Ultrasonic dispersion of nanoparticles for environmental, health and safety assessment issues and recommendations

Abstract

Studies designed to investigate the environmental or biological interactions of nanoscale materials frequently rely on the use of ultrasound (sonication) to prepare test suspensions. However, the inconsistent application of ultrasonic treatment across laboratories, and the lack of process standardization can lead to significant variability in suspension characteristics. At present, there is widespread recognition that sonication must be applied judiciously and reported in a consistent manner that is quantifiable and reproducible; current reporting practices generally lack these attributes. The objectives of the present work were to: (i) Survey potential sonication effects that can alter the physicochemical or biological properties of dispersed nanomaterials (within the context of toxicity testing) and discuss methods to mitigate these effects, (ii) propose a method for standardizing the measurement of sonication power, and (iii) offer a set of reporting guidelines to facilitate the reproducibility of studies involving engineered nanoparticle suspensions obtained via sonication.

Full text

Nanotoxicology, December 2011; 5(4): 711–729
Ultrasonic dispersion of nanoparticles for environmental, health and
safety assessment –issues and recommendations
JULIAN S. TAUROZZI
1
, VINCENT A. HACKLEY
1
, & MARK R. WIESNER
2
1
Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, and
2
Department of Civil and Environmental Engineering, Duke University, Durham, North Carolina, USA
(Received 9 July 2010; accepted 28 September 2010)
Abstract
Studies designed to investigate the environmental or biological interactions of nanoscale materials frequently rely on the use of
ultrasound (sonication) to prepare test suspensions. However, the inconsistent application of ultrasonic treatment across
laboratories, and the lack of process standardization can lead to significant variability in suspension characteristics. At present,
there is widespread recognition that sonication must be applied judiciously and reported in a consistent manner that is
quantifiable and reproducible; current reporting practices generally lack these attributes. The objectives of the present work
were to: (i) Survey potential sonication effects that can alter the physicochemical or biological properties of dispersed
nanomaterials (within the context of toxicity testing) and discuss methods to mitigate these effects, (ii) propose a method for
standardizing the measurement of sonication power, and (iii) offer a set of reporting guidelines to facilitate the reproducibility
of studies involving engineered nanoparticle suspensions obtained via sonication.
Keywords: Environmental assessment, toxicology, nanoparticle, nanomaterial, ultrasonics, sonication, dispersion, suspension
Introduction
Nanotechnology, and more specifically the emer-
gence of engineered nanoscale particles (ENPs),
shows promising potential for the development of
advanced materials and devices in the fields of med-
icine, biotechnology, energy, and environmental
remediation, among others (Zhang 2003; Salata
2004; Raimondi et al. 2005). Similarly, ENPs are
migrating into a wide range of consumer products,
many of which are now in the global market place
(Project on Emerging Nanotechnologies 2010). How-
ever, some of the properties that make ENPs attractive
may also pose environmental and health hazards
(Colvin 2003; Gwinn and Vallyathan 2006;
Kreyling et al. 2006; Dobrovolskaia and McNeil
2007; Kahru and Dubourguier 2010). As both public
and private funding for application-oriented nanoma-
terial research continues to grow and nanoparticle-
based consumer products reach the market, there is
an increasing demand for a more comprehensive
scientific understanding of the interactions of ENPs
in biological and environmental systems (Maynard
2006; Barnard 2006).
Moreover, the assessment of potential risks associ-
ated with ENPs requires the evaluation of their behav-
ior in environmentally and biologically relevant
matrices (e.g., cell media, serum, whole blood, stan-
dardized seawater). When ENPs are not already in a
fully dispersed form, the test suspensions are typically
obtained by dispersing dry powders or stock suspen-
sions of the source material into the desired test
medium prior to in vitro or in vivo evaluation
(Murdock et al. 2008; Jiang et al. 2009; Labille
et al. 2009). In these situations, the use of ultrasonic
energy, a process commonly referred to as ‘soni-
cation’, is arguably the most widely used procedure.
Sonication refers to the application of sound energy
at frequencies largely inaudible to the human ear
(higher than »20 kHz), in order to facilitate the
disruption of particle agglomerates through a process
known as cavitation. Ultrasound disruption is more
energy efficient and can achieve a higher degree of
powder fragmentation, at constant specific energy,
Correspondence: Dr. Vincent A. Hackley, National Institute of Standards and Technology, 100 Bureau Dr. Mail Stop 8520, Gaithersburg, 20899, MD, USA.
E-mail: vince.hackley@nist.gov
ISSN 1743-5390 print/ISSN 1743-5404 online 2011 Informa UK, Ltd.
DOI: 10.3109/17435390.2010.528846
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than other conventional dispersion techniques (Park
et al. 1993; Hielscher 2005; Mandzy et al. 2005).
Sonication is a convenient, relatively inexpensive tool
that is simple to operate and maintain. Consequently,
sonication has found extensive application in toxico-
logical and environmental studies, where it is used to
break down powders or re-disperse stock suspensions
to their nanoscale constituents and allow for the
evaluation of the properties and behavior of the dis-
persed nanoparticles in relevant systems.
However, the inconsistent application of ultrasonic
treatment across laboratories and between individual
operators, combined with the lack of accepted process
standardization, has likely contributed to variability in
observed results. Furthermore, the complex physical
and chemical phenomena that occur during sonica-
tion –and which may significantly alter the dispersed
material’s properties –are frequently underappreci-
ated, perhaps resulting in the use of suspensions that
may not meet the intended experimental design, or
potentially leading to artifacts that could compromise
conclusions as to how the observed ENP’s ecological
or toxicological behavior relates to the material’s
inherent properties. It is the authors’contention
that the inconsistent application and reporting of
sonication is a potential leading contributor to vari-
ability in test results in which sonication is utilized;
this conclusion has been echoed by the results of
recent interlaboratory studies (Roebben et al. 2010).
Moreover, while not the main focus of this work, it
is important to note that many of the sonication-
induced physicochemical effects and recommenda-
tions addressed herein are equally relevant to other
ENP research areas where sonication is utilized, in
particular those involving the fate and transport of
nanomaterials in environmental systems.
The objectives of the present work were to: (i)
Highlight salient aspects of the ultrasonic disruption
process and survey potential sonication-induced
effects that may alter the physical, chemical or bio-
logical properties and behavior of aqueous nanopar-
ticle suspensions (within the context of toxicological
testing), (ii) propose a method for standardizing the
measurement of sonication power, and (iii) offer a set
of reporting guidelines to facilitate the reproducibility
of studies involving engineered nanoparticle suspen-
sions obtained via sonication.
The authors aimed not only to indentify and discuss
key sonication issues relevant to ENP dispersion, but
also to address them by offering practical recommen-
dations. For this reason, some discussions will follow
a traditional literature review format, while others are
more prescriptive in style. The authors also recognize
that additional sample-specific preparation steps are
often required to achieve the desired state of
dispersion, but their consideration is beyond the
scope of the present work.
Terminology
We define at the outset several critical terms, which
are used throughout the text, in order to avoid ambi-
guity, as these terms can have different meanings in
different scientific and technical circles. For the most
part, we follow definitions relevant to nanotechnology
as set forth in the standard E2456 (2006) by ASTM
International (formally the American Society for
Testing and Materials). Additional guidance is
derived from recommendations of the International
Union of Pure and Applied Chemistry (IUPAC
2009).
For the purposes of this publication, a nanoparticle
is defined as a sub-classification of ultrafine particle
that is characterized by dimensions in the nanoscale
(i.e., between 1 and 100 nm) in at least two dimen-
sions, with the recognition that the more rigorous
definition of nanoparticle also requires that these
particles exhibit novel size-dependent properties.
Aprimary particle is the smallest discrete identifiable
entity associated with a particle system; in this con-
text, larger particle structures (e.g., aggregates and
agglomerates) may comprise primary particles. An
aggregate is a discrete assemblage of primary particles
strongly bonded together (i.e., fused, sintered, or
metallically bonded), which are not easily broken
apart. While the distinction is often blurred, aggre-
gates might also be differentiated from agglomerates,
which are assemblages of particles (including primary
particles and/or smaller aggregates) held together by
relatively weak forces (e.g., Van der Waals, capillary,
or electrostatic), that may break apart into smaller
particles upon processing (e.g., using sonication or
high intensity mixing). Commonly, agglomerates
exhibit the same overall specific surface area (SSA)
of the constituent particles, while aggregates have a
SSA less than that of the constituent primary particles.
