United States
Environmental Protection
^^0^ LhI # % Agency
Laser Detection of
Nanoparticles
In the Environment
APM 31
RESEARCH AND DEVELOPMENT
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EPA/600/R-11/097
September 2011
www.epa.gov
Laser Detection of
Nanoparticles
In the Environment
APM 31
Tammy Jones-Lepp, Research Chemist1
Kim Rogers, Research Chemist2
Richard Snell, Chemist IV3
1U.S. Environmental Protection Agency
National Exposure Research Laboratory
Environmental Sciences Division
Environmental Chemistry Branch
Las Vegas, NV 89119
2U.S. Environmental Protection Agency
National Exposure Research Laboratory
Human Exposure and Atmospheric Sciences Division
Las Vegas, NV 89119
3Senior Environmental Employee Program - Contractor to
National Exposure Research Laboratory
Environmental Sciences Division
Environmental Chemistry Branch
Las Vegas, NV 89119
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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The information in this document has been funded by the United States Environmental
Protection Agency. It has been subjected to the Agency's peer and administrative review and
has been approved for publication as an EPA document. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
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CONTENTS
Section pg #
FOREWORD iii
EXECUTIVE SUMMARY v
LIST OF ACRONYMS AND ABBREVIATIONS vi
1.0 INTRODUCTION 1
2.0 RESEARCH GOALS 2
3.0 NANOPARTICLE DETECTION METHODS 2
3.1 General Discussion
3.2 Assessment Criteria
3.2 Initial Candidate List
4.0 EXPERIMENTAL MATERIALS AND METHODS 4
4.1 AgNP Materials
4.2 Dispersion Preparation
4.3 Methods and Instrumentation
5.0 RESULTS and DISCUSSION 6
5.1 Uncertainties in Measurement
5.2 Effects of Sample Matrix
5.3 Measurement Methods - Limitations
5.4 Detection of ENMs under pristine conditions
5.5 Environmental Media
5.6 Powders vs. Suspensions
5.7 Additional Measurement Methods
6.0 CONCLUSIONS 12
7.0 FUTURE RECOMMENDATIONS 13
Appendices
Tables 15
Figures 18
References 23
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FOREWORD
The United States Environmental Protection Agency (EPA) is charged by Congress to
protect the nation's natural resources. Under the mandate of national environmental laws, the
EPA strives to formulate and implement actions leading to a compatible balance between human
activities and the ability of natural systems to support and nurture life. To meet this mandate, the
EPA's Office of Research and Development (ORD) provides data and scientific support that can
be used to solve environmental problems, build the scientific knowledge base needed to manage
ecological resources wisely, understand how pollutants affect public health, and prevent or
reduce environmental risks.
The National Exposure Research Laboratory (NERL) is the Agency's center for
investigation of technical and management approaches for identifying and quantifying exposures
to human health and the environment. Goals of the laboratory's research program are to:
1) develop and evaluate methods and technologies for characterizing and monitoring air, soil,
and water; 2) support regulatory and policy decisions; and 3) provide the scientific support
needed to ensure effective implementation of environmental regulations and strategies.
This report presents the experimentation, results and findings to date, relating to methods
of characterization of sample solutions containing nanoparticles of silver and polystyrene, using
nanoparticle tracking analysis (NTA). Also, this report is directed at confirming the efficacy of
NTA and comparing it to other methods to determine nanoparticle sizes and size distributions in
water samples. Included in the report are recommendations for additional work to address issues
identified herein.
NTA technology allowed for the direct visualization nanoparticles and has a unique
software tracking system to determine particle sizes. The goal of this research was to confirm
the applicability of NTA as a screening method for nanoparticles in environmental water
samples. An additional potential goal would be to support site remediation programs (e.g.,
Department of Defense and EPA Office of Solid Waste and Emergency Response) and other
applications to identify possible engineered nanomaterials contamination and to track the
transport and fate of these nano-contaminants.
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Acknowledgement
The authors would like to acknowledge the support of Dr. Manomita Patra for her
laboratory and technical support, and day-to-day operations.
Dr. Patra's research was supported through EPA funding of a National Research Council
(NRC) Post-doctoral Fellow grant. Mrs. Tammy Jones-Lepp was the EPA-NRC advisor for her
laser detection of nanoparticles research project.
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EXECUTIVE SUMMARY
This report covers efforts to investigate a relatively new technology, nanoparticle tracking
analysis (NTA) for use in identifying nanoparticles. NTA is a laser-based instrument capable of
real-time tracking of nanoparticles in solution. NTA was evaluated as a stand-alone screening
method to identify and calculate particle size distributions of metal-based, engineered
nanomaterials (ENMs) in situ and in laboratory synthesized environmental water samples.
Based on the information considered here, overall NTA sensitivity appeared to be good for
particle sizes in the 20 - 40 nm range, and larger sizes, including the ability to follow the real-
time formation of aggregates. Reliable data was more difficult to obtain for particle sizes below
the 20 nm size range. Benefits of the NTA technology were that the instrumentation allowed for
visualization of nanoscale particles in liquids on an individual basis, provided nanoparticle size
and distribution data for each sample, and was capable of measuring large numbers of individual
particles in substantially less time than required by microscopy techniques, i.e., transmission
electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy
(AFM). Also included in this report are comparisons of NTA with a number of other
nanoparticle characterization technologies including ultraviolet-visible spectroscopy (UV-Vis),
dynamic light scattering (DLS), TEM, and ultra small angle X-ray scattering (USAXS).
In addition to size distribution, there are several other aspects of nanoparticle tracking to be
considered. These include sensitivity/selectivity to discriminate between metal-bearing ENMs
and other background naturally occurring nanomaterials in water samples, influence of particle
surface coatings and particle tendencies to agglomerate, among others. These issues are
discussed and if NTA is used as a screening technique it is suggested that a complementary
nanodetection method, e.g., SEM, TEM, AFM, be considered as a part of the screening process.
