United States
Environmental Protection
Agency
U.S. EPA NANOTECHNOLOGY
GRANTEES MEETING
—REPORT-
IN CONJUNCTION WITH
SETAC NORTH AMERICA 31ST ANNUAL MEETING
BRIDGING SCIENCE WITH COMMUNITIES
NOVEMBER 8-9, 2010
OREGON CONVENTION CENTER
ROOMS D135 AND D136 ON LEVEL 1
PORTLAND, OREGON
SOQ8
National Center for Environmental Research
Science To Achieve Results (STAR) Research Program
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The Office of Research and Development's National Center for Environmental Research
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U.S. EPA Nanotechnology Grantees Meeting
Table of Contents
Agenda (presenter's name is linked to his or her presentation) vii
Meeting Summary 1
Abstracts and Presentations (listed in the same order as in the Agenda)
Day 1, Monday, November 8, 2010
AM Session 1: Systems Approaches
An Integrated Approach Toward Understanding the Impact of Aggregation and Dissolution of Metal
and Metal Oxide Nanoparticles 47
Vicki Grassicm, University of Iowa
Life Cycle Analysis and Nanostructured Materials 52
Thomas Theis, University of Illinois at Chicago
Platinum-Containing Nanomaterials: Sources, Speciation, and Transformation in the Environment 57
Martin Shafer, University of Wisconsin-Madison
Role ofNLRP3 Inflammasome and Nickel in Multi-Walled Carbon Nanotube-Induced Lung Injury 64
Andrij Holian, The University of Montana
AM Session 2: Effects of Nanoparticle Surface Properties
Microbial Bioavailability of Polyethylene Oxide Grafted to Engineered Nanomaterials 67
Gregory Lowry, Carnegie Mellon University
Surface Oxides: Their Influence on Multi-Walled Nanotubes Colloidal, Sorption, and Transport
Properties 72
Howard Fairbrother, Johns Hopkins University
Development of Hyphenated and "Particle Counting" ICP-MS Methods Exposure Assessment
of Inorganic Nanoparticles 79
James Ranville, Colorado School of Mines
Controlled Release of Biologically Active Silver From Nanosilver Surfaces 85
Jingyu Liu, Brown University
Effects of Polyethyleneimine Surface Modifications of Multi-Walled Carbon Nanotubes: Their
Toxicity, Sorption Behaviors, and Ecological Uptake by Earthworms and Daphnia Magna 89
Roger Pinto, University of Michigan, Ann Arbor
PM Session 1: Characterization Methods
A Biological Surface Adsorption Index for Characterizing Nanomaterials in Aquatic Environments
and Their Correlation With Skin Absorption of Nanomaterials 92
Xin-Rui Xia, North Carolina State University
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U.S. EPA Nanotechnology Grantees Meeting
Flexible Nanostructured Conducting Poly(amic) Acid Membrane Captures, Isolates, and Simultaneously
Detects Engineered Nanoparticles 97
Wunmi Sadik, State University of New York at Binghamton
Fate and Effects of Nanosized Metal Particles Examined Along a Simulated Terrestrial Food Chain
Using Genomic and Microspectroscopic Techniques 99
Jason Unrine, University of Kentucky
Determination of Manufactured Nanoparticle Toxicity Using Novel Rapid Screening Methods 105
John J. Rowe, MaqusoodAhamed, Ryan Posgai, Tiling Hong, Jayne Robinson,
and Mark Nielsen
PM Session 2: Environmental Effects on Nanoparticles
Influence of Natural Organic Matter on the Behavior and Bioavailability of Carbon Nanoparticles
in Aquatic Ecosystems 112
Stephen Klaine, Clemson University
Environmental Photochemical Reactions of nC6o and Functionalized Single-Walled Carbon Nanotubes
in Aqueous Suspensions 117
ChadJafvert, Purdue University
Impact of Photochemical Oxidation on the Stability of nC6o and Multi-Walled Carbon Nanotubes
in Aqueous Solutions 126
Qilin Li, Rice University
The Environmental Behaviors of Multi-Walled Carbon Nanotubes in Aquatic Systems 132
Quingguo Huang, University of Georgia
Day 2, Tuesday, November 9, 2010
AM Session 1: Effects on Cells
Functional Effects of Nanoparticle Exposure on Airway Epithelial Cells 138
Amiraj Banga, Indiana University-Purdue University at Indianapolis
Toxicity Assessment of Nanomaterials in Alveolar Epithelial Cells at the Air-Liquid Interface 144
Galya Orr, Pacific Northwest National Laboratory
Interactions of Nanomaterials With Model Cell Membranes 146
Jonathan Posner, Arizona State University
Development of an In Vitro Test and a Prototype Model To Predict Cellular Penetration of Nanoparticles 154
Yongsheng Chen, Georgia Institute of Technology
AM Session 2: Effects at Sub-Cellular Level
Impacts of Quantum Dots on Gene Expression mPseudomonas aeruginosa 161
Shaily Mahendra, University of California, Los Angeles
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U.S. EPA Nanotechnology Grantees Meeting
Thiol Redox-Dependent Toxicity and Inflammation Caused by TOPO-PMAT Modified Quantum Dots 164
Terrence Kavanagh, University of Washington
Bioavailability and Fates of CdSe and Ti02 Nanoparticles in Eukaryotes and Bacteria 168
Patricia Holden, University of California, Santa Barbara
Using Zebrafish Embryos To Test Phototoxicity of Ti02 Nanoparticles 174
Warren Heideman, University of Wisconsin-Madison
PM Session 1: Effects on Fish and Oysters
Effects of Subchronic Exposure to Nanoparticulate Silver in Zebrafish 180
David Barber, University of Florida
Refinements to the Use of Zebrafish for Nanomaterial-Biological Interaction Assessments 185
Robert Tanguay, Oregon State University
Impacts of Functionalization of Fullerenes and Carbon Nanotubes on the Immune Response
of Rainbow Trout 192
Devrah Arndt, University of Wisconsin-Milwaukee
Characterization of the Potential Toxicity of Metal Nanoparticles in Marine Ecosystems Using
Oysters - Silver Nanoparticle Studies With Adults and Embryos 198
Amy Ring-wood, University of North Carolina-Charlotte
PM Session 2: Nanoparticles and Waste Treatment
Bioavailability of Metallic Nanoparticles and Heavy Metals in Landfills 206
Zhiqiang Hu, University of Missouri
Biological Fate and Electron Microscopy Detection of Nanoparticles During Wastewater Treatment 212
Paul Westerhoff Arizona State University
Analysis and Fate of Single-Walled Carbon Nanotubes and Their Manufacturing Byproducts
in Estuarine Sediments and Benthic Organisms 217
P. Lee Ferguson, Duke University
Safety/Toxicity Assessment of Ceria (A Model Engineered NP) to the Brain 225
Robert Yokel, University of Kentucky
Handouts on Centers for Environmental Implications of Nanotechnology (CEIN)
University of California 233
Duke University 235
Speaker List 237
Participants List 241
Links to Information on Federal Agency Nanotechnology Programs 251
E-announcement Flyer 252
The Office of Research and Development's National Center for Environmental Research v
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VI
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U.S. EPA
In Conjunction with tie
Science with
(Session and presentation times in this agenda are the same as in the SETAC agenda)
Meeting Contacts: shapiro.paul@epa.gov and conley.tina@epa.gov
Registration Contact: dhoffman@scgcorp.com
DAY1, Monday, November 8, 2010
7:30-7:45 a.m.
7:45- 8:00 a.m.
8:00-9:35 a.m.
8:00- 8:20 a.m.
8:25- 8:45 a.m.
8:50-9:10 a.m.
9:15- 9:35 a.m.
9:35- 10:15 a.m.
Registration (Rooms D135 and D136)
Welcome and Ground Rules
AM Session 1: Systems Approaches
An Integrated Approach Toward Understanding the Impact of
Aggregation and Dissolution of Metal and Metal Oxide Nanoparticles
Vicki Grass!an, University of Iowa
Life Cycle Analysis and Nanostructured Materials
Thomas Theis, University of Illinois at Chicago
Platinum-Containing Nanomaterials: Sources, Speciation, and
Transformation in the Environment
Martin Shafer, University of Wisconsin-Madison
Role of NLRP3 Inflammasome and Nickel in Multi-Walled Carbon
Nanotube-Induced Lung Injury
Andrij Holian, The University of Montana
BREAK
10:15 - 11:50 a.m. AM Session 2: Effects of Nanoparticle Surface Properties
10:15 - 10:35 a.m. Microbial Unavailability of Polyethylene Oxide Grafted to Engineered
Nanomaterials
Gregory Lowry, Carnegie Mellon University
10:40 - 11:00 a.m. Surface Oxides: Their Influence on Multi-Walled Nanotubes Colloidal,
Sorption, and Transport Properties
Howard Fairbrother, Johns Hopkins University
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DAY 1, Monday, November 8, 2010 (Continued)
11:05-11:25 a.m. Development of Hyphenated and "Particle Counting" ICP-MS
Methods Exposure Assessment of Inorganic Nanoparticles
James Ranville, Colorado School of Mines
11:30 - 11:50 a.m. Controlled Release of Biologically Active Silver From Nanosilver
Surfaces
Jingyu Liu, Brown University
Note: Additional Presentation that could not be presented at the meeting:
Effects of Polyethyleneimine Surface Modifications of Multi-Walled
Carbon Nanotubes: Their Toxicity, Sorption Behaviors, and Ecological
Uptake by Earthworms and Daphnia Magna
Roger Pinto, University of Michigan, Ann Arbor
11:50 a.m. - 1:45 p.m. LUNCH
1:45 - 3:30 p.m. PM Session 1: Characterization Methods
1:45 - 2:15 p.m. A Biological Surface Adsorption Index for Characterizing
Nanomaterials in Aquatic Environments and Their Correlation With
Skin Absorption of Nanomaterials
Xin-Rui Xia, North Carolina State University
2:20 - 2:40 p.m. Flexible Nanostructured Conducting Poly(amic) Acid Membrane
Captures, Isolates, and Simultaneously Detects Engineered
Nanoparticles
Wunmi Sadik, State University of New York at Binghamton
2:45 - 3:05 p.m. Fate and Effects of Nanosized Metal Particles Examined Along a
Simulated Terrestrial Food Chain Using Genomic and
Microspectroscopic Techniques
Jason Unrine, University of Kentucky
3:10 - 3:30 p.m. Determination of Manufactured Nanoparticle Toxicity Using Novel
Rapid Screening Methods
John Rowe, University of Dayton
3:30-4:10 p.m. BREAK
4:10 - 5:45 p.m. PM Session 2: Environmental Effects on Nanoparticles
4:10 - 4:30 p.m. Influence of Natural Organic Matter on the Behavior and
Bioavailability of Carbon Nanoparticles in Aquatic Ecosystems
Stephen Klaine, Clemson University
4:35 - 4:55 p.m. Environmental Photochemical Reactions of nCeo and Functionalized
Single-Walled Carbon Nanotubes in Aqueous Suspensions
Chad Jafvert, Purdue University
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DAY 1, Monday, November 8, 2010 (Continued)
5:00 - 5:20 p.m. Impact of Photochemical Oxidation on the Stability of nCeo and Multi-
Walled Carbon Nanotubes in Aqueous Solutions
Qilin Li, Rice University
5:25 - 5:45 p.m. The Environmental Behaviors of Multi-Walled Carbon Nanotubes in
Aquatic Systems
Quingguo Huang, University of Georgia
5:45 - 6:30 p.m. Open Discussion
6:30 p.m. Adjournment
DAY2, Tuesday, November 9, 2010
7:30 - 7:45 a.m. Registration (Rooms D135 and D136)
7:45 - 8:00 a.m. Review of Monday and Plans/Ground Rules for Today
8:00 - 9:35 a.m. AM Session 1: Effects on Cells
8:00 - 8:20 a.m. Functional Effects of Nanoparticle Exposure on Airway Epithelial Cells
Amiraj Banga, Indiana University-Purdue University at Indianapolis
8:25 - 8:45 a.m. Toxicity Assessment of Nanomaterials in Alveolar Epithelial Cells at
the Air-Liquid Interface
Galya Orr, Pacific Northwest National Laboratory
8:50 - 9:10 a.m. Interactions of Nanomaterials With Model Cell Membranes
Jonathan Posner, Arizona State University
9:15 - 9:35 a.m. Development of an In Vitro Test and a Prototype Model To Predict
Cellular Penetration of Nanoparticles
Yongsheng Chen, Georgia Institute of Technology
9:35 - 10:15 a.m. BREAK
10:15 - 11:50 a.m. AM Session 2: Effects at Sub-Cellular Level
10:15 - 10:35 a.m. Impacts of Quantum Dots on Gene Expression in Pseudomonas
aeruginosa
Shaily Mahendra, University of California, Los Angeles
10:40 - 11:00 a.m. Thiol Redox-Dependent Toxicity and Inflammation Caused by TOPO-
PMAT Modified Quantum Dots
Terrence Kavanagh, University of Washington
11:05-11:25 a.m. Bioavailability and Fates of CdSe and TiOi Nanoparticles in
Eukaryotes and Bacteria
Patricia Holden, University of California, Santa Barbara
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DAY 2, Tuesday, November 9, 2010 (Continued)
11:30 - 11:50 a.m. Using Zebrafish Embryos To Test Phototoxicity of TiOi Nanoparticles
Warren Heideman, University of Wisconsin-Madison
11:50 a.m. - 1:45 p.m. LUNCH
1:45-3:30 p.m. PM Session 1: Effects on Fish and Oysters
1:45 - 2:15 p.m. Effects of Subchronic Exposure to Nanoparticulate Silver in Zebrafish
David Barber, University of Florida
2:20 - 2:40 p.m. Refinements to the Use of Zebrafish for Nanomaterial-Biological
Interaction Assessments
Robert Tanguay, Oregon State University
2:45 - 3:05 p.m. Impacts of Functionalization of Fullerenes and Carbon Nanotubes on
the Immune Response of Rainbow Trout
Devrah Arndt, University of Wisconsin-Milwaukee
3:10 - 3:30 p.m. Characterization of the Potential Toxicity of Metal Nanoparticles in
Marine Ecosystems Using Oysters - Silver Nanoparticle Studies With
Adults and Embryos
Amy Ringwood, University of North Carolina-Charlotte
3:30-4:10 p.m. BREAK
4:10-5:45 p.m. PM Session 2: Nanoparticles and Waste Treatment
4:10 - 4:30 p.m. Bioavailability of Metallic Nanoparticles and Heavy Metals in Landfills
Zhiqiang Hu, University of Missouri
4:35 - 4:55 p.m. Biological Fate and Electron Microscopy Detection of Nanoparticles
During Wastewater Treatment
Paul Westerhoff, Arizona State University
5:00 - 5:20 p.m. Analysis and Fate of Single-Walled Carbon Nanotubes and Their
Manufacturing Byproducts in Estuarine Sediments and Benthic
Organisms
P. Lee Ferguson, Duke University
5:25 - 5:45 p.m. Safety/Toxicity Assessment of Ceria (A Model Engineered NP) to the
Brain
Robert Yokel, University of Kentucky
5:45 - 6:30 p.m. Open Discussion
6:30 p.m. Adjournment
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U.S. EPA Nanotechnology Grantees Meeting
U.S. EPA Nanotechnology Grantees Meeting
Oregon Convention Center
Rooms D135 and D136
777 NE Martin Luther King Jr. Boulevard
Portland, OR
November 8-9, 2010
MEETING SUMMARY
The U.S. Environmental Protection Agency held this meeting in conjunction with the Society
of Environmental Toxicology and Chemistry's (SETAC) North America 31st Annual
Meeting: Bridging Science with Communities.
The Office of Research and Development's National Center for Environmental Research
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U.S. EPA Nanotechnology Grantees Meeting
The Office of Research and Development's National Center for Environmental Research
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U.S. EPA Nanotechnology Grantees Meeting
NOVEMBERS, 2010
OVERVIEW
The U.S. Environmental Protection Agency (EPA) currently funds research that focuses on what happens
to nanoparticles, and what impacts on aquatic organisms the particles have, when they enter water
environments. EPA holds an annual meeting at which its nanotechnology grantees present their research.
There may also be presentations by researchers who have been funded by other Federal agencies with
which EPA co-sponsored a Request for Applications. The purpose of the 2010 meeting was to provide a
forum for the researchers to share their findings, problems, solutions, and project plans, and to address
issues of common concern.
The meeting was held in conjunction with the Society for Environmental Toxicology and Chemistry
(SETAC) North America 31st Annual Meeting: Bridging Science with Communities so that EPA
researchers could attend the SETAC meeting and people attending the SETAC meeting could attend the
EPA meeting. The meetings were coordinated so that the EPA meeting was held at the beginning of the
week and the SETAC nanotechnology sessions were held later in the week. As a result, there were 117
attendees from academia, industry, and government at the EPA meeting. For information concerning the
SETAC meeting go to: http://portland.setac.org/
The meeting was organized by Paul Shapiro of the EPA Office of Research and Development (ORD)
National Center for Environmental Research (NCER). The leader of the NCER nanotechnology research
program is Nora Savage. Mitch Lasat and Michael McKittrick are also members of the NCER
nanotechnology team.
Welcome
Paul Shapiro, EPA
Mr. Shapiro called the meeting to order at 7:45 am and welcomed the participants. He introduced Nora
Savage, Mitch Lasat, and the contractor support staff. He explained the logistics of the meeting. He
emphasized the need to stick to the schedule because it matched the SETAC schedule, which set the length
of each presentation at 20 minutes and the time between each presentation at 5 minutes.
Mr. Shapiro said that in the past attendees have requested an opportunity to have an open discussion of
issues that come up during the presentations. He said that the schedule for this meeting includes an open
discussion session at the end of each day. There was an easel at the front of the room to serve as a "parking
lot" for attendees to write down topics they would like to discuss during these open sessions.
Meeting participants were asked to complete evaluation forms of the sessions each day and to submit them
to the meeting staff at the registration table. Mr. Shapiro noted that those presentations for which the
presenters give permission will be published on the Web site following the meeting.
Dr. Savage explained that the National Nanotechnology Initiative (NNI) is in the process of finalizing its
2010 Strategic Plan; public comment currently is being accepted. She announced that a Gordon Research
Conference focused on environmental nanotechnology will be held at the Waterville Valley Resort in New
Hampshire from May 29 to June 3, 2011. The conference steering committee is accepting abstract sub-
missions. Every accepted oral presentation also will be required to have an accompanying poster presented
during the conference. EPA also is working with the Organisation for Economic Co-operation and
Development (OECD) on a research strategy to understand fate and transport of nanomaterials to ultimately
understand toxicity. The next Nanotechnology Grantees Meeting will be held at Duke University in May
2011 in conjunction with a meeting sponsored by the Duke University Center for the Environmental
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U.S. EPA Nanotechnology Grantees Meeting
Implications of NanoTechnology (CEINT) and the University of California, Los Angeles Center for the
Environmental Implications of Nanotechnology (commonly known as CEIN). Dr. Savage asked
participants who have ideas for future meetings to submit them to her or Mr. Shapiro.
MORNING SESSION 1: SYSTEMS APPROACHES
An Integrated Approach Toward Understanding the Impact of Aggregation and Dissolution of Metal
and Metal Oxide Nanoparticles
Vicki Grassian, University of Iowa
This project aims at understanding the environmental and health implications of nanotechnology from the
perspectives of air, water, and soil. The researchers are interested in the toxicity of nanomaterials and have
partnered with other researchers to examine inhalation exposure to nanomaterials. Also of interest are
particles with size-dependent properties and quantifying their effects as they relate to toxicity in water, air,
or in vivo conditions. Particle dissolution impacts particle size and can impact aggregation by causing
deaggregation as the particles within the aggregate dissolve. Particle aggregation impacts size, shape,
density, available surface area, and surface chemistry. The researchers have chosen an experimental
approach that integrates macroscopic and microscopic measurements and methods to better understand the
implications of nanomaterials and are performing toxicity and biological interaction studies. The
researchers synthesize or purchase commercial nanomaterial powders and perform bulk and surface
characterization of these nanomaterials to determine their fate and transformation in water and aerosol and
inhalation toxicity.
Titanium dioxide (TiO2) nanoparticles from nanostructured and amorphous materials are some of the
smallest commercially manufactured oxide nanoparticles, and although they are sold at a primary size of
5 nm, characterization shows them to be 4 nm in size. The researchers determined that these nanoparticles
aggregate but do not dissolve in water at a temperature of 293 K. Aggregation and sedimentation in
aqueous suspensions will depend on nanoparticle-to-nanoparticle interactions. Research also indicates that
there is a switch in stability of TiO2 nanoparticle suspensions in the presence of citric acid. Derjaguin,
Landau, Verwey, and Overbeek (DLVO) calculations along with zeta potential measurements of the
surface charge show that TiO2 nanoparticle suspensions are stable at low pH in the absence of citric acid
and at near neutral pH in the presence of citric acid. Surface speciation suggests thatpKa values are lower
for surface adsorbed citric acid; less adsorption at higher pH is a result of the surface charge becoming
more negative with increasing pH. Thus, mobility in the environment of nanoscale TiO2 will depend on
surface coatings, coverage, and charge and pH in a complex manner.
The researchers compared the dissolution of nanorods to microrods and found that nanorods showed
increased surface density of hydroxyl groups compared to microrods. Nanorods can extensively aggregate
under certain conditions and form tight bundles. Nanorods have enhanced dissolution but aggregate more
readily than microrods in some conditions, but different chemical behavior is seen in different conditions.
Enhanced dissolution on the nanoscale is quenched in the aggregated state; therefore, dissolution depends
on aggregation and the aggregation state, and nanoparticle aggregation and dissolution are connected in
ways that are not fully understood. When researchers compared the inflammatory response of mice to
various metal and metal oxide nanomaterial aggregates, the greatest inflammatory response was found for
copper-based nanoparticles, and copper nanoparticles showed a higher propensity for dissolution in
simulated biological media. Differences between iron and copper nanoparticles are a result of different
chemical reactivity in biological media. Lung tissues show no evidence of copper nanoparticles, suggesting
that the nanoparticles dissolve, which may increase the inflammatory response.
The results of environmental fate and transport studies indicate that metals and metal oxides show unique
reactivity and physicochemical behavior on the nanoscale, and this behavior will be impacted by
aggregation. Surface area and chemistry impact aggregation, and aggregation impacts surface reactivity
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U.S. EPA Nanotechnology Grantees Meeting
(e.g., dissolution). Some ongoing environmental fate and transport studies in the laboratory include those of
size-dependent dissolution of zinc oxide (ZnO) nanoparticles and nanorods as well as aggregation and
dissolution of copper nanoparticles in aqueous media as a function of pH and in the presence of citrate
aggregation and dissolution. Inhalation toxicity studies indicate that chemical composition, size, and the
ability to undergo dissolution and translocation are important to toxicity in ways that have not been
discerned previously. Additional studies on silver, ZnO, and copper nanoparticles currently are underway.
Discussion
Warren Heideman (University of Wisconsin-Madison) asked, in terms of metal toxicity, particularly in the
cases of silver and copper, whether the effects of the nanoparticles can be distinguished from those of the
carrier ion. Dr. Grassian responded that her laboratory currently is exploring this with follow-up studies.
Bonnie Blazer-Yost (Indiana University-Purdue University Indianapolis) asked how reagents react with
mucin in the airway. Dr. Grassian replied that these experiments had not been performed.
Boris Jovanovic (Iowa State University) asked from which company the laboratory ordered the 4 nm TiO2
nanoparticles. Dr. Grassian responded that they had been supplied from Nanostructured and Amorphous
Materials, Inc., but laboratories using them should be sure to characterize them because some were up to
10 nm in size.
Qilin Li (Rice University) asked whether the pH adjustment and nanoparticle contact with citric acid were
simultaneous and about the reversibility of absorption. Dr. Grassian responded that the researchers set the
pH with citric acid and then added the nanoparticles. Then, pH was measured, and pH changes were not
seen. In terms of reversibility, this is a good question, and these studies were not performed.
Mr. Shapiro asked what types of products use copper nanoparticles. Dr. Grassian replied that they are used
as catalysts in electronics, and they also are beginning to be used for agricultural applications.
Life Cycle Analysis and Nanostructured Materials
Thomas Theis, University of Illinois at Chicago
Many of the topics discussed during this presentation were addressed at the National Science Foundation
(NSF)/EPA Life Cycle Aspects of Nanoproducts, Nanostructured Materials, and Nanomanufacturing:
Problem Definitions, Data Gaps, and Research Needs Workshop, which 60 individuals attended. Life cycle
assessment (LCA) is a systems methodology for compiling information on the flow of materials and energy
throughout a product chain. LCA evolved from industry needs to understand manufacturing and market
behavior and make choices among competing designs, processes, and products. It defines four general
sections of the product chain: (1) materials acquisition, (2) manufacturing/fabrication, (3) product use, and
(4) downstream disposition of the product. LCA is standardized by ISO 14040 and 14044 in a framework
whose four steps (goal and scope definition, inventory analysis, impact assessment, and interpretation) can
be described as "improvement analysis" and whose outcomes are expressed in common units to allow a
comparative systems tool.
EPA's LCA includes potentials for exposure to workers and consumers and disposal exposures. The
Agency imposes a risk assessment paradigm on LCA, which is difficult to accomplish. This adaptation of
LCA is a method by which to gather information on waste production, energy demand, and the potential for
risk to exposed populations. It works best when risks are nonlocal and the population is nonspecific. It is
not a substitute for regulatory risk assessment. The nanomaterial health/materials paradox was discussed at
the above-mentioned workshop. Those attributes of nanomaterials that are prized for commercial
development and application are the same ones that cause toxic reactions.
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U.S. EPA Nanotechnology Grantees Meeting
EPA's nanotechnology research is a two-pronged approach that focuses on environmental applications and
implications. This is a worthy approach for environmental regulation but does not apply to LCA. Elements
of an LCA-inspired interdisciplinary research program for nanotechnology include use of less toxic and
more available components, focus on structures that are less bioavailable, lowering the life cycle energy of
manufacturing, design for recovery of nanocomponents at end- of-life, understanding the social contexts
in which nano-based products are used and disposed, and application of LCA methodology to the entire
product chain. The topic of nanotechnology LCA is not well published and data are lagging. Additionally,
manufacturing of nanomaterials causes a variety of impacts including low process yields, significant energy
requirements, use of toxic and organic solvents, and high water consumption.
There also is an energy paradox to nanomaterials: Although nanomaterials are some of the most energy-
intensive materials known, they currently represent less than 1 percent of manufacturing costs. The costs
are low at this point because these materials are not yet commodities, but energy costs may not remain low
as they become commodities. Current estimates of world production of various nanomaterials appear to be
close to actual amounts, but carbon nanotubes and quantum dots are difficult to mass produce with their
current energy requirements. The potential U.S. energy savings from eight nanotechnology applications is
approximately 15 percent, but the stability of nanomaterials in the environment is a challenge that must be
overcome. Another challenge is that composite materials are not recycled.
In summary, engineered nanomaterials and products are already in use, not widely understood by
consumers, often energy intensive and materially inefficient to make, and often difficult to recover once
placed in commerce. They have increasingly complex functionalities and provide high added value,
although they often are composed of toxic and/or scarce chemicals or use such chemicals in processing.
The comparative benefits and impacts of nanoproducts are unknown, and LCA research and applications
for nanomaterials are lagging.
Discussion
Gregory Lowry (Carnegie Mellon University) noted that an issue in regard to risk assessment is developing
reasonable and reliable numbers for inputs and sources of nanomaterials into the environment. He asked
how the annual production figures for nanomaterials are determined and whether they are reliable. Also,
can information on potential product types and their release be distributed? Dr. Theis responded that the
figures are an estimate based on patents and the open literature. It is too speculative to release information
by potential product types, and potential demand is too difficult to predict.
Dr. Grassian noted that many consumer products state that they are "nano" when they actually are "micro."
Dr. Theis agreed and stated that the accepted definition of nanomaterials is those smaller than 100 nm in
size. When reviewing the literature, only those products identified as smaller than 100 nm were included.
Platinum-Containing Nanomaterials: Sources, Speciation, and Transformation in the Environment
Martin Shafer, University of Wisconsin-Madison
The work on platinum was motivated by several factors, including the increase in platinum levels in many
environmental receptors during the past 40 years as a result of platinum use in automobile exhaust catalysts
and industrial catalysts, the toxicity of certain platinum species, and the ability of platinum to transform in
environmental matrices. Platinum is likely to continue to be used because of a lack of other suitable
substances. The toxicological responses of many metals, including platinum, are determined by the specific
chemical and physical speciation in the primary source or environmental receptor. Extant modern
methodologies, however, provide little relevant speciation information, and traditional techniques that are
speciation capable lack the required sensitivity. The specific objectives of the study are to refine analytical
tools for measurement and chemical speciation of platinum in environmentally relevant sources and
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U.S. EPA Nanotechnology Grantees Meeting
receptors and integrate source and environmental sampling with advances in platinum analytical speciation
tools.
The study examines automobiles, diesel engines, roadside dust and soils, and ambient aerosol from urban
centers. Roadway and tunnel dust, which is an excellent integrated receptor from emissions and mobile
sources, from Milwaukee, Los Angeles, Atlanta, and Denver was studied. Roadside soils and catalyst
materials also were studied, and air sampling was performed adjacent to heavily trafficked roads. The
diesel engine dynamometer studies focused on platinum-cerium amended fuel, and the researchers
completed a good deal of roadside and ambient aerosol sampling and characterization. Extraction-based
and solid-phase speciation and electronic microscopy were used to characterize particulate matter (PM).
Physiologically relevant fluids were used for the extraction-based characterization.
The researchers measured the levels of platinum in road dusts and determined that it was from an anthropo-
genic source. A small fraction of platinum in road dust from the Los Angeles site was found to be soluble,
and it is much more soluble in macrophages. Sampling at the Milwaukee site indicated that there is a
significant difference in platinum and palladium aerosol mass-size distributions from week to week. The
Milwaukee road dust also showed increased solubility in the macrophage of platinum in roadside aerosol;
the levels approached the critical range established by EPA. Researchers also noted a potential dilution
effect with cerium. The extractable fraction of speciated water-soluble platinum in diesel PM was
approximately 3 percent. Studies showed that, in terms of gasoline vehicle catalyst, the modeled fraction of
oxidized platinum is significant. Significant contributions from oxidized platinum species are evident in the
spectrum in primary vehicle emissions. Early data suggest that oxide and metal are the two dominant
platinum species. Additionally, the laboratory is targeting two documented toxic/allergenic chloroplatinate
compounds and their hydrolysis products because only very limited information on the concentrations of
chloroplatinates in potential environmental sources and receptors is available and environmental fate and
transport data are lacking. The laboratory is developing an isocratic and gradient method to examine the
toxic form of platinum and will continue this work; once complete, it will apply the methods to engine PM,
road dusts, and airborne PM samples. Researchers also will study various environments to examine the
transformation state in different environments.
Discussion
Dr. Lowry asked where the chloroplatinate was found in the samples. Dr. Shafer responded that it has the
potential to form during the combustion process, so it sits in road dusts and attaches to surfaces. It is more
soluble than oxide species. Dr. Lowry asked whether it was possible to distinguish between adsorbed
species and others. Dr. Shafer explained that this was not possible with the tools that the laboratory uses.
Dr. Grassian asked whether different regions had specific chloroplatination profiles. Dr. Shafer replied that
the method had not been developed to the point that it could be quantitatively applied to field samples.
Quingguo (Jack) Huang (University of Georgia) asked what "SF" stood for in one of the mentioned
methods. Dr. Shafer explained that it meant "sector field." Dr. Huang asked whether using solids would
return original speciation to the particles. Dr. Shafer answered that the laboratory is collecting a large
volume of presize-fractionated aerosols so that species can be associated.
Role O/NLRP3 Inflammasome and Nickel in Multiwalled Carbon Nanotube-Induced Lung Injury
Andrij Holian, The University of Montana
The researchers have focused on determining the central mechanism to explain how engineered nano-
materials cause pathology and developing a high throughput in vitro screening tool to separate bioactive
from nonbioactive nanomaterials. The alveolar macrophage was chosen as a vehicle for study because it is
the front-line defense against inhaled particles and plays a major role in both the innate and adaptive
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immune responses. The alveolar macrophage is responsible for particle clearance from the lung and
contributes to the regulation of the inflammatory response. The research focuses on the NLRP3 inflamma-
some, which is present in alveolar macrophages and plays an important role in mediating the inflammatory
response to various danger signals, including crystalline particles. The inflammasome is activated by
cathepsin B, which signals assembly of the NLRP3 inflammasome and results in active caspase 1, which in
turn activates gene transcription of pro-inflammatory cytokines (e.g., interleukin [IL]-lp, IL-18). The
laboratory tested 24 different multiwalled carbon nanotubes and evaluated cytotoxicity and the
inflammasome in THP-1 cells and alveolar macrophages in mice.
Results indicated that the type of metal, diameter, purity, and length were not important following
histopathological analysis 7 days postexposure by two blind scorers. Pathology only correlated with nickel
content. At 56 days postexposure, multiwalled carbon nanotubes still were present as were granuloma
formations. The 7-day and 56-day pathology data are well-correlated; therefore, the 7-day data can be used
to predict the 56-day outcomes. There is significant correlation between nickel and various inflammatory
response markers (e.g., IL-lp, IL-18, percent viable cells). The increased correlation with in vivo cell
viability compared to in vitro was probably a result of the heterogeneity of the alveolar macrophages versus
the cell line. The work has not answered the question of whether there is a relationship between the effect
on cell viability and inflammasome activation, which are occurring by separate mechanisms. Additionally,
in vitro assays are predictive of pathology. There was an excellent correlation between IL-lp production
and percent viable cells with prediction of pathology, indicating that measurements of the inflammasome
can be used to predict pathological outcomes. Inflammasome production of IL-lp is critical to the inflam-
matory response.
In summary, the NLRP3 inflammasome is important in the bioactivity of engineered nanomaterials, and IL-
ip is central to initiating inflammation. Nickel on multiwalled carbon nanotubes appears to be a good
predictor ofNLRPS inflammasome activation, and activation of the NLRP3 inflammasome provides a good
explanation of in vitro and in vivo observations for both multiwalled carbon nanotubes and TiO2 nanowires.
Also, activation of the NLRP3 inflammasome, which can utilize alveolar macrophages or THP-1 cells, is a
good predictor of nanoparticle bioactivity. Disruption of lysosomes, which can be caused by bioactive but
not nonbioactive engineered nanomaterials, is required forNLRPS inflammasome activation.
Discussion
In response to a question by Dr. Blazer-Yost, Dr. Holian explained that the test materials were selected
because there was a clear difference among them in nickel content but not in size, which minimized the
variables; therefore, the main variable tested was nickel content. Dr. Blazer-Yost asked whether the nickel
was being taken up with the nanotubes, to which Dr. Holian replied that this was definitely the case.
Dr. Blazer-Yost asked about the concentration of nanomaterials in the lungs. Dr. Holian responded that
each mouse received 100 jog. Agglomeration, suspension, and singlets are critical determinants in the
process, and this is what the next phase of the research will study.
Dr. Jovanovic noted that this is an important field of immunotoxicology that has not been explored enough
in the past. He asked whether the researchers had considered additional work with neutrophils, especially
considering the recent Nature article indicating that nanoparticles are important inducers of neutrophil
interactions at environmentally relevant concentrations. Dr. Holian agreed that neutrophils are first
responders and contribute to cleanup, but he did not think that they contribute to chronic inflammation and
injury.
Wen Zhang (Georgia Institute of Technology) asked why the researchers chose a 7-day timeframe to
observe pathology. Dr. Holian responded that the time was chosen for practical considerations (e.g.,
expense), and many publications have indicated that multiwalled carbon nanotubes are able to cause
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distinct pathology within 7 days. Because it would be advantageous to perform shorter experiments, the
researchers then determined whether this timeframe was a valid predictor.
Howard Fairbrother (Johns Hopkins University) asked whether the correlation with nickel could have been
predicted a priori. Dr. Holian replied that nickel is more reactive and a better catalyst in redox reactions
than iron. A 2009 paper indicated that nickel was capable of activating the inflammasome. Dr. Holian's
theory is that nickel is being released by the multiwalled carbon nanotubes, or it has unique bioactive
properties and/or catalytic activities. Therefore, the results possibly could have been predicted, but the
study provides a deeper understanding. Dr. Fairbrother asked whether toxicological effects of nanotubes are
a result of nickel rather than the nanotubes themselves. Dr. Holian thought that contaminants would be an
important predictor, and pure nanotubes have less bioactivity. The idea is that nanotubes can interact with
lysosomal proteins and cause lysosomal permeability. Dr. Fairbrother noted that the data that Dr. Holian
showed indicated that there were nickel subsets that did not correlate with pathology. Dr. Holian responded
that the correlation occurs with those multiwalled carbon nanotubes that are composed of at least 2 percent
nickel.
MORNING SESSION 2: EFFECTS OF NANOPARTICLE SURFACE PROPERTIES
Microbial Bioavailability of Polyethylene Oxide Grafted to Engineered Nanomaterials
Gregory Lowry, Carnegie Mellon University
The goal of the research was to determine the effect of surface coatings on the environmental and microbial
fate of nano-iron and iron oxide (FeO) nanoparticles. The specific objectives were to determine the: (1) fate
of nanoscale zero valent iron (nZVI) in the environment, (2) effects of nZVI and its coatings on
biogeochemistry, and (3) fate of the coatings. To understand nanoparticle fate and transport, it is necessary
to understand coating fate; coatings affect aggregation, deposition, and biological interactions. Therefore,
the researchers asked whether nanomaterial coatings are bioavailable. Because nanomaterials must be 5 nm
or smaller to enter bacteria, the researchers focused on this size.
The researchers placed polystyrene covalently bound with polyethylene glycol (PEG) in water to determine
whether microbes could remove the coating in an aqueous environment and demonstrated that PEGs are
nontoxic, provide a permanent coating, and do not hydrolyze in water. Next, water from an urban river with
PEG degraders was run through enrichment culture to select for these PEG degraders, and species of
Novosphingobium, Pseudomonas, and Hydrogenophaga were found. These bacterial species were provided
with PEG, and their growth correlates with the addition of PEG. The same analysis was performed with
copolymers, and the same growth was seen, which is evidence that bacteria are able to remove PEG from
copolymers. Additionally, the researchers determined that microbes induced PEG copolymer aggregation
via a change in surface properties.
The researchers concluded that covalently bound PEG on nanoparticles is bioavailable, and
microorganisms can change nanoparticle stability, which in turn changes environmental fate and transport.
Bioavailability depends on coating attachment and degradability. The next step is to determine what
happens to coatings in the environment. The researchers faced several challenges, including the difficulty
of tracking coating fate in real environmental samples, recovering engineered nanomaterials from
environmental samples, and measuring the process and effects at realistic nanomaterial concentrations.
Discussion
Robert Yokel (University of Kentucky) asked whether similar results were received with citrate coatings.
Dr. Lowry replied that the researchers have not performed extensive studies regarding the bioavailability or
biodegradation of citrate. Free citrate would expected to be readily biodegradable, but if it is bound, then
Dr. Lowry was unsure of its ability to biodegrade.
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Dr. Heideman noted that the opportunity is present to measure molar amounts of carbon with the
microsystem and asked how much of the coating is removed. Dr. Lowry responded that the mass balance
on the particles indicated that percent levels are converted to carbon dioxide (CO2), and it clearly is
changing the character of the particles. This information has been included in a paper that will be submitted
to Nano Letters shortly.
Wunmi Sadik (State University of New York at Binghamton) asked whether the researchers performed
structural characterization. Dr. Lowry answered that this was difficult for these particular particles. They
are uncharged, so measuring zeta potential does not make sense. Dr. Lowry was unaware of any analytical
tools available to answer this question, so to indirectly address this, the laboratory examined the nature of
the particles postexposure. The mechanism by which the bacteria are removing the coating is interesting
but not fully known at this point. Dr. Sadik suggested that one method might be to look at the nuclear
magnetic resonance or mass spectrometry of the solution. Dr. Lowry responded that the laboratory would
have to restructure its approach to use these methods because of the concentrations involved.
Dr. Grassian asked about the quantitative aspects of surface chemistry and absorption. Dr. Lowry said that
the researchers had performed static light scattering on the particles, and this analysis showed that some of
the material was removed and converted to CO2.
Qilin Li (Rice University) asked, because the particles were not taken up by the bacteria, whether enzymes
in the extracellular matrix are responsible. Dr. Lowry replied that the next step is to determine the process
by which the bacteria are removing the coating.
Elijah Petersen (National Institute of Standards and Technology [NIST]) suggested the use of thermal
gravimetric analysis to examine carbon amounts that are released as the nanoparticles are released.
Dr. Lowry replied that this method could not be used because of the polystyrene core.
Surface Oxides: Their Influence on Multiwalled Nanotubes' Colloidal, Sorption, and Transport
Properties
Howard Fairbrother, Johns Hopkins University
This study focuses on the role that oxygen functional groups play in regulating the properties of
multiwalled carbon nanotubes. The laboratory performs physicochemical characterization to develop the
functional relationships related to material properties to create models to predict environmentally relevant
behavior. Surface analysis is a key component of the research; x-ray photoelectron spectroscopy (XPS) is
used to determine surface oxygen concentration because it is the most reliable and convenient method to
control the amount of oxygen grafted to the sidewalls. Aggregation properties are examined in a laboratory
setting. Surface oxygen may be a predictive metric as stabilization correlates with the amount of surface
oxygen. Other properties that the researchers measured were poor metrics for colloidal stability for carbon
nanotubes.
The researchers also are interested in studying turbidity, organisms, and natural organic matter to determine
the environmental aggregation behavior. To understand complex environmental behaviors, the researchers
study colloidal stability and correlate it with adsorption properties to ultimately determine whether surface
chemistry of the underlying particle plays a role after natural organic matter adsorption. Surface
concentration reduces the adsorption of natural organic matter onto the multiwalled carbon nanotubes'
surface. Results clearly indicate inversion of properties in environmental conditions and that surface
chemistry plays a significant role in how the multiwalled carbon nanotubes interact in the environment. The
researchers designed a column transport experiment to determine how surface oxygen affects the ability to
transport in the environment. Results indicated that as the amount of salt increases, multiwalled carbon
nanotubes show decreased transport ability. The researchers used a standard calculation method to
determine behavior and also found that pH plays a fairly important role in transport; an increase in pH
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causes an increased ability of the multiwalled carbon nanotubes to transport. The researchers also
determined the optimal conditions under which to obtain reliable and reproducible results.
Future work in the laboratory will focus on the effect of different oxidation on the deposition of surface-
oxidized multiwalled carbon nanotubes, the effect of particle sizes on deposition of surface-oxidized
MWCNTs, and facilitated transport.
Discussion
Dr. Grassian asked about the morphology of the multiwalled carbon nanotubes in water. Dr. Fairbrother
stated that they could be described as floppy rods.
Dr. Li asked Dr. Fairbrother to explain the fact that pulse results were larger than the researchers observed.
Dr. Fairbrother said that the confusion might be a result of the order in which he presented his slides, as
some of the results were obtained prior to the researchers determining how to consistently reproduce the
results. The plan is to return to these experiments now that this is known. Dr. Li asked about the shape of
the ethyl concentration profile, which was not typical, and whether it could have been caused because the
average was measured. Dr. Fairbrother agreed that this was possible.
A participant asked whether the researchers examined other nanotube-to-natural organic matter ratios
besides 10:1. Dr. Fairbrother responded that they studied ratios from zero to 30, and there is a systematic
evolution of the particle stability as a function of the amount of natural organic matter.
Hyphenated and "Particle Counting" ICP-MS Methods for the Detection and Characterization of Metal
and Metal Oxide Nanoparticles
James Ranville, Colorado School of Mines
The research focuses on risk assessment of nanotechnology. There are many factors that can be identified,
and the researchers initially focused on effects (e.g., uptake, toxicity). To understand exposure, it is
necessary to understand stability, for which aggregation and dissolution are important. Additionally, to
study exposure better metrology (e.g., quantitation, detection, characterization) must be developed. The
researchers observed the optical properties overtime, which may indicate that reactivity may be changing.
Questions to be addressed regarding detection and characterization are: How much sensitivity and
selectivity are needed? How can methods be applied to complex matrices? What is exposure? Are
researchers studying what they think that they are studying?
With respect to nanosilver, material flow indicates that surface waters and sewage treatment plants should
be studied, and environmentally relevant concentrations must be assessed at the parts per trillion (ppt) level
although toxic effects are seen at the parts per billion and parts per million levels in the laboratory. The
standard hypothesis is that inductively coupled plasma (ICP) mass spectrometry (MS) can be used to
detect, count, and size individual silver nanoparticles. The approach is to use element-specific "pulse"
counting (e.g., real-time single-particle [RTSP]-ICP-MS; time-resolved ICP-MS; single-particle ICP-MS).
The researchers chose to examine health food supplements, but these are polydispersed in size, so the
laboratory used nanoComposix, which is monodispersed.
Results indicated that silver nanoparticles up to 100 nm in size could be quantitatively detected by ICP-MS.
If the particle counting approach is valid, then the number of pulses will increase with increasing silver
nanoparticle concentration, the number of pulses will be reduced by filtration or acidification, and the
intensity of the pulse will be related to nanoparticle size. The results correlated with this. The time data can
be used to determine the difference between dissolved and particulate materials. Disk centrifuge is another
method to analyze particle size, and these data are in agreement with the ICP-MS particle counting method.
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The researchers than performed a proof-of-concept study to quantify silver in wastewater, and the results
were comparable to estimates from a previously completed materials flow analysis.
Another focus of the project was to determine whether nanotechnology researchers in general are studying
what they expect. The researchers examined the biovailability of the cadmium selenide (CdSe) quantum dot
core and whether it is toxic to Daphnia magna to help answer this question. Field-flow fractionation (FFF)-
ICP-MS can be used to sort the nanoparticles by size to allow further analysis of the nanoparticles. Tests
indicated that the cadmium-to-selenium ratio was not 1:1. The cadmium was associated with the quantum
dot but not with the core, possibly because cadmium associated with the polymer coating as a result of poor
washing during synthesis. The tests appeared to study the cadmium on the surface rather than in the core,
highlighting the fact that good characterization techniques are needed to ensure that researchers indeed are
studying what they expect.
In summary, RTSP-ICP-MS: (1) can be used to detect silver nanoparticles at environmentally relevant
concentrations (i.e., ppt levels) with high specificity; (2) can distinguish between dissolved and particle
silver, which provides the potential for the method's application in stability and exposure/toxicity
laboratory studies; and (3) has limitations in that there is a 40 nm size limit, and it cannot identify
nanoparticle type. FFF-ICP-MS can be used to more fully characterize complex nanoparticles and provide
information to interpret the results of experiments in which mixtures are used, manufacturing impurities are
present, and/or transformation/degradation products are present.
Discussion
Patricia Holden (University of California, Santa Barbara) asked how the method will enable researchers to
track mobile particle association. Dr. Ranville answered that the researchers plan to perform experiments to
simulate the processes occurring in wastewater at each step. Coupling FFF with particle counting may lead
the researchers forward.
Kim Rogers (EPA) stated that crystallography experiments were being performed to determine the
association of silver chloride with silver nanoparticles. Dr. Ranville acknowledged the limitations of the
current methods and noted that complementary techniques will be performed to obtain more information.
Dr. Huang asked how applicable the method is to other materials. Dr. Ranville replied that it could be used
element-by-element to build correlations between silver and other elements.
Controlled Release of Biologically Active Silver From Nanosilver Surfaces
Jingyu Liu, Brown University
Silver is a broad-spectrum antibiotic that has relatively low toxicity in humans and is being manufactured
in large quantities and incorporated into consumer and medical products. Is it a risk to the environment and
human health? It is known to be more toxic to aquatic organisms than any other metal except mercury. It
bioaccumulates quickly, and some organisms have a low toxicity threshold to nanosilver. Silver has
potential toxic effects on beneficial soil bacteria. An important research question is whether nanosilver
interacting in biological and environmental systems is the particle or the ion. Metal ions may coexist in
metal-containing nanoparticle suspensions. Silver ion is a known toxicant that binds to thiol groups in
enzymes, such as NADH dehydrogenase, which disrupts the bacterial respiratory chain and generates
reactive oxygen species (ROS) that can lead to oxidative stress and cell damage. Nanosilver particles
themselves may also contribute by binding to or passing through cell membranes and generating ROS
through surface reactions. There is some controversy about the role of particle-based mechanisms, but there
is broad agreement that silver ion is an important toxicant. Previous work regarding ion release kinetics and
particle persistence in aqueous nanosilver clouds indicate that the reaction produces active peroxide
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intermediates, is inhibited by natural organic matter, and leads to complete particle dissolution in aerobic
environments.
The researchers are interested in controlled-release nanosilver and application of the drug delivery
paradigm. Two questions that are being considered are: Can ion release rate be systematically increased or
decreased? Can nanosilver materials be engineered for optimal ion release? Specific benefits of controlled
release nanosilver formulations might include: (1) dose control to achieve desired bactericidal or
bacteriostatic effects; (2) dose limitation to avoid eukaryotic toxicity; (3) control of product lifetime, before
dissolution and diffusion end antibacterial activity; (4) minimization of environmental release through
excess ion production beyond that necessary for product performance; or (5) optimization of release profile
for targeted delivery to specific tissue or intracellular targets. The researchers use ultrafiltration and atomic
absorption to study particle-ion partitioning in aqueous nanosilver colloids. The results indicate that bulk
silver oxidatively resolves but much more slowly than nanosilver. Visual MINTEQ software was used to
determine the effects of chloride and thiol. The results showed that biological thiol can drive silver
equilibrium in a biological system. Nanosilver causes the gradual release of ionic silver because of its
affinity to thiol.
Functionalized nanosilver in the presence of citrate, sodium sulfide, or mercaptoundecanoic acid was
studied, and all three methods were found to inhibit ion release from silver nanoparticles. Pre-oxidation
shows a distinct two-stage release (i.e., fast then slow). The first stage is a result of the rapid dissolution,
and the second is because the remaining metal reacts with dissolved oxygen. Other results indicated that
antioxidants can inhibit silver ion release. Different surface treatment methods induce different release
rates. The primary release mechanism appears to be oxidative dissolution, which can be inhibited through
ROS. Other mechanisms are reversible surface binding, inhibition by insoluble silver sulfide, surface
passivation, and pre-oxidation. Future work will focus on the biological and environmental implications of
ion release kinetics and control.
Discussion
John Rowe (University of Dayton) asked whether this was tested in vitro or in tissue culture; he asked
because ion effects should be differential, with different toxic effects on prokaryotic and eukaryotic cells.
Ms. Liu responded that the researchers plan to perform this type of work in the future, but the current focus
is on the basic chemistry of ion release. Dr. Rowe commented that this type of work would be important to
perform because there may be two different toxic methods depending on whether the organism is
prokaryotic or eukaryotic.
AFTERNOON SESSION 1: CHARACTERIZATION METHODS
A Biological Surface Adsorption Index for Characterizing Nanomaterials in Aquatic Environments and
Their Correlation With Skin Adsorption of Nanomaterials
Xin-Rui Xia, North Carolina State University
Currently, most methods to characterize nanomaterials in aqueous environments measure physical
parameters. Surface chemistry and core material compositions are the only measurable chemical
information on nanomaterials, but these cannot be used directly for quantitative analyses. The octanol-
water partition coefficient has been used widely for predictive model development for small molecules, but
it is difficult to use for nanomaterials because most nanomaterials form stable suspensions in water or oil
but not both. Efforts have been made to understand the chemical interactions between nanoparticles and
biological or environmental components. Researchers have demonstrated that lipophilicity is a significant
factor in the nanoparticle adsorption of small chemicals. To date, there is no generally applicable approach
to quantitatively measure the molecular interactions of nanoparticles with biological or environmental
components, which is crucial information needed to develop a quantitative structure-activity relationship
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for nanomedicine research and risk assessment and safety evaluation of nanomaterials in occupational and
environmental exposures. Many researchers have focused on nanocharacterization of pure nanomaterials in
industrial applications, nanoprotein coronas in biological systems, and the nanohumic acid complex in the
environment.
The researchers have identified that the adsorption property at the solid-liquid interface is key to
understanding the behavior of nanoparticles in aqueous environments. The researchers also have developed
a biological surface adsorption index (BSAI) approach to characterize the molecular interaction strengths of
nanoparticles with small molecules and macromolecules in biological and environmental systems. The
BSAI approach is based on the molecular interaction similarity between nano-small molecule interactions
and nano-macromolecule interactions. Forces that govern the chemical and biological behavior of
nanoparticles are the Coulomb force, London dispersion, hydrogen bonding, dipolarity/polarizability, and
lone-pair elections. Results indicate that nanodescriptors derived from the BSAI approach provide better
prediction. The predictive model was cross-validated and determined to be robust.
The BSAI database is the final product of the approach, and it is composed of the five nano-descriptors for
each of the nanomaterials. The nanodescriptors are free energy -related quantities quantitatively describing
the molecular interaction potentials of the nanomaterials at the nano-water interface. Biological activities
are free energy-related quantities; their logarithmic values can be predicted directly via the similar
predictive model shown for multiwalled carbon nanotubes. The development of the BSAI approach could
open a quantitative avenue toward predictive nanomedicine development, particularly for developing
integrated physiologically based pharmacokinetic models and for quantitative risk assessment and safety
evaluation of nanomaterials.
The researchers also studied the impact of physicochemical properties on skin absorption of manufactured
nanomaterials. Pristine fullerene (Ceo) in different solvents is used in many industrial and pharmaceutical
manufacturing processes; therefore, human exposure to C6o could occur in various solvents. Currently, the
impact of solvents on its skin penetration is unknown. The laboratory studied four types of representative
industrial solvents. The laboratory developed a novel method to prepare nC6o nanoparticles with a narrow
size distribution. nC6o and most of the unprotected nanomaterials have a very narrow window in their
colloidal stability, and biological electrolytes will cause their aggregation. The researchers determined that
once the nanoparticles aggregate, they cannot get through the skin. Aqueous colloidal nanomaterials with
coatings did not penetrate intact skin regardless of particle size. Ion-pairing agents did not promote skin
penetration. Skin penetration of C60 was observed in different industrial solvents. Significant solvent effects
were observed; toluene and chloroform promote skin penetration of C6o, whereas mineral oil does not
promote skin penetration. The same results were found when the researchers examined deeper skin layers
as well.
The laboratory performed short-term studies, but long-term studies also are needed. Skin absorption into
aquatic animals should be studied because of their different skin structure (e.g., amphibian skin is very
permeable to small molecules). Additionally, more work is needed to make the BSAI approach a generally
useful tool for quantitative correlation and risk assessment of various nanomaterials.
Discussion
Mr. Shapiro asked whether the results could be used to design nanoparticles to have specific impacts on the
skin. Dr. Xia answered that tailor-made nanoparticles may be possible in the future.
Dr. Lowry expressed concern about applying an equilibrium system to a system so far from equilibrium.
Dr. Xia replied that this is a general question for the field. For example, quantitative structure -activity
relationship can be used as a driver, but then the kinetics of the actual model are used. Dr. Lowry still had
concerns about applying kinetics in this situation. Dr. Xia said that the approach was to correlate
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equilibrium parameters. Dr. Lowry asked whether the approach had been applied to macromolecules.
Dr. Xia replied that much more work was needed at the current level before moving into macromolecules.
Flexible Nanostructured Conducting Poly(amic) Acid Membrane Captures, Isolates, and Simultaneously
Detects EngineeredNanoparticles
Wunmi Sadik, State University of New York at Binghamton
Two types of sensors have been defined by an EPA white paper. Category 1 includes sensors that are
nanoscale or have nanoscale materials or components, and category 2 includes sensors that are used to
measure nanoscale properties. The overall project objective is to develop novel category 2 nanosensors for
application in complex environmental matrices. Nanoparticles must be isolated from complex matrices, and
there are several current characterization techniques. Environmental matrices require ultrafiltration of free-
engineered nanoparticles. The researchers used functional groups on poly(amic) acid (PAA) to isolate
nanomaterials. The researchers have studied nanoparticle crosslinking with PAA for years, as well as the
chemistry of the materials used for crosslinking. Additionally, ultrafiltration often is used for the separation
of suspended solids, colloids, bacteria, and viruses. If the porosity of the membrane is controlled, then the
ions and particles that pass through the membrane can be controlled. The researchers used the phase-
inverted membrane method to create several types of flexible PAA membranes. Phase-inverted membranes
allow control of pore size and are stable to most organic solvents, conductive, and flexible.
The researchers successfully filtered quantum dots directly from aqueous solution with 99 percent
efficiency and were able to control porosity. Next, the researchers analyzed commercially available
products, including food supplements and beverages. Nanosilver in food supplements can cause permanent
bluish-gray discoloration of the skin and eyes; nanosilver can be toxic at a dose of as low as 15 ppm and is
50 percent more toxic than asbestos. PAA coordinates different nanomaterial functionalities and separates
nanosilver, TiO2 nanoparticles, and quantum dots. The researchers compared the developed membranes to
commercially available membranes and found that the membranes developed by the laboratory show
superior performance.
In summary, the laboratory has developed a new class of polymeric materials that exhibit spatio-selection
via three-dimensional binding interaction with engineered nanomaterials, control porosity, provide
accessibility to the underlying transducer, and enable the removal of major interferences. PAA membranes
can be regenerated by exposure to fresh solvents or acid washing, and the laboratory successfully filtered
nanosilver and quantum dots directly from commercial products with greater than 99 percent efficiency.
Future work will focus on improving the fabrication process and testing other nanoparticle combinations to
correct defects of the PAA membrane and functionalize the surface of the PAA membrane to improve
selectivity.
Discussion
Dr. Huang asked whether the researchers had differentiated between silver ions and other nanoparticles.
Dr. Sadik responded that this had not been examined yet.
Dr. Li asked whether the main method of interaction between nanoparticles and the membrane was size or
chemical interactions. Dr. Sadik replied that both size exclusion and selective chemistry were occurring.
Dr. Li asked what the advantages of the membrane developed by Dr. Sadik's laboratory were compared to
commercial membranes. Dr. Sadik answered that the ability to control functional groups on the surface of
the membrane allowed for selectivity. Commercial membranes only offer physical selectivity. Dr. Li noted
that it would be beneficial to create a membrane that allowed for separation of particles of different sizes.
Dr. Sadik agreed and stated that the laboratory currently was working on this.
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In response to a question from Mr. Shapiro, Dr. Sadik explained that the researchers had not considered
commercializing the membrane that had been developed.
Fate and Effects of Nanosized Metal Particles Examined Along a Simulated Terrestrial Food Chain
Using Genomic andMicrospectroscopic Techniques
Jason Unrine, University of Kentucky
The researchers are examining the fate, transport, and effects of manufactured nanoparticles in the
environment by focusing on uptake of nanoparticles by soil invertebrates, microbes, and plants and their
subsequent transfer to higher trophic levels. The worm Eisenia fetida is a semimodel organism that is
important to the toxicity testing model; the test medium is natural sandy loam, and gold nanoparticles are
used as a probe for particle uptake. Nanoparticles up to 50 nm in size can be absorbed by earthworms. The
researchers examined the effect of source on bioavailability and determined that primary particle size alone
does not determine uptake in complex media, such as soil. The researchers next hypothesized that
nanoparticles are more bioavailable through trophic rather than direct exposure and added frogs to their
experimental procedures. Transformation appears to occur during the first few weeks of exposure that
affect uptake; therefore, future studies should examine this. Results indicated that there was slow
elimination of the gold by the earthworms with no significant decrease of gold particles. Frogs that were
exposed to gold via ingestion of earthworms showed much higher levels of gold accumulation than those
that were directly exposed through gavage. Therefore, the hypothesis is correct, and persistence has
significant implications for the food chain. Although there was no difference in frog growth between the
two experimental groups, frogs exposed via earthworms showed greater gold concentrations in kidney,
liver, and muscle tissues compared to those directly exposed. One alternative hypothesis is that once
particles enter earthworm tissues, they acquire a protein corona and become more bioavailable, and another
alternative is that earthworms absorb only the most bioavailable particles from the total population of
particles, thus enriching the transferable fraction.
Next, the researchers tested various silver nanoparticles with different coatings in two different media; the
sandy loam increased oxidation compared to artificial soil media, and the percentages correlate well with
the toxicity seen. Results also indicated transient changes in gene expression, so studies should be
performed in a time-result manner to observe changes while the organism is adapting. Studies involving
protein carbonyl showed an increased amount of protein carbonyl, which correlates with downregulation of
catalase gene expression. Catalase transcription is complex and context dependent. There is a cascade of
effects leading to the downregulation of catalase, and what most likely is being observed is accumulation of
peroxide, which can accelerate the dissolution of particles; therefore, this could be a self-feeding cycle.
Following molecular exploration, the researchers examined integrated orgamismal response to nano-
particles. Initial avoidance was seen in soil, but there are intact particles. It may be that dissolution is
occurring close to the biological surfaces, but the researchers did rule out that it was the result of changes in
microbial community composition.
The researchers concluded that nanoparticles are bioavailable from soil and can be transferred to higher
trophic levels, and particle size and redox properties are important for uptake and toxicity. Silver particles
cause a variety of adverse effects in earthworms translating from the molecular level to the population
level, some at concentrations similar to those expected in sewage sludge. Environmental variables are
probably more important than particle variables for silver toxicity.
Discussion
Christian Andersen (EPA) asked whether the differences seen between the two experimental frog groups
exposed directly or trophically were an experimental artifact from gavage. Dr. Unrine responded that this
was not the case; the doses and their confidence levels are known. Dr. Andersen asked whether the waste
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products were collected. Dr. Unrine explained that this was not possible because the frogs live in water, and
the waste products disperse.
Dr. Heideman asked Dr. Unrine to explain why the removal phase occurred more rapidly than the outflow
phase. Dr. Unrine replied that the mass of the worm at different time points needs to be examined, and this
study has not been completed yet. Worms can detach part of their body, which could be one possibility, but
it is puzzling.
Maria Victoria Peeler (Washington State Department of Ecology) asked whether the researchers had
examined sediments. Dr. Unrine answered that this type of work had not been completed, but there are
plans to collaborate with laboratories that work with sediments.
In response to a comment from Dr. Grassian, Dr. Unrine explained that the redox potentials listed in one of
his slides were for illustration purposes only.
