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Copyright 2014 American Scientific Publishers

All rights reserved

Printed in the United States of America

Journal of
Colloid Science and Biotechnology
Vol. 3, 130, 2014

Nanoparticle Properties, Behavior, Fate in Aquatic

Systems and Characterization Methods
Mohd Omar Fatehah1 , Hamidi Abdul Aziz2 , and Serge Stoll1

F.-A. Forel Institute, University of Geneva, 10 route de Suisse, Versoix, 1209, Switzerland
School of Civil Engineering, Universiti Sains Malaysia, Nibong Tebal, 14300, Pulau Pinang, Malaysia


The global demand for a wide variety of applications based on engineered nanoparticles (ENPs)
has expanded the worldwide industrial scale production and inevitably released these materials into
the environment. The increasing existence of NPs and its impact towards human health and the
environment especially the aquatic system has sparked a great concern among both the scientific
community and the public. It is therefore crucial to gain an in depth understanding of the properties
the manufactured nanoparticles possess along with the various transformations they undergo that
determine their behaviour and mobility. This review begins by addressing the fundamental physicochemical aspects of manufactured oxide nanoparticles with detailed attention given specifically to
ZnO as a representative example in a separated section. The literature collected is summarized and
focused on the essential point of view to evaluate their occurrence, fate and transport in the natural
aquatic environment as a result of their interactions with other nanoparticles or natural colloids. Key
methods and principles of nanoparticle characterization are also presented.

Keywords: Nanoparticles, ZnO, Fate, Transport, Transformation, Nanoparticle Stability, pH

Effects, Nanoparticle Characterization, Aquatic Systems.

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Zinc Oxide Nanoparticles and Environmental Risk . . . . . .
1.2. Influence of Natural Organic Matter on Fate and
Transport of ZnO Nanoparticles . . . . . . . . . . . . . . . . . . . . .
1.3. Influence of pH and Zeta Potential on Stability of
ZnO Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.4. Nanoparticles and Their Removal
from the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.5. Scope and Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Occurrence, Fate and Transport of
Nanoparticles in Aquatic Systems . . . . . . . . . . . . . . . . . . .
2.3. Zinc Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Methods and Principles of Nanoparticle
Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Challenges of Nanoparticle Characterization
in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Engineered nanoparticles (ENPs) are produced by human
activities on a relatively large scale and have at least one

Author to whom correspondence should be addressed.

J. Colloid Sci. Biotechnol. 2014, Vol. 3, No. 2

dimension in the size range of 1 to 100 nm.1 2 These

nanoparticles exist in groups of carbon-based materials
and inorganic nanoparticles including metal oxides, metals and quantum dots.3 The increasing use of nanoparticles
in a gamut of applications comprehending industrial and
households, will inadvertantly see large release of these
nanomaterials into the environment. This is supported by
an increasing body of scientific evidence which suggest
that nanoparticles have been found to end up in the environment and that their fate and transformation processes
are difficult to evaluate and control.4 5 As a result of their
nanometric dimensions and interactions with the surrounding environment, these manufactured nanoparticles will
become mobile due to their dissolution and disaggregation
behaviour.2 6 7 Abiotic factors that affect the mobility and
transport of nanoparticles are pH, ionic strength, particle
surface chemistry, interactions of nanoparticles with other
pollutants7 and natural organic molecules.1 810
1.1. Zinc Oxide Nanoparticles and
Environmental Risk
Some archetypes of nanoparticles are iron oxide, titanium
dioxide, fullerene, cerium oxide, carbon nanotubes and
others.11 12 One of the most studied manufactured nanoparticles, in particular for its characteristics and behavior
is zinc oxide.1317 Nanosized ZnO has shown potential



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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

1.2. Influence of Natural Organic Matter on Fate and

Transport of ZnO Nanoparticles
The role of NOM in the fate and transport of ZnO nanoparticles have been broadly studied.3842 NOM in natural
aquatic systems mostly comprise of humic substances (HS)
and polysaccharides.43 HS are macromolecule structures44
and consists of 3050% of dissolved organic carbon
(DOC), humic and fulvic acids. DOC is found naturally
in water with a concentration rarely exceeding 5 mg/L.
The typical DOC molecular weight ranges from 102 to
106 Da.45 They carry potentially important functions in the
environment as they can control the pH balance, govern
the mobility of contaminants through absorption, aggregation, and disaggregation and they can coat other surfaces
to give them an overall negative charge through charge
stabilization.39 46
J. Colloid Sci. Biotechnol. 3, 130, 2014

Humic acid (HA), on the other hand, is insoluble

and will precipitate out in water under acidic conditions,
especially below pH 2. It is then otherwise water soluble
at alkaline pH.45 The presence of HA is found ubiquitous
in the natural environment47 and has entailed with several investigations conducted on nanoparticles to observe
their aggregation and disaggregation behavior with HA
adsorption. The interaction of HA and negatively charged
ions is mainly due to the van der Waals interactions with
the NPs in the solution. Electrostatic and steric stabilizations have also been demonstrated in other independent studies involving NPs and NOM when they are in
suspensions.1 9 45 The NOM surface coatings around the
NPs indicate disaggregation through charge and steric stabilization mechanisms.4148
Polysaccharides constitute 1030% of the NOM in natural waters49 and in marine environments. Alginates are
naturally occurring polysaccharides, released by microorganisms such as algae, bacteria and plant roots50 51 commonly found in the marine environments.52 Some alginates
may also be found in nature as components of some algae
cell walls, and are likely to be excreted by the algae in the
form of extracellular organic matter.53 Alginates have the
tendency to promote and enhance particle aggregation and
deposition via bridging process.54 The interaction caused
by these macromolecules will have a profound effect on
the surface chemistry and transport of nanoparticles in
aquatic systems.55 56
1.3. Influence of pH and Zeta Potential on
Stability of ZnO Nanoparticles
ZnO is an amphoteric oxide and can easily dissolve in
both acids and bases.57 At acidic pH values of <63,
ZnO is hydrated to form Zn2+ cations and subsequently
forms hydroxide layers in water at basic pH values, where
Zn(OH)2 is in equilibrium with the Zn2+ , Zn(OH)
3 , and
become the dominant species in solution.58 The major
problem of ZnO nanoparticles arises from their poor stability in water59 60 which leads to the formation of aggregates as it approaches the point of zero charge (PZC)
or pHPZC .61 In aqueous suspensions of ZnO, certain pH
regions can strongly affect the stability electrostatically
due to the transformation of colloidal Zn(OH)2S particles
to Zn(OH)2aq as the suspension stability is highly dependent on the surface charge of the constituent oxides.62 63 It
is unknown to which extent that nanoparticles will agglomerate depending on the processing conditions and the balance between the attractive and repulsive forces among the
nanoparticles as well as in between them.41
pH has a huge influence on ZnO nanoparticles and has
led researchers to further investigate its rheological and
electrophoretic properties based on measuring the viscosity versus the pH and amount of dispersant.58 One of the
earliest studies on zeta potential and pH was done by


toxicity with its existence in the environment which has

sparked a great concern from both the scientific community and the public. In the past few years various adverse
effects of nanosized ZnO on plants, phytoplanktons, mammals, and even human cell lines have been reported.1822
The mechanism of ZnO toxicity has been discussed by
Djuriic et al.23 ZnO NPs in the aquatic systems have
been revealed to potentially cause harm to aquatic organisms, especially if dissolved Zn2+ ions are released.24 The
solubilized ZnO NP can exert stress on cells and have
adverse impacts on different organisms.2527 This is evident in the ecotoxicity studies on ZnO NPs conducted
on bacteria such as Escherichia coli,21 28 Bacillus subtilis,
Streptococcus aureus,29 and marine algae.30 It is therefore essential to comprehend the behaviour of ZnO NPs
because their fate, transport, behaviour and ecotoxicology are closely related to their intrinsic properties such
as particles in suspension, surface energy and colloid
stabilisation.5 31 Additionally, understanding how external factors such as physicochemical conditions e.g., pH,32
physicochemistry of the particles,8 and interactions with
other molecules10 32 will provide a clearer view on the complex system of ZnO NP and its behaviour. This is relevant
because the NP mobility is dependent on the physicochemical transformation they undergo such as surface modification, aggregation, disaggregation and dissolution.2 6 The
transformation is a function of abiotic factors including pH,
ionic strength, particle surface chemistry, the interactions
of nanoparticles with other pollutants and natural organic
molecules.78 10 3234
Based on previous research on NP environmental toxicology, the above factors like redox conditions, light,
natural organic matter (NOM), and the presence of
microorganisms may result in chemical and/or biological
transformations of ZnO nanomaterials and induce their
mobilization in the environment where they can potentially exert noxious effects on aquatic organisms and
humans.12 3537


Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Logtenberg and Stein64 who discovered that zeta potential

is distinctly influenced by changes in acidity and alkalinity and the chemisorptions of Cl and K+ ions. More
recent scientific literature also addressed the zeta potential behaviour of ZnO nanoparticles.58 62 6569 Research on
the effects of pH and time on nanoparticle zeta potential,
agglomerate size, and cellular viability have been done
by Berg et al.65 The ambient conditions surrounding the
nanoparticles have a close relationship with zeta potential
and this relationship remains a largely unexplored area.
The zeta potential is affected by pH and represents the
charge of a nanoparticle with respect to that ambient surroundings. Zeta potential cannot be taken as the actual
measurement of the individual molecular surface charge,
on the contrary, it is considered as a measurement of the
electric double layer produced by the surrounding ions
in solution (i.e., counter ions). Stability of the ZnO NPs
depend on the pH of the system, where ZnO can form
aggregation or remain in colloidal form. There have been
few comprehensive reports on the aggregation behaviour
of ZnO.17 70 71 Sadowski and Polowczyk66 reported that
without adjusting the pH of the suspension (pH 7.47.6),
and by adding cationic surfactants, will cause a positive
increase in zeta potential. In another similar study, specific
adsorption of carbonate ions in ZnO solution caused a shift
of the pH at pHPZC to 8.3 as its concentration increases67
while adsorption of anionic sodium dodecyl sulfate (SDS)
and propylene glycol coating in ZnO NPs significantly
shifts the pHPZC to pH 3.70 A pH study from pH 7 to
pH 11 was conducted by Tang et al.58 to see the effect on
the zeta potential of ZnO nanoparticles with the addition
of cationic polyelectrolyte-polyethylenimine (PEI). Subsequent to that, Tang et al.69 also explored the effect of
adding anionic polyelectrolyte, ammonium polyacrylate
(PAA) on the ZnO zeta potential. Other researchers examined the dissolution behaviour of ZnO nanoparticles as
a function of pH, ionic strength and addition of natural
organic matter, its role in the acute or chronic toxicity of
aquatic organisms and the chemical etching effect.24 7173
1.4. Nanoparticles and Their Removal
from the Environment
The exact amount of manufactured nanoparticles that
are released into the environment has yet to be determined. Nonetheless, numerous studies have justified that
it does occur on a relatively large scale.7477 This poses
a huge dilemma on environmental, health and safety
issues.19 7881 The occurrence of nanoparticles that undergo
physical transformation such as disaggregation39 82 will
form suspended sediment particles which are known to
be important in sequestering and transporting contaminant
chemicals over significant distances. The hydrodynamic
and characteristics of bodies of water and morphology
of coastal zones will largely determine the distribution
of these nanoparticles in the environment.22 83 A research

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by Zhang et al.84 revealed that chemical treatment and

sedimentation in water treatment is still inadequate to
remove NPs and requires microfiltration. Albeit the extensive efforts in both water and wastewater treatments to
remove NPs,8589 it is likely the unintentional release of
NPs will enter the water bodies in undetermined volumes.
Therefore it is crucial to first understand the NP characteristics such as the surface charge and how it is affected by
the aggregation and disaggregation to assess their behavior, fate and transport.
Nanoparticles, also defined as colloids, are construed
by environmental processes and are largely dominated
by aggregation behavior. Aggregates forming larger than
1 m conventionally subjugates to the course of sedimentation, sometimes termed as colloidal pumping, a process that has been well characterized to understand trace
metal behavior. The general tendency of metals to sorb
to high-specific-surface area small colloids will aggregate and deposit themselves. This physical transformation transfers metals from the water matrix to sediments
and is analogous to the behavior where water bodies
undergo self-purification, resulting in pollutant loss from
surface waters. The ultimate fate of NP aggregation and
subsequent sedimentation is an important process in the
1.5. Scope and Objectives
The overall objective of this review is to discuss the
physicochemical interactions that actually govern the
particle-surface and the particleparticle interactions that
represent conditions of aquatic environments.