In a related fashion, agglomeration and aggregation
refer, respectively, to the process by which agglom-
erates or aggregates form and grow. It should be noted
that, although here we define them as distinct entities,
the terms aggregate and agglomerate are often used
interchangeably to denote particle assemblies. Our
usage is consistent with previous recommendations
published in this journal (Oberdörster et al. 2007),
but is by no means universally adopted.
Additionally, in the present work, a suspension is
defined as a liquid (typically aqueous) in which par-
ticles are dispersed. The term dispersion is used in the
present context to denote the process of creating a
liquid in which discrete particles are homogeneously
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distributed throughout a continuous fluid phase (e.g.,
to obtain a suspension), and implies the intention to
break down agglomerates into their principal compo-
nent particles. Dispersion is also used as an adjective in
combination with terms relating to this process or
relating to the state of the resulting product.
Sonication basics and equipment
This brief overview highlights the basic aspects of
ultrasonic generation and propagation relevant to
particle dispersion, and serves as background for
the ensuing discussions; while it is not a comprehen-
sive review of the topic, readers can refer to the cited
references for a more in-depth treatment of the sub-
ject. During the process of ultrasonic disruption,
sound waves propagate through the liquid medium
in alternating high and low pressure cycles at fre-
quencies typically in the 20–40 kHz range. During
the low-pressure cycle (rarefaction), microscopic
vapor bubbles are formed in a process known as
cavitation (Figure 1). The bubbles then collapse dur-
ing the high pressure cycle (compression) producing a
localized shock wave that releases tremendous
mechanical and thermal energy.
Ultrasonic waves can be generated in a liquid
suspension either by immersing an ultrasound probe
(transducer horn) into the suspension (direct sonica-
tion), or by introducing the sample container into a
liquid that is propagating ultrasonic waves (indirect
sonication). This is shown schematically in Figure 2.
In a sonication bath or a so-called cup horn soni-
cator (indirect sonication), the sound waves must
travel through both the bath or cup liquid (typically
water) and the wall of the sample container before
reaching the suspension. In direct sonication, the
probe is in contact with the suspension, reducing
the physical barriers to wave propagation and there-
fore delivering a higher effective energy output into
the suspension. Bath sonicators typically operate at
much lower energy levels than are attainable using a
probe or cup horn. In the case of bath sonicators, the
transducer element is directly attached to the outside
surface of the metal tank, and the ultrasonic waves are
transmitted directly to the tank surface and then into
the bath liquid. In a cup horn, the radiating surface of
the horn is inverted and sealed into the bottom of a
(typically) transparent plastic cup into which the
sample container is immersed.
At ultrasound frequencies, the alternating forma-
tion, growth and subsequent shock waves produced
by the collapse of bubbles result in extremely large
localized temperatures up to 10,000 K, rapid temper-
ature changes (>10
5
Ks
1
), pressure bursts on the
order of several MPa, and liquid jet streams with
speeds reaching 400 km h
-1
(Mason 1989; Hielscher
2005). Such massive, local energy output is the basis
of the disruptive effect of sonication. It must be noted
that these considerable pressure and temperature
differentials are a result of the cavitation process
and occur at the local interface of the exploding
bubbles; consequently, these effects are inherent to
the sonication process and will occur whether the
sonicated container is cooled or not, or the treatment
performed using a bath, cup horn, or a probe
sonicator.
In all cases, the ultrasonic device transforms elec-
trical power into vibrational energy by means of a
piezoelectric transducer that changes its dimensions
in response to an applied AC electric field. In direct
sonication, the transducer horn serves to transmit and
focus the ultrasonic waves into the targeted liquid
sample. For direct sonication, the following equation
relates acoustic vibrational energy to probe and
medium parameters (Contamine et al. 1995):
P= cA f a
1
2)
22
ρπ(()21
Bubble
Low
pressure
cycle
High
pressure
cycle
Implosion
shock waves
Agglomerate
fracture points
Figure 1. Schematic illustration of ultrasonic wave-induced
cavitation and agglomerate fracture.
Probe Bath Cup
Figure 2. Schematic illustration of direct (left) and indirect (middle
and right) sonication configurations as described in the text.
Dispersion of nanoparticles 713
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where Pis the acoustic power (W) of the ultrasound
source, ais the ‘emission area’(m
2
), which is the
surface area of the emitting ultrasound source, ris the
liquid density (kg m
-3
), Ais the amplitude (m) of
oscillation of the ultrasound probe, cis the speed of
the acoustic wave in the liquid medium (m s
-1
) and fis
the vibration frequency (Hz).
The amplitude of oscillation per unit time deter-
mines the pressure difference between cavitational
rarefaction and compression cycles. Larger oscillation
amplitudes yield larger high-low pressure gradients
and consequently greater energy outputs. The probe’s
oscillation amplitude is in turn determined by the
amount of energy transferred by the instrument’s
generator to the ultrasonicator probe, which can be
regulated using the sonicator power settings.
Conventional direct sonicators operate by self-
adjusting their power consumption from the electrical
source in order to maintain the vibration frequency of
the transducer (e.g., quartz crystal) at a constant value
(usually 20 kHz). As it oscillates, the sonicator probe –
connected to the vibrating transducer –will experi-
ence a resistance from the sonicated medium that will
be transmitted back to the vibrating element and
detected by the internal control unit of the instru-
ment. The instrument’s control unit will in turn
adjust the power consumption of the instrument to
maintain a constant vibration frequency. Highly vis-
cous media will exert a greater resistance to the
oscillating probe, and will therefore require greater
power consumption to maintain a constant oscillation
frequency. For example, to produce an oscillation
with an amplitude of 3 mm at 20 kHz in deionized
water, a sonicator may consume ‘N’watts, while to
produce the same amplitude at 20 kHz in oil, it will
consume considerably more power.
The oscillation frequency value is usually fixed for a
given instrument and cannot be changed. Changing
the instrument power setting translates to a change in
the vibrating amplitude –not the frequency –of the
probe; that is, increasing the power setting results in
larger probe oscillation amplitudes. To maintain a
constant oscillation frequency, larger oscillation
amplitudes, as well as higher medium resistances,
require greater power consumptions from the electric
source.
A sonicator’s stated maximum power (e.g., 600 W)
refers to the maximum theoretical power it would be
able to consume, should it require so; it does not reflect
the actual amount of ultrasonic energy delivered to
the suspension. That is, for the same frequency and
amplitude (and thus the same sonicator setting), the
sonicator would need to consume more power to treat
a high viscosity suspension than it would for a low
viscosity suspension, and if so, it could consume up to
a maximum of 600 W. It is therefore erroneous to
assume that selecting the highest setting value will
result in the delivery of the instrument’s nominal
maximum power to the sonicated suspension. For
low viscosity media, even at the highest setting value,
the delivered power will be a significantly lower frac-
tion of the instrument’s maximum power rating.
Similarly, the power value (measured in W) usually
shown on the instrument display reflects the power
that the instrument is consuming from the electrical
source to produce the desired oscillation amplitude
(from the selected setting value) in the sonicated
medium. The consumed (i.e., displayed) power, how-
ever, does not necessarily reflect the power that is
actually delivered to the sonicated suspension, which is
affected by the probe’s oscillation amplitude in the
medium (see Equation 1). Herein lies the deficiency
with reporting either the manufacturer’s stated max-
imum power, the adjustable output power reading
provided by the instrument, or the chosen setting
level. The flowchart in Figure 3 illustrates the energy
transformations that occur in a conventional probe
sonicator.
The net fragmentation effect from applying ultra-
sonic energy to a suspension is dependent on the total
amount of energy delivered to the sonicated medium.
However, not all of the produced cavitational energy
is effectively utilized in disrupting particle clusters.
The delivered energy is consumed or dissipated by
several mechanisms, including thermal losses, ultra-
sonic degassing, and chemical reactions, such as
the formation of radical species. Only a portion of
the delivered energy is actually used in breaking
Electrical, mechanical and
thermal losses
Coupling, friction,
thermal losses
Suspension
Sonication
probe
Displayed
power
Generator
&
Transducer
Electric
source
power
Figure 3. Flowchart illustrating the energy transformation process in a conventional probe (direct) sonicator.
