Science of the Total Environment 438 (2012) 225–232

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Short Communication

The complexity of nanoparticle dissolution and its importance in

nanotoxicological studies
Superb K. Misra a, b,⁎, Agnieszka Dybowska b, Deborah Berhanu b,
Samuel N. Luoma c, Eugenia Valsami-Jones a, b
a
School of Geography, Earth & Env. Sciences, University of Birmingham, B15 2TT, UK
b
Natural History Museum, Mineralogy, Cromwell Road, London SW7 5BD, UK
c
John Muir Institute of the Environment, University of California, Davis, CA 95616, USA

H I G H L I G H T S G R A P H I C A L A B S T R A C T

Various physicochemical factors affecting dissolution of nanoparticles.

► In this study we discuss dissolution of
nanoparticles.
► Physicochemical properties of nano-
particles influence dissolution.
► Measuring dissolution of nanoparti-
cles can help to understand their bio-
logical response.

a r t i c l e i n f o a b s t r a c t

Article history: Dissolution of nanoparticles (NPs) is an important property that alters their abundance and is often a critical
Received 25 May 2012 step in determining safety of nanoparticles. The dissolution status of the NPs in exposure media (i.e. whether
Received in revised form 22 July 2012 they remain in particulate form or dissolve — and to what extent), strongly affects the uptake pathway, tox-
Accepted 16 August 2012
icity mechanisms and the environmental compartment in which NPs will have the highest potential impact.
Available online 19 September 2012
A review of available dissolution data on NPs demonstrates there is a range of potential outcomes depending
Keywords:
on the NPs and the exposure media. For example two nominally identical nanoparticles, in terms of size and
Nanoparticles composition, could have totally different dissolution behaviours, subject to different surface modifications.
Dissolution Therefore, it is imperative that toxicological studies are conducted in conjunction with dissolution of NPs
Nanotoxicology to establish the true biological effect of NPs and hence, assist in their regulation.
Toxicology © 2012 Elsevier B.V. All rights reserved.
Physicochemical properties
Solubility

1. Introduction

1.1. Importance of dissolution

Dissolution of NPs is an important property that influences their

⁎ Corresponding author. mode of action (e.g. antimicrobial properties, toxicity, medicinal appli-
E-mail addresses: s.misra@nhm.ac.uk, s.misra@bham.ac.uk (S.K. Misra). cations and environmental impact). Dissolution is a dynamic process in

