vvEPA
United States
Environmental Protection
Agency
Scientific, Technical, Research,
Engineering and Modeling Support
Final Report
State of the Science
Literature Review:
Everything Nanosilver
and More
RESEARCH AND DEVELOPMENT
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EPA/600/R-10/084
August 2010
www.epa.gov
Scientific, Technical, Research, Engineering
and Modeling Support
Final Report
Contract No. EP-C-05-057
Task Order No. 95
State of the Science
Literature Review:
Everything Nanosilver and More
Prepared for
Katrina Varner, Task Order Manager
U.S. Environmental Protection Agency
National Exposure Research Laboratory
Environmental Sciences Division
Las Vegas, NV
Submitted by
Jessica Sanford, Task Order Leader, Battelle
Raghuraman Venkatapathy, Project Manager, Pegasus
Prepared by
Amro EI-Badawy
David Feldhake
Raghuraman Venkatapathy
Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official
Agency policy. Mention of trade names and commercial products does not constitute endorsement or
recommendation for use.
U.S. Environmental Protection Agency
Office of Research and Development
Washington, DC 20460
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TABLE OF CONTENTS
List of Figures Hi
List of Tables v
List of Abbreviations vi
Executive Summary xii
1. Introduction 1
2. Historical and Current Applications of Silver and Silver Nanomaterials 5
2.1 Elemental Silver Characteristics and Sources 5
2.2 Chemistry of elemental silver 5
2.3 Historical and Current Applications of Elemental Silver and Silver compounds 7
2.4 Nanosilver: History and Applications 10
2.5 Silver Regulations in the US 11
3. Uses of Silver Nanomaterials 14
3.1 Properties of nanosilver 14
3.1.1 Antibacterial properties 14
3.1.1.1 Antibacterial mode of action 15
3.1.2 Antifungal properties 16
3.1.3 Antiviral properties 17
3.1.4 Anti-inflammatory properties 17
3.1.5 Anti-glycoprotein film properties 17
3.1.6 Anti-biofilm properties 18
3.1.7 Surface plasmon resonance properties 18
3.1.8 Plasmonic heating properties 18
3.1.9 Metal-enhanced fluorescence properties 19
3.1.10 Properties of silver nanomaterials that promote its biosynthesis 19
3.2 Scientific Applications 20
3.3 Industrial Applications 21
3.3.1 Catalysis 21
3.3.2 Electronics 22
3.3.3 Other Industrial Applications 22
3.4 Applications in Consumer Products 22
3.5 Medical Applications 23
3.6 Proposed and Projected Applications 24
4. Synthesis and Properties of Silver Nanomaterials 27
4.1 Methods of Synthesis 27
4.1.1 Synthesis Categories 27
4.1.1.1 Top-Down versus Bottom-Up 28
4.1.1.2 Synthesis Reactants in Bottom-Up Techniques 30
4.1.2 General Discussion on Nanosilver Synthesis 32
4.2 Silver Nanocomposites and Bimetallic Nanoparticles 35
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4.3 Environmental Perspective 35
4.4 Characteristics of the Silver Nanomaterials Products 37
4.5 Characterization Methods, Detection and Speciation 54
4.5.1 Methods for Measuring Ionic Silver in Nanosilver Suspensions 56
4.5.2 Methods for Isolating Ionic Silver from Nanosilver Suspensions 56
4.5.3 Novel Detection and Characterization Techniques for Environmental Samples 57
5. Potential Magnitude of Silver Nanomaterial Utilization and Environmental Exposure. 67
5.1 Inventory of Silver Nanomaterials: Industrial and consumer products 68
5.2 Routes of Release and Exposure, Ecological 72
5.3 Routes of Exposure, Human 77
5.3.1 Exposure via food 80
5.3.2 Exposure via consumer products 83
5.3.3 Exposure via Medical Applications 88
5.3.4 Exposure via occupation 92
5.4 Projected Quantities, Geographic and Demographic Distribution in the US 94
6. Toxicity and Health Effects 98
6.1 Silver Toxicity 98
6.2 Nanosilver Toxicity 100
6.2.1 Toxicity of Nanosilver to Organisms 102
6.2.2 Ecological or Multispecies Studies of Nanosilver Toxicity 105
6.2.3 Studies Concerning Human Health Including Mammalian Models 109
6.2.3.1 Respiratory Tract Toxicity 113
6.2.3.2NeuronalUptake 118
6.2.3.3 Dermal Toxicity 118
6.2.3.4 Gastrointestinal Tract Toxicity 120
6.2.3.5 Other Organ Toxicity 121
6.2.3.5.1 Kidney Toxicity 122
6.2.3.5.2 Liver Toxicity 123
6.2.3.5.3 Immune system Toxicity 123
6.2.3.5.4 Other blood effects 124
6.2.3.5.5 Reproductive system Toxicity 125
6.2.3.5.6 Genotoxicity, carcinogenicity 125
6.2.4 Cell Culture Nanosilver Toxicity 125
6.3 Conclusions on Nanosilver Toxicity 131
7. Life Cycle Analysis for Comprehensive Environmental Assessment 134
7.1 Nanosilver life cycle assessment 136
7.2 Nanosilver comprehensive environmental assessment 145
8. Data Gaps 147
9. Bibliography 150
Appendix A - List of US Companies Producing Nanosilver Containing Compounds 198
Appendix B - Woodrow Wilson Database Containing Nanosilver Products 207
Appendix C - List of Companies Producing Raw Nanosilver in the US 355
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List of Figures
Figure 2.1: Nanomaterials dimensions on the metric scale (in nm) 11
Figure 2.2: Analysis of FIFRA registered products containing nanosilver for the period 1950-
2010 12
Figure 4.1: Top-down and Bottom-up synthesis approaches 30
Figure 4.2: Schematic for an organometallic approach of synthesis of silver nanoparticles 34
Figure 4.3: Schematic of a spinning disc processor for synthesis of silver nanoparticles 36
Figure 4.4: Characterization of silver nanoparticle size and morphology with scanning electron
microscopy (SEM) 38
Figure 4.5: Schematic diagram of the Electrospray-Scanning Mobility Particle Sizer (ES-SMPS)
system 58
Figure 4.6: Cross section of a small part of theFlFFF channel 59
Figure 5.1: Forms of nanosilver incorporated in consumer products 69
Figure 5.2: Categories of nanosilver-containing products 70
Figure 5.3: Overview of silver flows triggered by biocidal plastics and textiles 75
Figure 5.4: Process simulated in the model developed by Blaser et al. (2008) 76
Figure 5.5: Routes of exposure, uptake, distribution, and degradation of nanomaterials in the
environment 78
Figure 5.6: Fresh Box, manufactured by FinePolymer, Inc. (South Korea), is a nanosilver
antimicrobial food container 81
Figure 5.7: The Nano Tea Pot - Aroma manufactured by Top Nano Technology Co., Ltd
(Taiwan) 81
Figure 5.8: Samsung's Silver Wash washing machine 85
Figure 5.9: Illustration showing production flow process and measurement locations in the
Korean silver nanoparticle manufacturing facility 88
Figure 5.10: Anti-microbial burn dressing manufactured by Anson Nano-Biotechnology
(Zhuhai) Co., Ltd., China 91
Figure 5.11: Conjuctival-corneal argyrosis in the craftsman occupationally exposed to silver.. 93
Figure 5.12: Number of companies, universities, laboratories and/or organization working in
nanotechnology across the US 95
Figure 5.13: Nanosilver flows during high emission scenarios 96
Figure 6.1: Systemic argyria of the skin from ingestion of colloidal silver 99
Figure 6.2: HAADF image of an HIV-1 virus 104
Figure 6.3: A schematic of the human body with pathways of exposure to nanoparticles 110
Figure 6.4: Deposition of particles in the respiratory tract as a function of their size 114
Figure 6.5: A simplified depiction of potential factors that may influence the effects of
engineered nanoparticles on the respiratory system 115
Figure 6.6: The proposed mechanism of nanosilver toxicity based on the experimental data.. 131
Figure 7.1: Comprehensive environmental assessment (CEA) 136
Figure 7.2: Choices associated with a nanotechnology throughout its life cycle as proposed by
Meyer et al. (2009) 137
Figure 7.3: Choices associated with a nanotechnology throughout its life cycle as proposed by
Gill (2007) 138
Figure 7.4: The Nanomaterial Database maintained by Nanowerk 142
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Figure 7.5: Quantified mass flows of silver triggered by the use of biocidal products and by
other silver uses 144
Figure 7.6: Nanosilver flows from the nanomaterial containing products to various
environmental compartments 145
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List of Tables
Table 3.1: Emerging applications of nanosilver in medical products 26
Table 4.1: Description of Evidence for Silver Nanoparticle Synthesis for General Applications39
Table 4.2: Description of Evidence for Silver Nanoparticles Synthesis for Specific Applications
45
Table 4.3: Description of Evidence for Silver Nanocomposites 49
Table 4.4: Description of Evidence for Bimetallic Silver Nanoparticles 51
Table 4.5: List of Acronyms used in Tables 4.1 to 4.4 52
Table 4.6: Possible Conventional Characterization and Detection Techniques for Nanosilver.. 62
Table 5.1: Main characteristics for human exposure to nanomaterials from food, consumer and
medical products 78
Table 5.2: Ranking of potential human exposures to nanosilver 79
Table 5.3: Summary of applications of nanotechnology in the food production chain 82
Table 5.4: Product categories with examples of products containing nanosilver 86
Table 5.5: Medical devices containing nanosilver 90
Table 5.6: Emerging applications of nanosilver in medical products 91
Table 5.7: Predicted environmental concentrations in Rhine River 96
Table 7.1: Companies selling nanosilver as listed in the Nanowerk database 139
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List of Abbreviations
-CN
-COOH
-NH2
-SH
AAS
ACGffl
AFM
107Ag, 109Ag
Ag
Ag+, Ag2+, Ag3+
Ag20
AgBr
AgCl
AgF
Agl
AgNO3
AgCN
AgN3
AgNP
AgOH
AgONC
AgS
ALP
ASTM
ATP
ATSDR
Au
BBB
B.C.
BET
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Cyano group
Carboxyl Group
Amine Group
Thiol Group
Atomic Absorption Spectroscopy
American Conference of Governmental Industrial Hygienists
Atomic Force Microscopy
Silver Isotopes
Elemental Silver
Ionic Silver
Silver Oxide
Silver Bromide
Silver Chloride
Silver Fluoride
Silver Iodide
Silver Nitrate
Silver Cyanide Complex
Silver Azide
Silver nanoparticle
Silver Hydroxide
Silver Fulminate
Silver Sulfide
Alkaline Phosphatase
American Society for Testing and Materials
Adenosine Triphosphate
Agency for Toxic Substances and Disease Registry
Gold
Blood Brain Barrier
Before Christ
Brunauer-Emmett-Teller Analysis
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BPEI
BSA
C2H5OH
Ca
CA
Cal/EPA
CE
CEA
Cl-
em
CNT
CO
CTAB
Cu
Cu(NO3)2
DI
DLS
DMA
DMF
DNA
DOC
DPR
DISC
E. coli
EDX
EFSA
EMEA
ES-SMPS
ESEM
Fe3O4
FFF
Branched Polyethyleneimine
Bovine Serum Albumin
Ethyl Alcohol
Calcium
California
California Environmental Protection Agency
Capillary Electrophoresis
Comprehensive Environmental Assessment
Chloride Ion
Centimeter
Carbon Nanotube
Carbon Monoxide
Cetyltrimethylammonium Bromide
Copper
Cupric Nitrate
Deionized
Dynamic Light Scattering
Differential Mobility Analyzer
N,N-Dimethyl Formamide
Deoxyribonucleic Acid
Dissolved Organic Carbon
Department of Pesticide Registration
California Department of Toxic Substances Control
Escherichia coli
Energy Dispersive X-ray Spectroscopy
European Food Safety Authority
European Medicines Evaluation Agency
Electrospray Scanning Mobility Particle Sizer
Environmental Scanning Electron Microscopy
Magnetite
Field Flow Fractionation
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FIFRA
F1FFF
FTIR
GE
GFAA
GI
GLP
H2
HAADF
HDA
HEK
HIV
HNO3
HR
1C
ICP
IL
IR
ISE
kg
L
LCA
LCST
LDH
M
m3
MA
mg
g
M
MS
Federal Insecticide, Fungicide and Rodenticide Act
Flow Field Flow Fractionation
Fourier Transformed Infrared Spectroscopy
Gel Electrophoresis
Graphite Furnace Atomic Absorption Spectroscopy
Gastrointestinal
Good Laboratory Practice
Hydrogen Gas
High Angle Annular Dark Field
Hexadecylamine
Human Epidermal Keratinocytes
Human Immunodeficiency Virus
Nitric Acid
High Resolution
Integrated Circuits
Inductively Coupled Plasma
Interleukin
Infrared
Ion Selective Electrodes
Kilogram
Liter
Life Cycle Analysis
Lower Critical Solution Temperature
Lactate Dehydrogenase
Molar concentration
Cubic Meter
Massachusetts
Milligrams
Micrograms
Micromolar concentration
Mass Spectrometer
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MTR
N2O
Na
NaBH4
NaCl
NaN3
NASA
NC
nm
NMR
NCV
NOEC
NOM
OECD
OES
OH'
OPP
ORD
OW
Pd
PEC
PEN
PNEC
PNIPAM
POTW
ppb
PVA
PVP
QA/QC
QSAR
RNA
Mass Transit Railway
Nitrous Oxide
Sodium
Sodium Borohydride
Sodium Chloride
Sodium Azide
National Aeronautics and Space Administration
North Carolina
Nanometer
Nuclear Magnetic Resonance
Nitrate Ion
No Observable Effect Concentration
Natural Organic Matter
Organisation for Economic Cooperation and Development
Optical Emission Spectroscopy
Hydroxide Ion
USEPA's Office of Pesticide Program
USEPA's Office of Research and Development
USEPA's Office of Water
Palladium
Predicted Environmental Concentration
Project of Emerging Nanotechnologies
Predicted No-Effect Concentration
Poly(N-isopropylacrylamide)
Publicly Owned Treatment Works
Parts Per Billion
Polyvinyl Alcohol
Polyvinylpyrrolidone
Quality Assurance/Quality Control
Quantitative Structure-Activity Relationship
Ribonucleic Acid
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ROS
rpm
Ru
SDS
SEC
SEM
SERS
SiO2
SMPS
Sn
SNCI
SNOMS
SNWG
SPR
SRHA
STP
TEM
TGA
TiO2
TNF
TSCA
TWT
UCPC
US/USA
USEPA
USFDA
USGS
UV
UV-Vis
WIP
X-EDS
Reactive Oxygen Species
Revolutions Per Minute
Ruthenium
Sodium Dodecyl Sulfate
Size Exclusion Chromatography
Scanning Electron Microscope
Surface Enhanced Raman Spectroscopy
Silicon Dioxide
Scanning Mobility Particle Sizer
Tin
Silver Nanotechnology Commercial Inventory
Single Nanoparticle Optical Microscopy and Spectroscopy
Silver Nanotechnology Working Group
Surface Plasmon Resonance
Suwannee River Humic Acids
Sewage Treatment Plant
Transmission Electron Microscopy
Thermal Gravimetric Analysis
Titanium Dioxide
Tumor Necrosis Factor
Toxic Substances Control Act
Thermal Waste Treatment
Ultrafine Condensation Particle Counter
United States of America
United States Environmental Protection Agency
United States Food and Drug Administration
United States Geological Society
Ultraviolet light
Ultraviolet-Visible light
Waste Incineration Plant
X-ray Energy Dispersive Spectrometry
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XANES X-ray Absorption Near Edge Structure
XPS X-ray Photoelectron Spectroscopy
XRD X-ray Diffraction
ZnO Zinc Oxide
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Executive Summary
Silver has been known to be a potent antibacterial, antifungal and antiviral agent, but in recent
years, the use of silver as a biocide in solution, suspension, and especially in nano-particulate
form has experienced a dramatic revival. Due to the properties of silver at the nano level,
nanosilver is currently used in an increasing number of consumer and medical products. The
remarkably strong antimicrobial activity is a major reason for the recent increase in the
development of products that contain nanosilver.
Of the more than 1000 consumer products that claim to contain nanomaterials, more than a
quarter of them contain nanosilver. Examples of consumer products that contain nanosilver
include food packaging materials, food supplements, textiles, electronics, household appliances,
cosmetics, medical devices, water disinfectants, and room sprays. While most of these
nanosilver-containing products were in the past manufactured in North America, manufacture of
nanosilver-containing products is shifting to the Far East, especially China, South Korea, Taiwan
and Vietnam. Currently, tracking products that contain nanosilver is getting to be difficult
because the products are almost always packaged under numerous brand names, and current
labeling regulations do not require that the nanomaterial be listed as an ingredient.
Knowledge of silver nanomaterials synthesis methods is important from an environmental
perspective. This information allows for the identification of characteristics and morphologies of
the produced silver nanomaterials that are crucial for a more focused approach when evaluating
their environmental fate, transport and toxicity. The main challenge in nanomaterials synthesis is
the control of their physical properties such as obtaining uniform particle size distribution,
identical shape, morphology, chemical composition and crystal structure. There are an extensive
number of synthesis methods of silver nanoparticles that are readily available in the literature.
All reported methods can be classified and categorized since they all follow common approaches
and the differences are limited to the specific reactants used and the reaction conditions.
Categories such as top-down versus bottom-up, green versus non-green and conventional versus
non-conventional have been reported. Physical methods such as milling or attrition, repeated
quenching and photolithography are usually involved in the top-down strategies while bottom-up
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techniques start with silver salt precursor that is reduced in a chemical reaction. Synthesis
methods can also be grouped under conventional and unconventional methods. Conventional
synthesis methods include the use of citrate, borohydride, two phase (water-organic) systems,
organic reducers, and inverse micelles in the synthesis process. Unconventional methods include
laser ablation, radiocatalysis, vacuum evaporation of metal, and the Svedberg method of
el ectrocondensati on.
Increased manufacture and use of nanosilver in products will lead to an inevitable increase in the
release of these particles into the environment at each and every step of its life starting from the
cradle (raw materials) to its grave (disposal/reuse). The availability of methodologies for the
detection and characterization of silver nanoparticles are thus essential in order to investigate
their fate, transport and toxicity. Current literature is focused on either the manufacture or testing
the toxicity of nanosilver. There is a lack of information on the characterization and detection
especially in environmental samples. There is a need for developing methods to measure the
nanosilver concentration, size, shape, surface charge, crystal structure, surface chemistry and
surface transformations. Some important questions to answer: Does nanosilver leach from
consumer products? If so, in what form? Is it aggregated or still in the nanoscale size? What are
its surface properties and chemistry? Does nanosilver dissolve or convert to ionic silver with
time or under different conditions such as pH? What is the speciation of silver? Is nanosilver
toxic? What are the toxicity mechanisms? Under what conditions do the mechanisms occur? Do
particles aggregate inside the testing media? Do particles aggregate inside the tested cells? In
order to answer these questions, characterization tools are needed. Possible characterization and
detection techniques for nanosilver include transmission electron microscopy, scanning electron
microscopy, electrospray scanning mobility particle sizer, atomic force microscopy, dynamic
light scattering, Brunauer-Emmett-Teller analysis, x-ray diffraction, x-ray photoelectron
spectroscopy, thermal gravimetric analysis, nuclear magnetic resonance spectroscopy, x-ray
absorption near edge structure, fourier transformed infrared spectroscopy, zeta size analysisr,
inductively coupled plasma mass spectroscopy, atomic absorption spectroscopy, and flow field
flow fractionation, among others.
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Nanomaterials have many potential benefits to society with their development and deployment in
science, engineering and technology. Their benefits, however, need to be weighed with any
potential cost to the environment and public health. The unknown health effects and risks
associated with these materials have drawn considerable attention from researchers, consumers
and regulators. As a result, scientists at the U.S. Environmental Protection Agency (USEPA) and
elsewhere have recognized the need to develop risk assessment processes to study the potential
health and environmental impacts of manufacturing nanomaterials as well as using these
materials in other products. In addition to the toxicological concerns, there are other aspects that
have to be considered during the risk assessment process. For example, the cost of transportation
must be evaluated, including the amount of emissions that are released from trucks, trains and
other vehicles that transport nanomaterials.
To address these issues, researchers have begun implementing more comprehensive assessment
tools such as Life Cycle Assessment (LCA) and Comprehensive Environmental Assessment
(CEA) to assess the cradle to grave cost/risk associated with any given product. A CEA
combines LCA with the risk assessment paradigm, which includes hazard identification, dose-
response assessment, risk characterization and exposure assessment. A CEA can establish the
comparative impact of products or processes in terms of specified impact categories including
the life cycle stages, environmental pathways, transport, transformation, exposure and effects
using a well-defined and documented methodology. Typical impact categories include global
warming/climate change, stratospheric ozone depletion, primary and secondary contaminants,
exposure, human toxicity, ecotoxicity, photo-oxidant formation, acidification, eutrophication,
land use, and resource depletion. The potential advantages of CEA-based evaluations for
nanomaterials are that they can address both the health and environmental consequences
associated with the inclusion of nanocomponents. The ultimate goal is to ensure that the potential
benefits of nanocomponents are realized in a manner that is safe for both consumers and the
environment without resulting in unintended consequences.
An LCA for nanomaterials generally has four main aspects: material selection, manufacturing,
application, and disposal/recycle. The material selection aspect of nanosilver LCA involves both
the composition (organic such as polymers, dendrimers, etc.; inorganic such as metals, metal
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oxides, etc.; carbon such as carbon tubes or a combination of any of these) and geometry of the
nanocomponents, which can be a variety of shapes (sphere, rod, etc.) and is dependent on the
synthesis methods. The manufacturing aspect of nanosilver LCA involves synthesis techniques,
while the application aspect of nanosilver LCA involves using the nanomaterials in either
naturally dispersive or composite form for a range of applications. The disposal/recycle aspect of
nanosilver LCA involves incineration, disposal in a landfill or removal during wastewater
treatment, among others.
To perform a CEA on nanoparticles, it is important to have some knowledge of the methods for
their synthesis. This information allows for the identification of the characteristics and
morphologies of the silver nanomaterials that are crucial for a more focused approach when
evaluating their environmental fate, transport and toxicity. These characteristics and
morphologies of the particles are determined by the methods of synthesis and the reactants that
are involved. The nanomaterials that are produced are known to aggregate unless the particle
surface is capped with a stabilizing agent, or unless the particles are suspended in a dispersant to
prevent their aggregation. Depending on the use of dispersants in the manufacturing process, or
lack thereof, different particle morphologies (e.g., size, shape, texture, phase, etc.) and surface
properties will emerge resulting in diverse characteristics that affect the fate, transport and
toxicity of the produced silver nanoparticles. Generally, aggregates of nanoparticles pose a lesser
risk to the environment than smaller nanoparticles.
Once information on the four aspects of an LCA have been determined (material selection,
manufacturing, application, and disposal/recycle), and information on environmental pathways
such as air, water, soil and food web, transport and transformation of primary and secondary
contaminants, exposure through inhalation, ingestion and dermal absorption, and toxicity is
collected, a CEA of the nanomaterials may be performed. The CEA determines the risk
associated with using a particular nanomaterial in a particular product, which is a function of
both exposure potential and toxicity. In some cases, risk may be low because the exposure
potential is low or the toxicity is low, or both. On the other hand, risk may be relatively high
even when exposure potential is low if the toxic potency is high, or vice versa. Calculating this
risk may be stymied by the fact that a large number of data gaps exist when considering the
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application of CEA to nanomaterials. Finding adequate data to model the potential fate and
effects of unintended releases of nanomaterials into the environment may be difficult. Minimal
data detailing the material inputs and environmental releases related to the manufacture, release,
transport, and ultimate fate of nanomaterials exist in the literature. Studies have mainly focused
on cradle-to-gate assessments (as opposed to the more extensive "cradle-to-grave" assessments
that look at the whole life cycle of the product, including disposal in a landfill or recycling into
raw materials for other products). Cradle-to-gate analyses investigate the production of either
nanocomponents or nanomaterials up to the point these materials leave the "gate" or the
manufacturing source. The usefulness of many nanomaterials has been demonstrated in
laboratory studies, and is yet to be implemented in consumer products. As a result, much of the
data must be estimated before a CEA can be performed.
Once nanosilver has been synthesized at a manufacturing facility, part of it may be used to
produce a final product, part of it may be shipped to a second manufacturing facility where it is
turned into a final product, and the remaining part may either be stored at the manufacturing
facility, lost due to leaks in the manufacturing process or disposed. It is necessary to know the
products that contain nanosilver, the amount each product contains, the process that is being used
to manufacture the product, the location of the product, the demographics of the end users of the
product, and the amount that is going to waste among other variables to perform a nanoparticle
LCA. Information on the amount that goes directly to waste or the amount that gets released
from a manufactured product due to interactions with its surrounding environment (e.g.,
nanosilver being released from socks during wash cycles) is necessary to determine the routes of
release of nanoparticles from its products as well as to determine routes of exposure to humans
and the ecosystem. The routes of release and exposure depend on the fate and transport of silver
nanomaterials, as well as the factors that affect transport (aggregation, capping agents and
environmental conditions such as pH, ionic strength, natural organic matter (NOM), etc.). In
addition, exposure will depend on whether nanomaterials interact with various environments
including soil, sediment, freshwater, groundwater, wastewater and marine environments.
There is evidence that silver, and in particular nanosilver, is toxic to aquatic and terrestrial
organisms, a variety of mammalian cells in vitro, and may be detrimental to human health. While
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undoubtedly silver and nanosilver have useful applications in the medical arena (for instance as
coatings for medical devices or as wound care for severe burns victims), their use may need to be
strictly controlled. Bacterial resistance to antibiotics is an ever increasing problem globally, and
indiscriminate use of biocidal silver in numerous consumer products is not only unnecessary, but
may further increase bacterial resistance to a dangerous level (Muhling etal., 2009). There are
preliminary indications that in nanoparticle form, the toxicity of ionic silver may be increased, or
that the nanoparticles may exert their own toxicity. The disposal of biocidal silver products into
wastewater raises a number of concerns as the resulting sewage sludge may be used on
agricultural soils, disposed as solid waste in landfills or be incinerated. Biocidal silver may also
disrupt the functioning of key soil microbial communities.
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1. Introduction
Silver has been valued throughout history for many of its properties that are useful to humans. It
is used as a precious commodity in currencies, ornaments, jewelry, electrical contacts and
photography, among others. One of the most beneficial uses of silver has been as a potent
antibacterial agent that is toxic to fungi, viruses and algae. Silver has long been used as a
disinfectant; for example, the metal has been used in treating wounds and burns because of its
broad-spectrum toxicity to bacteria as well as because of its reputation of limited toxicity to
humans.
In nanotechnology, a nano particle is defined as a small object or particle that behaves as a whole
unit in terms of its transport and properties. Nanotechnology takes advantage of the fact that
when a solid material becomes very small, its specific surface area increases, which leads to an
increase in the surface reactivity and quantum-related effects. The physical and chemical
properties of nanomaterials can become very different from those of the same material in larger
bulk form. Nanomaterials (such as nanotubes and nanorods) and nanoparticles are particles that
have at least one dimension in the range of 1 to 100 nm. Nanoparticles are classified solely based
on their size, and may or may not exhibit size-related properties that differ significantly from
those observed in bulk materials (ASTM, 2006; Buzea et a/., 2007). Due to the properties of
silver at the nanoscale, nanosilver is nowadays used in an increasing number of consumer and
medical products. Nanomaterials are nanoparticles that have special physicochemical properties
as a result of their small size (Buzea etal., 2007).
One important use of silver nanoparticles is to give products a silver finish. Nanosilver's strong
antimicrobial activity is a major reason for the development of nanosilver containing products.
Of the more than 1000 consumer products that contain nanomaterials, roughly 25% are claimed
to contain silver nanoparticles. Widely available consumer products that contain nanosilver
include food contact materials (such as cups, bowls and cutting boards), odor-resistant textiles,
electronics and household appliances, cosmetics and personal care products, medical devices,
water disinfectants, room sprays, children's toys, infant products and 'health' supplements
(Fauss, 2008).
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Some of the applications of nanosilver have resulted in government concern and discussions
among the public because, once released into the environment, the mobility, bioavailability and
toxicity of nanosilver on any ecosystem is determined in part by its stability in the environment.
An example of this is the addition of silver nanoparticles to socks in order to kill the bacteria
associated with foot odor. Several studies have shown that silver can easily leach into wastewater
during washing, thus, potentially disrupting helpful bacteria used in wastewater treatment
facilities or endangering aquatic organisms in lakes and streams. Some brands of socks were
shown to lose nearly all of their silver content within a few washings (Benn & Westerhoff,
2008). There is clear evidence that silver, and in particular nanosilver, is toxic to aquatic and
terrestrial organisms, a variety of mammalian cells in vitro, and may be detrimental to human
health. Surprisingly, there is little to no information on the behavior of silver nanoparticles in the
environment.
The stability of silver nanoparticles in the environment may be a function of many factors
including the type of capping agent (chemicals used in the synthesis of nanoparticles to prevent
aggregation) that is used, and surrounding environmental conditions, such as the pH, ionic
strength, nutrient levels, the presence of binding agents, etc. Because an extensive number of
capping agents are being used to manufacture silver nanoparticles and because it is almost
impossible to predict the behavior of silver nanoparticles in different environments,
understanding the implications of silver metal in the environment may provide an important
context for understanding the implications of nanosilver in the same environment. Nanosilver
may dissociate to form silver ions in the presence of moisture so at least part of the risk from
nanosilver will stem from release of these ions into the environment. The environmental risks
from silver itself may be mitigated by a tendency of the silver ion to form strong complexes that
are apparently of very low bioavailability and toxicity. In particular, the formation of complexes
with sulfides may strongly reduces the bioavailability of silver ions under some circumstances
(Luoma, 2008). It is not yet clear to what extent such speciation reactions will affect the toxicity
of nanosilver. If organic/sulfide coatings or complexation in natural waters similarly reduce the
bioavailability of nanosilver particles, the risks to natural waters will be reduced. It is also
possible that nanoparticles shield silver ions from such interactions, delivering free silver ions to
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the membranes of organisms or into cells. In that case, an accentuation of environmental risks
would be expected beyond that associated with a similar mass of silver itself.
To conduct a risk assessment of nanosilver under different environmental conditions, it is
important to characterize the nanoparticles, perform dose-metrics as well as quantify the
physicochemical properties of the nanomaterial. Nanoparticles have novel properties compared
to conventional chemicals. The characterization of these properties is important in order to
enable realistic estimations of exposure to humans and the ecosystem. This information is also
important to establish dose-response relationships for estimating the toxicity of these
nanoparticles. The determination of nanoparticle dose necessitates the development of analytical
tools to isolate and quantify these nanoparticles. Other analytical tools will be needed to quantify
these nanomaterials in order to obtain an accurate estimate of the risk due to exposure to these
particles.
Once a risk assessment of silver nanoparticles is performed, the regulatory policy challenge that
emerges is how to match the antiquated air-water-land basis of existing laws with the inherently
cross-media nature of the problem. Nanosilver can go from a manufacturing plant to a waste-
treatment plant to sludge to crops to the human-food chain. It is considered primarily a water
problem in the environment but primarily an air problem in the workplace. Like climate change,
acid rain and genetically modified crops, nanosilver is a problem that fits poorly into the old
boxes of the existing regulatory system. A cross-media approach is necessary as it allows a
policy maker to consider which sources of pollution or exposure are most important and which
can be most efficiently and effectively addressed. Current US government efforts to address
nanosilver are using the few cross-media tools available. Specifically, policy makers in the US
use the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) and the Toxic Substances
Control Act (TSCA) to regulate nanosilver in different ways. TSCA is broad, and potentially
could cover most nanomaterials. FIFRA, by contrast, is limited to pesticides, which are defined
to include antimicrobials. Since nanosilver is used primarily as an antimicrobial agent, most
nanosilver products may fall under the regulation of FIFRA. The acts also differ in the degree of
public protection and product oversight they offer. FIFRA is quite stringent and puts the burden
of proof for safety on the manufacturer. TSCA has a number of loopholes and exemptions that
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are perceived as lessening public protection and puts the burden of proof on the U.S.
Environmental Protection Agency to show that a substance is harmful.
This review is primarily intended to summarize available information that can be used to perform
a silver nanomaterial exposure assessment based on routes, quantities and effects of exposure.
This information can be used to perform a comprehensive environmental assessment or an LCA
of silver nanomaterials, which traces the path of silver nanomaterials from production to ultimate
disposal. This review presents the current state of knowledge or beliefs concerning these topics
and indicates what additional information is required to develop a thorough and effective risk
assessment paradigm for use in silver nanomaterial risk management. The nature of this
document requires that peer-reviewed literature and "grey" literature (e.g., posters, slide
presentations, proceedings, web pages, and personal communications) be used from industry,
consumer, academic and government sources of information. As more information becomes
available in the literature, this document will incorporate that information, and will, therefore, be
a living document that will adjust with knowledge and time.
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2. Historical and Current Applications of Silver and Silver
Nanomaterials
2.1 Elemental Silver Characteristics and Sources
Elemental or metallic silver (Ag) is a malleable and ductile transition metal with a white metallic
luster appearance (Brooks, 2010; Lenntech, 2010; Wikipedia, 2010). Of all metals, silver has the
highest electrical conductivity (higher than copper that is currently used in many electrical
applications) and thermal conductivity and has the lowest contact resistance (Brooks, 2010;
Lenntech, 2010; USGS, 2010). Silver has high optical reflectivity compared to other metals
(Edwards & Petersen, 1936). Silver is stable in pure air and water; the presence of ozone or
hydrogen sulfide or sulfur in the air or water may result in silver tarnishing (Hammond, 2000)
due to the formation of silver sulfide. The most common oxidation states of silver are 0 and +1,
but other oxidation states (+2 and +3) are also known. Silver has many isotopes with 107Ag being
the most common (Smith & Carson, 1977). To date, 28 radioisotopes of silver have been
characterized, with a majority of them having a half life of less than 3 minutes. Silver occurs
naturally in its pure form, and as an alloy along with gold and other metals. In addition, it is also
found in ores containing arsenic, sulfur, antimony and chlorine such as argentite, horn silver,
chlorargyrite and pyrargyritein (Helmenstine, 2010; Smith & Carson, 1977; Wikipedia, 2010).
The average concentration of silver in water is 0.5 ppb while its concentration in soil is
approximately 10 ppb. Silver is mainly produced as by product of copper, gold, lead and zinc
refining. Silver is generally extracted by amalgamation and displacement using metals such as
mercury, or by smelting. The top silver producing countries are Peru, Mexico, China, Australia,
Poland and Siberia. In the US, the state of Alaska leads the silver production through the Greens
Creek Mine followed by Nevada through the Comstock Lode Mine (Silver Mining, 2010).
2.2 Chemistry of elemental silver
Silver is the sixty-third most abundant metal in Earth's crust, and exists as two isotopes, 107Ag
and 109Ag, roughly occurring in the same proportion. The chemistry of silver was not well-
known before 1980, although silver nitrate was used medicinally in the 1800s. Recent research
has recognized the highly reactive nature of the silver ion and its ability to form numerous
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inorganic and organic complexes (halide, sulfide, nitrate, oxide, and acetylide compounds,
cyano-derivatives, olefm complexes, etc.). Ag(II) complexes are less stable than those of Ag(I)
and Ag(III), but unlike many other silver compounds are brightly colored red or blue. Silver ion
binds readily to proteins in the human body (including albumins and metallothioneins) and
interacts with trace metals in metabolic pathways.
Silver metal readily dissolves in nitric acid (HNOs) to form silver nitrate (AgNOs). Silver nitrate
is a transparent crystalline solid that is readily soluble in water, and is photosensitive. It is also
used as the starting point for the synthesis of many other silver compounds.
Ag + HN03 -> AgN03 + /2H2(t)
Silver nitrate can react with copper to form silver crystals and a blue-green solution of copper
nitrate. Alkaline solutions of silver nitrate can also be used to reduce silver nitrate to silver metal
in the presence of reducing sugars such as glucose. This reaction is used to silver glass mirrors
and the interior of glass Christmas ornaments.
2AgNO3 + Cu(s) -> Cu(NO3)2 + 2Ag(-l)
Silver or silver nitrate precipitates as silver chloride (AgCl) in the presence of chloride ions.
Silver chloride and other silver halides are used in the manufacture of photographic emulsions.
AgNO3 + Cl~ -> AgCl +
Silver nitrate reacts with bases to form silver oxide (Ag2O), which is used as a positive electrode
in watch batteries.
2AgNO3 + 2OH~ -> 2AgOH + NO; -> Ag2O + 2NO~3 + H2O
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Silver does not react with sulfuric acid; it reacts with sulfur or hydrogen sulfide to form silver
sulfide, which is the tarnish that is commonly observed in silver jewelry, utensils or coins.
2Ag H S - Ag2S
Silver metal reacts with nitric acid in the presence of ethyl alcohol (^HsOH) to form silver
fulminate (AgONC). Silver fulminate is a powerful touch-sensitive explosive used in percussion
caps. Silver nitrate reacts with sodium azide (NaNs) to form silver azide (AgNs), which is also
used as an explosive. Silver, in the presence of excess cyanide, forms cyanide complexes
(AgCN) that are soluble in water; these complexes are used in silver electroplating.
2.3 Historical and Current Applications of Elemental Silver and Silver
compounds
Besides elemental silver, other silver containing compounds that are found in the Earth's crust
include silver halides (AgBr, AgCl, Agl, and silver fluorides), silver fulminate, silver nitrate and
silver oxide (ATSDR, 1990; Greenwood & Earnshaw, 1997; Hammond, 2000; Romans, 1954)
among others. These compounds vary in solubility from readily soluble to barely soluble in
water. Throughout history, silver and its compounds have been used extensively for many
applications as a result of their useful properties. It is believed that silver was known and used
longer than what is recorded in history. Archeological evidence suggests that civilizations have
been using silver since at least 3000 B.C. Ancient Egyptians and Persians used silver vessels to
keep their water clean and safe. Romans and Greeks knew its powerful bactericidal effect and
used it for healing wounds. During World War I, silver compounds were used to prevent wound
infection before the emergence of antibiotics. In the American Old West, pioneers traveling
along Oregon trails used to toss silver coins into their water storage barrels to keep their water
fresh (Information and History, 2010; Russell & Russell, 1995; History of Silver, 2010;
Wijnhoven et a/., 2009). During the 19th century, beyond home remedies, silver was applied in
practical medicine such as eye treatment and the treatment of skin ulcers (Foot Defense, 2010).
Other uses of silver include making currency coins, ornaments, jewelry, tableware and utensils.
The US Food and Drug Administration approved silver solutions in the 1920s to be used as
antibacterial agents (Wikipedia, 2010).
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Silver and its compounds have an extensive number of applications in the 20th century including
electrical conductors, electrical contacts, catalysis, photography, electronics, mirrors, drinking
water filtration systems, swimming pool filtration systems, healthcare products and medical tools
(Clement et al., 1994; Luoma, 2008; Wikipedia, 2010). Since soluble silver compounds are toxic
to some bacteria, viruses, algae and fungi, various applications have emerged based on the strong
germicidal impacts of silver compounds. Silver is incorporated in textiles to inhibit the growth of
bacteria and to keep odor at minimum (Clement et al., 1994). In 1954, silver was registered in
the US as a pesticide for use in disinfectants, sanitizers and fungicides. Various diseases ranging
from mental illness to gonorrhea have been reported to be treated using silver compounds
(Panyala, 1996). Silver was used in 2007 to make the first antibacterial glass used in hospitals to
fight infections (AGC Glass, 2007). Silver is also used in catheters in order to make them more
effective for reducing bacteriuria (a urinary trace infection) in adults at hospitals while having
short term catheterization (Sanjay et a/., 2009). Not all silver compounds are known to have the
same impact on infections; silver alloy catheters are significantly more effective in preventing
urinary tract infections than are silver oxide catheters. NASA selected silver for purifying the
drinking water in space shuttles (Information and History, 2010).