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particle-particle bonds to produce smaller particle
aggregates, agglomerates, and primary particles.
Moreover, an excessive energy input can potentially
result in agglomerate formation or re-agglomeration
of previously fragmented clusters, as well as induce a
variety of physicochemical alterations on the materi-
al’s surface or to the constituents of the suspending
medium. Such sonication-specific side-effects are
addressed in the following sections, but first we
examine the particle disruption process as it is the
primary objective of ultrasonic treatment.
Sonication effects on particle disruption and
aggregation
The properties associated with ENPs, and conse-
quently their distinct biological and environmental
interactions, are strongly dependent on the particle
size of the processed material. For instance, the size of
the particles in suspension dictates –along with the
particles’surface chemistry –their biodistribution,
elimination, cellular uptake, toxicity, and environ-
mental transport behavior (Rejman et al. 2004;
Lanone and Boczkowski 2006; Pan et al. 2007;
Powers et al. 2007; Li and Huang 2008; Sonavane
et al. 2008). Additionally, the agglomeration state
determines whether the particles will settle out or
remain in suspension, which directly impacts assay
efficacy and delivered dose. Numerous other exam-
ples of size-dependent behavior can be found in the
recent literature.
Dry powders consist of particles that are bound
together into macroscopic structures by both weak
physical Van der Waals forces and stronger chemical
bonds including particle fusion (Zachariah and
Carrier 1999; Mandzy et al. 2005; Jiang et al.
2009). Commonly, these powders contain micro-
meter –and submicrometer –scale aggregates, which
are in turn held together by physical forces to con-
stitute larger agglomerates. This is depicted schemat-
ically in Figure 4. For powders consisting of nanoscale
particles and aggregates, and therefore having sub-
stantial interfacial contact areas per unit volume, the
nominally weaker Van der Waals forces can be
extremely large, requiring the use of techniques
such as sonication to effectively break down or disrupt
powder agglomerates. In contrast, micrometer size
primary particles can often be dispersed with moder-
ate mixing or stirring.
Many of the commercially available metal oxide
ENP source powders (e.g., silica, titania and alumina)
are synthesized by high temperature vapor phase
Average particle size
Average particle size
Sonication energy
(power, time, energy density)
Sonication energy
(power, time, energy density)
Aggregate
primary particles held
together by sinter necks
or strong bonds Primary particle
Sinter neck
Asymptotic
trend
Delivered sonication energy
Agglomerate
aggregates held together
by Van der Waals or
other weak forces
Smaller
agglomerates &
aggregates
Dispersed
aggregates and/or
primary particles
Sonication
induced
aggregation
(coagulation)
No further
disruption
A
B
Peaking trend
A
B
Figure 4. Schematic depiction of particle structures referred to in the text, and illustration of the typical effects of sonication on particle size asa
function of delivered sonication energy: (A) Asymptotic behavior (solid line) versus (B) peaking behavior (dashed line).
Dispersion of nanoparticles 715
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processes (Kraft 2005; Isfort and Rochnia 2009). In
such processes, chemical precursors react at high
temperatures to produce small ‘hot’nanoscale dro-
plets of the desired material. As these hot droplets
pass through the reaction zone, they collide and may
coalesce fully or partially, depending on the local
temperature. Full coalescence leads to a larger pri-
mary particle, while partial coalescence leads to the
formation of an aggregate of fused primary particles
(bound via sinter necks), which later become ‘solid
bridges’upon particle solidification (Figure 4). Par-
ticle coalescence and growth continue until the reac-
tants are consumed or the process temperature is
quenched (Singhal et al. 1999; Zachariah and Carrier
1999; Teleki et al. 2008; Isfort and Rochnia 2009).
Metal oxide and other ceramic powders frequently
show this configuration, wherein the ‘primary
particle’represents the solidified globules that con-
stitute powder aggregates (frequently chain-like struc-
tures). Yet, it must be noted that the solid bridges that
hold the primary particles together in the dry powder
aggregates are often too strong to be broken via
sonication or other moderate dispersion processes.
Therefore, in some cases, it can be unrealistic to
expect sonicated powders to break down to their
reported, nominal primary particle size. As a result,
powders will be effectively de-agglomerated to aggre-
gates of several primary particles (i.e., ‘primary
aggregates’), rather than isolated primary particles.
These aggregates should be thought of as the effective
primary size for the ENP. Although the underlying
formation processes in solution phase synthesis (e.g.,
via sol gel or precipitation) differ from the vapor phase
process, the resulting structures often express similar
physical properties to the above described structures
(i.e., particle fusion, presence of aggregates and
agglomerates), though typically to a lesser extent
(Brinker and Scherer 1990).
During sonication, shock waves from cavitational
collapse are principally responsible for powder break-
age. The particles, in fact, serve as nuclei to initiate the
cavitation process, which partly explains why sonica-
tion is so effective (Suslick 1988). When ultrasound
energy is applied to powder clusters in suspension,
fragmentation can occur either via erosion or fracture.
Erosion or ‘chipping’refers to the detachment of
particles from the surface of the parent agglomerates,
while fracture or ‘splitting’occurs when agglomerates
partition into smaller agglomerates or aggregates due
to the propagation of cracks initiated at surface defects
(Figure 1). These fragmentation effects may occur
simultaneously or in isolation, depending on the pow-
der, its environment and the energy levels involved.
For a given material in powder form, depending on
the physicochemical properties of the solid material
and method of manufacture, the occurrence of sur-
face defects in the powder clusters, the material’s
surface toughness, and the bonding nature (e.g.,
neck diameter or interfacial contact area) of aggre-
gates and agglomerates, there is a system-specific
acoustic energy threshold that must be crossed in
order to achieve agglomerate and aggregate breakage.
It is thus worth noting that two powders of the same
material, but obtained following different synthetic
routes, may show significantly different breakage
behavior. Material properties also affect the rate at
which fragmentation occurs, and, consequently, the
amount of time it takes at a given ultrasonic output
power and for a given amount of material to reduce all
of the powder to the smallest achievable dimensions.
Thus, in principle, the cavitation process can effec-
tively break down powder agglomerates. Yet, under
certain conditions, the applied acoustic energy can
conversely induce particle agglomeration or even lead
to the formation of aggregates. Coagulation in ultra-
sonic fields can occur from enhanced particle-
particle interactions due to the increased collision
frequency as well as the favorable reduction in free
energy that accompanies the resulting reduction in the
liquid-solid interface. As noted previously in discuss-
ing the basics of ultrasonic disruption, cavitation gives
rise to extreme local pressure and temperature gra-
dients, as well as shock waves and jet streams of the
order of hundreds of meters per second. Under such
conditions, sonicated particles in the neighborhood of
a collapsing bubble collide with each other while
simultaneously experiencing localized intense heating
and subsequent cooling cycles. Depending on the
effective energy delivered to the particles and the
thermal properties of the material and medium, these
concomitant effects can lead to re-agglomeration or
even thermally induced inter-particle fusion; that is,
sonication-induced aggregate formation.
Published studies have demonstrated the existence
of this phenomenon, which is often observed as an
increase in particle size beyond a given sonication
energy threshold (Aoki et al. 1987; Vasylkiv and Sakka
2001). In other cases, an asymptotic behavior is
observed, wherein sonicated powders break down
to particles of a given size value, beyond which no
further significant size decrease is obtained even at
higher ultrasound energy inputs (Bihari et al. 2008;
Murdock et al. 2008). The latter behavior further
confirms that, for some powders, primary particles
can be held together by bonds that cannot be broken
by sonication.
Interestingly, both asymptotic and coagulation
behaviors (see Figure 4) as a function of different
sonication parameters have been observed for the
same source material (e.g., TiO
2
, Mandzy et al.
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2005; Jiang et al. 2009), illustrating the system-
specific dependence (i.e., source powder, dispersing
medium, ultrasound input) of powder sonication
procedures. Asymptotic and peaking (coagulation)
behaviors upon sonication have been reported for a
wide variety of engineered nanomaterials. The fol-
lowing are some examples, where the respective
asymptotic or peaking energy thresholds are also
indicated in each case: TiO
2
(rutile) –60 s at 7 W
(Bihari et al. 2008); TiO
2
(anatase and rutile) –(100–
300) s at 750 W (Jiang et al. 2009); ZrO
2
–100 s at
160 W (Vasylkiv and Sakka 2001); Au –60 min at
40 W cm
-2
(Radziuk et al. 2010); Ag –10 s at (35–40)
W (Murdock et al. 2008). It must be noted that in
these cases the reported delivered energies do not
conform to the guidelines specified herein and there-
fore may not accurately reflect the actual energy
densities to which the ENPs were subjected.