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LIST OF ACRONYMS AND ABBREVIATIONS
APS Advanced Photon Source
AFM Atomic Force Microscopy
AF4 Asymmetric Field Flow Fractionation
AgNP silver nanoparticle
DI deionized
DLS Dynamic Light Scattering
ECs emerging contaminants
EDX energy dispersive X-ray
ENM engineered nanomaterials
EPA Environmental Protection Agency
ESD Environmental Sciences Division
eV electron volts
g gram
HEASD Human Exposure and Atmospheric Sciences Division
HDC hydrodynamic chromatography
h hour
ICP-MS Inductively coupled plasma-mass spectrometer
k 1000
kg kilogram
LIBD Laser-Induced Breakdown Detection
L liter
mg milligram
MHRW moderately hard reconstituted water
mL milliliter
MS mass spectrometer
ng nanogram
nm nanometer
NERL National Exposure Research Laboratory
NIST National Institute of Standards and Technology
NIST-NCL National Institute of Standards and Technology-Nanotechnology
Characterization Laboratory
NOM natural organic matter
NP nanoparticle
NRC National Research Council
NTA Nanoparticle Tracking Analysis
ORD Office of Research and Development
OSWER Office of Solid Waste and Emergency Response
PI polydispersity
ppb part-per-billion
PPE personal protective equipment
PPCP pharmaceutical and personal care product
PVDF polyvinylidene
PVP polyvinylpyrrolidone
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LIST OF ACRONYMS AND ABBREVIATIONS (continued)
s
second
SEC
Size exclusion chromatography
SEM
Scanning Electron Microscope
SP
single particle
SPR
Surface Plasmon Resonance
TEM
Transmission Electron Microscopy
|iL
microliter
UNLV
University of Nevada - Las Vegas
US
United States
USAXS
Ultra Small Angle X-Ray Scattering
UV
ultraviolet
UV-Vis
Ultraviolet Visible Spectroscopy
V
volts
WWTP
wastewater treatment plant
Z-avg
nanoparticle equivalent hydrodynamic diameter
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1.0 INTRODUCTION
Naturally occurring nanoparticles have always been around, created either by the weathering of
minerals (e.g., gold, silver, copper), forces of nature (e.g., forest fires, volcanoes), or incidentally
(e.g., emissions from combustion sources). However, anthropogenically engineered
nanomaterials are recent inventions (Owen et al. 2007; Lubick 2008; Farre et al. 2009).
"Nanotechnology" involves the manipulation and engineering of chemicals for structures in the
size range of 1 to 100 nm in at least one direction (one nanometer is 10"9 meters). These
nanomaterials can be considered as emerging contaminants (ECs), and are engineered from
nanometallic (e.g., silver, gold, iron) and nanocarbon (e.g., fullerenes) materials that are sized
between 1 nm and 100 nm. The Woodrow Wilson Institute, since 2006, has kept an on-line
database of the number of consumer nanomaterials products currently being offered on the
market (WWIC 2011). The number of nanomaterial-containing products has grown substantially
from 212 products listed in 2006 to 1317 products as of September 2011 (WWIC 2011).
It can be difficult to obtain information on total production volumes of nanoparticles (much of
which would be potentially available to the environment), because the production information is
often proprietary. Publicly available sources (which vary considerably) provide estimates of
nanoparticle Ti02 production exceeding 50,000 tons per year in 2010 in the United States (US)
(BusinessWire 2011). Worldwide production of silver nanoparticles is much lower, but has been
estimated at approximately 500 metric tons per year in 2008 (Mueller et al. 2008). The current
trend seems to be for increased production of all nanomaterials.
It can be expected that a large portion of nanomaterials will eventually find their way into the
environment, especially through wastewater treatment plants (WWTPs) (USEPA 2010). This
can be either through WWTP effluent, or via land-applied biosolids (the solid material leftover
during WWTP processes)(Geranio et al. 2009). As of 2002, WWTP biosolids usage, in the US,
was on the order of 3 million dry tons per year as amendments for soil treatment (NRC 2002).
However, the effects of nanoparticles on humans and the environment are not yet fully
understood, and there are mixed views on the risks. While there is evidence of nanosilver
toxicity to aquatic organisms found in the environment (Kennedy et al. 2010), there are currently
few known harmful effects on humans, and some possibly beneficial effects, from exposure to
certain nanoparticles. For example, nanosilver is presently used in medical applications as a
disinfectant in wound dressings and in catheters and breathing tubes to reduce incidents of
infections (NCCAM 2010). Moreover, in considering these risks, it is necessary to go beyond
just the particulate form of silver because, when released to the environment, the nanoparticles
can be converted to the ionic form of silver which can be more toxic than the particulate form
(Liu et al. 2010). Even less is known about human and environmental effects from other
nanomaterials (e.g., nanotitanium, nanoaluminum, nanocarbon) that are gaining wider usage.
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Given anticipated increases in total nanomaterials usage, the widespread and varied applications
for these materials, and a current lack of consensus standards and scientific opinion regarding
associated human and environmental risks, it is becoming ever more important to be able to
characterize nanomaterials in the environment. The development of a relatively quick and
reliable screening test(s) to correctly identify and characterize nanomaterials in various
environmental media would be beneficial. The ability to obtain such data more efficiently is
expected to aid researchers in focusing their efforts in evaluating the potential exposures and
risks posed by nanomaterials. To provide that ability was the objective for this effort on laser
detection of nanoparticles in the environment.
2.0 RESEARCH GOALS
The primary goal of this research effort was to evaluate the effectiveness and efficiency of a
relatively new instrumentation technology, nanoparticle tracking analysis (NTA). The NTA
technique (NanoSight, Ltd LM-20) uses a laser detection method to highlight nanoparticles,
which can then be viewed using a conventional optical microscope. NTA uses sophisticated
particle tracking software, based on Brownian motion and the Stokes-Einstein equation, to
calculate the sizes of the particles. NTA was evaluated to see if it could provide relative
concentrations and size distributions of metal-based nanoscale particles in liquid suspensions on
an individual basis.
The secondary goal of this research effort was intended to provide data for suitability
comparisons between the NTA technology and several other available detection methods (i.e.,
DLS, TEM, UV-Vis, and USAXS) which are detailed later in the report (Section 4.4). This
phase of research consisted of a lab-based study to initially establish the suitability of the
technology as a stand-alone method, or complementary to other engineered nanomaterial (ENM)
detection methods, to screen laboratory synthesized environmental waters for ENM particles and
potential ENM contamination.
