Health
and Safety Risk Management Information Needs and Practices among Nanotechnology
Firms in the Massachusetts Region." PEN Research Brief Number 1. http://www.
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Science Underpinning the Art of Communication for Health and Safety." http://www.
osha.gov/SLTC/hazardcommunications/otherresources.html.
8. Kolp, P., Williams, P., and R. Burtan. (1995). "Assessment of the Accuracy of Material
Safety Data Sheets. Journal of the American Industrial Hygiene Association 56:178-183.
9. Kolp, P., Sattler, B., Blayney, M. & T. Sherwood. (1993). "Comprehensibility of Material
Safety Data Sheets." Am. J. of Industrial Medicine (23) 139.
10. Phillips, C., Wallace, B., Hamilton, C., Pursley, R., Petty, G. and C. Bayne. (1999).
"Efficacy of MSDSs and worker acceptability." J. Safety Research (30) 113-122.
11. Printing Industries of America. (1990). Comments on the OSHA Hazard Communications
Standard. Docket H-022G.
12. Nicol, A. M., Hurrell, A.C., Wahyuni, D., McDowall, W., and W. Chu. (July, 2008)
Accuracy, Comprehensibility, and Use of Material Safety Data Sheets: A Review" Am. J.
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Industrial Medicine (51) 11 861-876.
13. Public Citizen. (2008). "But Nothing Would Have Happened Without Public Citizen,
PACE Lawsuit" http://www.citizen.org/pressroom/release.dm
14. Chemical Asbstract Service. "CAS Registry." http://www.cas.org;.
15. Frey, T. (October, 2008). "Nanotech and the Precautionary Principle." http://www.
futuristspeaker.com/2008/10/nanotech-and-the-precautionary-principal/
16. Pritchard, D. K. (2004). "Literature Review - Explosion Hazards Associated with
Nanopowders." HSL/2004/12. Health and Safety Lab. Health and Safety Executive.
Harpur Hill, Buxton, UK.
17. Oberdorster, G., Oberdorster, E. and J. Oberdorster. (July 2005). "Nanotoxicology: An
Emerging Discipline Evolving from Studies of Ultrafine Particles." 113 (7) 823-839.
18. Schulte et al. (2008) 244.
19. Perry, W.G. (May 31, 2006). "OSHAandNanotechnology: Current Activities and
Regulatory Considerations." Presentation to the National Response Team Technical
Conference. Washington, D.C.
20. Levine, D. (15 September 2006). Personal communications.
21. Lippy, B. (September, 2004). "Technology Safety Data Sheets: Tools Perceived as
Valuable by Workers, Technology Developers and Regulators." Proceedings of ICEM
03: International Conference on Environmental Remediation and Radioactive Waste
Management, Oxford, England.
Conference Questions and Answers
Question:
Material Safety Data Sheets (MSDSs) are based on Chemical Abstracts Service (CAS) Registra-
tion Numbers (RNs). How will nanomaterials be organized-as a Technology Safety Data Sheet
(TSDS) for hazardous communications (HAZCOM)?
Answer:
A change to a TSDS is probably not necessary. However, an easily accessible document is
needed that uses standard phrases. This approach has been adopted by the Europeans and the
United States' National Institute of Occupational Safety and Health (NIOSH). Standard phrases
incorporated in a safety communication document will help, but the CASRN will probably need
to be added.
Question:
How is the term "nanotechnology" used in a product description?
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Answer:
Some manufacturers use the term "nanotechnology," but there are no nanomaterials in the prod-
uct. I do not know of any regulation affecting the use of the term.
Question:
How is risk communicated in the absence of information?
Answer:
We do not want to lose the potential of the field by being overly cautious, but standard industrial
hygiene practice-for example, do not dump nanomaterials; use HEPA filters to keep particles
down-can be used.
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Q and A: Perspectives on Nanotoxicology
Panelists:
Dr. Christi Sayes, Dr. Igor Linkov, Dr. Bruce Lippy, Dr. Stacey Harper
Question:
We (Emory University Rollins School of Public Health) have been working with NIOSH
nanotechnology industry field team studies looking at occupational exposure to carbon
nanotubes, but have no characterization of different job titles that would help characterize
exposure.
Answer (Lippy):
This is a key problem. In the past, we characterized the work force by who's doing what. Now,
nobody seems to be doing this, but it should be done. NIOSH should be taking a lead.
Question:
This is a question for Igor Linkov. Your case studies look at many different types of
nanomaterials, metals and metal oxides, various forms of carbon. If you just focused on one,
aluminum oxide for example, what factors or features would you look at to determine toxicity as
compared to looking at a range of materials?
Answer (Linkov):
We have already done this. To determine a range of toxicity, you can do the same "weight-
of-evidence" procedure to develop a matrix, use professional judgment to weight the lines of
evidence, and analyze the data.
Question:
U.S. EPA and the Organization for Economic Cooperation and Development (OECD) are
working together to understand how to test nanomaterials for ecotoxicity, so that we can tell
industry how to test materials before they are marketed. In Stacey Harper's presentation there
appeared to be a mixture of exposure and response, but little characterization of the materials in
the exposure medium. Could a nanomaterial be misrepresented if it appeared to be potent in a
screen, but was, in fact, agglomerated in an exposure medium and, as a result, was less toxic?
Answer (Harper):
For many nanomaterials, we have moved on to the next level of investigation. We characterize
the materials as received and characterize them again in the exposure medium, so we are
addressing this point.
(Sayes):
It needs to be understood that nanoparticles will aggregate, agglomerate, but over time 95
percent of the nanoparticles I work with will de-aggregate.
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(Harper):
However, are the characterizations we can do at the present time sufficient? Do we have
sufficient data to be predictive? I do not think so.
(Sayes):
The methods we and the DuPont laboratory are using are adequate. However, there is a need to
be open about data, and research should be collaborative.
Question:
Where is the European Union on assessing the toxicity of nanomaterials? Are they taking the
same approach as the United States?
Answer:
The EU is taking a basically similar approach. There is a framework for judgment of toxicity,
but a verbal framework may not translate into a quantitative risk assessment. Further discussions
between the U.S. and the EU Commission are to be held in Brussels later his fall. An OECD
member-country has made a strong call for regulation and enforcement of manufacturers in
the area of product labeling. Products containing nanomaterials should be labeled with what
nanomaterials and coatings they actually contain. Some studies suggest that the Europeans are
taking a management approach and are spending more effort on looking at the implications of
nanomaterials rather than on the applications.
Question:
Given where we are now in our understanding of the toxicity of nanomaterials, how do we
communicate hazards to workers? How are hazards communicated and managed in research and
engineering laboratories?
Answer:
Many laboratories engaged in the production of nanomaterials take no precautions, and
engineering laboratory staff have been observed picking up nanomaterials with their bare hands.
We must get the message out about uncertainty, and communicate "our best guess," until we
have precise information.
(Lippy):
The National Institute for Occupational Safety and Health (NIOSH) is working on best practices
for these materials. In the meantime, good work practices are still important, and local exhaust
ventilation should be used in the work area. Uncertainty should be emphasized, and we must be
honest about what we do not know.
Question:
Given that risk is a product of hazard and exposure, are we looking at the wrong end of this? We
have little information on the raw materials, what they eventually turn into in the environment,
and what happens in the event of a release.
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Answer:
None.
Question:
What happens to these materials at the end of their life cycle, when they are cycling in the
environment?
Answer:
This is being studied, but as yet there is little information in this area.
Question:
There is a term "over engineering." Are we in danger of "over-toxicity" for nanomaterials?
Answer:
I do not think so. What we learn from nanotoxicity could be applied to nanomedicine.
Comment:
There is a fundamental difference between the nanotoxicity of consumer products and
nanomedicine that is regulated by the Food and Drug Administration (FDA). The FDA demands
that materials are safe and effective, and that you know what happens to nanomaterials in the
body after use. There is a requirement that nanomaterials are tracked through the body. However,
consumer products containing nanomaterials may degrade and end up in a landfill, subject to the
exotic chemistry of that location, and may end up in water streams.
(Linkov):
You cannot stop industrial progress, but you can go ahead with structured hypothesis testing. We
will learn from mistakes. We need to continue studies and collect information.
Comment:
It is important to assess risk over the life cycle of nanomaterials. As pharmaceuticals are now
turning up in drinking water, the question of where nanomaterials will end up is not irrelevant.
Worker exposure is probably the most important current exposure at the moment.
Question:
Nanotechnology is a great source of federal funding. Not all nanotechnology is recent; it has
been termed colloid science and catalytic science in the past. Colloidal gold was used before the
Romans in the production of colored glass, and nanomaterials were released when the first log
was set on fire. How do you assess the risk from nanomaterials, given that we are bombarded by
them all the time? How do you adjust the risk assessment to take them into account?
Comment:
At last year's Society of Toxicology (SOT) meeting, a member from the National Aeronautics
and Space Administration (NASA) stated that all human cancers are due to nanoparticles. That
was an outlandish statement. Perhaps if you can isolate antibodies to specific nanomaterials and
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look at their interactions, you might be able to isolate cause and effect.
Comment:
There is a need to work with industry. They could be given quick information on toxicity in
return for their maintenance of a national database on nanomaterial toxicity.
Comment:
That is a beautiful idea, but there is a problem with proprietary information. Regulators want full
information, but industry is usually unwilling to provide it. Maybe confidentiality agreements
could be signed. There is a need for a database that will enable us to be predictive.
Comment:
A database could be constructed using metadata analysis of the characterization and effects
of nanomaterials, without being explicit about the precise nature of the materials. Proprietary
information need not be revealed, and QSARs (quantitative structure-activity relationships) need
not be tied tightly to a particular product.
Question:
Are there any messages for average consumers that can be distilled down to help them estimate
their own risk?
Answer:
We can divide nanomaterials into broad categories-benign and more risky. It may then be
possible to engineer materials with properties that make them less toxic. We can try to integrate
risk within set properties and ranges.
Question:
Then this is a development process to aid industry-this material may cause too much risk to
continue to develop?
Answer:
Igor Linkov's models may be used to help industry to decide what to use or proceed with
by providing "GO'VNO GO" indications. Eventually consumers may be able to use this
information.
Linkov:
The drug companies exploit QSAR to engineer and design drugs. It should be relatively
inexpensive for a manufacturer to perform some up-front toxicity tests.
Question:
Is this what the nanorisk framework is about, "GO'VNO GO" decisions?
Answer:
Yes.
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Question:
How do nanomaterials affect consumers? We need to look first at potential health effects for
workers and then move towards consumers.
Answer:
Look at work in Europe by Peter Wick. Look at sensitive sub-populations, such as pregnant
women. Nanomaterials move across the placenta. That's where some real progress can be made.
Question:
What are we looking at for an exposure metric-concentration, chirality, surface charge?
Answer:
We should look at all of them. We need someone to perform a search of the current literature.
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Bioavailability and Toxicity of Nickel in Metallic Nanoparticles
Ashley Smith, Department of Pathology and Laboratory Medicine, Brown University,
Providence, Rhode Island, U.S.A.
Xinyuan Liu, Division of Engineering, Brown University, Providence, Rhode Island, U.S.A.
Kevin McNeil, Department of Pathology and Laboratory Medicine, Brown University,
Providence, Rhode Island, U.S.A.
Robert Hurt, Division of Engineering, Brown University, Providence, Rhode Island, U.S.A.
Agnes Kane, Department of Pathology and Laboratory Medicine, Brown University, Providence,
Rhode Island, U.S.A.
Abstract
Catalytic routes are used for arc synthesis of commercial carbon nanotubes and nickel is a
common metallic catalyst. Nickel and nickel oxide nanoparticles are also used commercially
in nanomagnetic devices, batteries, fuel cells, catalytic converters, and solar cells. Nickel is
classified as a known human carcinogen and is a common industrial and environmental pollutant.
Recent studies have shown that metallic nickel nanoparticles induce greater acute lung toxicity
and inflammation than micron-sized nickel particles following intratracheal instillation in
rodents that has been attributed to elevated surface area and reactivity of nanoparticles. Lung
epithelial cells are a primary target for nickel-induced toxicity following inhalation of poorly
soluble nickel particles. The proposed mechanism responsible for nickel toxicity is phagocytosis
or endocytosis of particulate nickel by target cells. It is postulated that intracellular release
of Ni (II) ions from the acidic environment of endosomes could interact with cytoplasmic or
nuclear protein targets. In acellular assays, Ni (II) ions were mobilized from metallic nickel
nanoparticles and mobilization was enhanced at acidic pH. Human lung epithelial cells (H460)
internalized metallic nickel nanoparticles within 24 hours in vitro. Intracellular mobilization of
Ni (II) ions was greater from metallic nanoparticles than from micron-sized particles. Metallic
nickel nanoparticles also induced dose-dependent toxicity in parallel with intracellular Ni (II)
ion mobilization as assessed by morphology, Syto-10/ethidium homodimer viability assay,
and Pico Green fluorescence to quantitate cellular DNA. These experiments support a role for
mobilization of Ni (II) ions from nickel nanoparticles in the enhanced toxicity of these metallic
nanomaterials.
Introduction
Carbon nanotubes and metallic nanoparticles have great potential as novel chemical sensors
and for new remediation technologies at Superfund and other toxic waste sites. Adverse
human health effects due to occupational and environmental exposure to nanomaterials are a
major concern and a potential threat to their successful commercialization and environmental
and biomedical applications (Duffin et al., 2007). Realization of their commercial potential
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will require a better understanding of the interactions of nanomaterials with biological
systems and the development of new strategies to minimize human health risk. Manufactured
nanomaterials are highly variable with respect to chemical and physical properties, state of
aggregation, and purity. Toxicological screening is urgently needed to identify potentially
hazardous nanomaterials and to re-engineer or post process these materials to minimize adverse
environmental and health impacts (Borm et al., 2006).
Transition metal catalysts including Fe, Ni, Y, Co, or Mo are used in the manufacture of
commercial carbon nanotubes (Donaldson et al., 2006). Transition metals in the form of
metallic nanoparticles or metal oxides are also useful as high-efficiency catalysts for chemical
remediation of contaminated ground water (Kanel et al., 2005), nanomagnetic devices, and the
next generation of energy technologies including biomass gasification (Li et al., 2008), fuel cells,
catalytic converters, and solar cells (Irwin et al., 2008). Transition metals in various chemical
and physical forms are known to be toxic and in the case of nickel and cobalt, known human
carcinogens (Lison, 1996; Salnikow and Zhitkovich, 2008). Recent studies have shown that
metallic nickel nanoparticles induce greater acute lung toxicity and inflammation than micron-
sized nickel particles following intratracheal instillation in rodents that has been attributed to
elevated surface area and reactivity of nanoparticles (Zhang et al., 2003; Monteiller et al., 2007).
Human lung epithelial cells (H460) were used as target cells to investigate bioavailability and
toxicity of nickel in metallic nanoparticles. These experiments had three objectives: 1) to assess
whether nickel (II) ions could be mobilized extracellularly from metallic nickel particles, 2) to
determine whether human lung epithelial cells internalize metallic nickel particles and whether
nickel (II) ions are mobilized intracellularly, and 3) to assess the acute toxicity of metallic nickel
particles relative to soluble MC12 in this model system.
Methods
Characterization of Nickel Particles
Nickel (II) chloride hexahydrate and metallic nickel particles were purchased from Sigma-
Aldrich and sterilized at 400°C for 15 minutes under nitrogen gas. Surface areas were measured
by the Brunauer-Emmett-Teller (BET) method and were determined to be 0.8 m2/g for nickel
microparticles and 4.4 m2/g for nickel nanoparticles. Zeta potential was measured as 27 mV
for the microparticles and -29 mV for the nanoparticles suspended in phosphate buffered saline,
pH 7.2. Samples were sonicated for one hour in a Branson 2510 sonicating water bath before
exposure to cells.
Mobilization of Nickel (II) Ions
Nickel (II) mobilization into cell culture medium was determined directly using ICP-OES
analysis as described in Liu et al., 2007 over a range of doses equivalent to 1-10 ug/cm2 used
in the cell toxicity assays. Intracellular mobilization of soluble nickel (II) ions was visualized
by Newport Green fluorescence as described previously (Ke et al., 2007). Newport Green
dichlorofluorescein diacetate (Molecular Probes) is a cell permeant fluorescent probe that was
diluted in dimethylsufloxide and Pluronic F127 detergent at a 1:1 ratio prior to loading at a final
concentration of 5 um for 30 minutes at 37C. The cells and all cell culture labware were washed
with Hank's balanced salt solution/lmM EDTA, pH 7.2 to chelate any extracellular metal ions
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prior to loading. Fluorescence was visualized using a Nikon Eclipse E800 microscope and
digital images were recorded.
Cell Culture and Toxicity Assays
The human lung epithelial cell line NCI-H460 was purchased from ATCC and cultured in
monolayer in RPMI 1640 (GIBCO) containing 2% fetal bovine serum (Atlanta Biologicals) and
1% penicillin/streptomycin at 37°C in 6% CO2, 94% air.
Cell Toxicity
Cell toxicity was evaluated by transmission electron microscopy, phase contrast microscopy,
Syto-10/ethidium homodimer viability assay (Molecular Probes), and PicoGreen (Invitrogen)
fluorescence induced by binding to cellular DNA.
Results
Previous studies (Liu et al., 2007) demonstrated mobilization of nickel at pH 5.5 in acetate
buffer from commercial nickel particles at a dose of 200 ug nickel/ml (Figurel). As particle
size decreases, significantly more nickel is mobilized from nickel nanoparticles compared to
nickel microparticles. Based on this acellular assay, it is hypothesized that metallic nickel
nanoparticles would lead to intracellular nickel mobilization and produce greater toxicity than
nickel microparticles. Lung epithelial cells are a primary target for nickel-induced toxicity and
carcinogenicity in humans following inhalation of poorly-soluble nickel particles (Oiler et al.,
1997). A human lung epithelial cell line (H460) was used to test this hypothesis. Cellular uptake
of <100 nm and 3 um nickel particles was demonstrated by transmission electron microscopy
after 24 hours of exposure (Figure 2). Agglomerates of metallic nickel nanoparticles were
seen in cytoplasmic vacuoles. It is hypothesized that enhanced release of nickel (II) ions from
nanoparticles would occur in the acidic environment of endosomes or phagolysosomes (Costa
Figure 1. Nickel mobilization from carbon nanotubes and nickel particles (Liu et al., 2007)
35
t? 30
Q.
Q-25
20 -
T3
g15
= 10
SWNT I—pure Ni powder, various size
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et al., 2005). After 48-72 hours of exposure to metallic nickel nanoparticles, there was a dose-
dependent increase in Newport Green fluorescence that was not observed following exposure to
1 um metallic nickel particles (Figure 3).
Nickel (II) ions were also mobilized from nickel nanoparticles after 72 hours in cell culture
medium; however, at the highest dose equivalent to 10 ug/cm2 used in the cell toxicity assay,
only 0.45-0.68 ppm of nickel was mobilized. This extracellular level of nickel is too low to
induce toxicity in human lung epithelial cells. Direct exposure to 50 uM -100 uM NiCl2 is
required to induce necrosis in H460 cells after 24 hours (Figure 4). Exposure to H460 cells to
metallic nickel nanoparticles induced dose-dependent toxicity after 48-72 hours as assessed by
Syto-10/ethidium homodimer viability assay (Figure 3), phase contrast microscopy (Figure 3),
and PicoGreen fluorescence (Figure 4). No toxicity was induced by exposure to H460 cells to
metallic nickel microparticles over this range of doses.
Discussion and Conclusions
These studies show enhanced mobilization of nickel (II) ions from metallic nanoparticles in
comparison with metallic micoparticles. Intracellular nickel (II) mobilization occurred after 24
hours of exposure of human lung epithelial cells to metallic nickel nanoparticles, followed by
cell toxicity and necrotic cell death after 48-72 hours.
The proposed mechanism responsible for nickel toxicity is phagocytosis or endocytosis of
poorly-soluble nickel compounds by target cells (Oiler, 2002). Nickel may be delivered to cells
following uptake of metallic particles, either as single particles or as respirable agglomerates.
Intracellular mobilization of nickel (II) ions is postulated to occur in the acidic environment of
endocytic or phagocytic vacuoles leading to release of nickel (II) ions into the cytoplasm and
Figure 2. Internalization of metallic nickel nanoparticles by human lung epithelial cells in
monolayer culture.
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Newport Green
fluorescence - 72 hours-
(200x)
Phase Contrast
Microscopy - 72 hours
Untreated
Syto-10/Ethidium Homodimer
Viability Assay - 72 hours (1 OOx)
10[Ag/cm
Figure 3. Exposure of human lung epithelial cells to metallic nickel nanoparticles leads to
intracellular release of nickel (II) and toxicity after 48-72 hours.
nucleus (Costa et al., 2005). This nickel-ion hypothesis has been proposed as the mechanistic
basis for nickel toxicity and carcinogenicity (Kasprzak et al., 2003). Nickel (II) ions have been
shown to have both genetic and epigenetic effects that contribute to development of lung cancer
(Denkhaus and Salnikow, 2002; Salnikow and Zhitkovich, 2008).
Bioavailability of nickel has been demonstrated in metallic nickel nanoparticles using acellular
assays (Liu et al., 2007) and has been confirmed in a human lung epithelial cell assay in these
120 1
|
"3 um
-90nm
-NiO
Dose (ug/cm2)
Figure 4. Toxicity of metallic nickel nanoparticles and microparticles after exposure of
human lung epithelial cells for 72 hours.
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studies. These results raise concern regarding potential toxicity and carcinogenicity of metallic
nickel catalyst residues mobilized from carbon nanotubes or nickel nanoparticles following
inhalation during manufacture and use of these nanomaterials.
Acknowledgements
This research was supported by a Superfund Basic Research Program Grant from the National
Institutes of Environmental Health Sciences (P42 ES013550) and aNanoscale Interdisciplinary
Research Team Grant from the National Science Foundation (NSF DMI-05066. Although
this research was funded by NIEHS and NSF, it does not necessarily reflect the views of these
agencies.
References
M. Costa, T.L. Davidson, H.Chen, Q. Ke, P. Zhang, Y. Yan, C. Huang, and T. Kluz, (2005)
"Nickel carcinogenesis: epigenetics and hypoxia signaling." Mut. Res. 592:79-88.
K. Donaldson, R. Aitken, L. Tran, V. Stone, R. Duffin, G. Forrest, and A. Alexander, (2006)
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M.D. Irwin, D.B. Buchholz, A.W. Hains, R.P.H. Chang and T.J. Marks. (2008) "p-Type
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C. Monteiller, L. Tran, W. MacNee, S. Faux, A. Jones, B. Miller and K. Donaldson. (2007) "The
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Conference Questions and Answers
Question:
How can dose best be represented? You compared nano and microsize particles. Did you apply
the same mass as concentration for both of these?
Answer:
Yes. All volumes, concentrations, and surface areas were the same.
Question:
Was this true if dose is expressed as particle concentration?
Answer:
A larger number of nanoparticles were used, and the number of microparticles was smaller, but
the amount of nickel was the same for both.
Question:
How did you choose the dose and the dose response curves?
Answer:
Dose response curves were performed to optimize the dose. We also found mobilization in the
cell at 200 micromoles (umol) soluble nickel, so we used this information as well.
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96
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Partial Oxidation (Aging) and Surface Modification Decrease the Toxicity of
Nano-Sized Zero-Valent Ironz
Tanapon Phenrat, and Gregory Lowry, Carnegie Mellon University, U.S.A.
Thomas Long, U.S. EPA National Center for Environmental Assessment, U.S.A.
Bellina Veronesi, U.S. EPA National Health and Environmental
Effects Research Laboratory, U.S.A.
Abstract
Zero-valent iron (nZVI) is a redox-active nanomaterial used for the in-situ remediation of
contaminated groundwater. To assess the effect of "aging" and surface modification on its
potential neurotoxicity, rodent microglia and neurons were exposed in vitro to fresh nZVI,
"aged" (>11 mo) nZVI, magnetite, and polyaspartate surface-modified (SM) nZVI. Increases in
oxidative stress occurred in BV2 microglia in the following rank order: nZVI > "aged" nZVI
> magnetite = SM nZVI. ATP levels were reduced in N27 neurons in the following rank order
nZVI > SM-nZVI >"aged" nZVI = magnetite. nZVI and SM-ZVI nanoparticles produced
ultrastructural changes in exposed neurons. Physicochemical properties of each material,
measured under exposure conditions indicated that all had electronegative zeta potentials. nZVI
sedimented and agglomerated more rapidly than SM-nZVI or other materials. Correlating
these properties with toxicity indicates that oxidation of nZVI decreases its redox activity,
agglomeration, sedimentation rate and toxicity to mammalian cells.
Introduction
Nano size, zero valent iron (nZVI) rapidly degrades contaminants relative to iron filings because
of its high surface area, high redox activity and unique catalytic activity. nZVI also generates
reactive oxygen species (ROS) though Fenton chemistry. In aqueous environments, nZVI
oxidizes over time (i.e., "ages") to magnetite (Fe3O4), and other oxides such as hematite and
goethite. For in situ applications, concentrated (-10 g/L) slurries of nZVI are injected directly
into the ground at or near the source of contamination where they rapidly form immobile
agglomerates due to attractive magnetic forces. Since the mobility of bare nZVI is limited to a
few centimeters, "second generation" nZVI particles are being developed that can be surface-
modified (SM) with polymers or surfactants to increase their migration and therefore proximity
to the pollutant materials. This increased mobility, as well as nZVFs direct application to
groundwater also increases the likelihood that that nZVI will disperse more widely in the
environment and at low concentrations, could enter the ecosystem and food chain and impact
biological systems.
Several studies indicate that ingested or inhaled nanoparticles can cross biological barriers (i.e.,
alveolar, intestinal, testes, dermal) and migrate in small numbers to various organs and tissues
where they can potentially damage organ systems sensitive to oxidative stress (OS) such as brain.
To examine the possible neurotoxicity of nZVI and its related products, OS_sensitive rodent
brain cells (BV2 microglia and N27 neurons) were exposed in vitro, to fresh nZVI, "aged"
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nZVI, magnetite, and polyaspartate (poly AA)SM-nZVI. Measures of OS and cytotoxicity were
collected and related to their particle size distribution, zeta potential, and dispersion stability of
material under exposure conditions.
Methods
Fresh nZVI and SM-nZVI (MRNIP) were purchased from Toda Kogyo (Onada, Japan). The
preparation of the nZVI formulations, cell culture assays, morphological preparations and
methods for PC measurements have been previously reported.
Results
OS and BV2 Microglia. Intracellular H2O2 generated from the oxidative burst, depolarization of
the mitochondrial membrane, and increases in caspase 3/7 activity occurred only in response to
fresh nZVI and/or "aged" nZVI. Reductions of ATP occurred in the following rank order: fresh
nZVI > "aged" ZVI > magnetite. SM-nZVI did not produce OS or apoptosis. Ultrastructurally,
large nZVI agglomerates, housed in membrane-bound vacuoles, occurred in close proximity to
populations of disrupted mitochondria in B V2. Light microscopy indicated that their nuclei
were swollen and centrally located, a morphology suggestive of apoptosis.
Neurotoxicity in N27 neurons. ATP levels of were reduced after a 6 h exposure in the
following rank order: Fresh nZVI > SM-nZVI > "aged" nZVI = magnetite. Ultrastructurally,
the nuclei of nZVI exposed N27 neurons displayed a perinuclear and pericellular distribution
of floccular material. The cytoplasm of N27 neurons, treated with SM-nZVI appeared normal
Ultrastructurally, although small agglomerates (-200-300 nm) and single nanosize particles of
electron dense nZVI appeared throughout and within the cell's nuclei and mitochondria. Frequent
examples of electron dense membrane invaginations, suggestive of clathrin-lined endocytotic
vesicles were also noted in SM-nZVI treated neurons.
Particle Characterization. Fresh nZVI, and MRNIP contained 35±1 and 24±2% FeO content,
respectively and indicated that both were redox active. In contrast, the FeO content of "aged"
nZVI was negligible or absent for magnetite. The zeta potential measured in exposure vehicle,
was consistently electronegative. In physiological buffer,, the zeta potential ranged from -13.8
mVto-18.6mVandinBV2 and N27 media they ranged -7.1 mVto-10.1 mV and -S.OmVto
-13.3 mV, respectively. The particle size distributions for the different nZVI materials, measured
under exposure conditions indicated a bimodal distribution in all exposure vehicles and
contained both small (hydrodynamic radius, (RH)<1 um) and large (RH>2 um) agglomerates.
The sedimentation of particles measured in each exposure vehicle, indicated that fresh nZVI
agglomerated and sedimented faster relative to the other materials. The slow agglomeration and
sedimentation rate of polyAA-SM-nZ VI was due to the electrosteric stabilization of its poly AA-
coating.
Discussion
The present data show that nZVI materials (i.e., fresh, "aged" and SM-nZVI) are differentially
toxic to mammalian nerve cells. In every instance, fresh nZVI was more toxic relative to its
"aged", oxidized or surface modified counterpart. Fresh nZVI produced higher levels of ROS
in microglia and was more cytotoxic to N27 neurons. Interactions between nanoparticles and
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the biological response of cells are influenced by physicochemical properties, including size,
aggregation state, surface charge, and the presence of surface coatings. Our data indicated that
differences in bulk chemistry, redox activity surface chemistry and dispersion contribute to the
differential toxicity of the nZVI formulations. The poly AA surface coating affected both particle
agglomeration and surface charge as well as cellular internalization. The agglomeration and
subsequent sedimentation plausibly affected the exposure of the particles to cells in culture by
modifying the amount and rate at which the materials physically contacted the cells.
Conclusions
These results have important implications on using nZVI materials for ground water remediation.
The unmodified nZVI particles are relatively immobile, oxidize in water and "age" over months
into the less toxic magnetite and/or maghemite. This indicates that bare nZVI particles have a
relatively low risk to ecosystems. Surface coating increases nZVI's dispersion to the subsurface
and also appears to decrease its toxicity. However, the coating facilitates the particle's physical
entry into into cultured neurons, a finding that may have long term neurotoxic consequences.
Conference Questions and Answers
No. Questions.
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100
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Ecotoxicological Evaluation of Carbonaceous and Metal Nanoparticles
Through Bioassays Relevant to Environmental Fate
AlanJ. Kennedy, Jacob K. Stanley, Jessica G. Coleman, DavidR. Johnson, and
Jeffery A Steevens, Environmental Laboratory, US Army Engineer
Research and Development Center, Vicksburg, Mississippi, U.S.A.
Abstract
This proceeding summarizes nanomaterial (NM) fate and toxicity investigations conducted at the
U.S. Army Engineer Research and Development Center (ERDC) on several Army relevant NMs
(fullerenes, nanotubes, aluminum oxides).
Introduction
As the novelty of nanoscience evolves into product development, ecotoxicologists are challenged
to proactively gain fate and toxicology information for a diverse suite of nanomaterials (NMs).
Unique NM properties may require flexibility in existing risk assessment (RA) approaches (EPA
1998, Davis 2007). Environmental RA includes key components (e.g., hazard identification,
exposure and effects, risk characterization) that necessitate identification of NM specific research
tools and information (Dale et al. 2008). Hazard identification uses a conceptual model to
identify relevant pathways, media, and receptors; we provide a generic conceptual model for
NM (Figure 1) to guide research towards relevant NM sources, media, and exposure pathways
(Metcalf et al., 2008). It is hypothesized that NM surface and suspension chemistry are
controlling factors determining exposure pathways in biphasic systems. The learning curve is
steep for establishing appropriate quantification, dosimetry and presumably unique fate, transport
and biological uptake information needed for RA. In the present paper, we summarize our study
of the partitioning and toxicity of raw fullerenes (C60), raw and functionalized multi-walled
nanotubes (MWNTs) and aluminum oxides (A12O3) in aquatic and terrestrial systems, to begin to
gain the type of information needed for RA.
Methods
Test materials. Dry fullerene (C60) powder (99.5% purity) was obtained from SES Corp (CAT
No. 600-9950, Lot BT-6977). Raw MWNT, MWNT-OH and MWNT-COOH (Cheaptubes, Inc.,
Brattleboro, VT, USA) and reference carbon (carbon black, activated carbon) are described in
Kennedy et al. (2008). Nano A12O3 (11 nm) was obtained from Nanostructures and Amorphous
Materials (Houston, TX, USA).
Analytical andfate. Dispersions of C60 were created by adding 200 mg/L dry powder to
various waters (Milli-Q, moderately hard reconstituted water or MHRW, 3 %o and 20 %o) and
magnetically stirring for four weeks (Oberdorster et al. 2006). Three replicates were sampled
at various timepoints, allowed to settle for 24-h and analyzed for concentration by extracting
C60 particles in toluene after H2O2 oxidization (Deguchi et al 2001; Oberdorster et al 2006) and
analyzing by photospectrometry (X = 336 nm) against a standard curve of known concentration.
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1. Sources
Engineered
Nanostructures
Engineered
Nanodevices
Reaction
Intermediates
Production
Waste
Product
Degradation
Product use, Spills
Intentional application,
disposal
2. Media and
Transport Processes
3. Exposure Pathways
and Receptors
Air
Aggregation/
LIV Degradation
Settling /suspension
Sorption / desorption
Water
Aggregation/ '-.
Degradation '
Settling/suspension
Sediment
Degradation
Figure 1. Conceptual model for potential nanoparticle and nanomaterial sources, fate, media
and transport processes, and exposure pathways and receptors.
NMs were analyzed at ERDC using a Nova 3200e (Quantachorme Instruments, Boynton
Beach, FL, USA), energy-dispersive X-ray (EDX) spectrometer (Quantax system, Bruker
AXS, Ewing, NJ, USA) a 90Plus/BI-MAS (Brookhaven Instruments, Holtsville, NY, USA) and
ZetaPALs (Brookhaven Instruments, Holtsville, NY, USA) for exposed surface area, elemental
composition, aqueous particle diameter and particle charge, respectively. Particle concentrations
(A12O3) in tissue, sediment, and soil were quantified by inductively coupled plasma-atomic
emission spectrometry (ICP-AES) or inductively coupled plasma-mass spectrometry (ICP-MS).
Bioassay. The cladoceran, Ceriodaphnia dubia (Aquatic Biosystems, Fort Collins, CO,
USA) was the selected model for water toxicity studies on stable C60 and MWNT sols,
tested at 25 ± 1° C for 48-h (U.S. EPA2002). Methods for spiking sediments with MWNTs
for Leptocheirusplumulosus and Hyalella azteca sediment tests (U.S. EPA 1994, 2000) are
described in Kennedy et al. (2008). Nano and bulk A12O3 were comparatively tested in sediment
(H. azteca, Tubifex tubifex, Lumbriculus variegatus, Corbicula flumined) and soil (Eisenia
fetidd). Hyalella response to nano or bulk A12O3 was characterized in a 10-d exposure (survival,
growth, bioaccumulation) in sediment homogenates (1-h rotary mixing at 1200 rpm) and a
14-d exposure to a thin surficial layer (0.625 or 2.5 g) on sediment or sand. A 10-d T. tubifex
sediment toxicity (survival) bioassay and 28-d L. variegates and C.fluminea bioaccumulation
bioassay were performed according to US EPA/US ACE (1998). A12O3 was homogenized in
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sediment for the T. tubifex, L. variegatus, and C.fluminea assays as above. Terrestrial exposures
of E. fetida included a 28-d bioaccumulation, 48-h soil avoidance bioassay, and dermal uptake
screening study. Bioaccumulation studies followed ASTM (1999) guidelines, with modifications.
Soil (Grenada Loring (GL) Silty Loam, Learned, MS, USA) was spiked with increasing
concentrations of bulk (tested range 0-13 g/kg) and nano (tested range 0-10 g/kg) A12O3 tumbled
for 24 h and hydrated prior to earthworm addition. Soil avoidance bioassays were conducted
according to Environment Canada (2004). The acute (72-h) dermal uptake study followed a
modified version of OECD guideline 207 (1984) using 1 ml of bulk and nano A12O3 solutions
(tested range 0-10,000 mg/L) to spike Whatman #1, 9 cm filter paper in 20 mL scintillation vials.
Statistical significance was determined using one-way analysis of variance and Dunnett's test
(SigmaStat 3.0, SSPS, Chicago, IL, USA). Lethal median concentration values (LC50) were
determined by trimmed Spearman Karber (Toxstat, Gulley, 1996).
Results
Analytical and fate. For C60, we determined suspension characteristics such as high ionic
strength (tested range: 0-20 %o), low pH (tested range: 4-10) and low natural organic matter
(NOM) concentrations (tested range: 0-100 mg/L) resulted in less stable C60 aggregates and
reduced repulsive forces, measured as zeta potential (£,). Dispersions of C60 were more stable
in Milli-Q water relative to MHRW. Primary particles were not observed in any dispersion and
concentration and aggregate size were charge related (Figure 2). The MWNT and reference
carbon agglomerated and settled rapidly in fresh, estuarine and marine waters, and DLS indicated
smaller effective diameter after sonication (Figure 3). However, NOM served as an effective
surfactant, resulting in stable dispersions (£, = -20 to -23 mV). The charge of A12O3 particles in
the dechlorinated tap (DTW) water used in sediment bioassays at neutral pH was negligible.
Aqueous particle size characterization (DLS) of the nano A12O3 in DTW yielded a bimodal
particle distribution (120-170, 300-500 nm).
Bioassay. Water column exposure of stable C60 concentration (5 mg/L) did not result in toxicity
to C. dubia. Neither A12O3 particle size resulted in toxicity to T. tubifex. The growth of//.
azteca was reduced only at the highest treatment of nano A12O3 (100 g/kg). However, measured
sediment Al concentrations from the bulk- and nano-spiked sediments differed substantially
(55.1 ± 0.6 and 66.2 ± 0.6 g/kg, respectively). In thin layer exposures, nano A12O3 was more
toxic to H. azteca than bulk, with greater nano A12O3 toxicity in sand relative to sediment. In
28-d bioaccumulation studies, biota-sediment accumulation factors were similar between nano
and bulk A12O3 for Corbiculafluminea but two-fold greater in Lumbriculus variegatus exposed
to bulk A12O3. Acute and chronic terrestrial toxicity and bioaccumulation studies with E. fetida
determined nano A12O3 reduced reproduction relative to controls and bulk A12O3. Body burdens
were higher in nano A12O3 exposures relative to control and bulk exposures in chronic soil
studies. Filter paper exposures resulted in greater uptake in both nano and bulk A12O3 relative to
control, although uptake was not dose dependent. Behavioral results suggested E. fetida preferred
control to both nano and bulk amended soils (5, 10 g/kg).
Discussion
Particle dispersions with charges (£,) greater than +20 or lower than -20 mV may be most
appropriate for short- to long-term (> 48-h) water exposures while particle dispersions inside
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(a)
70 -,
60 -
50 -
40 -
30 -
20 -
10 -
0 -
MilliQ
MHRW
3ppt
19ppt
10 20
Time (days)
30
2500 -,
_ 2000 -
E
1500 -
1000 -
500 -
-50 -
E -40 -
§ -30--
o
-20 -
-10 -
(b)
2 day/1 day 8 day /1 day 16 day/1 day 35 day /1 day 35 day / 7 day
(c)
li-QDMHRWD3%o
< -30 mV "stable"
Stirred 10 days, settled Stirred 35 days, settled Stirred 35 days, settled
1 day 1 day 7 days
Figure 2. Aqueous fullerene (C60) dispersion characterization in milli-Q, moderately hard
reconstituted water (MHRW), estuarine (3 %o) and marine (19 %o) waters. Panel (a) illustrates
suspended concentration (C60), panel (b) shows the population size distribution in MHRW as
determined by dynamic light scattering and panel (c) shows particle charge as zeta potential.
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700
600
500
"3
n 400
^
0)
~ 300
I
m 200
100
0
AC
CB
MWNT
Figure 3. Effective diameters (nm) of carbon particles after magnetic stirring or probe
sonication as indicated by dynamic light scattering. AC = activated carbon, CB = carbon black,
MWNT = multi-walled nanotube.
this range may be less stable and more likely to partition to sediment. While fullerenes were
not toxic at stable concentration (5 mg/L), it is likely higher stable C60 concentrations can be
obtained by stirring concentrations less than 200 mg/L (Duncan et al. 2008). Additionally,
exposure to C60 at higher, albeit unstable, concentrations (settling within 48-h) or longer
exposures induce significant C. dubia mortality. Raw MWNT in NOM were toxic to C. dubia at
40-50 mg/L and 18 mg/L after 48-h and 96-h but MWNT-OH/COOH were not toxic up 80 mg/L
(Kennedy et al 2008).
Since MWNTs in absence of NOM and A12O3 dispersions were near the iso-electric point,
sediment or soil bioassays were conducted. While MWNTs were toxic at very high
concentrations (> 60 g/kg), they were less toxic than reference carbon (LC50: 19 - 27 g/kg)
(Kennedy et al., 2008). This may occur for different reasons (e.g., micro-sized length dimension,
stirring does not fully de-agglomerated MWNTs). Current studies in our laboratory indicate
that sonication of MWNT in carriers allows for better dispersed, homogenous sediment. For
A12O3 sediment exposures, 10-d H. azteca growth was more sensitive to nano (100 g/kg) than
bulk A12O3 but substantial (-11 g/kg) differences in concentrations of bulk- and nano-spiked
sediment precluded direct particle size toxicity comparison. A size-related toxicity effect was
potentially indicated as nano A12O3 reduced survival more than bulk in both sand and sediment
in the thin layer exposure; greater toxicity in sand relative to sediment may indicate nano A12O3
bioavailability to H. azteca may change with substrate type. Species-specific differences in
relative bioaccumulation of bulk and nano A12O3 were observed, potentially related to differing
life characteristics and feeding (H. azteca, epibenthic detritivore; L. variegates, infaunal
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sediment ingester, C.fluminea, facultative filter feeder). While the terrestrial fate of A12O3 is yet
to be fully characterized, Doshi et al (2007) reported that dissolution, formation of A1(OH)3, and
surface charge characteristics of nano Al coated with A12O3 in a water/sand mixture may indicate
increased transport in soil mixtures relative to pH. Nano A12O3 chronic soil exposures resulted
in decreased reproduction. Dermal (filter paper) exposures were difficult to interpret, as the high
bulk exposures resulted in greater tissue concentrations than nano A12O3 Aluminum recovery in
high treatment bulk exposures may suggest ingestion of material or adherence of particles to the
dermis. Earthworms avoided soils containing the highest concentrations for both nano and bulk
A12O3 (5, 10 g/kg). In combination with the chronic study, these data may indicate that impacts
from nano A12O3 on earthworms and aquatic benthos may occur at very high concentrations,
unlikely to be found in the environment, in soil (2.5 g/kg) and sediment (66.2 g/kg Al). More
studies are needed to determine the potential for longer term exposure, different NM preparation
methods and dermal uptake to satiate risk assessment needs.
References
ASTM (1999) Standard Guide for Conducting a Laboratory Soil Toxicity or
Bioaccumulation Tests with the Lumbricid Earthworm Eisenia fetida and the Enchytraeid
Potworm Enchytraeus albidus. ASTM Standards 11.04: E 1676-97.
Dale, VH, GR Biddinger, MC Newman, JT Oris, GW Suter, T Thompson, TM Armitage, JL
Meyer, RM Allen-King, GA Burton, PM Chapman, LL Conquest, IJ Fernandez, WGLandis, LL
Master, WJ Mitsch, TC Mueller, CF Rabeni, AD Rodewald, JG Sanders, and IL van Heerden.
(2008) "Enhancing the Ecological Risk Assessment Process." Integr Environ Assess Manag. 4:
306-313.
Davis, JM. (2007) "How to Assess the Risks of Nanotechnology: Learning from Past
Experience." Journal of'Nanoscience and Nanotechnology. 7:402-409.
Deguchi, S., R.G. Alargova and K. Tsujii. (2001) "Stable dispersions of fullerenes, C60
and C70, in water. Preparation and characterization." Langmuir 17: 6013-6017.
Doshi, R, W. Braida, C. Christodoulatosa , M. Waznea, and G. O'Connor. (2007)
"Nano-aluminum: Transport through sand columns and environmental effects on plants and soil
communities." Environmental Research 106:296-303.
Duncan, L.K., J.R. Jinschek, and PJ.Vikesland. (2008) "C60 Colloid Formation in
Aqueous Systems: Effects of Preparation Method on Size, Structure, and Surface." Charge
Environ Sci Tech 42: 17
Environment Canada. (2004) "Biological Test Method: Tests for Toxicity of Contaminated Soil
to Earthworms (Eisenia Andrei, Eisenia fetida, or Lumbriculus terrestris)." Method Development
andAppl Sec Environ Tech Center 3-178.
Kennedy, A.J., M.S. Hull, J.A. Steevens, K.M. Dontsova, M.A. Chappell, J.C. Gunter,
C.A. Weiss, Jr. (2008) "Factors influencing the partitioning and toxicity of nanotubes in the
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aquatic environment." Environ Toxicol Chem 27: 1932 - 1941.
Metcalfe, C., Bennett, E., Chappell, M., Steevens, J., Depledge, M., Goss, G., Goudey,
S., Kaczmar, S., O'Brien, N., Picado, A., and A.B. Ramadan. (2008). "SMARTEN: Strategic
Management and Assessment of Risks and Toxicity of Engineered Nanomaterials." In:
Nanomaterials: Risks and Benefits. 2008. Eds. I. Linkov and J. Steevens eds. Springer Press,
Dordrecht, The Netherlands. 93-108.
Oberdorster, E., S. Q. Zhu, T. M. Blickley, P. McClellan-Green and M. L. Haasch.
(2006). "Ecotoxicology of carbon-based engineered nanoparticles: Effects of fullerene (C-60) on
aquatic organisms." Carbon 44: 1112-1120.
Organization for Economic Cooperation and Development. (1984) Earthworm acute toxicity
tests Guideline 207.
U.S. Environmental Protection Agency (EPA) /Army Corps of Engineers (ACE). (1994)
Methods for assessing the toxicity of sediment-associated contaminants with estuarine and
marine amphipods. US Environmental Protection Agency, EPA/600/R-94/025, Washington, DC
U.S. Environmental Protection Agency / US Army Corps of Engineers. (1998)
Evaluation of material proposed for discharge to waters of the U.S. - testing manual (Inland
Testing Manual). EPA/823/B-98/004. U.S. Environmental Protection Agency, Washington DC,
USA.
U.S. Environmental Protection Agency. (1998) Guidelines for ecological risk assessment.
Washington DC: Risk Assessment Forum. EPA/630/R-95/002R Washington, D.C.
U.S. Environmental Protection Agency (EPA). (2000) Methods for measuring the
toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates.
U.S. Environmental Protection Agency, EPA/600/R-99/064, Washington, D.C.
U.S. Environmental Protection Agency (EPA). (2002) Methods for Measuring the
Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms.
EPA/812/R/02/012, Office of Water, Washington, D.C., 20460.
Conference Questions and Answers
No. Questions.
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108
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Impact of Nanomaterial Structure and Composition on the Ecotoxicology of
Nanomaterials on Aquatic Species
Rebecca Klaper, Jordan Crago, and Devrah Arndt, Great Lakes WATER Institute,
University of Wisconsin-Milwaukee, U.S.A.
Jian Chen, University of Wisconsin-Milwaukee, U.S.A.
Abstract
There is a question as to how to predict the impact of exposure to nanomaterials on organisms,
and specifically how changes in the structure of a particle will impact the physiological
response of an organism to exposures. Our lab is using two models, Daphnia spp. and trout
(Oncorhynchus mykiss ) to examine the impacts of nanoparticle exposures on immune function,
behavior, mortality and genomics of aquatic species. We investigated the impact of fullerene-
based nanomaterials with different side chains and solubilities, as well as titanium dioxide,
and gold nanomaterials on toxicity, physiology and gene expression response in Daphnia
pulex. We have determined that core particle structure has a significant impact on toxicity and
an affects the interaction of nanomaterials and Daphnia. Side chains and solubility have an
impact on toxicity and physiological response that may provide a mechanism to mitigate the
impacts of nanomaterials on aquatic species. However the impact of side chains may be tissue
or assay dependent and in some cases the chemicals attached to the nanomaterials may have an
independent impact. From this data we can summarize what characteristics of a particle may be
the greater cause of toxicity so we can begin to make predictions about other types of particles to
better inform risk assessment.
Conference Questions and Answers
Question:
Does Daphnia become a vector for C60s? If it is behaviorally inhibited from avoiding predation,
it may be preyed upon more.
Answer:
Previous studies with gold nanoparticles show little tissue uptake by Daphnia. We may assume
little tissue uptake of C60, but if Daphnia have C60 in their gut when eaten they could still be a
vector.
Comment:
Other studies have shown cardiac effects in zebra fish.
Response:
We have not seen heart defects in Daphnia, as the heart is not visible, but hopping behavior may
be related to C60.
Comment:
Tetrahydrofuran (THF) modified work with C60 and negated work on genetox (genetic toxicity).
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Response:
THF does not negate the work, but it complicates the issue and needs further discussion. We still
see genetox effects, and THF is important as it is still used to get C60 into products.
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Evaluation of In Vitro Toxicity of Fullerene nC60 Derivatives Formed in
Conditions that Simulate Disinfection Processes
AllaL. Alpatova, Department of Civil and Environmental Engineering, Michigan State
University, East Lansing, Michigan, U.S.A.
Pavel Babica, Department of Pediatrics and Human Development, Michigan State University,
East Lansing, Michigan, U.S.A.
SyedA. Hashsham, Department of Civil and Environmental Engineering, Michigan State
University, East Lansing, Michigan, U.S.A.
BradL. Upham, Department of Pediatrics and Human Development, Michigan State University,
East Lansing, Michigan, U.S.A.
Susan J. Mastenl and Volodymyr V. Tarabara, Department of Civil and Environmental
Engineering, Michigan State University, East Lansing, Michigan, U.S.A.
Abstract
The progress in the development of facile methods of producing water-soluble fullerene
aggregates (nC60) brings about a higher likelihood of these materials' entering natural water
reservoirs and drinking water supplies. Existing literature data on the potential toxicity of
fullerenes and their derivatives show that the toxicity, if observed, is a strong function of
the surface chemistry of fullerenes species. Disinfection processes such as ozonation and
chlorination could, under certain conditions, modify surface properties of solubilized C60
nanoparticles and alter their toxicity. The study to be presented was aimed at the assessment of
the toxicity of derivatized nC60 nanoparticles formed in conditions that simulate disinfection
processes in a water treatment plant.
Conference Questions and Answers
Question:
C60 has oxidative stress ability. Why do you say you see no cytotoxicity to E.coli?
Answer:
The cytotoxicity of C60 depends on the method of preparation and is usually seen with solvent
preparation.
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Carbon nanoparticle exposure alters protein expression and cell function in
mouse renal principal cells
F.A. Witzmann, Cellular & Integrative Physiology, Indiana University School of Medicine,
Indianapolis, Indiana, U.S.A.
A.D. Amos, Biology, Indiana University.-Purdue University. Indianapolis,
Indianapolis, Indiana, U.S.A.
X. Lai, Cellular & Integrative Physiology, Indiana University School of Medicine,
Indianapolis, Indiana, U.S.A.
B.L. Blazer-Yost, Biology, Indiana University.-Purdue University. Indianapolis,
Indianapolis, Indiana, U.S.A.
Introduction
Carbon nanoparticles (CNP) are currently used in many industries, and their future application
is likely to increase. Three common types include single wall carbon nanotubes (SWCNT),
multiwall carbon nanotubes (MWCNT), and fullerenes (C60). SWCNT consist of covalently
bound carbon atoms arranged in a long, thin tube-like structure with a diameter of approximately
1.4 nm [1]. MWCNT have a similar structure, but they are longer than SWCNT and consist
of several complex layers of nanotubes inside each other with a diameter of 10-20 nm [1]. C60
consist of 60 carbon atoms covalently linked together to form a spherical molecule.
Recent research has revealed diverse effects of CNPs on biological systems. One study indicated
that SWCNT and MWCNT inhibit growth by apoptosis and loss of cell adhesion [2], while other
studies suggest that carbon nanotubes seem to increase the growth of mesenchymal cells, cause
fibrogenesis, and cause granuloma formation [3]. We have previously shown that MWCNT alter
expression of genes for cellular transport, metabolism, cell cycle regulation, and stress response
[4]. MWCNT are of special interest because of their structural similarity to asbestos [5]. Early
experiments with fullerenes have shown them to be cytotoxic, and they have been shown to bind
to ion channels [6]. Various types of nanoparticles are endocytosed and can alter the cytoskeletal
organization [7].
The cell model used in the current study is the mouse principal cell type of the kidney cortical
collecting duct, clone 4 (mpkCCDd4) cell line. mpkCCDd4 cells grow to form a confluent
monolayer that simulates the barrier epithelial function and hormone responsiveness found in
vivo in renal collecting ducts. These cells are of particular interest because they are responsible
for much of the hormonally-regulated ion transport in the kidney. If the CNP exposure alters
these cells, salt homeostasis could be modulated, resulting in changes in blood pressure.
We hypothesized that CNP exposure alters mpkCCDd4 cells resulting in abnormal cellular
function. Experiments were conducted to determine functional, structural and proteomic changes
induced by application of CNP to the renal barrier epithelial cells. Electrophysiological studies
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were used to determine the effect of CNPs on transepithelial electrical resistance (TEER).
Imaging studies were conducted to observe changes in specific cytoskeletal components and
nuclear proliferation. Quantitative proteomic studies were conducted to correlate the observed
structural and functional studies with CNP-induced changes in the expressed cellular proteome.
Methods
For the electrophysiological studies, SWCNT, MWCNT, and C60 were first sterilized by mixing
in ethanol. After evaporation of ethanol under an ultraviolet germicidal lamp, the CNP were
then diluted in fetal bovine serum to 5 mg/mL. Initially, CNP were diluted for each experiment
in tissue culture media by vortexing. Later, to increase the efficiency of the CNP suspension,
CNP/media mixing used sonication for electrophysiological, imaging, and LFQMS studies.
Specifically, CNP were sonicated in FBS, sterilized via autoclave and diluted to 2% FBS-CNP in
media and then diluted for each experiment. Cells were exposed to non-sonicated CNP at doses
of 200 jig/cm2 for 24 h in initial 2-DE studies and, in later experiments, exposed to sonicated
CNP for 48 h at 20 jig/cm2 or at 4 jig/cm2 three times over 7 d.
Electrophysiological techniques were used to monitor TEER. Cells were grown to confluency
over a period of 14 days on Transwell filters with CNP treatment as indicated in the figures. The
filters were excised, mounted in a Ussing chamber, as described in detail previously [8]. The
spontaneous transepithelial potential difference across the monolayer was measured and clamped
to zero. The resulting short circuit current is a measure of net ion flux. Every 200 seconds, the
zero holding potential was changed to a different holding potential and the resulting deflection in
the short-circuit current (SCC) was measured and used to calculate the TEER by Ohm's law.
For imaging, replicate cellular monolayers were washed, blocked, exposed to mouse monoclonal
PCNA antibodies overnight at 4°C, washed, and exposed to goat anti-mouse Alexofluor 488
secondary antibody. Another set of cells were exposed to Rhodamine-phalloidin and DAPI. All
cells were visualized using a Nikon Eclipse TE2000-U Microscope fitted with a Nikon Digital
Camera.
Proteins from duplicate samples exposed to non-sonicated SWNT and MWNT at 200 ug/cm2 for
24 h, were analyzed by two-dimensional electrophoresis (2-DE) and altered proteins identified
via mass spectrometry as described [4], and those from cells used in the TEER experiments
(sonicated CNP at 20 ug/cm2 for 48 h) by label-free quantitative mass spectrometry (LFQMS)
[9]. Briefly, cells were lysed and solubilized in appropriate lysis buffers, and in the case of
LFQMS, the resulting cell lysates then reduced, alkylated, and tryptically digested. Peptide
concentration was determined by Bradford Protein Assay. Peptides were subjected to LC/MS
analysis in random order by eluting with a linear gradient from 5 to 45% ACN developed over 2
h at a flow rate of 50 uL/min, and the effluent electro-sprayed into the LTQ mass spectrometer.
The acquired data were filtered and analyzed, and database searches against the IPI (International
Protein Index) mouse database used both the X! Tandem and SEQUEST algorithms. Protein
quantification was carried out using a proprietary protein quantification algorithm licensed from
Eli Lilly.
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Results
A 48 hour exposure to 20 ^g/cm2 of either sonication suspended SWCNT or MWCNT
significantly decreased the transepithelial resistance of the cellular monlayer (Figure 1). At this
concentration, C60 had no significant effect.
Imaging studies revealed that CNP prepared by sonication and applied at a concentration
of 20 ng/ml for 48 hours tended to agglomerate before settling on the monolayer. PCNA
imaging showed that MWCNT and SWCNT agglomeration induced nuclear proliferation in
cells surrounding the agglomerations (Figure 2, top). In a separate experiment, an increase in
actin filaments was seen in cells surrounding agglomerations. Chronic, low-level exposure to
MWCNT and SWCNT (5 ug/cm2 thrice weekly) induced an increased number and size of large
Effect of 48 hr CNP exposure on
transepithelial resistance in mpkCCD ,, cells
2500
2000 -
1500
1000
500 -
c
Figure 1. Effect of 48 hour CNP exposure on transepithelial resistance in mpkCCDd4 cells.