2.1. Nanoparticles
Nanoparticles are generally defined as particles smaller
than 100 nm in at least one dimension91 and have existed
for a long time in all mediums, water, air and soil.90
This definition puts them in a similar size domain as that
of ultrafine particles (air borne particulates) and places
them as a sub-set of colloidal particles.31 These materials with nanoscale sized structures and components exhibit
novel and significantly improved physical, chemical and
biological properties. Nanoparticle properties are used
extensively in various fields including medicine, pharmaceuticals, manufacturing technologies, electronics and
telecommunications.70 91 92 In Figure 2, nanoparticles can
be divided into natural and anthropogenic origin and the
two are distinguished according to their nature. The former is naturally occuring whereby it is generated through
any number of natural processes (e.g., mineral weathering)
or it may be an unintended by-product from technological processes while the latter results from target-oriented
manufacture.31 Both natural and manufactured NPs can be
sub-grouped into environmentally relevant carbonaceaous
J. Colloid Sci. Biotechnol. 3, 130, 2014

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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Fig. 1. Schematic illustration displays the possible interactions and behavior of ENPs in the aquatic environment. (A) Chemical transformation due to
abiotic factors such as pH, light or ionic strength can lead to dissolution, redox reaction etc.; (B) Biological transformation from biological degradation
of polymer coatings on nanoparticles can affect their surface properties and lead to aggregation; (C) Physical transformation include aggregation and
dispersion which will affect the mobility of the nanoparticles.

and inorganic nanoparticles based on their chemical composition which are later discussed.
2.1.1. Natural Nanoparticles
Natural nanoparticles have long existed on earth and
can be found in all three main mediums of earth i.e.,
atmosphere, soil and water. Sources of natural NPs in
the atmosphere include volcanic eruptions, forest fires,
hydrothermal vent systems, physical and chemical weathering of rocks, precipitation reactions and biological


Found in atmosphere, soil and water.
Divided into biogenic, geogenic,
atmospheric and pyrogenic NPs
Examples: organic colloids (i.e.
humic acid, fulvic acid), organisms
(viruses), soot, organic acids
(i.e. sea salt), CNTs

Materials purposely produced by
human activities-nanoproducts.
At least one dimension (1-100 nm)
Classified based on chemical
Examples: metals (Au, Ag, Fe),
metal oxides (i.e. TiO2, ZnO, Al2O3,
CeO2), quantum dots

Fig. 2. Nanoparticles are generally categorized into two groups, natural and engineered NPs. Natural NPs are commonly formed due to
environmental processes and eventually end up as biogenic, geogenic,
atmospheric and pyrogenic products. Examples are soot, organic acid
etc. ENPs are manufactured to create nanoproducts for human use and
applications such as paint, biomedical, cosmetics etc.

J. Colloid Sci. Biotechnol. 3, 130, 2014

processes93 including sea salt in the form of airborne

nanocrystals as a result of evaporation from sea water
sprays.20 In soil, colloids are known to constitute silica clay minerals, iron- or aluminium oxides/-hydroxides
or humic organic matter, including black carbon. There
are also forms of nanominerals i.e., ferrihydrite and natural organic-mineral aggregates. A complex matrix containing particles and colloids in pore water can be found
that may adsorb and bind pollutants within the matrix
while freshwater contains very complex colloid material which includes inorganic minerals and organic matter
such as humic substances.35 In the aquatic environment,
natural NPs comprise different forms of colloids (e.g.,
metal-sulfide nanoclusters from hydrothermal systems, and
hydrous iron and manganese oxides).93 Ocean surface
microlayer also contains colloids, sub-micron components
of phytoplankton, and carbon particles.
Carbon based natural NPs are divided into biogenic,
geogenic, atmospheric and pyrogenic NPs. Some examples
are fullerenes and CNT or geogenic or pyrogenic origin,
biogenic magnetite or atmospheric aerosols (both organic
such as organic acids and inorganic such as sea salt).12
However, the natural background of NPs in the atmosphere
is low compared to the levels caused by the combustion
processes, diesel and gasoline-fueled vehicles and stationary combustion sources, which have for many years contributed to the particulate material in the atmosphere.94


Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Here, we shall deliberate on two examples of natural

NPs. Soot, for instance, is one of the results of natural combustion processes which emit a wide variety of
particles from both stationary and mobile sources. Only
ultra-fine particles of these natural combustion processes
correspond to the standard definition of NP. In this case,
the term soot is used to represent nanosize Black Carbon
(BC) combustion continuum. Re-condensation processes
during incomplete combustion of fossil and renewable
fuels mainly emits soot as a product into the atmosphere
from where it distributes hemisphere-wide and is deposited
onto soils and water bodies. Another example is fullerenes.
Natural sources are known to have brought this nanomaterial to earth by comets or asteroids. However, majority of
fullerenes is believed to have been formed from polycylic
aromatic hydrocarbons (PAH) derived from algal matter
during metamorphosis at temperatures between 300 and
500  C and in the presence of elemental sulfur or during
natural combustion processes.12 Fullerene C60 has low solubility in water,95 however they are relatively soluble in
a number of organic nonpolar solvents, such as benzenes,
alkanes or naphtalenes.11
2.1.2. Manufactured Nanoparticles
Anthropogenic NPs, often referred to as engineered or
manufactured nanoparticles are materials purposely produced by human activities which have at least one dimension in the size range 1100 nm and can be classified
according to their chemical composition and properties.2
Manufactured NPs, can be either inadvertently formed
by a by-product, mostly during combustion, or produced
intentionally due to their particular characteristics. They
represent an intermediate supramolecular state of matter
between bulk and molecular material. Manufactured NPs
cover a wide spectrum of substances such as fullerenes
and CNTs, both pristine and functionalized, including elemental metals (e.g., silver, gold and iron), metal oxides
(e.g., titanium dioxide, iron oxide and aluminum oxide),
metal salts, quantum dots and fullerenes.11 The mentioned
examples are elaborated.
Fullerenes are made up of pure carbon. The simplest
fullerene, C60 , is a ball made up of 60 C atoms and resembles a football. Fullerenes are also examples of NPs that
can be present as a consequence of nanotechnology development. Of the large family of fullerenes, the buckminsterfullerene C60 is by far the most widely investigated.12
Carbon nanotubes (CNTs) are fibrous fullerenes consisting of rolled up graphene sheets that may or may not be
capped at the ends by a half fullerene sphere.20 CNTs exist
in two main manufactured forms,
(i) the single-walled or SWCNT and
(ii) multi-walled or MWCNT.
Carbon nanotubes are generated by arc evaporation, laser
ablation, pyrolysis, and electronic methods. SWCNTs possess important mechanical, thermal, photochemical and

Fatehah et al.

electrical properties which are industrially useful. MWCNTs contain several SWCNTs in their structure, and
therefore, they possibly have analogous physicochemical
properties to those corresponding to SWCNTs.3
Metal oxides nanoparticles are known to have the
unique ability to promote faster electron transfer kinetics
between the electrode and the active site of the desired
enzyme.96 Metal oxide NPs are widely used in a number
of applications i.e., food, material, chemical and biological
sciences. Among of the most important commercial metal
oxide NPs are elaborated below.
Nanoparticulate iron oxides (e.g., magnetite Fe3 O4 ,
maghemite Fe2 O3 , hematite Fe2 O3  are one of the most
abundant forms of anthropogenic nanomaterials as they are
found in soil, water and the cytoplasm of living cells. The
behavior of iron oxide NPs in aqueous media is largely
governed by the size, shape, oxidation state and stability of the iron oxide, all varying according to the specific synthesis procedure and conditions used. Iron oxide
has a positive surface in most environmentally relevant pH
conditions which means that these materials will interact
favourably with the majority of negatively charged natural
components in aqueous environments.11
The bulk structure of CeO2 is made of Ce(IV) atoms,
eight oxygens and four ceriums. Based on the respective equilibrium constants for proton adsorption, Ce3+ ions
may be more mobile and bioavailable than nanoparticles,
which raises the need to consider and quantify the contribution of cerium reduction and dissolution in the risk
assessment of cerium nanoparticles. CeO2 dispersion is
highly stable from below pH 6, since these surfaces are
strongly positively charged in this region. Self-aggregation
occurs at above pH 6, where the CeO2 surface is less
charged as the pH approaches the IEP, which results in
the interfacial interaction between CeO2 surfaces becoming more attractive. This phenomenon displays kinetics
that are correlated with the distance of the pH from the
Nano-TiO2 is produced on a large scale in the applications of paints and coatings (self-cleaning, antifouling,
and antimicrobial properties) and in cosmetics as a UV
adsorber.77 Titanium dioxide (TiO2  is one of the most
widely used nanomaterials in the industry.96
Metal nanoparticles such as silver NPs are nanoscale
clusters of metallic silver atoms, Ag0 . Metallic silver is
relatively nonreactive. However, in aqueous environments,
silver ions (Ag+  are released from the bulk metal and into
solution. Ag+ ions are used for a variety of antimicrobial
applications and sterile applications due to its antimicrobial, antifungal and partially antiviral properties.7798 Gold
nanoparticles are typically inert but become catalytic as
their size decrease to a few nanometers.39
2.1.3. Natural Aquatic Colloids
Natural aquatic colloids in surface waters are a composition of environmental complex mixtures and heterogenous
J. Colloid Sci. Biotechnol. 3, 130, 2014

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J. Colloid Sci. Biotechnol. 3, 130, 2014

complexity and heterogeneity of the colloidal structure,

and how this relates to their environmental function, is still
poorly understood.74 Humic Acid. Humic acid (HA) or its standard
name Suwannee River Humic Acid (SRHA) belongs to
one of the major classes of natural organic matter other
than fulvic acids, humins and polysaccharides.44 47 They
are naturally occurring, biogenic, heterogeneous organic
material, generally polydisperse and contains functional
groups such as carboxylic acid, amine, carbonyl, alcohol
and carboxylate (COO). The natural pH of humic acid
is pH 3.447 and its molecular weight ranges from 1000 to
greater than 106 amu. HA is insoluble at low pH (<2) but
easily dissolves at alkaline pH. In aqueous solutions, HA
exist as dissolved macroligands at low concentrations, and
as aggregates at higher concentrations.20
Though categorized as a supramolecular, HA has a
poorly defined structure and is characterized by aromatic
and aliphatic structures in which hydrogen bonding plays a
significant role in the aqueous phase structure. Site binding
in humic acid usually involves an electrostatic interaction
where one of the HAs functional groups (i.e., COO, carboxylate) and a cation (Ca2+ , Mg2+ , etc.) form an ionic
bond. The main factors affecting the structure and controlling the size are pH, the cation type and concentration,
and residence time. It is also well-known that HA can
significantly modify the surface properties (e.g., electric
charge, size, chemical nature of the exposed surface sites)
of natural aquatic colloids, significantly influencing their
transport, often due to increased electrostatic repulsion.100
Under most environmental conditions, small amounts
of HA can coat other surfaces to give them an overall
negative charge that results in reduced aggregation and
promote disaggregation through charge stabilization of the
complexes under the right conditions.3446 Collectively, HA
tends to aggregate as the ionic strength increases. This
aggregation phenomenon is more important at higher pH
values, because of the higher quantity of the dissociated
functional groups, and thus, the higher negative charge on
HA. It is obvious that the behavior of HA under variable pH and ionic strength is tremendously complex. This
behavior could be completely inversed depending on
(i) the pH and ionic strength,
(ii) the balance between the surface charges of HA developed by the dissociation of functional groups and the concentrations of cations present in the solution,
(iii) the preparation method: i.e., fixing the ionic strength
and varying the pH or fixing the pH and varying the ionic
strength. Consequently, different trends could be obtained
under different conditions as stated by other researchers. Biopolymers. Biopolymers can be divided
into two types which are protein and polysaccharides
nanoparticles.101 Protein nanoparticles are naturally occurring materials such as albumin, collagen, gelatin, silk protein from sericin and fibroin nanoparticles, sericin and


phases defined as solid-phase materials having at least one

dimension within the size range of 1 nm1 m.31 94 Colloids in the environment are formed from processes that
have taken place for several millions of years. Natural
aquatic colloids are produced (weathering, microbial processes) and lost (aggregation and sedimentation, microbial
action) by several processes.46 Based on the size scale,
manufactured nanomaterials which has a dimension less
than 100 nm is overlapped and thus included in the colloidal category.2 Colloids may originate from both natural
and anthropogenic sources. However in this case, we shall
focus more on the natural origin. Natural colloids can be
divided into two groups, organic and inorganic natural colloids. Organic colloids can be explained in terms of colloidal components such as small organic macromolecules
(such as humic acid and fulvic acids, 12 nm in diameter), fibrillar polysaccharides (110 nm wide and up to
several um long), biocolloids (i.e., bacteria, viruses and
fungi) while inorganic colloids comprise of silicates (e.g.,
clays, chlorites, kaolinite), oxides, carbonates and metal
sulphides.2 31 Natural aquatic colloids can also be classified by their particle size and are generally fractal with
a 3-D network type structure. Using the small-angle neutron scattering (SANS), three characteristic length scales
were determined-primary particle size with ca. 310 nm,
small aggregates of 2050 nm and transient networks of
aggregates with a length scale of 50200 nm.99
Besides manufactured nanoparticles, natural aquatic colloids too have significant effects on pollutant, nutrient
and pathogen chemistry, transport and bioavaibility. The
nature and morphology of major aquatic colloids have
been described elsewhere.49 By apprehending their chemistry and environmental impact will provide a better understanding the fate and behaviour or trace elements and trace
organic pollutants as these natural NPs can cause a deleterious effect.3
Colloids provide a molecular milieu into and onto
which chemicals can escape from the aqueous solution
and whose environmental fate is predominantly affected by
coagulation-breakup mechanisms, as opposed to removal
by settling.12 The physicochemical transformation of naturally occurring nanoparticles in the environment include
particle aggregation, disaggregation, and surface modification; processes which usually take place in the presence of
NOM, and that respond to changes in temperature, concentration, pH or ionic strength. Natural aquatic colloids
are of small size and large surface area per unit mass
makes them important in pollutant binding as the morphology, composition and structure of these colloids determine
their role in the environment.90 Recent studies have applied
the colloid science principles based on the DerjaguinLandau-Verwey-Overbeak (DLVO) theory to gain a better
understanding of NP aggregation under various conditions
which will lead to further information of the fate and
behavior of trace pollutants.34 In addition, the profound


Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

keratin.102 103 Polysaccharide nanoparticles are also naturally occurring with nanostructured surfaces which are
designed for the administration of peptides, proteins and
nucleic acids.104106 They are able to help to improve
biocompatibility of cell toxic material, which are currently being developed for novel bionanoparticle-derived
pharmaceutical formulations. Examples are alginate and
chitosan.107110 Alginate. Alginate is a natural occurring polymer and represents one of the common polyelectrolytes
found in suspensions comprising of colloidal particles used
in biomedical, environmental, and industrial applications.54
It is composed of linear unbranched polysaccharides
consisting of two types of uronic acids, -L-guluronic
acid (G) and (1,4)-linked -D-mannuronic (M). The
sequence and molecular weight of the monomeric units are
grouped in three ways: blocks of alternating guluronic and
mannuronic residues (MG-blocks), blocks of guluronic
acids (G-blocks) and of mannuronic acids (M-blocks)
(Fig. 3). The source of alginates also varies i.e., brown
seaweed,111113 commercial sources extracted from marine
algae i.e., Laminaria hyperborea, Ascophyllum nodosum
and Macrosystis pyrifera114 or even bacteria.54 Among the
alginate characteristics that are determined by the prevalence and sequence of the alginate block types that control
its chemistry in solution are water soluble, mucoadhesive,
biocompatible and non-immunogenic.112115

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Alginates are also known to undergo dissolution and

biodegradation under normal physiological conditions.113
However, they have poor mechanical properties and processing difficulties when compared with the synthetic
polymers. Alginates are negatively charged from the carboxyl groups located on the ring structure of both the M
and G monomers.112 116 The polymer chains of alginates
with carboxyl (COO  groups exists in the form of a
stretching conformation due to the repulsion between the
deprotonated carboxyl groups and have a high hydrophilicity in basic and neutral solutions. However, in an acidic
solution, the polymer chains tend to aggregate because
of the protonation of carboxyl groups which leads to the
decreased hydrophilicity. The aggregation behavior of alginate was studied by Yu et al.117 They found it difficult
to precisely control the hydrophilic/hydrophobic balance
of the polymer chains of natural polymers to induce selfassembly and to form stable aggregates in aqueous media.
Alginate has become a subject of academic as well as of
industrial interest because of their renewability and biodegradibility. It has been used as a chelating agent to enhance
interaction with Zn ion to form ZnO nanostructures.118
In another study, zinc alginate beads were prepared by
dropping aqueous solution of sodium alginate into a zinc
solution containing zinc nitrite or zinc acetate.109 Chen
et al.54 studied enhanced aggregations rates demonstrated
by nanoparticles coated with alginate. The aggregation

Fig. 3. Chemical structures of alginates consists of (1 4) linked -D-mannuronic acid (M) and -L-guluronic acid (G) residues. The structures
above depicts G-block, M-block, and alternating blocks in alginates.

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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

kinetics of alginate-coated iron oxide (hematite) increased

with the presence of divalent ions of Ca2+ , Sr2+ and Ba2+ .
In another study, alginate was used to control the synthesis of ZnO nanoparticles by microwave treatment and
produced mostly spherical in shape and hexagonal crystal structure and showed strong antibacterial activity with
99.9% reduction for S. aureus and 100% reduction for
E. coli after 2 hours of exposure.115

Other NP properties that could be studied are dispersibility, agglomeration/aggregation, dissolution rate and reactivity (e.g., catalytical activity, sorption capacity).119 These
properties are very useful in toxicological studies,8 food
production i.e., emulsification, gelation, foaming, waterbinding capacity,120 processing, packaging, additives and
safety,121 complex food and environmental samples.122 123 Zeta Potential and Surface Charge. The zeta
potential represents the charge of a nanoparticle in relation to the surrounding conditions. Nevertheless, the zeta
potential is not an actual measurement of the individual
molecular surface charge; rather, it is a measurement of
the electric double layer produced by the surrounding ions
in solution (i.e., counter ions).66 These counter ions play
a role in the calculation of zeta potential measurement.
All particle systems in an aqueous media carry an electric charge which may be positive, negative, or neutral.
For surface-derived nanoparticles, dissociation of an acidic
group, such as a carboxylic acid moiety on a nanoparticle surface will yield a negatively charged surface; while
dissociation of a basic group on a nanoparticle surface
will yield a positively charged surface. For unmodified
nanoparticles, the individual atoms on the surface of the
particle dictate its charge. The addition of HCl affects the
shift of the zeta potential of the ZnO dispersions to more
J. Colloid Sci. Biotechnol. 3, 130, 2014


2.1.4. Nanoparticle Properties Physicochemical Properties. With emphasis
that nanoparticles are typically engineered or formed post
processed for specific applications, their physico-chemical
properties and reactivity therefore vary considerably.21
Many investigators have outlined the key characteristics
of manufactured NPs that are believed to exert important controls on their environmental behavior, fate and
ecotoxicity,35 93 119 as well as uptake and distribution
within organisms, and the interactions of nanoparticles
with other pollutants.32 Due to their small size and homogeneous composition, structure, shape or surface characteristics, these manufactured NPs often exhibit a range of
special physico-chemical properties and reactivities that
are expected to deviate from bulk behaviour.12 35
The intrinsic properties of manufactured NP include
(i) physical characteristics, particularly size and shape,
surface area, electrical conductivity, state of dispersion;
(ii) chemical characteristics such acid-base character of
the surface charge, chemical composition, surface chemistry and the aqueous solubility of the NPs.8 32

positive values, while addition of KOH results in a similar

shift to more negative values.64
Surface charge is defined as an electric charge present at
an interface of a NP where the NPs propensity to interact
with charged surface and ions can be measured. Surface
charge results in the formation of an electrical double layer
containing ions attracted from the solution to the particle
surface in response to the charge. The electrical potential
at the interface of the diffuse layer and the bulk solution
can be measured, and its variation with solution chemistry
can effectively be used as a surrogate for the variation in
particle surface charge with solution chemistry.119
Surface charge of NPs may be either pH dependent,
as in oxide materials (due to protonation and deprotonation of functional groups).124 They may also be fixed,
as in clays, where this charge results from crystal lattice
defects and atomic substitution.20 62 The chemistry of the
medium will influence the electrostatic surface charge of
the particles, thereby affecting agglomeration/aggregation
rates and particle stability.119 In systems formed by aqueous solutions and oxides, hydroxides, or oxide hydroxides, the hydronium H3 O+ and hydroxyl OH ions are the
potential-determining species; therefore the surface charge
depends on the pH of the solution.125 Varying the solution
pH resulted in a significant change in the particle surface
and consequently, the hydrodynamic diameter.8 In another
study, guar gum adsorbed on the surface enhanced the
mobility of nano zerovalent iron (NZVI) in sandy porous
media regardless of the solution chemistry for instance,
pH and ionic strength.126 Surface charge is responsible
for colloidal NP properties causing particle repulsion and
attractions. Polyelectrolytes are able to modify the surface
charge of NPs by giving a surface coating.69 It is closely
related to hydrophobicity, which is a particles incapacity
to interact with water. Surface charge and hydrophobicity
is presumably an important factor which determines NP
behavior in the environment.20 Particles can acquire charge
when they adsorb ions present in the liquid.68 A study
by El Badawy et al.125 revealed that the particles can
acquire charge when they adsorb ions present in the liquid.
Adjusting the surface charge can be an effective method to
modify the cytotoxicity, cellular uptake, and specificity of
targeting of NPs.128 In systems formed by aqueous solutions and oxides, hydroxides, or oxide hydroxides, the
hydronium, H3 O+ and hydroxyl OH are the potentialdetermining species of the charges.129 Electrical Double Layer. The interface between
a metal oxide and aqueous solution is of great importance
in many fields of chemistry. The surface complexation of
the metal oxide is strongly influenced by the development
of the surface charge which results in an electrostatic double layer that forms around the oxide surface of each particle in the electrolytic solution.129 The development of a
net charge at the particle surface affects the distribution of
ions in the surrounding interfacial region, resulting in an

Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Fatehah et al.

Hydrodynamic plane of
shear (slipping plane)

Diffuse layer
lons are diffused
more freely
around the

Charges beyond the slipping plane will

not move with the particle as an entity.
ions within this boundary will move with
particle as one entity.

Negatively charged
Stern layer
The particle will attracrt ions of the
opposite charge. positive ions will
move closer to the surface. these
ions are tightly bound immediately
around the surface.

Potential enery curve

Surface potential



Stern potential
Zeta potential
Distance from particle surface

Fig. 4. The electrical double layer on the surface of a nanoparticle is based on the Gouy-Chapman-Stern model. The energy potential curve is depicted
below where the surface potential is the electrical potential surrounding the particle while the stern potential is the electrical potential at the stern layer.
The zeta potential is the electrical potential at the hydrodynamic plane of shear (slipping plane).

increased concentration of counter ions (ions of opposite

charge to that of the particle) close to the surface. The
liquid layer surrounding the particle forms two regions.
The inner region, called the Stern layer, where the ions
are strongly bound and an outer, diffuse region where they
are less firmly attached. Within the diffuse layer there is
a notional boundary inside which the ions and particles
form a stable entity. When a particle moves (e.g., due to
Brownian motion), ions within the boundary move with
it, but any ions beyond the boundary do not travel with
the particle. This boundary is called the surface hydrodynamic shear or slipping plane. The potential that exists at
this boundary is known as the zeta potential.130 Figure 4
depicts a schematic diagram of the electrical double layer
(EDL) on the surface of a particle, with the different potentials to be considered and the Debye length 1/k which is
the length where the potential has fallen to a value of 1/e
of the Stern potential. Particle Size and Shape. The most evident
parameter to describe nanoparticles is size, as it can influence a wide range of material properties such as electronic
properties, and interactions with light and other types of
electromagnetic radiation. When the particle diameter is
between 1020 nm, the number of atoms on a particle surface starts to constitute a significant fraction of the total
number of atoms in a particle, which may lead to a number
of changes related to free surface energy (lattice properties, cell parameters etc.).20

The surface charge of a nanoparticle is closely related to

its size and shape. Adjusting the surface charge is generally accompanied by size and shape variations, and therefore a combined effect is achieved as both the particle
size and shape greatly affect the NP properties.128 The fact
that the size of the nanoparticles itself can be a factor
in direct toxicity and pathology is extremely important,
and biodegradability may be a further significant factor in
governing harmful biological effects. Nanoparticles have
a proportionately very large surface area and this surface
can have a high affinity for metals (e.g., iron) and organic
chemical combustion products such as polycyclic aromatic
hydrocarbons, PAHs. Apart from the particle size (one or
more dimensions of the order of 100 nm or less) which
provides a very large surface to volume ratio, their biocompatibility surface properties depend on the charges carried
by the particle and its chemical reactivity.22 Size also has
important control over other physical and chemical properties such as zeta potential and metal binding.35 Surface Area. The surface area of a nanoparticle is the total area of both external and internal surfaces available from the outside of a particle. This is an
important parameter because all interactions between NPs
and other surfaces or solutes will relate to (or is quantitatively proportional to the particle surface). The specific
surface area instead is the ratio of the surface area to the
mass of a particle. Consequently, the reactivity of the NP
will strongly increase with increasing surface area or with
decreasing particle size.20
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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Table I. Point of zero charge (PZC) for various types of engineered nanoparticles (ENPs).
Type of ENP
Al2 O3
Fe2 O3
-Fe2 O3
Fe2 O3


Size (nm)



Sigma Aldrich, St Louis, USA

Sigma Aldrich, St Louis, USA
Synthesized via flame synthesis
Synthesized by forced hydrolysis
Synthesized by forced hydrolysis
Nanostructured and Amorphous Material Inc.
Evonik, Hanau-Wolfgang, Germany
Sigma Aldrich, St Louis, USA
Synthesized via polyol process
Sakai Chemical Industry Co., Ltd., Sakai, Japan
NanoAmor, Houston, USA

95 3
200 0.5


Berg et al.65
Berg et al.65
Berg et al.65
Palomino and Stoll41
He et al.15
Loosli and Stoll48
Berg et al.65
Domingos et al.100
Berg et al.65
Brayner et al.32
Tang et al.58
Mohd Omar et al.9

J. Colloid Sci. Biotechnol. 3, 130, 2014

nanoparticle, known as the shell, has a completely different chemical structure from the core. These layers may
be prepared intentionally in the lab for research purposes.
For example, quantum dots containing a shell layer of cadmium selenide with a core made of zinc sulfide. Generally
nanomaterials containing shells do not occur through other
processes and are unlikely to form serendipitously. The
final and most essential part of the NP is the core, located
at the centre. The centre holds the composition and properties of the nanoparticle that becomes the main focus of
studies by researchers.31
The morphology of a nanoparticle and its size has a
significant influence on the physical and chemical properties which determines their interaction with the environment and biological systems.17 113 Nanoparticles can
exist in fused, aggregated or agglomerated forms with various forms of morphology including spherical, tubular or
irregular shaped.12 132 Certain nanostructures such as ZnO
could also have novel applications in optoelectronics, sensors, transducers and biomedical sciences.133 134 An example of primary particles would be ZnO in the hexagonal
wurtzite phase where its form is spherical with sharp peaks
that indicate its high crystalline nature.66
2.2. Occurrence, Fate and Transport of
Nanoparticles in Aquatic Systems
The study on the fate and transport of manufactured
nanoparticles in the environment is becoming important
due to the current discharges to the environment.93 A fuller
understanding of the nanomaterial domain requires an
evaluation of the matrix of source materials, their transformation in the natural aquatic environment, and their
physical/chemical behavior that is specific to the water
medium.135 In order to assess the risks associated with
manufactured NPs, it is necessary to first understand their
mobility, bioavailability, interactions with other materials
and toxicity.93
2.2.1. Sources and Routes of Nanoparticles
Into the Aquatic System
The expansion of nanoparticles in a diverse range of products and applications including paints, fabrics, personal