Other medical applications of silver include its use in the manufacture of bone prostheses,
cardiac implants and replacement valves, needles used in ocular surgery, peritoneal catheters,
and wound sutures. It is an antiseptic ingredient used in wound management. While silver has
been used as an antiseptic for many years, new products that time-release silver in a sustained
manner are starting to be available in the market. These products are showing promise in the
treatment of skin wounds, skin ulcers, and burns. In these new products, which may contain
elemental or nanosilver, silver ions are released from the dressings (Acticoat™, Actisorb™, etc.)
in the presence of wound fluids, exudates, and the products are activated to keep the wounds
clean. Activated silver ion is toxic to bacteria and yeasts. Silver is toxic to bacteria at low
concentrations (10~5 to 10~7 Ag ions per cell). Although silver itself is not considered toxic, most
of its salts are poisonous, due to the anions involved. Exposure to silver (metal and soluble
compounds, as Ag) in air should not exceed 0.01 mg/m3 (8-hour time-weighted average for a 40-
hour week) (LANL, 2010). Silver compounds can be absorbed into the circulatory system, with
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the deposition of reduced silver in body tissues. This may result in argyria, which is
characterized by a grayish pigmentation of the skin and mucous membranes. Silver absorbed
through the skin is deposited in the liver and kidney and complexes with albumin and cellular
proteins. A potential hazard of using silver in jewelry, medicinal products, coins, and antiseptics
is allergies, which may result in red rashes, blisters, welts, hives, and itching or burning skin.
Sterling silver (i.e., 92.5% silver) is usually used for silverware and jewelry and some high-end
musical instruments such as flutes. When alloyed with mercury, tin and other metals at room
temperature, silver is used to make amalgams for use in dental filling. Silver is used in printed
circuit boards and keyboards as an electrical contact and as wires in some high end audio
hardware. Silver is also used as a catalyst in industrial processes such as catalyzing the
conversion of ethylene to ethylene oxide or the production of formaldehyde from methanol.
Applying a thin layer of silver on surfaces is also known to increase the galling resistance and
reduce the wear of surfaces under heavy loads (ATSDR, 1990; Hammond, 2000; Wikipedia,
2010).
Silver nitrate is widely used in photography and in the synthesis of other silver compounds (see
Section 2.2 for more information on the chemistry of silver) (Clement et a/., 1994; Wikipedia,
2010). Silver nitrate drops are used to prevent infections in infants' eyes, as an antiseptic, and in
stained glass. Silver halides are used in gravimetric analytical methods and are extensively used
in photography. Silver oxide is used as cathodes in batteries used for small devices. Silver azide
and silver fulminate are powerful explosives. To produce rain, silver iodide is used in cloud
seeding. Silver chloride can be made transparent and used in glass electrodes for pH and
potentiometric measurements. It is also used as cement for glass.
The catalytic properties of silver make it ideal for use as a catalyst in oxidation reactions.
Formaldehyde is produced from methanol and air in the presence of silver screens or crystallites
that contain a minimum of 99.95% silver by weight. Silver-coated catalysts are probably the only
catalysts currently available to convert ethylene to ethylene oxide. Ethylene oxide is ultimately
used in the production of polyesters and other polymers that have multiple industrial
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applications. Because silver readily absorbs free neutrons, it is commonly used to make control
rods that regulate the fission chain reaction in pressurized water nuclear reactors.
2.4 Nanosilver: History and Applications
Silver nanomaterials are fine particles of metallic silver that have at least one dimension less than
100 nm (Figure 2.1). Nanosilver is not a new discovery; it has been known for over 100 years
(USFDA, 2010). Previously, nanosilver or suspensions of nanosilver were referred to as colloidal
silver. To produce colloidal silver, a positive electrical current is applied through pure silver bars
suspended in water resulting in colloidal silver particles with a size range of 15-500 nm
(Lindemann, 1997). Before the invention of penicillin in 1928, colloidal silver had been used to
treat many infections and illnesses (Nano Health Solutions, 2010). By converting bulk silver into
nanosized silver, its effectiveness for controlling bacteria and viruses was increased multifold,
primarily because of the nanomaterials' extremely large surface area when compared to bulk
silver, thus resulting in increased contact with bacteria and fungi. Nanosilver, when in contact
with bacteria and fungus, adversely affects the cellular metabolism of the electron transfer
systems, and the transport of substrate in the microbial cell membrane. Nanosilver also inhibits
multiplication and growth of those bacteria and fungi which caused infection, odor, itchiness and
sores (Nanotech Pic, 2010).
In 1951, Turkevich et al. reported a wet chemistry technique to synthesize nanosilver using silver
nitrate as a silver ion source and sodium citrate as the reducing agent for the first time
(Turkevich etal., 1951). Recent advances in nanomaterials science in the last two decades have
enabled scientists to engineer silver nanomaterials by controlling their size, shape and surface
properties. This has been motivated by the unique chemical, physical and optical properties of
nanosilver compared to the parent silver metal. The unique properties of nanosilver are mainly
attributed to the high surface area to volume ratio, leading many industrial sectors to incorporate
silver nanomaterials into their products. Nanosilver is being incorporated in plastics, fabrics,
paper, paint, and surface coatings. More than 200 products containing nanosilver are now
available for public use. Numerous other applications have been reported for silver nanoparticles
in areas such as electronics, bio-sensing and surface enhanced Raman spectroscopy (SERS)
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(Tolaymat etal., 2010). More detailed information regarding the various applications of
nanosilver is provided in Chapter 4.
The Scale of Things - Nanometers and More
1
Things Natural
k*
DMA
-2-1/2 nm diameter
10-im
1tt-!m
ID
o
10-Sm
1 cm
10mm
— 1 milllmeler(mrn)
100 uJn
0,01 mm
10 urn
1.000 nanometers =
1 micrometer (/irti)
3
O
10-"m
ro
Z
10-'m
10-'° m
__ 0.01 urn
10 nm
r~ 1 nanometer (nm)
-I
Things Manmade
Head of a pin
1-2 mm
MicroEleclroMechanlcal
(MEMS) devices
10-100 fim wide
Zone plate x-ray icns
Outer ring spacing -35 nm
Self-assembled,
Nature-inspired structure
Many 10s of nrn
The Challenge
Fabricttlc
nuiiHM'uif
ktiitli\ (a mat? H
JcviVrt, e.g., a
i'HyniHFiic rtttclittn
center witli integral
tcmit't'tttltit'ttfr Mar fl^
Nanotube electrode
Carbon nanotube
-1,3 nm diameter
Quantum corral of 48 iron atoms on copper surface
positioned one at a lime with an STM tip
Corral diameier 14 nm
Figure 2.1: Nanomaterials dimensions on the metric scale (in nm) courtesy of the Office of
Basic Energy Sciences, Office of Science, U.S. Department of Energy
2.5 Silver Regulations in the US
Silver was registered in the United States as a pesticide in 1954. Nanosilver products registered
since 1950 are presented in Figure 2.2 (SNWG, 2009). The USEPA designated silver as a
priority pollutant in natural waters in 1977. The studies that formed the basis for the USEPA
regulation of silver were based on toxicity data from colloidal silver and not bulk silver (SNWG,
2009). A secondary maximum contaminant level was issued by USEPA's Office of Water (OW)
for silver in 1991 based on the ability of silver to cause argyria. In 1991, the USEPA established
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an oral reference dose of 0.005 mg/kg/day for silver. Between 1970 and 1990, all USEPA
registered silver products were either colloidal nanosilver or nanosilver-composite products. The
first product containing conventional silver was registered in 1994. Over 50% of USEPA's
registered silver in recent years is based on nanosilver (Rosalind, 2009).
• NANQSILVERs registered per year (left axis)
—Totd ALL siber product registered (f ighl aa
Total NANOSILVER products regsBred (light ans)
First silver registration (FIFRA) is nano
1054
Year
Figure 2.2: Analysis of FIFRA registered products containing nanosilver for the period 1950-
2010 (SNWG, 2009). Reprinted with permission from the Silver Institute. Copyright © 2004 The
Silver Institute
As a result of the expanding usage of nanosilver, USEPA has great concerns regarding its
environmental fate, transport and toxicity. USEPA is currently conducting and/or funding
fundamental research to help understand the potential human health and ecological implications
from exposure to manufactured nanomaterials including nanosilver. The USEPA's Office of
Research and Development (ORD) issued a nanomaterials research strategy in June 2009, in
which silver was one of the seven materials selected to be investigated. The USEPA's Office of
Pesticide Programs (OPP) plans to regulate certain consumer products containing nanosilver
under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (Rosalind et a/., 2009).
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Under FIFRA, products containing silver nanoparticles with the aim of killing microbes will be
classified as pesticides. An example of a product that is classified as a pesticide under FIFRA
would be washing machines that release silver nanoparticles to kill bacteria on clothes (Peabody,
2006). The regulations applicable to nanosilver containing products are yet to be issued. Under
FIFRA, if a product is claimed to release nanosilver to kill bacteria, the manufacturer must
submit product data to the USEPA, which is authorized to prohibit particular products that pose
unreasonable health effects on the environment or humans (Peabody, 2006).
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3. Uses of Silver Nanomaterials
Knowledge of the applications of silver nanomaterials in consumer products is crucial for an
accurate prediction of release pathways, exposure, LCA, and risk assessment. During the last two
decades, an extensive number of methods have been reported for the synthesis of silver
nanoparticles with different particle sizes, shapes and surface properties. This advancement in
their manufacturing techniques attracted the attention of many industries wanting to exploit the
unique properties of silver nanomaterials for beneficial use. The applications of silver
nanomaterials are scattered but they can be classified under three main categories: scientific,
industrial, and consumer products.
3.1 Properties of nanosilver
Two primary factors cause nanomaterials to behave significantly differently than bulk materials:
surface effects and quantum effects (Roduner, 2006). These factors affect the chemical reactivity
of materials as well as their mechanical, optical, electric, and magnetic properties. Nanosilver
has chemical and biological properties that are appealing to the consumer products, food
technology, textiles/fabrics, and medical industries. Nanosilver also has unique optical and
physical properties that are not present in bulk silver, and which are claimed to have great
potential for medical applications.
3.1.1 Antibacterial properties
Nanosilver is an effective killing agent against a broad spectrum of Gram-negative and Gram-
positive bacteria (Burrell et al., 1999; Wijnhoven et al., 2009; Yin et al., 1999), including
antibiotic-resistant strains (Percival et al., 2007; Wright et al., 1998). Gram-negative bacteria
include genera such as Acinetobacter, Escherichia, Pseudomonas, Salmonella, and Vibrio.
Acinetobacter species are associated with nosocomial infections, i.e., infections that are the result
of treatment in a hospital or a healthcare service unit, but secondary to the patient's original
condition. Gram-positive bacteria include many well-known genera such as Bacillus,
Clostridium, Enterococcus, Listeria, Staphylococcus, and Streptococcus. Antibiotic-resistant
bacteria include strains such as methicillin-resistant and vancomycin-resistant Staphylococcus
aureus, and Enterococcusfaecium.
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Silver nanoparticles (diameter 5-32 nm, average diameter 22.5 nm) enhance the antibacterial
activity of various antibiotics (Shahverdi etal., 2007). The antibacterial activities of penicillin G,
amoxicillin, erythromycin, clindamycin, and vancomycin against Staphylococcus aureus and
Escherichia coli increase in the presence of silver nanoparticles (Wijnhoven et a/., 2009). Size-
dependent (diameter 1-450 nm) antimicrobial activity of silver nanoparticles has been reported
with Gram-negative bacteria (Baker et al., 2005; Morones et al., 2005; Panacek et al., 2006) and
Gram-positive bacteria (Panacek etal., 2006). Small nanoparticles with a large surface area to
volume ratio provide a more efficient means for antibacterial activity even at very low
concentration.
In addition to size and concentration, shape-dependent antimicrobial activity of silver
nanoparticles has been shown with Gram-negative bacteria (Pal et al., 2007). Silver
nanoparticles of different shapes (spherical, rod-shaped, truncated triangular nanoplates) have
been developed by synthetic routes. Truncated triangular silver nanoplates display the strongest
antibacterial activity (Wijnhoven etal, 2009). The top basal plane of truncated triangular silver
nanoplates is a high-atom-density surface, i.e., a {111} facet. Generally, spherical silver
nanoparticles (generally with cubo-octohedral, multiple-twinned decahedral, or quasi-spherical
morphology) have {100} facets along with a small percentage of {111} facets, whereas rod-
shaped silver nanoparticles (e.g., pentagonal rods) have side surfaces with {100} facets and end
with {111} facets (Wijnhoven et al., 2009; Wiley et al. 2005). Silver reactivity is favored by
{111} facets (Hatchett & White, 1996). Spherical silver nanoparticles with {111} facets attach
directly to the bacterial surface of the cell membrane and are located inside bacteria (Morones et
al. 2005). The strong anti-bacterial activity of truncated triangular silver nanoplates could be due
to their large surface area to volume ratios and their crystallographic surface structures.
3.1.1.1 Antibacterial mode of action
Bacteria have different membrane structures, which are the bases of their general classification
as Gram-positive or Gram-negative. Structural differences reside in the organization of the key
component of the cell wall, peptidoglycan, which is located immediately outside the cytoplasmic
membrane. The cell wall of Gram-positive bacteria contains a peptidoglycan layer that is -30 nm
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thick. Unlike the Gram-positive cell wall, the Gram-negative cell wall has only a thin
peptidoglycan layer that is -2-3 nm thick. In addition to the peptidoglycan layer, the Gram-
negative cell wall also contains an additional outer membrane composed of phospholipids and
lipopolysaccharides, which face into the external environment.
Although the antimicrobial effect of silver ions has been studied extensively, the effects of
nanosilver on bacteria and the bactericidal mechanism are only partially understood. Based on
studies that show that silver nanoparticles anchor to and penetrate the cell wall of Gram-negative
bacteria (Morones et al, 2005; Sondi & Salopek-Sondi, 2004), it is reasonable to suggest that the
resultant structural change in the cell membrane could cause an increase in cell permeability,
leading to an uncontrolled transport through the cytoplasmic membrane, and ultimately cell
death. It has also been proposed that the antibacterial mechanism of silver nanoparticles is
related to the formation of free radicals and subsequent free radical-induced membrane damage
(Danilczuk et al, 2006; Kim et al, 2007). Hwang et al. (2008) performed a study of stress-
specific bioluminescent bacteria, based on a synergistic toxic effect of the silver nanoparticles
and the silver ions that they produce. The ions move into the cells and lead to the production of
reactive oxygen species. Because of the membrane damage caused by the nanoparticles, the cells
cannot effectively extrude the silver ions and limit their effect. Based on the greater tendency of
silver ions to strongly interact with thiol groups of vital enzymes and phosphorus-containing
bases (Hatchett & White, 1996) and on the presence of silver nanoparticles inside the cells
(Morones et al., 2005), it is likely that further damage could be caused by interactions with
compounds such as DNA. This interaction may prevent cell division and DNA replication from
occurring, and also ultimately lead to cell death. No DNA damage was found by Hwang et al.
(2008). Other studies have suggested that silver nanoparticles may modulate the phosphotyrosine
profile of putative bacterial peptides that could affect cellular signaling and, therefore, inhibit the
growth of bacteria (Shrivastava etal, 2007).
3.1.2 Antifungal properties
Nanosilver is an effective, fast-acting fungicide against a broad spectrum of common fungi
including genera such as Aspergillus, Candida, and Saccharomyces (Wright et al., 1999). The
exact mechanisms of action of silver nanoparticles against fungi are still not clear, but
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mechanisms similar to that of the antibacterial actions have been proposed for fungi (Wright et
a/., 1999). Silver nanoparticles (diameter 13.5 ± 2.6 nm) are effective against yeast isolated from
bovine mastitis (Kim etal., 2007).
3.1.3 Antiviral properties
Silver nanoparticles (diameter 5-20 nm, average diameter -10 nm) inhibit HIV-1 virus
replication (Sun, Chen, et a/., 2005). Gold nanoparticles (average diameter -10 nm) showed
relatively low anti HIV-1 activity (6-20%) when compared to silver nanoparticles (98%). Size-
dependent antiviral activity of silver nanoparticles has been shown with HIV-1 virus
(Elechiguerra etal., 2005). Interaction of silver nanoparticles with HIV-1 was exclusively within
the range of 1-10 nm.
3.1.4 Anti-inflammatory properties
Nanosilver dressings as well as nanosilver-derived solutions proved to have anti-inflammatory
activity (Nadworny et al, 2010). In animal models, nanosilver alters the expression of matrix
metallo-proteinases (proteolytic enzymes that are important in various inflammatory and repair
processes) (Kirsner et al., 2001), suppresses the expression of tumor necrosis factor (TNF)- ,
interleukin (IL)-12, and IL-1 , and induces apoptosis of inflammatory cells (Bhol & Schechter,
2005, 2007). Silver nanoparticles (diameter 14 ± 9.8 nm) modulate cytokines involved in wound
healing (Tian et al., 2007). The results indicate the possibility of achieving scar-less wound
healing even though further studies using other animal models are required to confirm this.
3.1.5 Anti-glycoprotein film properties
Glycoproteins are proteins that contain oligosaccharide chains that are covalently attached to
polypeptide side-chains. These proteins are important for normal immune system function such
as white blood cell recognition, and often play a role in cell-cell interactions. Examples of
glycoproteins in the immune system include molecules such as antibodies that interact directly
with antigens. In the case of impregnation of medical-grade silicone with silver nanoparticles
(diameter 10-100 nm) there is both a depot effect and a diffusion pressure available to equilibrate
the silver concentration and to push silver through the glycoprotein conditioning film (Furno et
a/., 2004). This unexpected finding has obvious clinical implications, because silver is known to
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have a high avidity to protein and the presence of a glycoprotein film has been assumed to
inactivate any silver ions released (Schierholz etal., 1998). Surfaces of implanted devices
immediately and rapidly become coated with patient-derived glycoproteins from tissue and blood
plasma (Green etal., 1999). Once protein adhesion has occurred, proliferation leads to the
development of a biofilm which is insusceptible to most therapeutic agents.
3.1.6 Anti-biofilm properties
Nanosilver inhibits the formation of biofilms (Percival et a/., 2007). Biofilms are complex
communities of surface-attached aggregates of microorganisms embedded in a self-secreted
extracellular polysaccharide matrix. Biofilm forming bacteria act as efficient barriers against
antimicrobial agents and the host immune system, resulting in a persistent colonization and/or
infection at the site of the biofilm formation.
3.1.7 Surface plasmon resonance properties
Noble metal nanoparticles can be deposited onto a glass matrix and exhibit a very intense color,
which is absent in bulk material as well as in individual atoms. Their origin is attributed to the
collective oscillations or fluctuations in electron density with an interacting electromagnetic
field. These resonances are denoted as surface plasmons. These oscillations are very sensitive to
adsorption of molecules to the metal surface. The plasmonic coupling of metal nanoparticles
with light enhances a broad range of useful optical phenomena which have application potential
in ultra-sensitive biomolecular detection and lab-on-a-chip sensors (Moores & Goettmann,
2006). The effect of the size of silver nanoparticles on the surface plasmon resonance, i.e.,
plasmon band width and peak position, has been demonstrated (Thomas etal., 2008). Decreasing
nanoparticle size (diameter < 10 nm) is associated with a red-shift and broadening of the
plasmon-related absorption peak. The impact of silver nanoparticle shape on plasmon surface
resonance has been less studied.
3.1.8 Plasmonic heating properties
Plasmonic photo activation of hollow poly electrolyte-multilayer capsules incorporating silver
nanoparticles and containing drug models has been demonstrated as proof-of-principle (Skirtach
et a/., 2004). Silver nanoparticles were remotely activated using laser irradiation, causing not
only absorption of photons but also heat transfer from the nanoparticles to the surrounding
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polymer matrix. The local heating disrupts the polymer matrix and allows the encapsulated
material/drug to leave the interior of the capsule. The concept of remote opening of
polyelectrolyte-multilayer capsules incorporating silver nanoparticles (diameter > 20 nm) has
been demonstrated in living cells (Skirtach et al., 2006). The duration of laser treatment to open
polyelectrolyte multilayer capsules is dependent on the size of silver nanoparticles (diameter 10-
23 nm; Radziuk et al, 2007).
3.1.9 Metal-en ha need fluorescence properties
Metallic nanostructures (size range 30-80 nm) alter the intrinsic spectral properties (i.e., emission
intensity and photostability) of fluorophores. The proximity of silver nanostructures results in an
increase in intensity of low-quantum-yield fluorophores. The effects of metallic surfaces include
fluorophore quenching at short distance (-0-5 nm), spatial variation of the incident light field
(-0-15 nm), and changes in the radioactive decay rate (-0-20 nm). Applications include
immunoassays and DNA/RNA detection (Asian et al., 2005).
3.1.10 Properties of silver nanomaterials that promote its biosynthesis
Despite the antibacterial properties of nanosilver, the feasibility of biosynthesis of silver
nanoparticles using bacteria has been demonstrated (Klaus et al., 1999). Silver nanoparticles
(sizes up to 200 nm) were synthesized using Pseudomonas stutzeri and found to be mostly
located at the periplasmic area of the bacteria. Another silver-resistant bacteria used in the
biosynthesis of silver nanoparticles (diameter 5-32 nm, average diameter 22.5 nm) is Klebsiella
pneumoniae (Shahverdi, Fakhhimi, et al., 2007). Fungi have also been used to biosynthesize
silver nanoparticles. Intracellular silver nanoparticles (diameter 25 ± 12 nm) were produced in
Verticullium fungal cells (Mukherjee et al., 2001) and extracellular silver nanoparticles (diameter
5-25 nm) using pathogenic filamentous fungi such as Fusarium oxyspomm (Ahmad et al., 2003)
andAspergillusfumigatus (Bhainsa & D'Souza, 2006). Extracellular silver nanoparticles
(diameter 13-18 nm) have been biosynthesized using non-pathogenic fungus Trichoderma
asperellum (Mukherjee et al., 2008). In addition to microbial organisms, plant extracts can be
used in the biosynthesis of metallic nanomaterial (Mohanpuria etal, 2008). The widespread and
increasing use of nanosilver within healthcare settings raises issues concerning bacterial and
fungal silver resistance. Whether resistance is a threat in the clinical setting needs to be
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elucidated (Chopra, 2007). Standardization for silver antimicrobial testing methods is lacking.
This is partly due to the complex solubility issues affecting the bioavailability of silver.
3.2 Scientific Applications
The remarkable physical, chemical and optical properties of silver nanomaterials allows for their
utilization in various scientific applications. These properties significantly depend on the size,
shape and surface chemistry of the nanomaterials. Metallic nanoparticles, including nanosilver,
exhibit surface plasmon resonance (SPR) upon irradiation with light giving rise to SPR peaks in
the UV-Vis wavelength range (Luoma, 2008; Tolaymat et a/., 2010). The SPR is a result of the
interactions between the incident light and the free electrons in the conduction band of the
nanomaterials. The width and location of the SPR peaks are dependent on the size, shape and
surface properties of the nanomaterials (Ju-Nam & Lead, 2008). Silver nanomaterials are widely
used for surface enhanced Raman scattering (SERS). Raman scattering by molecules could be
enhanced if the analyte molecules are adsorbed on rough metal surfaces. The enhancement factor
can be as much as 1014-1015 which allows for enough sensitivity to detect single molecules
(Doering & Nie, 2002). As a consequence of the SPR and SERS, silver nanomaterials are a
promising tool for sensing applications, including detection of DNA sequences (Jacob etal.,
2008), laser desorption/ionization mass spectrometry of peptides (Hua etal., 2007), colorimetric
sensors for Histidine (Xiong et a/., 2008), determination of fibrinogens in human plasma
(ZhiLiang etal., 2007), real-time probing of membrane transport in living microbial cells (Xu et
a/., 2004), enhanced IR absorption spectroscopy (Huo et a/., 2006), colorimetric sensors for
measuring ammonia concentration (Dubas & Pimpan, 2008a), biolabeling and optical imaging of
cancer (Wiley et a/., 2007), optical sensors for zeptomole (Nikolaj et a/., 2006), biosensors for
detection of herbicides (Dubas & Pimpan, 2008b), and glucose sensors for medical diagnostics
(Mishra et a/., 2007). SERS using nanosilver can be used for biological imaging, trace analysis
of pesticides, anthrax, prostate-specific antigen glucose, nuclear waste, identification of bacteria,
genetic diagnostics and detection of nitro-explosives (del Rocio et a/., 2006).
Silver nanomaterials are also known to be used for metal enhanced fluorescence applications.
The intrinsic spectral properties of fluorophores can be altered by metallic nanostructures. The
proximity of metallic nanosilver results in an increase in the intensity of low quantum yield
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fluorophores. The effects include fluorophore quenching at short distances, spatial variation of
the incident light field, and change in the radioactive decay rate (Wijnhoven et a/., 2009). These
characteristics enable nanosilver to be used in applications such as immunoassays and
DNA/RNA detection.
As previously mentioned, the characteristics of the silver nanomaterials are greatly influenced by
their surface properties. Modifying the surface of silver nanoprisms by alkanethiol makes them
potential candidates for streptavidin and anti-biotin sensing and may also aid in the diagnosis of
Alzheimer's disease (Pastoriza-Santos & Liz-Marzan, 2008). Para-sulfonatocalix modified silver
nanoparticles are used to probe histidine down to a concentration of 5 x 10"6 M (Xiong et a/.,
2008). This is important since histidine is needed for the growth and the repair of tissue, as well
as for maintenance of the myelin sheaths that act as the protector of nerve cells. It is
manufactured in sufficient quantities in adults, but children may develop a shortage of this
important amino acid (Xiong et a/., 2008).
3.3 Industrial Applications
3.3.1 Catalysis
The high surface area to volume ratio of silver nanomaterials provides high surface energy,
which promotes surface reactivity such as adsorption and catalysis. This has resulted in the use
of silver nanomaterials and silver nanocomposites to catalyze many reactions in industrial
processes such as CO oxidation, benzene oxidation to phenol, photodegradation of gaseous
acetaldehyde and the reduction of the p-nitrophenol to p-aminophenol (Tolaymat etal., 2010).
SiC>2 supported Ag catalysts (5 wt% Ag) exhibit good activity toward the decomposition of N2O.
Silver nanoparticles immobilized on silica spheres are used to catalyze the reduction of dyes by
sodium borohydride (NaBH4) (Nikolaj etal., 2006). Ag nanoparticles synthesized in
polyethylene glycol with simple bubbling of H2 gas have been used to catalyze the three-
component coupling reaction of aldehyde, alkyne, and amine with good to excellent yields in one
reaction vessel (Yan et a/., 2006), thus saving time and materials.
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3.3.2 Electronics
The high electrical and thermal conductivity of nanosilver along with the enhanced optical
properties result in various applications in electronics. Nanosilver is used in electronic
equipment, mainly in solder for circuit connections (DiRienzo, 2006). Silver nanowires are used
as nanoconnectors and nanoelectrodes for designing and fabricating nanoelectronic devices (Kim
et a/., 2007). Other applications include the preparation of active waveguides in optical devices
(Roldan etal., 2007), inks for printed circuit boards, optoelectronics, nanoelectronics (such as
single-electron transistors, and electrical connectors), subwavelength optics, data storage
devices, nonlinear optics, high density recording devices, intercalation materials for batteries,
making micro-interconnects in integrated circuits (1C) and integral capacitors (Tolaymat et a/.,
2010). Silver inks are used to replace wires and act as flat wires in printed circuit boards. In
addition, silver inks are also used to repair circuit breaks in printed circuit boards, thus
preventing their premature disposal in landfills (DiRienzo, 2006).
3.3.3 Other Industrial Applications
Nanosilver is being utilized in the paper industry. DocuGuard uses silver-based paper to protect
hospital case notes and medical files against the proliferation of bacteria (DiRienzo, 2006). The
company proposes future applications to include business stationery, envelopes, brochures and
book-binding materials. Nanosilver is used in commercial water purification systems. The
industry makes use of the antibacterial properties of nanosilver in the interior of automobiles
such as steering wheels and in building materials such as sanitary tubing and coverings (Blaser et
al., 2008). Nanosilver is also used for wood preservation to resist mildew and mold. MTR
Corporation in Hong Kong reports the use of silver nanoparticles in combination with titanium
dioxide coating to enhance hygiene by spraying it onto surfaces in MTR train stations, inside
train compartments, as well as MTR managed shopping malls, staff offices and recreational
facilities (Senjen, 2007).
3.4 Applications in Consumer Products
Nanosilver is one of the most widely used nanomaterials that are incorporated in consumer
products. Silver nanoparticles are used as antibacterial/antifungal agents in a diverse range of
applications including air sanitizer sprays, socks, pillows, slippers, face masks, wet wipes,
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detergent, soap, shampoo, toothpaste, air filters, coatings of refrigerators, vacuum cleaners,
washing machines, food storage containers, cellular phones, and even in liquid condoms (a liquid
containing spermicides that solidifies and becomes a protective condom when sprayed into the
female genital area; the condom is designed to transform back to a liquid in the presence of
semen, which releases the spermicide). The major reason for this prevalence is its strong
antibacterial effect for a wide array of organisms (Tolaymat et a/., 2010). Samsung produced a
version of washing machines (AG plus) that generate silver nanoparticles to disinfect clothes
rather than using hot water and detergents (DiRienzo, 2006). Samsung and GSH are using
nanosilver coating in their new refrigerators and air purifiers for the same purpose. Nanosilver
can be found in personal-grooming kits, female-hygiene products, beauty soaps, cleansers and
fabric softeners (Luoma, 2008). Nanosilver spraymist products are used to disinfect and
deodorize surfaces in kitchens, bathrooms and baby clothes. It is used in cosmetics, lotions,
creams, toothpastes, laundry detergents, soaps, surface cleaners, room sprays, toys, antimicrobial
paints, home appliances, automotive upholstery, consumer electronics (e.g., cell phone covers),
shoe insoles, brooms, food storage containers, tableware, slippers and shoe liners. Nanosilver is
widely incorporated in textiles and fabrics such as outerwear, sportswear, underwear, socks, and
bedding materials such as comforters, sheets and mattress covers (Luoma, 2008; Tolaymat et a/.,
2010). More detailed information on consumer products containing nanosilver is available at the
http ://www.nanoproj ect. org website.
3.5 Medical Applications
Nanosilver has many medical applications including diagnosis, treatment, drug delivery, coating
tools and medical devices. Nanosilver is used for coating medical tools and materials used in the
areas of surgery, anesthesiology, cardiology and urology (Wijnhoven et a/., 2009). It is also
incorporated in wound dressings, diabetic socks, scaffolds, sterilization materials in hospitals,
medical textiles, medical catheters and contraceptive devices. Nanosilver is used in orthopedics
in areas such as additives in bone cement, coating of implants for joint replacement and bone
prostheses (Tolaymat et a/., 2010). Nanosilver is used in dentistry for making artificial teeth and
in eye care for coating contact lenses (Senjen, 2007; Wijnhoven et a/., 2009). Advanced silver
nanotechnologies are used to improve battery performance in next-generation active implantable
medical devices. The use of nanosilver in combination with vanadium oxide in battery cell
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components is one example of advanced silver nanotechnology that has resulted in improved
cathode material homogeneity. Nanosilver is used for imaging of cell cancers (Boxall et a/.,
2007). It is also used for treating dermatitis, ulcerative colitis and acne. By using wound
dressings containing nanosilver, doctors found that not only did silver inhibit the growth of
bacteria, the wounds actually healed more quickly (DiRienzo, 2006). Dressings have also been
designed to release nanosilver slowly, depending on the presence of wound fluids. Nanosilver is
used in diet supplements. One company's website recommends ingesting a teaspoon of silver
colloid per day "to help maintain health," and one tablespoon four times per day to "help fortify
the immune system" (Mesosilver® - Nanoparticle Colloidal Silver from Purest Colloids, Inc.,
Westhampton, NJ; http://www.purestcolloids.com/mesosilver.htm). Another website claims that
"the number of people using colloidal silver as a dietary supplement on a daily basis is measured
in the millions" (http://www.silver-colloids.com/Tables/Experiment.PDF). Silver nanoparticles
are used as antibacterial/antifungal agents in a diverse range of applications including air
sanitizer sprays, socks, pillows, slippers, face masks, wet wipes, detergent, soap, shampoo,
toothpaste, air filters, coatings of refrigerators, vacuum cleaners, washing machines, food storage
containers, cellular phones, and even in liquid condoms.
Silver has possible applications in the treatment of cancer (Asharani etal., 2009). HIV-1 virus
was reported to be inhibited from binding to the host cells through the use of silver nanoparticles
(Elechiguerra et a/., 2005). Nanosilver is also used in drug delivery through plasmonic
photoactivation of hollow poly electrolyte-multilayer capsules incorporating silver nanoparticles
and containing drug molecules (see Section 3.1.8).
3.6 Proposed and Projected Applications
All applications reported for bulk and ionic silver could be projected for silver nanoparticles,
since nano-scale silver has more enhanced properties than the parent materials. Bulk silver is
registered as a pesticide and is used in public hygiene. AmeriSwiss, a provider of public
restroom equipment, is employing AglON-based silver ion antimicrobial products in a protective
finish for door pulls and plates, which minimizes bacterial growth on the surface of the finished
product. Bulk silver is being utilized in food processing and preservation. Commercial ice
machines are using silver embedded hoses, clamps, pipe fittings, and in other places where
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deposits (gunk) can build up and harbor bacteria. Meat processors are also using silver embedded
tables, grinders, tools, refrigerators and hooks. Silver is also used in specialty packaging,
occupational clothing worn by food processing workers, prevention of pathogen build-up in
climate control systems and on the floors, walls, and ceilings of food processing and storage
facilities. Silver is used to keep fruit, vegetables and cut flowers fresh while in transit. Silver is
also used in wood preservation to withstand exposure to aggressive brown-rot fungus and resist
molds. Home Water Purification Floatron, a small solar-powered ionization product, uses silver
and copper ions to purify and soften water in swimming pools. Nanosilver is incorporated into
nanocomposites and bimetallic nanoparticles; this will be an important gate for nanosilver
applications. All of the previously mentioned applications can be projected for nanosilver,
especially with advances in the nanomaterials industry (DiRienzo, 2006). Table 3.1 lists
emerging applications of nanosilver in medical products.
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Table 3.1: Emerging applications of nanosilver in medical products. Reprinted from
Nanotoxicology, Vol. 3 (2), Wijnhoven, S.W.P., Peijnenburg, W.J.G.M., Herberts, C.A., Hagens,
W.I., Oomen, A.G., Heugens, E.H.W., Roszek, B., Bisschops, J., Gosens, I, van de Meent, D.,
Dekkers, S., de Jong, W.H., van Zijverden, M., Sips, A.J.A.M., Geertsma, R.E., Nanosilver - a
review of available data and knowledge gaps in human and environmental risk assessment,
pp 109-138, Copyright 2009 with permission from Informa Healthcare.
Medical domains
Anesthesiology
Cardiology
Dentistry
Diagnostics
Drug delivery
Eye care
Imaging
Neurosurgery
Orthopedics
Patient care
Pharmaceutics
Surgery
Urology
Wound care
Examples
Coating of breathing mask
Coating of endotracheal tube for mechanical ventilatory
support
Coating of driveline for ventricular assist devices
Coating of central venous catheter for monitoring
Additive in polymerizable dental materials
Silver-loaded SiO2 nanocomposite resin filler
Nanosilver pyramids for enhanced biodetection
Ultrasensitive and ultrafast platform for clinical assays for
diagnosis of myocardial infarction
Fluorescence-based RNA sensing
Magnetic core/shell Fe3O4/Au/Ag nanoparticles with
turnable plasmonic properties
Remote laser light-induced opening of microcapsules
Coating of contact lens
Silver dendrimer nanocomposite for cell labeling
Fluorescent core-shell Ag@SiO2 nanoballs for cellular
imaging
Molecular imaging of cancer cells
Coating of catheter for cerebrospinal fluid drainage
Additive in bone cement
Implantable material using clay -layers with starch-
stabilized silver nanoparticles
Coating of intramedullary nail for long bone fractures
Coating of implant for joint replacement
Orthopedic stockings
Superabsorbent hydrogel for incontinence material
Treatment of dermatitis
Inhibition of HIV-1 replication
Treatment of ulcerative colitis
Treatment of acne
Coating of hospital textile (surgical gowns, face mask)
Coating of surgical mesh for pelvic reconstruction
Hydrogel for wound dressing
References
Patent
-
Patent
Jia et al., 2008
Walt, 2005
Asian & Geddes, 2006.
Asian et al., 2006
Xue/a/.,2007
Skirtache/a/.,2006
Weisbarthe/a/.,2007
Lesniake/a/.,2005
Asian et al., 2007
Tai et al., 2007
Bayston et al., 2007;
Galianoe/a/.,2007
Alt et al., 2004
Podsiadloe/a/., 2005
Alt et al., 2006
Chen et al., 2006
Pohlee/a/.,2007
Lee et al., 2007
Bhol et al., 2004; Bhol &
Schechter, 2005
Elechiguerra et al., 2005;
Sun et al., 2005
Bhol & Schechter, 2007
Patent
Uet al, 2006
Cohen et al, 2007
Yuetal.,2007
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4. Synthesis and Properties of Silver Nanomaterials
4.1 Methods of Synthesis
Knowledge of silver nanomaterials synthesis methods is important from an environmental
perspective. This information allows for the identification of characteristics and morphologies of
the produced silver nanomaterials that are crucial for a more focused approach when evaluating
their environmental fate, transport and toxicity. Not all silver nanomaterials are the same and the
characterization and morphology dictate physical properties such as solubility, diffusion
beharvior, and reactivity, which, in turn, dictate their environmental fate, transport, and toxicity.
The immense leap in the applications of nanotechnology is mainly attributable to advances in the
synthesis techniques of nanomaterials during the last two decades. The main challenge in
nanomaterials synthesis is the control of their characteristics such as particle size distribution,
shape, morphology, chemical composition and crystal structure. There are an extensive number
of synthesis methods of silver nanoparticles that are readily available in the literature. All
reported methods can be classified and categorized since they follow common approaches and
the differences are limited to the specific reactants used and the reaction conditions. Categories
such as top-down versus bottom-up, green versus non-green and conventional versus non-
conventional have been reported.
4.1.1 Synthesis Categories
Top-down techniques rely on the generation of isolated atoms from the bulk materials using
various distribution techniques. Physical methods such as milling or attrition, repeated quenching
and photolithography are usually involved in the top-down strategies (Gao, 2004; Ju-Nam &
Lead, 2008). Bottom-up techniques start with silver salt precursor (dissolved in solvent) that is
reduced in a chemical reaction and the nanoparticles are formed through nucleation and growth
(Tolaymat etal., 2010). With the bottom-up strategies, the use of capping agents is crucial to
control the particle size and shape, and to provide stability for the synthesized nanomaterials
(Balan et a/., 2007). Figure 4.1 presents a schematic for the top-down versus bottom-up
techniques.
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Synthesis approaches can be classified as either green or non-green. Green approaches use
environmentally friendly agents such as sugars and plant extracts to form and stabilize nanosilver
(Sharma etal., 2009). The weakness of the green approaches is the lesser amount of control a
researcher or synthesizer has over the morphology of the produced nanosilver compared to the
non-green methods.
Synthesis methods can also be grouped as conventional and unconventional methods.
Conventional synthesis methods include the use of citrate, borohydride, two phase (water-
organic) systems, organic reducers, and inverse micelles in the synthesis process.