0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2012.08.066
226 S.K. Misra et al. / Science of the Total Environment 438 (2012) 225–232
which constituent molecules of the dissolving solid migrate from the
surface to the bulk solution through a diffusion layer (Borm et al.,
2006). The thermodynamic parameter that controls this process is
described as solubility and along with the concentration gradient
between the particle surface and the bulk solution acts as the driving
force of particle dissolution. Both solubility and speed (rate) of dissolu-
tion are dependent on a particle's chemical and surface properties, as
well as size, and are further impacted by the surrounding media. The
potential for NPs to dissolve can effectively influence their persistence
in the environment and act as a critical control on their biological
response. In some cases, dissolution can lead to delivery of highly
toxic ions, for example when NPs are composed of elements which,
in solution, are known to be toxic (Zn2+, Cu2+, Cd2+ and Ag+)
(Brunner et al., 2006; Xia et al., 2008). As a result, in a nanotoxicological
context, an important challenge is to ascertain whether the toxicity ob-
served is due to the NPs, perhaps as a result of their novel properties, or
caused by the release of ions, or a combination of both. It is most likely
that in many circumstances dissolution of NPs in media will produce a
complex suspension containing partially dissolved NPs, free/
complexed ions derived from the NPs and adsorbed ions on the NP
surfaces. This complex “mix” of potential toxicants needs to be compre-
hensively assessed for a toxicity study to be robust. Dissolution can
significantly change the exposure route, dose, and nature of the
toxicant (ions/NPs) and therefore merits a systematic study in order
to (i) correctly interpret the biological response triggered by exposure
to NPs; (ii) establish the likely timescales during which there is a risk to
NP exposure in different biological/environmental compartments and
(iii) postulate the cellular uptake mechanisms NPs may undergo.
2. Factors affecting dissolution
It is generally assumed that solubility may increase as particle size
decreases, something that has been shown theoretically to hold true
(Ostwald–Freundlich equation) (Borm et al., 2006). However, exper-
imentally, it is often difficult to demonstrate a size effect in isolation,
particularly in cases where the mechanism for size control involves
surface modification. Although size is considered as the primary
physicochemical property affecting solubility of NPs, various other
parameters such as, surface area, surface morphology, crystallinity
and crystal structure also need to be considered, as the strength of
the surface bonds, their spatial arrangements and the presence of
impurities or adatoms, and storage conditions may influence NPs'
dissolution. As summarised in Table 1, it is clear from the data
available that most of the dissolution studies have been performed
on silver, zinc oxide and copper oxide NPs, which also reflect the
increased use of these particles in various applications. The experi-
ments have been performed on particles with various size ranges
and in a variety of exposure media, with varying results. In the case
of CuO NPs, there seems to be enough experimental data showing an in-
crease in dissolution (rate of dissolution and equilibrium concentration)
with reduction in particle size (refer to Table 1). For ZnO NPs, there does
not seem to be any significant difference in dissolution between
nanoparticles and bulk particles (micron sized particles) (refer to
Table 1). Ag NPs, on the other hand present a more complex scenario,
which is primarily because size control in the case of metallic Ag NPs
is often achieved using surface modifications and therefore the effect
of such surface modifications on solubility of the NPs also needs to be
taken into account. Nevertheless, emerging results show that for parti-
cles of a given surface chemistry, particle size has an inverse effect on
dissolution of Ag NPs (Zhang et al., 2011; Ma et al., 2012; Xiu et al.,
2012). Apart from the above mentioned NPs, there are also some stud-
ies performed on assessing solubility of TiO2 NPs (Schmidt and
Vogelsberger, 2006), SiO2 NPs (Roelofs and Vogelsberger, 2004;
Diedrich et al., 2012), hydroxyapatite NPs (Tang et al., 2004) and
quantum dots (Liu et al., 2008; Siy and Bartl, 2010). To highlight the
complexity of solubility of NPs; by just taking size as one of the