Determination of Manufactured Nanoparticle Toxicity Using Novel Rapid Screening Methods
John Rowe, University of Dayton
The focus of this project is to devise biological systems to rapidly assess the potential toxic effects of
nanoparticles and correlate in vitro results with in vivo outcomes. The approach is multidomain, using
viruses, plants, bacterial assays, mammalian in vitro cells, and Drosophila melanogaster as an in vivo
model, and examines the biogeochemical cycle and its effects on plants. D. melanogaster, which has a fast
life cycle, is used to study acute toxicity, and studies have moved to examine chronic toxicity. The overall
objective of the project is to establish D. melanogaster as an in vivo model system for rapid assessment of
nanoparticle toxicity. The current project objective is to study the effects of nanoparticle ingestion on
D. melanogaster growth and development.
Nanoparticle behavior is function of size, shape, and surface reactivity, and the researchers compare the
effects of different sizes and coating of nanoparticles on D. melanogaster development and reproduction.
Polysaccharide-coated silver nanoparticles were used in the experiments that were characterized by
transmission electron microscopy (TEM) and dynamic light scattering. Food was treated with uncoated
silver 10 nm in size, resulting in a linear effect on survivorship up to 30 |o,g/ml of silver. Survivors showed
a significant increase in pupation time and had a phenotype significantly different than untreated
D. melanogaster. The same toxic effects, although less, were seen with coated silver nanoparticles and
silver nanoparticles that were 60 nm in size. Nanoparticles have been shown to increase ROS, which may
result in oxidative stress, inflammation, and consequent damage to proteins, membranes, and DNA. The
researchers tested whether oxidative stress occurs in vivo using the model system and determined the effect
of treatment with ascorbic acid, which is a protector against oxidative stress through the antioxidant
defense mechanism, a pathway that provides protection against the harmful effects of ROS. Silver
nanoparticles induced superoxide dismutase activity, which is part of the antioxidant defense mechanism.
Results also indicated that ascorbic acid has protective effects. Additionally, results showed that silver
nanoparticles induced oxidative stress, which may be a mechanism of silver nanoparticle toxicity.
In summary, the researchers established an in vivo D. melanogaster model for studying nanoparticle
toxicity and demonstrated induction of oxidative stress by silver nanoparticles and the protective effect of
ascorbic acid treatment. Future directions will include elucidation of the pathway of oxidative stress
involved in the process and evaluation of the efficacy of an array of antioxidants.
Discussion
Paul Westerhoff (Arizona State University) asked whether the researchers had examined the first
generation offspring for the presence of nanoparticles. Dr. Rowe responded that other laboratories have
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demonstrated this, and researchers are studying an inhalation model to determine whether nanoparticles
enter the system completely via respiration.
Dr. Rogers asked whether the researchers knew how ROS production occurs in response to silver
nanoparticles. Dr. Rowe replied that the activation appears to be direct because it can be reversed with an
antioxidant.
Dr. Blazer-Yost asked whether the researchers tested lower concentrations of nanoparticles. Dr. Rowe
explained that this would be one of the next steps of the laboratory. The researchers used high
concentrations to ensure that an effect was seen before moving to lower concentrations. This is an
important question because the ultimate fate of nanoparticles is unknown.
Dr. Lowry asked whether the researchers experimented with silver nitrate in food. Dr. Rower answered that
they had not, but it may be worthwhile to do so.
AFTERNOON SESSION 2: ENVIRONMENTAL EFFECTS ON NANOPARTICLES
Influence of Natural Organic Matter on the Behavior and Bioavailability of Carbon Nanoparticles in
Aquatic Systems
Stephen Klaine, Clemson University
The researchers are examining how water quality parameters (e.g., natural organic material) influence the
bioavailability of carbon nanoparticles, and a major goal of the research is to examine food chain uptake. A
standard D. magna bioassay was used to measure the toxicity of a variety of carbon nanomaterials.
Multiwalled carbon nanotube toxicity did not change as a function of natural organic material. Natural
organic material-stabilized C6o, C70, and single-walled nanotubes were nontoxic.
The researchers explored whether carbon nanomaterials (multiwalled carbon nanotubes, carbon dots,
single-walled carbon nanotubes) are absorbed from the intestinal tract. Results indicated that there was no
movement of nanotubes in between the microvilli, and they do not appear to be biochemically toxic but are
physically toxic because they clog the intestinal tract. Acidified single-walled carbon nanotubes, however,
are found in between and in the microvilli, indicating that they have moved into the organism. Aggregation
inside the tissue also was observed. Raman spectroscopy was used to determine where the single-walled
carbon nanotubes were located within biological tissues, and results showed that they were within the
intestinal tract. Movement outside of the intestinal tract also was seen, and acidified single-walled carbon
nanotubes stabilized by natural organic material move farther outside of the tract than other single-walled
carbon nanotubes.
Carbon dots are useful for examining where nanomaterials travel after digestion by D. magna. The carbon
dots used by the researchers possess a carbon core with a PEG coating and have the same fluorescence as
quantum dots. Confocal microscopy was used to observe movement outside of the intestinal tract and
showed that there was a buildup around various organs and organ systems outside of the tract.
The researchers observed that multiwalled carbon nanotubes are acutely toxic to D. magna, and this is not a
function of natural organic material but appears to be a result of interference with orgamismal food
processing. It took 29 hours for D. magna to clear multiwalled carbon nanotubes from the intestinal tract,
compared to 30 minutes for clearance of clay. Multiwalled carbon nanotubes are not taken up from the
intestinal tract. Carbon dots migrate from the intestinal tract and appear to be associated with organelles.
Hydroxyl-functionalized single-walled carbon nanotubes may migrate from the intestinal tract, whereas
PEG-coated single-walled carbon nanotubes do not. The next steps are to continue to examine uptake from
the intestinal tract using fluorescent-labeled single-walled carbon nanotubes and employing other surface
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modifications. Food chain studies will use labeled carbon nanoparticles that are known to be bioavailable to
determine their movement through the aquatic food chain.
Discussion
Dr. Petersen asked about any impurities present in the original single-walled carbon nanotubes. Dr. Klaine
replied that they were pure by the time that they were treated with acid. Dr. Petersen suggested that the
researchers use electron energy loss spectroscopy to gain more definitive results.
Dr. Fairbrother asked why the acidified single-walled carbon nanotubes were more likely to move through
the organisms. Dr. Klaine answered that he was unsure, but possibly it was because items that are more
hydrophilic better associate with the microvilli. Dr. Fairbrother asked if similar results were seen with
multiwalled carbon nanotubes. Dr. Klaine answered that multiwalled carbon nanotubes are very stable.
Galya Orr (Pacific Northwest National Laboratory) asked about the zeta potential. Dr. Klaine was unsure
whether the laboratory had obtained these data. Dr. Petersen added that disbursement of nanotubes could be
the result of bundling and stronger interactions among the single-walled carbon nanotubes, which probably
are easier to disperse following acid treatment. Dr. Klaine agreed that this correlated with the data.
Environmental Photochemical Reactions ofnC60 and Functionalized Single-Walled Carbon Nanotubes
in Aqueous Suspensions
Chad Jafvert, Purdue University
Dr. Jafvert described published results of the grant, which is coming to an end. A paper focusing on the
photochemical transformation of aqueous C6o clusters in sunlight was the first paper to report on C6o
photochemical decay, measured by high-performance liquid chromatography (HPLC), in aqueous media
under sunlight. Results indicated that smaller clusters result in faster loss of C6o, and the photo-
transformation rate is not pH dependent. There is a negligible rate change with humic acids present, and
molecular oxygen is required for the process. A paper reporting on the photochemistry of aqueous C60
clusters highlighted that singlet oxygen forms during solar irradiation of nC6o, consistent with known
reaction mechanisms involving singlet oxygen. The photo-transformation of nC6o is mediated by singlet
oxygen, and the rate of singlet oxygen production is auto-catalyzed by nC60 water-soluble products formed
during irradiation. The singlet oxygen production rate increases with decreases in the size of nC6o. The
concentration of singlet oxygen induced by nC6o in sunlight is four- to 65-fold higher than the average
concentration typically found in sunlit natural surface waters.
A paper focusing on wavelength dependency and product characterization in terms of the photochemistry
of aqueous C6o clusters showed that several laboratory methods indicate that oxidation of C6o occurs in
aqueous suspensions of nC60 under sunlight, and C60 photo-transformation and singlet oxygen production
occur in visible light. Another paper focused on the photoreactivity of carboxylated single-walled carbon
nanotubes in sunlight and ROS production in water. In oxic aqueous solutions under sunlight, carboxylated
single-walled carbon nanotube dispersions generate singlet oxygen, superoxide anion, and hydroxyl
radicals. Reactions with probe molecules were corroborated, and photo-induced aggregation occurred at a
low pH. Another paper highlighted projects focusing on solar light-induced ROS production by single-
walled carbon nanotubes in water and the role of surface functionalization. Results indicated that oxic
aqueous colloidal dispersions of both types of functionalized nanotubes generated ROS in sunlight, and
Type I and Type II photochemical pathways occur by the functionalized single-walled carbon nanotubes in
sunlight. It appears that the functionalized single-walled carbon nanotubes can act as the electron donor
directly, resulting in a change in their properties, or can shuttle electrons from other electron donors to form
ROS.
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Discussion
Richard Zepp (EPA) asked whether the researchers attempted to generate ROS by nonphotochemical
methods to observe how they react with substrate. Dr. Jafvert answered that others have performed this in
organic solvents, and it would be worthwhile to attempt.
Dr. Jovanovic asked whether the researchers exposed the C6o species from the Materials and Electro-
chemical Research Corporation to sunlight or ultraviolet (UV) light to determine if it generated ROS.
Dr. Jafvert said that the compounds being generated could not be quantified. C6o was not used as the
starting material in any of the experiments.
Dr. Petersen asked whether multiwalled carbon nanotubes had been tested. Dr. Jafvert replied that this is
planned for the future.
Impact of Photochemical Oxidation on the Stability ofnC60 and Multiwalled Carbon Nanotubes in
Aqueous Solution
Qilin Li, Rice University
The main objectives of the study are to understand the changes in physicochemical properties of carbon-
based nanomaterials, specifically C6o and carbon nanotubes, in natural aquatic systems as a result of
interactions with NOM and sunlight and determine the subsequent changes in their aggregation and
deposition behaviors. To simulate the particle structures that may form when C60 and multiwalled carbon
nanotubes are released into the natural aqueous environment, the researchers prepared the nanoparticle
suspensions using a direct sonication method without using any organic solvent. Carboxylated multiwalled
carbon nanotubes were used for easier dispersal; C60 and the carboxylated multiwalled carbon nanotubes
were well dispersed in water. Sunlight irradiation was simulated with a photoreactor equipped with UV
lamps, and samples taken at various times of irradiation were characterized for their physicochemical
properties.
Results indicated that the outer surface layer of nC6o particles was oxidized following 7 days of irradiation.
When the aggregation of these surface-oxidized nC6o particles was examined, it was found that they were
significantly more stable than the pristine nC60 particles, as demonstrated by the reduced aggregation rate.
Comparison of stability curves shows that the surface oxidation caused by irradiation increased the critical
coagulation concentration by more than fivefold. Additionally, irradiated nC6o responds to humic acid
differently from the pristine nC6o, showing no change in particle stability, and shows differences in calcium
chloride (CaCl2) as well. The steric hindrance effect of humic acid in CaCl2, however, did not seem to be
affected by UVA irradiation. An adsorption experiment confirmed that this was a result of significant
humic acid adsorption on the irradiated nC6o surface, aided by calcium.
A similar study used carboxylated multiwalled carbon nanotubes. Contrary to the nC6o results, irradiation
reduced multiwalled carbon nanotube stability, and the surface negative charge decreased after irradiation,
suggesting changes in surface chemistry. Carboxylated multiwalled carbon nanotubes also appear to lose
surface hydroxyl and/or carboxyl groups following irradiation. In CaCl2 solutions, however, the stability
before and after irradiation was very similar. Multiwalled carbon nanotubes are unstable in the presence of
calcium, so it is important to remember that these nanotubes most likely will aggregate and settle quickly in
most natural aquatic systems.
In conclusion, sunlight irradiation and humic acid sorption mediate nC6o and carboxylated multiwalled
carbon nanotubes aggregation, and specific and nonspecific interactions are involved. Nanocarbon surface
chemistry plays a key role in its environmental fate and transport. Ongoing and future work in the
laboratory focuses on the impact of irradiation and natural organic material on sorption/deposition and
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transport in a subsurface porous medium as well as on the impact of irradiation and natural organic material
on bioavailability and bioaccumulation of nanoparticles.
Discussion
P. Lee Ferguson (Duke University) asked whether the researchers had plans to examine truly pristine
nanotubes. Dr. Li responded that the laboratory is interested in this, but original attempts with pristine
nanotubes did not allow for high enough concentrations.
Dr. Fairbrother noted that the techniques used were meant for solids rather than powders, and the method
may need to be modified. Dr. Li acknowledged the limitations of the technique and explained that to
counteract this the laboratory analyzed the XPS spectrum. Dr. Fairbrother cautioned that there still might be
issues, and the two researchers agreed to discuss the specifics later.
The Environmental Behaviors of Multi-Walled Carbon Nanotubes in Aquatic Systems
Quingguo Huang, University of Georgia
The objective of the research project is to examine solubilized carbon nanotubes currently under
development for a variety of applications. Their mobility and exposure also are being examined. The
project focuses on sorption, transformation, bioaccumulation, and trophic transfer. Because sediments
affect dispersed carbon nanotubes and dissolved organic matter, it is necessary to design experiments to
better understand each of these situations. The three treatments applied were control, peat with dissolved
organic matter, and solid peat. Results indicated that in peat, sodium is necessary for sorption in a dose-
dependent manner; in shale, there is strong sorption.
Dr. Huang noted that an inner nanotube core may slide, almost without friction, within its outer nanotube
shell, thus creating an atomically perfect linear or rotational bearing. Additionally, studies show that C6o
can be degraded by microbes via an enzyme. The researchers examined whether white rot fungus, used in
bioremediation, could degrade multiwalled carbon nanotubes and found that it could not. Bacterial
degradation was evidenced by multiwalled carbon nanotube mineralization, so the researchers attempted to
determine the method of degradation using DNA extraction, propagation, isolation, sequencing, and
comparison. Three bacteria (Burkholderia, Delftia, and Stenotrophomonas) were identified, all of which are
Gram-negative aerobes involved in the degradation of organic contaminants. These field bacteria probably
work in concert to degrade. Bacterial degradation has implications for nanotube behavior and sequestration.
The researchers also examined chronic exposures using Ceriodaphnia dubia with the goal of evaluating
reproductive toxicity and accumulation of multiwalled carbon nanotubes by adult and neonate C. dubia
under two different solubilization protocols. Results indicated that sonication increased toxicity, whereas
natural organic material stabilized the nanotubes. Sonicated multiwalled carbon nanotubes adhered to adult
organisms and prevented molting and release of neonates. Natural organic material was protective against
reproductive toxicity, with no observed adherence to adults. There was significant accumulation of natural
organic material-solubilized multiwalled carbon nanotubes in neonates. The next step is a feeding study
that will determine whether there is trophic transfer from C. dubia exposed to multiwalled carbon
nanotubes following ingestion by Artemia and fathead minnows. Also, full life cycle exposures of
multiwalled carbon nanotubes will be evaluated in fathead minnows.
Discussion
Dr. Zepp asked about the strategy used to locate the bacteria. Dr. Huang replied that the bacteria were
found attached to samples that were being examined for the white rot fungus. Dr. Zepp asked how the
researchers synthesized the labeled materials. Dr. Huang stated that he used a common method that has
been described in many papers.
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A participant asked about the purity of the carbon-labeled material. Dr. Huang responded that amorphous
carbon is not seen because it has been removed. The participant asked about the size distribution.
Dr. Huang answered that a variety of sizes had been examined. Dr. Petersen added that these sizes were
100 to 420 nm. The participant asked whether the researchers examined distribution following aggregation.
Dr. Huang explained that this would be difficult to accomplish using scanning electron microscopy with the
mixed system; the researchers will be examining the chemistry, however.
OPEN DISCUSSION
Mr. Shapiro explained that in the past, participants have asked for an open discussion session to be included
in the meeting to discuss issues that are introduced throughout the day's presentations. Four issues were
introduced during the Day 1 discussions: (1) How can more be done toward LCA? (2) Is there a substitute
for platinum as a catalyst? (3) Should research focus on plumes or far lower concentrations? (4) EPA
Administrator Lisa Jackson is interested in examining methods by which to treat groups of drinking water
contaminants, including nanoparticles.
Mr. Shapiro asked Dr. Grassian to discuss her concern, which is item 3 above. Dr. Grassian wondered what
is the "right" concentration to study. Sometimes, research is conducted at high concentrations that may be
relevant to plumes. Dr. Westerhoff agreed that this is an important consideration and noted that it does not
make sense to study concentrations that are orders of magnitude lower than those at which an effect is seen.
Concentrations studied should be environmentally relevant. Dr. Lowry commented that lower doses may be
relevant to chronic toxicity. Dr. Orr agreed that bioaccumulation is important. Dr. Holden added that what
constitutes a dose also is an important question. Mr. Shapiro asked Dr. Holden's opinion on what consti-
tutes a dose, and she responded that it depends on the mechanism. Dr. Xia said that it was hard to
generalize, and the approach to determining this should include dose response, exposure, and screening.
Dr. Petersen noted that there could be a wide range of doses (e.g., plume vs. environmental concentrations).
It is necessary to be cautious when interpreting toxicity results, which must be placed in context and
compared to other compounds in the environment.
Dr. Savage asked the best method for writing a solicitation that would address chronic toxicity of
nanomaterials, which requires a significant amount of time and money. Dr. Lowry stated that this issue
cannot be confined to nanotechnology. There is an EPA model that addresses similar issues; therefore, the
uncertainty already has been dealt with. Dr. Grassian noted that the National Institutes of Health fund long-
term studies (greater than 20 years). Dr. Savage asked whether a reasonable approach would be to focus on
fate and transport and then examine chronic toxicity after the fate and transport studies have yielded results.
A participant noted that extrapolating from acute toxicity is difficult. Terrence Kavanagh (University of
Washington) agreed that this extrapolation was difficult because often the targets of acute and chronic
toxicity are completely unrelated (e.g., organophosphate [OP] acute toxicity results in cholinesterase
inhibition, whereas OP chronic toxicity results in neurotoxicity).
A participant asked whether carbon nanomaterials, reactive metal nanomaterials, and so forth could each be
grouped together for study. Dr. Savage explained that the Request for Applications (RFA) should focus on
mixtures because the compound-by-compound approach is not working. Dr. Orr suggested that libraries of
data be created that focus on an array of compound modifications to develop the whole picture.
Dr. Petersen commented that some trends may be emerging (e.g., carbon nanotubes and physical effects).
Classification could be based on how nanoparticles cause toxicity generally to organisms. A participant
stated that a good deal of research has focused on the toxicology of chemicals and pharmacology of
toxicity. From a pharmacological point of view, receptors are important. Some nanotubes may have
properties and interact in ways for which researchers have no foundation; this is an infant science.
Dr. Lowry noted that even though this is an infant science, there are 20 years of toxicological PM research,
and particle science is not new.
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Dr. Rogers suggested considering nanotechnology from a purpose or functional standpoint. Nanomaterial
designers do not think about toxicity; they focus on making the best material for their purpose. If
manufacturers are encouraged or required to consider toxicity, then they will be more receptive to hearing
recommendations from the research community. The perception that nanotechnology toxicological research
is performed for the benefit of industry so that manufacturers avoid potential disasters should be
perpetuated. Dr. Kavanagh agreed that industry would appreciate feedback from nanotechnology
researchers.
The participants discussed persistence. A participant noted that even if a compound degrades quickly,
chronic exposures are possible if there is continual loading. Dr. Yokel pointed out that ceria nanoparticles
can persist for at least 3 months.
In response to a question from Mr. Shapiro, Dr. Lowry explained that there were two different thoughts: to
either make nanomaterials safe or minimize exposure. His opinion is that both approaches should be
implemented. Dr. Savage noted that it is difficult to design green nanotechnology because often a
nanomaterial appears safe, but in the aquatic environment it is not. Dr. Lowry stated that it was necessary to
obtain as much information as possible to make informed decisions. A participant noted that some sources
can be controlled, but it is difficult for other source streams; there is no one solution.
Dr. Orr commented that positively charged nanoparticles increase toxicity in mammalian cells, so
positively charged nanomaterials should be avoided. Research can start building similar "rules."
Mr. Shapiro asked what the participants thought about performing joint research with industry. Dr. Yokel
noted the example of the Health Effects Institute, which was cofounded by EPA and the automobile
industry. Any nanoparticle research that relates to combustion could have a funding source in place.
Dr. Savage added that the National Nanotechnology Initiative aims to increase private-public partnerships
(e.g., CEINT). Dr. Lowry stated that BASF Corporation and IBM Corporation would like to engage the
nanotechnology research community, although they are not interested in providing funding. He suggested
that making research relevant to the needs of industry may increase private funding. Industry has some
answers, but they are not publicized because of the nature of their confidential business materials. Industry
will release research and development materials. A participant cautioned that patents must be considered
when dealing with industry.
Mr. Shapiro asked whether it would be helpful if EPA emphasized, assisted, or encouraged the commer-
cialization of research products; this is another manner in which to partner with industry. Dr. Lowry noted
that NSF has programs that require grantees to have industry partners. Also, there are Small Business
Innovation Research grants available from various federal agencies.
Mr. Shapiro thanked the participants for attending and recessed the meeting at 6:22 p.m.
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NOVEMBERS, 2010
Review of Day 1 and Plans/Ground Rules for Day 2
Paul Shapiro, U.S. EPA
Mr. Shapiro called the meeting to order at 7:54 a.m. and welcomed the participants to the second day of the
meeting. He reiterated the ground rules and expectations for the meeting and discussed logistical issues. He
reminded the participants that the schedule is based on the SETAC schedule, and it was important to adhere
to this schedule. He pointed out the "parking lot" for the open discussion session at the end of the day.
During breaks between presentations, Mitch Lasat explained that EPA has partnered with other agencies
and international organizations to facilitate the flow of ideas and increase innovation. The resulting
program will support innovative nanotechnology research co-funded by EPA and a partner in the United
Kingdom. Each project is funded for 4 years at $1 million per year. Dr. Savage provided information about
the nanotechnology RFA that closed in February 2010. There were more than 100 submissions, and five
were funded by EPA, five by NSF, and four by the U.S. Department of Agriculture. The two research
categories are: (1) environmental matrices and (2) biological matrices with a food focus. A new $4 million
center will attempt to understand environmental matrices.
MORNING SESSION 1: EFFECTS ON CELLS
Functional Effects of Nanoparticle Exposure on Airway Epithelial Cells
Amiraj Banga, Indiana University-Purdue University at Indianapolis
Nanoparticles are being scrutinized as a health hazard, and humans are exposed to nanoparticles in various
ways. Workers handle nanoparticle materials in many industrial jobs, and nanoparticles can enter the body
via inhalation, ingestion, and penetration through the skin. Complete information about health effects of
nanoparticles is lacking. The research used three different unpurified and as-manufactured carbon
nanoparticles: multiwalled carbon nanotubes, single-walled carbon nanotubes, and C6o. The hypothesis is
that manufactured, nonfunctionalized carbon nanoparticles, when exposed to barrier epithelia, exert a
biological effect on the cell membrane and may alter the cell function. Dr. Banga explained the laboratory
approach and noted that all concentrations are expressed in micrograms per square centimeter.
Results indicated that both nanotubes significantly decreased the resistances of cells over a wide range of
concentrations, but C6o did not. It is interesting to note that the effects of these low concentrations have not
been reported in the literature; the laboratory hypothesizes that these concentrations are physiologically
more relevant. Additional experiments highlighted the fact that chloride moves in a secretory direction,
causing water to follow and leading to hydration of the passageway. Exposure to different types and
concentrations of carbon nanoparticles showed a variable response, but the effect of nanoparticles still is
observed at the lowest concentration (0.004 |o,g/cm2). Because the initial increase in chloride secretion is
mediated predominantly by an increase in intracellular cyclic adenosine monophosphate (cAMP), resulting
in activation of protein kinase A and consequently phosphorylation and activation of CFTR, the researchers
examined cAMP in the treated and control cells. After epinephrine stimulation, the rise in cAMP was found
to be the same in nanoparticle-exposed and control monolayers. These results suggest that the ion transport
element affected by the nanoparticles lies beyond the basolateral membrane epinephrine receptor and
intracellular cAMP production.
In summary, low-dose nanotube exposures decrease the barrier function of airway epithelial cells. Low-
dose nanotube exposures affect the ability of the airway epithelial cells to secrete chloride. These data
suggest that the levels of nanotubes found in the workplace, particularly during chronic exposures, are
likely to have physiological effects that can cause or exacerbate respiratory problems.
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Discussion
Dr. Holian asked if the researchers had examined nanotubes of varying metal content or considered that the
nanotubes might be conducting. He also asked about the purity of the materials. Dr. Banga responded that
the purity was 99 percent, and the nanotubes contain certain metals (nickel, cobalt, iron), but as 1-hour
experiments showed no significant effects, the nanotubes were not conducting. Dr. Holian noted that
nanotubes must enter the cells to conduct. He asked whether the researchers had tried carboxylation.
Dr. Banga responded that they had not yet done so.
Toxicity Assessment of Nanomaterials in Alveolar Epithelial Cells at the Air-Liquid Interface
Galya Orr, Pacific Northwest National Laboratory
The rationale of the project is that airborne nanomaterials that enter the respiratory tract are likely to be
deposited in the alveolar region, where alveolar epithelial cells are found at the interface with ambient air.
To date, the majority of in vitro studies characterizing the interactions and impact of engineered
nanomaterials in these cells have been carried out in cells submersed under growth media. To more closely
mimic in vivo exposures, the researchers have established the growth of alveolar type II epithelial cells at
the air-liquid interface, enabling realistic exposures to aerosolized nanoparticles. Type II cells play critical
roles in the function of the alveoli by secreting pulmonary surfactants, and by differentiating into type I
epithelial cells when these are damaged. Importantly, type II cells participate in the immune response to
certain particles and pathogens by releasing chemokines. By collecting the particles on millimeter-size
grids placed randomly over the cells and visualizing them using electron microscopy, it is possible to
accurately quantify the number of particles delivered per square centimeter or per cell. This approach also
enables physical and chemical characterizations of the collected nanoparticles, providing properties that are
relevant to airborne nanoparticles and the actual exposure at the air-liquid interface.
The project studies manufactured amorphous silica nanoparticles, which are used extensively in a wide
range of industrial applications. The results did not show decreased membrane integrity or proliferation of
alveolar type II epithelial cells following exposure to 50 nm bare amorphous silica nanoparticles. The
researchers estimated equivalent doses in submersed and air-liquid interface conditions using the
computational In Vitro Sedimentation, Diffusion, and Dosimetry (ISDD) Model. The ISDD model
integrates the influence of particle properties and cell culture conditions to calculate the actual deposited
cellular dose (particles per cell). Using estimates from the particokinetics model, cells were exposed to
submersed conditions, and no membrane compromise, toxicity, or decrease in proliferation was observed.
Next, the researchers focused on ZnO nanoparticles, which can be highly toxic, an effect that might
originate from the dissolved molecules. Large aggregates were created in two different solutions. Following
exposure to aggregates, toxicity in cells emerged at a concentration of 9-10 |o,g/ml (300 aggregates per
cell). Under submersed conditions, ZnO toxicity was observed at a concentration of 25 |o,g/ml. Therefore,
ZnO toxicity can be induced by intact particles or dissolution of the molecule in a local area, which
provides insight into ZnO toxicity. Testing the same outcome but in a different manner still showed ZnO
toxicity at a concentration of 25 |o,g/ml.
In conclusion, exposures of alveolar type II epithelial cells to 50 nm bare amorphous silica nanoparticles at
the air-liquid interface elicit no significant cytotoxic response at concentrations ranging from 10 to 1,000
particles per cell. These observations agree with the response of submersed cells exposed to equivalent
doses as estimated by a computational particokinetics model. Dose-response evaluations of 300 nm ZnO
aggregates (25 nm primary particle size) in alveolar type II epithelial cells exposed at the air-liquid
interface show a toxic response starting at approximately 300 aggregates per cell (10 (ig/ml) 24 hours
following exposure. Toxicity evaluation of these aggregates in submersed cells elicits a toxic response at
approximately 25 (ig/ml, indicating that they might be slightly more toxic at the air-liquid interface. These
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findings support the idea that ZnO aggregate toxicity can originate from the intact nanoparticles or from
molecules dissolved locally at the cell membrane or inside the cell.
Discussion
Dr. Yokel asked whether surfactants had any effect on the nanomaterials. Dr. Orr replied that the laboratory
is attempting to obtain artificial surfactants prescribed to premature infants to study next.
Dr. Sadik asked how background was accounted for by the researchers and whether the researchers were
aware that flow cytometry often introduces artifacts into results. Dr. Orr responded that controls always are
included to manage these types of issues. In terms of the flow cytometry, filters are applied or oxidative
trace studies are performed.
Interactions of Nanomaterials With Model Cell Membranes
Jonathan Posner, Arizona State University
This project attempts to measure particle properties and perform toxicity assays to develop global
descriptors that predict bioaccumulation for use in models. The main global descriptor is the octanol-water
partition coefficient, which is a ratio of concentration of solute in between two immiscible phases,
generally octanol and water. It is used in water quality models to predict fate, accumulation, and aquatic
toxicity of organic pollutants in the environment. It is not defined for particles, however. To examine
octanol-water partitioning of engineered nanomaterials, the researchers studied a variety of materials and
determined that surface charge is important, but it is difficult to identify trends. The researchers attempted
to quantify partitioning at various interfaces and conditions. Partitioning occurs because of the minimi-
zation in Helmholtz free energy. Although zeta potential correlates with pH, it cannot be used to predict
partitioning.
Challenges with determining octanol-water partitioning of engineered nanomaterials include importation
into EPA models and treatment of mass at the interface. Additionally, partitioning provides no information
on the state of engineered nanomaterials (e.g., aggregation, dissolution), is path dependent, does not
correlate with bioaccumulation, and is dependent on the poorly defined interfacial area. Therefore,
researchers have taken an analogous approach using lipids. The lipid bilayer is an important interface
between life and its environment and a potential exposure route for engineered nanomaterials. The lipid
bilayer-water distribution has been shown to be a more appropriate indicator than octanol-water
partitioning for bioaccumulation of ionizable organic molecular and surface active compounds, with which
engineered nanomaterials share some properties. Lipid bilayer-water distribution is being used increasingly
in environmental research regarding molecular pollutants. Lipid bilayers are the primary constituent of
many biological cellular membranes and often are used to model passive transport into cells.
The researchers used commercially available lipid bilayers noncovalently bound to silica in their
experiments. The engineered nanomaterials used were aqueous C6o aggregates, fullerol, and gold nano-
particles. The concentration of nC6o was determined by HPLC, fullerol concentration by UV-visible
absorption spectroscopy, gold nanoparticle concentration by ICP-optical emission spectroscopy, lipid
concentration by malachite green dye assay, and the sizes and zeta potential of the liposomes and
engineered nanoparticles by dynamic light scattering. Fullerols and nC6o were found to have similar size
distributions and charge. The researchers quantified all of the mass in the system and determined that there
was no loss to the glass walls at pH 7.4 and that distribution of nC6o and fullerol in lipid-water is pH
dependent. The next goal was to compare isotherms in environmentally relevant situations, and the
laboratory found qualitative agreement with other studies that suggest higher bioaccumulation and toxicity
of nC6o compared to fullerol. Lipid-water distribution isotherms of gold nanoparticles suggest that number
of particles appears to be a reasonable metric.
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In summary, the lipid bilayer-water distribution of the selected engineered nanomaterials is a pseudo-
equilibrium process that can be described by isotherm behaviors. The distribution behavior and
accumulation to lipid bilayers are pH dependent. Size dependency studies show that 20 nm gold
nanoparticles exhibit the highest propensity to accumulate in lipid bilayers. Comparisons with
bioaccumulation and toxicity studies using organisms suggest that the lipid bilayer-water distribution is
promising for assessing the bioaccumulation and toxicity potentials of engineered nanomaterials.
Bioaccumulation data (i.e., bioconcentration factor) data are needed for a variety of engineered
nanomaterials to verify whether lipid-water distribution can be used to predict the fate of engineered
nanomaterials.
Discussion
Mr. Shapiro asked about the next steps. Dr. Posner responded that the laboratory will examine a variety of
particles and collect additional bioconcentration factor data to determine trends.
In response to a question from a participant, Dr. Posner explained that when nanoparticles and electrolytes
are mixed, zeta potential is modified slightly in the final solution. When asked if this would occur with all
nanoparticles, Dr. Posner replied that it depended on the nanoparticle. For example, the researchers did not
observe gold absorption to glass; therefore, he did not want to generalize.
In response to a question from Dr. Huang, Dr. Posner explained that particles have ionizable surfaces;
therefore, the rationale is that the surface chemistry is similar.
Development of an In Vitro Test and a Prototype Model To Predict Cellular Penetration of
Nanoparticles
Yongsheng Chen, Georgia Institute of Technology
Surface interactions are the first step for nanomaterials to act in a beneficial or detrimental manner.
Governing parameters that contribute to interactions include nanoparticle properties, cell properties, and the
environment; these parameters lead to biological consequences (e.g., interfacial forces, sorption processes,
cellular damages). The researchers addressed the question of how particle size impacts biological
interactions and focused on hematite as a reference material because it is relatively stable and displays
uniform size distribution in culture media. Escherichia coll is used because it is a common model for
toxicity tests and ubiquitous in the environment. A model epithelium cell line for human intestinal cells was
used as well. The researchers evaluated surface property changes of E. coll, adsorption kinetics, size effects
on the adsorption kinetics, and DNA binding with ultrafine nanoparticles.
Results indicated that hematite accumulates on the surface of E. coll, causing deformity, death, and flagella
damage. Surface disruption can disrupt cellular respiration without nanoparticle entry into the cell.
Adsorption kinetics of hematite nanoparticles on E. coll cells also show the dependency on particle size,
with adsorption rates being faster for small nanoparticles compared to large ones. The contradiction in the
trend of size effects on adsorption kinetics caused by concentration expressions can be interpreted via the
Interaction Force Boundary Layer (IFBL) Theory. IFBL and DLVO are combined to interpret the size
effect on the adsorption kinetic, and the model agrees with the experimental observations.
Results of DNA binding experiments following E. coll exposure to quantum dots indicate that ultrafine
quantum dots can permeate into E. coll cells and unintentionally bind with DNA. Results of human cell line
experiments indicate that bio-nano interactions cause microvillus disruption, including structural damage
and decreased cellular integrity and nutrient absorption, and adhesion junction disruption. Cells lose their
integrity and eventually die. Adsorption kinetics on the human cell line show similar features to hematite
nanoparticle adsorption in E. coll. Large particles adsorbed faster by mass-based concentrations, and small
particles adsorbed faster in number-based concentrations. In terms of the size effects on the disruption of
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the adhesion junction and cell penetration, small nanoparticles penetrated cell lines faster and led to more
severe junctional disruption.
The researchers concluded that hematite nanoparticles are ideal for use as a reference nanomaterial because
of their high stability, uniform size distribution, and low aggregation. Adsorption kinetics are size
dependent, which can be interpreted by IFBL Theory. The exposure to hematite nanoparticles induced
reorganization and distortion of surface structure damages and cell penetration. Challenges include
determination of the role of interfacial forces and diffusion in the transport of nanoparticles toward
biological systems and DLVO theory versus mass transport mechanisms. During the next year, the
laboratory will continue to extend its developed methodologies (e.g., models) to evaluate other types of
nanoparticles regarding their environmental and biological behaviors. The researchers plan to develop
sophisticated imaging and quantifying techniques for the surface characterization of nanoparticles and their
interactions with the biological system at the nanoscale. The laboratory has published 15 papers in peer-
reviewed journals, submitted six manuscripts, and presented 20 invited talks at national and international
conferences.
Discussion
Mr. Shapiro thought that the presentation related to the previous day's discussion regarding providing
industry with recommendations and information.
Dr. Holian asked how the researchers took agglomeration into account. Dr. Chen replied that the
researchers verified that the small particles still were stable in the cell culture media.
Dr. Petersen asked how the researchers differentiated between adsorption and absorption because both
could be occurring; this is important to consider in a model based primarily on surface interactions.
Dr. Chen agreed that this was a good point.
Dr. Heideman asked how the researchers distinguished between live and dead E. coll with atomic force
microscopy. Dr. Chen replied that they mobilized E. coll cells on a silicone chip and verified whether they
were alive or dead via colony numbers. Dr. Heideman asked how the researchers could tell whether the
particles bound DNA in vivo. Dr. Chen dispersed quantum dots into the suspension for a 1-hour exposure,
extracted the DNA, and observed changes in the DNA, some of which may not be conformational. He
agreed, in response to a comment by Dr. Heideman, that this could have occurred following the opening of
the cells.
Dr. Unrine noted that receptor-mediated endocytosis occurs in eukaryotes. He asked whether the
researchers considered the strength of the interactions with cell surface receptors in the model. Dr. Chen
answered that this was difficult, and the laboratory is providing compelling evidence that penetration
cannot be controlled.
MORNING SESSION 2: EFFECTS AT THE SUBCELLULAR LEVEL
Impacts of Quantum Dots on Gene Expression in Pseudomonas aeruginosa
Shaily Mahendra, University of California, Los Angeles
Quantum dots are semiconducting nanocrystals that have biomedical and electronics applications.
Biocompatible quantum dots have a hydrophobic core, often containing toxic metals, surrounded by an
inorganic shell. Because of the hydrophobic core, these quantum dots can be stabilized in water by
derivatizing the surface with amphiphilic organic coatings. In terms of quantum dot weathering, the
laboratory's hypothesis is that the toxicity of quantum dots primarily is a result of free metal, and
environmental weathering of the coating will increase their toxicity to cells. They are safe for intended
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uses; therefore, decreasing exposure and/or degradation will eliminate, to the extent possible, quantum dot
toxicity. Degraded quantum dot cores increase bioavailability to cells and microbes.
Laboratory results indicate that coated quantum dots retard cell growth, and weathered quantum dots kill
bacteria. Additionally, cadmium and selenite are toxic to cells. Pseudomonas aeruginosa tolerated high
concentrations of cadmium from quantum dots; the bacterial species apparently has mechanisms by which
to expel quantum dots. Therefore, this may be a good candidate at the molecular (genetic) level to under-
stand the impact of sublethal doses, which would allow proactive predictions of risk. Dr. Mahendra
outlined several mechanisms of bacterial toxicity, including protein oxidation by nC6o, disruption of cell
membranes by single-walled carbon nanotubes, generation of ROS by TiO2, DNA damage by multiwalled
carbon nanotubes, and release of toxic ions by ZnO and nanosilver.
The researchers use microarrays to provide a snapshot of genome expression following exposure and
analyze global gene responses to quantum dot exposure. The researchers analyzed differences in gene
expression, functional genes and pathways affected by coated quantum dots, and functional genes and
pathways affected by weathered quantum dots. Results indicated that metal resistance genes were induced
by weathered quantum dots but not coated quantum dots.
In summary, coated and weathered quantum dots affected gene expression in P. aeruginosa, and the
functional categories of amino acid metabolism, energy production, and carbohydrate metabolism were
primarily regulated. Metal-resistance genes were upregulated following weathered quantum dot exposure.
Results also indicated that there is an apparent change from ammonium-assimilating aerobic metabolism
toward anaerobic, denitrifying metabolism in response to stress.
Discussion
Dr. Rowe stated that P. aeruginosa tends to switch to anaerobic respiration after sitting, so this may be the
cause of that observation. He also recommended that the researchers examine the proteosome to determine
whether there is translation, which is more relevant, in addition to transcription.
Dr. Petersen asked whether the researchers examined amounts of cadmium and selenium in the cells
following exposure to weathered and coated quantum dots. Dr. Mahendra replied that the laboratory used
TEM to image cells exposed to quantum dots and identified cadmium ions and zero-valent CdSe, which
were associated mostly with the surface membrane. Dr. Petersen asked whether the researchers performed
ICP-MS. Dr. Mahendra answered that they had, and the data are in a manuscript under review.
Dr. Lowry asked whether the researchers had examined different kinds of particles (e.g., FeO, silica) that
do not injure the bacteria to ensure that the bacteria are responding to the nanoparticle. Dr. Mahendra
responded that these types of experiments had been performed; bacterial and fungal responses were
compared, and bacteria responded only to the cadmium in the nanoparticles. The ion appears to be more
important than the nanoparticle.
Thiol Redox-Dependent Toxicity and Inflammation Caused by TOPO-PMATModified Quantum Dots
Terrence Kavanagh, University of Washington
Dr. Kavanagh explained that there was a recent review in Science regarding activities of nanoparticles in
the environment and how important surface chemistry is to induce the various forms of oxidative stress.
The hierarchical model of oxidative stress induced by exposure to nanoparticles consists of tiers: (1) antiox-
idant defense mechanisms, (2) inflammation, and (3) cytotoxicity. These increase as oxidative stress
increases. The researchers used quantum dots, which have multiple uses, including gene and drug delivery.
Because uncoated quantum dots often have poor solubility and are unstable in biological systems, the
researchers chose manufactured quantum dots that are exceptionally stable in aqueous solution and display
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red-orange fluorescence for use as in vivo tracers. The project examined the interactions of cell types with
macrophages, and results indicated that the effects (changes in NADPH, thiols, and viability) were
relatively minor after 24 hours, but colony forming efficiency decreased after 7 days. Researchers also
observed an increase in hemoxygenase and glutamate-cysteine ligase (GCLM), which is involved in
glutathione production. Hemoxygenase induction and necrosis are highly correlated with quantum dot
uptake, and a number of proinflammatory cytokines are upregulated as well.
Quantum dots can release heavy metals, causing oxidative stress and toxicity in biological systems.
Glutathione is important in preventing oxidative damage to cellular macromolecules and has been shown to
be an important modulator of the immune response. Therefore, glutathione could be an important
determinant of quantum dot-induced toxicity and inflammation. Glutathione, a heterodimer, is important in
scavenging free radicals, and its levels are controlled by cysteine availability, synthesis, and utilization, and
organ import and export. The researchers found that glutathione depletion does not increase the toxicity of
the quantum dots to the mouse macrophage cell line, and, unexpectedly, glutathione depletion suppresses
cytokine responses in this cell line. To more thoroughly investigate this phenomenon, researchers used a
Gclm-null mouse as an in vivo model of glutathione depletion. Humans are known to have polymorphisms
in GCLM, which predispose them to heart disease, lung diseases, schizophrenia, and heavy metal body
burden.
The researchers tested the susceptibility of mice with varying amounts of GCLM production to
nanoparticle-induced lung injury by exposing them to quantum dots. Gclm-null mice have low GCLM
activity and low levels of glutathione in most tissues. The researchers exposed the mice to quantum dots via
nasal instillation. There is correlation between neutrophil influx and protein in bronchoalveolar lavage fluid
8 hours postexposure. Surprisingly, nasal instillation of quantum dots increases neutrophils in the airways
of wild-type and Gc/m-heterozygous mice but not Gclm-null mice, and quantum dots increase
inflammatory cytokine levels in the bronchoalveolar lavage fluid of wild-type and Gc/w-heterozygous mice
but not Gclm-null mice. Possible reasons for the lack of inflammation in Gclm-null mice could be the
failure of their macrophages to take up the quantum dots, produce and/or secrete chemotactic peptides and
cytokines, or produce ROS. Alternatively, perhaps the lack of glutathione has resulted in an adaptive
response (e.g., upregulation of protective genes), which acts to squelch oxidative stress or the immune
response. Researchers also found that Gclm-null mice have attenuated myeloperoxidase activity but not
matrix metalloproteinase activity in their lungs after quantum dot exposure, and quantum dot-induced
cytokine responses are attenuated in cultured peritoneal macrophages from Gclm-null mice. Glutathione
depletion enhances nuclear factor-kappa B translocation induced by quantum dots in the mouse
macrophage cell line.
Ongoing studies focus on the mechanisms of quantum dot uptake by macrophages, markers of oxidative
stress in lung tissue and bronchoalveolar lavage cells and fluid, chronic effects of exposure to quantum
dots, DNA microarray analysis of gene expression for additional biomarkers of lung injury, systemic
inflammation/markers of lung injury, translocation of quantum dots and cadmium to other organs, and
effects on the olfactory epithelium and brain.
Discussion
Dr. Rowe asked if the phenotype of the knockout mouse was generally healthy. Dr. Kavanagh responded
that they are relatively healthy but have compromised fertility, and one research group saw behavior similar
to schizophrenia. Dr. Rowe asked whether the researchers examined weathered dots. Dr. Kavanagh replied
that various coatings and stability were examined.
David Barber (University of Florida) stated that a theme in the literature is that dramatic toxicity is not seen
until mitochondrial glutathione is depleted. He asked whether the researchers had examined the difference
between cytosolic and mitochondrial glutathione in the knockout mice. Dr. Kavanagh answered that they
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had; the knockout mice have depleted glutathione in the mitochondria (30% of normal) but not to the extent
of cytosolic glutathione depletion (10-15% of normal).
Bioavailability and Fates ofCdSe and TiO2 Nanoparticles in Eukaryotes and Bacteria
Patricia Holden, University of California, Santa Barbara
Through manufacturing and use, nanoparticles will enter the environment, particularly via waste streams,
where they will be taken up by individual cells, especially bacteria. The bioavailability continuum
(agglomeration, adhesion, entry, accumulation) is simplistic. Four questions to consider are: (1) When do
nanoparticles enter cells? (2) Do the particles stay intact? (3) What are the cellular effects? (4) What are the
variables? The hypothetical framework of the interactions of nanomaterials and cells has become more
complex as more research is completed.
The researchers used laboratory-synthesized CdSe/zinc sulfide (ZnS) quantum dots and laboratory- and
industrial-synthesized TiO2 nanoparticles and varied light and dark conditions. Certain laboratory methods
were selected to determine whether electron transfer is occurring between nanoparticles and cells that could
contribute to cell oxidation and generation of free radicals that ultimately could allow nanoparticles to enter
cells. Other methods were used to characterize and quantify the nanomaterials and measure exposure to and
effects on cells. Previous studies have shown that CdSe quantum dots enter planktonic cells in light
conditions. Quantum dot fluorescence lifetime is examined to study energy transfer to quantum dots to
understand how energy transfer may ultimately be linked to the generation of free radicals that could affect
cells with which quantum dots are associated. These quantum dot lifetimes vary with different cores, caps,
and conjugates.
Results indicated that CdSe/ZnS quantum dots photosynthesized with dopamine increased in superoxide
dismutase, intracellular ROS, and reactivity and decreased in metabolism in the cells. Bare CdSe quantum
dots enter and are toxic to Pseudomonas in dark conditions, and cadmium telluride quantum dots
differentially bind and transfer electrons to bacterial strains. As a result of electron transfer, Gram-positive
bacterial membranes are depolarized, but bacterial growth is not slowed. The researchers concluded that
quantum dots can enter cells with ROS-mediated membrane damage, but the ROS form varies. Quantum
dots can enter cells intact, but caps slow dissolution. Cells show consequences of uptake of quantum dots
(e.g., slow growth rate and lower yield), but membrane depolarization does not appear to be fully toxic.
When the laboratory examined the consequences for the next trophic level, it showed that CdSe quantum
dots can be trophically transferred from Pseudomonas to Tetrahymena, a protozoan. Furthermore, Pseudo-
monas binds and disagglomerates TiO2.
In summary, quantum dots can damage and enter cells and activate electron transfer. TiO2 binds to cells but
does not enter. Variables include light versus dark conditions, strain, specific nanoparticle, cap, conjugate,
and oxygen. The next steps of the laboratory are to perform high throughput studies on membrane effects
and quantify cell loading and bioprocessing.
Discussion
Dr. Rowe asked about the size distribution used when uptake was observed. Dr. Holden responded that it
was 5 nm. Dr. Rowe asked if the researchers used a size curve, to which Dr. Holden replied that they did
not. Dr. Rowe thought that bacterial surfactants might be involved, but Dr. Holden explained that the
researchers had proven that they were not by measuring surface tension; dispersion in citrate also did not
occur. Dr. Rowe asked whether the size and shape of the bacteria were taken into account in terms of
surface binding. Dr. Holden answered that for the quantum dot experiments, the researchers quantified the
amount of cadmium that was associated with the cell using cadmium as a tracer and used microscopy to
observe orientation. Therefore, the size and shape were not taken directly into account.
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Using Zebrafish Embryos To Test Phototoxicity ofTiO2 Nanoparticles
Warren Heideman, University of Wisconsin-Madison
The laboratory is examining the theory that light causes ROS production in vivo, and the question is
whether this matters in vivo. Zebrafish show cardiotoxicity following exposure to nanoparticles. Testing
TiO2 nanoparticles in vivo requires TiO2 nanoparticles, zebrafish embryos, and light. Because coral is
photodependent, the aquarium hobby industry has developed aquarium lights that mimic sunlight; the
researchers used these lights for their experiments. Results indicated that zebrafish embryos exposed to
TiO2 nanoparticles and illumination do not survive. When zebrafish embryo survival at various times and
TiO2 nanoparticle concentration were examined, the researchers found that decreased nanoparticle
concentrations increased survival time. There are several phenotypic defects associated with TiO2
nanoparticle exposure and illumination, including malformed head and tail, stinting, edema, and extended
yolk.
Because the researchers realized that it was possible that toxicity unrelated to the nanoparticles might have
been caused by a new reactive species of chemical created as a result of the plastic well in which the
experiments were conducted, they illuminated the TiO2 nanoparticles prior to exposing embryos, which did
not cause toxicity. Embryos pre-exposed to TiO2 nanoparticles, washed, and then illuminated showed
toxicity. TiO2 nanoparticles have a pronounced tendency to aggregate, and TiO2 nanoparticle exposure adds
measurable titanium to the fish. TiO2 nanoparticles are found throughout the zebrafish embryo. TEM
determined that the egg chorion shields the embryos from toxicity. Dehydroergosterol fluorescence was
used to detect superoxide production, and the yolk showed autofluorescence under all experimental
conditions. Additionally, DNA adducts are formed when TiO2 exposure is combined with illumination.
Fish have a clear defense mechanism to protect cells from oxidative stress; it involves transcription factors
that bind to a canonical sequence called "ARE" that drives production of enzymes that protect the organism
from oxidative stress. Using green fluorescent protein as an ARE reporter shows activation by TiO2
nanoparticles combined with illumination; therefore, this is the normal response to oxidative stress.
Preloading embryos with N-acetyl cysteine (NAC) can prevent some of the effects of TiO2 nanoparticle
exposure.
The photochemistry of TiO2 nanoparticles predicted that the nanoparticles might cause phototoxicity as a
result of ROS production. The uncertainty was whether this occurs in vivo. Using zebrafish embryos, the
researchers showed that TiO2 nanoparticles cause light-dependent toxicity associated with uptake and ROS
production. The findings in zebrafish may be relevant to humans because many biological systems are
strongly conserved, and mechanisms that work in zebrafish often are found in humans.
Discussion
Dr. Savage asked whether oxidative stress was seen in all of the same cells. Dr. Heideman responded that
he was unsure which cells are being affected, but the pattern is the same.
Dr. Jovanovic was concerned about the environmental relevance of the study because of the artificial nature
of the lights designed for coral, despite the in vivo construct. Many studies show that the amount of energy
needed to cause photoactivation is much higher than the particles can receive from sunlight. Dr. Heideman
replied that the response tends to be seen with high concentrations of nanoparticles. It is difficult to
determine whether this is environmentally relevant because zebrafish are relatively hearty. The artificial
light was developed by scientists in a very scientific manner. The illumination used likely is lower than
sunlight received on a sunny day, but it is necessary to remember that sunlight changes throughout the day,
so it is difficult to equate the two types of illumination.
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Xinyu Yang (Duke University) noted that NAC is a chelator and wondered whether it was possible that
NAC is chelating the metals in TiO2. Dr. Heideman said that he had not considered this, and another set of
controls would need to be added to test this.
AFTERNOON SESSION 1: EFFECTS ON FISH AND OYSTERS
Effects of Subchronic Exposure to Nanoparticulate Silver in Zebrafish
David Barber, University of Florida
Nanosilver is not as toxic as other nanomaterials in terms of gill proliferation in zebrafish, but accumulation
of nanosilver could cause chronic toxicity. The experimental design incorporates zebrafish exposed to
various concentrations of 25 nm nanosilver. Results indicated that accumulation was approximately
50 percent of nominal, and levels dropped to zero 4 days following the removal of nanosilver exposure.
Silver levels on day 3 following exposure were similar despite the exposure concentrations being different
by orders of magnitude. This may be because the researchers are not measuring soluble silver or because
the system is being saturated with particulate silver. Carcass tissue burden was found to be dose and time
dependent, indicating absorption and accumulation of particulate and dissolved silver in tissues outside of
the intestinal tract. After nanosilver exposure was discontinued, tissue silver concentrations remained
stable. Gill silver concentrations were greater than tissue concentrations, which is expected because gills
have increased accumulation compared to other organs. This concentration, however, decreased after
2 weeks, possibly indicating an adaptive response.
The bioconcentration factor decreased as concentration increased, indicating that the bioconcentration
factor is concentration dependent. There also is significant correlation between carcass/gill burden and
nanosilver concentration but a lack of correlation with soluble silver. Gill morphology (i.e., cell
proliferation in the interlamellar space) appears unchanged following the 28-day exposure. Although
accumulation of silver is seen in the skin and nasal epithelium, there is no evidence of morphological
injury; the same is true for several other tissues and organs (e.g., liver, heart). The researchers examined
transcriptional effects on the gill following 28 days of exposure, and a cluster analysis found three distinct
clusters: control, solubility, and high concentration. Although hundreds of genes were up- or
downregulated in response to the various concentrations, only 55 genes were common to all concentrations.
Increases in nanosilver concentrations increase the number of genes, but the genes differ by treatment. A
pathway analysis indicated that ribosomal and organ development effects were significant pathways.
The researchers concluded that zebrafish accumulate significant silver tissue burdens, gill levels are 10
times greater than carcass levels, and nanosilver remains for up to 4 days in the absence of additional
nanosilver exposure. There is a significant correlation between nanosilver concentration and tissue burden,
and soluble silver is not significant. There is no observable effect on epithelial morphology. Microarray
data indicate significant alterations in gene expression patterns and that there is a dose response pattern for
the number of genes affected. Pathway analysis indicates two pathways: organ development and ribosome
biogenesis.
Discussion
Dr. Westerhoff asked about the experiments in which bioconcentration decreased as a function of
concentration and whether on a nanogram per gram basis of tissue the results were similar. Dr. Barber
responded that tissue concentrations at later times were similar between the nominal and high doses.
Dr. Heideman suggested that the researchers examine a subset of genes affected by nanosilver versus
soluble silver. Dr. Barber answered that these types of studies have been performed in the past, and there
definitely is such a subset, but it is not annotated very well, so further work is needed to characterize it.
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Dr. Kavanagh asked whether the researchers looked specifically for widely recognized genes that might be
responsive (e.g., metallothionein). Dr. Barber answered that the researchers examined metallothionein, but
there was very little success in obtaining reproducibility with silver. Past studies indicate that there is
induction of a number of metal transport factors, heat shock proteins, and ROS.
Dr. Petersen asked about the laboratory's definition of nanosilver. Dr. Barber replied that finding soluble
silver was a challenge, and centrirugation yields more reproducible results compared to filtration at this
size. The researchers are aware, however, that complete dissolution probably is not being measured.
Refinements to the Use of Zebrafish for Nanomaterial-Biological Interaction Assessments
Lisa Truong, Oregon State University
Physicochemical properties influence nanoparticle behavior. Nanoparticle exposure to air, water, and
ground result in a variety of responses, including agglomeration, accumulation, aggregation, dissolution,
and so forth. Interaction of nanoparticles with environmental and biological systems remains largely
unknown. The research community is missing toxicological data to understand biocompatibility and needs
to identify the risk associated with nanoparticle exposure. The goal of the research is to determine what
influence each nanoparticle parameter has on biological activity. The hypothesis is that more than one
parameter (size, surface charge, functional group) activates different biological responses. Researchers used
a zebrafish model to test the hypothesis because zebrafish embryos develop within 120 hours. Zebrafish are
continuously exposed to various nanoparticles from 6 to 120 hours postfertilization. The researchers
assessed more than 200 nanoparticles via high throughput screening and found that a large portion did not
induce a biological response. Whether there are false negatives has not been established.
Nanoparticle properties change depending on the aqueous environment and conditions, aggregation can
occur in high-ionic-strength media, biological response can be altered, and it is necessary to characterize
aggregation in test media and throughout the exposure period. Therefore, the laboratory assessed nanoparti-
cle aggregation in aqueous media using gold nanoparticles. The three research questions were: Does ionic
strength play a role in aggregation? Can zebrafish develop and behave normally in low- or no-ion media?
Will suspension of gold nanoparticles in low-ionic-strength media induce biological activity? Results
regarding the first question indicate that high-ionic-strength media cause gold nanoparticle aggregation. In
terms of the second question, zebrafish morbidity, mortality, and phenotype were similar in all media.
Additionally, there was no statistical difference in the biological media following a period of darkness. The
researchers concluded that zebrafish develop normally in low-ionic-strength media. In answering the third
question, results indicated that decreasing the ionic concentration increased mortality and behavioral
effects. The researchers concluded that low-ionic-strength media favor dispersion of gold nanoparticles,
which are more toxic when dispersed.
The implications of these results are that every parameter must be taken into consideration when per-
forming nanomaterial-biological interaction studies and that refinement of the current high throughput
screening to include avoid false negatives and assess nanoparticles was deemed problematic. Finally,
zebrafish are a versatile model.
Discussion
Dr. Jovanovic asked whether the original medium was egg water or embryo water. Ms. Truong replied that
it was E2 embryo medium. Dr. Jovanovic asked how the low-ionic-strength medium was derived.
Ms. Truong responded that it was E2 embryo medium diluted with reverse osmosis water.
Dr. Zhang commented that this is an open carbon system that should allow CO2 to transfer to liquid, which
results in carbon speciation that could contribute depending on pH. He asked whether the researchers
considered this. Ms. Truong answered that they did.
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Dr. Holden stated that the point of using high throughput screening is to survey many different types of
nanoparticles and asked how the researchers plan to approach this issue with other nanoparticles.
Ms. Truong replied that most collaborations are characterizing media to determine whether nanoparticles
are available prior to assessment so that false positives and negatives are eliminated or at least minimized.
Dr. Heideman asked how the researchers buffered the system to prevent the pH from turning acidic.
Ms. Truong explained that embryo medium is not made with buffering capacity, and removal of ions
further decreases buffering capacity. The pH was measured throughout the experiment to ensure that it
remained neutral.
Dr. Xia asked about the ability of various coatings to aggregate depending on ionic strength. Ms. Truong
replied that all of the coatings were screened, and none of them aggregated.
Impacts of Functionalization ofFullerenes and Carbon Nanotubes on the Immune Response of
Rainbow Trout
Devrah Arndt, University of Wisconsin-Milwaukee
The immune system of all vertebrates is designed to recognize something as foreign, pathogens in parti-
cular. The immune system recognizes molecular patterns on the outside of pathogens, which then triggers
inflammatory and other biochemical responses. Different pathways are stimulated in the primary immune
system depending on the type of pathogen.
The hypothesis of the laboratory is that nanomaterials may instigate the same pathways and some unique
responses from the immune system. The laboratory's specific hypotheses are that: (1) nanoparticles should
be considered foreign and will stimulate the immune system, (2) core structure will impact the ability to
stimulate the immune system, (3) functionalization will impact the ability to stimulate the immune system,
and (4) nanomaterials will cause unique gene expression patterns that differ from each other and from
traditional stimulants. The researchers chose macrophages to assess the primary immune response to
nanomaterials because they are key to the innate primary immune response. The laboratory produces
carbon based nanoparticles of different types with various functionalizations, testing the impacts of these
particles first on cell viability. Next they chose nontoxic concentrations to evaluate key gene expression;
currently, the researchers are evaluating global gene expression in macrophage cells. Particles and their
suspensions were characterized using TEM, ICP-MS, and dynamic light scattering. Additionally, the re-
searchers have examined single-walled carbon nanotubes with carboxyl, amide, PEG, and other functional
groups.
Results indicated that cell viability does not decline with nanomaterial exposures when not suspended with
surfactants. Phagocytosis was initiated following 24-hour exposure to nanomaterials. Macrophage
responses to nanomaterials were more similar to that following bacterial exposure rather than viral expo-
sure. The researchers determined that the surfactants that were used stimulated an immune response by
themselves, so suspensions were created through sonication or stirring to eliminate surfactant use. The
results also indicated that multiwalled carbon nanotubes appear to be slightly more stimulatory than single-
walled carbon nanotubes. The researchers found that C6o appears to be equally as stimulatory to the
immune system as multiwalled carbon nanotubes with anionic functional groups. IL-lp also increased in
response to carbon nanotube exposure.
Current work compares nanomaterials in terms of global gene expression profiles. The researchers plan to
use the data to determine whether these profiles are similar to those of known pathogens and identify any
unique signatures these materials have on the immune system. The goal is to begin to group nanomaterials
by their toxicity based on these gene expression patterns. RNA from control and exposed fish were
replicated in the arrays and then compared to a database of more than 200 different exposures that have
been carried out on this platform. Preliminary results indicate that C6o causes a change in the total number
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of genes that is similar to a major component of the outer membrane of Gram-negative bacteria. Single-
walled nanotubes cause a change in approximately 20 percent of the genes responsive to the C6o treatment.
There is great similarity between the bacterial membrane component and C6o exposures but some difference
in the genes expressed and the extent of the fold-change, indicating a potentially different mechanism for
dealing with these nanomaterials.
In conclusion, trout macrophages are a sensitive tool to investigate the effects of nanoparticles on the
immune system. Nanomaterials stimulate the immune system without complete cell toxicity, and the level
of stimulation depends on the core structure and surface chemistry of nanomaterials. Functionalization may
increase toxicity, and C6o may bind RNA and influence total gene expression in cells. Finally, nano-
materials have unique gene expression signatures.
Discussion
Dr. Petersen asked whether the researchers looked for fullerols inside the cells. Dr. Arndt responded that
they had not, but it would be interesting.