Unconventional methods include laser ablation methods, radiocatalytic methods, vacuum
evaporation of metal and the Svedberg method of electrocondensation (Krutyakov et a/., 2008).
The top-down or bottom-up approaches are commonly used to synthesize silver nanoparticles;
typically, the bottom-up approaches involve wet chemistry techniques. It has to be mentioned
that there is plenty of overlap between all the previously-mentioned categories. For instance,
using plant extracts to synthesize nanosilver is a convent!onal/green/bottom-up synthesis
method.
4.1.1.1 Top-Down versus Bottom-Up
Typical top-down fabrication techniques for the production of nanosilver are cutting, grinding
and etching of a bulk piece of the material. Although small particle sizes ranging from 10 to 100
nm can be obtained, defects in the surface structure are likely to be present (Nikolaj et a/., 2006).
The imperfection of the surface structure is one of the disadvantages that can significantly impact
the properties of the manufactured nanoparticles. The bottom-up techniques overcome this
deficiency by producing homogenous and stable nanosilver suspensions with the ability to tune
their particle size and shape as well as functionalizing the nanosilver with capping agents that
makes it suitable for specific applications. Top-down techniques are the methods of choice for
the synthesis of highly complex structures. The scalability of production is an issue with the
bottom-up techniques since not all methods are feasible for the massive production of enough
quantities of nanomaterials for industrial use (Tolaymat et a/., 2010). Another concern with the
wet chemical techniques is the accumulation of residual chemicals in the nanoparticles
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suspension at the end of the synthesis processes. These impurities may have an impact on their
applications in medicine, catalysis, microelectronics and sensing devices. The impurities usually
include ionic silver since the reduction efficiency is not 100 % (El Badawy et a/., 2010). The
presence of impurities disables the capability of determining the actual concentration of
nanosilver in suspension. Most of the published scientific literature on synthesis of silver
nanomaterials deals with the bottom-up (wet chemistry) techniques and thus more information is
available for that method when compared to the top-down techniques (Tolaymat et a/., 2010).
The reactants included in the wet chemistry techniques mostly contain a silver salt precursor, a
reducing agent, a solvent and a capping agent. The morphology and surface chemistry of the
synthesized silver nanoparticles are governed by the chemical nature of the capping agents, the
molar ratio of that agent to the silver salt, the redox potential of the reducing agent, the stirring
speed and the temperature of the synthesis reaction (Gulrajani et a/., 2008). Of all mentioned
parameters, the concentration of the stabilizer has the highest influence. The solvent or the
capping agents are occasionally used as reductants for the silver salt precursor (Tolaymat et a/.,
2010).
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Physical methods:
•Photolithography
•Laser-beam processing
•Mechanical techniques
(grinding and polishing)
Top-down Strategy
0.1 ran
1 ran
10 ran
100 ran
1 ran
10 ran
100 ran
1 mm
Bottom-up Strategy
Wet chemical methods:
•Organic synthesis
•Self assembly
•Colloidal aggregation
Figure 4.1: Top-down and Bottom-up synthesis approaches. Reprinted from Sci. Tot. Environ.
Vol. 400 (1-3), Ju-Nam, Y. and Lead, J.R., Manufactured nanoparticles: An overview of their
chemistry, interactions and potential environmental implications, pp396-414, Copyright 2008
with permission from Elsevier.
4.1.1.2 Synthesis Reactants in Bottom-Up Techniques
Silver nitrate (AgNOs) is the most widely used silver ion precursor for the production of
nanosilver. This is a result of its low cost and chemical stability compared to the other available
silver salts. The use of silver nitrate makes it likely that nitrate (NCV) will be the dominant anion
associated with the silver nanomaterial synthesis processes. The reducing agents can refer to any
chemical agents, plant extracts, biological agents or irradiation methods that provide free
electrons to reduce silver ions and form silver nanoparticles. For the production of silver
nanoparticles, various reducing agents are reported such as H2 gas (Evanoff et a/., 2004), sodium
borohydride (Lee & Meisel, 1982), hydrazine (Kim etal., 2007), ethanol (Amendola etal.,
2007), ethylene glycols (Iyer et a/., 2007), Tollen's reagent (Fernandez et a/., 2008), ascorbic
acid (Kashiwagi & Nakamoto, 2006) and aliphatic amines (Rao & Trivedi, 2006). Depending on
the strength of the reducing agents, the particle size can be controlled. The strong and fast
reducing agents cause the formation numerous silver seeds at the beginning of the synthesis
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process, reducing the time of growth and preventing the formation of larger particles (Nikolaj et
a/., 2006). Although many organic solvents are being used for the synthesis of nanosilver, water
is the most frequently used solvent (Tolaymat et a/., 2010). An identified limitation to the use of
water as a solvent is the difficulty to remove the stabilizer residues from the surface of the
synthesized particles, leading to the use of organic solvents while synthesizing silver
nanomaterials. This is observed when there is a need to produce relatively high particle
concentrations coupled with predefined shapes and sizes (Dorjnamjin etal., 2008; Yang etal.,
2005).
Solvents such as N, N-dimethyl formamide (DMF) and polyethylene glycol act as a reducing and
capping agent. The capping agents are used to provide stability for the nanoparticles
suspensions. By changing the ratio of the capping agent to the silver salt, different particle sizes
and shapes are obtained. The molar concentration of the capping agent is usually significantly
higher than that of the silver salt; this results in low nanoparticle concentrations. The capping
agents include surfactants (such as sodium dodecyl sulfate [SDS]) or ligands and polymers that
contain functional groups such as thiol (-SH), cyano (-CN), carboxyl (-COOH) and amine (-
NH2) groups that act as the stabilizer (Olenin et a/., 2008; Si & Mandal, 2007). Polymers are
used extensively for the purpose of nanoparticles stabilization. The efficiency of a certain
polymer is linked with the solvent properties. Good solvent permits the polymeric stabilizer
attached to the particle surface to stretch away from the nanoparticles (Nikolaj et a/., 2006).
The selection of a capping agent may be driven by specific necessities. The catalytic properties
of silver nanomaterials depend on the type of stabilizers used (e.g., Cetyltrimethylammonium
bromide [CTAB] and SDS), which may decrease the adsorption of reactants to the silver surface
(Jiang et a/., 2005). Citrate and polyvinylpyrrolidone are the most widely used stabilizers for
nanosilver (Tolaymat et a/., 2010). Poly(N-isopropylacrylamide) (PNIPAM) is commonly used
as a temperature-sensitive polymer that has a lower critical solution temperature (LCST). Below
the LCST, PNIPAM is hydrophilic and soluble in aqueous solution, but upon raising the
temperature above LCST, the polymer becomes hydrophobic and insoluble, and aggregates in
solution. Because of this phenomenon, silver nanoparticles capped by (PNIPAM) allow for s
Final Report dated 07/15/2010 3 \
State of the Science - Everything Nanosilver and More
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novel application that combine surface plasm on and thermal switching applications (Guo et a/.,
2008).
4.1.2 General Discussion on Nanosilver Synthesis
Of the conventional methods for producing silver nanomaterials, the borohydride and citrate
methods are the most common. The main reasons are the relatively high reactivity of sodium
borohydride (compared to citrate and other reductants), moderate toxicity (compared to
hydrazine and hydroxylamines) and greater lab safety when compared to hydrogen gas and other
physical methods (i.e., dangerous steps involved in other synthesis methods such as pressurizing
hydrogen at relatively high temperature are avoided while using sodium borohydride)
(Krutyakov et al., 2008). The size of the nanosilver produced is fairly small (1-15 nm). Sodium
borohydride does not provide strong stability for the produced nanosilver and, therefore,
stabilizing agents are needed to keep the nanoparticles in suspension. Citrate is a weaker
reducing agent, and the reaction requires energy that is generally applied by heating the solution
(Krutyakov et al., 2008). The particle size produced by the citrate method is relatively larger, but
the nanosilver suspensions are stable due to the presence of the citrate layer coating the
nanoparticle surfaces.
Other conventional methods include biosynthesis, synthesis in two-phase systems, inverse
micellar systems and polyol processes (Krutyakov et a/., 2008). Biosynthesis methods could be
performed intra- or extracellularly. The extracellular methods are more advantageous because of
the large scale processing and ease of control over the environment. Gurunathan et al. (2009)
synthesized nanosilver extracellularly, through the reduction of silver ions by Escherichia coli.
Many other organisms have been used for the synthesis of nanosilver such as E. coli, Bacillus
licheniformis, Aspergillus fumigatus and Klebsiellapneumoniae (Gurunathan et al., 2009).
Synthesis in two-phase (water-organic) systems follows mainly the steps of the Brust-Schiffrin
method that is generally used for making gold nanoparticles, but the process details are different
for the two types of nanoparticles. The nanoparticles are prepared using reactants separated in
two immiscible phases. The interphase between the two liquids controls the reaction between the
metal salt precursor and the reducing agents. The process is also limited by the reactant transfer
from aqueous to organic medium. Inverse micellar systems can be considered a set of nanoscale
Final Report dated 07/15/2010 32
State of the Science - Everything Nanosilver and More
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chemical reactors formed by surfactant molecules. Two inverse emulsions are used in which one
contains a silver salt dissolved in water and the surfactant contains the reducing agent. The
diameter of the nanoparticles can easily be controlled by changing the molar ratio of water to
surfactant.
The formation of nanoparticles occurs in four main stages: 1) coalescence of water cores of
colliding micelles, 2) chemical reaction between the components 3) nucleation and 4) the
intermicellar growth of nanoparticles nuclei (Krutyakov et al., 2008). Krutyakov et a/., 2008
produced stable silver nanoparticles suspensions (5-6 nm) through the reduction of silver nitrate
by cetyltrimethylammonium bromide (CTAB) and non ionogenic surfactants. Polyol processes
for the synthesis of silver nanoparticles are also common. Polyvinyl alcohol (PVA) is used to
reduce the ionic silver to form metallic nanosilver; PVA also acts as a stabilizer for the silver
nanoparticles that are produced (Gautam etal., 2007).
Organometallic methods for nanosilver synthesis are also reported (Fernandez et a/., 2008) such
as treating the complex NBu4[Ag(C6P5)2] with AgClO4 to form the nanoparticles precursor
[Ag(CeF5)], which is further converted to 10 nm silver nanoparticles by adding hexadecylamine
(HDA) and refluxed for 5 h in toluene (Figure 4.2). Nanosilver embedded paint has been
obtained in a single step by the naturally occurring free radicals generated in situ during the
drying process of oils (Kumar et a/., 2008). These radicals reduce silver salts without the use of
any external reducing or capping agents. This system does not require an external heating source
since there is no heating step included. It is nontoxic and inexpensive. The nanosilver embedded
paint shows excellent antimicrobial impact against Gram-positive human pathogens
(Staphylococcus aureus) and Gram-negative bacteria (Escherichia colt).
Final Report dated 07/15/2010 33
State of the Science - Everything Nanosilver and More
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Et20
- NBu4CIO4
[AgfCflF5)1 + HDA — [Ag]
2
|AgfC6F5XHDA)] TolWne
reflux .fth
N = NDA
-------
Plant extracts (such as tea and coffee) and microorganisms such as bacteria, yeast, fungi and
actinomycetes are also used for the reduction of silver ions to produce nanosilver (Tolaymat et
a/., 2010). The characteristics of the produced nanoparticles can be manipulated by controlling
parameters such as pH, temperature, substrate concentration and the exposure time to the
substrate (Mohanpuria etal., 2008).
4.2 Silver Nanocomposites and Bimetallic Nanoparticles
When silver nanomaterials are integrated into polymer matrices to form nanocomposites or when
they are combined with other metal nanoparticles acting as a shell or as a core to form bimetallic
nanoparticles, a combination of useful properties is achieved. Nino-Martinez et ol. (2008) report
an enhancement in the antibacterial activity of silver nanoparticles when it is incorporated into an
Ag/TiO2 nanocomposite. Jiang et al. (2005) report that the successful catalytic reduction of
specific dyes is achieved using silver nanoparticles incorporated on silica spheres. Because of its
unique characteristics when in the form of metallic nanoparticles, silver is often used as a
substrate for magnifying surface enhanced Raman spectroscopy (SERS). Using Ag/Au
(core/shell) as a substrate for SERS provides stronger spectra than data obtained from using each
individual nanoparticle (Pande et al., 2007). Some nanomaterials incorporated with nanosilver
such as multiwalled carbon nanotubes and metallic nanoparticles (Sn, Ru and Pd) have been
reported (Britz & Glatkowski, 2010). The integration of silver into nanocomposites and
bimetallic nanoparticles is continual. With each combination, silver is being utilized to generate
nanoparticles with new characteristics. The environmental impacts of nanocomposites and
bimetallic nanoparticles should be examined on a case by case basis.
4.3 Environmental Perspective
The characteristics of the chemicals involved in the synthesis will affect the fate, transport and
toxicity of these nanomaterials in the environment. Tan et al. (2007) report that the use of
sodium citrate as a reducing agent generates negatively charged silver nanomaterials, which may
behave differently than the positively charged nanosilver generated using branched
polyethyleneimine (BPEI). The stability of the produced nanoparticles suspensions indicates the
Final Report dated 07/15/2010 35
State of the Science - Everything Nanosilver and More
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potential for mobility of these nanoparticles in the environment. One of the issues associated
with the synthesis of silver nanoparticles that has to be considered in their risk assessment is the
scalability and reproducibility of the synthesis techniques. Scalability is one of the major factors
that limit the commercialization of nanoparticles. Some of the synthesis processes under specific
reaction conditions are not reproducible if they are scaled up. This might indicate that massive
production is not feasible and, therefore, trace amounts of these materials will be released into
the environment. One of the techniques that are reported to be scalable is the use of spinning disc
processing with continuous flow (Iyer etal., 2007; Figure 4.3). With this technique, silver
nanoparticles with flexibly tuned characteristics can be produced. Industrial, large-scale
synthesis of silver nanoparticles uses controlled thermolysis of silver alkyl carboxylates to
produce nanosilver without the use of solvents (Kashiwagi et a/., 2006).
Co/rtrolfaf Feed Jets
Ttmptntun
H«r Exchange FtuM
Figure 4.3: Schematic of a spinning disc processor for synthesis of silver nanoparticles.
Reprinted from Lab Chip, Vol. 7, Iyer, K.S., Raston, C.L., Saunders, M., Continuous flow nano-
technology: manipulating the size, shape, agglomeration, defects and phases of silver nano-
particles, pp 1800-1805, Copyright 2007 with permission from RSC Publishing.
Final Report dated 07/15/2010 35
State of the Science - Everything Nanosilver and More
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4.4 Characteristics of the Silver Nanomaterials Products
A wide range of sizes are reported for the synthesized silver nanomaterials; the dominant particle
size ranges from 1-20 nm (Tolaymat et a/., 2010). This is expected to be the target size,
especially since the use of nanoparticles, in general, is driven by the unique properties that are
observed at the nanoscale level. The optimum combination of these properties is obtained for
particles in the range of 3 to 10 nm in size (Olenin et a/., 2008). The reasons behind the
emergence of these new properties are the high surface area to volume ratio as well as the
quantum confinement effect (the electrons are squeezed into a small area) caused by the
extremely reduced size. Cataleya (2006) reports that the highest toxic effect for nanosilver on rat
alveolar macrophages is obtained for 15 nm size when compared to larger nanoparticles with 30
and 55 nm. Navalandian et al. (2008) report that silver nanoparticles with 1-10 nm size
demonstrates interaction with HIV by inhibiting the virus from binding to the host cells. Smaller
particle size does not always translate to an enhancement in the particle properties. SERS
applications, for example, show enhanced signals with larger particle sizes (Dong etal., 2007).
The thermodynamic properties of silver nanomaterials (e.g., melting point, molar heat of fusion)
are directly proportional to particle diameter (Luo et a/., 2008). That is the reason nanosilver
(<10 nm) is utilized in the semiconductor industry as well as printed electronics products for its
lower melting point. Silver nanoparticles are synthesized in various shapes. They can be
grouped as ID objects (e.g., thin films), 2D objects (e.g., nanowires and nanorods) and 3D
objects (e.g., spheres). Spherical particles are the most prevalent among nanosilver particles
(Tolaymat et a/., 2010). The shape of silver nanomaterials might have an impact on some of its
properties. Triangular silver nanoparticles are found to pose stronger biocidal action against
Gram-negative bacterium E. coli than the spherical and rod shaped nanoparticles (Pal et a/.,
2007). This is attributed to the arrangement of the atoms within the crystal structure reported.
Final Report dated 07/15/2010 37
State of the Science - Everything Nanosilver and More
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Figure 4.4: Characterization of silver nanoparticle size and morphology with scanning electron
microscopy (SEM). Images of silver nanoparticles from Nanotechnologies, Inc., show the
spherical morphologies that increase in average size between samples. (A) Ag-15nm, (B) Ag-
30nm, and (C) Ag-55nm. Scale bars are 100 nm. Reprinted from J. Phys. Chem. B, Vol. 112
(43), Carlson, C., Hussain, S.M., Schrand, A.M., Braydich-Stolle, L.K., Hess, K.L., Jones, R.L.,
Schlager, J.J., Unique cellular interaction of silver nanoparticles: size dependent generation of
reactive oxygen species, pp!3608-13619, Copyright 2008 with permission from American
Chemical Society.
Tolaymat et al. (2010) carried out a review on the bottom-up synthesis methods of nanosilver
and the chemical agents used. Based on their review of approximately 200 scientific articles
dealing with synthesis, it was concluded that the chemical agents used most frequently for the
synthesis of nanosilver are sodium borohydride or sodium citrate as reducing agents and silver
nitrate as the metal salt precursor dissolved in water as the solvent and citrate and PVP as the
stabilizing agents. The most produced shape is spherical with a size of less than 20 nm. Table 4.1
provides a description of nanoparticles synthesized for general applications, while Table 4.2
provides a description of nanoparticles synthesized for specific applications. Table 4.3 provides a
description of silver nanocomposite production, while Table 4.4 provides a description of
bimetallic silver nanoparticle production. The four tables summarize the chemical agents used
for the synthesis of nanosilver as well as the morphology of the produced nanoparticles. The
tables also show which techniques are scalable and/or green and which of these techniques
generate stable particles. Table 4.5 provides a list of acronyms used in Tables 4.1 - 4.4.
Final Report dated 07/15/2010 38
State of the Science - Everything Nanosilver and More
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Table 4.1: Description of Evidence for Silver Nanoparticle Synthesis for General Applications (Adapted from Tolaymat et al., 2010)
Ag Salt
AgN03
Category *
Sodium
Borohydride
reduction
Sodium citrate
reduction**
Irradiation
reduction
Reducing Agent
NaBH4
Trisodium citrate
Trisodium Citrate &
Nd:YAG laser
Trisodium Citrate &
UV irradiation d
UV irradiation d
Solvent
Water
Water/ Chloroform
Methanol
Ethanol
Ethanol/Toluene
Toluene/BAm
Water
Water
Water
Water
Ethanol
Stabilizing Agent
P(NIPAM-co-AA)
P(NIPAM)
PVA
P(NIPAM)
P(SS-co-M)
OUDPPA
Trisodium Citrate
Trisodium Citrate
NaBH4
Na-MPS or TOAB or
CTAB or w-heptane
Oleic acid
MBA-AEPZ, MDA-AEPZ
PVP, Trisodium Citrate
Carboxylates
hexadecyl amine
SBEHS
MBTZ
H-GSC
Trisodium Citrate +
Dodecylamine
dodecanoic acid (DDA)
Trisodium citrate
Trisodium Citrate &
Nd:YAG laser**
Trisodium Citrate & UV
irradiation d
Laponite
Poly-
methylmethacrylate
Size (nm)
6-11
16-20
Varies0
Varies0
Varies °
10
4.4-5
NA
12
Varies0
8
Varies0
<3
Varies0
Varies0
4
Varies0
Varies0
7
7
varies
Varies0
NA
100-120
5-8
Source
Dong et al, 2008
Quo et al, 2008
He & Kunitake, 2006
Morones & Frey, 2007
Limsavarn et al., 2007
Hee/a/.,2007b
Wang et al., 1999b
He & Zhu, 2008b
Solomone/ al., 2007
Olenin et al., 2008
Seoetal., 2006
Sun et al., 2008
Song et al., 2008
Wang et al., 1999a
Kuilaetal., 2007
Setuae/a/., 2007
Tan et al., 2002
Kuoe/a/.,2004
Yange/a/.,2007a
Lee et al., 2007
Lee & Meisel, 1982
Pyatenko et al., 2007
Jia et al., 2006
Yang & Huang, 2007
Courrolrfa/,,2007
Notes
s, sph
s, sph
s, sph
s, sph
s, sph
s, sph
s, sph
s, o
s, sph
s, sph
s, sph
s, sph
0, S
s, sph
s, sph
s, sph
s
s, sph
s, sph
a, s, sph
S, sph
s, sph
s, sph
s
s, sph
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
39
-------
Table 4.1 continued
Ag Salt
AgN03
Category
Irradiation
reduction
Organisms and
plant extract
reduction
Reducing Agent
UV irradiation d + BPEI
UV irrad (Arg) + TSA
y- irradiation d
Microwave irrad.d
E irradiation d
Enterobacteriacae
Bacterium Bacillus
licheniformis
Enzyme nitrate
reductase
Fusarium semitectum
Trichoderma
asperellum
Fungus Aspergillus
flavus
Verticillium (Fungi)
Aspergillus fumigatus
(Fungus)
Emblica Officinalis
(Plant)
Pelargonium graveolens
(Plant)
Pseudomonas
stutzeriv(Bacterium)
Capsicum annuum L.
extract
Spent mushroom
substrate
Geranium leaf extract
Solvent
Water
Water
Water
Acetic Water
Ethylene Glycol
Water
Water
Stabilizing Agent
UV irradiation d + BPEI
NA
PVP
oligochitosan GlcN
Chitosan
PVP
PVA
NA
Bacterium Bacillus
licheniformis
Enzyme nitrate reductase
Fusarium semitectum
Trichoderma asperellum
Fungus Aspergillus flavus
Verticillium (Fungi)
Aspergillus fumigatus
(Fungus)
Emblica Officinalis (Plant)
Pelargonium graveolens
(Plant)
Pseudomonas
stutzeriv(Bacterium)
Capsicum annuum L.
extract
Spent mushroom substrate
Geranium leaf extract
Size (nm)
Varies0
Varies °
Varies0
5-15
4-5
Varies °
Varies0
52.5
50
10-25
10-60
13-18
8.92
25
5-25
10-20
16-40
Up to 200
10
30.5
16-40
Source
Tan et al, 2007
Yang et al., 2007c
Uetal, 2007b
Long et al., 2007
Chene/a/.,2007b
Navaladiane/a/.,2008
Bogle et al., 2006
Shahverdie/a/.,2007
Kalimuthu et al., 2008
Kumar et al., 2007
Basavaraja et al., 2008
Mukherjeee/a/., 2008
Vigneshwaran et al.,
2007a
Mohanpuria et al., 2008
Li et al., 2007a
Vigneshwaran et al.,
2006a
Shankare/a/.,2003
Notes
s, sph, o
a, sph
a, o, s
s, sph
s, sph, o
o, s, sph
s, sph
g, sph
g, s, sph
g, s, sph
g, s, sph
a, g, s,
sph
a, g, s,
sph
g
g, sph
a, g, s,
sph
g, s, sph
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40
-------
Table 4.1 continued
Ag Salt
AgN03
Category
Organisms
and plant
extract
reduction
Amines
reduction
DMF
reduction
Ethylene
Glycol
reduction
Hydrazine
reduction
Reducing Agent
Aloe vera Extract
heparin
(polysaccharide)
green tea (Camellia
sinensis) extract
linear polyethylenimine
Dodecylamine &
Formaldehyde
Octadecylamine
Alkylamines («-
Dodecylamine
(C12NH2), n-
octadecylamine)
Alkylamine
Poly(allylamine)
(PAAm)
DMF
EG
Hydrazine
Hydrazine
hydrazine hydrate
Solvent
Water
Water/Cyclohexane
Octadecylamine
Alkylamines (n-
Dodecylamine
(C12NH2), n-
octadecylamine)
Alkylamine
Water
Water
DMF
EG
Water
Toluene
Water/Cyclohexane/
IAA
Stabilizing Agent
Aloe vera Extract
heparin (polysaccharide)
green tea (Camellia
sinensis) extract
linear polyethylenimine
Dodecylamine &
Formaldehyde
SDS or CTAB
Alkylamines (n-
Dodecylamine (C12NH2), n-
octadecylamine)
Alkylamine
Poly(allylamine) (PAAm)
PVP
PMMA
—
PVP
PVP
SDS
—
SDS microemulsion
Size (nm)
15.2
20
NA
Varies0
4
4.7
Varies0
2.9-5.3
4.4
5-12
10
20-75
Varies0
Varies0
Varies0
Varies0
10-20
6
6.5
Source
Chandran et al, 2006
Huang et al, 2004
Vilchis-Nestor et al.,
2008
Sun & Luo, 2005
Chen & Wang, 2008b
Wange/a/., 2008a
Kashiwagi et al, 2006
Kashiwagi et al, 2006
Sardare/a/., 2007
Muthuswamy et al,
2007
Deng et al, 2008
He et al, 2007a
Yang et al, 2007b
Bregado-Gutierrez et
al, 2008
Kim et al, 2006
Wiley et al, 2007
Choi et al, 2005
Kim et al, 2007
Zhang et al, 2008
Notes
g, sph
a,g, s,
sph
g, s, sph
s, sph
s, sph
a, sph
s, sph, o
a,s, sph,
0
s, sph
a, ,o s,
sph
s, sph
0
s, sph
s, sph
0, S
s, sph
0
s, sph
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41
-------
Table 4.1 continued
Ag Salt
AgN03
Category
Ascorbic acid
reduction
Aldehyde
reduction
Hydrogen gas
reduction
Sugars
reduction
Other
reducing
agents
Reducing Agent
Ascorbic acid
Formaldehyde
Formaldehyde
or TEAm or DEFAm
Acetaldehyde
H2Gas
D-glucose
Fructose, Glucose,
Sucrose
PVP
PVA
Vitamin E
Vitamin B2 (Riboflavin)
Glycyl glycine
NaBH(OAc)3
Oligopeptide (NH2-
Leu-Aib-Trp-OMe)
Genamin T020
WPU
Sodium carboxymethyl
cellulose
Solvent
Water
Water
Water/Ethanol
Benzene or Toluene
Water
Water/Toluene
Water
Water
Stabilizing Agent
CTAB
SDS
Daxad 19
Soluble starch
P-4PV
PVP
Thiosalicylic acid
Stearic acid or Palmitic acid
or lauric acid
—
PVA/PVP/Starch
HTAB
Fructose, Glucose, Sucrose
PVP
PVA
NA
Vitamin B2 (Riboflavin)
Glycyl glycine
di-HCF6/Sc CO2
Oligopeptide (NH2-Leu-
Aib-Trp-OMe)
NA
NA
Sodium carboxymethyl
cellulose
Size (nm)
35
Varies0
Varies0
Varies0
Varies0
8
25-40
67
Varies0
Varies0
Varies0
Varies0
10-30
3-14
6.1
NA
3-5.2
13.6
3-9.6
NA
15
Source
Slawinski & Zamborini,
2007
Chaudharie/a/.,2007
Sondie/a/.,2003
Iyer et al, 2007
Hsu & Wu, 2007
Wu & Hsu, 2008a
Rao & Trivedi, 2006
Wang et al., 2007
Haselle/'a/.,2007
Yu & Yam, 2005
Panigrahi et al., 2005
Hopped al., 2006
Fernandez^ al, 2008
Zhang & Lakowicz,
2006a
Nadagouda & Varma,
2008
Yange/a/.,2006b
Shervanie/a/., 2007
Si & Mandal, 2007
Fauree/a/.,2003
Shen et al., 2007
Chene/a/.,2008c
Notes
0
0
a, o, s
a, g, s,
sph
0, S
o, s, sph
s, sph
0, S
s, sph
0
g, s, sph
s
s, sph
g, s, sph
g, s, sph
s
s, sph
s, sph
o, sph
—
g, s, sph
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42
-------
Table 4.1 continued
Ag Salt
AgN03
Ag2S04
AgC104
Category
Other
reducing
agents
Sodium
borohydride
reduction
Irradiation
reduction
Other
reducing
agents
Ethylene
glycol
reduction
Reducing Agent
Soluble Starch
Poly (2,6-dimethy 1-1, 4-
phenylene oxide)
Poly(o-methoxy aniline)
Auto reduction of AgTh
N\N2-
diphenylbenzamidine
benzyl mercaptan &
Ultrasound irrad. d
Superhydride
Amphiphilic Polyester
t-BuONa-activated
NaH
NEt3
Trioctylphosphine
Thermal decomposition
of(AgC02(CF2)«CF3
NaBH4
X ray irradiation d
Polyoxometalates
3 -pentadecylphenol
Ethylene Glycol
dihydroxy benzenes
Solvent
Water
Chloroform/methanol
Water/Chloroform
Chloroform/Ethanol
Ethanol
Ethanol/Toluene
Benzene
THF
NEt3
Trioctylphosphine
N.A
Water
Water
Water
Water/Chloroform
Water/EG
Water
Stabilizing Agent
Soluble Starch
Poly (2,6-dimethy 1-1, 4-
phenylene oxide)
Poly(o-methoxy aniline)
NA
N1 ,N2-diphenylbenzamidine
benzyl mercaptan &
Ultrasound irrad. d
DTC10
Amphiphilic Polyester
t-BuONa-activated NaH
Carboxylate group
Trioctylphosphine
Thermal decomposition of
(AgCO2(CF2)«CF3
AOT & SDS
NA
Polyoxometalates
3 -pentadecylphenol
Ethylene Glycol
dihydroxy benzenes
Size (nm)
10-34
Varies0
Varies0
16.1
10-30
1.7-10.4
2.5-5
<20
3.3
Varies0
6-10
5
20-90
28
40
11.8
Varies0
30
Source
Vigneshwaran et al.,
2006b
Dorjnamjine/a/., 2008
Dawn et al., 2007
Mishraetal., 2007
Sharmae/a/.,2007
Yang & Li, 2008
Tong et al., 2006
Voronov etal., 2007
Lee et al., 2007
Yamamoto et al., 2006
Chen et al., 2007c
Lee et al., 2002
Mandale/a/.,2005
Remita et al., 2007
Zhang et al., 2007
Swamie/a/.,2004
Jacobs al., 2007
Jacobs al., 2008
Notes
g, s, sph
s, sph
s, sph
a, o
s, sph
s, sph
s, sph
s, sph
a, s, sph
s, sph
s, sph
0, S
a, s
o, s, sph
g, s, sph
o, s, sph
s, sph
s, sph
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43
-------
Table 4.1 continued
Ag Salt
Ag20
Ag acetate
Ag
mysitrte
Category
Hydrogen
gas
reduction
Irradiation
reduction
DMF
reduction
Reducing Agent
H2Gas
e- irradiation d
DMF
Solvent
Water
Water
DMF
Stabilizing Agent
NNA
NA
NA
Size (nm)
50-200
15-40
10
Source
Mergaetal., 2008
Li et al, 2005
Khannae/a/.,2008
Notes
sph
sph
* Categorized based on the type of reducing agent
Table Key: a = Scalable Technique, g = Green Synthesis, c = Varies by varying reaction conditions, d = Irradiation is not the reducing agent but it assist providing
free electrons for reduction, s = Stable, sph = Particle shape is: Sphere, o = Other Chemicals or compounds are included in the synthesis process, NA = Not
Applicable
Reprinted from Sci. Tot. Environ., Vol. 408 (5), Tolaymat, T., El Badawy, A., Genaidy, A., Scheckel, K., Luxton, T., Suidan, M., An evidence-based environmental
perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and critical appraisal of peer-reviewed scientific papers, pp999-
1006, Copyright 2010 with permission from Elsevier.
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
44
-------
Table 4.2: Description of Evidence for Silver Nanoparticles Synthesis for Specific Applications (Adapted from Tolaymat et al., 2010)
Ag Salt
AgN03
Category
Sodium
borohydride
reduction
Reducing
Agent
NaBH4
Solvent
Water
Stabilizing Agent
NA
NA
NA
PVP, PAM
NA
NaBH4
NA
Trisodium Citrate-
PVP
NA
Trisodium Citrate
NaBH4
Trisodium Citrate
NaBH4
Trisodium Citrate
NA
Size (nm)
5
8
10,18,23
3-5
Varies °
1-8
<10
Varies °
5
18
N.A
<60
15
Varies °
2
Source
Dubas & Pimpan,
2008b
He & Zhu, 2008b
Radziuke/a/.,2007
Murthy et al., 2008
Nino-Martinez et al.,
2008
Xiong et al., 2008
Liu et al., 2005
Junior et al, 2003
Zhang & Lakowicz,
2006a
Wiley et al, 2007
Pergolese et al., 2004
Wen et al, 2007
Jiang et al, 2007a
Haselle/a/.,2007
Lee et al, 2007
Notes
s, sph
o, s, sph
sph
0, S
o, sph
o, s, sph
o, sph
S
s, sph
s, sph
s, sph
s, sph
s, sph
o, s, sph
s, sph
Applications
Biosensor for herbicides
Bio-sensors
Remote Opening of
Polyelectrolyte
Microcapsules
Antibacterial
Antibacterial Ag/Ti\O2
Colorimetric sensor to
probe histidine
Nanocatalyst: CO
oxidation
Effect of Ag NPs on the
photophysical properties of
cationic dyes
DNA Detection
Enhanced IR Absorption
Spectroscopy
Adsorption of 1,2,3-
Triazole for SERS
Growth of Human
Fibroblasts on Ag NPs
Growth of selenium
nanowires
Effect of shape on
antibacterial activity
Interaction with HIV-1
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
45
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Table 4.2 continued
Ag Salt
AgN03
Category
Citrate
reduction
Reducing
Agent
Trisodium
Citrate
Solvent
Water
Stabilizing Agent
Trisodium Citrate
Size (nm)
20-30
160
24-30
8
60-80
48
NA
10
10
NA
NA
NA
Source
Thompson et al.,
2008
Huaetal., 2007
Thomas et al, 2007
ZhiLiange/a/.,2007
Xuetal., 2004
Balaguera-Gelves,
2006
Doering & Nie, 2002
Wenseleers et al.,
2002
Kim et al., 2004
Xingcanrfa/.,2003
Gryczynski et al.,
2006
Wuetal., 2005
Notes
sph
o, s, sph
o, sph
s, sph
s, sph
s, sph
s, sph
s, sph
s, sph
s, sph
s, sph
s, sph
Applications
Detection of DNA
sequences
Mass spectrometry of
peptides
Antibacterial (hydrogel-
silver nanocomposites)
Determination of
fibrinogen in human
plasma
Detection of Nitro-
explosives
Probing of Membrane
Transport in Living
Microbial Cells
Surface Enhanced Raman
(SERS)
Absorption of Dyes
Decomposition of Benzyl
phenyl sulfide (EPS)
absorbed on the surfaces of
Ag NPs
Interaction between serum
albumins and Ag NPs
Depolarized light
scattering from Ag NPs
Adsorption for p-
Hydroxybenzoic Acid on
Ag Nanoparticles
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
46
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Table 4.2 continued
Ag Salt
AgN03
Category
Citrate reduction
Irradiation
reduction
Organisms and
plant extracts
reduction
DMF reduction
Ethylene Glycol
reduction
Hydrazine
reduction
Other reducing
agents
Reducing
Agent
Ferric
ammonium
citrate (FAC)
Triammonium
citrate (TAC)
Trisodium
citrate
dihydrate
PMA & UV
irradiation d
Fusarium
oxysporum
P.
chrysosporium
(white rot
fungus)
DMF
DMSO
EG
PEG & H2
Hydrazine
PAA
Solvent
Water
Water
Water
DMF
EG
Water
EG
Stabilizing Agent
Ferric ammonium
citrate (FAC)
Triammonium citrate
(TAC)
Trisodium citrate
dihydrate
NA
Fusarium oxysporum
P. chrysosporium
(white rot fungus)
NA
p-cyclodextrin
DMF
DMSO
PVP
PEG & H2
PVP
PAA
Co-sputtering of Ag and silica produce Ag NPs
embedded in silica
Size (nm)
Varies0
8
20-50
50-200
80
Varies
Varies
60-100
8-10
10-35
30
2.5-5.2
Source
Rashid & Mandal,
2007
Dubas & Pimpan,
2008b
Carlson, 2006
Vigneshwaran et al.,
2007b
Lakowicz &
Sabanayagam, 2007
Patakfalvi et al.,
2008
Patakfalvi et al.,
2008
McLellane/a/.,
2006
Yan et al., 2006
Gulrajani et al., 2008
Lee et al., 2006
Mishra et al., 2007
Notes
s
s, sph
sph
a, g, s, sph
sph
s, sph
s, sph
s
g, sph
s, sph
a, s, sph
sph
Applications
Catalysis
Ammonia Sensing
Antibacterial
Antibacterial
DNA microarrays by
metal-enhanced
fluorescence
Interaction with
pollutant gases NO and
SO2
Surface Enhanced
Raman (SERS)
Catalysis
Antibacterial
Silver Inks for printed
electronics
Medical diagnostics
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
47
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Table 4.2 continued
Ag Salt
AgN03
AgClO4
Ag20
Ag(acetate)
Ag(ethx)
AgBF4
NA
Category
Other
reducing
agents
Irradiation
reduction
Other
reducing
agents
Hydrogen gas
reduction
Hydrazine
reduction
Other
reducing
agents
DMF
reduction
DMF
reduction
Other
reducing
agents
Reducing
Agent
H2, UHMWPE
ATS
Thermal
decomposition
HEPES Buffer
Direct current of
100 uA
UV irradiation d
Thermal
Decomposition
H2gas
Phenylhydrazine
PAMAM/
photoreduction
DMF
DMF
Alkyd Paint
Solvent
NA
Ethanol
Water
Water
Water
Diethyl
Ether/Toluene
Water
toluene
Water
DMF
Ethanol / DMF
NA
Stabilizing Agent
NA
NA
NA
HEPES Buffer
NA
NA
HDA
NA
NA
NA
Trisodium Citrate
NA
NA
Size
(nm)
NA
5
Varies0
5-20
NA
6
10
30
<10
NA
Varies0
18-27
12-14
Source
Morleye/a/.,2007
Roldane/a/., 2007
Shim et al, 2008
Muthuswamy et al.,
2007
Parfenov et al.,
2003
Henglein & Meizel,
1998
Fernandez et al.,
2008
Merga et al., 2007
Liet al., 2005
Baloghe/a/., 2001
Patakfalvi et al.,
2007
Hamoudae/a/.,
2007
Kuoetal., 2004
Notes
o, sph
o, s, sph
s, sph
o, sph
s
s, sph
s, sph
o, s, sph
o, sph
g, s, sph
Applications
Biomedical applications
Applications in
Waveguides
Ink-jet printing
Interaction with HIV-1
Enhanced Fluorescence
from Fluorophores
Adsorption of
Organosulfur Compounds
on Ag NPs
Antibacterial
Catalysis
Printed electronics
Antibacterial silver-
PAMAM dendrimer
nanocomposite
Catalysis
Olefin/paraffin separation
in the oil industry
Antimicrobial paints
Table Key: a = Scalable Technique, g = Green Synthesis, c = Varies by varying reaction conditions, d = Irradiation is not the reducing agent but it assist providing
free electrons for reduction, s = Stable, sph = Particle shape is: Sphere, o = Other Chemicals or compounds are included in the synthesis process, NA = Not
Applicable
Reprinted from Sci. Tot. Environ., Vol. 408 (5), Tolaymat, T., El Badawy, A., Genaidy, A., Scheckel, K., Luxton, T., Suidan, M., An evidence-based environmental
perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and critical appraisal of peer-reviewed scientific papers, pp999-
1006, Copyright 2010 with permission from Elsevier.