REVIEW Point of Zero Charge and Isoelectric Point. The

point of zero charge (PZC) is a parameter identified at the
pH at which the particle surface charge sums up to zero
due to the absence of both positive and negative charges.131
The determination of PZC on a given manufactured NP
can indicate if it is charged or uncharged (i.e., hydrophilic
in an aqueous suspension). The isoelectric point (IEP) is
an equally important parameter which characterizes a state
of a particle surface sorbed by an equal balance of positive and negative charges (by the disassociation of H+ and
OH ions in an embedding liquid) such that the electrical
potential inside the double electric layer is equal to zero.
Both the terms PZC and IEP clearly represent different
conditions eventhough the sum of charges on the colloidal
particles is nullified.20
The pHPZC is a very important value for adsorption measurements and surface characterization due to several reasons. First, a crucial role in the sorption of protons and
hydroxyl groups is played by the acid-base properties of
the surface. Second, the electrostatic sorption of electrolyte
ions made complicated by the chemical interaction changes
at both the PZC and IEP, however, these values change
in opposite directions. The pH value of PZC is an intensive property, which depends on the surface chemical and
physical structure rather than on the specific surface area
(SSA).125 Studies emphasizing the importance of PZC of
ENPs have been vastly conducted. For example, the PZC
for iron oxides has been generally reported to be between
pH 7.2 and pH 9.5 while it is theoretically predicted the
PZC of CeO2 is at pH 7.92.11 Table II lists the values of
point of zero charge for various types of ENPs at different
sizes. Nanoparticle Structure and Morphology. The
structure of a nanoparticle can be divided into two or
three layers comprising of (i) a surface that is often functionalized; (ii) a shell material that may be intentionally
added; and (iii) the core material. The layer of the surface is typically known to interact with a range of metal
ions, small molecules, surfactants or polymers. The charge
at the surface will determine which interaction will govern its behavior and the type of bonding it forms with
other nanoparticles or molecules. The second layer of the

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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Fig. 5. The routes of nanoparticles entering all three mediums (air, water, soil) of the environment.

health care products, electronics, biomedicine, pharmaceuticals, cosmetics have increased significantly in the past
few decades.3 35 90 The incorporation of nano ingredients into products will increase the occurrence of manufactured nanoparticles in the environment as they may
be released during the life cycle of those products.46 76 136
The important processes and pathways of nanoparticles in
the environment are presented in Figure 5. Release of NPs
may come from point sources such as production facilities, production processes, landfills or wastewater treatment plants or from nonpoint sources such as wear from
materials containing NP.11 75 93 Accidental release during
production or transport is also possible. In addition to the
unintentional release there are also NPs released intentionally into the environment. NPs that are released will
inevitably end up in groundwater aquifers. Their dependence on the transformation and transport via a variety
of pathways will eventually bring them to their ultimate
destiny, the water/sediment interface.12 77
2.2.2. Interactions of Nanoparticles in Aquatic Systems
There is still a lot to understand about the fate and behavior of nanoparticles and their interactions with each other,
natural colloids and pollutants. Manufactured nanoparticles are considered to represent a special case, since they
may be designed to have particular surface properties and
(surface) chemistry that are less likely to be found in natural particles. Initial data on a number of studies suggest
that manufactured NPs interact with other contaminants

hence influencing their toxicity.8 18 25 137 138 The ecotoxicity effect imposed by NPs can be altered by dispersion process.7 The understanding of particle chemistry in
order to correctly interpret ecotoxicological data is necessary. These include the influence of particle size, shape
and surface area and the interactions of the NPs with
other material in the water or environmental matrix.35
Other factors such as microorganisms, naturally occurring
colloids, biomacromolecules (e.g., protein and polysaccharides), sunlight, and oxidants/reductants complicate particle behavior in the natural environments. In hard water and
seawater, nanoparticles are prone to aggregate as they are
greatly influenced by the specific type of organic matter
or other natural particles (colloids) present in freshwater.
However, conclusions are normally made after taking into
account abiotic factors that may influence this, such as pH,
salinity and the presence of organic matter. In the next subsection, we described the interactions NPs may undergo as
they enter the aquatic environment, the physicochemical
characteristics that govern their behavior and the transformations, the NPs experienced that will lead/determine their
fate and transport. Because the particles are of nanosize
(<100 nm), their interaction with solid surface, bio interfaces or other particles can be quite different from larger,
microsized particles.7 ParticleParticle and Particle-Surface Interactions. When released into the aquatic environments,
nanoparticle behaviour is dependent on particle-specific
properties (e.g., size, shape, chemical composition, surface
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J. Colloid Sci. Biotechnol. 3, 130, 2014

organic macromolecules (e.g., NOM) in the environment

can significantly alter the aggregation behaviour of NPs.
Even without fully defined structure, due to its macromolecular nature, NOM is expected to prevent aggregation
and deposition presumably due to electrostatic stabilization. Natural organic matter consists of mainly fulvic and
humic substances.
Manufactured nanoparticles entering aquatic systems
will thus become components of these colloids and their
subsequent behaviour and transport will depend both on
physicochemical characteristics of the aqueous media and
interactions with other colloidal components. The stability
of colloidal suspensions is determined by the interaction
between attractive and repulsive forces, which are governed by surface charges of the colloidal material. Colloids carry an electrical charge, which produces a force
of mutual electrostatic repulsion between adjacent particles. If the charge is high enough, the colloids will remain
discrete and are stabilized in suspension. Reducing or
eliminating the charge causes the colloids to agglomerate and settle out of suspension or form interconnected
matrices.90 Studies showed that the presence of added
humic acid altered the aggregation mechanism compared
with the nanoparticle alone as this may promote or reduce
aggregation, depending on conditions.9 41 48 The information available suggests a complex interaction between natural (organic) colloids and manufactured NPs of different
types. The observed effects include aggregation, disaggregation and surface film formation and are all dependent on conditions.3 Stabilization usually results from
NOM forming a charged stabilizing layer on the outside of the particle. Destabilization, on the other hand,
results from particles being bridged by larger NOM
molecules, such as rigid biopolymers. This occurs for relatively large-sized NOM (10100 kDa) and can dominate interactions between nanoparticles. Large molecular
weight biomolecules and biomacromolecules, including
polypeptides also affect aggregation of NPs in aquatic
environments.76 Colloidal material from natural waters
has been found to be coated by films of organic material and since particle surface charges and force interactions between particles are dominated by adsorbed layers.
This has important implications in understanding mechanisms by which colloids might bind trace elements and
pollutants.151 It has been shown that adsorption of humic
acid to various metal oxide nanoparticles (TiO2 , aluminium oxide [Al2 O3 ], and zinc oxide [ZnO] can result in
a decrease in particle zeta potential, suggesting that HAcoated nano-oxides could be more easily dispersed and
suspended and more stable in solution than uncoated ones
because of their enhanced electrostatic repulsion.152 Interaction with Organisms and Pollutants.
Nanoparticles entering waterways from industrial products and wastes and its interactions with the aquatic biota
has been highlighted as a major concern in previous


charge, and coating), particle state (free or matrix incorporated), the surrounding solution chemistry (e.g., pH, ionic
strength, ionic composition, natural organic matter content), and hydrodynamic conditions.139141 Such factors are
important in determining whether particles aggregate with
other particles or deposit onto various environmental surfaces. Recognizing which interactions particles experience
under different conditions enables the prediction of their
fate in the environment.7 Interactions leading to aggregation greatly influences particle behavior under natural environment. The nanomaterial reactivity, toxicity as well as
transport potential may be significantly altered due to the
aggregation as a consequence of change in particle size
and shape.142 143 Nanoparticle transport through aquatic
environment is also expected to be dominated by random Brownian diffusion where there will be an increase
in particle size created by aggregation that may result in
gravitational sedimentation.144 Overall it is essential to elucidate which physicochemical interactions govern particlesurface and particleparticle interactions under conditions
representative of aquatic environments.
There are two conditions to be considered. The first
would be favourable (non-repulsive) particle-surface interactions where nanoparticles will be less likely to travel
extensive distances. The second would be unfavourable
(repulsive) deposition conditions. At any rate, particle
particle interactions and particle-surface interaction play
key-roles in controlling the aggregation and deposition
behaviour of nanoparticles in aquatic environments.145 146
These interactions have traditionally been described by
the DLVO theory of colloidal stability.7 35 However, nonDLVO forces such as steric, magnetic and hydration forces
can also play an important role in the aggregation and
deposition of engineered nanomaterials.147 This will be
further elaborated in Subsection 2.2.3.
Iron-based nanomaterials, exhibit a magnetic dipole
moment, even in the absence of an applied magnetic
field. The magnetic force may dominate the total particle
particle interaction energy, leading to aggregation. In other
cases, some nanoparticles may carry hydrophilic material,
functional groups, or biomolecules (e.g., proteins, polysaccharides) at their surface that can have significant amounts
of bound water that may play a role in the interaction of
such particles. The approach of two particles with hydrated
surfaces will generally be hindered by an additional repulsive interaction. The range of this interaction is significant
compared to the range of electric double layer repulsion
and is expected to have an affect on nanoparticle stability,
particularly at high strength. Interaction with Natural Colloids. Colloids are
usually defined as material with one dimension between
1 nm and 1 m and in natural aquatic systems are a complex aquatic mixture including viruses and bacteria, natural
organic matter, protein and polysaccharide exudates from
inorganic matter such as oxides of iron, manganese, aluminium and silicon.2 9 41 92 127 148150 Naturally occurring


Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

reports.22 27 Though novel properties of nanoparticles are

increasingly studied, little is known of their interations
with aquatic organisms.153 The small size of ENPs has
created opportunities for them to interact with biological entities (i.e., cells, cellular components, bacteria and
viruses).154 At the cellular level, prokaryotes like bacteria
are more likely to be protected against the intrusion of
most ENPs as they are incapable of undergoing the mechanism for the bulk transport of supramolecular and colloidal
particles throught the cell wall. In contrast, when it comes
to eukaryotes (i.e., protists and metazoans) they have a
highly developed process for the cellular internalisation of
nanoscale (<100 nm) including microscale such as endocytosis and phagocytosis with a range of 100100000 nm
particles.22 In the case of microalgae Pseudokirchneriella
subcapitata, the interaction of aggregates of TiO2 entrapping algal cells played a major role in toxicity effect.153
Other studies have proven that nanoparticles undergoing
aggregation will sediment, thus becoming less mobile and
subsequently may be ingested by organisms.7 155 156 Other
interactions could involve the absorption of dissolved ions
due to dissolution of metal oxides.30 89 157
In the case of ENPs interacting with pollutants existing
in the aquatic system, it can either amplify or alleviate the
toxicity of the compounds contained within.12 There are
many ways the pollutants can interact with ENPs. Pollutants can be adsorbed on to the surfaces of ENPs, or either
adsorbed into the ENPs, co-precipitate during formation of
a natural NP or probably trapped by aggregates containing
a mixture of ENPs and adsorbed pollutants. The sorption
of pollutants onto ENPs depends on their properties such
as composition, size, purity, structure and solution conditions such as pH and ionic strength.31 However, ENPs have
also proven to have an advantageous role in the environment by adsorbing toxic organic compounds as a treatment
method as elaborated elsewhere.31 46 158
2.2.3. Nanoparticle Stability
The classical DLVO theory of colloidal stability describes
the total interaction energy experienced by a nanoparticle
when approaching another particle (in the case of aggregation) or a collector surface (in the case of deposition).159
The stability of nanoparticle suspended in an aqueous environment can be evaluated as the sum of van der Waals and
electrical double layer interactions, likely known as the
interaction energy. The interaction energy determines the
particle stability as the two surfaces approach one another.
VdW forces result from electrical and magnetic polarizations, yielding a varying electromagnetic field within
the media and in the separation distance between the two
surfaces. The dispersion interactions evaluation is proposed to be based on the assumption that the potential
between two surfaces could be represented as the sum
of the interactions between pairs of atoms located within

Fatehah et al.

the two surfaces (particle or collector). In aqueous environments, when particles approach each other (aggregation) or a surface (deposition), the overlap of the diffuse
electric double layers results in electrostatic double layer
interactions.160162 Widely used equations are given for the
most commonly encountered interaction geometries (i.e.,
two spherical particles or a spherical particle interacting
with a planar surface).7 The stability of a nanoparticle suspension can also be influenced by non-DLVO forces.163
The most significant forces encountered by engineered
nanomaterials in aqueous media include steric interactions,
magnetic forces (for iron-based nanomaterials) and hydration forces.84 159 Steric forces have been derived with generalized expressions for particles with adsorbed layers of
polymers or surfactants that might lead to steric repulsions. Steric interactions can be particularly important for
nanoparticles in natural and engineered aquatic environments, as most particular adsorb natural organic matter
that is known to stabilize colloids.139 164
The high surface area to volume ratio of the NPs results
in high reactivity which leads to particle aggregation and
settling.127 Based on the classical Derjaguin, Landau, Verwey and Overbeek (DLVO) theory, colloidal particles are
surrounded by a diffuse electrostatic double layer (EDL)
(Fig. 4) and the balance between the van der Waals attraction forces and the electrostatic repulsion forces determines the colloidal stability.31 However, the DLVO theory
does not take into account the effects of particle shape,
surface roughness which among other factors also influence the collision efficiency. The DLVO theory is only
applicable if there is no interference with such diffusive or
attractive forces.35
Aggregation may occur as homoaggregation (particles
of the same type aggregating together), or heteroaggregation (particles attaching to other particle types present).
Aggregation of NPs reduces the surface area to volume effects on NP reactivity. This physical transformation will cause an increase of aggregate sizes which in
turn affects their transport in soil, sedimentation, reactivity, uptake by organisms and toxicity. When aggregation
occurs, the number of concentration of NPs in the suspension decreases, with a concomitant increase in their effective (aggregate) size.158 This will decrease their mobility
in the environment especially in the water compartment.39
2.2.4. Nanoparticle Dissolution
Dissolution is an important process as the nanoparticle
solubility is a significant factor in determining NP properties, toxicity and persistence.39 136 This strongly affects
the uptake pathway, toxicity mechanisms and the environmental compartment in which NPs will have the highest potential impact.25 73 166 Dissolution is one among the
many important environmental transformations that affects
the form and concentration of NPs6 28 159 and may be a
critical step for some NPs in determining their fate in the
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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