Confluent monolayers of mpkCCDd4 cells were incubated for 48 hours with 20 ^g/cm2 CNP as
indicated. Cells were removed from the Transwell chambers and mounted in Ussing chambers to
monitor transepithelial electrical resistance. Bars indicate S.E.M. Significantly different from
matched control cultures ( p < 0.02).
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Control
SWCNT
MWCNT
I nil. KM,
PCNA
DAPI
Qualitative analysis ofchanges in cell division via CNIJ exposure. mpkCCDI4 cells
were grown to confliiciicy and t'Nl'S applied at O. Img/mL for 48hrs. The cells were
fixed and stained for UNA (L5AIJ[) and 15IMA replication (P("NA). Oils undergoing
nuclear replication appear as intermediate green. Bright green spots appeared as an
artifact of the staining procedure (arrowheads). Cells surrounding nanoparticle
aplomcratioiis snowed an increase ill DNA replication (red boxes).
Figure 2. Top panel: mpkCCDd4 cells were grown to confluence in 6-well plates and then treated
with sonication-suspended CNP at 20 ug/cm2 for 48 hours. The cells were fixed and stained
with proliferating cell nuclear antigen (PCNA) to indicate the nuclei of proliferating cells and
with DAPI to stain all cell nuclei. The red boxes depict areas of CNP agglomeration and are the
same in all fields. Bottom panel: mpkCCDd4 cells were seeded in 6-well plates and exposed for
seven days to low levels (4 ug/cm2) of the indicated nanopartical. The medium (containing fresh
CNP) was renewed 3 times. The cells were fixed and actin was visualized using rhodamine-
phalloidin (red) while the nuclei were stained with DAPI (blue). Cells treated with CNP showed
an increased number of large, multinucleated cells.
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multi-nucleated cells. Staining with rhodamine-phalloidin also revealed a general increase in
expression of polymerized actin.
Differential protein expression at high dose SWCNT and MWCNT exposures determined by
2-DE is shown in Table 1. The expression of 11 proteins deemed either directly or indirectly
associated with cell proliferation and function were altered by exposure. LFQMS analysis of
CNP exposure for 48 h resulted in differential expression (via ANOVA) of 43 proteins variably
effected by the different CNPs. Those whose expression differed from control are listed in Table
2.
Discussion
The electrophysiological studies and imaging studies indicate changes in cell function in cells
treated with CNP. To more closely model in vivo exposures, functional changes were measured
in mpkCCDd4 cells treated with CNP suspended via sonication. Previous studies have indicated
that coated nanoparticles remain in solution, while unaltered nanoparticles agglomerate
and settle out of solution. In the present study, visual observation confirmed that sonication
increased the solubility and decreased agglomerate particle size although there is still a degree of
agglomeration and precipitation onto the cellular monolayer.
TEER is a measure of monolayer integrity and is also a very sensitive measure of cellular
viability. As cellular viability decreases, TEER falls precipitously. In the experiments shown in
Figure 1, the decrease in TEER is substantial after treatment with either single- or multi-wall
carbon nanotubes. However, these changes do not represent a decrease in cell viability. Control
monolayers had an average TEER of 2370 ± 815 Q • cm2. A decrease to 1477 ± 530 (SWCNT
treated) or 1274 + 465 (MWCNT treated) still exceed 1000 Q - cm2 which is considered a high
resistance, intact epithelium. The changes in resistance indicate more subtle changes within
the cells. Examples of cellular alterations which could be manifested as changes like these
would be minor modifications of the cytoskeleton which is a major component in determining
the impermeability of the junctional complexes or changes in the composition of the cellular
membrane which would be sufficient to alter permeability.
Studies have suggested that CNP may have carcinogenic properties [10]. MWCNT have been
specifically implicated due to their structural similarity to chrysotile asbestos that is widely
accepted to cause carcinogenic responses in humans. After acute exposure to MWCNT and
SWCNT, we observed changes in cells surrounding agglomerations of SWCNT and MWCNT.
Cells seemed to exhibit proliferating nuclei as indicated by PCNA staining. In normal cells, once
confluence is reached, cells no longer actively divide. These results indicate that SWCNT and
MWCNT agglomerations cause cells to replicate abnormally, suggesting possible carcinogenic
properties. Chronic treatment with CNP (especially MWCNT) suspended via sonication also
revealed an increased number of large, multinucleated cells. Polyploidy, as observed here, is an
indicator of genotoxicity and suggests that CNP (at these exposure levels) may be mutagenic.
mpkCCDd4 cells treated acutely with SWCNT and MWCNT as well as cells treated chronically
with any of the three CNP types seemed to exhibit an increased expression of actin filaments.
Previously, such changes in actin expression have contributed to a decrease in cell viability [11].
Our observed trends in cell function and abnormal nuclear proliferation seen via PCNA staining,
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Table 1. Proteins altered by ethanol-sterilized, non-sonicated SWNT and MWNT at 200 ug/cm2
for 24 h, separated and analyzed by 2-DE and identified via mass spectrometry.
Increased expression of the proteins below, a response that may be directly or indirectly associated with
cell proliferation and function
Name Function
stathmin-like 2 regulator of microtubule dynamics; renal expression is j in with uranium
toxicity
pseudouridine synthase 1 involved in protein synthesis, serves to stabilize required RNA
conformations during translation
flotillin-2, isoform 1 scaffolding protein within caveolar membranes, functionally
participating in formation of caveolae or caveolae-like vesicles; tethers
growth factor receptors linked to signal transduction pathways, may also
be involved in cell adhesion
Ran-binding protein 1 bi-directional transport of proteins and ribonucleoproteins through the
nuclear pore complex, spindle formation, reassembly of the nuclear
envelope; expressed at sites of mesenchymal/epithelial induction
enolase, 1 alpha energy metabolism for proliferation; non-neuronal enolase is a
diagnostic marker for many tumors
Decreased expression of the proteins below, a response that may be directly or indirectly associated
with cell proliferation
Name Function
gap junction alpha-8 may contribute to minor changes observed in TEER
cyclin G2 acts as cell cycle inhibitors in certain cell types and may contribute in
inducing cell cycle arrest
myotubularin-related protein 9 protein-tyrosine phosphatase that acts on the 2nd messenger IPS,
localized on endosomes, and regulates intracellular vesicle trafficking
and autophagy; dysregulation can effect trafficking (see stathmin-like 2)
olfactory receptor 586 part of the cell surface receptor mediated signal transduction process
involving G-protein coupled receptors, including cyclic AMP and IPS
mediated processes
zona pellucida glycoprotein 4 cell adhesion molecule; intracellular matrix; may be involved in j TEER
protein kinase, cAMP dependent in PRKAR1A mutant cells (j functional kinase) there is an increase in
regulatory, type I, alpha DNA transcription and/or activation of other pathways leading to
abnormal growth and proliferation
support this. However, the proteomic results do not support differential expression of total actin.
Taken together, these results suggest that the changes we have observed in the filamentous
(phallodin-stained) actin are the result of changes in the filamentous/globular actin ratio in the
cells.
Conclusion
The present study has shown that CNP induced significant alterations in renal collecting duct
cell function, histology, and protein expression. CNP suspended via sonication cause histological
changes including increased nuclear proliferation, elevated filamentous actin expression, and
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Table 2. Proteins altered by autoclave sterilized, sonication-suspended C60, SWCNT, or
MCWNT at 20 ug/cm2 for 48 h versus Control, analyzed by label-free quantitative mass
spectrometry.
Name Cfin SWCNT MWCNT
acyloxyacyl hydrolase I - |
alpha-kinase 3 - I I
catenin, (3 like 1
chaperonin containing Tcpl, subunit 3 (y) t ~
creatine kinase, mitochondrial 1, ubiquitous | -
cytoskeleton associated protein 5 - t ~
F-box and leucine-rich repeat protein 13 - t
ferritin light chain 1 - I -
ferritin light chain 2 - I -
GrpE-like 1, mitochondrial | -
GTPase activating RANGAP domain-like 3 - t ~
hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A . .
thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), (3 subunit
isocitrate dehydrogenase 3 (NAD+) (3 - t t
olfactory receptor 584 t ~
peptidylprolyl isomerase B - I -
phosphate cytidylyltransferase 1, choline, a isoform t ~
prostaglandin E synthase 3 - I -
proteasome subunit, |3 type 7 ~ t t
protein phosphatase 2, catalytic subunit, a isoform ~ t
protein phosphatase 2, catalytic subunit, (3 isoform - t
protein tyrosine phosphatase, receptor type, B I - I
protein tyrosine phosphatase, receptor type, R - - I
Ras-GTPase-activating protein SH3-domain binding protein 1 ~ ~ t
ribosomal protein L30 - I -
serine (or cysteine) peptidase inhibitor, clade B (Serpin BIO) - t t
similar to DEAD (Asp-Glu-Ala-Asp) box polypeptide 43 - | -
sorting nexin 1 t ~ ~
SWI/SNF related, actin dependent regulator of chromatin cl - t t
THO complex subunit 4, isoform 1 t ~ ~
transmembrane protein 202 t ~
ubiquitin-conjugating enzyme E2 D2 I I |
ubiquitin-like modifier activating enzyme 6 III
voltage-dependent anion channel 2 III
zinc finger, C3HC type 1 - t ~
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multinucleation. The observed changes are subtle and likely represent cellular alterations that
would have physiological effects over a prolonged time-course.
References
[1] Jia, G, H. Wang, L. Yan, X. Wang, R. Pei, T. Yan, Y Zhao and X. Guo. (2005). "Cytotoxicity
of carbon nanomaterials: single-wall nanotube, multi-wall nanotube, and fullerene." Environ Sci
Technol 39(5): 1378-1383.
[2] Cui, D. X., F. R. Tian, C. S. Ozkan, M. Wang and H. J. Gao. (2005). "Effect of single wall
carbon nanotubes on human HEK293 cells." Toxicology Letters 155(l):73-85.
[3] Donaldson, K., R. Aitken, L. Tran, V. Stone, R. Duffin, G. Forrest and A. Alexander. (2006).
"Carbon nanotubes: a review of their properties in relation to pulmonary toxicology and
workplace safety." Toxicol Sci 92(l):5-22.
[4] Witzmann and Riviere Monteiro. (2006). "Multi-walled carbon nanotube exposure alters
protein expression in human keratinocytes." Nanomedicine: Nanotechnology, Biology, and
Medicine 2(3): 158-168.
[5] Poland, Craig A., Rodger Duffin, Ian Kinloch, Andrew Maynard, William A. H. Wallace,
Anthony Seaton, Vicki Stone, Simon Brown, William MacNee and Ken Donaldson. (2008).
"Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like
pathogenicity in a pilot study." NatNano 3(7):423-428.
[6] Park, K. H., M. Chhowalla, Z. Iqbal and F. Sesti. (2003). "Single-walled carbon nanotubes
are a new class of ion channel blockers." J Biol Chem 278(50):50212-50216.
[7] Gupta, A. K. and M. Gupta. (2005). "Cytotoxicity suppression and cellular uptake
enhancement of surface modified magnetic nanoparticles." Biomaterials 26(13): 1565-1573.
[8] Shane, M. A., C. Nofziger and B. L. Blazer-Yost. (2006). "Hormonal regulation of the
epithelial Na+ channel: from amphibians to mammals." Gen Comp Endocrinol 147(l):85-92.
[9] Fitzpatrick, D. P. G., J. S. You, K. G. Bemis, J. P. Wery, J. R. Ludwig and M. Wang. (2007).
"Searching for potential biomarkers of cisplatin resistance in human ovarian cancer using a label-
free LC/MS-based protein quantification method." Proteomics Clinical Applications 1(3):246-
263.
[10] Murr, L. E., K. M. Garza, K. F. Soto, A. Carrasco, T. G. Powell, D. A. Ramirez, P. A.
Guerrero, D. A. Lopez and J. Venzor, 3rd. (2005). "Cytotoxicity assessment of some carbon
nanotubes and related carbon nanoparticle aggregates and the implications for anthropogenic
carbon nanotube aggregates in the environment." Int J Environ Res Public Health 2(1):31-42.
[11] Shvedova, A. A., V. Castranova, E. R. Kisin, D. Schwegler-Berry, A. R. Murray, V. Z.
Gandelsman, A. Maynard and P. Baron. (2003). "Exposure to carbon nanotube material:
assessment of nanotube Cytotoxicity using human keratinocyte cells." J Toxicol Environ Health A
66(20): 1909-1926.
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Conference Questions and Answers
Question:
Does that system normally come into contact with natural particles?
Answer:
No. We will be running silica controls. We did not see evidence of inflammation. We are looking
at lactate dehydrogenase (LDH) in the media and ILA (the human homologue of murine 4-1BB),
but these data are not available yet. This system is not exposed to particles. The kidney does not
filter large particles, and the filtrate is not exposed to particles, but is exposed to small molecules
and metabolites.
Question:
Could the effects on endothelium-derived hyperpolarization (EDH)-induced ion channels be
caused by an effect on the channels themselves or an indirect effect due to signaling?
Answer:
Perhaps with the fullerenes that might be the case. Changes in transport may be due to signaling
mechanism change if the cell is injured or irritated.
Question:
Is the cell proliferation response due to toxicity or a signaling response?
Answer:
This remains to be seen.
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122
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Can Nanoscale Particles Affect Plant Growth - Alfalfa Case Study
Sylvia Chan-Remillard, Golder Associates Ltd.; Canada, HydroQualLaboratories Ltd., Canada;
Alberta Ingenuity Fund, Canada
Larry Kapustka, Golder Associates Ltd., Canada
Stephen Goudey, Golder Associates Ltd., Canada; HydroQual Laboratories Ltd., Canada
Abstract
Nanotechnology is a phenomenon that has the potential to change existing technologies and the
ability to create new technologies that were previously unattainable. However, along with this
potential is the added responsibility to understand and manage the hazards and risk associated
with nanotechnology exposure. Regardless of the intended end use of nanotechnology, the
environment will become the ultimate recipient of the products at the end of their life cycle. The
impact of these nanoscale particles on the environment is not very well understood. What is the
fate of these particles once they are in the environment? Do they biotransform, biomagnify or
bioaccumulate? Or do they simply react with organic matter and become benign? Are these
particles toxic to different ecological receptors? Currently very little data exists on the fate and
effects of these particles once they are in the environment. Ecological receptors may be exposed
to nanoscale particles in freshwater, saltwater, soil or sediment. Aquatic studies are beginning to
demonstrate that nanoscale particle toxicity is highly organism and nanoscale particle dependent.
However, within the terrestrial compartment there is still very little information on the toxicity
of nanoscale particles on receptor organisms. We examined the effect several nanoscale particles
had on plants using alfalfa inoculated with rhizobium. Measurement endpoints used were root
growth, shoot elongation and nodule formation. The results of this study will be discussed. We
will also discuss the development of a life cycle based risk assessment framework in the context
of managing environmental risks associated with nanotechnology.
Conference Questions and Answers
Question:
What were your controls for plant growth?
Answer:
We used boric acid as a control.
Comment:
Based on the data presented today, you cannot argue there was an effect or no effect of
nanomaterials on plant growth, as there were no controls for particle size, for the metals
themselves, or for impurities left after material synthesis. Both positive and negative controls are
needed.
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Question:
You presented a slide showing theoreticians, researchers, engineers, producers and marketers,
politicians, and the possible loss of public confidence due to an unplanned release. Do we have
political or public opposition?
Answer:
We do not think we are at the point of political or public opposition, but we need to learn lessons
from the nuclear power industry about managing concern.
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Potential Toxicity of Nanomaterials and their Removal
Xiaoshan Zhu, Xuezhi Zhang, Wen Zhang, Yung Chang, andHu Qiang, Department of
Civil and Environmental Engineering, Arizona State University,
Tempe, Ariaona, U.S.A.
Yongsheng Chen, School of Civil and Environmental Engineering, Georgia Institute of
Technology, Atlanta, Georgia, U.S.A.
Abstract
The potential effects of chronic exposure of the aquatic organism—zebra fish embryo toFerric
Oxide nanoparticles (nFe2O3) was investigated at the concentrations gradient (0 to 100 mg/L).
The preliminary results snowed that the survival rate of Zebra fish embryos were reduced when
dosed 50 mg/L or higher nFe2O3. The hatch time was significantly delayed by the exposure of
high concentrations (50 mg/L or higher) of nFe2O3. Zebra fish embryo test could be a potential
biomarker or bioindicator to assess the effects of exposure of the fish to nFe2O3. Removal of
manufactured nanomaterials (MNMs) from water was investigated by coagulation, flocculation,
sedimentation, and membrane ultrafiltration of nFe2O3 ranging in size from 53 nm to 240
nm. The coagulation, flocculation, or sedimentation process alone or in combination, could
not remove all the nFe2O3 at a very high alum dose (60 mg/L). A lower alum concentration
of 20 mg/L resulted in removal of ca. 90% of nFe2O3 after 24 hours treatment. However, the
ultrafiltation process completely removed nFe2O3 and the resulting permeate was essentially free
of nanoparticles (NPs), suggesting that ultrafiltration can be used as an effective way to remove
nFe203.
Introduction
The rapid growth of nanotechnology is stimulating the research on potential environmental
impacts of manufactured nanomaterials (MNMs). Unlike larger particles, MNMs can probably
accumulate or even penetrate the cell membrane which is the last protection barrier of living
cells from the exotic intrusion. Our project is to focus on evaluation of the potential toxicity of
MNMs, and investigation of potential treatment technologies that effectively remove MNMs
from water. This paper investigated the potential effect of nFe2O3 (nanoparticles of Fe2O3) on
zebra fish embryo and the removal efficiency by water treatment processes.
Materials and methods
Preparation ofn Fe2O3
Stock solutions (100 mg/L) of nFe2O3 were prepared by stirring nFe2O3 vigorously in ISO
standard culture medium (consisting of 64.75 mg/LNaHCO3, 5.75 mg/L KC1, 123.25 mg/L
MgSO4 7H2O, and 294 mg/L CaQ2 2H2O) using a magnetic agitator at room temperature for
2 h. The morphology and the actual size of nFe2O3 in the culture medium were determined
using a scanning electron microscopy (SEM, Philips XL30, FEI Company). Test solutions were
prepared immediately prior to use by diluting the stocks of nFe2O3 with culture medium. During
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the preparation of diluted solution, the stock solution/mixture was continuously stirred with a
magnetic stirrer to maintain the suspension at as stable of a concentration as possible.
Exposure test
Exposure test began as soon as the healthy eggs were selected. 24 eggs (blastula stage) were
transferred to the test wells of a 24-well multi-plate (Costar® 24Well Cell Culture Cluster,
Corning Incorporated, USA) at 1 embryo/well, in which twenty wells contain 2 mL nFe2O3 test
solution and four wells contain 2 ml of culture medium per well. The concentration gradients of
nFe2O3 solution tested in this study were 100, 50, 10, 5, 1, 0.5, 0.1 mg/L and water control. The
experiment was performed in triplicate (i.e., a total of 12 embryos were used in the water control
and 60 embryos in the exposure group) for each treatment. The plates containing experimental
embryos were placed in fish room with a controlled light and temperature (i.e., 28±0.5 °C
with a 14h/10h light/dark cycle). At the end of the experiment, water samples were collected
immediately for measuring the concentration of nFe2O3.
NPs removal
nFe2O3 solution was prepared using nano pure water for studying the removal of NPs with
conventional coagulation-flocculation and sedimentation. The experiments were evaluated
using standard jar test. Aluminum sulfate with a concentration of 20 and 60 mg/L were used
and pH of the water was adjusted into 6.5. APVC UF membrane was employed to evaluate the
removal efficiency of NPs by membrane process. Molecular weight cut off (MWCO) of the Ultra
filtration (UF) membrane is 50000 Dalton and the filtration area of the membrane is 0.125 m2.
Water samples was taken and dried and digested by concentrated nitric acid. GF-AAS was used
to analyze the nFe2O3 concentration.
Results
The state ofnFe2O3 in water phase
The addition of nFe2O3 to the culture medium resulted in a formation of aggregates that settled
down in the water column very quickly. Being observed by SEM, the nFe2O3 aggregates look
like floccules with variable sizes from a few hundred nanometers to several microns in diameter
(Figure 1). In this study, nFe2O3 with a primary particle size of 205 nm was observed to form
large aggregates with average sizes more than 1 um in diameter (Figure 1). This aggregation
phenomenon has also been reported in other NPs, including Cu, TiO2, NiO, fullerene NPs and
SWCNTs [1, 2, 3]. These findings revealed that aggregates or agglomerates of NPs are likely to
settle out of the solution and sink into sediments rather than remain in a suspension. Thus, the
highest concentrations of such MNMs in the environment could be found in sediments.
The toxic effect on Zebra fish embryo
These nFe2O3 aggregates were found to be toxic to zebrafish embryos and larvae, causing a
dose-dependent mortality and hatching inhibition, as shown in Figure 2. The survival rate of
Zebra fish embryos were reduced when dosed 50 mg/L or higher nFe2O3. The hatch time was
significantly delayed by the exposure of high concentrations of nFe2O3. The development of
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Figure 1. SEM images of nFe2O3 aggregates (10 mg/L) in zebrafish culture medium.
120
100
g 80
60
40
20
0
>
£
100 mg/L
-10 mg/L
• 1 mg/L
• 0.1 mg/L
-•—50 mg/L
—K- 5 mg/L
—•— 0 5 mg/L
-B- water contro
24 48 72 96 120 144 168
hpf(hours)
100
-o—100 mg/L
=»=• 50 mg/L
-A—10 mg/L
—K— 5 mg/L
-*- 1 mg/L
-•-05 mg/L
——0.1 mg/L
•- 40 D water contr
£ 80
« 60
O)
20
0 24 48 72 96 120 144 168
hpf (hours
Figure 2. Survival and Hatching rate (%) of zebrafish embryos exposed to nFe2O3 over 168 h.
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the zebrafish embryos and larvae was observed with an inverse microscope (Olympus, Japan)
equipped with a digital camera and was documented photographically at specified time points (t
=6, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144 and 168 hours post fertilization (hpf)). The endpoints
used to assess developmental toxicity included embryo/larvae survival and embryo hatching rate.
Malformations and pericardial edema were described and documented among the embryos and
larvae from both control and treated groups. Developmental abnormality, e.g., pericardial edema,
malformation and tissue ulceration, was found in 50 mg/L and 100 mg/L nFe2O3 exposure group
(Figure 3), affecting more than 13% of the surviving fish by 168 hpf.
Interactions between nFe2O3 and embryo surface
The interactions between nFe2O3 and embryo surface were studied through Atomic force
microscopy (AFM) to explore the possible underlying cause of toxicity of nFe2O3 to embryos.
Adhesion force results indicated that the chorion of the embryo at an early stage had adhesion
forces distributed from 2000 to 2800 pN. There was also a lower level of adhesion force domain
from 800 to 1600 pN. After 8 hours the force distribution moved slightly to the low force range
which indicated the average adhesion force decreased slightly and the chorion became less
adhesive and more hardened.
Evaluation ofNPs removal using conventional water treatment technology
Removal of nFe2O3 using conventional coagulation processes is shown in Figure 4. When alum
concentration was 20 mg/L, concentrations of hematite decreased gradually with time. 80% of
NPs was removed after 12 h sedimentation. There were still about 7% of hematite NPs remaining
in the solution even after 24 h sedimentation. When alum concentration increased to 60 mg/L,
the concentrations of hematite decreased sharply. More than 90% removal was achieved after 3
hour's sedimentation. However, it still took 12 hours to removal 97% of the NPs.
Evaluation of NPs removal using UF membrane
Figure 5 shows the removal efficiency of nFe2O3 NPs using membrane. The concentration
in permeate is less than the detection limit (0.005 mg/L) and their removal rate is more than
99.95%. Figure 5 also shows the EDX mapping images of the fiber after filtration. NPs deposited
on the filter layer of the hollow fiber and there were no particles in the inner pores of membrane,
indicating that no NPs can penetrate the active filtration layer.
Conclusion
The environmental behaviors and the toxicity of MNMs learned from this study provided the
insight information that may help scientists and manufacturers to design and manufacture more
environmentally benign MNMs and avoid environmental disasters such as DDT and PCB
occurred in the past. The survival rate of Zebra fish embryos reduced when dosed 50 mg/L or
higher nFe2O3. The hatch time was significantly delayed by the exposure of high concentrations
of nFe2O3. Conventional treatment process is not efficient for nFe2O3 removal while UF is very
effective for nFe2O3 NPs removal. No nFe2O3 NPs in permeate were detected and their removal
efficiency was more than 99.95%.
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• Tissue ulceration
Q Pencardial Edema
Q Malformation
100mg/L 50 mg/L 10mg/L Control
Iron Oxide Nps Concentration
Control at 168hpf SOmg/Ldead SOmg/Ldead lOOmg/Ldead
Figure 3. Developmental abnormality induced by nFe2O3 exposure at 168 hpf.
1.0-
0.8-
U° 0.6-
u
0.4-
0.2-
0.0-
Aluminium 20 mg/L
Aluminium 60 mg^L -
0 10 20 30 40
Time (h)
50
Figure 4. Removal of nFe2O3 using conventional coagulation processes.
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Feed
Permeate
ta&sfcS&ftSSK
Figure 5. Removal efficiency of nFe2O3 by UF membrane (a) and deposition of NPs on mem-
brane fibers.
References
1. Cheng, J.; Flahaut, E.; Cheng, S. H. (2007). Effect of carbon nanotubes on developing
zebrafish (Danio rerio) embryos. Environ. Toxicol. Chem., 26, 708-716.
2. Zhang, Y; Chen, Y; Westerhoff, P.; Hristovski, K.; (2008). Crittenden J. C. Stability of
commercial metal oxide nanoparticles in water. Water Res., 42, 2204-2212.
3. Adams, L. K.; Lyon, D. Y; Alvarez, P. J. J. (2006). Comparative eco-toxicity of nano-scale
TiO2, SiO2 and ZnO water suspensions. Water Res, 40, 3527-3532.
4. Hund-Rinke, K.; Simon, M. (2006). Ecotoxic effect of photocatalytic active nanoparticles
(TiO2) on algae and daphnids. Environ. Sci. Pollut. Res., 1-8.
Conference Questions and Answers
Comment:
Other studies have shown that, if silver nanoparticles are removed from the water column, they
may be left in the sediment. This may limit the use of the sediment if it is intended for soil
amendment.
Comment:
It would be interesting to consider the flow rate that can be achieved with an ultra filtration (UF)
filter.
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Endotoxin Contamination of Engineered Nanomaterials
R. Keith Esch, Li Han, DavidEnsor andKarin Foarde, RTIInternational, U.S.A.
Abstract
Endotoxins are bacterial cell wall components that occur naturally in soil, water and air; and
routinely contaminate other materials. Endotoxin exposure is associated with respiratory
symptoms as well as fever, septic shock, impaired organ function and death. Endotoxin is a
potential confounding factor in engineered nanomaterial (ENM) toxicity studies as induction
of inflammatory response and oxidative stress are observed for both endotoxin and ENM. The
established method for quantifying endotoxin relies on its activity in a complex biochemical
assay system. Because of their physical and chemical properties, examination of many ENM
under these conditions presents nontrivial technical challenges. We have made progress
in identifying and implementing methods for analysis of ENM with respect to endotoxin
contamination. An examination of a series of carbon-based ENM reveals varying levels of
endotoxin. The physical association of ENM and endotoxin and their shared physiological
effects suggest the possibility that contaminating endotoxin may represent a health risk and
contribute to the toxicity that is ascribed to ENM.
Introduction
The decreased size, and greatly increased surface area, of nanomaterials carries the potential
for much more activity, for a given mass, compared to larger particles of the same chemical
composition. These qualities, while highly desirable in industrial, analytical, consumer product
and medical applications also raise concerns for impact on human health.
Endotoxins are components of Gram-negative bacterial cell walls that contain both lipid and
polysaccharide components (Fig. 1) and vary somewhat depending upon the species of origin.
Endotoxins may be released upon cell death as well as during growth and division. They
are nearly ubiquitous in the environment, present on surfaces and in particles made up of
diverse materials (Douwes et al, 1995). Exposure to endotoxin is associated with respiratory
symptoms and pulmonary inflammation. Airflow restriction in those with allergic asthma can
be exacerbated by airborne endotoxin (Michel et al 1996). Repeated wheeze in infants has
been linked to low level exposure (Park et al 2001). Acute endotoxin exposure is also linked to
systemic response resulting in fever, septic shock, impaired organ function and death (Banner et
al, 1991; Wanderley et al, 1996).
Examination of a series of ENM reveals levels of endotoxin contamination that vary over nearly
4 orders of magnitude (Kayo Inaba, personal communication). An investigation by Vallhov et
al (2006) aimed at addressing the use of gold particles in therapeutic approaches found that gold
nanoparticles became contaminated with endotoxin, producing altered immune responses in
cell-based assays. These findings suggest the possibility that contaminating endotoxin may be
contributing to the toxicity that is ascribed to ENM.
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"Man—Abe
P,ha
Gal
Man—Abe
Rha
Qal
Glc— N4G
Qal
Glc—Gal
Hep
Hep—P —P—ethanolamine
KDO
K DO—K DO-P—©thanobmine
-OH
Figure 1. Endotoxin (LPS) is approx. lOkDa and has an amphiphilic structure consisting of a
lipid portion, an anionic core polysaccharide and a variable polysaccharide-rich O-Antigen.
We sought to investigate ENM endotoxin contamination in order to characterize associated
risks. Because of their hydrophobicity, many carbon-based ENM have limited dispersion in
the aqueous suspensions in which endotoxin assays are conducted. Therefore, methodologies
allowing accurate, representative assessments of endotoxin contamination need to be developed.
We describe here the studies initiated to address this complex problem as well as endotoxin
contamination findings for a set of carbon-based ENM from different sources.
Materials and Methods
We used Cryogenic gas sorption (BET) analysis to obtain ENM surface area information. In
this analysis, Nitrogen served as the adsorptive gas with analysis bath temperature at 77.3K. It
is known that the material diameter properties vary with different manufacturers and batch-to-
batch production processes. We obtained diameter analysis results from the manufacturers of
the carbon materials for individual production lots. Transmission Electron Microscopy (TEM)
and Scanning Electron Microscopy (SEM) verified the diameter information from vendors. As
Carbon Nanotube/C60 synthesis requires metal catalyst materials such as Ni, Fe, Co, Y and Mo,
metal residue remains in the final product. The weight percents of total metal composition were
obtained from manufacture lot analysis results. Scanning electron microscopy (SEM) combined
132
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with energy dispersive x-ray analysis (EDX) verified the information from vendors.
PyroCLEAN™ used for decontaminating endotoxin on surfaces was purchased from
ALerCHECK Inc. and used according to the manufacturer's directions. Vitamin E d-a-
tocopheryl polyethylene glycol - 1000 succinate (VETPGS), a water soluble vitamin E-based
surfactant was acquired from Eastman Chemical. All glassware used to conduct the endotoxin
assays was depyrogenated by exposure to 190 deg. C for at least 3 hours. All remaining labware
used for extractions and assays was designated as pyrogen free by the manufacturer.
Liquid suspensions of each ENM were prepared for determination of endotoxin contamination.
The mass of each sample was measured using a balance in a containment hood following
decontamination with pyroCLEAN. Depyrogenated implements and containers were used in
gravimetric procedures. Each preparation was agitated by a minimum of 30 seconds of vortexing
and 40 minutes of sonication in a Branson 2200 bath sonicator.
Preparation A: All nanomaterials were transferred to depyrogenated glass tubes and endotoxin-
free water was added to produce each at a concentration of 10 mg/ml. The nanomaterial particles
were allowed to settle/separate from the liquid and the liquid extracts were removed for assay of
endotoxin content.
Preparation B: Triethylamine (TEA) was added to each 10 mg/ml nanomaterial sample in
water to a concentration of 0.01%. Each sample was then diluted 2-fold in 0.01% TEA, resulting
in 5.0 mg/ml nanomaterial samples. The nanomaterial particles were allowed to settle/separate
from the liquid and the liquid extracts were removed for assay of endotoxin content.
Preparation D: A 1% VETPGS solution and endotoxin free water were added to the preparation
B samples to produce each nanomaterial sample at 1.0 mg/ml in 0.1% VETGPS. Samples of
each mixture were removed without a settle/separation period such that representative amounts
of nanomaterials remained in the portions removed for endotoxin testing.
Preparation E: Preparation D samples were centrifuged in a model 5415 D microcentrifuge
(Eppendorf, Westbury, NY) at maximum speed for 5 minutes. Supernatants were transferred
from the pelleted material, to minimize any ENM content, into endotoxin-free tubes for analysis.
Endotoxin levels were quantified using a kinetic chromogenic Limulus amebocyte lysate (LAL)
assay (Associates of Cape Cod, Woods Hole Mass.) following the manufacturer's instructions.
The level of endotoxin activity in a sample was determined by the reaction of endotoxins in the
specimen with the lysate and a substrate, producing a color change over time, and comparing rate
of color change to similar reactions of known endotoxin reference standards.
In preparation for endotoxin assay, the samples were put at R.T., vortexed for 30 seconds and
sonicated for a minimum of 30 minutes. Dilutions of tests samples and standards were made
using the solution used to generate the test samples. For samples that include endotoxin spikes,
indicated known quantities of standard endotoxin were added directly to samples at the time
the assay was conducted. VETGPS was included in a series of endotoxin standards of varying
concentrations. Assay interference was assessed by comparing the resulting endotoxin values of
these samples to those produced from the same endotoxin standards lacking this surfactant.
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Results and Discussion
Visual observation indicated that the mixing of the ENM at 10 mg/ml in endotoxin-free water
or 0.01% TEA generally failed to produce stable homogeneous suspensions. The ENM did not
extensively disperse in the liquid, separating out by settling or clinging to the inside surface
of the glass tubes. Accordingly, significant endotoxin was not detected in extracts from these
preparations. Dilution in 0.01% TEA to 5 mg/ml (preparation B) produced minor increases in
dispersion of the ENM. Assay of these suspensions produced detectable endotoxin for some
ENM, but the values were generally far lower than later determined values, indicating significant
interference by the ENM in the suspensions.
Because the ENM were largely refractory to dispersal in either water or TEA, the water soluble
vitamin E surfactant (VETGPS) was considered for use as a dispersing reagent and accordingly
was tested for interfering effects in the endotoxin assay. No significant interference was
observed for the vitamin E surfactant up to 0.1%, the highest concentrations tested. Addition
of VETGPS to 0.1% produced a readily visible increase in dispersion. These suspensions
(preparation D) produced quantifiable levels in some cases. For ENM 2, the value obtained
was reasonably close (within about two-fold) to the final estimates despite the presence of ENM
in the assay mixtures. However, there was considerable variation in individual assay samples,
perhaps due to particle interference with the colorimetry light path.
Since improved suspension had been realized by the addition of VETGPS, we reasoned that
an increase in interaction between the ENM and the liquid likely facilitated more complete
extraction of endotoxin from the ENM surfaces into the aqueous phase. Therefore, extracts were
produced (preparation E) that employed centrifugation to remove the ENM. This extraction
resulted in detection of quantifiable endotoxin for all five ENM (Fig. 2). The levels of endotoxin
extracted from this set of ENM varied by more than an order of magnitude, depending on
the material tested. Extracts from two of the ENM tested (#2 and 3) had considerably higher
endotoxin contamination than the others.
The results from the physical characterization studies are summarized in Table 1. No striking
similarities or commonalities are evident among the two ENM that were most contaminated with
endotoxin. While ENM #2 is a multi-walled carbon nanotube (MWCNT), ENM #3 is a C60
fullerene. Whereas ENM #2 has relatively high metal composition and surface area values, those
for ENM #3 are quite low. Each is manufactured by a different organization and thus in different
environments.
Conclusions
Significant progress toward meaningful determinations of endotoxin contamination of carbon
based ENM has been achieved. Once established, these methods can be employed to address
the extent of ENM-endotoxin associations in commercial preparations, laying a foundation for
assessing the risk presented by endotoxin contamination. The current work, in conjunction with
the successful completion of further studies, will allow examination of relationships between
specific ENM properties and the degree of endotoxin adsorption, producing critical information
for understanding factors determining contamination. These studies will also illuminate our
understanding of endotoxin as a confounding factor in nanoparticle toxicity investigations and as
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4.00
Estimates of Endotoxin Contamination
3 4
Engineered Nanomaterials
10
Figure 2. Determinations of extracted endotoxin from preparations of 5 commercially available
carbon-based ENM. Error bars are based on standard deviations from multiple independent
measurements.
Materials
2 MWCNTs
3 Fullerene
4 MWCNTs
8 Fullerene
10 Carbon
Black
Vendor
information
SES Research
MER
Corporation
Sigma -Aldrich
Sigma -Aldrich
Cabot Corp
Catalog #
900-119
MR6LP
636843
572500
Vulcan XC 72R
Surface
area
(m2/g)
194
0.07
97
0.22
221
Diameter (nm)
<10nm
20 nm
40-70 run
ND
30 nm- primary
particle size;
250 nm-
Aggregates size
Total Metal
composition
wt%
<5%
0.001%
>2.23%
0.1%
<0.006%
Table 1. Physical characteristics of ENM used in endotoxin contamination studies. (ND signifies
value was not determined). The surface areas of the ENMs are determined by BET measurement and
the low surface area number for ENM#3 and ENM#8 is likely due to the large agglomeration of the
fullerene particles, as indicated by TEM imaging of these samples (not shown).
135
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a contributor to ENM-based adverse health effects.
References
Douwes, J.D., Versloot, P., Hollander, A., Heedrik, D., and G. Doekes. (1995). Influence of
various dust sampling and extraction methods on the measurement of airborne endotoxin.
Applied and Environmental Microbiology 61, 5: 1763-1769
Michel O, Kips J, Duchateua J, Vertongen F, Robert L, Collet H, Pauwels
R, and R. Sergysels. (1996). Severity of asthma is related to endotoxin in house dust.
Am JRespir Crit Care Med. 154:1641-1646.
Park J, Gold D, Spiegelman D, Burge H, and D. Milton. (2001). House dust endotoxin
and wheeze in the first year of life. Am J Respir Crit Care Med. 163:322-328.
Danner, R.L., Elin, R.J., Hosseini, J.M., Wesley, R.A., Reilly J.M., and I.E. Parillo. (1991).
Endotoxemia in human septic shock. Chest. 99; 169-175
Wanderley, A. and C. Wanderley. (1998). Clinical Sepsis and Death in a Newborn Nursery
Associated with Contaminated Parenteral Medications - Brazil, 1996 CDC Morbidity and
Mortality Weekly Report July 31, 1998. 47(29);610-2
Vallhov, H., Jian Q., Johansson, S.M., Ahlborg, N., Muhammed, M.A., Scheynius, A. and S.
Gabrielsson. (2006). The Importance of an Endotoxin-Free Environment during the Production
of Nanoparticles Used in Medical Applications. NanoLett. 6, No. 8.
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Effects of Ingested Engineered Carbon Nanomaterials on Zooplankton
Aaron P. Roberts, and Leigh M. Taylor, Department of Biological Sciences, University of North
Texas, Denton, Texas, U.S.A.
Aaron E. Edgington, and Stephen J. Klaine, Institute of Environmental Toxicology, Clemson
University, Pendleton, South Carolina, U.S.A.
Abstract
Nanotechnology is a rapidly growing industry, and increased manufacturing and use of
engineered nanoparticles will likely increase their deposition into aquatic ecosystems. However,
relatively little is known about the potential impacts of engineered nanoparticles on aquatic
biota. Particularly relevant to aquatic ecosystems are those particles which display increased
solubility either through specialized coatings or through an ability to interact with water column
constituents such as natural organic matter. Previous research indicated that grazing zooplankton
(Daphnia magna) were able to ingest lipid-coated single walled carbon nanotubes (SWNTs)
from the water column during their normal feeding behavior. While SWNTs were observed to
fill the gut of the zooplankton, they were easily egested, and acute mortality was observed only
at high concentrations (>5mg/L). The purpose of this research was to examine the potential for
sublethal effects to occur at lower concentrations following ingestion of solubilized engineered
carbon nanomaterials. D. magna and C. dubia were exposed to a range of concentrations of
multiwalled carbon nanotubes (0.1 -Img/L) suspended in water using natural organic matter.
Survival was monitored in each species for the duration of the test period (7 days for C. dubia
and 4 days for/), magna). In order to assess sublethal effects, reproduction was monitored in
C. dubia. We hypothesized that the accumulation of nanotubes in the gut tract of zooplankton
would decrease their ability to take up normal food (algae) and, thus, growth (dry mass per
individual) was measured in both species using an electromicrobalance. No significant effect on
survival of either species was observed at any of the concentrations tested. However, C. dubia
reproduction was significantly decreased by 50% at concentrations > 0.25mg/L. Growth in both
species was inhibited in a concentration dependent manner. Although we observed no evidence
that the MWNTs were taken up across the gut membrane, we have shown that simply ingesting
the materials can lead to significant toxic effects in zooplankton through the inhibition of normal
feeding activity.
Introduction
Nanotechnology is a rapidly growing industry, and increased manufacturing and use of
engineered nanoparticles will likely increase their deposition into aquatic ecosystems. However,
while some authors have reported deleterious effects of nanomaterials on fish and plankton,
relatively little is yet known about the potential impacts of engineered nanoparticles on aquatic
biota (Oberdorster 2004, Lovern and Klaper 2006, Oberdorster et al. 2006, Cheng et al. 2007,
Lovern et al. 2007, Roberts et al. 2007). Particularly relevant to aquatic ecosystems are those
particles which display increased solubility either through specialized coatings (Wu et al. 2006,
137
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Roberts et al. 2007) or through an ability to interact with water column constituents such as
natural organic matter (NOM) (Hyung et al. 2007, Roberts et al. 2007). Previous research
indicated that grazing zooplankton (Daphnia magnet) were able to ingest lipid-coated single
walled carbon nanotubes (SWNTs) from the water column during their normal feeding behavior
(Roberts et al. 2007). While SWNTs were observed to fill the gut of the zooplankton, they were
easily egested, and acute mortality was observed only at high concentrations (>5mg/L). While
ingestion of nanotubes may not result in acute mortality, filter feeding or non-specific grazing
organisms might accumulate large amounts of the materials in their guts and inhibit the uptake
or digestion of normal food items resulting in toxicity following more chronic exposures. The
purpose of this research was to examine the potential for sublethal effects to occur at lower
concentrations following ingestion of solubilized engineered carbon nanomaterials, specifically
multiwalled carbon nanotubes (MWNTs). We hypothesized that the accumulation of nanotubes
in the gut tract of zooplankton would decrease their ability to take up normal food (algae)
resulting in decreased growth and reproduction.
25
20
0.05 0.1 0.15 0.2
Log(NP-H)
0.25
0.3
0.35
Figure 1. C. dubia reproduction.
138
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Methods
MWNT-NOM Solutions
Suwannee River Natural Organic Matter (SR-NOM) was purchased from International Humic
Substances Society (St. Paul, MN, USA) and used as the NOM source throughout this study.
NOM solutions were made by weighing out the desired amount of SR-NOM and placing it in a
volumetric flask filled with the appropriate amount of EPA Moderately Hard Water (MHW). The
solution was then stirred with a Teflon stir bar on a mixing plate before being filtered with a 0.2
cellulose membrane filter prior to use.
Multi-walled nanotubes (MWNTs) were made by Dr. A. Rao's Laboratory at Clemson University
(Clemson, SC, USA) using the thermal chemical vapor deposition method. MWNTs had an
approximate diameter of 25 nm, length of approximately 50 ^m, and a purity of >95%. In
order to suspend the MWNTs in NOM, they were first weighed on waxed weigh paper and
then placed in a 100 mL glass centrifuge tube. Twenty -five mLs of NOM solution was added
to the centrifuge tube and the solution was sonicated with a Fisher model 300 dismembrenator
with a 1/8" microtip for 15 min. Twenty -five mL aliquots of dilution water (containing NOM)
were added and the solution sonicated for additional 15 min intervals after each aliquot until
the solution reached a total volume of 100 mL and total sonication time of 1 hr. The solutions
were allowed to settle for approximately 24 hrs before the supernatant (stable solution) was
removed with a glass pipette. Test concentrations of NOM-MWNT were achieved by sonicating
appropriate volumes of NOM-MWNT supernatant stock solution in dilution water containing
NOM.
Bioassays
Bioassays were conducted according to US EPA methods (EPA 1993, 2002) with slight
modification using a dilution series of MWNTs in NOM. NOM solution (without MWNTs) and
MHW were used as controls. Fifteen mLs of stable MWNT solutions was added to 30 mL glass
beakers that served as test chambers (six concentrations 0.625-20 mg/L; n = 3 replicates per
concentration). Equal volumes of control waters were poured into 30 mL glass beakers to serve
as test controls (n = 3 replicates per control). Test solutions were renewed daily.
D. magna and Ceriodaphnia dubia were obtained from existing cultures at the Dept. of Biology,
University of North Texas (Denton, TX) maintained in MHW. Growth in D. magna neonates (<
24 hrs old) were measured following exposure to a range of MWNT concentrations for 96 hrs
(0-1 mg/L MWNT; n = 5 replicates per concentration). C. dubia reproduction was monitored
over a seven day exposure period and growth was measured at the end of the 7 day test (n = 5
replicates per treatment). Organisms were fed a mixture of green algae- YTC and test solution
renewed daily. Growth was measured as dry weight on a Kahn electromicrobalance.
Results
MWNTs were observed in the gut tract of C. dubia within hours of exposure initiation.
However, MWNTs in NOM were not acutely toxic (lethal) to C. dubia at the tested
concentrations (0-lmg MWNT/L). Mean survival was greater than 85% in all treatments, and no
relationship was observed between MWNT concentration and mortality.
139
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Chronic toxic effects at sublethal concentrations of MWNTs in NOM were observed in C. dubia.
A negative relationship between reproduction and MWNT concentration between 0 and 1 mg/L
was found (p < 0.01) (Fig. 1). Reproduction was significantly inhibited by approximately 50%
at concentrations >0.25mg/L MWNTs. Growth was also inhibited at concentrations >0.25mg/L
(Fig. 2).
0.08
0.07
0.06
3 0.05
O)
'3
0.04
0.03
0.02
0.01
0
0.05 0.1 0.15 0.2
Log(NP+1)
0.25
0.3
0.35
Figure 2. C. dubia growth.
A negative relationship was found between D. magna growth and MWNT concentrations
between 0 and Img/L (r2= 57%; p < 0.01) (Fig. 3). Mean dry weight was significantly reduced
by 25% in organisms exposed to 0.25 mg/L MWNTs, a trend which increased in a dose-
dependent manner. No acute mortality was observed.
Discussion and Conclusions
We were able to create relatively stable aqueous suspensions of MWNT using NOM, a
constituent of all surface waters. We observed that grazing zooplankton were able to ingest the
suspended MWNTs during their normal feeding behavior in a manner similar to previous reports
of the ingestion of coated SWNTs (Roberts et al. 2007). MWNTs were visible in the guts of
exposed animals at concentrations
-------
Interestingly, although we observed no evidence that the MWNTs were taken up across the gut,
it does appear that simply ingesting the materials can lead to significant toxic effects. Growth
was reduced in both of the test species and reproduction was inhibited in the C. dubia model.
We hypothesize that this is due to a decreased ability to take up normal food items such as algae
as a result of the physical agglomeration of MWNTs in the gut rather than through increased
oxidative stress or other biochemical mechanism. Future studies will examine these mechanisms
more closely as well as the potential for the materials to be passed up the food chain to other
planktivorous species.
0.3
0.25
0.2
0.15
0.1
0.05
0
0.05 0.1 0.15 0.2 0.25 0.3 0.35
Log(NP + 1)
Figure 3. D. magna growth.
References
Cheng, J. P., E. Flahaut, and S. H. Cheng. 2007. Effect of carbon nanotubes on developing
zebrafish (Danio rerio) embryos. Environmental Toxicology and Chemistry 26:708-716.
EPA, U. S. 1993. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters
to Freshwater and Marine Organisms.
EPA, U. S. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Freshwater Organisms. Page 350. US EPA.
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Hyung, H., J. D. Fortner, J. B. Hughes, and J. H. Kim. 2007. Natural organic matter stabilizes
carbon nanotubes in the aqueous phase. Environmental Science & Technology 41:179-184.
Lovern, S. B., and R. Klaper. 2006. Daphnia magna mortality when exposed to titanium dioxide
and fullerene (C-60) nanoparticles. Environmental Toxicology and Chemistry 25:1132-1137.
Lovern, S. B., J. R. Strickler, and R. Klaper. 2007. Behavioral and physiological changes in
Daphnia magna when exposed to nanoparticle suspensions (titanium dioxide, nano-C-60, and
C(60)HxC(70)Hx). Environmental Science & Technology 41:4465-4470.
Oberdorster, E. 2004. Manufactured nanomaterials (Fullerenes, C-60) induce oxidative stress in
the brain of juvenile largemouth bass. Environmental Health Perspectives 112:1058-1062.
Oberdorster, E., S. Q. Zhu, T. M. Blickley, P. McClellan-Green, and M. L. Haasch. 2006.
Ecotoxicology of carbon-based engineered nanoparticles: Effects of fullerene (C-60) on aquatic
organisms. Carbon 44:1112-1120.
Roberts, A. P., B. Seda, A. S. Mount, S. J. Lin, P. C. Ke, R. Qiao, and S. J. Klaine. 2007. In vivo
biomodification of a lipid coated carbon nanotube by Daphnia magna. Environmental Science &
Technology.
Wu, Y, J. S. Hudson, Q. Lu, J. M. Moore, A. S. Mount, A. M. Rao, E. Alexov, and P. C.
Ke. 2006. Coating single-walled carbon nanotubes with phospholipids. Journal of Physical
Chemistry B 110:2475-2478.
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Toxicity of CdSe/ZnS nanocrystals to D. magna.
H. P. Pace, Colorado School of Mines, Department of Environmental Science
and Engineering, Golden, Colorado, U.S.A.
J. F. Ranville, Colorado School of Mines, Department of Chemistry
and Geochemistry, Golden, Colorado, U.S.A.
Abstract
With the rapid expansion of the nanotechnology industry, quantum dots (QDs) are situated to
become a prominent new source of metal contamination. We are currently researching the acute
toxicity of CdSe/ZnS QDs on Daphnia magna using 48hr exposure studies. Toxicity of QDs
was hypothesized to be directly related to either one of two scenarios: 1) the solubilization of the
QD and subsequent release of toxic metals (Zn, Cd) or 2) the physical or chemical impairment
of key physiological functions by the nanoparticle itself. To test this hypothesis we investigated
QDs with two different CdSe core diameters, 2nm green emitting QDs and 5nm red emitting
QDs. To investigate potential particle effects we also examined two separate surface coatings,
polyethylene oxide (PEO) and 11-mercaptoundecanoic acid (MUA), which are polar and anionic
respectively (Evident Technologies, Troy NY and NN-Labs, Fayetteville AR). These coatings,
which serve to render the QDs water stable, increase the hydrodynamic diameter of all QDs to
approximately 25nm. Thus, while the metal content of the red and green emitting QDs (2nm vs.
5nm) was substantially different, the total particle size was the same. Using a fluorescence scan
of the QDs (400-800nm) we monitored the QD concentrations during exposures. We found that
PEO coated QDs remained well-dispersed throughout the 48hr exposures with no significant
change in QD concentration whereas MUA coated QDs had a higher tendency to aggregate.
In addition, we characterized the QDs before and after exposure via filtrations and ICP-OES
metal analysis (unfiltered, 0.02|im and 3kDa filtrations). Finally, fluorescence microscopy and
synchrotron micro-XRF showed accumulation of nanoparticles within exposed daphnids.
Introduction
Nanomaterials, due to their small size, differ from their bulk material counterparts. Accordingly,
when evaluating potential toxicity of nanomaterials, one must address not only their chemical
composition, but also the unique physiochemical properties associated with their extremely
small size. Quantum dots (QDs) are nanoparticles that frequently contain toxic heavy metals.
With numerous potential applications in the biological imaging industry as well as in optics
and electronics, QDs are situated to become a new source of metal contamination. Originally,
many reports indicated that QDs did not produce any significant cellular toxic effects [1-3].
However, it was recognized that these studies were designed to answer questions concerning
the QD's novel physiochemical properties such as fluorescence, detectability and stability, and
were not designed to specifically investigate QD toxicity [4]. Recently, a group at the University
of California San Diego reported that CdSe-core quantum dots can produce cytotoxic effects
under certain conditions [5]. It was found that these cytotoxic effects correlated with the release
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of free Cd2+, which arose from surface oxidation due to exposure to air or UV light. Various
surface coatings were found to resist oxidation, thus producing reduced cytotoxic effects. This
study suggests that it is the release of the metals from the nanoparticle that causes a toxic effect.
While further investigating QDs, Kirchner et al. found that besides the release of Cd2+ ions,
the aggregation and precipitation of the nanoparticles on the cell surface could impair cellular
function [6]. These results suggest that the particle itself, independent of QD metal release, can
play a role in QD toxicity. An additional study by Lovric et al. [7] found that green emitting
CdTe-core QDs produced increased cytotoxic effects over that of red emitting QDs. Since
emission wavelength of QDs is directly related to QD core diameter, these results further suggest
that particle effects are playing a role in QD toxicity.
Much of the research done on QD toxicity has been related to cytotoxic or in vivo effects.
However, questions concerning the environmental effects of QDs are also important to
characterizing potential risks of these nanoparticles and these questions remain largely
unanswered. Based on the results of previous studies, QD toxicity was hypothesized to be
directly related to either one of two scenarios: 1) the solubilization of the QD and subsequent
release of toxic metals (Zn, Cd) or 2) the physical or chemical impairment of key physiological
functions by the nanoparticle itself. The goal of this study was to use the well-established
framework of acute toxicology for dissolved metals to glean a basic understanding of the
potential risks associated with metal-containing nanocrystals in aquatic environments.
Methods
Green (2nm core) and red (5nm core) polyethylene oxide (PEO) coated CdSe/ZnS QDs (Evident
Technologies, Troy NY) as well as green and red mercaptoundecanoic acid (MUA) coated CdSe/
ZnS QDs (NN-Labs, Fayetteville AR) were investigated during this study. The two surface
coatings, which both serve to render the QDs water stable, increase the hydrodynamic diameter
of all four QDs to approximately 25nm. Thus, while the metal content of the red and green
emitting QDs (2nm vs. 5nm) was substantially different, the hydrodynamic diameter of all QDs
was approximately the same.
We performed 48 hr acute toxicity tests for the four QDs using Daphnia magna according to
USEPA Standard Test Protocol with a mortality (i.e. immobile) endpoint [8]. Daphnia magna
were obtained from cultures maintained in USEPA hard water medium in an incubator at 20 °C
with a 16:8 hr day: night cycle. QD solutions were made immediately preceding the test through
simple dilution into hard water with minor stirring and no additional sonication. Synthetic
natural waters were made from nanopure water and analytical grade chemicals according to
USEPA guidelines for hard water. Statistical analysis on acute toxicity data was done with
Priprobit (Ver 1.63, Kyoto Japan).
We characterized the nanoparticle concentrations during tests via fluorescence, which was
measured at 0, 24 and 48 hours after exposure (FluoroMax 4, Joriba Yorn). In addition, we
measured total metal concentrations suspended in solution (i.e. no stirring prior to sample
collection) at 0 and 48 hours using Inductively Coupled Plasma-Optical Emission Spectroscopy
(ICP-OES). Finally, 3000 Dalton nitrations were done at 0 and 48 hours to separate dissolved
metals from whole and partial nanoparticles (Millipore Bioseparation Spin Filters).
144
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Micro X-ray fluorescence (jiXRF) imaging for elemental mapping was performed at the National
Synchrotron Light Source (Beamline X27A) at Brookhaven National Laboratory, Long Island
NY. Daphnia were air dried and mounted on Kapton tap for image scans.
Results and Discussion
The decline of fluorescence along with the emergence of dissolved Zn and Cd in the 3kDa
filtration samples indicate that the MUA coated QDs are unstable during a 48hr toxicity test.
In addition, visible aggregation (Red MUA QDs specifically) and a significant drop in total
metals in solution after the 48hr exposures suggests MUA QDs not only undergo dissolution,
releasing dissolved metals, but they also fall out of solution as aggregates. We monitored actual
concentrations of MUA QDs in solution using fluorescence. In contrast, the characterization data
for the PEO coated QDs indicate that they remain stable in solution throughout the acute test.
Dose-response curves were established for each of the four QDs tested. The dose for each
curve was represented four different ways: 1) particle number (nmol QDs/L), 2) mass basis (mg
QDs/L), 3) equivalent Cd concentration (mg Cd/L), and 4) equivalent Zn concentration (mg
Zn/L). See Figure 1.