parameters, it seems that there are examples encompassing three sce-
narios, i.e. size dependent solubility of NPs (Bian et al., 2011), no differ-
ences in dissolution between nano and micrososcopic particles
(Franklin et al., 2007) and inhibition in dissolution by reduction in par-
ticle size (Tang et al., 2004).
Surface modification, achieved through the use of capping agents
to arrest the growth, or surfactants to promote dispersion can also
have a non-trivial effect on NPs' solubility depending on the type of
the surface species and the mode of attachment on the NP surface.
There are numerous capping agents that modify the surface to gain
size and shape tuning of NPs and this modification can bring out
changes in the NPs' dissolution behaviour. Surface chemistry depen-
dent solubility has been studied extensively for Ag NPs, wherein it
is shown that capping agents can considerably influence the dissolu-
tion of Ag NPs (Table 1). Polyvinylpyrrolidone (PVP) and citrate
capped Ag NPs have been extensively used for toxicity studies and
from the available data, both capping agents can have varying effects
on the Ag NPs' dissolution. For example, 50 nm Ag NPs, PVP capped
particles showed 50% dissolution at 25 °C in water compared to 14%
dissolution for citrate-Ag NPs (Kittler et al., 2010). On the
contrary, An Huynh and Chen (2011) showed that citrate-Ag NPs
(71 nm) had a higher dissolution compared to PVP-Ag NPs (67 nm)
in water over a 30 min period. For some surface modifications,
the amount of material dissolved does not seem to vary and instead
the rate of dissolution differs, as shown in the case of 80 nm Ag NPs
(uncoated, citrate capped, and Tween coated) (Li et al. 2012). Beyond
the structural termination of the NP, the stabilising mechanism of the
surfactants and capping agents can affect NPs' dispersion and, as a re-
sult, their dissolution (Liu et al., 2010). As an example, electrostatically
stabilised NPs are more sensitive to changes in ionic strength and pH of
the media and this in turn affects their behaviour in solution with re-
spect to aggregation; temperature, on the other hand, has more impact
on sterically stabilised suspensions (El Badawy et al., 2010; Sperling
and Parak, 2010; Yokoyama et al., 1990). Shape and surface morpholo-
gy (e.g. mesoporosity, hollow/compact) are some of the other factors
that can bring about significant changes in the specific surface area
and affect particle solubility. Particles having surface features with
smaller radius of positive curvature (convex) tend to be energetically
unstable and can thus have preferential dissolution and higher equilib-
rium solubility (Borm et al., 2006). A recent study on spherical and rod
shaped CuO NPs showed the spherical NPs to dissolve to a greater ex-
tent and faster than rod shaped NPs (Misra et al., 2012). Although
NPs are often described as spherical, they are actually highly faceted,
with different surface tensions in different surface planes. There have
been some studies which show that certain crystal faces can be more
susceptible to dissolution. For example, Liu et al. (2008) showed that
for galena (PbS) NPs {1 1 1} and {1 1 0} faces with lower coordination
numbers dissolve faster than {1 0 0} faces of nanocrystals. The dissolu-
tion behaviour of different crystal faces of minerals is a well studied
topic, bringing a lot of information about the effect of different surface
atomic configurations from the field of mineralogy to that of
nanoscience.
Apart from the intrinsic properties of the NPs discussed above,
characteristics of the surrounding media (pH, ionic strength, water
hardness) and presence of organic components (natural organic
matter, polysaccharides, proteins) can also affect suspension stability
and lead to NP agglomeration/aggregation, further affecting the
exposed surface area and dissolution of the particles. Both, the kinetics
of dissolution and the saturation concentration may vary for the same
NPs when suspended in different exposure (environmental, biological)
media. Increasing number of studies suggest the presence of organic
(viz. proteins, natural organic matter) and/or inorganic (viz. artificial
waters, simulated body fluids, buffers) components in the exposure
media can catalyse the process of NPs' solubility. In the case of CuO, it
is shown that the presence of amino acid rich media such as tryptone,
yeast extract can lead to almost complete dissolution of NPs, whereas
Table 1
Selected dissolution studies performed on nanoparticles pertaining to nanotoxicological studies.