2.2.5. Nanoparticle Toxicity

Extensive usage of engineered nanomaterials in multiple
applications has brought its existence into the environment, sparking a great concern from both the scientific
community and the public. This has lead ecotoxicity studies to be majorly performed on inorganic nanoparticles
such as titanium dioxide, copper oxide, zinc oxide and silver nanoparticles as well as carbon based nanomaterials
(namely fullerenes and carbon nanotubes).18 173177
Ecotoxicity experiments have been demonstrated
on various aquatic organisms,178 inverterbrates,79 156 179
bacteria,29 180 181 and microalgae.19 30 153 Several reviews
J. Colloid Sci. Biotechnol. 3, 130, 2014

related to nanoparticle key aspects such as physicochemistry and the transformations it undergoes including ecotoxicological impacts on the environment have
been published within the past few years.27 35 82 97 It is
known that the mechanism of the ecotoxicity is still at its
infancy stage and requires further research. To this date
investigations have demonstrated that the potential toxicity of nanoparticles does not depend on one, but several
characteristics of the nanoparticle system. For example,
Berg et al.65 hypothesized the cellular viability was influenced by both zeta potential and agglomeration state. In
a study by Hsiao and Huang174 nanorod ZnO particles
were found more toxic than the spherical shaped at a
fixed nanoparticle size and surface area, suggesting that
size and shape of ZnO NPs influence their cytotoxicity.
This clearly indicates that factors such as small particle
size, large surface area, shape and the ability to generate reactive oxygen species play a major role in toxicity
of nanoparticles. In addition, based on exposure modeling
studies on ENP effects yielded from textile product and
faade coatings, the following criteria for the environment
has been established(i) indication for hazardous effects,
(ii) dissolution in water increases/decreases toxic effects,
(iii) fate during wastewater treatment, (iv) stability during
One of the prominent engineered nanoparticles, ZnO,
has drawn a huge interest among researchers and been
reported on its behaviour and potential toxicity in the
environment.9 17 24 46 Zinc oxide nanoparticles are known
to exhibit antimicrobial properties, cell selective toxicity
toward potential disease causing cells and photocatalytic
ability against chemical and biological species. High concentrations of ZnO nanoparticles in biological wastewater treatment has the capability to induce the inhibition
of nitrogen and phosphorus removal and increase reactive
oxygen species (ROS) production.73 182
The mechanism of ZnO nanotoxocity has been discussed and is basically attributed to the release of zinc ions
caused by ZnO nanoparticle dissolution.23 The dissolution
properties of ZnO has been associated with higher toxicity
of nano ZnO which is most likely related to the physicochemical properties of the nanostate with respect to the
bulk material.30 183 However, there always exists a debate
whether the observed toxicity is caused by the effects
of Zn2+ ions, the non-dissolved ZnO particles or some
combination thereof. Solubilized ZnO nanoparticles have
shown that Zn2+ ions exerts stress on cells and can cause
adverse impacts on different microorganisms2527 such as
Escherichia coli21 28 Bacillus subtilis, Streptococcusaureus
and marine algae Dunaliella tertiolecta.29 30 Table II summarizes the impact of ZnO towards various organisms and
2.3. Zinc Oxide
Zinc oxide is categorized as a metal oxide nanoparticle which has a bulk, direct band gap of 3.3 eV at


environment.160 This type of environmental transformation

occurs when an ion detaches from the particle surface and
migrates through the electrical double layer into the solution. One of the significant effects of dissolution on metalbased NPs is that it can cause the release of ionic species
that are toxic towards humans and aquatic organisms.119
When nanoparticles undergo dissolution, dispersed colloids may enhance the mobility of environmental contaminants adsorbed to the colloid surfaces.167 Metal NP
leaching in contaminated soil water has been reported to
transport in dissolved forms.119 Some metal oxides such as
ZnO dissolve easily in acids.169 Anions from weak acids
form complexes with Zn2+ ions that will accelerate the
dissolution.71 The dissolution rate increases proportionately with the H3 O+ concentration.14 Parameters such as
pH and particle size have shown to have strong influence
on the dissolution rates.6 24 26 71 72 The rate of dissolution
is considered proportional to particle surface area137 and
consequently NPs should dissolve faster than large-sized
bulk materials, for the same mass, on surface area considerations alone. Studies have proven that the smaller the
particle size, the faster the dissolution rate.24 71 82 However, denser aggregates have smaller overall surface area
and subsequently slows down the dissolution rate.119 This
impedes dissolution by reducing the average equilibrium
solubility of the particle system and by introducing kinetic
Nanoparticles may be insoluble or have low solubility in
water but are soluble in other types of solvents. Mukherjee et al.170 studied the dissolution of TiO2 in ascorbic
acid and oxalic acid with an oxide:acid molar ratio of 1:2.
Another type of NP insoluble in water is fullerene which
is only soluble in several organic solvents.95 Nano CuO
have low solubility in water with about 12% of copper
ions release.18
The solubility of nanoparticles have been measured
using various methods including centrifugal ultrafilters
combined with inductively coupled plasma mass spectrometry, or employing the method of isothermal solution saturation using temperature as a variable as well as evaluation
via atomic force microscopy by monitoring the changes in
particle morphology and dissolution.6 138 171 172

Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Table II.

Summary of ecotoxicological studies on nano ZnO on various organisms and microorganisms.

RAW 264.7 murine macrophage


Daphnia magna

Primary particle size

100 20 nm

30 nm

E. coli

<100 nm

E. coli

185 nm

E. coli, P. aeruginosa; S. aureus

1030 nm

Variety of Gram-positive and Gram

negative bacteria

12307 nm

Daphnia magna

Vibrio fischeri Daphnia magna

Thamnocephalus platyurus
B. subtilis; E. coli

<200 nm <1000 nm

5070 nm

67 nm 820 nm



Low concentration ZnO (LC-ZnO) significantly

generated ROS higher than high concentration
ZnO (HC-ZnO) and exerted cell apoptosis
yielded from the secondary size-effect, size
dependent cellular uptake and ion solubility.
Chronic test (of 21 days) was tested to determine
the accumulation of zinc. Toxicological effects
of ZnO is majorly attributed to the dissolved
fraction of free ZnO ions compared to
nanoparticles or aggregates.

Tripathy et al. (2014)184

The interaction between ZnO NPs with E. coli

yielded ROS formation and was the key
phenomenon for antibacterial effect due to
ZnO NPs.

Dutta et al. (2012)185

Low concentrations of ZnO induced decrease in

cell viability.
The antibacterial effect towards Gram-positive
bacteria was stronger compared to the
Wide range of bacterial effects on both
Gram-positive and Gram-negative in light UV
enhances ROS generation and antibacterial

Kumar et al. (2011)186

Acutes toxicity tests (48-h) resulted EC50 values

1 mg/L (within zinc toxicity range) for
Daphnia magna
ZnO in all formations (bulk ZnO, nanoZnO and
ZnSO4 ) were highly toxic due to the solubilized
Zn ions.
Under dark conditions, bacterial growth inhibition
was observed.

Wiench et al. (2009)189

room temperature with a free exciton binding energy of

60 meV.190 191 The morphology of ZnO nanostructures can
exist in many forms such as nanobelt and nanolasers,
which have spurred an intensive research interest in studies
on the ZnO nanostructure synthesis and their applications.
The recent demonstrations of ZnO nanorods, nanowires
and nanotubes have shown extensive development in electronic and electical applications.192 193 ZnO are highly
crystalline in nature and has a wurtzite crystal structure
with an average particle size of 5.2 nm.194
ZnO has unique properties which are technologically
important for semiconducting materials including optical
transparency, electric conductivity, piezoelectricity, nearUV emission,195 photocatalytic, electronic, dermatological,
photonic,17 196198 antimicrobial activity,199 direct bandgap,
large exciton binding energy, excellent chemical, mechanical, thermal stability, and biocompatibility.200203 ZnO also
possesses techno-economic attributes making it readily
available with a reasonable cost have ensured its use in an
exceedingly wide range of industries.204
Among the large variety of applications include
textile materials,196 commercial suncare products,205
lasers and light-emitting diodes, field effect transistors (FETs), field-emission devices, piezoelectric

Fatehah et al.

Adam et al. (2014)157

Premanathan et al. (2011)187

Raghupathi et al. (2011)188

Heinlaan et al. (2008)173

Adams et al. (2006)78

nanogenerators, bio-imaging agents, biosensors, in drug

delivery vehicles, in ointments including coatings and
pigments.206 207 ZnO are also widely used nanomaterials
in commercial products with applications in catalysis, dyesensitized solar cells, sensors, sunscreens, cosmetics, coatings, optics, electronic materials and medicine.28 208
Zinc oxide has emerged as one of the most extensively used nanomaterials in various fields and commercial
applications such as semiconductor in microelectronics,
catalysis, optoelectronic devices, sensors, photovoltaic,
main component varistors, field-effect transistors, blue
light-emitting diodes, ultraviolet laser diodes, solar cells,
acousto-electrical devices and detectors, tranduscers, electrical and optical devices, preparing solar cells, gas
sensors, varistors, acoustic, electrostatic dissipative coatings, luminescent devices, and could a promising component for phosphor for low-voltage luminescence in flat
panel displays. ZnO nanoparticles are used as photocatalysts to degrade water pollutants. With its antimicrobial activity, it is used as an oxide in cosmetic industry.
i.e., sunscreens, facial creams, and is an active material
for bioapplication as antimicrobial agents in cholesterol
biosensors, biomedicine, dietary modulators for hydrolases activity, cell imagine, magnetic resonance imaging.
J. Colloid Sci. Biotechnol. 3, 130, 2014

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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

It is also used in products such as paints, coatings,

ceramics.145 178 191 193 209 210
2.3.1. Amphoteric Nature of ZnO
ZnO is an amphoteric oxide and can easily dissolve in
both acids and bases.68 Like other oxides of metal, ZnO
in the presence of water undergoes hydrolysis creating a
hydroxide coating on its surface (MOH). The hydroxide surface of these hydrolysed ZnO particles may experience increase in charge from the chemical and physical
adsorption of water molecules. This happens because of
the amphoteric nature of the hydroxide surface that can
react with both H+ or OH ions (Eqs. (1) and (2)).58 62
M OH + H+ M OH2+

with protons


M OH + OH M O + H2 O
with hydroxides


ZnOs Znaq
+ O2

metal oxide dissolution (3)

ZnOs + Haq

metal oxide hydroxylation


As the pH is lowered from pH 7.7 to 6.4, followed by

pH 5.5 and 4.3, the following equations are known to be


+ OHaq


ZnOH2s ZnOH+
aq + OHaq


ZnOH2s ZnOH2aq



+ 2OH

aq + 2H H2 Oaq


According to Degen and Kosec,62 the series of the following reactions that take place in the region 7.7 < pH <
11.6 are:
+ OH ZnOH+

aq + OH


ZnOH2s ZnOH2aq




ZnOH2s + 2OH ZnOH2



ZnOH2s + OH

J. Colloid Sci. Biotechnol. 3, 130, 2014

In Figure 6, within the pH region of 6.6 < pH < 7.7,

the zinc species that are present in the suspension are
aq and Zn(OH)aq which are in equilibrium with the
surface hydroxide ZnOHs or Zn(OH)2s . At 8 < pH <
8.7, Zn(OH)+
aq dominates the suspension. At pH 9, there
is equilibrium between Zn(OH)+
aq and Zn(OH)2aq . The
Zn(OH)2aq will be the dominating species with the concurrent reduction in the Zn(OH)+
aq as it is fully converted
to Zn(OH)2aq at pH 10. When the pH values are higher
than pH 10.5, more zincate ions, Zn(OH)
3aq are formed
and as pH increases, Zn(OH)
to Zn(OH)2
2.3.2. ZnO Behavior as a Nanoparticle
In aquatic systems, the intrinsic properties of the ZnO
nanoparticles in suspension, in particular, the surface
energy and nanoparticle stabilization will determine their
fate, transport, behaviour and ecotoxicology.5 31 Abiotic
factors that affect the mobility and transport of nanoparticle are pH, ionic strength, particle surface chemistry, and
the interactions of nanoparticles with other pollutants.7
Factors that influence aggregation or disaggregation are
dissolved NOM, ionic strength, type of electrolyte, aggregation state, and pH which can all affect the stability of
nanoparticles in water. Under most environmental conditions, small quantities of humic substances can coat
other surfaces to give them an overall negative charge
depending on the NPs point of zero charge and solution pH that result in reduced aggregation through charge
stabilization.1 46 Clearly, dissolved NOM enhances the stability of Zn NPs in solution as NOM adsorption significantly increases the electric double layer repulsive energy
and produces a net energy barrier between NPs. However,


The pH increase is due to the dissolution phenomenon

that takes place at these pH values. The reactions began
immediately after immersion of ZnO in deionized water
with the hydrolysis of the ZnO particle surface that took
place as a result of the adsorption of water molecules
with the simultaneous formation of Zn(OH)2s layer. It is
also established that zinc hydroxide is soluble in water,
and becomes more soluble as pH is lowered or increased.
The predominant reactions are the dissolution of ZnOs
to Zn2+
aq and Oaq and surface hydroxylation to Zn(OH)s
which takes place based on the following Eqs. (3)(4).