Dr. Jovanovic asked whether there is proof that fullerols bind to everything. Dr. Arndt replied that this is a
hypothesis that the laboratory will investigate. Dr. Jovanovic added that a design for Parkinson's disease is
to bind the second messenger to stop second messenger pathways.
Characterization of the Potential Toxicity of Metal Nanoparticles in Marine Ecosystems Using
Oysters—Silver Nanoparticle Studies With Adults and Embryos
Amy Ringwood, UNC Charlotte
Oysters are coastal estuary organisms. Filter-feeding bivalves are good models because they are highly
effective at removing particles, have high filtration rates, and sample the water column and surface and
resuspended sediments. Additionally, there is extensive information regarding their toxic responses to
metals and organic contaminants. Oyster nanoparticle studies in adults indicate that lysosomal destabili-
zation, lipid peroxidation, antioxidant responses, and tissue and cellular accumulation occur. Embryo
exposure to nanoparticles results in antioxidant responses and normal development. The researchers used
lysosomal destabilization assays extensively and determined that lysosomal endpoints have biological and
ecological relevance. The researchers worked with a variety of nanoparticle types and shapes.
Results showed a dose-dependent lysosomal destabilization response to fairly low concentrations of
nanosilver "seeds" in a citrate-based preparation. The researchers attempted to work with environmentally
relevant concentrations. Lipid peroxidation is significant in the hepatopancreas at higher doses of
nanosilver seeds, but no significant effect is seen in gills, suggesting that oyster gill responses differ from
those offish gills. Glutathione was not significantly upregulated, as was expected. There was a threshold
response in terms of embryo development at the highest concentration. Data indicated an increase in
metallothionein gene expression, particularly in embryos.
Similar experiments were carried out with polyvinylpyrrolidone (PVP)-coated "spheres" and "prisms."
Spheres significantly increased lysosomal destabilization compared to control, and prisms significantly
increased lysosomal destabilization compared to control and other treatments. There was no significant
increase in lipid peroxidation as a result of sphere or prism exposure. Prisms appear to increase toxicity,
showing a shape-based effect. Exposed oyster embryos showed a threshold-based response, and prisms
were toxic at lower concentrations compared to other shapes. Seeds, prisms, and "plates" significantly
increased embryo ROS production compared to control, and prisms significantly increased embryo ROS
production compared to other treatments.
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In summary, nanosilver prisms were found to be more toxic than spheres and plates in adult and embryo
oyster studies. Mechanisms of toxicity were associated with lysosomal dysfunction and oxidative stress.
PVP-coated particles may be slightly less toxic than citrate-based preparations. Oysters and other filter
feeding bivalves are valuable model organisms for characterizing potential nanoparticle toxicity.
Discussion
In response to a question from Dr. Lowry, Dr. Ringwood explained that oyster hepatopancreas cells have a
pH of 7.3. Dr. Lowry asked if this is related to differences in dissolution and amounts of available ion.
Dr. Ringwood responded that this is a good question, and she was unsure of the answer, but there is
evidence of a shape-based effect. Dr. Lowry asked whether the researchers had used prism forms of other
nanoparticles. Dr. Ringwood answered that they have completed some studies with titanium, and they will
continue to explore this.
Dr. Barber asked about the strength of the seawater, to which Dr. Ringwood responded that it is about
25 parts per thousand, which is not full strength. Dr. Barber asked about the solubility and effects of silver
toxicity at various concentrations of chloride. Dr. Ringwood answered that the researchers have not
examined the range of salinities, but they have worked with dynamic light scattering and TEM analysis,
which suggest increased aggregation in the distilled water and lowest salinity preparations.
Dr. Xia thought that the shape-based effect was interesting and asked whether the different geometries were
prepared by the same chemical process. Dr. Ringwood explained that seeds are a precursor for prisms,
which in turn are precursors for plates. The effect was geometric and not chemical.
Dr. Chen was surprised that PVP was found to be less toxic, and Dr. Ringwood agreed.
AFTERNOON SESSION 2: NANOPARTICLES AND WASTE TREATMENT
Bioavailability of Metallic Nanoparticles and Heavy Metals in Landfills
Zhiqiang Hu, University of Missouri
Silver ions and nanoparticles are commonly used in consumer products, and predicted silver concentrations
in sludge in wastewater treatment plants range from 7 to 39 mg/kg. In North America, approximately
2,200 mg of silver per year are wasted through landfill, accounting for approximately one-half the total
wasted silver. Nanosilver flows from products to the environment with potentially high exposure, and a
significant amount ultimately goes to landfills. Silver ion has been found to affect bacterial growth, interact
with thiol groups, deactivate vital enzymes, and inhibit DNA replication. Silver nanoparticles inhibit
autotrophic bacterial growth and are highly toxic to zebrafish, daphnids, and algal species. Silver
nanoparticles less than 10 nm in size may enter cells directly to release silver ions. There are two types of
sanitary landfills. Conventional landfills are based on the storage/containment concept and offer slow and
natural degradation with no recirculation. Bioreactor landfills offer leachate recirculation, increased
degradation rates, improvement of the setting ability of solids, and recovery of landfill space; they also
enhance methane generation in the leachate. Major biological processes in bioreactor landfills are
hydrolysis, acidogenesis/acetogenesis, and methanogenesis. Methanogens are important microorganisms
for final biogas production and good indicators of functional anaerobic bioreactor landfills.
The experimental design utilized municipal solid waste from a bioreactor landfill site in Columbia,
Missouri. Results indicated that there was a significant difference in gas volume between the control and
each of the reactors treated with the low and high concentrations of nanosilver. Solids treated with a low
concentration of nanosilver showed no inhibition of anaerobic process, whereas those treated at the higher
concentration affected biogas generation rate and volume. The pH drop resulting from volatile fatty acid
accumulation and the changes of leachate chemical oxygen demand in the bioreactor treated with the higher
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nanosilver concentration confirmed the inhibitory effect of nanosilver on anaerobic biodegradation of solid
waste. The dynamic changes of volatile fatty acids and acetic acid from the reactor containing the higher
nanosilver concentration confirmed the accumulation of volatile fatty acids and acetic acid, resulting in a
consistently low pH in the leachate; these results are consistent with the biogas production profile.
During the early stage of anaerobic decomposition, Methanobacteriales was the dominant organism (greater
than 90%) in the control and low-nanosilver-concentration bioreactors. By comparison, Methanosaeta
accounted for 40 percent of the bacterial species present in the high-nanosilver-concentration bioreactor.
Additionally, the methanogenic bacterial population continues to evolve in bench-scale bioreactor landfills.
Results of experiments focusing on total silver in leachate indicate that silver could be precipitated or
absorbed in landfill solid waste.
In summary, there was no significant difference in the cumulative gas production between the low-
nanosilver-concentration bioreactor and the control, whereas the high-nanosilver-concentration bioreactor
resulted in reduced biogas production, volatile fatty acid accumulation, and lower pH in the leachate. Other
results demonstrated a dominant population shift from acetoclastic methanogens to hydrogenotrophic
methanogens at the early stage of anaerobic solid degradation. These results could be useful to regulatory
agencies and landfill operators for decision-making and remedial actions.
Discussion
In response to a question by Dr. Holden, Dr. Hu replied that methane was not measured initially because of
CO2. At the early stages, there is no methane present.
Dr. Zhang asked how the researchers inoculated the methanogens and whether they used organic sludge.
Dr. Hu answered that the source of the methanogens was the municipal solid waste landfill, so they already
were inoculated. The food source was the organic waste from the landfill.
Dr. Mahendra asked Dr. Hu to clarify whether he thought that the acetoclastic methanogens were more
sensitive to silver than the hydrogenotrophic methanogens and whether this was the reason for the
metabolism shift. Dr. Hu replied that examining the substrate at the earliest stages was beneficial to attempt
to answer this question. The predominant reactions at early stages of the process favor the
hydrogenotrophic methanogens. Dr. Mahendra commented that metals comprised 1 percent of the waste at
municipal solid waste landfills and asked whether the researchers had characterized what metals are
present, as methanogens are susceptible to copper. Dr. Hu responded that silver was the only metal
measured, but the controls helped to determine that copper susceptibility is not the cause of the results.
Biological Fate and Electron Microscopy Detection of Nanoparticles During Wastewater Treatment
Paul Westerhoff, Arizona State University
The goal of the project is to quantify interactions between manufactured nanoparticles and wastewater
biosolids. The laboratory hypothesizes that dense bacterial populations at wastewater treatment plants
should effectively remove nanoparticles from sewage, concentrate nanoparticles into biosolids, and/or
possibly biotransform nanoparticles. The relatively low nanoparticle concentrations in sewage should have
negligible impact on the wastewater treatment plant biological activity or performance. The researchers aim
to develop mechanistic models for nanoparticle removal in wastewater treatment plants. Dr. Westerhoff
highlighted three papers that examine the release of nanosilver in consumer products and noted that his
laboratory submitted a paper that examines detection of fullerenes in cosmetic products. The dominant
removal mechanisms at wastewater treatment plants are settling and biosorption; therefore, the research
evaluated batch sorption to biomass, continuous loading bioreactors, and occurrence at full-scale treatment
plants.
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Previous research indicated that surface properties were important to biosorption of nanoparticles on
heterotrophic wastewater biomass. The researchers confirmed that the EPA sorption method is not valid for
nanosilver, and it is likely that it is not valid for other nanomaterials as well. Data also indicate that the
primary mechanism of the removal of nanomaterials is the interaction of the nanomaterials with wastewater
biomass. Results also indicated that freeze-dried biomass has different morphology. Next, the researchers
performed a continuous nanomaterial loading study. The removal of functionalized silver is a function of
the amount of biosolids present in the system. The same pattern is seen with titanium (i.e., biosolids
concentration decreases).
In terms of occurrence at full-scale wastewater treatment plants, nanoscale, microscale, and mixed element
titanium already are found in biosolids at these plants. The researchers evaluated the presence of TiO2 at
several wastewater treatment plants, as well as membrane technologies to characterize or remove
nanomaterials. Data indicate that titanium is well removed at wastewater treatment plants in Arizona. Other
experiments showed that nanomaterial surface properties were more important than membrane material
properties. Tighter ultrafiltration rejection was high, but recovery indicates significant absorption.
In summary, nanomaterials will accumulate in biosolids. Approximately 60 percent of wastewater
treatment plant biosolids are land applied, 22 percent are incinerated, and 17 percent are sent to landfills.
Better tools are needed to differentiate engineered from "other" nanoparticles in wastewaters, and pollutant
removal models for wastewater treatment plants currently are not suitable for predicting the fate of
nanoparticles. Better relationships between surface charge and core composition versus biosorption are
needed. Finally, the fate of nanomaterials in biosolids is poorly understood.
Discussion
Mr. Shapiro asked what the most cost-effective treatment would be for wastewater treatment plants. Dr.
Westerhoff responded that the best goal would be to design wastewater treatment plants to stop all
pollutants via a membrane bioreactor and tighter membranes.
A participant asked whether the biosolids were returned to the anaerobic bioreactor directly from sludge.
Dr. Westerhoff answered that all activated sludge is returned from the aeration basin; there are plans to
perform anaerobic digester sampling.
Dr. Holden asked whether settling characteristics are affected by the affinity to biomass. Dr. Westerhoff
replied that it was much more difficult with nanosilver compared to C6o to control the sequencing batch
reactors.
Dr. Huang asked how the researchers determined TiO2 plus and minus. Dr. Westerhoff explained that the
researchers did not determine these factors as the TiO2 was acquired from a commercial source.
Analysis and Fate of Single-Walled Carbon Nanotubes and Their Manufacturing Byproducts in
Estuarine Sediments and Benthic Organisms
P. Lee Ferguson, Duke University
Single-walled carbon nanotube composites have made their way into the marketplace, and numerous
companies now supply single-walled carbon nanotubes on a kilogram scale. Annual worldwide production
of single-walled carbon nanotubes is estimated to be greater than 1,000 tons by 2011. Currently, there are
no reliable methods to detect single-walled carbon nanotubes in complex mixtures at low concentrations.
The laboratory takes advantage of unique structural properties of single-walled carbon nanotubes that
create unique electronic properties. The overall research objective is to implement and apply near-infrared
fluorescence (NIRF) spectroscopy for qualitative and quantitative analysis of single-walled carbon
nanotubes in complex environmental media. The specific objectives are to: (1) develop sample preparation
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methods for isolating single-walled carbon nanotubes from sediment and tissue prior to NIRF spectroscopy,
(2) explore asymmetric flow field flow fractionation coupled with NIRF spectroscopy for separating single-
walled carbon nanotubes and reducing interferences, and (3) apply asymmetric field flow fractionation
(AFFF)-NIRF spectroscopy to analysis of single-walled carbon nanotube uptake and accumulation in
sediment-dwelling organisms. Researchers use a multilaser near-infrared spectrofluorometer to excite the
samples and measure emissions at defined excitation. The laboratory method is quantitative with little
matrix effect and is reproducible.
Results indicated that single-walled carbon nanotubes are detectible in complex sediment extracts using
AFFF-NIRF spectroscopy, and single-walled carbon nanotubes do not degrade in sediments during a
1-month timescale. Single-walled carbon nanotubes were undetectable in sediment-exposed amphipods and
mysid shrimp. When the researchers measured single-walled carbon nanotube body burden in sediment-
and/or food-exposed organisms, they found that the nanotubes are present in nondepurated amphipods.
Accumulation of single-walled carbon nanotubes in benthic macroinvertebrates and single-walled carbon
nanotube bioaccumulation and trophic transfer using worm and clam species also were examined. No
internal filter artifacts were present in the NIRF analysis of clam extracts. Additional microcosm-based
experiments will track the uptake of single-walled carbon nanotube manufacturing byproducts in sediment-
dwelling organisms as well as degradation in sediments, investigate chirality and diameter-dependence of
single-walled carbon nanotube interaction with sediment and organisms, and survey environmental media
for contamination with single-walled carbon nanotubes.
The researchers concluded that a novel and highly sensitive method based on NIRF spectroscopy for
analysis of single-walled carbon nanotubes in sediments has been developed. NIRF spectral features of
single-walled carbon nanotubes were retained after extraction from sediment, allowing diameter and chi-
rality characterization for dilute solutions. AFFF can be used as a clean-up tool prior to NIRF analysis.
Single-walled carbon nanotubes do not appear to be highly bioaccumulative in estuarine invertebrates
exposed via sediment or dietary routes.
Discussion
Dr. Zhang asked whether the extraction procedure was sensitive to the sample matrices and whether there
was a positive control to show nanotubes in biomass. Dr. Ferguson responded that calibration curves are
used to compare sediment extract spiked with nanotubes at different concentrations and clean surfactant
solution at the same concentration to ensure that there is no matrix effect. In terms of the second question,
positive control experiments always are performed to ensure that they can matrix spike and recover.
Dr. Zepp asked whether there was a faster, less expensive method to clean up the samples other than AFFF.
Dr. Ferguson replied that the nanotubes are "sticky." The researchers tried ultrafiltration, which did not
work. XAD is a possibility that the laboratory could try.
Dr. Holden asked whether the developed method could be used to identify rare earth nanomaterials.
Dr. Ferguson answered that it was possible if the excitation and emission pairs could be matched, but time-
resolved fluorescence might be more appropriate for rare earths.
A participant from EPA asked about the recovery with the AFFF and noted that there appeared to be
bimodal distribution in the sediment extract based on the results that were presented. Dr. Ferguson agreed
that there was bimodal distribution. The sediment matrix type makes a difference in peak shapes of the
AFFF. Natural organic material sorption to nanotubes is important. There is significant recovery of
nanotubes on membranes.
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Safety/Toxicity Assessment ofCeria (A Model Engineered Nanoparticle) to the Brain
Robert Yokel, University of Kentucky
The objective of this research is to characterize the physicochemical properties of a model engineered
nanomaterial that influence its biodistribution and effects, including distribution across the blood-brain
barrier, effects on oxidative stress endpoints in the brain, uptake into selected peripheral organs, and
persistence over time. The researchers studied ceria (also known as cerium dioxide or cerium oxide)
because it is an insoluble metal oxide that can be readily observed and quantified in tissue. Also, ceria has
current commercial applications and has been reported to be cytotoxic as well as neuroprotective,
representing the controversy about nanoscale materials. The laboratory prepared and characterized citrate-
coated ceria of five different sizes.
When the researchers assessed the influence of size on engineered nanomaterial distribution, persistence,
translocation, and toxicity, highest concentrations were found in the spleen and liver. Cerium found in the
brain did not necessarily cross the blood-brain barrier. Liver and spleen showed little decrease in cerium
concentration during a 30-day time period. The researchers concluded that brain cortex cerium always was
less than 1 percent of the dose, and ceria was seen only in brain vasculature. Spleen cerium concentration
was greater than liver cerium concentration, although liver had the greatest mass amount of the ceria dose.
There was little decrease in liver and spleen cerium up to 30 days.
Oxidative stress markers and antioxidant enzyme levels and activities were determined following exposure
to 5, 30, and 65 nm ceria, and significant changes were seen. In vivo exposure to ceria indicated that cerium
concentrations in the blood decreased over time. The 15 and 30 nm ceria predominantly associated with
blood cells, whereas the 5 and 65 nm ceria were generally evenly distributed between the two
compartments. The greatest association of the 30 nm citrate-coated ceria with blood cells in the clot
fraction is consistent with reports showing that this size is optimal for protein wrapping of engineered
nanomaterials. A 90-day survival study to assess longer term distribution, persistence, and effects revealed
that exposure resulted in modestly decreased body weight gain, and ceria was retained primarily in reticulo-
endothelial tissues. No significant decrease of the mass amount of ceria in liver and spleen was seen during
the 90-day period. Liver pathology was examined 30 and 90 days postexposure for 5 and 30 nm ceria, and
results 30 days after exposure to 5 nm ceria showed nonuniform granuloma formations that contained ceria-
loaded Kupffer cells and mononucleated cell infiltration among the hepatic parenchyma and at perivascular
sites. Mononucleated cells appeared to encircle Kupffer cells, and there was no evidence of fibrosis or
abscess formation. Live pathology 90 days after exposure to 30 nm ceria showed granulomatous
formations.
Ultimately, the researchers concluded that citrate-coated 5 to 65 nm ceria do not enter the brain to any
significant extent, and ceria primarily is cleared by reticuloendothelial organs and sequestered in
intracellular agglomerates. The cerium valence does not change in situ during the first 30 days. There is
little clearance of 5 to 65 nm ceria from reticuloendothelial organs. The smaller the ceria, the longer it
remains in blood before being cleared. Maximal distribution into blood cells was seen with 30 nm ceria,
and granulomatous formations were seen. Ceria and the cerium ion are very slowly eliminated, and ceria
does not always behave in a manner similar to the cerium ion in its distribution in blood or tissues. These
results further support the concern about the long-term fate and adverse effects of inert nanoscale metal
oxides that reach systemic circulation, from which they can distribute throughout the body, resulting in
persistent retention and potential adverse effects in multiple organs.
Future plans are to complete the histopathology, agglomeration extent and localization, cerium valence, and
oxidative stress marker analyses as a function of time following 30 nm ceria infusion; assess the
biodistribution and effects of a noncubic/nonpolyhedral ceria in the rat; and perform more direct
assessment of the physicochemical properties of ceria that influence brain uptake and blood-brain barrier
effects.
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Discussion
Vishal Shah (Bowling College) noted that most of the results appeared to be primarily dependent on the
stimulation and asked whether this might have more to do with stabilization than the nanoparticles.
Dr. Yokel replied that surface properties probably are the most important variables.
OPEN DISCUSSION
Mr. Shapiro opened the discussion regarding all issues that were introduced during the day. Dr. Xia thought
that the lipid bilayer-water partitioning approach was a better technique than octanol-water partitioning, but
he was concerned about the size and thickness of the lipid bilayer. Dr. Posner stated that the thickness of
the lipid bilayer is 4 nm, so it is small relative to the particle. One of the models that the researchers use is a
biologically relevant surface, another model that examines passage is extruded, and the third measures
conductance. Dr. Xia asked whether the effective surface was used when the partition coefficient was
measured. Dr. Posner responded that examination of particle numbers showed a nice trend. The surface
area does not deform in any way, so the surface area properties of the biological surface are well known.
Dr. Xia asked whether this would be published, and Dr. Posner replied that it would be.
Dr. Huang thanked EPA for a very informative meeting and asked the EPA staff members to share their
view of future directions. Dr. Savage replied that the next RFA will be released by February 2011. The
specific details are unknown, but the general theme will pertain to the lifecycle of nanomaterials. There was
a suggestion for EPA to request preproposals, and the EPA team still is working on the details.
Mr. Shapiro asked the participants what they thought should be the focus of the solicitation. A participant
noted that the literature does not allow all results to be compared. Another participant noted that no study
had demonstrated a nanoparticle that exerts acute toxicity at environmentally relevant levels. More research
is needed on understanding mechanisms of factors other than acute toxicity. Dr. Yokel stated that it is acute
versus chronic in terms of compensatory changes to repeated or prolonged exposure; therefore, regulation
should be function of exposure. There could be a significant difference between acute and chronic toxicity.
Mr. Shapiro asked if the typical 3- to 4-year grant cycle would be enough time to study chronic toxicity.
Dr. Yokel replied that it depends on the model.
A participant thought that, in terms of susceptibility factors, acute toxicity beyond cell death and gene
expression changes should be examined. How can researchers interpret an adaptive response? These types
of data should be incorporated into risk assessment.
Dr. Heideman commented that there is a significant diversity regarding what people see as a potential risk
and the best methods by which to examine these risks. He cautioned not to "put all of the eggs in one
basket" so that this diversity is not missed. Dr. Savage noted that the strategy of EPA' s Office of Research
and Development identified classes of materials, which were included in the last solicitation; examining
susceptible populations may go beyond populations currently known to be susceptible (e.g., children,
elderly) when the genomic databases are established.
Dr. Heideman thought that preselecting a theme might prevent the submission of proposals that are too
broad. Dr. Savage replied that the RFA must have a research topic. The current thought is that the RFA will
focus on understanding nanomaterials throughout their lifecycles. Possibly, materials could be tiered. The
compound-by-compound approach is not working, so a better method is needed to examine outcomes.
Dr. Heideman thought that each proposal should demonstrate a clear and present danger via preliminary
results so that it is plausible that the research is addressing a truly hazardous situation. Dr. Hu commented
that the purpose of EPA-funded research is to help the Agency with its regulatory needs, and decision-
makers need to know the critical numbers to allow them to make informed decisions. Dr. Ringwood had
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concerns about focusing on acute toxicity. Sublethal effects are different than chronic effects and can be
important. She did not think that the analytical tools currently are available to definitively declare
something a clear and present danger. It is more important to examine different kinds of potential receptors
to identify those that increase susceptibility. Researchers must continue to examine diverse systems
because different species may have different responses. She thought that there still was too much
uncertainty to eliminate any systems because important risk issues may be missed.
Dr. Barber said that a missing element was how to apply the 5 years' worth of data that have been collected
and begin to synthesize them into a product to help with risk assessment, which would help identify
knowledge gaps. A participant noted that the National Institute of Environmental Health Sciences released
a solicitation with a risk assessment core and agreed that the ability to apply data would be useful.
Dr. Savage explained that EPA is under more scrutiny than any other federal regulatory agency.
Solicitations must be released for the sake of science, although program offices do supply feedback. All of
the NNI agencies are realizing that data must be assimilated, but the question is which agency will maintain
the resulting database. There has been some discussion that OECD will maintain it, but this is not con-
firmed. The Woodrow Wilson International Center for Scholars no longer is maintaining its Nanotech-
nology Consumer Products Inventory. These are issues with which federal agencies are struggling as
budgets decrease.
Dr. Huang commented that a good use of the data would be to be able to generalize key issues in an
intercorrelated manner. The various nanotechnology research groups could determine relationships.
Dr. Yokel asked the EPA staff members how useful data that have not been extensively characterized are in
terms of risk assessment. Dr. Savage responded that characterization of data is very important to risk
assessment. The new environmental health and safety strategy emphasizes characterization as a key issue.
Unfortunately, many manufacturers incorrectly characterize their nanomaterials or cannot divulge
characteristics because of their confidential business practices.
Dr. Petersen asked whether the EPA staff members had any recommended reading regarding what EPA has
done in the past regarding uncertainty of pH and metals; this possibly could be applied to this field. He
noted that other fields have had 30 years to work through these issues, and nanotechnology research is
expected to have answers after only 5 years. Dr. Savage responded that past approaches are not working, so
they should not be repeated. Nanomaterials are novel, but it may be possible to glean generalities that allow
use of traditional chemical knowledge. Dr. Petersen thought that the new approach involving tiering was
helpful in providing decision-makers with the best possible data to make informed decisions. Dr. Savage
said that it also would be helpful for NIST to provide characterization. Dr. Petersen said that there are some
options, but they require funding.
Dr. Heideman suggested the idea of identifying rules for nanomaterial groups so that it is not necessary to
investigate each new one as it is developed. This is the only reasonable manner by which to approach this
problem. His initial remark about determining a clear and present danger was intended to communicate the
fact that research is not ready to develop these rules. Information on characterization and compound
concentration are needed to compare results because there are so many potential hazards. Dr. Savage asked,
if the research is not ready, how to get it ready. Dr. Heideman said that chemical companies constantly
develop new products and could be a model for how to proceed in terms of nanotechnology.
Dr. Xia stated that Dr. Savage and the EPA team did an excellent job in developing the last RFA; the
diversity of researchers and the amount of results and data presented during the meeting were impressive.
He thought that it was beneficial to have many researchers to increase the diversity of the research.
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Dr. Orr thought that the research community is "drowning" in too much data. The science is good, but
researchers need to focus the data to develop practical guidelines and predictions. There needs to be a
compromise so that the focus is not too narrow or broad. She suggested that EPA choose a common
nanomaterial and have all of the researchers focus on it.
Dr. Yokel asked whether OECD was assembling the data to synthesize U.S. collaborations. Dr. Savage
explained that currently OECD is gathering its own data. Dr. Yokel asked whether there were enough data
to start a data-gathering effort. Dr. Savage stated that there are enough data but no one to maintain a
database. Dr. Yokel thought that regulatory agencies would have a stake in this and, therefore, would be
interested in funding an effort. Dr. Savage agreed and said that the Consumer Product Safety Commission,
Occupational Safety and Health Administration, Food and Drug Administration (FDA), and EPA are the
agencies with the most interest, but it is falling to EPA, the agency with the smallest budget.
Dr. Ringwood asked whether industry could be pressured to provide funding. Dr. Savage explained that
public-private partnerships were being explored; this is in the strategic plan, but the data would not be
available to the general scientific public. Dr. Kavanagh noted that in addition to the example of the Health
Effects Institute, the Superfund Basic Research Program and a training effort funded via oil taxes also serve
as examples. The latter recognize that many problems were the result of synthetic manufacturing based on
petroleum. Although taxation should not be used to the extent that it stifles innovation, it is one possibility.
Another example is that pharmacological companies pay FDA for each new investigative drug. Dr. Savage
agreed that taxation was not favorable in the current political and economic climate, and Dr. Xia suggested
that industry be charged a "registration fee." Eric Grulke (University of Kentucky) added that many of the
U.S. companies that manufacture nanomaterials are small businesses and would not be viable if taxes were
leveraged against them. Much of the value added for nanomaterials is how they are used in various media;
therefore, functionalization is critical. This is an important clash that needs to be addressed. Dr. Savage
explained that many small businesses came to EPA and were very proactive regarding potential problems.
Dr. Holden thought that larger federal agencies that benefit from the research (e.g., Department of Defense)
should be lobbied to increase funding for nanomaterial research. Environmental toxicologists could partner
with these agencies' researchers. Dr. Savage agreed that some agencies have dedicated more funding since
2003, and they could increase funding significantly, but it is not their mission. Even the Department of
Energy should be more interested because it is in their best interest and the interest of the United States.
A participant said that the training of young scientists should be a priority. Dr. Savage explained that EPA's
People, Prosperity, and the Planet (commonly known as P3) Program accomplishes this.
Mr. Shapiro thanked the participants for attending on behalf of himself and Drs. Savage and Lasat and
adjourned the meeting at 6:44 p.m.
The Office of Research and Development's National Center for Environmental Research
-------
Abstracts and Presentations
-------
Day 1, Monday, November 8, 2010
AM Session 1: Systems Approaches
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Vicki H. Grassian
An Integrated Approach Toward Understanding the Impact of Aggregation and
Dissolution of Metal and Metal Oxide Nanoparticles
Vicki H. Grassian
Department of Chemistry and Nanoscience and Nanotechnology Institute,
University of Iowa, Iowa City, IA
Nanoparticles, the primary building blocks of many nanomaterials, may become suspended in air or get
into water systems (e.g., drinking water systems, ground water systems, estuaries and lakes, etc.). 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 in characterizing the nature and state of nanoparticles in air, water, or in vivo. For the
nanoparticles of interest in these studies, metals and metal oxides, it can be asked: (1) will metal oxide and
metal nanoparticles be present in air or water as isolated particles or in the form of aggregates?; (2) will metal
oxide and metal nanoparticles dissolve in aqueous solution or in vivo?; and (3) 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 the
studies discussed here, macroscopic and molecular-based probes that include quantitative solution phase
adsorption measurements, ATR-FTIR spectroscopy, dynamic light scattering techniques and zeta-potential
measurements are used to investigate the physicochemical properties including nanoparticle interactions as a
function of important environmental variables such as pH, presence of organic ligands, surface chemistry,
nanoparticle concentration, and solar irradiation. We have focused on several different metal and metal oxide
nanoparticles in aqueous environments, including those that contain Fe, Ag, Zn, Cu, Ce, and Ti. Results for
these different metal-containing nanomaterials will be presented with a focus on aggregation and dissolution in
the presence of citrate, a common organic ligand found in the environment. This research is beneficial as it
significantly contributes to the growing database as to the potential environmental and health implications of
nanoscience and nanotechnology and how nanomaterials will behave in the environment and impact human
health.
EPA Grant Number: R833891
The Office of Research and Development's National Center for Environmental Research 47
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An Integrated Approach Toward Understanding the
Impact of Aggregation and Dissolution on the Fate,
Behavior and Toxicity of Metal and Metal Oxide
Nanoparticles
Vicki H. Grassian
Departments of Chemistry, Chemical and Biochemical Engineering and
Occupational and Environmental Health
and Nanoscience and Nanotechnology Institute at the
University of Iowa
EPA Grantees Meeting - 2010
Portland, Oregon
Motivation
Interest in understanding the environmental and health
implications of natural and engineered nanomaterials
• Environmental Fate and Transport of Nanoparticles in Air and
in Water Systems
-Nano-
-Manu
use ani
rticle Fe oxi-. • ;,- ireacd -T ..>"."£ituenlinair, water am: so .!
Occupational Hazards and Toxicity of Airborne Nanoparticles
-Majority of reports indicate that exposure by inhalation is the greatest hazard faced by w-
orkers in the nanotechnology industry. Furthermore, it is well known that ultrafrne particles
are associated with health problems. Therefore, in occupational settings, there may be
associated risks with the production of nanomaterials.
Nanomaterials composed of metals and metal oxides are a large
percentage of the commercially developed nanomaterials on the
market and a focus of these studies.
Nanoparticles Less Than ca. 20 nm Are of Particular Interest
Quantum Size Effects and Other Size-Dependent Properties Become Increasingly Important
Reactive Edge and Corner Site Density Increase
Most inorganic nanoparticles are not spherical in shape but in fact more cubic
or octahedral in nature.
Defect sites which include edges and corners are more reactive - many corner
and edge sites for nanoparticles.
" "corner
- edge
Stun TIO2 particle
courtesy of MJ Shalt?.
Literature Studies Suggest Unique Surf ace Reactivity for
Smaller Metal and Metal Oxide Nanoparticles
Differences in reactivity due increase number of edge and corner sites?
Does this make smaller nanoparticles more or less toxic, or behave differently in the
environment compared to larger particles?
OneL
Na
Fora.
in e.g.
>sue is Related to the State of Metal and Metal Oxide
noparticles in Different Environments and Under
Different Conditions?
Water, Air and In Vivo
S1MU- ill Vin..|^KI,kv
DlitnltfU Ii.nV." hnlitlrJ I'lirlkliV.1 * unn-l!" !«l I'nrlirNV.'
-- H .. ll'l. 1 III--.. Ml :ll T \JH..[..llhl 1. \.."JI. '.'.li<
I-. 1 .1,.! \jniipirlldt «^
INtwihnl km ttw twklrd prlnun c*nkji- *l/r .\mrttt4l* Ufi
-Inn l-HIUnni 100-lllflOnni
Si«. Ki-hiu.
^cience that is "all about size " modeling these size regimes
transport and lung deposition models will be very different
Size Issues Beyond Primary Particle Size
Particle Dissolution and Aggregation From the
Particle Perspective
Particle Dissolution Particle Aggregation
Effect Each Other
-Impacts Particle Size
Specifically, With the Formation of Metal
Ions and There is Also the Formation of
Smaller Nanoparticles
-Dissolution Can ImpactAggregation By
Causing Deaggregation as the Particles
Within the Aggregate Dissolve
*
-Impacts Size, Shape and Density
-Impacts Available Surface Area
-Impacts Surface Chemistry
Including Nanoparticle Dissolution
An Experimental Approach That Integrates Macroscopic and Microscopic
Measurements and Methods Taken From Surface Science, Surface
Chemistry, SolidState and Materials Chemistry, Colloid Science andAerosol
Science to Better Understand the Implications of Nanomaterials
X-Ray Diffraction and Microscopy
SEM,TEMandAFM
Surface Area
BET
Metal and Metal Oxide
anomaterials in Gas and Liq>
Phase Environments
'uid }
Particle Sizing
SMPS
DLS
Surface Spectroscopy
ATR FTIR
TransmissionFTIR
X-Ray Photoelectron Spectroscopy
Quantitative Reactor Studies
Adsorption Measurements
Reactivity Studies
Dissolution Measurements
along with studies oftoxicity and biological interactions
48
-------
Combined Integrated Approach to Better Understand
Implications of Nanomaterials
Synthesize or Purchase Commercial Nanomaterial Powders
Bulk and Surface Characterization of Nanomaterials
Fate and Transformation in Water Air/Aerosol and Inhalation Toxicity
Molecular, Particle and Batch
Reactor Studies of Surface
Adsorption and Dissolution in
the Aqueous Phase
pH, ionic strength,
organic ligands
Inhalation Toxicity with
Aerosol Characterization,
Inflammatory Response and
Studies of Dissolution in
Simulated Biological Fluids
Environmental Fate and Transport
Examples of the Physicochemical Properties and Conditions that
Influence the Aggregation and Dissolution of Nanomaterials in
Aqueous Environments
TiO2 Nanoparticles
and a-FeOOH Nanorods
Titanium Dioxide Nanoparticles - ca. 4 nm
TiO2nanoparticles from Nanostructured and Amorphous Materials are
some of the smallest commercially manufactured oxide nanoparticles and
is sold as having a primary particle size of 5 nm.
Characterization of Bulk and Surface Properties
XRD anatase phase
TEM of isolated particles
3.5+ 1.0 urn
(sonicated in methanol before deposition
n to the TEM grid)
H
Surface Spectroscopy -
Surface Functionalization
am
-I:
^T-^
*> (\lsft '"*
Surface Area - BET measured
219 + 3 m2^1
Will they dissolve in water, aggregate or
remain as isolated particles?
-No dissolution observed at 293 K
-Aggregation is observed at 293 K
Aggregation and Sedimentation in Aqueous Suspensions
Will Depend on Nanoparticle-Nanoparticle Interactions
Q-KD
and whether that interaction is overall net repulsive or attractive
(Vtot = Vrep + Vll •:•!•, EJ m:f .ILIUM in . 9H
DLVO Calculations Along with Zeta Potential Measurements of the
Surface Charge Show that TiO2 Nanoparticle Suspensions Are
Stable at Low pH in the Absence of Citric Acid and at Near Neutral
pH in the Presence of Citric Acid
DLVO Calculations
49
-------
Surface Speciation and Surface Coverage of Adsorbed
Citric Acid as f(pH)
Aqueous Phase - Speciation andpKa values
oaVDI\j
JCO
HOT ^*^gT
'ns\ •••'•
- v
i
\
:
1 H.' ' "
Citric Acid
PK,,=3.I3
OH pKaI=4.76
pK^s = 6.40
; *:^J'
Surface Coverage asf(pH)
pH Maximum Surface Coverage"
H (molecules cm'2)
lo
4.0
6.0
7.5
9.7±0.4xl013
7.5±0.3x 101J
6.3±0.5xlOu
3.1±0.4xl013
1
Surface Speciation suggests pEa values
Surface
Speciation
«J^^,
are lower for surface adsorbed citric
acid. Less adsorption at higher pH
a result of surface charge becoming
more negative with increasing pH.
Summary of the Behavior of Some of the Smallest
Commercially Available TiO2 Nanoparticles
Thus mobility in the
environment of
nanoscale TiO2 will
depend on surface
coatings, surface
coverage, surface
charge andpH in
relatively complex
ways.
Comparison of the Dissolution of of a-FeOOH Nanorods (7 nm
x 80 nm) to Microrods (25 nm x 700 nm)
1.5 ml of 1 g/L suspension dried on ZnSe AIR
£<.
Surface OH stretch
Wavenumbers (cm" )
Consistent with a greater density of surface
hydroxyl groups found for nanorods.
But even greater than surface area
considerations
Surface Hydroxyl Groups
T
Wavenumbers (cm )
Nanorods Can Extensively Aggregate Under
Certain Conditions
Enhanced Dissolution on the Nanoscale Is Quenched in the
Aggregated State
a-FeOOH Acid Assisted Dissolution atpH 2 Nanorods vs Microrods
Isolated Rods Aggregated Rods
Increased Ionic Strength
Enhanced dissm
-------
Integrated Approach To Nanoparticle Inhalation
Toxicity: Research Design
Goal of this
approach is to
determine
which
p hy sic och emic a)
properties are
Ximport ant in
tianop article
toxicity
Comparison of Inflammatory Response of Mice to
Different Metal and Metal Oxide Nanomaterial
Aggregates on the order of 100 - 200 nm
Greatest Inflammatory Response Found for Cu-BasedNanoparticles as Determined
by Elevated Cell Count in BAL Fluid and Greater Percentage ofNeutrophils and
Lymphocytes. Copper Nanoparticles Showed a Higher Propensity for Dissolution in
Simulated Biological Media (Which Contain CitricAcid).
Nanoparticles" Handbook of Systems Toxicology, John Wiley and Sons 2010 (in press).
Fe and Cu Nanoparticle and Aerosol Characterization
Lung Tissues Show No Evidence for Cu particles
Controls - Staining Alone
Fe (blue stain) present in macrophages Cu (red stain) not present in macrophages
'•.-•• - . ''''-: H ::'^'-
•
, • •
* -
Suggesting Dissolution and/or
Translocation of Cu particles
Conclusions and Acknowledgements
Environmental Fate and Transport: Metal and metal oxides show unique
reactivity and physicochemical behavior on the nanoscale and this behavior
will be impacted by aggregation. Surface chemistry and surface impacts
aggregation and aggregation impacts surface reactivity (e.g. dissolution).
Some ongoing studies include: size-dependent dissolution of ZnO nanoparticles
and nanorods; aggregation and dissolution of copper nanoparticles in aqueous
media as a f(pH) and presence of citrate aggregation and dissolution
EPA
Imali Mudunkotuwa, Thillini Rupasighege,
Gayan Rubasinghege, Dr. Shaowei Bian
Inhalation Toxicity: Chemical composition, size and ability to undergo dissolution
and translocation are important in the toxicity in ways that have not been
discerned previously through inhalation toxicity studies. Additional studies on
Ag, ZnO and Cu nanoparticles are currently underway
NIOSH
Professors Peter Thorne and Patrick O'Shaughnessy,
Drs. John Pettibone, Andrea Adamacova-Dodd and Larissa Stebonouva
51
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2010 U.S. EPA Nanotechnology Grantees Meeting
Thomas Theis
Life Cycle Analysis and Nanostructured Materials
Thomas Theis1, Bhavik Bakshi2, Delcie Durham3, Vasilis Fthenakis4, Timothy Gutowski5,
Jackie Isaacs6, and Thomas Seager7
1 University of Illinois at Chicago, Chicago, IL; 2Ohio State University, Columbus, OH;
University of South Florida, Tampa, FL; Columbia University, New York, NY;
Massachusetts Institute of Technology, Cambridge, MA; Northeastern University,
Boston, MA; Arizona State University, Tentpe, AZ
The term nanotechnology is now widely employed to describe the unique properties and applications of
materials in the nm size range, typically taken to be 1-100 nm. Advances in our understanding of molecular
events at the atomic or near-atomic level, coupled with new methods of measurement and observation, have
led to the development of new products and manufacturing processes that comprise the domain of
nanotechnology. Nanoproducts are defined as small structures of controlled shape, size, composition, and
function (e.g., nanoparticles, carbon nanotubes, nanowires, nanofilms, quantum dots). Examples of industries
or sectors where nanoproducts or nanomanufacturing methods are being used today include ceramics,
membranes, coatings, composites, skin care products, biotechnology, semiconductors, and thin films.
However, this area is growing rapidly, thus new applications and products will undoubtedly be developed in
the near term.
Present environmental research on nanotechnology appears to be proceeding along two separate pathways;
one as a receptive view recognizing nanotechnology as an enabling force providing benefits such as innovative
remediation alternatives, improved catalysts and membranes, and better sensors for detection of contaminants,
and the other as a precautionary view seeking to identify fate and transport, potential toxicity, risk, and health
effects of nanostructured materials and resultant products. Significant research efforts on human health impacts
are underway; however, there are comparatively few studies that have focused on the application of life cycle
concepts.
This presentation will review the findings from a U.S. Environmental Protection Agency/National Science
Foundation-sponsored workshop on life cycle analysis and nanostructured materials and products. It will
examine the function and composition of nanostructured materials, their manufacture, and explore ways in
which a life cycle approach can be used to guide research on their environmental and health properties,
manufacturing methods, and end-of-life disposition.
EPA Grant Number: R831521
The Office of Research and Development's National Center for Environmental Research 52
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Life Cycle Analysis and
Nanostructured Materials
Thomas L. Theis
Institute for Environmental Science and Policy
University of Illinois at Chicago
Nano Grantees Meeting
8 November 2010
NSF/EPA Workshop
Life Cycle Aspects of Nanoproducts,
Nanostructured Materials, and
Nanomanufacturing:
Problem Definitions, Data Gaps, and
Research Needs
Life Cycle Assessment
•A systems methodology for compiling information on
the flow of materials and energy throughout a product
chain
•LCA evolved from industry needs to understand
manufacturing, and market behavior, and make
choices among competing designs, processes, and
products
•Defines four general sections of the product chain:
•materials acquisition
•manufacturing/fabrication
•product use
•downstream disposition of the product
ISO 14040 & 14044
Life cycle assessment framework
Interpretation
Major Impact Categories
HH (cancer)
HH (non cancer)
Global Warming
Eutrophication
Ecotoxicity
Acidification
Smog Formation
Ozone Depletion
Land Use
kg benzene eq/unit
kg toluene eq/unit
kg CO2 eq/unit
kg N eq/unit
kg 2,4 D eq/unit
eq H+/unit
kg NOX eq/unit
kg CFC eq/unit
(in progress)
Life Cycle Assessment Stages
(USEPA)
Human Population anrt Ecotegicaf Exposure
53
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LCA and Environmental
Regulation
•Adaption of LCA as a way to gather
information on waste production,
energy demand, and the potential for
risk to exposed populations
•Works best when risks are non-local,
and the population is non-specific
•Not a substitute for regulatory risk
assessment
The Health/Materials Paradox
Why might nanostructured materials be toxic?
size
shape
composition
photoactivity
redox activity
solubility
environmental instability
potential for exposure
Those attributes of NSM's that are prized for commercial
development and application, are the same ones that
cause toxic reactions
EPA Nanotech Research Focus
Environmental Applications
- Membranes, remediation, etc.
Environmental Implications
- End-of-pipe
- Toxicity
- Fate, transport, transformation
- Focus on NPs already in commercial production
• CNTs
• Ag°
• Fe°
• TiO2
• CeO,
Elements of a LCA-lnspired Interdisciplinary Research
Program for Nanotechnology
• Use of less toxic, more available components (eg.
Cd, Pb-free, AIP)
• Focus on structures that are less bioavailable
(e.g. coatings, solubility, stability, kinetics)
• Lowering of life cycle energy of manufacturing
• Design for recovery of nano-components at end-
of-life
•Understanding the social contexts in which nano-
based products are used and disposed of
•Application of LCA methodology to the entire
product chain
Nanotechnology Publication Trends
1
1
YEAR
• Total nano
.EHSnano
Nano LCA
: : ]
Embodied Energy (Cradle-to-Gate)
D) 5
O>
o
EAF Steel Aluminum Poly Si Wafer Si Nanotubes Quantum dots
Material
Adapted from Gutowski et al. 2007, and Sengul and Theis 2008.
-------
Sources of Impacts During
Manufacturing of NSMs
Strict purity requirements and less tolerance for
contamination during processing (up to "nine nines")
Low process yields
Significant energy requirements
Batch processing (post-processing, reprocessing), or very
low-yield continuous processing
Use of toxic/basic/acidic chemicals and organic solvents
High (or low) temperatures, pressures
High Water COnSUmptiOn SengulandTheisJIE,2008
Cumulative energy demand
(embodied energy) CdSe q-dots
Q 100
Ul
o
10 -
D
The Energy Paradox
Some of the most energy intensive
materials known to humankind
Less than 1% (currently) of the mfg cost
(Healy, Isaacs, 2008)
Current and Forecast World Production of Various Materials
990 1995 2000 2005 2010 2015 2020 2025 2030
Forecast Embodied Energy of Various Materials
1975 1980 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030
Nano-based Energy Savings
Table 3. Potential I'.S. Energy Saving? from Eight >",inotechnology Application?
(Adapted from Brown. 2005 a)
>*anuietliuulu«j Applkaiiuii
Strong, lightweight materials in transportation
Solid statp liglitiiip (flirt 31 white Itriir T.FD's)
Self-optimizing motor systems (smart sensors)
Smart roofs [temperature-dependent reflectivity)
Novel energy-efficient separation membranes
Energy efficient distillation tnrougn iupercomputing
MolccuLfl-kvcl toiitiul of iiidubuial talalyab
TraiMttiiiuon line conductance
Total
Reduction in Total
Annual I'.S, Energy
6.2*
ii
2.1
1.2
O.S
0.3
0.2
o.:
*Aj3uraJus: 1 MiHionE - Sarrel COQTOSIO (conespsmding to refonmilated gasoline -ftoraEIAmcsithly
**Bfljcd on L" S. onEtial cuctn1 conramf GOB from 2CC- $?- '- Q-j.iirillivs ETUTCU) from the Encrev Information
Adffiiniitrstbn Anniul En*r^- Esriew 2004
55
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CdSe in aquatic environments
PH
Composite materials are not recycled
To summarize...
Engineered nano-materials and products
• are already in use (how are they actually used?)
• are not widely understood by consumers
• are often energy intensive and materially inefficient to
make
•have increasingly complex functionalities
• have very high "value added"
• often use or are composed of toxic and/or "scarce"
chemicals in processing (availability?)
• are often difficult to recover once placed in commerce
(recyclability of bulk matrices)?
• comparative benefits and impacts of nanoproducts?
• LCA research and applications for NSMs are lagging
56
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2010 U.S. EPA Nanotechnology Grantees Meeting
Martin Shafer
Platinum-Containing Nanomaterials: Sources, Speciation, and Transformation
in the Environment
Martin Shafer , James Schauer , and Brandy Toner
1University of Wisconsin-Madison, Madison WI; 2University of Minnesota-Twin Cities, Minneapolis MN
Platinum is the archetypal element where chemical and physical speciation is essential for valid toxicology
assessments, yet critical basic information on environmental pools, speciation, and reactivity is lacking.
Anthropogenic platinum emissions to the environment have dramatically risen over the past 2-3 decades and
consumptive use, particularly in nano-catalytic applications, is projected to increase. Nano-particulate species
of platinum represent a major fraction of total platinum in most primary emissions, though it was thought to be
present in relatively benign elemental species. Recent evidence, however, indicates that primary emissions
may contain a significant oxidized platinum component and some studies suggest that the speciation of nano-
platinum can change rapidly after release into the environment—a factor that must be considered in
fate/transport and toxicology modeling. Information on environmental levels of the recognized toxic species of
platinum (chloroplatinates) is essentially absent.
Our research addresses three major questions: (1) what are the primary sources and environmental
receptors of platinum and nano-platinum? (2) what are the chemical forms of platinum introduced into the
environment from current and potential major sources? and (3) how does the speciation of platinum change
within specific environmental reservoirs after release? Our focus is on aerosol-mediated emissions, transport,
and exposure in non-occupational settings. Emissions from vehicles (exhaust catalysts [e.g., Three-Way-
Catalysts, TWC] are a major source of environmental platinum) are being addressed using roadside aerosol
sampling and a synoptic program of roadway dust sampling. Engine dynamometer experiments are being
conducted to evaluate platinum emissions from platinum-cerium based fuel-borne catalysts (FBC). High-
volume air samplers are used to collect ambient aerosols in several urban environments. Concentrations and
chemical speciation of platinum in particulate and "soluble" phases of these samples is being determined with
a suite of analytical tools. Synchrotron XAS (sXAS) is applied to solid phases. "Soluble" species, as defined
with physiologically relevant fluid extractions, including Gamble's Saline and Alveolar Macrophage Vacuole
Fluid, are characterized for particle size (Ultrafiltration and STEM), and charge (Ion Chromatography). The
presence of the particularly toxic chloroplatinate species is being probed using an HPLC-IC-ICPMS method.
Platinum species transformation will be evaluated in controlled laboratory experiments with both
environmental and model samples.
Road dust collections from multiple sites in cities across the country (including Atlanta, Denver, Los
Angeles, Milwaukee) exhibit elevated levels of total platinum (200-800 ng/g). Significant (8-23% of total)
soluble pools of platinum, with measureable anionic character, were measured in these vehicle emission
receptor samples. Our sXAS studies (ANL-APS, 20-BM) of aerosol emissions (PM) from diesel engines
burning a Pt/Ce-based FBC reveal a large fraction of oxidized platinum. Spectral fitting suggests that a
platinum(IV)oxide-hydrate is the dominant oxidized platinum species in the engine PM. Similarly, a
substantial component of the platinum pool in used TWCs was found to be oxidized. The majority of the
primary emissions of platinum from diesel engines burning a Pt-FBC was present in fine and ultra-fine
particle-size fractions. We have advanced the HPLC-IC-SFICPMS analytical methodology for separation and
detection of hexa- and tetra-chloroplatinate to achieve quantification limits of lower than 10 ng/L—an order-
of-magnitude better than reported in the literature—and we are working to further improve these limits.
Through our multidisciplinary approach, we expect to substantially advance our understanding of the
sources, speciation, transformation, and potential human exposures to nano-platinum materials in the
The Office of Research and Development's National Center for Environmental Research 57
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2010 U.S. EPA Nanotechnology Grantees Meeting
environment. We expect to provide some of the first measurements of the recognized toxic species of platinum
in environmental media. Vital information on the concentrations and chemical species of platinum in mobile
source emissions and important environmental receptors will be provided. Fundamental data on rates of
species transformation will be acquired. The chemical speciation and exposure data will enable enhanced
assessments of the toxicological relevance of environmental nano-platinum species.
EPA Grant Number: R833892
The Office of Research and Development's National Center for Environmental Research 58
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Platinum-Containing Nanomaterials:
Sources, Speciation and Transformation
in the Environment
Martin Shafer1, James Schauer1, Brandy Toner2
University of Wisconsin-Madison
Environmental Chemistry & Technology
Program and State Laboratory of Hygiene
2University of Minnesota-Twin Cities
U.S. EPA Nanotechnology Grantees
Meeting
November 08-09, 2010
Portland, OR
| General Motivation
1 Platinum is an archetypal element where speciation is essential
for valid toxicological assessments, yet relevant information on
environmental pools, speciation and reactivity is lacking.
1 Certain platinum species (most notably the chloroplatinates) are
toxic (allergenic).
1 Platinum levels in many environmental receptors has increased
over the past 40 years due to platinum use in automobile exhaust
catalysts and industrial catalysts.
1 Platinum-based catalysts are likely to remain a key strategy for
reduction of regulated pollutants from mobile sources.
1 Though platinum in most primary emission sources was thought
to be present in relatively benign elemental [Pt°] species,
evidence is mounting that the speciation of platinum (particularly
nano-sized platinum) can change rapidly after release.
Motivation (Mobile Sources)
1 Controlling emissions from mobile sources are critical for
continued reduction in health impacts of air pollution, and for
addressing regional and global climate impacts.
1 Most current and proposed emission control strategies for
diesel and gasoline engines employ metal catalysts to reduce
tailpipe emissions of regulated species.
' Gasoline Three-Way-Catalysts (Pt, Pa, Rh)
' Diesel Fuel-Borne Catalysts (Pt-FBC)
• • Diesel Particulate Filters (Pt-Catalyzed)
' Diesel Selective Catalytic Reactors (V-SCR)
' The use of these metals raises concerns about environmental
dissemination.
MOTIVATION - Chemical Speciation ~~
The toxicological responses of many metals (e.g. Cr, Cu, Mn, Pt, V)
are determined by the specific chemical & physical speciation in
the primary source or environmental receptor.
Extant modern methodologies provide little relevant speciation
information.
Traditional techniques that are speciation capable lack the
required sensitivity, particularly in the context of (a) ambient
aerosols, and (b) lower emissions from vehicles equipped with
modern control devices.
• OSHA-PEL/ACGIH-TLV
•:• Soluble salts- 0.002 mg/ms(Pt)
•:• Metal -1 mg/m3
• EPA-Toxicological Review (2009)
* NOAELam IxlO'6 mg/ms
* RfC (halogenated Pt salts) IxlO'9 mg/ms
Oxidized, halogenated (e.g.
chloroplatinic acids) species
(H, NH4, K, Na) are very
soluble and are 500-fold more
toxic than metallic species.
Specific Objectives of study
Refine analytical tools for measurement and chemical speciation
of platinum in environmentally relevant sources and receptors.
Integrate source and environmental sampling with advances in
platinum analytical speciation tools.
• Determine the physical and chemical forms of platinum introduced into
the environment from selected current and potential major sources.
• Evaluate changes in the speciation of platinum within specific reservoirs
after release to the environment.
••'Aerosol Sources
•/Soluble Species
•/Toxic Species
•/Nano-sized Species
;--'/.' •:^as&
Soluble Species
Halogenated Pt Salts
(chloroplatinates)
Cisplatin, Carboplatin
PtCI4
Pt(S04)2
Pt(NH3)4CI2
PtBr4
"Insoluble" Species
Pt metal
Pt oxides (PtO, PtO2)
PtCI2
Pt sulfides (PtS2)
Pt(cyclo-octadiene)
PtO2-H2O
Primary Platinum Sources and Receptors Under Study
a Automobiles: Three-Way-Catalysts (TWIG).
a Diesel Engines: Platinum-Amended Diesel Fuel (FBC1
and Platinum-Catalyzed Particulate Filters (DPF1
a Roadside Dust/Soils
Q Ambient Aerosol from Urban Centers
Gasoline Engine Catalytic Convene!
59
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Mobile Source Emissions: Pt-Catalyst Equipped Vehicles
Roadway and Tunnel Dust
•Several Urban Centers (Milwaukee, Los Angeles, Atlanta, Denver)
•Excellent integrated receptor for emissions from mobile sources
•Sieved and resuspended -> PM10and PM2 5
•PGE concentrations 50-500x background
Roadside Soils
Air Sampling adjacent to heavily trafficked roads.
•J'Size-fractionated recently emitted PM.
Catalyst Materials New and Used.
Lough, G.C., J.J. Schauer, J-S. Park, M.M. Shafer,
J.T. Deminter, and J.P. Weinstein. 2005. Emissions
of metals associated with motor vehicle roadways.
Environ. Sci. Techno!., 39:826-836.
(I Fuel
Diesel Engine Dynamometer Studies:
Our sampling train installed at MATC Engine Research Laboratory. Platinum speciation
was examined as a function of engine load, hot/cold start, particle size, and [Pt/Ce].
^FBC • Catalyst dosed directly into diesel fuel
- Pt/Ce fuel-soluble bimetallic catalyst
- delivered in situ
Active in high temperature combustion zone
- higher efficiency of fuel HC combustion
Liquid . FBC intimate contact with PM
Hydrocarbons _ more comp|ete combustion of solid C, HC
Delivers Catalyst to DOC / DPF
Atmospheric Aerosol Sampling
Roadside Aerosol Sampling &
Characterization
Ambient Aerosol Sampling &
Characterization
Size-resolving impactor (PCIS) sampler
25 nun PCIS Substrate
Majestic B.J., 1.1. Schauer, M.M. Shafer, P.M. Fine, M.
Singh, and C. Sioutas. 2008. Trace metal analysis of
and ambient samplers. 1. Environ. Eng. & Sci. 7{4):289-
298.
Ntziachristos L, Z. Ning, M. D. Getter, R. 1. Sheestey, 1.1.
Schauer, and C. Sioutas. 2007. Fine, Ultrafine and
Nanopartide Trace Element Composition Near A Major
Freeway With Heavy Duty Diesel Traffic. Atmospheric
Environment 41(27): 5684-5696.
Paniculate Matter Characterization
• Total Elemental and Isotopic: SF-ICPMS
• Extraction-Based Speciation
• Solid-Phase Speciation: XAS (XANES & EXAFS)
• Electron Microscopy: STEM
Applied to each of our target source and receptor samples:
a) PM from diesel engines burning Pt-amended fuels
b) Roadway dusts and roadside soils
c) Urban atmosphere aerosols Total pt (+48 additional elements) by SF-ICP-MS
d) Catalyst materials after microwave-assisted mixed acid digestion in
micro-Teflon bombs.
^V90gME
Extraction-Based Characterization Strategy
Solubility -Biochemically re levant fluids
I. Gambles Saline (pH=7.4)
II. Macrophage Vacuole Cytoplasm Fluid (pH=4.6)
III. MQ
IV. 1 M HCI
V. Methanol (access binding sites sequestered in hydrophobic
soot matrix)
Each extract filtered at 0.22 urn
Time points of 2, 6, 24, and 48 hours (kinetics of release)
Three solid-solution ratios (200, 500, and 2000 mg L1)
Room temperature and protected from light
Soluble ions (nitrate, chloride, sulfate & ammonium) and TOC determined.
Each filtered extract subjected to the following separations:
• Colloid/Nano-Particle Charge! Joe Chromatography(DEAE
chromatography). Anionic versus Cationic+ Neutral.
Fractions •* SF-ICP-MS.
• Colloid/Nano-Particle Size: Ultrafiltration (1O kDa). Nano-
particulate versus "dissolved". Fractions -> SF-ICP-MS
;--'/.' ':^as&
Complementary Total and Extractable Methods
60
-------
R
T
[
cad Dust: Los Angeles
otal Pt = 720 ng/g
1 1 < 10 kDa
^H Anionic
n* fin.
CCL
1
Site
JL
Gambles Saline Billi-Q Macrophage VF 1 M HCI
Speciation of Platinum
in
Extracts of Road Dust
Los Angeles CCL Site
Century Avenue Exit of I-110
Platinum & Palladium Aerosol Mass-Size Distributions: Milwaukee, July - August 2010
Speciation of
Platinum in Extracts
of Road-Side Aerosol
Road-Side Aerosol: Milwaukee Site
Total Pt= 146 ng/g (5.4 pg m"3) -
eVF 0.07 M HCI
Influence of FBG on Chemical Composition of PM
Okuda, T., J.J. Schauer, M.R. Olson, M.M. Shafer.A.P. Rutter, K.A. Walz, and P.A. Morschauser.
2009. Effects of a platinum-cerium bimetallic fuel additive on the chemical composition of diesel
engine exhaust particles. Energy&Fuels 23:4974-4980.
—*— EC emission I
--0--OC emission
5 10
1
E 5
T--4
0.0 0.2 0.4 0.6 0.8 1.0
Additive cone (ppm-Pt)
Additive cone (ppm-Pt)
54% reduction in PM25 EC; 23% reduction in PM25 OC; 34%
reduction in PM25 MASS at 0.1 ppm Pt and 7.5 ppm Ce (Hot Start)
Engine out platinum fraction = 7% (1.7 ug bkW1h-> at 0.1 ppm Pt)
Engine out platinum fraction = 14% (3.4 ug bkW'fr1 at 0.7 ppm Pt)
"Typical" E/O% = 2-22% of added Pt
Particle Size Distribution of
Platinum and Cerium in PM
Emissions from Engines Burning
Pt-Amended Fuel
SpeciatedWaterSoluble
Platinum in Diesel PM
1. Extractable fraction = < 3%.
2. Large colloidal fraction (44% of
extractable species).
3. Dissolved (<10 kDa) species
exhibit significant anionic
character on DEAE (42%).
Total Platinum Emission Rates
5,000-50,000 ng/km Pt (w/o DPF)
50-500ng/km R (w DPF)
TWC Vehicles: 10-100 ng/km Pt
61
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Synchrotron XAS: EXAFS Spectra of Platinum Reference Materials
EXAFS Spectra of 18
Platinum Reference
Compounds
Measured to Date
LBL-Aduanced Light Source
Pt-foil = platinum foil; Pt-alumina = 5% platinum on alumina; Pt-C = 5% platinum on graphite; Pt(ll)CI2 =
platinum(ll) chloride; TCP = potassium platinum(ll) tetrachloroplatinate; PtO2 = platinum(IV) oxide;
Pt(IV)CI4 = platinum(IV) chloride; HCP = potassium platinum(IV) hexachloroplatinate.
Majestic, B.J., J.J. Schauer, M.M. Shafer. 2007. Application
of Synchrotron Radiation for Measurement of Iron Red-Ox
Speciation in Atmospherically Processed Aerosols.
Atmospheric Chemistry and Physics 7:2475-2487.
Gasoline Vehicle
Catalyst (TWO).
J,
J
J
Pt02
^ K2Pt(ll)CI4
\ 5% Pt-Alumina
J
|/\^S^— 1^*--S— •"-— «
88% Pt-alumin
KmKjPtdllCI,,
2%PtO2
NSS 5.2E-5
5
_
5J
Pt speciation was studied in a 4year old 3-way automobile catalyst. A 30
um thick, quartz slide mounted, longitudinal section of the center of
the catalyst was prepared.
(A) Light microscope image; outlined area was examined with XRF
mapping.