•Green MUA * Red MUA o Green PEO A Red PEO
—Green MUA -Red MUA —Green PEO —Red PEO
• Dissolved Cd • Dissolved Zn
Dissolved Cd Dissolved Zn
EC50
Green MUA 5.14(4.32-5.19}
RedMJA 0.10(0.05-0.13)
Green PEO 1.28(1.18-1.45)
Red PEO 1.44( - , + )
EC3«
Green MUA 0.42(0.33-049)
Red MUA 0.13(0.08-0.17)
Green PEO 0.78(0.70-0.89)
RedPEO 4.111 - , + )
10 15 23 25
QD Concentration (nmol/L)
30
1234
QD Concentration (mg dots/I)*
EC50
Green MUA 034(0.03-0-04]
RsdMUA 0.009(0.006-0.011]
Gram PEO 0.39(035-0.«]
RfldPEO 124( - , t )
amoved Zn 1.3^11.03-185
0 0.5 1 15 2 0
EquivalentZn Concentration (mg/L)
•M3ssofQDs{m$'L.! »«
-------
On a particle concentration basis (nmol/L): 1) MUA coated QDs are more toxic than PEO coated
QDs, 2) Red (5nm) MUA QDs are more toxic than Green (2nm) MUA QDs, and 3) Green and
Red (2 and 5 nm) PEO QDs are similar in toxicity. On a mass concentration basis (mg/L): 1)
MUA QDs are still more toxic than PEO QDs, however there is a greater similarity in toxicity
between MUA QDs and Green PEO QDs, and 2) Red PEO QDs are significantly less toxic than
other QDs tested. On an equivalent Zn basis, QDs are more toxic than dissolved Zn, indicating
that Zn is not solely responsible for QD toxicity. This excludes Red PEO where the toxicities
are similar suggesting Zn as the cause of QD toxicity. Finally, on an equivalent Cd basis, all
QDs are less toxic than dissolved Cd, indicating that the Cd in the CdSe core is not entirely
bioavailable.
Finally, synchrotron images show presence of QDs in gut of the daphnid, however current
images show no evidence of QD metals migrating to other daphnid organs within the 48hr
exposure. Future work will include further synchrotron imaging, including 3D tomography to
further explore potential exposure routes and target organs for QD toxicity.
References
Dubretret, B., Skourides, P., Norris, D.J., Noireaux, V, Brivanlou, A.H., and A. Libchaber.
(2007) "/« vivo imaging of quantum dots encapsulated in phospholipids micelles." Science
298,1759-1762.
Parak, W.J., Baoudreau, R., Le Gros, M., Gerion, D., Zanchet, D., Micheel, C.M., Williams, S.C.,
Alivisatos, P., and C. Larabell. (2002) "Cell motility and metastatic potential studies based on
quantum dot imaging of phagokinetic tracks." Adv Mater 12, 882-885.
Lidke, D.S., Nagy, P., Heintzmann, R., and IN. Arndt-Jovin. (2004) "Quantum dot ligands
provide new insights into erbB/HER receptor-mediated signal transduction." Nat Biotechnol
22,1-6.
Hardman, R. (2006) " A toxicologic review of quantum dots: Toxicity depends on
physiochemical and environmental factors." Environ Health Perspect 114(2), 165-172.
Derfus, A.M., Chan, C.W., and S.N. Bhatia. (2004) "Probing the cytotoxicity of semiconductor
quantum dots." Nano Lett 4(1),11-18.
Kirchner, C.K., Liedt, T., Kudera, S., Pellegrino, T., Javier, A.M., Gaub, H.E., Stolzle, S., Fertig,
N., and WJ. Parak. (2005) "Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles." Nano
Lett 5(2), 331-338.
Lovric, J., Bazzi, H., Cuie, Y, Fortin, G., Winnik, F., and D. Maysinger. (2005) "Differences in
subcellular distribution and toxicity og green and red emitting CdTe quantum dots." J Mol Med
83, 377-385.
U.S. Environmental Protection Agency. 2002. Methods for measuring the acute toxicity of
effluents and receiving waters to freshwater and marine organisms, 5th ed. EPA/82l/R-02/012.
Final Report. Office of Water, Washington, DC.
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Acute and Chronic Toxicity of TiO to Freshwater Aquatic Organisms
Scott Hall, and Tina Bradley, ENVIRON International, Tennessee, U.S.A.
Joshua T. Moore, Department of Chemistry, Tennessee State University,
Nashville, Tennessee, U.S.A.
Tunishia Kuykindall, and Lauren Minella, ENVIRON International,
Brentwood, Tennessee, U.S.A.
Abstract
Preliminary testing of TiO2 particles indicated the fathead minnow is much less sensitive to TiO2
than Ceriodaphnia dubia and Daphniapulex. The most chronically sensitive species tested
was the green algae Pseudokirchneriella subcapitata. Total organic carbon (TOC) appears to
decrease TiO2 acute toxicity to C. dubia.
Introduction
The aquatic toxicity of metals-based nanoparticles is relatively unknown, but the limited data
available indicate it is not highly toxic to aquatic life, with effects concentrations in the range
of approximately 5 to above 100 mg/L (e.g., Velzeboer et al., 2008; Lovern and Klaper, 2006).
Aquatic testing of nanoparticles has indicated concerns related to test material preparation
techniques, and the general applicability of conventional toxicity test methods for evaluation
of nanoparticles. The effects of water quality parameters on the toxicity of nanoparticles is
unknown. This study evaluated the acute and chronic toxicity of TiO2 nanopowder to freshwater
aquatic organisms as determined in standard USEPA toxicity tests, and assessed the effects of
organic carbon on TiO2 acute toxicity.
Methods
The test material was 99 percent titanium dioxide nanopowder (10 nm) from American Elements.
TiO2 stock solutions were prepared by adding known masses to moderately hard water (USEPA,
2002a) or algae culture water (USEPA, 2002b) and stirring for a minimum of 30 minutes before
addition to test waters. Test exposures were prepared by serial dilution of TiO2 stocks with
respective test waters: moderately hard water for fish and cladocerans, and algae culture medium
for algal tests. Fish (fathead minnow, Pimephalespromelas) and cladocerans (Ceriodaphnia
dubia and Daphnia pulex) were cultured in moderately hard or similar water, and green algae
(Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum) was cultured in USEPA
culture water. Acute (48 hours for cladocerans, 48 and 96 hours for fathead minnow) and
chronic (96 hours for P. subcapitata, 1 days for fathead minnow and C. dubia) toxicity tests
followed methodologies outlined by USEPA (2002a, 2002b). Acute median lethal concentrations
(LC50) and chronic 25 percent inhibition concentrations (IC25) were calculated as recommended
by USEPA (2002a, 2002b). Nominal TiO2 concentrations were used in the calculation of LC50
and IC25 values. A wheat grass food component of the cladoceran foods was used to establish
147
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total organic carbon (TOC) levels of 1.5 mg/L in test waters to evaluate the effects of TOC on
TiO2 toxicity. Assessments of the forms of TiO2 in toxicity test solutions were performed using
powder x-ray diffraction (XRD) techniques (Klug and Alexander, 1974).
Results and Discussion
All toxicity tests met control and test acceptability criteria specified by USEPA for survival
and growth (fathead minnow), survival and reproduction (C. dubia), and cell production (P.
subcapitata). The addition of TiO2 to test waters did not alter general test water chemistry
(e.g., pH, conductivity). Table 1 summarizes the results of TiO2 acute toxicity tests. For
tests conducted in the absence of TOC additions, the cladocerans (C. dubia and/), pulex)
demonstrated high sensitivity to TiO2 relative to the fathead minnow: cladoceran LC50 values
approximately 3 to 16 mg/L, fathead minnow LC50 values 500 mg/L and above. Although C.
dubia at times demonstrated higher sensitivity to TiO2 than D. pulex, the range of C. dubia and
D. pulex LC50 values was similar: C. dubia 3.0 to 15.9 mg/L, and D. pulex 6.5 to 13.0 mg/L,
and no between-species values were statistically different given that 95 percent confidence
intervals overlapped. Fathead minnow LC50 values were consistently greater than 1,000 mg/L.
These data confirm the relatively high sensitivity of the cladocerans (as compared to fish) as has
been reported in the literature for conventionally-tested compounds such as salts and metals.
In an initial acute toxicity test conducted in the presence of approximately 1.5 mg/L TOC, C.
dubia demonstrated much lower sensitivity to TiO2 as compared to tests in the absence of TOC.
The C. dubia LC50 value for TiO2 in the presence of organic carbon was above 100 mg/L. This
indicates that organic carbon decreases the bioavailability of TiO2, likely by complexation (e.g.,
chelation or sorption) with the TOC and/or suspended solids in the wheat grass preparation added
to increase the organic carbon content of the test water. Decreases in toxicant bioavailability due
to complexation mechanisms have long been recognized for both metal and organic toxicants. It
is unlikely that the decrease in TiO2 toxicity observed in this study is due to enhanced nutritional
status of C. dubia in test waters to which organic carbon was added. In tests in our laboratory
with salts (e.g., NaCl), which should not be markedly complexed by organic carbon, toxicity
decreases due to wheat grass additions were typically on the order of 20 percent, not upwards of
6-fold as observed for TiO2.
Table 2 summarizes the results of TiO2 chronic toxicity testing. Although the C. dubia IC25
value was among the lowest observed, P. subcapitata demonstrated the highest sensitivity to
TiO2, with an IC25 value of 1 mg/L TiO2. These data indicate that algae and cladocerans are
much more sensitive to TiO2 than the fathead minnow on a chronic toxicity basis. It should also
be noted that the ratio of the acute to chronic toxic effects levels for C. dubia and the fathead
minnow are small (on the order of two to three). This indicates that acute and chronic toxicity to
water column organisms occurs at similar concentrations of TiO2. The food additions in chronic
toxicity tests may play a role in this phenomenon given the results of the TOC-addition tests.
Powder XRD scans of test solutions indicated that the only detectable TiO2 phase present was
the anatase form. No traces of crystalline rutile or brookite were observed. The average particle
size of the TiO2 nanoparticles determined from XRD peak widths was 11.2 ± 3.0 nm, very
similar to the nominal particle size of 10 nm listed by the manufacturer. TiO2 samples isolated
from the moderately hard test waters showed no significant changes in average particle size due
148
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Table 1. Results of TiO2 acute toxicity tests
Species
Cladocerans
Ceriodaphnia dubia
Ceriodaphnia dubia
Ceriodaphnia dubia
Ceriodaphnia dubia - + TOC
Daphniapulex
Daphniapulex
Fish
Fathead Minnow- 48 hour
Fathead Minnow- 48 hour
LC50 (mg/L TiO2)
3.0
13.4
15.9
>100
6.5
13.0
500
>1,000
95% Confidence Intervals
(mg/L)
1.6 to 6.4
9.5 to 18.4
13.4 to 19.0
NC
4.4 to 12.9
2.8 to 24.0
NC
NC
NC = Not calculable.
Table 2. Results of chronic toxicity tests with TiO2
Species
Cladoceran
Ceriodaphnia dubia
Ceriodaphnia dubia
Fish
Fathead Minnow
Green Algae
Pseudokirchneriella
subcapitata
IC25 (mg/L TiO2)
2.5
9.4
342
1.0
95% Confidence Intervals
(mg/L)
NC
2.8 to 15. 3
283 to 430
0.33 to 5.2
NC = Not calculable.
to agglomeration.
Conclusions
The green algae P. subcapitata was shown to be most sensitive to TiO2, followed by cladocerans
(daphnids), then the fathead minnow. In comparison to the acute and chronic toxicity of many
environmental contaminants, TiO2 is of moderate toxicity. This study also demonstrated that
the widely-applied USEPAtest protocols are appropriate for evaluation of the ecotoxicological
effects of TiO2. A key study finding was that TOC can decrease TiO2 toxicity. Thus, toxicity
tests utilizing reconstituted test waters likely overestimate TiO2 toxicity in natural receiving
streams containing TOC. The TiO2 acute to chronic ratio was shown to low, indicating that acute
and chronic toxicity occurs at similar concentrations of TiO2.
References
Lovern, S.B. and R. Klaper. 2006. Daphnia magnaMortality when Exposed to Titanium
Dioxide andFullerene (C6g) Nanoparticles. Environ. Toxicol. Chem. Vol. 25, No. 4. pp. 1132-
1137.
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USEPA, 2002a. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters
to Freshwater and Marine Organisms. Fifth Ed. EPA-821-R-02-012. USEPA Office of Water.
Washington, D.C.
USEPA, 2002b. Short-term Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to Freshwater Organisms. Fourth Ed. EPA-821-R-02-013. USEPA Office of
Water. Washington, D.C.
Velzeboer, I, A.J. Henriks, A.M. Ragas, D. van de Meent, 2008. Aquatic Ecotoxicity Tests of
Some Nanomaterials. Env. Toxico. Chem. In Press.
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Toxicity of Multi-Walled Carbon Nanotubes in Water to Sediment-Dwelling
Invertebrates
Joseph N. Mwangi, Department of Civil and Environmental Engineering,
University of Missouri, Columbia, Missouri, U.S.A.
Ning Wang, Christopher G. Ingersoll, Doug K. Hardesty, and Eric L. Brunson, U.S.Geological
Survey, Columbia Environmental Research Center, Columbia, Missouri, U.SA.
Li Hao, Department of Mechanical and Aerospace Engineering, University of Missouri,
Columbia, Missouri, U.SA.
Baolin Deng, Department of Civil and Environmental Engineering, University of Missouri,
Columbia, Missouri, U.SA.
Abstract
Carbon nanotubes (CNTs) are relatively insoluble in water and are likely to accumulate in
sediments if released into the aquatic environment. The potential impacts of CNTs released
into the environment are largely unknown. The objective of this study was to evaluate the
potential toxicity of commercially available multi-walled carbon nanotubes (MWCNTs) to
sediment-dwelling invertebrates. Short-term 14-d water-only tests were conducted by exposing
the amphipod (Hyalella azteca), the midge (Chironomus dilutus), the oligochaete (Lumbriculus
variegates), and rainbow mussels (Villosa iris) to a thin layer of two MWCNT samples with
periodic replacement of water. The survival of the invertebrates was significantly reduced in both
MWCNT samples relative to the control, and in most cases the growth of the test organisms was
also significantly reduced. Photographs and light microscopy images of surviving organisms
at the end of the tests showed presence of MWCNTs in the guts of the amphipods, midge and
oligochaete. The MWCNTs appear capable of smothering the organisms and may interfere
with their ability to feed. Other mechanisms may exist for the demonstrated toxicity such as by
dissolution of toxic metals from the MWCNTs. Further tests are planned to evaluate the toxicity
of the MWCNT in sediment to the sediment-dwelling invertebrates.
Introduction
Carbon nanotubes (CNTs) have high electro-optical, thermal conductivity, mechanical strength
and large surface area. Applications of the CNTs include aerospace and fiber industries,
electronics and semiconductors, hydrogen-based fuel cells, environmental sensors and medical
fields. With increasing commercial interests, the supply and demand of CNTs is expected to grow
rapidly (Hyung et al. 2007). The CNTs have shown toxicity to living biological cells and tissues
(Panessa-Warren et al. 2006, Pulskamp et al. 2007) and to rainbow trout (Oncorhynchus mykiss)
and cladoceran (Daphnia magna) (Nowack and Bucheli 2007) and to developing embryos of
zebra fish (Danio rerio) (Chen et al. 2007). The potential impact of CNTs released into the
environment however is largely unknown. Since the CNTs are relatively insoluble in water, the
materials would likely be associated with sediment in the aquatic environment. The objective
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of this preliminary screening study was to evaluate the potential toxicity of MWCNTs in water
using four sediment-dwelling invertebrates.
Method
Two samples of MWCNTs for toxicity testing were obtained from Helix Material Solutions
Inc., TX, USA (Sample 1) and Shenzhen Nanotech Port Inc., Shenzhen, China (Sample 2). The
amphipod (Hyalella Azteca), the midge (Chironomus dilutus), the oligochaete (Lumbriculus
variegates), and rainbow mussel (Villosa iris) were exposed to the two MWCNTs samples in
water adjusted to 100 mg/L hardness with 4 replicates/treatment and 10 organisms/ replicate
chamber. The treatments included 200 mg of sonicated or non-sonicated MWCNTs added to each
300-ml glass beaker containing 200 ml of water. The MWCNTs formed a thin layer at the bottom
of the beakers. The control treatment received 200 ml of water and a 5-ml fine sand substrate
except for mussel tests with no sand. The tests were conducted for 14 d in static conditions
with aeration of overlying water and 50% water replacement every Monday, Wednesday and
Friday. Juvenile mussels were fed 2 ml of a non-viable algal mixture twice daily, amphipods
were fed 0.5 ml of Yeast-Cerophyl-Trout Chow (YCT) while oligochaete and midge were fed
1.0 ml of Tetrafin® flake fish food every Monday, Wednesday and Friday immediately after
water replacement. Other test conditions generally followed the standard conditions outlined
in ASTM (2007) and USEPA (2000) with test water maintained at 23±1°C, ambient laboratory
illumination, wide-spectrum fluorescent lights at about 200 lux, and photoperiod of 16L: 8D.
Results and Discussion
The survival or growth of all four species exposed to the MWCNT Sample 1 was significantly
reduced relative to the control except for the growth of mussels in non sonicated or sonicated
MWCNTs (Table 1). The survival of amphipods exposed to non sonicated or sonicated MWCNT
Sample 2 was significantly reduced relative to the control. The survivals of oligochaete exposed
to non-sonicated MWCNTs Sample 2 and mussel exposed to sonicated MWCNTs Sample 2
were significantly reduced relative to the control (Table 1). The midge survival of 63% in control
from test with MWCNT Sample 2 was below the established acceptability criteria (>70%)
but a general decline in survival between the controls, sonicated or non sonicated MWCNT
treatments was observed. These results indicate the two MWCNTs samples tested were toxic to
the invertebrates.
The MWCNTs were observed in the gut of midge, oligochaetes and amphipods (e.g., Figure
1 for amphipods) and was adsorbed onto the surface of these invertebrates including the shell
of mussels. Light Microscopy images illustrated MWCNTs clumped in the gut of midge
and amphipods. The MWCNTs may obstruct the passage of food through the gut leading to
starvation. The coating of the MWCNTs on the surface of the organisms may impair respiration
through blockage of the gills or possibly offered an entry route of the MWCNTs into the body
of the invertebrates. Other mechanisms may exist for the demonstrated toxicity such as by
dissolution of toxic metals from the MWCNTs and will be documented. Toxicity studies are
planned spiking MWCNTs and other nanomaterials into sediment under environmentally realistic
exposure concentrations (perhaps up to 1% MWCNTs added to sediment by weight).
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Figure 1. Amphipod Hyalella azteca (x!8) on 6-d of exposures in: (A) control (water only), (B)
non-sonicated MWCNT treatment, showing MWCNTs in the gut.
Acknowledgements
Support of this research was provided in part by the United States Environmental Protection
Agency (USEPA) STAR program (Grant #RD 83331601-0) and Missouri Alliance for Graduate
Education and the Professoriate Fellowship to JM through the University of Missouri-Columbia.
References
American Society for Testing and Materials (ASTM). 2007. ASTM International standard
test method for measuring the toxicity of sediment-associated contaminants with freshwater
invertebrates (ASTM E1706-05). Annual Book of ASTM Standards Volume 11.06, West
Conshohocken, PA.
Cheng JP, Flahaut E, Cheng SH. 2007. Effect of carbon nanotubes on developing zebrafish
(Danio rerio) embryos. Environmental toxicology and Chemistry 26 (4):708-716.
Hyung H, Former JD, Hughes JB, Kim J. 2007. Environ. Sci. Technol. 41:179-184.
Panessa-Warren BJ, Warren JB, Wong SS, Misewich JA. 2006. Biological cellular response to
carbon nanoparticle toxicity. J Physics: Condensed Matter 18: S2185-S2201.
Pulskamp K, Diabate S, Krug FH. 2007. Carbon nanotubes show no sign of acute toxicity
but induce intracellular reactive oxygen species in dependence on contaminants. Toxicology
Letters. 168:58-74
Nowack B, Bucheli TD. 2007. Occurrence, behavior and effects of nanoparticles in the
environment, a review. Environment Pollution 150:5-22
United States Environment Protection Agency (USEPA). 2000. Methods for measuring the
toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates,
second edition, EPA/600/R-99/064, Washington, DC.
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Table 1. Mean (n=4) survival or grcnvth ofamphipods (Hyalella azteca), rainbow mussels (Villosa iris), midge (Chirunomus dilutus),
and oligochaetes (Lumbriculus variegalus) in 14-d water-only toxicity test with two multi-walled carbon nanotube (MWCNT)
samples. Standard deviations in parenthesis.
Test organism Treatment
Amphipod
Midge
Mussel
Oligoehaete
Control
Non-sonicated MWCN T
Sonicated MWCNT
Control
Non-sonicated MWCNT
Sonicated MWCNT
Control
Non-sonicated MWCNT
Sonicated MWCNT
Control
Non-sonicated MWCNT
Sonicated MWCNT
•MWCNT Sample 1
Survival (%)
88(5.0)
5.0(10)*
2.5 (5.0)'
80 (8.2)
60 (8.2)*
43 (9.6)*
98(5.0)
20(14)*
43(19)*
16(1.5)
14(1.0)*
13(1.7)*
Biomass (mg) a
0.41 (0.07)
0.02 (0.03)*
0.01 (0.01)*
5.4 (0.2)
0.4(0.1)*
0.2(0.1)*
Shell length (mm)
2.2(0.1)
1.6(1.0)
2.4 (0.3)
Biomass (nig)1
15(1.3)
13(1.0)*
11(1.7)*
MWCNT Sample 2
Survival (%)
100(0)
7.5 (9.6)*
5.0(10)*
63(15)b
55 (6)
7.5 (9.6)
80 (28)
35 (25)
5.0(10)*
3.3(1.8)
0.8 (0.2)*
2.8(1.7)
Biomass (mg) c
ND
ND
ND
2:i (1.0)
1.2(1.0)
ND
ND
ND
ND
2.7(1.8)
0.2(0.2)
2.2(1.3)
* Dry weight of individual amphipods was estimated from the measurements of the lengths of individual amphipods using a length-
weight relationship equation generated from a linear regression of amphipod length to the cube root of amphipod dry weight
(unpublished data, Nile Kemble, USGS, Columbia, MO).
h Less than 70%, the lest acceptability criteria established for the control and statistical analysis was not performed.
L Ash free biomass.
ND - Not determined because the surviving organisms were used for light microscopy imaging.
* Significantly different from control (t- test, p<0.05).
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Chapter 7 - Introduction
Environmental Fate & Transport of Nanomaterials
Barbara Karn and Madeleine Nawar, United States Environmental Protection Agency
As more manufactured/engineered nanomaterials are produced, their release into the
environment becomes more probable. Since some of these materials have exhibited intrinsic
toxicity in laboratory tests, it is important to understand their transformation, pathways, and
potential hazards of exposure to public health and the environment. Some nanomaterials may
persist, bioaccumulate, or become mobile in the ecological matrices of air, water and land.
This understanding can lead to mitigating and minimizing exposure and possible adverse effects.
For example, magnification and accumulation of nanomaterials through the food chain and other
routes of exposure may lead to effects that are not evident in short-term toxicity tests. While
the short-term effects of a toxic nanomaterial may result from a single exposure, the long-term
effects due to bioaccumulation and persistency may be more severe, ranging from lasting health
problems to organ damage.
The fate and transport session consisted of 17 presentations of multidisciplinary research
papers. The plenary addressed the importance of using studies and techniques involving natural
nanoparticles in order to inform research on manufactured nanomaterials. In particular, studies
on ultrafines in air and natural colloids or nanoparticles in water and soil in the environment can
help inform work on manufactured nanomaterials. Existing techniques may be interchangeable.
However, because of the novel properties of nanomaterials, new or modified test methods for
environmental fate and transport endpoints, and applications of new or existing air dispersion,
soil transport, groundwater models, may be needed. Changes that nanoparticles undergo due to
environmental factors such as pH, ionic strength, organic matter, or calcium need to be taken into
account.
Characterization of the nanomaterials under study is essential to understand their fate, transport,
toxicity and other factors. An integrated approach involving characterization of bulk and surface
properties of nanoparticles, the effects of different environmental factors and combining both lab
and field studies is necessary. Characterizing aggregation and agglomeration is also important.
Several talks focused on measuring the movement of nanomaterials in various media.
For example, quantum dot movement can be traced by their fluorescence; gold nanoparticles can
be used to detect bacteria density using X-ray computed tomography.
Standard column tests showed that multi-walled carbon nanotubes, fullerenes, and aluminum
nanoparticles behaved differently from normal chemical flows, indicating the need for different
models. Columns were also used to compare movement of lactate-coated and uncoated particles
of nanoscale zero valent iron.
Transformations and aging played a role in changing iron nanoparticles in water. Humic
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acids caused aggregation of boron nanoparticles by different mechanisms dependent on the
natural background. Natural organic matter was also studied as it affects quantum dot phase
transfer, enhanced C60 dispersion, movement of iron oxide nanoparticles, and multiwall carbon
nanotubes.
Models can be used to inform the movement of nanoparticles through the soil. A commercial
model was used to predict nanoparticulate zero valent iron (nZVI) concentration and determine
injection rates needed to clean up hexavalent chromium. Mesocosms can also be used to
determine the movement, bioavailability, and toxicity of nanomaterials in various environmental
compartments of water systems. For example, TiO2 has been studied as a sample nanomaterial
in mesocosms. These mesocosm systems then can provide data for building models.
Fate, transport, and transformation could be affected by bacteria in the environment. For
example, composition of quantum dots is not the only aspect of these materials that could change
their toxicity. Bacteria can change the aggregation state which may change their environmental
toxic effects, indicating that toxicity is controlled by aggregation as well as composition.
The papers in the session were indicative of the diverse approaches and the diverse nanomaterials
being studied to determine fate and transport of nanoparticles in the environment. In general,
the papers highlighted the differences in behavior of nanomaterials when using conventional
environmental tests, the importance of characterization of the nanomaterials, and the different
environmental interactions nanomaterials can undergo.
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Interactions Between Natural and Manufactured Nanoparticles.
Jamie R Lead, Mohammed Baalousha, and Emilia Cieslak, School of
Geography, Earth and Environmental Sciences, University of Birmingham,
Birmingham, United Kingdom
Abstract
Manufactured nanoparticles have huge potential in a number of areas. However, there is also
potentially large but unknown risk from their use both to human and environmental health.
Understanding their potential exposure and hazard requires that their chemistry and transport
in the environment is better understood. Some research has been performed in these areas,
especially on the impacts of pH and ionic strength. A few studies have investigated the role
of natural colloids and nanoparticles and these are reviewed in this study and the effects this
interaction may have manufactured nanoparticle effects and behaviour.
Manufactured nanoparticles (NPs) are usually denned as being between 1-100 nm in size and are
an important product of both nanoscience and nanotechnology i.e. the science and technology of
the nanoscale (1-100 nm). There is huge current investment in nanotechnology and the expected
market for nanoparticles is already large and set to grow massively. Evidence of the scientific
interests and outputs is abundant in the literature (Ju-Nam and Lead, 2008). Nanotechnology
has the potential to enable, support and develop current industries and has the potential to
revolutionise fields such as environmental remediation, health care, computing and electronics.
Nevertheless, despite this potential, there are concerns that nanotechnology and nanoparticles
could pose serious risks to the environment, although both exposure and hazard are poorly
understood; indeed a key problem is that our direct understanding is extremely poor. Innovation
and the synthesis of new materials are currently expanding near-exponentially from a large
base of current research mass and research spending. However, the communities investigating
the possible effects of nanoparticles is small and relatively poorly funded. Greater funding
in this area is starting to occur but is still inadequate given the development and changes in
nanotechnology.
Understanding the environmental behaviour of NPs can be performed fully only if their
behaviour at realistic conditions is considered. Such conditions include ionic strength, pH
and, importantly, NP concentration. Another important consideration, often overlooked is the
importance of natural organic colloids and particles. Naturally occurring colloids and particles
are usually defined as materials between 1 nm - 1 um and > 1 um, respectively (Lead and
Wilkinson, 2006). These materials are naturally produced by microbial action, weathering,
hydrolysis and other processes and are important in manufactured NP fate and behaviour for two
reasons:
1) As with the study of atmospheric ultrafine particles, natural aquatic and terrestrial colloids
offer a large background of data and understanding which can be used to help understand what
might happen to NPs in environmental systems.
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2) Natural colloids/nanoparticles and manufactured nanoparticles directly interact in the
environment and this results in changes in their dispersion, bioavailability and potential mobility.
Below, we will discuss briefly the two points raised above: what we can learn from the behaviour
of natural colloids that is relevant to manufactured nanoparticle behaviour and the direct
interactions which affect nanoparticle behaviour and effects.
Natural colloids and nanoparticles. There is a long history of research in aquatic and terrestrial
systems which has developed for two reasons. Firstly, the development of often cheap, readily
available and robust technology during the twentieth century enabling their study (membrane
technology, humic substances, HS, extraction, electron microscopy etc) and the realisation
that these solid-phase material were important in pollutant, particularly metal, transport and
bioavailability. This area has been thoroughly reviewed recently (Lead and Wilkinson, 2006).
Areas of particular relevance are:
1) The ability of some small nanoparticles such as HS to form nanoscale films on all
surfaces (Lead et al, 2005) which stabilise surfaces by imparting a highly negative charge at
environmental concentrations and also by steric mechanisms, giving further stability. HS of
a few nm to tens of nm have also been shown to sorb onto biological cell surfaces (Lead and
Wilkinson, 2006), changing cell membrane charge and permeability and also to act as an energy
source for bacteria, indicating uptake through cell walls
2) The secondary role of fibril-like polysaccharides and proteins which result in increased
aggregation via bridging.
3) The large specific surface area of natural colloids and nanoparticles allowing them to bind
pollutants strongly while aggregating and settling over short time periods, removing pollutants
from the water column and causing a build-up in the sediments, sometimes termed colloidal
pumping.
4) In porous media, an increased pollutant transportation rate and distance due to binding with
small colloids. This reduces pollutant uptake onto the immobile solid phase rock or soil, while
enabling transport though small pores, the whole system being analogous to a chromatography
system.
Colloid-nanoparticle interactions. In the absence of direct information of nanoparticles,
this data is one of the main sources for consideration of nanoparticle effects on environmental
human health. The points above have provided the background to generate the on-going debate
on nanoparticle dispersion and aggregation, nanoparticle sedimentation or retention in the
water column and nanoparticle transport in soils and groundwater. Nevertheless, most current
research in this area has looked directly at point 1 and the ability of HS to stabilise nanoparticles
(Baalousha et al, 2008; Diegoli et al, 2008). The general conclusion is that stabilisation is
effected to a large degree, due to both charge and steric stabilisation. However, some studies
have shown that ionic strength, Ca concentration and pH are all important and may result in
further aggregation. Our own data (manuscript in prep) further shows that the dispersion of
strongly bound, sterically stabilised NPs (PVP stabilised 7 nm old) are unaffected by any of these
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conditions, although small changes in surface chemistry were apparent, indicating the importance
of binding and stability mechanisms of capping agents to environmental behaviour.
References
Baalousha, M.A., A. Manciulea, S. A. Cumberland, K. Kendall, K. and J. R. Lead (2008).
'Aggregation and surface properties of iron oxide nanoparticles; influence of pH and natural
organic matter'. Environ. Toxicol. Chem., 27, 1875-1882.
S. Diegoli, A. L. Manciulea, S. Begum, I. P. Jones, J. R. Lead and J.A. Preece. (2008)
'Interactions of charge stabilised gold nanoparticles with organic macromolecules'. Science of
the Total Environment, 402, 51-61.
Y. Ju-nam, J.R.Lead (2008) 'Manufactured Nanoparticles and Natural Aquatic Colloids: An
Overview of their Chemical Aspects, Interactions and Potential Environmental Implications'.
The Science of the Total Environment, 400, 396-414.
S. J. Klaine, P.J.J. Alvarez, G. E. Batley, T. F. Fernandes, R. D. Handy, D. Lyon, S. Mahendra,
M. J. McLaughlin, and J. R. Lead (2008). 'Nanomaterials in the Environment: fate, behaviour,
bioavailability and effects'. Environmental Toxicology and Chemistry, 27, 1825-1851.
J. R. Lead and K. J. Wilkinson (2006). 'Natural aquatic colloids: current knowledge and future
trends'. Environmental Chemistry, 3, 159-171.
Figure l.TEM micrograph showing iron oxide particles coated with a layer of HA (white ar-
rows) and surrounded by a network of humic substances molecules (black arrows).
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Figure 2. TEM micrograph showing single walled carbon nanotubes in the presence (top) and
absence (bottom) of a filtered natural water (Vale Lake, Birmingham, UK).
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An Integrated Approach Toward Understanding the Environmental Fate,
Transport, Toxicity and Occupational Health Hazards of Metal and Metal
Oxide Nano materials
VickiH. Grassian, Department of Chemistry, and the Nanoscience and Nanotechnology Institute
at The University of Iowa, Iowa City, Iowa, U.S.A.
Abstract
There are many questions related to the environmental fate and transport of nanomaterials as
well as the environmental health and safety of nanomaterials on living systems. Nanoparticles,
the primary building blocks of many nanomaterials, may become suspended in air during
production, distribution, use and disposal, or get into water systems, e.g. drinking water systems,
ground water systems, estuaries and lakes. Therefore, manufactured nanoparticles can become
a component of the air we breathe or the water we drink. One important issue in understanding
the environmental fate, transport, toxicity and occupational health hazards of nanoparticles is the
characterization of the nature and state of nanoparticles in air, water or in vivo.
Introduction
Studies directed toward understanding the environmental and biological fate of nanoparticles and
the potential transformation of nanoparticles have just begun in recent years (Elzey et al. 2009).
It is becoming increasingly clear that the environmental and biological fate as well as the toxicity
of nanoparticles depends on nanoparticle size, shape, bulk composition and phase, and surface
area and composition. Therefore, it is imperative that an integrated approach that combines
extensive nanomaterial characterization along with any investigation of the environmental fate,
transport, toxicity or environmental health and safety of nanomaterials be employed (Pettibone et
al. 2008a).
For the nanomaterials of interest in these studies, metal oxide and metal nanoparticles, it can
be asked: (i) will metal oxide and metal nanoparticles be present in air or water as isolated
particles or in the form of aggregates?; (ii) will metal oxide and metal nanoparticles dissolve
in aqueous solution or in vivo?; and (iii) under what conditions will metal oxide and metal
nanoparticles aggregate or dissolve? As the size regime will be very different depending on the
state of the nanoparticles, as dissolved ions, isolated nanoparticles or nanoparticle aggregates,
these questions are important to address as it impacts the size regime that needs to be considered
or modeled in, for example, environmental transport or lung deposition models. Furthermore,
the effect on biological systems including nanoparticle-biological interactions and toxicity will
depend on the state of nanoparticles in vivo.
In the studies discussed here, an integrated approach is used to address these questions and
issues. The approach combines state-of-the-art characterization of the bulk and surface
properties of nanoparticles, studies of the state of nanoparticles in different environments as
determined by aggregation measurements and dissolution measurements in laboratory, field and
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toxicity studies. The data from these studies are expected to provide for a better understanding
of the environmental fate, transport, toxicity and occupational health hazards of metal and metal
oxide nanomaterials.
Methods
Nanoparticle Characterization Methods. Unlike molecules with discreet molecular formulas,
nanoparticles are often of varying size with size distributions that can be quite narrow and
monodispersed or in some cases much more polydispersed with wider distributions. Since both
the macroscopic and microscopic behaviors of metal and metal oxide nanomaterials depend on
size, phase and surface properties, it is essential that these properties be characterized using a
suite of complementary techniques that include X-ray diffraction, microscopy, surface area and
surface composition measurements.
Aggregation in Air and Water. Since there is a propensity for nanoparticles to aggregate
under certain conditions, measurements of aggregate size are important in the characterization
of nanoparticles. Aggregation measurements in solution with two types of light scattering
measurements are used to investigate nanoparticle aggregation size and nanoparticle aggregation
kinetics, dynamic light scattering and sedimentation kinetics using light scattering. These
techniques provide information about the size of the aggregates and the stability of the
aggregates in solution. In air, a scanning mobility particle sizer is used to measure the size of
nanoparticles formed once generated as an aerosol.
Surface Chemistry. Surfaces play an important role in the properties of nanomaterials. Surfaces
of nanomaterials can be functionalized either in the manufacturing process or from the
adsorption of molecules from the ambient environment. In these studies, spectroscopy is used to
measure surface adsorption and surface chemistry of nanoparticles from both solution and gas-
phase environments for nanoparticles of different size. In addition, since dissolution by its very
nature is a surface phenomena, dissolution studies in various media provide essential data on the
stability of metal oxide and metal nanoparticles.
Inhalation and Instillation Toxicity Studies. As inhalation is expected to be a major route of
exposure, especially in occupational settings, a summary of some recent toxicity studies will be
discussed. Details of the experimental protocols can be found in Grassian et al. (2007a,b) and
Pettibone et al. (2008c).
Field Measurements. There is a great deal of interest in characterizing nanomaterials in outdoor
and indoor environments, especially in occupational settings. Samples were collected on filter
media in a manufacturing facility (Peters et al. 2008). These samples were then analyzed further
with electron microscopy techniques including: transmission electron microscopy, scanning
electron microscopy and energy dispersive X-ray analysis.
Results and Discussion
Metal Oxide and Metal Nanoparticles - Laboratory Studies. Laboratory studies of metal oxide
nanoparticles have focused on metal oxide and metal nanoparticle aggregation and dissolution as
well as the surface chemistry of these nanoparticles. One example is a study of the adsorption
of oxalic acid and adipic acid, on TiO2 nanoparticles (Pettibone et al. 2008b). Solution phase
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measurements were used to quantify the extent and reversibility of oxalic acid and adipic acid
adsorption on anatase with primary particle sizes of 5 and 32 nm. At all pH values considered,
there were minimal differences in measured Langmuir adsorption constants between the two.
Although macroscopic differences in the reactivity of these organic acids as a function of
nanoparticle size were not observed, ATR-FTIR spectroscopy show some distinct differences in
the infrared absorption bands present for oxalic acid adsorbed on 5 nm particles compared to 32
nm particles, suggesting different adsorption sites or a different distribution of adsorption sites
for oxalic acid on the 5 nm particles. Furthermore, it is clear that particle aggregation occurs at
all pH values and that organic acids can destabilize nanoparticle suspensions.
Although metal nanoparticle aggregation is evident in air and water, the focus of our most recent
studies is on the propensity of metal nanoparticles to dissolve under different environmental
conditions. We have investigated the dissolution of Ag, Fe and Cu nanoarticles using particle
sizing measurements as well as more conventional solution phase studies. The results of these
studies provide insights into the stability of nanoparticles in different environments and media.
Nanoparticle Toxicity - Size and Composition Comparisons. Using murine models for
inflammation, size effects of inflammatory response in instillation and acute inhalation
exposures of TiO2 nanoparticles with manufacturers' average particles sizes of 5 and 21 nm were
investigated. The properties of the primary nanoparticles, aerosol and instillation solution for
both sized nanoparticles were evaluated. Results show the larger TiO2 nanoparticles were found
to be moderately, but significantly, more toxic. The nanoparticles had different agglomeration
states which may be a factor as important as the surface and physical characteristics of the
primary nanoparticles in determining toxicity. Furthermore, we recently investigated using
similar methods copper and iron nanoparticles. Copper nanoparticle-exposed mice had
significantly higher levels of inflammation and response. At biologically relevant pHs, in vitro
studies showed that copper nanoparticles displayed a propensity for dissolution. We conclude
that the presence of dissolved ions and the concomitant formation of smaller nanoparticles play
major roles in copper nanoparticle toxicity.
Characterization of Manufactured Nanomaterials Collected in a Manufacturing Facility.
Analysis of airborne particles collected from a manufacturing facility that produces titanium
oxide-based nanomaterials provides insight into the potential exposure in an occupational setting
(Peters et al. 2008). In particular, particles collected on filter media by electron microscopy and
energy dispersive X-ray analysis were used to distinguish airborne engineered nanomaterials
from incidental particles. The analysis showed that during the production of engineered
nanomaterial relatively large particles are liberated into the air. In particular, microscopy
shows that formation of several different types of particles including large 200-nm to 10-|im
spheres. Further analysis showed that the large spheres were composed of smaller fused 10-80
nm nanoparticles. These large spherical aggregates contained titanium and were thus positively
identified as related to the production of engineered nanoparticles and distinct from other particle
types that were incidental to production.
Conclusions
A number of conclusions come about from these studies and include: (i) dissolution and
aggregation of metal and metal oxide nanoparticles will play a large role in the environmental
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fate, transport and toxicity of these nanomaterials; (ii) smaller TiO2 nanoparticles below 10 nm
in diameter are statistically less toxic than larger ones indicating that the nanoparticle surface
area alone does not account for nanoparticle toxicity; (iii) characterization of nanoparticles in
air and water in both laboratory and field studies should remain a high research priority if the
environmental fate, transport, toxicity and occupational health hazards of nanomaterials are to be
well understood.
Acknowledgements
The studies conducted at the University of Iowa and described here involve collaborations with
colleagues in the College of Public Health and include: Professors Peter S. Thorne, Patrick T.
O'Shaughnessy and Thomas M. Peters. I also acknowledge the contributions of John Pettibone,
Dr. Andrea Adamacova-Dodd, Sherrie Elzey, Dr. Heaweon Park and Dr. Jonas Baltrusaitis.
The work was supported in part by the National Institute of Safety and Occupational Health,
the Environmental Protection Agency and the Center for Health and Environmental Effects of
Contaminants.
References
Elzey, S., C. Howe, R.G. Larsen annd V.H. Grassian (2009). "Nanoscience and Nanotechnology:
Environmental and Health Impacts" in Nanoscale Materials in Chemistry, 2nd Edition, K.J.
Klabunde and R. M. Richards, Hoboken, NJ, John Wiley and Sons.
Grassian, V. H., P. T. O'Shaughnessy, J.B. Pettibone, J., A. Adamcakova-Dodd and P. S. Thorne
(2007a) "Inhalation Exposure Studies of Nanoparticulate Titanium Dioxide with a Primary
Particle Size of 2 to 5 nm" Environmental Health Perspectives 115, 397-402.
Grassian, V.H., A. Adamcakova-Dodd, J.B Pettibone, PT. O'Shaughnessy, PS. Thorne (2007b).
"Inflammatory response of mice to manufactured titanium dioxide nanoparticles: comparison of
size effects through different exposure routes" Nanotoxicology 1, 211-226.
Pettibone J.B., S. Elzey and V.H. Grassian (2008a). "An Integrated Approach Toward
Understanding the Environmental Fate, Transport, Toxicity and Health Hazards of
Nanomaterials" in Nanoscience and Nanotechnology: Environmental and Health Impacts, Vicki
H. Grassian, Hoboken, NJ, John Wiley and Sons.
Pettibone, J. B., D. M. Cwiertny, M. Scherer and V.H. Grassian (2008b). "Adsorption of Organic
Acids on TiO2 Nanoparticles: Effects of pH, Nanoparticle Size and Nanoparticle Aggregation"
Langmuir, 24, 6659.
Pettibone, J. B., A. Adamcakova-Dodd, PT O'Shaughnessy, P. S. Thorne, and V H. Grassian
(2008c)."Inflammatory Response of Mice Following Inhalation Exposure to Iron and Copper
Nanoparticles." Nanotoxicology, 2, 189-204.
Peters, T., S. Elzey, R. Johnson, H. Park, V.H. Grassian, PT. O'Shaughnessy (2008).
"Airborne Monitoring to Distinguish Engineered Nanomaterials from Incidental Particles for
Environmental Health and Safety" Journal of Occupational & Environmental Hygiene, 6, 73-81.
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Conference Questions and Answers
Question:
In my research, we see that metal oxides in water tend to aggregate to a certain size, but we do
not see aggregation behavior with the metals alone or the carbonaceous metal materials. We
know it has to do with the surface charge, and maybe the smaller particles have more quantum
effects than the larger particles. Could you comment on this?
Answer:
The steps necessary to get the nanomaterial to the chamber may have some effect on the
aggregation process. I do not think I have the data to answer the question. These aggregates
are really densely packed; I think it may be related to edge and corner effects. I have not done
experiments to investigate quantum effects. The aggregates made tend to be about 125 nm, and I
believe it may be due to the way they are made. A different process might yield a different size.
Question:
Are the chemical properties of, for example, titanium dioxide or copper different?
Answer:
Copper is different from titanium dioxide, and in terms of toxicity, copper is more toxic. Copper
is used in different ways, for example as a catalyst, as compared with titanium dioxide. Copper
tends to have a copper oxide overlayer. In that way, they are similar; there are no organic
functional groups.
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Fate of Quantum Dots Nanomaterials in Unsaturated and Saturated
Porous Media
Christophe J. G. Darnault, SolideaM. C. Bonina, and Burcu Uyusur, University of Illinois at
Chicago, Department of Civil and Materials Engineering, Chicago, Illinois, U.S.A.
Preston T. Snee, University of Illinois at Chicago, Department of Chemistry,
Chicago, Illinois, U.S.A.
Introduction
Nanomaterials are at the leading edge of the rapidly growing field of nanotechnology. Their
unique size-dependent properties make these materials superior and indispensable in many
areas of human activity. Nanotechnology has considerable global socio-economic value, and is
expected to have significant impacts on everyday life. Nanomaterials have numerous commercial
and technological applications in chemical, biomedical, energy, electronics and space industries.
A wide range of nanomaterials such as carbon nanotubes, fullerene derivatives, and quantum dots
are used in almost all industries and all areas of society and the prevalence of these materials in
society will be increasing, as will the likelihood of exposures (Roco and Bainbridge, 2001; Roco,
2003; Hardman, 2006). Once nanomaterials are released into the environment via manufacturing,
use or disposal, their transport is the critical parameter in assessing their exposure and impact
on the public health and the ecosystem, therefore understanding the fate of nanomaterials in the
environment is critical (Colvin, 2003; Biswas and Wu, 2005; Weisner et al., 2006; Sayre, 2007;
USEPA, 2007). Among the various types of nanomaterials, the semiconductors, quantum dots
are key enablers in nanosciences, engineering and technology. Since they were discovered in
early 1980's they have a longer impact on nanotechnology compared to the other nanomaterials
such as carbon nanotubes and composites emerged in 1990's. Currently, the data and literature
on the fate and transport of quantum dots, currently is sparse and there is a great need for
knowledge and detailed information. Quantum dot nanomaterials are a potentially new source
of contaminants, and because of the broad suite of physcial-chemical properties, could exhibit a
wide range of transport properties. Futhermore, their unique fluorescence properties make them
an excellent material to use for the investigation of the transport of nanomaterials in porous
media as it greatly facilitate their detection and quantification through visualization. Therefore,
our research goal aims at developing a visualization method and imaging process to investigate
the fate and transport of quantum dot nanomaterials in variably saturated porous media using a
non-intrusive high spatial and temporal visualization technique based on white light transmission
and UV fluorescence detection.
Method
Visualization of quantum dot nanomaterials in variably saturated porous media
The visualization method was derived from a light transmission method developed by Darnault
et al., 2001. The visualization technique selected to investigate transport of quantum dot
nanomaterials in two-dimensional variably saturated porous media is a non-intrusive method
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based on fluorescence resulting from the quantum dots optical properties. The visualization
procedure consists of exciting fluorescent quantum dots in porous media by using a UV light on
front side of the chamber and by using a light emitted devices (LEDs) as a light source in the
back of the chamber and detecting the light transmitted through the porous media to characterize
the water content. Images were acquired through a Q-IMAGING MicroPublisher RTV camera
located in front of the chamber. The visualization, calibration and image analysis was performed
using IPLab software.
To calibrate the fluorescence intensity to the quantum dots concentration in variably saturated
sand, calibration cells were used. A stock solution of quantum dot nanomaterials was prepared
and diluted by 4, 6.6 10, 13.3, 20, 50 and 100 to obatin a wide range of concentrations
(corresponding to 25%, 15%, 10%, 7.5%, 5%, 2% and 1% respectively of the stock solution
concentration). Calibration cells consist of plastic cuvettes (1.1 x 1.1 x 4.5 cm) filled with
sand as porous media and with various degree of water saturations to obtain both saturated and
unsaturated systems as well as a wide range of quantum dots concentrations. The saturated
cells were filled by 5 steps. Dry sand was poured into the cuvette and then the quantum dots
solution was added for saturation. 1.45 g of sand and 0.32 ml of quantum dots solution were
used in each step. For the unsaturated cells, the quantum dots solution and the dry sand were
first mixed in a beaker. Afterwards, the mixture was put into the cuvettes. Two calibration curves
were obtained to determine the relationship between water saturation and intensity; as well as
quantum dots concentration and hue. Each experiment includes a two-step process. In the first
step, the light source placed behind the calibration cells is switched on in a dark room and the
resulting transmitted light from the cells is recorded with the camera. In the second step, the UV
light is placed in front of the calibration cells (about 25 cm away) and the fluorescence resulting
from the cells is recorded. Both images are recorded in RGB format and processed with IPLab
software as follow: the image resulting from the light transmission is converted in intensity
format to relate the intensity parameter to water saturation, and the image resulting from the cell
fluorescence is converted in hue format to relate the hue parameter to quantum dot nanomaterials
concentration in variably saturated porous media.
Results
Water content of each calibration cell was obtained from the intensity image of the light
transmitted and quantum dot concentration of each calibration cell was obtained from the hue
image representing the fluorescence detected with the UV light (Fig. 1). Calibration curves
where developed to establish relationships between intensity versus water content and hue
Figure 1. Hue image of the calibration cells under UV light.
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versus quantum dots concentrations (Fig. 2 and 3). The procedure to quantify the quantum dots
concentration in variably saturated porous media includes two steps: first the water content in
porous media is quantified using intensity values resulting from the image obtained through
light transmission through porous and then, once the water content is determined, the quantum
dots concentration is obtained from hue values resulting from the image obtained through
fluorescence using the UV light. A linear relationship is observed between hue values and
quantum dots concentration for a constant water content (Fig. 3).
-»-25%QD
-»-15%QD
10%QD
7.5%QD
-*-5%QD
-»-2%QD
-1—1 %QD
20%
40% 60%
% Saturation
80%
100%
Figure 2. Water Saturation versus intensity values for various quantum dot concentrations.
200
180
160
» 100
• 75
A 50
• 25
Linear (25)
Linear (50)
Linear (75)
Linear (100)
0% 5% 10% 15% 20%
QD Concentration
25%
30%
Figure 3. Quantum dots concentrations versus hue values for various degrees of saturation of
porous media (25%, 50%, 75% and 100%).
Application
Quantum dots transport through variably saturated porous media
Vadose zone processes play a pivotal role in the fate and transport of subsurface contaminants
as it is typically the first subsurface environment encountered by contaminants before reaching
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the groundwater (Looney and Falta, 2000; Witherspoon, 2000). Groundwater contamination is
influenced by the hydrodynamics of vadose zone system, and the two main processes controlling
water in the vadose zone are gravity which moves water downward and capillary process that
moves water in all directions, stores it and releases it (Looney and Falta, 2000; Faybishenko
et al., 2000). In this context, a two-dimensional flow experiment in homogeneous sand was
designed to assess the role of preferential flow - fingered flow - on the transport of quantum dot
nanomaterials in vadose zone. This experiment was analyzed and processed by the visualization
technique and imaging procedures. The experimental system consisted of a two-dimensional
chamber - height: 30 cm, width: 20 cm - with 1 cm thick inner compartment that was filled with
sand porous media and various degree of water saturation were achieved through saturation and
drainage. The resulting initial experimental conditions simulated both vadose zone and aquifer
system. A quantum dots solution was applied as a point source on the sand surface to simulate
the release of nanomaterials in the subsurface environment. This simulation resulted in the
formation of a fingered flow phenomena. The fate and transport of quantum dot nanomaterials
in the vadose zone were observed and analyzed with the visualization method. The image
obtained under the UV light exposure were converted to hue system to visualize and quantify the
quantum dots nanomaterials in porous media (Fig. 4a, b). The mobility and transport of quantum
dot nanomaterials through the vadose zone by preferential flow phenomena - fingered flow -
were demonstrated (Fig 4a). The role of gas-water interfaces on the retention of quantum dot
nanomaterials at the capillary fringe was also established (Fig. 4b).
Figure 4. Fate and transport of quantum dots nanomaterials in vadose zone in Hue format
(Quantum dots are visualized in red color). Transport of quantum dots by fingered flow in
vadose zone (a). Retention of quantum dots nanomaterials by gas-water interface located at the
capillary fringe (b).
Acknowledgements
This research was funded by the University of Illinois at Chicago, U.S.A. and Regione Puglia
and High Cultural Activities, Italy (Assessorato al Lavoro, Copperazione e Formazione
Professionale FOR Puglia 2000-2006, Asse III; Misura 3.7).
References
Biswas, P., and Wu, C.-Y, 2005, Nanoparticles and the Environment, J. Air and Waste Manage.
Assoc., 55:708-746.
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Colvin, V. L., 2003, The potential environmental impact of engineered nanomaterials, Nature
Biotechnology, 21(10): 1166-1170.
Darnault, C. J. G., DiCarlo, D. A., Bauters, T. W. J, Jacobson, A. R., Throop, J. A., Steenhuis,
T. S., Parlange, J. -Y, and Montemagno C. D., 2001, Measurement of fluid contents by light
transmission in transient three-phase oil-water-air systems in sand, Water Resources Research,
37:1859-1868.
Faybishenko, B., Bandurraga. M., Conrad, M., Cook, P., Eddy-Dilek, C., Everett, L., FRx Inc. of
Cincinnati, Hazen, T., Hubbard, S., Hutter, A. R., Jordan, P., Keller, C., Leij, F. J., Loaiciga, N.,
Majer, E. L., Murdoch, L., Renehan, S., Riha, B., Rossabi, J., Rubin, Y, Simmons, A., Weeks, S.,
and Williams, C. V, 2000, Vadose Zone Characterization and Monitoring. Current Technologies,
Applications, and Future Developments, p 133-509, in Vadose Zone Science and Technology,
Brian B, Looney and Ronald W. Falta eds., Volume 1.
Hardman, R., 2006, A Toxicology Review of Quantum Dots: Toxicity Depends on
Physicochemical and Environmental Factors, Environmental Health Perspectives, 114:165-172.
Looney, B., and Falta, R., 2000, Vadose Zone What It Is, Why It Matters, and How It Works,
p 3-59, in Vadose Zone Science and Technology, Brian B, Looney and Ronald W. Falta eds.,
Volume 1.
Roco, M. C. and W. Baunbridge, eds., 2001, Societal implications of nanoscience and
nanotechnology. National Science Foundation Report
Roco, M. C., 2003, Broader societal issues of nanotechnology, Journal of Nanoparticle
Research, 5:181-189
Sayre, P., (June 30, 2007), Nanomaterials and the Environment. Priority Research Areas http://
www.nano.gov/html/meetings/ehs/uploads/Sayre_EHS_20070104.pdf.
Tindall, J. A., and Kunkel, J. R., 1999, Unsaturated Zone Hydrology for Scientists and
Engineers, Prentice Hall, NJ.
USEPA, ( June 30, 2007), Nanotechnology White Paper, http://es.epa.gov/ncer/nano/
publications/whitepaper 12022005.pdf
Wiesner, M. R., Lowry, G. V, Alvarez, P., Dionysiou, D., and Biswas, P., 2006, Assessing the
Risks of Manufactured Nanomaterials, Environmental Science and Technology, 40(14):4336-
4345.
Witherspoon, P. A., 2000, Forewords, p xxii-xxv, in Vadose Zone Science and Technology, Brian
B, Looney and Ronald W. Falta, eds., Volume 1.
Conference Questions and Answers
Question:
In the cells that you homogenized, do you have any concerns that the air-water interface is
different in comparison to the sand box where there is flowing water?
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Answer:
The cells were 1 centimeter (cm) thick. We input that by matrix and container type. By doing
that, you have a better distribution of the gas interface in the cell. As you fill the cells up the
particles want to settle, which you want to avoid. We have done comparative experiments using
the two techniques and have found no difference between them in results.
Question:
How do you dispose of waste?
Answer:
We follow EPA guidance but do not have a formal policy.
Question:
Do you have plans to conduct these experiments in soil (mixed texture as opposed to sand)?
Answer:
Yes.
Question:
Have you characterized the interactions between your nanoparticles and the porous media itself?
Answer:
No.
172
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Assessing Transport of Gold Nanoparticles and Bacteria in Porous Media
Using X-ray CT Scanning
Subhasis Ghoshal, Department of Civil Engineering, McGill University,
Montreal, Quebec, Canada
Nathalie Tufenkji, Department of Chemical Engineering, Department of Civil Engineering,
McGill University, Montreal, Quebec, Canada
Gregory McKenna, Department of Civil Engineering, McGill University,
Montreal, Quebec, Canada
Abstract
X-ray computed tomography (CT) scanning is used in medical research for non-invasive tracking
of nanoparticle tracers or drug delivery agents in animal and human bodies. We have recently
demonstrated that a medical X-ray CT scanner can be used to quantitatively determine the spatial
distribution of gold and other metallic nanoparticles in environmental porous media. The ability
to non-invasively detect gold nanoparticles using X-ray CT was used to develop a technique for
assessing bacterial density distributions in a saturated porous media column by labelling bacterial
cells with gold nanoparticles.