Fig. 6. Speciation of Zn (II) species existing as Zn2+
aq , Zn(OH)aq ,
Zn(OH)2aq , Zn(OH)3aq and Zn(OH)2
at 25  C. Calculations were made by using the MINTEQA2 software
(Developed by Allison Geoscience Consultants Inc. and hydroGeologic
Inc.) which applies the thermodynamic and mass balance equations to
solve geochemical equilibria and calculate the ion speciation/solubility.


Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

the stabilizing effect of NOM on NP disaggregation will

not only cause the particle size to decrease and resuspend but will also enhance the NPs toxicity on aquatic
life as they become more mobile and dispersed in natural
waters.32 178
The pH has a huge influence on ZnO nanoparticles
that has lead researchers to further investigate its rheological and electrophoretic properties and dispersion in the
presence of surfactants.58 Pradhan et al.193 reported that
pH influences the ZnO structures between a pH range 5
to 10 i.e., disk, rod, spindle or flower-like. Research has
also been conducted to understand the transport properties under saturated flow conditions in the presence and
absence of stabilizing agents36 167 211213 as well as the
synthesis methods that affects the morphology of ZnO
structures.133 204 One of the earliest studies on zeta potential and pH was by Logtenberg and Stein64 who discovered
that zeta potentials are distinctly influenced by changes in
acidity and alkalinity of the suspension and the chemisorption of Cl and K+ ions. More recent scientific literature also addressed the zeta potential behaviour of ZnO
nanoparticles.58 62 6569 On the other hand there have been
a few comprehensive reports on the aggregation behaviour
of ZnO.17 26 71 Sadowski and Polowczyk66 reported that
without adjusting the pH of the suspension (pH 7.47.6),
adding cationic surfactants caused a positive increase in
zeta potential. In another similar study, specific adsorption
of carbonate ions onto the ZnO NPs caused a shift of the
pHPZC to 8.3 as its concentration increases.67 Adsorption
of anionic SDS and propylene glycol coating on ZnO NPs
was found to significantly shift the pHPZC to pH 3.26 pH
effect was studied from pH 7 to 11 by Tang et al.58 on
the zeta potential of ZnO nanoparticles with the addition
of cationic polyelectrolyte-polyethylenimine (PEI). Subsequent to that, Tang et al.69 also explored the effect of
adding anionic polyelectrolyte, ammonium polyacrylate
(PAA) on the ZnO zeta potential. Other researchers examined the dissolution behaviour of ZnO nanoparticles as
a function of pH, ionic strength and addition of natural
organic matter, its role in the acute or chronic toxicity of
aquatic organisms and the chemical etching effect.24 7173
2.4. Methods and Principles of Nanoparticle
A large number of nanoparticle parameters such as size,
size distribution, specific surface area, surface charge,
degree of agglomeration, shape and chemistry have
been used by previous researchers involving investigations such as ecotoxicological,8 32 synthesis of ZnO
nanoparticles,196 198 nanocomposites,194 215217 nanosphericals and nanobundles,134 nanofilms, nanostructures,218
environment,1 100
3 84 97
and biopolymers.113
Characterization is one of the main challenges to
quantify the concentration and changes to the physicochemical form of these nanoparticles especially fate,

Fatehah et al.

transport and toxicity studies.96 219 Several state-of-theart analytical techniques are able to determine the particle morphology (i.e., transmission electron microscopy,
TEM; scanning electron microscopy, SEM; atomic force
microscopy, AFM), particle size and particle size distribution (i.e., dynamic light scattering, DLS, nanoparticle
tracking analysis, NTA), structure (i.e., X-ray diffraction)
as well as spectrometry and chromatography techniques
(i.e., flow field flow fractionation, FIFFF; size exclusion
chromatography, SEC; fluorescence correlation spectrometry, FCS).8 123 135 220223
Identifying different types of ENPs may vary in analytical methods and techniques. In the case of characterizing engineered nanoparticles in the environment,
no single ideal technique is able to perform a complete characterization analysis to get an insight of its
true nature in the form of natural colloidal suspension. An overall look can be obtained by combining different instrumental methods.94 120 121 For example,
small angle X-ray scattering (SAXS) in combination with TEM to provide superior information with
regard to shape and size of nanoparticles in dispersions or powders.224 Techniques for the characterization of colloid structure and colloid-pollutant interactions
have been developed and applied to environmental
colloids including FIFFF-ICP-MS, FIFFF-LIBD and
Many research studies involving laboratory work
on the aggregation or dissolution of ENPs simulating
environmental aqueous systems utilizes a combination of
analytical techniques to evaluate the behaviour of the
nanomaterials. This is summarized in a table by Petosa
et al.7 enlisting various types of nanoparticles characterized with different techniques. It must be noted that the
techniques available for the detection and characterization may overlap to a great extent as most are based on
different fundamental principles.100 225 This section aims
to introduce the basic principles, descriptions of methods and some examples for the characterization of ENPs
including natural colloids. At the end of this section, the
principles, advantages and limitations of the techniques
discussed here are summarized.
2.4.1. Particle Morphology
The size and morphology of nanoparticles have a significant influence on the physical and chemical properties
which determines their interaction with the environment
and biological systems.113 Electron microscopy (EM) techniques such as TEM, SEM and AFM are among the techniques which enable visualization and characterization of
nanosized objects. The great resolving power of EM is a
result of the application of an electron beam with a wavelength well below the nm range. Optical microscopy, operating at wavelengths in the range 400800 nm gives
a resolution of 200 nm. Sample types such as emulsions, turbid and clear liquids can be freeze-dried prior
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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

TEM, with appropriate detectors and software, gives a

range of other data such as fractal dimensions, elemental
composition and chemistry bonding and redox activity.100
Figure 7 displays an example of a TEM image. Atomic Force Microscopy. Atomic force
microscopy (AFM) is among the powerful tool for determining surface topography. It is a very high-resolution
type of scanning probe microscopy that obtains 3D images
with a resolution of 10 nm.93 The high resolution of AFM
is able to discern particles of 0.1 nm and has been utilized to view directly single atoms, molecules and hard
spheres that have dimensions of a few nm (soft objects
are difficult to analyze). AFM images provide information on the surface structure of biomolecular systems
J. Colloid Sci. Biotechnol. 3, 130, 2014

Fig. 7. TEM image of zinc oxide aggregates formed at pH 7.7 (initial pH) with dark zones representing dense aggregates and lighter areas
which are aggregate branches. The ZnO individual nanoparticles reveal
both hexagonal-like and circular shapes. Samples were prepared by drying a drop of ZnO nanoparticle suspension (100 mg/L) on a copper grid at
room temperature for 3 minutes. TEM image was captured using CM12
equipped with analysis Docu Version 3.2 image analysis [Philips Electron
Optics, Eindhoven, Netherlands] [Soft Imaging System GmbH, Munster,

that is complementary to other established techniques (e.g.,

light microscopy and EM, nuclear magnetic resonance and
X-ray crystallography).120 AFM is also able to directly
observe NP aggregation behaviours.6
AFM has the advantage of imaging almost any type of
surface, including polymers, ceramics, composites, glass
and biological samples, and produces a high-resolution,
three-dimensional profile of the surface under study. AFM
measurements may not always be in accordance with other
electron microscopy techniques. For example, the same silicon nanoparticles were determined at 100 nm by SEM
and 94.2 nm by TEM. However, this was not achieved by
AFM which measured it at 50.9 nm.26
In a study by Baalousha,2 AFM was proven useful to
confirm the role of HA in the disaggregation process by
demonstrating the sorption and the formation of surface
coating of HA at the iron oxide NP aggregates. Environmental Scanning Electron Microscopy.
Environmental scanning electron microscopy (ESEM) is a
technique capable of imaging micron and submicron particles. Generally ESEM has been used to study flocs in
wastewater treatment. Redwood et al.45 applied it to image
and quantify natural organic matter (standard Suwanee
River humic acid) to observe humic aggregate structures
as a function of humidity and pH, the first study ESEM
application to natural environmental particles and colloids.
Fractal analysis of ESEM images was performed to probe
the change in aggregate structure as a function of pH.
ESEM has also served as an important complementary
technique to other analytical methods. Though ESEM cannot be used to image nonperturbal natural samples, it can
evaluate ENPs in foodstuff which does not require any
sample preparation enabling its image to be captured in
an unperturbed state.121 However, the method is ideal for


to applying electron-microscopy techniques.121 Other electron imaging techniques are atmospheric scanning electron
microscope (ASEM), WetSEM, FESEM and others.226 227 Scanning Electron Microscopy. Scanning electron microscopy (SEM) is one of the most popular electron
microscopic techniques. SEMs today achieve resolutions
of 1 nm by using high-resolution imaging instruments
whereas 3 nm by using conventional instruments.3 121
Before an SEM analysis, the nanoparticle solution is
freeze-dried to convert them into dry powder. An alternative method, is when the SEM samples are prepared by
drying several drops of 10 mg/L nanoparticle suspensions
(prepared in nanopure water) on a thin slab.84 The sample is then placed on a sample holder followed by coating
with a conductive metal (gold) using a sputter coater. The
sample is then scanned with a focused fine beam of electrons. An image of the sample scanned from the sample
surface with a low-energy beam of electrons (130 keV)
is created by detecting the electrons scattered off by the
sample. SEM also allows us to estimate the sample composition roughly with energy-dispersive X-ray spectroscopy
(EDS).93 Transmission Electron Microscopy. TEM is
used as an approach to understand the aggregation and
behaviour of ENPs, where the images can provide a lot
of information. The breadth of the NP clustering can be
calculated using the radial distribution function (RDF), a
probability of measuring a particle at radial distances from
the centre of the representative sample.228
TEM operates on a different principle as it offers greater
resolution. Four protocols are initially employed to prepare
the TEM grids:
(1) air-drying a small portion (a droplet) of suspension
directly onto a grid which had previously been covered by
an electron-transparent film:
(2) fast-freezing a droplet, then subliming away the water;
(3) fast-freezing a droplet, then sectioning it and placing
the sections on a grid;
(4) adding a droplet of sample to a tiny portion of
Nanoplast (a hydrophilic melamine embedding resin),
polymerizing the Nanoplast, then sectioning it and placing
then ultrathin sections on a grid.


Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

probing the changes in colloid structure as function of

hydration state and has the potential to perform fully quantitative and nonperturbing analysis of colloidal structure.
Poorly and electron dense material such as natural organics
do not require staining because of the amplication of signal due to the interactions of the electron beam and vapor
within the ESEM sample chamber. The resolution is similar to, but slightly reduced compared with atomic force
microscopy (AFM), conventional SEM, and transmission
electron microscopy (TEM). Ideally, resolution limits are
ca. 510 nm but will likely be lower for nonideal environmental materials. ESEM also has the unique capability
of allowing the hydration state to be altered under conditions between 0 and 100% humidity through the use of
controlled pressure and temperature conditions, allowing
the impacts of dehydration and rehydration processes on
organic structure to be probed.
Theoretically, ESEM is able to overcome the precluding
of imaging wet samples in their native state as required
in SEM.93 227 A short working distance of a few mm at
the sample stage is at a pressure of 110 Torr. Using a
water-cooled Peletier stage to choose the correct temperature (ca. 210 C), the sample can be maintained under
liquid water and the water content varied by altering pressure or temperature. There is no requirement for staining
poorly electron dense material such as humic substance.
The interactions of the secondary electrons and the water
vapor around the sample produces a cascade of ionized
gas atoms, which amplify the signal allowing even natural
organic material to be imaged.
2.4.2. Nanoparticle Stability
Assessing the stability of nanoparticles in the environment requires evaluating their ability to aggregate and
to interact with other particles.122 Electrophoretic mobility, related to the formation of the electric double layer
around the nanoparticle, is a function of the zeta potential of the nanoparticle. The zeta potential can provide
information regarding the nature of material encapsulated
within the nanocapsule or coated onto the surface.113 The
zeta potential of the nanoparticles can be determined
from their electrophoretic mobility according to Smoluchowskis equation.84 Since zeta potential is affected by
the nanoparticle surface charge, use of modifying agents
in the mobile-phase buffer can alter the rate of migration
towards the electrode.135 Laser Doppler velocymetry is the
usual technique used to measure the zeta potential. It is
based on the evaluation of the velocity of particles by the
shift caused in the interference fringe, which is produced
by the intersection of two laser beams. The electrophoretic
mobility is then transformed into zeta potential. Most natural colloidal particles have negative zeta potential values
ranging from about 100 to 5 mV. Surface charges prevent the agglomeration of nanoparticles in the dispersions
due to the strong electrostatic repulsions, thereby enhancing the stability of the nanoparticles.

Fatehah et al.