(B) Red (Pt)-green (Cu)-blue (Ce)XRF-derived tricolor map. PtLIII-
edge extended-XANES spectra were collected at spots 0-2.
(C) The e-XANES spectra (11,466-12,077 eV) were fit with reference
spectra - Pt foil, 5% Pt in alumina matrix, 5 % Pt in carbon matrix,
Pt(ll)CI2, Pt(IV)CI4, Pt02, K2Pt(IV)CI6'H20, and K2Pt(ll)CI4 - by linear least
squares method.
(D) Select reference spectra and an example fit shown in C and D.
Modeled fraction of oxidized Pt is significant.
XRF Map, and Extended-XANES Fitting Results, of Diesel Exhaust Particulates
Trapped on a Diesel Paniculate Filter [engine running with Pt-FDCl
X-ray Fluorescence Map
Particle -Pi Speciation
Large heterogeneities in particle composition
are observed with many particles exhibiting a
significant oxidized platinum component.
Strong evidence for PtO2 (14-25% in many spots, up to 40% when
associated with Ca and S).
•• "*";x;!ffh
Platinum XAS
XAS Spectra of Diesel Engine Exhaust
PM (Pt-FBC Fuel at 0.7 ppm Pt; PM8)
Significant contributions from oxidized
platinum species are evident in spectrum.
(in primary vehicle emissions)
•Platinum (IV) oxide-
K-space Spectrum of PM from Pt-amended fuel
Quality of EXAFS spectrum will support shell-by-
shell fitting. Early data suggests that oxide and
metal are the two dominant platinum species.
;--'/.' ':^as&
LARGONNK
Ghloroplatinate Method Development and Application
We are targeting two documented toxic/allergenic chloroplatinate compounds and
their hydrolysis products.
* hexa-chloroplatinate (PtCI6-2) Pt(IV) (H+, K+, Na+, NH4+)
* tetra-chloroplatinate (PtCI4-2) Pt(ll) (H+, K+, Na+, NH4+)
Only very limited information on the concentrations of chloroplatinates in potential
environmental sources and receptors is available.
Environmental fate and transport data is lacking.
It is unknown whether chloroplatinates may form from environmental processing of
other platinum compounds in the environment.
Very sensitive analytical techniques are required as the levels in environmental
samples are expected to be very low (ng g-1, pg mr1, pg nr3).
Few published methods. [Nachtigall, Nischwitz : HPLC-ICPMS (0.3 ug/L (12 pg)).
X'
tetra-chloroplatinate
a
01*1*01
ci"'7~a
ci
hexa-chloroplatinate
62
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HPLG-SF-IGP-MS Isocratic Method
HPLG-SF-IGP-MS Gradient Method
A gradient elution method
(total run time = 25 min.) was
developed to help elucidate
the identity of chloroplatinate
transformation products.
PtClx2' + H2O -» PtClx..,(l-l2O)- + Cl-
* \2gE7 35
Tetrachloroplatinate (PtCI42', "Tetra")
and hexachloroplatinate (PtCI62-,
"Hexa") are separated isocratically
using a Dionex AG11 guard column
containing an alkanol quaternary
ammonium stationary phase and a
mobile phase consisting of 0.1 M Na-
pe re h brat e/HC I at pH= 1.9 and
detected with magnetic sector ICP-MS.
Chromatogram displaying
response at LOD.
, T«ra Hesa
post turbovap blowdown of
MeOH/EDTA extract.
and peak-capture we have
achieved LODs of 0.2 pg.
With an 80 ul injection (sample loop) the
current limit of detection of the method is
approximately 15 parts-per-trillion (ppt) [1
pg] in both standard mixtures and in
spiked tunnel dust (CRM 723) extracts (1M
HCI). (10-fold improvement over published
methods). We are working toward another
10-fold improvement. Our goal is <0.05%
of total Pt (<50 pg/g, <0.5 pg/10 mg).
Tetra-chloroplatinatepeak
Ghloroplatinate MethodDevelopmentPlans
1 Continued method development on chloroplatinate speciation
*J* Further improvement in already achieved sub-pg detect ion limits
••'volume reduction (turbo-vap)
•/off-line peak capture and concentration
•/Br-PADAP ortrioctylamine ligands (in Ml KB) to selectively complex (and
preserve) chloroplatinates
*J* Further validation of extraction methods (MeOH/HCl and MeOH/EDTA) for
target environmental matrices
*J* Synthesize stable-isotopically enriched (194Pt and 196Pt) target compounds
for isotope dilution and tracer experiments
Apply methods to engine PM, road dusts and airborne PM samples
*J* Determination of ambient chloroplatinate concentrations
*** Investigation of transformation and degradation of chloroplatinates in the
environmental matrices
Follow-up with our previoi
workwith tandem mass
spectrometry (hydrolysis
Environmental Transformation Studies
University of Wisconsin-Madison Biotron
• Environments
- Aerosol in contact with air
- Soil-sediment system
- Aquatic suspension
• Samples
- PM from Pt-FBC-treated diesel exhaust
- Tunnel /road dust and roadside aerosol
- Size-resolved PM from urban air
• Variables
- Time
- Humidity
- Light
- Oxidant
Acknowledgments
MATC
;--'/.' ':^as&
63
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2010 U.S. EPA Nanotechnology Grantees Meeting
Andrij Holian
Role of NLRP3 Inflammasome and Nickel in Multi-Walled Carbon
Nanotube-lnduced Lung Injury
Andrii Holian, Teri Girtsman, Mary Buford, and Raymond Hamilton, Jr.
Center for Environmental Health Sciences, The University of Montana, Missoula, MN
There is insufficient information on what characteristics of engineered nanomaterials (ENM) result in the
greatest health risk. Significant questions regarding chronic inflammation and the subsequent development of
fibrosis as observed in animal models need to be addressed. In addition, discrepancies in study outcomes for
the same class of materials makes risk assessment difficult. Specifically, carbon nanotubes have been reported
by some to have minimal effects while others have reported significant pathological outcomes following
exposure. It is likely that variations in the manufacturing methods of these materials are responsible for the
inconsistent results in the literature. For example, multiwall carbon nanotubes (MWCNT) are prepared by a
variety of methods using different metals as catalysts. This variability in manufacturing method results in tubes
that not only vary in size, but also metal content.
The molecular mechanism of action where ENM such as MWCNT causes lung inflammation leading to
lung fibrosis has not been elucidated. Studies with other particles such as silica and asbestos indicate that
activation of the NLRP3 inflammasome resulting in the release of potent inflammatory cytokines such as IL-
1(3 is important in the resulting pathogenesis. Furthermore, we have reported that long Ti02 nanobelts activate
the NLRP3 inflammasome and generate an inflammatory response in vivo. Therefore, the current study utilized
the availability of a family of MWCNT that were provided by the National Toxicology Program and
characterized by the Research Triangle Institute to test the hypothesis that the inflammatory potential of
MWCNT correlated with activation of the NLRP3 inflammasome. These studies were conducted in vitro using
primary alveolar macrophages (AM) isolated from C57B1/6 mice and human macrophage like THP-1 cells. In
vivo studies were conducted to examine the pathology at 7 and 56 days. All MWCNT were suspended in
dispersion medium and administered by pharyngeal aspiration.
Pathology varied from little to no evidence of lung injury to significant inflammation and pathology.
Correlations were made depending on contaminants. When a subset of MWCNT was evaluated with similar
diameters, there was an excellent correlation between pathology and Ni content, but not Fe, Co or Mo. The
correlation held for pathology at 7 and 56 days, although there was a tendency towards resolution at 56 days
compared to 7 days. Also, there was significant correlation between Ni content and inflammasome stimulation
(IL-1(3 and IL-18 release) in both primary AM and THP-1 cells. Furthermore, inflammasome activation
correlated with in vivo pathology using both primary AM and THP-1 cells. Activation of the NLRP3
inflammasome required lysosomal rupture and release of cathpesin B. In summary, the bioactivity of a broad
range of MWCNT could be predicted from NLRP3 inflammasome activation using either primary AM or
THP-1 cells. For MWCNT, Ni content was an excellent predictor of lysosomal rupture, NLRP3 activation and
pathology.
EPA Grant Number: R828602
The Office of Research and Development's National Center for Environmental Research 64
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Principal investigator did not authorize publication of the presentation.
65
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AM Session 2: Effects of Nanoparticle Surface
Properties
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Gregory V. Lowry
Microbial Bioavailability of Polyethylene Oxide Grafted
To Engineered Nanomaterials
Teresa Kirschling ' , Kelvin Gregory , Robert Tilton , and Gregory V. Lowry
1 Department of Civil and Environmental Engineering, 2Department of Chemical Engineering,
Carnegie Mellon University, Pittsburgh, PA
Coatings are an integral part of nanoparticle design, imparting changes in particle reactivity, stability, and
toxicity. These coatings frequently consist of polymers adsorbed or grafted to particle surfaces. The fate of
these polymeric coatings will affect the long-term fate, partitioning, and ecological impact of engineered
nanoparticles in the environment. Depending on the polymer composition and method of surface attachment,
some coatings may be removed from particle surfaces by desorption or non-biological hydrolysis processes.
Direct microbiological removal or degradation of nanoparticle coatings has not been demonstrated. In this
study, we synthesized 70 nm diameter star copolymers consisting of 2000 molecular weight polyethylene
oxide (PEO) arms emanating from dense, cross-linked polystyrene-like cores via atom transfer radical
polymerization (ATRP). These serve as model engineered nanomaterials with a covalently grafted polymer
brush coating for a study of direct coating degradation by microbes. Because the arms are covalently linked to
the cores, the possibility of PEO desorption and subsequent microbial degradation of free polymers in solution
is eliminated. A consortium of PEO degrading microorganisms was enriched from Monongahela River
(Pittsburgh, PA) water. Cultures were grown on either a 2000 molecular weight PEO homopolymer solution or
a PEO star polymer solution as the sole carbon source. Cultures grew on both carbon sources indicating that
the covalently attached PEO coatings on these nanoparticles are bioavailable. PEO star copolymers aggregated
after microbial degradation, demonstrating the loss of colloidal stability caused by PEO arm degradation. Such
microbiological processing of nanoparticle coatings would have significant implications for the long-term
mobility of engineered nanoparticles in the environment. Furthermore, because a growing body of evidence
shows that toxicity of engineered nanoparticles to cells and organisms is decreased by aggregation, microbial
processing also may impact the long-term ecotoxicity of engineered nanoparticles.
EPA Grant Number: R833326
The Office of Research and Development's National Center for Environmental Research 67
-------
Microbial Bioavailability of Polyethylene Oxide
Grafted Nanomaterials
Carnegie Mellon University Departments of 'Chemical Engineering. 2Chemistry.
3Civil and Environmental Engineering and 4Biomedical Engineering
• N
CEIN1
EPA STAR Grantees Meeting
November 8-9, 2010
Carnegie Mellon
The Effect of Surface Coatings on the
Environmental and Microbial Fate of Nanoiron
and Fe-Oxide Nanoparticles
• Objectives
• Determine the fate of NZVI in the environment
• EXAFS characterization after aging (Reinsch et al, K5T2009)
Effects of NZVI and coatings on biogeochemistry
• Effect of coatings on NZVI toxidty (Li et al. EST20W, Phenrat et al, EST
2010
• Examine shifts in native nucrobial populations and delialococcoid.es spp.
upon exposure to NZVI (3 g/L)
• Kirschlinget al., EST2010, Xiu et al., Biotech Bioproc., 2010, KST2011
• Determine the fate of the coatings
• Desorption of coalings from NZVI (Kim et al., _E^T2009)
• Biodegradation of covalently bound polymers on ENMs
Carnegie Mellon
Impacts of Nanoparticle Coatings
Manufactured
Coatin
are
Nanomateria
In order to understand nanoparticle fate and
transport, we must understand coating fate
Carnegie Mellon
Biological
Interactions
CElNi
5nm cutoff
ARE NANOMATERIAL
COATINGS BIOAVAILABLE?
C'arncgie \1ellun
CEIN
Bioavailability experiments
•Changes in nanoparticles
l/\
\^_ X,
•Growth of culture
Carnegie Mcllnn
Model nanoparticles: PEO star copolyrners
i 10 100 1000 10000 -Nontoxic
Hydrodynamic Diameter (nm) -Permanent COating
•Does not hydrolyze in water
Carnegie Mdlo 11
EIN
68
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Enrichment culture
Camcgic Mellau
Enrichment culture
Otrncgjc Mellon
CEIN
Enrichment Culture
Carnegie Mellon
CEI
Enrichment culture
C'arncgie Mvlluii
Growth on PEO star copolymers
Carnegfc Melliin
Growth on PEO star copolymers
Carnegie Meilon,
69
-------
I
8
q
11
a ^
>~.
T3
ffi
Microbial induced PEO star copolymer
aggregation
140 -i
130 -
120 -
110 -
100 -
90
80
70 -
60 •
^^^*^^
^^*^*\^^^*
-•-Sterile Control
-•-Stars Culture 1
^ , , * -*-Star Culture 2
0 100 200 300 400
Time (hours)
CEIN
Carnegie Mellon ,7
Microbial induced PEO star
aggregation
Carnegie Mellon
Conclusions
Covalently bound PEO on nanoparticles is
bio available
Microorganisms can change nanoparticle
stability which will change the fate and
transport in the environment.
Availability will depend on
Coating attachment
Degradability of coating
Carnegie Mellon
Next Steps
What happens to coatings in the environment?
Desorption Displacement Hydrolysis Photodegradation Multilayers
Carnegie Mellon
cO
CEIN
Problems Encountered
Difficult to track coating fate in real environmental
samples
14C labeled coatings
Recovering ENMs form real environmental samples
Measuring processes and effects occurring at
realistic concentrations of ENMs
Carnegjc Melliin
CEIN
• Questions?
Carncgje Mellon,
CEIN
70
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Microbial induced PEO star
aggregation
Camcgic Mellon
CEIN
Availability of polymer end groups
Otrncgjc Mellon
How coatings influence interactions
Carnegie Mellon
dfer
71
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2010 U.S. EPA Nanotechnology Grantees Meeting
Howard Fairbrother
Surface Oxides: Their Influence on Multi-Walled Nanotubes Colloidal, Sorption
and Transport Properties
Howard Fairbrother, William Ball, Billy Smith, Jin Jang, Kevin Wepasnick, and Julie Bitter
Johns Hopkins University, Baltimore, MD
Nanomaterials are being produced and integrated into consumer products and specialized applications at
an accelerating rate, and concern has increased about their environmental fate and effect. Fueling this
apprehension, in part, is the fact that many nanomaterials are being deliberately surface functionalized to
enhance their aqueous colloidal stability and biocompatibility. As a consequence, these surface modified
nanomaterials are likely to exhibit different behaviors in aquatic environments as compared to the pristine
nanomaterials. In our research group, we have focused on understanding how oxygen-containing functional
groups (surface oxides) influence the environmental properties (e.g., colloidal stability, transport through
porous media, and sorption) of multi-walled carbon nanotubes (MWNT), a prominent class of engineered
nanomaterials. In doing so, we hope to provide the information that can be used to predict and rationalize the
effect of surface chemistry on the environmental fate of MWNTs.
Our scientific approach has been to develop structure-property relationships between the MWNT's surface
oxygen concentration and their colloidal, transport and sorption properties. To accomplish this task, we have
used a suite of wet chemical-treatments that allow us to controllably vary the extent of MWNT surface
oxidation. Typical oxidants include HN03, KMn04, and mixtures of H2S04-HN03. To determine the
concentration of surface oxides imparted by these treatments, we have used X-ray photoelectron spectroscopy.
Additional characterization of our as-received and oxidized CNTs has been carried out using the techniques
listed in Table 1.
Analytical Technique Information Obtained on MWNTs
Transmission Electron Microscopy (TEM)
Atomic Force Microscopy (AFM)
BET Isotherm
Chemical Derivatization
Dynamic Light Scattering (DLS)
Potentiometric Titration
Electrophoretic Mobility
Structural Integrity
Length Distribution - Before/After Oxidation
Surface Area
Surface Concentration Hydroxyls, Carbonyls and
Carboxyls
Spherically Equivalent Particle Size
Surface Charge
Sense of Surface Potential
Table 1. Analytical techniques used to characterize oxidized MWCNTs and the information acquired
Colloidal Stability: To examine the aqueous colloidal stability and aggregation properties of oxidized
MWNTs, sedimentation and time-resolved dynamic light scattering (TR-DLS) experiments were conducted on
single component suspensions prepared by prolonged sonication of MWNTs in Milli-Q water. Over a range of
environmentally relevant pH values (4-9) and electrolyte (NaCl, CaCl2) concentrations (0.001-1.000 M), the
aggregation and colloidal properties of MWCNTs were found to agree with the basic tenants of DLVO theory,
in that (1) more highly oxidized, negatively charged MWNTs remained stable over a wider range of solution
conditions than lowly oxidized tubes, (2) oxidized MWNTs adhered to the empirical Schulze-Hardy rule, and
The Office of Research and Development's National Center for Environmental Research
72
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2010 U.S. EPA Nanotechnology Grantees Meeting
(3) MWNTs exhibited reaction- and diffusion-limited aggregation regimes. To complement investigations
conducted under ideal solution conditions, the effect that natural organic matter (NOM) had on the MWNT's
colloidal properties also was examined. Due to steric stabilization, the colloidal stability of MWNTs was
greatly enhanced in the presence of NOM, as expected. However, bench-top sedimentation and TR-DLS
studies indicated that the colloidal stability of less oxidized MWNTs was greater than that of more highly
oxidized MWNTs at environmentally relevant NOM concentrations (~3 mg/L). This effect was due to the fact
that although the presence of negatively charged surface oxides increases the colloidal stability of MWNTs,
they also decrease their sorption capacity towards NOM. Consequently, surface oxidation has the effect of
increasing the colloidal stability of MWNTs in the laboratory but decreases the relative colloidal stability of
MWNTs in the natural environment.
Transport: Studies examining the transport properties of MWCNT through model columns are underway.
Suspensions of MWCNTs are prepared by prolonged sonication in Milli-Q water and model columns have
been prepared using spherical glass beads (0.355-0.425 mm diameter). Current results show that the transport
of MWCNTs through idealized porous media obeys traditional DLVO and clean bed filtration theory.
Specifically, the deposition rate of colloidal MWCNTs increases with increasing ionic strength until reaching a
diffusion-limited deposition regime. For a set of highly oxidized MWCNTs, critical deposition was found to
increase significantly with pH. Having completed these initial studies, the next step in this investigation is to
examine the role that MWCNT surface oxidation plays in transport.
Next Steps: Two new avenues of research are being undertaken to further examine the role that surface
oxides play in regulating the environmental fate of CNTs. In one study, solid phase MWCNT powders are
being continuously stirred in water to mimic natural currents. The goal of these experiments is to examine the
rate of CNT transfer from one phase to another (solid to colloidal) and to determine how releases rates are
influenced by particle and aqueous phase conditions. The other avenue of research is to determine the extent to
which results from our MWCNT studies apply to single-walled CNTs (SWCNT). While seemingly
straightforward, issues associated with purity arise when using pristine and oxidized SWCNTs. According to
TEM analysis, purity issues are predominantly associated with amorphous carbon. Methods to purify as-
received and oxidized SWCNTs are currently being investigated. One method that has shown some promise is
rinsing SWCNTs with strong NaOH.
EPA Grant Number: R828771
The Office of Research and Development's National Center for Environmental Research 73
-------
Effect of Surface Oxygen on
Environmentally Relevant
Properties of Carbon Nanotubes
(Aggregation, Transport and
Sorption)
Howard Fairbrother
Department of Chemistry
Johns Hopkins University
Surface Oxides and Their
Effect on MWCNT Properties
Questions
Research Questions
Develop functional
relationships related to
Create models used to
predict
RELEVANT BEHAVIOR
Surface Oxidation of MWCNTs
MWCNT
Reflux at Clean by
140°C,2hours DI water
Dry at 70°C Ball mill
Oxidants:
H2SO4/HNO3, HNO3
KMnO_,03, H2O2
Prevalent Oxidative
— Method
Surface Analysis
Surface Analysis
X-ray photoelectron Spectroscopy
Determine Surface Oxygen
Concent!;'!)on i-jj. "b i
Controllable Oxidation
Jl
Aggregation Properties
74
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Surface Oxygen and Colloidal Stability
Laboratory vs. Environment
NOM & CNTs - Our Approach
PHYSICOCHEMICAL CHARACTERIZATION
MWCNT Sofption Properties:
Influence of Surface Oxygen
Aggregation in NOM: Observations
4.2mg/LMWCNT@pH6
: 4.2 mg/L MWCNT® pH 6 after 13 ho
fe
•mMNaCljNoNOM 60mMNaCl; lOxNOM 290mMNaCl; lOxNOM
m
TR-DLS: 0.8 mg/L MWCNT@ pH 6
75
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Transport Properties
Transport: Column Transport Experiment
Step-Input Method
Glass beads
0.3 55-0.42 5mm
Dispersed
MWCNTs
Transport: Column Transport Experiment (cont/d)
Pulse-Input Method
Glass beads
0.355-0.425mm
. M
laCl soln
2.5 (ig
CNTs
I DI water 3.5 mL/ml
Transport: Experimental Parameters
Flow parameters
7 ml/min
length
10.2cm
Volumetric
flow rate
Linear 0.0238cm/s Intersection 4.91cm2
velocity area
Superficial 0.0624cm/s Porosity 0.38
velocity
Pore volume 18.67cm3 9.73cm3
5.2cm
4.91 cm2
0.38
Transport: Breakthrough curves
Short columns
Long columns
0.25
0.20
J
If 0.15
C 0.10
0
0.05
0.00
- OmMNaCl
SmMNaCl
- 7mMNaa
lOmMNaCl
- MmMNaCl
•WmMNaCl
SOmMNaCl
- lOOmMNaCl
- ISOmMNaCl
- SOOmMNaCl
0.20
I
f 0.15
! 0.10
0 mM NaCl
5 mM NaCl
7 mM NaCl
20 mM NaCl
5OmMNaCl
200 mM NaCl
=00 mM NaCl
Breakthrough curves of 53% HNO3 treated MWCNTs at different ionic
strengthes at pH 5.6-5.8
Transport;Calculation method
One-dimensional advection-
disuersion eauati on with a sink
dc d c fh~
For step inputs
Colloid deposition rate coefficient k
76
-------
Transport: CDC curves at different pH
10 100
[NaCl], mM
pH4.0
CDC=5.2mM
pH5.8
CDC=28.6mM
pH 10.0
CDC = 114.8mM
Transport Issues
(& The Solutions)
Increasing plateau and irreproducible
results in step-input experiments
Poremlmre
[^Breakthrough curve of 30p HNO3 oxidized
MWCNTs at 15 mM NaCl
Breakthrough curve of 30p HNO3 oxidized
MWCNTs at 64 mM NaCl in different runs
Problems &
Solutions
The Importance of Column Treatment -
Sanitation
a
I
S 0.1
s
g
•S
I
Beads sonicated in 250 ml beaker
Beads sonicated in 600 ml beaker
Problems &
Solutions
The Influence of Sorricatiori Time in
Creating a Reliable Porous Media
30 min 60 mln 90 min
Attachment efficiency of O-MWCNTs through
porous media treated for different sonication times.
(pH 5.8, 10 mM NaCl)
Problems &
Solutions
Preparation of columns for reliable data
> Acid & base wash glass beads.
> Pack a quartz column with wet-pack
method.
> Take the column apart, sonicate
beads for 1 h and repack the column.
77
-------
Sorption Properties
Adsorption experiments
Adsorption: Effect of Surface Oxidation
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Ce, mmol/!
Effect of oxidation degree (oxygen concentration) on the
adsorption of Zn (II) onto MWCNTs, total ionic strength is 30 mM,
pH = 6.0 was buffered by MES.
Adsorption: Effect of solid-to-liquid ratio
0.6
0.5
0.4
0.3
0.1
0.0
s/l = 8.78 * 10 V1
s/l = 4.09 * 10 3 all
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Ce, nimol/1
Niz+ adsorption affinity of colloidal O-MWCNTs at different solid-to-liquid
(s/l) ratios; pH = 7.0 buffered by NaHCOS.
Adsorption: colloidal versus powdered phases
0.6
0.5
"'4
0.3
°'2
0.1
0.0
•
*
A
t
o s/l = 2.5 g/1 (powdered)
« s/l - 8.78"104 g/1 (colloidal)
A s/l - 4.09"10S g/1 (colloidal)
°
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Ce, inmol/1
Niz+ adsorption affinities of colloidal (open symbols) versus powdered
(solid phase, closed symbols) O-MWCNTs. pH = 7.0 buffered by
NaHCO,.
Future plans
The Future
k Effect of different oxidation on deposition of O-
MWCNTs
> Effect of particle sizes on deposition of O-MWCNTs
t Facilitated transport
78
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
James Ranville
Development of Hyphenated and "Particle Counting" ICP-MS Methods Exposure
Assessment of Inorganic Nanoparticles
James Ranville and Christopher Higgins
1 Department of Chemistry and Geochemistry, 2Environmental Science and Engineering Division,
Colorado School of Mines, Golden, CO
Quantifying environmental loadings and organism exposures is critical for the development of
nanoparticle (NP) risk assessment models. Development of detection, characterization, and quantitation
methods could lead to direct measures of organism exposure, both in laboratory and field settings. Inductively
coupled plasma (ICP) techniques, which are generally capable of achieving ppt detection limits, are well suited
to the analysis of metal-containing NPs. The power of ICP for detecting trace amounts of the elemental
constituents of the NP must be combined with a means of discriminating between dissolved and NP-associated
elements. Furthermore, techniques that provide size distribution information for NPs greatly increase our
ability to understand their environmental transformations and implications. Although generally an accepted
technique for NP size characterization, serial filtration and ultrafiltration are prone to numerous artifacts. New
methods for using ICP-mass spectrometry (ICP-MS) include hyphenated techniques such as field flow
fractionation (FFF-ICP-MS) and hydrodynamic chromatography (HDC-ICP-MS). Use of ICP-MS as an
element specific single particle counter (SP-ICP-MS) can be achieved by using the ICP-MS in a non-
traditional mode of operation. Recent developments in these methods and their potential for use in
environmental fate and effects studies will be the central topics of the presentation.
EPA Grant Number: RD-83332401-1
The Office of Research and Development's National Center for Environmental Research 79
-------
Hyphenated and "Particle Counting" ICP-MS
methods for the detection and characterization
of metal and metal oxide nanoparticles
Dr James F Ranville
Department of Chemistry & Geochemistry
Dr. Chris Higgins
Environmental Science & Engineering
Colorado School of Mines
Golden CO
Presented at
EPA PI meeting, Portland, Nov 8th 2010
Risk Assessment of Nanotechnolo
4,911-917.
• , 1338-1344.
B. Lee & J. Ranville, Poster RP059, Thursday
Detection & Characterization
Questions to be addressed
- Detection & Quantification (counting methods)
• How much sensitivity & selectivity do we need?
• How do we apply methods to complex matrices (waters,
tissues, sediments)?
• Example: Nano Ag in wastewater
- Characterization (hyphenated methods)
• What is the exposure (form, and amount)?
• Are we studying what we think we are?
• Example: FFF applied to QD toxicity on D. magna
Detection: How much sensitivity & selectivity do we need?
Material Flow Analysis: Ag
c
Gottschalket al, ET&C, 29, 1036-1048, 2010
Detection: How much sensitivity & selectivity do we need?
Predicted Environmental Concentrations (PECs)
Mode
Sinu-Ai
Air 0.021
Surface wiur 0172
S'n3 cfllurnl W.7
S'lTstadjt l.B!
Sediment 1203
Soil 11.2
0.56
ms
1,46
IJ».,..
— 0.074
— 127
6J4
— IOIW
ng L-'
«f.L-'
mg kg''
'«
'
Parts per trillion
Gottschalket al, ET&C, 29, 1036-1048, 2010
Environmental levels unknown, likely ppt
From laboratory toxicity testing, effects seen at ppb, ppm
Hypothesis: We can use ICP-MS to:
•detect
•count
•size
individual Ag nanoparticles
Approach is to use element specific "pulse" counting
Real time single particle ICP-MS
or
Time resolved ICP-MS
or
Single particle ICP-MS
-------
Silver Nanomaterials
ASAP (polydisperse)
Nanocomposix
(monodisperse)
How does it work?
ICP-MS of dilute solutions
- Dissolved metals produce
relatively constant signal
- NP appear as a pulse which
deviates from the baseline or
"dissolved" background
ICP-MS Parameters
- Collect individual data points
10 to 20 ms dwell times
- Optimize parameters (flow
rate, neb. Gas flow, etc.) for
best Ag detection
Analyze Signal Intensity
- Ideally in dilute solns only one
NP will be ionized and will
appear as a packet of ions
- Compare pulse height to
dissolved standards
What the data look like.
Are Ag Nanoparticles quantitatively
detected by ICP-MS ?
• Acicfied Samples
GDI water
Phosphate Buffer
NanoComposix Silver Size
Defining NPs
Run blanks (a) and
dissolved
standards (b)
Determine background
concentration (c )
Find appropriate way
to differentiate
between
background and
NP(d)
Determine number
concentration and
mass of NP (e)
If the particle counting approach is valid:
• Number of pulses will increase with increasing nano Ag
concentration
• Number of pulses will be reduced by filtration or
acidification
• The intensity of the pulse will be related to N P size
81
-------
ASAP Results
I:
*»
1:
• Haw
K FlRCSCd •
' Aodrficd 1
^ * *
* •* * ofe 1*0
What is the minimum size that can be detected?
Dissolved Ag
100nm NanocomposixAg
» •™
: -
0.5 ppb
Total counts = J06.?;5
What is the minimum size that can be detected?
Disk Centrifuge
SP-ICP-MS
Quantitation (Estimation) of Ag in wastewater
J SO IUO I* aK ZK ti * TO 150
11ire (SecondsI
•Summation of baseline signal
= "dissolved" Ag
•Summation of pulses =
concentration of Ag-NP
•Raw Wastewater influent:
Dissolved Ag = 520 ppt Ag-NP
= 200 ppt
•Final Effluent: Dissolved Ag =
60 ppt Ag-NP =100 ppt
•Results comparable to
estimates from materials flow
analysis (Nowack)
•In what form is the Ag-NP?
Need complimentary analysis
(TEM)
Characterization: Are we testing what we think we are?
Question: Is the CsSe core toxic to D. magna
Quantum Dots (QDs)
• Basic Structure
- Metalloid core (1-5 nm in diameter), usually with
protective shell
- Can add coating to make Hydrophilic
hydrophilic (e.g. PEO or MUA) Coatim
Intense fluorescence
Fluoresce 490-680nm
- Determined by CdSe core diameter
ZnS Shell
CdSe Core
httpMamp. tu-graz.ac. at/~hadley/nanoscienceAfl/eel<2/l\lano-CdSe.png
Sizing Hydrodynamic Radius by Fl FFF - ICP - MS
82
-------
Size analysis by FFF theory or calibration
Field: 0.9 ml/min
Carrier flow: 1.0 ml/min
PSS standards (Duke)
20 microliter injection
Fl detector
Elemental Size Distribution by Fl FFF-ICP-MS
Non 1:1 Cd: Se ratio
Possible explanation: Cd associated with polymer coating due to poor
washing during synthesis
Low Zn indicates thin ZnS shell
Elemental Size Distribution by Fl FFF-ICP-MS
Rod EvtTaflc PEG coated
(.7
c
Io.4
= "
ta
0.1
fn
"H It/tiff °^.
^**^
u
0.7
u!
E
°J| Thick ZnS shell
OL3
».! Large Zn peak at early
0.1 time
Possible exolanation:Zn
o sw 6W MO izw isoo associated with free
lime.scc polymer
No dissolved Zn detected in
a 3K Dalton filtrate
Dissolved metals (mg/L) and % dissolved metals for a 7.5 nmol/L
QD solution
2.5 i
3 2-
g
§ '
U 0.5 •
0 -
Ohrs|mg/L)
48hrs (mg/L)
61%
Cd
86%
Zn
Green MU A
BDL
0.222
0.014
0.044
33%
• Ohrs
• 48 hrs
14%
Cd Zn Cd Zn Cd
7%
Zn
Red MU A Green PEO Red PEO
BDL 0.006 BDL BDL BDL
2.3 0.083 BDL BDL BDL
0.013
0.045
MUANPs release Cd to
solution.
"Excess" Cd source of
toxicity?
Are we testing CeSe
core bioavailablity or
so me other form?
PEO NP stable during
test
Appears to be a size
affect when comparing
mass dose
No Size effect when
comparing number
dose
15 20 25
:entration (nmolfL)
Summary
RTSP-ICP-MS can be used to:
• Detect NPAg at environmentally relevant concentrations
(ppt levels)
•High specificity (contrast to DLS)
• Distinguish between "dissolved" and NPAg
•Potential for application in stability and exposure/toxicity
laboratory studies
• Current limitations
• About 40 nm size limit
•Cannot identify NP type
FFF-ICP-MS can be used to:
• More fully characterize complex NPs
• Provide information to interpret results of experiments where:
• Mixtures are used
• Manufacturing impurities are present
• Transformation/ degredation products are present
83
-------
Acknowledgments
Collaborators
Dr. Anthony Bednar: USAGE
Dr. Nicola Rodgers: CSIRO
Dr. Antonio Nogueira: U. of Aveiro
Dr. Brian Jackson: Dartmouth College
Funding
US Environmental Protection
Agency
US Army Corps of Engineers
Students
E. Lesher, D. Mitrano.J. Monserud, H. Pace
Unexpected metal ratios
Cor
Mem! A/o/*- Rat
a
PEO t
a
Mi: A 1
* Red
o Cd'Sc Zn/Cii
2. 1 1.-1
3.2 U
2J 0-14
22 0.14
Orcco
Cd'Sc ZtL/Cd
1.3 5.6
ND 5.6
ND 0.23
1 1 0.23
a. ICP-A iS: Q1J in hard walcr
b. ICP-AES: QD in DI water
•Cd:Se ratio not 1:1
• Excess Cd in MUA QDs, especially high for red MUA QDs
Sizing of Core by UV-Vis
Polar, non-ionic
Anionic (carboxyl functionality)
500 550 600
Wavelength (nm)
84
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Jingyu Liu
Controlled Release of Biologically Active Silver from Nanosilver Surfaces
Jinsvu Liu1, David Sonshine2, Saira Shervani2, and Robert Hurf'3
Department of Chemistry, 2Division of Engineering, 3Institute for Molecular andNanoscale Innovation,
Brown University, Providence, RI
Major pathways in the antibacterial activity and eukaryotic toxicity of nano-silver involve the silver cation
and its soluble complexes, which are well-established thiol toxicants. Through these pathways, nano-silver
behaves in analogy to a drug delivery system, in which the particle contains a concentrated inventory of an
active species, the ion, which is transported to and released near biological target sites. Although the
importance of silver ion in the biological response to nano-silver is widely recognized, the drug delivery
paradigm has not been well developed for this system, and there is significant potential to improve nano-silver
technologies through controlled release formulations. This work applies the drug delivery paradigm to nano-
silver dissolution and presents a systematic study of chemical concepts for controlled release. After presenting
thermodynamic calculations of silver species partitioning in biological media, the rates of oxidative silver
dissolution are measured for nanoparticles and macroscopic foils and used to derive unified area-based release
kinetics. A variety of competing chemical approaches are demonstrated for controlling the ion release rate over
four orders of magnitude. Release can be systematically slowed by thiol and citrate ligand binding, formation
of sulfidic coatings, or the scavenging of peroxy-intermediates. Release can be accelerated by pre-oxidation or
particle size reduction, while polymer coatings with complexation sites alter the release profile by storing and
releasing inventories of surface-bound silver. Finally, the ability to tune biological activity is demonstrated
through bacterial inhibition zone assay carried out on selected formulations of controlled release nano-silver.
EPA Grant Number: R833862
The Office of Research and Development's National Center for Environmental Research 85
-------
Controlled Release of Biologically Active Silver
from Nanosilver Surfaces
Jingyu Liu, David A. Sonshine, Saira Shervani, Robert H. Hurt
Department of Chemistry, Division of Engineering,
Institute for Molecular and Nanoscale Innovation
Brown University, Providence, Rl
-, :•• .
Nanosilver- Two Faces of Janus
A new generation of antimicrobials
Silver is a broad spectrum antibiotic
* has relatively lowtoxicity in humans;
* is being manufactured in large quantities and incorporated into
consumer and medical products.
A risk to the environment and human health?
Silver is a known toxicant to aquatic organisms
* is more toxic than any other metal except mercury;
* bioaccumulates quickly;
* nanosilver has toxicity threshold as low as 10 ng/L (zebrafish embryos).
Silver has potential toxic effects on beneficial bacteria in soil
laipiiln] fti Itrfiari ^
Nanosilver in Biological and Environmental Systems
- Is It the Particle or the Ion?
:• Metal ions may coexist in metal-containing nanoparticle suspensions.
:• Silver ion is a known toxicant that binds to thiol groups in enzymes, such
as NADH dehydrogenase, which disrupts the bacterial respiratory chain
generating ROS that can lead to oxidative stress and cell damage.
:- Nanosilver particles themselves may also contribute by binding to or
passing through cell membranes, and generating ROS through surface
reactions.
:•• There is some controversy about the role of particle-based mechanisms,
but there is broad agreement that silver ion is an important toxicant.
What determines particle/ion partitioning?
Ion Release Kinetics and Particle Persistence
in Aqueous Nano-Silver Colloids
Liu J.; Hurt R.H. Environ. Sci. Technol. 2010, 44, 2169-2175
O2, H+
This reaction produces
r Ag+(slow) active peroxide intermediates
Is inhibited by
H2O2, -O2- natural organic matter
Leads to complete particle dissolution in
aerobic environments
Imtttirt* (DI MotecuUr •
"Controlled Release" Nanosilver - application of the drug delivery paradigm
Can we systematically increase
or decrease ion release rate?
Can we engineer nanosilver materials
for optimal ion release profile?
Specific benefits of controlled release nanosilver formulations might include:
(i) dose control to achieve desired bactericidal or bacteriostatic effects;
(ii) dose limitation to avoid eukaryotic toxicity that can, for example, slow wound
healing in bandage applications;
(iii) control of product lifetime, before dissolution and diffusion end antibacterial activity;
(iv) minimization of environmental release through excess ion production beyond that
necessary for product performance;
(v) optimization of release profile for targeted delivery to specific tissue or
intracellular targets.
Particle-Ion Partitioning
in Aqueous nAg Colloids
Basic Experiment
Ultrafiltration + Atomic absorption
nAg Amlcon cellulose membrane
Ag+ 3K Da, (1-2 nm pore size)
Separation ^^^"--' Quantification
-------
If nano-silver oxidatively dissolves, why doesn't bulk silver?
Incubation time (li| incubation time (h)
Answer: it does! (but slowly - about 2 nm/day)
Speciation in Biological Media - Effect of Chloride and Thiol
(By Visual MINTEQ)
A
I-
III Ml 01
Total Ihlollnpul(mM)
Liu, Sonshine, Shervani, Hurt,
"Controlled Release of Biologically Active Silver from
Nanosilver Surfaces" ACS Nano, in press
Nanosilver Behavior in Biological Environments
- Nanosilver vs. Silver Salts
V.IMNI
Controlled Release Approaches: Coatings and Ligands
Original nAg
ft t «
Irscubali on lime (Ci)
Controlled Release Approaches: Pre-Oxidation
Two-Stage (fast/slow) Release , •
•/ IMNI
Irtcubaiion lime |h)
Controlled Release Approaches: Media Composition
Total silver (2 mg/L)
Acetate
buffer
nAg
/1E-4 1E-J C.
-------
Comparison of Surface Treatment Methods for Release Control
I
Biological Response to
Controlled Release nAg
£. co//; ?S hr incubation
With 10 mm nAg-doped fitter papers
Experimental assistance from Dr. Charles Vaslet, Kane laboratory
Future work - Biological and Environmental Implications
of Ion Release Kinetics and Control
Hurt lab - dissolution kinetics and controlled release formulations
How can the transformations of nAg be engineered and controlled?
Role of sulfides and the role of photochemistry
Pennell - coupled reactive dissolution kinetics laws with environmental material flow modeling
What are the ultimate amount, fate, and form of nAg in the environment?
Kane lab - uptake and biodistribution in Xenopus
What is the fate and form of nAg
in whole biological organisms?
Approaches for Controlling Biologically Active Silver
Release from Nanosilver Surfaces
Liu, Sonshme, Shervani, Hurt
-"•CS 'Vsra. :n D'fi^f.
Financial support from the US EPA Science to Achieve Results
Program and the NIEHS Superfund Research Program grant at
Brown is gratefully acknowledged
The Laboratory for Environmental and Health Nanoscience
At Brown University
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Roger A. Pinto
Effects of Polyethyleneimine Surface Modifications of Multi-Walled Carbon
Nanotubes: Their Toxicity, Sorption Behaviors, and Ecological Uptake by
Earthworms and Daphnia magna
Roger Pinto , Elijah Petersen , and Walter Weber, Jr.
Department of Chemical Engineering, University of Michigan, Ann Arbor, MI; Chemical Science and
Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD
In support of the mission of the U.S. Environmental Protection Agency to provide substantive information
for ecological risk assessments, this research focuses on investigation of the environmental fate,
bioaccumulation potential, and toxicity of surface-modified carbon nanotubes (CNTs) in terrestrial and aquatic
ecosystems. Carbon-14 multi-walled carbon nanotubes (MWNTs) were synthesized by a CVD process, grafted
with polyethyleneimine (PEI) polymers, and further modified to render them with a range of different surface
charges and resultant higher stability in aqueous suspensions. Assessments of the extent to which these
modifications influence CNT ecotoxicity, accumulation, and elimination behaviors were performed using the
earthworm Eisenia foetida and the water flea, Daphnia magna.
Liquid scintillation counting of residual 14C in vivo provided insights on the uptake and elimination
behaviors for the organisms tested. D. magna exposed to PEI-coated and acid-modified MWNTs at
concentrations of approximately 25 or 250 ^g/L indicated that the PEI surface coatings did not appear to
substantially impact nanotube accumulation or elimination rates. Microscopy observations revealed substantial
aggregation in the guts of D. magna similarly to previous studies with acid-treated MWNTs and fullerenes.
Algae feeding to Daphnia was necessary to achieve almost complete elimination in 48 h, whereas the absence
of algal amendments caused minimal CNT elimination. Immobilization studies allowed for the determination
of EC50 values and indicated that PEI modifications increased MWNT acute toxicities, though this trend
corresponded to the overall size of the grafted polymers.
Phase distribution experiments with soils measuring a combined effect of sorption and attachment to
particles indicated linear sorption isotherms for the regular MWNTs and non-linear trends for the PEI-
modified MWNTs. Differences in uptake behaviors by earthworms were not apparent among the different
types of PEI-modified and MWNTs, results that indicated limited interaction of these carbon nanotubes with
the organism tissues. In contrast to previous results for unmodified MWNTs, elimination patterns for the
grafted PEI-MWNTs were well fit by an exponential decay. To determine whether earthworm exposure to
these MWNTs elicits a stress response, two biomarkers of oxidative stress (glutathione-S-transferase (GST),
catalase) and two biomarkers of neurological stress (monoamine oxidase, cholinesterase) were measured in
whole-body samples. A dose-response relationship was not observed within the concentration range of the
exposure treatments (3-1,000 mg MWNT kg" soil). However, positively (PEI-amino) and neutrally (PEI-
acetate) charged nanotubes consistently revealed a toxic response with catalase, monoamine oxidase, and
cholinesterase, while negatively charged CNTs (PEI-succinic and acid-treated MWNTs) had no effect.
EPA Grant Number: RD-833321
The Office of Research and Development's National Center for Environmental Research 89
-------
Principal investigator did not authorize publication of the presentation.
90
-------
PM Session 1: Characterization Methods
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Xin-Rui Xia
A Biological Surface Adsorption Index for Characterizing Nanomaterials
in Aquatic Environments and Their Correlation With Skin Absorption
of Nanomaterials
Xin-Rui Xia, Nancy Monteiro-Riviere, and Jim Riviere
North Carolina State University, Raleigh, NC
As nanoparticles are increasingly being used in commercial products, it becomes more and more important
to understand how they interact with living organisms and the environment. The behavior of nanomaterials in a
biological or environmental system is governed by the molecular interactions of their surface species with the
biological or environmental components. Quantitative assessment of the adsorption properties of nanomaterials
is a crucial step for developing predictive structure-activity relationship in nanomedicine and risk assessment
of nanomaterials.
We have developed a biological surface adsorption index (BSAI) approach to characterize the surface
activity of nanomaterials in biological systems. A set of small molecules having diverse physicochemical
properties was used as probe compounds. The adsorption coefficients (k) of the probe compounds were
obtained by measuring the quantities of the probe compounds adsorbed on the surfaces of the nanomaterials
and the equilibrium concentrations of the probe compounds in the media. The log (k) values were scaled to a
set of solvation molecular descriptors of the probe compounds via multiple linear regressions to provide a set
of five nano-descriptors representing the contributions of the five types of molecular interactions
(hydrophobicity, hydrogen-bond acidity and basicity, dipolarity/polarizability, and lone pair electrons). The
nano-descriptors for multi-walled carbon nanomaterials (MWCNT) with different surface chemistries
(unmodified, -OH and -COOH modified) and fullerenes were measured; for example, the regression model
obtained for MWCNT (-OH modified) was log(£) = 0.77^ + 2.55n - 0.14a - 2.36(3 + 4.90F; n = 30, R2 = 0.89.
The measured nano-descriptors can be used to develop predictive structure-activity relationships in
nanomedicine and nanomaterial risk assessments.
We have prepared a series of hydroxylated fullerenes, C6o(OH)x. Their hydrophobicity was adjusted by
controlling the number of hydroxyl groups (e.g., x = 16, 20, 30, 40). After characterization using conventional
techniques such as particle size, zeta-potential and solubility, the nano-descriptors were measured using the
BSAI approach for each of the C6o(OH)x nanomaterials. Then, the adsorption coefficients of different
C6o(OH)x into the stratum corneum, the primary barrier of skin, were measured by in vitro adsorption
experiments. The equilibrium adsorption coefficients were correlated with the physicochemical parameters and
the nano-descriptors to establish quantitative correlations.
The following findings will benefit EPA: (1) The BSAI approach measures five molecular descriptors for
each of the nanomaterials. The BSA indexes are free energy-related physicochemical parameters that can be
used for predictive models developments in EPA guidelines for environmental health of nanomaterials; (2) The
hydroxylated fullerenes with different hydrophobicity can be used to study the environmental transport and
fate of fullerene nanomaterials; (3) The quantitative approach to correlate the adsorption coefficients of
stratum corneum with the BSAI nano-descriptors of the C6o(OH)x nanomaterials could be a useful approach
for developing predictive models for safety evaluation and risk assessment of nanomaterials.
EPA Grant Number: R833328
The Office of Research and Development's National Center for Environmental Research 92
-------
gical Surface Adsorption Index
acterizing Nanomaterials in Aauatic
aqueous environments
Adsorption coefficients (logk) and solute descriptors of the probe compoun'
93
-------
94
-------
Absorption of Nanomatenals
3l method to prepare nC60 nanoparticle with a narrow size
distribution. This method does not use THF while provides nC60 concentration in
water 100 times higher than the THF method. The nC60 nanoparticles are formed in
a SDS aqueous solution, then SDS is removed via dialysis. After exhaustive
dialysis, the nC60 nanoparticles were stable in water for years.
After 26 Tape-Strips
alyzed using the improved sLLE-
HPLC method.
Nanomaterial residues were detected
in the skin tissues even after 26
tape-strips.
The amount of fullerene residues
was greater when dosed in
chloroform than dosed in toluene or
cyclohexane.
No C60 was detected it „
tissues when dosed in mineral oil;
this is consistent \
jllerene residues in skin tissues
95
-------
Further Research Needed
Acknowledgements
96
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Wunmi Sadik
Flexible Nanostructured Conducting Poly(amic) Acid Membrane Captures,
Isolates, and Simultaneously Detects Engineered Nanoparticles
Wunmi Sadik1, Nian Du1, Michael Feurstein1, Veronica Okello1, Cheuk Wong2, and Howard Wang2
Center for Advanced Sensors and Environmental Systems (CASE), Department of Chemistry;
Department of Mechanical Engineering and Materials Science Flagrant,
State University of New York-Binghamton, Binghamton, NY
The goal of this project is to develop nanocavity sensor (category II) arrays for the isolation, detection and
quantitation of engineered nanoparticles (ENPs) in complex environmental matrices. There is urgent demand
for rapid screening methods to isolate, detect, and monitor engineered nanomaterials in the environment.
Conventional methods for characterizing nanomaterials such as transmission electron microscopy, scanning
electron microscopy, and atomic force microscopy tend to be bulky and inadequate for field and rapid
screening of free nanomaterials.1 At SUNY-Binghamton, we have developed a new class of nanostructured
poly(amic acid) -PAA-membranes that are conductive and electroactive by preventing its imidization to
polyimide, while retaining its carboxylic acid and amine functionalities.2"4 We have studied the effect of
composition and micro structure on the optical and electrochemical properties of PAA hybrid composites. The
uniqueness of PAA lies in its excellent physical and chemical properties: transparency, flexibility, electrical
conductivity, and accessibility to forming a large-area device. During the reporting period, our group
discovered that this new class of flexible, stand-alone membranes could be successfully used as both sensors
and nanofilters. A new nanofilters device based on PAA membranes is hereby introduced. The nanofilters were
derived from phase-inverted, copolymers of PAA and other polymers, with the surface and pore sizes
systematically controlled by varying the conditions of the synthesis. This presentation will focus on the use of
PAA membranes for simultaneous removal and electrochemical detection of silver nanoparticles, quantum
dots, and titanium dioxide nanocrystal from food supplements and environmental samples.
References:
1. Sadik OA, Zhou AL, Kikandi S, Du N, Wang Q. Sensors as tools for quantitation, nanotoxicity and
nanomonitoring assessment of engineered nanomaterials, Journal of Environmental Monitoring (Critical
Review) 2009;! 1:1782-1800.
2. Du N, Wong C, Feurstein M, Sadik 0, Umbach C, Sammakia B. Flexible conducting polymers: effects of
chemical composition on structural, electrochemical and mechanical properties. Langmuir 2010. DOI:
10.102 l/la!01314j
3. Andreescu D, Wanekaya A, Sadik OA, Wang J. Nanostructured polyamic membranes as electrode
material. Langmuir 2005;21(15):6891-6899.
4. Breimer M, Yevgheny E, Sadik OA. Incorporation of metal particles in polymerized organic conducting
polymers - a mechanistic insight. Nano Letters 2001;1(6):305.
EPA Grant Number: R834091
The Office of Research and Development's National Center for Environmental Research 97
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Principal investigator did not authorize publication of the presentation.
98
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2010 U.S. EPA Nanotechnology Grantees Meeting
Jason Unrine
Fate and Effects of Nanosized Metal Particles Examined Along
a Simulated Terrestrial Food Chain Using Genomic
and Microspectroscopic Techniques
Jason Unrine, Aaron-Shoults Wilson, Olga Tsyusko, Sarita Hardas, D. Allan Butterfield, and Paul Bertsch
Department of Plant and Soil Science, University of Kentucky, Lexington, KY
Terrestrial environments are likely to serve as the ultimate sink for a significant fraction of manufactured
nanomaterials (MNM) from accidental releases, use in agriculture, and through land application of sewage
sludge as biosolids. Risk from exposure to MNM in terrestrial food webs partly depends on their propensity for
uptake and retention by detritivorous soil organisms and subsequent trophic transfer to higher trophic levels as
well as inherent toxicity. Our research is investigating interactions between chemical composition and particle
size in determining bioavailability and adverse effects of Cu, Ag, and Au nanoparticles (NPs). Our results
indicate that uptake of nanoparticles from soil by earthworms does not vary systematically with primary
particle size on a mass concentration basis; however, on a number concentration basis, smaller particles are
much more bioavailable than larger ones. Also, we have found that nature of surface coating (PVP versus oleic
acid) has little effect on Ag uptake. It is clear that the redox behavior of metal NPs in soil varies considerably.
Although Cu NPs oxidize immediately upon exposure to the air, Au NPs are completely stable and resistant to
oxidation. Ag NPs are resistant to oxidation in the air, but they can be readily transformed in natural soil. We
have confirmed that either reduced or oxidized metal NPs can be absorbed from soil and taken up into internal
tissues using a combination of X-ray microspectroscopy, laser ablation-inductively coupled plasma mass
spectrometry, asymmetrical field flow fractionation-multidetection (AF4), and expression of the metal specific
gene, metallothionein. We have observed decreased reproductive success associated with exposure to Au and
Ag NPs as well as evidence of soil avoidance for Ag NPs. We also have obtained evidence for oxidative
damage of proteins as a result of exposure to Ag ions and Ag NPs. In our toxicity tests, Au and Ag NPs were
significantly less toxic than their corresponding metal salts; however, NPs, including oxidized NPs, may be
bioavailable and cause adverse effects at relatively high concentrations. Behavioral avoidance of Ag NPs is the
most sensitive endpoint investigated to date and occurs at concentrations of Ag NPs that are similar to those
expected in sewage sludge. The next phase of our research investigated trophic transfer of NPs along a
simulated food chain consisting of soil, earthworms, and bullfrogs. We obtained evidence that Au NPs can be
transmitted from soil to earthworms to bullfrogs, although the rate of transfer is somewhat limited. It is
important for future studies to investigate how aging processes influence the stability and surface chemistry of
metal NPs over longer time periods and how this impacts toxicity.
This research is likely to be of great benefit to the U.S. Environmental Protection Agency, which is
charged with regulating the land application of biosolids and pesticides. The results will be beneficial for
making predictions about how chemical composition and particle size relate to biogeochemical transformations
of NPs in soil and NP bioavailability as well as providing information on potential adverse effects on soil
invertebrates and by extension ecosystem functions. These predictions will be necessary components of
ecological risk assessments for MNMs and for deriving models that predict MNM behavior in the environment
based on physicochemical properties.
EPA Grant Number: R833335
The Office of Research and Development's National Center for Environmental Research 99
-------
Bioavailability and Toxicity of Nanosized Metal
Particles Along a Simulated Terrestrial Food Chain
Pis: Jason Unrine1, Olga Tsyusko1, Paul Bertsch1, Andrew Neal2
Postdocs: W.Aaron Shoults-Wilson1,Simona Hunyadi13
Undergraduate Students: Alison Willis4, OksanaZurbich5
Collaborators: Brian Reinsch6, Greg Lowry6
1 .University of Kentucky Department of Plant and Soil Sciences, Lexington, KY
2.Rothamsted Research Harpenden, UK.
3.Savannah River National Laboratory Aiken, SC
^.Toxicology Excellence for Risk Assessment, Cincinnati, OH
S.University of Kentucky Department of Chemistry, Lexington, KY
e.Carengie Mellon University, Dept. of Civil and Environmental Engineering, Pittsburgh, PA
UK
KENTUCKY
Fate, transport, and, effects of manufactured
nanoparticles in the environment
HIGHER TROPHIC LEVELS
s omnivores herbivore
Half
Reaction
Particle
Size
n -a
** 5,
Eisenia fetida
semi-model organism
Important soil toxicity testing model
OECD/EPA test media (7O% quartz, 2O %
kaolin, 1O % sphagnum peat)
Natural sandy loam
Using Au N Ps as a probe for particle uptake-LA-ICPMS
Animals
exposed to 25
mg kg-1 HAuCI4
or 50 mg kg-1
Au NPs in OECD
soil media for
28 d.
Unrine et al., 2010, ES&T
al., in press
ES&T
100
-------
Trophic transfer of Au NPs
B
1 X
2 X
3 X
4 X
5 X
X
14 X
Treatment 1 = Control Gavage, Control worm
Treatment 2 = Au Gavage, Control Worm
Treatment 3 = Control Gavage, Control Worm
• Gavage dose adjusted each time to
maintain similar cumulative dose
as earthworm dose
Earthworms
Accumulation of Au NPs in Tissues -
Effect of source on bioavailabilH
Accumulation in liver
Alternative hypotheses
Once particles enter the earthworm tissues,
they acquire a protein corona and thus
become more bioavailable
Earthworms absorb only the most bioavailable
particles from the total population of
particles, thus enriching the transferable
fraction (analogous to trophic enrichment of
methylmercury).
Ag nanoparticles -size and coating
1 Particle Diameter (nm)
35.23 ±0.81
56.35 ±1.16
50.60 ±1.02
27.37 ±0.36
1 Name pH™ CEC
Yaeger
Sandy 5.17 9.18
OECD 7.00 14.45
Coating
PVP
PVP
Oleic acid
Citrate
Soils
Sand Silt
76.34% 16.53%
79.12% 6.71%
Properties ^^1
Hydrophilic
Hydrophilic
Amphiphilic
Hydrophilic
Clay OM 1
7.13% 1.77%
14.17% 7.65%
101
-------
«*')
Stored in
glovebox
ls'X'v-v\A/\/M
„- • ^,0.11(4)11
UK
28 d in artificial soil
medium
28 d in sandy loam
Shoults-Wilson et al. in pres
7.0 -I
E
O 6.0 •
~£ 50-.
8 •
3 "•<)_:
|
ja
E
3 2.0 •
z
£
3 i.o •
i
Ir "
ft
1 *
1
f
»AgN03 t ,
QlOnmAgNP(PVP) 5 *
A30-50nmAgNP(PVP) , !•
O 30-50nm AgNP (Oleic Acid) I
— Control T
10 100 1000
[Ag] in soil* (mg kg'1)
Shoults-Wilson et al. in press, SSSAJ
UK .0ECD,r.,,,c,,,,o,,medl,
' *•
10 nm
PVPAg
Nps
30-50
nm PVP
Ag Nps
Ag ions
protein carbouyl
Hardas, Butterfield
et al., in progress
Possible mecha
Agnp
HspTO »t
Hsp60 *
Ubq •«— Pfotei"
dysfunction
t
Protein
t r
t H202 <
nisms of toxicity
of lipid
*1, peroxidation
*| (HNE)
Cat transcription
factors
of nitrosative
stress
Cat j (4-NT)
102
-------
Avoidance of Ag NPs
Sensitive
- Initial avoidance
— Final avoidance
• 48 h
High ecological
relevance
o o
o o o o
O o O °
Shoults-Wilson et al. in review
Initial Response
I -,
Final Response
3
Shoutts-Wilson et al. in review
Avoidance at 10 mg kg"1
Shoults-Wilson et al. in
Conclusions
• Nanoparticle are bioavailable from soil and can
be transferred to higher trophic levels.
• Particle size and redox properties are important
for uptake and toxicity.
• Ag particles cause a variety of adverse effects in
earthworms translating from the molecular level
up to the population level, some at
concentrations similar to those expected in
sewage sludge.
• Environmental variables are probably more
important than particle variables for Ag toxicity.
Publications
103
-------
CEIN
• Antonio Lanzirotti -U. Chicago/NSLS
• William Rao -UKY/NSLS
• Melissa Lacey-UKY
• Jonathan Judy -UKY
• Greg Joice - UKY
• Diane Addis —Medical College of Ge-'-r^in
• Ellen Harding - Transylvania Univ>-r~: s>
• The Kim Lab- Chapman University
• Sam Webb, John Bargar, Joe Rogers -SSRL
104
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2010 U.S. EPA Nanotechnology Grantees Meeting
John J. Rowe
Determination of Manufactured Nanoparticle Toxicity Using Novel Rapid
Screening Methods
John Rowe , Saber Hussain , Rajender Varma , Ryan Posgai, Caitlin-Cipolla McCulloch , Tim Gorey,
and Mark Nielsen1
Department of Biology, University of Dayton, Dayton, OH; 2Biosciences and Performance Division Human
Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Dayton, OH; 3Clean
Processes Branch, Sustainable Technology Division, National Risk Management Research Laboratory,
U.S. Environmental Protection Agency, Cincinnati, OH
We have developed a first tier rapid screening system to determine the toxicity of nanoparticles (NPs) that
includes a broad spectrum of organisms, including plants, bacteria, tissue culture, and the fruit fly. The
individual organismal (Posgai et al., 2009; Ahamed et al., 2010) and in vitro (Ahamed et al., 2008) models
have been used to address not only toxicity but also the molecular mechanisms behind toxicity. We will focus
in this presentation on Ag NPs and the correlation of surface properties with toxicity. Special emphasis will be
placed on the effects of sublethal concentrations on development and reproduction in the fruit fly. The results
will be prefaced with a brief review of our earlier molecular findings implicating excess ROS production after
exposure to Ag NPs (Ahamed et al., 2008, 2009; Posgai et al., 2009).
Fruit flies provide a powerful model for investigating human health and nanotoxicity. Counterparts of
genes responsible for more than 700 different human genetic diseases, including neurological, immunological,
cardiovascular, auditory, visual, developmental and metabolic disorders, are found in Drosophila (Koh et al.
2006; Wolf et al., 2006; Khurana et al., 2006; Rieter et al., 2001; Sykiotis and Bohmann, 2008). Flies are
particularly amenable to investigations of chronic exposure health effects and ecotoxicology, two particularly
understudied aspects of NP toxicology. Invertebrates lie at the bottom of food webs and thus are likely to
interact with and potentially bioaccumulate environmental NP pollution. Their cost-effectiveness, experimental
flexibility, and short generation time permit rapid assessment of the vast number of NPs being produced,
including chronic, reproductive, and genotoxic effects less accessible in mammalian systems.
In generating a fly NP toxicity model, we first developed models for different uptake modes (ingestion,
Ahamed et al., 2010; inhalation, Posgai et al., 2009). Herein, we report long-term chronic exposure effects.
Survival (LD50), developmental rate, reproductive effort, gene expression, and cell physiology (Ahamed et al.,
2010) will be assessed, with fully characterized particles generated using different coatings, dispersants and
agglomeration states, and particle sizes. Co-exposure with anti-oxidants, phenocopying NP toxicity with
known oxidants, and tests in mutant fly backgrounds will be used to experimentally dissect mechanisms of NP
toxicity.
The results of our ingestion studies with Ag NPs demonstrate very clear toxic effects on viability,
development, and reproduction at levels as low as 10^g/mL. At sublethal concentrations, development was
retarded and pupation rate significantly lower than the control. There were also clear differences in phenotype,
especially size and coloring at all stages of development. The effects of Ag NPs on development was not
reversed by vitamin E but was almost completely reversed by vitamin C.