Size (nm) Capping agent Media Method (analytical technique) Observation References

Copper/copper oxide nanoparticles

30 Osterhout's medium Recombinant metal sensor bacteria - 2% dissolution for CuO NPs compared to 0.12% for bulk CuO particles Mortimer et al. (2010)
(Escherichia coli MC1061)
25 Gastric fluid Centrifugation (AAS) - 1.2% of particles dissolved immediately within 5 min. Lei et al. (2008)
- Thereafter, the dissolution rate showed a slow, gradual rise to 2.1% at 2 h.
20 Ultra pure water Centrifugation (AAS) - 95% dissolution at pH 5.5 Studer et al. (2010)
- 0.3% dissolution at pH 7 and b0.1% dissolution at pH 7.4
14 - LB Broth Membrane filters (ICP-AES) - Dissolution is enhanced by the presence of the organic tryptone and yeast Gunawan et al. (2011)
- Water extract (80–95% dissolution)
- NaCl (5 g/L) - Deionised water or NaCl has little effect on the dissolution.
- Tryptone (10 g/L) - CuO NPs dissolved much more in the LB Broth medium compared to CuO
- Yeast extract (5 g/L) microparticles.
- Starting concentration affects the apparent equilibrium concentration.

S.K. Misra et al. / Science of the Total Environment 438 (2012) 225–232
20–30 - LB Broth Membrane filters (ICP-AES) - Dissolution rate reduced with increasing NP concentration Baek and An (2011)
- 2.16% dissolution
7 spheres 1 mM NaNO3 Dialysis (ICP-AES) - Spherical 7 nm NPs showed a higher dissolution compared to rod shaped NPs Misra et al. (2012)
7 × 40 rods and micron sized CuO particles
- Up to 3.5% and 1% dissolutions for spherical and rod shaped NPs

Silver nanoparticles
55 Nano lithography Buffer solution containing AFM - Dissolution rates varied linearly from 0.4 to 2.2 nm/d. Kent and Vikesland
of NPs 10–550 mM NaCl - Strong linear correlation between Ag dissolution rate and the concentration of NaCl (2012)
39 ± 9 PVP 0.01 M NaNO3, Ultra centrifugation (ICP) - 2% dissolution over a one month period. Levard et al. (2011)
- Sulfidation of the Ag-NPs caused a significant reduction in dissolution rates.
19 ± 13 Citrate Buffer solution containing 7 mM Membrane filters (ICP-MS) - Less than 10% dissolution of Ag NPs in the absence of cysteine Gondikas et al. (2012)
8±2 PVP NaHCO3, 10–500 mM NaNO3, - Presence of cysteine increased the amount of dissolved silver from the NPs. Up
and 400 μM cysteine to 36% for citrate-Ag NPs and 47% for PVP-Ag NPs
82 Bare (uncapped) Natural river water Centrifugal ultrafiltration (GF-AAS) - Ag dissolution of up to 3% after just 6 h in Tween-Ag NPs and similar level Li et al. (2012)
82 Citrate in 15 days for citrate and bare-Ag NPs.
88 Tween - Ag release was not impacted by the artificial sun light.
6, 25 Gum arabic 1 mM NaHCO3 Centrifugal ultra-filtration - Solubility was not affected by the synthesis method and coating as much as by Ma et al. (2012)
5–38 PVP (GF-AAS) their size.
10–200 Carbonate MOPS media in the presence Centrifugal ultrafiltration (ICP-MS) - 0.7–0.8% of dissolution over 3 h period Navarro et al. (2008)
median = 40 of cysteine - No increases of dissolved Ag in presence of cysteine (10–500 nM).
30–50 Oleic acid 1 or 100 mM NaNO3 medium Centrifugation (ion selective - Dissolved Ag decreased with increased EDTA loading Chappell et al. (2011)
containing varying amounts of electrode and ICP-MS) - In the presence of Brij 35, Ag NPs is considered to be in a supersaturated state as
EDTA, Brij-35 and alginic acid both dissolved Ag+ and total dissolved Ag.
- Brij-35 and alginate enhanced Ag solubility, whereas for EDTA enhanced
solubility was only observed at 1 mM.
20 Stabilised in 2 mM 2.5 mM (NH4)2SO4, 30 mM Centrifugal ultrafiltration (ICP) - After 3 h experiment 30% dissolution for 20 nm Ag NPs and 5% dissolution for Radniecki et al. (2011)
phosphate buffer HEPES buffer 80 nm Ag NPs.
80 Citrate Ultra pure water Dialysis membrane (atomic - Citrate-Ag NPs: up to 14% and 37% dissolutions at 25 °C and 37 °C, respectively. Kittler et al. (2010)
50 ± 20 PVP absorption spectroscopy) - PVP-Ag NPs: up to 50% and 90% dissolutions at 25 °C and 37 °C, respectively.
- Starting concentration significantly affects the apparent equilibrium concentration.
4.8 ± 1.6 Citrate Deionised water Centrifugal ultrafiltration - Ag dissolution is dependent on pH and dissolved oxygen. Liu and Hurt (2010)
(GF-AAS) - Existence of three distinct forms of silver: Ag NPs dissolved Ag+ and adsorbed Ag+.
- Temperature and starting concentration have a significant effect on apparent
equilibrium concentration.
71 ± 19 Citrate Media containing NaCl, Centrifugal ultrafiltration - Over the 30 min interval both the types of NPs showed higher degree of dissolution An Huynh and Chen
67 PVP MgCl2, CaCl2 (ICP-MS) in all electrolyte solutions than in deionised water. (2011)
5 and 11 PEG-thiol Deionised water, 2 mM Centrifugal ultrafiltration - 5 nm NPs released more dissolved silver under aerobic conditions than 11 nm NPs. Xiu et al. (2012)
NaHCO3 buffer (ICP-MS) - The dissolution of Ag NPs reduced under anaerobic conditions.