3.0 NANOPARTICLE DETECTION METHODS
There are many techniques that have been reported in the literature that are used to detect and
size nanoparticles. Some are microscopy-based, like TEM, SEM and AFM (Bootz et al. 2004;
Buhr E et al. 2009; Boyd et al. 2011; Klein et al. 2011). Others are based on light and
absorbance or emission of light through back-scattering of the light and use of software to track
the Brownian motion of the light using the Stokes-Einstein equation, such as DLS and NTA
(Filipe et al. 2010; Boyd et al. 2011). Other techniques are based on size exclusion with a
detector at the end, for example, size exclusion chromatography (SEC), gel electrophoresis,
asymmetrical flow field-flow fractionation (AF4), ultracentrifugation, and hydrodynamic
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chromatography (HDC) [coupled with inductively coupled plasma-mass spectrometry (ICP-
MS)](Bootz et al. 2004; Brown et al. 2006; Messaud et al. 2009; Pergantis et al. 2011)
The research reported herein includes comparisons of NTA with several of the characterization
technologies: UV-Vis, DLS, TEM, and USAXS.
3.1 General Discussion. The NTA, UV-Vis and TEM research was conducted by EPA: Dr.
Manomita Patra, a National Research Council (NRC) post-doctoral research fellow [Mrs.
Tammy Jones-Lepp (National Exposure Research Laboratory-Environmental Sciences
Division, NERL-ESD) was the EPA-NRC advisor for Dr. Patra] collected the NTA and UV-
Vis data, and the TEM data was collected by Dr. Kim Rogers (NERL-Human Exposure
Atmospheric Sciences Division, HEASD). The DLS, AFM, USAXS data were collected by
Dr. Robert MacCuspie, Andrew Allen, Matthew Martin, and Vincent A. Hackley at National
Institute Standards and Technology (NIST), Gaithersburg, MD.
3.2 Assessment Criteria. To provide a suitable evaluation of available methods for
characterizing nanoparticles in the environment, a multi-step evaluation was used. This
evaluation included examining the following criteria:
1. Ability to detect particles in the 1-100 nm size range.
2. Allow visualization and counting of nanoparticles on an individual basis.
3. Detection of particles in pure water, natural organic material water, and solvents.
4. Detection in concentrations in the 106 to 109 particles per mL range.
5. Sensitivity to differentiate between ENM and natural nanoparticles.
3.2 Initial Candidate List. Only nanosilver (AgNP), and polystyrene beads, with particle
sizes of 1-100 nm, were evaluated. The intent was to identify a screening test with wide
applicability and the comparisons discussed herein included both laser and non-laser detection
methods. Following is a list of each method evaluated and primary equipment used.
Method
EauiDment and Laboratory testing the eauioment
UV-Vis Spectroscopy
Perkin Elmer Lambda 750 spectrophotometer
Hewlett Packard Model 8453 (NERL-EPA) *
Dynamic Light Scattering
Malvern Instruments Zetasizer Nano (NIST)1'
Nanoparticle Tracking Analysis
NanoSight LM20 (NERL-EPA)
Transmission Electron Microscopy
FEI Technai TEM (NERL-EPA using UNLV n
equipment)
Atomic Force Microscopy
Dimension 3100 AFM (NIST)
Ultra Small Angle X-Ray Scattering
Advanced Photon Source (APS) (NIST)
"3R 5
National Exposure Research Laboratory-Environmental Protection Agency; ' National Institute
of Standards and Technology; ^ University of Nevada-Las Vegas
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4.0 EXPERIMENTAL MATERIALS AND METHODS
4.1 AgNP Materials. AgNPs were selected to provide as broad of a range as possible of
sizes, capping agents, and powder/suspension states. The vendor-reported nominal diameters
were relied upon as accurate and were used as the diameters for AgNPs. Two separate batches
of AgNPs were obtained for use at NERL-Las Vegas and NIST. Figure 1 illustrates a flow chart
of samples received and subsequent preparation techniques.
Citrate-capped AgNPs were obtained from several sources, including (10, 20, and 100) nm
from the NanoXact product line (Nanocomposix, San Diego, CA, USA), (20, 40, 60, and 80)
nm from the unconjugated silver colloids BBI product line (Ted Pella, Redding, CA, USA),
and 20 nm from Microspheres Nanospheres (Cold Spring, NY).
Starch-capped (10 to 15) nm AgNPs were obtained from Strem Chemicals (Newburyport,
MA, USA).
Polyvinylpyrrolidone (PVP)-capped AgNPs (10 and 50) nm were obtained from the
NanoXact product line (Nanocomposix, nominal, San Diego, CA, USA) and (10, (30 to 50),
and 50) nm from NanoAmor (Houston, TX, USA).
Oleic acid capped (30 to 50) nm AgNPs were obtained as powders from NanoAmor
(Houston, TX, USA). No capping agent specified AgNPs (1 to 10) nm were obtained from
Vive Nano (Toronto, Canada).
4.2 Dispersion Preparation. AgNPs were analyzed as-received, diluted with deionized
(DI) water, diluted with EPA moderately hard reconstituted water (MHRW) (Hackley et al.
2007), or MHRW plus Suwannee River Fulvic Acid Standard I or Suwannee River Humic
Acid Standard II (1S101F and 2S101H, respectively, International Humic Substances
Society, St. Paul, MN, USA) at a natural organic material (NOM) concentration of
10 |ig mL"1. AgNPs were added to diluents, shaken by hand for 2 s, then allowed to stand for
at least 1 h before measurements began. Stock dispersions of AgNPs received as powders
were prepared by adding 1.0 mg of AgNP powder to 1.000 mL of DI water and sonicating in
a bath sonicator for 10 min.
Before some measurements of the reconstituted AgNPs were made, the suspensions were
passed through a 0.45 |im polyvinylidene fluoride (PVDF) syringe filter. DI water was
produced using 18.2 MQ*cm water obtained from a Aqua Solutions (Jasper, GA, USA) Type
I biological grade water purification system outfitted with an ultraviolet lamp to oxidize
residual organics and a low relative molecular weight cut-off membrane, then passed through
a 0.1 |im PVDF syringe filter before use. All AgNPs were stored at 4 °C in the dark in their
original containers, and were characterized within 6 months of receipt from vendors.