References:
Ahamed M, Karns M, Goodson M, Rowe J, Hussain SM, Schlager JJ, Hong Y. DNA damage response to
different surface chemistry of silver nanoparticles in mammalian cells. Toxicology and Applied Pharmacology
2008;233:404-410.
The Office of Research and Development's National Center for Environmental Research 105
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Ahamed M, Posgai,R, Gorey T, Nielsen MG, Hussain SM, Rowe JJ. Silver nanoparticles induced heat shock
protein 70, oxidative stress and apoptosis in Drosophila melanogaster. Toxicology and Applied Pharmacology
2010;242:263-269.
Koh K, Evans JM, Hendricks JC, Sehgal A. A Drosophila model for age-associated changes in sleep:wake
cycles. Proceedings of the National Academies of Science USA 2006;103:1383-1384.
Khurana V, Lu Y, Steinhilb ML, Oldham S, Shulman JM, Feany MB. TOR-mediated cell-cycle activation
causes neurodegeneration in ^Drosophila tauopathy model. Current Biology 2006;16:230-241.
Posgai R, Ahamed M, Hussain SM, Rowe JJ, Nielsen MG. Inhalation method for delivery of nanoparticles to
the Drosophila respiratory system for toxicity testing. Science of the Total Environment 2009;408:439-443.
Reiter LT, Potocki L, Chien S, Gribskov M, Bier E. A systematic analysis of human disease-associated gene
sequences in Drosophila melanogaster. Genome Research 2001; 11:114-1125.
Sykiotis GP, Bohmann D. Keapl/Nrf2 signaling re3gulates oxidative stress tolerance and lifespan in
Drosophila. Developmental Cell 2008;14:76-85.
Wolf M, Amrein H, Izatt JA, Choma MA, Reedy MC, Rockman HA. (2006) Drosophila as a model for the
identification of genes causing adult human heart disease. Proceedings of the 'National Academies of Science
f/&4 2006; 103:1394-1399.
EPA Grant Number (vis NSF): CBET-0833953
The Office of Research and Development's National Center for Environmental Research 106
-------
Model Systems for Rapid
Assessment of Long and Short Term
Effects of Nanomaterials on Biological
Systems
John Rowe, Ph.D.
Department of Biology
University of Dayton
UD/AFRL Nanotoxicitv Research Group
John Rowe Ph.D., Jayne Robinson Ph.D., Mark Nielsen Ph.D.,
Saber Hussain Ph.D., Maqusood Ahamed, Ph.D., Tracy Collins
Ph.D., Ryan Posgai, Brittany Demmitt, Caitlin Cipolla-McCulloch,
Timothy Gorey, Kyle Murphy
UD/AFRL Nanotoxicity Group
Multi-Domain approach
Standardized approach
Coupled in vitro/in vivo
models
- Long term studies
- Reproductive effects
- Development
- Multi-dimensional
assays
Life history toxicity effects and vitamin C
reversal: a novel in vivo Drosophila model
for chronic nanoparticle exposure
Department of Biology
University of Dayton
WPAFB/AFRL
Drosophila melanogaster. Life Cycle
Overall Objective:
Establish D. melanogaster as a model system for rapid assessment
of nanoparticle toxicity, in vivo
Current Project Objective:
• Study the effects of nanoparticle ingestion on D. melanogaster
growth and development
Method:
Supplement fly food with NPs
Assay for:
Allow fly larvae to feed
on the N Placed food
1) Survivorship
2) Development
3) Fecundity
4) Mechanism(s) of
toxicity
NP Parameters Investigated
NP behavior is function of:
> size
> shape
> surface reactivity
• Compare the effects of different sizes and
coating of NPs on Drosophila development
and reproduction
- Uncoated or polysaccharide coated
107
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Silver Nanoparticles (Ag NPs)
Gift: Dr. Dan Goia, Center for Advanced materials,
Clarkson University
Size: 10 nm, 60 nm Shape: Almost Spherical
Surface Coating/ Stabilizing Agent: Polysaccharide
(Starch) The 10 nm coated Ag NPs were synthesized by
the reduction of silver ions in a solution of a
polysaccharide (acacia gum), which leads to surface
coating.
Transmission Electron Microscopy Characterization
s
4.5» (n=l»SJ
5.00-10.0 >10.0-15.0 >15.0-20.0 >20.0
Dynamic Light Scattering Characterization
DLSMean=48
-
Zeta Potential = -38.6
Quantification of NP effect on
Drosophila larvae
50
Uncoated 20nm Titanium Oxide (ug/mL)
•% Survivorship OAvgTime Until Pupation (hours)
200
if! -=
30 •
0 10 IS 20 25 30 35 40
UiiLi--aLKU lOrllil S live I (ijyrllL'l
•% 6«ivivoRiiip *Avy. Time Until Pupalkm thoursj
Utnfr.
0 10 15 20 25 30 35 50
Coaled 10inn Silv
_
1C 20 30 W 50 60 75 00 W 105
d COnm Silver tH9<'<"L]
SSSRSSSS2ESS8S
Coated 60iKii Silver (pg.'DhL)
% Sui-vworjhip »Av3. Tune Uirtil Puputioti (hwus)
108
-------
Mechanism of NP Toxicity
NPs have been shown to increase ROS
- may result in oxidative stress, inflammation,
and consequent damage to proteins,
membranes and DMA
We tested whether oxidative stress occurs
in vivo using our model system
Determined effect of treatment with
ascorbic acid (Vitamin C)
- Protector against oxidative stress
Oxidative Stress
•DEFINITION:
Oxidative stress occurs when generation of reactive oxygen species (ROS)
exceeds the capacity of antioxidant defense mechanisms of cells.
•LIPID PEROXIDATION (LPO):
The process whereby ROS "steal" electrons from the lipids in our cell
membranes, resulting in cell damage and increased production of ROS
•REACTIVE OXYGEN SPECIES (ROS):
Superoxide ion: O2'
Hydroxyl radical: OH'
Hydrogen peroxide H202
•ANTIOXIDANT DEFENSE MECHANISM
Pathway that provide protection against harmful effects of ROS.
Antioxidant Molecule: e.g. Glutathione (GSH)
Antioxidant Enzymes: e.g. Superoxide dismutase (SOD) and Catalase (CAT)
Ag NPs Enhanced Membrane Lipid Peroxidation
Malondialdehyde (MDA), an end product of lipid peroxidation
was quantified to see the extend of membrane lipid
peroxidation
Data represented are
Control 50|jg/ml lOOugM
^^H AgNPs lOnm
)an±SD (n = 3). Significance is ascribed as "p < 0.05 vs. contrc
Ag NPs Induced Superoxide Dismutase (SOD) Activity
SOD CM
2 + °z H,0,
NPs Induced Catalase (CAT) Activity and
Depletes Glutathione (GSH) Content
^Wt^^i AgNP,10m
sented are mean±SD (n = 3). Significance is ascribed as fp < 0.05 vs. control
! oip')pio-,if. 1:1 I.S'UK'PIl:!.:!
% Survivors
1 1
m
n
;nt
ilver (Ag) Uncoated
*Avg. Time Until Pupation (hours)
109
-------
Protection by Vitamin Cfrom Reproductive
Effects of Uncoated 60 nm Silver Ingestion
Oxidative Stress as Mechanism of Silver NP
Toxicity?
Ag NPs Induced Oxidative Stress
Impaired Oxidant/Antioxidant Status I
1
Summary
Conclusions
• Established in vivo D. melanogaster model for
studying NP toxicity
• Demonstrated
- Induction of oxidative stress by silver NPs
- Protective effect of Vitamin C treatment
Future Directions
Elucidate pathway of oxidative stress involved
Evaluate efficacy of an array of antioxidants
UD/AFRL
Nanotoxicity Research Group
John Rowe Ph.D.
Jayne Robinson Ph.D.
Mark Nielsen Ph.D.
Saber Hussain Ph.D.
Tracy Collins Postdoctoral
Maqusood Ahamed Postdoctoral
Ryan Posgai Ph.D. Candidate
Undergraduates:
Timothy Gorey
Caitlin Cipolla-McCulloch
Brittany Demmitt
Kyle Murphy
Funding: EPA STAR program via NSF CBET-0833953, Air Force Research
Laboratories, Naval Research Laboratories, and Consortium of Universities
Research Fellows Program WPAFB/AFRL
Effect of Larval Uncoated and Coated 60 nm
Silver NP Ingestion on Reproductive Success
110
-------
PM Session 2: Environmental Effects on
Nanoparticles
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Stephen J. Klaine
Influence of Natural Organic Matter on the Behavior and Bioavailability of Carbon
Nanoparticles in Aquatic Ecosystems
Stephen Klaine , Aaron Roberts , Sharmila Mukhopadhyay , G. Allen Burton , Pu-Chun Ke ,
and
E. Michael Perdue5
1Clemson University, Clemson, SC; 2University of North Texas, Denton, TX; 3 Wright State University,
Dayton, OH; 4 University of Michigan, Ann Arbor, MI; 5 Georgia Institute of Technology, Atlanta, GA
The overall goal of this research was to characterize the interaction between carbon nanoparticles and
natural organic matter (NOM) and the influence this interaction might have on nanoparticle bioavailability.
Further, our goal also was to characterize movement of these particles through an aquatic food chain. This
research is approximately 18 months into the 36-month project. We have examined the behavior of carbon
nanoparticles in solutions of natural organic matter. Suwannee River NOM was obtained from the International
Humic Substances Society. This research has utilized transmission electron microscopy, dynamic light
scattering, and infrared analysis of tubes before and after NOM adsorption. Stability of multi-walled nanotubes
is not influenced by NOM concentrations over 2 mg/L as carbon suggesting that these nanoparticles could be
stable in most surface waters. As expected, increased ionic strength decreased the stability of these
nanoparticle suspensions. Similar results were obtained with C6o and C70 fullerenes. However, single-walled
carbon nanotubes were not stable in the NOM solution.
Also, we have examined the bioavailability of surface modified carbon nanomaterials. For this research,
we conducted static renewal bioassays with the aquatic filter-feeding invertebrate, Daphnia magna.
Methodology for both rearing organisms and bioassays was as described in the EPA methods. We used
transmission electron microscopy to examine the fate of the nanotubes within the organism. Multi-walled
carbon nanotube toxicity to D. magna was not influenced by the concentration of NOM. The 96 hr LC50 value
was 2.2 ± 0.2 for concentrations of NOM ranging from 2-20 mg/L carbon. These nanotubes did not appear
to aggregate in the gut tract of the organism. Further, these nanotubes appeared to stay within the gut tract and
were ultimately eliminated when transferred to clean medium. Toxicity appeared to be due to gut tract
clogging and interference with food uptake and processing. This is an energetics effect and similar to that
which we described previously for suspended clay particles.
The maximum concentration of fullerenes that we were able to achieve was 15 mg/L in NOM solutions.
These suspensions, while stable, did not exhibit sufficient toxicity to generate an LC50 value. However, C70,
surface modified with gallic acid (a phenolic acid) was not only stable, but also acutely toxic to D. magna with
a 96 hr LC50 value 0.4 ± 0.1 mg/L. In a 21-day chronic study, the NOEC was 0.02 mg/L.
Because there was no indication that either multi-walled carbon nanotubes or fullerenes entered the D.
magna body from the gut tract, we examined small, highly fluorescent carbon dots (4 nm diameter). These
particles are very hydrophilic with a polyethylene glycol surface coating. These particles were non-toxic to D.
magna, and we were able to detect migration out of the gut tract and into the organisms.
Because one of our ultimate goals is to examine the fate of these materials in an aquatic food chain we
have begun focusing on which parameters facilitate the movement of nanoparticles accross the D. magna gut
tract.
EPA Grant Number: R834092
The Office of Research and Development's National Center for Environmental Research 112
-------
Influence of Natural Organic Matter
on the Behavior and Bioavailability
of Carbon Nanoparticles in Aquatic
Ecosystems.
Stephen J. Klaine
Clemson University
sklaine@clemson.edu
Collaborators
Aaron Roberts, University of North Texas
Sharmila Mukhopadhyay, Wright State
University
G. Allen Burton, University of Michigan
Pu-Chun Ke, Clemson University
E. Michael Perdue, Georgia Institute of
Technology
How do Water Quality Parameters such as NOM
Influence the Bioavailability of Carbon Nanoparticles ?
NOM stabilizes most carbon nanoparticle suspensions
-
A: Water
B: 100 mg/L NOM
C: 100 mg/L NOM + Cj,
D: 100 mg/L NOM + C,,
E: 100 mg/L NOM + SWNT
II.Ill
•400 mg/L
nanoparticles
• F: 100 mg/L NOM + MWNT
• G: 100 mg/L NOM + Nanocoil
• H: 100 mg/L NOM + Nanowire
**Sonicated in small quantities
for 30 min
Toxicity of Carbon Nanomaterials
(96hrLC50 values)
MWNT (NOM stabilized) 2.2 mg/L
C60 (NOM stabilized) >15 mg/L
C70 (NOM stabilized) >15 mg/L
C70-Gallic acid 0.4 mg/L
OH-SWNT (NOM stabilized) no toxicity at 2mg/L
PEG-SWNT (NOM stabilized) no toxicity at 2mg/L
Carbon dots >20 mg/L
Are Carbon Nanomaterials Absorbed
from the Intestinal Tract?
*?
113
-------
PEG Functionalized
,
PEG Functionalized
OH-SWNT
Raman Spectroscopy
KBM
*
D-band
I. II..I..I
/
SWNTs posses resonant Raman spectral features. Atypical
spectra is on the left.
We can track G-band, D-Band for mapping SWNTs in any
biological tissue.
OH-SWNTs Raman Spectroscopy
D. magna 96 hr exposure to 2 mg/L OH-SWNTs
8 um section, strong G and D Band signal within
gut tract
114
-------
OH-SWNTs Raman Spectroscopy
Carbon Dots
Emission from
pass vated surface
Optical effects due to
size
Available in range of
sizes
« 8,40, 113nm
Observations
MWNTs are acutely toxic to D. magna
• Not a function of NOM
• Appears to be a result of interfering with food processing.
- Gut tract clearance: 29 hrsfor MWNT; 30 min for clay
MWNTs are not taken up from the gut tract
Carbon dots migrate from the gut tract and
appear to be associated with organelles
OH-SWNT may migrate from the gut tract
PEG-SWNT do not migrate from the gut tract
Next Steps
Continue to examine uptake from the gut tract
— Fluorescent labeled SWNT
- Other surface modifications
Food chain studies
- Carbon nanoparticles that are bioavailable will be
13C-enriched and run through our aquatic food chain
115
-------
Acknowledgments
Aaron Edgington, Brandon Seda
U.S. Environmental Protection Agency's STAR
program
Clemson University Public Service Actvities
Clemson University Office of Research
116
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2010 U.S. EPA Nanotechnology Grantees Meeting
Chad T. Jafvert
Environmental Photochemical Reactions of nC60 and Functionalized
Single-Walled Carbon Nanotubes in Aqueous Suspensions
Chad T. Jafvert, Wen-Che Hou, and Chia-Ying Chen
School of Civil Engineering and Division of Environmental and
Ecological Engineering, Purdue University, West Lafayette, IN
Risk assessment of engineered nanomaterials necessitates the need for information on the reactivity (or
conversely, persistence) and transformation pathways of these materials in the natural environment. To this
end, we have characterized the reaction rates and products formed when aqueous C6o clusters (nC6o) are
exposed to natural sunlight, and have initiated studies on the photochemical reactivity of functionalized and
unfunctionalized single-walled carbon nanotubes (SWCTs). Using furfuryl alcohol (FFA) as a singlet oxygen
(:02) scavenger, we have shown that aqueous suspensions of nC6o clusters produce singlet oxygen (:02) upon
exposure to sunlight. Mass loss of molecular C60 occurs within these suspensions over a period of days in
summer sunlight (40° 26' N lat), whereas mass loss does not occur in dark control samples or in samples
containing no 02. A combination of 13C-NMR analysis of 13C-enriched nC6o, X-ray photoelectron
spectroscopy, and FTIR analysis indicates that photoproducts have olefinic carbon atoms as well as a variety of
oxygen-containing functional groups, including vinyl ether and carbonyl or carboxyl groups, whose presence
destroys the native Tt-electron system of C6o. Thus, the photoreactivity of nC6o in sunlight leads to the
formation of water soluble C6o derivatives. Laser desorption ionization time-of-flight (LDI-TOF) mass
spectroscopy indicated that most of the photoproducts formed after 947 hours of irradiation in natural sunlight
retain a 60-atom carbon structure. Long-wavelength visible light (A, > 400 nm) isolated from sunlight, was
shown to be important in both the photo transformation of nCeo and in the production of 02.
Unlike molecular C6o that can be analytically separated and quantified by HPLC methods, the reactivity of
carbon nanotubes in sunlight must be studied by examining: (1) formation of indirect photochemical products,
(2) changes in spectroscopic properties from which functional group distributions can be deduced, and (3)
changes in other bulk physicochemical properties, such as length, colloidal stability, electrophoretic mobility,
etc. As a start, we have investigated the production of reactive oxygen species (ROS) (i.e., indirect
photochemical product formation) in aqueous suspensions of commercial preparations of carboxylic acid
functionalized SWNTs (SWNT-COOH), polyethylene glycol functionalized SWNTs (SWNT-PEG), and
unmodified (i.e., pristine or unfunctionalized) SWNTs. Using FFA, a tetrazolium salt, and />-chlorobenzoic
acid as molecular probes for 102, superoxide anion (02~), and hydroxyl radial (-OH), respectively, photo-
production of all three reactive oxygen species occurred in aqueous suspensions of both types of functionalized
tubes, but not to any significant degree over the time period of our experiments in aqueous suspensions of
unfunctionalized SWNTs containing sodium dodecylsulfate, used to facilitate disaggregation and dispersion.
Defects in the fullerene surface caused by functionalization may facilitate ROS production, as well as
differences in amorphous carbon and metal impurity content within the different SWNT preparations.
Experiments suggest that the metal impurities may especially contribute to -OH generation.
These results suggest that functionalization, even with moieties that do not contain sunlight-active
chromophores, and/or surface defects strongly influence the environmental photoreactivity of SWCTs, and
potentially the environmental persistence of carbon nanotubes in general.
References:
1. Hou Wen-Che, Jafvert Chad T. Photochemical transformation of aqueous C6o clusters in sunlight.
Environmental Science and Technology 2009;43:362-767.
The Office of Research and Development's National Center for Environmental Research 117
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
2. Hou Wen-Che, Jafvert Chad T. Photochemistry of aqueous C6o clusters: evidence of 102 formation and its
role in mediating Ceo phototransformation. Environmental Science and Technology 2009;43:5257-5262.
3. Chen Chia-Ying, Jafvert Chad T. Photoreactivity of carboxylated single-walled carbon nanotube in
sunlight: reactive oxygen species production in water. Environmental Science and Technology
2010;44:6674-6679.
4. Hou Wen-Che, Kong Lingju, Wepasnick Kevin A, Zepp Richard G, Fairbrother D. Howard, Jafvert Chad
T. Photochemical transformation of aqueous C6o clusters: wavelength dependency and product
characterization. Environmental Science and Technology (in press, 2010).
5. Chen Chia-Ying and Chad T. Jafvert. Photoinduced reactive oxygen species production by single-walled
carbon nanotubes in water: role of surface functionalization. (to be submitted, 2010).
EPA Grant Number: R8333401
The Office of Research and Development's National Center for Environmental Research 118
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Environmental Photochemical Reactions of
nC60 and Functionalized Single-Walled
Carbon Nanotubes in Aqueous Suspensions
Chad T. Jafvert, Wen-Che Hou, Chia-Ying Chen
Division of Environmental & Ecological Engineering
and School of Civil Engineering
Purdue University, West Lafayette, IN 47907
PURDUE
Previous Studies with C60*
4 "Solubility of C60 in solvent mixtures"
(Env. Sci. Technol. 42: 845-851, 2008)
4 "C60's Kow and Aqueous Solubility"
(Env. Sci. Technol. 42: 5945-5950, 2008)
4 "Sorption of C60 to Saturated Soils"
(Env. Sci. Technol. 43: 7370-7375, 2009)
'Funded by NSF
Previous Results
4 Solvated crystals occur
* Kow m 106-7 ; Koc m 106-2 - 107-1
* Aqueous Solubility limit* 8 ng/L
Excess free energy of mixtures (ATOL-ACN, o
THF-ACN, D TOL-THF OTOL-EOH). For the
TOL-THF dala-eiv the abscissa s X,,o, Ir.oi X™
Current EPA-funded Study
Project period: May 2007-April 2009 (currently in extension)
4 "Photochemical transformation of aqueous C60 clusters (nC60) in
sunlight" (Env. Sci. Technol. 2009, 43:362-367)
4 "Photochemistry of aqueous C60 clusters: Evidence of 1O2 formation and
its role in mediating C60 phototransformation" (Env. Sci. Technol. 2009,
43:5257-5262)
4 "Photochemistry of aqueous C60 clusters: Wavelength dependency and
product characterization" (Env. Sci. Technol. 2010, 8121-8127)
4 "Photoreactivity of carboxylated single-walled carbon nanotubes in
sunlight: Reactive oxygen species production in water" (Env. Sci.
Technol. 2010, 6674-6679,)
4 "Solar light induced reactive oxygen species production by single-
walled carbon nanotubes in water: Role of Surface Functionalization"
(under review, Env. Sci. Technol.)
Photochemical transformation of aqueous C60 clusters
(nC60) in sunlight" (Env. Sci. Technol. 2009, 43:362-367)
Irradiation time
under lamps (day)
[nC60] (mg/L)
Color
TEM image*
Mean diameter**
(nm)
After
Centrifugation***
0 10 30 65
65 19.5 2.6 0.47
III!
•"•"" • .'-*v * " '•
•*-$?••.•-. :-
500 350 250 160
• III
*Scale bars indicate 1000 nm.
**Mean hydrodynamic diameters by DLS.
***Samples after centrifugation (ISOOOxg, 1 h) and
filtration (nylon membrane, 0.2-u.m pore size
Summary
* First paper to report on C60 photochemical decay in
aqueous media under sunlight.
* C60 measured quantitatively by HPLC
4 Smaller clusters result in faster loss of C60
* son/nC60 and THF/nC60 react at similar rates
» Photo-transformation rate is not pH dependent (3-11)
4 Negligible rate change with humic acids present
« Molecular Oxygen (02) is required.
"•=• Q Q O
.*]„ 0 0 © D ® ©.
.
® © ® ® ©©
© © ©
119
-------
^2 measurement
• The production of 1O2 during irradiation of nanomaterials in
sunlight and solar-simulated light is monitored by the loss of
furfuryl alcohol (FFA) as a trapping indicator.
• [FFA] is analyzed by HPLC with UV detection at 219 nm.
d[FFA]
dt
d[FFA]
Jt
fo2],,=-
Photochemistry of aqueous C60 clusters:
Evidence of *O2 formation and its role
in mediating C60 phototransformation"
(Env. Set. Technol. 2009, 43:5257-5262)
deoxygenated (•}
deoxygenated (D)
air-equilibrated (A)
air-equilibrated (A)
Irradiation of 0.8 mg/LnC60 with 0.2 mM FFA at pH = 7 in lamp light,
showing (a) C60 and (b) FFA measured in deoxygenated, air-
equilibrated, and dark control samples.
*,»
».? '
!""
!«
Ml
-1130
a [soo
& f,
i * fl
i
• *
•
(a) FFA •
im«)L
im (A) |
I 2
S 1.5
£ 1
0.5
0 i 10 11 20 21
Ihsftnl
(b) Estimated pOJ "
•
•
A
A
: "
5 10 15 20
Time(hrs)
fOJ calculation
Steady-state [1O2]SS (Haag et al., 1986): Non-steady-state [1O2]t :
—*• • *^ZfJ _ cf[FFA]^
-2£-M.» ['°a]c- MEMfr
,.ci_;» -^
2
5
Photochemical production of 1O2 by 130-nm (•) and 500-nm (A)
diameter nC60 (1 mg/L) under sunlight from July 23 to August 1 1 , 2008
at pH = 7, showing (a) FFA loss, and (b) the calculated [1O2] in the
irradiated samples, and the recovery of FFA in dark control samples.
Comparison of [1O2] measured in this study to
values reported for surface waters.
nCg,3
FAa
Swiss surface
waters'5
Municipal
wastewate rsb'c
US surface and
coastal watersd
Dutch surface
Sunlight
intensity
(W/m2)
525
525
1000f
1000f
800
800
DOC
(mg/L)
5
2.6
3.2-13
8.6-31
4-77
8-21
1 0 g
1 2\ ss
(x 10" M)
71. 111-'
5.6'
5.9-28'
11-15'
6-71'
0.4-7.6'
^his study. bData from Haag etal. (18). influents and second
1 0
1 2\ ss/
(x IO14 M per
mg/L)
14.211
2.1
0.8-3.2
0.3-1.1
0.7-2.9
0.22
ary effluents,
and the inflow and outflow of a waste stabilization pond in Switzerland. dData
from Zepp et al. (17). eData from Wolff et al. (29). \ = 280-2800 nm in
summer-noon sunlight. ^Corrected to a flat surface water body (18). Sjwas
calculated at 400 nmfor nCg,. hValue after 10 h of sunlight irradiation.
'Measured by the FFA method. 'Measured by 2, 5-dimethylfuran (DMF)
method using k, = 6.3 x io8 M^s1 (18).
Summary
102 forms during solar irradiation of nC60.
(Loss of FFA (as the probe molecule) in D20 and in the
presence of NaN3 is consistent with known reaction
mechanisms involving 102)
The photo-transformation of nC60 is mediated by 102.
The rate of 102 production is auto-catalyzed by nC60
water-soluble products (formed during irradiation).
102 production rate is higher when nC60 size is smaller.
[102] induced by nC60 in sunlight is 4-65 fold higher
than the average concentration typically found in
sunlit natural surface waters.
Photochemistry of aqueous C60 clusters: Wavelength dependency
and product characterization" (Env. So. Technol. 2010, 8121-8127)
Wen-Che Hou, . , Kevin Wepasnick,
RichardZepp, HowardFairbrother, ChadJafvert
due University, U.S. EPA, Athens GA, Johns Hopkins U.
a) Dark control sample
b) Irradiated sample (780 h)
c) Fullerenol, Cfin(0)v(OH)v, x + y=22
Wavenumbers (cm1)
C-0 stretch (1060 cm-1)
C-O-H in-plane bending or carboxylate
asymmetric stretching (1390 cm4)
C=C stretching or carboxylate symmetric
stretching (1600 cm-1)
FTIR spectra of AQU/nC60 showing (a) the dark control sample, (b) the irradiated
sample (780 h), and (c) a commercial fullerenol [C60(O)x(OH)y, where x + y = 22,
(MER Corp.)]. Spectrum (c) is reproduced from Fortner, 2007.
120
-------
fw/ Carl Wood, Campus-Wide Mass Spectrometry Center)
500 1000 1500 20OO 2500 500 1000 1500 2000 2500
Laser desorption ionization (LDI)-time-of-flight (TOP) mass
spectroscopy of AQU/nC60, showing (a) the dark control
sample, (b) the irradiated sample (780 h) in sunlight
176 ppm
C=O
(Carboxyl or
Carbonyl)
161 ppm
C=C-O
(vinyl ether
Carbon)
135 ppm
C=C
(w/ Zepp and Kong, EPA)
113 days of
27 days of
exposure
200 150 100 50
0.0 mg/L
5 mg/L
25 mg/L
13C NMR spectra of 13C-enriched (25%) THF/nC60 (58 mg/L)
exposed to sunlight from February 12 to June 15, 2009.
(parent peak occurs at 143 ppm)
(w/ Weposniek and Fairbrother, JHU)
(A)
(B)
A..
Binding Eneigj (eV)
C(ls)
y-v
""""""
jv^
*y
""•™
.11
h
r\
Binding Energy (eV)
XP spectral envelopes of the
O(1s) and C(1s) regions for(a)
as-received C60, (b) dark control
AQU/nC60, and (c) AQU/nC60
irradiated in sunlight for 947 h.
For each sample the integrated
area under the C(1 s) spectral
envelope has been normalized
and the O(1 s) signal intensity
adjusted accordingly. In the
C(1 s) spectra of (b), the binding
energy regions for C-C/C=C and
oxidized carbon atoms (i.e., COK
species) has been shown. The
inset in the C(1s) region shows
the change in the TT-TT* shake-up
feature centered at 291 eV for
each C60 sample; in each insert,
the vertical arrow indicates the
binding energy below which
spectral intensity is observed.
Summary
t NMR
4 FTIR
t LDI-TOF-MS
t XPS
- all indicate oxidation of C60 occurs
in aqueous suspensions of nC60
under sunlight (i.e., destruction of n-bonds)
(vinyl ethers, carbonyl and/or corboxyl groups)
* Experiments with 400 nm cut-off filters and with
monochromatic light at A = 436 nm indicate that C6(
photo-transformation and 1O2 production occur in
visible light (A > 400 nm).
"Photoreactivity of carboxylated single-walled carbon
nanotubes in sunlight: Reactive oxygen species
production in water" (Em. Sci. Technol. 2010, 6674-6679;
H2O
50% (v/v) D2O
D2O
0 20 40 60 80
Time (hrs|
FFA loss at pH 7 under lamp light in 0.01 mg/mL COOH-ARC
dispersed in 100 % (v/v) D2O, 50 % (v/v) D2O, and H2O, and FFA
recovery in the corresponding dark control samples
| 0.12 •
S
Are other Reactive Oxygen Species involve in
COOH-ARC phototransformation in sunlight?
(a)
• NT+NBT
iNBT alone
x NT alone
IS
•0.04
(b)
•NT+NBT
ANBTalone
x NT alone
40 60
Time (hrs)
20 40
Time (hrs)
Evidence of O2-~ production, via NBT2+ (0.2 mM) product formation
induced by 10 mg/L COOH-ARC at pH 7 under (a) lamp light and (b)
sunlight.
121
-------
Superoxide anion (O2•-) measurement
• Nitro bluetetrazolium salt (NBT2*) has been one of the
most widely used reagent for the detection of O/
[Bartosz, 2006]: NBT2* reacts with O2'" producing
products that absorbs light at 530 nm.
• XTTforms a water-soluble reduction product in the
presence of O2'" [Ukeda, 1997; Bartosz, 2006]. The
concentration of superoxide was measured by comparing
XTT (0.1 mM) reduction with and without superoxide
dismutase (40 U/mL)
XTT = 3'-{l-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)-benzenesLilfonic acid
•OH Measurement
•p-Chlorobenzoic acid (pCBA) was used as a reactive
•OH radical scavenger.
•pCBA concentrations were measured by HPLC with a
UV/Vis detector set at 230 nm.
d[pCBA]
= *,
, -T OH ,pCBA
d[pCBA]
[*OH]ss[pCBA]
- = k[pCBA]
[Elovitzetal., 2000]
pH effect?
j"s
1 •
+
1
0
A
i
• 5
xg
X11
+ W/0
4
X
0 20 40 60 80 100 120
Time (hrs|
FFA loss under lamp light in 0.01 mg/mL COOH-ARC at pH
3, 5, 7, 9, 11, and without buffering , and FFA recovery in
the corresponding dark control sample at respective pH
0.01 mg/mL COOH-ARC in water after 6 hours (A = 350 ± 50 nm):
(a) without buffer and at pH = 11, 9, 7, 5, 3 (left to right), and
(b) and the same irradiated pH 3 sample (left) and dark control
sample (right).
Summary
In oxic aqueous solutions under sunlight,
carboxylated-SWNTs dispersions generate
singlet oxygen (102), superoxide anion (02"),
and hydroxyl radicals (-OH).
Reactions with probe molecules were
corroborated with experiments using D20 and
azide (for 102), superoxide dismutase (for 02-~),
and tert-butanol (for -OH).
Photo-induced aggregation occurred at pH 3.
"Solar light induced reactive oxygen species production by single-
walled carbon nanotubes in water: Role of Surface Functionalization"
(under review, Env. Sci. Technot.)
Sample
SWNT-ARC
SWNT-ARC-P
COOH-ARC
PEG-ARC
SWNT-CVD-P
Synthesis method
Electric arc discharge
Electric arc discharge
Electric arc discharge
Electric arc discharge
Chemical vapor
deposition
Functionalization
Nofunctionalization
No functionalization
Carboxylation
PEGylation
Nofunctionalization
"Vender specification determined by thermal gravimetric analys
The supplier of "ARC" tubes was Carbon Solutions, Inc.
The supplier of "CVD" tubes was NanoLab, Inc.
Metal Carbonaceous
residue purity
content"
30% ~53%
5% >90%
5.9% >90%
5.2% >90%
>95%
s(TGA)at9005Cinair.
122
-------
ARC-PEG
ARC-COOH
SWNT-ARC
SWNT-ARC-P
%3 *fc
1000 1500 2000 2500
Raman shift (crrr1)
Raman spectra confirms the degree of functionalization:
ID/IG increases through the following sequence:
SWNT-ARC-P < SWNT-ARC « ARC-COOH < ARC-PEG.
0.25
0.2
S0.15
it 0.1
0.05
0
X PEG standard
1 PEG-ARC
• COOH-ARC
20 40 60
Time (hrs)
FFA loss indicating 1O2 production at pH 7 in lamp light by aqueous
COOH-ARC, PEG-ARC, and 100 mg/mL polyethylene glycol, and the
corresponding dark control samples
0.25
i
0.2 •
50.15 •
* SWNT-ARC-P
O SWNT-ARC-P (high)
ASWNT-CVD
XSDS control
• COOH-ARC
D COOH-ARC (high)
50 100
Time (hrs|
'Oj detection by FFA loss under lamp light in 1% SDS,
and FFA recovery in the corresponding dark control
samples at the last sampling time
A PEG-ARC
• COOH-ARC
XXTT alone
20 40
Time (hrs)
Evidence of O2 production via XTT (0.1 mM) product
formation, under lamp light in aqueous suspensions
• COOH-ARC
XXTT alone
« SWNT-ARC-P
• SWNT-ARC
20 40 60
Time (hrs)
Evidence of O2 production via XTT (0.1 mM) product
formation under lamp light in 1% SDS suspensions
2.5
2
1.5
ii i
» SWNT-ARC-P
XSDS control
• SWNT-ARC
• COOH-ARC
i PEG-ARC
0 20 40 60 80
Time (hrs)
Detection of -OH using pCBA (2 |iM) as the -OH scavenger at pH 7
under lamp light, in aqueous suspension of COOH-ARC and PEG-
ARC, and 1% SDS suspension of SWNT-ARC-P, SWNT-ARC, and SDS
alone
123
-------
0.2
§"0.15
£ 0.1
0.05
0 '
(a)* .
R £1 X
A
xpure water at pH 7
* COOH-ARC filtrate
• • COOH-ARC
•
•
U. 13 '
|
§ 0.1 •
«
8
1
•§ 0.05 •
1
(b) . -
•
•
A
X
* *
•
• SWIMT-COOH
it i filtrate at pH 7
x XXTT alone
0 15 30 45 60 0 10 20 30 40
Time (hrs) Time (hrs)
(a) 1O2 and (b) O2~- production - at pH 7 in lamp light by
aqueous COOH-ARC, COOH-ARC filtrate, and pure water.
+ COOH-ARC w/ catalase
Xpure water at pH 7
ACOOH-ARC filtrate
• COOH-ARC
(Catalase reacts with H ,0 ?
0 20 40 60 80
Time (hrs)
Detection of -OH using pCBA (2 |iM) as the -OH
scavenger at pH 7 under lamp light, in aqueous
suspension of COOH-ARC, COOH-ARC filtrate, COOH-
ARC and catalase (500 U/mL), and pure water control.
1 I
0.8 •
0
y
0 °-61
g 0.4 •
a
0.2 •
0 •
* * * " *
• x m
• • •
•
XNi2+
* filtrate at pH 7 + deferoxamine
• filtrate at pH 7
0 20 40 60 8
1 I
•g 0.8 •
§
Si 0.6 •
<
to
*i 0.4 •
0.2 •
4 D n D
A
t
i
•
•
•
n H2O2 dark control
A H2O2@ Sunlight
• H2O2@ lamp light
0 0 10 20 30 40 50
Time (hrs) Time (hrs)
Detection of -OH using pCBA (2 uM) as the -OH scavenger at pH 7 under
lamp light and/or sunlight in (a) aqueous suspension of COOH-ARC,
COOH-ARC filtrate, COOH-ARC w/deferoxamine, and NiCI2 (2 mg/L), and
(b) H2O2 100 uM solution, and the corresponding dark control samples
COOH-ARC+NADH
NADH
«SWNT-ARC-P+NADH ,
/ & a ft &
1 2
Time (hrs)
Effects of NADH (0.2 mM) on O2 production
detected via XTT (0.1 mM) product formation at
pH 7 under lamp light and dark controls
Reactions that may occur upon sunlight
absorption by functionalized SWNTs
Summary
Oxic aqueous colloidal dispersions of both types
of functionalized nanotubes generated ROS (1O2,
O2 ,and -OH) in sunlight.
Both Type I and Type II photochemical pathways
occur by the functionalized SWNTs in sunlight.
It appears that the functionalized SWNTs can act
as the electron donor directly (resulting in a
change in their properties) or can shuttle
electrons from other electron donors to form
these reactive oxygen species.
124
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PURDUE
125
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Qilin Li
Impact of Photochemical Oxidation on the Stability of nC60 and Multi-Walled
Carbon Nanotubes in Aqueous Solutions
Xiaolei Qu, Yu Sik Hwang, Pedro J.J. Alvarez, and Qilin Li
Department of Civil and Environmental Engineering, Rice University, Houston TX
In natural aquatic systems, various environmental factors including natural organic matter and sunlight can
interact with engineered carbon nanomaterials and influence their transport. The main objective of this study
was to investigate the impact of natural organic matter and sunlight on the aggregation and deposition
behaviors of aqueous C6o fullerene nanoparticles (nC60) and multiwalled carbon nanotubes.
Suwannee River humic acid (SRHA) standard (II) and Elliot soil humic acid (ESHA) were used as the
model aquatic and soil organic matter, respectively, and the UVA fraction of sunlight was simulated with UV
lamps with output wavelength and intensity of 350 ± 50 nm and 1.66 mW/cm2, respectively. Initial aggregation
rates of nC6o and carboxylated multiwalled carbon nanotubes (COOH-MWCNTs) before and after irradiation
in solutions of different ionic strength, ionic composition, and humic acid concentration were determined from
time resolved particle size measurement using dynamic light scattering. Deposition onto Si02 surfaces was
characterized using a quartz crystal microbalance with dissipation (QCMD) and compared to results from
traditional column experiments; the impact of soil organic matter was investigated using ESHA coated Si02
crystals or quartz sand.
Our study revealed that UVA irradiation in the presence of dissolved oxygen introduced oxygen-
containing function groups on nC6o surface, but reduced the oxygen content of the COOH-MWCNTs. Such
changes in surface chemistry greatly altered the humic acid adsorption capacity and aggregation and deposition
behavior of these carbon nanomaterials. In NaCl solutions, UVA irradiation induced surface oxidation
remarkably increased nC6o stability by increasing the negative surface charge and reducing surface
hydrophobicity. On the contrary, UVA irradiation reduced nC6o stability in CaCl2 solutions due to specific
interactions of Ca + with the oxygen-containing functional groups on the UVA-irradiated nC6o surface and the
consequent charge neutralization. In the absence of Ca2+, the surface photochemical oxidation greatly reduced
the adsorption of SRHA on nC6o surface, resulting in weak dependence of nC6o stability on SRHA; Ca2+, on
the other hand, facilitated SRHA adsorption on the UVA-irradiated nC6o surface by neutralizing surface
charges of both UVA-irradiated nC6o and SRHA as well as forming intermolecular bridges, leading to
enhanced stability in the presence of SRHA. Deposition of nC6o onto silica surface was found to be controlled
by electrostatic interactions. The attachment efficiency increased with increasing ionic strength due to surface
charge screening. ESHA adsorbed on the quartz crystal and sand surfaces hindered nC6o deposition at NaCl
concentrations between 5 and 40 mM. However, at lower NaCl concentrations, enhanced deposition was
observed on ESHA coated quartz crystal and sand.
UVA irradiation affected stability of the COOH-MWCNTs differently. Unlike nC60, the stability of
COOH-MWCNTs in NaCl solutions decreased with increasing UVA irradiation time. Meanwhile, the
deposition rate of the COOH-MWCNTs onto silica surface increased by 2.6 times after 1 week of UVA
irradiation. Particle electrophoretic mobility measurements suggested that UVA-irradiated COOH-MWCNTs
were less negatively charged than the pristine COOH-MWCNTs, consistent with their higher aggregation and
deposition rates. Based on the reduced oxygen content observed in XPS analyses, we speculate that the
COOH-MWCNT surface underwent decarboxylation during the UVA irradiation, but the underlying
mechanism remains unclear and requires further study.
The Office of Research and Development's National Center for Environmental Research 126
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Immediate future study will focus on two tasks to: (1) determine the impact of UVA irradiation on aquatic
NOM adsorption to nC6o particle surface. NOM sorption will be investigated via batch adsorption and QCMD
experiments under various solution conditions and (2) investigate the effect of organic matter content in
sediment and soil on nC6o deposition/sorption. Batch sorption and column experiments will be conducted.
References:
1. Hwang YS and Li QL. Characterizing photochemical transformation of aqueous nC(60) under
environmentally relevant conditions. Environmental Science and Technology 2010;44:3008-3013.
2. Qu X, Hwang YS, Alvarez PJJ, Bouchard D, and Li Q. UV irradiation and humic acid mediate
aggregation of aqueous fullerene (nC60) nanoparticles. Environmental Science and Technology (DOI:
10.1021/esl01947f).
EPA Grant Number: R834093
The Office of Research and Development's National Center for Environmental Research 127
-------
Impact of Photochemical
Oxidation on the Stability of nCE
and Carboxylated MWCNTs
Qilin Li
Department of Civil & Environmental Engineering
Rice University
RICE
Research Objectives
Q Exposure
MWPNT ^^ Aqueous suspension ^^ Living organisms
Interactions with Environmental Component:
(e.g. NOM and sunlight)
a Changes of physicochemical properties in nature
aquatic systems (e.g. interacts with NOM and sunlight )
Q Resulting changes of transport pattern (e.g. aggregation
and deposition)
ication
biochemical Iran
Sonication/
(2um Filtration)
MWCNT-COOH
Sonication
UVA-irradiation
Characterization
0 100 200 300 400
Particle Size (nm)
400 600
Wavenumber (nm)
• 3.8-4.0mg/L
• pH : 5.5-6.0
Luzchem reactor
Four Hitachi lamps
(300-400 nm, 8W)
2 mW/cm2
UVA-1week
295 290 285
C(1s) binding energy
Peak
1
2
3
Position
284.3
285.6
288.0
% C(1s)
66%
19%
15%
Carbon
Underivatized C(C=C)
Monooxygenated C
(e.g., C-0)
Di-oxygenated C
(e.g.,O-C-OandC=O)
After etching
290 285 280
C(1s) binding energy
Hwang, Y. S.; Li, Q. L. ES&T, 2010,
44, 3008-3013.
radi
Stability in NaCI
8.
o
I
240
220
200
180
160
140
120
100
,3,Op,rpMiN9Cf
200 400 600 800 1000 1200 1400 1600
Time (s)
60
Qu et al, ES&T, 2010
128
-------
ITradiatfon
Stability in NaCI
m
60
S 0.1
E
0.01 0.1 1
NaCI Concentration (M)
E -2
0.001 0.01 0.1
NaCI Concentration (M)
Qu et al., ES&-T, 2010
>y Surface Oxidation
• 7DUV
• 7DUV+1 mg/LSRHA
* 7DUV+10mg/LSRHfl
449 mM
563 mM
NaCI Concentration (M)
Dristine
7DUV
nC60
(ma/L)
192
192
SRHA
added
(ma/L)
0.934
0.947
SRHA after
sorption
(ma/L)
0.702
0.971
q (ma/ka)
1209
-
Qu et al., ES&T, 2010
Stability in CaCI.
SHU
'60
Stability in CaCI*
ti
U
c
u
i
£ 0.1
U
1
. //
* /
3.2 mM
C" •
• Pristine
4.2 mM • 7DUV
0.001 0.01 0.1
CaCI Concentration (M)
NE -0.5
°?
E -1
t
S -1.5
0 0.001 0.002 0.003 0.004 0.005 0.006
CaCI2 Concentration (M)
til
• Pristine
• Pristine •
1 mg/L SRHA
4.2 mM
=F
12.8mM
0.001 0.01 0.1
CaCI Concentration (M)
• 7DUV
• 7DUV+1 mg/LSRHA
0.001 0.01 0.1 1
CaCI2 Concentration (M)
Qu et al., ES&-T, 2010
Qu et al., ES&-T, 2010
ianc
on UVA irradiated nC
60
Pristine nC60 Dl water
JV nC60 Dl water
UV nC60 3 mM CaCI2
na
Stability in NaCI
™ 0.1
52 mM
175mM
• Prjistine MWCNT
• UV MWCNT
°?
o
t-2
n
o
S
— -3
£
o
0 4
• »*
t :
i * *
^
• Pristine MWCNT
• UV MWCNT
NaCI Concentration (M)
10 100
NaCI Concentration (mM)
129
-------
Loss of Oxygen Functional Groups
450 400 350
Binding Energy (eV)
Original MWCNT-COOH (14% O)
Irradiated MWCNT-COOH (11% O)
fnchanged Stability in CaCI2
CaCI2 Concentration (M)
CaCI2 Concentration (M)
including Remar
a Sunlight irradiation and humic acid sorption
mediate nC60 and MWCNT-COOH aggregation
a Both specific and nonspecific (i.e., DLVO)
interactions are involved
a nanocarbon surface chemistry plays a key role in
its environmental fate and transport.
ingoing Research am
Directions
:uture
a Impact of UVA irradiation and NOM on
sorption/deposition and transport in subsurface
porous medium
aNature of NOM: aquatic vs. soil
a Properties of suspended solids/sediment/ aquifer
media
a Impact of UVA irradiation and NOM on
bioavailability and bioaccumulation
nC60 Deposition on SiO2
Acknowledgement
.3! 1
u
I
10
NaCI (mM)
o
-5
E -10
£-20
-25
-30
10 100
NaCI (mM)
a NSF Center for Biological and Environmental
Nanotechnology (Award EEC-0647452)
a USEPASTAR program (Grant No. 834093)
130
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I
Fullerene cosmetics
131
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2010 U.S. EPA Nanotechnology Grantees Meeting
Qingguo Huang
The Environmental Behaviors of Multi-Walled Carbon Nanotubes
In Aquatic Systems
Qinssuo Huang , Marsha C. Black , Liwen Zhang , Emily R. Roberts , and Elijah Petersen
1 Department of Crop and Soil Sciences, University of Georgia, Griffin, GA;2Department ofEnvironmental
Health and Science, University of Georgia, Athens, GA; 3Chemical Science and Technology Laboratory,
National Institute of Standards and Technology, Gaithersburg, MD
Our study was designed to investigate the environmental behavior of water-dispersed carbon nanotubes in
natural aquatic systems (i.e., water-sediment phase distribution, possible degradation, ecological exposure and
toxicity), thereby providing useful information for environmental risk assessment and potential waste
treatment. We have used C14-labeled multi-walled carbon nanotubes (14C-MWNTs) in our experiments to
unambiguously identify and quantify carbon nanotubes from various natural materials, including water,
sediments and organisms. Our experiments have yielded important information regarding three important
behaviors of MWNTs in aquatic systems: water/solid phase distribution, biotic degradation, and possible
toxicity and exposure to aquatic organisms. The results on each topic are briefly summarized below,
respectively.
We conducted experiments to examine the phase distribution of 14C-MWNTs in aqueous systems
containing peat, shale, or clay as model solid phases under a series of varying pH and ionic strength
conditions. Our results suggest that solid matter interacts with water-dispersed MWNTs via three interactive
processes: (1) dissolved cations tend to promote MWNT aggregation via double layer compression; (2)
dissolved organic matters released from the solid phase tend to stabilize MWNT dispersion; and (3) MWNTs
sorb to the solid phase, primarily driven by hydrophobic interaction. All processes are variously influenced by
aqueous conditions (e.g., pH, electrolytes) and their interplay governs the phase distribution of MWNTs.
Recent studies have discovered biotic degradations of fullerols and single-walled carbon nanotubes
(SWNTs), but there has not been a report on microbial degradation of MWNTs. We in our study found an
enrichment culture that is capable of mineralizing 14C-MWNTs into 14C02. Our initial study indicates that the
mechanism involved in MWNT degradation seems to differ from that of fullerols and SWNTs. The
microorganisms responsible for MWNTs degradation may not be fungi, but a consortium of bacteria, and the
peroxidases that were found responsible for SWNT and fullerol degradation were absent in the MWNT-
degrading culture. Currently, we are conducting a systematic study to identify and characterize the
microorganisms that are responsible for degrading MWNTs and the biochemical pathways that are involved in
MWNT metabolism.
We studied chronic effects of 14C-MWNTs on Ceriodaphnia dubia, an aquatic invertebrate, in 8-day
exposures. For chronic exposures, 14C-MWNTs were solubilized in moderately hard water (MHW) by four
different methods: bath sonication (Branson) for 2 h; probe sonication for 2 h with 50 sec. pulses (Cole-
Parmer 500-Watt Ultrasonic Homogenizer); bath sonication followed by addition of Sewanee River natural
organic matter (NOM; final concentration = 4.5 mg/L); and by stirring nanotubes overnight in 4.5 mg/L
Sewanee River NOM dissolved in MHW. Ceriodaphnia exposed to bath-sonicated MWCNTs had
significantly smaller brood numbers and size at the 2.5 mg/L concentration (LOEC), compared with controls.
Chronic exposures with probe-sonicated nanotubes showed less reproductive toxicity, with a LOEC of 5 mg/L.
No reproductive toxicity was observed for nanotube exposures with added NOM. Reproductive toxicity of the
bath-sonicated nanotubes may be related to association of the MWNTs onto the body surfaces of the adults,
which likely interfered with molting and prevented neonate release.
EPA Grant Number: R834094
The Office of Research and Development's National Center for Environmental Research 132
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flic University of Georgia
The Environmental Behaviors of Solubilized
Multi-walled Carbon Nanotubes
in Aquatic Systems
Qingguo Huang1, Marsha C Black2, Liwen Zhang1, Emily R. Roberts2, Elijah Petersen
Department of Crop and Soil Sciences, University of Georgia, Griffin, GA 30223
department of Environmental Health Science, University of Georgia, Athens, GA
3Chemical Science and Technology Laboratory, NIST, Gaithersburg, MD
Objective
Solubilized CNTs
-> Mobility -> Exposure
Taste
- 1) Sorption
- 2) Transformation
- 3)Toxicity, accumulation and transfers
Contents
1. Objective
2. Sorption
3. Transformation
4. Toxicity/Bioaccumulation
Phase Distribution
gregation
Sorption
| Dissolved Species |
| Sediments |
Three Treatments
|Trea 2: peat DOM + CNTs
Treatment 3: Peat + CNTs
Effect of Peat
133
-------
Effect of Shale
Contents
^ 1. Objective
{§> 2. Sorption
3. Transformation
4. Toxicity/Bioaccumulation
Reactivity
Biotransformation: Fullerene
Schmrsf K H, 3 at Environ So Jtchnol 2009.43 (9), 3t62 3168
134
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Mineralization
1 10-
1
1-
Mineralized F
3 ^
• ^H noSR NOM
\ i i with 8 ppm SR NOM
\ — •— Percentage of MWNTs Mineralized
\
1
\ »
Hfi
• 6
•5 i
-------
Chronic Exposures with Ceriodaphnia dubia
Goal: Evaluate reproductive toxicity and accumulation of
MWCNTs by adult and neonate Ceriodaphnia dubia in
solutions that are prepared by two methods
• Bath sonication
• Sewanee River NOM (4.5 mg/L)
• 7-day chronic test
- C. dubia <24 h old; 3 brood test (US EPA)
- 14C-MWNT concentrations: 1.25-5 mg/L (in MHW)
- Solubilization procedures
- Daily renewal (exposure water + food)
- Endpoints: # of broods, # of offspring
• NOEC, LOEC calculated by ToxCalc®
- Accumulation measured by LSC (14C)
Results
Reproductive Effects of MWNTs in C. dubia
Accumulation of MWNTs in C. dubia neonates
Discussion and Conclusions
Control (40x)
2.5 mg/L MWNT (40x) 2.5 mg/L MWNT+NOM (40x)
Sonicated MWNTs adhered to adult organisms
- Prevented molting/release of neonates = fewer broods
NOM protective against reproductive toxicity
- No observed adherence to adults
Significant accumulation of NOM-solubilized MWCNTs in neonates
- NOM-MWCNTs were consumed (vs. diffusion)?
- Clumping of sonicated MWCNTs prevented consumption?
What's Next?
Feeding studies
- C. dubia fed Artemia exposed to MWNTs
- Fathead minnows fed C. dubia exposed to MWCNTs
-Trophic transfer?
Full lifecycle exposures of MWNTs
- Fathead minnow
- Maternal transfer?
Acknowledgements
• Major Participants
Liwen Zhang; Emily Roberts; Dr. Marsha Black; Dr. Zhengwei Pan;
Dr. Elijah Petersen; Dr. Mussie Habteselassie
• Major Collaborator
Roger Pinto; Yenjun Zhuang; Vijaya Mantri; Wen Zhang;
Dr. Yongsheng Chen; Dr. Aaron Thompson
• EPA STAR support
136
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Day 2, Tuesday, November 9, 2010
AM Session 1: Effects on Cells
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Amiraj Banga
Functional Effects of Nanoparticle Exposure on Airway Epithelial Cells
Amiraj Bansa1, Frank A Witzmann2, and Bonnie L Blazer-Yost1'2
Department of Biology, Indiana University Purdue University at Indianapolis, Indianapolis, IN;
2Department of Cellular and Integrative Physiology, Indiana University School of Medicine,
Indianapolis, IN
Nanotechnology, the creation and manipulation of structures and systems at a nanoscale level (< 100 nm),
significantly alters fundamental properties from large-scale materials. With nanotechnology being a focused
area of exponential scientific and industrial growth in the last few decades, concerns have arisen regarding the
potential biological effects of nanoscale materials. These effects remain poorly understood, especially with
regard to occupational and environmental hazards. Populations exposed to increasing levels of nanomaterials
include not only workers exposed during the production, recycling, and disposal, but also to the general
population that uses commercially available nanomaterial-containing products and is exposed to them via
environmental contamination.
The unique physico-chemical properties of these nanoscale products cause them to interact with cellular
systems in an unknown and undefined manner. Demonstrated effects include oxidative stress, inflammatory
cytokine production, DNA mutation membrane damage, and even cell death.1 Although the nanotechnology
industry holds great promise in the future, its darker side has to be explored to obtain the maximum benefits
from this industry in a safe manner.
Carbon-based nanoparticles are one group of widely produced nanomaterials both industrially and
environmentally. These include fullerenes and nanotubes (Single-wall carbon nanotubes [SWCNT] and Multi-
wall carbon nanotubes [MWCNT]). Fullerenes or Buckyballs are the most stable and are composed of 60
carbon atoms with an average diameter of 0.72 nm. Carbon nanotubes are graphite sheets rolled to form
seamless tubes or cylinders. Whereas SWCNT consist of a single layer with diameters ranging near 1 nm,
MWCNT are larger and consist of many single-walled tubes stacked one inside the other with diameters
reaching 100 nm. Because of their nano sizes, fibrous shapes, and carbon base, CNTs are expected to behave
differently than the large-sized particles. They are potentially toxic like other small fibers (asbestos and silica)
and biopersistent because of their stability.
One primary route of nanoparticle uptake in the body is through inspiration of airborne nanoparticles.
Combustion-derived nanoparticles have been shown to cause lung cell injury and inflammation due to
oxidative stress2 that may manifest itself as airway disease, cardiovascular disease, fibrosis, or cancer.3 Using
quartz and carbon nanoparticles at equal mass dose, it was concluded that SWCNT in the lungs were far more
toxic than carbon black and even quartz.4 Following inhalation, ultrafme carbon particles can travel through the
circulatory system and invade the brain.5 Nanoparticles can have prothrombotic effects in vivo and demonstrate
platelet activation in vitro, as has been shown in response to SWCNT exposures.6 They also demonstrated that
MWCNTs can reach the subpleural tissue in mice with a single inhalation dose of 30 mg/m3 for 6 h. A stable
C6o suspension has been shown to produce genotoxicity as a result of DNA damage in human lymphocytes.7
One of the respiratory cell lines commonly used for tracheobronchial epithelial cell studies is Calu-3.
Although it is adenocarcinoma in origin, it is one of the few cell lines that form tight junctions in vitro and
produces features of a differentiated, functional human airway epithelium. Calu-3 cell line is a human airway
epithelial cell line that responds to epinephrine with an increase in Cl" secretion via Cystic Fibrosis
Transmembrane Regulator channel (CFTR). Water follows Cl" and together with mucous helps to clear the
airways of any foreign substances. The cells show a high resistance phenotype after 13 days of growth. The
The Office of Research and Development's National Center for Environmental Research 138
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2010 U.S. EPA Nanotechnology Grantees Meeting
current studies utilize this well-characterized model to study the effects of unpurified, as manufactured,
nanoparticles that are most likely to be found as environmental and occupational pollutants.
We hypothesized that exposure of epithelial cells to unpurified, as manufactured CNPs such as C6o, SWNT
and MWNT, may alter the function of barrier epithelia. The effect of exposure of each of three different CNPs
was studied in air-interface cultured Calu-3 cell model over seven orders of magnitude (4 ug/cm2-0.004
ng/cm ). Electrophysiological techniques were used to study transepithelial ion transport and the barrier
function expressed as Trans Epithelial Electrical Resistance (TEER). After 48 h of exposure to CNPs,
fullerenes did not show any effect on TEER, whereas the nanotubes significantly decreased TEER over a wide
range of concentrations (4 ug/cm2-0.004 ng/cm2). The ion transport response to epinephrine also was
significantly decreased by the nanotubes but not by fullerenes. To look at the effect of exposure times, cells
were exposed to same concentrations of CNPs for 24 and Ih time periods. Although the 48 h and 24 h time
period exposures exhibited same effects, there was no effect seen after 1 h in terms of TEER or hormonal
responses. In cells exposed to either of the nanotubes, the TEER was not statistically different from control
after treatment with 4 ng/cm2 concentration, whereas in the case of hormonal responses, the nanotubes,
especially multi-walled, still showed significant inhibitory effect. To examine which step of the epinephrine
stimulated intracellular pathway is affected by CNPs, cAMP assays were performed. The cAMP levels for the
exposed cells vs. the control cells were not different, suggesting an effect manifested after the epinephrine-
induced increase in cAMP.
Our results indicate that there are changes in response to physiologically significant nanoparticle
concentrations that could, in vivo, be manifested as changes in transcellular permeability and hormone
responsiveness. Such effects could alter airway function, emphasizing the need of further study on the effect of
these nanoparticles.
References:
1. Oberdorster G, Maynard A, Donaldson K, Castranova V, Fitzpatrick J, Ausman K, Carter J, Karn B,
Kreyling W, Lai D, Olin S, Monteiro-Riviere N, Warheit D, Yangvv H. Principles for characterizing the
potential human health effects from exposure to nanomaterials: elements of a screening strategy. Particle
and Fibre Toxicology 2005;2:8.
2. Donaldson K, Tran CL. Mutation Research 2004;53:5-9.
3. Mauderly JL, Snipes MB, Barr EB, Belinsky SA, Bond JA, Brooks AL, Chang IY, Cheng YS, Gillett NA,
Griffith WC, et al. Part I: neoplastic and nonneoplastic lung lesions. Research Report/Health Effects
Institute 1994;68(1): 1-75.
4. Lam CW, James JT McCluskey R, Hunter RL. 2004. Pulmonary toxicity of single-wall carbon nanotubes
in mice 7 and 90 days after intratracheal instillation. Toxicological Sciences 2004;77:126-34.
5. Oberdorster G, Sharp Z, Elder AP, Gelein R, Kreyling W, Cox C. Inhalation Toxicology 2004;16:437^5.
6. Bihari P, Holzer M, Praetner M, Pent J, Lerchenberger M, Reichel CA, Rehberg M, Lakatos S Krombach
F. Toxicology 2010;269(2-3): 148-154.
7. Dhawan A, Taurozzi AS, Pandey AK, Shan W, Miller SM, Hashsham SA, Tarabara W. Environmental
Science and Technology 2006;40:7394-7401.
NIH/NIGMS Grant Number: R01GM085218
The Office of Research and Development's National Center for Environmental Research 139
-------
Functional Effects of Nanoparticle
Toxicity on Airway Barrier Epithelial
Cell Function
Amy Banga
Blazer-Yost/Witzmann Group
IUPUI, Indianapolis
Health Hazard of Nanoparticle
Workers handle nanoparticle materials ir
many industrial jobs to produce consumers'
items
Nanoparticles can enter the body by:
-Inhalation
-Swallowing
-Penetration through the skin
Complete information about health effects is
lacking
MWCNT
(diameter ~2-25 nm;
length: few nm to microns)
SWCNT
(diameter as small as 1 nm;
length: few nm to microns)
C60 (fullerene)
(Avg diameter 0.72 nm)
CNP purchased from SES Research, Inc., Houston, TX
(http://www.sesres.com)
ircsjp-: rro'T.1 i-i.ir'.v'.v'."1.'.; photon.: u- i~c;
-------
Approach
CNP preparation - CNP suspended in Fetal
Bovine Serum (FBS), sonicated, autoclaved and
added to serum free media. The amount of CNP
was regulated so as to obtain a desired final
concentration of CNP in media when FBS was
added.
CNP exposure - The cells were incubated
with CNP-FBS containing media at a normal
concentration of 15% for last 1, 24 or 48 h of
growth to simulate in vivo CNP exposure.
Conversion equation
X ug/cm2 = 25X ug/ml.
Effect on the TEER of C
types and cone
e«
9
1™
|
1
a u-3 cells by exposure to different
entrations of CNPs for 48 h
48h
i
fffl1
n. 33;
ffft Jfi ffl
Fullerenes Single wdl nun wall
* indicates that the value was statistically different from the control value
CP<0.05) using a Students' t-test.