Furthermore, while the disruptive effect of sonica-
tion is in most cases instrumental to the achievement
of nanoscale particles, it can also result in either
desired or undesired physical changes that may alter
the inherent properties of the dispersed material.
A mainstream example of this phenomenon is the
sonication induced scission of carbon nanotubes
(Forrest and Alexander 2007; Huang et al. 2009;
Lucas et al. 2009).
While it may not be possible to completely elimi-
nate all of the aforementioned phenomena, it is crit-
ical to optmimize the sonication procedure in order to
minimize the undesirable effects, while attaining the
desired level of particle disruption. The recom-
mended optimization steps are detailed in a subse-
quent section.
Chemical effects of sonication
As with size, surface chemistry –and consequently
surface charge –plays a critical role in governing the
environmental and biological interactions of ENPs.
Surface chemistry, whether it is that of the pristine
material or includes the effects of a surface coating or
capping agent, largely determines the adsorption
behavior at the solid-solution interface, the organic/
inorganic phase partitioning, colloidal stability in
aqueous media, reactivity with other medium com-
ponents, and affinity towards cell membrane walls
(Guzman et al. 2006; Li and Huang 2008; Jiang et al.
2009; Kittler et al. 2010; Walczyk et al. 2010), to
name just a few examples. Surface chemistry is thus a
key parameter that affects the fate, transport, bioavail-
ability, and bioaccumulation behavior of ENPs. It is
therefore critical to consider the potential for changes
in surface chemistry in ENP suspensions arising from
the application of ultrasonic energy.
The extreme localized temperatures and pressures
generated by the cavitation process can yield highly
reactive species within a sonicated medium, in a
process known as ‘sonic activation’. As an indication
of the importance, variety, and complexity of sonica-
tion induced physicochemical effects in liquid media,
in recent years, sonochemistry has emerged as a distinct
discipline devoted to the study of a wide range of
sonication induced chemical reactions in both homo-
geneous and heterogeneous solid and liquid systems
(Mason 1989; Mason and Peters 2003).
An example of sonic activation of particular rele-
vance to environmental and toxicological studies is
that of water sonolysis. Sonicated water partly dis-
sociates into hydrogen and hydroxide radicals, with
concentration and lifetime dependent on the sonica-
tion conditions (e.g., sonication time and energy
density) (Makino et al. 1983; Yanagida et al.
1999), that subsequently recombine to form hydrogen
peroxide (Brown and Goodman 1965; Mason and
Peters 2003). Any materials or chemical species pres-
ent in sonicated water will thus be exposed to low (and
variable) concentrations of highly reactive species,
and as a result may undergo oxidative and other
chemical transformations.
Uncoated, sonicated ENPs may undergo changes
in their surface chemistry due to the formation of
sonolysis radicals. As previously mentioned, the soni-
cation of water can yield reactive radicals that can
initiate a variety of chemical reactions. The generated
hydrogen and hydroxyl radicals, and peroxide mole-
cules, can interact with the material’s surface, chang-
ing its oxidation state or inducing the hydroxylation of
the materials’surface, potentially altering the materi-
al’s hydrophilicity and stability, as well as its chemical
compatibility with other medium components.
Chemisorbed hydrogen radicals generated during
sonication could potentially lead to the irreversible
disintegration of sonicated carbon nanomaterials,
such as fullerenes and nanotubes, via ‘hydrogen-
induced exfoliation’(Berber and Tomanek 2009).
Other changes in the material’s surface chemistry,
such as oxidation or hydroxylation, may result in
an increased aqueous solubility and result in an
enhanced leaching of ionic or soluble species into
the medium. The potentiality of sonication-
enhanced leaching should be given special consider-
ation when testing the biological interactions of ENPs
with known toxicity in their molecular or ionic form; a
case in point is nanoscale silver (Wijnhoven et al.
2009; Elzey and Grassian 2010), in which the oxi-
dized Ag
+
form is well documented to exhibit biocidal
properties.
Moreover, if the dispersion medium is not just
water, but incorporates other chemical species, the
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extent and degree of formation of radicals from both
water and other medium components, and the com-
plexity of their interactions with the dispersed material
can be further magnified. Such potentiality for soni-
cation induced changes in the surface chemistry, and
in particular its oxidation state or the hydroxylation of
the material’s surface, should be given special con-
sideration in light of recent findings that attribute
ENP’s toxic effects to oxidative stress and reactive
oxygen species (ROS)-mediated events, among other
mechanisms (Unfried et al. 2007; Wang et al.
2009a; Kim et al. 2010).
While radical induced changes in the pristine mate-
rial’s surface might be restricted to the period of
sonication treatment and be potentially reversible –
although to the best of our knowledge this has
yet to be determined –it is unquestionable that
sonication-specific, radical-mediated changes have
been observed as a result of sonication, as discussed
below, and can significantly alter the suspension
medium properties or the dispersed material’s surface
chemistry, and consequently influence its environ-
mental and biological behavior.
If organic molecules are present when sonicating
ENP suspensions, either as components of the dis-
persing medium or as a surface coating on the ENP
itself, they can be irreversibly degraded via direct
sonolysis from the dissipated cavitational energy
(Williams 1983; Wang et al. 2009c). Furthermore,
sonication-induced active radicals, such as those pro-
duced from water sonolysis, have been shown to
cleave polymers creating smaller oligomers and to
activate polymerization reactions (so called sonopo-
lymerization), producing new polymeric species and
irreversibly modifying the organic molecules present
in the suspension (Brown and Goodman 1965;
Mason 1989). Sonication can also induce covalent
bonding of organic functionalities to the ENP surface
(Liu et al. 2005). The irreversible degradation
and/or transformation of organic molecules due to
sonication is a potentiality that should be considered
when preparing ENP suspensions for environmental
or biological testing.
As a result of sonolysis, the sonicated medium may
not retain its native properties. Degraded medium
components, such as denatured proteins, will likely
interact differently with the ENPs or the intended
assay, than they would in their pristine, non-
sonicated form. Sugars and proteins are two major
biological and environmental media components.
Basedow and Ebert studied the molecular degrada-
tion of dextran as a function of exposure time
under 20 kHz, low intensity (10 W cm
-2
) sonication
(Basedow and Ebert 1979), while in a later
work Lorimer et al. (1995) extended the study to
account for the effect of sonication intensity, temper-
ature, and sugar concentration. In all cases, a signif-
icant decrease in the sugars’molecular weight at
increasing sonication times and powers was observed.
Protein and enzyme sonolytical degradation has
been broadly studied as well. Wang et al. (2009c)
examined the sonication-induced damage to bovine
serum albumin (BSA) both in the presence and
absence of metal complexes. In all cases, even at
sonication powers as low as 20 W, ultrasonic irradi-
ation was shown to damage the BSA structure, the
extent of which increased with increasing sonication
time. Coakley et al. (1973) measured the sonolytical
inactivation of alcohol dehydrogenase and lysozyme
exposed to a 20 kHz ultrasonic field as a function of
sonication time (at 90 W), and measured a 70%
inactivation of alcohol dehydrogenase and lysozyme
after 10 and 25 min of sonication, respectively.
A more comprehensive review and discussions on
sonication induced alterations of relevant biological
compounds (e.g., enzymes, DNA, biological mem-
branes) can be found in the works of Williams (1983)
and Riesz and Kondo (1992).
Alterations in the molecular structure of common
organic compounds (e.g., sugars, proteins) as a result
of sonication may elicit specific biological responses in
the animal model or assay upon inoculation with the
sonicated suspension, thereby yielding false positives
or negatives that could incorrectly be attributed to the
ENPs, rather than the degraded medium. To account
for this possibility, we recommend using a superna-
tant control in which the medium, in the absence of
ENPs, is subjected to the sonication procedure and
tested in parallel with the ENP suspensions.