2.4.3. Nanoparticle Structure X-Ray Diffraction. X-ray diffraction (XRD) is
a primary tool for investigating the structure of crystalline
materials, from atomic arrangement to crystallite size and
imperfections.63 184 190 XRD also analyzes phase composition, crystallite size and shape, lattice distortions and
faulting, composition variations, orientation and in situ
structure development of the nanoparticles.196 229 230 The
broadening of the peaks in the XRD patterns of solids is
attributed to nanoparticle size effects. Usually, the XRD
pattern is obtained by illuminating the sample with an
X-ray source (Copper K line) with wavelength of 1.54
and scanning the diffraction within a certain range at an
angle of 2
.113 196 Fourier Transform Infrared Spectroscopy.
Another technique to supplement XRD is Fourier transform infrared spectroscopy (FTIR). FTIR is capable to provide information about the structural details of substances
in solution with greater spatial and temporal resolution.
Samples commonly characterized are usually lyophilized
nanoparticles in minute quantities. The basic principle that
governs is that the bonds and groups of bonds vibrate at
characteristic frequencies. A molecule that is exposed to
infrared rays absorbs infrared energy at frequencies which
are characteristic to that molecule.
Principally, FTIR analysis is carried out by illuminating
the sample with a modulated IR beam. The sample transmittance and reflectance of the infrared rays at different
frequencies is translated into an IR absorption plot, which
is then analyzed and matched with known signatures of
identified materials in the FTIR library.113 In another study
by Bitenc and Orel,198 FTIR was used to characterize and
determine the shapes of ZnO at wavebands in a region
from 680 up to 300 cm1 . It shows spherical particles were
obtained at 458 cm1 . On the other hand, when ZnO was
modified with polymethacrylic acid (PMAA), the carboxyl
groups, COO interacted with the surface of ZnO, causing a shift on the absorption bands of the FTIR indicating the formation of poly(zinc methacrylate) complex on
the surface of nano-ZnO. This was due to polymer chains
of PMAA creating covalent bonds onto the nanoparticle
surface.231 ATR-FTIR Spectroscopy. Attenuated total
reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is generally conducted by using a horizontal
liquid cell. The sample of NPs is prepared by drying a
suspension in methanol (1 mL) on the ATR element for
quantification. The cells are crimp sealed, covered in foil
to inhibit photoinduced chemistry. It is then mixed endover-end on a circular rotater (Cole-Palmer) for at least
two hours. Aqueous samples are then extracted so that the
dissolved concentration of the sample can be quantified
with a HPLC instrument. Wavelengths of the instrument
are adjusted accordingly to the type of samples analyzed
and the concentration is then measured.24 150
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Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

2.4.4. Separation Methods Flow Field-Flow Fractionation. Flow fieldflow fractionation (FIFFF) is chromatography-like, flowassisted hydrodynamic separation technique that permits
physical separation of small quantities (injected mass in
the ng-ug range) of macromolecules or nanoparticles.1 220
Separation is achieved by retention of particles in the FlFF
channel compared to the average flow velocity of the carrier. Retention is caused by balancing the diffusion movement of the particles by an externally generated field force,
which acts on the particles perpendicular to the carrierdriven flow through the FFF channel.123 The particles are
accumulated by being pushed towards the back wall, from
which they diffuse back into the channel because of the
concentration gradient. The more diffusive (smaller) particles migrate farther into the channel at higher flow rates
and thus, elute first. When fractograms are coupled with
absorbance detection, the diffusion coefficients (mode values) are determined from the fractograms. When coupled
with absorbance detection, the diffusion coefficients (and
their derived sizes) corresponded most closely to a mass
based diffusion coefficient, Dw (or diameter dw .100
FIFFF using an F1000 model Universal Fractionator,
Postnova Analytics Europe, Landsberg, Germany, was performed to examine the aggregation process between iron
oxide nanoparticles and standard Suwannee River Humic
Acid (SHRA) under a restricted set of conditions (pH 26)
relevant to environmental conditions.1 In another study
using the same model, natural particles from freshwater
J. Colloid Sci. Biotechnol. 3, 130, 2014

sites were determined with size ranges <4.2 nm, 4.2

15.8 nm and 15.832.4 nm as accordance to FIFFF theory
(<25 nm).74 Size Exclusion Chromatography. Size exclusion chromatography (SEC) is applicable for particles
smaller than 100 nm. A porous packing-material column is required, and unspecific adsorption can cause
unwanted interactions. So it is necessary to add additives to block active sites. SEC combined with detection techniques (e.g., voltammery, inductively coupled
plasma mass spectrometry (ICP-MS), DLS, multi-angle
laser light scattering (MALLS) can be successfully applied
to characterize nanoparticles.46 93 To determine the effectiveness of SEC for isolating NPs, the pore size of the
stationary phase must be able to reject those particles
with diameters exceeding the targeted ENPs. However, as
applied ENPs may be presented in functionalized forms,
targeting unmodified ENPs could possibly miss a significant fraction of a more generic ENP group in aquatic
systems. In the case for aquatic matrices analysis, the
application of SEC requires a pre-treatment step in order
to prevent pore blockage.135 Fluorescence Correlation Spectroscopy. Fluorescence Correlation Spectroscopy (FCS) is a technique
that is based on diffusion coefficients where samples such
as fluorescent molecules and particles need to pass through
a defined optically, laser illuminated volume. The analysis
process involves an autocorrelation curve that is derived
from measured fluorescence intensity due to temporal fluctuations. The translational diffusion of the fluorophore
can be determined from the autocorrelation curve in the
absence of chemical reactions that may affect sample fluorescence over the time scale of the measurement. A number average diffusion coefficient, Dn , is provided for a near
single molecule detection. In the case of singly labelled
particles or a weight average diffusion coefficient, Dw , is
provided for conditions of several fluorescent labels are
bound to each nanoparticle.100
In a study by Domingos et al.100 FCS measurements
were performed on a Leica TCS SP5 laser scanning microscope using an argon ion laser fluorescence excitation at
488 or 514 nm. Fluorescence emission due to adsorbed
humic substances (TiO2 , ZnO) was followed at 500
530 nm while quantum dots (QD) particle fluorescence
was measured in the range 607682 nm. Another example of an FCS analysis was conducted by Lead et al.233
to determine the diffusion coefficients of humic and fulvic acid in order to measure the effects of various environmental factors. The advantages of FCS is that it can
be used to measure diffusion coefficients of fluorescent
macromolecules under a wider range of solution conditions, including low concentration (0.5 mg/L). The diffusion times in solution were measured directly to eliminate
the possibility of interactions with a solid phase. Due to
the fact that only a fraction of humic macromolecules are


The principles of spectrum collection is based on the IR

light which is directed into the crystal element at an angle
that it is totally reflected at each point of incidence inside
the crystal. The evanescent waves, known as the reflection part of the light, pass beyond the crystal interface and
interacts with the sample, which is placed in close contact
with the crystal. The light ultimately exits the crystal and
is directed to the IR detector, from which a signal is then
directed to the data processing system and then measured
by the analytical instrument.232
Studies applying ATR-FTIR were used to compare the
adsorption of adipic and oxalic acid on the two different
sizes of anatase nanoparticles. It showed significant differences in the adsorption bands present for oxalic acid
adsorbed on both nanparticle sizes, 5 nm and 32 nm. This
clearly indicates that different adsorption sites or different
distribution of adsorption sites exists for oxalic acid.150 In
another study, the adsorption of humic acid on ZnO NPs
were investigated and revealed a spectrum of ZnO NPs
with adsorption bands at 1400 and 1586 cm1 due to carboxylate which are assigned to symmetric and asymmetric
stretching modes. In correspondence to CH2 rocking mode
and CC stretching mode, the adsorption bands were at
1333 and 1022 cm1 the surface adsorbed acetate.24

Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Fatehah et al.

Table III(a). Summary of advantages and disadvantages of microscopy techniques on nanoparticles.


Technique and parameters




Scanning electron
Particle morphology
Determine aggregation,
dispersion, size, structure
and shape of NPs.

Image magnification up to 10 nm.

Provides a better overall visual image
of the sample: detailed 3D and
topographical images, versatile
information collected from different
Easy to operate and more flexible in
variety of samples. Sample does not
need to be nearly as thin as with
Can analyze samples such as larger
wear debris particles and distressed
machine surfaces.

Preparation of samples may result in

Samples types are limited to solid,
inorganic with small size sufficient to
fit inside the vacuum chamber and
can handle moderate vacuum
Lower resolution than TEM.
Samples condition: Dry or will disturb
the efficiency of SEM measurement.
Incurs huge cost, large in size, requires
free interference of any possible
electric, magnetic or vibration.

Farr et al. (2011)93

Peters et al. (2011)120
Gontijo et al., (2004)248

Transmission electron
Determine aggregation,
dispersion, size, structure
and shape of NPs.

Powerful in assessing NP structure.

Delivers direct images and local
information on size and shape of
Greater resolution than SEM.
Magnification approximately 10
times that of an SEM (objects as
small as three to 10 for TEM.
Easy to operate.
Allows also physical characterization of
shape, size, structure, fractal
dimensions, elemental compositions,
chemistry, aggregation and internal
structure (when they are sufficient in
size and electron density).

Data accuracy depends on suitable

sample preparation rather than correct
use and choice of instrumentation.

Ju-Nam and Lead

Farr et al. (2011)93
Domingos et al. (2009)100
Peters et al. (2011)120
Weinberg et al. (2011)135
Pabisch et al. (2012)224

Atomic Force Microscopy

Determine aggregation,
dispersion, size, structure
and shape of NPs.

Obtains 3D images from the tip

movement with a resolution of
10 nm.
High resolution (0.1 nm) is able to
view directly single atoms or
molecules with few nm dimensions
and hard spheres.
Wide range of NPs characterization,
different media (i.e., ambient air,
controlled environments, liquid
conditions), any type of surface (i.e.,
polymers, ceramics, composites, glass
and biological samples).
Elemental composition of a sample can
be gained if AFM is paired with
analytical electron microscopy
Structural characterization of proteins,
polysaccharides and liposomes.
Can measure lateral distances.

Soft objects are difficult to analyze.

Measurements are only possible for
NPs that are sufficiently attached to
the substrate.
Bias could occur due to the preferential
collection of smaller material
(<50 nm).
Alteration of the AFM derived signal
due to the uptake of nanoparticles to
the AFM cantilever.

Lead and Wilkinson

Farr et al., (2011)93
Domingos et al. (2009)100
Peters et al. (2011)120
Weinberg et al. (2011)135

Environmental scanning
electron microscopy

Captures NP images in more natural

Yields best results for imaging natural
aquatic colloids.
Allows imaging samples under ambient
conditions at lower relative humidity.
Does not require any sample
preparation, enabling the specimen to
be visualized in an undisturbed state.

Sample chamber and detector cannot

achieve atmospheric pressure (max.
1050 torr).
Has difficulties analyzing smallest NPs.
Does not work at real atmospheric
pressures which is very important for
environmental studies.


Ju-Nam and Lead

Farr et al., (2011)93
Peters et al. (2011)120
Dudkiewicz et al.
Luo et al. (2013)227

J. Colloid Sci. Biotechnol. 3, 130, 2014

Fatehah et al.

Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Table III(b). Summary of advantages and disadvantages of techniques measuring nanoparticle morphology and structure.
Technique and parameters



The XRD technique has a

non-destructive nature.

It is more accurate for measuring large

crystalline structures compared to small

Fourier transform infrared

Surface particle

Offers continuous real-time

Able to provide information about
structural details of substances in
solution with greater spatial and
temporal resolution compared to
other crystallographic techniques.

Spatial resolution with a synchrotron source

using FTIR imaging in transmission is
inherently worse than ATR-FTIR.
Lower sensitivity and lower signal-to-noise
ratio compared to ATR-FTIR.
Need to apply complex mathematical
procedures i.e., Kramers-Kronig

Attenuated total
transform infrared
Adsorption studies

Requires minimal or no sample

preparation prior to spectral
Procedures: easy and time efficient
to obtain pure samples.
A powerful tool for analyzing
biomedical samples or substances
with strong infrared absorption
i.e., water. Measurements only
require small sample areas.
Good contact and corresponding
reliable FTIR images by simply
placing the sample directly on the
diamond of ATR crystal.
Spatial resolution achievable with a
synchrotron source by micro
ATR-FTIR with a Ge crystal.
Better signal-to-noise ratio and
increased sensitivity, no
significant spectral distortion.

Lower spatial resolution of about 100 m,

compared with about 20 m achievable
by transmission or reflectance methods.
Sample contact is necessary and this may
damage the condition of the samples

fluorescent, thus FCS measurements cannot fully represent

humic acid during the brief time (<1 ms) the experiments
were conducted.233
2.4.5. Dynamic Light Scattering
Dynamic light scattering (DLS), also known as photoncorrelation spectroscopy (PCS), is currently the fastest and
most popular method of determining particle size and size
distribution of ENPs in aqueous suspensions.113 120 The
major advantage of this instrument is that it can monitor the nanoparticle aggregation behavior in situ over a
wide size range (10 nm6 m). The zeta potential of
ZnO nanoparticles can be calculated concomitantly from
the electrophoretic mobility measurement according to the
Smoluchowski equation.165 In a capillary cell, the electrophoretic mobility of the sample is measured directly
when an electric field (E) is applied. The suspended
charged nanoparticles in the sample are attracted towards
the electrode of opposite charge. As these nanoparticles
move, their velocity (v) is measured and expressed as a
unit field strength (  as their mobility. The electrophoretic
mobility (e  is related to the velocity through e = v/E.
The zeta potential (  is then calculated via the Smoluchowski equation e = r 0 / , where r is the static
J. Colloid Sci. Biotechnol. 3, 130, 2014

Gontijo et al., (2004)248

Sundar et al. (2010)113

Li et al. (2007)232
Doyle (1991)249
Kazarian and Chan (2006)250
Bian et al., (2011)24
Pettibone et al. (2008)150
Li et al. (2007)232
Kazarian and Chan (2006)250


X-Ray Diffraction
Crystalline structure


dielectric constant of the medium (water), 0 is the permittivity of free space, and is the dynamic viscosity of
the medium.
Depending on the type and model of the analytical
instrument, particle sizes and size distributions are normally operated with a He-Ne laser at a wavelength of
633 nm using back scattered light.1 100 234 In DLS, the
autocorrelation of the temporal fluctuations in scattered
light intensity due to Brownian motion of particles in a
dilute suspension is evaluated in order to determine the
intensity-weighted average translational diffusion coefficient D of the particles. From the diffusion coefficient, the
average hydrodynamic radius Rh can then be calculated
using the Stokes-Einstein equation: Rh = kB T /6 D,
where kB is the Boltzmann constant, T is the absolute
temperature, and is the viscosity of the medium. This
method yields a hydrodynamic diameter that is a calculated particle diameter of a sphere that has the same measured motion in the solute as the actual particle.167
DLS has been widely used to estimate the particle size of different nanoparticle suspensions and
determine the aggregation behavior at different ionic
strengths,24 26 43 47 70 126 149 235
natural organic matter2 41 48 95 236 and in colloidal aqueous
environments.228 234 237

Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Table III(c).