Introduction
Traditionally, the transport of microbes and other colloids has been studied in experiments with
packed columns filled with a porous medium, and the changes in effluent concentration are
monitored as a function of time and compared to the influent concentration (Tufenkji, 2007).
Non-invasive visualization of columns could potentially provide additional valuable data for
improving our understanding of colloid transport and deposition behaviour in granular porous
media.
Non-invasive characterization of bacterial density distributions in porous media has been
performed using magnetic resonance imaging (MRI) (Olson et al., 2004). Those studies
employed iron-oxide labelled bacteria. However, MRI cannot easily characterize environmental
porous media, and thus it is difficult to correlate the bacterial distributions with respect to the
porous media features. X-ray CT can be used to characterize porosity and provide 3-D images of
the interior features of packed columns based on density differences. Colloid transport has been
visualized in X-ray CT, but only at the microscale (Li et al., 2006), and, the use of X-ray CT for
the imaging of bacterial density distributions in granular porous media has not been previously
reported.
The overall objective of this study was to quantify the bacterial density distribution within a
saturated sand column using X-ray CT. This was achieved by labelling bacterial cells with
gold nanoparticles which served as an X-ray contrast agent for the detection of bacteria in
CT scanning. Because bacterial cells have a density similar to that of water, they cannot be
173
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identified by X-rays in aqueous phases.
Methods
The Gram-positive bacterium, Bacillus subtilis (ATCC 6633) was used in this study. Gold
nanoparticles labelled on the B. subtilis cells served as a contrast agent to facilitate the
determination of bacterial density distributions within saturated sand columns by X-ray
computed tomography (CT). Methods for synthesis of the gold nanoparticles and its labelling on
to B. subtilis cells have been described by Berry et al. (2005).
Quartz sand (Unimin #2040) was used as the granular porous medium in the column
experiments. The sand was milled and fractionated into coarse and fine sands, and by sieving
the sand the resulting mean grain size for coarse and fine sands was 775 ^m and 200 ^m,
respectively. Traditional column experiments were carried out to analyze the deposition of the
gold nanoparticles alone and gold-labelled bacteria using X-ray CT. These were conducted by
pumping either a gold-labelled bacteria or gold nanoparticle suspension through a Plexiglas
column (diameter 25 mm, height 40 mm) with adjustable Teflon end fittings (Ace Glass). Half
the column (20 mm) was wet-packed with coarse sand, and half the column (20 mm) was wet-
packed with fine sand. This created an interface where changes in the deposition behaviour
could be observed.
The columns were pre-equilibrated with de-ionized (DI) water and placed horizontally on the bed
of the CT scanner (Toshiba XVision medical scanner) and were scanned to provide the baseline
scan for the CT data analysis. After scanning, the column was removed from the scanning bed
and replaced in the horizontal position. Forty mL (~ 5 PV) of gold nanoparticles or gold-labelled
cells in DI water were then pumped through the column followed by 40 mL of DI water. The
effluent at the end of the outlet tube was collected every 2 min for a duration of 1 min. The
effluent samples were then analyzed using a UV-Vis spectrophotometer (HP Model 8453) at
a wavelength of 535 nm. At the end of the DI water injection, the column was returned to its
horizontal position on the CT scanning bed and was scanned. Techniques for CT scanning have
been discussed elsewhere (Goldstein et al, 2007; McKenna 2008).
Results and Discussion
Cell Labelling
Gold nanoparticles were successfully synthesized and labeled onto B. subtilis cells in suspension
as shown in Figure 1. Gold nanoparticles capped with CTAB molecules were synthesized with
a diameter of 94.3 ± 12 nm and a positive surface charge (^-potential ~ +40 mV) in DI water.
These gold nanoparticles were attached to suspended B. subtilis cells (average length = 2.0 ^m,
dia = 0.75 ^m, ^-potential of approximately -10 mV in DI water) in a single step. The gold-
labelled cells retained their structural integrity over time, remained viable, and suspended in
solution, throughout the time period used for the column experiments.
The gold nanoparticles and labelled cells showed predictable X-ray attenuation properties
that permitted quantification of their mass using X-ray CT. The CT scanning signal (CT
number) for the gold nanoparticles and labelled cells increased linearly with particle and cell
concentration. By measuring the CT number at various concentrations, the detection limits for
the gold nanoparticles and labelled cells were determined to be 4.5 x io10 particles/mL and 3.3
174
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McGill 2.0kV 12.0mm x20.0k SE(M) 2.00um
Figure 1. SEM image of a gold-labelled B. Subtilis cell.
x 107cells/mL, respectively. The detection limit for cell concentration is one order of magnitude
less than the cell concentrations quantified by MRI in another study that employed iron oxide
nanoparticles for cell-labelling (Sherwood et al., 2003).
Characterization of Cell Distribution in Sand Columns
Experiments were conducted in a packed column containing a 20 mm layer of fine quartz sand
overlaid with a 20 mm layer of coarse grains of the same quartz sand. The gold-labelled cells
were pumped in the direction from the coarse sand to the fine sand. A column breakthrough
curve obtained for gold-labelled cells pumped through the layered sand column is shown
in Figure 2 with the normalized effluent concentration, C/C0, plotted as a function of time.
The influent concentration of labeled cells was 1.0 xio8 cells/mL. The maximum effluent
concentration for the gold-labelled cells was 0.19 and occurred after 14 min followed by a
gradual decline suggesting that a high concentration of gold-labelled cells had been retained
within the column. After the first forty min, the injection was switched to DI water for a further
40 min.
The profiles showing the spatial distribution of the retained gold-labelled cells for the same
experiment were obtained from the X-ray CT data with the retained particle or cell concentration
plotted as a function of distance (Figure 3). The concentration of retained gold-labelled cells
shows a sharp increase in the retained concentration at the interface, followed by a region of very
low retention. The data confirms that there was a relatively high concentration of gold-labelled
cells retained in the column.
The average porosity for this experiment was 0.42. From the CT data, the porosity for each slice
was found to be consistent with the average porosity. The porosity in the coarse and fine sand
layers are not expected to be different because both layers are comprised of grains of relatively
175
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o
U
^
u
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
n
10 20 30 40 50
Time (min)
60
70
80
Figure 2. Representative breakthrough curve for gold-labelled cells in a sand column with coarse
and fine sand layers. Key experimental conditions: (pH =6.5, flow rate = ImL/min, porosity =
0.42, mean coarse grain diameter = 800 um, mean fine grain diameter = 200 um, interface at 20
mm.
uniform size.
At the interface of the two sand layers, the pore size is reduced due to a decreased grain size,
which increased the effect of the straining on the gold-labelled cells. As the bacteria are removed
from the aqueous phase by the collector grains in this region, the pore spaces become smaller
and the labelled bacteria can no longer pass through (Dunmore et al., 2004). This resulted in a
high concentration of gold-labelled cells being retained within the column. In the coarse sand
region of the column, the particle to collector diameter (dp/dc) ratio is 0.002 for the gold-labelled
cells. After the interface, in the fine sand region of the column, the mean grain diameter is
reduced to 200 um and the dp/dc ratio increases to 0.008 for the gold-labelled cells. Experimental
evidence has shown that straining can be important at ratios above 0.002 (Bradford and Bettahar,
2006) and was most likely the governing mechanism in the removal of the gold-labelled cells.
The breakthrough curves, as well as the spatial distribution of the retained CTAB-capped gold
nanoparticles (with no cells attached) were significantly different from the gold-labelled cells,
suggesting that gold nanoparticles did not detach from the cells during the column transport.
Conclusions
To the best of our knowledge, this study is the first to examine the transport and deposition
176
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^uuuu -
1 pfinn
•D
C
M
Ol
ft
n -
tin
D U
D n n
n
n
10 15 20 25
Slice Number
30
35
40
Figure 3. Profile for gold-labelled cells in a packed column with coarse and fine sand
layers determined by X-ray CT scanning. Key experimental conditions: (pH =6.5, flow
rate = ImL/min, porosity = 0.42, mean coarse grain diameter = 800 um, mean fine grain
diameter = 200 um, interface at 20 mm (slice 20).
behavior of bacteria in porous media using X-ray CT. In this study, the density distribution of
gold nanoparticles and labelled cells was quantified at a spatial resolution of 1 mm. Furthermore,
X-ray CT permitted the non-invasive calculation of porosity in situ as well as observing changes
in deposition behavior due to sand heterogeneities such as the accumulation of gold-labelled
bacteria at the interface between coarse and fine sands. This knowledge on spatial distribution of
bacterial density distributions could not be otherwise determined from analyses of the effluent
concentrations or without sampling the porous medium destructively. Ongoing work is aimed at
labeling cells with gold nanoparticles inside the cell rather than on the cell surface. If successful,
this technique will allow use of cells with relatively unaltered surfaces in studies aimed at
characterizing their deposition behavior using X-ray CT scanning.
References
Bradford, S. A.; Bettahar, M. (2006) "Concentration dependent transport of colloids in saturated
porous media." Journal of Contaminant Hydrology, 82, (1-2), 99-117'.
Berry, V; Gole, A.; Kundu, S.; Murphy, C. 1; Saraf, R. F. (2005) "Deposition of CTAB
terminated nanorods on bacteria to form highly conducting hybrid systems." Journal of the
American Chemical Society, 127, (50), 17600-17601.
Dunsmore, B. C.; Bass, C. J.; Lappin-Scott, H. M. (2004) "A novel approach to investigate
177
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biofilm accumulation and bacterial transport in porous matrices." Environmental Microbiology,
6, (2), 183-187.
Goldstein, L.; Prasher, S. O.; Ghoshal, S., (2007) "Three-dimensional visualization and
quantification of non-aqueous phase liquid volumes in natural porous media using a medical
X-ray Computed Tomography scanner." Journal of Contaminant Hydrology, 93, (1-4), 96-110.
Li, X.; Lin, C. L.; Miller, I. D.; Johnson, W. P. (2006) "Pore-scale observation of microsphere
deposition at grain-to-grain contacts over assemblage-scale porous media domains using x-ray
microtomography." Environmental Science and Technology, 40, (12), 3762-3768.
McKenna, G. (2008) "Visualization of bacteria density distributions in saturated sand columns
using X-ray computed tomography." M.Eng. Thesis. Department of Civil Engineering, McGill
University.
Olson, M. S.; Ford, R. M.; Smith, J. A.; Fernandez, E. J. (2004) "Quantification of bacterial
chemotaxis in porous media using magnetic resonance imaging." Environmental Science and
Technology, 38, (14), 3864-3870.
Sherwood, J. L.; Sung, J. C.; Ford, R. M.; Fenandez, E. J.; Maneval, J. E.; Smith, J. A. (2003)
"Analysis of bacterial random motility in a porous medium using magnetic resonance imaging
and immunomagnetic labeling." Environmental Science and Technology, 37, (4), 781-785.
Tufenkji, N. (2007) "Modeling microbial transport in porous media: Traditional approaches and
recent developments." Advances in Water Resources, 30, (6-7), 1455-1469.
Conference Questions and Answers
No questions.
178
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Impact of Size on Carbon Nonotube Transport in Natural Media
Xueying Liu, and Denis M. O'Carroll, Department of Civil & Environmental Engineering, The
University of Western Ontario, London, Ontario, Canada
Elijah Peter sen, Department of Civil and Environmental Engineering, University of Michigan,
Ann Arbor, Michigan, U.S.A.
Qingguo Huang, Department ofCrop and Soil Sciences, University of Georgia, Georgia, U.S.A.
Lindsay Anderson, Biological andEnv. Eng., Cornell University, Ithaca New York, U.S.A.
Abstract
Carbon nanotubes are becoming increasingly common in commercial applications. However,
there is evidence that carbon-based nanoparticles could cause cell damage. As a result
their impact to humans and the ecosystem has become a concern with the growing use of
manufactured carbon nanotubes. Therefore, it is essential to understand the factors that control
the transport of carbon nanotubes in the environment, and of particular interest to this study, their
transport in porous media. An assessment of the mobility of carbon nanotubes in porous media
would facilitate the determination of the ability of drinking water treatment facilities to remove
them from source waters as well as assist in the prediction of their fate in subsurface aquifers. In
this work, the transport behavior of multi-walled carbon nanotubes (MWCNTs) is investigated
in sand packed column experiments. To determine the importance of MWCNT shape and
aspect ratio experiments were conducted using four commercially available MWCNTs with
differing diameters and lengths. MWCNT diameter was varied within one order of magnitude
and MWCNT length was varied two orders of magnitude. Results suggest that under the
experimental conditions tested smaller diameter MWCNTs are less mobile than larger diameter
MWCNTs and that MWCNT length is less important than MWCNT diameter for the prediction
of MWCNT mobility in porous media. Experiments were also conducted at ionic strengths
typical of groundwater environments and at very low ionic strengths. Results from these column
experiments suggest that the transport of MWCNTs is not solely governed by mechanisms
traditionally associated with filtration theory (ie: sedimentation, diffusion and interception) and
other transport mechanisms also control their transport.
Introduction
Carbon nanotubes have been the subject of intense research since their discovery in 1991 (lijima,
1991). Their unique properties (e.g., light weight, significant strength, excellent conductivity,
and outstanding chemical resistance) have lead to their application in a wide variety of industries
such as composite material and electrical field emission applications (Andrews et al., 2002;
Wang et al., 2001). However, there is concern that carbon-based nanoparticles could cause
cell damage, and with the growing use of manufactured carbon nanotubes, their impact to
humans and the ecosystem has become a concern to researchers, regulators, manufacturers, and
consumers. It is therefore essential to understand the factors that control the transport of carbon
179
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nanotubes in the environment, and of particular interest to this study, their transport in porous
media. An assessment of the mobility of carbon nanotubes in porous media would facilitate a
determination of the ability of drinking water treatment facilities to remove them from source
waters as well as assist in the prediction of their fate in subsurface aquifers.
MATERIALS AND METHODS
Multi-Walled Carbon Nanotubes
Multi-walled carbon nanotubes (MWCNTs) were purchased from Cheap Tubes Inc. (Brattleboro,
VT). These MWCNTs were synthesized using the chemical vapor deposition (CVD) method
with metal catalysts. Energy dispersive X-ray spectroscopy found that these purified MWCNTs
are over 97% by weight carbon (Cheap Tubes Inc.). Detailed dimension information is listed
in Table 1. Purchased carbon nanotubes were further functionalized using a concentrated
aggressive acid mixture containing a 3 to 1 ratio by volume of sulfuric and nitric acids (95-
97% and 70%, respectively) to enhance the MWCNT hydrophilicity through the addition of
carboxylic groups on the MWCNT surface (Liu et al., 1998). An ultrasonic probe was used
to produce stable carbon nanotube suspensions in aqueous solutions at a pH of 10 and at two
ionic strengths. The first aqueous phase solution (SS I) had an ionic strength of 10 mM and was
buffered to pH 10. 1 mM NaBr or NaCl was added to SS I as a conservative tracer. For the
low ionic strength aqueous phase solution sodium hydroxide (NaOH) was added to de-ionized
water to make a 0.1 mM ionic strength solution with a pH around 10.2 mg of carbon nanotubes
were added to a 250 ml beaker containing 200 ml aqueous solution and sonicated at 210 watts
in an ice-water bath for 45 minutes. These suspensions were stable in the aqueous phase for
months. Concentrations of aqueous phase MWCNT suspensions were quantified using a UV-Vis
spectrophotometer at a wavelength of 400 nm.
Table 1. Average Length and Diameter of theCommercial Multi-Walled Carbon Nanotubes
CNT-a CNT-b CNT-c CNT-d
Outer diameter (nm) 30-50 30-50 <8 <8
Length (|im) 0.5-2.0 10-20 0.5-2.0 10-30
Porous Media
Quartz sand (d50 = 476 jim, U: = 1.5; Barco 32, BEI Pecal, Hamilton, ON, CA) was used as the
representative porous medium. Purchased quartz sand was cleaned alternately with hydrochloric
acid (0.1M) and hydrogen peroxide (5%) to remove all impurities and grease. De-ionized
water was used to rinse the sand between steps and afterwards until a neutral pH was achieved.
Washed sand was dried in an oven (105°C) over night. Particles smaller than 152 |im were
removed by sieving.
Aluminum columns, 5 cm in diameter and 10 cm in length, were dry packed with clean sand.
A stainless steel mesh (150 jim openings) was placed at both ends of the column to support the
sand as well as distribute the aqueous flow. Carbon dioxide gas was flushed through the column
upwards for at least 15 minutes to displace air. De-ionized water was then pumped upwards
through the column for at least 30 pore volumes to saturate the sand column. This packing
180
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procedure yielded an average column porosity of 0.347 (ranging from 0.335 to 0.368). The stock
solution and MWCNT suspensions were delivered by two or three 60-ml plastic syringes to the
column via a syringe pump at a pore water velocity of 0.42 m/d. The MWCNT suspensions were
injected downwards through the column.
Results and Discussion
A series of column experiments were conducted to investigate the impact of size on the
transport of MWCNTs. The selected pore water velocity (0.42 m/d) represents typical natural
groundwater conditions. Representative breakthrough curves are presented in Figures 1 to 4.
In these figures the normalized effluent concentrations of MWCNTs are plotted as a function of
pore volumes (Vp) flushed. For CNT-a the carbon nanotubes exited the column at the same time
as the conservative tracer for the lower ionic strength solution and were retarded to a very small
extent at the higher ionic strength solution. The maximum normalized effluent concentrations
0.5
Figure 1. Breakthrough Curves of CNT-a.
were similar at both ionic strengths. CNT-b was retarded to a more significant extent at the
higher ionic strength in comparison to CNT-a. Similar behaviour was observed for CNT-c and
CNT-d but to a more significant extent. It would appear that for the experiments conducted with
the higher ionic strength solution a similar maximum effluent concentration to the lower ionic
strength solution would be achieved if sufficient pore volumes were flushed. Unfortunately not
enough pore volumes were flushed for cases CNT c and d. These results indicate that the lower
ionic strength aqueous solution impeded MWCNT deposition on the porous medium.
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0.5
1.5
2
V/Vp
2.5
3.5
Figure 2. Breakthrough Curves of CNT-b.
In low ionic strength systems it is generally assumed that the energy barrier is too large for
colloid removal due to deposition (i.e., interception, sedimentation or Brownian diffusion)
(Tufenkji and Elimelech, 2004). At the lower ionic strength all of the carbon nanotubes
generally exited the column with the conservative tracer. However they all achieved a maximum
normalized effluent concentration of less than 1.0, with CNT-a achieving a maximum normalized
effluent concentration of 0.77 and the other carbon nanotubes achieving a maximum normalized
effluent concentration of approximately 0.7. Given that deposition on the sand surface should
be minimal at the lower ionic strength this suggests that other removal mechanisms are also
important in carbon nanotube transport. One removal mechanism that is achieving significant
attention is straining, which is the removal of particles at grain to grain intersections (Bradford
et al., 2006). If the carbon nanotube major axis is oriented perpendicular to the flow direction
straining is more likely than if the minor axis is oriented perpendicular to the flow direction.
This is due to the larger dimension attempting to pass through the pore space. Therefore, CNT-a
& c, with shorter lengths, are expected to be less susceptible to straining. At the lower ionic
strength the results from CNT-a would support this hypothesis as the maximum normalized
effluent concentration is higher than the longer carbon nanotubes (CNT-b & d). The results
from CNT-c, however, do not support this observation as the maximum normalized effluent
concentration is similar to the longer carbon nanotubes.
Experiments at the higher ionic strength are used to evaluate carbon nanotube removal
182
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•-l=10mM
*-|=0.1mM
•I- Br
- Cl
0.5
1.5
2
V/Vp
2.5
3.5
Figure 3. Breakthrough Curves of CNT-c.
mechanisms due to mechanisms associated with classical filtration theory (ie: interception,
sedimentation and Brownian diffusion) and straining. At the higher ionic strength the pore
volumes flushed prior to the normalized effluent concentration achieving 50% of its maximum
value increases from CNT-a, to CNT-b, to CNT-d and finally CNT-c. For these experiments it
would appear that the smaller diameter carbon nanotubes are retained to a greater extent than
the larger diameter carbon nanotubes (ie: CNT-c & CNT-d have smaller diameters than CNT-a
& CNT-b). It would appear that carbon nanotube length is less important than diameter, as the
pairs CNT-a and CNT-c or CNT-b and CNT-d have similar lengths but very different retardation
behaviour. Finally CNT-c was removed to the greatest extent in these experiments at the higher
ionic strength. This carbon nanotube had the smallest diameter (< 8 nm) and shortest length
(500 to 2000 nm). With decreasing particle size Brownian diffusion becomes a dominant
classical filtration removal mechanism. Due to its smaller size classical filtration predicts that a
larger fraction of these smaller carbon nanotubes that approach the sand grain would strike the
sand grain. For example r|0 (both based on carbon nanotube diameter and surface area based
equivalent diameter) is greatest for CNT-c. Based on r|0 alone it is unclear why CNT-a is more
mobile than CNT-b as r|0 is equivalent to or smaller for CNT-b. At both ionic strengths CNT-a
was removed to a lesser extent than the other carbon nanotubes. This carbon nanotube had a
larger diameter than CNT-c and CNT-d and had a shorter length than CNT-b and CNT-d. Carbon
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0.5
1.5
2
V/Vp
2.5
3.5
Figure 4. Breakthrough Curves of CNT-d.
nanotube removal, however, is not solely related to r|0 but also related to a, which is the fraction
of particles that strike the sand surface that are removed and is generally treated as an empirical
parameter. Even though the surface functional groups of these carbon nanotubes should be the
same the differing carbon nanotube sizes could impact a. For example larger carbon nanotubes
may have more difficulty finding appropriate deposition sites. Other factors that may influence
a include the species and the concentrations of electrolyte in solution, pH value, surface
characteristics of MWCNTs and the grain collectors (Lecoanet and Wiesner, 2004).
Summary
A series of column experiments were conducted to determine the impact of MWCNT dimensions
on their transport in porous media. This work suggests that mechanisms associated with
traditional colloid filtration theory cannot solely be used to predict MWCNT transport in porous
media and other removal mechanisms are important. In addition MWCNT diameter appears
to be more important than MWCNT length in the prediction of the fate of carbon nanotubes in
porous media.
References
Andrews, R.; Jacques, D.; Qian, D.; Rantell, T. Multiwall Carbon Nanotubes: Synthesis and
Application. Ace. Chem. Res. 2002, 35, 1008-1017.
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Bradford, S. A.; Simunek, J.; Bettahar, M.;van Genuchten, M. T.; Yates, S. R. Significance
of straining in colloid deposition: Evidence and implications. Water Re sour. Res. 2006, 42,
W12S15, doi:10.1029/2005WR004791.
lijima, S. Helical Microtubules of Graphitic Carbon. Nature, 1991, 354(6348): 56-58.
Lecoanet, H.F.; Wiesner, M.R. Velocity Effects on Fullerene and Oxide Nanoparticle Deposition
in Porous Media, Environ. Sci. Technol. 2004, 38, 4377-4382.
Liu, J.; Rinzler, A. G.; Dai, H.; Hafner, J. H.; Bradley, R. K. ; Boul, P. J.; Lu, A.; Iverson, T.;
Shelimov, K.; Huffman, C. B.; Rodriquez-Marcias, R; Shon, Y.-S.; Lee, T. R.; Colbert, D. T.;
Smalley, R. E. Fullerene Pipes. Science, 1998, 280, 1253.
Tufenkji, N.; Elimelech, M. Correlation Equation for Predicting Single-Collector Efficiency in
Physicochemical Filtration in Saturated Porous Media. Environ. Sci. Technol. 2004, 38, 529-536.
Wang, Q.H.; Yan, M.; Chang, R.P.H. Flat panel display prototype using gated carbon nanotube
field emitters. Appl Phys. Lett. 2001, 78, 129
Conference Questions and Answers
Question:
You are basing your experiment on the shape and size of individual carbon nanotubes. Would
you care to comment on how your method affects bundling in suspension?
Answer:
We did transmission electron microscopy (TEM) images, which dry the sample and are not
exactly representative of what is in the aqueous solution. When you do TEM you reduce the
coiling environment of the carbon analysis.
Question:
Did you try standardized settling?
Answer:
We did, and we identified two peaks-one for the diameter, and one for the length.
Question:
How do they compare with particles in suspension?
Answer:
They compare reasonably well.
Question:
What is the reason for using a solution with a pH of 10?
185
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Answer:
When the work began several years ago, there was a great deal of difficulty in getting nanotubes
stable in suspension. We found that the carbon nanotubes were more stable at a higher pH.
Since then, we have found other techniques to stabilize the nanotubes at a lower pH; however,
we decided to continue to use a pH 10 solution for consistency with the earlier research. The
governing principles would be the same for a pH of 7.
Question:
Are you going to conduct further experiments with a different soil matrix and nanoparticle?
Answer:
Yes, we plan on expanding the research. One interest is what happens in clay. We are considering
an in-field experiment at Canadian Forces Base Borden.
Question:
In addition to your looking at the diameter and length of the carbon nanotubes and their effects
on transport, have you looked at chirality?
Answer:
No.
186
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Evaluating the Clean-Bed Filtration Theory for Modeling Transport of
Fullerene C60 Aggregates in Saturated Porous Media
Yusong Li, Department of Civil and Environmental Engineering, Tufts University, Medford,
Massachusetts, U.S.A.
Yonggang Wang, Department of Neurology, Emory University School of Medicine,
Atlanta, Georgia, U.S.A.
Linda M. Abriola, Department of Civil and Environmental Engineering,
Tufts University, Medford, Massachusetts, U.S.A.
Kurt D. Pennell, Department of Neurology, Emory University School of Medicine, Atlanta,
Georgia, U.S.A. and Department of Neurology, Emory University School of Medicine, Atlanta,
Georgia, U.S.A.
Abstract
A coupled experimental and mathematical modeling investigation was undertaken to evaluate
the applicability of the clean-bed filtration theory for modeling transport of fullerene aggregates
(nC60) in water-saturated porous media. nC60 transport experiments were conducted in the
columns packed with 40-50 or 100-140 mesh Ottawa sand. The clean-bed filtration model failed
to reproduce both the observed asymmetric breakthrough curves and flat retention profiles. A
model that incorporated a maximum retention capacity term provided improved simulation
of nC60 transport and retention. The collision efficiency factor values calculated based on the
clean-bed filtration model were orders of magnitude smaller than the values calculated by the
maximum retention capacity model.
Introduction
World wide production of fullerenes (C60) is expected to exceed 300 tons/year in year 2010
(UNEP 2007). Widespread application and production of C60will inevitably lead to its release
into the environment. Once released, the bioavailability and potential exposure pathways of
the fullerene nanoparticles will be strongly influenced by transport and retention processes. In
aqueous systems, C60 is capable of forming stable nano-scale aggregates (nC60). Although the
transport of nC60 in porous media has typically been analyzed using the clean-bed filtration
model (Yao, Habibian et al. 1971), its appropriateness for this application has not been carefully
assessed. The goal of this research was to evaluate the applicability of the clean-bed filtration
model for simulating nC60 transport and retention in water saturated quartz sand.
Experimental Methods
Aqueous suspensions of C60 were prepared following procedures in (Wang, Li et al. 2008 ).
The resulting suspension contained 3.0 mg/L of nC60 in 1.0 mM CaCl2 solution with an average
diameter of 120 nm as determined by dynamic light scattering (DLS). Transport experiments
were conducted in borosilicate glass columns packed with either 40-50 or 100-140 mesh water
187
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saturated Ottawa sand (U.S. Silica, Berkeley Springs, WV). A 10 pore volume (PV) pulse of
nC60 suspension was introduced at a flow rate of ca. 1.0 mL/min, followed by a 3 PV pulse
of nC60-free solution at the same flow rate and ionic strength. Column effluent samples were
collected continuously and the nC60 concentration was monitored using UV spectroscopy. At the
conclusion of each experiment, the column was dissected into 1.5-cm increments and retained
nC60 aggregates were extracted by addition of deionized water, agitation for 3 h on an oscillating
shaker (Labquake, Barnstead International, Dubuque, IA), and ultrasonication for 1 min (Model
FS20H, Fisher Scientific). A summary of the conditions for each column experiment is provided
in Table 1.
Table 1. Experiment conditions of nC60 column experiments.
Column
OT1
OT2
OT3
dca
(mm)
0.335
0.335
0.125
vpb
(m/d)
1.04
1.04
1.03
c c
<-/>
(mg/L)
3.05
3.12
3.07
Retained
(%)
27.0
37.2
99.8
MB"
(%)
91.7
97.4
99.7
«0
0.00045
0.00048
--
«i
0.086
0.084
0.074
k.y
on
2.50
2.44
10.10
Smax
Og/gsand)
1.93
2.44
13.7
a mean sand grain diameter, pore water velocity. ° influent n-C60 concentration, mass balance.
Clean-bed Filtration Model
According to clean-bed filtration theory (Yao, Habibian et al. 1971), the transport of particles
through a water-saturated homogeneous porous medium can be described by advection,
hydrodynamic dispersion, and retention (filtration) processes. A one dimensional advection-
dispersion equation with first-order particle retention kinetics is typically employed to simulate
particle transport:
^ + A^ = jD
-------
Results and Discussion
Effluent breakthrough curves (BTCs) and retention profiles for nC60 columns packed with
either 40-50 or 100-140 mesh Ottawa sand are shown in Figures 1A and IB, respectively.
Transport of nC60 in columns packed with 40-50 mesh sand yielded asymmetrical BTCs that
gradually increased to a maximum value before declining sharply to relative concentrations (C/
C0) approaching zero. The retention profiles of nC60 in 40-50 mesh Ottawa sand columns were
relatively uniform over the entire length of the column. On the other hand, no breakthrough of
nC60 was observed in the column packed with 100-140 mesh sand. Retained nC60 concentrations
in this column were higher close to the column inlet and then declined to lower values near the
column outlet. These results are consistent with those reported in (Wang, Li et al. 2008 ).
Steady State Analysis
The clean-bed filtration model is often applied by assuming steady-state conditions and
negligible hydrodynamic dispersion effects. Under such conditions, the collision efficiency can
be calculated as (Espinasse, Hotze et al. 2007):
2d
«o = -:
ln(C/C0)
L l ' o!
(4)
Here, L is the length of the column. aQ values were calculated as 0.00045 and 0.00048 (Table
1) for the two duplicate experiments conducted in Ottawa 40-50 sand, where a relatively
20
70
I
o'
15 •
10 -
5 •
OT1-M1
OT2-M1
OT1-M2
OT2-M2
OT3-M1
OT3-M2
OT1
OT2
OT3
468
Pore Volumes
10 12 14
4 6 8 10 12
Distance from Inlet (cm)
14
16
Figure 1. Measured and simulated breakthrough curves (A) and retention profiles (B). Symbols
represent experimental data, and lines are modeling results. Ml refers to filtration model under
transient conditions; M2 refers to maximum retention capacity model.
189
-------
stable effluent concentration was achieved after injecting 10 PV of nC60 suspension. Since no
breakthrough occurred in the column packed with 100-140 mesh sand, ag cannot be calculated
for this experiment. Note that, at steady state, an exponential decay of retained particle
concentration with distance is predicted, which obviously is not consistent with the observed
flat retention profile for nC60 transport. Thus, the validity of these calculated ag values can be
questioned. This contradiction is further discussed below.
Transient Analysis
Under transient conditions, we employed clean bed filtration model (eq. 1-2) to simulate nC60
transport. The attachment rate coefficient, £aftwas obtained by minimizing the sum of the squares
residuals between measured and modeled effluent concentration data and the retention profile
data. Comparisons of the measured and simulated nC60 breakthrough curves and retention
profiles are presented in Figure 1. As illustrated here, application of the clean-bed filtration
model to simulate nC60 transport under these experimental conditions failed to reproduce both the
observed asymmetric BTCs and flat retention profiles.
Maximum Retention Capacity Model
The poor performance of the clean-bed filtration model demonstrated above could be attributed
to the assumptions that attachment depends only on the aggregate concentration in the aqueous
phase and follows a first-order kinetics. Relatively flat retention profiles of the nC60, however,
are indicative of a maximum retention capacity. The attachment rate, thus, may depend on the
retained particle concentration. Therefore, a modification of Eq.2 was proposed (Li, Wang et al.
2008 ):
Smax ~ S (5)
Here, Smax is the particle maximum retention capacity. Eqs. 1 and 5 were applied to simulate the
BTCs and retention profile, with katt and Smax as fitting parameters. As illustrated in Figure 1, this
model provides a markedly improved simulation to the asymmetrical BTCs and relatively flat
retention profiles observed in the column experiments. Collision efficiency factors a1 calculated
based on eq.3 and fitted values of katt ranged from 0.074 - 0.086 (Table 1), which is more than
two orders-of-magnitude larger than aQ. Since the a1 values were obtained from a model that
more accurately captures the transport and retention of nC60 in porous media, these data provide
a more accurate representation of nC60 collision efficiency.
References
Espinasse, B., E. M. Hotze, et al. (2007). "Transport and retention of colloidal aggregates of
C60 in porous media: effects of organic macromolecules, ionic composition, and preparation
method." Environmental Science & Technology 41(21): 7396-7402
Li, YS., Y. Wang, et al. (2008 ). "Investigation of the Transport and Deposition of Fullerene
(C60) Nanoparticles in Quartz Sands under Varying Flow Conditions." Environmental Science &
Technology (accepted).
Tufenkji, N. and M. Elimelech (2004). "Correlation equation for predicting single-collector
efficiency in physicochemical filtration in saturated porous media." Environmental Science &
190
-------
Technology 38(2): 529-536.
UNEP (2007). Emerging Challenges - Nanotechnology and the Environment, Geo Yearbook
2007
Wang, Y, YS. Li, et al. (2008 ). "Transport and Retention of C60 Nanoparticles in Saturated Soil
Columns." Environmental Science & Technology 42(10): 3588-3594
Yao, K. M., M. M. Habibian, et al. (1971). "Water and Waste Water Filtration - Concepts and
Applications." Environmental Science & Technology 5(11): 1105-1112.
Conference Questions and Answers
Question:
What was the method for coating the sand with the surfactant?
Answer:
Ten pore volumes of surfactant solution were flushed through the sand column, and this was
followed by a surfactant-free water flush.
Question:
Do you know how far nanoparticles might travel during a remediation, and how this work would
inform that?
Answer:
Surfactants are being used to increase the mobility of the nanoparticles. The increase in mobility,
however, is very site-specific.
191
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192
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Aging of Iron Nanoparticles in Water: Effects on Structure and Reactivity
Paul G. Tratnyek, Vaishnavi Sarathy, and James T. Nurmi, Division of Environmental and
Biomolecular Systems, Oregon Health and Science University, Portland, Oregon, U.S.A.
Donald R. Baer and James E. Amonette, Environmental Molecular Sciences Laboratory,
Pacific Northwest National Laboratory, Richland, Washington, U.S.A.
Chanlan Chun andR. Lee Penn, Department of Chemistry, University of Minnesota,
Minneapolis, Minnesota, U.S.A.
EricJ. Reardon, Department of Geology, University of Waterloo, Waterloo, Ontario, Canada
Abstract
We studied the aging of Fe°-core/oxide-shell nanoparticles in water with a focus on changes in
(/') the composition and structure of the particles (by XRD, TEM, XPS, and bulk Fe° content),
and (/'/') the reactivity of the particles (by carbon tetrachloride reaction kinetics, electrochemical
corrosion potentials, and H2 production rates). The results show that nano-Fe° becomes more
reactive between 0 and ~2 days exposure to water, and then gradually loses reactivity over the
next few hundred days. The behavior of unaged nano-Fe° in our laboratory experiments may be
similar to that in field-scale applications for source-zone treatment due to the short reaction times
involved. However, nano-Fe° that has been aged in water for >3 days acquires properties that are
relatively stable over weeks or even months.
Introduction
Most nanoparticles that are used in environmental applications are reactive in ways that alter
the particle's properties over time. This process is responsible for the apparent aging of these
materials and is a primary determinant of their longevity. Aging (or longevity) and transport
in aqueous media are among the most important and potentially limiting factors in the use of
nano-Fe° to reduce contaminants in groundwater remediation. However, while transport of nano-
Fe° in the subsurface is now receiving a great deal of investigation, few studies have explicitly
addressed the issue of aging.
From a priori considerations—and results from the few prior studies that have significant
bearing on the aging issue (Liu and Lowry 2006; Sohn et al. 2006)—we anticipate the following
fundamental processes will be responsible for nano-Fe° aging under environmental conditions:
(/') breakdown of the oxide shell by hydration, autoreduction, etc.; (/'/') oxidation of the exposed
surface coupled with reduction of solutes; and (Hi) aggregation of particles and subsequent
cementation. Two additional considerations are (/v) potential feedbacks between the above
primary effects (e.g., between /' and /'/', which alter solution chemistry of the medium, or between
//-///, which are affected by solution chemistry) and (v) the relative timing (i.e., kinetics) of the
above primary effects, which vary so widely that some processes are essentially independent of
others.
193
-------
The multiplicity of processes that contribute to aging—combined with uncertainties regarding
their relative significance, relative timing, and interactions—makes the pursuit of a complete
and balanced understanding of the problem quite challenging. To achieve this goal, we have
employed expanded and novel set of complementary characterization methods for both particle
structure and reactivity. The scope covers the whole range of aging regimes (concentrated slurry
and dilute suspensions, with and without contaminants present), and therefore the results have
implications for laboratory and field-scale applications of nano-Fe° in any aquatic media.
Methods
We used two preparations of Toda RNIP-10DS (FeH2): one that had never been exposed to water
(FeH2(D)) to represent short-term aging effects, and one that had been in an aqueous slurry for
approximately a year (FeH2(w)) and therefore reflected long-term aging. For both of these types of
materials, we characterized the time dependent changes in (/') the structure of the iron particles,
using spectroscopy and microscopy (XPS, XRD and TEM); (/'/') the Fe°-content and rate of H2
production by reaction of Fe° with H2O (using manometry of H2 pressure); (/'/'/) the kinetics and
pathway of reaction (focusing on carbon tetrachloride, CT); and (/v) corrosion potential of the
iron-oxide particles using electrochemical experiments. Details of the treatment regimes and
analytical methods are given in (Sarathy et al. 2008).
Results and Discussion
While the Fe°-content of the nanoparticles decreased with aging time, as has been reported
previously by others (Liu and Lowry 2006; Sohn et al. 2006), most of the other properties show
more complex behavior, with a period between 0 and a few days exposure to water where the
FeH2(D) becomes more reactive followed by a gradual decline in reactivity of the next few hundred
days. In Figure 1, this can be seen in the kinetics of CT reduction, yield of chloroform from CT,
corrosion potential, and hydrogen production rate (i.e., &CT, 7CF, Ecorr, and,RH2, respectively).
Between 7CF and Rm, nearly all the data fall on a line because both properties peak at the same
time (1 day) and the rates of change on the sides of the peaks are similar. In contrast, kCT and
RH2 gives a correlation with marked hysteresis, even though both of these parameters also
peak at 1 day, because the rate of change in kCT is less than that for Rm. The time series are
less complete for correlations involving Ecorr because aging data were only collected up to 2
days. Nevertheless, the correlations between E and k~T or 7_ have similar features to those
•> ' corr CT CF
involving H2 production rate because the data for FeH2(w) apparently are sufficient to represent the
effect of long term aging. In this case, the correlation between k~T and E shows no hysteresis
O O O J CT corr •>
(and also is nonlinear); whereas the correlation between 7CF and Ecorr shows modest hysteresis. In
general, correlations without hysteresis imply a more direct relationship between two variables.
Therefore, it appears that Rm is better at describing the effect of aging on 7CF, whereas Ecorr
comes closer to explaining changes in kcr These relationships are mechanistically plausible
given that (/') Ecorr reflects the particle's potential to donate electrons (Nurmi and Tratnyek 2008),
and accepting electrons is generally regarded as determining kCT, and (if) H2 production involves
formation of reduced forms of hydrogen (Reardon et al. 2008), and the availability of reduced
forms of hydrogen probably control 7CF.
194
-------
-COAT
R
H2
WtA.
®*
^*
9
©
00
•©
time
-con
Figure I. Matrix of scatter plots showing all combinations of four measured properties of Fe°
(pseudo first order rate constants for carbon tetrachloride disappearance, kCT; yield of chloroform
from carbon tetrachloride, Ycp, corrosion potential, £corr; and the rate of hydrogen production due
to reduction of water, RH2) and aging time. Open symbols are for FeH2(D) and solid symbols are for
peH2(w) Symboi color scales from black to gray with increasing age. Data adapted from (Sarathy
et al. 2008).
Conclusions
In general, decreasing Fe° content, and concomitant shrinking of the Fe° core, are the primary
characteristics of aging nano-Fe°. However, while these changes in structure eventually must
result in the loss of reactivity, the short- and medium-term effects of aging on two types of
reactivity—hydrogen production or contaminant degradation—seem to vary with the type
of aging regime and other reaction conditions. These changes in reactivity correlate with
evidence for rapid destruction of the original Fe(III) oxide film on FeH2 during immersion and
the subsequent formation of a new passivating mixed-valence Fe(II)-Fe(III) oxide shell. These
dynamics have implications for in situ remediation applications of nano-Fe°, because the oxide
shell must mediate reaction of the core with all solution species, including contaminants (Scherer
etal. 1998).
195
-------
Acknowledgements
This work was supported by grants from the Nanoscale Science, Engineering, and Technology
Program (DE-AC05-76RLO 1830) and the Environmental Management Sciences Program (DE-
FG07-02ER63485) of the U.S. Department of Energy (DOE), Office of Science. It has not been
subject to review by DOE and therefore does not necessarily reflect the views of the DOE, and
no official endorsement should be inferred.
References
Liu, Y. and G. V. Lowry (2006). "Effect of particle age (Fe° content) and solution pH on nZVI
reactivity: H2 evolution and TCE dechlorination." Environ. Sci. Technol. 40(19): 6085-6090.
Nurmi, J. T. and P. G. Tratnyek (2008). "Electrochemical studies of packed iron powder
electrodes: Effects of common constituents of natural waters on corrosion potential." Corros. Sci.
50(1): 144-154.
Reardon, E. J., R. Pagan, J. L. Vogan, and A. Przepiora (2008). "Anaerobic corrosion reaction
kinetics of nano-sized iron." Environ. Sci. Technol. 42(7): 2420-2425.
Sarathy, V, P. G. Tratnyek, J. T. Nurmi, D. R. Baer, J. E. Amonette, C. Chun, R. L. Penn, and
E. J. Reardon (2008). "Aging of iron nanoparticles in aqueous solution: effects on structure and
reactivity." J. Phys. Chem. C 112(7): 2286-2293.
Scherer, M. M., B. A. Balko, and P. G. Tratnyek (1998). The role of oxides in reduction reactions
at the metal-water interface. Mineral-Water Interfacial Reactions: Kinetics and Mechanisms.
Washington, DC, American Chemical Society. ACS Symp. Ser. 715: 301-322.
Sohn, K., S. W. Kang, S. Ahn, M. Woo, and S. -K. Yang. (2006). "Fe(0) nanoparticles for nitrate
reduction: Stability, reactivity, and transformation." Environ. Sci. Technol. 40(17): 5514-5519.
Conference Questions and Answers
No questions.
196
-------
Effects of Humic Acid on Aggregation of Boron Nanoparticles in Various
Electrolyte Solutions
XuyangLiu, Mahmoud Wazne, and Christos Christodoulatos, Keck Geotechnical/
Geoenvironmental Laboratory, Center for Environmental Systems, Stevens Institute of
Technology, Hoboken, New Jersey, U.S.A.
Kristin L. Jasinkiewicz, U.S. Army, Environmental Technology Division, Picatinny, New Jersey,
U.S.A.
Abstract
Nano boron is a promising new propellant and is being considered for military and civilian
applications; however, the impact of its release on the environment is largely unknown.
Aggregation studies help to assess the fate, transport, and exposure pathways of various
nanopartcles in aquatic environment. The aggregation kinetics of boron nanoparticles was
investigated in the presence of monovalent (NaCl) and divalent electrolytes (CaCl2 and MgCl2),
and Suwannee River humic acid (SRHA) through time- resolved dynamic light scattering (DLS).
In the presence of SRHA, the attachment efficiency of the boron nanoparticles decreased for
the reaction- limited regime. The presence of SRHA caused the boron nanopartciles to stabilize
and resulted in greater critical coagulation concentrations (CCC). It appeared that for the
sodium, magnesium and calcium solutions, the surface charge became more negative due to the
adsorption of SRHA on the surface of the boron nanoparticles.
Introduction
Studies on fate and toxicity of engineered nanomaterials are being reported more frequently
due to the potential risk of these materials to human safety [1-7]. However, due to the different
surface characteristics of nanomaterials, the potential impact of these particles is being evaluated
on a case-by- case basis [8-10]. Due to its desirable heat of combustion and fast energy release
rate, boron nanoparticle is being considered as a promising solid fuel for rocket and gun
propellants [11-14]. Even though boron is beneficial to plants in small amounts, excessive
amounts are injurious and even lethal [15]. Upon release to the environment, natural organic
matters (NOMs) are expected to play a critical role in the stabilization and transport of these
particles as has been recently reported [16]. The purpose of this paper is to investigate the
influence Suwannee River humic acid (SRHA) on the aggregation of boron nanoparticles in three
common electrolyte solutions.
Materials and Methods
The boron nanoparticles were obtained from Alfa Aesar with an average particle size of 10-
20 nanometers. The boron nanoparticles were dispersed in DI water and ultrasonicated for 30
minutes, to break up aggregates before aggregation experiments. The particle size distribution
and surface charge were measured by a Nano Zetasizer (Malvern, Worcestershire, UK). All
197
-------
Dynamic light scattering (DLS) measurements were conducted at 25 °C at pH 5.6± 0.2 unless
otherwise specified.
The electrolyte stock solutions (NaCl, CaCl2, and MgCl2) were prepared separately and filtered
through 0.2 um filters before use. SRHA (standard II, International Humic Substances Society)
solutions were made by dissolving 22.9mg dry powder into 50 mL DI water and were stirred
overnight. The solutions were then filtered through 0.2 um filters and pH was adjusted from 3.4
to 10.2 by addition of NaOH. The total organic carbon content was measured at 232.76 mg/L.
For experiments in the absence of NOM, various electrolytes were added into 1 mL boron
dispersion in cuvettes. The dispersions were then shaken by hands and were placed into the zeta-
sizer immediately. For experiments in the presence of NOM, 70 uL SRHA stock solution was
added to the nanoparticle dispersions following the addition of the electrolytes. The aggregation
rate constant kn is proportional to the slope of the hydrodynamic radius R^ versus time as t-*0 at
each salt concentration, divided by the initial nanoparticle number concentration N0 [16-19]
(!)
dt
The attachment efficiency a (the inverse of the stability ratio W) is defined as the aggregation
rate constant of interest normalized by the rate constant derived under diffusion-controlled (fast)
aggregation conditions (in the absence of an energy barrier).
1 ,drh(t)N
dt
Results and Discussion
The boron nanoparticles suspensions in various electrolytes displayed similar aggregation
behavior as shown in Figure 1 . There appears to be two regimes; a reaction controlled regime
and a diffusion controlled one. The two regimes are separated by the critical coagulation
concentration (CCC). When ionic strength (IS) is smaller than the CCC, the attachment
efficiency increased with IS due to the screening of the electrostatic forces. As the IS increased
and became greater than CCC, the attachment efficiency kept constant (diffusion- controlled
regime) because electrostatic repulsion was screened completely. The CCC for the Na+ ions was
determined at -0.2M much greater than those for the divalent ions -- ImM for Ca2+ and Mg2+.
Boron nanoparticles suspensions were stabilized in the presence of SRHA in various electrolyte
solutions. As shown in Figure 2, the attachment efficiency in the presence of SRHA was smaller
than that in the absence of SRHA in the reaction-controlled regime. However, in the diffusion-
controlled regime, the aggregation rates were similar to those in the absence of SRHA. At the
same time, the CCC for NaCl increased from 0. 1 8 M in the absence of SRHA to 0.22 M in the
presence of SRHA. The CCC for CaCl2 increased from 1 mM in the absence of SRHA to 2.5 mM
198
-------
-
fr
5
10
1
*»»»++»*
0.1
10
2 0.1 -
0.01
0.1
10
P 0.1 -
0.01
0.1
0.1 0.2 0.3 0.4
Ionic stienyth (M N.iCII
»**++* ** * *+»««,
10
Ionic stienyth (mill CaGI;)
»**+++ * » +**<
10
Ionic stienyth (mill MyCI2l
Figure I. Attachment efficiencies as a function of (a) NaCl (b) CaCl2 and (c) MgCl2
concentration.
in the presence of SRHA. Although the CCCs for MgCl2 did not changed greatly, the aggregation
rates decreased ostensibly in the presence of SRHA. Therefore, it appears that SRHA stabilizes
the boron nanoparticles suspensions.
The surface charge of boron nanoparticle suspensions was measured to delineate the
mechanism of stabilization induced by SRHA. As shown in Figure 3, the surface charge was
more negative in the presence than that in the absence of SRHA at low IS. This indicates that
electrostatic repulsion increased probably due to the adsorption of SRHA on the surface of boron
nanoparticles. With the increase of ionic strength surface charge became less negative, probably
due to the neutralization effect [20]. Interestingly, it was also observed in Figure 3, that when IS
was greater than 0.22 M, the surface charge was similar in the presence and absence of SRHA.
It is consistent with the fact that attachment efficiencies were unity in the diffusion- controlled
regime whether in the absence or presence of SRHA as shown in Figure 2- a.
For the CaCl2 electrolyte, the surface charge in the presence of SRHA was greater (more
negative) than those in the absence of SRHA. More interesting as shown in Figure 4, the zeta-
199
-------
10
1 -
0.1 -
n ni
»»»»»»»»»*»!
* * *
+
D
* + No HA
o 15 mg/L TOC
0.1 0.2 0.3
Ionic strength (M NiGIl
0.4
10
'o 1 -
I
£
E
0.1-
< 0.01
**+**? ** »*••••;:
0°
+ NOHA
° 15 mg/L TOC
0.1
10
Ionic strength (mM CaCI2)
10
£ 0.1
a
» D
* O
*NoNOM
n 15 mg/L TOC
0.1
10
Ionic strength (mM MgCI;)
Figure 2. Attachment efficiencies as a function of (a) NaCl (b) CaCl2 and (c) MgCl2
concentration in the presence of SRHA compared with in the absence of SRHA.
potential in the absence of SRHA at CCC (16.3 mV at ImM CaCl2) was, almost the same as that
in the presence of SRHA at CCC (2.5 mM CaCl2). It reveals that at the CCC, boron nanoparticles
and aggregates have similar surface charge in the presence and absence of SRHA. Therefore, it
can be speculated that the increased electrostatic repulsion in presence of SRHA is the reason
for the stabilization of boron nanoparticles in calcium or sodium electrolytes solutions. Similar
phenomenon was also observed in the presence of SRHA and magnesium ions.
Conclusions
The behavior of boron nanoparticles aggregation changed in the presence SRHA. It appears
that in the presence of SRHA boron nanoparticles suspensions were stabilized due to increased
surface charges in the presence of sodium or calcium ions, however, more investigation is needed
to confirm this observation. In natural aquatic environments, the aggregation process will be
more complicated due to the presence of various ions and NOMs. It is therefore important to
200
-------
0
-5
|-10
£ -15
c
0)
•5 -20
Q.
I -25
N
-30
-35
*NoHA
• 15mg/LTOC
0.1
.-::-.
0.4
0.2 0.3
Ionic Strength (M NaCI)
Figure 3. Study of surface charge of boron nanoparticles in the presence of SRHA and NaCI.
> -5-
+ NOHA
-HSmgyLTOC
IS(mMCaCI2)
Figure 4. Study of surface charge of boron nanoparticles in the presence of SRHA CaCl
conduct more studies to predict the fate and transport of emerging nanomaterials on a case- by-
case basis.
References
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national nanotechnology initiative funding, 2000-2004." Environ. Sci. Technol. 40 (5), 1401-
1407.
201
-------
-5
£ -10
0)
t-15
as
"8 -20
N
-25
0.1
* No HA
• 15 mg/L TOC
* **•
1
IS (mM MgCI2)
10
Figure 5. Study of surface charge of boron nanoparticles in the presence SRHA and MgCl2.
2. K. L. Chen, M. Elimelech. (2006) "Aggregation and deposition kinetics of fullerene (C60)
nanoparticles." Langmuir 22 (26), 10994-11001.
3. K. L. Chen, M. Elimelech. (2007) "Influence of humic acid on the aggregation kinetics of
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interface sci. 309 (1), 126-134.
4. H. F. Lecoanet, M. R. Wiesner. (2004) "Velocity effects on fullerene and oxide nanoparticle
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15. E. Huertas, M. Herzberg, G. Oron, M. Elimelech. (2008) "Influence of biofouling on boron
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18. J. Kleimann, C. Gehin-Delval, H. Auweter, M. Borkovec. (2005) "Super-stoichiometric
charge neutralization in particle-polyelectrolyte systems." Langmuir 21 (8), 3688-3698.
19. A. M. Puertas, F. J. Nieves. (1999) "Colloidal stability of polymer colloids with variable
surface charge." J. colloid interface sci. 216 (2), 221-229.
20. C-H. Ko; M. Elimelech. (2000) "The "shadow effect" in colloid transport and deposition
dynamics in granular porous media: measurement and mechanism." Environ. Sci. Technol.
34(17), 3681-3689.
Conference Questions and Answers
Question:
If your hypothesis that steric tendency may play in the formation of aggregates with MgCl2 is
correct, could you say the same for sodium chloride?
Answer:
Maybe, but I cannot say for certain. For the steric tendency to take place you must have some
absorption on the surface of the boron nanoparticle. Maybe you can do some experiments for
these effects without calcium or magnesium to characterize the particle surface. Calcium and
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magnesium are known to complex, so it is possible that, when you have a higher concentration
of calcium with the same concentration of NOM, you show less effect for the humic acid. It is
possible that the humic acid and the boron nanoparticle are competing for calcium, thus reducing
its ionic strength and leading to less effectiveness.
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Predictive Numerical Model of Post-Injection Distribution of
Nano-Size ZVIin the Ringold Aquifer for Mending an Existing
Permeable Reactive Barrier in the 100-D Area at the Hanford Site
MarekH. Zaluski, Gilbert M. Zemansky, Adam Logar, Kenneth R. Manchester,
MSE Technology Applications, Butte, Montana, U.S.A.
Akshai K Runchal, Analytic and Computational Research, Inc., Bell Air, California, U.S.A.
DavidReichhardt, Montana Tech, Butte, Montana, U.S.A.
Scott Peter sen, Fluor Hanford, Washington, U.S.A.
Abstract
MSE Technology Applications, Inc. has conducted investigations associated with the injection
of nano-size zero-valent iron (nZVI) into the subsurface of the 100-D Area at the U.S.
Department of Energy (DOE) Hanford Site in Washington State. The purpose of this work was
to demonstrate the feasibility of using nZVI to repair portions of the In Situ Redox Manipulation
(ISRM) installed at the site to intercept a hexavalent chromium plume moving towards the
Columbia River. The investigation identified RNTP-M2 (RNTP), produced by Toda Kogyo
Corporation, as most suitable for mending the ISRM barrier. Since Toda nZVI will be emplaced
in the Ringold aquifer through injection wells, the PORFLOW™ computer model was used
to optimize injection parameters and predict the post-injection distribution of nZVI material
in the Ringold aquifer. The model used an empirically developed mathematical expression for
deposited nZVI as a function of injection time, distance from the injection point, and nZVI-fluid
velocity. Modeling results provided information on the predicted concentration of deposited
nZVI within the model domain and optimized the injection rate. This work was conducted
through the support of Fluor Hanford, a subcontractor to the DOE under Contract Number
30994.
Introduction
We have conducted investigations associated with the injection of nano-size zero-valent iron
(nZVI) into the Ringold aquifer beneath the 100-D Area at the U.S. Department of Energy (DOE)
Hanford Site in Washington State. The purpose of this work was to demonstrate the feasibility of
using nZVI as a source of electrons to repair portions of the In Situ Redox Manipulation (ISRM)
barrier. The ISRM barrier was installed at that site to intercept a hexavalent chromium (Cr6+)
plume moving towards the Columbia River. The barrier was installed from 1999 to 2002 (DOE,
2006) by injecting sodium dithionite to the Ringold Formation aquifer and creating persistent
reducing conditions by converting native Fe3+ to Fe2+. Although laboratory and field tests
indicated the barrier would effectively treat Cr64" for nearly 20 years, a few of the barrier wells
exhibited signs of breakthrough after less than two years. The work reported here was performed
to support testing an alternative technology to mend the ISRM barrier by injecting nZVI into the
Ringold aquifer through the existing injecting well.
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We conducted comprehensive investigations on commercial ZVI materials (Zaluski et al. 2008),
and identified an nZVI manufactured by Toda as most suitable for mending the ISRM barrier.
This investigation included geochemical and injectability lab studies and the computer modeling
described in this paper.