Furthermore, sonication-induced transformations
in the medium could potentially result in a loss of
the medium’s stabilizing properties, therefore reduc-
ing its ability to maintain the ENPs in suspended non-
agglomerated form. For example, species-specific
albumin homologs, such as BSA, have been utilized
as stabilizing components in dispersion media
intended for toxicity testing of ENPs (Bihari et al.
2008; Porter et al. 2008; Kittler et al. 2010;
Tantra et al. 2010), while humic acid is commonly
used as a medium component and organic stabilizer
added to ENP suspensions to simulate environmental
matrices (Chen and Elimelech 2007; Pallem et al.
2009; Saleh, Pfefferle and Elimelech 2010). Typi-
cally, the albumin or humic acid is present when
the suspension is sonicated. Naddeo et al. (2007)
demonstrated that humic acid is degraded (either
via oxidative mechanisms or physical aggregation)
when subject to 20 kHz sonication, the extent of
which was found to increase with sonication intensity
and organic concentration. Preliminary studies in our
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own laboratory on the stabilization effect of BSA on
sonicated TiO
2
powders have shown a measurable
loss of nanoparticle stability against agglomeration
when BSA is sonicated together with the source
powder, as opposed to when BSA is added post-
sonication. In the latter case, the particles did not
exhibit agglomeration over the same time frame.
These results will be published as part of a separate
research article currently in preparation.
Moreover, the ultrasonic degradation of organic
molecules has the potential to irreversibly modify
the chemical structure of ENP functional surface
coatings (e.g., polyethylene glycol [PEG], and poly-
vinylpyrrolidone [PVP]), or even a complete loss of
the coating itself. Kawasaki et al. (2007) studied the
ultrasonic degradation of PEG as a function of son-
ication time at 28 kHz and 20 W, over a wide range of
molecular weights. In all cases, radical mediated
sonolysis caused C-O bond scission and molecule
breakage. Taghizadeh and Bahadori (2009) measured
the degradation kinetics of PVP when subject to
24 kHz 100 W sonication, revealing a dependence
of the degradation rate on molecular weight and
polymer concentration. Such potential sonication-
specific alterations of organic surface coatings may
adversely impact the ENP’s capacity to remain in
suspension, as particle coatings often provide protec-
tive electrostatic or steric stabilization. Ultrasonic
degradation of organic surface coatings could also
modify the ENP’s interactions with other medium
components, interfering with the ENP’s transport,
adsorption, or toxicological behavior.
Additionally, ENPs may act as sonolysis catalysts,
synergistically enhancing the aforementioned effects
when sonicated in the presence of organics.
Wang et al demonstrated the sonocatalytic effect of
nano-sized silica and titanium dioxide towards the
degradation of BSA, showing an enhanced degrada-
tion of BSA when the ENPs were present in the
sonicated solution, with respect to ENP-free controls
(Wang et al. 2005, 2009b).
For these reasons, the chemical complexity of the
sonicated suspension and the particle coating should
be reduced to the greatest possible extent. Whenever
possible, organic compounds should be spared from
sonication and the formation of radicals minimized.
A proposed approach for the preparation of ENP
suspensions is, when applicable, to first sonicate the
pristine, uncoated powder in de-ionized water (or
other appropriate pH adjusted aqueous solution)
and then mix the sonicated suspension into the
desired, non-sonicated, dispersion medium, or add
the stabilizing agent and other organic additives to the
suspension after sonication. This may require signifi-
cant additional effort to properly formulate and
disperse the ENPs, but the extra effort will result in
less perturbation of the chemical species present. The
formation of radicals can also be mitigated by mini-
mizing the sonication power, again prompting the
need for optimizing the dispersion procedure to min-
imize undesired effects while obtaining the desired
dispersion properties. Furthermore, nitrogen or car-
bon dioxide bubbling, or the addition of dry ice or
other radical scavengers, can quench free radicals
during the sonication process and minimize their
potential impact (Kondo et al. 1986; Honda et al.
2002).
Measuring and reporting delivered power
A critical parameter for the reproducible preparation
of sonicated ENP suspensions is the delivered soni-
cation power. As previously mentioned, only a por-
tion of the total output power is effectively consumed
in particle disruption.
The energy loss and consequently the efficiency of
the energy transformation (or the ‘energy yield’)
from the electrical source to the acoustic power
effectively received by the sonicated suspension is
heavily instrument dependent (on the instrument’s
particular driving circuitry, self-adjusting feedback
loops, and its various electrical components). Fur-
thermore, the displayed power omits thermal and
mechanical energy losses, which are also instrument
specific, that occur as the output power is transferred
‘downstream’through the sonication probe and into
the suspension (Figure 3).
Therefore, simply reporting the sonicator’spower
setting value (e.g., ‘setting 5’or ‘60%’)orthedis-
played power does not provide an accurate indica-
tion of the effective acoustic power delivered to the
sonicated suspension, and are as such reporting
parameters that do not allow for transferable or
reproducible results. Two different instruments
operating at a ‘level 5’,ora‘60%’setting can deliver
significantly different effective acoustic powers to the
same suspension, as they may translate into different
oscillation amplitudes for different instruments.
Alternatively, instrument ‘A’may consume –and
display –50 W from the electrical source to produce
a probe oscillation amplitude of 3 mm, while instru-
ment ‘B’may produce a probe oscillation amplitude
of 2 mm by consuming 50 W, thereby delivering
different powers to a suspension even when showing
the same displayed power. Published works have
discussed and demonstrated that the power reading
displayed by the instrument bears poor correlation
with the measured power received by the sonicated
suspension (Contamine et al. 1995; Kimura et al.
1996; Raso et al. 1999). This issue is broadly known
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in the sonochemistry field as ‘the reproducibility
problem’(Mason 1991; Contamine et al. 1995;
Kimura et al. 1996).
While there is yet no available standardized proce-
dure for reporting the effective acoustic energy deliv-
ered to liquid suspensions, we hereby propose the
calorimetric method as a simple procedure to
establish a standardized, instrument-independent,
transferable and reproducible method for the mea-
surement and reporting of sonication power. It is
our belief that by implementing such an approach,
variabilities derived from the inconsistent applica-
tion and reporting of ultrasonic disruption will be
reduced.
The calorimetric method
We propose a simple and easily transferable set of
experimental guidelines for the determination of calo-
rimetric curves. The calorimetric method has been
proposed to standardize sonochemistry studies and
used to calculate the amount of acoustic energy deliv-
ered to a liquid medium subject to direct sonication
(Kimura et al. 1996; Zhu et al. 2009). Calorimetry is a
relatively simple, rapid, and inexpensive procedure
that allows for the direct measurement of the acoustic
energy delivered to a sonicated liquid (Mason 1991).
The method is based on the measurement of the
temperature increase in a liquid medium over time,
as a bulk effect of the cavitational process induced in a
liquid by an ultrasound probe.
At a given sonicator setting, the temperature
increase in the liquid is recorded over time and the
effective delivered power can then be determined
using the following equation:
P=dT
dt MCp,()2
where Pis the delivered acoustic power (W), Tand t
are temperature (K) and time (s), respectively, C
p
is
the specific heat of the liquid (J g
-1
K
-1
) and Mis the
mass of liquid (g).
By obtaining calorimetric curves following a stan-
dardized procedure, users can report and reproduce
the power level applied to a sonicated suspension in a
manner that is easily transferrable and instrument
independent.
The following protocol is proposed for the deter-
mination of direct sonication calorimetric curves
(Taurozzi et al. 2010a):
(1) Fill a 600 mL cylindrical borosilicate beaker
with 500 mL of de-ionized water (resistivity
>18 MWcm);
(2) Determine the mass of the liquid using a top load-
ing balance. First tare the empty 600 mL beaker;
(3) Immerse the sonicator probe (horn) approxi-
mately 2.5 cm (1 inch) below the liquid surface;
(4) Immerse a temperature probe connected to a
temperature meter and data logger (e.g., an
Extech HD 200
1
temperature meter and data-
logger coupled to a Type K immersion temper-
ature probe). The temperature probe tip should
be about 1 cm from the sonicator probe (see
Figure 5);
(5) Select a sonicator output power setting and,
operating in continuous mode, record the water
temperature increase for the initial 5 min. Dur-
ing sonication, ensure that the beaker does not
shift position, especially when operating at high
power settings; this can be accomplished for
instance, using a clamp attached to a ring stand;
(6) Using the recorded values, create a temperature
vs. time curve and obtain the best linear fit for
the curve using least squares regression.