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4.3 Methods and Instrumentation
In this section only those instalments that were used at EPA-NERL-Las Vegas to evaluate
alongside NTA will be discussed, i.e., NTA, UV-Vis and TEM.
4.3.1 Nanoparticle Tracking Analysis (NTA). Solutions were diluted to an approximate
AgNP concentration range of 109 AgNPs per mL, and injected via a 1 mL disposable syringe
into aNanosight LM20 (Nanosight, Amesbury, United Kingdom) equipped with a 633 nm laser
and low sensitivity detector. Rinsing with filtered DI water between samples cleaned the liquid
cell. Fresh DI water, post-rinsing, was checked to ensure no cross-contamination of AgNPs
occurred. The optics were adjusted by finding the immobile area of diffraction from the laser
beam, or the so-called fingerprint area, and moving the liquid cell so that the volume closest to
the fingerprint without interferences was the volume observed. Camera settings were adjusted
empirically by maximizing the brightness of the AgNPs while minimizing any background light.
Videos were collected for 30 s. Post-collection analysis parameters were adjusted empirically to
maximize the number of particles correctly identified by the proprietary software (NTA version
2.0) while simultaneously minimizing the number of noisy pixels incorrectly identified.
As with most measurement techniques, incorrect use of the instrument can produce incorrect or
misleading data. Thus, video analysis parameters including blur, detection threshold, gain,
brightness, and number of completed tracks were adjusted independently, and systematically, to
maximize the number of completed tracks obtained during a video analysis before reporting
sizing results using those processing conditions.
4.3.2 UV-Vis Spectroscopy. UV-Vis spectra were collected on both a Perkin Elmer
(Waltham, MA, USA) Lambda 750 and a Hewlett Packard (HP) 15 Model 8453
spectrophotometers, using UV-transparent disposable plastic semi-micro cuvettes (Brandtech,
Inc., Essex, CT, USA), with a 1-cm path length, requiring 1 mL sample volumes. The Perkin
Elmer spectrometer uses a split-beam configuration equipped with an 8 + 8 cell changer and
water-jacketed 20 temperature control; measurements were performed at 25.0 ± 0.2° C. The HP
instrument has a single beam configuration, and measurements were taken at 25 °C.
Concentrated AgNP solutions were diluted such that the initial absorbance was approximately
1.0 at the surface plasmon resonance (SPR) absorbance peak wavelength (X 25 max).
4.3.3 Transmission Electron Microscopy (TEM). NIST-Nanotechnology
Characterization Laboratory (NCL) Joint Assay Protocol PCC-7 was broadly followed
(Bonevich et al. 2010). Briefly, amine-functionalized silicon TEM windows (Dune Sciences,
Eugene, OR, USA) were immersed into an AgNP solution, incubated for 1.0 min, and then
rinsed by immersion into filtered DI water. Images were acquired at 300 kV using a Technai
TEM (FEI, Hillsboro, OR, US) with a 2k Gatan camera. The imaged structures were also
analyzed by energy dispersive X-ray (EDX) spectroscopy for elemental composition using an
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EDX detector. At least five locations on the TEM window (grid) were examined, and between
50 and 90 images were recorded. Image J software was used for image analysis, freely available
on the internet (Rasband 1997-2011; Abramoff et al. 2004). Sizes were measured by using the
line distance-measuring tool across the diameter of the AgNP, calibrated to the scale bar
imprinted on the TEM.
5.0 RESULTS AND DISCUSSION
5.1 Uncertainties in Measurements. The reported values from microscopy
measurements are often the mean of all NPs measured with an uncertainty of one standard
deviation about this mean. The uncertainty in this case reflects the width of the size distribution.
Uncertainties in the mean of number-based particle sizing measurements for NTA can be
reduced by increasing the number of particles sampled. This can be easily done by increasing
the number of tracks recorded. On the other hand, techniques like DLS typically report the mean
of several measurements performed under repeatability conditions with an uncertainty of one
standard deviation about the mean of those measurements. Wherein, the uncertainty in this case
represents the repeatability or precision of the measurements, and does not reflect the width of
the distribution.
5.1.1 NTA. NTA mean and mode diameters were based on the number of AgNPs sized,
or the number of completed tracks. Analysis of each sample yielded 100-500 individual track
tracings that were used to determine mean, mode and relative standard deviation measurements.
For this type of measurement, the variation about the mean is not an indication of the precision
of multiple measurements of a single value, but is rather representative of the particle size
distribution. For this reason, standard deviation values may be misleading and were not reported
for this technique. Reported means for each particle type (Table 1) represent the average of five
mean values from separate sample measurements. A useful indication of the relative size
distribution measured using the NTA technique may be derived by comparison of mean and
mode measurements (Table 2). The mode was typically associated with a peak (most commonly
measured particle size to the nearest nm) close to the nominally reported particle diameter.
5.1.2 TEM and AFM. Error bars for TEM and AFM represent one standard deviation
about the mean for all particles counted in a sample. Therefore, the error bars are representative
of the width of the distribution and are not reflective of the uncertainty associated with
determining the mean size.
5.1.3 DLS. For DLS, the mean of five replicate Z-avg measurements under repeatability
conditions is reported with error bars of one standard deviation about the mean of the Z-avg
values. The DLS error bars represent the repeatability of the DLS measurement (the ability to
achieve the same measurement result consecutively) and do not indicate the width of the size
distribution.
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5.1.4 USAXS. For USAXS, an estimated measurement uncertainty of ± 10 % is
assigned to all values reported, based on previous experience measuring NIST AuNP reference
materials RM8011, RM8012, and RM8013 (NIST 2008; NIST 2008; NIST 2008).
5.2 Effects of sample matrix. The conditions under which the materials are
"initially" characterized can impact their reported size and size distributions, which in turn are
used as the basis for interpretation of test results. To compare the strengths and weaknesses of
each measurement technique, the AgNPs selected for the studies were analyzed as received or
diluted with DI water (these are referred to as pristine conditions; Table 1). Alternatively, the
samples were diluted into either MHRW, or MHRW with NOM standard I or II (MHRW+I or
MHRW+II, respectively). The sizing results for each dispersion condition are summarized in
Tables 1 and 2.