Problem Definition
Hydrogeologic setting comprises an unconfmed aquifer of 4.9 m (16 feet) saturated thickness
and 26.2 m (86 ft) thick unsaturated zone. The aquifer is stratified with respect to hydraulic
conductivity (K) that ranges from T.lxlO'5 m/s to 2.3xlQ-3 m/s (20 ft/d to 652 ft/d), with the
highest K present in the bottom 0.3 m (1 ft) of the aquifer (Figure 1). At the location of injection
(Well 199 D4-26) the aquifer is intercalated by a 0.6 m (2 ft) thick layer of very low K sediment.
Because of proximity to the Columbia River the hydraulic gradient of the aquifer is controlled by
river stage, and for the injection time (August 2008) is expected to be negligible in magnitude.
RNIP is provided by Toda as a solution containing 80 % water, 17-18 % solids, and 2-3 %
Hydraulic conductivity of Ringold aquifer at 199 D4-26 well
O.OE-K30 5.0E-04 10E-03 1.5E-03
Hydraulic conductivity (m/s)
2.0E-03
2.5E-03
Figure 1. Hydraulic Conductivity of Ringold Aquifer.
olefin maleic copolymer (all by weight). The solids, which include 65 % ZVI and 35 % Fe3O4,
come from the production line at an average particle size of 70 nanometers (nm), but promptly
agglomerate to 2 micrometer particles (Jazdanian, 2008). Though the injected nZVI fluid
contains 1 % (weight) of solids and only 0.14 % of maleic copolymer, the presence of the
latter precludes use of Stokes' law to define deposition of the solids. This phenomenon was
demonstrated in our laboratory experiments with 3-m long flow cells into which nZVI fluid was
injected at four different flow velocities.
Amassing of nZVI particles, defined as an increase in concentration of suspended particles
(nZVIs) above influent nZVI concentration, was observed during flow cell tests. The results
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of our tests indicated that unlike transported solute, which partition to an adsorbed portion and
that remaining in the solution, suspended particles may partition in three ways: (1) a significant
portion of particles drops out of suspension and deposits within the medium, possibly due to
gravity, electromagnetic forces, adsorption etc. - an equivalent of adsorption for the solute
transport, (2) a small portion of particles remains suspended moving at the same velocity as
the water, and (3) particles (nZVIs) that have some mobility, thus can be measured via aqueous
sampling, but move at a velocity much slower than that of the water.
Our observation of nZVI amassing may be mathematically described by the following advection-
dispersion-deposition-reentrainment equation (Johnson et al 2007):
(3C/a)0 = -v(3C/ac)0 + D(3C2/cFx)0 -KfC9 + KCspb (1)
Where C is the aqueous concentration of the constituent, Cs is the reversibly retained stationary
phase concentration of the constituent, Kf and Kr are the forward (removal from the aqueous
phase) and reverse (addition to the aqueous phase) coefficients, D is the dispersion coefficient,
6 is volumetric water content, and pb is a bulk density of the stationary phase. The amassing
phenomenon that we observed in our flow cell experiments is related to the last components of
Equation 1.
Since we were limited to conducting the flow cell experiment using only one concentration of
the nZVI in influent, it was impossible to define the Kf and Kr coefficients. Instead, we used a
statistical application of multiple linear regression (applied through MSExcel) to develop the
following mathematical expression for deposited mZVI (nZVId) as a function of injection time,
distance from the injection point, and nZVI-fluid velocity:
nZVId = 0.0322 + 3.77E-7 x Time - 0.0192 x Distance + 24.12 x Darcy Velocity (2)
Where nZVId, time, distance, and Darcy velocity are expressed in Kg of nZVI per Kg of soil,
seconds, meters, and meters per seconds, respectively.
The objective of the investigations was to deposit at least 0.001 Kg/Kg or 1 g/Kg of nZVI at the
distance of 7m (23 ft) from an injection well, which is half of the ISRM barrier's width.
Modeling
For the computer modeling we used the PORFLOW™ model and focused on prediction
of spatial distribution of nZVId emplaced in the Ringold aquifer by injecting nZVI fluid.
PORFLOW™ is a software tool for solution of multi-phase fluid flow, heat transfer, and mass
transport problems in variably saturated porous or fractured media. The PORFLOW™ model
was developed by Analytic & Computational Research, Inc. (ACRI 2008).
Because PORFLOW™ is a highly flexible, modular, and user-oriented software package we were
able to develop a special subroutine (Figure 2) for solving Equation 2 based on the simulated
flow field, which resulted from the injection of nZVI fluid, and the distribution of K. While
solving Equation 2 for a given element of the model domain flow velocity, elapsed injection
time and distance of the given element from the injection point were used to calculate nZVIe i.e.
the concentration of nZVId for each element. These values were then normalized with respect
to K to obtain nZVIKe, i.e. the concentration of nZVIe in each element. Finally, for mass
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Flow-call lab
experiment w\[k
nZVI Injected
Hydrogological
data
Analyses for total
suspended Fe
(nZVIs)
Calculation of F
(nZVId/nZVI)
Flow field
simulation by
FOR FLOW
Calculation of nZVIe
(nZVId for elements)
nZVI
{injection
rasa)
Calculation of nZVIKe
(i.e normalization whl
respect to K)
Calculation of ZVIK
=I(nZVIe)"F/nZVIi
Figure 2. Modeling Logistics.
conservation, we calculated ZVIK, i.e. concentration of deposited nZVI for each element as the
following:
ZVIK = nZVIKe * I(nZVIKe) * F / nZVIi
(3)
Where nZVIi is the total mass of injected nZVI through the injection well at the given time, and
F is a dimensionless laboratory-defined factor calculated as a ratio of nZVId to nZVI.
We used a simplified 3D approach, i.e., cylindrical coordinates, with the injection well being
the axis of the cylinder. The model domain encompasses a cylindrical block, 31.1m (102 ft) of
height (X-axis) and of 30 m (98.4 ft) radius set along the Y-axis. The domain was discretized to a
109x82-structured grid with irregular (progressively larger) spacing along the Y-axis.
By using different injection rates (nZVIi) of nZVI fluid we defined the optimal injection rate
of 0.00089 m3/s (14 gpm) that was related to nearly maximum concentration of ZVIK in the
highest-K strata at the distance of 7 m (Figure 3). This nZVI concentration was 4.7 g/Kg as
illustrated in Figure 4.
Conclusions
Because of the observed amassing phenomenon of colloidal nZVI, modeling of its transport and
deposition can only be achieved by flexible modular computer codes like PORFLOW™.
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Concentration of RNIP at 7m from the Injection Well D4-26
1.0
2.0 3.0 4.0
g of RNIP/Kg of soil
5.0
Figure 3. Concentration of RNIP at 7m from the Injection Well D4-26.
6.0
ZVIK
O.OOB45
0.00760
0.00676
0.00591
0.00507
0.00422
0.00338
0.00253
0.00169
0.000045
0.000
X: 150.9E-3 :: Y: 7.0441 EO :: Element 5458 :: ZVIK: 4.7497E-3
5.21
X
Figure 4. Distribution of ZVIK - Concentrations in Kg/Kg.
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Intensive laboratory efforts are required to develop empirical constants needed for predicting
nZVI deposition within the model domain.
To increase the reliability of the prediction, flow-cell experiments need to be conducted using
materials of various K and several nZVI concentrations in the influent, so an isotherm for
amassing of nZVI can be developed. This would allow for solving nZVI transport equation
internally in the model, rather than using a deposition function in a post-processor manner.
Acknowledgements
The authors appreciate the insight of and helpful discussions with Drs. P. Tratnyek, G. Lowry, C.
Palmer, and A. Jazdanian during execution of the investigations.
References
Analytic and Computational Research, Inc. (2008). PORFLOW Users Manual (Version 6.0,
Revision 3, p. 21).
DOE (2006). "The Second CERCLA Five-Year Review Report for the Hanford Site." DOE/RL-
2006-20, Revision 1
Jazdanian , A. of Toda America (2008). Personal communication.
Johnson, W., and P. M. Thong and X. Li (2007). "On Colloid Retention in Saturated Porous
Media in the Presence of Energy Barriers: The Failure of" and Opportunity to Predict 0." Water
Resources Research, Vol. 43, W12S13
Zaluski, M.H., G. Wyss, A. Logar, N. Jaynes, M. Foote, G. M. Zemansky, K. R. Manchester,
S. Antonioli, M. A. Harrington-Baker, D. Reichhardt, M. Ewanic, S. Petersen. (2008).
"Comprehensive Investigations on Nano-Size ZVI for Mending an Existing Permeable Reactive
Barrier in the 100-D Area at the Hanford Site". Proceedings of International Environmental
Nanotechnology Conference, EPA, Chicago, Illinois.
Conference Questions and Answers
Question:
What do you think about this amassing function with different kinds of ZVI, and you said you
were also testing the polymetals?
Answer:
We investigated six ZVIs. Some of them were micro, and some were nano. We observed
advancement of the particles, and we collected samples and measured how much iron was
forming. After a number of pour volumes, the iron in the effluent was greater than the iron in
the influent. This build up of suspended iron particles was unexpected. The different iron types
behaved in different fashions, but we only ran one of the ZVIs in the 3-meter column. We did,
however, observe the "amassing" effect for other ZVIs in earlier experiments.
Question:
You said earlier that you would be using geophysics. What type?
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Answer:
Electromagnetic for sure and maybe others.
Question:
There will be a lag between the injection time and the geophysics measurements. Will this lag
affect the results?
Answer:
Yes.
Question:
Do you think the effective life of the ZVI will be longer than the lag time in performing the
geophysics?
Answer:
Absolutely. Why inject the iron if it is only going to last a few months.
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Fate and Transport of Titania Nanoparticles in Freshwater Mesocosms
Ann L. Miracle, andAmoret L. Bunn, Environmental Sustainability Division,
Pacific Northwest National Laboratory, Richland, Washington, U.S.A., and
Marine Sciences Division, Pacific Northwest National Laboratory,
Sequim, Washington, U.S.A.
JillM. Brandenberger, Marine Sciences Division,
Pacific Northwest National Laboratory, Sequim, Washington, U.S.A.
Dan Caspar, Energy and Efficiency Division,
Pacific Northwest National Laboratory, Richland, Washington, U.S.A.
Abstract
Titania nanoparticles are currently associated with air, soil, and water and with numerous
products directed at human use and consumption (e.g., sunscreen, cosmetics, and food coatings).
The environmental fate and transport of TiO2, or any nanomaterials entering dynamic aquatic
environments are largely unknown. Because the physical and chemical properties of TiO2
are variable (size, surface chemistry, and composition), the movement, bioaccumulation, and
toxicity of these materials are difficult to study in a complex ecosystem. Many metal oxide
materials are durable and recalcitrant, and the accumulation of TiO2 in the environment could be
significant over time and cause unforeseen impacts on ecosystems. Fate and transport of TiO2
nanomaterials in a bench-scale mesocosm system was assessed through nanomaterial partitioning
and complexation in water, sediment, and tissue media characterized using inductively coupled
plasma mass spectrometry and scanning electron microscopy with energy dispersive X-ray
spectroscopy, respectively. Research data sets like these will build the foundation for future
use in fate and transport of other nanomaterials in different water systems (fresh, estuarine, and
marine) and in building empirical and process models that investigate environmental fate and
transport and relevant freshwater ecological impacts of nanomaterials.
Introduction
The environmental fate and transport of TiO2 nanoparticles (NPs) or any NPs entering
dynamic aquatic environments has never been studied in a quantified manner (Maynard, 2006;
USEPA, 2006). Because the properties of TiO2 are dependent on size, surface chemistry, and
composition, the movement, toxicity, and bioaccumulation of these materials are difficult to
study in a complex ecosystem. Titania materials are likely to enter an aquatic environment
through waste water treatment plants or directly into waters used for recreation. Because these
materials are durable and recalcitrant, the accumulation of TiO2 in the environment could be
significant over time and cause unforeseen impacts on ecosystems. Research that demonstrates
the capability to characterize and quantify TiO2 distribution in different environmental
compartments (i.e., water, sediment, and biota) will be relevant to setting guidelines for risk
analysis.
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Titania nanoparticles are currently associated with air, soil, and water and with numerous
products directed at human use and consumption (e.g., sunscreen, cosmetics, and food coatings).
What little research that has been done on TiO2 has been primarily focused on in vitro exposure
or air exposures (Bermudez et al. 2004; Gurr et al. 2005; Hussain et al. 2005; Peters et al.
2004; Ramires et al. 2001; Warner et al. 1997; Warheit et al. 2005; Zhang and Sun 2004), all
demonstrating the induction of oxidative stress. Recently, Lovern and Klaper (2006) reported the
first study of TiO2 on an environmental sentinel organism. In this study, the lethal concentration
of TiO2 (avg. size 30 nm) was only 10 ppm for Daphnia magna following a 48-hour water
exposure. Federici et al (2007) investigated the toxicity of titania nanoparticles following
exposures of 0.1, 0.5, and 1.0 mg/L to rainbow trout (Oncorhynchus mykiss) and while there was
no acute toxicity to dispersed TiO2 at the concentrations tested, there was a range of sub-lethal
effects observed related to biochemical changes and respiratory distress. These aquatic exposure
studies were carried out in artificial water systems used for organism culture and maintenance.
A gap remains in understanding the likely exposure scenarios or probabilities under real
environmental conditions. For instance, pH, dissolved solids, flow, and other chemical/physical
parameters exist in an environmental setting. NP properties and bioavailability will depend
on the surrounding aquatic environment. This study investigates the partitioning of nano-
sized titanium dioxide using Columbia River (WA) water and a constructed mesocosm with a
homogenous sediment and biota that have life histories associated with the sediment or surface
waters. A flow-through, benchtop, riverine mesocosm was used to examine the fate, transport,
and association of titania NPs to determine relevant exposures for each of the examined
compartments and biota.
Methods
Two different types of titanium dioxide were used for fate and transport experiments; pure
anatase form, 5-30 nm in water dispersion (Nanostructured and Amorphous Material, Los
Alamos, NM) and an anatase/rutile mixture, <75 nm in water dispersion (Sigma-Aldrich,
St. Louis, MO). Dispersion and size were confirmed using scanning electron microscopy
and ImageJ (Rasband, 2007) software analysis for particle sizing. The titania solutions were
added to separate triplicate mesocosms with continual 50 mL/min flow rate of Columbia River
water (CRW) at a concentration of 5 ppm for 12 hours, followed by an additional 36 hours of
unamended CRW at the same flow rate. Water samples of the dosing solution, inlet, and outlet of
the mesocosms were collected prior to titania addition, and at 1, 8, 12, 13, 24, 36, and 48 hours
post titania addition for TiO2 mass analysis.
Mesocosms were constructed using sterile Accusand at 2 cm deep sediment in 15 x 15 cm mesh
baskets. Baskets were placed in chambers designed for flow-through aquatic exposures and
flow rate was regulated by peristaltic pumps. Six Asiatic clams (Corbiculaflumined) and 25
amphipods (Hyallela azteca) were added to each of the 9 total mesocosms to provide 3 replicates
per treatment (2 forms of titania and control). Mesocosm systems were allowed to run for biota
acclimation for seven days prior to beginning exposures. Biota and sediment samples were
collected following the conclusion of the 48 hour exposure and analyzed for TiO2 uptake.
To assess mass of TiO2 associated within a sample, inductively coupled plasma optical emissions
spectroscopy (ICP-OES; Perkin Elmer 4300 DV) was used. All matrices (including water
214
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samples) were lyophilized in trace metal cleaned polypropylene vials. The remaining dried
material for the biota and river water samples were dissolved using a 70:30 mixture of nitric/
sulfuric acids followed by heating in a hot block at 95°C for 4 hours. The instrument was
calibrated over a range of concentrations using certified Ti standards. Sediment samples required
leaching using a dissolution method containing a mixture of hydrochloric, nitric acid, and
hydrofluoric acids to solubilize all minerals (adapted from Wu et al. 1996). The experimental
data generated from all compartments was used to parameterize and validate a physically based
process model based on conservation of mass for each phase (i.e., water, sediment, biota).
Results and Discussion
The different titania solutions changed dispersion characteristics upon dilution in Columbia
River water (CRW). The primarily single particle suspensions quickly formed large aggregates
on the order of several microns (Figure 1). Water concentrations of titania were close to nominal
dose through the first twelve hours of dosing, and then rapidly fell upon the change to depuration
with unamended CRW (Table 1). The concentration of titania decreased 90% for the anatase
material and 85% for the anatase/rutile mixture from the inlet to the outlet during the initial 12
..'••-*••.•*". • -•
Figure 1. Scanning electron micrographs of titania solutions. A. Anatase titanium oxide, 5-30
nm, 15% w/v dispersion in water. B. Anatase-rutile titanium oxide, <75 nm, 10% w/v dispersion
in water. C. Titanium oxide material shown in A following dilution to 5 ppm in Columbia River
water. D. Titanium oxide material shown in B following dilution to 5 ppm in Columbia River
water.
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Table 1.
Titania Nanomaterial
Anatase
Anatase/Rutile
CRW
1-12 hr exposure
inlet (ppm)
8. 196 (±0.88)
5. 138 (±0.27)
0.008 (0)
outlet (ppm)
0.833 (±0.05)
0.777 (±0.09)
0.001 (0)
13-48 hr depuration
inlet (ppm)
0.147 (±0.01)
0.013 (0)
0.007 (0)
outlet (ppm)
0.037 (0)
0.078 (±0.01)
0.002 (0)
ICP-OES results of titanium dioxide concentrations in water samples. Concentrations are
given as means in parts per million (ppm) with standard deviations in parentheses from
triplicate samples. Two types of titanium oxide are listed as Anatase and Anatase/Rutile,
and controls are listed as Columbia River water (CRW).
hours of continuous exposure. For both nanomaterials, there were visible floes throughout the
dosing portion of the experiment and the material was observed to settle out over the sediment,
thus accounting for the rapid loss of titania from the inlet to the outlet of the mesocosms. The
sediment analysis indicated that indeed the majority of sediment-associated titania was present
in the inlet third, with a gradient in decreasing concentration for the middle and outlet thirds.
However, more of the anatase titania nanomaterial solution associated with the sediment (0.5%)
than the anatase/rutile titania nanomaterial (0.08%).
A greater percentage of anatase titania associated with the amphipods (48 mg/g) than the
clams (0.55 mg/g). This difference was visible in that the amphipods appeared coated with
the nanomaterials while the clams were observed to flush out visible titania floes through their
excurrent siphons and through deposition in fecal material. Although the same observations
were also made with the anatase/rutile exposures, a greater concentration of titania nanomaterial
was associated with amphipods (67.8 mg/g) and clams (1.2 mg/g). Mass measurements of the
anatase/rutile mixture were more difficult to accomplish than the anatase material alone due to
increase insolubility. The greater association of the anatase/rutile material with the biota may
be attributed to lower overall solubility as well. Complexation of either material with dissolved
solids in the CRW was not examined. The observations and mass concentration data are being
explored with sedimentation rates for each titania nanomaterial for assessing association rates
using a physically based process model.
Acknowledgements
A portion of the research described in this paper was performed in the Environmental Molecular
Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's
Office of Biological and Environmental Research and located at Pacific Northwest National
Laboratory."; http://www.emsl.pnl.gov/using-emsl/terms.shtml
References
Bermudez E, JB Mangum, B A Wong, B Asgharian, PM Hext, DB Warheit, and JI Everitt. 2004.
"Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium
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dioxide particles." Toxicol Sci 77:347-357.
Federici, G, BJ Shaw, and RD Handy. 2007. Toxicity of titanium dioxide nanoparticles to
rainbow trout (Oncorhynchus mykiss): Gill injury, oxidative stress, and other physiological
effects. Aquat Toxicol 84:415-430.
Gurr J-R, AS Wang, C-H Chen, and K-Y Jan. 2005. "Ultrafine titanium dioxide particles in the
absence of photoactivation can induce oxidative damage to human bronchial epithelial cells."
Toxicology 213:66-73.
Hussain SM, KL Hess, JM Gearhart, KT Geiss, and JJ Schlager. 2005. "In vitro toxicity of
nanoparticles in BRL 3A rat liver cells." Toxicol. In Vitro 19:975-983.
Loven SB, and R Klaper. 2006. "Daphnia magna mortality when exposed to titanium dioxide
and fullerene (C60) nanoparticles." Environ. Toxicol. Chem. 25(4): 1132-1137.
MaynardA. 2006. Nanotechnology: A Research Strategy for Addressing Risk. Woodrow
Wilson International Center for Scholars, Washington D.C.
Peters K., RE Unger, CJ Kirkpatrick, AM Gatti, and E Monari. 2004. "Effects of nanoscaled
particles on endothelial cell functioning vitro: studies on viability, proliferation and
inflammation." J. Mater. Sci. Mater. Med. 15:321-325.
Ramires PA, ARomito, F Cosentino, and E Milella. 2001. "The influence of titania/
hydroxyapatite composite coatings on in vitro osteoblasts behaviour." Biomaterials 22:1467-
1474.
U.S. Environmental Protection Agency (EPA). 2006. Nanotechnology: An EPA research
perspective. EPA Fact Sheet. Available at: www.epa.gov/ord (accessed 08-02-08).
Warner WG, JJ Yin, and RR Wei. 1997. "Oxidative damage to nucleic acids photosensitized by
titanium dioxide." Free Radical Biol. Med. 23:851-858.
Warheit DB, WJ Brock, KP Lee, TR Webb, and KL Reed. 2005. "Comparative pulmonary
toxicity inhalation and instillation studies with different TiO2 particle formulations: impact of
surface treatments on particle toxicity." Toxicol. Sci. 88:514-524.
Wu, S, YH Zhao, X Feng, and A Whittmeier. (1994). Application of inductively coupled plasma
mass spectrometry for total metal determination in silicon-containing solid samples using the
microwave-assisted nitric acid-hydrofluoric acid-hydrogen peroxide-boric acid digestion system.
Journal of Analytical Atomic Spectrometry 11: 287-296.
Zhang AP, and YP Sun. 2004. "Photocatalytic killing effect of TiO2 nanoparticles on Ls-174-t
human colon carcinoma cells." World J. Gastroenterol. 10:3191-3193.
Conference Questions and Answers
Question:
How long did you let the titania sit in the river water before you exposed it? Did you vary that
parameter? Did you see any settling out?
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Answer:
The carboys had stir plates and stir bars to provide a uniform distribution. We took samples
before and during the experiment. The material was mixed overnight before starting.
Question:
What was the pH of the water? Did you vary it?
Answer:
The pH of Columbia River is around 8, so it's basic. It was not changed over time. The pH of the
titania material is around 4, so when it was added there was precipitation.
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Natural Organic Matter-Mediated Phase Transfer of Quantum Dots in the
Aquatic Environment
DivinaA. G. Navarro, David F. Watson, Diana S. Aga, and Sarbajit Banerjee,
Department of Chemistry, University at Buffalo,
State University of New York, Buffalo, New York, U.S.A.
Abstract
The increasing interest in quantum dots (QDs) raises a concern with regard to their
environmental impact. With the eventual commercialization of these materials for applications
such as in solar energy conversion and as fluorophores in biomedicine, their release in the
environment is inevitable. One way by which the fate and transport of QDs will be influenced
is through their interactions with Natural Organic Matter (NOM). This study examined the
NOM-mediated phase transfer of TOPO-capped CdSe quantum dots in water. Results from our
study indicates that humic and fulvic acids (HA and FA) could facilitate the solubilization of
the organic QDs in water with kinetics that is measurable in less than 24 hours. Solution pH and
Ca2+ ion concentration also influenced the rate of phase transfer, favoring lower pH and absence
of Ca2+. Dynamic light scattering, transmission electron microscopy and infrared spectroscopy
studies showed that the interaction between HA/FA and the QD surface capping groups, as
opposed to metal coordination, is the primary mechanism for transfer. Whether HA or FA forms
aggregates with random coil conformations or as micelles when they facilitate QD transfer
remains inconclusive and needs further investigation. The results observed with the Suwannee
River HAs and FAs translated to the natural surface water samples collected from local creeks.
This study presents the first evidence of stabilization of QDs in water by humic substances in real
environmental samples, illustrating that NOM will have a significant role in the fate and transport
of QDs in aquatic systems.
Introduction
Quantum dots (QDs) are semiconductor nanocrystals with diameters in the 2-100 nm size range
that exhibit remarkable size-dependent optical properties. [1, 2] These properties, not present in
their bulk counterparts, have made QDs ideal for applications like solar energy conversion, [3-6]
and medical diagnostics. [7, 8] QDs are typically prepared as colloidal solutions by
organometallic synthesis. These nanocrystals consist of an inorganic crystalline core surrounded
by a shell of organic ligands; the ligands serve to passivate the nanocrystal surfaces, prevent
the agglomeration of the particles, and impart solubility in various dispersion media. Typically,
hydrophobic ligands and organic solvents are used. [9-11] However, for biological applications,
water-soluble QDs are prepared and obtained by various methods involving exchange or
modification of the initial hydrophobic capping ligands. [8] The most well characterized QDs
synthesized to date are CdSe and CdS, [12, 13] owing to their bandgap tunability through the
visible region of the electromagnetic spectrum. [2, 12, 14]
With increasing interest and their eventual large-scale production, release of these materials to
the environment is inevitable and human exposure is likely from several sources including air
219
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and the aquatic environment. This raises concern as to their potential harmful effects to humans
and to the environment. Although several cell culture and animal studies of QDs have been
reported in the literature, very little is known about the fate, transport, and bioavailability of
these particles. Thus, there is an urgent need to evaluate the partitioning of QDs in air and water
and to study the mobility and persistence of these materials in different phases. [15, 16]
In the environment, humic substances play a key role in the biogeochemical cycling of various
metal species. Humic substances are ubiquitous NOM with highly complex molecular structures
that allow metal chelation. Earlier studies have identified that metal-NOM complex formation
involves coordination between the metal ions and the carboxylic or phenolic groups of NOM.
[17, 18] In addition, humic acids are also able to change their aggregation states and act as
amphiphilic systems in aquatic environments.
Our objectives in this study were to determine the mechanisms of interactions between TOPO-
capped CdSe QDs and NOM, and to examine how NOM-mediated phase transfer of QDs
between organic and aqueous phases is affected by pH and ionic strength. Finally, results based
on the use of reference humic and fulvic acids have been compared with systems using natural
water samples to demonstrate environmental relevance.
Methods
QD suspensions (in hexane) (3-nm TOPO-capped CdSe) were mixed with the same amount of 20
ppm HA, 20 ppm FA, deionized water, or Creek water in a clear vial. In between measurements,
set-ups were continuously stirred and protected from light. This was stopped after 7 days by
separating the different layers into individual vials. Phase transfer was also monitored as a
function of the pH of the aqueous solutions, ionic strength and HA concentration. Absorption and
emission spectroscopy were used to monitor the transfer of the QD particles from the organic
solvent into the aqueous phase on an hourly/daily basis. Sample aggregation was monitored
over time using dynamic light scattering (DLS) while transmission electron microscopy (TEM)
and infrared spectroscopy (TR) were used to characterize the mechanisms of interactions.
Approximately 1.0-10.0 uM of QD solutions were used in the experiments.
Results and Discussion
Evidence of phase transfer is clearly visible in the digital photographs shown in Figure 1 and is
also apparent from the absorption spectra of the organic and aqueous phases. Figure 2 shows a
strong diminution in the intensity of the QD optical absorptions in the organic phase alongside
an increase in the optical absorbance of the aqueous phase after 24 hours of equilibration.
As seen from the increased absorption baseline and DLS results, this aqueous solubilization
involves transfer of QD aggregates instead of individual QDs. The DLS correlation curves
shows that the correlogram for the FA—QD composites decays to the baseline over a much
longer period of time as compared to FA molecules alone, indicating slower diffusion and thus
greater aggregation in these samples.[19] In addition, when transferred to the aqueous phase,
the structural integrity of the QDs is still retained. This is supported by the photoluminescence
spectra which show that the band edge emission is still clearly visible. This is significant because
leaching of Cd2+ ions into the environment is a major concern.[20] However, the quantum yield
of the transferred QDs is significantly diminished. This maybe a result of quenching from the
220
-------
A
Oh
1 h
3h
5h
24 h
B
Oh ih 3h 5h 24 h
Figure 1. (A) Digital photographs illustrating transfer of 3-nm TOPO-capped CdSe in
hexane (top layer) to the aqueous phase (bottom layer) containing 20 ppm HA. (B)
Control set-ups for the phase transfer experiments with deionized water only.
0.2500,
A
0.2500,
zi 0.2000
ro
§ 0.1500
ro
0.1000
0.0500
0.0000
O.OOOOJ-
B
(HA+QD)aq
400 450 500 550 600 650 700
Wavelength, nm
400 450 500 550 600 650 700
Wavelength, nm
Figure 2. A) Hexane layer: UV-Vis absorption spectra of the 3-nm CdSe-TOPO after phase
transfer, (HA+QD)org, in comparison with the QD spectra. B) Aqueous layer with 20 ppm HA
solution: UV-Vis absorption spectra of the 3-nm CdSe-TOPO that transferred from the hexane
layer, (HA+QD)aq, in comparison with the HA spectra.
NOM components and/or alternatively, a result of the immobilization of QDs within the solid
humic matrix.
The size and morphology of aggregated and flocculated structures were characterized by
TEM. TEM images of the QDs, HA, and the phase-transferred QDs are given in Figure 3.
QD-HA aggregation, which is consistent with the increase in the absorption baseline and the
DLS measurements discussed above, is shown clearly in Figure 3D with several embedded
QDs demarcated in white clearly shows the lattice planes of several QDs residing within the
amorphous humic matrix. Furthermore, surface-related interactions were determined by FTIR
221
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70 nm
A
B
.lOOOnm
*? '-.
XK '
>
4 nm
Figure 3. Low-resolution (A) and High-resolution (B) TEM image of the 3-nm CdSe-TOPO
particles. Low-resolution (C) and High-resolution (D) TEM image showing phase-transferred
QDs embedded in HA.
spectroscopy which provided more information corroborating the proposed model involving
possible encapsulation of QDs by humic acid micelles. Considering the spectral signatures of
the TOPO-capped CdSe and the HA, Figure 4, shows that the most noteworthy feature in the
spectrum for the transferred QDs is that the TOPO peaks are essentially preserved, P=O stretch
at -1150 cm"1 and C-H stretch at -2900 cm"1. This indicates that TOPO ligands are definitely part
of the phase-transferred QD which strongly suggests interaction between hydrophobic segments
of the HA and hydrophobic alkyl chains of TOPO ligand on the QD. In addition, the asymmetric
stretching bands of the carboxyl groups of HA undergo noteworthy changes which show possible
coordination of the carboxylate groups of HA to Cd2+ surface sites that may play a role in the
QD-NOM interaction.
Additional information on NOM-QD interactions comes from the phase transfer experiments
performed by varying the solution pH or ionic strength of the aqueous phase. Both pH and ionic
strength influence the structural conformation of the HA and FA moieties. Adjusting the pH of
222
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(HA+QD)aq
QD
4)
o
in
4000 3500 3000
2500 2000 1500
Wavenumber, cm1
1000 500
Figure 4. IR spectra fpr TOPO-capped CdSe QD, Suwannee River HA and the phase-transferred
QD.
the aqueous solutions to low pH values results in HA and FA which are aggregated. At this range,
the rate of phase transfer was observed to be fastest. In the same manner, the presence of cation,
such as Ca (II), engenders aggregation of component HAs and FAs through its metal chelating
ability. However, the phase transfer experiments using varying ionic strengths showed that the
presence of CaCl2 only increases the rate at which the QDs flocculate at the aqueous/organic
interface and not the actual solubilization of the HA-QD composites.
To determine how results using the model NOM could translate into the behavior of QDs in the
natural environment, experiments were performed using natural surface water samples collected
from Tonawanda (TON) and Buffalo (BUF) Creeks, containing 11 and 5 ppm dissolved organic
carbon (DOC), respectively. Results showed that only TON water sample was able to exhibit
significant transfer of QDs into the aqueous phase in one day whereas in the BUF set-ups the
QDs remain at the interface and do not transfer. This slow transfer of QDs settling at the hexane/
water interface in the BUF set-up may indicate occurence of other factors affecting phase transfer
which were not investigated in this paper.
Conclusions
This study presents the first evidence of NOM-QD interactions based on simple phase transfer
experiments. HA and FA systems are able to engender the phase transfer of TOPO-capped QDs
from hexane to water. Remarkably, NOM is able to stabilize in aqueous phase, hydrophobic
systems that otherwise have very little tendency to dissolve in water. These results clearly
illustrate that NOM present in the aqueous environment will have a strong influence on the
partitioning and transport of these novel manufactured nanomaterials. Our spectroscopic
measurements and control experiments point to the mechanism where the humic substances
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essentially form micelles that can encapsulate the hydrophobic quantum dots with the polar
functional groups on the humics engendering solubility in water.[21, 22] The precise structural
conformations, however, are difficult to discern as it could essentially comprise of aggregates
with random coil conformations, micelles with hydrophobic cavities, or a mixture of other such
conformations. Furthermore, coordination of humic substances with QD surfaces following
displacement of surface capping groups remains a possibility. The results here demonstrate
the importance of the surface capping ligands on the QD surfaces and support the shift to
biocompatible ligands based on poly(ethylene glycol).[23]
References
1. Trindade, T., O'Brien, P., and Pickett, N.L. (2001). Nanocrystalline Semiconductors:
Synthesis, Properties, and Perspectives. Chem. Mater. 13, 3843-3858.
2. Murray, C.B., Kagan, C.R., and Bawendi, M.G. (2000). Synthesis and characterization of
monodisperse nanocrystals and close-packed nanocrystal assemblies. Annu. Rev. Mater. Sci.
30, 545-610.
3. Plass, R., Pelet, S., Krueger, 1, Graetzel, M., and Bach, U. (2002). Quantum Dot
Sensitization of Organic-Inorganic Hybrid Solar Cells. J. Phys. Chem. B 106, 7578-7580.
4. Luque, A., Marti, A., and Nozik, AJ. (2007). Solar cells based on quantum dots: multiple
exciton generation and intermediate bands. MRS Bull. 32, 236-241.
5. Robel, I, Kuno, M., and Kamat, P.V. (2007). Size-Dependent Electron Injection from Excited
CdSe Quantum Dots into TiO2 Nanoparticles. J. Am. Chem. Soc. 129, 4136-4137.
6. Robel, I, Subramanian, V, Kuno, M., and Kamat, P.V. (2006). Quantum Dot Solar Cells.
Harvesting Light Energy with CdSe Nanocrystals Molecularly Linked to Mesoscopic TiO2
Films. J. Am. Chem. Soc. 725, 2385-2393.
7. Bruchez, M., Jr., Moronne, M., Gin, P., Weiss, S., and Alivisatos, A.P (1998). Semiconductor
nanocrystals as fluorescent biological labels. Science (Washington, D. C.) 257, 2013-2016.
8. Yu, W.W., Chang, E., Drezek, R., and Colvin, V.L. (2006). Water-soluble quantum dots for
biomedical applications. Biochem. Biophys. Res. Commun. 348, 781-786.
9. Murray, C.B., Norris, D.J., and Bawendi, M.G. (1993). Synthesis and characterization of
nearly monodisperse CdE (E = sulfur, selenium, tellurium) semiconductor nanocrystallites. J.
Am. Chem. Soc. 775, 8706-8715.
10. Peng, Z.A., and Peng, X. (2002). Nearly Monodisperse and Shape-Controlled CdSe
Nanocrystals via Alternative Routes: Nucleation and Growth. J. Am. Chem. Soc. 124, 3343-
3353.
11. Peng, Z.A., and Peng, X. (2001). Formation of high-quality CdTe, CdSe, and CdS
nanocrystals using CdO as precursor. J. Am. Chem. Soc. 723, 183-184.
12. Murphy, CJ. (2002). Optical sensing with quantum dots. Anal. Chem. 74, 520A-526A.
13. Klostranec, J.M., and Chan, W.C.W. (2006). Quantum dots in biological and biomedical
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research: recent progress and present challenges. Adv. Mater. (Weinheim, Ger.) 18, 1953-
1964.
14. Alivisatos, A.P. (1996). Perspectives on the physical chemistry of semiconductor
nanocrystals. J. Phys. Chem. 100, 13226-13239.
15. Wiesner, M.R., Lowry, G.V., Alvarez, P., Dionysiou, D., and Biswas, P. (2006). Assessing the
risks of manufactured nanomaterials. Environ. Sci. Technol. 40, 4336-4345.
16. Maynard, A., Aitken, R.J., Butz, T., Colvin, V, Donaldson, K., Oberdorster, G., Philbert,
M.A., Ryan, J., Seaton, A., Stone, V, Tinkle, S.S., Iran, L., Walker, N., J., and Warheit, D.B.
(2006). Safe handling of nanotechnology. Nature 444, 267-269.
17. Schnitzer, M., and Skinner, S.I.M. (1965). Organo metallic interactions in soils. IV. Carboxyl
and hydroxyl groups in organic matter and metal retention. Soil Sci. 99, 278-284.
18. Gamble, D.S., Schnitzer, M., and Hoffman, I. (1970). Cu2+-fulvic acid chelation equilibrium
in 0.1M potassium chloride at 25.0.deg. Can. J. Chem. 48, 3197-3204.
19. van de Hulst, H.C. (1981). Light Scattering by Small Particles (NY: Dover).
20. Lewinski, N., Colvin, V, and Drezek, R. (2008). Cytotoxicity of nanoparticles. Small 4, 26-
49.
21. Clapp, C.E., and Hayes, M.H.B. (1999). Sizes and shapes of humic substances. Soil Sci. 164,
777-789.
22. Zara, L.R, Rosa, A.H., Toscano, I.A.S., and Rocha, J.C. (2006). A structural conformation
study of aquatic humic acid. J. Braz. Chem. Soc. 17, 1014-1019.
23. Yu, W.W., Chang, E., Falkner, J.C., Zhang, J., Al-Somali, A.M., Sayes, C.M., Johns,
J., Drezek, R., and Colvin, V.L. (2007). Forming Biocompatible and Nonaggregated
Nanocrystals in Water Using Amphiphilic Polymers. J. Am. Chem. Soc. 129, 2871-2879.
Conference Questions and Answers
Question:
Did you differentiate the effect of capping agent interactions with the humic materials? You said
there was displacement of the surface capping groups as well as interactions of the humic/fulvic
acids with the capping groups themselves.
Answer:
We have not done this and have not determined what technique could be used.
Question:
Do you have any quantitative information on the partition coefficient? Did you do a mass balance
to see exactly how much there is in each phase?
Answer:
We measured the cadmium concentration in the organic and aqueous phases by ICP/OES
225
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(inductively coupled plasma/optical emission spectrometry), but we cannot associate this with
the actual transfer, since there may have been leaching from the quantum dot.
Question:
On the transmission electron microscopy (TEM), you show aggregates; however, they are often
seen due to evaporation. Did you do cryo-TEM to correct for this potential?
Answer:
We did not do cryo-TEM, but the overall evidence does point to aggregation.
Question:
When you showed images of quantum dots or aggregates at the interface between hexane and
water you used the word "settling." When the dots or aggregates are in hexane, they are at 3 nm
and unlikely to settle. Did you mean "settling" or something else?
Answer:
The solutions are continually stirred and the settlement is perhaps due to the interaction of the
quantum dots with humic acids that allows them to aggregate and then settle out.
226
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Natural Organic Matter Enhanced C60 Fullerene Dispersion
in the Aqueous Phase
Qilin Li, and Bin Xie, Department of Civil and Environmental Engineering,
Rice University, Houston, Texas, U.S.A.
Steven Xu, Department of Chemical and Biomolecular Engineering,
Rice University, Houston, Texas, U.S.A.
Abstract
Assessing exposure and risk of engineered nanomaterials requires accurate prediction of
their concentrations and physicochemical properties in the natural environment. Although C60
fullerene is virtually insoluble in water, stable aqueous suspension of C60 nanoparticles (nC60)
can form when C60 powder is mixed with water for an extended period of time. In this study, we
investigate the effect of natural organic matter (NOM) on the dispersion of C60 in water as well as
the properties of nC60 particles formed. We used Suwannee River humic acid (SRHA) and fulvic
acid (SRFA) standards as model NOM compounds and tested a range of solution conditions (i.e.,
pH, total ionic strength and ionic composition) to simulate realistic natural aqueous environment.
NOM was found to greatly increase C60 dispersion in water, and the dispersed C60 concentration
increased with NOM concentration. Total ionic strength and calcium ion concentration also
played a role in C60 dispersion. The effect of NOM was further enhanced under sunlight. UV/
Vis spectra of the nC60 suspensions formed in the presence of NOM under sunlight suggest
photochemical transformation of C6Q. Further investigation is necessary to reveal the reaction
mechanisms and to identify the products.
Introduction
Carbon-based nanomaterials have received increasing attention for their potential application
in electronics, optics, and pharmaceuticals 1-3. In particular, C60 fullerene is being produced at
industrial-scale in tons per year 4. Potential non-regulated discharge and incidental spill of C60
into the environment raises growing concern on its impact on the ecosystem as well as human
health.
Although virtually insoluble in water5, C60 can form stable aqueous suspensions of nanoparticles
(nC60) when mixed with water for an extended period of time 6"n. The process typically took
weeks to months6'7-9'10. The amount of C60 that can be dispersed in water, i.e. "solubility" of
nC60, has not been quantified. In addition, almost all previous studies used organic-free water.
The impact of natural organic matter (NOM), which has been shown to alter particle properties
of preformed nC6012 and stabilize nC6013 as well as multi-walled carbon nanotubes 14, is unknown.
In this study, we investigated the effect of NOM on direct dispersion of C60 in water under typical
natural water conditions. The "solubility" of nC60 was quantified at different concentrations of
NOM under systematically varied solution and light conditions. The physicochemical properties
of the nC60 particles formed including particle size, morphology, and surface charge were
carefully characterized.
227
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Experimental Methods
Dry C60 powder was mixed with test solutions containing 0 to 20 mg/L of Suwannee River humic
acid (SRHA) or fulvid acid (SRFA) in dark, with room light or sunlight. The total ionic strength
of the test solutions ranged from 0.1 to 10 mM, adjusted using NaCl and CaCl2. Concentration of
C60 in all nC60 suspensions was determined by total organic carbon (TOC) measurement using a
high-sensitivity TOC analyzer (Shimadzu Scientific Instruments, Columbia, MD). Experiments
in sunlight were performed on 10 consecutive sunny days during the summer. Mixing in dark
or room light went on for up to 29 days. Samples were retrieved from each test suspension at
predetermined times, filtered through 2-um and 0.45-um- pore-size membrane filters, and stored
in darkness at 4°C before analysis. All samples were analyzed shortly after collection.
Hydrodynamic diameter and electrophoretic mobility of nC60 were measured by dynamic
light scattering and phase analysis light scattering, respectively, using ZetaPALS (Brookhaven
Instruments, Holtsville, NY). nC60 particle morphology and structure were analyzed using a
JEOL-2010 TEM (JEOL Inc., Peabody, MA). UV/Vis absorbance spectra of nC60 suspensions
were obtained using a dual beam, high resolution UV/Vis spectrophotometer (UV-2550,
Shimadzu Scientific Instruments, Columbia, MD).
Results and Discussion
C60 dispersion kinetics. NOM significantly increases the rate of C60 dispersion in water (Figure
1), and the amount of C60 dispersed in water increased with NOM concentration. The rate of
dispersion strongly depends on the total ionic strength (Figure 2). The amount of C60 dispersed
increases significantly with decreasing total ionic strength. Although Ca2+ usually reduces
colloidal stability due to its more efficient charge screening compared to monovalent cations,
the presence of Ca2+ did not seem to affect C60 dispersion. In the presence of sunlight, C60
dispersion is greatly enhanced (Figure 3): In the presence of 1 mM Ca2+ and 10 mg/L SRHA
with a total ionic strength of 10 mM, 9.9 mg/L of C60 was found in the aqueous phase after only
72 hours of mixing. This nC60 concentration is well above the minimal inhibitory concentration
(MIC) or median lethal dosage (LC ) of several bacteria according to literatures8'15> 16. Under
<
\
*
1
>
'
5 0 5 10 15 20 2
SRHA concentration (mg/L)
0.1 mM(w/oCa)
1.0mM(w/oCa)
Ionic strength
10mM(w/oCa)
Figure 1. Effect of NOM concentration on
fullerene dispersion. Samples were mixed for
72 hrs with sunlight; ionic strength = 1 mM.
Figure 2. Effect of solution condition on
fullerene dispersion. Samples were mixed
for 72 hrs in 10 mg/L SRHA with sunlight
228
-------
- " C60 cone, room light
v> C60 cone, dark
• C60 cone, sunlight I
\\vi \\\
\\X9994 \\\>9Q9
....
Figure 3. Fullerene dispersion under dark and
fluorescent room light conditions. Samples were
mixed for 72 hrs in 10 mg/L SRHA; ionic strength :
dark or fluorescent room light conditions, however, C60 dispersion rate was substantially lower.
Increasing NOM concentration again increased C60 dispersion.
Dynamic light scattering measurement showed a rapid decrease in nC60 particle size during the
dispersion process under sun light. At the same time, nC60 particle surface zeta potential became
more negative with mixing time. Consistent with the classic colloidal theory, nC60 particle size
increased with the total ionic strength and Ca2+ concentration. It is noteworthy that extremely
small nC60 particles (less than 5 nm in diameter) formed after 72 hours of mixing at the lowest
ionic strength tested (0.1 mM). These particles are much smaller than those previous reported in
studies using organic free water.
Potential photochemical derivatization ofC6g. Figure 4 compares the UV/Vis absorbance
spectra of the nC60 suspensions prepared by direct dispersing in NOM solutions and NOM-free
water, and nC60 formed through the solvent exchange method 13. nC60 particles preformed using
the solvent exchange method maintains C characteristic absorbance peaks at 266 nm and 343
o
in
.a
< CD
—•— Readily formed nC60 in 10 mg/L
-•—NOM-free, 0.1 mM IS (3
-9—Dispersed in 10 mg/L SRHA, 10 mM ISw/o Ca (3
--•--Dispersed in 10 mg/L SRHA, 0.1 mM IS (3
_.* Dispersed in 10mg/LSRHA, 10 mM IS with 1 mM Ca (3
0.
0
25 30 35 40 45
Wavelength
Figure 4. UV/Vis spectra of C6o aqueous
suspensions prepared in NOM solutions
50
55
229
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nm in the presence and absence of NOM, indicating that adsorption of NOM on nC60 particle
surface does not interfere with its UV absorbance. Similarly, nC60 dispersed in NOM-free water
showed characteristic peaks at 273 nm and 355 nm. The slight red shift is probably the result of
aggregate formation. These results are in consistency with previous reports 8>9> 17~19. However,
nC60 directly dispersed in NOM solutions did not show any of the C60 characteristic absorbance
peaks in all solution conditions tested. The absorbance spectra remained featureless after
removal of free NOM molecules using a dialysis membrane with molecular weight cutoff of
5000 Dalton. These results indicate that C60 may have been photochemically derivatized when
dispersed in NOM solutions under sun light.
References
1. Guildi, D. M.; Martin, N., From synthesis to optoelectronic properties. Kluwer Academic
Publishers, Dordrecht, The Netherlands: 2002.
2. Osawa, E., Perspective offullerene nanotechnology. SPringer, Berlin, Germany: 2002.
3. Tagmatarchis, N.; Shnohara, H., Fullerenes in medical chemistry and their biological
applications. M/'w/' Reviews inMed. Chem. 2001, 1, 339-348.
4. Tremblay, J., Mitsubishi aims at a breakthrough. Chem. Eng. News 2002, 80, 16-17.
5. Heymann, D., Solubility of C60 and C70 in water. Lunar & Planetary Sci. 1996, 27, 543-544.
6. Cheng, X. K.; Kan, A. T.; Tomson, M. B., Naphthalene adsorption and desorption from
Aqueous C-60 fullerene. Journal of Chemical and Engineering Data 2004, 49, (3), 675-683.
7. Dhawan, A.; Taurozzi, J. S.; Pandey, A. K.; Shan, W. Q.; Miller, S. M.; Hashsham, S. A.;
Tarabara, V. V, Stable colloidal dispersions of C-60 fullerenes in water: Evidence for
genotoxicity. Environmental Science & Technology 2006, 40, (23), 7394-7401.
8. Lyon, D. Y; Adams, L. K.; Falkner, J. C.; Alvarez, P. J. J., Antibacterial activity offullerene
water suspensions: Effects of preparation method and particle size. Environmental Science &
Technology 2006, 40, (14), 4360-4366.
9. Brant, J. A.; Labille, J.; Bottero, J. Y; Wiesner, M. R., Characterizing the impact of
preparation method on fullerene cluster structure and chemistry. Langmuir 2006, 22, (8),
3878-3885.
10. Labille, J.; Brant, J.; Villieras, R; Pelletier, M.; Thill, A.; Masion, A.; Wiesner, M.; Rose,
J.; Bottero, J.-Y, Affinity of C60 fullerenes with water. Fullerenes, Nanotubes, and Carbon
Nanostructures 2006, 14, 307-314.
11. Tseluikin, V. N.; Chubenko, I. S.; Gun'kin, I. R; Pankst'yanov, A. Y, Colloidal dispersion of
fullerene C-60 free of organic solvents. Russian Journal of Applied Chemistry 2006, 79, (2),
325-326.
12. Xie, B., Xu, Z., Guo, W. and Li, Q. Impact of Natural Organic Matter on the Physicochemical
Properties of Aqueous C60 Nanoparticles. Environmental Science and Technology., 2008,
42(8): 2853-2859.
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13. Chen, K. L.; Elimelech, M., Influence of humic acid on the aggregation kinetics of fullerene
(C-60) nanoparticles in monovalent and divalent electrolyte solutions. Journal of Colloid and
Interface Science 2007, 309, (1), 126-134.
14. Hyung, H.; Fortner, J. D.; Hughes, J. B.; Kim, J. H. Natural organic matter stabilizes carbon
nanotubes in the aqueous phase. Environ. Sci. & Technol. 2007, ¥7, 179-184.
15. Lyon, D. Y; Adams, L. K.; Falkner, J. C.; Alvarez, P. J. J., Antibacterial activity of fullerene
water suspensions: Effects of preparation method and particle size. Environmental Science &
Technology 2006, 40, (14), 4360-4366.
16. Sayes, C. M. R, J. D.; Guo, W.; Lyon, D.; Boyd, A. M.; Ausman, K. D.; Tao, Y. J.;
Sitharaman, B.; Wilson, L. J.; Hughes, J. B.; West, J. L.; Colvin, V. L., The Differential
Cytotoxicity of Water-Soluble Fullerenes Nemo Lett. 2004,4, (10), 1881-1887.
17. Andrievsky, G. V; Klochkov, V. K.; Bordyuh, A. B.; Dovbeshko, G. L, Comparative analysis
of two aqueous-colloidal solutions of C60 fullerene with help of FTIR reflectance and UV-
Vis spectroscopy. Chem. Phys. Letters. 2002, 364, 8-17.
18. Rivelino, R.; Maniero, A. M.; Prudente, F. V; Costa, L. S., Theoretical calculations of the
structure and UV-vis absorption spectra of hydrated C-60 fullerene. Carbon 2006, 44, (14),
2925-2930.
19. Scharff, P.; Risch, K.; Carta-Abelmann, L.; Dmytruk, I. M.; Bilyi, M. M.; Golub, O. A.;
Khavryuchenko, A. V; Buzaneva, E. V; Aksenov, V. L.; Avdeev, M. V; Prylutskyy, Y. L;
Durov, S. S., Structure of C-60 fullerene in water: spectroscopic data. Carbon 2004, 42, (5-
6), 1203-1206.
Conference Questions and Answers
Question:
What was the filter size used to measure the C60 in the aqueous solution?
Answer:
We used two filter sizes. For particle size analysis, we used a 2-micrometer (um) filter followed
by a .45-micrometer filter, because we wanted to include all non-separatable particles in the
suspension. There was very little difference in the two methods, because most of the particles
are smaller than .45 micrometers. Before sampling we let particulate in the sample water settle,
because we found that particulate buildup (filtercake) on a filter had the potential to block the
flow of particles smaller than .45 micrometers.
Question:
You used TEM. Could you describe how you sampled?
Answer:
At the beginning of the project we compared dried samples and ones prepared cryogenically
and found that there was no real difference between the techniques. For sample preparation, we
placed a 3-microliter (uL) sample on a grid in an evaporating environment and allowed it to dry
231
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overnight.
Question:
For the dynamic light scattering (DLS) measurements, you had a choice between per-mass/per-
number or scattered cross-section. Why did you choose one over the other?
Answer:
When using DLS you can get number-based averages, intensity-based averages, or mass-based
averages. We chose to use number-based averages, because particle population was more
important for our project. Intensity is proportional to the diameter of the particle to the power of
6, so if you have some larger particles the results are shifted towards the larger population.
Question:
With time mixing and the source of the light, are you saying that you have degradation in terms
of carboxylation of materials?
Answer:
We are not sure it is carboxylation. We have some hypotheses about what is happening, but we
have not done any work to identify the reaction.
Question:
Do you have increasing negative charge with time?
Answer:
There is increasing negative charge in the de-ionized water, but we have not identified why.
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Interactions of Bacteria with Engineered Metal, Metalloid, and Metal Oxide
Nanomaterials
Patricia Holden, Allison Horst, John Priester, and Andrea Neal,
Donald Bren School of Environmental Science & Management,
University of California, Santa Barbara, California, U.S.A.
Abstract
Engineered nanomaterials in the environment can have many interactions with bacteria,
including toxicity, breakdown, and accumulation. Such interactions will have consequences
for the transport and fate of nanomaterials, and thus the broader environmental and societal
outcomes. This talk presents laboratory research results regarding interactions of environmental
strains of Pseudomonas with several metal, metalloid, and metal oxide nanomaterials. We ask:
how do engineered nanomaterials affect bacteria, and how do bacteria affect the nanomaterials?
The focus is on bacterial physiological effects and nanomaterials fates with follow-on to toxicity
mechanisms where appropriate. Three projects are discussed: 1) comparative effects and fates of
Cd(II) ions versus bare CdSe quantum dots with Pseudomonas aeruginosa PG201, a relatively
cadmium-resistant bacterial strain, 2) effects of several industrial metal oxide nanomaterials on
growing/! aeruginosa and bacterial effects on nanomaterials, and 3) effects of various TiO2 on
P. putida growth and bacterial effects on nanomaterial structures. Our research reveals several
outcomes of interest, i.e. that nanoparticles can have specific particle effects on bacteria that
are not explainable by the toxic metal content of the nanoparticles, that bacteria can change
nanoparticle aggregation states, that aggregation may preclude toxicity of some nanoparticles to
bacteria, and that photoactive nanoparticles can be toxic to bacteria in the dark. These results are
applicable to envisioning possible outcomes of nanomaterial release into the natural environment
where bacteria are abundant, readily colonize surfaces, and catalyze essential reactions.
Introduction
The fates of engineered nanomaterials (ENMs) in the environment could depend substantially
on their interactions with bacteria. Laboratory research with bacterial cultures and introduced
ENMs can reveal potential interactions that are environmentally-relevant such as ENM binding
to the cell envelope 1-3, uptake of ENMs either nonspecifically4 through damaged membranes 5>6
or via specific receptors in the light7, and dose-dependent reductions in population growth l- 8~10.
In some cases, bacterial inhibition appears to arise from toxic metal ions released from metallic
ENMs n, yet there is also evidence for ENM-specific effects in that ENM shape altered inhibition
patterns 9 and ENM capping with another material did not7. Extrapolating such findings to the
environment is challenging, in part because ENMs can readily aggregate under environmental
conditions and possibly lose ENM-specific characteristics. However, bacteria may in fact alter
ENM aggregation states in, for example, wastewater treatment systems 12. Our research regards
bacterial interactions with pre-aggregated as well as dispersed ENMs, with particular interests
in bacterial effects on aggregation states and the toxicity of ENMs that are either stable in
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suspension or become dispersed.
Methods
Two gram negative bacterial species, both widely-distributed in nature and known to be
metabolically-versatile and resistant to many toxic substances, were studied in separate pure
culture experiments in the laboratory: Pseudomonas aeruginosa and P. putida. Rich media
was used to simulate one type of environment, i.e. an environment rich in reduced organic
nutrients and salts; rich media (Luria Bertani or LB broth) was also used to avoid starvation
stress associated with nutrient-limiting conditions. Several metal oxides from industrial sources,
including TiO2, were amended to cultures at the inoculation stage. Separate experiments were
also performed with CdSe quantum dots. As described elsewhere (Priester et al., manuscript in
preparation; Horst et al., manuscript in preparation), growth was monitored by optical density
and DNA analysis. Cell morphologies and associations with NMs were visualized with electron
microscopy (EM), and in the case of CdSe QDs, the distribution of metals was quantitatively
analyzed using various spectrometric methods. Evidence for intracellular oxidative stress was
also acquired.
Results
As described elsewhere, (Priester et al., manuscript in preparation; Horst et al., manuscript in
preparation), a range of observations were made including: 1) variations in bacterial colonization
of NM aggregates, 3) variations in NM disaggregation in the presence of bacteria, 4) NM
inhibition of bacterial growth concurrent with disaggregation, and 4) both ion and NM-specific
growth inhibition. The observations are in the forms of EM images, quantitative growth data,
and quantitative analyses of NM integrity as well as metal distribution.