Figure 5. Set-up for the measurement of calorimetric curves. Inset
shows sonicator tip and temperature probe immersion depths.
Sound protection enclosure was omitted for clarity of the image.
1. Certain trade names and company products are mentioned in the text or
identified in illustrations in order to specify adequately the experimental
procedure and equipment used. In no case does such identification imply
recommendation or endorsement by National Institute of Standards and
Technology, nor does it imply that the products are necessarily the best
available for the purpose.
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With the obtained slope, calculate the delivered power
using Equation 2. The procedure should be repeated
for all desired power settings, relating each setting
level to the measured power. The power obtained via
calorimetry is the value we recommend for reporting
power used in sonication-based dispersion proce-
dures. By doing so, laboratories using different com-
mercial ultrasonic disruptors should be able to
reproduce a similar delivered power by selecting the
appropriate calibrated setting. It is important to note
that the intended use of calorimetry curves is not to
measure the actual fraction of power utilized for
powder disruption under specific dispersion condi-
tions, but simply to establish a standardized proce-
dure to relate instrument setting levels to measurable
powers under a fixed set of controlled conditions, and
thus allow for the reporting and transference of son-
ication power levels between laboratories and users.
The robustness of the method was tested under
different scenarios, including variations in tempera-
ture probe location, water volume, and the use of
stirring. While variations in temperature probe loca-
tion and stirring only resulted in minor variations of
the measured power (<5%), it should be emphasized
that using a different water volume, a container of a
different material or a different type of horn tip than
those recommended here can result in changes in the
measured temperature versus time slope for a given
power setting due to variations in the rate of heat
transference between the sonicated water and the
environment. If conditions other than those recom-
mended here are used to measure calorimetric curves,
these modifications to the protocol would need to be
reported.
Figure 6 shows calorimetric curves and calculated
delivered power at different sonicator settings,
obtained using the above method for a Branson
450 analog probe sonicator with a standard
1
/
2
-inch
probe and tip.
Additional considerations
As noted previously, sonication is a highly system
specific dispersion procedure, involving a variety of
complex concomitant physicochemical interactions in
both the sonicated ENP and the suspension medium.
Therefore, for a given system, optimal sonication
conditions can only be determined by considering
the effects of a variety of sonication parameters on
the dispersion state of the suspension under a broad
range of relevant conditions. As follows, a discussion
of the effects of these parameters is offered along with
recommendations for implementation of ultrasonic
disruption as a preparatory tool. Two parameters,
namely powder properties and sonication power,
are not discussed here as they have already been
addressed in previous sections.
Temperature
During sonication, the extreme local heating cycles
that take place at the micro-scale bubble interface due
to cavitation will result in bulk heating of the liquid
over time. Excessive bulk heating can cause
1.9
Setting 1, 8.85 W
Setting 3, 32.71 W
Setting 6, 60.50 W
Setting 8, 104.1 W
Setting 10, 134.7 W
1.8
1.7
1.6
1.5
1.4
Normalized temperature (T/T0)
1.3
1.2
1.1
1
0 50 100 150
Time (s)
200 250 300
Figure 6. Calorimetric curves, linear fits, and corresponding calculated power values for different operational settings, obtained using a
Branson 450 analog sonicator. For ease of illustration, the y-axis shows the recorded temperatures normalized to the initial temperature (T0) in
each case.
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evaporative loss of liquid, resulting in changes in the
sample volume after sonication. Moreover, high sus-
pension temperatures may lead to degradation of the
ENP or other medium components if proper precau-
tions are not employed.
A common approach to minimize temperature
driven side effects is to keep the sample from expe-
riencing substantial high-temperature excursions by
immersing the suspension container in a cooling bath.
The container should be immersed to a level roughly
equal to that of the contained suspension.
An ice-water bath is often sufficient to keep suspen-
sions from overheating. If, however, ice water is not
sufficient to cool the sample under intense sonication
(e.g., the sample volume is too low, the sonication
time is too long, or high sonication powers are used)
an ice-salt bath could be considered as an option.
Working with containers made of materials with
high thermal conductivities ensures a rapid release of
the generated heat from the suspension into the
surrounding cooling bath. With regard to thermal
properties, the following container materials work
best, in order of decreasing thermal conductivity:
aluminum, stainless steel, glass, and plastic (e.g.,
polyethylene). When selecting the container, consid-
eration must also be given to the chemical compat-
ibility between the container material and the
suspension components, especially under intense
local heating conditions; for instance, aluminum is
incompatible with acidic suspensions, and glass is
incompatible with alkaline solutions.
Additionally, an aluminum foil or thin thermoplas-
tic film with an opening just large enough for the
probe to enter untouched, will reduce the evaporative
loss of liquid content, especially when sonicating in
volatile solvents or for substantial durations. Cooling
the sample will also reduce evaporative loss. This
cover will also minimize the potential release of aero-
sols generated by the sonication process.
Sonication time and operation mode
The total amount of energy (E) delivered to a sus-
pension not only depends on the applied power (P)
but also on the total amount of time (t) that the
suspension is subject to the ultrasonic treatment:
E=P t×()3
Consequently, two suspensions treated at the same
power for different times can show significantly dif-
ferent dispersion states.
Ultrasonic disruptors typically can operate in either
continuous or pulsed mode. In pulsed mode, ultra-
sonic intervals are alternated with static (sonication
off) intervals. The duration of on and off intervals can
be regulated. Operating in pulsed mode retards the
rate of temperature increase in the medium resulting
from the cavitation process, minimizing unwanted
side effects and allowing better temperature control
than with continuous mode operation.
Sample volume and concentration
While sonication power and time describe the amount
of energy delivered to the suspension, the work done
on samples of different volumes and particle con-
centrations can differ. At constant volume, higher
particle concentrations result in an increased particle
collision frequency. In principle, an increased colli-
sion frequency can enhance particle breakage due to
an increase in particle-particle impact events. How-
ever, if sufficient local activation and sintering ener-
gies are achieved, increased collision frequencies can
also induce agglomerate or aggregate formation as
particles collide and coalesce (Figure 4). The effect of
concentration is thus dependent on both the energy
delivered into the suspension and the physiochemical
properties of the suspension.
Particle concentration can also alter the acoustic
properties of the suspension, affecting the medium’s
viscosity and acoustic conductivity and thus the
effectively delivered power. However, at the concen-
trations commonly used for environmental and
toxicological evaluations, typically in the 50–
2000 mgmL
-1
range, a significant impact on the
medium’s acoustic properties is not anticipated.
Where particle concentration may alter acoustic prop-
erties is when working with concentrated stock sus-
pensions prior to dilution.
The effect of suspension volume (at constant par-
ticle concentration) is often measured as energy density
(W s mL
-1
). This magnitude expresses the amount of
delivered energy per unit of suspension volume. In
principle, at equal power and particulate concentra-
tion, higher energy densities (i.e., smaller suspension
volumes) will result in a higher disruptive effect. It
should be emphasized that when working with small
volumes the temperature of the suspension will rise at
faster rates and more intense cooling conditions may
be required in this case.
Sonicator probe, container geometry, and tip immersion
The sonicator probe is the acoustic element that
conducts the acoustic energy from the transducer
into the sonicated suspension. The amount of acous-
tic energy transferred to the suspension will heavily
depend on the shape of the sonicator probe and its
immersion depth in the suspension.
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The vibrational amplitude of the horn at a given
power is dependent on the horn taper (Berlan and
Mason 1992). Tapered probes act as vibration ampli-
tude magnifiers, and consequently allow for higher
delivered powers (see Equation 1). For example,
stepped horn probes allow for up to 16-fold power
amplifications, while exponentially tapered probes
can yield power densities on the order of hundreds
of W cm
-2
(Mason 1989).
At equal sonicator power settings, microtip probes
vibrate with larger amplitudes than conventional flat tip
horns. However, microtips are less mechanically robust
and often limited in terms of the maximum power
setting at which they can be used. The manufacturer of
the sonicator will typically specify a maximum power
setting (based on a percentage of the maximum power
output setting) for use with microtips.