227
(continued on next page)
 228
Table 1 (continued)
Size (nm) Capping agent Media Method (analytical technique) Observation References

Zinc oxide nanoparticles

50–70 LB Broth Membrane filters (ICP-AES) - Dissolution rate reduced with increasing NP concentration Baek and An (2011)
- 3.2% dissolution
50–70 Osterhout's medium Recombinant metal sensor - No difference in dissolution between nano and bulk ZnO particles, with both showing Mortimer et al. (2010)
bacteria (E. coli MC1061) up to 80% dissolution.
30 Algal test medium Dialysis membrane (ICP-AES) - No difference in dissolution rate and equilibrium Zn concentration for both Franklin et al. (2007)
bulk and nano ZnO.
- 16 mg/L representing 19% dissolution
13 - Ultrapure water Centrifugation (ICP-MS) - Dissolution followed the order: DMEM (90 μM) > BEGM (80 μM) > H2O (60 μM). Xia et al. (2008)
- DMEM with 10% FBS - Organic components enhance dissolution, with to 80% of total dissolved Zn within 3 h.
- BEGM (growth factors,

S.K. Misra et al. / Science of the Total Environment 438 (2012) 225–232
cytokines and supplements)
19 - Ultrapure water with -Centrifugation and filtration - Equilibrium solubility followed the order: LB (68 mg/L) > DM (38 mg/L) > NaCl Li et al. (2011)
0.85% v/w NaCl (AAS) (14 mg/L) > H2O (6.9 mg/L) > PBS (0.57 mg/L)
- 5 mM PBS - Centrifugal - Nano and bulk ZnO dissolution nearly the same in ultrapure water. Dissolved Zn
- Minimal Davis medium Ultrafiltration (AAS) increased with increasing nano ZnO concentration in all media except PBS
- LB medium - No difference reported in the measurement of dissolved Zn by
ultracentrifugation and centrifugal filtration.
- 6 and 16 spheres Artificial seawater Centrifugation and filtration - Dissolution kinetics did not differ significantly between particle types. Peng et al. (2011)
- 240 × 50 and (GFAAS) - 4.1–4.9% dissolution, with highest equilibrium solubility for smallest spherical particles
860 × 300 rods and lowest for largest rods
- No significant effect of starting NPs concentrations on dissolution kinetics and
equilibrium Zn concentrations.
20–30 Natural seawater Centrifugal filtration (ICP-AES) - 70% dissolution at initial concentration in the range 0.1–1 mg/L and 32% at initial (Miller et al., 2010)
concentration of 10 mg/L
4 Aqueous solutions with Centrifugation ( ICP-AES) - Size dependent dissolution with equilibrium Zn concentrations of 57 mg/L for 4 nm, Bian et al. (2011)
15 varying pH and humic acid 22 mg/L for 15 nm and 10 mg/L for 241 nm particles.
241 contents - Dissolution is pH dependent (higher at lower pH) and the presence of humic
acid affects dissolution (increase) at high pH (>9.0)