Figure 2 illustrates the differences between the reported nominal size and observed mean size for
the citrate-capped AgNPs for the pristine materials (Figure 2a), and dilution into environmental
media, MHRW (Figure 2b). The range of deviations from the nominal vs. measured line (dashed
lines in Figure 2) illustrates the complexity of intercomparing results. Among the citrate- and
PVP-capped AgNPs from various sources, certain trends were noted.
For example, for those particles that the nominal size was reported at < 20 nm, the dry-state
measurements provided by AFM and TEM showed average diameter values that differed by less
than 10 nm from the nominal reported values in pristine conditions. However, the average
hydrodynamic diameter measurements from NTA and DLS showed a difference of > 200 nm,
for those particles <10 nm. In pristine waters, in general AgNPs that were nominally < 20 nm
(Samples A, I, J, M, and N), mean diameter measurements by AFM and TEM ranged from 7.9
nm to 12.4 nm and 3.6 nm to 13.2 nm, respectively. By contrast, hydrodynamic diameters, as
measured by DLS and NTA, ranged from 22.7 nm to 547 nm and 28.8 nm to 195 nm,
respectively. The larger increases (> 300 % for DLS) for the hydrodynamic diameter
measurements of these AgNPs suggest agglomeration of AgNPs. This would suggest an
influence from surface coatings, a few large-sized outliers, and/or the presence of small
agglomerates.
In pristine conditions, for most of the citrate- or PVP-capped AgNPs that were > 20 nm DLS and
NTA still followed a measurement method trend of measuring larger than the nominally reported
sizes, but not as severe as at the smaller, < 20 nm, particle sizes. TEM showed the most
accuracy overall for all sizes of AgNPs, while AFM showed a trend of measuring less than the
nominally reported size for those AgNPs that were > 60 nm.
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In solutions with MHRW, again we see that DLS and NTA trended toward measuring larger than
the reported nominal size for those AgNPs that were < 10 nm. For those particles > 20 nm, NTA
performed better than DLS in measuring closer to the nominal reported sizes, but both NTA and
DLS still trended toward measuring larger than the nominal reported size, for all AgNPs. Again,
suggesting an influence from surface coatings, a few large-sized outliers, and/or the presence of
small agglomerates.
For those AgNPs in MHRW that were > 40 nm, the AFM measurement technique trended
toward measuring less than the nominal reported value.
It is important to note that for multimodal distributions of AgNP sizes, as measured by DLS and
NTA, the average size of all AgNPs observed in a number distribution may not be the most
informative way to report the size. Identification of the mean or mode of each peak in the size
distribution may be more appropriate (this will be discussed later in Sections 5.4 and 5.5). From
a biological perspective, when attempting to assess the risk of a specific size ENM crossing
certain biological barriers, or the available AgNP surface area per unit volume of solution, the
small AgNPs in a sample may become critical to successful interpretation of data.
5.3 Measurement Methods - Limitations. All of the methods considered: NTA,
UV-Vis, DLS, TEM, AFM and USAXS; can provide one or more measures of characterization
of ENMs in liquid dispersions. However, no one method provides all of the capabilities that
might be useful and/or desired. Therefore the following comments are only intended to provide
insights into the relative merits of the methods considered for comparison to NTA in this report.
5.3.1 NTA provides data on nanoparticle size (hydrodynamic diameter) and distribution.
This technique requires a somewhat narrow concentration range in order to be effective,
and data for nanoparticles in sizes below 20 nm becomes questionable except for
particles with a high refractive index (e.g. Au or Ag).
5.3.2 DLS provides data on size (hydrodynamic diameter) and distribution. Since it
depends on Raleigh scattering proportional to particle diameters; larger particle diameters
will tend to dominate the intensity signal. DLS does not collect intensity (brightness)
data and may not be able to distinguish between particles of comparable size but of
different composition.
5.3.3 UV-Vis method is typically used to provide quantitative data on total concentration
of nanoparticles, but only qualitative information regarding size and size distribution of
nanoparticles.
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5.3.4 TEM and AFM provide size and distribution data over all size ranges but are
sensitive to sample preparation methods which can impact results. Both TEM and AFM
tend to respond more to particle core sizes and may not fully reflect particle coatings
and/or associated molecules that would increase the hydrodynamic diameter.
5.3.5 USAXS (similar to TEM) provides size and distribution data but is apparently not
as effective in detecting small AgNPs (e.g. < 5 nm), especially in sample populations
which also contain substantial numbers of large diameter NPs.
Since NTA and DLS both measure hydrodynamic diameters (reflecting coatings and aggregates),
and AFM and TEM both measure diameters based on particle core size (not reflecting coatings
or associated surface elements), these techniques are consequently complementary and one of
each would be required to adequately characterize particle and aggregate size distributions.
Consideration should also be given to the use of Scanning Electron Microscopy (SEM) in lieu of
TEM. The SEM resolution is perhaps an order of magnitude less than TEM, but is still adequate
for particles of about 1 nm in size. The SEM can image large bulk samples, has good depth of
field for 3D image representations, and can be set up to produce adequate quality and resolution
for environmental ("wet") samples.
5.4 Detection of ENMs under Pristine Conditions. Figure 2a illustrates the range of
mean sizes that could potentially be reported for AgNPs under pristine conditions, and Table 1
details the numerical sizing results.
The hydrodynamic diameters, as measured by DLS and NTA range from 22.7 nm to 547 nm and
18.2 nm to 195 nm, respectively. In contrast, the nominal sizes, reported by the vendors, ranged
from 10 nm to 100 nm. The larger hydrodynamic diameters, sometimes greater > 300% than the
nominal range, suggest the presence of AgNP agglomerates even in pristine water.
Agglomerates can be common for some of these particle types in solution in that clusters of
nanos combine to be seen as single particles. Figure 4a, sample O shows that this agglomeration
is probably what occurred in measuring the sample with NTA. The nominal size of sample O is
30-50 nm, but the hydrodynamic diameter mean measured by NTA is around 75 nm in pristine
water. The NTA mode values, however, trended toward a more accurate representation of the
nominal size of the AgNPs, than the mean values, Figures 3 and 4, but still misrepresented the
nominal sizes.