Response of Calu-3 cells to epinephrine
Hcnrcrd response of Cafci-3 cdls eqnsed to dfftrrt types of
rtarra^respcnsecfCau3 cells eposedtodlfererttipescf
*e^W-^-e-fl-e
2 4 S B 10
Tlrre(rrin)
141
-------
Effect on the TEER of Calu-3 cells by exposure to different
types and concentrations of CNPs for 24 h
« 400
!
Fullerenes Single wall Multiwall
* indicates that the value was statistically different from the control value
(P<0.05) using a Students' t-test.
Hormonal response of Calu-3 cells exposed to different types of
nanoparddesfor 24 h at concentration of 4 jjo/cnf to epinephrine
Hormonal response of Calu-3 cells exposed to different types of
nanopaitides for 24 h at concentration of 4 pgfcnf to epinephrine
Effect on the TEER of
types and cone
„- 800
I
i MO
1
.« 400 •
1
I 200
I
2
"- o J
/
1 If,
3 3
J //
^ &
Ca u-3 cells by exposure to different
entrations of CNPs for an hour
1h
J IITT flf.
33 5533 i i
// //// //
fa
3 t
//
Fullerenes Single wall Multi wall
* indicates that the va ue w
(P<0.0
as statistically different from the control value
5) using a Students' t-test.
150
"|
50
0
Horn
nan
EpnEphnne
tonal response of Calu-3 cells ex|
ipaitides for 1 h at concetration c
150
' 1
1 100
8
IP^^^^ffi\
8~8~8~? ^8—0 i
used to different types of
f 4 jj^cm2 to epinephrine
I
Time (min) Tlme(nin)
-•- control (n=10|
-O- Fullerene(n=3)
-•- Sinojewdled (n=3)
-O-FVUtiwdled(n=3|
Hormonal response of Calu-3 cells exposed to different types of
nanopartidesfoM hat concetration of4pgfcm2 to epinephrine
142
-------
H2p Apical (filtrate)
Aquapori
Epinephrine
Increase in cAMP concentration after epinephrine stimulation
Fold increase in cAMP after epinephrine stimulation
Concentration Control
of
CNP(Mg/em2)
4
4
0.004
0.004
6.5
8.3
11.3
5.3
*CNP treated
7.6
6.6
11.8
7.8
*CNP Treated = 48 hn= Carbon nanoparticle treated cells at concentrations indicated
Summary
Low dose nanotube exposures decreases the
barrier function of airway epithelial cells.
Low dose nanotube exposures affects the
ability of the airway epithelial cells to secrete
chloride.
These data suggest that levels of nanotubes
found in the workplace, particularly during
chronic exposures, are likely to have
physiological effects that can cause or
exacerbate respiratory problems.
hanks
Preliminary Imaging study
0.4 ng/cm2
4 ng/cm2
40 ng/cm2
143
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2010 U.S. EPA Nanotechnology Grantees Meeting
Galya Orr
Toxicity Assessment of Nanomaterials in Alveolar Epithelial Cells
at the Air-Liquid Interface
Galya Orr, YumeiXie, Nolann Williams, Ana Tolic, Justin Teeguarden, and Alexander Laskin
Pacific Northwest National Laboratory, Richland, WA
Airborne nanomaterials that enter the respiratory tract are likely to be deposited in the alveolar region,
where alveolar epithelial cells are found at the interface with ambient air. These cells provide a vulnerable
target for particles that escape the first line of defense by the alveolar macrophages. To date, the majority of in
vitro studies characterizing the interactions and impact of engineered nanomaterials in these cells have been
carried out in cells submersed under growth media. To more closely mimic in vivo exposures, we have
established the growth of alveolar type II epithelial cells (CIO cell line) at the air-liquid interface (ALI),
enabling realistic exposures to aerosolized nanoparticles. This approach supports accurate quantification of the
delivered particles per cm2 (or particles per cell) by collecting the particles on millimeter-size grids or glass
cover-slips, placed randomly over the cells and visualized using electron or fluorescence microscopy,
respectively. This approach also enables physical and chemical characterizations of the collected nanoparticles,
providing properties that are relevant to airborne nanoparticles and the actual exposure at the air-liquid
interface. The cells have been cultured on membrane inserts and initially grown under submersed conditions
until reached confluence. The apical surface of the cell monolayer was then exposed to ambient conditions for
24 hours, and the integrity of a representative subset of the cells has been monitored using propidium iodide,
quantified by fluorescence microscopy. Exposures to aerosolized nanoparticles, generated using a vibrating
membrane nebulizer, have been done over 10-minute sessions using an enclosed exposure chamber that
ensures uniform particle delivery. The cells have been maintained at the ALI until assayed for lactose
dehydrogenase (LDH) release to evaluate cell damage at 6 and 24 hours post exposure, and for proliferation
rate (MTS) to evaluate viability at 24 hours post exposure. Using the above conditions, we found that
exposures to 50 nm bare amorphous silica nanoparticles, containing embedded fluorescent dye molecules,
elicit no significant cytotoxic response at concentrations ranging from 10 to 1000 particles per cell. These
observations agree with observations we obtained in submersed cells exposed to equivalent doses, as estimated
by a computational particokinetics (sedimentation, diffusion) model. Studies with surface modified animated
50 nm amorphous silica nanoparticles and other particles that have shown to elicit toxic responses in
submersed conditions are currently being pursued at the ALI.
EPA Grant Number: R833338
The Office of Research and Development's National Center for Environmental Research 144
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Principal investigator did not authorize publication of the presentation.
145
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2010 U.S. EPA Nanotechnology Grantees Meeting
Jonathan D. Posner
Interactions of Nanomaterials With Model Cell Membranes
Jonathan Posner1'2, Wen-Che Hou13, Steve Klein1'2, Babak Moghadam12, Charles Corredor1'2,
Kiril Hristovski4, and Paul Westerhoff
1 Chemical Engineering, 2Mechanical Engineering, 3Environmental Engineering, 4 School of Sustainable
Engineering and the Built Environment, Arizona State University, Tentpe, AZ
Toxicological studies of engineered nanomaterials (ENMs) have primarily focused on the toxicity and
uptake of ENMs by a variety of organisms, including human cell lines, microbes, plants, or aquatic organisms
such as fish and Daphnia magna. Because the reported results vary depending on the organisms and test
conditions, it is difficult to draw a comprehensive conclusion of ENMs' environmental impact based on these
empirical studies, especially considering the ecological diversity and wide range of ENMs' properties. The
partitioning between the organic solvent phases (typically n-octanol) and water (K0w) has traditionally been
used as an empirical approach to evaluate the bioavailability of organic pollutants and is used extensively in
current EPA models. For colloids, mechanistic and dynamic fate models in aqueous matrices are more
complex than for organic pollutants and require multi-parameter input to describe the colloid transport and
interactions with soils and biota. Characterization of ENMs often involves numerous physical measurements of
size distribution, surface area, porosity, aqueous zeta potential, surface chemistry, and stability. However, it is
challenging to transition from these precise measurements to models suitable to assess fate and bioavailability
of ENMs in the environment, especially in complex matrices. Analogous partition type global descriptor
methods have not been used extensively for nanomaterials; therefore, there is a need to develop empirical
model approaches for predicting bioaccumulation of ENM that account for the collective influence of ENM
properties, in a similar way as Kow depends on multiple parameters of organic pollutants (molecular weight,
conformation, hydration states, ionic charge, etc.).
In this talk, we quantify the lipid-water distribution coefficients for ENMs and use them as a global
descriptor that captures the critical interactions between ENM and biological interfaces, which may be used to
predict the bioaccumulation potential of ENM. The lipid-water distribution ratio has been shown to be a more
appropriate descriptor than Kow partitioning for biological uptake and bioaccumulation of hydrophobic
ionizable compounds and surfactants, which ENMs share similar properties (e.g. charged, resident at
interfaces). Lipid bilayers' mass is nearly all at the interface that eliminates the difficulty encountered in the
octanol-water partitioning of surface-active compounds and some types of ENMs that also partition to the
interfaces.
We evaluate the lipid-water distribution of aqueous C6o clusters (nC6o) and polyhydroxylated C6o
aggregates (i.e., fullerol) using lipid bilayers that mimic biological membranes. The kinetic studies indicate
that the distributions of nC6o and fullerol between lipid bilayers and aqueous phases reach pseudo-equilibrium
after 2 days and 2 h of equilibration, respectively. The pseudo-equilibrium distribution of nC6o and fullerol can
be described by isotherm-like behaviors that fit reasonably with Langmuir isotherms. Although both nC6o and
fullerol exhibit pH-dependent distribution behaviors with accumulation in lipid bilayers increasing
systematically with the decrease in pH from 8.6 to 3, the distribution coefficients of nC6o is up to 1 order of
magnitude larger those of fullerol. The pH-dependent distribution trend is consistent with the decrease in the
electrostatic repulsion between lipid bilayers and fullerene aggregates, as the zeta potentials of both increased
(i.e., became less negative) as pH decreased.
The lipid bilayers that make up cellular membranes are believed to be impenetrable to ions and
unfunctionalized macromolecules; however, epidemiological studies have shown that unfunctionalized ENMs
can, under some conditions, cross or disrupt the cell membrane through passive, unmediated routes causing
The Office of Research and Development's National Center for Environmental Research 146
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2010 U.S. EPA Nanotechnology Grantees Meeting
acute cellular toxicity and cell death. The unmediated ENM disruption of cellular membranes is poorly
understood. Also, we are developing methods for understanding the mechanisms and conditions under which
engineered nanomaterials can cause disruption of, and passive transport through, simplified model cell
membranes, namely lipid bilayers. We will show that some ENM disrupt membranes allowing the flux of ions
across the membranes.
Distribution of ENM between the aqueous phase and biologically relevant interfaces and disruption of
bilayers by ENM may ultimately be used as high-throughput global descriptors for predicting bioaccumulation
and toxicity of ENM. Future work includes (1) extending current work to include ENMs of other core
compositions, sizes, shapes, and surface functionalities as well as water chemistry parameters such as ionic
strength and ion species; (2) correlate with existing or future bioaccumulation studies using aquatic organisms;
and (3) translating methods to high-throughput formats.
DOE Grant Number: DE-FG02-08ER64613
The Office of Research and Development's National Center for Environmental Research 147
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Interactions of Nanomaterials with
Model Cell Membranes
Jonathan D. Posner1, Wen-Che Hou1 2,
Steve A. Klein1, BabakY Moghadam1,
Charles Corredor1, Kiril Hristovski2, Paul Westerhoff2
1Chemical Engineering, Mechanical Engineering
2School of Sustainable Engineering and the Built Environment
Arizona State University, Tempe, AZ 85287-6106
ASU
DOE BER: DE-FG02-08ER64613
Using Global Descriptors to Predict ENM Fate and Transport
Properties
Octanol-Water Partitioning Coefficient
• Ratio of concentration of solute in between two
immiscible phases, generally octanol and water.
• Used in water quality models (WASP, QUAL2K,
Aquatox, EPD-RIV1) to predict fate, accumulation,
aquatic toxicity of organic pollutants in the
environment.
• Required for high-volume chemicals
• Methods published in OPPTS
• Methods most appropriate for unionizable chemicals
such as many organic chemicals
• More difficult to interpret for ionizable substances
• Not defined for particles
Octanol
Water
ASU
Octanol-Water Partitioning of ENM
• 60 mL Teflon cap glass vials
• 1 mM NaHCO3 buffer (bicarbonate)
• Mixed for 3 days at 30 rpm
• Phases separated
• Vary pH, ionic strength
•high pH > 11 (NaOH)
•lowpH<3 (HN03)
Partitioning of Hematite Fe2O3
ASU
Octanol-Water Partitioning of ENM
oclsnol
Interface
water
Partitioning of Hematite Fe2C
•Some particles at interface
•Minimization in Helmholtzfree energy
•Not quantified or treated in classical partitioning theory
ASU
Octanol-Water Partitioning of ENM
ENM at Octanol-Water Interface
•Some particles at interface
•Minimization in Helmholtz free energy
•Not quantified or treated in classical partitioning theory
ASU
148
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Octanol-Water Partitioning of ENM
Minimum in Helmholtz Energy
E.
•Some particles at interface
•Minimization in Helmholtz free energy
•Not quantified or treated in classical partitioning theory
•Basis for Pickering emulsions
J5U
Octanol-Water Partitioning
Observe that ENM partitioning experiments result in combination of three
primary, path and solution chemistry dependent scenarios:
A- in the aqueous phase
B - In the octanol
C-at interface
J5U
Challenges with ENM Octanol-Water Partitioning
Importing into EPA models
How do we treat mass at interface?
Partitioning gives no information on state on ENM (aggregation
and settled in water, dissolved, suspended, emulsion)
Partitioning is path dependent
Does not correlate with bioacumulation*
Partitioning dependent on poorly defined interfacial area
;tersen et al. Relevance of Octanol-Water Distribution Measurements to the potential Ecological Uptake of
Multi-Walled Carbon Nanotubes, Environmental Toxicology and Chemsitry, 2010
HSU
ENM Distributions in Lipid-Water Systems
• Lipid bilayer is an important interfaces between life and its
environment and a potential exposure route to EN Ms.
• The lipid bilayer-water distribution (K,j,w) has been shown to be a
more appropriate indicator than (Kow) for bioaccumulation of
ionizable organic molecular and surface active compounds, which
ENMs share some properties (e.g., charged surface and residence
in interface).
• Klpw is increasingly used by the pharmaceutical industry and
environmental research for drugs and molecular pollutants.
• All mass is at the surface
• Surface area can be controlled and quantified
J5U
Lipid Bilayers
Primary constituent of many biological cellular membranes.
Often used to model passive transport into cells.
JSU
Lipid bilayer-water distribution on Solid Supported Lipid Bilayers
Lipid bilayer
Solid-supported lipid £NMs
membrane (SSLM)
not drawn to scale
Lipid Bilayer noncovalent bond
to silica
Bilayer is fluid
Bilayer robust over wide range
electrolyte conditions
JSU
149
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Lipid Bilayer-Water Distribution of ENMs Method
Calculation of lipid bilayer-water distribution coefficients |
flee IHMt. £,„
X-
EHMt,C,c..,
Validated using a reference
compound (2,4,6-trichlorophenol).
^^, * [ENMs] ui ilw cont
.** = fr«c[F-NMs] in
m>,9 =• lipid cotKMittaii
log Kipw = 3.89 ± 0.03 (L/kg), close to
3.90 reported by Escher et al. (ES&T,
it equilibrium
J5U
ENMs and Lipid Sources and Preparations
ENMs:
• Aqueous C^ aggregates (nCgo): dry C60 powders (MER, Tucson, AZ) were
pulverized and then mixed with Dl water for 2 weeks prior to passing through
0.7 and 0.45 urn filters sequentially (Hou et al., 2009).
• Fullerol (CeotONaJ^OH)^ x + y = 24): dry fullerol powder (MER, Tucson, AZ)
was directly mixed with water and then passed through 0.7, 0.45, and 0.2 urn
filters sequentially.
• Gold nanoparticles (nAu) were tannic acid coated and well-characterized
gold colloids at 5, 10, 20, 50, and 70 nm, purchasing from nanoComposix (San
Diego, CA).
Lipid bilayers:
• SSLMs were purchased from Sovicell (Leipzig, Germany). The lipid
composition was 100% phosphatidylcholine from chicken egg.
• Unilamellar lipid bilayer vesicles (i.e., liposomes) were prepared using the
same lipid composition as SSLMs by the extrusion method (Hope et al.,
1985). Liposomes were used to determined the effective zeta potential of
lipid bilayers.
Analytical methods
nCgo concentration was determined by high performance liquid
chromatography (HPLC) with UV detection at 336 nm. Because HPLC is only
applicable to molecules, molecular C60 was extracted from the aqueous phase
to toluene in the aid of 0.1 M Mg(CIO4)2. The toluene extract was injected to
HPLC (Hou etal., 2009).
Fullerol concentration was determined by UV-visible absorption spectroscopy
using UV at 254 nm.
nAu concentration was determined by inductively coupled plasma-optical
emission spectroscopy (ICP-OES). Prior to ICP, nAu was dissolved in aqua
regia (i.e., 1 part of HNO3 and 3 parts of HCI).
Lipid concentration was determined by the malachite green dye assay (Petitou
et al., 1978). Before assay, lipid was digested, adding concentrated H2SO4 and
H2O2 under heating.
The sizes and zeta potential of liposomes and ENMs were determined by
dynamic light scattering (DLS) (NICOMP 380 ZLS, Particle Sizing Systems,
Santa Barbara, CA).
HSU
Size and Effective Zeta
20
10 -
_ 0
1 -10
1 -2°
o
0 -30
a,
"* -40
-50
-60
, , ,,,
• 50 100150200250
Size (nm)
Bi
'*,,
Lipid bilayer vesicles (•)
Fullerol 650 mg/L (»)
nC60 6.5 mg/L (A)
nC60 and fullerols
have similar size
distributions and
charge
2 3 4 5 6 7 8 9 10 11 12 13 14
PH ^J
Fi
1.2
1
- 0.8
S o.s
~ 0.4
D.2
0
illerol and nC60 Lipid
(a) nCu
L
* * AD
n
•f
1
• •
10 20 30 40 S
Time (h)
Bilaye
1.2
1
f 0.8
|;0.6
30.4
0.2
0
0
Interaction Kinetics
(b) fullerol
1 $ •> i
' '
* * •
Tim« [HI
pH=3 («) and control (0) pH=5 (•) and control (n) pH=7.4 (A) and control (A)
[lipid] = 0.47 mM — Controls are vial without SSLM — [nC60]0 = 6.5 mg/L at pH = 7.4 and
5. [fullerol]0=8.0 mg/L (pH 7.4); 11 .0 mg/L (pH 7.4), and 11 .0 mg/L (pH 3)
JGU
Ui/.«\ivmi
nC60 Mass Balances
1
7
S6
(a) PH=7.4
* ' *** 0137 20.6 29.6 43.5
Tima(h> Tim* |h)
Black: free nC60 —Grey: nC60 in SSLMs —White: nC60 lost to walls
Serial ENM extraction from SSLM by original electrolyte then toluene
ASU
150
-------
Distribution of nC60 and Fullerol in Lipid-Water
PH=5(.)
pH=7.4(A)
1
I- 10000
• Lines are Langmuir isotherms
• Bars indicate one standard deviation.
ASU
Comparisons with Daphnia Bioaccumulation
& lonizable Organic Compounds
lonizable compound
Show pH dependent
partitioning
Compare with chlorophenol
compounds (Escher 1996)
0.1 1 10 100
C,,,, (mg/L)
*Bioaccumulation of nC60 in aquatic organisms (Daphnia magna)
(Tervonen et al., 2010)
J5U
Comparisons with Daphnia Bioaccumulation
& lonizable Organic Compounds
Studies suggest higher bioaccumulation and toxicity of nCg0 than fullerol:
•Kiseret al., H/aferRes., 2010. (biosorption using wastewater biomass)
•Sayes et al., Nano Lett., 2004. (toxicity to human cell membranes)
•Zhu etal., Environ. Toxicol. Chem., 2007. (toxicity to fish)
•Fang et al., Environ. Sc/. Technol., 2007. (toxicity to bacteria membranes)
Lipid-Water Distribution Isotherms of nAu
10000 •
1
£1000 •
1
o
0.
AlOnm
•20 nm
A
by mass
0.1
i
-0.01
1
•5nm
•50 nm
ATOnm
fl
"**.'
by surface area
1 10 10C O-001 °-01
C,via, (mg/L)
Measure
mol/mol
area/area
particle #/kg
J'LE+I?
(fe
s
1.E+15
•
by particle #
-->' '° "'" ''E'^'r "'
[nAu]04lipid]
0.03 to 0.32
0.01 to 0.476
1016to1019
Also observed for cells.
Chithrani et al. Nanoletters
2006
[nAu]0=3-30 mg/L, [lipid]=0.47 mM, and pH = 7.4) ISU
SEM Images of 50 nm nAu Adsorbed on SSLMs
-.
-* -41 \J+
1
Summary OF Lipid-Water Distributions
The lipid bilayer-water distribution of the selected ENMs is pseudo-
equilibrium process that can be described by isotherm behaviors.
The distribution behavior is pH dependent
Accumulation to lipid bilayers increasing as pH dependent (electrostatics)
Analogous distribution behaviors of ionizable organic pollutants such as
chlorinated phenols.
Size dependency studies show that 20 nm gold nanoparticles exhibit the
highest propensity to accumulate in lipid bilayers.
Comparisons with bioaccumulation and toxicity studies using organisms
suggest that the lipid bilayer-water distribution is promising for assessing
the bioaccumulation and toxicity potentials of ENMs.
Need bioaccumulation data (BCF) data on variety of ENM to verify if the
lipid-water distribution can be used for predicting ENM fate.
ASU
151
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backup
25,
J5U
Lipid bilayers as model cell membranes
Lipid bilayers are the
primary constituents of
many biological cellular
membranes. Arguably the
most important interface
between cellular life and its
surrounding environment.
Lipid - amphiphilic
molecule that can
spontaneously arrange in
aqueous solution to have a
hydrophobic interior and
hydrophilic exterior.
26
Focus
• Assess the affinity of ENPs to model cell membranes by
quantifying ENP distribution between lipid bilayer and water.
• ENPs: C60 and polyhydroxylated C60 (i.e., fullerol) aggregates
•S Applications in cosmetics, energy production, catalysts, etc.
S Focus of recent ecotoxicological studies
•S Fullerol-like materials is potential transformation products of C60 in the
environment.
• pH dependency: pH = 3, 4, 5, 7.4, and 8.6 using phosphate
electrolytes (5-20 mM), covering the pH range of physiological
and environmental conditions,
• Interaction kinetics
• Pseudo-equilibrium isotherms: Langmuiror Freundlich model
• Comparisons with existing aquatic organism bioaccumulation
and toxicity studies and with lipophilicity of ionizable organic
pollutants. BS1I
T7 WiFMSTMl
£. / I^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^H Usivtitsm
Mass Balance
J5U
Mass Balance
Extract w/original
upernatants electrolytes at pH =
drawn for 7.4 or 5 and then w/
nalysis (Cw) electrolyte at pH = 8.6
JSU
Mass Balance
Extract w/ original
upernatants electrolytes at pH =
drawn for 7.4 or 5 and then w/
nalysis (CJ electrolyte at pH = 8.6
Supernatants
drawn for analysis
(^electrolytes)
AS1I
152
-------
Mass Balance
Extract w/original
electrolytes at pH =
7.4 or 5 and then w/
electrolyte at pH = 8.6
Resuspended in H2O
and transferred to
Supernatants new clean vials
drawn for analysis
(^electrolytes)
JSU
Mass Balance
Extract w/ original
Extract w/ toluene
analysis (CJ electrolyte at pH = 8.6 (Electrolytes)
*
Extract w/toluene
Ctotal = Cw + Celectrolytes (Combined) + CNp + Cwa||
Table 1. Parameters of the Freundlich and Langmuir models
fitted to nCfin and fullerol isotherm data.
Freundlich model:
Langmuir model:
? = ^Hp,max'^ails'^w,eil
logKF
(Ln/mg"-f -kg lipid)
(mg/kg lipid) (L/mg)
O.SS 0.95
0.74 0.98
4.21
4.02
0.94 0.95
0.54 0.95
0.37 0.98
0.54 0.76
4.70 0.2 0.91
5.05 0.009 1.00
5.70
4.70
3.52
3.22
0.90
0.98
0.94
0.98
0.85
Gold nanoparticles size measurements
in Dl water
J5U
3: Wtir vaiK. tfecn .markt^: h>cafc rtijt
153
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2010 U.S. EPA Nanotechnology Grantees Meeting
Yongsheng Chen
Development of an In Vitro Test and a Prototype Model To Predict
Cellular Penetration of Nanoparticles
Yonsshens Chen , David Capco , and Zhongfang Chen
1 School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA;
2School of Life Sciences, Arizona State University, Tempe, AZ;
3Department of Chemistry, University of Puerto Rico, San Juan, PR
The aim of this study is to gain fundamental insight into the nanoscale properties of engineered
nanomaterials and the relationships with environmental and biological impacts. The overarching questions we
are trying to answer are how nanoparticles interact with the environment and cells and how these interactions
will impact the environment and biological systems. To answer these questions, we have been focusing on
characterizations of NPs in the environment and investigating the nano-bio interactions over the past 1 year.
Characterizations. We are trying to obtain the physicochemical parameters of various nanomaterials of
interest by experiments and theoretical computations. One reason is that the environmental behaviors (e.g.,
fate, transport, and biological impacts) are significantly influenced by their inherent nanoscale properties and
characterization of these properties is critical for better understanding their environmental impacts. For
experimental research, we focused on Fe203, Ce02, and Ag to conduct substantial experiments. Our results
indicated that except hematite (a-Fe203), Ce02, and Ag NPs had larger hydrodynamic diameters than the
diameter measured by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Our
AFM images revealed that particle aggregation occurred instantly when they were dispersed, especially at the
presence of electrolyte (e.g., KC1) in the solution. The particle size and particle size distribution influenced by
the solution chemistry show significant effects on their fate and biological impacts. Thus, we conducted
aggregation kinetics experiments, which were studied by time resolved-dynamic light scattering (TR-DLS)
technique. Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and the attachment efficiency (or inverse
stability ratio) was used to distinguish the aggregation into two regimes, diffusion-limited and reaction limited
regimes, which were both observed for the aggregation kinetics of Ce02 and Ag NPs under ionic strengths
between 0.005 and 0.1 M. Based on DLVO theory, we developed a combination of Arrhenius equation,
extended DLVO theory, and von Smoluchowski's population balance equation for modeling the aggregation
kinetics, which was not only suitable for interpreting the aggregation kinetics in the initial aggregation stages,
but also applicable in the transition and post-aggregation stages. In particular, this work lays the groundwork
for developing appropriate mathematical descriptions of nanoparticle behaviors and provided insight into
aggregation kinetics mechanisms. For example, particle aggregation is dominated by the interplay of van der
Waals, electrostatic, and acid-base forces and particle size as well as particle surface charge (indicated by zeta
potential) that contribute to the magnitude of energy barrier, which governs the aggregation kinetics rate. In
quantum calculation, we obtained theoretical predictions of various nanomaterials (e.g., ZnO) on their
structural, electronic, and magnetic properties, and these predictions can not only help our experimental work,
but also provide potential physico-chemical properties to develop our prototype model.
The biological interactions. In this aspect, we carried out our studies through two approaches; one is
quantifying the cellular exposure to NPs to establish the quantitative description and correlation in the
nanotoxicity database, and the other is developing imaging techniques to provide a visualization of the
exposure impacts on cells at the nanoscale. For cellular exposure, we conducted experiments with Caco-2
cells, which are a model epithelium for human intestinal cells and Escherichia coli (E. coli) cells, which are
one of the most common microorganisms widely present in the environment and widely used in toxicological
tests. Cellular impairment of Caco-2 cells was evaluated by measuring transepithelial electrical resistance
(TEER). Cell surface disruption, localization, and translocation of NPs through the cells were further analyzed
The Office of Research and Development's National Center for Environmental Research 154
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2010 U.S. EPA Nanotechnology Grantees Meeting
with immunocytochemical staining and confocal microscopy. Our results showed that hematite NPs reached
adsorption equilibriums after approximately 5 min but adsorption kinetics were size dependent. After the
adsorption equilibrium and a longer exposure time (> 3 hr), severe cellular effects were observed from the drop
of TEER compared to the control cells. Hematite NPs triggered a dynamic reorganization and detachment of
microvilli structures from Caco-2 cell surfaces. Particularly, the confocal microscopy revealed that the
exposure to 26 nm disrupted the cellular junctions more severely than larger sizes. Similarly, the adsorption
kinetics of hematite NPs upon E. coli cells was also found size dependent. The adsorption rates expressed as
mg Fe-L" -s" decreased in the order of 98 nm > 76 nm > 53 nm > 26 nm. However, adsorption rates expressed
as the number of adsorbed hematite NPs per unit cell surface area (#-m~2-s~l) were faster for small NPs than
those for large NPs. To interpret the size effects on adsorption kinetics, the extended DLVO theory was
combined with interfacial force boundary layer (IFBL) theory. The theories divided the adsorption into two
regimes, one is dominated by interfacial forces and the other is dominated by diffusion. Faster kinetics for
smaller NPs could be attributed to faster particle mobility and lower energy barriers in the total interaction
energy according to the derived adsorption rate from EDLVO-IFBL theories. To visualize the exposure
impacts on E. coli cells, we developed an AFM-based imaging technique as a novel tool to investigate the NP-
cell interactions. Our results demonstrated for the first time AFM's superior performance in resolving the
individual hematite NPs interacting with live E. coli cells, which provided a striking visualization of the
adsorption of hematite NPs onto E. coli cells and the subsequent disruption in their extracellular appendages
(flagella).
The major challenge we encounter for exposure experiments, also recognized by other groups, is the
difficulty in maintaining a stable dispersion of NPs in the biological relevant solutions. In most cases, NPs
aggregate rapidly and transit to colloidal particles (amorphous and large size clusters). The biological impacts
observed from such toxicity experiments may not be representative of what real NPs exhibit. Another issue we
identified through adsorption experiments is the concentration of NPs, which is used to establish the
relationship of dose-response in risk evaluation. However, NPs, due to the size distribution, may require a
number-based concentration rather than the mass-based concentration alone for establishing dose-response risk
assessment.
Next year, we will continue to extend our developed methodologies to other types of NPs (besides,
hematite, Ce02, Ag, and QDs) to study the environmental behaviors such as aggregation and ion release.
Furthermore, we will establish AFM-based methods for quantifying the surface characteristics of NPs and their
interactions with the biological system.
References:
1. Wen Zhang, Madhavi Kalive, David G Capco, Yongsheng Chen. Effect of nanoparticle size on adsorption
onto E. coli and Caco-2 cells and the role of adhesion force. Nanotechnology 21 355103.
2. Wen Zhang and Yongsheng Chen. Effect of nanoparticle size on bacterial cell adhesion force. Colloids
and Surfaces B: Biointerfaces, 10.1016/j.colsurfb.2010.09.003
3. Wen Zhang, Kungang Li, Yongsheng Chen. A novel approach for analysis of aggregation kinetics of Ce02
nanoparticles. Environmental Science and Technology (under review).
4. Wen Zhang, Joe Hughes, Yongsheng Chen. Imaging and quantifying nanoelectric properties of hematite
nanoparticles interacting with E. coli cells. Environmental Science and Technology (under review).
5. Wen Zhang, Ying Yao, Yongsheng Chen. Quantifying and imaging the morphology and nanoelectric
properties of soluble quantum dot nanoparticles interacting with DNA. Journal of Physical Chemistry
(under review).
6. Lu Wang, Yiyang Sun, Kyuho Lee, Damian West, Zhongfang Chen, Jijun Zhao, Shengbai Zhang. Stability
of graphene oxide phases from first-principles calculations. Physical Review B (Rapid Communication)
2010, accepted.
7. Xingfa Gao, De-en Jiang, Yuliang Zhao, Shigeru Nagase, Shengbai B. Zhang, Zhongfang Chen.
Theoretical insights into the structures of graphene oxide and its chemical conversions between graphene.
Journal of Computational and Theoretical Nanoscience 2010, accepted
The Office of Research and Development's National Center for Environmental Research 155
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2010 U.S. EPA Nanotechnology Grantees Meeting
8. Qing Tang, Yafei Li, Zhen Zhou, Yongsheng Chen, Zhongfang Chen, Tuning electronic and magnetic
properties of wurtzite ZnO nanosheets by surface hydrogenation. ACS Applied Materials & Interfaces
2010;2:2442-47.
9. Yiyang Sun, Kyuho Lee, Lu Wang, Yong-Hyun Kim, Wei Chen, Zhongfang Chen, SB Zhang. Accuracy
of density functional theory methods for weakly bonded systems: the case of dihydrogen binding on metal
centers. Physical Review B. 2010;82:073401.
10. Jijun Zhao, Lu Wang, Fengyu Li, Zhongfang Chen. B80 and other medium-sized boron clusters: core-
shell structures, not hollow cage. Journal of Physical Chemistry A. 2010;! 14:9969-72.
11. Jijun Zhao, Zhongfang Chen. Highlighted by Chem. Eng. News as Science & Technology Concentrates
(August 16 issue, Page 40) Highlighted as a cover picture, Forward, A Special Issue on Structures,
Properties, and Applications of Nanomaterials: a Computational Exploration. Journal of Computational
and Theoretical Nanoscience (editorial) 2010, accepted
12. Yafei Li, Zhen Zhou, Peng Jin, Yongsheng Chen, Shengbai Zhang, Zhongfang Chen. Achieving
ferromagnetism in single-crystalline ZnS wurtzite nanowires via Cr-doping. Journal of Physical Chemistry
C.2010;114:12099-103.
13. Kyuho Lee, Yong-Hyun Kim YY, Sun D. West, Yufeng Zhao, Zhongfang Chen, SB Zhang. Hole-
mediated hydrogen spillover mechanism in metal-organic frameworks. Physical Review Letters
2010;104:236101.
14. Chang Liu, Zhongfang Chen, Chen-Zhong Li. Surface engineering of graphene-enzyme nano composites
for miniaturized biofuel cell. IEEE Transactions on Nanotechnology 2010, accepted
15. Peng Jin, Fengyu Li, Kevin Riley, Dieter Lenoir, Paul v. R. Schleyer, Zhongfang Chen. Meisenheimer-
Wheland complex between sym-Triaminobenzene and 4, 6-Dinitrobenzofuroxan. Journal of Organic
Chemistry 2010;75:3761-65.
16. Peng Jin, Zhen Zhou, Ce Hao, Zhanxian Gao, Kai Tan, Xin Lu, Zhongfang Chen. NC unit trapped by
fullerenes: a density functional theory study on Sc3NC@C2n (2n=68, 78 and 80) PCCP 2010, DOI:
10.1039/B923106D. Highlighted as a cover picture
17. Yafei Li, Zhen Zhou, Zhongfang Chen. Polyphenylene: experimentally available porous graphene as
hydrogen purification membrane. Chemical Communications 2010;46:3672-3674. Highlighted by Nature
China.
18. Tristram Chivers, Robert W. Hilts, Peng Jin, Zhongfang Chen, Xin Lu, Anne Pichon. Separation materials:
An ace in the hole. Published online: 2 June 2010, doi:10.1038/nchina.2010.71
19. Yafei Li, Zhen Zhou, Guangtao Yu, Wei Chen, Zhongfang Chen. CO catalytic oxidation on iron-embedded
graphene: a computational quest for low-cost nanocatalysts. Journal of Physical Chemistry C.
2010;! 14:6250-6254. Highlighted as cover picture
20. Wei Chen, Yafei Li, Guangtao Yu, Chenzhong Li, Shengbai Zhang, Zhen Zhou, Zhongfang Chen.
Hydrogenation: a simple approach to realize semiconductor—half-metal—metal transition in boron nitride
nanoribbons. Journal of the American Chemical Society 2010;132:1699-1705. Highlighted by
www.nanowerk.com (http://www.nanowerk.com/spotlight/spotid=15647.php).
EPA Grant Number: R83385601-1
The Office of Research and Development's National Center for Environmental Research 156
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Development of an In Vitro Test and a
Prototype Model To Predict Cellular
Penetration of Nanoparticles
o
PI: Yongsheng Chen*'1, Co-Pi: David Capco2, and Zhongfang Chen3
* Current Affiliation: School of Civil and Environmental Engineering, Georgia
Institute of Technology, GA 30332; Email: yongsheng.chen@ce.gatech.edu; Phone:
(+1) 404-894-3089;
1 Previous Affiliation: Department of Civil and Environmental Engineering,
Arizona State University, Tempe, AZ 85287;
2 School of Life Sciences, Arizona State University, Tempe, AZ 85287;
3 Department of Chemistry, University of Puerto Rico, San Juan, Puerto Rico
00931-3341.
Tfcch
Bio-nano Interface
Governing parameters:
Nanoparticles (NPs): Size,
aggregation state, surface
chemistry, hydrophobicity,
surface geometry, surface
roughness, surface charge, etc
Cell: Protein, lipo-polysaccharide,
hydrophobicity,
metabolic/signaling response, etc
Environment: pH, temperature,
ionic strength, organic content,
etc
Biological consequences:
>lnterfacial forces;
>Sorption processes;
>Surface accumulation, permeation,
penetration, and cellular
accumulation;
> Cellular damages (membrane
disruption, machinery malfunction,
genetic mutation)
KtmdKOI * I li^tMm* fjff^- urt*'
/\ Mr^^^
f -rfrjJrr*.
limillllllinir, I'ftfl '.llUIMIlllllMIIKI ^S
Andre E. Nel, et al., Nature tumotechnology. 2009
Today's talk
Characterizing, Imaging, and Quantifying the
Biological Interactions with Engineered
Nanomaterials.
Questions to answer
How particle size impacts the biological
interactions (e.g., interfacial forces, adsorption
kinetics, cell surface disruption)?
Nanomaterials
a-Fe2O3
Ce02
Ti02
ZnO
A12O3
CuO
Si02
QDs
Au
Ag
and their Characterizations
^•Morphology, size, nanoelectric, and
adhesive properties (TEM, SEM, and
AFM);
^•Surface energy, hydrophobicity, and
crystallographic analysis (contact
angle and HRTEM);
>Hydrodynamic sizes and zeta
potential (DLS);
^•Environmental behaviors:
aggregation, metal ion release, and
bioaccumulation (DLS and ICP-MS);
Escherichia coli (E. coin cells
Bio-nano interactions: hematite NPs versus Caco-2 cell
line
Caco-2 cell line
>Adsorption kinetics
^•Membrane disruption by
scanning electron
microscopy
>Transepithelial Electrical
Resistance (TEER)
measurement and confocal
microscopy to indicate cell
penetration
157
-------
Results: Bio-nano interactions using Caco-2 Cell line
Microvillus disrupt!
> Structural damage
>Cellular integrity
^nutrient absorptio
Adhesion junctional
disruption
Green network:
y catenin antibody
stained junctional
structure
Blue dots: nucleus
1511
j Zhai
;, Wen, et al2010 Nanotechnology 21 355103 doi: 10,1 ^ •-?*''" •• ;B4/21/35/355103
Structural disruption of microvilli on the cell surface
Intact microvilli
Affer the exposure
^The left SEM image shows the morphological changes of microvilli on
Caco-2 cell surfaces after the exposure to hematite NPs;
>The right Carton shows the possible mechanism of depletion attraction by
which the structures of microvilli were affected.
technology21 355103doi: 10.1088/0957 •?'-< ,J 35 '.= :,>•
m
Results: Adsorption kinetics on Caco-2 cells
^Adsorption kinetics
show similar features
(e.g., size dependency)
as hematite NFs did
with E.coli cells;
>Large particles
adsorbed faster by
mass-based
concentrations;
^Small particles
adsorbed faster in
number-based
concentrations.
^ettd2010 Nanotechnology 21 355103 doi: W.IQSK^ '-4484 .1 , &55103
Results: Size affects the disruption of adhesion junction
and cell penetration
Small NFs penetrated cell lines faster and led to severer junctionai
disruption.
Zhang, Wen, et(d2QlQNanotechnology21 355103 doi: 10.1088/0957-4484/21/35/355103
Conclusions
> Hematite NPs is ideal for use as a reference
nanomaterial due to the high stability with
uniform size distributions and low aggregation;
^ Adsorption kinetics is size dependent, which can
be interpreted by IFBL theory;
^ The exposure to hematite NPs induced
reorganization and distortion of surface structure
damages (e.g., microvilli and flagella), and cell
penetration.
Challenges and problems
What are the roles of interfacial forces and diffusion in the
transport of nanoparticles toward biological system?
DLVO theory VS mass transport mechanisms
Combined effects are
incorporated in IFBL
theory
158
-------
Next steps
^•Next year we will continue to extend our developed
methodologies (e.g., models) to evaluate other types of
NPs (e.g., CeO2, Ag, and QDs) in their environmental
and biological behaviors.
^•Furthermore, we will develop sophisticated imaging
and quantifying techniques for the surface
characterizations of NPs and their interactions with
biological system at nanoscale .
Achievements
• 15 papers published in journals, such as JACS, Nano
letters, and ACS Nano in 2009 to 2010;
• 6 manuscripts have been submitted.
• 20 invited talks or presentations in international wide
conferences.
Dr. Yongsheng Chen's
Group members
PhD students:
Wen Zhang, Kungang Li,
Jia Yang, Wei Zhang,
Nicole Sullivan.
Research engineers:
Ying Yao, Hao Jiang.
Visiting scholars:
Ying Huang,
Rongjun Su,
Yang Li.
Acknowledgements
^•U.S. Environmental Protection Agency Science
to Achieve Results Program Grant RD-
83385601;
> Semiconductor Research Corporation
(SRC)/ESH grant (425.025).
Semiconductor
Research Corporation
159
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AM Session 2: Effects at Sub-Cellular Level
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Shaily Mahendra
Impacts of Quantum Dots on Gene Expression in Pseudomonas aeruginosa
Shaily Mahendra1'2, Yu Yang2, Huiguang Zhu3, Vicki Colvin2'3, and Pedro J.J. Alvarez2
Department of Civil and Environmental Engineering, University of Calif ornia, Los Angeles, CA;
2Department of Civil and Environmental Engineering, Rice University, Houston, TX; 3Department
of Chemistry, Rice University, Houston, TX
Gene expression studies are valuable techniques for characterizing cellular responses to toxic substances
as well as identifying mechanisms of toxicity. Global gene expression of Pseudomonas aeruginosa exposed to
quantum dots (QDs) was investigated using whole transcriptome microarrays. Following exposure to 20 mg/L
Qdot 655 ITK carboxyl-coated QDs, 54 genes were downregulated while 25 genes were upregulated. QDs
artificially weathered by exposure to low pH, released Cd, Se, and Zn into the medium and caused repression
of 100 genes and induction of 40 genes. Forty-four downregulated genes and 11 upregulated genes were found
in both treatments. Quantitative PCR of impacted genes validated the microarray results (R2 = 0.92).
Gene ontology analyses revealed that classes of inorganic ion transport and metabolism, energy production
and conversion, nucleotide transport and metabolism, and DNA replication and repair were primarily
upregulated. On the other hand, in the categories of carbohydrate, coenzyme, fatty acid and lipid transport and
metabolism, cell motility, transcription, translation, and post-translational modification, the downregulated
gene numbers were higher than those upregulated.
P. aeruginosa is a Gram-negative bacterium, which contains cation-antiporter Cd and Zn efflux pumps.
Exposure to weathered QDs, which released heavy metals, caused relatively more negative impacts on gene
expression. The categories of fatty acid, phospholipid, inorganic ion and coenzyme transport and metabolism,
as well as transcription, cell motility, and energy production included more downregulated than upregulated
genes. Weathered QDs also restrained aerobic respiration and energy production genes, amino acid synthesis,
citric acid cycle, and acetyl-CoA assimilation. Although ammonium assimilation was inhibited, pathways of
anaerobic respiration, fermentation, and denitrification were induced. This suggests that significant changes in
cellular metabolism occurred in response to toxic stress.
This research will develop and disseminate critical data central to EPA's mission of environmental risk
assessment and management. Characterizing ecotoxicological impacts of engineered nanomaterials such as
QDs also will enhance future modeling efforts to support regulatory decisions, evaluate mitigation and cleanup
strategies, and the development of durable QDs. Thus, this project will contribute to strengthening our
scientific, engineering, research, education, and human resource base.
EPA Grant Number: R833858
The Office of Research and Development's National Center for Environmental Research 161
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Portland, OR, Nov. 8, 2010
Impacts of Quantum Dots on Ge
Expression in Pseudomonas aeruginosa
Shaily Mahendra
Civil and Environmental Engineering
University of California, Los Angeles
Acknowledg
Yu Yang, Huigu
Vicki Colvin, & Pedn
Quantum Dots
Biomedical Applications:
in-vivo imaging, immunoassays,
targeted gene and drug delivery
LED displays, solid state lig
OOO
Biocompatible Quantum Do
Hydrophobic core/shell contains toxic
metals (e.g., Cd and Se, Pb) surrounded by
inorganic shell (e.g., ZnS)
Can be stabilized in water by derivatizing
the surface with amphiphilic organic
coatings (may be elliptical)
Cadmium and Selenite are Toxic
Coated
loos: ; :
&
m
Weathered QDs
^ynw i^i i-^»fl nip 1 1 ]•• i in i
j *• ^
Cd2* + Se032-
|>ffi1|Cd2+
3 50 100 150 2C
Cacfrnium (mcyi_)
162
-------
Summan
Quantum dots exhibited antibacterial activity.
Microarrays were used to understand QD toxicity
mechanisms.
Coated as well as weathered QDs affected gene
expression in P. aeruginosa.
Functional categories of amino acid metabolism,
energy production, and carbohydrate metabolism
were primarily regulated.
163
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2010 U.S. EPA Nanotechnology Grantees Meeting
Terrance J. Kavanagh
Thiol Redox-Dependent Toxicity and Inflammation Caused by TOPO-PMAT
Modified Quantum Dots
Terrance Kavanash , Dianne Botta , Collin White , Chad Weldy , Lisa McConnachie ,
Jasmine Wilkerson1, Sean Gill2, XiaogeHu3, William Parks,2 andXiaohu Gao3
Department of Environmental and Occupational Health Sciences, 2Department of Medicine,
3Department of Bioengineering, University of Washington, Seattle, WA
Quantum dots (QDs) are semi-conductor fluorescent nanoparticles with potential uses in a variety of
applications. Concerns have been expressed regarding their potential toxicity, specifically their capacity to
induce oxidative stress. In this study, we assessed the effects of CdSe/ZnS core/shell QDs with atri-n-
octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coating on the respiratory
tract of mice. In vitro data in macrophages had shown that these QDs cause mild oxidative stress and secretion
of pro-inflammatory cytokines, but this was dependent on the levels of the antioxidant tripeptide glutathione
(GSH). We therefore investigated the pro-inflammatory effects of TOPO-PMAT QDs in vivo in mice
genetically engineered to have deficiencies in GSH synthesis (GCLM null mice). Mice were exposed to QDs
via nasal aspiration. Neutrophil counts in broncho-alveolar lavage fluid (BALF) increased in both wild-type
(WT) as well as GCLM heterozygous (HT) mice, whereas GCLM null (KO) mice exhibited no increase in
neutrophils. HT mice had a significantly higher level of neutrophils than WT mice. TOPO-PMAT gold
nanoparticles had no effect on neutrophil influx in either WT or HT mice. Lung cadmium (Cd) levels peaked at
1 hr in HT mice, but were similar in WT mice at 0.5 hr, 1 hr and 3 hr. Cd levels in KO mice peaked at 0.5 hr.
Levels of the pro-inflammatory cytokines KC and TNFa in BALF increased in the WT and HT mice, but not in
KO mice. There was no change in matrix mettaloproteinase (MMP) activity in the lungs for any genotype.
Neither WT nor HT mice had increased levels of myeloperoxidase (MPO - neutrophil marker). Interestingly,
there was a decrease in MPO in the KO mice relative to untreated WT mice. We conclude that TOPO-PMAT
QDs are pro-inflammatory in the respiratory tract of mice and are modulated by GSH-status. Because people
are known to carry functional polymorphisms in GCLM which compromise GSH synthesis, GCLM HT and
KO mice may be good models for investigating genetic susceptibility to QD-induced lung-inflammation in
humans.
NIH/NIEHS Grant Numbers: P50ES015915, P30ES07033, T32ES07032, andR01ESO16189
The Office of Research and Development's National Center for Environmental Research 164
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Thiol Redox-Dependent Toxicity and
Inflammation Caused by TOPO-PMAT
Modified Quantum Dots
Terrance J Kavanagh1, Dianne Botta1, Collin C White1, Chad S Weldy1, Lisa
A McComachie1, Jasmine Wilkerson1, Sean E Gill2, Jfiaoge Hu3, William
C Parks -.I'ltl ^iaohuGao3
Departments of Environmental and Occupational Health Sciences, 2Medicine,
and 3Bioengineering, University of Washington, Seattle, WA 98195
Overview
Interactions of nanoparticles with biological systems,
especially oxidative stress
Role of glutathione (GSH) in preventing oxidative
stress
Quantum dot (QD) nanoparticles as a model system
Effects of amphiphilic polymer coated QDs on
multiple cultured mouse and human cell types
Inflammatory response in normal and GSH depleted
macrophages and in mice treated with QDs
Ongoing studies
Quantum Dots (Qdots)
• Semiconductor nanocrystals
* Highly resistant to photobleaching
* Narrow fluorescence emission pattern
• Range in size from approximately 2 -150 nm
• Metalloid crystalline core
* Cadmium (Cd)
•:• Selenium (Se)
* Tellurium (Te) TIFFO-A)
•:• Indium (In) are needed t
•:• Mercury (Hg)
•:• Lead (Pb
•:• Arsenic (As)
• Cap or shell covering core
*ZincSulfide(ZnS)
• Coatings
* Biocompatible coatings
* Amphiphilic polymer w/functional groups
• Multiple uses
* Biological imaging, photo-optronics, smart dyes
* Gene and drug delivery
"Tunable" Fluorescence
Synthesis of Stable Aqueously Soluble Functionalized QDs
• Uncoated QDs often have poor
solubility and are unstable in biological
systems.
• We thus decided to mfg stable QDs for
use as in vivo tracers.
• CdSe core
•ZnS shell
• TOPO: Tri-n-octylphosphine oxide capping ligand
• PMAT: Poly (maleic anhydride-alt-1-tetradecene);
polymer with functional groups
• Exceptionally stable in aqueous solution (pH 7)
and display red-orange fluorescence
Quantum dot nanoparticles,
oxidative stress and GSH
QDs can release heavy metals (e.g. Cd, Zn) causing
oxidative stress and toxicity in biological systems
GSH is important in preventing oxidative damage to
cellular macromolecules
GSH has also been shown to be an important modulator
of the immune response (lymphocyte proliferation; antigen
presentation; T-helper cell polarization)
GSH could thus be an important determinant of QD
induced toxicity and inflammation
Glutathione (GSH)
• Tripeptide thiol (y—glutamylcysteinylglycine)
• Antioxidant properties
* Important in scavenging free radicals
• Xenobiotic conjugation reactions (GSTs)
• Other important functions:
Amino acid transport
Protein thiol redox status
Cysteine storage
• Cellular GSH levels are controlled by:
Cysteine availability
Synthesis and utilization
.
Organ export/import
165
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GLUTATHIONE METABOLISM
AMINO ACID
TRANSPORT
XENOBIOTIC
CONJUGATION
t __ . ^
-*T ^~ NADPhT^ ^-GSSGT*^ ^~ "2°
RIBULOSE
5-PHOSPHATE
Glutathione Biosynthesis
Cysteine
GCLC 73kDa
0
GCLM 31kDa
y-OC
• GCLC possesses all catalytic activity
• GCLC is feedback inhibited by GSH
• GCLM: lowers the Km for glutamate
increases the K, for GSH
possesses no catalytic activity
• GCL is inhibited by buthionine sulfoximine (BSO)
The Gclm null mouse: an in vivo model of GSH depletion
1 To more thoroughly characterize the role of GCLM in
GSH biosynthesis and oxidative stress, we made a
Gclm knock-out mouse model.
• Humans are known to have polymorphisms in GCLM
which predispose to heart disease, lung diseases,
schizophrenia, and heavy metal body burden
1 We tested the susceptibility of mice with varying
amounts of GCLM production to nanoparticle induced
lung injury by exposing them to Qdots
Gclm null mice have low GCL activity and low levels of GSH in most tissues
GSH levels
GCL activity
.1,.1,1.1 .1.1, ,
McConnacllieet al., Tox Sci, 2007
Nasal Instillation of PMAT QDs
Lightly anesthetize mouse (ketamine/xylazine)
Instill 0.4 ul/gm of a 20 nM solution intranasally (~6 ug/kg Cd)
Sacrifice 8 or 24 hrs post instillation.
Collect BALF cells and fluid, serum, lungs, heart, spleen, kidney
Stain BALF cells for MO (F4/80+) and neutrophils (GM+)
Analyze cells by FACS for % Gr1 + cells
Measure total protein in BALF fluid
Possible reasons for lack of inflammation
in Gclm null mice
• Failure of their MO to take up Qdots?
• Failure of their MO or airway epithelium to produce
and/or secrete chemotactic peptides and cytokines?
• Failure of their MO to produce ROS (NADPH oxidase
activity compromised?)
• Perhaps the lack of GSH has resulted in an adaptive
response (up regulation of protective genes) which
acts to squelch oxidative stress or the immune
response?
166
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Ongoing studies
Mechanisms of PMAT Qdot uptake by MO
- Scavenger Receptors; SRA, MARCO, LOX1
- Calveoli, clathrin, endocytosis/macropinocytosis
Examine markers of oxidative stress in the lung tissue,
and BAL cells and fluid
Chronic effects of exposure to PMAT-Qdots (e.g.
pulmonary fibrosis?)
DMA microarray analysis of gene expression for
additional biomarkers of lung injury
Systemic inflammation/markers of lung injury (plasma
cytokines/chemokines; CC16; SPD; KL-6)
Translocation of QDs and Cd to other organs
Effects on olfactory epithelium and brain
Many Thanks To:
Xiaohu Gao
Emily Hu
Dave Eaton
Francois Baneyx
Mike Yost
Elaine Faustman
Bill Parks
Sean Gill
Carol Ware
Warren Ladiges
Sengkeo Srinouanprachanh
Pat Stapleton
Jasmine Wilkerson
Fred Farin
Theo Bammler
Dick Beyer
Funding
NIIMNIEHS Grants P50ES015915, P30ES07033, T32ES07032, R01ES01618'
Dianne Botta
Collin White
Kellie Fay
Tao Lin
Chad Weldy
Dave Cox
Lisa McConnachie
Shelly Hsiao
Mike Dabrowski
Megan Zadworny
Erin Peck
Wes Smith
167
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2010 U.S. EPA Nanotechnology Grantees Meeting
Patricia A. Holden
Bioavailability and Fates of CdSe and TiO2 Nanoparticles in Eukaryotes
and Bacteria
Patricia Holden , Galen Stucky , and Jay Nadeau
1Bren School of Environmental Science and Management, 2Department of Chemistry and Biochemistry,
University of California, Santa Barbara, CA; 3Biomedical Engineering, Faculty of Medicine,
McGill University, Montreal, Canada
This research addressed questions regarding interactions between specific engineered nanoparticles and
cellular organisms, including: (1) what are the characteristics of nanoparticle uptake into cells?, (2) what is the
stability of nanoparticles in association with cells?, (3) how do nanoparticles, including intact materials and
products of instability, affect cells?, and (4) how do uptake, stability, and cellular effects vary with
nanoparticles, cells, and environmental conditions? To address these questions, laboratory experiments were
designed and performed using a variety of cells and nanoparticles. Research activities included nanoparticle
synthesis and characterization, cell culturing, quantifying nanoparticle effects on growth and other cellular
outcomes, analysis of nanoparticle and chemical constituent state during experiments, and using microscopy
and spectroscopic methods as needed to assess cellular spatial interactions with nanoparticles at various levels
of resolution. This research was aimed primarily at assessing the bioavailability of nanoparticles to cells and
cellular outcomes of bioavailable nanoparticles, given that cellular effects including toxicity and cell-mediated
nanoparticle alteration are predicated upon cells and nanoparticles interacting physicochemically. Major
findings included that quantum dots (QDs) were toxic to bacteria, with oxidative stress and membrane damage
explaining much of the observed nanoparticle-specific toxicity. Effects levels varied with gram positive versus
gram negative bacteria, and between CdSe and CdTe-composed QDs.1 Cadmium ions did not fully account for
cellular effects of Cd-containing QDs.2 Similarly to non-toxic gold nanoparticles that had been developed in
this project as phylogenetic probes using oligonucleotide conjugation3, QDs entered planktonic bacteria;
however, differently, the QDs decomposed intracellularly plus generated cell-damaging reactive oxygen
species (ROS).2 Although cell exopolymers afforded some differential protective effects in planktonic culture1,
the exopolymer matrix of biofilm bacteria both in natural system mixed communities4 and in laboratory pure
cultures (manuscript in preparation) did not impede delivery of QDs to cells as assessed by staining for
fluorescence and electron microscopy (EM) imaging4 and by toxic effects to biofilm bacteria (manuscript in
preparation). Photosensitization5 of illuminated QDs and photoenhancement6 of QDs using specific conjugates
each led to ROS formation which damages cells in the light. Cellular toxicity also was shown to occur with
direct electron transfer between cells and QDs; irradiated particles generated cell-damaging hydroxyl radicals.
Differences between gram positive and gram negative bacteria were observed7, yet questions remain regarding
the role of nano-bio interfacial charge transfer to cellular toxicity under dark conditions.
Intracellularization and bioaccumulation of QDs by bacteria2 resulted in QD-containing bacterial prey that
were subsequently studied for trophic transfer and QD biomagnification by protozoa. These studies showed
that QDs were trophically transferred intact and were differently toxic to protozoan predators as compared to
cadmium ions also packaged within bacterial prey. In contrast to QDs, nano-Ti02 particles did not enter cells
but associated externally on bacterial membranes, which led to the disagglomeration of large nanoparticle
agglomerates outside of cells.8 Association occurred in the dark, as did growth inhibition that appeared to scale
inversely with nanoparticle size. Studies with these and other metal oxide nanoparticles are continuing to
address outstanding questions regarding origins of apparent toxicity in dark conditions, including membrane
damage and potentially cellular oxidation, plus indirect effects owing to the frequently observed affinity of
metal oxide nanoparticles for cell envelopes. Overall, this research provides new and important insights into
mechanisms of select nanoparticle toxicity to cells. This research suggests that nanoparticles are frequently
growth-inhibitory, particularly under conditions that promote direct cell contact. Although not directly studied
in this research, there may be broader implications to bacterially-driven processes in the environment as most
The Office of Research and Development's National Center for Environmental Research 168
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2010 U.S. EPA Nanotechnology Grantees Meeting
biogeochemical processes in nature are catalyzed through bacterial population growth that was studied in this
research. The importance of this research to the U.S. Environmental Protection Agency is that it provides
insights into the effects of nanoparticles on microbes that are responsible for biogeochemical processes,
including pollutant transformations; this research also addresses fates of nanoparticles in the environment as
affected by microbes that are omnipresent in soil, sediment, and water. The research is similarly important to
the National Science Foundation with its focus on mechanisms of effects on microbes that could be important
targets for rapid screening or biomarker development.
References:
1. Dumas EM, et al. Toxicity of CdTe quantum dots in bacterial strains. IEEE Transactions on
Nanobioscience 2009;8(l):58-64.
2. Priester JH, et al. Effects of soluble cadmium salts versus CdSe quantum dots on the growth of planktonic
Pseudomonas aeruginosa. Environmental Science & Technology 2009;43(7):2589-94.
3. Ehrhardt CJ, et al. An improved method for nanogold in situ hybridization visualized with environmental
scanning electron microscopy. Journal of Microscopy 2009;236(1):5-10.
4. Clarke S, et al. Bacterial and mineral elements in an Arctic biofilm: a correlative study using fluorescence
and electron microscopy. Microscopy andMicroanalysis 2010;16(2): 153-65.
5. Cooper DR, NM Dimitrijevic, JL Nadeau. Photosensitization of CdSe/ZnS QDs and reliability of assays
for reactive oxygen species production. Nanoscale 2010;2(1): 114-21.
6. Cooper DR, et al. Photoenhancement of lifetimes in CdSe/ZnS and CdTe quantum dot-dopamine
conjugates. Physical Chemistry Chemical Physics 2009;11(21):4298-4310.
7. Dumas E, et al. Interfacial charge transfer between CdTe quantum dots and gram negative vs gram
positive bacteria. Environmental Science & Technology 20\Q;44(4): 1464-70.
8. Horst AM, et al. Dispersion of Ti02 nanoparticle agglomerates by Pseudomonas aeruginosa. Applied and
Environmental Microbiology 2010 (in press).
EPA Grant Number: R833323
The Office of Research and Development's National Center for Environmental Research 169
-------
Bioavailability and Fates of
CdSe and TiO2 Nanoparticles
in Eukaryotes and Bacteria
P. A. Holden
Bren School of Environ. Sci. & Mgmt., University of CA, Santa Barbara
J. L. Nadeau
Dept. Biomedical Engineering, McGill University
G. D. Stucky
Dept. Chem. &Biochem.. Materials Research Laboratory. University of CA. Santa Barbara
^^r5^
manufacturing f\ \ •;_
Allison Horst © 2007
Bioavailability Continuum
agglomeration
adhesion
entry
accumulation
Questions
When do nanoparticles enter cells?
Do the particles stay intact?
What are the cellular effects?
What are the variables?
Hypothetical Interactions: Nanomaterials (NMs) and cells
Nanparticles in this Research
CdSe quantum dots (QDs)
- laboratory-synthesized
- bare and core shell (ZnS)
- various conjugates
- also CdTe
Ti02
- Industrial (Evonik P25)
and laboratory
synthesized
- 80% anatase / 20%
rutile
IQQnm
(Horstetal. 26TO. AEM)
170
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Methods
Nano materials
• Characterization
- Microscopy (optical,
TEM/EDS, STEM/EDS)
- DLS, EPM (TiO2)
- ROS(DCFDA;SOSGfor
1O2;XTTforO2-; Na
terephthalatefor'OH; EPR
spectroscopy)
- TCSPC (lifetime
fluorescence)
• Quantification
- ICP-AES;AA
- Dialysis / ultrafiltration
- XANES (Se oxidation
state)
Cells
• Exposure
- Planktonic (growth, short
term) & biofilms
- Light / dark; oxygen/anoxic
• Effects
- Growth (rate, extents)
- Membrane integrity (as
above); LIVE/DEAD
- Membrane potential
(BacLight: DIOC2)
- Eukaryotic specific (e.g.
MMP)
- Metabolism
(dehydrogenase; MTT).
Background: CdSe/ZnS QDs Enter
Planktonic Cells in Light Conditions
Mammalian A9 cells w/
green QD-dopamine.
6. subtilis w/ yellow
QD-adenine.
Adenine auxotrophic £.
co// w/ green QD-adenine.
—» Photoactivated uptake and fluorescence
—» Conjugate and receptor mediated
—» External binding prerequisite
—» Transient membrane damage
—'Cellular processing
—»Toxicity not from Cd(ll)
(Kloepfer et al., 2003; Kloepfer et al., 2005; Clarke et al., 2006)
QD fluorescence lifetimes vary
with core, cap, conjugate
A CdSe/ZnS
(DA= dopamine conjugate)
CdTe
'0 20 30 «
Tlm*(ni)
(Cooper etal. 2009. Phys. Chem. Chem. Phys).