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
48
-------
Table 4.3: Description of Evidence for Silver Nanocomposites (Adapted from Tolaymat etal., 2010)
Ag Salt
AgN03
Category
Sodium
borohydride
reduction
Citrate
reduction
Irradiation
reduction
DMF
reduction
Hydrazine
reduction
Aldehyde
reduction
Sugar
reduction
Other
reducing
agents
Reducing Agent
NaBH4
Trisodium Citrate
HMEM, UV
irrad.
Ultrasonic Irradd
UV Irrad. d
DMF
DMA
Hydrazine
Formaldehyde
gas or Irrad. d
Formaldehyde
Glucose
Calcination
500 °C
Heat, (NH4)2S2O8
Thermolysis, H2
gas
Solvent
Cyclohexane
Water
NA
DMF
Water
DMF
Water/Ethanol
Water
Ethanol
Water
Water/Ethanol
Water
NA
Template
AOT-TMH
Isostearic acid
MCAP, APS
Graphite Oxide
o-Toluidine
SDS
PAN-6-PEG-6-
PAN, PEA
Rubber Latex
MoS2 / citrate
PSS
PVP, MWCNT
TSPP
TSD, TEOS,
NH3.H2O
[C14mim]BF4
Titanium(IV)
isopropoxide
MB Am,
acrylamide
Monolithic silica
aerogel
Size (nm)
2-4
20
20
o
J
12-20
4-10
Varies °
Varies °
18
Varies °
2-8
25
Varies °
4-7
Varies °
Source
Anande/a/.,2006
Jiange/a/.,2007b
Cassagneau &
Fendler, 1999
Reddye/a/.,2008
Lue/a/.,2007
Lei & Fan, 2006
AbuBakare/a/.,
2007
Patakfalvi etal.,
2008
Huang & Wen,
2007
Dai etal., 2007
Liu et al., 2008
Chene/a/.,2007d
Wu & Hsu, 2008
Hamal&
Klabunde, 2007
Saravanane/a/.,
2007
Ameen et al.,
2007
Notes
s, sph
sph
s, sph
s, sph
s, sph
s, sph
s, sph
s, sph
o, sph
sph
sph
o, sph
sph
s, sph
s
Nanocomposite
Ag- AOT-TMH
Ag/polypyrrole
Ag-GO
POT-Ag
PS-PAA-Ag
—
—
Ag/C, Ag/MoS2
PDMA-PSS-Ag
Ag/MWCNT
—
Ag/Si02
Ag/C
Ag/(C, S)-Ti02
Ag/polyacrylamide
—
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
49
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Table 4.3 continued
Ag Salt
AgN03
Ag(acetate)
AgBF4
AgCF3S03
AgSbF6
Category
Other
reducing
agents
Sodium
borohydride
reduction
Amine
reduction
Other
reducing
agents
Irradiation
reduction
Other
reducing
agents
Reducing Agent
NH4VO3
Thermolysis
NaBH4
Oleylamine
Poly(n-
hexylsilane)
UV Irrad.d
DMPA, UV irrad.
Solvent
Water
MIBK
Water
Toluene
Cyclohexane
THF
Template
NA
PMMA, HFA
PAH & PAA
Poly(n-
hexylsilane)
Pvf-co-ctf-gp-
omec
Pvf-co-ctf-gp-
omec
EEC
Size (nm)
Varies0
Varies0
Varies0
6-12
8.4
4-8
5
NA
Source
Li et al, 2008
Deshmukh &
Composto, 2007
Logare/a/.,2007
Zhang et al.,
2006b
Shankar et al.,
2003
Kohe/a/.,2008
Lee et al., 2008
Sangermano et al.,
2007
Notes
sph
sph
s, sph
s, sph
sph
sph
s, sph
Nanocomposite
—
Ag-HFA-PMMA
Ag-Fe304
—
—
Ag/Epoxy
Table Key: a = Scalable Technique, g = Green Synthesis, c = Varies by varying reaction conditions, d = Irradiation is not the reducing agent but it assist providing
free electrons for reduction, s = Stable, sph = Particle shape is: Sphere, o = Other Chemicals or compounds are included in the synthesis process, NA = Not
Applicable
Reprinted from Sci. Tot. Environ., Vol. 408 (5), Tolaymat, T., El Badawy, A., Genaidy, A., Scheckel, K., Luxton, T., Suidan, M, An evidence-based environmental
perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and critical appraisal of peer-reviewed scientific papers, pp999-
1006, Copyright 2010 with permission from Elsevier.
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
50
-------
Table 4.4: Description of Evidence for Bimetallic Silver Nanoparticles (Adapted from Tolaymat et al., 2010)
Ag Salt
AgN03
Ag2S04
AgC104
Ag acetate
Category
Sodium
borohydride
reduction
Organisms
and plant
extracts
reduction
Sugars
reduction
Other
reducing
agents
Hydrazine
reduction
Other
reducing
agents
Amine
reduction
Reducing Agent
NaBH4
NaBH4&
Ascorbic acid
Neem
(Azadirachta
indica) leaf broth
Fructose
Beta-cyclodextrin
OS-CD)
Hydrazine
Ethanol
Dodecylamine,
OLEA
Solvent
Water/toluene
Methanol
Water
Water
Water
Water
NA
Ethanol/Water
Dodecylamine
Stabilizing Agent
Sodium Citrate-
Dodecylamine
1,10-
phenanthroline
Trisodium Citrate
&TDDM
Neem
(Azadirachta
indica) leaf broth
Fructose
Beta-cyclodextrin
OS-CD)
N.A
PVP
NA
Size (nm)
Varies °
10
36.3
NA
10
13&11
Varies °
Varies °
6
Source
Yang et al, 2005
Jiang et al.,
2007b
Bakshie/a/.,
2007
Shankar et al.,
2003
Panigrahi et al.,
2005
Pande et al., 2007
Damle et al.,
2002
Toshima et al.,
2005
Wang et al.,
2008b
Notes
o, s, sph
o, s, sph
o, s, sph
o, s, sph
o, sph
o, s, sph
o, sph
o, s, sph
o, sph
Bimetal (core/shell)
Ag/Au
Sn/Ag
Au/Ag
Au/Ag
Au/Ag
Au/Ag, Ag/Au
Ag/Pd
Ag/Pt, Ag/Rh
Ag/TiO2
Table Key: a = Scalable Technique, g = Green Synthesis, c = Varies by varying reaction conditions, d = Irradiation is not the reducing agent but it assist providing
free electrons for reduction, s = Stable, sph = Particle shape is: Sphere, o = Other Chemicals or compounds are included in the synthesis process, NA = Not
Applicable
Reprinted from Sci. Tot. Environ., Vol. 408 (5), Tolaymat, T., El Badawy, A., Genaidy, A., Scheckel, K., Luxton, T., Suidan, M, An evidence-based environmental
perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and critical appraisal of peer-reviewed scientific papers, pp999-
1006, Copyright 2010 with permission from Elsevier.
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
51
-------
Table 4.5: List of Acronyms used in Tables 4.1 to 4.4 (Adapted from Tolaymat etal., 2010)
Acronym
P(MMA)
P(NIPAM-co-
AA)
CTAB
EG
SBEHS
TSPP
P(SS-co-M)
Na-MPS
TOAB
di-HCF6 di-HCF6
MBA-AEPZ
MDA-AEPZ
AOT
Daxad 19
ATS
TDDM
HMEM
AOT-TMH
Definition
Polymethyl Methacrylate
poly[(N-isopropylacrylamide)-co-(acrylic acid)]
Cetyltrimethylammonium bromide
Ethylene Glycol
Sodium bis(2-ethylhexyl)sulfosuccinate
5,10,15,20-tetra-4-oxy(2-stearic acid) phenyl
porphyrin
poly(styrene sulfonate-co-maleic)
Sodium 3-mercaptopropanesulfonate
Tetraoctylammonium bromide
(sodium
bis( \H, \H,1H ,dodecafluoroheptyl)sulfosuccinate)
poly (N,NO-methylene bisacrylamide N-aminoethyl
piperazine)
poly(N,NO-dodecyl diacrylamide N-aminoethyl
piperazine)
Aerosol OT (sodium bis-2 -ethylhexyl-sulfosuccinate)
sodium salt of naphthalene sulfonate formaldehyde
condensate
Aminosilane N-[3-
(trimethoxysilyl)propyl]diethylenetriamine]
Trimethylene- 1,3 -bis (dodecyldimethylammonium
bromide)
2-[p-(2-Hydroxy-2-methylpropiophenone)]-
ethyleneglycol methacrylate
Sodium bis(3,5,5-trimethyl-l-hexyl)sulfosuccinate
Acronym
THF
E irrad.
PVP
PVA
SDS
DMF
TEAm
DBF Am
IAA
AgTH
MBTZ
BAm
P-4PV
Arg
TSA
PAA
PAM
DMSO
Definition
Tetrahydrofuran
Electron Irradiation
poly(vinyl pyrrolidone)
Poly -vinyl Alcohol
Sodium Dodecyl Sulfate
N,N-dimethyl
formamide
Triethanolamine
Dimethylformamide
Isoamylalcohol
silver thiolate
2-mercaptobenzothiazole
n-butylamine
poly(4-vinylpyridine)
arginine
Tungstosilicate acid
poly(acrylic acid)
poly(acrylamide)
Dimethyl sulfoxide
Acronym
tiopronin
PAH
PAA
TEOS
PMMA
HTAB
MIBK
DMA
PSS
TSD
Genamin
T020
H-GSC
HFA
OUDPPA
UHMWPE
PTA
MB Am
POT
Definition
7V-(2-Mercaptopropionyi)glycine
poly ally lamine
polyacryllic acid
Tetraethylorthosilicate
poly(methyl methacrylate)
w-Hexadecyltrimethylammonium
bromide
methyl-isobutylketone
2 , 5 -dime htoy aniline
poly(styrene sulfonic acid)
N-[3-
(Trimethoxysilyl)dropyl]ethylene
diamine
mixture of fatty ethoxylated amines
Hydrophile-Grafted Silicone
Copolymers
1, 1, 1,5,5,5-hexafluoroacetylacetone
O/-di(10-
undecene)dithiophosphonic acid)
ultra-high molecular weight
polyethylene
phosphotungstic acid [PTA,
H3(PW12O40)
7V,7V_-methylene-bisacrylamide
poly(o-toluidine)
Final Report dated 07/15/2010
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52
-------
Table 4.5: continued
Acronym
([C14mim]BF4)
EEC
Pvf-co-ctf-gp-
omec
APS
WPU
PAN-6-PEG-6-
PAN, PEA
Definition
imidazolium ionic liquid l-n-tetradecyl-3-
methylimidazolium tetrafluoroborate
3 ,4-epoxycyclohexylmethyl-3 ,4-
epoxycyclohexanecarboxylate
poly(vinylidenefluoride-co-chlorotrifluoroethylene)-
graftpoly (oxyethylene methacrylate)
ammonium peroxydisulfate
Segmented copolymer of waterborne polyurethane
Polyacrylonitrile-block-poly(ethylene glycol)-blcok-
Polyacrylonitrile
Acronym
PEG
BSA
PAMAM
PMA
OLEA
NEt3
Definition
polyethylene glycol
Bovine Serum Albumin
poly(amidoamine)
poly(methacrylic acid)
Oleic acid
triethylamine
Acronym
DMPA
MCAP
MWCNT
GO
Definition
2,2-dimethoxy-2-phenyl
acetophenone
Mercaptocarboxylic acid Pyrrole
Multiwalled carbon nanotube
Graphite Oxide
Reprinted from Sci. Tot. Environ., Vol. 408 (5), Tolaymat, T., El Badawy, A., Genaidy, A., Scheckel, K., Luxton, T., Suidan, M., An
evidence-based environmental perspective of manufactured silver nanoparticle in syntheses and applications: A systematic review and
critical appraisal of peer-reviewed scientific papers, pp999-1006, Copyright 2010 with permission from Elsevier.
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
53
-------
4.5 Characterization Methods, Detection and Speciation
The increasing use of manufactured silver nanoparticles makes their release into the environment
inevitable. Various environmental streams such as water systems, ground water, wastewater and
landfills might be influenced. Methodologies for the detection and characterization of silver
nanoparticles are thus essential in order to investigate their fate, transport and toxicity. Current
literature is focused on the manufacture or toxicity testing of nanosilver. There is a lack of
information on characterization and detection, especially in environmental samples. There is a
need for developing methods to measure nanosilver concentration, size, shape, surface charge,
crystal structure, surface chemistry and surface transformations. Some important questions to
answer: Does nanosilver leach from consumer products? If so, in what form? Is it aggregated or
still in the nanoscale size? What are its surface properties and chemistry? Does nanosilver
dissolve to form ionic silver with time or under different conditions such as pH? What is the
speciation of silver? Is nanosilver toxic? What are the toxicity mechanisms? Under what
conditions do the mechanisms occur? Do particles aggregate inside the testing media? Do
particles aggregate inside the tested cells? In order to answer these questions, characterization
tools are needed.
There are plenty of techniques to characterize pure silver nanomaterials. The majority of these
techniques will, of course, work for pure nanomaterials suspensions, but there are still challenges
to fully characterize these pure suspensions. One of the major challenges that inhibit
characterization is that it is difficult to determine the speciation of silver compounds in the
nanosilver suspensions. The mechanisms of formation and the interactions between the capping
agents and the surface of silver nanoparticles are not fully understood. The characterization and
detection of nanomaterials become more complicated when dealing with real environmental
samples such as wastewater or landfill leachate, which contains a variety of different impurities,
colloids and organic materials. The sample matrix will make it almost impossible to determine
aggregation state or speciation. Based on an extensive search of the literature, there are no
methods available to answer any of the questions mentioned in the previous paragraph for
environmental samples. It may be necessary to use a combination of many different known
techniques to detect and estimate nanomaterials in the environment. This section will highlight
Final Report dated 07/15/2010 54
State of the Science - Everything Nanosilver and More
-------
conventional techniques that are currently available for characterization and detection in hopes
that researchers can make modifications to existing methods or combine two or more methods to
help characterize the nanosilver in environmental samples. Table 4.6 (at the end of the chapter)
provides a summary of these techniques and the limitations of each technique.
At the end of the synthesis processes, nanomaterials suspensions usually contain residual
chemicals and reaction byproducts. The ratio of the capping agent to silver ions is usually high in
order to achieve stabilization, leaving an excess of capping agents in the synthesized nanoparticle
suspensions. Another major impurity that is present is residual ionic silver that is not reduced to
metallic silver. El Badawy et al. (2010) report that the conversion efficiency for ionic silver to
the metallic form is not always 100%. The presence of ionic silver in the nanoparticles
suspension has two major impacts. First, it will prohibit the ability to determine the concentration
of nanosilver in suspension. The reason is that the conventional methods for determining the
silver concentration in a sample rely on digesting silver in concentrated acid and then analyzing
the total amount of silver using Inductively Coupled Plasma (ICP) or Atomic Absorption (AA)
Spectroscopy. The concentration of the nanosilver can not be determined since it completely
dissolves to ions during the digestion step. Another possibility for determining the nanosilver
concentration in pure nanosilver suspensions is using UV-Vis spectroscopy. Nanosilver has a
distinct SPR peak in the UV-Vis region (Amendola et al., 2007). In order to construct a
calibration curve to determine the concentration of silver nanoparticles in unknown samples, a
stock solution of known concentration of the specific nanoparticles suspension is prepared. Some
of the nanosilver in the stock solution will break down to form silver ions, which again fails to
address the problem of having a method to determine the nanosilver concentration in a
suspension containing silver ions. Second, the toxicity of nanosilver cannot be accurately
determined because silver ions are also toxic. During toxicity tests, it will not be clear which
compound is the source of toxicity. The contradictory results obtained by Buzea et al. (2007)
from toxicity of nanosilver compared to silver ions supports that point. This leads to the
conclusion that methods for quantifying ionic silver in nanosilver suspensions are needed or
otherwise purification methods for removal of ionic silver and other chemical impurities are
crucial.
Final Report dated 07/15/2010 55
State of the Science - Everything Nanosilver and More
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4.5.1 Methods for Measuring Ionic Silver in Nanosilver Suspensions
Conventional methods exist for determining ionic silver concentration in solution without acid
digestion and analysis of total metals. These methods might be utilized with refinement to
measure ionic silver concentration in environmental samples containing nanosilver. A mass
balance can then be obtained and the difference between the total metal concentration as
determined by TCP or AA analysis and the silver ion concentration is the nanosilver
concentration. Titration methods using sodium chloride or sodium thiocyanate have been
commonly used for the detection of silver ion concentration (Kraemer & Stamm, 1924). These
are colorimetric methods that depend on color change when the endpoint is reached. Since the
nanosilver suspensions are colored, the endpoint cannot be easily determined leading to incorrect
quantification of ionic silver. The use of ion selective electrodes (ISE) for measuring free silver
ions in solutions and in nanosilver suspensions is also reported (Benn & Westerhoff, 2008). The
limitations of using this technique are possible matrix interference and the need for ionic strength
adjustment for the tested suspensions in order to match the ionic strength of the calibration
standards. Dileen et al. (2008) developed a method for measuring silver ions in solution at
concentration as low as 0.2 g/L in surface water using square-wave stripping voltammetry at a
carbon paste electrode. Sample matrix was found to be an important factor affecting the
measurement results. Because of these matrix effects, the peak shape of the voltammograms
varied and multiple stripping peaks for silver were observed. These techniques were not tested
previously for measuring the ionic silver in nanosilver suspensions or environmental samples
containing both the ionic and the metallic forms, but they might be a good start.
4.5.2 Methods for Isolating Ionic Silver from Nanosilver Suspensions
Using ion exchange resins is a conventional method for isolating silver ions present in the
wastewater of film processing plants. This water is rich in ionic silver. Cerjan-Stefanovi et al.
(1991) used lonenaustauscher I, II and IV resins to isolate ionic silver and then eluted it using
highly concentrated solutions such as nitric acid or sodium hydroxide. To date, there are no
reported studies for testing this technique for isolating ionic silver from nanosilver suspensions.
Another method that was reported to clean nanoparticle suspensions is centrifugation and
washing. The samples are centrifuged under specific conditions (e.g., 15000 rpm for 20 min,
[Benn & Westerhoff, 2008]). The supernatant is removed and replaced with clean water and this
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process is repeated until clean nanosilver suspensions are obtained. Theoretically, the
supernatant is free of nanosilver and other impurities and by analyzing the supernatant using ICP
or AA, the ionic silver can be determined. This procedure is not proven to be accurate and is time
consuming. It is also not scalable for large quantities of purified nanosilver suspensions. Dialysis
can also be used to purify nanosilver suspensions or environmental samples. In this method,
dialysis tubing with a specific size is used to separate particles from ions. It could be a suitable
purification technique but is time consuming, and the accuracy level is not controlled (Sweeny et
a/., 2006). None of the techniques mentioned in this paragraph are proven, and researchers
should investigate and improve these techniques for isolating nanosilver from silver ions.
One of the promising techniques for purification of nanoparticle suspensions is the use of
ultrafiltration membranes. Hollow fiber tangential flow filtration was successfully used for the
purification of gold nanoparticles from both organic and inorganic impurities (Sweeny etal.,
2006). It is an efficient, rapid and scalable alternative to traditional methods of nanoparticle
purification such as ultracentrifugation, ion exchange resins and dialysis. In this technique, the
membrane pore size determines the retention or transmission of solution components. The
permeate contains the rejected impurities and the retentate contains the nanoparticles. The
nanoparticle suspension is continuously recirculated through the membrane while it is exchanged
with clean buffers (such as water) until the impurities are completely removed. This technique is
more efficient and scalable compared to the other separation techniques. Dialysis is effective in
removing excess salts but leaves free ligands in the solution. Size exclusion chromatography is
useful for removing both salts and free ligands; but, the nanoparticles tend to irreversibly
precipitate on the chromatography supports, leading to decreased yields at the end of the process
(Sweeny et al., 2006). Sweeney et al. (2006) developed a purification method using diafiltration
to overcome the limitation of the other purification techniques, which yields highly pure
nanoparticles compared to other methods.
4.5.3 Novel Detection and Characterization Techniques for Environmental
Samples
Using an electrospray atomizer coupled to a scanning mobility particle sizer (ES-SMPS) (Figure
4.5) is a novel method investigated by Elzey et al. (2009) for determining whether commercially
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manufactured silver nanoparticles form agglomerates, behave as isolated particles, or dissolve as
ions in neutral or low pH (6.5-0.5) aqueous suspensions. By drawing a liquid nanosilver
suspension through a capillary tip, the electrospray generates an aerosol of nanoparticles and the
droplets are sprayed into a steam of dry air. The exiting nanoparticles flow through a diffusion
dryer and the remaining liquid diffused away from the nanoparticles is captured by silica beads.
The nanoparticle aerosols are then charged using a radioactive source and enter the SMPS for
size determination. The particles flow down the high voltage column of the nano differential
mobility analyzer (DMA) where their size is sorted based on their electrical mobility. The nano-
DMA allows only particles within a narrow mobility diameter to exit, which implies that the
stream consists only of monodisperse particles that enter the ultrafine condensation particle
counter (UCPC) for the determination of particle concentration. The combination of the nano-
DMA and the UCPC allows the SMPS to provide particle size distribution of a nanosilver
suspension. This is a promising technique; however, the analysis of environmental samples is
still challenging because other colloidal particles may be present.
SMPS
Figure 4.5: Schematic diagram of the Electrospray-Scanning Mobility Particle Sizer (ES-SMPS)
system Reprinted from J. Nanoparticle Res., doi:10.1007/sll051-009-9783-y, Elzey, S.,
Grassian, V.H., Agglomeration, isolation and dissolution of commercially manufactured silver
nanoparticles in aqueous environments, Copyright 2009 with permission from Springer.
Flow Field Flow Fractionation (F1FFF) is probably the most promising technique for nanosilver
characterization in environmental samples. Ranville (2009) suggested that the development of
F1FFF-ICP-MS based tools will facilitate the future studies of nanoparticle behavior in
environmental systems. The difference in diffusion coefficients of the colloids in a sample is the
basis for size separation using F1FFF technique (Stolpe et a/., 2005). The sample is eluted
through a thin channel by a laminar hydrodynamic flow. A cross flow is applied perpendicular to
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the channel flow forcing the sample colloids towards the lower wall of the F1FFF channel which
is covered by an ultrafiltration membrane with specific cutoff size. The ability of a colloid to
diffuse against the cross flow determines the average position of the colloid. Smaller colloids are
transported with faster flow elements in the channel flow (Figure 4.6). The hydrodynamic
diameter of the colloids can be determined from the retention time and the channel dimensions.
Using ICP-MS as an online detector for F1FFF allows its use for various applications especially
with the low detection limits, high sensitivity and the ability of ICP-MS to detect large number
of elements (Lyven etal., 2003). F1FFF has also been coupled with other detectors such as UV,
light scattering, graphite furnace atomic absorption spectroscopy (GFAA), transmission
electronic microscopy (TEM), scanning electron microscopy (SEM) and atomic force
microscopy (AFM).
Cross flow
Channel flow with
parabolic profile
channel
thickness
-0,2 mm
UltrafJIter
membrane
Porous frit
Figure 4.6: Cross section of a small part of the F1FFF channel. Reprinted from Anal. Chim.
Acta, Vol. 535 (1-2), Stolpe, B., Hassellov, M., Andersson, K., Turner, D.R. High resolution
ICPMS as an on-line detector for flow field-flow fractionation; multi-element determination of
colloidal size distributions in a natural water sample, pp!09-121, Copyright 2005 with
permission from Elsevier.
FIFFF ICP-MS has been used for the elemental characterization of colloidal materials (1-50 nm
hydrodynamic diameters) in natural fresh water samples (Lyven et a/., 2003; Staple et a/., 2005).
F1FFF-AFM was used to quantify the structure of the smallest size fraction (< 5 nm) of aquatic
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colloids in surface water samples. Baalousha etal. (2006) used F1FFF-ICP-MS and TEM
coupled to X-ray energy dispersive spectrometry (X-EDS) in series to characterize the physical
properties, surface chemical composition and colloid-trace elements association for the colloidal
materials present in surface water samples. Gimbert et al. (2007) used F1FFF to determine the
particle size distribution of ZnO nanoparticles spiked in soil suspensions.
Although the literature lacks information on using F1FFF for nanosilver detection and
characterization in environmental samples, it is promising and more attention should be paid to
developing methodologies for nanosilver detection using this technique coupled with suitable
detectors. The main challenge for this technique is sample preparation and preconcentration that
increases the potential for particle aggregation.
A number of approaches have been proposed for the detection and characterization of
nanoparticles in aquatic samples, including microscopy-based techniques, light scattering
methods and several based on chromatography. The most promising of these involve the use of
separation techniques such as field flow fractionation (FFF; Baalousha etal., 2005a, 2005b,
2006a, 2006b; Chen & Beckett, 2001; Siepmann et al, 2004; von der Kammer et al, 2005a,
2005b), liquid chromatography (Arangoa et al, 2000; Jiminez et al, 2003; Saridara & Mitra,
2005; Sivamohan etal, 1999; Song etal, 2003, 2004; Wilcoxon etal, 2000, 2001, 2005), size
exclusion chromatography (SEC; Bolea etal, 2006; Bootz etal, 2005; Huve et al., 1994;
Krueger etal, 2005; Liu & Wei, 2004; Wang etal, 2006; Wei & Liu, 1999), gel electrophoresis
(GE; Bruchert & Bettmer, 2005), and capillary electrophoresis (CE; Feick & Velegol, 2000; Lin
et al, 2007; Schmitt-Kopplin & Junkers, 2003; Schnabel et al, 1997). Where these have been
combined with element-specific detectors, such as ICP-MS (Giusti etal, 2005; Hassellov etal,
1999; Helfrich et al, 2006); Metreveli et al, 2005; von der Kammer et al, 2004), an additional
degree of selectivity is gained, thereby, increasing the quality of the data obtained. To our
knowledge, only FFFICP-MS has been successfully applied to samples in environmental media
(e.g., Gimbert et al, 2007; Stolpe et al, 2005), the others having only been used on standards
and/or simple solutions.
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Tiede et al. (2009) evaluated the methods listed in this section, and came to the following
conclusions as to the limitations of detection and characterization techniques in environmental
samples: (a) complexity and time demands of FFF; membrane interactions and membrane cut-off
effects, (b) limited size separation range of SEC columns, (c) intricate post-separation sample
handling of gels, and (d) GE complex interpretation of migration times, i.e., distinguishing size-
based from non-size-based interactions (CE and GE).
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Table 4.6: Possible Conventional Characterization and Detection Techniques for Nanosilver
Technique
Function
Limitations
Particle size and morphology
Transmission Electron
Microscopy (TEM)
&
Scanning Electron
Microscopy (SEM)
TEM and SEM offer nanometer resolution for
measuring nanoparticle size.
High resolution TEM (HRTEM) provides more
information regarding the lattice fringes and
crystal structure.
HRTEM sometimes provides information on the
thickness of the capping agent layer.
TEM, with appropriate detectors and software,
also gives a range of other data such as fractal
dimensions, elemental composition and chemistry
(bonding and redox activity).
Proper sample preparation is needed.
The sample has to be dehydrated before analysis. This
might cause major structural changes for the
nanoparticles.
Drying may cause shrinkage for the capping agent
molecule which affects the size measurement. The PVP
molecule coating the nanosilver was reported to be
shrunken after drying.
For environmental samples containing nanosilver both
techniques cannot be used since these techniques do not
distinguish between different particles unless the analysis
was performed after a separation step or there was an
EDX attached to the instrument to differentiate various
elements and it will still be difficult.
In order to ensure no bias in the size analysis, several
images representing diverse regions of the TEM grid
have to be acquired and several hundreds to thousands of
nanoparticles are taken. This helps to avoid artificial size
separation or skewing as a result of drying effects or
aggregation.
Electrospray atomizer
coupled to a scanning
mobility particle sizer
(ES-SMPS)
ES-SMPS can be used to determine particle size
distribution of AgNPs in suspensions.
Samples are analyzed in aqueous form. This give
the ES-SMPS advantage over TEM and SEM
which needs a drying step that might impact the
particle size.
Samples can be centrifuged and the supernatant is
further analyzed for the ionic silver using ICP or
AA.
The presence of other types of colloidal particles might
prohibit the applicability of this technique to determine
the size distribution in environmental samples.
BET (Brunauer-Emmett-
Teller) analysis
Provides the ability to measure the surface area of
nanosilver.
Not applicable for environmental samples which contain
various impurities.
Samples have to be dry. Thus dehydration must be
considered, as major structural changes can occur due to
this process.
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Table 4.6: continued
Technique
Function
Limitations
Atomic Force
Microscopy (AFM)
The AFM is an instrument capable of measuring
the topography of a given sample.
A nanosized tip attached on a cantilever is traced
over the sample and a 3D image of the sample
topography is generated on a computer.
AFM is used for investigating the size and shape
of silver nanoparticles in three dimensions.
AFM provides higher resolution than SEM.
Dehydration is not a concern with AFM.
Visualization can be achieved by using AFM
under humid or fully hydrated conditions.
Sample preparation is a crucial issue in order to obtain
the required results.
The choice of the right tip is also a limitation to obtain
good images.
AFM is slower than SEM and the scanning area is
smaller than SEM.
Works for silver nanoparticle suspensions and most
probably would not work for environmental samples.
Environmental Scanning
Electron Microscopy
(ESEM)
Allow imaging and determining the size of the
samples under ambient conditions without the
need for drying.
ESEM has difficulties analyzing the small nanoparticle
sizes.
Dynamic Light
Scattering (DLS)
DLS is a frequently used technique for the
characterization of manufactured silver
nanomaterials.
Provides information on the hydrodynamic
diameter of nanosilver in suspensions.
Used frequently for the aggregation studies of
manufactured nanomaterials under different
environmental conditions.
DLS is capable of measuring particles in the size
ranges from to a few nanometers to few
micrometers
Not suitable for analysis of environmental samples.
Larger particles or aggregates in the sample will 'mask'
the presence of nanoparticles and cause difficulty in
interpretation.
Even if the nanoparticles can be detected, there is no
method to ensure that the detected particles are nanosilver
and not other colloids.
Relatively high concentrations of nanosilver have to be
present in order to measure the particle size.
Flow Cytometery
Provides quantitative information regarding the
cellular interactions with nanoparticles.
Provide information regarding the physical
properties of the nanoparticles.
Measures both light scattering and fluorescence
from particles.
The reliance of this technique on light scattering may
limit its applicability for detection of nanoparticles in
environmental samples.
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Table 4.6: continued
Technique
Function
Limitations
X-ray Diffraction (XRD)
Determines the crystal structure of nanosilver and
the elemental composition.
Sample has to be dried.
It is a qualitative technique.
For environmental samples detection limit might prohibit
the ability to detect the presence of nanosilver with the
relatively low concentrations expected to be present in
the environment.
Centrifugation
Differential centrifugation can be used to separate
particles in a suspension based on size.
Centrifugation can be also used as a primary step
for the determination of nanosilver concentration
in a suspension using ICP or AA. Particles are
removed by centrifugation and the supernatant is
analyzed using ICP or AA. The concentration of
nanosilver is determined from the mass balance
between the total silver and the ionic silver
concentration.
Not applicable for environmental samples as a result of
the presence of other colloidal particles.
There is no guarantee for complete removal of nanosilver
from suspension after centrifugation. This limits the
applicability for using this technique as a primary step for
nanosilver concentration in aqueous suspensions.
Surface and Coating Layer Characterization Techniques
X-Ray Photoelectron
Spectroscopy (XPS)
XPS is a useful surface analysis technique for
analyzing solid state samples.
It gives information on oxidation states and
elemental composition on a sample surface.
XPS can be used to determine the concentration of
ligands bound to the surface of a nanoparticle as
well as determine how much free inorganic
impurities are associated with the sample.
Silver can be detected in environmental samples but the
oxidation state might be undistinguishable.
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Table 4.6: continued
Technique
Function
Limitations
Thermal gravimetric
analysis (TGA)
TGA is a method for monitoring mass changes of
a heated sample.
It can be used to determine the percentage of
volatile organic species associated with a sample
or the amount of organic capping agent coating the
nanoparticles surface.
The temperature at which mass loss occurs as well
as the mass loss profile can be correlated with the
identity of the ligand and the strength of ligand
binding.
Not applicable for environmental samples, which usually
contain lots of organics that will interfere with the
organic coating on the nanosilver.
Nuclear magnetic
resonance (NMR)
NMR can be used primarily to confirm the
composition of the ligand shell coating the
nanoparticles and to identify any impurities in the
suspension.
Used to study the mechanism of the nanosilver
formation and testing the type of intermediate
compounds formed.
Can be used to test the behavior of the polymers
and organic coating at different solution
chemistries
It is a challenge to use with environmental samples, but
more research should focus on this powerful technique,
especially with advances of NMR techniques such as 2D
NMR and solid state NMR.
X-ray Absorption Near
Edge Structure (XANES)
Of the various applications of XANES, it can be
used to determine surface transformation of
nanosilver in the presence of various anionic,
cationic and organic compounds.
Useful for aging studies.
Not easily accessible instrument.
Powerful technique but research is needed to develop
methodologies to handle the complexity of environmental
samples
Fourier Transformed
Infrared Spectroscopy
(FTIR)
Can be used to identify organic impurities in the
nanoparticle suspensions and the functional groups
on the nanosilver surface.
Not applicable for environmental samples
Zeta Potential
Measurement
Provide information on the surface charge of
nanosilver.
It is also used for the determination of point of
zero charge (or isoelectric point), which is the pH
at which the surface is neutralized (no charge).
Require relatively high concentration of nanosilver in the
sample.
Not applicable for environmental samples full of other
colloidal particles. Thus the charge obtained will be a
result of a combination of colloidal particles not only
nanosilver.
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Table 4.6: continued
Technique
Function
Limitations
Measurement of nanosilver concentration
Inductively Coupled
Plasma-Optical Emission
Spectroscopy (ICP-OES)
&
Atomic Absorption
spectroscopy (AA) and
Graphite Furnace AA
(GFAA)
UV-visible Spectroscopy
These are major techniques used to quantify the
total concentration of silver.
The selection of AA or ICP versus GFAA depends
on the concentration of silver in the sample. For
extremely low concentrations, the graphite furnace
is used.
Excellent technique for measuring nanosilver
concentration in pure nanosilver suspensions with
relatively low detection limits ( g/L range).
SPR contains information on size, aggregation and
surface chemistry since the peak shifts in response
to change in these parameters.
Since the sample is digested using concentrated acids
before analysis, the total silver in the sample is obtained.
There is no ability to distinguish between ionic silver and
nanosilver using these techniques unless the residual ions
are removed.
The nanosilver has to be stable. SPR does not exist for
aggregated form of nanosilver.
Not applicable for environmental samples because of the
possible interferences from the different compounds and
probably other nanoparticles present in the sample.
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5. Potential Magnitude of Silver Nanomaterial Utilization
and Environmental Exposure
The environmental fate and effects of silver has received considerable attention since the
early 1990s, in part, due to concerns about the potentially low silver effect levels that were
determined on the basis of toxicity tests in laboratory water. It was expected that the
environmental effect levels that were applicable to field conditions might differ markedly
from levels measured in lab waters as a result of the effect of site-specific water quality on
metal speciation and bioavailability. Between 1991 and 2006, a group of photographic
manufacturers (referred to as the Silver Coalition and later as The Silver Council, and first
managed by the National Association of Photographic Manufacturers (NAPM) during the
early 1990s, then the Photographic Imaging and Manufacturing Association (PIMA) in 1997,
and most recently by the International Imaging Industry Association (ISA) in 2001), working
in cooperation with regulatory agencies, provided support for a coordinated $10 million
silver research and development (R&D) effort (called the Silver Research program, SRP) that
was designed to gain an improved understanding of the environmental fate and effects of
silver. The SRP consisted of investigations of the fate and effects of silver in various
environmental media, including soil, water, and sediment. Chemistry investigations were
undertaken to review existing analytical techniques and to develop new and improved
methods to characterize and predict silver speciation in environmental media. Physiology and
ecotoxicology studies were undertaken to better understand the mechanisms of toxicity and
how effect levels varied with the characteristics of a particular medium. Finally, modeling
studies were performed with the aim of developing improved methods for quantitatively
evaluating silver speciation and the potential for effects. Paquin et al. (2007) published an
overview of the SRP, which had resulted in more than 200 peer-reviewed publications and
300 conference presentations, in a report entitled 'Overview of the Silver Research and
Development Program (1991-2006): Advancing the Science of the fate and Effects of Silver
in the Environment'.
Unlike silver, there is not much information on the fate and effects of nanosilver in the
environment. This report attempts to list potential methods for synthesizing and using silver
nanomaterials in commercial products, potential sources and routes of exposure, and toxicity
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information generated to date. The following subsections describe potential routes of
exposure from manufacturing consumer products that contain nanosilver.
5.1 Inventory of Silver Nanomaterials: Industrial and consumer
products
The project on emerging nanotechnologies that was established in April 2005 as a
partnership between the Woodrow Wilson International Center for Scholars and the Pew
Charitable Trusts has published a fair amount of information on the inventory of consumer
products containing nanomaterials including nanosilver. There is still a lack of information in
regards to the characteristics of the particles (shape, size and surface chemistry), synthesis
methods, production quantities, production losses, production consumption and the
geographic distribution of these nanosilver-containing products in the US. These data are
required in order to perform risk assessment and LCA. A survey was constructed to collect
data to fill the gaps highlighted above. The survey was sent to a total of 122 companies, 53 of
which are in the US producing various types of consumer products containing nanosilver, 32
are international companies that produce consumer products that contain nanosilver, and the
remaining 37 US companies produce raw silver nanomaterials. A list of these companies in
addition to the questionnaire is included in Appendix A. There was a very low response to
the survey (0.8% of the companies answered the survey). The websites of the companies
producing consumer products containing nanosilver (both domestic and international) were
visited in the hope that information on production rates, source(s) of raw materials, amount
used in each product, etc. could be found. Unfortunately, the amount of information available
on the company websites was very limited. The most common information found on the
websites is the concentration of nanosilver in the product. Since no new information was
obtained, this section is a review of inventory information available in the literature.
In 2008, Fauss compiled the Silver Nanotechnology Commercial Inventory (SNCI) during an
internship with the Emerging Nanotechnologies project (Fauss, 2008). A total of 65
companies (from 11 different countries) producing 240 products were listed (Appendix B):
214 were general commercial products and 26 were precursor products. The product
information was obtained through the websites, product listings, email and/or phone
conversations with customer service representatives. Out of 214 consumer products
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containing nanosilver, 45% reported the nanoparticle size used in the product ranged from
0.3-250 nm. The average particle size of nanosilver used was 24 nm. Fauss (2008) reported
the various forms of nanosilver incorporated in the surveyed consumer products and the
results are presented in Figure 5.1. Products that are coated during the manufacturing process
are listed under "coating".
7?,
20-
DGeneral Commercial Products
• Precursor
•iH
1C
Coating Coatlng.'Spray Powder Solid Liquid
Type/Form of Nanomaterial
Spun
N.'A
Figure 5.1: Forms of nanosilver incorporated in consumer products. (Fauss, 2008)
The products were categorized according to their applications. As presented in Figure 5.2,
nanosilver is most notably incorporated into health and fitness products, including sporting
goods, clothing, cosmetics, and personal care products.
Wijnhoven et al. (2009) reviewed the inventory of consumer products containing nanosilver.