5.5 Environmental Media. The diameter of the AgNPs in environmental media, just like
in pristine waters, can be expected to differ depending on several factors, including the
measurement technique, the thickness of the initial capping agent, and in the case of
environmental waters, the thickness of the natural organic matter (NOM) layer sorbed onto the
AgNPs. The capping agent and NOM density on the surface and ability of NOM molecules to
9
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competitively displace the initial capping agent also impacted the observed size by NTA and
DLS.
For example, when in MHRW, particle agglomeration, as seen by the NTA mean diameter, was
around 130 nm, when the nominal size of the nanoparticle was reported to be 10 nm (Figure 3b).
However, the NTA mode measured more accurately, in most cases, the nominal sizes of AgNPs
in MHRW (Figure 3b). With the exceptions to this observation in that the NTA mode size was
off significantly in samples M and O (Figure 4b). Again, the errors of the NTA mode
measurements were probably due to the formation of agglomerates between AgNPs and the
NOM in the water sample.
The results in Table 2 suggest that the types of NOM, as well as, the length of time between
dispersion preparation and examination, are critical experimental factors to report alongside size
distributions of "stock" AgNPs.
5.6 Powders vs. Suspensions. AgNPs received as aqueous suspensions more frequently
exhibited a narrow size distribution compared with the powder stock sources, which required a
subsequent dispersion step. The quality of dispersions formed from the dry powders varied
widely, depending upon many factors, including those associated with the application of
ultrasonic energy (e.g., input power, temperature, geometry of the sonicator and solution vessel),
the selection or inclusion of capping agents, and solution chemistry (Taurozzi et al. 2011).
Recently, protocols addressing how to prepare (Taurozzi et al. 2010), and report the preparation
of these powders (Taurozzi et al. 2010) has been published.
There can also be temporal changes to the quality or stability of powder dispersions. For
example, serial dilutions of a powder of PVP-capped AgNPs dispersed into DI water were
examined by collecting an absorbance spectrum every hour for 96 h after treatment in a bath
sonicator and vortexing for approximately one min. This experiment revealed what might
typically be reported for an absorbance spectrum over the wavelength range of 300 to 800 nm.
At longer wavelengths, there occurred a turnover to a broad absorbance spectrum, which was
nearly featureless. This suggested that the sample was very polydisperse, significantly
aggregated, in a suspension that settled below the beam-path nearly completely (approximately
90 % loss of absorbance) over a few days.
5.7 Additional Measurement Methods. In addition to the information provided above for
the methods of nanoparticle detection, other references provided further insights into the use of
NTA for screening test purposes.
5.7.1 Filipe et al., compared NTA with DLS for measurement of nanoparticles and
protein aggregates (Filipe et al. 2010). The evaluation was conducted using polystyrene beads
(size range of from 60 nm to 1000 nm) in addition to drug delivery and protein aggregate
10
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particles (minimum size of about 1 nm). The evaluation concluded that NTA could "...
accurately analyze the size distribution of monodisperse and polydisperse samples. Sample
visualization and individual particle tracking are features that enable a thorough size distribution
analysis. The presence of small amounts of large (1000 nm) particles generally does not
compromise the accuracy of NTA measurements, and a broad range of population ratios can
easily be detected and accurately sized."
However, the study also concluded that while the presence of large particles had little impact on
sizing accuracy it did reduce the number of small particles detected by the software. The study
further noted a lower detection limit for nanoparticles (protein samples) of about 30 nm for NTA
and remarked that the analysis can be relatively more time consuming than other nanoparticle
measuring techniques, and required optimization steps by a skilled operator.
5.7.2 Bundschuh et al., describes the Laser-induced Breakdown Detection (LIBD)
method for determination of mean particle size and concentration in aqueous samples
(Bundschuh et al. 2005). The method was described as capable of detecting particles sizes
ranging from about 10 to 1000 nm, and in concentrations in the range from about 1 ng/L up to
mg/L. This technique used a high-energy pulsed laser beam to selectively generate plasma on
particles and was described as being non-invasive, not requiring sample preparation and allowing
on-line measurements. This method did not provide for determination of size distributions and
did not allow for differentiation between inorganic, organic and biological particles. A typical
on-line measurement can be performed in several minutes.
5.7.3 Pergantis et al., coupled hydrodynamic chromatography (HDC) with inductively
coupled plasma-mass spectrometry (ICP-MS) for detecting metal-containing ENMs in
environmental matrices (Pergantis 2010). HDC is suitable for sizing nanoparticles within the
range of 5 to 300 nm, and has rapid analysis time. HDC coupled with the selectivity and
sensitivity of ICP-MS, makes this a promising analytical tool for investigating the fate of
nanoparticles.
However, one serious drawback with this technique was that it could not distinguish between a
large concentration of nanoparticles, containing a small metal fraction, and a low concentration
of the same size nanoparticles, with a high metal fraction. To address this, Pergantis et al.,
investigated the use of single particle (SP) ICP-MS coupled with the HDC (Pergantis et al.
2011). SP-ICP-MS demonstrated the capability being able to simultaneously determining the
concentration of metal-containing nanoparticles and measuring the metal mass in individual
AgNP
11
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6.0 CONCLUSIONS
6.1 Based on the results of the research, and reviews of current literature, no one testing
technique appeared to provide all of the desired measurement information for a screening test to
characterize nanoparticles in the laboratory and environmental water samples. The evaluations
suggest that the most effective approach would be to employ at least two complementary
methods, a microscopy-based (e.g., SEM, TEM, AFM), and a dynamic light-based method (e.g.,
NT A, DLS), to meet desired criteria.
6.2 The laser detection technique using NTA appeared to be a good candidate for one of
the two complementary methods noted above. The method described in this report used the
NanoSight Ltd LM-20 instrument system together with the NTA software to perform the
analyses. In this evaluation, the major advantages of this system were that it provided data on
nanoparticle sizes and distribution, and that it allowed direct/real-time visualization of the
nanoparticles in a sample.
Other observations regarding NTA are that it:
1. Does not appear to be materials-specific with regard to ENMs. It has been shown to perform
satisfactorily when analyzing metallic nanoparticles.
2. Is size-sensitive above approximately 20 nm and can analyze multi-sized particle dispersions.
Results are less reliable below 20 nm to 10 nm with best results only for particles with a high
refractive index (e.g., Ag and Au).