Size (nm) Type of NPs Media Method (analytical technique) Observation References

Other types of nanoparticles

2–3 ZnS EDTA (pH 9–10) Small-angle X-ray scattering - Particle size and pH play important roles in dissolution. Zhang et al. (2010)
- Lower the pH and the smaller the particle size, the higher the solubility of ZnS
nanoparticles
Range of sizes TiO2 (various range Water Filtration (ICP-MS and adsorptive - Concentration results obtained using both the analysis techniques agreed well. Schmidt and Vogelsberger
of phases) stripping voltametry) - The amount of titanium dissolved increases strongly with decreasing pH. (2006)
- For anatase, reducing particle size increased dissolution.
- Non significant effect of temperature (25–37 °C) on dissolution
6–12 SiO2 (amorphous) TRIS buffer Molybdic acid method (ICP) - The silica concentration slowly decreased after reaching maximum value. This Roelofs and Vogelsberger
Different types of NPs NaCl solution was attributed to Ostwald ripening of NPs. (2004)
- Dissolution showed a strong size effect, with smaller sized NPs leading to higher
dissolved concentration than bulk phase.
25, 39, 101, 177 SiO2 (amorphous) Water with various inorganic Dialysis (molybdate colorimetric - At far-from-equilibrium conditions when surface area normalised: dissolution rates Diedrich et al. (2012)
salts method) decrease with decreasing particle size at both neutral and basic pHs.
- At far-from-equilibrium conditions when mass normalised: dissolution rates are close
to independent of particle size.
 S.K. Misra et al. / Science of the Total Environment 438 (2012) 225–232
the presence of NaCl does not have any significant effect on the solubil-
ity of CuO NPs (Gunawan et al., 2011). Similarly, the presence of cyste-
ine can increase the dissolution of Ag NPs (Gondikas et al., 2012). The
interaction of NPs with the media components can also result in pre-
cipitation and complexation of free ions produced during the process
of dissolution from NPs, further affecting their bioavailability. The char-
acteristic and interaction of the media with NPs can in some case act as
a levelling factor in terms of particle solubility by either reducing the
dissolution through steric protection or increasing the dissolution
through ligand promoted processes. The inherent differences in solu-
bility observed in simple media between size/shape/surface chemistry
specific NPs can become insignificant when suspended in relevant ex-
posure media. Although dissolution studies in organic solvents are
limited, a recent study by Siy and Bartl (2010) reported the dissolution
of CdSe quantum dots to be a reversible process affected by size, con-
centration, temperature and presence of ligands. Fig. 1 and various ex-
amples cited above, highlight the complexity of dissolution of NPs and
the various intrinsic and extrinsic factors that can influence the dissolu-
tion process. By having a better understanding of these factors,
comprehensive studies can then be conducted to measure the dissolu-
tion of various NPs to help understand their fate and their biological
response.
3. Assessing dissolution
Experimental dissolution protocols for macroscopic materials can
generally be adapted to the study of NPs, but key challenges remain
in describing adequately the physicochemical properties and other
variables (e.g. contaminants, structural defects, dislocations etc.) of
NPs, as they all can affect the dissolution kinetics and the approach
to equilibrium. Due to the increasing awareness of NP solubility,
emerging body of literature on nanotoxicity is able to interpret better
the biological response of NPs. In the absence of dissolution data the
observed toxicity was often attributed to the NPs, but by factoring
Fig. 1. A simplistic representation showing the factors that can affect dissolution of nanoparticles and the possible interconnectivity among the factors, themselves.
229
in dissolution of e.g. ZnO NPs, results (Franklin et al., 2007; Xia et
al., 2008) indicate that ionic Zn may play a significant role in eliciting
a toxic responses. In fairness to the work published to date, where
dissolution has not been factored in, it should be noted that evaluat-
ing dissolution of NPs, is not trivial. There are difficulties in adapting
existing techniques for measuring dissolution of bulk materials to
NPs. There are two important factors in the assessment of dissolution,
(i) separation of particles from dissolved component and (ii) instru-
mentation available to measure dissolution. The separation of the
nanoparticulate form from the dissolved component is the pivotal as-
pect, as ineffective method of separation can lead to over/under repre-
sentation of NPs' dissolution. Traditionally, truly dissolved fraction has
been separated from the suspension by using filtration through 0.2 or
0.45 μm filters, centrifugation or dialysis methods. All of these tech-
niques are also used either in isolation or in combination to separate
NPs from the dissolved species. Membrane separation techniques are
based purely on physical separation due to size of membrane pores