They, too, concluded that there is a lack of data on the concentrations of nanosilver in these
products, as well as the size and form in which it is present. The authors specified the
missing information as a knowledge gap. In this review, tables including the products,
manufacturers, countries of manufacturing, form of nanosilver in the products and particle
size were constructed (Appendix C).
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13-
K M-
Product Category
Figure 5.2: Categories of nanosilver-containing products. (Fauss, 2008)
There is information on manufacturers of either raw nanosilver or products containing
nanosilver on the Internet and/or in the literature. However, there is a knowledge gap in the
characteristics and quantities of nanosilver that is produced and used in consumer products.
More attention needs to be paid to find this information and to enlist the companies in
providing this information in order to assess the potential risks associated with nanosilver.
The California Department of Toxic Substances Control ("DTSC") has announced its
intention to request information and data for "reactive nanometal oxides" and other
nanomaterials. The original announcement, on April 14, 2009, identified nanoscale
aluminum oxide, silicon dioxide, titanium dioxide, and zinc oxide as examples of possible
nanometal oxides to be reviewed. A recent update added nanoscale silver, nano zerovalent
iron, and cerium oxide. DTSC's formal, mandatory data call-in is the second call-in for
nanomaterials under California's Chemical Information Call-In law, A.B. 289, California
Health and Safety Code, Chapter 699, sections 57018-57020.
In January 2009, DTSC issued its first call-in for carbon nanotubes (CNTs); the call-in ended
in January, 2010. Through the proposed data call-in, DTSC intends to expand its current
knowledge of "analytical test methods, fate and transport in the environment, and other
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relevant information" for the nanomaterials selected. Information being requested in the call-
in includes the following (DTSC, 2010):
"Value chain for a company? For example, in what products are carbon nanotubes
used by others? In what quantities? Who are the major customers?
What sampling, detection and measurement methods are being used to monitor
(detect and measure) the presence of chemical in the workplace and the environment?
Provide a full description of all required sampling, detection, measurement and
verification methodologies. Provide full QA/QC protocol.
What is the knowledge about the current and projected presence of chemical in the
environment that results from manufacturing, distribution, use, and end-of-life
disposal?
What is the knowledge about the safety of chemical in terms of occupational safety,
public health and the environment?
What methods are being used to protect workers in the research, development and
manufacturing environment?
When released, does chemical constitute a hazardous waste under California Health
& Safety Code provisions? Are discarded off-spec materials a hazardous waste? Once
discarded, are the carbon nanotubes being produced a hazardous waste? What are the
waste handling practices for carbon nanotubes?"
DTSC issued the request to "manufacturers," defined to include businesses located in
California that produce these materials or import them for sale in California. After DTSC
issued the request, companies subject to the call-in had one year to respond to DTSC with the
required information, including generating any data that DTSC requires (DTSC, 2010).
DTSC and California Environmental Protection Agency's (Cal/EPA) Department of
Pesticide Registration (DPR) began collaborating on nanosilver in 2009. In May 2010, the
two agencies entered into an agreement to work together on this nanomaterial (DTSC, 2010).
The two agencies have received two pesticide registrations involving nanosilver use as of the
date of this report, and four additional registrations are pending. DPR and DTSC anticipate
an increase in applications to register pesticides and anti-bacterial containing nanosilver in
California. Through this collaborative partnership, the two agencies aim to share registration,
research and other information about nanosilver (DTSC, 2010).
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As part of the chemical information call-in under California's Chemical Information Call-In
law, A.B. 289, to assist DTSC on carbon nanotube call-in process, DTSC sought research
and analytical support from the Sustainable Technology & Policy Program at the University
of California, Los Angeles (UCLA). Pursuant to State Standard Agreement No. 08-T3631,
UCLA will review and evaluate responses received by DTSC in response to the formal
information request under A.B. 289 relating to carbon nanotubes. UCLA will also review the
process used by DTSC for this carbon nanotube call-in and will develop a written evaluation,
including recommendations for future chemical call-ins under A.B. 289 and other
information collection authorities available to DTSC. This work is a continued partnership
on nanomaterials research between DTSC and UCLA that has been in place for over three
years (DTSC, 2010).
5.2 Routes of Release and Exposure, Ecological
Once released into the environment, the mobility, bioavailability and toxicity of silver
nanoparticles in any ecosystem is largely determined by colloidal stability. Colloidal
stability is a function of many factors including the type of capping agent, the characteristics
of the surrounding environment such as the pH, ionic strength, presence/absence of humic
acids and other ligands, and the background electrolyte composition (Chen, 2006; Cosgrove,
2005; Tielemans et a/., 2006). An extensive number of capping agents have been
investigated to enhance the ability of nanoparticles to stay suspended in solution. Capping
agents are chemicals that are used in the synthesis of silver nanoparticles to prevent their
aggregation through electrostatic repulsion, steric repulsion or both. In the case of silver, the
most prevalent capping agents are citrate, sodium borohydride (NaBH4) and
polyvinylpyrrolidone (PVP) (Tan et a/., 2007; Tolaymat et a/., 2010). The mechanism and
functional groups involved in colloid stabilization differ with capping agents, which may
lead to varying particle size and stability. Colloidal interactions, mobility and toxicity may
differ.
Although numerous studies have investigated the effect of the colloidal surface properties on
the stability of various nanoparticles under various environmental conditions, there is little
information available in regards to silver nanoparticles. In a study by Jiang et al. (2009), the
size of TiC>2 nanoparticles increased 50 fold upon increasing the solution ionic strength from
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1 to 100 mM NaCl. The same study also showed that varying the solution pH resulted in a
significant change in the particle surface charge. In another study, guar gum adsorbed on the
surface enhanced the mobility of nano zero valent iron in sandy porous media regardless of
the pH and ionic strength (Tiraferri & Sethi, 2009). Saleh et al. (2008) reported that
uncoated nano zero-valent iron particles were immobile in water-saturated sand columns
while triblock copolymer-coated particles were highly mobile. Coating quantum dot
nanocrystals with polyethylene glycol suppressed agglomeration and stabilized the
suspension regardless of the ionic strength (Jiang et a/., 2009). Research has also shown that
increases in cation valence and surface adsorption of ionic species significantly impacts
suspension stability.
Gaiser et al. (2009) compared different sizes of nanosilver and cerium oxide nano particles
for their potential for uptake by aquatic species, human exposure via ingestion of
contaminated food sources and to assess their resultant toxicity. The results of their study
demonstrated the potential for uptake of nano and larger particles by fish via the
gastrointestinal tract, and by human intestinal epithelial cells, suggesting that ingestion is a
viable rout of uptake into different organism types.
Cumberland et al. (2009) studied the particle size distributions of silver nanoparticles under
environmentally relevant conditions. As part of the study, monodisperse 15 nm citrate-
stabilized silver nanoparticles were synthesized, characterized and then fractionated by flow
field-flow fractionation (F1FFF) at environmentally relevant conditions (pH 5 or 8, presence
of natural organic macromolecules (NOM) and presence of sodium or calcium). At low ionic
strength, nanosilver particle size increased as pH increased from 5 to 8. However, changing
the ionic strength from 10~3 to 10~2 M Na increased instability of the nanosilver. In the
presence of humic substance, a reduction in nanoparticle size was seen, most likely due to a
reduction in the diffuse layer. The presence of Ca2+ ions, at the higher ionic strengths caused
complete loss of the solution nanosilver, with or without humic acids, most likely due to
aggregation. The presence of humic acids improved stability of silver nanoparticles under
these conditions by forming a surface coating resulting in both steric and charge stabilization.
Cumberland et al. (2009) theorize that silver nanoparticles could have long residence times in
aquatic systems in the presence of humic substances, potentially resulting in increased
bioavailability.
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El Badawy et al. (2010) studied the impact of capping agent and environmental conditions
(pH, ionic strength and background electrolytes) on the surface charge and aggregation
potential of five nanosilver suspensions. The nanosilver suspensions examined were:
uncoated nanosilver (Hydrogen reduced AgNPs), electrostatically stabilized (Citrate-AgNPs
and NaBH/pAgNPs), sterically stabilized (PVP-AgNPs) and electrosterically stabilized
(Branched polyethyleneimine (BPEI) stabilized AgNPs). The uncoated and the
electrostatically stabilized nanosilver tended to aggregate at higher ionic strength and/or
acidic pH. The authors reported that the ionic strength, pH and electrolyte type had no
impact on the aggregation of the sterically stabilized nanosilver.
Muhling et al. (2009) tested whether silver nanoparticles released into estuarine
environments result in increased antibiotic resistance within the natural bacterial population
in estuarine sediments. A 50-day microcosm exposure experiment was carried out to
investigate the effects of nanosilver (50 nm average diameter) on the antibiotic resistance of
bacteria in sediments from an estuary in southwest England. Sediment samples were
screened at the end of the exposure period for the presence of bacteria resistant to eight
different antibiotics. The antibiotics used in the study were erythromycin, oxytetracycline,
sulfadiazine, trimethoprim, lincomycin, ceftazidime, amoxicillin and vancomycin.
Multivariate statistical analyses showed that there was no increase in antibiotic resistance
amongst the bacterial population in the sediment due to dosing of the microcosms with silver
nanoparticles. This study indicated that, under the tested conditions, nanosilver released into
the coastal marine environment did not increase antibiotic resistance among naturally
occurring bacteria in estuarine sediments.
Blaser et al. (2008) estimated the cumulative aquatic exposure and risk due to nanosilver
being released from plastics and textiles using the flow diagram presented in Figure 5.3. The
authors presented the analysis in four stages; (i) silver mass flow analysis and estimation of
emissions, (ii) assessment of the fate of silver in a river system and estimation of predicted
environmental concentrations (PECs), (iii) critical evaluation of available toxicity data for
environmentally relevant forms of silver and estimation of predicted no-effect concentrations
(PNECs), and (iv) risk characterization. The authors also estimated silver use in the year
2010, focusing on the Rhine river as a case study. The process simulated in the Rhine River
model is shown in Figure 5.4. In 2010, biocidal plastics and textiles were predicted by the
model to account for up to 15% of the total silver released into water in the European Union.
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The majority of silver released into wastewater was incorporated into sewage sludge and
were spread on agricultural fields. The amount of silver reaching natural waters depended on
the fraction of wastewater that was being effectively treated. The authors found that the
modeled PECs in the Rhine River were in satisfactory agreement with monitoring data from
other river systems. This agreement indicated that the silver mass fluxes entering the aquatic
system were reasonable estimates and that the emission scenarios provide a useful basis for
the exposure assessment of freshwater ecosystems.
Aquatic Environment
Effluent V* Freshwaters
— 71
TWT
("Bottom Ashesy^
V__ SlacL^x
(
Fly Ashes j-+
[\ "
Solid Waste
Landfills
Incinerator
Ash
Landfills
Residue
Landfills
Atmospheric Environment
Figure 5.3: Overview of silver flows triggered by biocidal plastics and textiles. Arrows
represent silver flows; dashed lines represent different environmental spheres. In the Figure,
TWT represents thermal waste treatment and STP represents sewage treatment plant.
Reprinted from Sci. Tot. Environ., Vol. 390, Blaser, S.A., Scheringer, M., MacLeod, M.,
Hungerbuhler, K., Estimation of cumulative aquatic exposure and risk due to silver:
contribution of nano-functionalized plastics and textiles, pp396-409, Copyright 2008 with
permission from Elsevier.
Final Report dated 07/15/2010 75
State of the Science - Everything Nanosilver and More
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mmm^^ Process includes dissolved & particle-bound fraction ot substance
nnjl> Process includes particle-bound fraction only
c^>- Process includes dissolved fraction only
W1
W1
W1
Water Now
mm
W2
•
*~ A li
Sedimentation
|
* 3 Diffusion
- " M
i"? D \/|
can Bed load shift V Burial Resuspension ~. V U Bed
ai£b^ g aw3
permanent
sediment
y permanent sediment
V (not modelled)
•
**-
W2
load shift Cgj
ate*
permanent
sediment
Figure 5.4: Process simulated in the model developed by Blaser et al. (2008). In the Figure,
Wl represents the moving water, W2 represents stagnant water and 'Sed' represents the top
layer of the sediment. Reprinted from Sci. Tot. Environ., Vol. 390, Blaser, S.A., Scheringer,
M., MacLeod, M., Hungerbuhler, K., Estimation of cumulative aquatic exposure and risk due
to silver: contribution of nano-functionalized plastics and textiles, pp396-409, Copyright
2008 with permission from Elsevier.
Nanoparticles released from various nanotechnology-enhanced consumer products will
inevitably enter sewers and wastewater treatment plants (WWTPs). A project funded by the
Water Environment Research Foundation (WERF) evaluated how silver nanoparticles would
affect wastewater treatment systems and anaerobic digestion. Under this project, researchers
set up several lab-scale wastewater treatment modular units using activated sludge processes
designed to remove organic matter and nutrients in wastewater. The results demonstrated that
nitrifying bacteria were especially susceptible to inhibition by silver nanoparticles. At a
concentration of 0.4 mg/L total Ag, a mixture of positively charged silver ions and silver
nanoparticles (50:50 in mass ratio, average size = 15-21 nm) inhibited the growth of
nitrifying bacteria from the modified Ludzack-Ettinger bioreactor by 11.5 percent. In an
experiment on shock loading of 100% silver nanoparticles (lasting for 12 hours), a peak
concentration of 0.75 mg/L total Ag in the activated sludge basin (more than 95% associated
with biomass) was detected, and about 50% nitrifying bacterial growth inhibition (or
nitrification inhibition) accompanied with a slight accumulation of nitrite concentration in
wastewater effluent was observed. Studies of anaerobic digestion, a commonly used solid
stabilization process in wastewater treatment plants, indicated that silver nanoparticles at
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concentrations of 19 mg/L (19,000 ppb) or above in biomass might inhibit anaerobic
microbial activities. Most of the silver particles were in the activated sludge. After
considering concentration factor and safety factor, the researchers suggested a threshold
concentration of 0.1 mg/L total silver including nanosilver in wastewater influent. A report
on the WERF study is expected to be published on April 1, 2011 (Hu et al., 2011). This study
suggests that accumulation of silver in activated sludge could have a detrimental effect on
wastewater treatment, if the concentration reaches threshold levels.
5.3 Routes of Exposure, Human
Humans are exposed to nanosilver primarily through the ingestion of drinking water and
food, and through dermal contact with various consumer products and/or medical
applications that contain nanosilver. Nanosilver is incorporated in everyday products such as
water filters and washing machines; the presence of nanosilver in these products easily leads
to leaching, which discharges into the aqueous environment. Once the nanomaterials reach
the environment, there are a myriad of ways through which these materials are transported to
media such as other water bodies, plants and sediments, which then get recycled back to
humans. Table 5.1 lists the main characteristics for human exposure to nanomaterials from
food, consumer and medical products. Figure 5.5 illustrates potential routes of exposure,
uptake, distribution, and degradation of nanomaterials in the environment.
Table 5.1: Main characteristics for human exposure to nanomaterials from food, consumer
and medical products. Reprinted from Nanotoxicology, Vol. 3 (2), Wijnhoven, S.W.P.,
Peijnenburg, W.J.G.M., Herberts, C.A., Hagens, W.I., Oomen, A.G., Heugens, E.H.W.,
Roszek, B., Bisschops, I, Gosens, I., van de Meent, D., Dekkers, S., de Jong, W.H., van
Zijverden, M., Sips, A.J.A.M., Geertsma, R.E., Nanosilver- a review of available data and
knowledge gaps in human and environmental risk assessment, pp!09-138, Copyright 2009
with permission from Informa Healthcare.
Characteristic
Type of nanomaterials
Exposure route
Physical form of product
Application of the
consumer product
Type, use of the
consumer product
Concentration of
nanomaterial in product
Comments
Free nanoparticles or integrated nano structures into larger materials
Inhalation, dermal or oral exposure.
Intravascular, intrathecal, intravesical, urethral, ophthalmic, intramedullary,
intraperitoneal exposure
Spray, powder, liquid, emulsion or solid (coating)
Applications with direct human exposure (e.g., sunscreen products, medical
applications) or indirect human exposure (e.g., food storage bags, computers).
Applications with direct emissions to an environmental compartment (e.g., tooth
paste) or without direct emissions to the environment (e.g., computers).
Widely used or rarely used product. Frequency and amount of product used
Unknown
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-
Figure 5.5: Routes of exposure, uptake, distribution, and degradation of nanomaterials in the
environment. Solid lines indicate routes that have been demonstrated in the laboratory or
field or that are currently in use (remediation). Magenta lettering indicates possible
degradation routes, and blue lettering indicates possible sinks and sources of nanomaterials.
Reprinted from Environ Health Perspect, Vol. 113 (7), Oberdorster, G., Oberdorster, E.,
Oberdorster, I, Nanotoxicology: an emerging discipline evolving from studies of ultrafine
particles, pp823-839, Copyright 2005 with permission from National Institute of Health.
The growing application of nanosilver in food products, medical applications, cleaning
sprays and other consumer products with increasing use and disposal to the environment
indicates that human exposure to nanosilver is expected to increase in the future. The
exposure to these nanoparticles depends on the way they were incorporated into the product
(free nanoparticles or nanomaterials integrated into larger scale structures) in combination
with the application of the product (with either direct or indirect human exposure). For
example, products containing free nanoparticles with direct human exposure (e.g., food
supplements or sunscreen products) are considered to have a high potential exposure, while
products in which nanomaterials are integrated into larger scale materials with indirect
human exposure (e.g., food storage bags or computers) are considered to have a low potential
exposure. The route of exposure seems to be important as well. Inhalation exposure via
sprays or oral exposure of food supplements are considered to have the highest risk. For
medical applications, especially for coated catheters and orthopedic implants, more specific
exposure routes are possible, depending on the location of application. Most of the time, this
is local exposure; however, intravascular catheterization can lead to intravenous and, thus,
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systemic exposure. Table 5.2 ranks the potential human exposure to nanosilver for common
products/applications that contain the nanomaterial. The fact that a product category is
ranked with either a high or low potential exposure in the previously-mentioned table should
not be seen as evidence for absolute high exposures or the lack thereof, but as an indication
of potentially high exposures (Dekkers et al., 2007b). A point of interest for the future could
be cumulative exposure; currently no information on this is available. To determine the risk
for exposure, more information is needed on the concentrations of nanosilver in the product,
the size and the form in which it is present (aggregates, agglomerates) as well as the
probability of release of nanosilver from the products.
Table 5.2: Ranking of potential human exposures to nanosilver. Reprinted from
Nanotoxicology, Vol. 3 (2), Wijnhoven, S.W.P., Peijnenburg, W.J.G.M., Herberts, C.A.,
Hagens, W.I., Oomen, A.G., Heugens, E.H.W., Roszek, B., Bisschops, J., Gosens, I., van de
Meent, D., Dekkers, S., de Jong, W.H., van Zijverden, M., Sips, A.J.A.M., Geertsma, R.E.,
Nanosilver - a review of available data and knowledge gaps in human and environmental
risk assessment, pp!09-138, Copyright 2009 with permission from Informa Healthcare.
Category
Food and
beverages
Personal care and
cosmetics
Textile and shoes
Electronics
Household
products/home
improvement
Filtration,
purification,
neutralization,
sanitization
Subcategory
Cleaning
Cooking utensils, coatings
Storage
Supplements
Skin care
Oral hygiene
Cleaning
Hair care
Baby care
Over the counter products
Clothing
Other textiles
Toys
Personal care
Household appliances
Computer hardware
Mobile devices
Cleaning
Coating
Furnishing
Furnishing/coating
Filtration
Cleaning
Exposure route
Inhalation/dermal
Dermal
Dermal
Oral
Dermal
Oral
Dermal
Dermal
Dermal
Dermal?
Dermal
Dermal
Dermal/oral
Dermal
Dermal
Dermal
Dermal
Inhalation/dermal
Dermal
Dermal
Dermal
Inhalation
Inhalation/dermal
Potential exposure*
High
Low
Low
High
High
High
High
Low? High? High?
?
?
?
Low
Low
Low
Low
High High?? Low
Low
?
High
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Table 5.2: continued
Category
Medical products
Subcategory
Breathing mask
Endotracheal tube
Gastrointestinal tube
Catheters
Contact lens
Incontinence material
Orthopedic implants
Orthopedic stockings
Pharmaceuticals
Sling for reconstructive
pelvic surgery
Surgical mask/textile
Wound dressings
Exposure route
Inhalation
Inhalation
Oral
Intravascular/intrathecal/intravesic
al/urethral Ophthalmic
Dermal
Intramedullary
Dermal
Oral/dermal
Intraperitoneal
Inhalation/dermal
Dermal
Potential exposure*
High
?
?
?
?
?
?
?
?
?
?
High
* 'High" indicates either a high probability of exposure or a possibility of high exposure or both. 'Low"
indicates a low probability of exposure, or a possibility of low exposure, or both. '?' indicates that there is no
sufficient information available.
5.3.1 Exposure via food
Nanotechnologies are being used throughout all phases of food production including
cultivation (for example, through the application of pesticides or providing nutrients to
plants), processing and packaging (Bouwmeester et a/., 2007). Nanotechnologies are also
being used to enhance the nutritional aspects of food by means of nanoscale additives and
nutrients and nanosized delivery of drugs. The way nanotechnology is used within the food
production leads to a first estimate of potential consumer exposure and, thus, can be used as a
ranking of risks. Nanotechnology used for food production without introducing/adding
nanoscale products or compounds in the food can be considered as low risk for the consumer
(for example, using storage containers that contain minute amounts of nanosilver; Figure
5.6). Direct consumer exposure may be expected when nanoparticles are included into food
directly or when the nanomaterials act directly on the food (for example, using water filters
or using a nanosilver coated teapot to brew tea; Figure 5.7). Table 5.3 lists a summary of
applications of nanosilver in the food production chain.
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Figure 5.6: Fresh Box, manufactured by FinePolymer, Inc. (South Korea), is ananosilver
antimicrobial food container. The manufacturer claims that the container "shows excellent
antimicrobial properties against various bacteria and fungus due to the effect of finely
dispersed nanosilver particles and hence it make a food fresh longer compared with
conventional food containers." (http://goodgary.en.ec21.com/Nanosilver Food Container—
1600253 1600254.html)
Figure 5.7: The Nano Tea Pot -
Aroma manufactured by Top Nano
Technology Co., Ltd (Taiwan). The
manufacturer claims that the teapot is
manufactured using patented precious
metal techniques and that it releases
tea flavor in 30 seconds.
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Table 5.3: Summary of applications of nanotechnology in the food production chain.
Reprinted from RIKILT/RIVM Report 2007.014, Bouwmeester, H., Dekkers, S., Noordam,
M., Hagens, W., Bulder, A., De Heer, C., Ten Voorde, S., Wijnhoven, S., Sips, A., Health
impact of nanotechnologies in food production, Copyright 2007 with permission from
RIKILT - Institute of Food Safety.
Chain phase
Agricultural
production
Production and
processing of
food
Conservation
'Functional food'
consumption
Applications
Nanosensors
Pesticides
Water purification
Soil cleaning
Food production
Refrigerators, storage
containers, food
production equipment
Food products
Packaging materials
Supplements
Nanotechnology
Nanospray on food
commodities
Hand-held devices
Incorporated in packaging
materials
Nanoemulsions,
encapsulates
Triggered release
nanoencapsulates
Filters with nanopores
Nanoparticles
Nanoceramic devices
Incorporated nanosized
particles, mostly silver,
occasionally zinc oxide
Nanosized silver sprays
Incorporated sensors
Incorporated nanoparticles
Incorporated active
nanoparticles
Colloidal metal
nanoparticles
Delivery system
'nanoclusters'
Nanosized clustered food
drinks (nutrients)
Function
Binds and colors
microorganisms
Detection of contaminants,
etc.
Detection of food
deterioration
Increased efficacy, water
solubility and crop adherence
Triggered (local) release
Pathogen contaminant
removal
Removal or catalyzation of
oxidation of contaminants
Large reactive surface area
Anti-bacterial coating of
storage and food handling
devices
Anti-bacterial action
Detection of food
deterioration, monitoring
storage conditions
Increasing barrier properties,
strength of materials
Oxygen scavenging,
prevention of growth of
pathogens
Claimed to enhance desirable
uptake
Protecting and (targeted)
delivery of content
Claimed enhanced uptake
Food/nutritional supplements that contain nanosilver are known to have statements such as
'Purifying and conservation of unknown targets', 'Supporting the immune system' and
'Helpful against severe illness' (Wijnhoven et a/., 2009). Since these statements have not
been evaluated by, for instance, the European Medicines Evaluation Agency (EMEA), the
European Food Safety Authority (EFSA) or the US Food and Drug Administration (FDA),
the products are not medical products and are not intended to diagnose, treat, cure or prevent
any disease. Appendix C lists some of the food-related products that contain nanosilver.
It is difficult to estimate the exposure of humans to products that contain coatings of
nanosilver to prevent bacterial growth. The expected human exposure remains low as long as
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the inert nanosilver particles are bound in the packaging materials or in the coatings on
surfaces of packaging materials and food preparation devices. When nanosilver particles are
bound to other materials, exposure to nanosilver is only expected to occur when there is a
risk of wear-off or migration of nanosilver particles in the free or aggregated form into the
food (SCENIHR, 2006). Since potential benefits of using nanosilver in consumer products
are due to the release of silver in some form (mostly as silver ions), exposure to nanoparticles
in consumer products cannot be prevented. The potential benefit of using nanosilver in paint
is to prevent the formation of mold on walls, whose spores presumably get destroyed when
they come into contact with the nanosilver in paint. Humans may thus get exposed to
nanosilver when they come into contact with walls painted with nanosilver-containing paint.
It is anticipated that humans will be exposed to the nanosilver throughout the use of the
products that contain the nanomaterial.
5.3.2 Exposure via consumer products
For nanotechnology consumer products, there are several existing inventories that contain a
wide variety of globally available products. The most extended publicly available inventory
is the database of the Nanotechnology project of the Woodrow Wilson International Centre
for Scholars (www.nanotechproject.org). Since this project on Emerging Nanotechnologies
launched the first online inventory of manufacturer identified nanotech goods in March 2006,
the number of items has increased 175%, from 212 to 580 products in December 2007. By
August 2009, the total number of products was further increased to 803, a rise of 279% when
compared to the first inventory in 2006. This clearly indicates how fast the market for nano
containing products is growing. However, the inventory database has also seen some
decreases because of proposed intentions of regulatory agencies in the US, Europe and
elsewhere to regulate the industries using nanomaterials in their products. Wijnhoven et al.
(2009) mentioned that companies based in the US produce most of the nano-containing
consumer products (317), followed by companies in Asia (127), Europe (92), and elsewhere
around the world (32). For nano-products produced outside Europe, including those produced
in the US, consumers in Europe are only allowed to obtain them through European
distributors or ordering the products directly from manufacturers or sellers online.
Several nanomaterials such as metal oxides (e.g., titanium dioxide, zinc oxide, silica), metals
(e.g., silver, gold, nickel) and organic nanomaterials (e.g., nanovitamins, nanoclays, carbon
nanotubes) (Dekkers et a/., 2007a, b) are used in consumer products. Nanosilver appears to
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be incorporated into the highest number of different products, with manufacturers claiming
nanosilver incorporation in 233 consumer products and in 33 food products as of 2008
(Wijnhoven et al., 2009). This was 30% of the Woodrow Wilson inventory, far more than
other materials such as carbon, gold or silica. As of August 2009 (the last time the inventory
was updated), 259 consumer products contained nanosilver out of 1015 products listed in the
database (25%). The application of nanosilver in these consumer products is mainly based on
the antibacterial property of silver. Apart from the Food and Beverages category, the product
categories in which nanosilver is represented include: electronics, filtration, purification,
neutralization, sanitization, personal care and cosmetics, household products/home
improvement, textiles and shoes. Table 5.4 lists a summary of products that contain
nanosilver. As can be concluded from this table, most nanosilver containing consumer
products are in the product categories textiles and shoes (34), personal care and cosmetics
(30) and electronics (29) with clothing, skin care and personal care as subcategories with the
largest number of products. Also the categories household products/home improvement (19)
and filtration, purification, neutralization, and sanitization (13) contain a substantial amount
of products with nanosilver. Some products are difficult to classify and can be categorized in
more than one group, therefore it is possible that discrepancies exist between this and former
inventories.
One of the earliest consumer products to include nanosilver was Samsung's Silver Wash
washing machine (Figure 5.8). Samsung claimed that the washing machine achieves 99.9%
sterilization, and kills 650 different types of bacteria. Samsung also states that the washing
machine coats silver nanoparticles onto the fabrics, which maintain antibacterial activity for
up to a month.
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Figure 5.8: Samsung's Silver Wash washing machine
(http://ww2.samsung.co.za/silvernano/silvernano/washingmachine.html).
Samsung also claims that the machine releases up to 400 billion silver ions per wash cycle.
Geranio et al. (2009) investigated the amount and the form of silver released during washing
from nine fabrics with different ways of nanosilver incorporated into or onto the fibers. The
effect of pH, surfactants, and oxidizing agents was also evaluated. Results from their study
indicated that little dissolution of silver nanoparticles occurred under conditions relevant to
washing (pH 10) with dissolved concentrations 10 times lower than at pH 7. Bleaching
agents such as hydrogen peroxide or peracetic acid could greatly accelerate the dissolution of
silver. The amount and form of silver released from the fabrics as ionic and particulate silver
depended on the type of nanosilver incorporated into the textile. The percentage of the total
silver emitted during one washing of the textiles varied considerably among products (from
less than 1 to 45%). In the washing machine, the majority of the silver (at least 50% but
mostly >75%) was released in the size fraction >450 nm, indicating the dominant role of
mechanical stress. A conventional silver textile did not show any significant difference in the
size distribution of the released silver compared to many of the textiles containing nanosilver.
A recent study (Benn & Westerhoff, 2008) revealed that the silver can easily leak into
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wastewater during washing, thus, potentially disrupting helpful bacteria used in wastewater
treatment facilities, or endangering aquatic organisms in lakes and streams. Benn and
colleagues found that some brands of socks lose nearly 100% of their silver content within
four washings, while two other brands lost less than 1% over the same number of washings
(Benn & Westerhoff, 2008).
Nanosilver is used in washing machines because of its antimicrobial activity (Chen &
Schluesener, 2008). Several Swedish Agencies, including the Swedish Environmental
Protection Agency, have protested against this application because wastewater may be
contaminated with nanosilver. The USEPA has recently decided to regulate this specific form
of nanotechnology (Federal Register Notice 73 Fed. Reg. 69,644, November 19, 2008).
In the US, silver-ion generating devices such as washing machines, with the declared aim to
kill bacteria, will no longer be considered a simple washing device, but a pesticide. This
notice is not an action to regulate nanotechnology; it is the silver's bactericidal effect rather
than the size that led to the decision. In view of potential effects in aquatic ecosystems, new
purification methods need to be developed to eliminate possible negative effect of nanosilver.
Table 5.4: Product categories with examples of products containing nanosilver. Values in
brackets indicate the number of subcategories. Reprinted from Nanotoxicology, Vol. 3 (2),
Wijnhoven, S.W.P., Peijnenburg, W.J.G.M., Herberts, C.A., Hagens, W.I., Oomen, A.G.,
Heugens, E.H.W., Roszek, B., Bisschops, J., Gosens, I., van de Meent, D., Dekkers, S., de
Jong, W.H., van Zijverden, M., Sips, A.J.A.M., Geertsma, R.E., Nanosilver - a review of
available data and knowledge gaps in human and environmental risk assessment, pp 109-13 8,
Copyright 2009 with permission from Informa Healthcare.
Categories
Personal care and
cosmetics (30)
Textile and shoes (34)
Electronics (29)
Subcategories
Skin care (14)
Oral hygiene (6)
Hair care (3)
Cleaning (2)
Coating (2)
Baby care (2)
Over the counter health
products (1)
Clothing (28)
Other textiles (2)
Toys (4)
Personal care (13)
Household appliances (8)
Computer hardware (6)
Mobile devices (2)
Examples
(Body) cream, hand sanitizer, hair care products,
beauty soap, face masks
Tooth brush, teeth cleaner, toothpaste
Hair brush, hair masks
Elimination wipes and spray
Make-up instrument, watch chain
Pacifier, teeth developer
Foam condom
Fabrics and fibers, socks, shirts, caps, jackets,
gloves, underwear
Sheets, towels, shoe care, sleeves and braces
Plush toys
Hair dryers, wavers, irons, shavers
Refrigerators, washing machines
Notebooks, (laser) mouse, keyboards
Mobile phones
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Table 5.4: continued
Categories
Household products/home
improvement (19)
Filtration, purification,
neutralization, sanitation (14)
Subcategories
Cleaning (9)
Coating (4)
Furnishing (3)
Furnishing/coating/flooring (3)
Filtration (8)
Cleaning (6)
Examples
Cleaning products for bathrooms,
kitchens, toilets, detergents, fabric
softener
Sprays, paint supplements
Pillows
Showerheads, locks, water taps, floors,
tiles
Air filters, ionic sticks
Disinfectant and aerosol sprays
Park et al. (2009) monitored and analyzed the exposure characteristics of silver nanoparticles
during a liquid-phase process (Figure 5.9) in a commercial production facility in Korea. The
facility produces approximately 3000 kg of silver nanoparticles month. Based on the
measured exposure data, the source of silver nanoparticles emitted during the production
processes was indentified and a mechanism for the growth of silver nanoparticle released was
proposed. The authors concluded that silver nanoparticles were released from the reactor
during the liquid-phase production process and agglomerates of silver nanoparticles were
formed in the atmosphere of the workplace. The increase in particle number concentration
during the liquid-phase process was higher than that during processes that involved the
handling of a dry powder. The data reported in this study could potentially be used in
conjunction with other similar studies to establish occupational safety guidelines in the
nanotechnology workplace, especially in a liquid-phase production facility.
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(a)
Synthesis of AgNO3
I
Reduction of AgNO3
I
(R) Aging for 24h
I
Fill ration for 2 h
I
Drying for 24h
I
Grinding
1
Packaging (Dp<200 nm)
(b)
. .Vent hood
Reactor
Tern. 24.3'C
RH 55%
11
(C)
Tern. 25.0DC
RH58%
Conditioner
v
»
Vent haod
[G)| Grinder
Tern. 25.2'C
RH55%
Figure 5.9: Illustration showing production flow process and measurement locations in the
Korean silver nanoparticle manufacturing facility, (a) Production flow process of the silver
nanoparticle. The points marked R, D and G were monitored, (b) Reaction room, (c) Drying
room. Reprinted from J. Nanoparticle Res., Vol. 11 (7), Park, J., Kwak, B.K., Bae, E., Lee,
I, Kim, Y., Choi, K, Yi, J. Characterization of exposure to silver nanoparticles in a
manufacturing facility, pp!705-1712, Copyright 2009 with permission from Springer.
5.3.3 Exposure via Medical Applications
Silver has been known for decades for its antimicrobial properties in curative and preventive
medicine. The most widespread uses of silver are silver salts, silver complexes, and metallic
silver in pharmaceutical and homeopathic products (e.g., ointments, suspensions). Silver
antibiotic salts such as silver sulfadiazide are used as prophylaxis of infections in patients
with burns (Church etal., 2006). Medical devices such as catheters, orthopedic implants,
heart valves and wound care products are prone to bacterial adhesion, colonization, biofilm
formation and adhesion of glycoproteins from tissue and blood plasma. Silver coatings, using
silver salts or ion beam implantation of metallic silver, have been devised to address these
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problems. Some coatings have shown disappointing clinical results as effective strategies to
prevent medical device-related infections. For instance, in 2000, a voluntary recall of a
silver-coated sewing cuff fabric for heart valve replacement was initiated due to elevated
rates of paravalvular leakage (Schaff et al., 2002). Advanced silver nanotechnologies have
substituted the use of bulk silver in curative and preventive medicine in an attempt to
engineer out the risks caused by medical device-related infections.
Several other medical devices and products for wound care management that incorporate
nanosilver are on the market. Table 5.5 provides a summary of consumer products with
medical applications. In general, nanosilver is deposited, impregnated or coated onto medical
devices or fabrics rendering them suitable for controlling infections. Advanced
nanotechnologies, such as physical vapor deposition, chemical vapor deposition, or ink-jet
technology, are used to create thin layers of nanosilver on a broad range of substrates, e.g.,
metals, ceramics, polymers, glass, and textiles. Silver nanotechnologies that have been
launched for antimicrobial coatings are Bactiguard® (Bactiguard AB, Sweden), HyProtect™
(Bio-Gate AG, Germany), Nucryst's nanocrystalline platform technology (Nucryst
Pharmaceuticals Corp., USA), Spi-Argent™ (Spire Corp. USA), Surfacine® (Surfacine
Development Company LLC, USA), and SylvaGard® (AcryMed Inc., USA). Nanosilver is
extensively used for wound management, particularly in medical devices for the treatment of
burns (Tredget et al, 1998; Figure 5.10), chronic wounds (Yin et al., 1999; Sibbald et al.,
2001), burns in children (Dunn & Edwards-Jones, 2004), burn injuries in neonates (Rustogi
et al., 2005), rheumatoid arthritis-associated leg ulcers (Coelho et al. 2004), diabetic ulcers
(Thomas, 2007), venous ulcers (Sibbald et al., 2007), toxic epidermal necrolysis (Asz et al.,
2006), for healing of donor sites (Innes etal., 2001), and for meshed skin grafts (Demling &
Leslie DeSanti, 2002).
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Table 5.5: Medical devices containing nanosilver. Values between brackets indicate the
number of devices. Adapted from Nanotoxicology, Vol. 3 (2), Wijnhoven, S.W.P.,
Peijnenburg, W.J.G.M., Herberts, C.A., Hagens, W.I., Oomen, A.G., Heugens, E.H.W.,
Roszek, B., Bisschops, I, Gosens, I., van de Meent, D., Dekkers, S., de Jong, W.H., van
Zijverden, M., Sips, A.J.A.M., Geertsma, R.E., Nanosilver- a review of available data and
knowledge gaps in human and environmental risk assessment, pp!09-138, Copyright 2009
with permission from Informa Healthcare.
Medical Domains
Anesthesiology
Cardiology
Nephrology
Urology
Wound care
Examples
Catheter for administration of local anesthetic (1)
Battery used in implantable
cardioverter-defibrillator (1)
Hemodialysis catheter (2)
Urinary catheter (2)
Battery used in implantable electrical pulse generator (1)
Burn and wound dressing, professional use (15)
Burn and wound dressing, over the
counter (2)
Bum glove (1)
Bum sock (1)
Tubular stretch knit (1)
(Adhesive) strip, professional use (2)
(Adhesive) strip, over the counter (2)
Gel(l)
Compress (2)
IV/catheter dressings (2)
The application of nanosilver in medical products is emerging in the field of medical devices
and pharmaceutical research and development (Table 5.6). Other potential applications of
nanosilver coated/deposited/impregnated medical devices are infusion ports, orthopedic
protruding fixation devices, endovascular stents, urological stents, endoscopes, electrodes,
peritoneal dialysis devices, subcutaneous cuffs, surgical and dental instruments. Silver
nanoparticles can be deposited on various natural and synthetic textile and fabrics which can
be useful in hospitals to control infection (Lee et a/., 2003). The incorporation of nanosilver
into medical products has been of great interest in recent years. Properties of nano-structured
silver can be controlled and tailored in a predictable manner and imparted with biological
properties and functionalities that bring new and unique capabilities to a variety of medical
applications ranging from implant technology and drug delivery, to diagnostics and imaging.