CdSe/ZnS QDs Photosensitized w/
Dopamine; PC12 Cells inhibited
O2' intracellular ROS metabolism
t t J,
(Cooperetal. Nanoscale. 20101
Bare CdSe QDs Enter & Toxic to
Pseudomonas in Dark Conditions,
si
i*.
(Priester et al. ES&T 2009)
CdTe QDs differentially bind,
transfer e- to Bacteria Strains
(Dumas etal. ES&T. 2010)
171
-------
G+ bacterial membranes
depolarized, but growth not slowed
(Dumas etal. ES&T. 2010)
Quantum Dot summary
QDs can enter cells
-With ROS-mediated membrane damage
- ROS form varies (light/dark; QD; conjugate)
- Binding prerequisite
QDs can enter intact
- Cap slows dissolution
Cells show consequences
- Slow growth rate and lower yield
- Membrane depolarization (not "toxic"?)
CdSe QDs trophically transferred
from Pseudomonas to Tetrahymena
(Werlin et al. 2010 in revision)
Pseudomonas binds & disagglomerates TiO2
- cells I
(Horstetal. 2010. AEM)
Summary & Next Steps
• SUMMARY:
- QDs can damage & enter cells; e~ transfer
- TiO2 binds, but didn't enter
- consequences to NP transport
-variables: light/dark, strain, NP,
cap/conjugate, oxygen
• NEXT STEPS:
- High throughput studies: membrane effects
- Quantify cell loading (dose)
- Quantify bioprocessing
DMA repair
ii Membrane repair
Efflux pump synthesis
Antioxidant synthesis
DMA damage (mutation)
Membrane damage (permeabilization)
Membrane depolarization (proton motive force)
e~ transfer impairment (dehydrogenase activity)
Starvation (nutrient sorption, cellular adhesion)
Poster WP190 Wednesday
172
-------
Acknowledgments
People: John Priester, Allison Horst,
Andrea Neal, Won Sun, Peter Stoimenov, Sam
Clarke, Sam Webb, Chris.Ehrhardt, Randy
Mielke, Rebecca Werlin , Ed Orias, Gary Cherr,
Suzy Jackson, Rachel Haymon, Stephan Kramer
Funding: U.S. EPA STAR Program
UC TSR&TP NSF /EPA UC CEIN
DOE (DE-FG02-06ER64250),
UC CEIN (NSF/EPA DBI-0830117)
1
173
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2010 U.S. EPA Nanotechnology Grantees Meeting
Warren Heideman
Using Zebrafish Embryos to Test Phototoxicity of TiO2 Nanoparticles
Warren Heideman1, Ofek Bar-Ilan2, Sarah Yang2, Joel Pedersen2, Robert Hamers2,
and Richard Peterson
School of Pharmacy, University of Wisconsin-Madison, Madison, WI
The properties of nano-scale Ti02 allow the production of an electron-hole pair in response to absorption
of a photon of sufficient energy. In aqueous solutions, this can lead to the generation of Reactive Oxygen
Species (ROS). Because ROS can react with a variety of essential cellular macromolecules, the production of
ROS can be cytotoxic. With these facts in mind, we hypothesized that Ti02 nanoparticles might produce
toxicity in vivo if an exposed organism is illuminated. To test this hypothesis, we conducted dose-response
experiments in which zebrafish embryos were exposed to a solution containing graded doses of commercially
available Ti02 nanoparticles. The fish were divided into two groups. In one group, the fish were illuminated
with a bright light source using a 14h/10h light/dark cycle. This illumination was designed to simulate the
slightly blue-shifted spectrum of sunlight, such as would be found at approximately 1 m below the surface of a
clear body of water. The other group was kept in dim tungsten filament lighting using the same light/dark
cycle. After 5 days of exposure, we observed toxicity that was clearly photo-dependent. In the illuminated
group, we observed lethality with an LC50 in the upper ppm range. The non-illuminated group showed almost
no lethality at any concentration tested. We found that embryos pre-exposed to Ti02 nanoparticle solution and
then washed into fresh water retained photosensitivity, consistent with a model in which the embryos absorb
the Ti02 nanoparticles internally.
The nominal individual particle size was 21 nm; however, the particles rapidly aggregated in solution to
produce aggregates of approximately 1 micron in size. Nonetheless, these particles were internalized by the
developing zebrafish. Studies of uptake using inductively coupled plasma optical emission spectrometry (ICP-
OES) and transmission electron microscopy (TEM) showed uptake throughout the tissues of the developing
zebrafish. Uptake and the potency of the nanoparticles were affected by hatching from the chorion, the
protective egg shell. Artificial dechorionation produced increased Ti02 uptake, and increased sensitivity to
toxicity.
To test the hypothesis that light exposure would induce the production of ROS in vivo, we used a
combination of chemical probes for ROS presence and measures of cellular damage induced by ROS. These
showed light-dependent ROS production. We also developed a transgenic zebrafish line in which a GFP
reporter is expressed from Antioxidant Response Elements (AREs). The Tg(are:eGFP) line showed elevated
reporter expression when the fish were exposed to both the Ti02 nanoparticles and illumination, but not in
response to either stimulus alone. Together, these results demonstrate an in vivo test of our hypothesis that
Ti02 nanoparticles activated by light produce ROS and phototoxicity in a developing vertebrate.
EPA Grant Number: RD-83386001
The Office of Research and Development's National Center for Environmental Research 174
-------
175
-------
176
-------
TiO2 Uptake
The TiO2
nanoparticles
have a
pronounced
tendency to
aggregate
TiO2 Uptake
( ^ I
11
TiO2 nanopart
exposure adds
measurable Tito
the fish.
Using DHE to
detect superoxide
production
I
j outofluoresce
177
-------
Preloading
embryos with N-
acetyl cysteine can
prevent some of the
effects of TiO2
nanoparticle
exposure
• The photochemistry of TiO2 nanoparticles predicted
that the nanoparticles might cause phototoxicity due to
ROS production. The uncertainty was whether this
occurs in vivo.
' Using zebrafish embryos we show
that TiO2 nanoparticles cause toxicity
that is dependent on light and
associated with uptake and ROS
production.
•Zebrafish are not humans. However biol'
systems are strongly conserved. Mechanisms that
work in zebrafish are often found in humans.
178
-------
PM Session 1: Effects on Fish and Oysters
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
David S. Barber
Effects of Subchronic Exposure to Nanoparticulate Silver in Zebrafish
Robert Griffitt1, Rachel Ryan, and David Barber
1 Center for Environmental and Human Toxicology, 2Particle Engineering Research Center,
University of Florida, Gainesville, FL
To examine the effects of subchronic exposure to nanoparticulate silver on zebrafish, we exposed adult
female Danio rerio to nominal concentrations of 5, 15, 25, or 50 jxg/L nanoparticulate silver for 28 days using
a flowthrough system. A soluble silver treatment (5 jxg/L nominal, ~2.5 ^g/L measured) also was included.
Samples were taken at days 7, 14, 21, and 28 for gene expression and tissue burden analysis, and at days 14
and 28 for histopathology analysis. Our results indicate that the use of flowthrough systems for chronic
nanometal studies is a viable concept, as we were able to maintain measured concentrations of approximately
60 percent of nominal values over the course of the 28 day exposure. Dissolution of nanoparticulate silver
were measured twice weekly throughout the exposure, with measured concentrations ranging from 0.5 to 1.0
Hg/L, and there were no significant differences between treatments. Silver burdens of gills at conclusion of the
study was concentration dependent with the 50 ppb nominal exposure producing burdens of 45 ± 9 ng Ag/mg
wet wt, which was similar to that produced by silver nitrate (28 ± 6 ng Ag/mg wet wt). Microarray analysis of
gills demonstrated that expression of 3,019 genes was significantly altered by silver exposure, mostly driven
by 50 ppb nominal exposure. Clustering using LSMEANS places the 50 ppb and 25 ppb exposures together, 5
ppb and 15 ppb together, and all treatments separate from controls. These data demonstrate that subchronic
exposure to nanosilver produces substantial effects on gill transcription, which does not appear to be driven
solely by bulk release of dissolved silver as dissolved silver concentrations were comparable in all treatments.
NSF Grant Number: 08340 75
The Office of Research and Development's National Center for Environmental Research 180
-------
Effects of subchronic exposure to
nanoparticulate silver in zebrafish
Joe Griffin, Rachel Ryan
University of Southern Mississippi, Gulf Coast Research Laboratory
Andrew Kane, David Barber
University of Florida, Center for Environmental and Human Toxicology
Gill proliferation - acute exposure
24 hours
48 hours
Silver accumulation - acute exposure
Gills +48 hours Carcass +48 hours
urn » » "^
to-
£
!
B \
I r:|
,ij i
"
IB
BSS^T*
• 4
c. t*, c« m
rox.5d.l0712]404-115.
Experimental Design
• QSI nanosilver (25nm primary particles)
• Stable suspension prepared by centrifugation
• 28d flow through exposures with 6 groups
- 4 concentrations of nanosilver
• 5 ug/L, 15 ug/L, 25 ug/L 50 ug/L
- 1 Ag+ exposure (5 ug/L)
- Control
• Sampled at Day 1, 14, 21, 28, 32
i:
Particle Dissolution
21 2* 2B HtH
Exposure Day
181
-------
Carcass tissue burden
Gill Tissue burden
Silver tissue accumulation
Jlj
a re* isppb 2ip
Particulate silver and tissue burden
5 10 15 20 25 30 35
Water NFS concentration (ug/L)
Soluble silver and tissue burden
Water soluble silver concentration (ugfL)
Gill morphology
Control Gill - 28 days
50 ppb gill -28 days
182
-------
Skin morphology
Control Gill-28 days
*
50ppbgill-28 days
—t —•* -
Nasal epithelium
Control Gill-28 days • 50 ppb gill — 28 days
Transcriptional effects in gill at 28 days
(
\
GO analysis
pseudouridine synthesis
RNA modification
skeletal system
development
ribosome biogenesis
rRNA processing
RNA processing
embryonic organ
morphogenesis
embryonic organ
development
DNA-dependent DNA
replication
0.0402
0.04442
0.0402
0.00003509
0.001303
0.006019
0.04442
0.001303
0.0402
Conclusions
Zebrafish accumulate significant silver tissue burdens
— Gill levels 10X higher than carcass levels
— Remains for up to 4 days in the absence of AgNP
Significant correlation between AgNP concentration and
tissue burden
— Not significant for soluble silver
No observable effect on epithelial morphology
Microarray data indicates significant alterations in gene
expression patterns
— Dose response pattern on number of genes affected.
— GO analysis indicates two pathways
• Organ development
• Ribosome biogenesis
183
-------
Acknowledgements
Nancy Brown-Peterson, Idrissa Boube, Steve
Manning (USM)
April Feswick, Cody Smith
National Science Foundation (BBS 0540920)
184
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Robert Tanguay
Refinements to the Use of Zebrafish for Nanomaterial-Biological
Interaction Assessments
Lisa Truong ' , Tatiana Zaikova ' , Jim Hutchison ' , and Robert Tansuay '
'Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon
State University, Corvallis, OR; 2The Oregon Nanoscience and Microtechnologies Institute and the Safer
Nanomaterials and Nanomanufacturing Initiative, Corvallis, OR; 3Department of Chemistry,
University of Oregon, Eugene, OR
With the increased usage and introduction of nanoparticles into industrial and consumer products, the
concern about environment and health impacts remains unclear. It is largely unknown what environmental
conditions and/or physico-chemical property influence how a nanoparticle will behave in complex
environments. Often, under standard aqueous exposure conditions, nanoparticles precipitate and/or agglo-
merate complicating hazard identification. Ionic concentrations in aqueous media have a major influence on
NP agglomeration rates and as a result, synthesis and storage of nanoparticles are often in ion-free water. This
is problematic, however, for biological systems requiring buffering ions. Necessary suspension of
nanoparticles in media appropriate for zebrafish embryo toxicity testing presents an assay development
challenge.
Our group has developed rapid methods to assess nanomaterial responses in embryonic zebrafish. To date,
we have assessed several classes of nanomaterials, including carbon nanotubes, fullerenes, silver and gold
nanoparticles. Zebrafish offer inherent advantages, including rapid external development, optical clarity,
genetic similarity to humans, and minimal material requirement (~1 mg). Additionally, zebrafish are native to
brackish water. Our focus was to define the minimum ion concentration necessary to support normal
embryonic development. We reasoned that if zebrafish could develop normally in low ionic concentration,
more classes of NPs could be assessed with this rapid in vivo model because of reduced NP agglomeration
during the exposure period. To determine the lowest tolerable salinity level ,embryos were developed in to 0,
0.16, 0.8, 4, 20, and 100 % zebrafish medium. Embryos in all groups developed normally when assessed at
120 hpf The embryos were enzymatically removed from their chorions at 4 hours post fertilization (hpf). We
anticipated that de-chorionated embryos would be more sensitive to developmental malformations. This was
not the case. To determine if more subtle changes occurred, we also assessed larval behavioral. Exposure to
both zero and 100% zebrafish medium did not induce statistically different behavior, reinforcing the
morphological finding that plain RO water supports normal zebrafish development.
A standard protocol was used to characterize nanoparticle agglomeration under the different ionic
concentrations. We selected a gold nanoparticle (AuNP) highly predisposed to agglomeration in 100% embryo
medium and used UV-Vis spectroscopy to characterize the percentage of either a 10 or 50 ug/mL AuNP
concentration remaining in 0, 0.16, 0.8, 4, 20, and 100 % medium over time. We found that at concentrations >
4% zebrafish medium, both the 10 and 50 ug/mL AuNP precipitated almost immediately. At < 4% embryo
medium, more than 80 percent of the nanoparticles remained in solution and monodispersed, confirming the
ionic effect on agglomeration. Increasing ion concentration, and the resultant agglomeration, predictably
affected AuNP toxicity. In the higher (4 - 100%) ionic strength medium, embryos exposed to 0.08, 0.4, 2, 10,
or 50 ug/mL did not exhibit behavioral or morphological aberrations. In the lower ionic strength medium,
embryos exhibited both developmental and behavioral responses to the range of AuNP concentrations. Our
findings of normal zebrafish development in RO water and reduced toxicity of AuNPs in higher ionic strength
medium have substantially refined the exposure conditions for more accurate nano-toxicology in the zebrafish.
EPA Grant Number: R833320
The Office of Research and Development's National Center for Environmental Research 185
-------
Lisa Truong1'2, Tatiana Zaikova2'3, Jim Hutchison23, Robert Tanguay1'
EPA Nanotechnology Grantee Meeting- Nov7-8, 2010
«? *«**
Physico-Chemical Properties Influence
"Behavior" of Nanoparticles
Precipitation
Agglomeration
Biological Interactions
Biological Response
Biological Fate
Environmental Fate
Environmental Interactions
Knowledge Gap
Interaction of nanoparticles with environment and
biological systems remains largely unknown
Missing toxicological data to understand
biocompatibility
Identify risk associated with nanoparticle exposure
Current Knowledge Target Knowledge
Research Goal
To determine what influence each NP parameter
has on biological activity
Differential
Biological
Responses
The Zebrafish Model
Vertebrates share many cellular, anatomical and
physiological characteristics with humans
Early development is the period most well-conserved
between species
Embryos are clear, which allows for non-invasive
assessments over the course of development
'
186
-------
High Throughput Screening
Experimental Design
5 Concentration ranges:
0.08 to 50 ug/mL
100 ML NP solution per well
1 embryo per well
High Throughput Screening
Results
Assessed and evaluated over 200 nanoparticles
A large portion did not induce a biological response
Are there false negatives?
Assessment of NP Aggregation in
Aqueous Media
NP properties change depending on aqueous
environment/condition
Aggregation can occur in high ionic strength media
Biological response can be altered
Necessary to characterize aggregation in test media
and over-exposure period
Gold Nanoparticles (AuNPs)
A diverse family of functionalized AuNPs has been
prepared for 0.8-nm, l.B-nm and 10-nm core sizes
US-
SR SR
Characterization of l.Snm 3-MPA in
Test Media
1
6
V)
£2
300 400 500 600 700
Wavelength (nm)
Research Questions
Question 1
Does ionic strength play a role in aggregatio
Question 2
Can zebrafish develop and behave normally i
low/no ion media?
Question 3
Will suspension of l.Snm 3-MPA-AuNP in low
ionic strength media induce biological activitv ?
187
-------
Research Question 1
Specific Aim 1
Does ionic strength play a role in aggregation?
Specific Aim 2
Can zebrafish develop and behave normally in
low/no ion media?
Specific Aim 3
Will suspension of l.Bnm 3-MPA-AuNP in low
ionic strength media induce biological activity?
Ql: Does ionic strength play a role in aggregation?
Experimental Design
Size analysis using UV-Vis Spectroscopy
Ql: Does ionic strength play a role in aggregation?
Results
Percentage of
Embryo Media (%)
100
20
4
0.8
0.16
0
10 (Mg/mL)
18 hr
5.4%
85.4%
78.5%
98.6%
94.9%
98.5%
114hr
4.8%
14.5%
63.2%
94.2%
88.83%
81.2%
50 (Mg/mL)
18 hr
3.4%
24.9%
90.4%
95.8%
93.5%
96.7%
114hr
1.8%
3.0%
88.1%
94.4%
93.3%
91.8%
15
Conclusions
High ionic strength media causes l.Bnm 3-MPA-AuNP
aggregation
Specific Aim 1
Does ionic strength play a role in aggregation?
Specific Aim 2
Can zebrafish develop and behave normally in
low/no ion media?
Specific Aim 3
Will suspension of l.Snm 3-MPA-AuNP in low
ionic strength media induce biological activity?
Q.2: What Ion Concentration is Necessary?
Experimental Design
6hpf
6 Ionic Concentration Media
0,0.16,0.8,4,20, 100% EM
100 ML solution per well
1 embryo per well
120 hpf
Exposure
High Throughput Evaluation
Behavioral Analysis
188
-------
Q2: What Ion Concentration is Necessary?
Results
Q2: What Ion Concentration is Necessary?
Results
20 40 60 80 100
Percent of Total (%)
I Mortality i 1 Malformation i 1 Unaffected
Q2: What Ion Concentration is Necessary?
Experimental Design
6 hpf
120 hpf
High Throughput Evaluation
Behavioral Analysis
Q2: What Ion Concentration is Necessary?
Experimental Design - Behavior
Q2: What Ion Concentration is Necessary?
Experimental Design - Behavior
Time (Alternating Dark/Light)
0.2: What Ion Concentration is Necessary?
Results
Dark Cycle
'/o EM ^320% EM i—id% EM
^3 0.16% EM ^m 0% EM
189
-------
High ionic strength media causes l.Snm 3-MPA-AuNP
aggregation
Zebrafish develop normally in low ionic media
Research Question 3
Specific Aim 1
Does ionic strength play a role in aggregation?
Specific Aim 2
Can zebra fish develop and behave normally in
low/no ion media?
Specific Aim 3
Will suspension of l.Snm 3-MPA-AuNP in low
ionic strength media induce biological activity?
Q3: Biological Responses when NP is bioavailable?
Results
Percent of Total (%)
Q3: Biological Responses when NP is bioavailable?
Results
•a '
i;
E
1
Illl.
i
i
d
Ik
Dark Cycle
Data parted ™th" donate statistically s,en,f,rant vdu=s (°n=V
ay ANOVA- Dunnetts Post HocTest, p<0 05)
High ionic strength media causes l.Snm 3-MPA-AuNP
aggregation
Zebrafish develop normally in low ionic media
Low ionic strength media favors dispersion of l.Snm
3-MPA-AuNP
l.Snm 3-MPA-AuNP are more toxic when dispersed
Implications
Every parameter must be taken into consideration
when doing nanomaterial-biological interaction
studies
Refinement of the current high throughput screening
to include avoid false negatives and assess NPs
deemed "problematic"
The zebrafish is a versatile model
190
-------
Acknowledgements
Tanguay Lab
Dr. Robert Tanguay
Dr. Mike Simonich
Dr.TamaraTal
Jane LaDu
Kate Sa ill
Jill Franzosa
Britton Goodale
Galen Miller
Joe Fisher
JiangFei Chen
Andrea Knecht
Collaborators
Dr. Jim Hutchison
Dr. Tatiana Zaikova
Funding Agency
• EPARD-833320
• NIEHSP3000210
• ES016896
• Air Force Research Laboratory
#FA86 50-0 5-1-5041
ONAMI
191
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2010 U.S. EPA Nanotechnology Grantees Meeting
Devrah Arndt
Impacts of Functionalization of Fullerenes and Carbon Nanotubes on the Immune
Response of Rainbow Trout
Rebecca Klaper , Devrah Arndt, Jian Chen , and Frederick Goetz
1 School of Freshwater Sciences, 2Department of Chemistry,
University of Wisconsin-Milwaukee, Milwaukee, WI
The overall objective of this project is to assess the innate immune reaction of an aquatic model, the
rainbow trout, to manufactured nanomaterials of varying chemistries at levels not inducing cellular toxicity.
This research will create a mechanism with which to test other nanomaterials, provide data to support
ecological risk assessments, and ultimately inform decisions as to which materials will be the safest to
industrialize and use with respect to aquatic environments.
We investigated how structure and type of functionalization of manufactured nanomaterials could impact
the immune response of the aquatic model species Onchorycus mykiss (rainbow trout). We examined cell
viability as well as gene expression of genes associated with a pro-inflammatory or antiviral response in a
well-studied trout macrophage primary cell culture system. There was a significant difference among different
carbon nanotube-based nanomaterials in their level of pro-inflammatory gene expression behavior in
macrophage cells and the dose at which they became stimulatory. All concentrations tested were sublethal to
cells, yet almost all nanomaterials were stimulatory at some concentration. Functionalization to create water
soluble particles caused a variable effect. Each nanotube type caused a dose-dependent response with the
lowest exposures (0.05 to 1.0 |ag/mL) having no stimulatory response and at the highest concentrations (5
|ag/mL and 10 |ag/mL) stimulating a response similar to the positive LPS positive control. Anionic
functionalized multi-walled nanotubes and zwitterionic single-walled nanotubes were stimulatory at the lowest
dose (0.5 |ag/mL. Surfactants often used to suspend nanomaterials also were as stimulatory to the immune cells
as the nanomaterials.
For fullerene-based particles, almost all nanomaterials we have tested caused an increase in candidate
proinflammatory genes that is equivalent to stimulation of positive controls at the highest concentration of 10 ^g/mL
but as we decreased the concentration to 1.0 or 0.1 ug/mL, we began to see differences in inflammatory responses in
a dose-dependent fashion. This would indicate that the dose that ultimately enters the organism will be extremely
important to determine potential immune responses. Our data indicate that chemicals used for functionalization also
may stimulate the immune response and that this response is equivalent to the nanoparticle alone.
We are now focusing on a broader suite of genes to monitor for each compound at these lower doses. These
include individual genes known to be important for immune function as well as others that have been identified
through microarray experiments.
This study is the first report of the effects of nanomaterials on the function of the immune system in a
nonmammalian vertebrate. Because the innate immune system is the first to respond to the intrusion of foreign
material, analysis of the effects of nanomaterials on cells of the innate immune system should provide valuable
information on how these materials are perceived and affect an animal. Ultimately, such research will provide
the means to determine which nanomaterials are most harmful to aquatic species and how particles may be
altered or functionalized to decrease their toxicity.
The Office of Research and Development's National Center for Environmental Research 192
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
References:
Klaper R, Arndt D, Setyowati K, Chen J, Goetz F. Structure and functionalization impacts the effects of carbon
nanotubes on the immune system of an aquatic vertebrate model. Aquatic Toxicology 2010; 100(2):211-7. Epub
2010Jul27.
Klaper R, Crago J, Arndt D, Goetz R, Chen J. Impact of nanomaterial structure and composition on the
ecotoxicology of nanomaterials on aquatic species. Proceedings of the International Environmental
Nanotechnology Conference-Applications and Implications, U.S. EPA, Chicago, IL, October 2008.
Klaper R. Ecological effects of nanomaterials: impacts from genomic to immune system inDaphnia and trout.
NanoECO Meeting, March 3-8, 2008, Ascona, Switzerland.
Klaper R, Chen J, Goetz F. The cellular and gene expression effects of manufactured nanoparticles on primary
cell cultures of rainbow trout macrophages. SETAC, November 11-15, 2007, Milwaukee, WI.
EPA Grant Number: R833319
The Office of Research and Development's National Center for Environmental Research 193
-------
Hypotheses:
• May act as P.
l)Nanoparticles should be considered foreign and will stilm
the immune system
2) Core structure will impact ability to stimulate immune sy
3) Functionlization will impact ability to stimulate immune system
4) Nanomaterials will cause unique gene expression patternb
differ both from each other and from traditional stimulants
(bacteria and viruses)
_. Produce nanoparticles of different types (fullerenes .„
tubes) and with different side groups (functionalizatio
anionic, cationic)
2. Test the nanoparticles directly on macrophages in culti1
• Cell viability
• Gene expression
- candidate genes
qi - microarrays
Nanoparticle Types
C60-X
ZP^42.2 mV
Z ave=103.7 nm
C60-OH(24)
ZP—54.2 mV
Z ave=171.1 nm
194
-------
Nanoparticle Types
SWNT-COOH
ZP=-61.1 mV
Z ave=227.5 nm
SWNT-CONH2
ZP= -52.4 mV
Z ave=177.1 nm
SWNT-PEG
ZP=-58.1 mV
Z ave=452.4 nm
________
Differentiation of monocytes to macrophages in vitro
Time in culture
monocytes '[
macrophages
Northern Blot
5 = LPS (lipopolysaccharide)
C = control (no LPS)
Specific Experimental Scheme
1. Plate trout macrophages - incubate 5 days
2. Remove medium and add nanoparticles
3. Incubate for 24 hours (proinflammatory) or 6 hours
(proviral)
4. Cell Viability: Add QBlue Reagent and measure
fluorescence
. Gene Effects: Remove medium, add 1.0 ml Trizol,
extract for RNA, prepare cDNA and QPCR for IL-lp,
TNFa (proinf lammatory) or IFNa, IP-10 (proviral)
Cell viability does not decline with nanomaterial exposures
when not suspended with surfactants
| Cell Viability
195
-------
Pathogen Associated Molecular Patterns (PAMPs)
V xr^ f
y' f, I, Gram negative Q
Bacteria • J
(LPS)
positive
bacteria
Expression versus Control
s S s w w S
196
-------
Macropnue response la dilfnem nanoiiarll
MacroDhage response to different naimiarfl
Conclusions
1. Trout macrophages are a sensitive tool to investigate
the effects of nanoparticles (NP) on immune system
2. Nanomaterials are stimulatory of the immune system
without complete cell toxicity
3. Level of stimulation depends on core st
chemistry of nanomaterials
4. Functionalization may increase toxicity
5. C60-OH may bind RNAand influence total gene expression in cells
6. Nanomaterials have unique gene expression signatures
197
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Amy Ringwood
Characterization of the Potential Toxicity of Metal Nanoparticles in
Marine Ecosystems Using Oysters - Silver Nanoparticle Studies
with Adults and Embryos
Amy Rinswood, Melissa McCarthy , Nicole Levi-Polychenko , and David Carroll
1 University of North Carolina, Charlotte, NC; 2Department of Plastic and Reconstructive Surgery, Wake
Forest University Health Sciences, Winston-Salem, NC; 3Wake Forest University, Center for
Nanotechnology and Molecular Materials, Winston-Salem, NC
The use of silver and other metal nanoparticles continue to be incorporated into numerous consumer
products. Metal nanoparticles may be introduced into aquatic environments during production processes and
also as a result of release following their use in electronic and biological applications. The purpose of these
ongoing studies is to characterize the toxicity of various metal nanoparticle preparations on oysters,
Crassostrea virginica, a common estuarine species. Filter-feeding bivalve mollusks such as oysters spend their
lives removing particles so they are very valuable as model species for characterizing nanoparticle
bioavailability and interactions with basic cellular processes. Moreover, the adults release their gametes into
the environment, so their embryos are also likely targets of nanoparticles. Therefore, the effects on lysosomal
integrity, antioxidants, and oxidative damage, as well as the responses of different life history stages, are being
investigated.
Adult oysters and newly fertilized oyster embryos were exposed to different preparations of silver (Ag)
nanoparticles and dissolved Ag (AgN03) for 48 hours. For one set of studies, silver nanoparticle spheres and
prisms were prepared with PVP; for another set of studies, silver nanoparticle spheres, prisms, and hexagonal
plates were prepared with citrate. Gill and hepatopancreas tissues of adult oysters (both whole animal and
isolated tissue exposures) were used to evaluate lysosomal destabilization, lipid peroxidation, and cellular
antioxidant and detoxification responses (e.g., glutathione, catalase, superoxide dismutase, and metallothionein
gene expression). Some studies with isolated hepatopancreas tissues also were conducted using an intracellular
fluorescent probe to visually evaluate the production of reactive oxygen species (ROS) by microscopy. For the
embryo studies, the percent normal development was determined. The intracellular fluorescent probe also was
used to visually evaluate the production of ROS in the oyster larvae. The results of the lysosomal
destabilization and lipid peroxidation assays with the adult oysters indicated differential toxicity with the
different Ag nanoparticles. The prism preparations were consistently more toxic than either the spheres or
plates. Based on the lipid peroxidation results, there was less toxicity with the PVP-coated particles. For the
embryo studies, the prisms also were more toxic than the spheres or plates. Furthermore, the results of the
fluorescent ROS studies with both oyster hepatopancreas cells and oyster larvae indicated higher levels of ROS
in the prism exposed organisms.
This research program is designed to address a number of important issues regarding metal nanoparticle
toxicity in marine organisms (e.g., nanoparticle characteristics associated with toxicity and adverse effects on
fundamental cellular responses). These kinds of basic studies are essential for characterizing the potential risks
and impacts of nanoengineered particles on estuarine and marine organisms.
EPA Grant Number: R833337
The Office of Research and Development's National Center for Environmental Research 198
-------
Characterization of the Potential Toxicity of Metal Nanoparticles
in Marine Ecosystems using Oysters -
Silver Nanoparticle Studies with Adults and Embryos
LNCCHARIOTn;
Amy H. Ringwood, Melissa McCarthy
University of NC-Charlotte,
Charlotte, NC USA
David Carroll, Nicole Levi-Polyachenko
Wake Forest University,
Center for Nanotechnology and Molecular Materials, and
Wake Forest University Health Sciences, Winston-Salem, NC USA
Lauren Marston, NC State University, Raleigh, NC
REU
NanoSure
UNCC
Nanoparticle Products
•••• •
Oysters - Crassostrea virginica
I MIHMMlllH illl T 'r
Filter Feeding Bivalves as Models
* Highly effective at removing particles
High filtration rates
Sample water column AND
surface / resuspended sediments
Extensive information regarding toxic
responses to metals and organic
contaminants
Oyster Nanoparticle Studies
^ Adult Exposures
• Lysosomal Destabilization
• Lipid Peroxidation
• Antioxidant Responses
• Tissue/Cellular Accumulation
r Embryo Exposures
• Normal Development
• Antioxidant Responses
199
-------
Oysters - Crassostrea virginica
Lvsosomal Destabilization Assay
Incubate in neutral red for 1 li
and score > 50 ceUs
Centrifuge gently thru 41 um nyli
Oyster Hepatopancreas Cells
Stable Lysosomes
Destabilized Lysosomes
Oyster Nanoparticle Studies
Embryo Exposures
• Normal Development
• Metallothioneins
• * « «
* '*
Oyster Veligers (48 hour)
100
80
60
40
20
r2 = 0.5
p <0.01
**
-•-
0 -I 1 1 1 « i
10 20 30 40 50 60 70 80
% Lysosomal Destabilization
200
-------
Ag Nanoparticle Studies
"Seeds" - Citrate
"Spheres" and "Prisms" - PVP (Polyvinylpyrrolidone)
"Seeds", "Prisms", "Plates" - Citrate
v.
"Seeds"
"Prisms"
"Plates"
Hepatopancreas Cells - Lysosomal Destabilization
"Seeds" - Citrate
Control 0.025
0.25
2.50
[Ag] Nanoparticles- ug/L
25.0
Lipid Peroxidation
"Seeds" Citrate
Control 0.025 0.25 2.5 25
Ag Nanoparticles (ug/L)
Lysosomal Destabilization
*
1
Control 0.025 0.25 2.50 25.0
Lipid Peroxidation
Control 0.025 0.25 2.5 25
Ag Nanoparticles (ug/L)
1600 -
1400
•ss 120° J
:§ 1000 -
S 800 -
•2- 600 -
% 400 n
O 200 -
0 -
Glutathione
hill
Contro 0.025 0.25 2.50 25
Ag Nanoparticles (ug/L)
Knihiyn l}cvcln[inicnt
• AgCI
CoDtrol 0.025 O.J5 2.5 25
Ag Exposures (ug/L)
201
-------
METALLOTHIONEINS (MT)
r Low molecular weight metal-binding proteins
(6000 - 7000 D)
> High cysteine content (30%), (Cys - X - Cys)
HSCPCHC lETSTCACSDSeWiTGCKCGPGCKCGDD- CKCXSCKVKC SCTSKGGCKCGRKCTGPATCKCGSGCSCXX
Metallothionein
§
'33
i
Si
X
H
Relative MT
30 -
25 -
20 -
15 -
10 •
5 -
o -
.1
1 '
Con 0.025
i
0.25
1
1
2.5
Adults
Gene Expression
T
1
1
n
• i i i
25 Con 0.2
Embryos
[Ag]NP-ug/L
Ag Nanoparticle Studies
> ,-eds" -
r "Spheres" and "Prisms"-PVP(Polyvinylpyrrolidone)
>
]
80 •
QJ
1 60.
VI
$
* *#
1 T
*
T
T
J
f. 'z £ £
S t £ i
5 • * *
O
a
fi
^g NPs (
*#
1
O
*E
CH
PVI
T
1
d
f^
O
')
*
I
I
O
fl
O
z
OB
Ag NPs (PVP)
(20ug/LAg)
Ag Nanoparticle Studies
-:>eds"
"Spheres" and "Prisms" - PVP (Polyvinylpyrrolidone)
"Seeds", "Prisms", "Plates" - Citrate
V,
"Seeds"
"Prisms"
"Plates"
202
-------
Lysosomal Destabilization - Ag NPs (Citrate)
2 1 E "J =
S 5
Seeds Prisms Plate*
Ag (•£/!,) NanapBrtictat
Lysosomal Destabilization
(Isolated Hepatopancreas Tissues)
Control
Seeds Prisms Plates
Ag (20 ug/L) Nanoparticles
Lipid Peroxidation
(Isolated Tissues)
Gills
Hepatopancreas
Ag (20 ug/L) Nanoparticles
Oyster Embryos
1
1
Seeds Prisms Pbtes
Ag (ug/L) Nanoparticles
Embryo ROS Studies
1: Very low 3: Moderate
5: High
Embryos (24 hr) were exposed to a 20 ppb concentration of
AgNPs for 2 hr.
Reactive oxygen species (ROS) production was assessed using a
fluorescent probe (Carboxy H2DFDA, Molecular Probes)
Embryo fluorescence was categorized as a 1, 3, or 5
Embryo ROS Studies
A #
Control TBHP
Seeds Prisms Plates
203
-------
Summary
V Ag Nano "Prisms" were more toxic than "Spheres", and
"Plates" in both adult and embryo oyster studies
*«* Mechanisms of toxicity associated with lysosomal
dysfunction and oxidative stress.
>» PVP coated particles may be slightly less toxic than
citrate-based preparations.
Oysters and other filter feeding bivalves are valuable
model organisms for characterizing potential
nanoparticle toxicity.
204
-------
PM Session 2: Nanoparticles and Waste
Treatment
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Zhiqiang Hu
Bioavailability of Metallic Nanoparticles and Heavy Metals in Landfills
Yu Yang, Meng Xu, and Zhiaians Hu
University of Missouri, Columbia, MO
Silver nanoparticles (AgNPs, nanosilver) released from the industry and consumer products will be likely
disposed in landfills. The objectives of this research are to determine the bioavailability of nanoparticles and
heavy metal species in bioreactor landfills as compared to traditional municipal solid waste landfills and to
elucidate the mechanisms governing bioavailability as well as the mode of antimicrobial action by
nanoparticles.
In this study, bioreactor landfill experiments were carried out to determine the impact of newly
synthesized AgNPs (average particle sized = 21 nm) on the anaerobic/fermentation process in bench-scale
bioreactor landfills. The solid waste taken from Columbia Sanitary Landfill (Columbia, MO) was exposed to
AgNPs at the concentrations ranging from 1 to 10 ppm (mg/kg). The time course of cumulative biogas volume
was recorded automatically, and the gas composition was determined by the gas tube method. At AgNPs
concentrations of 1 ppm, there was no statistically significant difference of the cumulative gas volume or gas
production rate between the nanosilver treated solid waste and the control. However, exposure of solid waste
to nanosilver at a concentration of 10 ppm resulted in the reduced cumulative biogas volume (p < 0.05).
Volatile fatty acid (VFA) accumulation and thereby consistently acidic condition (pH = 5.2) was observed in
the leachate from the 10 ppm nanosilver treated bioreactor. The results suggest that AgNPs at low
concentrations (1 ppm or below) have negligible impact on anaerobic waste decomposition and biogas
production, but the concentration of nanosilver at 10 ppm result in reduced gas production and changes of
methanogenic assemblages.
Quantitative PCR results demonstrated dominant methanogenic population shift from acetoclastic
methanogens to hydrogenotrophic ones with nanosilver concentration. The bioreactor exposed to 10 ppm
AgNPs had 40% acetoclastic methanogens in total, while the control bioreactor and the one treated with 1 ppm
nanosilver had above 90% hydrogenotrophic methanogens, mainly Methanobacteriales. Total silver in the
leachate decreased rapidly in 10 ppm nanosilver-treated bioreactor from 14.8 mg/L to below 2 mg/L. The
concentrations of silver in leachates from the control and 1 ppm nanosilver treated bioreactor were
approximately 2 mg/L.
Results of this project provide some of the first data on the bioavailability and risk assessment of metallic
nanomaterials in solid waste disposal systems, especially under anaerobic conditions. Considering the potential
release of nanomaterials in municipal landfills, the results of this project could help to better understand the
transport, partitioning, and toxicity of nanoparticles to syntrophic anaerobic communities in municipal
landfills.
EPA Grant Number: R833893
The Office of Research and Development's National Center for Environmental Research 206
-------
The Impact of Silver Nanoparticles on
Anaerobic Processes in Bench-scale
Bioreactor Landfills
University of Missouri
Columbia, MO 652 11
Outline
Introduction
Materials and Methods
Results and Discussion
Summary
AgNPs as An Antimicrobial Agent
Silver ions and silver nanoparticles AgNPs (nano silver): now both
commonly used in consumer products.
With a concentration factor of more than 100 in WWTP,the predicted silver
concentrations in sludge is from 7 to 39 mg/kg (Blaser et al. 2008).
In North America about 2200 Mg A;-:, year were wasted through landfill,
which was about 50% of the total wasted silver (Eckeiman and Graedd 2007).
Fate of Nanosilver as Solid Wastes
Nanosilver flows from product to environments at high exposure scenario.
WIP; waste incineration plants, STP; sewage treatment plants. The number is a value in tori:;\ *;;*
(Eavnon -~-o ^chuoi >003 C'. P2!. 4^7-44531'
Silver Ion and Nanosilver Toxicity
Ag+
-Affected bacterial growth at 200 ppb(1.9 uM) under some pH.
-Reduced bacterial growth entirely at 2000 ppb (19 uM) (Pabre^etai.20os)
-Interact with thiol groups of proteins, deactivate vital enzymes and inhibit DNA
replication (Klame et al. 2008).
AgNPs
-Inhibited autotrophic bacterial growth by 86% at 1 mg/L (Choi, 200S).
-Highly toxic to zebrafish, daphnids and algal species with 48-h median lethal
concentrations as 40 to 60 u,g/L (Gnffitt et al. 2008).
-Small size AgNPs (< 10 nm) may enter the cells directly to release silver ions
(Morones et al. 2005).
Sanitary Landfills
(i) Conventional Landfill:
Storage/containment concept
No recirculation
Slowly and naturally degradation
(ii) Bioreactor Landfill:
Leachate recirculation
Increased degradation rate
Improved the setting ability of solids, the recovery of landfill space
Enhance the methane generation in the leachate
207
-------
Major Biological Processes
in Bioreactor Landfills
Hydrolysis
Degrade long chain polymers such as cellulose and hemicelluloses to
simple organic molecules.
II Acidogenesis/Acetogenesis
Amino Acids, long chain fatty acids and simple sugars are degraded
during fermentation reactions, producing VFAs including acetic acids.
III. Methanogenesis
Convert primarily acetate and hydrogen plus carbon dioxide to methane.
a Hydrolysis and fermentation provide the substrate for methane generation.
Methogenesis is very sensitive to reactor conditions
a Inhibition on methanogenesis may result from the interruption of
hydrolysis and fermentation
Anaerobic Microorganisms:
-Methanogens
Methanogens: Important microorganisms for final biogas production;
Good indicator of functional anaerobic bioreactor landfill.
"•• •e'.hruioi.'rns' 'Mgi'has-H'-i.azla &>v\ Meiha^ij-ia,,
convert acetate to methane and carbon dioxide
CH3COOH^CO2+ CH4
Hydrogenotrophic Methanogens: Methanobacteriales, \uAanococci and
!\-'Iefh;monucrolnai2, Municipal Solid Waste (MSW): Fresh MSW from the sanitary
landfill site in Columbia, MO.
The solid composition (in weight): metal 0.9%, paper 14.1%, brick
17.7%, wood and shredding 4.7%, soil 36.7%, organic waste 15.6%,
plasticbags 10.2%.
II
Bioreactor Setup
•Total Volume: 9 L
•2.9 kg MSW +1 L anaerobic sludge + Control/ IppmAgNPs/10 ppm AgNPs.
•Temperature, 37 °C. Recirculationrate, 5% of reactor volume.
208
-------
Bench-scale Bioreactors
•The bioreactor landfills are operated with automatic biogas recording using
Challenge resiprometer.
•The leachate collection bottles on the ground are not shown.
Sampling & Chemical/Microbial Analysis
^ Gas Production and Chemical Analysis:
•Total volume of gas :AER-200 Respirometer
•Carbon dioxide : Gastec Tube 2HT (Gastec, Japan).
•20 mL leachate withdrawn every two weeks
(•jOd 20 mL DI water back after sampling)
-pH, COD and NH4+, NO2- and NO3-
-Total silver : ICP-AES
-Volatile fatty acids (VFAs) (HPLC)
>DNA extraction and real time PCR
•The extracted DNA samples from leachates were stored at -20°C
before use.
•Real time PCR assays were performed using ABI 7500 Real Time
PCR System formethanogens.
Primers and Probes for qPCR
Methanosaeta MSlb 585F (S'-CCGGd
Methanosacaa Mblb 586F (S'-CGGTT
;A-3') SYBR Green (Conklin, Stensel et al. 2006)
MMB 749-pro! -
Results and Discussion
Cumulative Biogas Production
*Abig difference of gas volume between the control, the reactors treated with 1
ppm AgNPs and the 10 ppm AgNPs.
* Solids treated with 1 ppm AgNPs show no inhibition on anaerobic process,
while those treated at 10 ppm did affect biogas generation rate and volume.
Leachate pH and COD Changes
30 60 Day 90 120 150
* The pH drop due to VFA accumulation in the bioreactor treated with 10 ppm
AgNPs and the changes of leachate COD in 10 ppm AgNPs reactor confirmed
the inhibitory effect of nanosilver on anaerobic biodegradation of solid waste.
209
-------
Changes of VFAs and Acetic Acid in the
Leachate
*The dynamic changes of VFAs and acetic acid from the reactor containing 10
ppm AgNPs confirmed the accumulation of VFAs and acetic acid, resulting in
consistently low pH (5) in the leachate.
* Results are consistent with biogas production profile.
Leachate Ammonic Concentration
— ICypn. AiNP, tnn A*.VP. —Canrt
*Ammonia-N appeared to be constant at relatively low concentrations (~50
mg/L).
S3 Changes of Methanogens Population in
Leachate
3.E+08
— !(to»A*Nh - Ipp-AfrMS C-urf
3.E+08 - T
| 2.E+08 - JXxX"^\ .S^
1 *r
5 2.E+08 •
I l.E+08
E§ 5.E+07 -i ^"^vX
* * ' "^^r
0 20 40 60 80
*B egiiming: similar methanogen numbers , mainly from the anaerobic sludge
added.
*At day 30, the control bioreactor had the highest methanogen numbers, about 8
and 2 times higher than 10 ppm AgNPs, 1 ppm AgNPs respectively.
*From day 40 to 70: almost the same total methanogenic population
14 Day 2S
II
• During the early stage of anaerobic
decomposition (from day 14 to day 42), in
control and bioreactor treated with 1 ppm
AgNPs, Me-,':;.,}---1:. '• --''ales dominated (at
90%).
•For comparison, in bioreactor treated with 10
ppm AgNPs. \f, .!. w '•-..ztawas still above
40%.
•The methanogenic population continues to
Total Silver in Leachate
oTotal Ag in leachate from
10 ppm AgNPs treated
bioreactor was about 14.8
mg/L, decreasing to 2 mg/L
after about 100 days of
operation
oTotal silver from the
bioreactor treated with 1
ppm AgNPs and the control
was around 1 mg/L, below
TCLP maximum value 5
mg/L.
o Results indicate that
silver could be precipitated
or absorbed in landfill solid
Summary
There was no significant difference of the cumulative gas production
between the bioreactor treated with 1 ppm (mg Ag/kg solid) AgNPs and the
control. But 10 ppm AgNPs resulted in the reduced biogas production,
VFA accumulation and lower pH ( around 5) in the leachate.
qPCR results demonstrated dominant population shift from acetoclastic
methanogens to hydrogenotrophic methanogens at the early stage of
anaerobic solid degradation.
G In early stage of solid degradation, leachate from bioreactor treated withlO ppm
AgNPs had 10% :-:':& r-elastic methanogens in total, compared to other reactors
including the control which had more than 90% hydrogenotrophic
methanogens-mainly \4ethcmobacteriales.
After about 100 days, total silver concentrations in the leachate were all
around 1-2 mg/L in bench-scale bioreactors.
The results could be useful to the regulatory agencies and landfill operators
for decision making and remedial actions.
210
-------
Acknowledgements
Funded by EPA STAR Program
(#83389301)
211
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Paul Westerhoff
Biological Fate and Electron Microscopy Detection of Nanoparticles
During Wastewater Treatment
Paul Westerhoff, Bruce Rittmann, and Terry Alford
Arizona State University, Tempe, AZ
The market for nanomaterials is increasing rapidly, and nanoparticles (NPs) present in consumer products,
industrial wastes, biomedical applications, and so on will become significant in the near future for wastewater
treatment, just as nutrients, pathogens, metals, and synthetic organic chemicals have been important for the last
few decades. Wastewater (WW) treatment plant (WWTP) discharges (treated effluent, biosolids, and possibly
aerosols) may become significant routes for NPs to enter the environment. Today, almost no information is
available on the fate of manufactured NPs during biological wastewater treatment.
The goal of this project is to quantify interactions between manufactured NPs and WW biosolids. The
objectives of this project are to: (1) quantify removal mechanisms and biotransformation of NPs by wastewater
biomass/biosolids under different operational regimes (aerobic, anoxic, anaerobic); (2) verify that low NP
dosages have minimal effect on WWTP operations; (3) develop microscopy techniques to rapidly scan for the
presence of NPs in biological matrices and develop extraction techniques to separate NPs from biosolids; and
(4) assess the relative significance of WWTP effluents and biosolids as significant environmental loadings of
NPs.
Over the past year, a series of sequencing batch reactors (SBRs) containing either heterotrophic or
nitrifying bacteria were operated with and without NP addition. The systems were operated for up to 120 days,
and both hydraulic and sludge residence times were managed. NPs were applied twice per day. Silver
(functionalized and non-functionalized), titanium dioxide (Ti02), fullerene, and fullerols had no effect on
heterotrophic bacteria performance (i.e., COD removal). At milligram per liter application dosages, silver NPs
and silver ions impaired nitrifying bacteria. Functionalized nano-silver was removed from the SBR
supernatant, compared with non-functionalized nano-sliver. Nano-Ti02 was well removed (> 90%). Fullerols
were removed less efficiently than aqueous fullerenes. In addition to SBRs, a number of batch isotherm-like
experiments also have been completed with the same NPs and show removal results comparable with SBR
findings, which suggest that simpler batch methods may be valuable for screening NP removal capability by
wastewater biosolids. This research has been published and additional manuscripts are in preparation.
Papers on sources of NPs from textiles, a wide range of household products and cosmetics have been
published or submitted. This is critical to provide NP content of commercial products that will enter sewage.
Imaging of NPs in commercial products and full scale WWTPs and their biosolids have been published. We
are currently analyzing titania solids in biosolids from the U.S. Environmental Protection Agency and U.S.
Department of Agriculture archives.
EPA Grant Number: R833322
The Office of Research and Development's National Center for Environmental Research 212
-------
^' Biological Fate & Electron IS11
Microscopy Detection of NPs During
Wastewater Treatment
Paul Westerhoff
Bruce Rittmann & Terry Alford
Ayla Kiser, Yifei Wang, Troy Benn
Kiril Hristovski, David Ladner
November 2010
sE
Projec
• Goal: to quantify
interactions between
manufactured NPs and WW
biosolids:
— We hypothesize that dense
bacterial populations at
WWTPs should effectively
remove NPs from sewage,
concentrate NPs into
biosolids and/or possibly
biotransform NPs.
- The relatively low NP
concentrations in sewage
should have negligible
impact on the WWTPs
biological activity or
performance.
— Develop mechanistic models
for NP removal in WWTPs
tGo
al
NM Sources &
Uses in Society
^
Wastewater
Treatment
Plants
]
E
JSU
Liquid Effluent to
Surface Waters 1
Biosolids to
Land
Application, etc
IB
xk
Release of Engineered Nanomaterials
1 Nanoparticle Silver Released into Water from Commercially Available Sock
Fabrics (Benn & Westerhoff, ES&T42:11:4133-4139 (2008))
- Observed release of silver materialsfrom nano-silver impregnated socks
— Six types of socks contained up to a maximum of 1360 ^.g-Ag/g-sock and
leached as much as 650 y.g of silver in 500 mi of distilled water.
' The Release of (Nano)Silver from Consumer Products Used in the Home (Benn
et al., J. Environmental Quality, 39:1-8 (2010))
— Silver was quantified in a shirt, a medical mask and cloth, toothpaste,
shampoo, detergent, a towel, a toy teddy bear, and two humidifiers.
— Silver concentrations ranged from 1.4 to 270,000 ug-Ag/g-product.
- Silver was released into water up to 45 u.g-Ag/g-product, and size fractions
were both > & < 100 nm
- TCLP tests conducted to simulate release to landfills (0.13 to 54 ug-Ag/g-
product)
ASU
Release of Engineered NMs
Detection of Fullerenes (C60 and C70) in Commercial Cosmetics
(Benn et al., submitted to JEM)
- Five cosmetic products were evaluated for their fullerene content.
— A common cosmetic formulation that disperses fullerenes using
polyvinylpyrrolidone (C^-PVP) was characterized TEM
- LC/SM was used to separate and specifically detect fullerenes (C60 and C^)
from interfering substances typically present in cosmetics (e.g., castor oil).
- Recovery of C60 from aqueous C60-PVP using LLE and SPE approached 100%
after accounting for LC-MS signal suppression caused by matrix interferences
(acetic acid)
- CgQ was detected in four commercial cosmetics ranging from 0.04 to 1.1 ug/g,
and C^ was qualitatively detected in two samples.
- A single-use quantity of cosmetic (0.5 g) may contain up to 0.6 u.g of C60 and
demonstrates a pathway for human exposure to engineered fullerenes.
- Fullerenes may enter the environment through wastewater systems after
being released from cosmetics.
m
Nanomaterial Removal
• Settling and Biosorption are
dominant removal mechanisms
• Research evaluated:
— Batch sorption to biomass
- Continuous loading bioreactors
— Occurrence at full-scale treatment Liqi.
plants Su
Primary Aeration Secondary Tertiary
Headworks clarify Basin clarify Filtration
sig, — ™-«aH#c=>-H=p-»s--
\ Handling
ASU
at WWTPs
NM Sources &
Uses in Society
*
Wastewater
Treatm ent
Plants
A 1
id Effluent to 1
-face Waters |
Biosolids to
Land
Application, etc
j 1 Treated
T__l * Effluent
~~| > Finished
| Biosolids
NM Sorption to Wastewater Biomass
Biosorption of
nanoparticles on
heterotrophic
wastewater biomass
(Kiser et al., Water
Research, 44:14:4105-
4114(2010))
Robust sorption
method developed
Surface properties were
very important
C6TPVP showed < 10% removal
213
-------
-m
V «^1 I 1 1*4 t^TVIuw h_ 1 t 1 MP ^^ 1
WM
• OPPTS 835. 1110
Activated Sludge m
Sorption Isotherm
I m
• Validated method £
for organic pollutant ! *»
(MB) using fresh and *
freeze-dried biomass
• Method not valid
for nanosilver, and
likely other NMs
^ h i *«^ i i -i
V 1 ^ W 1 1 Vrf' Srfl
Fw«»-fr»d (jonlss FmftbxnMI
1
— •
arUt Gum
torn
ft
.,
. •
MOT CUM H
JT C
.—
MHt
KK(IO«Hfc*d SIIVN
Materials courtesy of CEINt (Wiesner)
Freeze-Dried Biomass
has different morphology
J5U
Freeze-Dried
Biomass:
Fresh Biomass:
* Biosorption of fluorescent latex spheres (20 nm sulfate
coated) are far less on freeze-dried biomass
Continuous NM Loading Study
Sequencing batch reactors (SBRs)
operated for weeks to months with
daily renewal of simulated sewage +
NMs
Operated are realistic HRTand SRT
Trends in removal for different types of
NMsfollowed batch isotherms
Functionalized nanosilver resulted in
sludge bulking issues; removal
decreased as TSS fell
Varied nC60 loading and reduced
biomass -still achieved very high nC60
removals
No effect of heterotrophic removal of
COD
Minimal effect of nano-Ag on
nitrification, compared to major effect
from ionic silver
||2-- ^^V^****
0
•••Settled effluent flg
1
V
Time (days)
-•-Influent nC60
T -^Effluent nC60
0 20 40 60 80 100 120
Time (days)
Another Example (f-Ag)
0 5 10
Days of Operation
ISU
10 20 30
Days of Operation
Round 2 has 50% more biomass (TSS) than round 1
Nanomaterials Removed from Liquia
Go to Biosolids (fn-Ag example)
Nanomaterials Removed from Liqui
NP in Feed . No NP in Feed
T 1"
214
-------
Occurrence at Full-Scale WWTPs
Titanium is found in biosolids at full-scale WWTPs already
Titanium Nanomaterial
Removal and Release .3
from Wastewater c fjioooo ,.-
Treatment Plants (Kiser et ~ 6
al., ES&T, 43:17:6757-6763 S 5
,2009) | | 100°
>90% removal in coming o 1
titanium 11 10°
Threeforms of titanium | "z
present: "~ 5 10
— Nanoscale J£
— Microscale
— Mixed element (clays)
HSJ
Nanomaterials in WWTP Effluents
Evaluate TiO2
presence at several
WWTPs
Evaluate membrane
technologies to
characterize or
remove NMs
Liquid Effluent to
Surface Waters
Biosolids to
Land
Application, etc
Titanium well removed
at WWTPs in Arizona
Effluent Ti
concentrations are
similarto LCA
model predictions
Membrane
bioreactors(MBR)
have very low
effluent Ti
We isolated NPs in
effluents also using
roto-evap + dialysis
(under analysis)
Activated sludge
Act. Sludge + filter
Activated sludge
Activated sludge
Activated sludge
Activated sludge
Activated sludge
Trickling filter
MBR
MBR
4
NM surface properties trumped
membrane material properties in 0.22-u.m
syringe filtration experiments
57
£
I Agio
I TiOZH
i
flSU
Tighter Ultrafiltration Rejects NMs
(image from Sigma-Aldrich web site).
20 40 60 80 10Q
Rejection was high, but
recovery indicates
significant sorption
Nanomaterial in Biosolids
Nanomaterials will
accumulate in biosolids
What do we do with WWTP
biosolids:
- 60% land applied
— 22% incinerated
- 17%landfilled
Approximate content (not all
"nano"):
- 0.4 to 1 mgTi/gdrySS
- 0.004 to 0.03 mgAg/ g dry SS
Working with biosolids from
EPA Inventory and local
facilities
Biosolids to
Land
Application, etc
215
-------
& ^1
Summary & Needs
NM Sources &
Uses in Society
«•
Wastewater
Treatment
Plants
4
'
Liquid Effluent to
Surface Waters
• Need better tools to
differentiate engineered
from "other" NPs in
wastewaters
• Pollutant removal models
for WWTPs are current not
suitable for predicting NPs
• Better relationships
between surface charge
and core composition
Biosohds to
• . • Fate of NMs in biosolids is
Land ,
, .. .. , poorly understood
Application, etc
Biological Fate & Electron
Microscopy Detection ofNPs During
Wastewater Treatment
Gordon Conference
2011 Gordon Research Conference
v-(J
Environmental Nanotechnology
Waterville Valley Resort, Waterville Valley, NH,
May 29 - June 3, 2011
s used here were functionalized anc
small (even among nonoparticles.)
Ag(-)
Fluorescence Images
ISU
Freeze-Dried
Biomass:
Fresh Biomass:
* Freeze-dried biomass shows less fluorescence than fresh
biomass.
216
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
P. Lee Ferguson
Analysis and Fate of Single-Walled Carbon Nanotubes and Their Manufacturing
Byproducts in Estuarine Sediments and Benthic Organisms
P. Lee Ferguson and G. Thomas Chandler
1 Department of Civil and Environmental Engineering, Duke University, Durham, NC; 2Department of
Environmental Health Sciences, University of South Carolina, Columbia, SC
Single-walled carbon nanotubes (SWNT) have emerged as a promising material for commercial and
industrial applications due to their outstanding electrical, optical, mechanical, and thermal properties. It is clear
that as these nanomaterials become more commonplace, they will eventually reach the ambient environment
through waste discharge or disposal. Our recent work and that of others has shown that SWNT have high
affinity for natural particulates in aquatic systems and are thus expected to concentrate in sediments after
discharge to receiving waters. Any assessment of the occurrence and fate of SWNT in the aquatic environment
will thus necessitate development of sensitive and selective detection of these materials in sediments.
Near Infrared fluorescence (NIRF) spectroscopy has advanced as a highly selective and information-rich
technique for sensitive detection and structural characterization of SWNT materials. We have combined
asymmetric flow field flow fractionation (A4F) with NIRF spectroscopy as a promising tool for determination
of SWNT in the environment. Different purification, concentration, and separation methods are discussed to
reduce matrix complexity and improve the detection limit of SWNT. In addition to concentration, structural
information such as shape, length distribution, or agglomeration state of SWNT also must be identified and
quantified to describe behavior and transport processes as well as biological interactions. NIRF spectral
features of SWNT were retained after extraction from sediment, allowing diameter/chiral wrapping angle
characterization for dilute solutions. Furthermore, A4F was applied as a separation method prior to NIRF
spectroscopic analysis to determine SWNT length distribution and to reduce matrix complexity by separation
of NOM and SWNT. We have utilized this comprehensive analytical approach to assess the fate and biological
uptake of CoMoCAT SWNT in marine sediment microcosms and benthic deposit feeding organisms. SWNT
were extracted from sediments and meiobenthic copepods and polychaete worms by ultrasonication in 2%
surfactant solutions and individual surfactant-wrapped nanotubes were isolated from aggregates by
ultracentrifugation. SWNT extracted from sediment and tissue in 2% sodium deoxycholate could be quantified
down to 9 ng/mL, and detection was linear over > 3 orders of magnitude. Our results show that NIRF-
spectroscopy is a valuable method for detection and characterization of surfactant-stabilized SWNT at trace
concentrations in the aquatic environment.
EPA Grant Number: R833859
The Office of Research and Development's National Center for Environmental Research 217
-------
Analysis and fate of single-walled
carbon nanotubes in estuarine
sediments and benthic organisms
P. Lee Ferguson1'2, Ashley N. Parks1,
P. Ariette Schierz2, Kate Washburn, G. Thomas Chandler3,
Kay Ho4, and Rob Burgess4
'Nicholas School of the Environment and department of Civil &
Environmental Engineering, Duke University, Durham, NC
'Department of Environmental Health Sciences, University of South
Carolina, Columbia, SC
4Atlantic Ecology Division, NHEERL, Narragansett, Rl
• •lllBllrr
E|N
SWNT as potential environmental
contaminants
• •ITIBIIIT
SWNT composites have already made
their way into the marketplace
(composite sports equipment,
nanoelectronic devices).
Numerous companies now supply
SWNTs on kilogram scale.
Annual worldwide production of SWNT
is estimated > 1,000t by 2011.
There are currently no reliable methods
to detect SWNT in complex mixtures
(e.g. sediment, tissue, ambient waters)
at low concentrations.
IN
CE|H
SWNT have unique structural
characteristics
Each possible nanotube structure is labeled
by two integers, (n,m), that uniquely define
its diameter and chiral angle. Red- metallic,
black semi metal lie
• •1IIBIITT
CE
IN
RESEARCH OBJECTIVE:
Implement and apply near infrared fluorescence
spectroscopy for qualitative and quantitative analysis of
SWNT in complex environmental media
1. Develop sample preparation methods for isolating SWNT from
sediment and tissue prior to near infrared fluorescence
spectroscopy.
2. Explore asymmetric flow field flow fractionation coupled with NIRF
spectroscopy for separating SWNT and reducing intereferences.
3. Apply AFFF-NIRF spectroscopy to analysis of SWNT uptake and
accumulation in sediment-dwelling organisms.
CE|N
Multi-laser NIR spectrofluorometer
Qualitative characterization of CoMoCat
SWNT type SG65 by NIRF spectroscopy
/ = 0.0794V"2 + nm + m2
i
;
L
CEIH
218
-------
Detection of CoMoCat SWIS
estuarine sediment
• CoMoCat SWNT were spiked into _ m
estuarine sediment at 10 jj,g/g "•= ?..
concentration. I ....