Fatehah et al.

Summary of advantages and disadvantages of separation techniques on nanoparticle.

Technique and parameters




High size resolution. Does not

require a stationary phase for
ENP separation.
Wide application in ENP analysis
(soil suspensions and colloids in
freshwater and marine water
Enables further characterization of
NPs after fractionation.
Useful for separating the dissolved
phase metal from the NP metals
(i.e., inorganic NPs) with high
size resolution fractionation
technique (<100 nm).

The possibility of aggregation when sample

preconcentration is required which
contradicts that sample pre-concentration
can promote disaggregation.
Requires coupling with other
characterization techniques to quantify
ENPs in natural environment.
Combination with other analytical
instrument will only measure strong (i.e.,
non-labile) complexes due to a
continuous re-equilibration in the

Ju-Nam and Lead (2008)3

Lead and Wilkinson (2006)46
Peters et al. (2011)120
Weinberg et al. (2011)135

Size exclusion
Isolation of NPs.

Applicable for particles <100 nm.

Suitable for NP separation.
Can be a stand alone technique to
characterize nanosize particles
ranging from 320 nm.

Requires combination detection methods to

further characterize NPs.
Efficiency depends on pore size of
stationary phase to reject other particles
with bigger diameters than the targeted
Requires pre-treatment step to prevent
blockage of pores for analysis of aquatic
Unspecific adsorption can cause unwated
Irreversible adsorption of NPs to stationary
phase is still a challenge.

Farr et al. (2011)93

Peters et al. (2011)120
Weinberg et al., (2011)135
Wu et al., (2011)223

Fluorescence correlation
Diffusion coefficients
Hydrodynamic diameter

Measures diffusion coefficients of

fluorescent macromolecules under
a wide range of solution
conditions i.e., low concentration
(0.5 mg/L) and sample volumes
(<10 L).
Directly measures particle number
and signal amplication not
Analyzed by far standards of humic
substances and polysaccharides.

Single-molecule sensitivity.

Lead and Wilkinson (2006)46

Domingos et al. (2009)100


Flow field-flow fractionation

Separation and
characterization of
environmental colloids.

Multiangle laser light scattering (MALLS) is another

light scattering technique worth mentioning, which is able
to measure root mean square radius and average molecular weight of both polymers and biopolymers238 including
aggregation kinetics experiments.239
2.4.6. Nanoparticle Tracking Analysis
Nanoparticle tracking analysis (NTA) is the latest technique developed by NanoSight Ltd. to specifically characterize particles at nanoscale by providing size, count and
concentration measurements. It has a unique capability to
directly size and visualize nano-scale particles in liquid
suspension with high resolution, in real-time with minimal sample preparation for systems that are even complex and polydisperse.240 Individual particle trajectories are
tracked and the mean squared distances that are travelled
by the particles in two dimensions are recorded in order

to determine the number based on diffusion coefficients.

The technique is used in conjunction with an ultramicroscope which permits small particles in liquid suspension
to be observed moving under Brownian motion based on a
slight modification to photon-correlation spectroscopy.241
Since the scattering of small particles varies strongly with
particle radius, larger particles can still mask the signal
of smaller nanoparticles. However, the effect is less significant than in DLS. Using a specific computer software,
mean square displacements of single particles are able
to be tracked and recorded while concurrently estimating
their hydrodynamic radius, using the Stokes-Einstein equation. The mean size corresponds to the arithmetic average
of all particles sizes while the modal size corresponds to
the most frequently observed particle diameter.98 113
The NTA technique has been applied in diversified
research areas related to characterizing protein aggregations and drug delivery nanoparticles, metal oxide
J. Colloid Sci. Biotechnol. 3, 130, 2014

Fatehah et al.

Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods

Table III(d). Summary of advantages and disadvantages of light scattering techniques on nanoparticle.
Technique and parameters


Dynamic light
Particle size distributions
Hydrodynamic diameter

Analyze polydisperse samples.

Provides a more sensitve means of
detecting large aggregates.
Nanoparticles aggregation behaviour
can be monitored in situ over a
wide size range (10 nm6 m).
Accurate and simple to perform.
Frequently used for in situ NP
sizing and physical
characterization of aggregated
The signal is dominated by the
larger, more intensely scattering
particles, and the result is an
intensity weighted mean size.

Results may be influenced by the presence

of dust or agglomerated fractions in a
Capital and running costs.
Analysis turnaround time.
Limited ability to resolve particle size
distribution profiles.
Large ensemble of particles are analyzed
and only a z-average particle mean is
In some cases, relatively small number of
highly scattering larger particles (e.g.,
aggregates or contaminants) masks the
presence of the bulk of smaller particles,
leading to a limitation of intensity
weighted, z-average.

Peters et al. (2011)120

Weinberg et al. (2011)135
Pabisch et al. (2012)224
Mohd Omar et al. (2014)243
Gillespie et al. (2011)246
Carr and Malloy (2007)251

Nanoparticle Tracking Analysis

Particle size distributions
Number of particles

Requires minimal sample

Applicable for complex and
polydisperse systems.
Delivers insight of realtime, true
size distributions and state of
High resolution particle sizing of
individual particle.
Measurement is based on
concentration and particle count.
Minimum size to 10 nm, and
material dependent.
Each particle size class is given a
number versus intensity.
Unaffected from occasional
presence of an anomalous dust of
Possible to generate particle size
distribution profiles that reflect
the actual number of particles
seen on camera.
Concentration of particles present
and their size distribution can be
estimated based on the tracking
of individual particles.

Sensitive to ambient vibrations.

Sample concentrations should be within
105 1010 particles/ml for effective
Larger size particles become increasing
difficult to measure accurately.
Particles approaching or exceeding 600 nm
will restrict the Brownian motion and
hence reduce the measurement accuracy.

Malloy (2011)240
Walker (2012)241
Filipe et al. (2010)242
Mohd Omar et al. (2014)243
Gillespie et al. (2011)246
James and Driskell (2013)247
Carr and Malloy (2007)251

nanoparticle size distribution and aggregation behaviour

of catalyst nanoparticles in proton exchange membrane
fuel cells including virus particle determination.242244 The
NTA technique has also demonstrated as a new, feasible analytical instrument by furnishing additional information not offered by DLS in other studies related to
growth of particles of a poorly water soluble drug and
bio-conjugation of gold nanoparticles withal characterizing protein-protein interactions.240 245247 In respect to the
NTA-based methods that have been developed, NTA provides a more accurate determination in many novel aspects
for nanoparticle analysis.
All the techniques mentioned above are summarized in
the Tables III(a)(d) including principle mechanism, methods, limits and advantages.
J. Colloid Sci. Biotechnol. 3, 130, 2014


2.5. Challenges of Nanoparticle Characterization in

the Environment
Much is known about the diversity of engineered nanoparticles n the environment but their characterization and
detection in the natural aquatic environment are incompletely understood. We currently lack methods that
are able to directly collect or obtain data on occurrence levels, fate and transport of ENPs in aquatic
systems.46 94 120 135 227 Sequential steps need to be taken to
gain a better understanding of the nanomaterial domain.
Prior to analyzing the ENPs as it is, an evaluation of
(i) the matrix of source materials;
(ii) their transformation in the natural aquatic environment



Nanoparticle Properties, Behavior, Fate in Aquatic Systems and Characterization Methods


(iii) their physical/chemical behavior that is specific to the

water medium are required beforehand.
ENP exclusive properties are easily affected by the process of sampling, extraction and quantification.177 One
obstacle to overcome is the ability to detect and to quantify NPs in the environment, to distinguish natural from
engineered products contained in the same sample and
to differentiate between the toxicity of these materials.35
This remains as one of the most critical priorities for the
advancement of the nanomaterial industry as there is no
simple method to characterize the exact molecular structure of an unknown NP pollutant in a complex environmental matrix (e.g., water).226
Although efforts have been exhausted to analyze the
ENPs without perturbing their natural existence, for example by Tiede et al.252 who applied WetSEM on environmental samples for the first time by retaining the aqueous
samples in its liquid form, there were still limitations.
Another example of a first time application was attempted
by Jarvie and King99 who applied small-angle neutron
scattering (SANS) to quantitatively analyze the nanostructure and length scales in natural freshwater aquatic
colloidal dispersion from a number of different samples.
SANS was found to be an appropriate assessment technique of natural aquatic colloidal samples with minimal
The reasonable step to conduct analysis would be
involving multiple instruments in quantifying the nanoparticles in aqueous environmental samples accurately. Some
samples containing NPs are not initially separated from
larger particles, and depend on the analytical methods.
The requirement of individual component analysis in the
sample may need to be size fractionated which could
be destructive towards the original state of the sample.
The inability to determine the presence and/or chemical
structure of ENPs accurately severely hinders efforts to
evolve treatment technologies that use them or to investigate the impact of these materials once they are released
in an uncontrolled environment.135 For example, in a
study on the fate of Al2 O3 in a fresh water microcosm,
replicating a natural lake water ecosystem, a combination of analytical techniques was applied. X-ray diffraction analysis to analyze the crystalline properties of Al2 O3
nanoparticles, TEM analysis to evaluate the size and shape,
DLS to measure the hydrodynamic size distribution and
mean hydrodynamic diameter while aggregation of particles were tested using the DLS method.178 237 However
Majedi et al.228 and his group attested another time and
cost effective approach by employing orthogonal array
design (OAD). OAD is a multiparametric technique to
evaluate optimal conditions to investigate environmental
interactions among water chemistries (i.e., organic acid
type, organic acid concentration, NP concentration, pH,
salt content, and electrolyte type) and their effects on ZnO
aggregation behaviour.

Fatehah et al.

This review discusses the nanoparticle physicochemical
properties by adopting ZnO as an example, to obtain an
indepth sight on some possible uncertainties that lack fundamental knowledge for a deeper understanding of ENPs
environmental fate and behaviour. It is evident the driving
force that governs ENP behaviour is generally controlled
by their surface properties and formation of corona in presence of aquagenic compounds. However, there is still a
whole new domain to explore each nanoparticle type, in
various conditions with many factors to consider.
Individual analytical methods on ENPs elaborated earlier were more distinct on their principle mechanism,
advantages and limitations in respect to their physicochemical properties. It should be noted as well that the
analytical methods of evaluating ENP entities in complex environmental media particularly in aqueous systems,
which we currently are lacking, is gaining a huge interest among researchers. The consensus of reviews conclude
that due to the complexity of ENPs and their occurrence,
fate and behaviour in the environment, it is unlikely to
apply a single suitable technique in order to determine
certain physico-chemical characteristics. There are a number of analytical parameters which require equal attention in order to represent the in-situ state of the ENPs
in the aqueous environment. Among them include surface
area, surface charge, size distribution, particle morphology
and/or the mineral phase of the ENPs. Surrounding external factors that exist in aquatic systems may dictate the
interactions between the ENPs and natural water components. Though efforts have been made to analyze the ENPs
with minimal perturbance, there are still limitations which
require detailed attention for future work. For example,
to address the need for reproductible and accurate experiments, a large amount of work and effort is still needed
on the development of relevant dispersion protocols for
complex powdered samples.

AFM Atomic force microscopy
ATR-FTIR Attenuated total reflectance Fourier transform infrared
ASEM Atmospheric scanning electron microscopy
DLS Dynamic light scattering
DLVO Derjaguin-Landau-Verwey-Overbeek
EDL Electric double layer
EM Electron microscopy
ENP Engineered nanoparticle
ESEM Environmental
FCS Fluorescence correlation spectroscopy
FET Field effect transistors
FIFFF Flow field-flow fractionation
FTIR Fourier transform infrared red
HA Humic acid
J. Colloid Sci. Biotechnol. 3, 130, 2014

Fatehah et al.

Humic substance
Isoelectric point
Multi-angle laser light scattering
Natural organic matter
Nanoparticle tracking analysis
Orthogonal array design
Polycyclic aromatic hydrocarbons
Photo-correlation spectroscopy
Proton exchange membrane fuel cell
Point of zero charge
Radial distribution function
Surface charge
Size-exclusion chromatography
Scanning electron microscopy
Suwannee River Humic Acid
Specific surface area
Transmission electron microscopy
Van der Waals
Wet scanning electron microscopy
X-ray diffraction.

Acknowledgments: The authors gratefully acknowledges Arnaud Clavier for his assistance and contribution to the content of this publication. The authors also
acknowledge the research support received from the International Foundation of Science (Sweden) in part of the
IFS programme Individual Research Approach (Grant no.
W/5334) for the School of Civil Engineering, Universiti
Sains Malaysia. This work is additionally supported by the
Swiss National Foundation (200021_135240).

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Received: 25 June 2014. Accepted: 24 September 2014.


J. Colloid Sci. Biotechnol. 3, 130, 2014