to determine the effectiveness and extent of separation of dissolved
and particulate. Dialysis and ultrafiltration methods come under this
category with the difference between the two being the pressure ap-
plied in ultrafiltration as opposed to concentration gradient being a
driving force for dialysis. Since, the lowest size of filters currently avail-
able (20 nm pore size filters) cannot cater for quantum dots or various
other sub 10-nm particles. Ultracentrifugation and dialysis membrane
have seen increased use for the separation of NPs. In the case of certain
heavy elements (e.g. silver) prone to getting adsorbed, use of dialysis
membrane alone remains a non-trivial task, as the efficiency of recov-
ery of dissolved species is often compromised due to adsorption of
the metal ions onto the membrane. The use of centrifugal ultrafiltration
(combination of centrifugation and filtration) units has recently been
shown to become an effective way of separating NPs from the
dissolved species due to the low pore sizes (cut off value down to
3 kDa) of the membranes available. The technique also allows for
high recovery of dissolved species (Ma et al., 2012). Due to the nature
230 S.K. Misra et al. / Science of the Total Environment 438 (2012) 225–232
of the NPs and their dissolved species, the effective separation of partic-
ulates from truly dissolved species and maintaining an optimal recov-
ery of the dissolved fraction remains the main challenge.
Once the separation of NPs from dissolved species is ensured,
there are a range of direct/indirect analytical techniques (Table 2)
that can be used to measure the dissolution. In the case of direct
method, no separation process of NPs from dissolved species is
required and the measurement can be in situ, for example, atomic
force microscopy (AFM), ion selective electrodes (ISE) (Benn
and Westerhoff, 2008), spectroscopic methods (Zook et al., 2011),
single-particle-inductively coupled plasma mass spectrometry
(SP-ICP-MS). (Mitrano et al., 2012) For Indirect methods, a separation
of NPs from the dissolved species is essential, as the technique itself
cannot distinguish the NPs from the dissolved species, for e.g., induc-
tively coupled plasma atomic emission spectroscopy (ICP-AES),
inductively coupled plasma mass spectrometry (ICP-MS) and atomic
absorption spectrometry (AAS). Each of the mentioned methods has
their own sensitivity and detection limits and a detailed discussion
of them would be beyond the scope of this article. Nevertheless, it
seems relevant that the way forward to measure dissolution of NPs
should entail (i) developing standard protocol(s) to measure dissolu-
tion of NPs addressing the technical difficulties of separating NPs
from ionic species, (ii) better understanding of the complex role of
physicochemical properties on dissolution and (iii) accounting for in-
teraction of NPs with the exposure media and its effect on dissolution.
Although, the separation and analytical methods discussed here have
their applicability in measuring dissolution of NPs, it is recommended
that the following aspects are addressed while measuring dissolution
of NPs: (a) measuring the effectiveness of the method used to sepa-
rate the NPs from the dissolved species; (b) estimate the percentage
recovery of dissolved species using ionic standard for the separation
method in use. This is particularly relevant due to the loss of dissolved
components when working with metals such as Ag which are known
to be unstable in solution at neutral pH and have high affinity for
sorption to surfaces (membranes, sample containers). (c) Under-
standing the limiting factors for each analytical technique (for exam-
ple, ISE measures free ions as opposed to ICP-MS which will measure
total dissolved species).
4. Toxicological implications of dissolution
Depending on the NPs' status within the surrounding media with
respect to their dissolution behaviour (i.e. which of the following
combinations will be present: NPs/ions; ions/complexes; suspended/
agglomerated NPs) their bioavailability, uptake rates and toxicity will
vary, along with the risks posed by the NPs to different environmental
compartments (Fig. 2). For example, if NPs aggregate and sediment on
release, they become available to sediment dwelling organism and can
enter through dietary uptake. Similarly, if the NPs dissolve in the expo-
sure media, speciation of ions with other ligands can be prominent and
the uptake path and uptake mechanism of ions in organisms will be dif-
ferent from the NPs themselves (Fabrega et al., 2009). Although, in many
Table 2
Selected methods for separation of nanoparticles from the dissolved species along with
the analytical techniques currently used for measuring dissolution of nanoparticles.
Separation method Analytical method
Dialysis membrane ICP-AES, ICP-MS
Centrifugation method ISE
Diffusion AFM
Complexation/speciation method Spectroscopic
Filtration/ultrafiltration Metal sensing bacteria
Centrifugal ultrafiltration AAS
Adsorptive stripping voltametry