3. Has detection capabilities that do not seem to be hampered by surface coatings, with the
limitation that indicated particle sizes may be for individual particles or for particle
agglomerates.
4. Have detection capabilities that should not be affected by agglomeration (results would be
expected to show an increase of larger size particles in the sample distribution).
5. Has been tested for nanoparticles only (ionic form testing has not been addressed).
6. Has screening level capabilities that are satisfactory for two "reconstituted" (contaminated)
samples. Additional testing will be needed for a wider array of samples.
7. Have levels of sensitivity to discriminate between ENMs and other materials that will require
further evaluation to determine whether nanoparticle materials (besides Ag) can be differentiated
from other natural and/or man-made particles in solution.
In the final analysis, the selection of a complementary screening technique to NTA should be
made with regard to the additional considerations noted below.
12
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7.0 FUTURE RECOMMENDATIONS
For this report, detection of nanoparticles using NTA was the primary focus. There are a
substantial number of related items which were beyond the scope of this report which have to do
not only with the ability to detect ENMs, but also with the potential toxicity of detected materials
in environmentally relevant suspensions.
7.1 Additional Considerations. Related physical and chemical features that are important
to the full characterization of nanoparticles are as follows:
1. Nanoparticle Material Composition. The evaluations described in this report were
conducted using silver nanoparticles (AgNPs) because they are in widely used in commercial
products today. However, there are a number of other nanoparticle materials in various forms in
current use (e.g., Au, Ti, Al, C). Therefore, any nanoparticle screening test should address these
types of nanomaterials as well. Additional testing of the NTA method using nanoparticles other
than Ag (e.g., Au, Ti, Al, and/or C) should be performed. NTA performance for Ag samples has
initially been established for "pristine conditions" and for one NOM containing water. The NTA
method worked well for particles with a high refractive index (i.e., AgNP) and testing other
nanomaterials with different refractive indices should be instructive, particularly with regard to
detecting smaller nanos (in the range of 1 to 10 nm).
2. Other variables. For toxicity evaluation, nanoparticle sizes, concentrations and exposure
times may all be relevant variables.
3. Particle coatings and capping agents. Particle coatings combined with capping agents and
other surface coatings should be tested with NTA. As was seen in the data tables, capping agents
and other surface coatings, such as citrate, starch and polyvinylpyrrolidone can affect test
measurement results since some test methods react more strongly to particle core (uncoated) size
as opposed to overall size. There are data suggesting that the use of coatings can affect the
toxicity of the NPs compared to the pure form (Allen et al. 2010). Several of the nanoparticle
types tested have these properties, but further attention is warranted.
4. Aggregation and agglomeration tendencies. There are data available that indicates that the
AgNPs have a tendency to agglomerate in solution and settle out (Fabrega et al. 2011). This
could affect availability, transport mode and eventually the potential toxicity of the AgNPs, and
should be addressed.
5. Ag Nanoparticles vs. Ionic Form. There are indications that both forms can be toxic
(Kennedy et al. 2010) and any tendencies for changes in form due to chemical effects or other
factors need to be better understood.
6. Sample matrix composition. The samples tested by NTA were limited to two standardized
dispersions (i.e., DI water, MHRW). These provided a set of "baseline" conditions, but a wider
variety of contaminated waters (both field-obtained and laboratory samples) should be evaluated.
13
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7. Sensitivity and discrimination capabilities. The ability of a screening test to distinguish
between potentially toxic nanoparticles and other naturally occurring and/or man-made (non-
toxic) particles may be a significant challenge. Drinking waters, for example, contain substantial
nano-sized particulates in comparable concentrations that may, or may not, be toxic (Kaegi et al.
2008). Also, the possibility that particulates (other than those of specific interest in the
nanoparticle screening test) could be toxic should not be ignored.
8. Additional Testing. Testing of additional contaminated samples with a wider range of
contaminants (both sizes and types) is warranted. Results could be compared, using the same
samples, with results from SEM, TEM and/or AFM methods. The microscopy-based techniques
are effective for smaller nanoparticle sizes. They appear to be especially responsive to particle
core size and not necessarily to overall size, such that sample selection and/or preparation will be
important.
14
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TABLES
Table 1 Mean sizes of AgNPs under pristine conditions
Table 2 Mean sizes of AgNPs in MHRW
15
-------
Table 1. Mean sizes of AgNPs in DI water.
Sample Code
A
B
c
D
E
F
G
H
I
J
K
L
M
N
O
Nominal Size
(nm)
10
20
20
20
40
60
80
100
10
10
50
50
1-10
10-15
30-50
Capping Agent
citr.
citr.
citr.
citr.
citr.
citr.
citr.
citr.
PVP
PVP
PVP
PVP
unk.
starch
oleic
acid
Form Received
susp.
susp.
susp.
susp.
susp.
susp.
susp.
susp.
susp
powd
powd
susp.
susp
susp.
powd.
DLS mean Z-
avg (nm)
22.7
35.1
50.0
30.8
46.3
84.1
114
98.7
23.8
110
146
54.4
547
155
103.0
DLS std dev
(nm)[1]
1.9
0.2
1.6
1.2
0.6
0.9
1.9
1.2
1.0
6.7
1.0
0.8
7.5
1.2
1.3
DLS mean PI
0.377
0.536
0.354
0.095
0.292
0.155
0.113
0.073
0.374
0.416
0.231
0.195
0.597
0.280
0.332
AFM mean
height (nm)
10.8
21.3
15.6
23.6
39.5
44.7
56.3
39.5
11.0
12.4
104
46.4
7.9
10.2
97.3
AFM std dev
(nm)[2]
2.2
3.9
6.5
4.6
18.9
37.5
50.4
40.6
2.2
19.9
91.8
8.5
5.4
5.4
69.9
AFM # NPs
counted
107
102
100
100
131
71
44
100
102
100
102
100
102
106
105
NTA Mean Size
(nm) 131
28.8
44.0
48.2
28
74
106
111
130
54
...
...
39
195
18.2
75
NTA Mode Size
(nm)
20.0
33.2
39.0
51
62
87
106
122
26
...
...