Discussion and Conclusions
Our research reveals several outcomes of interest, i.e. that nanoparticles can have specific particle
effects on bacteria that are not explainable by the toxic metal content of the nanoparticles, that
bacteria can change nanoparticle aggregation states, that aggregation may relate to the toxicity
of some nanoparticles to bacteria, and that photoactive nanoparticles can be toxic to bacteria in
the dark. These results are applicable to envisioning possible outcomes of nanomaterial release
into the natural environment where bacteria are abundant, readily colonize surfaces, and catalyze
essential reactions.
References
1. Zhang, L. L.; Jiang, Y. H.; Ding, Y. L.; Povey, M.; York, D., Investigation into the
antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids). Journal of
Nanoparticle Research 2007, 9, (3), 479-489.
2. Thill, A.; Zeyons, O.; Spalla, O.; Chauvat, R; Rose, 1; Auffan, M.; Flank, A. M.,
Cytotoxicity of CeO2 nanoparticles for Escherichia coli. Physico-chemical insight of the
cytotoxicity mechanism. Environmental Science & Technology 2006, 40, (19), 6151-6156.
3. Lu, Z. S.; Li, C. M.; Bao, H. R; Qiao, Y; Toh, Y. H.; Yang, X., Mechanism of antimicrobial
activity of CdTe quantum dots. Langmuir 2008, 24, (10), 5445-5452.
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4. Hirschey, M. D.; Han, Y. J.; Stucky, G. D.; Butler, A., Imaging Escherichia coli using
functionalized core/shell CdSe/CdS quantum dots. Journal of Biological Inorganic Chemistry
2006, 11, (5), 663-669.
5. Stoimenov, P. K.; Klinger, R. L.; Marchin, G. L.; Klabunde, K. J., Metal oxide nanoparticles
as bactericidal agents. Langmuir 2002, 18, (17), 6679-6686.
6. Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fievet, F.,
Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO
nanoparticles colloidal medium. Nano Letters 2006, 6, (4), 866-870.
7. Kloepfer, J. A.; Mielke, R. E.; Nadeau, J. L., Uptake of CdSe and CdSe/ZnS quantum dots
into bacteria via purine-dependent mechanisms. Applied and Environmental Microbiology
2005, 71, (5), 2548-2557.
8. Shrivastava, S.; Bera, T.; Roy, A.; Singh, G.; Ramachandrarao, P.; Dash, D., Characterization
of enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology 2007, 18,
(22).
9. Pal, S.; Tak, Y. K.; Song, J. M., Does the antibacterial activity of silver nanoparticles depend
on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli.
Applied and Environmental Microbiology 2007', 73, (6), 1712-1720.
10. Sondi, L; Salopek-Sondi, B., Silver nanoparticles as antimicrobial agent: a case study onE1.
coli as a model for Gram-negative bacteria. Journal of Colloid and Interface Science 2004,
275, (1), 177-182.
11. Heinlaan, M.; Ivask, A.; Blinova, L; Dubourguier, H.-C.; Kahru, A., Toxicity of nanosized
and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and
Thamnocephalusplatyurus. Chemosphere 2008, 71, (7), 1308-1316.
12. Limbach, L. K.; Bereiter, R.; Mu; x; Her, E.; Krebs, R.; Ga; x; Hi, R.; x; Stark, W. J., Removal
of Oxide Nanoparticles in a Model Wastewater Treatment Plant: Influence of Agglomeration
and Surfactants on Clearing Efficiency. Environ. Sci. Technol. 2008, 42, (15), 5828-5833.
Conference Questions and Answers
Question:
How strongly does acetate have the ability to chelate cadmium (Cd)?
Answer:
I do not know.
Comment:
You might want to try the experiment again with a simple Cd salt that does not complex
heavily. The difference in toxicity may be due to the Cd being removed from availability by
complexation.
Question:
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Do you know why the Cd appeared to be associated with specific areas of the bacteria
membrane?
Answer:
These areas may have been where the efflux membrane pumps were.
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Structure of Iron Oxide Nanoparticles;
Influence of pH and Natural Organic Macromolecules
Susan Cumberland and Jamie R. Lead,
University of Birmingham, Birmingham, United Kingdom
Abstract
Metal oxide nanoparticles (defined as material with at least one dimension is between 1 and
100 nm) are being exploited in a host of industrial and commercial products and processes
remediation of ground and drinking water. Iron oxides in particular are used in vast quantities.
However, due to the small size, increased surface area and related effects nanoparticles differ
from their bulk counterparts in significant and unexpected ways. Studies have highlighted that
there may also be environmental risks as the nanoparticle industry grows. With nanoparticles
entering the environment, knowledge of fate and transportation in ground and surface waters is
essential.
Synthesized iron oxide nanoparticles were mixed with an aquatic fulvic acid and a peat humic
acid at different concentrations (0-25 mg L-l) and pH values (2-10). The suspensions were
analysed by particle size using flow field-flow fractionation (F1FFF), dynamic light scattering
(DLS) and electrophoresis. Primary particle size increased with both increased concentration
of natural organic macromolecules (NOM) and with pH. Particle aggregation occurred at low
pH and was extensive at pH 6 and higher. Some stabilisation with NOM occurred, although
aggregation was found to increase at higher NOM concentrations. Aggregation occurred as
surface charge approached zero, as no stabilising surface active agents (apart from NOM) were
added in this system. NOM were found to form surface coatings on the iron oxide nanoparticles
which were only 1-2 nm in thickness. The influence of pH and NOM concentration will affect
the fate and bioavailability of nanoparticles in the aquatic environment due to these changes in
surface properties, aggregation and subsequent sedimentation.
Introduction
In the past 20 years, there has been a marked increase in the application of nanotechnology
in industry. Nanoparticles consisting of carbon, metal, metal oxides and other compounds,
are defined as having one dimension measuring between 1 and 100 nm. They have unusual
properties that are not observed in the bulk material, including large specific surface areas. At
just a few nanometres, quantum effects begin to dominate affecting optical, conductive and semi-
conductive properties (Alivisatos, 1996, Owen and Depledge, 2005), a characteristic which has
been driving innovation in the electronics industry. Nanoparticles also have rheological, optical
and adhesion properties that make nanoparticles applicable in metallic paints and thin films
(Kendall, et al., 2004). Other applications include clear sun creams (Villalobos-Hernandez and
Muller-Goymann, 2006), self cleaning windows, solar panel technology, car tyres, hydrogen
fuel storage cells and anti-microbial laundry products (Colvin, 2003). The nanotechnology
sector has created a multibillion US dollar market, and is expected to grow to 1 trillion US
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dollars by 2015 (Aitken, et al., 2006). With huge investments and wide applications, release of
products containing nanoparticles to the aquatic environment is inevitable. While there are clear
benefits from the use of this technology, there is little understanding of their environmental fate,
behaviour and ecotoxicology. A few toxicological studies have revealed that some nano-sized
materials are more toxic than their bulk counter parts. Studies involving human and animal
cells, plants and aquatic fauna have found varying degrees of toxicity ranging from irritation
and aggression (Smith, et al., 2007), oxidative stress in fish (Oberdorster, 2004) and, ultimately,
death. Interference and damage to DNAhas been highlighted (Lewinski, et al., 2008).
Iron oxide (FeOx) nanoparticles are used in industry, particularly as pigments, catalysts, medical
devices, sensors, recording media, and thin films (Cornell and Schwertmann, 2003, Jolivet, et al.,
2006, Navrotsky, et al., 2008). Haematite is the most stable of all the nanoparticle iron oxides,
with strong absorption to water (Navrotsky, et al., 2008).
Iron reactive barriers use zero-valent iron (ZVI) as a soil and ground water decontaminant. It
is more effective at the nanoscale due to its greater reactivity and has shown to be an effective
treatment where there is arsenic contamination (Giasuddin, et al., 2007), but rapidly forms an
oxide layer resulting in a similar surface chemistry to the iron oxide (Sohn, et al., 2006). Iron
oxides are more stable than nanoscale ZVI and therefore a more suitable material to study under
laboratory conditions.
Despite the advantages that might be gained from using iron reactive barriers to remediate
ground water, oxygen depletion (Zhang, 2003), an increase in hydrogen gas coupled with an
increase in microbial population (Gu, et al., 2002) have been noted side effects. Moreover,
cytotoxicity of iron oxides (Brunner, et al., 2006, Lewinski, et al., 2008), raises questions
whether these releases of nanoparticle slurries is environmentally ethical.
The presence of humic and fulvic acids, a fraction of NOM, could stabilise nanoparticles in
aquatic systems and increase the rate and distance of transportation in groundwaters and surface
waters (Baalousha, et al., 2008, Chen and Elimelech, 2007, Diegoli, et al., 2008, Hyung, et al.,
2007). By binding to the surfaces of nanoparticles, thereby creating a surface film, humics and
fulvic acids may increase stability of the nanoparticles by charge and steric effects, increasing
residence times in the water column. To assess the impact on aggregation behaviour of metal
oxide nanoparticles of NOM, a number of laboratory experiments and measurements were
conducted by looking at particle size under different pH and humic substance concentrations.
Methods
Experiments were conducted on charge-stabilised iron oxide (haematite) particles (formed by
hydrolysis of iron chloride in dilute HC1 at 100 °C) (Kendall and Kosseva, 2006, Matijevic
and Scheiner, 1978). The resulting particles at pH 2 were used without further modification,
and were mixed with varying concentrations (0-25 mg L"1) of IHSS Suwannee river fulvic
acid (FA) or peat humic acid (PHA), as a representation of NOM. The solutions were then
analysed at different pH values (2-10). Particle sizes of the iron oxides and the iron oxides
plus FA or PHA were determined by flow-field flow fractionation (F1FFF ) (F1000 Universal
Fractionator (Postnova Analytics, Germany) and dynamic light scattering (DLS) (HPPS,
Malvern instruments). F1FFF is a chromatography-like technique which separates particles
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across a 1 kDa membrane using a low salt concentration eluent and field force applied at right
angles to the flow (Schimpf, et al., 2000). Particles are then detected with a UV spectrometer
at 254 nm. DLS, in contrast, uses laser to detect Brownian motion in situ and converts the data
to a Z-average particle size. Both techniques employ the Stokes- Einstein equation to convert
diffusion coefficients into particle diameter. Iron oxide particle were also examined under a
transmission electron microscope (TEM Tecnai F120) and characterised with F1FFF and was
determined to be ca 7 nm ±2 nm. FeOx was used at concentrations of 200 mg L"1. Electrophoretic
mobilities (Zetamaster, Malvern instruments) (charge measurements) were also conducted on all
the solutions and point of zero charge was determined to be at pH 7 for the FeOx, lowered by
the presence of Cl~ ions. Measurements were carried out across the entire pH range, to represent
pH in found in both natural and modified water bodies (such as acid mine drainage and acidified
catchments) and to determine the size and aggregation properties at all pH range
Results and Discussion
Results are in agreement with Cromeries et al (Cromieres, et al., 2002) for hematite and
Baalousha et al (Baalousha, et al., 2008) for FeOx with Suwannee River humic acid. Particle
size increased with increase in pH from pH 2-7 and with an increase in organic matter from
0-25 mg L"1. The particle size maxima from DLS coincided with the point of zero charge of the
iron oxide, both in the presence and absence of NOM. Our previous results have had to invoke
steric hindrance to explain stability of gold nanoparticles (Diegoli, et al., 2008) In this case,
aggregation and stability could be explained purely by charge effects. Increased particle size with
pH and NOM could be explained by: 1) a decrease in charge on the Fe oxide particles allowed
particle growth by coagulation and aggregation, 2) hydrolysis and precipitation of dissolved
iron as the pH rose and 3) by the humics forming a surface layer around the particle and thus
shielding the charge. All three processes may be operative, but measurement of truly dissolved
iron (using ultrafiltration) indicated that only (1) and (3) were important above pH 4.
FFF results at pH 2-6 allowed a greater discrimination between particles and for instance
quantified a surface layer sorbed onto the iron oxide aggregates of ca 1 nm in thickness, which
was responsible for charge (and potential steric) effects.
Conclusions
The addition of the fulvic or the peat humic acid and increase in pH caused an increase in
hydrodynamic diameter of the nanoparticles. The influence of pH and NOM concentration
will affect the fate and bioavailability of nanoparticles in the aquatic environment due to these
changes in surface properties by altering aggregation and subsequent sedimentation. The
increase in size with NOM may indicate that some stability may occur in the water column
allowing some degree of transportation before sedimentation would occur. However, NOM only
slowed aggregation and the production of large aggregates occurred rapidly. Implications for
environmental transport are clear.
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References
Aitken, R. I, M. Q. Chaudhry, A. B. A. Boxall and M. Hull. (2006). "Manufacture and use of
nanomaterials: current status in the UK and global trends." Occup Med (Lond) 56 (5), 300-306.
Alivisatos, A. P. (1996). "Semiconductor clusters, nanocrystals, and quantum dots." Science 271
(5251), 933-937.
Baalousha, M., A. Maniculea, S. Cumberland, J. Lead and K. Kendall. (2008). "Aggregation and
Surface Properties of Iron Oxide Nanoparticles: Influence of pH and Natural Organic Matter."
Environmental Toxicology and Chemistry 9 (29), 1875-1882.
Brunner, T. J., P. Wick, P. Manser, P. Spohn, R. N. Grass, L. K. Limbach, A. Bruinink and W. J.
Stark. (2006). "In Vitro Cytotoxicity of Oxide Nanoparticles: Comparison to Asbestos, Silica,
and the Effect of Particle Solubility." Environmental Science & Technology 40 (14), 4374-4381.
Chen, K. L. and M. Elimelech. (2007). "Influence of humic acid on the aggregation kinetics
of fullerene (C60) nanoparticles in monovalent and divalent electrolyte solutions." Journal of
Colloid and Interface Science 309 (1), 126.
Colvin, V. L. (2003). "The Potential Environmental Impact of Engineered Nanoparticles." Nature
21 (10), 1166-1170.
Cornell, R. M. and U. Schwertmann (2003) "The Iron Oxides; Structure, Reactions, Occurrences
and Uses." Wiley-VCH.
Cromieres, L., V. Moulin, B. Fourest and E. Giffaut. (2002). "Physico-chemical characterization
of the colloidal hematite/water interface: experimentation and modelling." Colloids And Surfaces
A-Physicochemical And Engineering Aspects 202 (1), 101-115.
Diegoli, S., A. L. Manciulea, S. Begum, I. P. Jones, J. R. Lead and J. A. Preece. (2008).
"Interaction between manufactured gold nanoparticles and naturally occurring organic
macromolecules." Science of The Total Environment 402 (1), 51.
Giasuddin, A. B. M., S. R. Kanel and H. Choi. (2007). "Adsorption of Humic Acid onto
Nanoscale Zerovalent Iron and Its Effect on Arsenic Removal." Environ. Sci. Technol. 41 (6),
2022-2027.
Gu, B., D. B. Watson, L. Wu, D. H. Phillips, D. C. White and J. Zhou. (2002). "Microbiological
Characteristics in a Zero-Valent Iron Reactive Barrier." Environmental Monitoring and
Assessment 77 (3), 293.
Hyung, H., J. D. Former, J. B. Hughes and J. H. Kim. (2007). "Natural Organic Matter Stabilizes
Carbon Nanotubes in the Aqueous Phase." Environ. Sci. Technol. 41 (1), 179-184.
Jolivet, J. P., E. Tronc and C. Chaneac. (2006). "Iron oxides: From molecular clusters to solid. A
nice example of chemical versatility." Comptes rendus - Geoscience 338 (6), 488.
Kendall, K. and M. R. Kosseva. (2006). "Nanoparticle aggregation influenced by magnetic
fields." Colloids and Surfaces A: Physicochemical and Engineering Aspects 286 (1-3), 112.
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Kendall, K., C. W. Yong and W. Smith. (2004). "Particle adhesion at the nanoscale." Journal Of
Adhesion 80 (1-2), 21-36.
Lewinski, N., V. Colvin and R. Drezek. (2008). "Cytotoxicity of Nanoparticles." Small 4 (1), 26-
49.
Matijevic, E. and P. Scheiner. (1978). "Ferric Hydrous Oxide Sols.3. Preparation of Uniform
Particles by Hydrolysis of Fe(III)-Chloride, Fe(III)-Nitrate, and Fe(III)-Perchlorate Solutions."
Journal of Colloid and Interface Science 63 (3), 509-524.
Navrotsky, A., L. Mazeina and J. Majzlan. (2008). "Size-Driven Structural and Thermodynamic
Complexity in the Iron Oxides." Science 319 (5870), 1635-1638.
Oberdorster, E. (2004). "Manufactured Nanomaterials (Fullerenes, C60) Induce Oxidative Stress
in the Brain of Juvenile Largemouth Bass." Environmental Health Perspectives 112 (10), 1058-
1062.
Owen, R. and M. Depledge. (2005). "Nanotechnology and the environment: Risks and rewards."
Marine Pollution Bulletin 50 (6), 609-612.
Schimpf, M., K. Caldwell and J. C. Giddings (2000) "Field-Flow Fractionation Handbook."
Wiley -Interscience.
Smith, C. J., B. J. Shaw and R. D. Handy. (2007). "Toxicity of single walled carbon nanotubes
to rainbow trout, (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies, and other
physiological effects." Aquatic Toxicology 82 (2), 94.
Sohn, K., S. W. Kang, S. Ahn, M. Woo and S. K. Yang. (2006). "Fe(0) Nanoparticles for Nitrate
Reduction: Stability, Reactivity, and Transformation." Environ. Sci. Technol. 40 (17), 5514-5519.
Villalobos-Hernandez, J. R. and C. C. Muller-Goymann. (2006). "Sun protection enhancement
of titanium dioxide crystals by the use of carnauba wax nanoparticles: The synergistic interaction
between organic and inorganic sunscreens at nanoscale." International Journal of Pharmaceutics
322(1-2), 161.
Zhang, W.-x. (2003). "Nanoscale Iron Particles for Environmental Remediation: An Overview."
Journal of Nanoparticle Research 5 (3 - 4), 323.
Conference Questions and Answers
Question:
Did you change the pH of all the carrier solutions?
Answer:
Yes. Keeping everything in equilibrium is very important.
Question:
Is the change in aggregation size due to pH reversible? What happens if you change it one way
and then change it back?
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Answer:
It is likely that there will be some hysteresis. The extent of this may be due to the sample
equilibrium, the advancement of the aggregation, and the final pH. Sample preparation may
also have an influence-e.g., how quickly the pH was raised initially, and the order in which the
nanoparticles were mixed with the natural organic matter. Irreversible effects may occur so that,
under some conditions, fully aggregated samples may not disaggregate.
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Surfactive Stabilization of Multi-Walled Carbon Nanotube Dispersions with
Dissolved Humic Substances
Mark. A. Chappell, Environmental Laboratory, Engineering Research & Development Center,
US Army Corps of Engineers, Vicksburg, Mississippi, U.S.A.
Aaron J. George, KaterinaM. Dontsova, and Beth E. Porter, SpecPro, Inc., Huntsville, Alabama,
U.S.A.
Cynthia L. Price, Environmental Laboratory, Engineering Research & Development Center, US
Army Corps of Engineers, Vicksburg, Mississippi, U.S.A.
Pingheng Zhou, and Eizi Morikawa, J. Bennett Johnston Sr. CAMD Louisiana State University,
Baton Rouge, Lousiana, U.S.A.
AlanJ. Kennedy, andJefferyA. Steevens, Environmental Laboratory, Engineering Research &
Development Center, US Army Corps of Engineers, Vicksburg, Mississippi, U.S.A.
Abstract
Soil humic substances (HS) stabilize carbon nanotube (CNT) dispersions, a mechanism we
hypothesized arose from the surfactive nature of HS. Experiments dispersing multi-walled
CNT in solutions of dissolved Aldrich humic acid (HA) or water-extractable Catlin soil
HS demonstrated enhanced stability at 150 and 300 mg L"1 added Aldrich HA and Catlin
HS, respectively, corresponding with decreased CNT mean particle diameter (MPD) and
polydispersivity (PD) of 250 nm and 0.3 for Aldrich HA and 450 nm and 0.35 for Catlin HS.
Analogous trends in MPD and PD were observed with addition of the surfactants Brij 35, Triton
X-405, and SDS, corresponding to surfactant sorption behavior. NEXAFS characterization
showed that Aldrich HA contained highly surfactive domains while Catlin soil possessed a
mostly carbohydrate-based structure. This work demonstrates that the chemical structure of
humic materials in natural waters is directly linked to their surfactive ability to disperse CNT
released into the environment.
Introduction
Research over the past decade has elucidated much about the functionality of CNT and the
many chemical derivatives possible, greatly expanding the potential uses of these materials.
One potential use involves the environmental application of CNT for removing contaminants.
Research was recently conducted in using CNT as a selective sorbent for organic/biological
contaminants in water streams, such as carcogenic cyanobacterial microcystins (Yan et al., 2006),
a variety of nitro- and chloro-substituted aromatics (Thomas, 1994), and methanol (Burghaus
et al., 2007). CNTs also effectively adsorb dissolved heavy metals and actinides, including
Cd(II), Cu(II), Ni(II), Pb(II), Zn(II), and Am(III) (Chen and Wang, 2006; Rao et al., 2007; Wang
et al., 2005). However, little is actually known regarding how CNT will interact with soil-
water systems once released into the environment. The poor water solubility of CNTs (unless
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chemically derivitized) makes it difficult to disperse these materials in aqueous solution. Yet,
CNT was successfully dispersed by the addition of ionic surfactants such as SDS, NaDDBS, and
Dowfax (Vaisman et al., 2006, and references therein). Hyung et al (2007) found that natural
organic matter served to stabilize CNT aqueous suspensions, yet there is no agreement on the
mechanisms by which this behavior occurs. Thus, it is difficult to predict whether some forms of
naturally occurring, biopolymeric substances may promote dispersion, while other may not. For
example, polysaccharides do not apparently promote CNT dispersion (Lead, 2008).
The purpose of this work was to demonstrate the mechanism by which humic materials stabilize
CNT dispersions in aqueous solution. Discerning this mechanism will facilitate a better
understanding of how HS promote CNT dispersion, as well as provide a means for making
qualitative assessments regarding the type of dissolved HS in the environment.
Materials and Methods
Aliquots of dissolved humic stock solutions were added to 50-mL test tubes containing 100
mg L"1 CNT suspension in 5 mM NaNO3 solutions. In separate experiments, dissolved HS
solutions were replaced with varying concentrations of the surfactants Brij 35, Triton X, or
SDS. The tubes were capped and then shaken for 24 hours. Suspension settling was analyzed
using a Varian Carey 50 UV-Vis-NTR spectrometer by reading the absorbance at 600 nm with
time (Mathangwane et al., 2008). Suspension particle size was measured using a Brookhaven
Instruments 90Plus/BI-MAS dynamic light scattering (DLS) spectrometer. Solution total organic
carbon (TOC) was analyzed by a catalytic combustion technique.
Composition of carbon functional group was investigated by near-edge x-ray absorption
spectroscopy (NEXAFS) at the carbon K edge. Measurements were carried out at the varied-
line-space plane-grating-monochromator (VLSPGM) beamline at the J. Bennett Johnston Sr.
Center for Advanced Microstructures and Devices (CAMD) synchrotron light facility, Louisiana
State University. The photon energy scale was calibrated for the C Is-rc* resonance peak using
a polystyrene sample (Sigma-Aldrich) which was fixed at 285.4 eV. Sample spectrum were IQ
normalized using the total yield of clean gold mesh placed in the incident beam before sample.
C-NEXAFS spectra was processed using the program Athena from the IFEFFIT software
package (Newville, 2001). Linear combination fits of the C-NEXAFS spectra were compared to
carbon reference standards also analyzed at VLSPGM beamline.
Results & Discussion
The settling behavior of CNT was studied in the presence of two different HS (Fig. 1). Settling
data showed a rapid reduction in the solution optical density within the first 15 min. Afterwards,
the suspension appeared to stabilize. Settling data showed that CNT suspensions demonstrated
enhanced dispersion stability with Aldrich HA additions beginning at 150 mg L"1 Aldrich HA,
with approx. twice the concentration of dissolved humics required for the Catlin soil HS. Data
from DLS measurements showed that CNT MPD readily dropped to 600 nm with the addition
of 5 mg L"1 Aldrich HA (Fig. 2). Further additions of Aldrich HA up to 150 mg L"1 and Catlin
HS up to 300 mg L"1 resulted in a minimized MPD of approx. 250 and 420 nm, respectively. PD
index also minimized to approx. 0.30 and 0.35 for the Aldrich HA and Catlin HS, respectively,
along with the MPD. Both trends correspond to enhanced dispersion stability and particle size
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9
I
Aldrich HA
— n—0 ppm
—o—5 ppm
- 20 ppm
-T—35 ppm
-50 ppm
- 150 pprn
-r>—300 ppm
1.0-
0.8-
0.4-
0.2-
0.0
Catlin soil HS
60
—n — 5 ppm
—o— 100 ppm
- 300 ppm
-400 ppm
- 500 ppm
- I—600 ppm
20 40
Settling time (min)
60
Figure 1. Settling data showing optical density (A/Ao for X = 600 nm) of a 100 mg L"1 CNT
dispersion, suspended in 5 mM NaNO3 background solution and varying initial concentrations of
dissolved humic substances (obtained from Aldrich humic acid and a Caitlin soil) with time.
homogeneity of CNT - a behavior particular to surfactive molecules.
To test this hypothesis, we conducted similar experiments investigating the effect of
surfactants on CNT suspension particle size characteristics (Fig. 3). The data show that
CNT MPD minimized to 210, 230, and 370 nm for SDS, Brij 35, and Triton X, respectively.
Correspondingly, particle size PD minimized to 0.27, 0.26, and 0.32 for SDS, Brij 35, and Triton
X, respectively. Note that CNT MPD and PD minimized in the presence of SDS and Brij 35
to values similar to the Aldrich HA system, indicating that the Aldrich HA exhibited strong
surfactive ability. Following this reasoning, the surfactive ability of the Catlin soil HS (like the
245
-------
| 2500-
& 2000-
0)
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1 1000-
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o 0 -
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0 50 100 150 200 250 300
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\
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0 100 200 300 400 500 600
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Added Aldrich HA (mg L"1)
Added Catlin HS (mg L1)
Figure 2. Effect of humic substances on the properties of CNT dispersions suspended in 5 mM
NaNO3 background solution. Mean particle diameter and polydispersivity measurements were
obtained by dynamic light scattering. Sorption of humic substances to CNT was calculated by
difference. Connecting lines are to guide the eye.
Triton X) was less capable of stabilizing CNT dispersions.
Minimization of MPD and PD values for CNT was compared to surfactant sorption isotherms
(Fig. 3). All surfactants exhibited a high affinity of sorption for CNT, with individual differences
in the sorption behavior. For Brij 35, minimization of CNT MPD and PD coincided with
the surfactant saturation on the surface. This behavior is consistent with surfactant behavior
in biphasic systems, where surfactant micelles tend to dissociate, and individual surfactant
molecules adsorb to the surface, until the surface is saturated with surfactant (Chappell, 2004;
Chappell et al., 2005). Surfactants tend to reach sorption maximum around its critical micelle
concentration (Chappell et al., 2005, and references therein). Such a trend for the SDS and
Triton X surfactants was more difficult to observe given the unexpected shapes of the sorption
isotherms. However, for SDS, CNT MPD and PD does appear minimized with the first change
in slope (perhaps an intermediate saturation point) of the biphasic sorption isotherm. Triton X
sorption quickly maximized, then became negative, indicating reduction of Triton X surface
coverage on CNT (supported by both TOC and MS measurements), but the relatively large error
246
-------
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VT_I i i
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•
i ' i • i '
5 S 0 200 400 o 2000 4000 0 1000 2000
CO
Equilibrium Surfactant in solution (mg L1)
Figure 3. Effect of surfactants on the properties of CNT dispersions suspended in 5 mM NaNO3
background solution. Mean particle diameter and polydispersivity measurements were obtained
by dynamic light scattering. Surfactant sorption on CNT was calculated by difference. Connecting
lines are to guide the eye.
associated with this data limits this interpretation.
Differences in surfactant sorption (and the resulting CNT MPD) are most likely attributed to
differences in the surfactant's structure. For example, CNT exhibited a much higher sorption
affinity for nonionic surfactants than the anionically charged SDS. However, the combination of
both bulkier hydrophilic and lipophilic moieties of Triton X may have contributed to the poorer
surfactive ability relative to Brij 35. Although anionic, SDS showed similar ability of Brij 35
to minimize CNT MPD. This ability may have been related to the simplicity in structure of the
surfactant's hydrophilic/lipophilic moieties as well.
We investigated the structure of the Aldrich HA and Catlin soil HS using C-edge NEXAFS
(Figure 4) to assess how the above relationships may influence their surfactive ability. Linear
combination analysis of the NEXAFS data (Table 1) revealed that the Aldrich HA possessed a
247
-------
240
270
alginic acid
300
330
D-fructose
LJJ
dextrose
diesel soot
T3
0
N
methylreserpine
o
Aldrich HA
Catlin soil
240
270
300
Photon energy (eV)
330
Figure 4. Carbon-edge NEXAFS for the Aldrich HA and Catlin soil HS compared to reference
standards. Red lines demonstrate the linear combination fit of the spectra from standards to
the Aldrich and Catlin samples. The peaks appearing at approx. 270 eV are due to 2nd order
contribution from oxygen K edge absorption.
248
-------
Table 1. Linear combination fits of the Aldrich HA and Catlin HS carbon-edge
NEXAFS spectra.
sample
Aldrich HA
Catlin soil HS
standard
alginic acid
diesel soot
methyl
reserpine
D-fructose
glucose
weight
0.191
0.179
0.629
0.359
0.641
X
0.18776
1.070372
R-factor
0.001191
0.017806
structure that was highly aromatic: 63 % analogous to an alkaloid reserpine, 18 % analogous to
a black carbon (diesel soot) material, and 19 % analogous to a polymeric polysaccharide (alginic
acid). The Catlin soil HS structure was dominated by simple sugars, consisting of glucose and
D-fructose-type analogs. Clearly, the superior surfactive ability of the Aldrich HA was linked
to the high aromaticity of the black carbon phase (representing the material lipophile), the high
polarity of the polymeric polysaccharide phase (representing the hydrophile), and "mixed"
alkaloid phase containing oxygen-rich aromatic groups. The saccharide polymer-rich Catlin
soil HS exhibited a limited ability to stabilize CNT dispersion because the material lacked a
significant hydrophilic domain necessary for surfactive activity.
Conclusion
In this work, the potential of humic substances to stabilize CNT dispersions was demonstrated.
This behavior was attributed to the surfactive nature of humics and their ability to promote the
smallest CNT particle sizes and homogeneities. As demonstrated with well-defined surfactants,
this stabilization is maximized when CNT is saturated with a monolayer of surfactant, which
corresponds to the sorption maximum of the sorption isotherm and closeness of the equilibrium
surfactant concentration in solution to the CMC value. The superior surfactive ability of the
Aldrich HA appeared to be linked to the mixture of strong hydrophilic and lipophilic domains,
compared to the Catlin soil HS, which appeared to be overwhelmingly hydrophilic. We conclude
from this work that the most natural humic materials should exhibit at least some ability to
stabilize CNT dispersions in aqueous environments.
References
Bohmer, M.R., L.K. Koopal, R. Janssen, E.M. Lee, R.K. Thomas, and A.R. Rennie. 1992.
Adsorption of nonionic surfactants on hydrophilic surfaces. An experimental and theoretical
study on association in the adsorbed layer. Langmuir 8:2228-2239.
Burghaus, U., D. Bye, K. Cosert, J. Goering, A. Guerard, E. Kadossov, E. Lee, Y. Nadoyama, N.
Richter, E. Schaefer, J. Smith, D. Ulness, and B. Wymore. 2007. Methanol adsorption in carbon
nanotubes. Chem. Phys. Lett. 442:344-347.
Chappell, M. A. 2004. Confounding factors and tertiary-phase control by a surfactive agent on
the sorption of atrazine. Ph.D. dissertation, Iowa State University, Ames.
249
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Chappell, M.A., D.A. Laird, M.L. Thompson, and V.P. Evangelou. 2005. Co-Sorption of atrazine
and a lauryl polyoxyethylene oxide nonionic surfactant on smectite. J. Agric. Food Chem.
53:10127-10133.
Chen, C., and X. Wang. 2006. Adsorption of Ni(II) from aqueous solutions using oxidized
multiwall carbon nanotubes. Ind. Eng. Chem. Res. 45:9144-9149.
Hyung, H., J.D. Former, J.B. Hugues, and J.-H. Kim. 2007. Natural organic matter stabilizes
carbon nanotubes in the aqueous phase. Environ. Sci. Technol. 41:179-184.
Lead, J. 2008. Interactions between natural aquatic colloids and manufactured nanoparticles:
Effects on chemistry, transport, and ecotoxicology Nanoparticles in the Environment:
Implications and Applications, Centre Stefano Franscini, Monte Verita, Ascona, Switzerland.
Mathangwane, B.T., M.A. Chappell, J.R.V. Pils, L.S. Sonon, and V.P. Evangelou. 2008.
Dispersion potential of selected Iowa lake sediments as influenced by dissolved and solid-phase
constituents. Clean 36:201-208.
Newville, M. 2001. IFEFFIT: Interactive EXAFS analysis and FEFF fitting. J. Synchotron
Radiat 8:322-324.
Rao, G.P, C. Lu, and F. Su. 2007. Sorpiton of divalent metal ions from aqueous solution by
carbon nanotubes: A review. Separation Purfication Technol. In Press.
Thomas, R.N. 1994. Effects of contaminants and charge transfer on the molar absorptivities of
fullerene solutions. Anal. Chim. Acta 289:57-67.
Vaisman, L., H.D. Wagner, and G. Marom. 2006. The role of surfactants in dispersion of carbon
nanotubes. Adv. Colloid Interface Sci. 128-130:37-46.
Wang, X., C. Chen, W. Hu, A. Ding, D. Xu, and X. Zhou. 2005. Sorption of 243Am(III) to
multiwall carbon nanotubes. Environ. Sci. Technol. 39:2856-2860.
Yan, H., A. Gong, H. He, J. Zhou, Y. Wei, and L. Lv. 2006. Adsorption of microsystins by carbon
nanotubes. Chemosphere 62:142-148.
Conference Questions and Answers
Question:
With humic acids in soils, do you know what will happen with rain or irrigation?
Answer:
Rain or irrigation increases the ionic strength of the solvent and causes the humic acids to swell.
This provides an ideal condition for dispersion of colloids in run off, whether they are engineered
or natural.
Question:
You added very different concentrations of surfactants, for example, 400 milligrams per liter
(mg/L) for Brij-35 (Polyoxyethyleneglycol dodecyl ether) and 4000 milligrams per liter for SDS
250
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(sodium dodecyl sulfate). Why?
Answer:
We wanted to scan above and below the critical micelle concentration (CMC) for each surfactant.
Brij-35 has a much lower CMC than the others and hence required a less concentrated solution.
251
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252
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Evaluation of Nanoparticle and Matrix Characteristics Affecting
Transport in the Environment
Arianne M. Neigh, Thomas K. Darlington, Oanh Nguyen, and Steven J. Oldenburg,
nanoComposix, Inc., San Diego, California, U.S.A.
Abstract
Concern is mounting over the potential for nanomaterials to enter the environment and cause
adverse effects to biota and human health. While in vitro and in vivo toxicity research has
progressed, there is a critical gap in the scientific literature linking release and exposure potential
to the current body of toxicity evaluations on nanomaterials. Few studies have evaluated an
ambient environment release scenario and attempted to determine how nanomaterials interact,
are transported, and may change physically and chemically. Aluminum nanoparticles are being
used in combination with metal oxides in propellants and have the potential to be released to the
environment through aerosol deposition. Although aluminum is abundant naturally in the soil
matrix, aluminum loading can lead to toxicity if it is transported to aquatic systems in soluble
forms. Aluminum chemistry is complicated and the unique characteristics of aluminum at the
nano-scale are not well understood. The objective of the study was to evaluate how aluminum
nanoparticles changed physically and chemically in different environmentally relevant scenarios
and how these changes affect transport. Aluminum nanoparticles were suspended in the different
media by sonication and eluted by a forced up-flow system through the soil matrix over 17 hrs at
a rate of 3 ml/hr. The type of media used to suspend the nanoparticles had a marked effect on the
surface charge, stability, and aggregation state of the nanoparticles prior to introducing to the soil
column. The properties of the suspension at the time of introduction and throughout the course
of the experiment were important in determining transport. Additionally, the properties of the
soil matrix including pore size, charge on the surface of the grains, salt content, and composition
further impacted transport. Suspended aluminum nanoparticles with negatively charged
surfaces had the highest rate of transport of the scenarios evaluated. Transport was also greater
for matrices composed primarily of sand compared to those containing greater proportions
of fine particulates. It is clear from these studies that many factors influence the transport of
nanoparticles in the environment and transport cannot be reliably predicted from one factor
alone, but evaluation should include many different physicochemical aspects of the nanoparticles
and soil.
Introduction
Aluminum nanoparticles are incorporated into energetics, alloys, coatings, incendiary devices,
and sensors by the Department of Defense (DoD) (Air Force Studies Board 2006). Their use in
propellants is increasing because of the high energy release obtainable upon oxidation relative
to micron-sized aluminum (Meda et al. 2007). In order to understand the impact of aluminum
nanoparticles on the environment, the physical and chemical properties of these materials
needs to be correlated to their fate and transport. To date, only studies on the transport of
253
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uncharacterized aluminum nanomaterials has been performed (Doshi et al. 2008). The work
presented here investigates the changes to aluminum nanoparticles that occur in different liquid
media and the effect of these changes on the transport of aluminum nanoparticles through porous
matrices.
Methods
Aluminum nanoparticle suspensions were prepared from powder (nominal 50 nm diameter
aluminum, Novacentrix, Inc., Austin, TX) in different room temperature solutions by probe
sonication (30 pulses for 1 s/cycle at 50% output [W-380 sonicator, CL4 375 watt converter
head, Heat Systems Ultrasonics, Farmingdale, NY]) at a concentration of 1000 mg/L.
Suspensions were then diluted to 50 mg/L in 50 mL conical vials. Aliquots were analyzed using
laser doppler electrophoresis and dynamic light scattering (DLS) with a Malvern Zetasizer Nano
ZS (Worcestershire, UK) DLS over a 168-hr time course.
Powdered particles were fixed on Formvar/carbon-coated copper transmission electron
microscope (TEM) grids 300 mesh size (Ted Pella, Redding, CA) by aspiration in a vacuum
chamber. Imaging was performed on a Jeol 1010 TEM and the diameters of randomly selected
individual particles were quantified with Image J software. Statistical analysis of TEM size was
conducted with SYSTAT statistical software package and Excel.
An automated system delivered nanoparticle suspensions by upflow to a borosilicate glass (6.6
mm bore size) liquid chromatography column (Biochem Valve, Inc., Boontown, NJ) containing
sand or soil packed to a final column density of 1.68 ± 0.01 g/cm3 and 1.60 ± 0.01 g/cm3,
respectively. Aluminum nanoparticle suspensions were delivered to the column at 3 ml/hr for
16.7 hr (50 ml total). Absorbance data from real time Ultraviolet-Visible spectrometry was
collected under temperature controlled conditions (25 ± 2°C) every 100 s.
Results
The arithmetic mean diameter of individual particles as measured by TEM was 48 nm, which
was comparable to the 50 nm nominal size stated by the manufacturer. The particle diameters
are polydisperse with a wide distribution of sizes ranging from 7 to 126 nm. Figure 1 presents
an image of the material in the powdered form and particle size distribution. When the size
distribution is log-transformed to better approximate a normal distribution (Kolmogorov-
Smirnov, n=433, p=0.044), the geometric mean of the sample is 41 nm, 18% less than the
manufacturer quoted size.
Figure 2 presents a comparison of the hydrodynamic diameter of materials in different solutions.
The hydrodynamic diameter of the particles is in all cases is larger (133 to 178% increase in
size) than the measured size of the powdered materials due to extensive agglomeration of the
nanoparticles. The rank order of particle hydrodynamic size immediately upon suspension is:
phosphate treated particles, water, fetal bovine serum (FBS) < phosphate buffer saline (PBS) <
salt solutions (RPMI, very soft reconstituted water [VSRW], moderately hard reconstituted water
[MHRW], and NaCl). In water or reconstituted water solutions, the aluminum has a positive
surface charge; however, in media that contain phosphate, the phosphate binds to the surface of
the aluminum particle and imparts a negative surface charge to the nanoparticle (Figure 2B). In
addition, solutions with higher ionic concentrations reduce the Zeta Potential of the nanoparticle
254
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B)
Figure 1. A) Transmission electron microscope image of powdered aluminum nanoparticles. B)
Particle size distribution for each size class is based on the relative percent of the total particle
diameters measured.
Al 9°° ]
r\j soo •
700 •
"B soo •
f 500 •
•5 400 •
g 300 •
200 •
100 •
III 1 1
III III
'/'/*/*/'/'
1 1
B)
^^^_
^
. NA
• • •
Figure 2. A) Hydrodynamic diameter (nm) by dynamic light scattering (DLS) of nominal 50
nm aluminum nanoparticles in different media. DLS measurements were performed in triplicate
at 25°C with each size measurement being the average of 20 runs. Average of the triplicates is
reported. Hydrodynamic diameter is based on the intensity weighted Z-average as calculated
using a cumulative fit with Malvern Dispersion Technology Software. Water = distilled water
was produced from a MilliQ-Plus filtration unit; PO3 = phosphate; FBS = fetal bovine serum;
PBS = phosphate buffered saline; MHRW = moderately hard reconstituted water by EPA Method
600/4-91/002; and VSRW = very soft reconstituted water by EPA Method 600/4-91/002. B) Zeta
potential by laser doppler electrophoresis of nominal 50 nm aluminum nanoparticles in different
media. Zeta potential measurements were performed in triplicate at 25° C with each size mea-
surement being the average of 10 runs. Average of the triplicates is reported.
255
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that result in larger aggregate sizes in solution.
The agglomerate size of aluminum nanoparticles was dependant on the suspending
fluid. In general, phosphate treated particles had not only the smallest starting diameter, but the
diameter stayed consistent over the study period (Figure 3). Particles suspended in a salt solution
(NaCl) had the largest diameter at the end of the experiment. Time course data is important as a
change in the hydrodynamic size of the particle will affect the settling rate and pore-size related
capture of the nanomaterials in the soil column.
-* -Water
•3.4mM
PO3
•PBS
•ISOmM
NaCl
•=• 1000 +
g
' 800 +
a
600
12 24 48 72 168
Hours
Figure 3. Hydrodynamic diameter (nm) by dynamic light scattering (DLS) of nominal 50 nm
aluminum nanoparticles in different solutions over time. Water = distilled water; PO3 = phos-
phate; PBS = phosphate buffered saline.
When three different suspensions of differing properties were eluted through a sand
column, phosphate treated particles had the greatest transportability compared to particles in
water and those in a MHRW (Figure 4). Phosphate treated particles had >95% transportability
and continued unimpeded through the column throughout the test. Particles in MHRW had less
than 6% of the starting material transported through the column. Particles suspended in water
had intermediate transportability; however, transport was delayed until approximately 27 pore
volumes. Phosphate treated particles were also eluted through soil as a comparison of the effect
of soil type on transport. Transportability was approximately 71% at breakthrough compared to
95% at breakthrough for the sand column.
Discussion
The size of nanoparticles relative to their bulk formulations have been implicated as a
potential cause of toxicity (Colvin 2003, Hoet et al. 2004, Warheit 2004, Nel et al. 2006);
therefore, describing size accurately of primary importance. However, for the transport of
nanomaterials through porous media, it is important to differentiate between the primary size
of the nanoparticles and the agglomerate size in the solution media. The vast majority of
dry nanopowders are highly agglomerated in suspension. For the aluminum nanopowders
investigated, the agglomerate size of the suspended material was over 133% larger than the
256
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1.2 T
10 20 30 40
Pore Volumes
50
— — —Water-Sand
MHRW-Sand
-3.4mM
Phosphate-
Sand
3.4 mM
Phosphate-
Soil
60
Figure 4. Transport of nominal 50 nm aluminum nanoparticles suspended in different solutions
through a sand and soil column over time. Pore volumes (ml) were calculated as the difference
between the total column volume and matrix volume and elution through the column was mea-
sured as the change in absorbance at 700 nm compared to the starting material (C/CO). Water =
distilled water; MHRW = moderately hard reconstituted water.
primary size. The phenomenon of agglomeration is a critically important factor in nanomaterials
research as it can change how the particles act in suspension. The formation of agglomerates
will increase settling rates in suspension (Brant et al. 2007) and may impact deposition on solid
surfaces from a colloidal suspension (Elimelech and O'Melia 1990). Since agglomerate size
is dependent on the suspension media, monitoring the size and charge as a function of time is
important. Additionally, the surface of the nanoparticle can be altered by the dispersion media.
For aluminum nanoparticles, the surface charge is made negative when phosphate containing
media is utilized. In water, the aluminum nanoparticles carry a positive charge and bind to the
sand column for 27 pore volumes until the sand is saturated with positively charged aluminum
after which transport occurs. For aluminum nanoparticles exposed to phosphate the surface
charge is negative and the nanoparticles have a much higher initial transport rate.
Conclusions
This study demonstrates that understanding the properties of nanomaterials is important in
predicting transport and that aluminum nanomaterials may be transported under certain but
environmentally relevant conditions. In areas where high phosphate levels are present in surface
waters, aluminum nanoparticles entering the system may be relatively small and stable, which
may facilitate transport. Other organic materials found in surface water such as tannic acids may
produce the similar results. However, if soils or surface water contain salts, the nanoparticles
have high site fidelity, or in the case of surface waters settle quickly, due to agglomeration. It is
therefore important to consider how other chemical components of the environment may interact
257
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with nanomaterials. Only by understanding the characteristics of nanomaterial and how those
characteristics change over time will researchers be able to develop meaningful models for fate
and transport.
References
Air Force Studies Board. (2006). "A Review of United States Air Force and Department of
Defense Aerospace Propulsion Needs." Committee on Air Force and Department of Defense
Aerospace Propulsion Needs, Division of Engineering and Physical Sciences, National Research
Council of the National Academies, Washington, D.C.: The National Academies Press.
Meda, L., Marra, G., Galfetti, L., Severini, F., and L. De Luca. (2007). "Nano-aluminum as
energetic material for rocket propellants." Mater. Sci. Eng. C 27, 1393-1396.
Doshi, R., Braida, W., Christodoulatos, C., Wazne, M., and G. O'Conner. (2008). "Nano-
aluminum: Transport through sand columns and environmental effects on plants and soil
communities." Environ. Res. 106, 296-303.
L.V. Colvin. (2003). "The potential environmental impact of engineered nanomaterials." Nat.
Biotechnol. 21, 1166-1170. Erratum in: Nat. Biotechnol. 22, 760.
Hoet, P.H.M., Briiske-Hohlfeld, I, and O.V. Salata. (2004). "Nanoparticles - known and
unknown health risks." Journal of Nanobiotechnology 2, 12.
D.B. Warheit. (2004). "Nanoparticles: health impacts?" Mater. Today 7, 32-35.
Nel, A., Xia, T., Madler, L., and N. Li. (2006). "Toxic potential of materials at the nanolevel."
Science 311, 622-627.
Brant, I, Labille, 1, Bottero, J.Y., and M.R. Wiesner. (2007). "Nanoparticle transport,
aggregation, and deposition." in Environmental Nanotechnology, M.R. Wiesner & J. Y. Bottero,
New York, NY: McGraw Hill.
Elimelech, M. and C.R. O'Melia. (1990). "Effect of particle size on collision efficiency in the
position of Brownian particles with electrostatic energy barriers." Langmuir 6, 1153-1163
Conference Questions and Answers
Question:
What were the dimensions of your column?
Answer:
It was a 6.6 micron bore, basically a chromatography column.
Question:
Did you look at the wall effect? The literature indicates that small diameter columns may affect
the movement of the particles.
258
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Answer:
I do not think there was a problem with wall effect in this experiment. The column setup is
commonly used at Rice University.
Question:
Did you look at the particle size distribution as particles came out of the column?
Answer:
Yes.
Question:
Did you monitor the pH? What was the column flow rate? Did you notice any generation of
hydrogen? Were there any changes?
Answer:
The flow rate was approximately 3 milliliters per hour. We did monitor the pH and did see some
hydrogen production.
259
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260
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Transport and Reactivity of Lactate-Modified Nanoscale Iron Particles in
PCP-Contaminated Field Sand
Krishna R. Reddy, Amid P. Khodadoust and Kenneth Darko-Kagya, University of Illinois at
Chicago, Department of Civil and Materials Engineering, Chicago, Illinois, U.S.A.
Abstract
In this study, the transport and reactivity of nanoscale iron particles (NIP) was investigated in
horizontal column experiments using field sand contaminated with pentachlorophenol (PCP).
Bare NIP and modified NIP with 10% aluminum lactate were investigated at two different slurry
concentrations of 1 g/L and 4 g/L. Lactate was found to prevent or slow agglomeration and
settlement of NIP. NIP slurry was introduced at the inlet of the soil column under a constant
hydraulic gradient. Visual observations revealed that the distribution of NIP was uniform in the
4 g/L modified-NIP experiment compared to all other experiments. Hydraulic conductivity of the
soil was measured during the course of each experiment and it remained approximately the same
in all the experiments except it reduced in the experiment with bare NIP at 4 g/L concentration.
Transport of NIP in experiments with bare NIP was not uniform and most of the PCP degradation
occurred near the inlet where NIP could be transported during the initial stages of testing. The
transport of NIP is enhanced by lactate, but the reactivity of NIP with PCP was decreased as
compared to the bare NIP experiments. Degradation and the removal of the PCP were found
higher (61.2% and 9.7%, respectively) for the 1 g/L lactate-modified NIP; while the degradation
and removal were lower (51.6% and 6.4%, respectively) for the 4 g/L lactate-modified NIP.
Overall, the results showed that 4 g/L lactate-modified NIP favors relatively uniform distribution
of NIP in the soil, but the extent of PCP reduction is lowered by the surface modification. Further
research is being performed to optimize the lactate-modified NIP that provides both efficient
delivery as well as enhanced reduction of PCP in the soil.
Introduction
Nanoscale iron particles (NIP) have the potential to effectively treat PCP contaminated soils
(Reddy and Karri, 2008). Despite the good reactivity, mobility of NIP in soils becomes restricted
due to their aggregation and settlement. Delivery of NIP uniformly in required amounts is
essential for successful in-situ remediation of soils. Schrick et al. (2004) revealed that the
transport of NIP through most environmental media such as soil is difficult or not possible if
the surface of iron particles is not modified. If the NIPs are modified with poly electrolytes and
polymers, their mobility increases through media such as soils (Saleh et al., 2007).
The objective of this study was to determine the transport of NIP and consequent PCP
degradation in a natural sand. Two column experiments were conducted on PCP-contaminated
sand using bare NIP slurry at two different concentrations (1 g/L and 4 g/L). Additional two
column experiments were conducted with the same NIP slurry concentrations, but modified with
10% (NIP w/w) aluminum lactate to investigate enhanced transport and corresponding effects on
degradation of PCP.
261
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Materials and Methods
NIP used in this study was obtained from Toda Kogyo (Japan). The particles had an average
diameter of 70 nm (with a range of 50-300 nm), pH of 10.7, and BET surface area of 37.1
m2/g. Natural sand was used for this study and PCP was used to spike the sand at an initial
target concentration of 100 mg/kg. PCP was chosen as the contaminant due to its toxicity and
presence at numerous sites, including the Superfund sites. 98% purity PCP was obtained from
Aldrich Company, CAS 87-86-5. The aluminum lactate used for surface modification of NIP
was obtained from Aldrich CAS-18917-91-4. Electrolyte was used to simulate groundwater
conditions. The electrolyte contained 0.006 M of sodium bicarbonate, 0.002 M of calcium
chloride and 0.002 M of magnesium chloride. The pH, total dissolved solids and electrical
conductivity of the electrolyte solution were 7.76, 500 mg/L and 1020 uS/cm, respectively.
For spiking of the soil, about 600 mL hexane was used to dissolve 100 mg of solid PCP. To
ensure all the PCP solids are dissolved, the PCP-hexane mixture was mixed on a magnetic stirrer
for about 45 minutes. Approximately one kilogram sand was weighed in a large glass beaker.
The PCP-hexane solution was added to the soil in the beaker and mixed well with a stainless
steel spoon continuously for about 30 minutes to ensure the PCP is distributed uniformly. The
soil-hexane-PCP mixture was placed in a ventilation hood nearly seven days for the mixture to
dry. During the drying period, the soil was mixed regularly to ensure uniform spiking and drying.
A horizontal column was used for this study. The column had an inside diameter of 3.81 cm and
a length of 14 cm. The column was made of Plexiglas. One end of the column was connected to
a reservoir made of Plexiglas with an inside diameter of 2 cm using a Tygon tube. The height of
the reservoir could be adjusted to apply desired constant hydraulic head conditions. Two different
concentrations (1 and 4 g/L) of bare NIP slurries were prepared using electrolyte and additional
two slurries were prepared with 1 and 4 g/L NIP containing 10% aluminum lactate (w/w NIP).
The spiked sand was placed in the cell in uniform layers and compacted using a tamper to ensure
uniform density. The initial hydraulic conductivity was calculated by measuring the outflow
volume in a given specified time interval under a constant hydraulic gradient. The electrolyte in
the reservoir was replaced with the selected NIP slurry and allowed to flush through the sand.
The effluent samples were collected in 120 mL bottles (i.e. approximately every 3 pore volumes)
for analysis. At the end of each experiment, the soil was extruded from the column and sectioned
into four parts. Soil sample from each section was visually observed and photographed, and
the pH, and iron and PCP concentrations were measured. The aqueous effluent samples were
analyzed for pH, total dissolved solids, electrical conductivity, and iron and PCP concentrations.
Results and Discussion
Figure 1 shows the residual PCP distribution within the soil at the end of testing. It can be
observed that the amount of PCP remaining in the soil of 4 g/L NIP with lactate experiment
was uniformly distributed, but at higher concentration levels as compared to the other three
experiments. This indicates that the presence of lactate provides uniform delivery of the NIP,
but reduces reactivity. In the experiments with bare NIP as well as with 1 g/L NIP with lactate,
PCP concentrations increased from the inlet to the outlet. This indicates that the NIP was not
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uniformly distributed. The presence of higher amount of NIP near the inlet caused higher
reduction of PCP. The presence of lactate at low concentration of 1 g/L NIP contributed slightly
better transport than that found in bare NIP tests, but the presence of small amount of lactate
slightly reduced reactivity of NIP.
The amount of PCP found in the effluent is shown in Figure 2 and it demonstrates that the
removal of PCP in the bare NIP experiments is approximately similar, while a decrease in
removal of PCP is observed in the experiments with lactate. The presence of higher lactate has
led to lower PCP removal. Based on the mass balance analysis, the amount of PCP reduced due
to the presence of NIP is shown in Figure 3. These results show that maximum PCP reduction
is observed in the experiment with 1 g/L NIP with lactate. This demonstrates that the presence
of low amount of lactate caused slightly enhanced transport of NIP through the soil without
significantly reducing the reactivity. The presence of higher amount of lactate caused enhanced
transport, but reduced the reactivity, thus causing the lower reduction of PCP. The bare NIP at
both concentrations was effective in reducing the PCP, but the transport of the NIP was limited
and most of the reduction occurred near the inlet region of the soil before the NIP agglomerated
and settled in the soil.
The outflow was carefully monitored during each experiment in order to determine changes in
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the hydraulic conductivity of sand as NIP is transported through the soil. As shown in Figure 4,
hydraulic conductivity was not impacted in the experiment with 4 g/L NIP with lactate; however,
it slightly reduced after 15 pore volumes in the experiments with 1 g/L NIP with or without
lactate and it reduced significantly in the experiment with 4 g/L bare NIP. Therefore, the results
suggest that higher amounts of bare NIP has potential to clog the pores and reduce the hydraulic
conductivity, while low concentration of NIP may not have significant affect on hydraulic
conductivity of sand. The use of lactate modified NIP prevents clogging and maintains the
hydraulic conductivity of the sand. This study shows that lactate enhances the transport of NIP in
the soil, but optimization of lactate and NIP concentrations is essential in order to achieve both
adequate transport as well as high reactivity of NIP.
Conclusions
The objective of this study was to determine the transport and reactivity of bare and lactate-
modified NIP for the remediation of PCP in field sand. Hydraulic conductivity was unaffected in
the 4g/LNIP with lactate experiment; however, it slightly reduced after 15 pore volumes of flow
and reduced in bare NIP concentration. Significant decrease in hydraulic conductivity was found
in the experiment with 4 g/L bare NIP. Degradation and the removal of the PCP were found
higher (61.2% and 9.7%, respectively) for the 1 g/L lactate-modified NIP; while the degradation
and removal were lower (51.6% and 6.4%, respectively) for the 4 g/L lactate-modified NIP.