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Figure 5.10: Anti-microbial burn dressing manufactured by Anson Nano-Biotechnology
(Zhuhai) Co., Ltd., China. The manufacturers claim that the core material is nanosilver
antibacterial granule. The manufacturer does not provide information on the size or amount
of silver nanoparticles in their products
(http://www.ansonano.com/productinfo.asp?newsid=156349068&lan=zh-
en&skin=5&open=0y
Table 5.6: Emerging applications of nanosilver in medical products. Reprinted from
Nanotoxicology, Vol. 3 (2), Wijnhoven, S.W.P., Peijnenburg, W.J.G.M., Herberts, C.A.,
Hagens, W.I., Oomen, A.G., Heugens, E.H.W., Roszek, B., Bisschops, I, Gosens, I., van de
Meent, D., Dekkers, S., de Jong, W.H., van Zijverden, M., Sips, A.J.A.M., Geertsma, R.E.,
Nanosilver - a review of available data and knowledge gaps in human and environmental
risk assessment, pp!09-138, Copyright 2009 with permission from Informa Healthcare.
Medical Domains
Anesthesiology
Cardiology
Dentistry
Diagnostics
Drug delivery
Eye care
Imaging
Neurosurgery
Examples
Coating of breathing mask
Coating of endotracheal tube for mechanical ventilatory support
Coating of driveline for ventricular assist devices
Coating of central venous catheter for monitoring
Additive in polymerizable dental materials
Silver-loaded SiO2 nanocomposite resin filter
Nanosilver pyramids for enhanced biodetection
Ultrasensitive and ultrafast platform for clinical assays for
diagnosis of myocardial infarction
Fluorescence-based RNA sensing
Magnetic core/shell FesCVAu/Ag nanoparticles with tunable
plasmonic properties
Remote laser light- induced opening of microcapsules
Coating of contact lens
Silver/dendrimer nanocomposite for cell labeling
Fluorescent core-shell Ag@SiC>2 nanoballs for cellular imaging
Molecular imaging of cancer cells
Coating of catheter for cerebrospinal fluid drainage
References
Patent
-
Patent
Jiaetal., 2008
Walt, 2005
Asian & Geddes,
2006
Asian et al., 2006
Xuefa/,,2007
Skirtachefa/., 2006
Weisbarthef al.,
2007
Lesniakef a/., 2005
Asian et al., 2007
Taiefa/,,2007
Baystonefa/,,2007
Galianoefa/,,2007
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Table 5.6: continued
Medical Domains
Orthopedics
Patient care
Pharmaceutics
Surgery
Urology
Wound care
Examples
Additive in bone cement
Implantable material using clay-layers with starch-stabilized silver
nanoparticles
Coating of intramedullary nail for long bone fractures
Coating of implant for joint replacement
Orthopedic stockings
Superabsorbent hydrogel for incontinence material
Treatment of dermatitis
Inhibition of HIV- 1 replication
Treatment of ulcerative colitis
Treatment of acne
Coating of hospital textile (surgical gowns, face mask)
Coating of surgical mesh for pelvic reconstruction
Hydrogel for wound dressing
References
Alt et al, 2004
Podsiadlo et al.,
(2005)
Alt et al, 2006
Chen et al., 2006
PohleefaZ.,2007
Lee et al., 2007
Bhol ef a/., 2004
Bhol & Schechter,
2007
Elechiguerra etal.,
2005
Sun et al, 2005
Bhol & Schechter,
2007
Patent
Li etal. ,2006
Cohen et al, 2007
Yuefa/,,2007
5.3.4 Exposure via occupation
Small quantities of silver and/or nanosilver are absorbed by humans through diet or
inhalation at occupational sites. Occupations that have a potential for exposure to nanosilver
include metallurgists, solderers, electroplaters, jewelers, cosmeticians, and individuals
working in a silver nanoparticle manufacturing facility or electronics manufacturing facility.
The most common clinical presentations due to occupational exposure are argyria and
argyrosis (Pala et al., 2008). Other clinical presentations involve exposure to soluble silver
compounds, which may cause liver and kidney damage, irritation of the eyes, skin,
respiratory and intestinal tract, and hematological changes (Drake & Hazel wood, 2005;
Lansdown, 2007).
Pala et al. (2008) presented a case study of a 71-year old craftsman, working from the age of
17, producing silver-containing items such as vases, plates, trays and frames by using cutting
tools, welding and hammering silver sheets. The craftsman's work bench was approximately
30-40 cm from his face, and he was exposed to silver at least 8 hours per day. Pala et al.
(2008) conducted an ocular examination of the craftsman, and diagnosed bilateral
conjuctival-corneal argyrosis (Figure 5.11) without systemic intoxication.
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Figure 5.11: Conjuctival-corneal argyrosis in the craftsman occupationally exposed to silver.
Reprinted from J. Occup. Health, Vol. 50, Pala, G., Fronterre, A., Scafa, F., Scelsi, M.,
Ceccuzzi, R., Gentile, E., Candura, S.M., Case Study: Ocular Argyrosis in a Silver
Craftsman, pp521-524, Copyright 2008 with permission from Japan Society for Occupational
Health.
Moss et al. (1979) studied ocular manifestations and functional effects of occupational
argyrosis. Thirty employees of an industrial plant involved in the manufacture of silver
nitrate and silver oxide underwent ophthalmologic evaluation in an effort to evaluate the
frequency and extent of ocular argyrosis. The most frequently noted ocular abnormality was
pigmentation of the conjunctiva, present in 20 workers; corneal pigmentation occurred in 15
workers. A direct relationship existed between the levels of pigmentation and duration of
employment. Ocular pigmentation was seen more frequently than cutaneous pigmentation.
Ten workers noted decreased night vision, but electrophysiologic and psychophysiologic
studies of seven of these ten workers demonstrated no functional deficits.
Tsai et al. (2009) studied airborne exposures associated with manual handling of
nanoparticles in three fume hoods operating under a range of operational conditions. The
handling tasks the authors studied included transferring particles from beaker to beaker by
spatula and pouring. Measurements studied included the room background, researcher's
breathing zone, and upstream and downstream from the handling location. The test results by
the authors found that the handling of dry powders consisting of nano-sized particles inside
laboratory fume hoods can result in a significant release of airborne nanoparticles from the
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fume hood into the laboratory environment and the researcher's breathing zone. Many
variables were found to affect the extent of particle release including hood design, hood
operation (sash height, face velocity), work practices, type and quantity of the material being
handled, room conditions, and the adequacy of the room exhaust.
5.4 Projected Quantities, Geographic and Demographic Distribution
in the US
Specific data on silver emissions to the environment and the distribution of the emitted
nanosilver across the US are not available. As previously mentioned in Section 5.1, the
survey was designed to collect information covering various aspects of the projected
quantities, geographic and demographic distribution. With the lack of response, the only
source from which information can be extracted is the Project of Emerging Nanotechnologies
(PEN). On their website (www.nanotechproject.org/121X an interactive map for the US
highlights the distribution of more than 1200 nanotechnology companies, universities,
research laboratories and other organizations working with nanotechnology could be found
(Figure 5.12). The map was launched in 2007 and updated in 2009 and it includes 955
companies, 182 university and government laboratories and 81 other types of organizations.
This map is not limited to nanosilver distribution but it is generic for all produced
nanomaterials. The main findings were: a) all the states in the US contain at least one
organization deal with nano materials, b) the top 4 states contain organizations working with
nano materials are California, Massachusetts, New York and Texas, c) the top 6 cities
(including more than 30 entries) are: Boston, MA; San Francisco, CA; San Jose, CA;
Raleigh, NC; Middlesex-Essex, MA, Oakland, CA, d) the top 3 sectors working in
nanotechnology are, materials, tools and instruments, and medicine and health and e)
California is the lead state, with more than double the entries in any other state. This
information is valuable for predicting source points for nanomaterials activities. But the final
destinations of nanomaterials as well as produced quantities still need to be addressed. This
information is greatly lacking and is as seen as a knowledge gap.
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Total Companies, Universities, Government Labs, and Organizations
Worldng on Nanotechr>ology
lei KKff All KiqhH Prserwd Wcodro w WUm Imr-wtlnral Cc nKT fnr Schnlan
Figure 5.12: Number of companies, universities, laboratories and/or organization working in
nanotechnology across the US. (http://www.nanotechproject.org/inventories/map/)
All available data related to quantities of released nanosilver are modeled data. Mueller &
Nowack (2008) modeled the release of three types of nanoparticles including (nanosilver)
into air, water and soil in Switzerland. The estimated worldwide production volume,
allocation of the production volume to product categories, particle release from products, and
flow coefficients within the environmental compartments were used as the model's input
parameters. The authors presented a schematic (Figure 5.13) for the flows of the nanosilver
to the environmental compartments (air, water and soil), Sewage treatment plant (STP),
Waste incineration plants (WIP) and the landfills for the high emission scenario. In the case
of nanosilver, the most prominent flows are between the products and the STP (3.27 t/year),
the STP and the WIP (2.65 t/year) and the WIP to landfills (3.26 t/year). The predicted
concentrations of nanosilver in all different environmental compartments were extremely low
(1.7*10"3 g m"3 in air, 0.03 g L"1 in water and 0.02 g kg"1 in soil). In another study,
Blaser et al. 2008, estimated the cumulative nanosilver release based on the estimated silver
use in plastics and textiles in the year 2010. It was estimated the nanosilver-containing
plastics and textiles account for up to 15% of the total nanosilver released into water in the
European Union. The Rhine River was used as a case study and the predicted concentrations
are presented in Table 5.7. In this study, Blaser et al. (2008) estimated that 9-20 tons of
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silver from biocidal uses would be discharged to the European environment in wastewaters,
with the remainder going to sewage sludge.
Figure 5.13: Nanosilver flows during high emission scenarios. Reprinted from Environ. Sci.
Techno!., Vol. 42 (12), Mueller, N.C., Nowack, B., Exposure modeling of engineered
nanoparticles in the environment, pp4447-4453, Copyright 2008 with permission from
American Chemical Society.
Table 5.7: Predicted environmental concentrations in Rhine River. Reprinted from Sci. Tot.
Environ., Vol. 390, Blaser, S.A., Scheringer, M., MacLeod, M., Hungerbuhler, K.,
Estimation of cumulative aquatic exposure and risk due to silver: contribution of nano-
functionalized plastics and textiles, pp396-409, Copyright 2008 with permission from
Elsevier.
Compartment
Sewage treatment plants
River water
River sediment
Interstitial water in sediment
Unit
g/L
ng/L
mg/kg
ng/L
Minimum scenario
2
40
2
9
Intermediate scenario
9
140
6
30
Maximum scenario
18
320
14
70
Luoma (2008) provided various important tables on estimates of environmental exposure
information, masses of silver discharged to the aquatic environment from different sources in
1978, discharges of silver into South San Francisco Bay from one waste treatment facility
and from the combined POTW discharges from the surrounding urban area in the 1980s and
in 2007, typical silver concentrations in water bodies of the world and a comparison of
discharges from silver nanotechnologies for several different near-term scenarios for the
USA and for San Francisco Bay. Some of the information in these tables revealed that no
individual product will release silver at rates equal to those released by photographic
development in the 1980s. The authors however state that the amount of silver released from
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all products containing nanosilver in the monitored areas in 2007 might significantly exceed
the release of silver from just the photographic industry in the 1980s. The author gave an
example by calculating the maximum predicted release scenario of nanosilver from 3
consumer products to be high as 457 metric tons/year for the USA and 128 metric tons/year
after wastewater treatment compared to 124 ton/year in 1978 from the photographic industry.
Luoma (2008) predicted similar quantities of nanosilver to be released for 100 consumer
products such as silver socks if used by 10% of the population in the US.
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6. Toxicity and Health Effects
6.1 Silver Toxicity
Among all forms of silver that can potentially be found in the environment, the majority of
toxicological data in the literature is only available for its two most common forms -
elemental silver (Ag(0)) and monovalent silver ion (Ag+). Even though silver has been used
widely for its medicinal, antibacterial and antiviral properties for hundreds of years, there is
relatively limited information available on its toxicity in the literature. Existing
environmental and human studies suggest that some forms of silver, especially those that
dissociate and release free silver ions (Ag ), are more toxic than others. Many researchers
have theorized that the toxic effects (especially acute) of silver-containing materials are
directly proportional to the rate of release of monovalent silver ions. Drake & Hazelwood
(2005) showed that metallic silver appeared to pose minimal risk to health, whereas soluble
silver compounds were more readily absorbed and, hence, had the potential to produce
adverse effects. Various studies have shown that 1-5 g Ag+/L is enough to kill sensitive
aquatic and marine species (Bryan & Langston 1992; Wood et al. 1994). Some studies have
shown that accumulation of silver in species exposed to a slightly lower concentration of
silver may lead to adverse effects on growth (Eisler, 1997). Other investigations have shown
that the concentrations of Ag+ ions, especially in the environment, are too low to lead to
toxicity (WHO, 2002).
The wide variety of uses of silver in everyday life allows for exposure through various routes
of entry into the body, including inhalation, ingestion and dermal exposure. Ingestion is the
primary route for entry for silver compounds and colloidal silver proteins (Silver, 2003).
Wijnhoven et al. (2009) estimate a dietary intake of 70-90 g of silver/day. Inhalation,
ingestion and dermal contact of dusts or fumes containing silver occurs primarily in the
manufacturing sector such as chemical plants that produce silver-containing compounds and
its surrounding communities (ATSDR, 1990; Drake & Hazelwood, 2005). Dermal contact
also occurs in medical settings such as from the application of burns creams, use of dental
amalgams and acupuncture needles, catheters, accidental punctures, and from contact with
jewelry and silverware (Catsakis & Sulica, 1978; Drake & Hazelwood 2005; Wan etal,
1991). The most common health effects associated with chronic exposure to silver are a
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permanent grey or blue grey discoloration of the skin (argyria; Figure 6.1) and other organs
(ATSDR, 1990; Drake & Hazelwood 2005; White et al., 2003). Lower-level exposure to
silver also results in the metal being deposited in the skin and other parts of the body such as
liver, brain, muscles and kidneys, and may cause changes in blood cells (Fung & Bowen,
1996; Venugopal & Luckey, 1978). Exposure to high levels of silver in the air can result in
breathing problems, lung and throat irritation, and stomach pains. Skin contact with silver
can cause mild allergic reactions including rashes, swelling, and inflammation in some
people. Since bulk silver in any form is not thought to be toxic to the immune,
cardiovascular, nervous or reproductive systems, and it is not considered to be carcinogenic,
many researchers and regulatory agencies consider silver to be relatively non-toxic except for
argyria and other symptoms mentioned previously (ATSDR, 1990; Chen & Schluesener
2008; Furst & Schlauder, 1978).
Figure 6.1: Systemic argyria of the skin from ingestion of colloidal silver (bottom hand)
when compared to normal pigmentation (top hand) (Wadhera & Fung, 2010).
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6.2 Nanosilver Toxicity
Even though silver is usually not available in concentrations high enough to pose a risk to
human health and the environment, nanosilver has physical and surface properties which
could pose a threat to human and environmental health (Lee et al., 2007). Nanosilver has
characteristics such as its size, surface area, solubility, ability to aggregate, chemical
composition and surface chemistry that are different from the characteristics of bulk silver.
Because of the different physicochemical properties and biological activities of nanosilver
when compared with the regular metal, it cannot be excluded that the increased reactivity of
nanosilver (because of the large surface area) leads to increased toxicity due to the activity of
free silver ions released by the nanoparticles. Nanomaterials including nanosilver, primarily
because of their extremely small size, which is comparable to the size of viruses, may have
the ability to enter, translocate within, and damage living organisms. Some nanoparticles
could penetrate the lung or skin and enter the circulatory and lymphatic systems of humans
and animals, reaching body tissues and organs, and potentially disrupting cellular processes
and causing disease.
The health effects of the nanoparticles used in consumer products are not yet known, though
various studies have revealed adverse health effects of materials previously considered safe.
Silver, however, has been shown to be toxic to humans or animal cells when in nanoparticle
form, with reported observations of a cytotoxic response nearly identical to that for chrysotile
asbestos (Soto et al., 2005). Inhalation of silver nanoparticles leads to their migration to the
olfactory bulb, where they locate in mitochondria, translocation to the circulatory system,
liver, kidneys, and heart (Oberdoster et al, 2005a, 2005b; Takenaka et al, 2001). Silver
nanoparticles have been found in the blood of patients with blood diseases and in the colon of
patients with colon cancer (Gatti, 2004; Gatti et al., 2004). There are contradictory studies on
silver nanoparticles and ion cytotoxicity from laboratories around the world. Silver is known
to have a lethal effect on bacteria, but the same property that makes it antibacterial may
render it toxic to human cells. Concentrations of silver that are lethal for bacteria are also
lethal for both keratinocytes and fibroblasts (Poon & Burd, 2004). In vitro studies have
demonstrated that nanosilver has effects on reproduction, development, and has an effect on
DNA among others.
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Recent research with zebra fish showed that highly purified, single silver 12 nm
nanoparticles affected early development offish embryos (Lee etal. 2007). Silver
nanoparticles have the potential to cause chromosomal aberrations and DNA damage and are
capable of inducing proliferation arrest in zebrafish cell lines (Asharani et al. 2007). More
nanosilver in vitro and in vivo toxicity studies have been performed in mammalian species
have shown that silver nanoparticles have the capability to enter cells and cause cellular
damage (Hussain et al., 2005; Ji et al., 2007).
Recent epidemiological studies have shown a strong correlation between particulate air
pollution levels, respiratory and cardiovascular diseases, various cancers, and mortality
(Brook et al., 2004). Adverse effects of nanoparticles on human health depend on individual
factors such as genetics and existing disease, as well as exposure, and nanoparticle chemistry,
size, shape, agglomeration state, and electromagnetic properties. Animal and human studies
show that inhaled nanoparticles are less efficiently removed than larger particles by the
macrophage clearance mechanisms in the lungs, leading to lung damage (Asgharian & Price,
2007; Card et al. 2008; Oberdorster et al, 2007). Nanoparticles can also translocate through
the circulatory, lymphatic, and nervous systems to many tissues and organs, including the
brain (Takenaka et al., 2001). The key to understanding the toxicity of nanoparticles is that
their minute size, smaller than cells and cellular organelles, allows them to penetrate these
basic biological structures, disrupting their normal function. Examples of toxic effects
include tissue inflammation, and altered cellular redox balance toward oxidation, causing
abnormal function or cell death (Samberg, etal., 2010; Udeaetal., 2002).
The same properties that make nanomaterials appealing also cause problems with studying
the toxicity due to exposure to such particles. Researchers have shown that while exposure to
nanosilver is toxic under certain experimental conditions, other researchers have shown that
nanosilver is non-toxic under similar experimental conditions. From a review of the
toxicological studies being conducted in the literature, it is becoming increasingly apparent
that the main difference in the outcome of the toxicity studies is due to variations in
physicochemical features of the nanosilver being used in various studies. The
physicochemical features of the nanoparticles must be characterized under the experimental
setting so that definitive associations between these parameters and any biological responses
observed may be identified. There are difficulties in monitoring nanomaterial behavior when
dispersed in physiological solutions as the latter often contain particulate and charged
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materials that will mask the true size distribution and charge measurements of the
nanomaterials themselves. Agglomeration can also be temperature dependent and so
measurements should be made at a constant temperature, which requires temperature-
controlled equipment. Many of the techniques currently available to assess surface area,
morphology and composition are reliant on dry samples and are difficult to apply to
nanomaterials in solution.
Due to the lack of reliable nanosilver toxicity data in the literature, it is impossible to assess
the environmental risks associated with the production and use of nanosilver. An important
research question is the validation of the hypothesis that toxic effects of nanoparticles are
proportional to the activity of the free silver ions released by the nanoparticles. Apart from
nanosilver toxicity assessment in the aqueous environments, more research is needed to
investigate the effects of nanosilver in terrestrial environments as no toxicity data for
nanosilver in soils were found in the literature. Additional research is also needed on
ecologically relevant species to investigate whether silver nanoparticles present a threat to
environmental health in general. It should be determined whether nanosilver in products is
actually capable of reaching the aqueous and terrestrial environment. Specifically, the
strength of the bonds between nanosilver and the product it is incorporated into should be
investigated. Additional questions to consider include release patterns and release kinetics of
nanosilver from specific applications and whether the physicochemical properties change
under certain circumstances leading to more/less release of nanosilver into the aqueous
environment.
6.2.1 Toxicity of Nanosilver to Organisms
There is a significant body of literature discussing the toxicity of silver nanoparticles to
various bacterial species; however, the diverse methods of synthesis, capping agents and
dispersants used may make direct and meaningful comparisons difficult. Results using
different bacterial strains, even if they are of the same species, may not be comparable.
Wang et al. (2010) studied the impact of silver on the metabolism of anaerobic cultures of
Shewanella oneidensis. The authors found that S. oneidensis MR-1 reduced toxic silver ions
in solution to elemental nanosilver particles, which was later confirmed using X-ray
diffraction analyses. Low silver ion concentrations (1 to 50 uM) had a limited impact on
growth, while higher ion concentrations (100 uM) reduced both the doubling time and cell
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yields. At the higher concentration, the authors determined that the silver nanoparticles were
accumulated within the cell, while at lower concentrations, the nanoparticles were
exclusively reduced and precipitated outside the cell wall. Whole organism metabolite
fingerprinting, using the method of Fourier transform infrared spectroscopy analysis of cells
grown in a range of silver concentrations, confirmed that there were significant physiological
changes at higher silver concentrations. Molecular analyses confirmed a dramatic drop in
cellular yields of both the phospholipid fatty acids and their precursor molecules at high
concentrations of silver, suggesting that the structural integrity of the cellular membrane was
compromised at high silver concentrations. The authors concluded that this was a result of
intracellular accumulation of the silver nanoparticles.
Elechiguerra et al. (2005) studied the interaction of silver nanoparticles with HIV-1 virus.
The authors demonstrated that the silver nanoparticles undergo a size-dependent interaction
with HIV-1 (Figure 6.2), with nanoparticles exclusively in the range of 1-10 nm attached to
the virus. Figure 6.2 shows high angle annular dark field (HAADF) scanning transmission
electron microscope image of a HIV-1 virus in the presence and absence of silver
nanoparticles. The regular spatial arrangement of the attached nanoparticles, the center-to-
center distance between nanoparticles, and the fact that the exposed sulfur-bearing residues
of the glycoprotein knobs would be attractive sites for nanoparticle interaction suggested that
silver nanoparticles interact with the HIV-1 virus via preferential binding to the gp!20
glycoprotein knobs. Due to this interaction, silver nanoparticles inhibited the virus from
binding to host cells.
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Figure 6.2: HAADF image of an HIV-1 virus that is (a) exposed to silver nanoparticles, (b)
without silver nanoparticle treatment. Reprinted from J. Nanobiotechnology, Vol. 3,
Elechiguerra, J.L., Burt, J.L., Morones, J.R., Camacho-Bragado, A., Gao, X., Lara, H.H.,
Yacaman, M.J., Interaction of silver nanoparticles with HIV-1, pp6, Copyright 2005 with
permission from BioMed Central.
Nanosilver has been recently recognized as a more potent antimicrobial form of silver (Alt et
al, 2004; Aymonier et al, 2002; Baker et al, 2005; Melaiye et al, 2005; Sondi et al, 2004;
Wright et al, 2002). As an example, Wright et al. (2002) demonstrated that wound dressing
coated with sputtered nanosilver reduced infections in burns. The antibacterial action of Ag+1
is thought to have several mechanisms. Recent observations have suggested the primary
mechanism of action is cell death due to the uncoupling of oxidative phosphorylation (Holt &
Bard, 2005), which confirms work from other investigators; however, others have reported
interaction with membrane-bound enzyme and protein thiol groups that may results in
compromised cell wall integrity that would lead to deterioration of proton gradient-driven
oxidative phosphorylation (Bragg & Rainnie, 1974; Liau et al., 1997; Silver, 2003; Zeiri et
al, 2004).
Lok et al. (2006) have recently examined the effect of nanosilver on E. coli using proteomics
and measurement of membrane properties. They have extended the previous observations
that silver mechanism of action is disruption of proton motive force and decoupling of
oxidative phosphorylation resulting in loss of intracellular ATP. They reported the effective
concentration of nanosilver was considerably lower than that for Ag+ ions.
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6.2.2 Ecological or Multispecies Studies of Nanosilver Toxicity
The same unique physical and chemical properties of nanomaterials that make them of
interest in industrial applications have also increased concern that nanomaterials may have
unique biological properties resulting in potential toxicity in the event of unintended use or
release into the environment (Lovern & Klaper, 2006; Moore, 2006). Following
environmental release, engineered nanomaterials are likely to deposit in aquatic systems and
represent a possible danger to aquatic life (Moore, 2006). Soluble forms of many of these
metals may be toxic to aquatic organisms, implying that the potential exists for
nanoparticulate formulations of these metals to induce toxicological effects in aquatic
species. For example, the toxicity of silver to aquatic organisms has been shown to be
primarily because of exposure to silver ions (Navarro et al, 2008). Although regulations such
as the Clean Water Act exist for protecting aquatic life from dissolved forms of these metals,
it is unclear if they are appropriate for use with nanomaterials as the mechanisms of toxicity,
if any, may be different from those due to exposure to bulk metals.
Griffitt et al. (2008) reported the effects of particle composition on the toxicity of metallic
nanomaterials to aquatic organisms. They used zebrafish, daphnids, and an algal species of
various trophic levels and feeding strategies as a model. To understand whether observed
effects were caused by dissolution, the particles used in their toxicity experiments were
characterized before testing, and particle concentration and dissolution were determined
during exposures. Organisms were exposed to silver, copper, aluminum, nickel, and cobalt as
both nanoparticles and soluble salts as well as to titanium dioxide nanoparticles. Results from
the toxicity experiments indicated that nanosilver and nanocopper caused toxicity in all
organisms tested, with 48-h median lethal concentrations as low as 40 and 60 g/L,
respectively, in Daphnia pulex adults, whereas titanium dioxide did not cause toxicity in any
of the tests. The authors reported that susceptibility to nanomaterial toxicity differed among
species, with filter-feeding invertebrates being markedly more susceptible to nanometal
exposure compared with larger organisms (i.e., zebrafish). The observed toxicity was found
to vary with the role of dissolution, with dissolution playing a minor role for studies
involving silver and copper, however, it played a major role in studies involving nickel. The
authors also observed that nanomaterial forms of metals were less toxic than soluble forms
based on mass added.
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Griffitt et al. (2009) also examined the interplay of nanoparticle composition and dissolution
on response of the zebrafish gill following exposure to toxic (nanocopper or nanosilver) or
nontoxic (nano-TiC^) nanometals. Female zebrafish were exposed to the 48-h no observable
effects concentration (NOEC) concentrations of nanocopper and nanosilver or to soluble Cu
and Ag that matched the concentration of dissolved metals released during nanoparticle
exposure. The authors observed that both nanocopper and nanosilver exposures increased
metal content associated with gill tissue. Silver concentrations were much higher following
nanosilver exposures suggesting that intact silver nanoparticles were associated with the gill.
Morphological and transcriptional responses of the gills differed among various
nanomaterials and between nanoparticulate and soluble species. Nanocopper increased mean
gill filament width three to fourfold between 24 and 48 hours, whereas nanosilver did not
alter gill filament width at either time point. Soluble silver and copper exposure both
increased gill filament widths by approximately twofold over control values. Gill filament
widths were higher in soluble silver exposures than in nanosilver exposures, despite both
tanks containing highly similar concentrations of soluble silver. Global gene expression
analysis performed by the authors demonstrated that the exposure to each nanometal or
soluble metal produced a distinct gene expression profile at both 24 and 48 h, suggesting that
each exposure was producing biological response by a different mechanism. The differences
in responses among the exposures indicated that each particle was having a distinct
biological effect that did not appear to be driven solely by release of soluble metal ions into
the water column.
The authors note that exposure to silver nanoparticles produced significantly higher levels of
silver associated with the gills than did exposure to only the soluble fraction. This suggested
that the nanoparticles themselves were contributing to the gill burden of silver through a
mechanism that did not involve the formation of silver ions. The authors theorize several
mechanisms by which nanoparticulates may increase the gill silver levels. Nanoparticles may
be trapped in the mucus layer of the gill as demonstrated for larger particles (Sanderson et
al., 1996; Tao et al., 1999). The authors reason that nanoparticles trapped in this manner may
not actually enter the cells, but mucus entrained particles can also increase intracellular metal
content by enhanced dissolution due to changes in water chemistry in the gill
microenvironment including mucus complexation (Tao et al., 2002). It is possible that
nanoparticles are actually taken up by gill epithelial cells. Martens & Servizi (1993)
demonstrated that sediment particles less than 500 nm were present intracellularly in
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salmonid gill epithelial cells. Though the results from their study demonstrated that not all
nanoparticles interacted with the gill in the same manner, the authors cautioned that further
work would be required to ascertain the mechanism by which silver and copper nanoparticles
interacted with the gill. Griffitt et al. (2009) noticed that exposure to silver nanoparticles also
significantly increased whole body silver content; however, it was not clear whether this is
due to translocation of silver from the gills to the rest of the body, as had been shown
previously in rainbow trout (Morgan et al., 2004), or due to ingestion of particulates and
gastrointestinal (GI) absorption.
Harris & Bali (2008) investigated the limits of uptake of metallic silver by two common
metallophytes, Brassica juncea wAMedicago saliva and assessed the form and distribution
of the metal once sequestered by the plants. B. juncea accumulated up to 12.4 wt.% silver
when exposed to an aqueous substrate containing 1,000 ppm AgNOs for 72 hours; silver
uptake was largely independent of exposure time and substrate silver concentration. M. sativa
accumulated up to 13.6 wt. % silver when exposed to an aqueous substrate containing 10,000
ppm AgNOs for 24 hours. In contrast to B. juncea, there was a general trend for M sativa to
show an increase in metal uptake with a corresponding increase in the substrate metal
concentration and exposure time. In both cases the silver was stored as discrete nanoparticles,
with a mean size of approximately 50 nm. Haverkamp et al. (2007) demonstrated a way for
plants to synthesize mixed metal nanoparticles by adding solutions containing the appropriate
metal ions. In their study, the authors demonstrated a way for B. juncea to synthesize
nanoparticles containing gold, silver and copper as an alloy. This work could potentially lead
to a biosynthetic process to force plants to produce nanoparticles of metal alloys for a range
of nanotechnology applications. Assuming that other commonly consumed plants have a
similar capacity for nanosilver uptake, animals and humans consuming such plants will be
susceptible to the toxicity caused by the intake of nanosilver.
Gao et al. (2009) conducted experiments that likely mimicked the introduction of
manufactured nanomaterials into aquatic systems to assess the effect of nanoparticle
dispersion/solubility and water chemical composition on nanomaterial toxicity. Aqueous
suspensions of fullerenes, nanosilver, and nanocopper were prepared in both deionized water
and filtered (0.45 um) natural river water samples collected from the Suwannee River basin,
to emphasize differences in dissolved organic carbon (DOC) concentrations and solution
ionic strengths. Two toxicity tests, the Ceriodaphnia dubia and MetPLATE bioassays were
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used in the experiments. Results obtained from exposure studies showed that water chemistry
affected the suspension/solubility of nanomaterials as well as the particle size distribution,
resulting in a wide range of biological responses depending on the type of toxicity test used.
MetPLATE results for nanosilver showed decreasing trends in toxicity with increasing DOC
concentrations and ionic strength. The biological responses in C. dubia was contrasting in
that increasing DOC concentrations reduced toxicity, while the latter increased with
increasing ionic strength. The results showed that laboratory experiments that use Dl-water
and drastic nanomaterial suspension methods may not be realistic as nanomaterial dispersion
and suspension in natural waters vary significantly with water chemistry and the reactivity of
the nanomaterials.
In a 96-hour acute exposure study with Daphnia magna, Gaiser et al. (2009) determined that
nanosilver caused more mortality than bulk silver. In Cyprius carpio, the authors mentioned
that silver was detected in the liver, intestine, gills and gall bladder after treatment with both
sizes of nanoparticles. However, the authors noted a trend towards higher uptake of the
nanosilver than the micro-sized particles.
Lee et al. (2007) synthesized silver nanoparticles using sodium citrate and sodium
borohydride as reducing agents, and evaluated their effects on zebrafish embryos. The
authors concluded that Brownian motion and transport into and out of embryos through
chorion pore canals occurred; however, restricted diffusion out of the embryos due to the
viscosity inside the embryo resulted in the accumulation of nanoparticles. The authors
determined the locations of individual nanoparticles (5-46 nm) within embryos using dark-
field single nanoparticle optical microscopy and spectroscopy (SNOMS), determining that
the embryonic response was dose-dependent. The authors showed that deformities increased
with nanoparticle concentration up to 0.19 nM, then decreased with increasing concentration
(up to the maximum concentration tested, 0.71 nM) based on an increase in dead zebrafish.
Specific deformities were correlated with the concentration of nanoparticles. All tested
concentrations of nanoparticles resulted in fmfold abnormality and tail/spinal cord defects,
while head edema occurred with 0.44-0.71 nM and eye deformity only occurred with 0.66-
0.71 nM nanoparticles. They concluded that the great sensitivity of zebrafish early embryos
to silver nanoparticles indicates that this species may be useful for in vivo toxicity assays to
screen other nanomaterials.
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Asharani et al. (2008) also investigated the toxicity of silver nanoparticle using zebrafish as a
model. Silver nanoparticles were synthesized using starch and bovine serum albumin (BSA)
as capping agents to study their deleterious effects and distribution pattern in zebrafish
embryos. TEM/EDX of the embryos showed that nanoparticles accumulated in the brain,
heart, yolk, and blood of embryos. They also conclude that silver nanoparticles effect normal
embryo development and have a dose-dependent toxicity in embryos.
Very little is known on the specific effects of nanosilver in the environment, especially its
fate and transport in the environment and its effect on biota and the ecosystem. It is currently
impossible to reliably assess the environmental risks associated with the production and use
of nanosilver, and its release into the environment.
6.2.3 Studies Concerning Human Health Including Mammalian Models
No specific mammalian models for nanosilver were available in the literature, although
several were identified for nanoparticles in general. The toxicity of nanoparticles to humans
and mammals depends on various factors such as the size, their composition, ease of
aggregation, physical surface characteristics, chemical surface characteristics such as
crystallinity, presence of functional groups, etc. The toxicity of the nanoparticle is also
heavily dependent on the mammal's genetic complement, its susceptibility and its ability to
adapt to changes in the environment, and to fight toxic substances. Diseases associated with
inhaled nanoparticles might include asthma, bronchitis, emphysema, lung cancer, and
neurodegenerative diseases. Nanoparticles in the gastrointestinal tract have been linked to
Crohn's disease and colon cancer. Nanoparticles that enter the circulatory system are related
to occurrence of arteriosclerosis, blood clots, arrhythmia, heart diseases, and ultimately
cardiac death. Translocation to other organs such as liver, spleen, etc., may lead to diseases
of these organs as well. Exposure to some nanoparticles is associated with the occurrence of
autoimmune diseases such as systemic lupus erythematosus, scleroderma, and rheumatoid
arthritis (Buzea et al., 2007). Figure 6.3 provides a schematic of a human body with
pathways of exposure to nanoparticles, affected organs, and associated diseases from
epidemiological, in vivo and in vitro studies.
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NANOPARTICLES INTERNALIZED
IN CELLS
Mithocondriwt •
Nucleus .
Cytoplasm
Membrane
Lipidvesicle w/mw,,Y,.v/.v.
system Kaposi X sarcoma>
I (Auto-immune diseases.
"Skin dermatitis)
Figure 6.3: A schematic of the human body with pathways of exposure to nanoparticles,
affected organs, and associated diseases from epidemiological, in vivo and in vitro studies.
Reprinted from Biointerphases, Vol. 2 (4), Buzea, C., Pacheco, 1.1., Robbie, K.
Nanomaterials and nanoparticles: Sources and toxicity, ppMR17-MR71, Copyright 2007
with permission from American Institute of Physics.
The increased biological activity of nanoparticles can be either positive or desirable
(e.g., antioxidant activity, carrier capacity for therapeutic penetration of blood-brain barrier,
and the stomach wall or tumor pores), and dispersed throughout the whole body including
entering the central nervous system, or negative and undesirable (e.g., toxicity, induction of
oxidative stress, or cellular dysfunction) or a mix of both (El-Ansary & Al-Daihan, 2009;
Oberdorster et a/., 2005b). Nanoparticles have been found to be distributed to the colon,
lungs, bone marrow, liver, spleen, and the lymphatics after intravenous injection (Hagens et
a/., 2007). Distribution in the human body is generally followed by rapid clearance from the
systemic circulation, predominantly by action of the liver and splenic macrophages
(Moghimi et a/., 2005). Clearance and opsonization, the process that prepares foreign
materials to be more efficiently engulfed by macrophages, occur under certain conditions for
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nanoparticles depending on size and surface characteristics (Moghimi etal., 2005). When
inhaled, nanoparticles are found to be distributed to the lungs, liver, heart, spleen, and brain
(Hagens et al., 2007). Nanoparticles are cleared in the alveolar region via phagocytosis by
macrophages facilitated by chemotactic attraction of alveolar macrophages to the deposition
site (El-Ansary & Al-Daihan, 2009; Curtis etal, 2006; Garnett & Kallinteri, 2006). The
average halflife (t/2) for nanoparticles in the respiratory tract is -TOO days in humans (El-
Ansary & Al-Daihan, 2009; Oberdorster et al, 2005b). After intraperitoneal injection,
nanoparticles were found to cross the transplacental membrane or cross the peritoneal cavity
into uterus. This affected the embryos cranial development and even caused embryo death
(Vega-Villa et al, 2008). After oral exposure, nanoparticles were found in the kidneys, liver,
spleen, lungs, brain, and the gastrointestinal (GI) tract (Hagens, et al, 2007). Some
nanoparticles passed through the GI tract and were rapidly excreted in feces and urine,
indicating that they can be absorbed across the GI barrier and into the systemic circulation
(Hagens, et al, 2007). There is some evidence, but no proof, that silver nanoparticles are
adsorbed within the first few feet of the small intestine, and do not proceed far enough into
the gastrointestinal tract to cause problems. However, some nanoparticle systems can
accumulate in the liver during the first-pass metabolism (El-Ansary & Al-Daihan, 2009;
Oberdorster et al, 2005a).
In sharp contrast to the emphasis on the application of silver nanoparticles, information on
the toxicological implication of the use of silver nanoparticles is limited (Chen &
Schluesener, 2008; Wijnhoven et al, 2009). Between the different toxicological studies that
are reported in the literature so far, the compositions of the silver nanoparticles vary widely.
Also the descriptions of used silver formulations diverge from detailed to very limited, with
variable attention paid to the size, solubility and aggregation of the nanoparticles. This
information may be highly relevant, since a good dispersion of the silver nanoparticles is
required for effective toxicological and/or antibacterial activities, and might influence its
subsequent toxicity (Lok et al, 2007; Wijnhoven et al, 2009). Toxicity determination of
nanosilver particles may be dependent on the size distribution of the particles (Ji et al, 2007;
Wijnhoven et al, 2009). Although the oxidation state of the silver nanoparticles may
influence their biological and/or toxicological activity, little attention has been paid to the
oxidation state of the silver nanoparticles in the literature. Other factors such as ionic
strength, pH and the presence/absence of other salts may also play a role in the oxidation
state of the silver nanoparticles. Only oxidized silver nanoparticles exert an antibacterial
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effect, most likely due to the combination of nanocarrier material (i.e., silver nanoparticle)
and the Ag+ ions which are tightly adsorbed/chemisorbed on the particle surface (Lok et al,
2007; Wijnhoven et al, 2009). It should be noted that reduced silver nanoparticles appeared
very unstable and can easily be oxidized (Lok et al, 2007; Wijnhoven et al, 2009). It is
because of the antimicrobial properties of oxidized nanosilver that nanosilver is mostly used
and studied. According to various studies in the literature, these properties are supposed to be
dependent upon the biological activity of silver ions (Ag+; Lansdown, 2007; Wijnhoven et
al., 2009). It may be assumed that most of the silver nanoparticles used in the studies
discussed in this report are in the oxidized form. Colloidal silver represents another
formulation with silver particles. The size of the silver particles in colloidal suspension is
assumed to be mainly in the range of 250-400 nm. These particles are
aggregates/agglomerates of smaller sized nanoparticles (-100 nm), that under certain
conditions can disaggregate/disagglomerate. Reports on colloidal silver have also been
included in this report as well.