The way in which the sonic energy is distributed
within the suspension is also heavily influenced by the
container geometry. For example, in one published
study (Pugin 1987) acoustic energy distribution pro-
files measured in round bottom flasks subject to
indirect sonication significantly differed from those
of flat bottom (e.g., Erlenmeyer) flasks subject to
similar sonication conditions. Conical bottom, flat
bottom, and round bottom flasks will also show
different energy maxima and minima distribution
profiles, which will in turn vary significantly for dif-
ferent probe tapers and probe tip immersion depths.
When possible, using the smallest diameter vessel
that allows for the probe to be inserted without
touching the container walls is probably the best
approach. Using smaller container diameters raises
the height of the liquid and maximizes the liquid-
probe surface area exposed to the acoustic waves, as
well as the container wall surface/volume ratio for
dissipation of heat by the cooling bath. Cylindrical
shaped beakers work well, especially for small
volumes.
Probe immersion depths between 2 and 5 cm are
generally recommended when operating with stan-
dard horns having a flat radiating surface (tip) or with
microtips. Probes should be placed no closer than
about 1 cm from the bottom of the sample container,
and contact between the probe and the container walls
should always be avoided.
Aerosoling and foaming
If the sonicator probe is not sufficiently immersed in
the suspension it can give rise to surface agitation
resulting in nebulization (i.e., the formation and
release of aerosols). This could pose a risk if ENPs
or other potentially harmful medium components are
released in this manner.
Aerosoling may be indicated by fluctuations in the
audible sound pitch during operation, fluctuating
power readings, or by the appearance of a fine spray
in the vicinity of the probe. If aerosoling is observed, it
can usually be eliminated by lowering the sonication
probe deeper into the suspension.
Additionally, if surfactants are present, the suspen-
sion could generate a foam during sonication, and the
presence of a stable foam in contact with the horn
surface will interfere with the delivery of ultrasonic
energy to the suspension in a self-limiting process.
Pulse mode operation with long off periods will help
avoid foaming in samples subject to this effect. The
appearance of foaming may require that operation is
ceased until the foam dissipates. Anti-foaming agents
can be effective, but would probably not be compat-
ible with toxicological studies.
Tip maintenance
As the source of ultrasonic waves, all sonicator probes
are subject to progressive erosion due to the intense
cavitation that occurs in the immediate vicinity of the
probe tip. Tip erosion is therefore an unavoidable side
effect in direct sonication. There are two effects of tip
erosion that are relevant to the present discussion.
During erosion, microscopic tip residues (typically
titanium metal) are released from the tip into the
sonicated suspension, introducing an impurity and
potentially contaminating the suspension. Operation-
ally, tip erosion also results in a reduced energy
output. The extent of potential contamination from
tip erosion can be evaluated by performing a control
experiment, or by measuring the amount of metal
released into the sonicated medium (e.g., via diges-
tion followed by ICP-MS analysis) under relevant
sonication conditions. Erosion contamination can
be minimized by adequate and frequent tip mainte-
nance procedures, as explained below, or by using
indirect sonication.
The extent and occurrence of tip erosion depends
on the intensity, duration, and frequency of sonica-
tion cycles, as well as the chemical properties and
concentration of the sonicated suspensions. If used on
a daily basis, tips should be inspected weekly for signs
of erosion; tip erosion can be recognized by the
appearance of a grayish matting, as opposed to the
shinny lustrous appearance of a non-eroded tip (see
Figure 7).
A mildly eroded (i.e., matted) tip or horn surface
can be reconstituted (dressed) by buffing with a very
fine grit carbide paper or emery cloth (refer to the tip
or sonicator manufacturer for the correct grit size; do
not use conventional sand paper) (Berliner 2010).
Substantially eroded tips or horn surfaces (i.e., with
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visible pitting) should be replaced, as these cannot be
reconstituted. Standard
1
/
2
-inch diameter horns typ-
ically have a removable threaded tip, but larger probes
are likely to consist of a solid horn and thus greater
care may be required to dress the horn’s active surface
if it becomes matted.
It is also essential to ensure that removable probe
tips are tightly coupled to the horn, and do not loosen
as a result of prolonged operation; a loose tip will
result in a reduced acoustic power conversion and
energy yield.
Medium properties
As previously noted, the ultrasonic energy delivered to
a suspension is partly attenuated and dissipated by the
suspending medium. The amount of energy lost to the
medium at a given acoustic frequency is governed by
its physicochemical properties and chemical
composition.
Generally, the attenuation of acoustic energy in a
given medium can be estimated using the following
equation (Brown and Goodman 1965):
I=Ie
x
x
0
24
−
α
()
where I
0
is the acoustic intensity (power/area) at the
source, I
x
is the intensity at a distance xfrom the
source and ais the absorption coefficient.
The magnitude of the absorption coefficient, a,is
dependent on the medium’s physicochemical prop-
erties and composition, as well as the acoustic fre-
quency. In principle, higher medium viscosities and
acoustic frequencies will result in larger avalues. For
a given liquid, acan be estimated by using the
following equation (Brown and Goodman 1965):
αμ
ρ
∝2
35
f2
()
where mis the dynamic viscosity of the liquid (kg m
-1
s
-1
), fis the acoustic frequency (s
-1
) and ris the
liquid’s density (kg m
-3
).
As shown by Equation 5, acoustic attenuation
increases with frequency for a given medium. For
this reason, to achieve equivalent energy densities,
higher power inputs are required when operating at
higher frequencies. For example, to achieve an acous-
tic intensity of 20 W cm
-2
at a distance of 10 cm,
would require approximately 54% greater source
intensity at 10 MHz compared with that required at
10 kHz (Mason 1989).
Additionally, higher viscosities will dampen the
cavitation process, requiring higher power inputs to
achieve dispersion. The medium’s density and acous-
tic wave speed (c) will also impact on the amount of
vibrational energy that is transformed into acoustic
energy (see Equation 1).
The medium’s viscosity, density, acoustic wave
speed, and chemical composition are all important
parameters that impact on the amount of delivered
energy that is effectively utilized to disrupt powder
clusters. For media relevant to environmental and
biological testing, the salt content is likely to have
only a very small impact on the acoustic properties
relative to pure water, as the presence of physiological
salt levels will slightly alter the density and viscosity of
the solution. Overall, at ultrasound frequencies, these
effects are probably insignificant compared to other
factors discussed previously.
Yet, as noted previously, the medium’s chemical
composition is of particular relevance in terms of the
occurrence of potential side reactions during sonica-
tion (e.g., radical activation, sonopolymerization) that
may alter the physicochemical properties of the son-
icated medium and/or the sonicated nanomaterial.
Optimizing sonication for dispersion of ENPs
As discussed in preceding sections, the treatment
conditions required to achieve complete ENP disper-
sion are highly system specific. It is therefore advi-
sable to conduct dispersion optimization studies (e.g.,
measure particle size as a function of sonication
parameters) for the test system (Bihari et al. 2008).
While it would be unrealistic and impractical to offer a
Figure 7. Active surface of removable
1
/
2
-inch flat tips, ranging from pristine (left) to matted (center) to eroded with extensive pitting (right).
724 J. S. Taurozzi et al.
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unique set of sonication conditions for all possible
systems, the ultimate goal of the optimization proce-
dure should be to achieve the desired degree of
particle dispersion with the least possible energy input,
in order to minimize unwanted side effects.
The process to determine such conditions is based
on a trial and error approach. The guidelines offered
below are intended as tools for the interpretation of
the observed behavior at a given set of sonication
conditions, and to determine the appropriate param-
eter modifications to be made in response.
As noted previously, the relationship between dis-
persion efficiency and the different sonication para-
meters is not linear. The convolution of delivered
energy input and suspension properties can result
in either a net agglomerate breakage or cluster re-
formation effect.
For this reason, the optimization process should
scan parameter values covering a range both above
and below a starting point. The first trial set of
sonication conditions for an untreated powder in
suspension should be intended to ensure that disper-
sion can in fact be achieved. Being the first trial and
therefore likely not the optimal set of conditions, it
should be designed to confirm that under reasonably
attainable sonication conditions, the powder in sus-
pension can be effectively dispersed. The starting
point of the optimization should therefore provide a
high energy input to the suspension. This is achieved
by selecting a relatively large sonication output power
level, a prolonged sonication time, and a high energy
density (small suspension volume).