• Sequential extractions were
performed with 2% sodium
deoxycholate (ultrasonication at 40 W
for 10 minutes).
SWNT Diameter Chiral Recovery % after
0.7S2 8, 79 1
0.757 6, 96 •£•
0.706 7, 74 | 000001.
0.829 1, 11 \
0.916 9, 79
O.S40 8, 62 °™°°'
O.S95 7, SI
O.S06 9, 75
T typeSG65 in
by NIRF
f
SWNT extracted
from sediment:
81 ± 5% \
_LJL
am ?™ em mo i™» noro mm
Ifete CE|N
Quantitative performance of NIRF
spectroscopy for SWNT in sediments
'E
1
o
c
.2
0
• SWNT ;n sec! extract
280 ng mM •
I f
i
1 01 • 10
JJlUCC ^EHUT
• •ITIBIIIT 1vC|n
Quantitative performance of NIRF
spectroscopy for SWNT in sediments
• •1IIBIITT
sediment extract
CE
IN
Challenges: Sample purification methods
extracts of natural sediments
SWNTcsww=31 ng/ml
-Oxidation e.g. H2O2, KMnO4, ..
- Ultrafiltration (Centriprep 100 kDa)
- Ultracentrifugation
- Asymmetric Flow Field Flow Fractionation
CE|N
Separation mechanism in asymmetric flow field flow
fractionation AF4
CEIHT
Field flow fractionation coupled
with NIRF spectroscopy
FFF channel
CEIH
219
-------
Field flow fractionation coupled
with NIRF spectroscopy
FFF channel
DAD detector
• •IIIBIIfT
E|N
Field flow fractionation coupled
with NIRF spectroscopy
• •ITIBIIIT
FFF channel
»
DAD detector
»
MALS detector
IN
CE|H
Field flow fractionation coupled
with NIRF spectroscopy
FFF channel
»
DAD detector
»
MALS detector
»
NIR Fluorescense
detector
• •1IIBIITT
CE
IN
AF4 of CoMoCat SWNT
CE|N
Qualita
2%S
^
I
live detection of CoMoCat SWNT in
iDC by AF4 -NIRF spectroscopy
SWNT SG65 in 2% SDC C0 = 46 \ig ml-1
UV at 320 nm
SLS at 90* / \
. Vv~_...
NIR Flourescence at A 638 nm excitation
638 nm excitation / \ BB10crn-.
K\ =;=;::,
/ HTSiw
-^~~>:-5::^1-in nil i -
" time (mm) "
ate cEJfei
Separation of CoM
coupled with NIF
6.00EJH3-,
J SWNT c0 = 10 |jg ml"1
E „„
1 ,..«J svmrc.-iOKimi1
2- _i ,>11-1— mr, ,- ,r
! Z] rnvrvvm*
1.20E-012 -I
e.ooE-013 J 9fit^ co = 20 |jg ml '
oCat SWNT by AF4
?F spectroscopy
A
A
A
638 nm excitation
L 5765 cm'1
,0,40 cm"
Ntawt
s^r
0 20 40 60 80 100 120
time [min]
DU& CElN
220
-------
Separation of CoMoCat SWNT by AF4
coupled with NIRF spectroscopy
] SWNTc.-IOKimf1 S\ BB10cm"
1 kA — ™™-',
1 «J ' • '
3 i...i,J svmrc.-iorami1 /A
1 —] «- — JX—
£
o fcocE-ottJ SWNTc =20|jgmr'
8 1
«..«,] svmrc0-2oramr'
1 ' 1 1 1—
0 20 40
iM Cross flow variation
*A
_^_
60 80 100 120
time [min]
Ifete CE|N
Detection of CoMoCat SWNT extracted
from sediment by UV
SWNTin2%SDC
5WNT SG65 C0 = 46 |jg mM
SWNT extracted from
sediment
SWNTSG65 C0 = 10|jgmM
Blank sediment extract
• •ITIBIIIT
IN
CE|H
Detection of CoMoCat SWNT in sediment by
static light scattering
• •1IIBIITT
W^-^*"**
SWNT in 2%SDC
avNTSC-Co^ =46Mgmi'
SWNT extracted from
sediment
SG65c0 = 10|jgmM
Blank sediment extract
CE
JN
Detection of C
ex
:::]_
°
'E
1 «„•»:
t „:
I «••"]
| o-l^v^^i-J*-
0 JLff^K^^ff^.
0
oMoCat SWNT in sediment
tracts by NIRF
1 SWNT in 2%SDC
f. SWNT SG65 ca - 46 |jg mM
2%SDC
SWNT extracted from
^-ir**fc/'**S-. ' sediment
i^™«Hrf ~V^^ ^ SWMTSG65c0 = 10|jgmM
Blank sediment extract
20 40 60 80
time [min]
RS™ CE|H
CoMaCat SWNT are detectible in complex
sediment extracts using AFFF-NIRF
sediment spiked 10 ng SWNT/g
NIRF static
Reference: sediment extract
CoMaCat SWNT are detectible in complex
sediment extracts using AFFF-NIRF
sediment spiked 10 ng SWNT/g
NIRF static
Reference: sediment extract
CEllT
221
-------
SWNT do not degrade in sediments over one
month timescale
• •IIIBIIfT
E|N
Detection of SWNT SG65 in sediments and
tissues by NIRF spectroscopy
E/j/iydrosoma propinquum
Sediment extracts
(2% SDC)
DL = 60 ng g~1 sed
Extracts of Enhydrosoma
propinquum (2% SDC)
LOD= 7 ng g1 fis:
• •ITIBIIIT
IN
CE|H
CoMoCat SWNT were undetectable in sediment-
exposed amphipods and mysid shrimp
CE
IN
SWN I body burden measurements in sediment
and/or food exposed organisms
V 10g SWNT/g sediment
B Algae
Depurated Amphipods
^H Non-depurated Amphipods
Sediment
Extract
Algae Extract Amphipod;
sediment
exposure
Amphipod; Amphipod;
sediment + algae algae
exposure exposure
•viriif i IT
CE|H
Accumulation of SWNT in benthic
macroinvertebrates
SWNT suspension
in 0.5% gum
arable
i Nominal
concentrations: 1
M9/g, 10|jg/g
Long Island
Sound sediment
14-day exposure following US EPA (1993}
CEINT
SWNT bioaccumulation and trophic transfer
SWNT suspensioi
in 0.5% gum
arable
SWNT-spiked
Isochrysis galbana
Mercenaria
mercenaria
14-day exposure
Nominal concentrations: 100 |jg/g, 1000 |
Mercenaria
mercenaria fed
SWNT-spiked
Isochrysis
c
Nereis
virens
CEIN
222
-------
SWNT-spiked sediment exposure to Nereis virens
Nominal SWNT-
spiked sediment
concentration
10 ug/g
NIRF
spike
l_
1
S 0.00004
I
| 0.00002
Measured SW
concentration:
Batch A
7.1+1.4 ug/g
analysis of SWNT in NIRF an
sediment depurat
I1
, i c
EO'OO robo eo'oo oolio lofooniooialoo eobo
MT Measured SWNT
concentration:
Batch B
10.4+1. 6 ug/g
alysis of SWNT in ,/ Nl
d worms no
a , i~"
^ J i"
/ I-
y> |_
^S^-— I
"anuIllT1 °12 °°
fit runt*
^F analysis of SWNT in
n-depurated worms
B .
j£
•*• "ii:i™ "" "! "
CE|N
Direct NIRF analysis of worm extracts
may be limited by internal filter effects
NIRF analysis of SWNT in
non-depurated worms
900 1000 1100 1200 1300 1400 1500 1600
Wavelength nm 3m
AFFF-NIRF analysis at 1 1-°°"1:
638nm excitation | 5-"»EJ
'1 OOOE +
NIRF analysis of SWNT in
depurated worms
CE
IN
SWNT-spiked Isochrysis galbana exposure to
Mercenaria mercenaria
Nominal SWNT-
spiked algae
concentration
1000 |jg/g
Measured SWNT
concentration:
7/30/10
94.5 ug/g
Measured SWNT
concentration:
8/2/10
63.2 ug/g
NIRF analysis of
SWNT in spiked
_ gae
= O.OOOW.
NIRF analysis of
SWNT in
Ited clan
NIRF analysis of
SWNT in non-
Ited clan
• viriif iIT
CEJN
No internal filter artifacts present in
NIRF analysis of clam extracts
NIRF analysis of
SWNT in
depurated clams
NIRF analysis of
SWNT in non-
depurated clams
CEIHT
OUTLOOK
Additional microcosm-based experiments
- Track uptake of SWNT manufacturing
byproducts in sediment-dwelling organisms
as well as degradation in sediments
- Investigate chirality/diameter-dependence
of SWNT interaction with sediment and
organisms
Survey environmental media (e.g. field
sediment) for contamination with SWNT
CEIH
223
-------
Conclusions
A novel and highly sensitive method for analysis of SWNT in sediments
based on near infrared fluorescence spectroscopy has been developed.
NIRF spectral features of SWNT were retained after extraction from
sediment, allowing diameter/chirality characterization for dilute
solutions.
AFFF can be used as a clean up tool prior to Nl RF analysis.
SWNT do not appear to be highly bioaccumulative in estuarine
invertebrates exposed via sediment or dietary routes.
• •IIIBIIfT
EjN
Acknowledgement
Ron Meyer and Dr. Sigrid Kuebler, Wyatt
Technology
Dr. Phil Wallis, Southwest Nanotechnolygy
Dr. Sergei M. Bachilo, Applied Nanofluorescense
CE1NT
• •ITIBIIIT
IN
CE|H
224
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
Robert A. Yokel
Safety/Toxicity Assessment of Ceria (A Model Engineered NP) to the Brain
Robert Yokel1, Mo Dan1, Rebecca Florence1, Jason Unrine1, Robert MacPhaif, Michael Tseng3,
Uschi Graham1, Rukhsana Sultana1, Sarita Hardas1, D. Allan Butterfield1 Peng Wu , and Eric Grulke1
University of Kentucky, Lexington, KY; 2U.S. Environmental Protection Agency,
Research Triangle Park, NC; University of Louisville, Louisville, KY
Nanoscale ceria has extensive commercial uses that can contribute to its environmental release, including
its use as a diesel fuel additive. We are studying it as a model metal oxide engineered nanomaterial (ENM).
Nanoscale ceria was nominated by the National Institute of Environmental Health Sciences for toxicological
consideration and is on the priority list of the OECD for measurement, toxicology, and risk assessment studies.
The purpose of this study is to characterize the physico-chemical properties of a representative ENM that
influences its distribution in blood, and into the brain compared to peripheral organs, biopersistence in those
organs, and resultant effects.
Studies were conducted with in-house produced and characterized ~ 5, 15, 30, and 65 nm citrate-coated
ceria ENM, compared to the cerium ion. Ceria ENM or the cerium ion was iv infused into rats to enable study
of its distribution in blood and translocation from systemic circulation, as would occur following absorption
into blood from any route of exposure. Blood was repeatedly sampled, an aliquot allowed to clot, and cerium
determined by ICP-MS in serum and clot up to 4 h and in whole blood for much longer. To extend our prior
work showing no appreciable reduction of cerium in mammalian reticuloendothelial tissues up to 30 days after
a single administration of nanoscale ceria; determine the routes and rate of its excretion; and further
characterize its distribution, persistence, and associated effects in the rat; a longer term study was conducted
with 30 nm ceria. Rats were terminated 1, 7, 30, or 90 days after a single iv ceria ENM infusion, compared to
cerium ion or vehicle controls. Rats were housed in metabolism cages for up to 2 weeks to quantify urinary
and fecal cerium output, cage-side observations were recorded daily, and they were weighed weekly. Nine
organs were weighed and samples of 14 tissues, blood, and CSF were collected for cerium determination by
ICP-MS. Oxidative stress markers (protein carbonyls [PC], 3-nitrotyrosine [3-NT], and protein bound 4-
hydroxy-2-trans-nonenal [HNE]), the glutathione antioxidant defense system (glutathione reductase and
peroxidase), and antioxidant enzymes (Mn-SOD and catalase) were measured.
Ten minutes after infusion, < 1 percent of 15 to 65 nm ceria ENM, < 2 percent of a mixture of 30 nm cubic
and rod ceria ENM, approximately 14 percent of the cerium ion, and approximately 33 percent of the 5 nm
ceria ENM remained in blood. For all 4 ceria ENMs the elimination from blood was biphasic, with an initial
half life of approximately 1 h and the second for the 5 to 30 nm ceria of approximately 100 to 200 h, and
approximately 12 h for the 65 nm ceria. The 15 and 30 nm ceria predominantly associated with blood cells,
whereas the 5 and 65 nm ceria and the cerium ion were approximately evenly distributed between serum and
the clot fraction of blood. The 5 nm ceria ENM was not seen in BBB or brain cells. The amount of 15 to 65 nm
ceria ENMs in brain samples was very small. Energy electron loss spectroscopy showed the ceria in situ to
have similar valence (considerable Ce(III)) to the dosing material up to 30 days. The 30 nm ceria ENM was
less acutely toxic than the cerium ion. Less than 1 percent of the ceria or cerium ion was excreted in the first
week, of which 98 percent was in feces. Ceria was primarily retained in the spleen, liver, and bone marrow.
Spleen weight was significantly increased in ceria-treated rats at several times after its infusion, and associated
with visual evidence of abnormalities.
Thirty nm ceria was associated with blood cells to a greater extent than larger or smaller ENMs, consistent
with reports showing this size is optimal for protein wrapping of ENMs. Ceria in blood is primarily cleared by
The Office of Research and Development's National Center for Environmental Research 225
-------
2010 U.S. EPA Nanotechnology Grantees Meeting
the reticuloendothelial tissues, in which it persists without significant decrease in mass amount for at least 3
months. Little enters the brain. Referring to nanoscale fiber-like structures, it has been stated: "The slower
[they] are cleared (high bio-persistence), the higher is the probability of an adverse response" (European
Parliament, Policy Department Economic and Scientific Policy "Nanomaterials in consumer products"). Our
results support the concern about the long-term fate and adverse effects of inert nanoscale metal oxides that
reach systemic circulation, from which they can distribute throughout the body, resulting in persistent retention
and potential adverse effects in multiple organs.
These results of ENM translocation, biopersistence, and hazard identification in the mammal provide data
for ENM risk characterization.
Reference:
Hardas SS, Butterfield DA, Sultana RL, Tseng MT, Dan M, Florence R, Unrine JM, Graham UM, Wu P,
Grulke EA, Yokel RA. Brain distribution and toxicological evaluation of a systemically delivered engineered
nanoscale ceria. Toxicological Sciences 2010;116(2):562-576, doi: 10.1093/toxsci/kfql37.
EPA Grant Number: R833772
The Office of Research and Development's National Center for Environmental Research 226
-------
Safety/toxicity assessment of
ceria (a model engineered NP)
to the brain
The research team
Robert A. Yokel and Mo Dan
- Department of Pharmaceutical Sciences, College
of Pharmacy & Graduate Center for Toxicology,
University of Kentucky, Lexington, KY
Jason Unrine
- Department of Plant and Soil Sciences, U KY
Michael T. Tseng
- Departments of Anatomical Sciences &
Neurobiology, University of Louisville, Louisville,
KY
The research team
Uschi M. Graham
-Center for Applied Energy Research, U KY
D. Allan Butterfield, Rukhsana Sultana,
& Sarita Hardas
- Department of Chemistry, U KY & (DAB)
Center of Membrane Sciences, U KY
Eric A. Grulke & Peng Wu
-Chemical & Materials Engineering
Department, U KY
Objective of this research
Characterize the physico-chemical properties
of a model engineered nanomaterial (ENM)
that influence its biodistribution and effects,
including:
- distribution across the blood-brain barrier (BBB)
- effects on oxidative stress endpoints in the brain
- uptake into selected peripheral organs
- persistence over time.
ENM studied
Ceria (CeO2, cerium dioxide, cerium oxide)
was selected because:
- it is an insoluble metal oxide that can be readily
observed and quantified in tissue (electron
microscopy, ICP-MS).
- it has current commercial applications (a catalyst
in diesel fuel and an abrasive in integrated circuit
fabrication).
- it has been reported to be cytotoxic as well as
neuroprotective, representing the controversy
about nanoscale materials.
ENM studied
We prepared citrate-coated ceria and
characterized them using:
- dynamic light scattering (size)
- transmission electron microscopy (TEM) with an energy
dispersive X-ray spectrometer (size and morphology)
- X-ray diffraction (composition & crystallinity)
- BET surface area analyzer (size)
- zeta potential (surface charge & stabilizing agent)
- FTIR (surface charge and stabilizing agent)
- scanning TEM with electron energy loss spectroscopy
(valence)
- thermogravimetric analysis with mass spectrometry
(surface citrate coating)
227
-------
ENMs studied
Ceria
ENM
size
(nm)
5
15
30
30
65
shape
polyhedral
polyhedral
cubic
cubic + rods
polyhedral
Zeta potential
-53±7mVatpH~7.35
-57±5mVatpH~7.3
-56±8mVatpH~7.3
-22±5mVatpH~7
Miller indices
(111), (210), (200)
(111), (210), (200)
(111), (210), (200)
(111), (210), (200)
Extent of
surface
citrate
coating
- 40%
- 27%
- 18%
~ 15%
HRTEM and STEM images of ceria ENMs
5 nm polyhedral
15 nm polyhedral
tf.
30 nm cube
•'ECa ''
30 nm cube & rod
0
Objective: To assess the influence of size
on ENM distribution, persistence,
translocation and toxicity
• Citrate-coated ceria i.v. infused into un-
anesthetized rats (0 or ~ 100 mg/kg);
terminated 1 or 20 h or 30 days later.
Blood and tissue [cerium]
• Brain cortex cerium was always < 1% of the
dose.
- We did not see 5 nm ceria in brain, only in brain
vasculature
• Spleen cerium concentration was greater than
liver cerium concentration.
• Liver had the greatest mass amount of the ceria
dose.
• There was little decrease in liver and spleen
cerium up to 30 days.
Electron energy loss spectrometry
characterization of ceria as synthesized and in
situ
There was no observable change in the M5/M4
peak ratio (Ce(lll)/Ce(IV) ratio) of 5 or 30 nm
ceria in spleen agglomerates 30 days after
ceria administration compared to the freshly
prepared ceria ENM.
Ceria distribution in and
elimination from blood
Rats were iv infused with 5, 15, 30 or 65 nm
citrate-coated ceria, an mixture of 30 nm cubic
and rod citrate-coated ceria, or the cerium ion.
After the infusion blood was drawn at 10, 30,
45,60, 120 and 240 min.
Cerium was determined in whole blood,
plasma and clot.
Whole blood cerium was determined in these
and other rats up to 90 days after ceria dosing.
228
-------
Ceria in blood
Ten min after completion of the 1 h ceria infusion
30% of the 5 nm ceria was in blood; < 1% of the 15,
30 and 65 nm ceria.
Compartmental pharmacokinetic analysis of whole
blood cerium generally showed an initial t,/2 of 1 h and
a beta phase half-life of ~ 100 h.
The 15 and 30 nm ceria predominantly associated
with blood cells, whereas the 5 and 65 nm ceria were
~ evenly distributed between the two compartments.
The greatest association of the 30 nm citrate-coated
ceria with blood cells in the clot fraction is consistent
with reports showing this size is optimal for protein
wrapping of ENMs.
A 90 day survival study to assess longer
term distribution, persistence and effects
Single iv dosing of 87 mg 30 nm ceria/kg, 50
mg cerium ion/kg, or vehicle.
Termination 1, 7, 30 or 90 days later.
Fecal and urinary Ce excretion (metabolic
cage).
Weekly body weight.
Weights and cerium concentration in multiple
organs and fluids.
Oxidative stress markers, histology (LM &
EM).
A single ceria ENM infusion resulted in
modest decreased body weight gain.
Less than 1% of the ceria ENM or cerium ion
dose was eliminated in a week.
Ceria was retained primarily in
reticuloendothelial tissues. The liver
contained ~ 20% and the spleen ~ 15% of
the dose 90 days after ceria administration.
No great decrease of the mass amount of
ceria in liver and spleen occurred over 90
days.
Spleen pathology 30 and 90 days after ceria
- Splenomegaly: ~ 2-fold with 5 and 15 nm and 1.5-fold with
30 nm at 30 days.
- The red pulp 30 days after 5 nm ceria showed numerous
densely stained lymphatic cells .
- The white pulp 90 days after 30 nm ceria showed ceria
containing cell clusters.
- Granulomatous formations were seen 90 days after ceria.
Liver pathology 30 and 90 days after ceria
• 30 days after 5 nm ceria:
- Non-uniform granuloma formations containing
ceria-loaded Kupffer cells.
- Mononucleated cell infiltration among the
hepatic parenchyma and at perivascular sites.
- Mononucleated cells appeared to encircle
Kupffer cells.
- No evidence of fibrosis or abscess formation.
• 90 days after 30 nm ceria:
- Granulomatous formations seen.
Conclusions
Citrate-coated 5 to 65 nm ceria does not enter the brain
to any significant extent.
It is primarily cleared by reticuloendothelial organs and
sequestered in intracellular agglomerates.
Some of these results are in Hardas et al Toxicological
Sciences 2010 116(2):562-576.
The Ce valence does not change in situ (in the first 30
days).
There is little clearance of 5 to 65 nm ceria from
reticuloendothelial organs, for 30 nm up to 90 days.
The smaller the ceria ENM, the longer it remains in
blood before being cleared.
Maximal distribution into blood cells was seen with 30
229
-------
Conclusions
Ceria ENM and the cerium ion are very slowly eliminated.
Ceria ENM does not always behave like the cerium ion, in
its distribution in blood or tissues.
Referring to nanoscale fiber-like structures, it has been
stated: "The slower [they] are cleared (high bio-
persistence) the higher is the probability of an adverse
response". (European Parliament, Policy Department
Economic and Scientific Policy "Nanomaterials in
consumer products").
These results further support the concern about the long
term fate and adverse effects of inert nanoscale metal
oxides that reach systemic circulation, from which they
can distribute throughout the body, resulting in persistent
retention and potential adverse effects in multiple organs.
Future Plans
Complete the histopathology, agglomeration
extent and localization, cerium valence, and
oxidative stress marker analyses as a
function of time (1, 7, 30 and 90 days) after
the 30 nm ceria infusion.
230
-------
Handouts on Centers for Environmental
Implications of Nanotechnology (CEIN)
-------
232
-------
UNIVERSITY
UC CEIN Predictive ami Midti-dlsdpltHry •RNdcetofy model
EWM
libraries
Center for Environmental
Implications of Nanotechnology
Mission Statement
The University of California Center for Environmental Implications of Nanotechnology (DC CEIN) was established to ensure
that nanotechnology is introduced in a responsible and environmentally compatible manner, thereby allowing the US and
International Communities to leverage the benefits of
nanotechnology for global economic and social benefit. This
mission is being accomplished by developing a series of decision
tools based on models of predictive toxicology and risk ranking
premised on selected nanomaterial property-activity
relationships that determine fate, transport, exposure, and
biological injury mechanisms at cellular, tissue, organism, and
population levels. Since its founding in September 2008, the UC
CEIN has successfully integrated the expertise of engineers,
chemists, colloid and material scientists, ecologists, marine
biologists, cell biologists, bacteriologists, toxicologists, computer
scientists, and social scientists to create the predictive scientific
platform that will inform us about the possible hazards and safe
design of engineered nanomaterials [ENMs] that may come into
contact with the environment.
The research of the UC CEIN is carried out by 46 distinct but highly interactive research projects across 7
interdisciplinary research groups (IRGs):
• ENM Standard Reference and Combinatorial Libraries and Physical-chemical Characterization [IRG 1 ]
• Studying ENM Interactions at the Molecular, Cellular, Organ, and System Levels [IRG 2]
• Organismal and Community Ecotoxicology [IRG 3]
• Nanoparticle Fate and Transport [IRG 4]
• High-Throughput Screening [HTS], Data Mining, and Quantitative-Structure Relationships for NM Properties and
Nanotoxicity [IRG 5]
• Modeling of the Environmental Multimedia NM Distribution and Toxicity [IRG 6]
• Risk Perception of Potential Environmental Impacts of Nanotechnology [IRG 7]
Key Center Accomplishments
• UC CEIN plays a national leadership role in Nano EHS
initiatives
• Compiled a nanomaterial library with 60+ different types of
NP [characterized and introduced into active research] -
Includes ZnO, CeO=, TiO=, CNT, Ag, Au, CdSe, Silica, Clays
• Developed a series of standardized dispersion protocols for
relevant environmental conditions
• Successfully demonstrated the oxidative stress heirarchical
paradigm for NPs with varying properties
• As an exercise of safe design, ZnO doped with Fe has been
found to significantly reduce ZnO NP toxicity in mammalian
cells
• Use of zebrafish embryos as an in vivo model for high-
content screening has shown that in vitro toxicity testing
results agree with HCS zebrafish in vivo studies, except
when using silver nanoparticles
Bacterial toxicity research shows that ZnO and Ce02 inhibit
growth more than Ti02, with gram positive bacteria found
to be relatively more sensitive
Exposure to Quantum Dots across trophic levels show
significant bioaccumulation in bacteria and biomagnification
in protozoa
Testing of ZnO across ecosystems has shown that ZnO is
consistently toxic, with toxicity resulting from exposure to
Zn ions following ENM aggregation and Zn ion shedding
Studies of mobility, persistence, bioavailability and reactivity
of ENMs in environmental conditions are providing key
characteristics of metal oxide NPs in seawater, freshwater,
and terrestrial environments
Efficient removal of ENM from aqueous systems can be
archived by pH destabilization, coagulant dosing,
sedimentation, and ultrafiltration
233
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Through validation of commercially available HTS
technology, we have implemented gene reporter assays
that provide readouts of known cellular signaling pathways.
Preliminary results identify genotoxicity in a subset of ENMs
A new efficient computer algorithm for feature selection
ranking was developed for screening and ranking
nanoparticle properties for the development of quantitative
property-structure relationships
• An international survey of Industry NanoEHS is providing
key insights into industry practices, perceived risks, and
gaps in understanding, with "lack of information" being a
key impediment to the implementation of Nano EHS
programs in industry
• A Summer 201 0 survey of nanotoxicology and regulatory
experts will provide a vital comparative framework for
future public and industry risk perception studies
Future Directions Include:
• Develop and characterize new libraries of Pt, Pd, SWCNT, Mesoporous Silica, and new derivations of metal oxides
• Continue ongoing cytotoxicity studies with ENM libraries and analyze data from in vitro and in vivo studies to rank NP toxicity,
assess predictive power of in vitro studies, and begin building expert system required to generate structure activity relationships
• Expand marine organisms, cellular, and bacterial studies beyond the initial metal oxide NPs [ZnO, Ti02, Ce02]
• Adapt HTS methods of toxicity screening to marine and bacterial cells, demonstrating and documenting performance and
challenges
• Results from ongoing HTS ecotoxicology experiments are being used to design mesocosm experiments in marine, terrestrial,
and freshwater ecosystem studies
• Incorporation of CNT, Ag, QDs, Pt as well as NP of different sizes and morphologies into ongoing Fate and Transport studies
• Advance HTS gene knockout studies with yeast and bacterial strains
• Development of an automated high content screening method to enhance zebrafish in vitro toxicity studies
• As experimental data from across Center projects enters the Central Data Management system, models for multimedia
transport and fate and nanoparticle structure activity relationship models will be refined and expanded. Development of a series
of NP decision tools will commence with an initial focus on establishing questions needed to design model pathways
• Data from industry survey, public environmental risk perception survey, and survey of nanotoxicology and regulatory experts will
help provide valuable knowledge about the societal implications and contexts for risk characterization
Education and Outreach
UC CEIN will serve to enhance our understanding of the environmental hazards of nanomaterials. Education and outreach programs to
train scientists, develop safe handling guidance for nanomaterials, and develop methods to communicate the implications of our research
to the public are key to the success of the Center. The knowledge generated by the Center will directly benefit scientists, public agencies,
industrial stakeholders, and the general population.
UC CEIN is housed within the California NanoSystems Institute at the University of California, Los Angeles, with a second major hub at the
University of California, Santa Barbara. Research partners include: UC Davis, UC Riverside, Columbia University, University of Texas, Nanyang
Technological University, Northwestern University, the Molecular Foundry at Lawrence Berkeley National Laboratory, Lawrence Livermore
National Laboratory, Sandia National Laboratory, University of Bremen, University of British Colombia, University College Dublin, Cardiff University,
and the Universitat Rovira I Virgili.
For more information: Please visit the UC CEIN website: http://www.cein.ucla.edu
UC CEIN, 6522 CNSI, Box 957227, Los Angeles, CA 90095-7227
Andre E. Nel, UCLA, Director/Pi; Arturo Keller, UCSB, Associate Director/Co-Pi; Hilary Godwin, UCLA,
Education Director/Co-Pi; Yoram Cohen, UCLA, Co-Pi; Roger Nisbet, UCSB, Co-Pi; David Avery, UCLA, CAD
UCLA
UCSB
University of California Center for Environmental Implications of Nanotechnology [UC CEIN] is supported by the
National Science Foundation and the U.S. Environmental Protection Agency [NSF: DBE-0830117]
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Center for the Environmental
Implications of NanoTechnology
CEINT Goals
• Elucidate the fundamental principles that
determine environmental behavior and
effects of nanomaterials
• Provide guidance in assessing existing and
future concerns surrounding the use of
engineered nanomaterials
• Educate students and the general public
regarding nanotechnology, nanoscale
science, and the environment
•*CEINT Member
CEINT Partner
Research Focus Areas
CEINT organizes a comprehensive effort looking
at the environmental implications of
nanotechnology with a focus on:
• Exposure: transport, fate, and
transformation
• Effects in complex, real environments
• Risk assessment to inform decision-making
CEINT Research Themes and Cores
CoreiA_jK^-^^ ^Core B
^~~~^ ~~"\ ^'" ~~~\ f^~ \ / \ i \
^engineered,) \ incidental )\ natural ) (v ecosystem ) ( organism )
Theme 3 \ ^^^^^fheme 2
DukeCarnegieMellonHViigniaTech i
CEINT Research Approach
Nanomaterial Properties
Engineered (Core A)
Natural and Incidental (Core B)
Theme #1
Exposure: Fate, Transport, and Transformation
I?
(D -i
3 I
m Qj
p* r+
O m
o- I
ij <
m -a
2 rt
Theme #2
Cellular & Organismal Responses
Theme #3
Ecosystem Responses
Modeling, Risk Assessment, and Societal Implications
(Core C)
Key Findings to Date
1. Laboratory experiments in simplified
systems were not sufficient to fully
evaluate NM risks
• Observed mesocosm effects from Ag
NP exposure were not predicted
based on findings from laboratory
experiments
2. Particle coatings have a substantial role
in all observed NP behaviors
• Coatings (particularly organic
macromolecular) directly impact
fate, transport, toxicity, and effects
observed for nanomaterials
3. NPs are ubiquitous in the environment
• Natural NPs are found in impacted
and unimpacted natural
environments and in engineered
environments
4. Different nanoparticle properties map to
different toxicological endpoints
UNIVERSITY OF KENTUCKY 1
STANFORD
UNIVERSITY
-------
Center for the Environmental
Implications of NanoTechnology
Significant Challenges and Approaches to Overcoming Them
1. Quantifying and speciating NMs in complex matrices at relevant environmental concentrations
o Distinguishing between effects from dissolved and particulate Ag species
• Working on methods to speciate Ag in complex media
o Difficult to quantify and locate C- and Fe-based nanomaterials in natural samples because of
background concentrations of these elements
• Developing new detection methods and using advanced techniques, including synchrotron
techniques (XANES and U.-XRF), and darkfield microscopy/hyperspectral imaging
o Difficult to characterize nanoparticle macromolecular coatings
• Developing methods to characterize the adsorbed macromolecules on NP surfaces in situ
2. Bioavailability and Toxicity
o Separating dissolved and particulate toxicity is difficult - using multiple techniques to improve
confidence in results
o Difficult to determine uptake mechanisms (i.e. dissolved vs. particulate)-using advanced techniques,
including synchrotron techniques (XANES and U.-XRF), and darkfield microscopy/hyperspectral imaging
paired with various ICP-MS methods (e.g. laser ablation and flow field-flow fractionation)
3. Providing NPs of sufficient quality and consistency for Center researchers and for mesocosms
o Avoiding commercially produced Ag NPs due to insufficient characterization and inconsistencies
between batches
o Dedicated personnel producing large quantities of materials with stringent QC protocols
o Dramatic differences in the behavior and toxicity of the "same" materials (similar size, composition,
coating) from commercial and internal sources
4. Insufficient and fragmentary information available for risk assessment
o Reducing variance of estimates of nanoparticle production and environmental emissions
o Using machine learning to identify nanoparticle properties responsible for toxicity and to identify the
most meaningful measurements (units, distributions) of these properties
o Prioritizing research needs based on value of information from research projects
Funding
This material is based upon work supported by the National Science Foundation (NSF) and the Environmental
Protection Agency (EPA) under NSF Cooperative Agreement EF-0830093, Center for the Environmental Implications
of NanoTechnology (CEINT). Any opinions, findings, conclusions or recommendations expressed in this material are
those of the author(s) and do not necessarily reflect the views of the NSF or the EPA. This work has not been
subjected to EPA review and no official endorsement should be inferred.
Contact Information
http://ceint.duke.edu
Mark Wiesner, Director, Duke University, wiesner(5)duke.edu
Greg Lowry, Deputy Director, Carnegie Mellon, glowrvffiandrew.cmu.edu
Cole Matson, Executive Director, Duke University, matsonffiduke.edu
Glenda Kelly, Assoc. Dir. for Assessment and Outreach, Duke University, glenda.kellvffiduke.edu
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U.S. EPA NANOTECHNOLOGY GRANTEES MEETING
In Conjunction with the SETAC North America 31st Annual Meeting
Bridging Science with Communities
November 8 - 9, 2010 • Oregon Convention Center • Portland, OR
Speaker List
Devrah Arndt
University of Wisconsin, Milwaukee
School of Freshwater Sciences
600 E Greenfield Avenue
Milwaukee, WI 53204
Telephone: (414) 382-1700
E-mail: arndtd@uwm.edu
Amiraj Banga
Indiana University-Purdue University
Indianapolis
Department of Biology
SL354
723 W Michigan Street
Indianapolis, IN 46202
Telephone: (317)517-2880
E-mail: abanga@iupui.edu
David Barber
University of Florida
Department of Physiological Sciences
Building 471, Mowry Road
PO Box 110885
Gainesville, FL 32611
Telephone: (352)294-4636
E-mail: barberd@ufl.edu
Yongsheng Chen
Georgia Institute of Technology
School of Civil and Environmental
Engineering
200 Bobby Doddy Way
Atlanta, GA 30332
Telephone: (404) 894-3089
E-mail: yongsheng.chen@ce.gatech.edu
Howard Fairbrother
Johns Hopkins University
Department of Chemistry
3400 N Charles Street
Baltimore, MD 21218
Telephone: (410)516-4328
E-mail: howardf@jhu.edu
P. Lee Ferguson
Duke University
Department of Civil and Environmental
Engineering
121 Hudson Hall, Box 90287
Durham, NC 27708
Telephone: (919) 660-5460
E-mail: lee.ferguson@duke.edu
Vicki Grassian
University of Iowa
Department of Chemistry
Chemistry Building Madison Street
Iowa City, IA 52242
Telephone: (319)335-1392
E-mail: vicki-grassian@uiowa.edu
Warren Heideman
University of Wisconsin
Department of Pharmaceutical Sciences
777 Highland Avenue
Madison, WI 53705
Telephone: (608) 276-7997
E-mail: wheidema@wisc.edu
237
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Patricia Holden
University of California
Bren School
3508BrenHall
Santa Barbara, CA 93106-5131
Telephone: (805)893-3195
E-mail: holden@bren.ucsb.edu
Andrij Holian
The University of Montana
Center for Environmental Health Sciences
280 Skaggs Building
32 Campus Drive
Missoula, MT 59812
Telephone: (406)243-4018
E-mail: andrij .holian@umontana.edu
Zhiqiang Hu
University of Missouri
Department of Civil and Environmental
Engineering
E2509 Lafferre Hall
Columbia, MO 65211
Telephone: (573) 884-0497
E-mail: huzh@missouri.edu
Jack Huang
University of Georgia
Department of Crop and Soil Sciences
1109 Experiment Street
Griffin, GA 30223
Telephone: (770) 229-3302
E-mail: qhuang@uga.edu
Chad Jafvert
Purdue University
Department of Civil Engineering
550 Stadium Mall Drive
West Lafayette, IN 47907
Telephone: (765)494-2196
E-mail: jafvert@ecn.purdue.edu
Terrance Kavanagh
University of Washington
Department of Environmental and
Occupational Health Sciences
Box 354695
Seattle, WA 98195
Telephone: (206) 685-8479
E-mail: tjkav@uw.edu
Stephen Klaine
Clemson University
Institute of Environmental Toxicology
509 Westinghouse Road
PO Box 709
Pendleton, SC 29670
Telephone: (864) 710-6763
E-mail: sklaine@clemson.edu
Qilin Li
Rice University
Department of Civil and Environmental
Engineering
6100 Main Street
Houston, TX 77005
Telephone: (713) 348-2046
E-mail: qilin.li@rice.edu
Jingyu Liu
Brown University
Department of Chemistry
324 Brook Street
Providence, RI 02912
Telephone: (401)489-8407
E-mail: jingyu_liu@brown.edu
Gregory Lowry
Carnegie Mellon University
Department of Civil and Environmental
Engineering
119 Porter Hall
Pittsburgh, PA 15213
Telephone: (412)268-2948
E-mail: glowry@cmu.edu
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Shaily Mahendra
University of California, Los Angeles
Department of Civil and Environmental
Engineering
5732 Boelter Hall
420 Westwood Plaza
Los Angeles, CA 90095
Telephone: (310)794-9850
E-mail: mahendra@seas.ucla.edu
Galya Orr
Pacific Northwest National Laboratory
Chemical and Materials Sciences Division
3335 Q Avenue
Richland, WA 99352-0000
Telephone: (509) 371-6127
E-mail: galya.orr@pnl.gov
Jonathan Posner
Arizona State University
Department of Chemical Engineering
PO Box 876106
Tempe, AZ 85287
Telephone: (480) 965-1799
E-mail: jposner@asu.edu
James Ranville
Colorado School of Mines
Department of Chemistry and Geochemistry
1400 Illinois Street
Golden, CO 80401
Telephone: (303) 273-3004
E-mail: jranvill@mines.edu
Amy Ringwood
University of North Carolina, Charlotte
Department of Biology
9201 University City Boulevard
Charlotte, NC 28223
Telephone: (704) 687-8501
E-mail: ahringwo@uncc.edu
John Rowe
University of Dayton
Department of Biology
300 College Park
Dayton, OH 45460-2320
Telephone: (937) 725-7308
E-mail: john.rowe@notes.udayton.edu
Wunmi Sadik
State University of New York, Binghamton
Department of Chemistry
Center for Advanced Sensors and
Environmental Systems
PO 6000
Binghamton, NY 13902-6000
Telephone: (607) 777-4132
E-mail: osadik@binghamton.edu
Martin Shafer
University of Wisconsin, Madison
Department of Environmental Chemistry
and Technology
660 N Park Street
Madison, WI 53706
Telephone: (608)217-7500
E-mail: mmshafer@wisc.edu
Robert Tanguay
Oregon State University
Environmental and Molecular Toxicology
Department
Sinnhuber Aquatic Research Laboratory
28645 E Highway 34
Corvallis, OR 97333
Telephone: (541)737-6514
E-mail: robert.tanguay@oregonstate.edu
Thomas Theis
University of Illinois, Chicago
The Institute for Environmental Science
and Policy
2121 W Taylor Street
Chicago, IL 60612
Telephone: (312)996-1081
E-mail: theist@uic.edu
239
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Jason Unrine
University of Kentucky
Department of Plant and Soil Sciences
N212N Agricultural Science Center North
Lexington, KY 40546
Telephone: (859)257-1657
E-mail: jason.unrine@uky.edu
Paul Westerhoff
Arizona State University
Department of Civil, Environmental, and
Sustainable Engineering
Box 5306
Tempe, AZ 85287-5306
Telephone: (480) 965-2885
E-mail: p.westerhoff@asu.edu
Xin-Rui Xia
North Carolina State University
Center for Chemical Toxicology Research
and Pharmacokinetics
College of Veterinary Medicine
4700 Hillsborough Street
Raleigh, NC 27606
Telephone: (919)513-6188
E-mail: xia@ncsu.edu
Robert Yokel
University of Kentucky
College of Pharmacy
Department of Pharmaceutical Sciences
511C Multidisciplinary Science Building
725 Rose Street
Lexington, KY 40536-0082
Telephone: (859) 257-4855
E-mail: ryokel@E-mail.uky.edu
240
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U.S. EPA NANOTECHNOLOGY GRANTEES MEETING
In Conjunction with the SETAC North America 31st Annual Meeting
Bridging Science with Communities
November 8 - 9, 2010 • Oregon Convention Center • Portland, OR
Participants List
Christian Andersen
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory
200 SW 35th Street
Corvallis, OR 93333
Telephone: (541) 754-4791
E-mail: andersen.christian@epa.gov
Joel Baker
University of Washington
Center for Urban Waters
326 E D Street
Tacoma, WA 98402
Telephone: (253) 254-7025
E-mail: jebaker@uw.edu
Ofek Bar-Han
University of Wisconsin, Madison
Division of Pharmaceutical Sciences
777 Highland Avenue
Madison, WI 53705
Telephone: (608) 262-4525
E-mail: barilan@wisc.edu
Marsha Black
University of Georgia
The College of Public Health,
Environmental Health Science
150 E Green Street
Athens, GA 30602-2102
Telephone: (706) 542-0998
E-mail: mblack@uga.edu
Bonnie Blalock
Western Washington University
Department of Environmental Toxicology
2514Peabody Street
Bellingham, WA 98225
Telephone: (360) 570-8652
E-mail: blalock@students.wwu.edu
Bonnie Blazer-Yost
Indiana University-Purdue University
Indianapolis
Department of Biology
723 W Michigan, SL358
Indianapolis, IN 46202
Telephone: (317)278-1145
E-mail: bblazer@iupui.edu
Raanan Bloom
U.S. Food and Drug Administration
Center for Drug Evaluation and Research
10903 New Hampshire Avenue
Silver Spring, MD 20993
Telephone: (301)796-2185
E-mail: raanan.bloom@fda.hhs.gov
Audrey Bone
Duke University
Nichols School of the Environment
Levine Science Research Center
308 Research Drive
Durham, NC 27707
Telephone: (843) 870-3752
E-mail: ajb62@duke.edu
241
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Ashley Bradley
Tennessee State University
Department of Chemistry
709 S 18th Street
Nashville, TN 37206
Telephone: (615)983-2409
E-mail: ashleybradleyroland@gmail.com
Amanda Carew
University of Victoria
Department of Biochemistry and
Microbiology
3800FinnertyRoad
Victoria, BC V8N6K8
Canada
Telephone: (250) 721-7086
E-mail: acarewl4@uvic.ca
Sung-Su Choi
Korea Environmental Industry and
Technology Institute
Green Technology Development Office
290, Jinheung-ro, Eunpyeong-gu
Seoul 122-706
Korea
Telephone: 82-2-3800-331
E-mail: choissu@keiti.re.kr
Rhett Clark
University of Alberta
Department of Chemistry
11227 Saskatchewan Drive
Edmonton, AB T2G 3E6
Canada
Telephone: (780) 492-3046
E-mail: rhett@ualberta.ca
Fiona Crocker
U.S. Army Engineer Research and
Development Center
Environmental Laboratory
3909 Halls Ferry Road
Vicksburg, MS 39180
Telephone: (601) 634-4673
E-mail: fiona.h.crocker@usace.army.mil
David Cwiertny
University of California, Riverside
Chemical Division
Department of Environmental Engineering
A242 Bourns Hall
Riverside, CA 92521
Telephone: (951)827-7959
E-mail: dcwiertny@engr.ucr.edu
John Davis
Dow Chemical
1803 Building
Midland, MI 48674
Telephone: (989) 636-8887
E-mail: jwdavis@dow.com
James Ede
University of Alberta
Department of Biological Sciences
11455 Saskatchewan Drive
Edmonton, AB TSJOB5
Canada
Telephone: (780)492-6162
E-mail: ede@ualberta.ca
Charles Eirkson
U.S. Food and Drug Administration
Center for Veterinary Medicine
Office of New Animal Drug Evaluation
7500 Standish Place
Rockville, MD 20855
Telephone: (240)276-8173
E-mail: charles.eirkson@fda.hhs.gov
Amro El Badawy
University of Cincinnati
Department of Environmental Engineering
3430 Telford Street, Apt. 9
Cincinnati, OH 45220
Telephone: (513)702-4117
E-mail: elbadaam@mail.uc.edu
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Jennifer Field
Oregon State University
Department of Environmental and
Molecular Toxicology
1007 Agriculture and Life Sciences Building
Corvallis, OR 97330
Telephone: (541) 737-2265
E-mail: jennifer.field@oregonstate.edu
Joe Fisher
Oregon State University
Division of Public Health
Department of Environmental and
Molecular Toxicology
PO Box 2875
Corvallis, OR 97339
Telephone: (831)915-9404
E-mail: fishejos@orid.orst.edu
April Ga
Northeastern University
Department of Civil and Environmental
Engineering
435 Snell Engineering Center
360 Huntington Avenue
Boston, MA 02115
Telephone: (617)373-3631
E-mail: april@coe.neu.edu
Gordon Getzinger
Duke University
Nicholas School of the Environment
Division of Environmental Sciences and
Policy
Box 2SRC
Durham, NC 27707
Telephone: (330) 328-1734
E-mail: gordon.getzinger@duke.edu
Greg Goss
University of Alberta
Department of Biological Science
11455 Saskatchewan Drive
Edmonton, AB T6H5N7
Canada
Telephone: (780)492-2381
E-mail: greg.goss@ualberta.ca
Eric Grulke
University of Kentucky College of
Engineering
Department of Chemical and Materials
Engineering
359 Ralph G. Anderson Building
Lexington, KY 40506-6097
Telephone: (859) 257-6097
E-mail: egrulke@engr.uky.edu
Raymond Hamilton
University of Montana
Center for Environmental Health Sciences
Department of Biomedical and
Pharmaceutical Sciences
32 Campus Drive
Missoula, MT 59812
Telephone: (406) 243-4542
E-mail: raymond.hamilton@umontana.edu
Heather Henry
National Institutes of Health
National Institute of Environmental Health
Sciences
Division of Extramural Research and
Training
Superfund Research Program
530 Davis Drive
Research Triangle Park, NC 27709
Telephone: (919) 541-5330
E-mail: henryh@niehs.nih.gov
Ashley Hinther
University of Victoria
Department of Microbiology and
Biochemistry
3800FinnertyRoad
Victoria, BC V8N 6K8
Canada
Telephone: (250) 721-7086
E-mail: ahinther@uvic.ca
243
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Kay Ho
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory
27 Tarzwell Drive
Narragansett, RI 02874
Telephone: (401)782-3196
E-mail: ho.kay@epa.gov
Ehsanul Hoque
Trent University
Environmental Resource Studies Program
CSBE108
1600 W Bank Drive
Peterborough, ON K9J7B8
Canada
E-mail: ehsanulhoque@trentu.ca
Wen-Che Hou
Arizona State University
School of Sustainable Engineering and the
Built Environment
Department of Chemical Engineering
501 E Tyler Mall, ECG 303
Tempe, AZ 85287
Telephone: (480) 727-9463
E-mail: whou4@asu.edu
Helen Hsu-Kim
Duke University
Department of Civil and Environmental
Engineering
121 Hudson Hall
Durham, NC 27708
E-mail: hsukim@duke.edu
Wesley Hunter
U.S. Food and Drug Administration
Center for Veterinary Medicine
Office of New Animal Drug Evaluation
MPNII(HFV-162)
7500 Standish Place
Rockville, MD 20855
Telephone: (240) 276-9548
E-mail: wesley.hunter@fda.hhs.gov
David Johnson
U.S. Army Engineer Research and
Development Center
Environmental Laboratory
Department of Risk Assessment
Building 6011
3909 Halls Ferry Road
Vicksburg, MS 39180
Telephone: (601) 634-2910
E-mail: david.r.johnson@usace.army.mil
Mark Johnson
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory
Western Ecology Division
200 SW 35th Street
Corvallis, OR 97333
Telephone: (541) 754-4696
E-mail: johnson.markg@epa.gov
Boris Jovanovic
Iowa State University
Division of Veterinary Medicine
Department of Biomedical Sciences
1090 College of Veterinary Medicine
Ames, IA 50011-1250
Telephone: (515)509-3274
E-mail: prcko@iastate.edu
244
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Kitae Kim
Oregon State University
Department of Environmental Molecular
and Toxicology
1007 Agriculture and Life Sciences Building
Corvallis, OR 97331
Telephone: (801) 503-5349
E-mail: kitae77@gmail.com
Jussi Kukkonen
University of Eastern Finland
Joensuu Campus
Department of Biology
PO Box 111
Joensuu Fl-80101
Finland
E-mail: jussi.kukkonen@uef.fi
David Lai
U.S. Environmental Protection Agency
Office of Pollution Prevention and Toxics
Ariel Rios Building (7403M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-7667
E-mail: lai.david@epa.gov
Mitch Lasat
U.S. Environmental Protection Agency
Office of Research and Development
Ariel Rios Building (8722F)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (703) 347-8099
E-mail: lasat.mitch@epa.gov
Eunkyung Lee
Gwangju Institute of Science and
Technology
Department of Environmental Science and
Engineering
261 Cheomdan-Gwagiro (Oryoug-dong)
Buk-gu, Gwangju 500-712
Republic of Korea
Telephone: 82-62-715-2449
E-mail: storyhil@gist.ac.kr
Shibin Li
Texas Tech University
Institute of Environmental Toxicology and
Human Health
Department of Environmental Toxicology
Reese Technology Center Building 555
1207 Gilbert Drive
Lubbock, TX 79416
Telephone: (806) 790-1927
E-mail: shibinli@tiehh.ttu.edu
Igor Linkov
U.S. Army Engineer Research and
Development Center
Environmental Laboratory
696 Virginia Road
Concord, MA 01742
Telephone: (617)233-9869
E-mail: igor.linkov@usace.army.mil
Stephen Lofts
Natural Environment Research Council
Lancaster Environment Centre
Library Avenue, Bailrigg
Lancaster LAI4AP
United Kingdom
Telephone: 44-1524-595878
E-mail: stlo@ceh.ac.uk
Cole Matson
Duke University
Center for the Environmental Implications
of NanoTechnology
124 Hudson Hall
Durham, NC 27708
Telephone: (919)660-5193
E-mail: matson@duke.edu
Heather McShane
McGill University
Division of Natural Resource Sciences
72111 Lakeshore Boulevard
Sainte-Anne-de-Bellevue, QC H9X 3V9
Canada
Telephone: (514)398-4306
E-mail: heather.mcshane@mcgill.ca
245
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He Miao
Tsinghua University of China
Department of Environmental Science and
Engineering
Beijing 100086
China
Telephone: 86-10-62796952
E-mail: hemiao@tsinghua.edu.cn
Denise Mitrano
Colorado School of Mines
Department of Chemistry and Geochemistry
1100 Illinois Avenue
Golden, CO 80401
Telephone: (603) 568-8324
E-mail: jmitrano@mines.edu
Wataru Naito
National Institute for Advanced Industrial
Science and Technology
16-1 Onogawa Tsukuba
Ibaraki 305-8569
Japan
Telephone: 81-29-861-8299
E-mail: w-naito@aist.go.jp
Kimberly Ong
University of Alberta
Department of Biological Sciences
CW315
Edmonton, AB T6G2E9
Canada
Telephone: (780)277-0355
E-mail: kjong@ualberta.ca
Emily Oostreen
Texas Tech University
Institute of Environmental Toxicology and
Human Health
Department of Environmental Toxicology
PO Box 41163
Lubbock, TX 79409
E-mail: e.oostreen@ttu.edu
Maria Victoria Peeler
Washington State
Department of Ecology
Department of Hazardous Waste
and Pollution Prevention
PO Box 47600
Olympia, WA 98504-7600
Telephone: (360) 407-6704
E-mail: mvpeeler@u.washington.edu
Elijah Petersen
National Institute of Standards and
Technology
Biochemical Science Division
100 Bureau Drive, MS 6311
Gaithersburg, MD 20899
Telephone: (301)975-8142
E-mail: elijah.petersen@nist.gov
Helen Poynton
University of Massachusetts, Boston
Department of Environment, Earth, and
Ocean Sciences
100 Morrissey Boulevard
Boston, MA 02125-3393
Telephone: (617)287-7323
E-mail: helen.poynton@umb.edu
Robert Reed
Colorado School of Mines
Department of Chemistry
1500 Illinois Street
Golden, CO 80401
Telephone: (360)434-1017
E-mail: roreed@mymail.mines.edu
Kim Rogers
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
944 E Harmon Avenue
Las Vegas, NV 89119
Telephone: (702) 798-2299
E-mail: rogers.kim@epa.gov
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Charlita Rosal
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
944 E Harmon Avenue
Las Vegas, NV 89119
Telephone: (702) 798-2179
E-mail: rosal.charlita@epa.gov
Nora Savage
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Research
Potomac Yards South (Building 1) (8722P)
2777 S. Crystal Drive
Arlington, VA 22202
Telephone: (703)347-8104
E-mail: savage.nora@epa.gov
Anne Sergeant
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Research
Potomac Yards South (Building 1) (8722P)
2777 S. Crystal Drive
Arlington, VA 22202
Telephone: (202) 343-9661
E-mail: sergeant.anne@epa.gov
Vishal Shah
Dowling College
Department of Biology
150 Idle Hour Boulevard
Oakdale,NY 11769
Telephone: (631)244-3339
E-mail: shahv@dowling.edu
Paul Shapiro
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Research
Potomac Yards South (Building 1) (8722P)
2777 S. Crystal Drive
Arlington, VA 22202
Telephone: (703)347-8106
E-mail: shapiro.paul@epa.gov
Heather Shipley
University of Texas at San Antonio
Department of Civil and Environmental
Engineering
One UTSA Circle
San Antonio, TX 78249
Telephone: (210)458-7926
E-mail: heather.shipley@utsa.edu
Babina Shrestha
Texas Tech University
Institute of Environmental Toxicology and
Human Health
Department of Environmental Toxicology
Reese Technology Center Building 555
1207 Gilbert Drive
Lubbock, TX 79415
E-mail: babina.shrestha@tiehh.ttu.edu
Ruth Sofield
Western Washington University
Huxley College of the Environment
Department of Environmental Sciences
516 High Street, MS 9181
Bellingham, WA 98225
Telephone: (360)650-2181
E-mail: ruth.sofield@wwu.edu
Jeff Stevens
U.S. Army Engineer Research and
Development Center
Environmental Laboratory
3909 Halls Ferry Road
Vicksburg, MS 39180
Telephone: (601) 634-4199
E-mail: jeffery.a.stevens.@us.army.mil
David Stewart
University at Buffalo
Department of Chemistry
261 Robert Drive, Apt. #4
North Tonawanda, NY 14120
Telephone: (716) 696-0382
E-mail: dts8@buffalo.edu
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Bikram Subedi
Baylor University
Department of Chemistry
2019 S Second Street
Waco, TX 76706
Telephone: (256)292-3513
E-mail: bikram_subedi@baylor.edu
Toshinari Suzuki
Tokyo Metro Institute of Public Health
Division of Water Quality
3-24-1 Hyakunin-Cho, Shinjaka
Tokyo 169-0073
Japan
Telephone: 81-3-3363-3231
E-mail:
toshinari-suzuki@member.metro.tokyo.jp
Claus Svendsen
Centre for Ecology and Hydrology
Department of Ecotoxicology
Maclean Building
Benson Lane
Crowmarsh Gifford
Wallingford, Oxon OX101BS
United Kingdom
Telephone: 44-7789920919
E-mail: csv@ceh.ac.uk
Soheyl Tadjik!
Postnova Analytics
230 S 500 E, #120
Salt Lake City, UT 84102
Telephone: (801) 521-2004
E-mail: st@postnova.com
Lisa Truong
Oregon State University
Environmental and Molecular Toxicology
Department
28645 E Highway 34
Corvallis, OR 97333
Telephone: (425)445-8182
E-mail: duongl@onid.orst.edu
Laxminath Tumburu
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
Research Laboratory
200 SW 35th Street
Corvallis, OR 97333-4902
Telephone: (541) 754-4632
E-mail: tumburu.laxminath@epa.gov
George Tuttle
Oregon State University
Environmental and Molecular Toxicology
Department
1007 Agriculture and Life Sciences Building
Corvallis, OR 97330
Telephone: (707)236-0918
E-mail: tuttleg@onid.orst.edu
Sascha Usenko
Baylor University
Department of Environmental Sciences
One Bear Place, 97226
Waco, TX 76655
Telephone: (541) 760-3855
E-mail: sascha-usenko@baylor.edu
Katrina Varner
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
PO Box 933478
Las Vegas, NV 89193-3478
Telephone: (702) 798-2645
E-mail: varner.katrina@epa.gov
Jiafan Wang
Texas Tech University
Institute of Environmental Toxicology and
Human Health
Department of Environmental Toxicology
PO Box 41160
Lubbock, TX 79410
Telephone: (806) 224-7080
E-mail: jiafan.wang@tiehh.ttu.edu
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Annie Whitley
University of Kentucky
Department of Plant and Soil Sciences
Agriculture Science Center
North Building (Office N-122G)
Lexington, KY 40546
Telephone: (904)864-8155
E-mail: annie.whitley@uky.edu
Paige Wiecinski
University of Wisconsin, Madison
Department of Molecular and
Environmental Toxicology
1525 Observatory Drive
Madison, WI 53706
Telephone: (608) 265-4849
E-mail: wiecinski@wisc.edu
Frank Witzmann
Indiana University School of Medicine
Department of Physiology
1345 W 16th Street, Room 308
Indianapolis, IN 46202
Telephone: (317)278-5741
E-mail: fwitzman@iupui.edu
Sarah Yang
University of Wisconsin, Madison
Department of Toxicology
1555 Observatory Drive
Madison, WI 53706
Telephone: (608) 265-4849
E-mail: klingbiel@wisc.edu
Xinyu Yang
Duke University
Nicholas School of the Environment and
Earth Sciences
Box 90328
Durham, NC 27705
Telephone: (919)475-8104
E-mail: xy20@duke.edu
Young-Hun Yoon
Korea Environmental Industry and
Technology Institute
Green Technology Development Office
290, Jinheung-ro, Eunpyeong-gu
Seoul 122-706
Korea
Telephone: 82-2-3800-345
E-mail: yhyoon@keiti.re.kr
Holly Zahner
U.S. Food and Drug Administration
7500 Standish Place, HFV-162
Rockville, MD 20855
Telephone: (240)276-8181
E-mail: holly.zahner@fda.hhs.gov
Richard Zepp
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
960 College Station Road
Athens, GA 30605-2700
Telephone: (706)355-8117
E-mail: zepp.richard@epa.gov
Wen Zhang
Georgia Institute of Technology
School of Civil and Environmental
Engineering
200 Bobby Dodd Way
Atlanta, GA 30332
Telephone: (480) 294-9782
E-mail: wzhang76@gatech.edu
Huajun Zhen
Rutgers University
Department of Environmental Sciences
14 College Farm Road
New Brunswick, NJ 08901
E-mail: zhenhuajun@gmail.com
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Contractor Support:
Mary Compton
The Scientific Consulting Group, Inc.
656 Quince Orchard Road, Suite 210
Gaithersburg, MD 20878
Telephone: (301) 670-4990
E-mail: mcompton@scgcorp.com
Denise Hoffman
The Scientific Consulting Group, Inc.
656 Quince Orchard Road, Suite 210
Gaithersburg, MD 20878
Telephone: (301) 670-4990
E-mail: dhoffman@scgcorp.com
Kristen LeBaron
The Scientific Consulting Group, Inc.
656 Quince Orchard Road, Suite 210
Gaithersburg, MD 20878
Telephone: (301) 670-4990
E-mail: klebaron@scgcorp.com
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U.S. EPA NANOTECHNOLOGY GRANTEES MEETING
In Conjunction with the
SETAC North America 31st Annual Meeting
Bridging Science with Communities
November 8 - 9,2010 • Oregon Convention Center • Portland, OR
REGISTRATION W LOGISTICS W AGENDA 1 f SETAC NANO
LINKS
For more information on EPA's Nanotechnology Research Program, go to:
www.epa.gov/nanoscience
www.epa.qotf/ncer/nano
www.epa.aQV/ncer/nanQ/Dublications/nano strategy iune2009.pdf
For more information on the National Science Foundation's Nanotechnology Programs,
go to:
http://www.nsf.gov/crssprgm/nano/
For more information on the National Nanotechnology Initiative and other Federal Agencies,
go to:
hrtF;.'/'-,vyvvv.nanc,gcv
http://www.nano.Qov/html/about/nniparticipants.html
251
National Center for Environmental Research
Science To Achieve Results (STAR) Research Program
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oEPA
United States
Environmental Protectio
Agency
U.S. EPA Nanotechnology
Grantees Meeting
Monday, November 8, and
Tuesday, November 9,2010
Where:
Oregon Convention Center
777 NE Martin Luther King, Jr. Blvd.
Rooms D135 and D136
Portland, OR 97232
Web Site:
http://www.scgcorp.com/nano2010
Meeting Contacts:
Paul Shapiro
shapiro.paul@epa.gov
Tina Conley
conley.tina@epagov
Registration Contact:
Denise Hoffman
dhoffman@scgcorp.com
In Conjunction With
SETAC North America 31st Annual Meeting
Bridging Science With Communities
The U.S. Environmental Protection Agency (EPA) Nanotechnology
Grantees meeting will provide a forum for EPA-funded researchers
to share their findings, problems, solutions and project plans, and
to discuss strategies for addressing issues of common concern.
The research focuses on what happens to nanoparticles and what
impacts on aquatic organisms the particles have when they enter
water environments.
This year, the EPA will hold its Nanotechnology Grantees Meeting
in conjunction with the Society of Environmental Toxicology and
Chemistry's (SETAC) North America 31st Annual Meeting, Bridging
Science With Communities. Participants in the SETAC meeting and
all others are welcome to attend.
Please register to attend.
http://www.scgcorp.com/nano2010/registration.asp
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