A case report was published regarding elevated liver enzymes following topical use of a
nanosilver preparation on a young burn victim (Trop et al., 2006). Six days after treatment
the patient developed grayish discoloration with bluish-lips (argyia) and elevated serum
aspartate aminotransferase, alanine aminotransferase, and y-galactosyl transferase without
elevation of bilirubin, lactate dehydrogenase, or cholinesterase. The patient had elevated
urinary (28 ug/kg) and serum (107 ug/kg) silver levels. Cessation of the nanoscale silver
treatment resulted in an immediate decrease of the clinical signs of hepatotoxicity, argyria,
and serum and urinary silver; however, serum and urinary levels of silver (42 and 2.3 g/kg,
respectively) were still elevated at 7 weeks. In preclinical studies with pigs, no elevated
plasma levels or adverse reactions were reported with the same nanosilver preparation
(Burrell, 1997). While clinical studies contrasted the efficacy of the nanosilver versus other
silver forms, there was no measurement of serum levels or reports of adverse reactions
(Tredget et al, 1998; Yin et al, 1999; Innes et al, 2001).
The plasma levels of silver in the patient (Trop et al. 2006) were higher than the modest
levels reported by Boosalis et al. (1987) and comparable to the rapid increase reported by
Coombs et al. (1992) following topical application of silver sulfadiazine; however, in the
silver sulfadiazine studies there were no reports of hepatotoxicity, although others have
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reported allergic reactions (McKenna et al, 1995), erythema multiform (Lockhart et al,
1983), mental deterioration (Iwasaki et al., 1997), and transient leucopenia (Caffee &
Bingham, 1982). It is not clear at this time if the report of hepatotoxicity by Trop et al.
(2006) with nanosilver is an isolated incidence or the beginning of a trend with nanoscale
versus other forms of silver. In the only report on carcinogenicity of silver (Furst &
Schlauder, 1978) a single intramuscular injection of 300 mesh (40-50 micrometer or smaller
particles) silver did not result in induction of any cancer in lifetime study with Fischer-344
rats.
6.2.3.1 Respiratory Tract Toxicity
Human exposure to inhaled ambient particles, including nanosilver, may have adverse health
effects (Buzea et al, 2007; Dockery, 2005; Donaldson et al, 2004; Lippmann et al, 2003;
Shah, 2007; Vermylen et al, 2005). Pulmonary and cardiovascular diseases may result when
inhaled particles interfere with the normal function of bodily systems (Peters et al, 1991,
2001 and 2005). The health consequences of particle inhalation vary greatly with particle
composition and concentration, among other factors. After inhalation, nanoparticles deposit
throughout the entire respiratory tract, starting from nose and pharynx, down to the lungs
(Buzea et al, 2007; Oberdoster, 2001; Elder et al, 2006). Lungs consist of airways, which
transport air in and out, and alveoli, which are gas exchange surfaces. Human lungs have an
internal surface area between 75 and 140 m2, and about 300 xlO6 alveoli (Hoet et al, 2004).
Due to their large surface area, the lung is the primary entry portal for inhaled particles.
Spherically shaped solid material with particle diameters smaller than 10 m can reach the
gas exchange surfaces (Buzea et al, 2007; Hoet et al, 2004; Oberdoster, 2001). Larger
diameter particles tend to be deposited further up in the respiratory tract as a result of
gravitational settling, impaction, and interception (Lippmann, 1990). Many larger diameter
fibers are deposited at saddle points in the branching respiratory tree. Smaller-diameter
particles are more affected by diffusion, and these can collect in the smaller airways and
alveoli. Fibers having a small diameter may penetrate deep into the lung, though very long-
aspect ratio fibers will remain in the upper airways (Buzea et al, 2007; Hoet et al, 2004).
The nasopharyngeal region captures mainly microparticles and nanoparticles smaller than 10
nm, while the lungs will receive mainly nanoparticles with diameters between 10 and 20 nm
(Buzea et al, 2007; Oberdoster, 2001). Figure 6.4 shows the deposition of nanoparticles in
the respiratory tract as a function of their size. A small fraction of the applied dose of
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nanosized particles can pass from the epithelial surface of the air space into blood, but the
fraction increases if the barrier is disrupted, for example, by an inflammatory stimulus. The
amount that gets into blood has also been shown to be size dependent (Chen et al., 2006),
with smaller (-55 nm) particles having greater fractional penetration than larger particles
(-200 nm).
Nasal Airway
Pharynx
Lymph
nodes
Bronchi
Bronchioles
Alveolar
ducts
Alveol
^Vasculature
1.0-1
0.6-
o 0.4-
9 •
IT 0.2-
3?
0.0
Nasal, pharyngeal, laryngeal
0.0001 0.001 0.01 0.1 1 10 100
Diameter (nm)
1.0-
| 0.8-
I
8-0.6-
-D
I 0.4-
H1
I 0.2-
T racheobronchial
0.0
0.0001 0.001 0.01 0.1 1
Diameter (nm)
10 100
1.0-
I 0.8-
O
tO.6-
10.4-
'en
(r 0.2-
I
Alveolar
0.0
0.0001 0.001 0.01 0.1 1
Diameter (nm)
Figure 6.4: Deposition of particles in the respiratory tract as a function of their size, with
inset illustrating the proximity of the air spaces (alveoli) to the vasculature (in pink).
Reprinted from WIREs Nanomedicine and Nanobiotechnology, Vol. 1 (4), Elder, A.,
Vidyasagar, S., DeLouise, L., Physicochemical factors that affect metal and metal oxide
nanoparticle passage across epithelial barriers, Copyright 2009 with permission from Wiley.
As summarized elsewhere (Asgharian & Price, 2007; Card et al., 2008; Oberdorster et al.,
2007), inhaled particles of different sizes exhibit different fractional depositions within the
human respiratory tract. Although inhaled ultrafme particles (<100 nm) deposit in all regions,
tracheobronchial deposition is highest for particles <10 nm in size, whereas alveolar
deposition is highest for particles approximately 10-20 nm in size (Asgharian & Price, 2007;
Card et al., 2008; Oberdorster et al., 2007). Particles <20 nm in size also efficiently deposit
in the nasopharyngeal-laryngeal region. Human studies of potential adverse pulmonary
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effects resulting from exposure to engineered nanoparticles appear to be limited, although a
number of investigations into pulmonary deposition patterns of inhaled nanoparticles in the
healthy and diseased lung have been conducted (Anderson et al., 1990; Card et al., 2008;
Chalupa et al., 2004; Daigle et al., 2003; Moller et al., 2008). Computational models predict
increased deposition of inhaled nanoparticles in diseased or constricted airways (Card et al.,
2008; Farkas et al., 2006), and consistent with this prediction, obstructive lung disease and
asthma have both been demonstrated to increase their pulmonary retention (Anderson et al.,
1990; Card et al, 2008; Chalupa et al). Nonetheless, Pietropaoli et al (2004) did not
observe differences between healthy and asthmatic subjects in respiratory parameters
assessed up to 45 h after a 2-h inhalation of ultrafme carbon particles (up to 25 g/m2), nor
was airway inflammation observed in either group (measured as exhaled nitric oxide). The
same study reported that exposure of healthy subjects to a higher concentration of ultrafme
carbon particles (50 g/m2 for 2 h) resulted in decreased mid-expiratory flow rate and carbon
monoxide diffusing capacity 21 h after exposure, albeit still in the absence of airway
inflammation (Card et al, 2008; Pietropaoli et al, 2004). Figure 6.5 shows a simplified
depiction of potential factors that may influence the effects of nanoparticles in the respiratory
system.
Route of delivery
Inhalation
Intranasal
Qropharyngeal aspiration
Systemic (oral, dermal, intravenous, etc.)
Particle characteristics
Shape, number, composition
Surface area and modifications
Charge
Solubility
Others
Host factors
Genetics
Health status
Environmental exposures
Pulmonary Response
Figure 6.5: A simplified depiction of potential factors that may influence the effects of
engineered nanoparticles on the respiratory system. Reprinted from Am. J. Physiol. Lung
Cell Mol. Physiol., Vol. 295, Card, J.W.; Zeldin, D.C., Bonner, 1C., Nestmann, E.R.,
Pulmonary applications andtoxicity of engineered nanoparticles, ppL400-L411, Copyright
2008 with permission from American Physiological Society.
There is a need to conduct inhalational studies of exposure to silver nanoparticles to ensure
the health of workers and consumers. The dispersion of inhalable ambient nano-sized
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particles evenly has been an obstacle in evaluating the effect of the inhalation of nano-sized
particles on the respiratory system. Ji et al. (2007) exposed Sprague-Dawley rats to silver
nanoparticles following OECD test guideline 412 (OECD, 1993), based on 28 days of
repeated inhalation with Good Laboratory Practice (GLP) application. Their study used a
device that generated silver nanoparticles by evaporation/condensation using a small ceramic
heater to distribute the desired concentrations of silver nanoparticles to chambers containing
experimental animals. The concentrations and distribution of the silver nanoparticles with
respect to the size were also measured directly using a differential mobility analyzer and
ultrafme condensation particle counter. Eight week old rats were divided into 4 groups (10
rats in each group): a fresh air control, a low dose group (1.73 x 104/cm3), middle dose group
(1.27 x io5 /cm3), and a high dose group (1.32 x 106/cm3, which equates to approximately 61
g/m3). The animals were exposed to the silver nanoparticles for 6 hours/day, 5 days/week,
for a total of 4 weeks. At the conclusion of the exposure period, the clinical and
histopathological parameters were examined; later, the tissue distribution of silver
nanoparticles in the blood, lungs, brain, olfactory bulb and liver was also investigated. The
male and female rats did not show any significant changes in body weight relative to the
concentration of silver nanoparticles. No distinct clinical and histopathological effects on the
respiratory system of silver nanoparticles were seen during the 28 days inhalation study in
rats (Ji et al. 2007). The authors report that the content of silver in the liver of male rats
increased in a concentration-dependent manner following inhalation of silver nanoparticles
for 5 days/week for 4 weeks, and concluded that exposure to silver nanoparticles at a
concentration near the current American Conference of Governmental Industrial Hygienists
(ACGIH) silver dust limit (100 g/m3) did not appear to have any significant health effects.
The study might not have been complete as it lacked specific examinations of the respiratory
system such as respiratory rate, airway resistance, tidal volume, hemoglobin oxygen
saturation as well as inflammation status.
In a similar study conducted by Takenaka et al. (2001), nanosilver accumulation was seen in
the lungs of the rats (1.7 mg) of which 4% was still left after seven days, but again additional
toxicity parameters were not included. The tissue distribution of inhaled silver nanoparticles
seemed to indicate that silver or silver nanoparticles can be translocated to other organs such
as the liver, olfactory bulb and brain, as seen in the previous acute silver nanoparticle (15 nm
modal diameter) inhalation study (Takenaka et al., 2001). The lung silver concentration
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exhibited a dose-dependent increase following silver nanoparticle inhalation exposure. Three
possible translocation routes to the blood were suggested by the authors: (1) translocation of
silver nanoparticles from the tracheobronchial region by a mucociliary escalator, with
subsequent ingestion into the gastrointestinal tract, (2) translocation of the particles into the
lymph nodes, and (3) entry into the blood via alveolar epithelial cells. Significant increase of
silver in the liver indicated the blood circulation of silver deposited in the lungs. Sung et al.
(2008) performed a 90 days rat inhalational study (18 nm sized silver nanoparticles 6
hours/day at concentrations of 0.7, 1.4 and 2.9 *106 particles/cm3). The authors showed lung
function decrease (including tidal volume, minute volume and peak inspiration flow),
inflammatory lesions in the lung morphology and effects of inflammatory markers.
One acute colloidal silver aerosol inhalation study of rabbits (whole-body exposure;
concentration was not provided) reports ultrastructural damage and disruption of the tracheal
epithelium (Konradova, 1968). The authors did not provide toxicity data on subacute or
subchronic exposure to silver dust. Respiratory effects have been observed in humans
following the inhalation of silver compounds (Rosenman et al., 1979, 1987), yet the causal
relationship is difficult to establish due to a lack of information on the concentration,
diameter, and chemical composition of silver in workplace air along with occupational
history of the workers.
Potential factors in the increased inflammatory profile observed for nanoscale materials in
some studies include their size, increased number, and higher surface area per unit mass
compared with that of larger particles of the same material (Borm et al., 2006; Card et al.,
2008; Nel et al., 2006; Oberdorster et al., 2005a). The increased ratio of surface area to mass
for nanoparticles meant that a greater percentage of the atoms or molecules of a given
particle were present on the surface of the particle; thereby, providing an increased number
of potential reactive groups at the particle surface that may influence toxicity. Although this
appeared to be a useful metric for assessing the toxic potential of some nanoparticles, there is
consensus among experts in the field that no single dose metric (i.e., particle number, size,
surface area, or other) has emerged to be useful for assessment of the reactivity and potential
toxicity of nanoparticles in general (Maynard & Aitken, 2007; Warheit et al., 2007).
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6.2.3.2 Neuronal Uptake
Inhaled nanoparticles are known to reach the nervous system via the olfactory nerves (Borm
et al, 2006; Oberdorster et al, 2004, 2005 a,b) and/or blood-brain barrier (Borm et al, 2006;
Buzea et al., 2007; Peters et al., 2006). Nanoparticles that reach the lungs are predominantly
cleared via the mucociliary escalator into the gastrointestinal tract and then eliminated in the
feces (Semmler et al., 2004) lymphatic system (Lui et al, 2006), and circulatory systems
(Buzea et al., 2007; Oberdorster et al., 2005b). From the lymphatic and circulatory systems,
nanoparticles may be distributed to organs, including kidneys from where partial or total
clearance may occur.
As mentioned in Section 6.2.3.1, silver was found in the brain of rats systemically exposed to
silver nanoparticles via inhalation (Takenaka et al. 2001; Ji et al. 2007), but no toxicity
endpoints were monitored in the brain. Passage of the blood brain barrier (BBB) was also not
investigated. According to a recent review on neurotoxicity of silver (Lansdown, 2007), most
animal studies indicate that after silver exposure, silver was contained within the blood brain
barrier but did not pass it.
In the only known human exposure study available, epileptic seizures and coma following
daily ingestion of colloidal silver for 4 months was reported (Mirsattari etal. 2004), along
with high levels of silver in plasma, erythrocytes and cerebrospinal fluid. The authors suggest
that silver caused signs of irreversible neurological toxicity which eventually led to death
after the patient remained in a persistent vegetative state for close to 6 months.
6.2.3.3 Dermal Toxicity
Though nanosilver-based dressing and surgical sutures have received approval for clinical
application and good control of wound infection has been achieved, their dermal toxicity is
still a topic of concern. Despite laboratory and clinical studies confirming the dermal
biocompatibility of nanosilver-based dressings, several other researchers have demonstrated
the cytotoxicity of these materials (Chen et al., 2006; El-Ansary & El-Daihan, 2009;
Limbach et al., 2007; Muangman et al., 2006; Oberdorster et al, 2005b; Supp et al, 2005;
Wright et al, 2002). Paddle-Ledinek et al. (2006) exposed cultured keratinocytes to extracts
of several types of silver containing dressings. The results showed that extracts of
nanocrystalline coated dressings are among those that are the most cytotoxic. Keratinocyte
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proliferation was significantly inhibited, and cell morphology was affected (El-Ansary & El-
Daihan, 2009; Paddle-Ledienk et al, 2006).
Acticoat® is a topical wound dressing consisting of a polyethylene mesh coated with
nanosilver (average size 15 nm). There is one reported case of silver poisoning after the use
of Acticoat® for treatment of severe burns to the legs (Trop et al. 2006; Wijnhoven et al.,
2009). On day 6 post-injury, the patient developed a grayish discoloration in the treated area,
complained of being tired and had no appetite. On day 7, silver levels in urine and blood
were found to be elevated (28 and 107 mg/kg, respectively). Acticoat® was removed and the
discoloration of the face gradually faded and liver function test returned to normal values.
Elevated blood silver levels were seen 7 weeks post-injury, but were negligible after 10
months. These observed adverse effects may be associated with the release of Ag+ ions from
the nanosilver dressing. Absorption of silver from Acticoat® was confirmed in 30 patients
treated in another study (Vlachou et al. 2007; Wijnhoven et al., 2009). Despite measurable
amounts of serum silver levels (median 59 g/1), very limited changes in hematological or
biochemical indicators of toxicity associated with the silver absorption were observed.
In a moist environment, silver is released from the Acticoat® dressing (possibly as
nanocrystals) and improves microbial control of the wound. Acticoat® has been tested in
small clinical trials (Innes et al., 2001; Tredget et al, 1998; Wijnhoven et al., 2009) with
contradictory results. No adverse effects were found in the Tredget et al. study, but silver
absorption was not assessed (Tredget et al., 1998). Innes et al. (2001) reported delayed re-
epithelialization and temporary scars while in another study, an increase in re-epithelization
was found in meshed skin grafts (Demling & Leslie DeSanti, 2002). A case of delayed
wound healing was also reported by Trop et al. (2006). All the studies mentioned in this
paragraph were small scale and used different controls; thus, inter-study comparison was not
possible.
In a porcine model of wound healing, nanosilver wound dressing promoted rapid wound
healing of full-thickness wounds on the back of pigs (Wright et al., 2002; Wijnhoven et al.,
2009). The proteolytic environment of the wounds treated with nanosilver was characterized
by reduced levels of metalloproteinases and enhanced cellular apoptosis. Application of
Acticoat® on cultured skin substitutes grafted on nude mice did not inhibit nor promote
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wound healing (Supp etal., 2005). In the mice, application of a 1% nanosilver cream (96.1%
is <50 nm) induced apoptosis of inflammatory cells but not of keratinocytes (Supp et al.,
2005).
The potential cytotoxicity of silver nanoparticles in human epidermal keratinocytes and their
inflammatory and penetrating potential into porcine skin in vivo was assessed by Samberg et
al. (2010). The authors used eight different types of silver nanoparticles [unwashed/uncoated
(20, 50, and 80 nm particle diameter), washed/uncoated (20, 50, and 80 nm), and carbon-
coated (25 and 35 nm)]. Skin was dosed topically for 14 consecutive days. Human epidermal
keratinocytes (HEK) viability was assessed by MTT, alamarBlue (aB), and CellTiter 96
AQueous One (96AQ). Release of the proinflammatory mediators interleukin (IL)-1(3, IL-6,
IL-8, IL-10, and tumor necrosis factor-a (TNF-a) were measured. The effect of the unwashed
silver nanoparticles on HEK viability after a 24-hr exposure indicated a significant dose-
dependent decrease (p < 0.05) at 0.34 ug/ml with aB and 96AQ, and at 1.7 ug/ml with MTT.
Both the washed silver nanoparticles and carbon-coated silver nanoparticles showed no
significant decrease in viability at any concentration assessed by any of the three assays. For
each of the unwashed nanoparticles, a significant increase (p < 0.05) in IL-1(3, IL-6, IL-8, and
TNF-a concentrations were noted. Macroscopic observations showed no gross irritation in
porcine skin, whereas microscopic and ultrastructural observations showed areas of focal
inflammation and localization of nanosilver on the surface and in the upper stratum corneum
layers of the skin. Samberg et al. (2010) showed that nanosilver was nontoxic when dosed in
washed nanosilver particle solutions or carbon-coated nanosilver particles. The authors also
concluded that toxicity of nanosilver in HEKs was influenced by the residual contaminants in
the nanosilver solutions, and that the particles themselves may not have been responsible for
an increase in cell mortality.
6.2.3.4 Gastrointestinal Tract Toxicity
Jeong et al. (2010) investigated the effects of silver nanoparticles on the histological structure
and properties of the mucosubstances in the intestinal mucosa of Sprague-Dawley rats. For
the experiment, the rats were divided into four groups (10 rats in each group): control group,
low-dose group (30 mg/kg), middle-dose group (300 mg/kg), and high-dose group (1,000
mg/kg). Silver nanoparticles (60 nm) were administered for 28 days, following OECD test
guideline 407 and using GLP. The control contained no silver nanoparticles; however, the
treated samples showed luminal and surface particles, and the tissue contained silver
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nanoparticles. A dose-dependent increased accumulation of silver nanoparticles was
observed in the lamina propria in both the small and large intestine, in the tip of the upper
villi in the ileum and in the protruding surface of the fold in the colon. The silver
nanoparticle-treated rats exhibited higher numbers of mucus granules than the controls,
resulting in more mucus materials in the crypt lumen and ileal lumen. The authors concluded
that silver nanoparticles induced the discharge of mucus granules along with an abnormal
mucus composition in the goblet cells of the intestines.
In a previous inhalation study, Ji et al. (2007) found no significant toxicity in Sprague-
Dawley rats that had been repeatedly exposed to silver nanoparticles via inhalation, based on
6 h each day, 5 days a week, for a total of 4 weeks. Plus, no significant changes were found
in the male and female body weights or hematology and blood biochemical values relative to
various concentrations of silver nanoparticles during the 28-day experiment (Ji et a/., 2007).
In another oral toxicity study using male and female Sprague-Dawley rats, Kim et al. (2008)
found that oral exposure to silver nanoparticles (60 nm) at concentrations of 30, 300, and
1,000 mg/kg for 28 days had no affect on micronucleated polychromatic erythrocytes or bone
marrow cells. However, the repeated oral doses of silver nanoparticles did induce liver
toxicity and had a coagulation effect on peripheral blood. The histopathologically evaluated
liver toxicity included dilatation of the central vein, bile duct, and hyperplasia (Kim et al.
2008).
6.2.3.5 Other Organ Toxicity
Tang et al. (2009) investigated the distribution and accumulation of silver nanoparticles in
rats with subcutaneous injection. Rats were injected with either silver nanoparticles or silver
microparticles at 62.8 mg/kg, and then sacrificed at predetermined time points. The main
organs of the experimental animals, including the kidney, liver, pancreas, brain, lung and
heart, were harvested for ultrastructural analysis by transmission electron microscopy (TEM)
and for silver content analysis by inductively coupled plasma mass spectrometry (ICP-MS).
Results indicated that the silver nanoparticles were carried to the organs by blood circulation,
and was distributed throughout the main organs, especially in the kidney, liver, spleen, brain
and lung in the form of particles. Silver microparticles, however, could not invade the blood
stream or organ tissues. Ultrastructural observations indicate that those nanoparticles that had
accumulated in organs could enter different kinds of cells, such as renal tubular epithelial
cells and hepatic cells. The nanoparticles also induced blood-brain barrier (BBB) destruction
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and astrocyte swelling, and caused neuronal degeneration. The results suggest that the
cellular uptake of silver nanoparticles is dependent on its size.
Death has been observed in rats following exposure to very high doses of colloidal silver
after intravenous administration (LD50, 67 mg/kg) (Schmaehl & Steinhoff, 1960) and after
oral ingestion (1680 mg/kg/day for four days; ATSDR, 1990). Following the intravenous
injection of colloidal silver, rats died from lung edema; while liver, spleen and kidney
showed signs of brown discoloration (Schmaehl & Steinhoff 1960). The cause of death
following oral intake was not reported. Chronic subcutaneous administration of colloidal
silver (1.75-2.5 mg weekly) appeared relatively well tolerated, apart from the development of
argyria. Very limited health effects were observed in a 28-day inhalation toxicity study (Ji et
al., 2007) and a 28-day oral toxicity study (Kim et al., 2008) with silver nanoparticles in
Sprague-Dawley rats despite absorption into major organs. In the Ji et al. study rats inhaled
12-16 nm sized silver nanoparticles 6 h/day, 5 d/week, for four weeks. Three exposure levels
were used, a low exposure of 1.73 x 104 /cm3, a medium exposure of 1.27 x 105/cm3, and a
high exposure of 1.3 x io6 nanoparticles/cm3 (approximately 61 g/m3, which is near the
American Conference of Governmental Industrial Hygienists (ACGIH) silver dust threshold
limit of 0.1 mg/m3). A study on the 28-day oral toxicity of silver nanoparticles (60 nm) at
doses of 30 mg/kg, 300 mg/kg, and 1,000 mg/kg, following OECD test guideline 407 and
good laboratory practices (GLP), indicated that exposure to over 300 mg of silver
nanoparticles could result in slight liver damage, plus a gender-related difference was noted
in the accumulation of silver in the kidneys, with a twofold higher accumulation in the
female kidneys when compared with the male kidneys (Kim et al. 2008).
6.2.3.5.1 Kidney Toxicity
Previously, Kim et al. (2008) reported gender differences in the accumulation of silver
nanoparticles in kidneys of Sprague-Dawley rats after subacute exposure. It is of interest that
subacute inhalation of silver nanoparticles did not produce gender differences with respect to
accumulation in various tissues. In a study by Kim et al. (2009), the tissue distribution of
silver nanoparticles showed a dose-dependent accumulation of silver in all the tissues
examined, including testes, kidneys, liver, brain, lungs, and blood. A gender-related
difference in the accumulation of silver was noted in the kidneys, with a twofold higher
concentration in female kidneys compared males after subacute exposure to silver
nanoparticles via inhalation or oral ingestion. To investigate the gender specific accumulation
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of silver nanoparticles in kidneys of Fischer 344 rats, detailed histopathological studies were
conducted by silver enhancement staining. Female rats showed a higher accumulation of
silver nanoparticles in all kidney regions, including cortex, outer medulla, and inner medulla.
In particular, the glomerulus in the cortex contained a higher accumulation in females than
males. The silver nanoparticles were also preferentially accumulated in the basement
membranes of the renal tubules in the cortex, middle and terminal parts of the inner medulla,
and outer medulla. Silver nanoparticles were detected in the cytoplasm and nuclei of
interstitial cells in the inner medulla of the kidney.
6.2.3.5.2 Liver Toxicity
Significant amounts of silver in the liver were observed after inhalation (Takenaka et al.,
2001; Ji et al., 2007; Wijnhoven et al., 2009). At each time point analyzed, 9-21% of the
nanosilver lung content was observed in the liver (Takenaka et al., 2001). Histopathology of
the liver revealed cytoplasmic vacuolization in both sexes with a clear dose dependent
increase in females. Several cases of hepatic focal necrosis were seen in the high dose groups
(Ji et al. 2007). No effect on the liver enzyme alkaline phosphatase (ALP) was observed. In
contrast, repeated oral doses of 60 nm silver nanoparticles during 28 days did induce liver
toxicity, as shown by increases in ALP and histopathological observations of dilatation of the
central vein, bile-duct hyperplasia and increased foci (Kim et al., 2008). In the case report of
Trop et al. (2006), elevated liver enzymes (aspartate amino transferase, alanine
aminotransferase and gamma-galactosyl transferase) after the use of Acticoat® were
reported. Levels returned to normal following cessation of exposure. The patient did not
receive any other potentially hepatotoxic medication.
6.2.3.5.3 Immune System Toxicity
No treatment related effects on hematology and blood cell subset distribution (%
lymphocytes, monocytes, etc.) was seen after inhalation of nanosilver particles (Wijnhoven et
al., 2009). Of note, nanosilver particles were detectable in the spleen in the Takenaka study
(Takenaka et al., 2001), but not in the Ji study (Ji et al., 2007). In the 90-day inhalation study
of Sung et al. (2008), the presence of nanosilver particles in the lung may have induced a
local inflammatory response in the high dose group. Parameters on potential systemic
immune effects were not monitored in this study (Sung et al., 2008). In mice, application of a
1% nanosilver cream (96.1% is <50 nm) inhibited DNB-induced allergic contact dermatitis
(Bhol & Schechter, 2005). It was found that the expression of two cytokines (TNF and IL-
12) was suppressed (histopathological staining) and apoptosis of inflammatory cells but not
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keratinocytes was induced. Similar concentration-dependent anti-inflammatory effects have
also been seen in guinea pigs by the same group (Bhol et al, 2004). These latter data may
suggest that silver nanoparticles are especially effective at inhibiting inflammations and may
thus be used to treat immunologic and inflammatory diseases (Shin et al, 2007). Further
studies may be necessary to determine effective doses since local inflammatory responses
may be induced when applying high doses of nanosilver particles (Sung et a/,2008).
At high concentrations, nanoparticles tend to cluster, forming aggregates often larger than
100 nm. Larger nanoparticles (>100 nm) can be readily phagocytized by alveolar
macrophages (Buzea e? a/., 2007; Oberdorster, 1988; Takenaka e? a/., 2001). Results of
studies involving inhalation or intratracheal instillation of high concentrations of nanoparticle
(silver, iron, India ink, or titanium dioxide) smaller than 100 nm, which aggregate in larger
particles, suggest that most nanoparticles are indeed stopped by alveolar macrophages
(Takenaka et al., 2001). Rat studies based on inhalation of low concentrations of 15 nm
diameter silver nanoparticles showed that soon after inhalation (30 min), nanoparticles are
distributed in the blood and brain, and subsequently, to organs, such as heart and kidney,
while the lungs are rapidly cleared off of the nanoparticles (Buzea et al., 2007; Takenaka et
al, 2001). Hence, minute concentrations of nanoparticles with size smaller than 100 nm can
have a higher probability of translocating to the circulatory system and organs than high
concentrations of the same particles, which are likely to form aggregates and will be stopped
from translocation by macrophage phagocytosis (Buzea et al., 2007).
Very limited changes in hematological or biochemical indicators of toxicity were associated
with the silver absorption from Acticoat® in humans (Vlachou et al., 2007), despite
measurable amounts of silver in serum. Another case report possibly involving uptake of
silver particles is the finding of small electron-dense particles, probably silver nanoparticles,
in mast cells following 20 years of local acupuncture (Kakurai et al., 2003). The mast cells
showed focal or partial loss of granule content suggesting degranulation (activation)
associated with pruritus (itching) and an inflammatory reaction.
6.2.3.5.4 Other blood effects
Apart from a small increase in blood calcium, no additional effects of systemic exposure of
nanosilver on hematology and blood chemistry parameters have been reported after
inhalation exposure (Ji et al., 2007). Oral administration of 60 nm silver nanoparticles
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induced some changes in the red blood cell compartment (increased red blood cell count,
hemoglobin, and hematocrit) and on coagulation parameters (decreased active partial
protrombine time) (Kim et al., 2008).
6.2.3.5.5 Reproductive System Toxicity
In the Ji et al. study (2007), no effect on the histopathology of the epididymis was noted. No
additional in vivo data on the potential toxic effect of nanosilver on female or male
reproductive function (e.g., female egg development or male sperm formation) is readily
available.
6.2.3.5.6 Genotoxicity, Carcinogen/city
In a chronic subcutaneous administration of colloidal silver (1.75-2.5 mg weekly), eight of
the 26 (31%) rats that survived longer than 16 months developed malignant tumors
(Schmaehl & Steinhoff, 1960; Wijnhoven et al., 2009). In six of the rats, the tumor arose at
the site of subcutaneous injection. This was significantly higher than the historical control
tumor levels that were between 1-3% (Schmaehl & Steinhoff, 1960; Wijnhoven et al., 2009).
In contrast, no tumor induction at the site of injection was found in rats after intramuscular
injection of a suspension of fine silver powder (<300 mesh) in trioctanoin (Furst &
Schlauder, 1978). Kim et al. (2008) investigated the in vivo genotoxicity using a bone
marrow micronucleus test after oral administration of 60 nm silver nanoparticles for 28 days
at various doses. They found no statistically significant effects.
6.2.4 Cell Culture Nanosilver Toxicity
In vitro cell line studies by Samberg et al. (2010) have shown decreased mitochondrial
function after exposure to silver nanoparticles in murine neuroblastoma cells (Schrand et al.,
2008), hepatic cells (Hussain et al, 2005), germline stem cells (Braydich-Stolle et al, 2005),
human skin carcinoma cells (Arora et al, 2008), and human epidermal keratinocytes (HEKs)
and fibroblasts (Burd et al, 2007). Although in vivo studies have not been performed with
silver nanoparticles, polyvinylpyrrolidone (PVP)-stabilized silver nanoparticles with a mean
size of 25 nm were shown to penetrate into the upper layers of the epidermis in excised
human skin in static diffusion cells (Larese et al, 2009). The in vitro cytotoxicity of silver
nanoparticles (15 nm diameter) in mammalian mouse C18-4 germline stem cells indicated
that a silver nanoparticle concentration of more than 5 g/ml reduced the mitochondrial
function and cell viability while increasing the LDH leakage (Braydich-Stolle et al, 2005). It
also suggested that the use of silver nanoparticles in bone cement or implantable devices as
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antimicrobial agents (Alt et al., 2004) could be toxic for the bone-lining cells and other
tissues.
Carlson et al. (2008) designed a study to evaluate size-dependent cellular interactions of
known biologically active silver nanoparticles (Ag-15nm, Ag-30nm, and Ag-55nm).
Alveolar macrophages provide the first defense and were studied for their potential role in
initiating oxidative stress. Cell exposure produced morphologically abnormal sizes and
adherence characteristics with significant nanoparticle uptake at high doses after 24 h.
Toxicity evaluations using mitochondria! and cell membrane viability along with reactive
oxygen species (ROS) were performed. After 24 h of exposure, viability metrics significantly
decreased with increasing dose (10-75 ug/ml) of Ag-15nm and Ag-30nm nanoparticles. A
more than 10-fold increase of ROS levels in cells exposed to 50 ug/ml Ag-15nm suggests
that the cytotoxicity of Ag-15nm is likely to be mediated through oxidative stress. Activation
of the release of traditional inflammatory mediators were examined by measuring levels of
cytokines/chemokines, including tumor necrosis factor (TNF- ), macrophage inhibitory
protein (MIP-2), and interleukin-6 (IL-6), released into the culture media. After 24 h of
exposure to Ag-15nm nanoparticles, a significant inflammatory response was observed by
the release of TNF- , MIP-2, and IL-1 . There was no detectable level of IL-6 upon
exposure to silver nanoparticles. A size-dependent toxicity was produced by silver
nanoparticles, and one predominant mechanism of toxicity was found to be largely mediated
through oxidative stress. The toxicity of silver exhibited in liver cells was also shown to be
mediated by oxidative stress (Hussain et al., 2005), and silver nanoparticles were found to
induce toxicity in germline stem cells (Braydich-Stolle et al., 2005)
There are various in vitro studies found in the literature on the effects of silver nanoparticles
with size varying between 1 and 100 nm (Hussain et al., 2005; Park et al., 2007; Skebo et al.,
2007; Suzuki et al., 2007). There is no consensus on the cytotoxicity of nanosilver. Most
publications do show reduced cell viability following exposure. Additional toxic effects seen
in the in vitro studies are glutathione depletion, mitochondria! deviations or destruction and
damage to cell membranes. In vitro exposure of human peripheral blood mononuclear cells to
silver nanoparticles (1-2.5 nm, 72 h) resulted in inhibition of phytohemagglutinin (PHA)
induced proliferation (at a concentration of 15 ppm) (Shin et al., 2007).
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Hussain et al. (2005) evaluated the in vitro toxicity of several nanoparticles, including
nanosilver (15 and 100 nm) on a rat liver derived cell line (BRL 3A). Following 24 h after
exposure, the mitochondrial function and membrane integrity were significantly decreased
(at > 5 mg/ml and >10 mg/ml, respectively). LDH leakage was dose dependent and more
severe for 100 nm than for 15 nm silver nanoparticles. Visual microscopic evaluation
indicated that not all nanoparticles accumulated in the cell, but some remained associated
with membranes. The observed cytotoxicity was attributed to be mediated by oxidative
stress, as indicated by the detection of GSH depletion, reduced mitochondrial potential, and
increased reactive oxygen species (ROS) levels. A similar concentration-dependent
cytotoxicity was observed when the effects of the same nanosilver particles on a mouse cell
line with spermatogonial stem cell characteristics was studied (Braydich-Stolle et al., 2005).
Here, a concentration dependent effect on mitochondrial function, cell viability and
membrane integrity was seen, albeit at somewhat lower concentrations. In another study,
nanosilver particles (-30 nm) were classified again to be amongst the most cytotoxic
nanoparticles when tested on a murine alveolar macrophage cell line, a human alveolar
macrophage cell line and epithelial lung cell line (Soto et al., 2005, 2007).
Using a human alveolar epithelial cell line (A549), Park et al. (2007) confirmed that various
metallic nanoparticles (Ag, TiC^, Ni, Zn, Al) induce variable extents of cellular toxicity in a
dose dependent manner. Nanosilver (mean diameter 150 nm, 24 h exposure, and
concentrations up to 200 mg/ml) were found to be among the least cytotoxic among the
metallic nanoparticles. Neuroendocrine cells were found to be sensitive to the cytotoxic
activity of silver nanoparticles (15 nm; Hussain et al., 2006). Inhibition of dopamine
production was only seen at the highest cytotoxic levels. Addition of 1.0% silver
nanoparticles (5-50 nm) to bone cement, a dose at which antibactericidal activity was seen,
did not result in additional cytotoxicity towards mouse fibroblasts (L929), or on growth of
human osteoblast cell line (hFOB 1.19) (Alt et al, 2004).
Acticoat® dressing was found to be cytotoxic to primary keratinocytes cultured on a pliable
hyaluronate derived membrane (Laserskin) (Lam et al, 2004). Reduced mitochondrial
metabolism, as well as reduced viability of human keratinocytes and fibroblast cultured on a
collagen substrate were detected (Supp et al, 2005). Similar effects (cytotoxicity and
disordered morphology) on keratinocytes were reported for extracts of various silver-
containing dressings (including Acticoat®) (Paddle-Ledinek et al, 2006). Fibroblasts appear
Final Report dated 07/15/2010
State of the Science-Everything Nanosilver and More
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to be more sensitive for these effects than keratinocytes (Poon & Burd, 2004). When the
complexity of the environment increased, e.g., after 3-d culture in collagen lattices, the toxic
effect of silver appears to decrease (Poon & Burd, 2004).
Carlson et al. (2008) reported size-dependent alveolar macrophage cells interactions with
silver nanoparticles. Their result shows that after 24 hr of exposure, viability significantly
decreased with increasing dose (10-75 /ug/mL) of 15 and 30 nm silver nanoparticles. There
was more than a 10* increase of ROS levels in cells exposed to 50 ug/mL of 15 nm silver
nanoparticles, which suggests that oxidative stress may be responsible for the observed
cytotoxicity.
Soto et al. (2005) characterized a range of manufactured nanoparticulate materials, including
Ag, TiC>2, Fe2Os, A12O3, ZrC>2, SisN^ and a range of carbonaceous nanoparticulate materials:
single-wall and multi-wall carbon nanotube aggregates and aggregated nanoparticles of black
carbon, as well as commercial (mineral grade) chrysotile asbestos nanotube aggregates, using
transmission electron microscopy. These nanoparticulate materials ranged in primary particle
sizes from roughly 3 to 150 nm (except for the nanotube materials with lengths in excess of
15 m). Aggregate sizes ranged from 25 nm to 20 m. Comparative cytotoxicological
assessment of these nanomaterials was performed utilizing a murine (lung) macrophage cell
line. Considering the chrysotile asbestos to be a positive control, and assigning it a relative
cytotoxicity index of unity (1.0), relative cytotoxicity indexes were observed as follows at
concentrations of 5 g/ml: 1.6 and 0.4 for Ag and TiC>2, respectively; 0.7-0.9 for the Fe2Os,
A12O3 and ZrO2, 0.4 for the Si3N4, 0.8 for the black carbon, and 0.9 to 1.1 for the carbon
nanotube aggregate samples. Observations of a cytotoxic response, nearly identical to that for
chrysotile asbestos, for multi-wall carbon nanotube aggregates which very closely resemble
anthropogenic multi-wall carbon nanotubes in the environment, raise some concern for
potential health effects, especially for long-term exposure.