Table I offers a general guideline of starting points
for power, time, and volume selection, which might
be utilized to investigate new ENPs or media/ENP
combinations (Hielscher 2005).
The choice for the starting powder concentration is
subject to the desired application. It is also possible to
start with concentrations higher than the target level,
and then if needed dilute the processed suspension
post-sonication.
The effect of different processing parameters on the
dispersion state of the suspension can be analyzed
independently by varying one parameter while keep-
ing the others constant. For each parameter, the
effectiveness of the applied sonication conditions
can be evaluated by measuring the particle size dis-
tribution (PSD) of the sonicated suspension. In prin-
ciple, a reduction in the average particle size and
polydispersity (breadth of the distribution) are indic-
ative of more effective dispersion. The parameter
optimization sequence is explained as follows.
Once a starting set of values for time, concentra-
tion, and volume has been selected, and keeping all
other processing parameters constant, sonication
power is varied. Sonication powers above and below
the starting point should be tested. As a general
guideline, the effect of power can be scanned in
increments of 10 W both above and below the starting
sonication power. Using the power value that yields
the smallest measured size and lowest polydispersity,
then sonication time should be scanned (e.g., in
increments of 30 s) above and below the starting
point. Operation in pulsed mode is recommended
for sonication times greater than 1 min, particularly
when using small volumes (i.e., below 50 mL).
For the optimized time, power, and selected vol-
ume, the effect of particle concentration both above
and below the concentration starting point and down
to the minimum acceptable concentration for the
desired application, may be evaluated if desired. If
the optimal concentration is above the desired value
for the particular application, the suspension’s PSD
should be measured following dilution to ensure there
has been no change.
The above described approach for optimizing the
sonication conditions for dispersion is both simple
and practical, but the authors recognize that there
may be other factors to consider or other optimization
approaches that work equally well; it should be con-
sidered as a recommendation not a prescription for
success.
Reporting guidelines
In light of the relevance of the above discussed para-
meters and their significance in dictating the state and
quality of suspensions obtained via sonication, we
propose that the following parameters be reported
in published work involving the use of ultrasonic
energy to disperse ENPs prior to environmental,
exposure, or biological testing (Table II). It should
be noted that many of these parameters or material
characteristics would likely be reported anyhow, or
certainly should be according to several recent pub-
lications and recommendations (Murdock et al. 2008;
Warheit 2008; Minimum Information for Nanoma-
terial Characterization [MinChar] Initiative 2009;
Boverhof and David 2010).
The lengthiness of this list may preclude full dis-
closure within a typical experimental section in a
Table I. Guidelines for sonication starting points.
Energy density
(W s/mL)
Sample
volume (mL) Power (W) Time (s)
<100 10 50 <20
100–500 10 50 20–100
>500 10 50 >100
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Table II. List of parameters and material characteristics recommended for reporting purposes.
Sonication ENP or source material Test medium Final suspension
Type of sonicator used
(bath, probe immersion,
inverted cup)
Description of powder or ENP as received
(to the extent relevant and known)
Composition including ionic strength or
molar salt concentrations
Order in which components were added
and whether or not they were present
during sonication
Sonicator make, model,
rated power output, frequency,
probe type/diameter (if used)
For commercial ENPs, product trade name
and/or product number together with
batch/lot number if available
For commercial media, source, product
and batch number(s), as applicable
Post-treatment storage conditions prior to
testing (e.g., container material, temperature,
exposure to light, exposure to oxygen, time)
Sample container volume and type Primary particle size and method of measurement
(e.g., TEM)
Initial pH (preferably measured, otherwise
nominal buffer value)
Whether endotoxin was evaluated or
procedures applied to avoid/remove endotoxins
(relevant if suspension is to be used for
toxicity testing)
Tip/probe immersion depth (if used) Gross particle morphology (e.g., spheroidal,
tubular, fibrous, platelet, aggregate)
Dispersing agent(s) and concentration(s),
if used
Conditions of filtration and/or centrifugation
used (where appropriate)
Operation mode (continuous or pulsed),
if pulsed indicate pulse duration
Specific surface area (for powders) and method
of measurement
Source and quality of water if test medium
was prepared or diluted on-site
Post-treatment mass concentration of
particle phase
Total cumulative treatment time Identity of coatings or surface functionality
if known
Any relevant details relating to storage,
dilution, or preparation of medium
Final pH
Power input to sample (as measured for
probe sonicators: See above and also
Taurozzi et al. (2010a)
Particle size distribution in the relevant
suspending medium and method
of measurement
Whether cooling bath was used; if not
temperature rise during sonication
Mass of powder and total volume of
suspension sonicated
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refereed journal, but could easily be accommodated
in the supplemental information supported by most
scientific journals. The recommendations may also be
viewed as a checklist for identifying issues that may
need to be addressed during the selection or devel-
opment of a test material or dispersion procedure. For
a more detailed and formal guidance document on
reporting, refer to Taurozzi et al. (2010b) or consult
the references cited above.
Additional parameters and conditions may need to
be reported in order to fully describe the test sample
and experiment in their entirety. For commercial
sources, steps should be taken, if possible, to ensure
that the as-received material reflects the man-
ufacturer’s description –for instance, comparison
of powder characterization to nominal specifications
and typical values. Examples might include moisture
content by TGA, primary particle or aggregate size by
electron microscopy, or purity determination using
analytical methods. Under certain circumstances it
may be prudent to test for the presence of contami-
nants arising from the dispersion treatment process
itself, such as additional dissolved species (e.g., metal
ions, organic carbon). It may be equally important to
test for the loss of species that should be present, such
as constituents of the original test medium. The fate
of dispersing agents (e.g., surfactants, proteins,
sugars) may be relevant for both environmental and
biological testing.
Other material-specific parameters that are com-
monly mentioned as reportable include aspect ratio,
crystalline form and content, amorphous content, and
zeta potential from electrophoretic mobility or other
measure (such as the isoelectric point or zero point of
charge) related to the particle surface charge proper-
ties; zeta potential is both material and medium
dependent, and typically varies with pH for materials
that contain acidic or basic moieties, so the pH and
ionic strength of the sample should always accompany
zeta potential results. It may also be necessary, and
certainly prudent to demonstrate that the post-
dispersion suspension is stable over the time period
of the experiment; for instance, particle size could be
monitored in situ (e.g., using dynamic light scatter-
ing) over a period of time congruent with the testing
protocol. The potential list of reportable physico-
chemical endpoints is nearly unlimited, so prioritiza-
tion within the context of the intended use is critical.
Closing remarks
Sonication has proven to be a simple and energy
efficient technique for the disruption of coarse
powders and, as such, has become a mainstream
tool for the preparation of ENP suspensions for
environmental and toxicological studies. While we
do not intend to discourage the use of ultrasonic
disruption to produce such suspensions, our goal
here is to promote a rational use of sonication, in
order to ensure that unintended side effects, such as
sonolysis, are minimized and that the results obtained
using such suspensions are accurately interpreted.
Additionally, by providing a set of guidelines for
the standardized measurement and reporting of rel-
evant sonication parameters, we seek to improve the
reproducibility of related studies and allow for a
comprehensive peer review process, which may be
otherwise hindered by insufficient information
regarding the preparation of the tested suspensions.
We do not attempt to address the nearly infinite range
of possible experimental scenarios, but instead offer a
starting point towards the reproducible preparation
and comprehensive evaluation of ENP suspensions.
Acknowledgements
The authors thank Fred Klaessig of Pennsylvania Bio
Nano Systems, LLC, for helpful suggestions and for
his critical review of the draft manuscript. The authors
would also like to thank participants of the Interna-
tional Alliance of NanoEHS Harmonization (IANH)
for useful discussions, some of which helped motivate
the present work.
Declaration of interest: This work was funded in
part by a cooperative research agreement
(70NANB9H9166) between NIST and the Center
for the Environmental Implications of Nanotechnol-
ogy (CEINT). The authors report no conflict of
interest. The authors alone are responsible for the
content and writing of the paper.
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NOTICE OF CORRECTION
The Early Online version of this article published online ahead of print on 15 November 2010 contained an error
on page 3. Equation 1 should have read
P= cA f a
1
2)
22
ρπ(()21
This has been corrected for the current version.
Dispersion of nanoparticles 729
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