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Overall, this study shows that lactate-modified NIP can be effective for in-situ remediation.
Acknowledgements
This project was funded by the U.S. National Science Foundation Grant CMMI #0727569 and
the support of this agency is gratefully acknowledged.
References
Reddy, K.R., and Karri, M.R. (2008). "Removal and Degradation of Pentachlorophenol
in Clayey Soil Using Nanoscale Iron Particles." Geotechnics of Waste Management and
Remediation, Geotechnical Special Publication No. 177, ASCE Press, Reston, Virginia, 2008,
463-469.
Saleh, N., K. Sark, Y. Liu, T. Phenrat, B. Dufour, K. Matyjaszewski, R.D. Tilton, and G.V. Lowry
(2007). ' Surface Modifications Enhance Nanoiron Transport and NAPL Targeting in Saturated
Porous Media.' Environ. Engr. Sci., 24(1), 45-57.
Schrick, B., B. Hydutsky, J. Blough, and T. Mallouk (2004). "Delivery Vehicles for Zerovalent
Metal Nanoparticles in Soil and Groundwater." Chemistry of Materials 16, 2187-2193.
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Figure 4. Effect of NIP transport on permeability of the soil
Conference Questions and Answers
Question:
Did you measure the concentrations of the iron in the influent and the effluent? Did you use the
same method?
Answer:
We measured the effluent iron concentrations but not the influent.
Question:
You coated the nano-iron with lactate and this improved its ability to stay in suspension. Did you
measure the activity of the coated iron?
Answer:
Yes. That was what the reactivity experiment did, and it showed that coated iron was less
reactive than uncoated iron. As time passes the lactate degrades, and the iron reactivity increases.
The coating does not affect the iron's innate activity.
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Question:
In the permeability experiment, why does the 1-gram-per-liter solution initially exhibit less
permeability than the 4- gram-per-liter solution?
Answer:
This is due to the initial packing of the cell.
Question:
Could you comment on the batch study for removal of pentachlorophenol (PCP) by bare iron and
coated iron?
Answer:
The study showed that the bare iron removed more PCP than the coated iron, and the higher
concentration bare iron (4 grams per liter) performed much better than the higher concentration
coated iron.
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Chapter 8
Report Backs and Panel Discussion: Implications
Session Report Backs
Fate & Transport
Reported by Michele Conlon, U.S. EPA Office of Research and Development
Dr. Conlon reported that the underlying goal of fate and transport research is to understand
nanoparticle interactions with their environment. One theme that came from the fate and
transport researchers was the need to verify that the material received is in fact what was ordered.
In many illustrations, a manufacturer specified nanoparticles to be within a given diameter
range when, in fact, they were not, or the particles were heterogeneous in shape and size due to
manufacturing procedures when they were supposed to be uniform.
All of the characterization and detection experiments reported on were in laboratory settings.
While the findings from these studies are important, they may not reflect what can be
achieved in a natural setting or predict the behavior of nanoparticles in that environment. The
characterization of nanomaterials is key to fate and transport. One cannot determine migration
unless the material found at point b can be identified as the same material found at point a.
As would be expected, the physical, chemical, and biological environmental context can strongly
affect the fate and transport of nanoparticles. Nanoparticle aging and environmental factors such
as water presence, ionic strength, and pH; humic and fulvic acid presence and concentrations;
sunlight presence, quality, and intensity; and microbe presence and types, all can influence
nanoparticle transformation and migration. Promotion or retardation of nanoparticle aggregation
is one of many important mechanisms by which environmental factors can influence nanoparticle
fate and transport. Analyzing the risks of nanoparticles in the environment are further
complicated by chemical transformations of nanoparticles in the environment, which can greatly
affect their bioreactivity and toxicity.
The laboratory studies have identified many challenges to understanding underlying principles
in nanoparticle fate and transport that will only increase when the studies are taken into the field.
This emphasizes the need for working together and sharing insights. The effort has to be a multi-
disciplinary one. We need to develop predictive tools and models, which we do not have now.
Mitigation of risk is not possible if the fate and transport of a material cannot be determined.
Toxicity & Risk Assessment
Reported by Barbara Walton, U.S. EPA Office of Research and Development
Dr. Walton reminded the audience that at the start of the meeting they had been challenged by Dr.
George Gray to "characterize and collaborate." Forty-four
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organizations were represented in the 23 platform presentations and nine posters related to
toxicity, which clearly showed inter-organizational collaboration. In addition, the
plenary session speakers posed challenges for nanomaterial toxicity. Jeff Morris asked what
properties and characteristics of nanomaterials contribute to toxicity, and questioned whether
toxic doses of nanomaterials are environmentally relevant. The numerous gaps in the knowledge
about nanomaterial toxicity to the health of human and ecological receptors were presented
by Dr. Anne Fairbrother. Dr. Jo Ann Shatkin discussed what constitutes realistic exposures
to nanomaterials, the need for cross-disciplinary collaboration, sources of uncertainty in risk
assessment, and the issues for risk assessment in an environment that is not highly regulated. Dr.
Martin Philbert called for adherence to the fundamental principles of rigorous scholarship and
scientific rigor.
Some overarching concerns emerged from the toxicity presentations and posters at the
conference. A heavy emphasis was placed on the characterization of nanomaterials, and on
life-cycle assessment of potential exposure. The relevance of the currently used test species
and endpoints to existing test guidelines for human health and ecological receptors also was
emphasized.
Many nanomaterials, including nanosilver, gold nanoparticles, a wide range of metals and metal
oxides, quantum dots, and the carbon-based nanomaterials such as the fullerenes and carbon
nanotubes, were evaluated in the presentations. The presentations indicated that ecotoxicological
risk is being assessed using a wide variety of test species, including micro-organisms, fish,
mollusks, macro-invertebrates, and plants. In vitro test systems discussed included mouse renal
and liver cells, rat liver cells and human lung epithelial cells, and dermal fibroblasts. Twenty
variables affecting toxicity and 28 endpoints were examined. Careful qualifications were given to
the findings of the toxicological investigations; for examples, that multi-walled carbon nanotubes
do not appear to be lethal to fish but are associated with histopathology of the gill, and that the
toxicity of nanosilver is dependant on size and related to reactive oxygen species. The presenters
were mindful of what is known and unknown, and were rigorous in their approach.
Dr. Walton distilled four nanotoxicity principles from the presentations:
Toxicity is often associated with reactive oxygen species.
• Characterization of nanomaterials under investigation is essential for data interpretation.
• Aggregation of nanoparticles typically reduces toxicity.
• Aggregation and agglomeration are dynamic processes, so dissociation will occur over time,
changing the initial associated toxicity.
Areas of concern noted by Dr. Walton:
• Nanomaterials - too many, too fast and too complex for conventional approaches.
• Material data safety sheets do not adequately communicate the hazards of nanomaterials to
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workers - hazard communication must be improved.
How to balance risks and benefits in hazard communication.
Environmentally relevant concentrations and metrics for dose response are unknown.
The validity of cross-species extrapolation (fish to mammal) is questionable for nanomaterial
toxicity.
Current studies and data are not standardized and thus are frequently incompatible.
Finally, broad areas were identified during the toxicity and risk assessment sessions, where
improvements can and should be made in approaches to nanotoxicology. For example, in the
absence of information, best practices including an honest appraisal of what is known and
unknown should be employed to manage potential hazards. Nanomaterials need to be better
characterized under a variety of conditions, in wet and dry states, in vitro, in vivo, and ex
vivo. Nanomaterial characterization should be hypothesis-driven. It was recommended that
toxicity assessment should be incorporated early in the research and development process of
nanomaterial applications in order to prevent a potentially costly rework at the end of a project.
Finally, in the absence of complete data, tools to enable decision-making are needed; these may
include expert judgment and ad hoc processes.
Panel Discussion
Moderator:
Charles Maurice
Panelists:
Michele Conlon, Steve Diamond, Mark Johnson,
Jamie Lead, Martin Philbert, Barbara Walton
Charles Maurice: I will ask the same question that my colleague Dr. Layne asked the previous
panel. Describe an important finding from your session and how you feel it has been affected by
discussions amongst the international audience here.
Michele Conlon: Although laboratory methods are available to detect nanomaterials, there are
no field portable/transportable instruments that can be employed. We need to detect them in
the environment, and will need another approach using a secondary indicator. Biota (daphnia,
fairy shrimp) can be collected and analyzed for nanoparticles. Results can be inferred back to a
location. There is a need for research and collaboration to find engineered nanoparticles in the
environment. At present they cannot be distinguished from natural materials.
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Jamie Lead: This area is relatively new, 5 or 6 years old, and there are many research needs.
There is a need for validated methods to quantify and determine their physico-chemical form to
facilitate further understanding nanomaterials in the environment. We do not have information
on the current situation, and modeling gives us the best guess available. We need to look to
the future and try to see which nanomaterials will be important over the next 5-10 years and
materials that will be more active than the passive materials, such as titanium dioxide, currently
available.
Stephen Diamond: Dr. Philbert said that current data will be of little or no use, and this may be
true in some cases. Why did he say this? There is an analogy with PCBs. When concern about
PCBs first arose, we were looking at them at the Aroclor level. Much later we realized that we
needed to understand what constitutes an Aroclor and the
concentrations of those constituents, so the early data were not useful. We need this vision
for nanomaterials. The sessions were representative of a highly scatter-gun approach to the
understanding of nanomaterials in the environment and nanotoxicity as a whole. Experimental
material selection ranged from those prepared in small academic laboratories, to those produced
by specialist laboratories making high quality products for research, to manufacturers producing
them in ton quantities. This makes it hard to generalize across materials and results, and
generalizations may not support risk assessment. Efforts to characterize nanomaterials face
difficulties. There is a growing awareness of the need to characterize nanomaterials, yet media
preparation is again a scatter-gun approach. There is an immense variety of preparation methods
and characterization in specific media. Food chain transfer is a gap in understanding. Tissue
uptake is probably limited, but guts of daphnia are full of C60 and metal nanoparticles. This
enables them to act as vectors for the sediment accumulation of nanomaterials. If information
was available from the producers of nanomaterials about what is being bought, used, or
amended, we would have a better understanding.
Mark Johnson: Jo Ann Shatkin gave an excellent overview of the challenges we face, and
endorsed the retention of the basic risk assessment framework for exposure to chemicals. It
may be futile to do this in most respects, due to the rapid evolution of the field and complexity
of the materials. In the face of such uncertainty it is daunting to translate and communicate
technical information to the public. Dr. Linkov's suggested characterization approach may be one
way to determine hazards semi-quantitatively and divide materials into low, medium and high
hazards. Manufacturers may use information to decide against the development of a material.
Worker exposure could also be appropriately managed. Christie Saye's approach could be used
to predict toxicity for industry and the public. This approach utilizes the physical properties of
nanomaterials and gives a score to a particular attribute (e.g., charge, mass, reactive oxygen
species, or concentration) to derive a composite score indicative of toxicity.
Martin Philbert: We are at the gateway of a new and potentially disruptive technology. We
have seen the first applications, but there are block-buster applications in development. There
is a need to think critically now. Nanotechnology will be applied to many pressing social
and environmental problems, but we are at the stage where the azo dye prontosil rubrum
was in World War II. This drug saved lives but was replaced by penicillin. Penicillin was in
turn replaced by safer and more useful derivatives, and by newer classes of antibiotics. The
point is that we do the best we can, but keep a vigilant eye towards re-inventing. Another
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underlying assumption is that the United States would be the world leader in the production
of nanomaterials. We should be challenged, however, by the idea that the major producers of
nanomaterials will be countries with lax regulatory frameworks, and we will be importing these
materials. There is a need for detection methods for these materials.
Question:
In addition to conducting research, I work with ASTM (American Society for Testing and
Materials) and ISO committees, and we need to recommend good practices for these materials
and communicate best practices to the community. How do you envision this?
Diamond:
I served on an OECD working party for manufactured nanomaterials, doing extensive reviews of
existing test guidelines used for regulatory purposes. Existing biotic effects test guidelines are
framed in terms of concentration and solutions, for example, and do not work for nanomaterials.
A guideline is in preparation for how to prepare a nanomaterial for one aspect of characterization,
particle size. Rather than using a prescriptive approach, a range of parameters for any aspect of
nanomaterial preparation should be devised, and results should be required to be reported. This
is no real answer, but much attention is being given to this by working parties associated with the
OECD.
Question:
Are state, local and national/international government doing enough to protect worker and public
health and the environment from nanomaterials?
Philbert:
Perhaps, but there are not enough data. Some nanotoxicity is going to take a long time to
develop. The current testing paradigm, loading a rat for two years, may not reflect what happens
with low level exposures over the long haul.
Johnson:
We are always exposed to naturally occurring nanoparticles. It is difficult to know if we are
doing enough if we do not know what is in the environment. Agnes Kane's work has shown
similarity of nanotubes to asbestos fibers. This is an important analogy as there are similar
mechanisms of action, but there are many aspects of these materials that we are just beginning to
understand.
Question:
Is it safe to use a precautionary principle, i.e., to list all nanomaterials as hazardous until they are
considered or known to be safe, especially on the MSDS data sheets?
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Philbert:
"Do not deploy until safe" is not a realistic position. There are countries that do not respect
intellectual property, but are going ahead with the technology and manufacturing nanomaterials.
Question:
Considering the current use of sunscreen, should it be labeled to show that it contains
nanoparticles?
Philbert:
Sunscreen is needed by the fair-skinned, as exposure to sun can cause skin cancers.
Comment:
Nanomaterials should be listed to give the user a choice.
Conlon:
From the regulatory perspective, EPA has authority through statutes (Clean Water Act, Resource
Conservation and Recovery Act), but does not have the authority to label materials as hazardous
without evidence. We cannot stifle innovation and useful products in production.
Comment:
The EPA is not the only regulatory agency.
Question:
We have seen a lot of researchers, regulatory agencies, and universities at this meeting, but no
speakers from industry are here. What are they using for best management practices to protect
their workers?
Answer:
When we were advertising this conference, we tried to advertise in as many venues as possible;
nobody was excluded.
Comment:
Two keynote speakers from the private sector cancelled, but for good reasons. Another thing to
consider is that when companies develop products they are very reluctant to disclose what might
be proprietary information. We have to understand the commercial side and the implications of a
competitive market.
Question:
Is EPA collecting information from industry?
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Answer:
EPA's Nanoscale Materials Stewardship Program is voluntary and is intended for the early
collection of data. The program is about a year old. You can find a description of it on the OPPT
website at http://www.epa.gov/oppt/nano/stewardship.htm.
Diamond:
For the OECD working party, industry was invited to participate in working/steering groups.
Some companies are generous with their participation, information, time, and, in the case of
Evonik Industries (formerly Degussa), with providing titanium dioxide for research.
Question:
Many nanotechnology companies are in the start-up phase. Many are very small and do not have
deep pockets for nanotoxicology. What is being done on the national government level to help
them?
Answer:
There are federal dollars for small business support, which includes support for research and
development. EPA's Office of Research and Development also offers Cooperative Research and
Development Agreements (CRADAs) that support collaboration between EPA and non-federal
organizations. This is a good program for all small businesses concerned about health effects on
their workers and waste streams that are emitted, and that are willing to collaborate.
Closing Comments
Charles Maurice
This conference has drawn 185 registrants from five continents and speakers from government,
the private sector and non-government organizations (NGOs). The fundamental goal has been to
get applications and implications people together, because a holistic, multidisciplinary approach
is needed to this new area. We wish to thank the University of Illinois at Chicago for registration
and catering this event. Credit is also given to the Hyatt Regency. Proceedings from the
conference will be published and PDF-format files will be posted on EPA websites. Participants
will be notified of the link. Thank you all for participating.
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Presenter Biographies
Dr. Linda Abriola is a Professor of Civil and Environmental Engineering and Dean of the School of
Engineering at Tufts University, and is a member of the National Academy of Engineering. Dr. Abriola
is a leading researcher in the area of flow and transport in porous media, and has developed several
numerical models to describe the contaminant fate and transport in heterogeneous soils and aquifer
materials.
Diana S. Aga is an Associate Professor at the Chemistry Department of the University at Buffalo, The
State University of New York. She obtained her degree in B.S. Agricultural Chemistry at the University
of the Philippines at Los Banos, Philippines, and her Ph.D. degree in Analytical and Environmental
Chemistry at the University of Kansas, Lawrence, KS. Dr. Aga was a postdoctoral fellow at the Swiss
Federal Institute of Science and Technology (ETH)/ Institute for Environmental Science and Technology
(EAWAG), Switzerland, and at the U.S. Geological Survey, Water Resources Division, in Lawrence,
KS. Recently, she received a research fellowship from the Alexander von Humboldt Foundation to
conduct research at the Bundesanstalt fur Materialforschung und -priifung, Berlin, Germany. Her research
interests include investigation of the fate and transport of contaminants in the environment, such as
persistent organic pollutants, pesticides, pharmaceuticals, and engineered nanomaterials.
Dr. Souhail Al-Abed is a Research Chemist at the National Risk Management Research Laboratory
of the U.S. Environmental Protection Agency in Cincinnati Ohio. His research activities include using
electrochemical methods and bimetallic nano-materials in the remediation of contaminated soils and
sediments, removal of heavy metals from aqueous waste streams, and development of methodical
leach tests for waste evaluation. His research contributed to the understanding of many challenging
environmental problems and developing cleanup strategies based on sound science. He authored and
coauthored more than 42 peer-review journal articles and five book chapters. He is member of the
American Chemical Society and the American Geophysical Union and serves in many national and
international research committees.
Clare Allocca is the Chief of The United States Measurement System (USMS) Office at NIST This
office is building upon a NIST-led assessment of the state of the USMS to transform the approaches
taken into an increasingly more effective and efficient USMS. Her responsibilities include leadership,
strategic planning, customer engagement, process development and program implementation. Ms.
Allocca previously served as Senior Scientific Advisor to the Director of the NIST Materials Science
and Engineering Laboratory; Senior Technical Advisor for the Automotive Sector in the NIST Industrial
Liaison Office; Program Analyst in the NIST Program Office, as advisory staff to the NIST Director; and
Program Manager for Materials in the Advanced Technology Program (ATP). Before joining NIST, she
was a Senior Materials Engineer for Pratt & Whitney engaged in the development of advanced ceramic
composites for jet engines. Ms. Allocca holds Bachelor of Science Degrees in Materials Science and
Engineering and Geochemistry from the Massachusetts Institute of Technology; a Master of Science
Degree in Ceramic Engineering from the University of Illinois at Urbana-Champaign; and an Executive
Master of Science Degree in the Management of Technology from the University of Pennsylvania
(Wharton Business School/School of Engineering).
Alia L. Alpatova received a M.Sc. degree (2004) in environmental diagnosis from Imperial College,
University of London, UK. After graduation, he worked for Anglian Water, UK, where he was responsible
for coordination of plumbsolvency trials and monitoring lead levels across domestic pipelines to establish
the most cost-effective strategy of lead control in portable water. He is currently a Ph.D. student at the
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Department of Civil and Environmental Engineering at Michigan State University, where he works within
the National Science Foundation-funded project "Self-cleaning ceramic membranes for the removal
of natural and synthetic nanomaterials from drinking water using hybrid ozonation-nanofiltration."
His research interests include: (1) advanced membrane processes such as combination of membrane
nitration with oxidation processes; (2) fate and transport of nanomaterials in environment; (3) toxicity of
engineered nanomaterials.
Publications:
Alpatova A.L; Shan W; Rogensues, A.R; Masten, S.J; Alocilja, E.A. and Tarabara, V.V. Biocompatibility
of single wall carbon nanotubes solubized by non-covalent functionalization technique. In preparation
Alpatova, A.L; Babica, P; Hashsham, S.A; Upham, B.L; Masten, S.J. and Tarabara, V.V. In vitro toxicity
evaluations of fullerene nC60 derivatives formed in conditions that simulate disinfection processes at
water treatment plant. In preparation
Dr. Pedro J. Alvarez is the George R. Brown Professor of Engineering at Rice University. He
previously taught at the University of Iowa, where he also served as Associate Director for the Center for
Biocatalysis and Bioprocessing and as Honorary Consul for Nicaragua. Prof. Alvarez's research focuses
on the environmental applications and implications of biotechnology and nanotechnology, including
bioremediation of contaminated aquifers, phytoremediation, fate and transport of hazardous substances,
and nanomaterial-bacterial interactions and related disinfection approaches. Dr. Alvarez received a B.
Eng. degree in Civil Engineering from McGill University and MS and Ph.D. degrees in Environmental
Engineering from the University of Michigan, and was a visiting professor at the Swiss Federal Institute
of Technology (EAWAG). Dr. Alvarez is a P.E., a Diplomate of the American Academy of Environmental
Engineers and a Fellow of ASCE. Dr. Alvarez currently serves on the editorial boards of Environmental
Science and Technology, Biodegradation, and the European Journal of Soil Biology. He is also an
honorary professor at Nankai University in China and adjunct professor at the Universidade Federal de
Santa Catarina in Florianopolis, Brazil, and UNAM in Mexico City.
James E. Amonette is a senior research scientist in the Fundamental and Computational Sciences
Directorate, Pacific Northwest National Laboratory, Richland WA.
Beth Anderson hails from the National Institute of Environmental Health Sciences (NIEHS) of the
National Institutes of Health where she shepherds research translation for the Superfund Basic Research
Program (SBRP). She began her long career at NIEHS in the molecular sciences studying prostaglandin
synthesis and later switched to science administration where she worked for the National Toxicology
Program (NTP). After a decade with the NTP, she joined the extramural program and SBRP. Here she
pursues her professional passion of advancing SBRP research findings with the goals of improving
human health and identifying better, faster and cheaper clean-up strategies for hazardous waste sites.
Ms. Anderson has an undergraduate degree from the University of North Carolina at Chapel Hill and a
masters degree from Duke University.
Dr. Anthony Andrady has more than 25 years of research and development experience in polymer
science and engineering, having served as program manager on numerous research programs funded
by US government agencies. Dr. Andrady is a polymer scientist with specialized research experience
in degradation of polymers in the environment. His main areas of research interest are fabrication of
electrospun nanofibers, biomedical applications of nanofibers and characterization of nanoscale particles
(particularly carbon nanotube materials). He has authored or co-authored about 100 peer-reviewed
publications including book chapters and two books.
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Donald R. Baer is the lead scientist interfacial chemistry for the Environmental Molecular Sciences
Laboratory, Pacific Northwest National Laboratory, Richland WA
Dr. Sarbajit Banerjee is an Assistant Professor at the Chemistry Department of the University at
Buffalo, The State University of New York. He received his undergraduate degree in B.S. Chemistry at
St. Stephen's College, University of Delhi, in 2000 and his Ph.D. degree at the State University of New
York at Stony Brook and Brookhaven National Laboratory under the supervision of Prof. Stanislaus S.
Wong. His graduate work was focused on the surface chemistry of carbon nanotubes and the use of X-ray
absorption spectroscopy to study nanostructures. From 2004 to 2007, he was a post-doctoral research
scientist in the group of Professor Irving P. Herman in the Department of Applied Physics and Applied
Mathematics and the Nanoscale Science and Engineering Center at Columbia University. Professor
Banerjee and his research group are interested in the broad areas of carbon and metal oxide nanostructures
for electronics and energy conversion.
Dr. Melissa Baumann is an associate professor in Department of Materials Science and Chemical
Engineering at Michigan State University. She obtained her Ph.D. from Case Western Reserve University
in 1988, after which she was postdoctoral fellow in the UKAEA Harwell Laboratories, Great Britain.
Dr. Neppolian Bernaurdshaw is a Research Scientist in the Department of Environmental Science
and Engineering at Gwangju Institute of Science and Technology in Korea. Dr. Bernaurdshaw earned
his bachelor's and master's degrees in chemistry at Government College, Kumbakonam, Bharathidasan
University, India, and his Ph.D. in chemistry from Anna University in India. Prior to joining Gwangju
Institute of Science and Technology, Dr. Bernaurdshaw served as a doctoral researcher at Osaka
Prefecture University in Osaka, Japan. His work involves preparation of semiconductor photocatalysts
(nanotubes, nanorods, etc.) using novel methods, as well as pollution abatement studies both in gas and
liquid phases. Dr. Bernaurdshaw works with visible light responsive photocatalysts and synthesized
hybrid PLEDs gold capped TiO2 polymer nanocomposites. He also studies Advanced Oxidation
Techniques (AOTs) for complete degradation of organic and inorganic pollutants.
Dr. Dibakar Bhattacharyya is the University of Kentucky Alumni Professor of Chemical Engineering
and a Fellow of the American Institute of Chemical Engineers. He received is B.S. in chemical
engineering from Jadavpur University, M.S. in chemical engineering from Northwestern University,
and Ph.D. in environmental engineering from the Illinois Institute of Technology. He has published
167 (mostly in water related area) refereed journal articles and 21 book chapters, and has recently
received five U.S. Patents (Functionalized Materials/Membranes for toxic metals capture from water
at ultrahigh capacity, and one on hazardous waste destruction technology). Dr. Bhattacharyya has
mentored many graduate and undergraduate students in the area of environmental research, membranes,
and separation/reactions. He and his graduate students pioneered the development of poly-ligand
functionalized material development for toxic metal capture, and synthesis of nanostructured metals
in polymers (nanocomposites) for toxic organic dechlorination from wastewater at room temperature.
He has worked with several industries in projects dealing with wastewater, material recovery, and
important separation problems. Dr. Bhattacharyya has received a number of awards for his research
and educational accomplishments, including the 2004 Kirwan Prize for Outstanding Research, Larry
K. Cecil AIChE Environmental Division Award, the Kentucky Academy of Sciences Distinguished
Scientist Award, Henry M. Lutes Award for Outstanding Undergraduate Engineering Educator, AIChE
Outstanding Student Chapter Counselor Awards, and the University of Kentucky Great Teacher (1984,
1996, and 2008) Awards. For his highly significant technical contributions in the area of environmental
separation (particularly water treatment) and polymer-nanoparticle composite materials development
for toxic organics degradation, he was recently honored (plenary/keynote lectures) at the NAMS
meeting (Orlando), European Chemical Engineering Meeting (Copenhagen), Inter-Federation Chemical
Engineering Congress (Buenos Aires), and in Indian Chemical Engineering Congress (Calcutta). In
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Feb 2007 he was the Technical Program coordinator and Chair of the ECI Water Treatment and Reuse
Conference in Tomar, Portugal.
Dr. Pratim Biswas is the Stifel and Quinette Jens Professor and Chair, Department of Energy,
Environmental and Chemical Engineering, Washington University, USA. He received his Ph.D. from
California Institute of Technology, and a M.S. from the University of California.
Claudio Cameselle is an Associate Professor in the Department of Chemical Engineering at the
University of Vigo, Spain. He teaches advanced chemical reactors, waste water treatment, and solid
waste management and treatment. His research expertise includes electrokinetic remediation of polluted
soils and wastes, and bio-production of organic acids and other metabolites of industrial interest. He was
awarded the fellowship from Xunta de Galicia (Spain) to perform research at the University of Illinois at
Chicago during 2007-2008.
Dr. Tom Campbell of ADA Technologies is an active researcher in the nanotechnology/ environmental,
health, and safety sector. He is currently under contract with NIST to support a national assessment of
measurement needs for determining the effects of nanomaterials on environmental health and safety. Dr.
Campbell received his B.E. in Mechanical Engineering from Vanderbilt University and his M.S./Ph.D.
in Aerospace Engineering Sciences from the University of Colorado at Boulder. Most recently, he has
worked as a Senior Research Scientist/Nanotechnology Program Manager within ADA Technologies, Inc.,
in Littleton, CO. He recently successfully completed a National Science Foundation (NSF) Phase I STTR
project, A Carbon Nanotube Metrology System for Industry and Research Environments, in which he
demonstrated the world's first quantitative, low cost, reproducible, and rapid means to characterize single
wall carbon nanotubes (SWNTs). Prior to joining ADA, he worked for six years researching advanced
materials at Saint-Gobain Crystals. This research had as its focus optical materials (CaF2, BaF2, MgF2)
for the 157nm and 193nm microlithography laser markets. Dr. Campbell has also held a post-doctoral
fellowship in Germany through the Alexander von Humboldt Foundation. His self-proposed, independent
research project was to study Gel-xSix crystal growth.
Barbara J. Carter is the Director of Research and Development for EcoArray, Inc. She is the Principal
Investigator on two Phase 2 SBIR grants awarded by NIEHS, "Microarrays in fathead minnows and
bass," in the process of completion, and "Developing and using sheepshead minnow microarrays for
ecotoxicology" which began August 2007. She is also P.I. of a Phase 1 SBIR awarded by the EPA in
March 2008, "Using fathead minnow microarrays to test toxicity of nanoparticles." She was hired in
2002 at the inception of EcoArray; providing laboratory expertise on two NIEHS Phase 1 SBIR grants, a
CRADA with the EPA, and a grant from Project Wild Dolphin. Ms. Carter graduated from Northwestern
University with a dual major in biological sciences and anthropology, and received her M.A. in
anthropology (archaeology) from the University of Washington. A career military officer, she retired as a
Captain, U.S. Navy Reserve.
Evrim Celik is a doctoral candidate in the Department of Environmental Science and Engineering at
Gwangju Institute of Science and Technology in Korea. Mr. Celik received his bachelor's degree from
Middle East Technical University in Ankara, Turkey, and his master's degree from Akdeniz University
in Antalya, Turkey. His areas of interest include reactive membrane synthesis, membrane filtration
processes, advanced oxidation processes, and water and wastewater treatment.
Maryam Zarei Chaleshtori holds a B.S. and a M.S. from Isfahan University of Technology, Iran. After
her bachelor's degree, she worked with the Textile Department of Isfahan University of Technology,
Iran, for almost 8 years as an expert and teacher of textile laboratories in dyeing and printing techniques,
natural fibers chemistry, and textile fibers and material identification labs. During her employment, she
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also continued her study toward an M.S. at the same university. As an undergraduate, she studied the
dyeing of wool with natural dyes. A paper was published from her work in the 6th National Conference
of Rug, Tehran, Iran, 1999, on which she received an award. Also, in her post-graduate research work,
she studied the treatment of wool and nylon with the sulfamic acid to improve their dyeability. A paper
was published from her work in 3rd National Conference on Textile Engineering in Isfahan University
of Technology, Isfahan, Iran, 1999. She came to the United States in 1999, attended El Paso Community
College, and then she started her Ph.D. studies in 2004 at the University of Texas at El Paso. Since then
she has been doing research with Professor G. Saupe on photochemical water decontamination. Also she
published a paper in Renewable Energy magazine in 2007.
Dr. Sylvia Chan-Remillard is an Alberta Ingenuity Industry R&D Associate awarded an Industrial
Post Doctoral Fellowship through the Alberta Ingenuity Fund. She is an Environmental Scientist within
the Strategic Risk Group in the Contaminated Sites Management Division of Golder Associates Ltd.,
Calgary and the Applied Sciences Group at HydroQual Laboratories Ltd. Calgary. Sylvia obtained her
undergraduate degree in Food Sciences and Nutrition and a Ph.D. in Food Science and Technology
from the University of Alberta. Her Ph.D. examined the ability of dairy derived probiotics and bioactive
peptides in altering intestinal microbial ecology in the treatment of gastrointestinal disorders such as
inflammatory bowel disease and colon cancer. She was nominated for the Governor General Gold Medal
Award for her Ph.D. research. Sylvia is currently examining the fate and effects of nanoscale particles
on ecological receptors and involved in developing a risk-based framework to assess the impact of
nanotechnology on the environment. She has presented her current and previous work at numerous
international and local conferences. Sylvia is a member of various ad-hoc nanotechnology working
groups (SETAC, ASTM and SRA) and is a fellow of the International College of Nutrition.
Dr. Mark Chappell is a Research Physical Scientist at the Engineer Research and Development Center,
US Army Corps of Engineers in Vicksburg, MS. He received his Ph.D. in Soil and Science in 2004 from
Iowa State University, a M.S. in Plant & Soil Science in 1998 from the University of Kentucky, and a B.S.
in Agronomy in 1995 from Brigham Young University. He was the ORISE Postdoctoral Research Fellow,
US Environmental Protection Agency, Cincinnati, OH, in 2005 and a Postdoctoral Research Associate at
Iowa State University in 2004. His research interests include metal-organic complexes in soil, solid-phase
in-situ speciation of metals and organics, and chemistry of formulations in soil
Dr. Sandip Chattopadhyay, TetraTech/EM, Inc., has more than 18 years of experience in environmental
fate and transport of emerging contaminants, sampling, handling, preservation techniques of samples in
various matrices, development of analytical methodologies, treatment and monitoring of contaminated
sediment, soil and groundwater and air. He has more than 10 years experience in managing numerous
task orders for U.S. EPA. In the past, he has collaborated with different national laboratories and
universities, such as Lawrence Berkeley Laboratory, Argonne National Laboratory, Stanford Synchrotron
Radiation Laboratory and Purdue University. He is organizing, presenting and chairing a session on
"Nanoscale ZVI" at the Sixth International Conference on Remediation of Chlorinated and Recalcitrant
Compounds at Monterey, California. He has participated as an Expert Panel Member on Water Security
Workshop organized by U.S. EPA and other federal agencies. He is a member of the Interstate Technology
Regulatory Council's (ITRC) technical team, and has prepared technical guidance documents for
scientists, engineers, regulators. Dr. Chattopadhyay published more than 50 peer-reviewed journal
articles and reports. He led various R&D effort on dispersion, aggregation and sampling of anthropogenic
(manufactured) nanoparticles (iron oxides and titanium oxides and other manufactured nanomaterials)
in treatment of groundwater. These studies resulted in successful application of dispersed nanoparticles
and control of aggregation in subsurface systems, and several reports for U.S. Navy and U.S. EPA.
He is interested in application of nanomaterials (natural or man-made) as decontamination agent for
chemical/biological/radiological- contaminated systems. He has received his Masters degree in Chemical
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Engineering from Ohio University and Ph.D. from the Ohio State University. Presently, he is Tetra Tech's
National Program Manager under the company's STREAMS contracts with ORD. Previously, he worked
at Battelle Memorial Institute, Columbus, Ohio; ManTech Environmental Research Services Corp.,
and U.S. EPA's Kerr Environmental Research Center in Ada, Oklahoma. He has authored over 60 peer-
reviewed publications and reports.
Dr. Heechul Choi is an Assistant Professor, Associate Professor, and Professor in the Department of
Environmental Science and Engineering at Gwangju Institute of Science and Technology in Korea. Dr.
Choi received his bachelor's degree in environmental engineering from National Fisheries University
in Busan, Korea, his master's degree from Asian Institute of Technology in Bangkok, Thailand, and his
Ph.D. in environmental engineering from Texas A&M University in the United States. Prior to joining
Gwangju Institute of Science and Technology, he worked as a senior researcher in the Department of
Environmental Engineering at the Korea Institute of Construction Technology. Dr. Choi's areas of interest
include using nanomaterials (e.g., metal oxides, mesoporous materials, carbon nanotubes, etc.) for water
purification and fate and transport of nanomaterials in ecosystems, advanced oxidation technologies
for water and wastewater, contaminant transport and modeling through porous media, remediation of
contaminated soil and groundwater, and water reuse and reclamation by natural purification.
Dr. Hyeok Choi is currently an Oak Ridge Institute for Science and Education research fellow at the U.S.
Environmental Protection Agency National Risk Management Research Laboratory in Cincinnati, Ohio,
USA. He obtained his Ph.D. degree at the Department of Civil and Environmental Engineering of the
University of Cincinnati in 2007. His general research area includes environmental nanotechnologies with
emphasis on the novel synthesis and environmental applications of nanostructured TiO2 photocatalysts
and reactive metallic nanoparticles, advanced oxidation technologies, and membrane separation
processes.
Okkyoung Choi is currently a Ph.D. student at the University of Missouri. She previously studied in the
Department of Botany and Microbiology, University of Oklahoma, USA. She earned her B.S. and M.S.
Degrees in environmental engineering from Korea in 2000 and 2002, respectively. Ms. Choi worked as a
Research Associate, Research Institute of Biological and Environmental Technology, Biosaint Co., Seoul
for one year before coming to the U.S. to pursue her Ph.D. degree. She has published several papers
related to silver nanoparticle research in Water Research and Environmental Science & Technology.
Chanlan Chun was a Postdoctoral Fellow in the Department of Chemistry, University of Minnesota,
Sue Cumberland is currently a third year PhD student at the University of Birmingham UK under
the supervision of Dr Jamie Lead. Her study area is the fate, transport and behaviour of manufactured
nanoparticles within the aquatic environment. To date she has investigated the aggregation behaviour
of synthetic iron oxide nanoparticles under conditions of pH and natural organic matter. Techniques
include light scattering, electrophoresis, TEM and flow-field flow fractionation separation techniques.
In addition she is also investigating in-house synthesized silver nanoparticles and bonding properties to
natural organic matter and trace metals. Her background as a research assistant has involved working in
areas of soil science, hydrology, water quality, and catchment studies of upland agricultural pollution in
Scotland and lowland ground water recharge and wetland sytems in the Midlands. Her research interests
include the role of humic substances in aquatic environment particularly with nanoparticles and pollution
pathways through riparian systems. She holds degrees from Plymouth and Reading University, UK.
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Kenneth Darko-Kagya: Kenneth Darko-Kagya is a doctoral graduate student in the Department of Civil
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and Materials Engineering at the University of Illinois at Chicago. His research focus is on the fate and
transport of nanoscale iron particles in soils and the remediation of contaminated sites.
Dr. Christophe Darnault is an Assistant Professor and the Director of the Burke Endowed Hydrology
and Hydraulic Laboratory in the Department of Civil and Materials Engineering at the University of
Illinois at Chicago since 2004. He is specialized in the hydrological, biochemical and environmental
processes impacting water dynamic, water quality and quantity, the fate and transport of contaminants in
the subsurface environment as well as water resources engineering and management. He obtained his PhD
in Environmental and Water Resources Engineering from Cornell University in 2000. He is the editor of
the book titled "Overexploitation and Contamination of Shared Groundwater Resources: Management,
(Bio)Technological, and Political Approaches to Avoid Conflicts" published by Springer in collaboration
with NATO in 2008. He is the author or co-author of more than 30 peer-reviewed book chapters and
journal articles and presented more than 50 conferences papers at national and international meetings.
Dr. Simon Davies is a research specialist in Department of Civil and Environmental Engineering at
Michigan State University. He obtained his Ph.D. from California Institute of Technology in 1985, after
which he was a post-doctoral fellow at the Swiss Federal Institute of Technology (ETH) and Department
of Physical Chemistry, University of Melbourne, Australia.
Dr. Dermot Diamond received his Ph.D. and D.Sc. from Queen's University Belfast (Chemical Sensors,
1987, Internet Scale Sensing, 2002), and was Vice president for Research at Dublin City University
(DCU), Ireland (2002-2004). He has published over 160 peer reviewed papers in international science
journals, is a named inventor in 13 patents, and is co-author and editor of three books 'Spreadsheet
Applications in Chemistry using Microsoft Excel' (1997), 'Principles of Chemical and Biological
Sensors', (1998) both published by Wiley, and 'Smart NanoTextiles', (Materials Research Society
Symposium Proceedings, Volume 20, (2006). Professor Diamond is currently director of the National
Centre for Sensor Research at DCU (www.ncsr.ie) which is one of the largest sensor research efforts
world-wide (>260 researchers) and a Principal Investigator with the Adaptive Information Cluster (AIC),
a major research initiative in the area of wireless sensor networks founded by Science Foundation Ireland
(see www.adaptiveinformation.ie). He was also formerly the director of the Centre for Bioanalytical
Sciences (www.cbas.ie). He is a member of the editorial advisory boards of the international journals 'The
Analyst' and 'Talanta'. In 2002 he was awarded the inaugural silver medal for Sensor Research by the
Royal Society of Chemistry, London. Details of his research can be found at http://www.dcu.ie/chemistry/
asg/.
Dr. Steve Diamond is a Research Biologist with the US EPA's National Health and Environmental
Effects Research Laboratory, within the Office of Research and Development. He currently coordinates
the EPA's nanomaterials ecological toxicology research. He is a contributing author of EPA's ORD
Nanotechnology Research Strategy, has coordinated reviews of standard test guidelines for their
adequacy for testing nanomaterials for both the EPA and the Organization for Economic and Cooperative
Development (OECD), and plays a leading role in the development of the OECD's nanomaterials research
program (OECD Sponsorship Program). He earned his Ph.D. at Miami University (Ohio) and has
worked in the area of Natural Resource Damage Assessments and phototoxicity of polycyclic aromatic
hydrocarbons.
Dr. Baolin Deng is currently C. W. LaPierre Associate Professor in the Department of Civil and
Environmental Engineering at the University of Missouri (MU). He completed his Ph.D. training from the
Johns Hopkins University in 1996. After a year of postdoctoral research as a National Research Council
research associate at the Air Force Research Laboratory, he began his academic career at New Mexico
Tech as an assistant professor in 1996 and moved to MU in 2001. His research concerns with important
environmental and geochemical processes relevant to contaminated site remediation, drinking water
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treatment, and chemical and biological transformation of contaminants in aquatic systems. More recently,
he has been exploring environmental applications of nanotechnologies and examining the aquatic toxicity
of nanomaterials. He has obtained funds from the Department of Energy, National Science Foundation,
and Environmental Protection Agency to support the research activities, and has authored ~50 journal
articles and book chapters. Dr. Deng teaches several undergraduate and graduate courses, including
Fundamentals of Environmental Engineering, Water Treatment Process Design, Water and Wastewater
Laboratory, Aquatic Chemistry, Environmental Chemical Kinetics, Physicochemical and Biological
Processes, and Hazardous Waste Management.
E
Aaron E. Edgington is a Ph.D. candidate at Clemson University in South Carolina. Aaron is a graduate
research assistant in Dr. Stephen Klaine's lab.
Debbie Elcock is a policy analyst with the Environmental Science Division of Argonne National
Laboratory in Washington, D.C. Among other things, she evaluates environmental regulatory approaches
and helps develop cost-effective alternatives. She helped develop a strategy for establishing a laboratory-
wide ES&H program for nanotechnology and has examined potential applications for nanotechnologies
in areas ranging from groundwater remediation to energy transmission corridors. She has also made
presentations on the ES&H concerns of nanotechnologies and the consequent challenges for regulation.
Ms. Elcock has taught courses on environmental management standards, conducted workshops with
various stakeholder groups on improved environmental regulatory approaches, authored more than
50 reports on environmental and energy topics, and spoken at numerous national and international
conferences. Her education includes a masters degree in Business Administration from Dartmouth
College and a bachelor's degree in mathematics from Connecticut College.
Dr. Daniel W. Elliott has more than 15 years of experience in the environmental industry 11 of which
were spent in industry and that past 4 in consulting. In industry, Dr. Elliott focused on environmental due
diligence assessments, the quantification of environmental liability, and internal compliance audits at
two Fortune 500 multinational industrial firms. In the consulting arena, he has significant experience in
leading and conducting environmental due diligence assessments as well as the management of various
complex remediation projects in accordance with NJDEP's Industrial Site Recovery Act. He also led
or supported several remediation projects, including one in NJ utilizing the innovative nanoscale zero-
valent iron (nZVI) technology. Dr. Elliott is a recognized expert in the application of the emerging nZVI
technology and has co-led implementation of numerous bench-scale and pilot scale assessments. He
has co-authored several articles on the nZVI technology and applications in peer-reviewed journals. Dr.
Elliott has significant experience in negotiating with regulators at all levels and has worked on technical,
regulatory, and technology-transfer aspects of environmental projects in the United States, Mexico,
and the Peoples Republic of China. Dr. Elliott holds a Ph.D. in Environmental Engineering, an M.S. in
Environmental Science and Engineering, and an A.B. in Chemistry.
Robert J. Ellis, L.G., is a Senior Scientist, based in the ARCADIS Novi, Michigan office. He received
a B.S. in Geology and a M.S. in Environmental Geosciences, both from Michigan State University.
Mr. Ellis has been in the environmental consulting industry since 1998 and has managed remedial
investigations and remedy selection/implementation at Resource Conservation and Recovery Act
(RCRA), Toxic Substances Control Act (TSCA), and state-lead project sites with soil, sediment, and/or
groundwater impacted with metals, chlorinated solvents, polycyclic aromatic hydrocarbons (PAHs), and
polychlorinated biphenyls (PCBs). Mr. Ellis is currently focused on performing geochemical evaluations
that enhance conceptual site models, design and management of effective bench scale and field pilot
studies for technology demonstrations, and development of remediation strategies that complement
ARCADIS' innovative in-situ remediation techniques and lead to effective site closure strategies for
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industrial and federal government clients.
Dr. David Ensor has 30 years of experience in aerosol and air pollution research as Director of the
Center for Aerosol Technology (CAT), Senior Program Director, and Department Manager at RTI and
as Manager of the Aerosol Science Department at Meteorology Research, Inc. Dr. Ensor has managed
programs in nanotechnology, aerosol research, nitration, air pollution control technology, particle
sampling and characterization, indoor air quality, pollution prevention, exposure research, surface
cleaning, protective garments, microcontamination control, instrumentation development, and test
methods development. These projects have been for the U.S. Environmental Protection Agency (EPA), the
U.S. Department of Energy (DOE), the U.S. Army, SEMATECH, Semiconductor Research Corporation,
universities, and numerous private organizations. Recently Dr. Ensor has been shifting his research
interests to nanotechnology.
Dr. R. Keith Esch is a research microbiologist now serving in RTI's Microbiology Department and
as adjunct faculty member in the Biochemistry and Biophysics department of the University of North
Carolina, Chapel Hill. Dr. Esch received his B.S. degree in genetics from the University of California,
Davis, and his Ph.D. in biology from the University of California, San Diego. He designs, and conducts
applied and basic research in environmental biotechnology and bioaerosol science. Some areas of
expertise include: method development, system evaluation, environmental monitoring, and exposure
assessment. He conducts research in environmental microbial assessment, biological particulate
matter analysis and antimicrobial/biocide efficacy evaluations. He supervises the sampling, isolation,
quantitation, identification, and inactivation of microorganisms (bacteria, fungi, viruses) and their
components in air, water, soils, industrial fluids and materials. His background in biochemistry and
molecular biology is applied to bacteriology; mycology; sampling and analysis of microbiological agents,
components or by-products; study design; quality assurance and quality control; and exposure assessment.
Dr. Anne Fairbrother, DVM, leads the Risk Assessment and Toxicology program at Parametrix, Inc. in
Seattle, WA. She provides services in ecological risk assessment and ecotoxicology, with an emphasis
on wildlife toxicology and terrestrial systems. Anne works in the areas of contaminated site assessment,
pesticide regulatory science and similar needs of the chemical and metals industries. A recent addition
to her practice has been regulatory support for companies that now need to comply with the European
REACH legislation. She also supports state or national agencies through development of guidance
documents e.g., for metals risk assessments and through technical support for site-specific soil and water
criteria development for wildlife protection. Anne received her D.V.M. from Univ. California, Davis
and her Ph.D. from Univ. Wisconsin. She has been the recipient of several honors and awards from
professional societies, and holds a courtesy appointment on faculty at Oregon State University. She
has authored more than 75 scientific papers and has delivered over 100 seminars, workshops, or other
technical presentations.
Karin Foarde is a Senior Research Microbiologist with 30 years of experience and is the Director of RTI
International's (RTFs) Microbial and Molecular Biology Department. She designs, directs, and conducts
applied and basic research in microbiology and aerobiology. Her research interests focus on bioterrorism
associated biological aerosols (bioaerosols) and the environmental causes of allergy and asthma. Her
bioterrorism research experience includes detection, decontamination, and protection from biowarfare
agents. Her asthma/allergy work focuses on researching the biological contaminants isolated from the
environment to identify environmental causes of illness and to recommend methods for preventing
such biological contamination and its associated adverse health effects. Some areas of expertise include
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isolating and characterizing environmental microorganisms, airborne allergens, and pathogens. She
directs analysis of samples for microorganisms, endotoxins, (3-(l-3) glucans, and a variety of antigens.
Dr. Glenn E. Fryxell is a member of the Materials Chemistry and Surface Research Group within the
Materials Division of ESTD, and has been a member of Materials since 1990. For the last 15 years,
his research has focused on organic synthesis, surface chemistry, silane chemistry and the interfacial
elaboration of self-assembled monolayers. He is a co-inventor of self-assembled monolayers on
mesoporous supports (SAMMS) and has developed these materials for a wide variety of environmental
applications, such as the sequestration of toxic heavy metals, radionuclides and oxometallate anions. Dr.
Fryxell is named as inventor in 11 patents, and has over 100 publications and 60 invited presentations.
He obtained his B. Sc. from the University of Texas in 1982, where he worked for two years in the
laboratories of Prof. Marye Anne Fox studying the photochemistry of enolates and carbanions. His Ph.
D. was award in 1986 from the University of North Carolina, where he worked with Prof. Paul J. Kropp
studying the photochemistry of phenylthio ethers. A two-year postdoctoral appointment with Prof. Albert
Padwa at Emory University was dedicated to the study of intramolecular dipolar cycloaddition and
heterocyclic synthesis.
Florin Gheorghiu, C.P.G. is Project Director and Principal in the Philadelphia Office of Golder
Associates. He is an expert in hydrogeologic testing, modeling and hydrogeologic designs and has
over 29 years of experience in engineering geology and hydrogeology. He has directed numerous
environmental projects at CERCLA and RCRA sites that required numerical groundwater flow and
solute transport modeling using computer codes such as MODFLOW, MODPATH and MT3D. Mr.
Gheorghiu served as Project Director and technical manager for the design and implementation of a large
bedrock remedial system at Modern Landfill that involved extensive hydrogeologic testing of fractured
bedrock, numerical modeling and deep bedrock blasting. This project received the Year 2000 Outstanding
Groundwater Remediation Award from the National Groundwater Association. His publications include:
"Hydrogeologic Characterization of Blasted Rock Mass," (2001 Key Note to the Geological Society of
Philadelphia, Philadelphia, Pennsylvania); "Enhanced Western Groundwater Control System," (2000
Key Note to the Regional Hydrogeologists Meeting of the Department of Environmental Protection,
Harrisburg, Pennsylvania); "Application of Analytical and Numerical Models for Natural Attenuation
Characterization," (1997 Presentation at the Golder Associates Natural Attenuation Seminar, Princeton,
New Jersey); "Advanced Test Analysis Methods for the Hydrogeological Characterization of Potential
Nuclear Waste Repositories in Switzerland and Germany," (1996 Presentation to the technical staff of the
U.S.EPA Region 3, Philadelphia, Pennsylvania); "Use of Derivative for Hydrogeologic Test Flow Model
Identification with Application in Deep Borehole Testing," (1995 Presentation to the technical staff of the
U.S.Geological Survey, Trenton, New Jersey).
Dr. Subhasis Ghoshal is an Associate Professor in the Department of Civil Engineering & Applied
Mechanics. He joined McGill as an Assistant Professor in 1997 after completing his Ph.D. at Carnegie
Mellon University and a postdoctoral fellowship at the University of Michigan at Ann Arbor. His research
is in the area of Environmental Engineering and currently focuses on bioremediation of polluted sites
and groundwater, and on carbon dioxide sequestration technologies for greenhouse gas mitigation.
Prof. Ghoshal has contributed substantially to the understanding of NAPL-water interfacial mass
transport processes and its impacts on remediation performance and groundwater quality. He has worked
extensively on NAPL dissolution, biodegradation and interphase mass transfer in NAPL-surfactant
systems. He has recently developed techniques for imaging of NAPL contamination in porous media
using a medical X-ray scanner which allows non-invasive, quantitative contaminant mass characterization
in soil columns and cores. Prof. Ghoshal received the PetroCanada Young Innnovator Award in 1998 and
was named as a Dawson Scholar in 2005. He is a founding member of the CT Scanning Laboratory for
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Agricultural and Environmental Research at McGill.
Michael Gill received his B.S. degree in electrical engineering from Northeastern University in Boston
and his MSEE from Renssalear Polytechnic Institute in Troy, NY. He practiced electrical engineering
in the 1980's with the U.S. Navy and government contractors until he made a career change to the
environmental field in 1992.
He is currently the EPA Office of Research and Development (ORD) Superfund and Technology Liaison
to EPA's San Francisco office (Region 9). This position is one of technical support and information
brokering. His customers are for the most part Remedial Project Managers in the Superfund Program,
but may include RCRA and other Regional EPA staff, state environmental staff, industry, and the public.
In this position since 1998, he provides hazardous waste technical support to his customers and he also
participates in research planning, environmental technology demonstrations, and workshop planning. He
has been with EPA since 1992, when he took a position as a Remedial Project Manager in Region 9's
Superfund program.
Dr. Vicki Grassian is currently a Full Professor in the Department of Chemistry and has secondary
appointments in the Departments of Chemical and Biochemical Engineering and Occupational and
Environmental Health. At the University of Iowa, Professor Grassian has been the recipient of a Faculty
Scholar Award (1999-2001) a Distinguished Achievement Award (2002), a James Van Allen Natural
Sciences Faculty Fellowship (2004), the Regents Award for Faculty Excellence (2006) and was named
a Collegiate Fellow in the College of Liberal Arts and Sciences in 2007. In 2006, she became the
Director of the Nanoscience and Nanotechnology Institute at the University of Iowa. She also serves as
an Associate Director for the Institute of Clinical and Translational Science. For the past several years,
a major research focus in her group has been on the applications and implications of nanoscience and
nanotechnology in environmental processes. Professor Grassian has edited three books including the most
recent one published by John Wiley and Sons entitled Nanoscience and Nanotechnology: Environmental
and Health Impacts. She has over 140 peer-reviewed publications. In 2003, Professor Grassian received
a US-National Science Foundation Creativity Award and in 2005, she was elected as a Fellow of the
American Association for the Advancement of Science.
Dr. George M. Gray was sworn in on November 1, 2005, to serve as the Assistant Administrator for the
Office of Research and Development, which is the 1,900-person, $600 million science and technology
arm of the Environmental Protection Agency. Dr. Gray was appointed to this position by President George
W. Bush and confirmed - by unanimous consent - by the U.S. Senate.
Prior to joining EPA, Dr. Gray was Executive Director of the Harvard Center for Risk Analysis and a
Lecturer in Risk Analysis at the Harvard School of Public Health (HSPH). In 16 years at HSPH, his
research focused on scientific bases of human health risk assessment and its application to risk policy with
a focus on trade-offs in risk management. Dr. Gray taught toxicology and risk assessment to both graduate
students and participants in the School's Continuing Professional Education program.
Dr. Gray holds a B.S. degree in biology from the University of Michigan, and M.S. and Ph.D. degrees in
toxicology from the University of Rochester.
Kimberly Guzan is an aerosol engineer at RTI. She earned a Masters of Science at University of Akron
School of Polymer Science and a Bachelor of Engineering Degree in Chemical Engineering. Her research
interests include polymer nanotechnology and spectroscopic characterization in materials fabrication,
aerosol filtration, chemical sensors and bio-materials research. Subsequently, her research work has
included metal/polymer interfacial adhesion in biomedical devises and organometallic-crystal synthesis.
H
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Scott Hall manages ENVIRON's Ecotoxicology Group and toxicity testing facilities in Nashville,
Tennessee. He serves on ENVIRON's Nanto-technology Task Force, and is conducting research related
to the effects of titanium dioxide on aquatic life. Mr. Hall received a bachelor's degree in Environmental
Protection from West Virginia University and a master's degree in Aquatic Toxicology from North Texas
State University. He has been a consultant to industry for over 20 years.
Dr. Li Han has more than eight years of research and development experience in nanoscience and
nanotechnology. Her original research findings have been published in 30 peer-reviewed journals, and
resulted in two patents and six RTI International invention disclosures. Dr. Han's research interests
include fabricating nanoscale materials, developing novel microscopic and spectroscopic characterization
techniques for nanoparticles and nanofibers, and exploring the application of nanoscale materials in
chemo- and biosensors, catalysis, and biomedical devices.
Dr. Stacey Harper leads the Nanotoxicology Division of the Tanguay laboratory at OSU where
she employs in vivo approaches to provide feedback on the biological activity and toxic potential of
nanomaterials. She has established a collaborative research group to develop the knowledgebase of
Nanomaterial-Biological Interactions (NBI). She received her B.S. in natural sciences and mathematics
from Mesa State College, Colorado in 1990; and earned her M.S. and Ph.D. in biological sciences from
University of Nevada Las Vegas in 1998 and 2003. From 2003 to 2005, she held a biology postdoctoral
position with the Exposure and Dose Research Branch of the EPA.
Dr. Ted Henry is a Research Assistant Professor in the Center for Environmental Biotechnology at
The University of Tennessee (Knoxville, TN) and a Research Council of the United Kingdom (RC-
UK) Academic Fellow at the University of Plymouth (Plymouth, UK). Investigating the characteristics
and toxicity of nanoparticles is major part of his research program at both institutions and presently his
work is supported by a U.S. EPA STAR grant to investigate the ecotoxicology of fullerenes in fish. A
primary objective is to link nanoparticle characteristics with toxic effects and his research aims to clarify
mechanisms at lower levels of biological organization with higher order effects at tissue and whole
organism levels. His role at The University of Tennessee and the University of Plymouth provides a
unique opportunity to integrate research in nanotoxicology among laboratories in the U.S. and the UK.