Kim et al. (2009) demonstrated the cytotoxicity induced by silver nanoparticles and the role
that oxidative stress plays in the process in human hepatoma cells. Toxicity induced by silver
(Ag+) ions was studied in parallel using AgNOs as the Ag+ ion source. Using cation exchange
treatment, the authors confirmed that the silver nanoparticle solution contained a negligible
amount of free Ag+ ions. Metal-responsive metallothionein Ib (MTlb) mRNA expression
Final Report dated 07/15/2010 128
State of the Science - Everything Nanosilver and More
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was not induced in nanosilver-treated cells, while it was induced in AgNOs-treated cells.
These results indicate that nanosilver-treated cells have limited exposure to Ag+ ions, despite
the potential release of Ag+ ions from silver nanoparticles in cell culture. The nanoparticles
agglomerated in the cytoplasm and nuclei of treated cells, and induced intracellular oxidative
stress. Nanosilver exhibited cytotoxicity with a potency comparable to that of Ag+ ions in in
vitro cytotoxicity assays. The toxicity of silver nanoparticles was prevented by use of the
antioxidant N-acetylcysteine, and nanosilver-induced DNA damage was also prevented by
N-acetylcysteine. AgNOs treatment induced oxidative stress-related glutathione peroxidase 1
(GPxl) and catalase expression to a greater extent than nanosilver exposure, but treatment
with AgNOs and silver nanoparticles induced comparable superoxide dismutase 1 (SOD1)
expression levels. These findings suggest that nanosilver cytotoxicity is primarily the result
of oxidative stress and is independent of the toxicity of Ag+ ions.
Foldbjerg et al. (2009) investigated the toxicity of silver nanoparticles in vitro. Silver ions
(Ag+) have been used in medical treatments for decades whereas silver nanoparticles have
been used in a variety of consumer products within recent years. This study was undertaken
to compare the effect of well characterized, PVP-coated nanosilver (69nm±3 nm) and Ag+ in
a human monocytic cell line (THP-1). Characterization of the nanosilver was conducted in
both stock suspension and cell media with and without serum and antibiotics. By using the
flowcytometric annexin V/propidium iodide (PI) assay, both nanosilver and Ag+ were shown
to induce apoptosis and necrosis in THP-1 cells depending on dose and exposure time.
Kawata et al. (2009) evaluated in vitro toxicity of nanosilver at non-cytotoxic doses in
human hepatoma cell line, HepG2, based on cell viability assay, micronucleus test, and DNA
microarray analysis. They also used polystyrene nanoparticles (PS-NPs) and silver carbonate
(Ag2COs) as test materials to compare the toxic effects with respect to different raw chemical
composition and form of silver. The cell viability assay demonstrated that silver
nanoparticles accelerated cell proliferation at low doses (<0.5 mg/L), which was supported
by the DNA microarray analysis showing significant induction of genes associated with cell
cycle progression. Only nanosilver exposure exhibited a significant cytotoxicity at higher
doses (>1.0 mg/L) and induced abnormal cellular morphology, displaying cellular shrinkage
and acquisition of an irregular shape. Only nanosilver exposure increased the frequency of
micronucleus formation up to 47.9 ± 3.2% of binucleated cells, suggesting that silver
Final Report dated 07/15/2010 \29
State of the Science-Everything Nanosilver and More
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nanoparticles appear to cause much stronger damages to chromosome than PS-NPs and ionic
Ag+.
In a study by Asharani et al. (2009), normal human lung fibroblast cells (IMR-90) and
human glioblastoma cells (U251) were exposed to different doses of nanosilver in vitro.
Uptake of nanosilver occurred mainly through endocytosis, accompanied by a time
dependent increase in exocytosis rate. The electron micrographs revealed a uniform
intracellular distribution of nanosilver both in cytoplasm and nucleus. Nanosilver treated
cells exhibited chromosome instability and mitotic arrest in human cells. There was efficient
recovery from arrest in normal human fibroblasts whereas the cancer cells ceased to
proliferate. Toxicity of nanosilver is mediated through intracellular calcium (Ca2+) transients
along with significant alterations in cell morphology and spreading and surface ruffling.
Down regulation of major actin binding protein, filamin, was observed after silver
nanoparticle exposure. Silver nanoparticle induced stress resulted in an increase in the
number of metallothionein and heme oxygenase -1 genes (upregulation). The results suggest
that cancer cells are susceptible to damage with lack of recovery from nanosilver-induced
stress. Silver nanoparticles are found to be acting through intracellular calcium transients and
chromosomal aberrations, either directly or through activation of catabolic enzymes. Figure
6.6 shows the proposed mechanism of nanosilver toxicity based on the results from this
study. The signaling cascades are believed to play key roles in cytoskeleton deformations and
ultimately to inhibit cell proliferation.
According to the criteria of the USEPA (U.S. EPA, 1994), silver is not classifiable as a
human carcinogen (group D). Silver powder and colloidal silver do not induce cancer in
animals, and silver chloride is considered nonmutagenic in the Ames assay. Silver
compounds have been generally considered not to have carcinogenicity in humans and
animals. No evidence of the carcinogenicity of silver nanoparticles has been reported despite
the growing commercialization of nanosilver. The upregulation (increase in quantity of cells
upon external stimulation) of a number of the genes associated with DNA repair and the
increase in micronuclei in the nanosilver exposed cells at relatively low doses (<1.0 mg/L)
clearly suggested the DNA damaging effects (chromosome aberration) of silver
nanoparticles. Both nanosilver and Ag+ contribute to the toxic effects of silver nanoparticles.
The nanosilver concentration assessed in the study by Kawata et al. (2009) would be higher
than those occurring in air and water environment. Since the internal kinetics of nanoparticles
Final Report dated 07/15/2010
State of the Science-EverythingNanosilver and More
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has not been elucidated, the local concentration in tissues might reach higher level as the
result of accumulation. Given that the physicochemical properties of nanosilver such as
particle size, particle agglomeration, and dispersibility significantly influence the degree and
actions of toxicity of silver nanoparticles, further research is necessary to assess the effects of
these variables.
Inhibition of
cell proliferation
Figure 6.6: The proposed mechanism of nanosilver toxicity based on the experimental data
obtained by Asharani et al. (2009). Reprinted from BMC Cell Biology, Vol. 10, Asharani,
P.V., Hande, M.P., Valiyaveettil, S. Anti-proliferative activity of silver nanoparticles, pp65-
79, Copyright 2009 with permission from BioMed Central.
6.3 Conclusions on Nanosilver Toxicity
Several factors influence the ability of a metal to produce toxic effects on the body. These
include the solubility of the metal, its ability to bind to biological sites, and the degree to
which the complexes formed are sequestered or metabolized and excreted. For nano-sized
particles, additional parameters such as size and surface area are recognized as important
determinants for toxicity (Ji et al. 2007; Wijnhoven et al., 2007). Nanoparticles can pass
through biological membranes. After administration, nanoparticles are small enough to
penetrate even very small capillaries throughout the body. Silver nanoparticles are used
because of the antibacterial activity of silver. The antibacterial action of silver (ions) may
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State of the Science-Everything Nanosilver and More
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have several mechanisms. It has been suggested that the primary mechanism of action is cell
death due to the uncoupling of oxidative phosphorylation (Holt & Bard, 2005) or the
induction of free radical formation (Kim et al., 2007). Interference with respiratory chain at
the cytochrome C level, and/or with components of microbial electron transport system, has
also been reported (Muangman et al., 2006). Interactions with membrane bound enzymes and
protein thiol groups that may result in compromised cell wall integrity have been postulated
(Bragg & Rainnie, 1974; Lok etal, 2006; Silver, 2003; Wijnhoven etal, 2007; Zeiri et al,
2004). It has also been suggested that silver ions bind to DNA and may cause DNA strand
breaks and DNA replication (ATSDR, 1990; Russell & Hugo, 1994). The reason why
eukaryotic (mammalian) cells appear less sensitive to this action of silver can be explained
by the higher structural and functional redundancy and size of eukaryotic compared to
prokaryotic (bacterial) cells. This may increase the silver concentration needed to achieve
comparable toxic effects on eukaryotic cells than for bacterial cells (Alt etal, 2004). There
may be a therapeutic window in which bacterial cells are successfully attacked, at which
harmful effects on eukaryotic cells cannot yet be observed. The effective concentration for
silver nanoparticles is much lower in comparison to Ag+ ions (nmol vs. mmol levels) (Lok et
al, 2006). Given the potential higher toxicity and the specific concerns associated with the
use of nano-sized materials particular attention to the toxicity of silver nanoparticles may be
warranted.
Information on the toxicological implication of the use of silver nanoparticles is limited
(Chen & Schluesener, 2008; Wijnhoven etal, 2007). Toxicity of silver nanoparticles is
mostly determined in vitro with particles ranging in size from 1-100 nm. The available in
vivo animal studies are generally relative short term (max 28 days), except for one 90 day
inhalation study of the use of one size of silver nanoparticles (Sung et al, 2008). Only
limited health effects of the use of nanosilver in humans have been documented. Argyria or
argyrosis was rarely reported, and appeared to occur only after intake of large amounts of
silver particles (usually colloidal, a suspension with nanosilver of different sizes). Potential
target organs for nanosilver toxicity may involve the liver, kidneys and the immune system.
Accumulation and histopathological effects were seen in the liver of rats systemically
exposed to silver 10-15 nm nanoparticles (Ji et al, 2007), while an effect on liver enzymes
was noted in one human case study with dermal exposure to particles with an average of the
same size (Trop etal, 2006). Accumulation, histopathological effects and increased liver
enzymes were reported after oral exposure to 60 nm nanosilver (Kim et al, 2008). It is not
Final Report dated 07/15/2010
State of the Science-Everything Nanosilver and More
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known if the silver reaches the liver as silver nanoparticles or as ions, nor has the location of
the silver (nanoparticles) within the liver been studied. Effects on the immune system,
especially cytokine excretion, have been noted in vitro and in vivo, where application of a 1%
nanosilver cream with <50 nm particles, inhibited DNB-induced allergic contact dermatitis
(Bhol & Schechter, 2005), and accumulation in the spleen has also been noted (Takenaka et
al., 2001). It has been suggested that silver nanoparticles are especially effective at inhibiting
inflammations and may be used to treat immunologic and inflammatory diseases (Shin et al.,
2007). There were only very limited changes in hematological or biochemical indicators of
toxicity associated with the systemic silver absorption from 15 nm nanosilver wound
dressings in humans (Vlachou et al., 2007), and after inhalation in rats (Ji et al., 2007). Oral
administration of 60 nm silver particles in rats induced some local inflammatory effects (Kim
et al., 2008). Whether silver nanoparticles indeed have a (systemic) effect on immune
function in vivo needs to be further explored.
No reports on effects of silver nanoparticles on the cardiovascular, renal/urinary or
gastrointestinal systems for humans have been found, however, specific studies addressing
these organs have not been identified. There are very limited well controlled human studies
on the potential toxicities of nanosilver. Additional long term, higher dosed studies,
preferably using multiple particle sizes, are needed to better characterize the risk of the use of
silver nanoparticles on humans.
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State of the Science-Everything Nanosilver and More
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7. Life Cycle Analysis for Comprehensive Environmental
Assessment
Materials that incorporate particles manufactured at the nano scale (nanomaterials) may have
many potential benefits to society with their development and deployment in science,
engineering and technology. The use of nanosilver in socks potentially benefits society by
preventing foot odor, and may reduce the number of washings, which results in less water
usage. Their benefits, however, need to be weighed with any potential cost to the
environment and public health. In the case of the above-mentioned example, there is plenty
of evidence that the nanosilver impregnated in the socks leaches out after a few wash cycles
(Benn & Westerhoff, 2008). The leached nanosilver makes its way into wastewater treatment
plants, and depending on its fate and transport characteristics, may ultimately wind up in
streams and sediments where it may cause some risk to the ecosystem. The unknown health
effects and risks associated with these materials have drawn considerable attention from
researchers, consumers and regulators. As a result, scientists at the USEPA and elsewhere
have recognized the need to develop risk assessment processes to study the potential health
and environmental impacts of manufacturing nanomaterials as well as using these materials
in other products. In addition to the toxicological concerns, there are other aspects that have
to be considered during the risk assessment process as well such as the cost of transportation,
including the amount of emissions that are emitted from trucks and trains. To address these
issues, researchers have begun implementing more comprehensive assessment tools such as
Life Cycle Assessment (LCA) to assess the cradle-to-grave cost/risk associated with any
nanomaterial (Khanna et al, 2007; Krishnan et al, 2008; Lloyd & Lave, 2003; Lloyd et al,
2005; Osterwalder et al., 2006; Roes et al., 2007). An LCA can establish the comparative
impact of products or processes in terms of specified impact categories using a well-defined
and documented methodology (Baumann et al, 2004; ISO, 2006; Meyer et al, 2009;
Rebitzer etal., 2004; U.S. EPA, 1993). Typical impact categories include global
warming/climate change, stratospheric ozone depletion, human toxicity, ecotoxicity, photo-
oxidant formation, acidification, eutrophication, land use, and resource depletion (Meyer et
al, 2009; Rebitzer et al, 2004). A potential advantage of LCA-based evaluations for
nanomaterials is that they can address both the health and environmental consequences
associated with the inclusion of nanocomponents. The ultimate goal is to ensure that the
Final Report dated 07/15/2010 ^34
State of the Science-Everything Nanosilver and More
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potential benefits of nanocomponents are realized in a manner that is safe for both consumers
and the environment without resulting in unintended consequences (Meyer et a/., 2009).
A second method that is commonly used to assess the total risk associated with
manufacturing nanomaterials as well as using these materials in other products is the use
comprehensive environmental assessment (CEA), which combines LCA with the risk
assessment paradigm (NRC, 1983) to examine the environmental impacts of technology in a
broad, systematic manner (Davis, 2007; Davis & Thomas, 2006). The general features of
CEA are highlighted in Figure 7.1. In the figure, as listed in Column 1, the life cycle of a
product typically comprises several stages, including feedstock production or extraction,
manufacturing processes, distribution, storage, use, and disposal of the product and waste
byproducts. At any stage of the life cycle, pollutants may enter one or more environmental
pathways: air, water, soil, and food web (Column 2). It is important to identify these primary
contaminants and the transport and transformation processes they undergo. The idea is to
characterize the primary as well as secondary or byproduct pollutants associated with the
entire life cycle for all relevant environmental media (Column 3). The potential for humans
and other organisms (biota) to become exposed or come into contact with primary and
secondary pollutants via all pertinent routes, for example, inhalation, ingestion, and dermal
absorption is also considered (Column 4). In this column, microenvironmental and high-end
exposure scenarios should be considered, not just "typical" or "average" exposure levels.
This scenario-driven approach to exposure assessment is a feature that distinguishes the CEA
approach from LCA. Once exposure potential has been characterized, the health and
ecological hazards associated with respective contaminants need to be described qualitatively
and quantitatively (Column 5). To characterize risk quantitatively, the dose-response
characteristics of a toxicant must be considered in relation to exposure potential. Some
pollutants may pose low risk because the exposure potential is low or the hazard potential is
low, or both. In other cases, risk may be relatively high when exposure potential is low but
hazard potential is high, or vice versa (Davis, 2007; Davis & Thomas, 2006).
Final Report dated 07/15/2010
State of the Science - Everything Nanosilver and More
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n/
f
£>
Primary
contaminants
Secondary /
contaminants
Inhalation
Ingestl*
Derma/
_ab$orpt]on
Ecological
Health
Figure 7.1: Comprehensive environmental assessment (CEA). CEA combines LCA and the
risk assessment paradigm. Reprinted from Ann. N.Y. Acad. Sci., Vol. 1076, Davis, J.M.,
Thomas, V.N., Systematic approach to evaluating trade-offs among fuel options. The lessons
of MTBE, pp498-515, Copyright 2006 with permission from Wiley Interscience.
7.1 Nanosilver life cycle assessment
Meyer et al. (2009) propose using life cycles with four main aspects (Figure 7.2): material
selection, manufacturing, application, and disposal/recycle. The material selection aspect of
nanosilver LCA involves both the composition (organic such as polymers, dendrimers, etc.;
inorganic such as metals, metal oxides, etc.; carbon such as carbon tubes or a combination of
any of these) and geometry of the nanocomponents, which can be a variety of shapes (sphere,
rod, etc.) and is dependent on the synthesis methods (Meyer et al., 2009; Nowack & Bucheli,
2007; U.S. EPA, 2007). The manufacturing aspect of nanosilver LCA involves the synthesis
techniques discussed in Section 4.1. The application aspect of nanosilver LCA involves using
the nanomaterials in either naturally dispersive or composite form for a range of applications,
most of which are described in Chapter 3. The disposal/recycle aspect of nanosilver LCA
involves incineration, disposal in a landfill or removal during wastewater treatment among
others (Mueller & Nowack, 2008; U.S. EPA, 2007).
Gill (2007) presented an alternate life cycle assessment/materials flow analysis (Figure 7.3)
at the Geology Symposium 2007 held in Sacremento, CA. Similar to the four aspects of LCA
Final Report dated 07/15/2010
State of the Science-Everything Nanosilver and More
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proposed by Meyer et al. (2009), the LCA/materials flow analysis presented by Gill (2007)
had the materials and disposal aspects of the LCA. Other aspects in the LCA proposed by
Gill (2007) include material processing, manufacturing, distribution, use and/or reuse. These
four aspects fall into the two main aspects (manufacturing and application) proposed by
Meyer et al. (2009). Much more inventory information will be required for the more complex
LCA proposed by Gill (2007).
Key Aspects of a Selected Nanotechnology for Life Cycle Assessment
Recycle
Material
Selection
1 Composition
Surface
FunctionaiizaHon/
Charge
Figure 7.2: Choices associated with a nanotechnology throughout its life cycle as proposed
by Meyer et al. (2009). Reprinted from Environ. Sci. Techno!., Vol. 43 (5), Meyer, D.E.,
Curran, M.A., Gonzalez, M.A., An examination of existing data for the industrial
manufacture and use of nanocomponents and their role in the life cycle impact of
nanoproducts, pp!256-1263, Copyright 2009 with permission from American Chemical
Society.
USEPA's Nanotechnology Research Program within the ORD is conducting a series of LCAs
on various products made from nanomaterials to gain knowledge about potential release into
the environment. The assessments are holistic and comprehensive and track a product from
its inception (cradle) through its final disposal (grave). LCAs are essential to analyze,
evaluate, understand, and manage the overall health and environmental impacts of products.
The LCAs are focused on answering questions which include: (i) what are the trade-offs
associated with nanomaterials? and (ii) is the large-scale production of an environmentally
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State of the Science-Everything Nanosiher and More
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taxing material justified if it has beneficial applications for society or if it can reduce costs or
enhance performance?
Raw Materials
(Energy, Renewable Resources,
Nonrenewable Resources')
Disposal
(Air Emissions, Liquid and
Solid Wastes')
Figure 7.3: Choices associated with a nanotechnology throughout its life cycle as proposed
by Gill (2007). (Adapted from Gill, 2007).
Characterization of nanomaterials on a life cycle basis is challenging because this is a new
field of study. A large number of data gaps exist when considering the application of LCA to
nanomaterials (Bauer et al., 2008; Khanna et al., 2007; Kloepffer et al., 2007; von Gleich et
al., 2008). Finding adequate data to model the potential fate and effects of unintended
releases of nanomaterials into the environment may be difficult to obtain. USEPA
researchers are working to locate and provide the necessary data. Specifically, only minimal
data exist detailing the material inputs and environmental releases related to the manufacture,
release, transport, and ultimate fate of nanomaterials (Meyer etal, 2009). Studies have
mainly focused on "cradle-to-gate" assessments investigating the production of either
nanocomponents or nanomaterials up to the point these materials leave the "gate," or the
manufacturing source. However, many nanomaterials are not yet in full production to create
a consumer product; therefore, much of the data must be estimated.
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As mentioned in Chapter V, the project on emerging nanotechnologies that was established
in April 2005 as a partnership between the Woodrow Wilson International Center for
Scholars and the Pew Charitable Trusts has published a fair amount of information on the
inventory of consumer products containing nanomaterials including nanosilver. There is still
a lack of information in regards to the characteristics of the particles (shape, size and surface
chemistry), synthesis methods, production quantities, production losses, production
consumption and the geographic distribution of these nanosilver-containing products. All
these data are required in order to perform risk assessment and life cycle analysis.
Additional data is available in a database maintained by Nanowerk
(http://www.nanowerk.com). The site lists 2437 items containing nanoparticles as of March
31, 2010 in nine categories. Figure 7.4 illustrates the number of items in each category, while
Table 7.1 lists the company with specifics on nanosilver in items containing elemental silver
nanoparticles. Even with this information, the database does not list the necessary
information to conduct an LCA of nanosilver either.
Table 7.1: Companies selling nanosilver as listed in the Nanowerk database (as of March 31,
2010).
Company
Shanghai Huzheng Nano
Technology
Vive Nano
nanoComposix, Inc.
Nanocs
Nanocs
Nano structured &
Amorphous Materials, Inc.
Quantum Sphere, Inc.
Shanghai Huzheng Nano
Technology
Shanghai Huzheng Nano
Technology
Argonide Corporation
Inframat Advanced
Materials LLC
Inframat Advanced
Materials LLC
Nano structured &
Amorphous Materials, Inc.
Nano structured &
Amorphous Materials, Inc.
Nano structured &
Amorphous Materials, Inc.
nGimat
APS
Inm
l-10nm
10-127nm
lOnm
lOnm
lOnm
10-60nm
lOnm
lOnm
lOOnm
100-
127nm
100-
127nm
lOOnm
lOOnm
lOOnm
lOOnm
Phase
suspension
suspension
suspension
suspension
suspension
powder
powder
suspension
suspension
powder
powder
powder
powder
powder
powder
powder
Specification
Ag content <10,000ppm
Carboxy-functionalized silver nanoparticles, sodium
as carboxy counterion. 1.5 mg/mL in water or
powder
Monodisperse
Dextran coated, 0.01% Ag
In aqueous, 0.01% Ag
P(99.9), w/~0.3% (PUP) surfactant (alcohol
dissolvable)
Ionic solution; mono silver, pH 7
Ionic solution; silver ion or nitrate ion, pH <4.5 or
>9
Exploding wire synthesis
P(99.95)
P(99.95)
Ag coated, SiO2 cored 40:60
Ag coated, SiO2 cored 30:70
Ag coated, SiO2 cored 20:80
Nanospray technology
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State of the Science-Everything Nanosilver and More
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Table 7.1: Continued
Company
Sky Spring Nanomaterials
Inframat Advanced
Materials LLC
Nano Technology Inc.
NanoMetal
ABC Nanotech
AgPure Nano silver
AgPure Nano silver
AgPure Nano silver
Nano-Vision Tech
Nano-Vision Tech
Shanghai Huzheng Nano
Technology
loLiTec
NTbase
Microspheres-Nanospheres
Nanocs
Nanocs
PlasmaChem GmbH
Sky Spring Nanomaterials
Sky Spring Nanomaterials
NaBond
Nanogap
NovaCentrix
Nanogap
NanoSonic, Inc.
Nanogap
Auto Fibre Craft
Chengdu Alpha Nano
Technology
Nanocs
Nanocs
Nanostructured &
Amorphous Materials, Inc.
Nanostructured &
Amorphous Materials, Inc.
loLiTec
Shenzen Junye Nano
Material
Sunano
Sunano
Nanocs
Nanocs
NanoDynamics, Inc.
Applied Nanotech
Holdings
Bio-Gate
nanoCompound
Nanogap
Polytech & Net GmbH
Nano-Size Ltd.
Nano-Size Ltd.
Nanocs
APS
lOOnm
127nm
127nm
127nm
15-35nm
15nm
15nm
15nm
15-25nm
15-25nm
15nm
150nm
150nm
2-250nm
20nm
20nm
20nm
20-30nm
20-30nm
25nm
25-45nm
25-30nm
28-57nm
28nm
3.5-7.5nm
30-54nm
30-50nm
30nm
30nm
30-50nm
30-50nm
35nm
35nm
35nm
35nm
40nm
40nm
40nm
45nm
5-50nm
5nm
5-10nm
5-20nm
50nm
50nm
50nm
Phase
powder
powder
powder
powder
suspension
suspension
suspension
suspension
suspension
suspension
suspension
powder
powder
suspension
suspension
suspension
suspension
powder
powder
powder
powder
powder
powder
suspension
powder
powder
powder
suspension
suspension
powder
powder
powder
powder
powder
suspension
suspension
suspension
suspension
powder
suspension
powder
powder
suspension
powder
suspension
suspension
Specification
P(99.95%) spherical
P(99.95)
Spherical
Spherical
dispersed in alcohol; solid content <10wt%
<5% Ag concentration
<10% Ag concentration
<20% Ag concentration
P(99.99); pH 8-9; Suspended in water, alcohol;
SOOppm
P(99.99); pH 7-8; Suspended in water, alcohol;
2000ppm
Colloid; Ag content 350ppm
P(99.95%)
P(99.99) spherical
Spheres sizes available from 2-250nm
Dextran coated, 0.01% Ag
In aqueous, 0.01% Ag
colloidal suspension 0.5 mg/ml
P(99.95%) spherical, partially passivated
P(99.95%) spherical, PVP coated
P(>99.9%)
P(85%)
Strong crystallinity, spherical
P(85%)
Suspended in water
P(85%)
Dry, uncoated, pure powder silver in elemental form
P(>99.9%)w/0.3%PVP
Dextran coated, 0.01% Ag
In aqueous, 0.01% Ag
P(99.9), w/~0.3% (PUP) surfactant (alcohol
dissolvable)
P(99.9), w/~0.3% (PUP) surfactant (alcohol
dissolvable), surface coated with 2%wt oleic acid
P(99.5%)
P(>99.9%)
P(>99.8%)
P(>99.9%) Aqueous Solution with Non-Ionic
Surfactant; 1.6wt%
In aqueous, 0.01% Ag
Dextran coated, 0.01% Ag
P(99.99), crystalline
P(>99%)
P(99.9)
P(>99.9);Spherical
P(85%)
Yellow color; PH 6-8; antibacterial rate 99.9%
Cubic
Cubic, suspended in benzyl alcohol
Dextran coated, 0.01% Ag
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Table 7.1: Continued
Company
Nanocs
Seashell Technology, LLC
Sky Spring Nanomaterials
MTI Corporation
Nano Ocean Tech
Nano Ocean Tech
Neo-Eco systems
Nano-Size Ltd.
NanoDynamics, Inc.
Nano structured &
Amorphous Materials, Inc.
Blue Nano
MKnano
Nano structured &
Amorphous Materials, Inc.
APS
50nm
50-1 27nm
50-60nm
55nm
6nm
6nm
70nm
80nm
80nm
80-127nm
90-110nm
90nm
90-127nm
Phase
suspension
powder
powder
powder
suspension
powder
suspension
powder
powder
powder
powder
powder
Specification
In aqueous, 0.01% Ag
Spherical
P(99.95%) spherical
Surface ligand dodecanethiol
In water/surface ligand COOH
P(99%)
Cubic, suspended in benzyl alcohol
P(99.99), crystalline
P(99.95), spherical
P(99.9%)
P(>99), spherical
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Nanomaterial Database
Search
(choose from one category only)
o so no plot
Fullerenes <129 items >:
Material: I Select One
v[Search
Carbon Nanotubes (580 items }:
Material: I Select One v | Configuration: | SelectOne "v][Search"|
Nanoparticles of Elements <454 items >:
Material: I Silver vj [Search]
Nanoparticles of Binary Compounds (673 items }:
Material: I Select One
v| [Search]
Nanoparticles of Complex Compounds |169 items ):
Material: SelectOne
^[Search"]
Quantum Dots (171 items ):
Material: SelectOne
[Search]
Biomedical Quantum Dots (209 items ):
Material: I SelectOne
Search
J
Nanowires (24 items ):
Material: SelectOne
[ Search]
Nanofibers (28 items ):
Material: Select One
[Search]
Figure 7.4: The Nanomaterial Database maintained by Nanowerk (www.nanowerk. com).
The database currently has 2437 items listed under nine categories.
To obtain information to perform an LCA, a survey was created and sent to a total of 122
companies. Response was low; only one company replied. This lack of information
identifies a data gap which must be filled to successfully perform an LCA. An alternative
may be to estimate the information necessary to perform the LCA.
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As an example, it may be possible to use the estimates generated by Blaser et al. (2008), who
conducted a risk analysis of nanosilver incorporated in textiles and plastics to freshwater
ecosystems after estimating the amount of silver use in 2010. Blaser et al. (2008) assessed the
risk in four stages. First, the system boundaries were defined, mass flows of silver were
quantified, and three emission scenarios were defined. Second, the behavior of silver in
natural freshwater was reviewed, and a mass balance model was applied to calculate
predicted environmental concentrations (PECs) for freshwater and freshwater sediments.
PECs were also estimated for sewage treatment plants (STPs) and sewage sludge. The
uncertainty of the results was assessed and predicted concentrations were compared to
empirical data. Third, toxicity data for environmentally relevant silver compounds were
compiled and predicted no-effect concentrations (PNECs) were determined where possible.
Finally, the potential for risk caused by the release of silver into freshwater was evaluated
using all available data. Figure 7.5 illustrates the quantified mass flows of silver triggered
mainly by the use of biocidal products with other silver uses. This information can directly be
used to estimate the disposal/recycle aspect of the LCA.
Mueller & Nowack (2008) used a life-cycle perspective to model the quantities of engineered
nanoparticles into the environment. The quantification was based on a substance flow
analysis from nanomaterials to air, soil and water in Switzerland. Production and product
distribution was estimated by multiplying the total nanoparticle production (based on the
Woodrow Wilson Center database; www.nanotechproject.org) by a weighting factor. The
weighting factor was determined by combining three sources: (i) the inventory of the Wilson
Center, (ii) a search of the www.products.ec21.com database, and (iii) a web search. Figure
7.6 illustrates the flow of nanosilver from the nanomaterial containing products to different
environmental compartments - waste incineration plants (WIP), sewage treatment plants
(STP) and landfill. As in the case of the data estimated by Blaser et al. (2008), the data from
the model generated by Mueller & Nowack (2008) can directly be used to estimate the
disposal/reuse aspect of the nanosilver LCA.
Final Report dated 07/15/2010 ^43
State of the Science-EverythingNanosilver and More
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Production
190
/•""BiocidaT'N
X^Products__x
Other Sources
i
i
i
i
i
270
Aquatic Environment
f — — — i
Atmospheric Environment
Figure 7.5: Quantified mass flows of silver triggered by the use of biocidal products and by
other silver uses. Reprinted from Sci. Tot. Environ., Vol. 390, Blaser, S.A., Scheringer, M.,
MacLeod, M., Hungerbuhler, K., Estimation of cumulative aquatic exposure and risk due to
silver: contribution of nano-functionalized plastics and textiles, pp396-409, Copyright 2008
with permission from Elsevier.
Sayes & Ivanov (2010) developed a method to obtain Quantitative Structure-Activity
Relationship (QSAR) models to predict cellular membrane damage caused by exposure to
two different types of nanomaterials - TiC^ and ZnO. It is hypothesized that nanosilver can
also be predicted in this manner. QSAR models are mathematical models that relate a
response to certain physicochemical properties of chemicals that can either be measured or
estimated. The authors use a mathematical modeling approach that uses physical properties
of the nanomaterials such as its engineered size and size in water (or buffer) and one
chemical property - zeta potential as descriptors (or independent variables) to describe
cellular membrane damage as measured by lactate dehydrogenase release. More elaborate
QSAR models can be developed to predict various toxicity endpoints of interest. Once these
models have been developed, such models can be used to estimate the toxicity of a
nanoparticle at any given site. This information can be used to estimate the toxicity
component of an LCA.
Final Report dated 07/15/2010 ^44
State of the Science-Everything Nanosilver and More
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Figure 7.6: Nanosilver flows from the nanomaterial containing products to various
environmental compartments (WIP, STP and landfill). All flows are in tons/year. The
thickness of the arrows is proportional to the amount of silver flowing between the
compartments. Dashed arrows represent the lowest volume. Reprinted from Environ. Sci.
Techno!., Vol. 42 (12), Mueller, N.C., Nowack, B., Exposure modeling of engineered
nanoparticles in the environment, pp4447-4453, Copyright 2008 with permission from
American Chemical Society.
7.2 Nanosilver comprehensive environmental assessment
The USEPA's mission and mandates call for an understanding of the health and ecological
implications of engineered nanomaterials, which necessitates conducting a comprehensive
environmental assessment on such nanomaterials. To serve as a foundation for creating a
long-term research strategy to provide the information needed for comprehensive
environmental assessments of nanomaterials, the USEPA completed two case studies that
focused on nanoscale titanium oxide (nanoTiO2) and released a preliminary draft report that
is currently undergoing external peer review (U.S. EPA, 2009). The first case study focused
on the use of nanoTiO2 as an agent for removing arsenic from drinking water while the
second case study focused on nanoTiO2 as an active ingredient in topical sunscreen. The
USEPA followed up on the two nanoTiO2 case studies with a case study that focused on
nanoscale silver as possibly used in aerosol disinfectant sprays, and released a preliminary
draft report that is currently undergoing internal (interagency) peer review (U.S. EPA, 2010).
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The process for selecting nanosilver in disinfectant spray as a case study involved a
workgroup representing several USEPA program offices, regional offices, and ORD
laboratories and centers. The USEPA workgroup considered several candidate nanomaterials
and voted for their preferences based on, among other things, apparent relevance of the
nanomaterial to USEPA programmatic interests. The organization of the draft report reflected
the CEA approach: Chapter 1 provided an overall introduction while Chapter 2 provided
introductory material on silver and nanosilver. Chapter 3 in the draft report highlighted stages
of the product life cycle (feedstocks, manufacturing, distribution, storage, use, disposal),
followed by fate and transport processes in Chapter 4, exposure-dose characterization in
Chapter 5, and ecological and health effects in Chapter 6. The draft report contained lists of
questions that reflect information gaps or possible research issues. This report is not expected
to be released to the public until 2011.
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8. Data Gaps
Nanosilver is being used in an extensive number of applications and this number is still
growing. Eventually, all known applications for metallic silver may involve the use of
nanosilver instead of silver to take advantage of nanosilver's unique properties. In spite of all
beneficial uses for nanosilver, its impact on the environment is of concern. For that reason,
research is being conducted in order to answer many questions such as the fate, transport and
toxicity of nanosilver. This report is a state of the art review that covers all available
information on various aspects of nanosilver research, including synthesis methods,
applications, fate and transport, exposure, toxicity, life cycle analysis (or comprehensive
environmental assessment) and risk assessment. Even though there is information available
on each individual aspect listed previously, there are many research gaps that have to be
filled to gain a comprehensive understanding of benefits and risks of using nanosilver. This
section highlights some of these data gaps that exist:
There is a plethora of synthesis methods for nanosilver, but the ones that are most
commonly utilized in the industry are not yet known. This is extremely important to
know since each synthesis method will result in nanoparticles with specific surface
properties that will govern their fate, transport, toxicity and environmental
interactions and impacts. These synthesis methods may also involve the use of
different raw materials and may yield reaction byproducts or waste that may be toxic.
Such information is also necessary while performing a cost-benefit analysis, life cycle
analysis or comprehensive environmental assessment. Thus, this is the first important
question that the researchers should answer in order to gear the efforts towards the
investigation of the right types of silver nanoparticles.
There are plenty of techniques to characterize pure silver nanomaterials. The majority
of these techniques will, of course, work for pure nanosilver suspensions, but there
are still there is lack of methodologies and analytical tools for the detection and
characterization of silver nanoparticles in environmental samples. Thus, there is a
need for developing methods to measure the nanosilver concentration, speciation (e.g.
ionic versus metallic), size, shape, surface charge, crystal structure, surface chemistry
and surface transformations in complicated matrices such as rivers, streams,
wastewater and/or landfill leachate, soils and sediments.
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It is important to characterize the nanoparticles, perform dose-metrics as well as
quantify the physicochemical properties of the nanomaterials. Nanoparticles have
novel properties compared to conventional chemicals. The characterization of these
properties is important in order to enable realistic estimations of exposure to humans
and the ecosystem. This information is also important to establish dose-response
relationships for estimating the toxicity of these nanoparticles.
There is still a lack of information in regards to the characteristics of the silver
nanoparticles used in consumer products (shape, size, surface chemistry and synthesis
methods), production quantities, production losses, production consumption and the
geographic and demographic distribution of these nanosilver-containing products in
the US and elsewhere. All these data are necessary in order to perform risk
assessment and life cycle analyses. The manufactures of nanosilver and nanosilver-
containing products are yet not cooperative in providing this necessary information.
State or Federal laws that mandate providing this information to regulatory agencies
may be necessary to obtain information from manufacturers.
There is limited number of studies on the teachability of silver nanoparticles (or other
forms of silver) from consumer products containing nanosilver under various
environmental conditions that a given product may potentially encounter. The
speciation of the leached silver is also unknown for many consumer products.
Additional questions to consider include the investigation of the release patterns and
release kinetics of nanosilver from specific applications and whether the
physicochemical properties change under certain circumstances leading to more/less
release of nanosilver into the aqueous environment.
There is no information available yet on the fate and transport of silver nanoparticles
in waste streams, solid waste landfills, soils and sediments.
There is a significant body of literature discussing the toxicity of silver nanoparticles
to various bacterial species; however, the diverse types of reducing agents, capping
agents and dispersants used to synthesize and stabilize the nanosilver may make
direct and meaningful comparisons difficult. Results using different bacterial strains,
even if they are of the same species, may not be comparable. Between the different
toxicological studies that are reported in the literature so far, the compositions of the
silver nanoparticles vary widely. The descriptions of used silver formulations diverge
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from detailed to very limited, with variable attention paid to the size, solubility and
aggregation of the nanoparticles.
Due to a lack of reliable nanosilver toxicity data in the literature, it is impossible to
assess the environmental risks associated with the production and use of nanosilver.
An important research question is the validation of the hypothesis that toxic effects of
nanoparticles are proportional to the activity of the free silver ions released by the
nanoparticles.
Various toxicity mechanisms of nanosilver have been proposed but the exact
mechanisms are yet to be determined.
Apart from nanosilver toxicity assessment in the aqueous environments, more
research is needed to investigate the effects of nanosilver in terrestrial environments
as no toxicity data for nanosilver in soils were found in the literature. Additional
research is also needed on ecologically relevant species to investigate whether silver
nanoparticles present a threat to environmental health in general. It should be
determined whether nanosilver in products is actually capable of reaching the
aqueous and terrestrial environment. Specifically, the strength of the bonds between
nanosilver and the product it is incorporated into should be investigated.
No specific mammalian models for nanosilver were available in the literature
although several were identified for nanoparticles in general.
Although the oxidation state of the silver nanoparticles may influence their biological
and/or toxicological activity, little attention has been paid to the oxidation state of the
silver nanoparticles in the literature.
Toxicity of silver nanoparticles is mostly determined in vitro with particles ranging in
size from 1-100 nm. The available in vivo animal studies are relatively short term
studies (maximum of 28 days) except for one 90-day inhalation study on the use of a
single size of silver nanoparticles.
Only limited health effects on the use of nanosilver in humans have been
documented.
Limited risk assessment studies for nanosilver are available in the literature as a result
there is a lack of adequate data to model the potential fate and effects of unintended
releases of nanosilver into the environment. Specifically, only minimal data exist
detailing the material inputs and environmental releases related to the manufacture,
release, transport, and ultimate fate of silver nanoparticles.
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