30
21.8
16.6
38
# completed
Tracks
125
520
645
38
428
371
620
175
106
106
150
125
10.16
TEM Mean size
(nm)
8.0
20.3
20.9
29.2
95.6
9.0
49.8
3.6
13.2
46.2
TEM std dev
(nm)[2]
2.1
2.2
3.1
3.0
11.8
2.3
5.2
1.4
6.8
10.4
TEM# NPs
counted
81
80
80
51
75
74
74
80
NA
82
SAXS Mean
Size (nm)[4]
13.6
21.3
27.9
112
98.4
17.0
48.7
11.1
SAXS Peak %
of Total Vol.
Frac. [5]
99
83
71.2
100
100
95
100
82
SAXS Total
Vol. Frac. of All
AgNPs (xlO"6)
2.34
1.90
6.86
3.87
8.49
4.29
0.25
0.38
2.23
33.2
UV-Vis Xmax
(nm)
389
403
401
407
432
489
471
486
394
432
424
430
408
423
UV-Vis peak
Abs [6]
3.42
2.2
NA
0.96
0.54
0.3
0.19
0.75
76.16
NA
79.42
17.64
3.86
7.38
Abs/mass Ag
(Hg mL4)
0.17
0.11
NA
0.048
0.027
0.015
0.094
0.037
0.076
NA
0.079
0.012
0.24
0.008
Nominal diameters or size ranges are provided by the vendor. [1] DLS Zavg diameters are the mean of no less than five
experiments performed under repeatability conditions, with one standard deviation uncertainty from the mean. [2] AFM
heights and TEM diameters are the mean and one standard deviation of all AgNPs sized. [3] NT A mean and mode
diameters are based on the number of AgNPs sized, or number of completed tracks. [4] USAXS diameters are the mean
size of the most significant size distribution peak, i.e., the peak with the greatest percentage of the total volume fraction of
all AgNPs. [5] An estimated uncertainty of ± 10 % should be applied to all USAXS values. [6] UV-vis peak absorbance is
calculated for the undiluted stock solution, using the dilution factor for solutions that provided measured absorbance values
between 0.05 and 2.0. UV-Vis lambda max is the wavelength in nm of peak absorbance, and the absorbance per mass of
AgNP is in units of |ig mL"1. Dashes indicate no measurements using that technique were made on that sample.
16
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Table 2. Mean sizes of AgNPs in MHRW.
Sample
Code
A
B
D
E
F
G
H
I
J
K
L
M
O
Nominal
Size (nm)
10
20
20
40
60
80
100
10
10
50
50
1-10
30-50
DLS mean
Z-avg
(nm)
33
49.9
39.0
51.6
90.8
121
196
146
228
111
43.8
127
251
DLS std
dev (nm)
7.3
0.7
0.5
0.5
0.7
3.2
17.4
133
38.1
3.5
0.6
3.0
233
AFM
mean
height
(nm)
13.3
21.3
24.3
33.0
29.1
35.7
111
11.4
32.7
37.5
38.0
4.8
149
AFM std
dev (nm)
4.6
5.3
4.6
23.0
24.8
44.1
22.6
4.0
38.8
49.8
14.1
3.8
87.6
AFM#
NPs
counted
101
100
102
37
26
18
4
100
56
8
55
102
82
NTA
Mean
Size (nm)
133
42
56
60
85
104
104
...
...
...
52
62.7
131
NTA
Mode
Size (nm)
20
52
53
56
81
100
97
...
...
...
46
52.7
114
#
completed
Tracks
40
1128
1829
1653
7
403
1134
1869
646
293
Samp
e codes and uncertainties fol
ow the same conventions described in Table 1.
17
-------
FIGURES
Figure 1 Illustration of common measurement points of "stock" AgNPs
Figure 2 Measured particle mean size vs. "nominal" size reported by manufacturer
Figure 3 Distribution of NTA mean and mode measurements in (a) pristine and (b) MHRW
Waters - Samples A to H
Figure 4 Distribution of NTA mean and mode measurements in (a) pristine and (b) MHRW
Waters - Samples L - O
18
-------
(l)AgNP
synthesis or manufacture
Time
->~ (2) As-received, pristine
Time
(3a) Dilution
into MHRW
Time
(3b) Dilution into
MHRW + NOM
(4) Use in Experiments
Figure 1 Illustration of common measurement points of "stock" AgNPs - Scheme illustrating
some of the common points that measurements of "stock" AgNPs could take place. The variables of
time between points and storage conditions can affect the stability and degradation kinetics of the
AgNPs and thus the observed size and size distribution of the same lot of material.
19
-------
„ 200
|
S 150
to
£
100
o 50
w
f Af M
¦ NT A
~ TEM
~ USAXS
— — Nominal '
20 40 60 80 100
Nominal Size (nm)
500
m
£ 100
| 50
o
8
Jk*'
.M
20 40 60 80
Nominal Size (nm)
100
Figure 2 Measured particle mean size vs. "nominal" size reported by manufacturer.
Observed mean size by measurement technique plotted against the "nominal" size reported by
manufacturer for (a) pristine conditions and (b) dilution into MHRW. Samples A-H are plotted in (a)
and (b). All panels use same legend of points and lines shown in (a). Vertical bars in observed size
(y-axis) follow conventions described in the experimental section, keeping in mind that error bars for
different techniques have different meanings, from precision of the measurement to width of the
distribution; refer to Tables 1-2 for values smaller than symbols. Horizontal bars in nominal size (x-
axis) represent the range provided by the source, where appropriate.
20
-------
NTA Mean size
¦ NTA Mode size
Vendor ABCDE FGH
NTA Mean size (nm)
I NTA Mode size (nm)
(b)
20 20 40 60 80 100
B D E F G H
Figure 3. NTA Mean and Mode sizes of citrate-capped AgNPs in (a) pristine water;
(b) MHRW.
-------
250
200
150
100
50
0
PVP
nm 50
Vendor L
¦ NTA Mean size (nm)
¦ NTA Mode Size (nm)
¦
¦
h
unk.
1-10
M
starch oleic acid
10 - 15 30 - 50
N O
140
120
100
80
60
40
20
0
nm
PVP
50
Vendor L
unk.
1-10
M
¦ NTA Mean size (nm)
¦ NTA Mode size (nm)
(b)
oleic acid
30-50
O
Figure 4. NTA mean and mode sizes of different capped AgNps in (a) pristine water and (b)
MHRW.
22
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United States
Environmental Protection
Agency
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