cases dissolution can help in explaining the observed nanotoxicological
response, recent studies seem to favour a complex interplay between
NPs and dissolved species, both potentially contributing to the biological
response (Navarro et al., 2008). Studies with Ag NPs (Liu et al., 2010)
highlight this complexity and bring a further aspect of the NPs' behaviour
to focus, in that Ag NPs' suspension can contain different forms of Ag in-
cluding Ag NPs, free/complexed Ag ions and adsorbed Ag+ on Ag NPs, a
factor that can significantly affect the biological response to these NPs. In
addition, some in vitro cytotoxicity assays may also be sensitive to metal
ions and thus may be perturbed in the presence of dissolving NPs (Kroll
et al., 2009). While physiological effects of dissolved metals have been ex-
tensively studied, the unique reactivity of NPs along with a potentially dif-
ferent uptake mechanism means the current equilibrium models used to
calculate free metal ion concentrations have limited use in the case of NPs.
A recent study by Li et al. (2011) demonstrated that the toxicity observed
for 19 nm ZnO NPs when suspended in various media followed the toxic-
ity order of ultrapure water>0.85%NaCl>minimal Davis>LB>PBS.
Although ZnO NPs have a high dissolution, the reason for the order of
toxicity was explained by the generation of precipitates (Zn3(PO4)2 in
PBS) and zinc complexes, which significantly decreased the concentration
of Zn2+ ions. Such studies illustrate that if dissolution of NPs is known
under experimental conditions, it can then be factored into existing biotic
ligand models.
Among the known major cellular entry mechanisms for NPs and
ions, the extent of NPs dissolution will play a key role to ascertain
whether free metal ion concentration, or endocytosis mediated NPs'
response or a combination of both mechanisms will be driving toxic-
ity. For example, NPs that undergo dissolution in the media before
being taken up by the organism will clearly have ion transporters/ion
channels as the preferred route for cellular entry. Whereas, NPs that
resist complete dissolution in the media can have a combination of
possible routes, viz. endocytosis of NPs, or ion transportation of the
dissolved components or a combination of both. There may be another
scenario, where NPs associated with the biological membrane can act
as a reservoir of metal ions, which are released at a variable rate
when NPs undergo dissolution. It is also worth noting that studying
NPs' dissolution is not only important for correct interpretation of
nano-toxicological data, but can also help in the current move towards
“safety by design of NPs”. For example, surface functionalisation of
carbon nanotubes (e.g. using polyethylene glycols) can facilitate their
solubility (Lin et al., 2004) and thus reduce their biopersistence and
any potential toxicity. Whereas, dopants such as iron is used to reduce
dissolution of ZnO NPs (George et al., 2010), and therefore, indirectly,
influence cytotoxicity.
5. Conclusions
Dissolution can often be the key in understanding the cellular
response of compositionally different NPs and has the potential to
become a key component of a screening process for categorising
NPs with common hazard potential based on their potential to release
ionic species. Simple correlations between dissolution and physico-
chemical properties of NPs are often difficult to make and this is
mainly because a change in certain aspect of NPs can often induce a
simultaneous significant change in another physicochemical proper-
ty. For example, change in size and shape affects the surface area,
change in surface chemistry affects size, change in shape affects the
exposed crystal faces etc. This implies that many existing techniques
will need adaptation and establishment of new standard protocol for
NPs' solubility measurements in appropriate exposure media. This
may sound like a lot of work but failing to proceed in this way will
allow perpetuation of comparing apparently identical NPs and
coming up with different conclusions. Given the importance of the dis-
solution, increasing number of nanotoxicology studies have started to
incorporate dissolution while interpreting the biological response of
 S.K. Misra et al. / Science of the Total Environment 438 (2012) 225–232 231

Fig. 2. (A) Schematic representation of fate of nanoparticles when exposed to environment and under different dissolution scenarios. (B) Cellular entry mechanisms based on the
fate of the nanoparticles.

NPs. A strategy, favourable in understanding the true biological re- RD-834557501-0) (for the work of AD). The authors also thank Dr.
sponse of nanoparticles. Farhan Khan for his valuable discussion on particle internalisation.

Acknowledgements
The research leading to these results has received funding from the
QNano Project (for the work of SKM) http://www.qnano-ri.eu from
the European Community Research Infrastructures under the FP7
Capacities Programme FP7/2007-2013 under grant agreement no.
262163 and (US-EPA and UK-NERC-ESPRC Research Program Grant
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