Mbhuti Hlophe is Head of the Department of Chemistry at North-West University (Mafikeng campus)
in South Africa. His major research area is water treatment, particularly for the provision of potable
water to rural communities. He is one of the principal researchers in water purification in the India,
Brazil and South Africa (IBSA) trilateral cooperation agreement on nanotechnology. He has authored
relevant conference papers, including a case study on a nanofiltration method for water treatment in
South Africa background paper for Meridian's workshop in Chennai (India), membrane nanotechnology
in water treatment (IBSA workshop in Kalpakkam, India), and the role of nanotechnology in the
provision of potable water to rural communities (IBSA workshop in Pretoria, South Africa). Papers that
have been published include: "Nanotechnology, Water and Development" (http://www.merid.org/nano/
waterpaper); "Nanotechnology and the challenge of clean water" (Nature Nanotechnology, 2 (11), 663
- 664); "Nitrogenous pollution in borehole water due to pit latrines and fertilizers" (submitted to Water
SA review for possible publication); and a chapter in a book for the U.S. EPA titled "Nanotechnology
Applications: Solutions for improving water quality.". He also has performed consulting work, the most
important of which was the development of water safety and security plans for the Department of Water
Affairs and Forestry in South Africa.
Dr. Patricia Holden is a Professor at the University of California, Santa Barbara in the Donald
Bren School of Environmental Science & Management. The Holden lab researches environmental
microbiology, focusing on questions in water, soil science and emerging pollutants. Holden's education is
in Civil & Environmental Engineering (B.S., M.S.) with 8 years of professional engineering experience
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followed by her Ph.D. and Postdoctoral Research in Soil Microbiology (U.C. Berkeley). Holden has
been on the faculty at UC Santa Barbara since 1997. Current projects in the Holden group are in coastal
bacteriological water quality with an emphasis on watershed processes, bacterial interactions with
engineered nanomaterials, and vadose zone microbial ecology. Her co-authors for this talk include Allison
Horst (doctoral student), John Priester (postgraduate researcher) and Dr. Andrea Neal (postdoctoral
researcher) who are all actively researching nanomaterials interactions with bacteria.
Dr. Zhiqiang Hu is an Assistant Professor of environmental engineering at the University of Missouri.
Dr. Hu has been studying biochemical processes for wastewater treatment and nutrient removal for
more than ten years. His recent research interest includes bioavailability and toxicity of nanoparticles in
wastewater treatment systems. One of his ongoing research projects entitled "Nitrification inhibition by
silver nanoparticles" was financially supported by the National Science Foundation. Dr. Hu has published
some of the nanotoxicity research findings by working with her Ph.D. student, Okkyoung Choi. The Water
Environment Research Foundation recently awarded Dr. Hu $150,000 to determine more precisely when
silver nanoparticles start to impair wastewater treatment. In that project, his research team will determine
how silver nanoparticles affect representative wastewater treatment processes by gradually releasing as
well as injecting a shock load of the nanomaterial into wastewater and sludge. Measuring subsequent
microbial growth will allow MU researchers to determine the nanosilver levels that will harm wastewater
treatment and sludge digestion.
De-Huang Huang is a senior environmental engineer in the Chinese Petroleum Corporation, Kaohsiung,
Taiwan, ROC. He received his M.S. degree in Environmental Engineering from the Graduate Institute of
Environmental Engineering at National Taiwan University. He is interested in applying novel technologies
for groundwater remediation including iron nanoparticles, chemical oxidation and thermal technology.
Dr. William D. Hunt is Professor of Electrical Computer Engineering at the Georgia Institute of
Technology and is Adjunct Professor in the Department of Hematology and Oncology at the Emory
University School of Medicine. He runs the Microelectronics Acoustics Group at Georgia Tech and has a
diverse collection of graduate students
Dr. Robert Hurt is Professor of Engineering at Brown University and Director of Brown University's
Institute for Molecular and Nanoscale Innovation (IMNI). Dr. Hurt has a hybrid technical background
in nanomaterials science and energy/environment. He has devoted the last four years to understanding
the fundamental biological interactions of nanomaterials and in developing new nanostructures for
environmental and biological applications. He has been involved in discussion of nanotechnology
environmental safety and health policy and regulation through talks at the National Research Council
of Canada (2007), the Environmental Business Council of New England (2006), the World Technology
Evaluation Center (2006), and participation in the 2007 NanoBusiness Alliance Public Policy Tour in
Washington D.C. Dr. Hurt received a Sc.B. from Michigan Technological University and a Ph.D. from
the Massachusetts Institute of Technology, both in chemical engineering. Prior to joining Brown in 1994
he held posts at Bayer AG in Leverkusen, Germany, and Sandia National Laboratories in Livermore
California. Professor Hurt is an Editor of the materials science and nanotechnology journal CARBON,
has served as the Graffin Lecturer of the American Carbon Society, and has won the Silver Medal of
the Combustion Institute for work on the high-temperature reactions of carbon materials. His current
research interests are in the applications and implications of nanotechnology for human health and the
environment, including nanosorbents for pollution abatement and the intelligent design and formulation of
nanomaterials to minimize health risks. He is a member of the scientific advisory board for the company
Nanotox.
Dr. Jim Hutchison is Professor of Chemistry and Director of the Materials Science Institute at the
University of Oregon. He also directs the Safer Nanomaterials and Nanomanufacturing Initiative of
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the Oregon Nanoscience and Microtechnologies Institute and has pioneered the University's Green
Organic Chemistry Laboratory program. A native of Oregon, he received his B.S. in Chemistry from the
University of Oregon in 1986 and a Ph.D. from Stanford University in 1991 (with James P. Collman).
He then did postdoctoral work with Royce W. Murray at University of North Carolina, Chapel Hill. He
has won numerous awards including a Postdoctoral Fellowship and a CAREER award from the National
Science Foundation, as well as awards from the Sloan and Dreyfus Foundations. His current research
interests include the design, synthesis and study of functional organic and inorganic materials, including
functionalized surfaces and nanoparticles, green chemistry and green nanoscience.
Nick Jaynes is a Geotechnical Engineer for MSE Technology Applications in Butte, Montana. Mr. Jaynes
holds degrees in Environmental Engineering and Civil/Geotechnical Engineering from Texas A&M
University. His previous experience includes consulting in the environmental and geotechnical fields in
Wyoming and Montana.
Dr. Gautham Jegadeesan is an Environmental Engineer with Pegasus Technical Services, Inc at
Cincinnati. A graduate in Chemical Engineering and a Ph.D in Engineering Science, Dr. Jegadeesan has
worked on diverse water remediation projects including the use of bimetallic nanoparticles for trace metal
remediation and electrolytic processes for contaminant reduction. He is currently working on the fate and
transport of engineered nanoparticles in the environment, speciation of trace metals in coal combustion
residues and mining wastes.
Vijay T. John is Professor and Chair of the Department of Chemical and Biomolecular Engineering at
Tulane University. He works on self-assembled nanoscale materials for environmental applications and in
targeted drug delivery. He has published 130 Journal articles and has supervised 19 Ph.D. dissertations.
He is funded by U.S. EPA, the NSF, U.S. Department of Energy, and NIH.
Jon Josephs' academic background includes degrees in chemical engineering from Rutgers University
(1971) and Stevens Institute of Technology (1973). He was selected for membership in Tau Beta Pi, the
national engineering honor society. In 1973, Jon joined the EPA Region 2 office in New York City where
he was employed in permitting industrial wastewater discharges, regulating hazardous-waste management
facilities and as a Superfund Remedial Project Manager. In 1994 Jon was reassigned from Region 2 to the
Office of Research and Development as the Superfund and Technology Liaison (STL) assigned to EPA
Region 2.
As an STL, Jon's activities have included: organizing a workshop on the natural attenuation of
chlorinated solvents in groundwater, managing the development of a compendium of methods for
monitoring the remediation of contaminated sediments, serving on Science Advisory Committees for the
Northeast Hazardous Substance Research Center and for the Center for Hazardous Substances in Urban
Environments, participating in the workgroup that developed the Office of Solid Waste and Emergency
Response's directive on monitored natural attenuation and serving as EPA project coordinator for a
research project on biodegradation of polychlorinated-dibenzo-p-dioxins. More routine activities include
coordinating technical support for Region 2 Superfund projects, identifying EPA Region 2 research needs,
serving on the EPA Region 2 Science Council and organizing technical presentations for Region 2 staff.
K
Dr. Agnes Kane is Professor and Chair of the Department of Pathology and Laboratory Medicine at
Brown University, and has devoted her career to the study of biological responses to particulate and
fibrous toxicants. She has served on scientific panels in environmental health sciences, including current
membership of the EPA Science Advisory Board and the ICON working group on nanomaterial safety.
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She has devoted the last four years to understanding the biological impacts of new nanomaterials. She has
served as scientific advisor and invited participant in workshops on fiber toxicology and nanotechnology
for NIOSH, US EPA, NAS, IOM, NTP and IARC.
Dr. Barbara Karn, a U.S. EPA scientist, built and managed a research grant program in nanotechnology
and the environment at EPA. She formed and sustained a community of researchers in nanotechnology
and the environment-both applications and implications-and brought nanotechnology into EPA's programs
and mission. Through the interagency Nanoscale Science and Technology subcommittee of the Office
of Science and Technology Policy, she led workshops to build consideration of the environment and
human health in other government agency research programs related to nanotechnology. She helped
provide leadership in international activities involving nanotechnology in the environment and human
health. Currently, she is the nanotechnology scholar at Georgetown University's Program for Science in
the Public Interest and recently returned from a detail at the Woodrow Wilson International Center for
Scholars Program in Emerging Nanotechnologies. Dr. Karn holds the Ph.D. from Florida International
University and a B.S. in chemistry from Ohio State.
Dr. Ian M. Kennedy joined the Department of Mechanical and Aeronautical Engineering at the
University of California Davis in 1986 after a period as a Research Staff member at Princeton University
and several years at the Aeronautical Research Laboratories in Australia. He has developed a major
aerosol research facility at the University of California Davis in which efforts are directed at varied
problems related to ultrafine particle synthesis and applications in technology. A major thrust of Dr.
Kennedy's efforts is directed towards understanding the impact of ultrafine aerosol particles on human
health. This interest is pursued via extensive multidisciplinary collaborations with colleagues in
Environmental Toxicology, Land Air Water Resources, Veterinary Medicine, Chemistry and Civil and
Environmental Engineering. He is also involved in applying nanoscale particles to detection technologies
in biology and biophotonics e.g., using nanoscale phosphors as labels of bio-molecules. This work
involves collaborative research with colleagues in the Departments of Entomology, Internal Medicine and
Land Air Water Resources.
Alan J. Kennedy is a Research Biologist with the U.S. Army Engineer Research and Development
Center in Vicksburg, Mississippi. His responsibilities include serving as project manager/principal
investigator for ecotoxicological exposure and effects assessment; conducting water column and whole
sediment toxicity and bioaccumulation testing in support of research, dredged material assessments, and
other client needs; writing manuscripts, proposals, technical reports and laboratory SOPs; and managing
laboratory technicians. His research has involved chemicals such as DDTs, PCBs, PAHs, explosives,
metals and nanoparticles. Mr. Kennedy received a M.S. in Aquatic Ecotoxicology in 2002 from Virginia
Polytechnic Institute and State University. His thesis work involved risk assessment methodologies to
gauge multiple levels biotic impairment caused by the total dissolved solids (TDS) toxicity of a treated
coal-mining effluent in southeastern Ohio. He received a B.S., with high honors, in Environmental
Biology/Zoology in 1999 from Michigan State University.
Dr. Amid P. Khodadoust is an Associate Professor of Environmental Engineering at the University of
Illinois at Chicago. He teaches environmental engineering, physico-chemical processes, waste water
treatment, and pollution prevention. His research expertise includes bioavailability of contaminants in
sediments, remediation of contaminated soils and sediments, and environmental nanotechnology.
Dr. Jeonghwan Kim is a research associate in Department of Civil and Environmental Engineering at
Michigan State University. He received his Ph.D. degree in Environmental Sciences and Engineering
from University of North Carolina at Chapel Hill in 2005.
Ayla Kiser received her bachelor of science in mechanical engineering and her master of science in
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environmental engineering from the University of Nevada, Las Vegas. In August 2006, she began
the environmental engineering Ph.D. program at Arizona State University. Under the guidance of her
advisors, Dr. Paul Westerhoff and Dr. Bruce Rittmann, Kiser is currently doing research on the biological
removal, environmental fate, and detection of engineered nanoparticles from wastewater. She is expected
to graduate in 2010.
Dr. Stephen J. Klaine is a professor in the Department of Biological Sciences at Clemson University
in Pendleton, SC. Dr. Klaine received his bachelor's degree in biology from University of Cincinnati
and his master's degree and Ph.D in environmental science from Rice University in Texas. Dr. Klaine's
research focuses on the fate and effects of contaminants in the environment. Specifically, he is interested
in contaminants that migrate from various land uses into aquatic ecosystems and their effects on aquatic
plants and animals. His laboratory studies contaminant effects on fish, aquatic invertebrates, plants,
and algae. Current research on nanomaterials includes work on their behavior in aquatic systems,
bioavailability, and food chain transport.
Dr. Rebecca Klaper received her Ph.D. in Ecology from the Institute of Ecology, University of Georgia.
She is currently a Shaw Scientist at the Great Lakes WATER Institute, an organization dedicated to
providing basic and applied research to inform policy decisions involving our freshwater resources. Dr.
Klaper studies the potential impact of emerging contaminants, such as nanoparticles and pharmaceuticals,
on aquatic organisms using traditional toxicology methods as well as investigations using genomic
technologies. Dr. Klaper has served as an American Association for the Advancement of Science-Science
and Technology Policy Fellow where she worked in the National Center for Environmental Assessment
at the US Environmental Protection Agency. She has served as an invited scientific expert to the
Organization for Economic and Cooperative Development panel on nanotechnology where she testified
on the potential impact of nanoparticles on the environment. She also was involved in writing the EPA
White Paper on the use of genomic technologies in risk assessment. She belongs to several scientific
societies including the Ecological Society of America, The Society for Environmental Toxicology and
Chemistry and the American Fisheries Society.
Dr. Sarah C. Larsen, is a Professor of Chemistry and the Associate Director of the Nanoscience
and Nanotechology Institute at the University of Iowa. Professor Larsen has research interests in the
applications of nanocrystalline zeolites to environmental remediation, decontamination and drug delivery.
Professor Larsen has expertise in the synthesis, characterization and functionalization of nanocrystalline
zeolites and hollow zeolite structures. Her research has been funded by the National Science Foundation
(NSF), the Environmental Protection Agency, the Army Research Office, the Department of Energy
and the Petroleum Research Fund. Professor Larsen has also been involved with educational efforts in
nanoscience and nanotechnology. Currently, she is the Director of an NSF Research Experiences for
Undergraduates (REU) program focused on nanoscience and nanotechnology. Professor Larsen is also a
senior editor for the Journal of Physical Chemistry.
Dr. Warren Layne has a BA in chemistry from Boston University, MS in inorganic analytical chemistry
from University of Massachusetts, and Ph. D. in medicinal chemistry from Northeastern University in
Boston, with postdoctoral training at Harvard School of Public Health in nuclear medicine. He also has
additional years of industrial experience in radiopharmaceutical research as an Assistant Professor at
University of Connecticut Medical Center, University of Texas at Galveston, and Baylor University in
Houston. Dr. Layne joined the EPA in 1991 as the Toxic Release Inventory (TRI) coordinator for Region
6 (Dallas, TX) and is the currently serves as a Quality Assurance Project Plan (QAPP) reviewer and
Regional Sample Coordinator for the Superfund Division as well as Nanotechnology expert in Region
5 (Chicago, IL). He was a coauthor of Nanotechnology White Paper, participated in EPA-sponsored
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National Nanotechnology Conferences, and is a member of Nanometers, the current co-chair of the EPA
National Nanotechnology Workgroup. He is co-chair of the multi-agency steering committee for the
International Environmental Nanotechnology Conference: Applications and Implications scheduled for
Chicago, October 7-9, 2008.
Dr. James M. Lazorchak is an aquatic biologist/toxicologist for the U.S. EPA National Exposure
Research Laboratory, Ecological Exposure Research Division, where he is Acting Chief of the Molecular
Indicators Research Branch. He received a B.S. in biology (1987) from Southeast Missouri State
University, a M.S. in aquatic ecology (1974) from Wright State University, and a M.S. in environmental
sciences (1978) from the University of Texas at Dallas. He received his Ph.D. in ecotoxicology (1986)
from the University of Texas at Dallas.
Research in Dr. Lazorchak's early career centered around developing fish, invertebrate, and plant
bioasssessment and ecotoxicology methods to assess the biological integrity of lakes, streams, rivers, and
estuaries. My current research activities are to bring genomic tools to bioassessments and ecotoxicity tests
to assess ecosystem health and develop water quality criteria and water quality standards and limits that
can be used in regulatory programs of emerging contaminants (i.e., EDCs and pharmaceuticals).
He has written 36 peer reviewed papers, 13 EPA manuals, 4 book chapters.
Dr. Qilin Li is an Assistant Professor in the Department of Civil and Environmental Engineering at Rice
University. Dr. Li obtained her B.E. in Environmental Engineering from Tsinghua University in China.
She received her M.S. and Ph.D. degrees in Environmental Engineering from University of Illinois at
Urbana-Champaign in 1999 and 2002, respectively. Before joining the faculty at Rice University, she
worked as a post-doctoral research associate at Yale University from 2002 to 2003 and an assistant
professor at Oregon State University from 2004 to 2005. Dr. Li's current research focuses on advanced
technologies for water quality control including adsorption and membrane separation, and environmental
application and impact of nanotechnology.
Dr. Yusong Li is currently a postdoctoral associate in the Department of Civil and Environmental
Engineering at Tufts University. She received her Ph.D. in Environmental Engineering from Vanderbilt
University in 2005. She will start as an Assistant Professor in the Department of Civil Engineering
at University of Nebraska-Lincoln. Her research area is numerical simulation of fate and transport of
contaminants in the subsurface system.
Dr. Hsing-Lung Lien is an associate professor in the Department of Civil and Environmental
Engineering at the National University of Kaohsiung in Taiwan. He received his Ph.D. in Environmental
Engineering in 2000 from Lehigh University, under the guidance of Dr. Wei-xian Zhang. He worked
as a research associate at the Ground Water and Ecosystems Restoration Division, an USEPA research
laboratory, in Ada, Oklahoma from 2000 to 2002. Dr. Lien has joined the National University
of Kaohsiung since 2002. His research interests include environmental nanotechnologies and
physicochemical processes for water treatments. He has published over 10 peer-reviewed papers on the
use of iron nanoparticles for groundwater remediation.
Dr. Igor Linkov is a Research Scientist at the US Army Engineer Research and Development Center
and Adjunct Professor of Engineering and Public Policy at Carnegie Mellon University. Dr. Linkov has
managed multiple risk assessments and risk management projects. Many of his projects have included
application of the state-of-the-science modeling and software tools (e.g., probabilistic and Bayesian
Monte-Carlo, spatially-explicit modeling) to highly complex sites and engineering problems (e.g.,
Hudson River, Dow Midland, Natick Soldier Systems Command, Elizabeth Mine, etc.) and projects
(e.g., insuring emerging risks, risk-based prioritization of remedial projects, developing performance
metrics for oil spill response). He was instrumental in developing an integrated risk assessment and
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multi-criteria decision analysis framework that is now being widely applied by the US Army Corps of
Engineers, including restoration planning for coastal Louisiana and Mississippi affected by the hurricane
Katrina where a multi-billion dollar budget is at stake. Dr. Linkov is currently involved in several
projects that examine factors responsible for nanotoxicology and nanomaterials risks. These projects
investigate fate and transport of nanoparticles in the environment, ecotoxicology, assessment of nano-
enabled product life cycle and risks. Dr. Linkov have organized three continuing education workshops
in the area of nanomaterials health and safety and an international conference on "Nanomaterials:
Environmental Risks and Benefits" (Portugal, April 2008). Dr. Linkov was part of international and
national panels on nanotechnology, including: EPA Nanotechnology White Paper Peer Review Panel
(2006), Nanotechnology Research Strategy (2008), and Nanotechnology Grants Review Panel (2007);
Environment Canada Nanotechnology Expert Panel (2007); and the City of Cambridge Nanotechnology
Ordinance Advisory Panel (2007-2008). The Governor of Massachusetts has appointed Dr. Linkov
as a Scientific Advisor to the Massachusetts Toxic Use Reduction Institute. He is the recipient of the
prestigious Chauncey Starr Award for exceptional contribution to Risk Analysis. Dr. Linkov has a BS
and MSc in Physics and Mathematics (Polytechnic Institute, Russia) and a Ph.D. in Environmental,
Occupational and Radiation Health (University of Pittsburgh). He completed his post doctoral training in
Biostatistics and Toxicology and Risk Assessment at Harvard University.
Dr. Bruce Lippy has a Ph.D. in policy from the University of Maryland, with coursework concentrated
in regulatory economics and quantitative measures of management. His doctoral research was on
communicating the hazards of operating and maintaining innovative environmental technologies for
cleaning up the Department of Energy's nuclear weapons complex. His work led to the development of
over 150 Technology Safety Data Sheets for the Department of Energy. His undergraduate degree is a
B.A. summa cum laude in biology from Western Maryland College. He is a Certified Industrial Hygienist
and Certified Safety Professional. While with the University of Maryland School of Medicine, he co-
authored an extensive review of the hazard communication literature on MSDSs, labels and warnings. He
has participated in the White House Office of Science and Technology Policy's Nanoscale Environment
and Health Initiative. Dr. Lippy has spoken on the worker health and safety issues of nanotechnologies at
the Mount Sinai School of Medicine, the University of Massachusetts at Lowell, the Society for Chemical
Hazard Communication, the American Society of Safety Engineers and the Community Colleges of
Baltimore.
Dr. Tom Long is a staff scientist in the U.S. Environmental Protection Agency's (US EPA) National
Center for Environmental Assessment (NCEA). Here, he prepares science assessments that evaluate
the scientific evidence that relates to the health effects of criteria air pollutants. Prior to joining NCEA,
he conducted research in the laboratory of Dr. Bellina Veronesi on the biological effects of titanium
dioxide and nZVI nanoparticles used in environmental remediation. He has published these findings in
Environmental Science & Technology and Environmental Health Perspectives. He recently received his
Ph.D. from the Department of Environmental Sciences and Engineering, School of Public Health
University of North Carolina at Chapel Hill (2007).
Dr. Gregory Lowry is an associate professor in the department of Civil and Environmental Engineering
at Carnegie Mellon University. He teaches Environmental Engineering, Water Quality Engineering,
Environmental Fate and Transport of Organic Compounds in Aquatic Systems, and Environmental
Sampling and Sample Characterization. His research interest is broadly defined as transport and reaction
in porous media, with a focus on the fundamental physical/geochemical processes affecting the fate of
inorganic and synthetic organic contaminants and engineered nanomaterials in the environment. He is
primarily an experimentalist and works on a variety of application-oriented research projects developing
novel environmental technologies for restoring contaminated sediments and groundwater. His current
projects include in situ sediment management using innovative sediment caps, DNAPL source zone
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remediation through delivery of reactive nanoparticles to the NAPL-water interface, and CO2 capture,
sequestration, and monitoring. The primary goal of most projects is to provide economical engineering
solutions to specific relatively well-defined environmental problems, but each step of engineering
development also provides the opportunity to make fundamental scientific contributions in the areas of
contaminant transport and fate.
M
Dr. Bettye L.S. Maddux is the assistant director of the Safer Nanomaterials and Nanomanufacturing
Initiative, a major research thrust of the Oregon Nanoscience and Microtechnologies Institute and a
member of the Materials Science Institute at the University of Oregon. In 1992, she earned her Ph.D. in
biological sciences with an emphasis in chemical carcinogenesis from the University of Texas at Austin.
Her postdoctoral work at the University of California, Santa Barbara involved elucidating nature's
mechanisms for creating environmentally benign nanomaterials. Previously, she has published peer-
reviewed research articles as 'Bettye L. Smith' in the fields of nanotechnology, biophysics and chemical
carcinogenesis.
Dr. Shaily Mahendra is CBEN Research Associate in the Department of Civil and Environmental
Engineering at Rice University. Dr. Mahendra earned her B.Tech. degree from Indian Institute of
Technology, Delhi, M.S. from Syracuse University, and Ph.D. from University of California, Berkeley.
Her research areas are environmental toxicology and applications of nanomaterials, applications of
molecular and isotopic tools in environmental microbiology, and biodegradation of emerging groundwater
contaminants.
Dr. Susan Masten is a professor in Department of Civil Engineering at McMaster University, Canada.
She obtained her Ph.D. in Environmental Engineering from Harvard University in 1986.
Bharat Mathur was appointed Deputy Regional Administrator of U.S. Environmental Protection
Agency Region 5 in 2002. In this role, he assists the Regional Administrator in implementing federal
environmental programs in the Great Lakes states of Illinois, Indiana, Michigan, Minnesota, Ohio and
Wisconsin.
Mr. Mathur served as Acting Regional Administrator twice — for 16 months, beginning in April 2004,
and again for six months, beginning in April 2006. During his second stint as the Region's acting leader,
he assumed the additional responsibilities of Acting Manager of the Great Lakes National Program. In
this role, he oversaw EPA's continued efforts to protect and clean up the Great Lakes, including advancing
the efforts of the Great Lakes Regional Collaboration and pushing forward with Great Lakes Legacy Act
cleanups.
Mr. Mathur came to EPA in January 2000 as director of the Air and Radiation Division after a lengthy
career with the state of Illinois, where he managed Illinois EPA offices dealing with air pollution,
hazardous and solid waste, and Clean Water Act programs.
He has served on numerous state and national committees to develop environmental policies and
programs, and has consulted with government agencies in India, China, Indonesia, Korea and Mexico
Dr. Charles Maurice has served as the U.S. EPA Office of Research and Development (ORD) Superfund
& Technology Liaison (STL) to Region 5 (Chicago, IL) since April 2004. As such, he holds a joint
appointment with the Office of Science Policy in ORD and with the Innovative Systems & Technology
Branch in the Region 5 Superfund Division. Chuck provides technical support regarding hazardous
substances both through his own expertise as an ecological risk assessor and by coordinating with other
scientists in the technical support centers and laboratories throughout ORD. He also communicates
Regional research priorities and needs to ORD.
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From 1995 to 2004, Chuck was an ecologist and ecological risk assessor the Region 5 Office of
Strategic Environmental Analysis (OSEA), both in the immediate office and on the Critical Ecosystems
Team. Chuck was an ecological risk expert, corrective action manager, and permit writer in the RCRA
Permitting Branch, Region 5 Waste Management Division from 1993 to 1995. Before joining EPA,
Chuck was a senior ecologist and ecological risk assessor for Ecology & Environment, Inc., a Superfund
contractor.
Chuck holds a B.S. degree (1980) in environmental biology from Eastern Illinois University, a M.S.
degree (1982) in biological sciences from Bowling Green State University, and a Ph.D. (1989) in plant
biology from the University of Illinois at Urbana-Champaign.
Dr. Ann Miracle is currently involved in research incorporating environmental biomarkers into
relevant remediation, monitoring, and risk assessment guidelines; and the environmental exposure of
nanomaterials to aquatic organisms. Dr. Miracle leads a team of scientists addressing anthropogenic
impacts to complex, ecological assemblages in freshwater communities using system biology
approaches. In previous employment with the US EPA, Dr. Miracle led a team of scientists in linkages of
chemical exposure and effects using 'omics technologies in small fish models as a part of that agency's
Computational Toxicology Initiative.
Jeff Morris is EPA's National Program Director for Nanotechnology, and is responsible for managing
EPA's Nanomaterials Research Program. Mr. Morris leads the U.S. delegation to the Organization of
Economic Cooperation and Development's Working Party on Manufactured Nanomaterials, and co-
chairs the Working Party's test guidelines steering group. He also co-chaired EPA's Nanotechnology
Coordinating Committee, which issued EPA's Nanotechnology White Paper in February 2007. Prior to
becoming National Program Director for Nanotechnology, Mr. Morris served as acting director of EPA's
Office of Science Policy. His academic training is in economics and environmental policy, and all of
the several positions he has held during his 16 years at EPA have focused on either regulatory issues or
science policy.
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Divina Angela G. Navarro is a graduate student at the Chemistry Department of the University at
Buffalo, The State University of New York, working towards her Ph.D. in Analytical Chemistry.
She obtained her undergraduate degree in B.S. Chemistry at the University of the Philippines at Los
Banos, Philippines. Currently, she is working on studying the fate and transport of quantum dots in the
environment, under the supervision of Dr. Diana Aga and Dr. Sarbajit Banerjee.
Dr. Arianne M. Neigh received her Ph.D. from Michigan State University in Environmental
Toxicology and Zoology. Her work focused on ecosystem-level studies to identify exposure and effects
of poly chlorinated biphenyls to wildlife in a riverine system. This work is to date the most detailed
evaluation of congener pattern changes of organochlorines in aquatic and terrestrial food webs. Dr. Neigh
then joined COM Federal Programs Corporation where she conducted human health and ecological
risk assessments, biological evaluations, remedial investigations, and feasibility studies at hazardous
waste sites for military and industrial clients. In 2007, she joined nanoComposix as a research scientist
to apply her knowledge in risk assessment, environmental fate and transport, and toxicity evaluations
to nanomaterials. Her work with nanomaterials includes evaluating assays for compatibility, high-level
characterization during the course of experiments, and detecting and evaluating nanomaterials in the
environment. Dr. Neigh's work is also focused on developing collaborations with a diverse group of
researches in the US and in Europe in a multi-disciplinary approach to understand nanomaterials and the
environment. She has authored or co-authored nine papers in the area of toxicology and risk assessment,
in addition to presenting her work at national and international scientific meetings.
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Dr. James T. Nurmi is a Senior Research Associate in the Department of Environmental and
Biomolecular Systems, Oregon Health & Science University, Portland, OR.
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Dr. Denis O'Carroll is an Assistant Professor in Civil and Environmental Engineering at the University
of Western Ontario. Dr. O'Carroll completed his Ph.D. at the University of Michigan where he was
awarded the 2004 Walter J. Weber, Jr. Student Prize. Upon completion of his Ph.D. Dr. O'Carroll
completed one postdoctoral fellowship at the University of Michigan and was awarded a Government of
Canada NSERC postdoctoral award to complete a postdoctoral fellowship at the University of Toronto.
He was recently awarded the Province of Ontario 2007 Early Researcher Award for his work in the
"Development of Nanomaterials and Hot Water Flooding for Enhanced Groundwater Remediation".
The goal of this award is to attract and retain the best and brightest research talent in the Province of
Ontario. Dr. O'Carroll has significant experience in laboratory studies developing innovative remediation
schemes in addition to site remediation consulting experience. His work has investigated the utility
of nanotechnology for contaminated site remediation, the impact of soil surface chemistry on NAPL
migration and remediation and the utility of hot water flooding for NAPL remediation. He has ongoing
research projects developing nanometals for contaminated site remediation and investigating the fate of
carbon based nanoparticles in the environment.
Pankaj J. Parikh has been with U.S.EPA over 25 Years. He is an environmental scientist. He worked
in EPA's Chicago Regional laboratory as a team leader/chemist, Asian Pacific Program manager, and
as a project officer for Superfund contracts. He also has been a commissioner on the Village of Mount
Prospect Solid Waste Commission and has served on the Village's Community Relations Commission for
over five years. Prior to joining, EPA, he worked in private industry as a qualty control manager.
Dr. R. Lee Penn is an Associate Professor in the Department of Chemistry, University of Minnesota,
Minneapolis, MN.
Dr. Kurt Pennell is a professor in the School of Civil and Environmental Engineering (CEE) at Georgia
Tech and an adjunct professor in the Department of Neurology at Emory University School of Medicine.
His expertise is in the areas of soil physics, contaminant fate and transport, and multiphase flow.
Tanapon Phenrat is a Civil and Environmental Engineering PhD candidate at Carnegie Mellon
University. His PhD research involves the application of nanoscale zerovalent iron (nZVI) particles
for groundwater and soil remediation. He has published multiple original papers on nanoparticle
characterization in peer-reviewed journals including Environmental Science & Technology, Nano Letters,
and Journal of Nanoparticle Research. In addition, he is involved in an EPA study on the fate, risk, and
toxicity of nanomaterials in the environment.
Dr. Jonathan D. Posner earned his Ph.D. degree in Mechanical Engineering at the University of
California, Irvine in 2001. In addition, he spent 18 months as a fellowship student at the von Karman
Institute for Fluid Mechanics in Rhode Saint Genese, Belgium and two years as a postdoctoral fellow
in the Stanford Microfluidics Laboratory. Dr. Posner is currently an assistant professor at Arizona State
University in the Department of Mechanical and Aerospace engineering and director of the ASU Micro/
Nanofluidics Lab. His interests include manipulation and self-assembly of nanomaterials, the physics of
nanoparticles at interfaces, and transport and fate of nanomaterials in the environment and within animals.
Dr. Posner was honored with a 2008 NSF CAREER award for his work on the physics of self-assembly
of nanoparticles at fluid-solid and fluid-fluid interfaces. He has also been recognized for his Excellence in
Experimental Research by the von Karman Institute for Fluid Dynamics.
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You Qiang is an Associate Professor of Physics, University of Idaho, Moscow ID.
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Eric J. Reardon is a Professor in the Department of Geology, University of Waterloo, Waterloo, ON.
Dr. Krishna R. Reddy is a Professor of Civil and Environmental Engineering at the University of
Illinois at Chicago. He teaches courses on environmental remediation, solid waste management and
landfill engineering, and groundwater flow and contaminant transport modeling. His research expertise is
remediation of contaminated soils, sediments and groundwater, environmental nanotechnologies, waste
containment and landfills, and beneficial reuse of waste materials.
Dr. Bruce Rittmann is a professor of the Department of Civil and Environmental Engineering and
director of the Center for Environmental Biotechnology at Arizona State University. Rittmann is an
international leader in the fields of biofilm kinetics, biological treatment of drinking water, detoxification
of hazardous organic chemicals, nitrification, the use of molecular techniques to study microbial
communities in natural and engineered processes, bioremediation, and mathematical modeling that
couples microbial kinetics to geochemical processes. His professional standing is evidenced by the
numerous research prizes he received and his selection to be a chairman of two National Research
Council committees (Water, Science, and Technology Board and Committee on Intrinsic Remediaion).
Rittmann was elected to the National Academy of Engineering in 2004, cited for pioneering the
development of biofilm fundamentals and contributing to their widespread use in the cleanup of
contaminated waters, soils and ecosystems. Other honors and awards include: Founders Award, USA
National Committee of IAWQ (1998), Fellow, American Association for the Advancement of Sciences
(1996), A.R.I Clarke Prize, National Water Research Institute (1994), Engineering-Science Award, AEEP
(1979, 1993), Montgomery-Watson Award, AEEP (1992, 1995).
Dr. Aaron P. Roberts is an Assistant Professor in the Department of Biological Sciences at University
of North Texas in Denton, TX. Dr. Roberts received his bachelor's degree in biological sciences from
the University of Missouri and his master's degree and Ph.D. in zoology from Miami University. His
laboratory studies the interactive effects of non-chemical and chemical stressors on aquatic organisms
including fish and zooplankton. He is primarily interested in the mechanisms by which these stresses
elicit effects as well as the adaptations organisms use to ameliorate those effects. Work conducted
in his laboratory on carbon nanomaterials has focused on dietary uptake, food chain transport, and
biomodification.
Anna Ryu has research experience in water purification using nanoscale zero-valent iron, DNAPL,
and water treatment of nitrate. She received a M.S. in environmental engineering, Gwangju Institute
of Science and Technology, Gwangju, Korea, and a B.X. in construction, urban, and environmental
engineering from Handong Global University, Pohang, Korea.
Dr. Vaishnavi Sarathy received her Ph.D.from the Department of Environmental and Biomolecular
Systems, Oregon Health & Science University, Portland, OR.
Dr. Christie M. Sayes is currently an Assistant Professor in the Department of Veterinary Physiology
and Pharmacology at Texas A&M University. Before her appointment at A&M, she held a post-doctoral
fellowship at DuPont Haskell Global Centers for Health and Environmental Sciences under the direction
of Dr. David Warheit. She is actively studying the health effects of various nanomaterials in animals,
tissues, and cultured cells. She has made significant correlations between in vitro and in vivo studies,
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which in turn have the potential to shape the landscape of nanotoxicology. Dr. Sayes earned her Doctorate
of Philosophy in Chemistry, specializing in nanotechnology, from Rice University in Dr. Vicki Colvin's
research group and earned her Bachelor's of Science in Chemistry from Louisiana State University,
magna cum laude. Dr. Sayes has authored numerous research publications, reviews, and book chapters.
She has ongoing collaborations and funding with academic, industry, and government. She has received
awards including a Welch Fellowship, the Harry B. Weiser Graduate Student Award for Research, the
Houston Livestock and Rodeo Endowed Scholarship, the International Toxicology of Nanomaterials:
Young Investigator Award, a Society of Toxicology Post-doctoral Award, and a Society of Toxicology
Best Publication Award. She is currently an active member of Texas A&M's Intercollegiate Faculty of
Toxicology as well as the Faculty of Material Sciences & Engineering.
Dr. Kirk Scheckel is a Research Soil Scientist in the Waste Management Branch of the National Risk
Management Research Laboratory at the US Environmental Protection Agency in Cincinnati, OH. Dr.
Scheckel received his Ph.D. from the University of Delaware in Soil Science and a BS in Agronomy
from Iowa State University. Dr. Scheckel professionally serves as an Associate Editor of the Journal of
Environmental Quality, as an Adjunct Assistant Professor of Soil Chemistry at the Ohio State University,
and as Chair-elect of the Division of Environmental Quality for the Soil Science Society of America as
well as other committee assignments. Kirk Scheckel actively participates in laboratory and field research
projects with the assistance of Postdoctoral Fellows and collaborators. The focus of his research program
is solving fundamental problems regarding metal speciation in soils, sediments, and water via advanced,
molecular-level spectroscopic techniques coupled with macroscopic kinetic and thermodynamic
laboratory studies to elucidate reaction mechanisms that influence fate, transport, reactivity, mobility,
bioavailability, and toxicity of metals in the natural environment leading to effective and economic
remediation strategies.
Hatice Sengiil is an Environmental Manufacturing Management fellow at the Institute for Environmental
Science and Policy (IESP) and a Ph.D. student at the Department of Civil and Materials Engineering at
University of Illinois at Chicago (UIC). She has a B.S. degree in environmental engineering from Middle
East Technical University and an M.Sc. degree in environmental engineering from Tulane University.
She worked at TUBITAK (Scientific and Technical Research Council of Turkey) and Simas Engineering
(a private engineering firm based in Ankara) as an intern engineer. At UIC, she has been involved in an
NSF funded research project under the direction of Thomas L. Theis concerning life cycle impacts of
nanomanufacturing techniques. Her research interests include nanotechnology, clean energy, sustainable
technology development, and natechs.
Dr. Virendra Sethi is Professor of Centre for Environmental Science and Engineering, Indian Institute of
Technology Bombay in Mumbai, India. She received her PhD in Environmental Engineering in 1996 and
a M.S. in Environemtal Engineering in 1990 from the University of Cincinnati in Cincinnati, Ohio.
Dr. Jo Anne Shatkin is Managing Director of CLF Ventures, a non-profit affiliate of the Conservation
Law Foundation, New England's most influential environmental advocacy organization. CLF Ventures
works at the intersection of business, stakeholder, and environmental issues to optimize environmental
and economic gain. Dr. Shatkin is a recognized expert in strategic environmental initiatives, human health
risk assessment, technical communications, and environmental aspects of nanotechnology. She leads and
provides expertise on projects and manages the day to day operations of CLF Ventures.
Her work focuses on approaches for evaluating new and emerging contaminants in the environment,
particularly on assessments of chemical and microbial concerns that inform policy development. She
recently developed NANO LCRA, an adaptive life cycle framework for identifying and managing
the risks of nanomaterials, described in her book, Nanotechnology Health and Environmental Risks,
published in 2008 (CRC Press). Dr Shatkin recently founded the Emerging Nanoscale Materials Specialty
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Group of the Society for Risk Analysis, with 130 international members from public and private
organizations. A Research Fellow at the George Perkins Marsh Institute at Clark University, she received
her Ph.D. in Environmental Health Science and Policy in 1994 and her MA in Risk Management and
Technology Assessment, both from Clark University, Worcester, Massachusetts and possesses a Bachelor
of Science degree from Worcester Polytechnic University in molecular biology.
Dr. Weiguo Song is Professor of Chemistry, Key Laboratory for Molecular Nanostructures and
Nanotechnologies, Institute of Chemistry, the Chinese Academy of Sciences (ICCAS) in Beijing, Chinia.
He received a Ph.D. in Physical Chemistry in 2001 from the University of Southern California, Los
Angeles, California, a B. Sc. In Chemistry in 1992 from Beijing University, Beijing, China. Prior to
joining ICCAS, he was a Postdoctoral Research Associate at the University of Iowa, Iowa City, Iowa,
from 2003 to 2005, and at the Loker Hydrocarbon Research Institute, University of Southern California in
Los Angeles, California, from 2001 to 2003. He was named to National Science Fund for Distinguished
Young Scholars (2007) and the Chinese Academy of Sciences One Hundred Talented Program (2005)
Dr. Desmond Dion Stubbs received a B.S. degree in Chemistry from Morris Brown College, Atlanta GA
in 1997. He later received his M.S. in Chemistry from Georgia Tech in 1999. After working in Georgia
Tech's School of Chemistry as a Demonstrations Teacher for two years he later returned to Georgia
Tech and received his doctoral degree in May, 2006. One of the highlights of his graduate career was a
publication in Analytical Chemistry entitled "Investigation of a Cocaine Plume Using Surface Acoustic
Wave Immunoassay Sensors". The paper was then flagged by the American Chemical Society interest and
later featured as a press release on their website. The story led to numerous media interviews including
an appearance on Fox News (cable service) and a feature in Time Magazine's new series Innovators
highlighting the "dog-on-a-chip" a chemical sensing electronic device. Desmond currently holds joint
positions at Oak Ridge Associated Universities (ORAU) and Battelle as a Senior Project manager and a
Scientist in Residence respectively.
Dr. Chunming Su is a Soil Scientist in the Subsurface Remediation Branch in the Ground Water and
Ecosystems Restoration Division (GWERD) of the USEPA's National Risk Management Research
Laboratory, Ada, Oklahoma. He received a B.S. degree from China Agricultural University, China, an
M.S. degree from University of Guelph, Canada, and a Ph.D. degree from Washington State University,
all in Soil Science. His former work experience includes a term soil scientist position with the U.S.
Department of Agriculture, a National Research Council Resident Research Associateship, and a project
scientist position with ManTech Environmental Research and Services Corporation. Dr. Su conducts
laboratory and field investigations in environmental geochemistry and nanotechnology. He is interested
in studying: (1) applications and implications of environmental nanotechnology with respect to fate
and transport of nanomaterials in the subsurface, (2) in situ treatment of organic (chlorinated solvents)
and inorganic (chromate, arsenic, nitrate, etc) contaminants in ground water and soils using permeable
reactive barrier technologies and monitored natural attenuation approaches, (3) arsenic sorption and redox
transformation processes using specimen iron minerals including green rusts and iron oxides, and (4)
organic contaminant degradation pathways using stable isotopes. Dr. Su is the principal author of more
than 30 peer-reviewed journal articles and book chapters, and a co-recipient of a US patent. He also has
served as a technical reviewer for numerous scientific journals including Environmental Science and
Technology, Geochimica et Cosmochimica Acta, Chemistry of Materials, and Soil Science Society of
America Journal; and on proposal review panels for the Department of Commerce, EPA, USDA, and
USGS. Dr. Su has received several EPA awards for his research and technical support activities (including
EPA Scientific and Technological Achievement Awards and an ORD Honor Award for Exceptional/
Outstanding Technical Assistance to the Regions and Program Offices).
Dr. Rao Y. Surampalli is the Engineer Director with United States Environmental Protection Agency
(USEPA, Region 7). He received M.S and Ph.D. degrees in Environmental Engineering from Oklahoma
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State and Iowa State Universities, respectively. He is a Registered Professional Engineer and has
authored more than 370 technical publications, including five books, 31 book chapters, 136 refereed
(peer-reviewed) journal articles, presented at more than 180 national and international conferences, and
given over 30 plenary, keynote or invited presentations worldwide. Currently, he serves on 39 national
and international committees, review panels, or advisory boards including the ASCE's National Energy,
Environmental and Water Resources Policy Committee. He is Editor of two well-known refereed journals
- the Water Environment Research Journal published by the Water Environment Federation (WEF), and
the Hazardous, Toxic, and Radioactive Waste Management Journal published by the American Society of
Civil Engineers (ASCE). He also serves on the Editorial Boards of three other Environmental Journals.
His main expertise includes, but is not limited to emerging contaminants including nanomatetrials, water/
wastewater treatment, hazardous/solid waste management, soil and groundwater treatment, and sludge
treatment/disposal.
Dr. Robert Tanguay received a B.A. degree in biology from California State University, San Bernardino
in 1988 and his Ph.D. degree in biochemistry from the University of California, Riverside in 1995. He
received postdoctoral training in molecular and developmental toxicology with Richard E. Peterson
at the University of Wisconsin between 1996 and 1999. He is currently an Associate Professor in the
Department of Environmental and Molecular Toxicology at Oregon State University and is the director of
the Sinnhuber Aquatic Research Laboratory. His current research interests include developmental biology,
nanotoxicology, developmental toxicology, regenerative medicine, and chemical genetics.
Dr. Volodymyr Tarabara is an assistant professor in Department of Civil and Environmental
Engineering at Michigan State University. He obtained his Ph.D. in Environmental Engineering and
Computational Science and Engineering from Rice University in 2004.
Leigh M. Taylor is an undergraduate student at the University of North Texas in Denton, TX. Leigh is an
undergraduate research assistant in Dr. Aaron Roberts' lab.
Dr. Thomas L. Theis is the director of the Institute for Environmental Science and Policy and a full
professor at the Department of Civil and Materials Engineering, University of Illinois at Chicago.
Professor Theis' areas of expertise include the mathematical modeling and systems analysis of
environmental processes, the environmental chemistry of trace organic and inorganic substances,
interfacial reactions, subsurface contaminant transport, hazardous waste management, industrial pollution
prevention, and industrial ecology. He has been principal or co-principal investigator on over forty
funded research projects totaling in excess of eight million dollars, and has authored or co-authored
over one hundred papers in peer reviewed research journals, books, and reports. He is a member of the
USEPA Science Advisory Board (Environmental Engineering Committee), is erstwhile editor of the
Journal of Environmental Engineering, and serves on the editorial boards of The Journal of Contaminant
Transport, and Issues in Environmental Science and Technology. From 1980-1985 he was the co-director
of the Industrial Waste Elimination Research Center (a collaboration of Illinois Institute of Technology
and University of Notre Dame), one of the first Centers of Excellence established by the USEPA. He
is currently Principal Investigator on the Environmental Manufacturing Management Program, one
of the Integrated Graduate Education Research and Training (IGERT) grants of the National Science
Foundation.
Dr. Vinay Tiwari is a Research Scholar at the Centre for Environmental Science and Engineering, Indian
Institute of Technology Bombay, Mumbai, India. He receive a M.E. in Chemical Engineering in 2004
from the Nirma Institute of Technology, Gujarat, India
Dr. Paul G. Tratnyek is Professor in the Department of Environmental and Biomolecular Systems,
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Oregon Health & Science University, Portland, OR.
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Dr. Ashok K. Vaseashta is Professor of physics and physical sciences in the Graduate Program
in physical sciences at Marshall University, Huntington, WV. Presently, he is on detail to the U.S.
Government. He received a B.S. and M.S. in Physics Honors from the University of Delhi, M.Tech.
from the Indian Institute of Technology, Delhi, and a Ph.D. from Virginia Polytechnic Institute and State
University, Blacksburg, VA. He directs research at the Nanomaterials Processing and Characterization
Laboratories at Marshall University. His current research interests include nanostructured materials
for energy generation and storage; development of chemical-bio sensors; and use of nanomaterials for
monitoring, detecting and remediation of environmental pollution. He is one of the leading researchers in
the field of green nanotechnology. He has authored over 170 research publications, edited/authored two
books on nanotechnology, presented many keynote and invited lectures worldwide, served as Director of
two NATO Advanced Study Institutes, and co-chair of an International Symposium on Nanotechnology in
Environmental Protection and Pollution in Ft. Lauderdale, FL. He is an active member of several national
and international professional organizations. He has earned several awards for his meritorious service
including 2004/2005 Marshall University Distinguished Artist and Scholar (MU DASA) award. His
experience spans the spectrum of academic and industrial positions. He has visiting positions at several
national laboratories and universities in Eastern Europe. He also serves on the Nanotechnology Standard
Committee of ISO/ANSI TAG-TC 229 and ASTM.
Dr. Bellina Veronesi is a senior scientist at the US EPA Division of Neurotoxicology (NHEERL). She
is an in vitro and in vivo experimentalist and has published extensively in the areas of in vitro modeling,
pesticide neuropathology and air pollution neurotoxicity. More recently, she has documented the oxidative
stress-mediated neurotoxicity of various nanomaterials used in environmental remediation. Currently,
she is developing in vitro models to examine how the physical properties of nanoparticles influence their
movement through biological barriers such as the intestines and blood brain barrier.
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Dr. T. David Waite is Director, Centre for Water and Waste Technology, and Director of Research,
School of Civil and Environmental Engineering, The University of New South Wales. He hold a Ph.D.
in environmental engineering from the Massachusetts Institute of Technology, Cambridge, MA (USA);
a M.App.Sci. from Monash University, Melbourne, Victoria ,(Australia); a Grad. Diploma of Electronic
Instrumentation from the Royal Melbourne Institute of Technology, Melbourne, Victoria (Australia); and
a Bachelor of Science (Honours) from the University of Tasmania, Hobart, Tasmania (Australia). His
research interests include chemical processes involving colloids and particles in aquatic systems; redox
chemistry at the solid-solution interface; photochemistry in aquatic systems; water and wastewater
treatment processes; hydrometallurgical techniques involving redox processes; hydrogeochemistry;
theoretical and experimental studies on the fate and effects of chemical pollutants; and interactions
between trace elements and microbiota in aquatic systems.
Barbara T. Walton is Assistant Laboratory Director for Emerging Programs, National Health
and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency. She has
responsibility for EPA's research on the health and ecological effects of nanomaterials.
Walton's 20-year career at the U.S. Department of Energy's Oak Ridge National Laboratory focused
on the ecotoxicology of organic, inorganic, and radioactive contaminants in terrestrial and aquatic
ecosystems. Before joining EPA, Barbara was Senior Policy Analyst for Environment, White House
Office of Science and Technology Policy, Washington, DC. She's a board-certified toxicologist
(American Board of Toxicology) and Adjunct Professor, Department of Environmental Science and
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Engineering, University of North Carolina-Chapel Hill. Walton is a former President of the Society of
Environmental Toxicology and Chemistry.
Yonggang Wang is currently a Ph.D. candidate in the School of Civil and Environmental Engineering
at Georgia Tech. His research area is experimental investigation of fullerene nanoparticle aggregates
transport in saturated and unsaturated soils.
Dr. Mahmoud Wazne is Assistant Professor, Department of Civil, Environmental and Ocean
Engineering, Stevens Institute of Technology in Hoboken, New Jersey. He received his Ph.D. in
Environmental Engineering in 2003 from Stevens Institute of Technology, a M.S. in 1991 and a B.S.in
1990 in Civil Engineering from Columbia University in New York. He was a Research Assistant
Professor, Department of Civil, Environmental and Ocean Engineering., Stevens Institute of Technology
from 2004 to 2005; a Postdoctoral Research Associate, Stevens Institute of Technology from 2003 to
2004; a Graduate Research Assistant, Stevens Institute of Technology from 2000 to 2003; a Licensed
Civil Engineer, Sands Contractors from 1992 to 1998; Lecturer, City University of New York from 199 Ito
1992; and a Graduate Research Assistant, Columbia University from 1990 to 1991.
Yu-Ting Wei is currently a Ph.D. student in the Graduate Institute of Environmental Engineering at
National Taiwan University (NTU). He works with Dr. Shian-Chee Wu for investigating the feasibility
of using iron nanoparticle for groundwater remediation in field tests. He has been working as a senior
engineer at Apoll Tech Environmental Consulting and Engineering Company in Taiwan for over 10 years.
Ryan Westafer is a doctoral candidate and NNCS fellow at the Georgia Institute of Technology. He was
previously awarded the President's Scholarship at Georgia Tech and subsequently graduated with Highest
Honor in Computer Engineering in 2005. After a brief stint in residential broadband at the Broadcom
Corporation, he received the MSECE degree from Georgia Tech in 2006. As a graduate researcher in the
Microelectronic Acoustics Group, Ryan has since authored multiple papers in the area of surface acoustic
wave devices and sensors.
Dr. Paul Westerhoff is a professor and chair of the Department of Civil and Environmental Engineering
in Arizona State University's Ira A. Fulton School of Engineering. His research focuses on water quality
and treatment, and he has led the department's environmental and water faculty group for the past six
years. He has earned some of the leading research awards from the American Society of Civil Engineers
and the Water Environment Federation. In 2006, the WERF Endowment for Innovation in Applied Water
Quality Research presented Westerhoff with the Paul L. Busch Award for his research investigating the
fate of commercial nanomaterials in drinking water and wastewater treatment plants, and their potential
human toxicity. More than 65 of his research articles have been published in peer-reviewed science and
engineering journals, and he has made more than 200 conference presentations. Westerhoff earned a
bachelor of science from Lehigh University, a master's degree from the University of Massachusetts-
Amherst, and a PhD from the University of Colorado-Boulder.
Dr. Frank A. Witzmann is Professor of Cellular & Integrative Physiology at the Indiana University
School of Medicine. He has applied gel and mass spec-based proteomic analyses in a variety of paradigms
for over two decades and currently directs the use of these proteomic approaches in projects concerning
various aspects of toxicology and cardiovascular, renal, and CNS physiology.
Dr. Shian-Chee Wu is a professor in the Graduate Institute of Environmental Engineering at National
Taiwan University (NTU). He received his Ph.D. in Environmental Engineering in 1987 from
Massachusetts Institute of Technology (MIT), under the guidance of Prof. Philip M. Gschwend. He
became a faculty member at the NTU in 1998. His research interests focus on environmental pollutants
fate and environmental hazard assessment. He serves as an Asia regional editor for Environmental
Engineering Science.
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Gary Wyss is a Senior Geochemist for MSB Technology Applications in Butte, Montana. Mr. Wyss
holds master's degrees in Chemistry and Geology from Montana Tech in Butte. His previous experience
includes serving as Laboratory Manager, Quality Assurance Officer, and Organic Chemist for HKM
Analytical Laboratory in Butte, Montana.
Weile Yan is currently a Ph.D. candidate at civil and environmental engineering, Lehigh University,
working on nano-engineered zerovalent-iron materials for environmental applications. She received her
bachelor's degree (B. Eng) in environmental engineering from the National University of Singapore, and
holds a master degree in molecular engineering for biological and chemical systems from the Singapore-
MIT Alliance.
Dr.Gordon C. C. Yang is Professor, Institute of Environmental Engineering, National Sun Yat-Sen
University, Taiwan. His specialties and research interests include Nanotechnology and the Environment;
Membrane Technology Preparation, Characterization, and Applications; Treatment and Reclamation of
Nanoparticles-Containing Wastewaters; Remediation of Contaminated Soil and Groundwater; Hazardous
Waste Management and Treatment; and Resources Recovery and Recycling. He received his Ph.D. from
the University of California, Berkeley in 1983, a S., University of Alaska in 1979, and a B.S. from the
National Cheng-Kung University in Taiwan in 1974. He was Editor, Journal of Hazardous Materials
(1998-2002), Editorial Board Member, Journal of Hazardous Materials (1994-1998), Guest Editor,
Journal of Hazardous Materials—Special Issue on Waste Management Technology in Taiwan '97 (1998).
Dr. Marek Zaluski holds Master and Ph.D. degrees in Hydrogeology. He is currently working for MSE
Technology Applications in Butte, Montana as a Staff Hydrogeologist. His previous experience includes
professorship at Montana Tech in Butte, consulting in hydrogeology and environmental sciences in
Michigan, Illinois, Wisconsin and in Libya, North Africa, as well as research for Geological Institute in
Poland.
Dr. Wei-xian Zhang is Professor of Environmental Engineering, Advanced Materials and
Nanotechnology at Lehigh University, Bethlehem, Pennsylvania. He teaches Introduction to
Environmental Engineering and Environmental Nanotechnology. His research is in the area of chemical
and biological transformation of environmental contaminants such as chlorinated organic solvents,
pesticides, PCBs and heavy metal ions. His research group has pioneered the research and development of
iron nanoparticles for environmental remediation.
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